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Sample records for cocktail enzyme-linked immunosorbent

  1. Development of a Novel Cocktail Enzyme-Linked Immunosorbent Assay and a Field-Applicable Lateral-Flow Rapid Test for Diagnosis of Contagious Bovine Pleuropneumonia.

    PubMed

    Heller, Martin; Gicheru, Nimmo; Tjipura-Zaire, Georgina; Muriuki, Cecilia; Yu, Mingyan; Botelho, Ana; Naessens, Jan; Jores, Joerg; Liljander, Anne

    2016-06-01

    Contagious bovine pleuropneumonia (CBPP) is a severe respiratory disease that is widespread in sub-Saharan Africa. It is caused by Mycoplasma mycoides subsp. mycoides, a bacterium belonging to the Mycoplasma mycoides cluster. In the absence of an efficient CBPP vaccine, improved and easy-to-use diagnostic assays for recurrent testing combined with isolation and treatment of positive animals represent an option for CBPP control in Africa. Here we describe the comprehensive screening of 17 immunogenic Mycoplasma mycoides subsp. mycoides proteins using well-characterized bovine sera for the development of a novel cocktail enzyme-linked immunosorbent assay (ELISA) for laboratory use. Two recombinant Mycoplasma immunogens, MSC_0136 and MSC_0636, were used to set up a standardized cocktail ELISA protocol. According to the results from more than 100 serum samples tested, the sensitivity and specificity of the novel cocktail ELISA were 85.6% and 96.4%, respectively, with an overall diagnostic accuracy comparable to that of the Office International des Epizooties (OIE)-prescribed serological assays. In addition, we provide a proof of principle for a field-applicable, easy-to-use commercially produced prototype lateral-flow test for rapid (<30-min) diagnosis of CBPP. PMID:27053669

  2. Proficiency monitoring of monoclonal antibody cocktail-based enzyme-linked immunosorbent assay for detection of allergen-specific immunoglobulin E in dogs.

    PubMed

    Lee, Kenneth W; Blankenship, Karen; McKinney, Brennan; Kern, Gerhard; Buch, Jesse; Greenwood, Janice; Brazis, Pilar; Drouet, Laurent; Tambone, Cecilia; Faas, Rebecca; Weaver, Gareth

    2015-07-01

    The purpose of our study was to document the continued comparative proficiency of different laboratories that perform a monoclonal antibody-based enzyme-linked immunosorbent assay (macELISA) for detection of allergen-specific immunoglobulin (Ig)E in dogs. Replicate samples of 18 different sera pools were independently evaluated in a single blinded fashion by each of 16 different operators functioning in 10 different laboratories. The average intra-assay variance among reactive assay calibrators in all laboratories was 6.0% (range: 2.7-16.1%), while the average intralaboratory interassay variance was 7.5% (range: 3.9-10.9%). The overall interassay interlaboratory variance was consistent among laboratories and averaged 11.4% (range: 8.5-12.5%). All laboratories yielded similar profiles and magnitudes of responses for replicate unknown samples; dose response profiles observed in each of the laboratories were indistinguishable. Considering the positive or negative results, interassay interlaboratory concordance of results exceeded 90%. Correlation of optical density values between and among all laboratories was strong (r > 0.9, P < 0.001). Collectively, the results demonstrated that the macELISA for measuring allergen-specific canine IgE is reproducible, and documents that consistency of results can be achieved not only in an individual laboratory by differing operators but also among laboratories using the same monoclonal-based ELISA. PMID:26069227

  3. Clickable enzyme-linked immunosorbent assay.

    PubMed

    Canalle, Luiz A; Vong, TuHa; Adams, P Hans H M; van Delft, Floris L; Raats, Jos M H; Chirivi, Renato G S; van Hest, Jan C M

    2011-10-10

    Click chemistry is explored as a potential cost-effective and selective immobilization method for the production of an enzyme-linked immunosorbent assay (ELISA). Coatings were formulated containing either a terminal alkyne or a bicyclo[6.1.0]non-4-yne (BCN) chemical handle, and a diagnostic peptide was subsequently immobilized onto these coatings by the copper-catalyzed azide-alkyne 1,3-dipolar cycloaddition (CuAAC) or copper-free strain-promoted azide-alkyne 1,3-dipolar cycloaddition (SPAAC), respectively. The terminal alkyne-containing coating showed high background levels in subsequent ELISA's due to the copper catalyst used in the immobilization step. The BCN-containing coating, however, was successfully employed and presents a cost-effective alternative to existing (strept)avidin-biotin immobilization methods. This technology was illustrated with an ELISA used for the diagnosis of rheumatoid arthritis (RA) but could be easily applied to a wide range of diagnostic tests. PMID:21866934

  4. Enzyme-linked immunosorbent assay for cercospora beticola in soil

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Soil mixed with naturally infected sugar beet residues during tillage after harvest can serve as inoculum for the leaf spot fungal pathogen Cercospora beticola when a new sugar beet crop is planted. An enzyme-linked immunosorbent assay (ELISA) was developed in an attempt to quantify the inoculum in ...

  5. Microwave-mediated enzyme-linked immunosorbent assay procedure.

    PubMed

    Nahar, Pradip; Bora, Utpal; Sharma, Gainda L; Kannoujia, Dileep Kumar

    2012-02-15

    Here we demonstrate a novel microwave-mediated enzyme-linked immunosorbent assay (MELISA) method that has dramatically reduced the enzyme-linked immunosorbent assay (ELISA) timing to less than 5 min with a result comparable to that obtained by 18-h conventional ELISA. Efficacy of the MELISA procedure is demonstrated by detecting human immunoglobulin G (IgG), rabbit IgG, human immunoglobulin E (IgE), human interleuken 1β (IL-1β), Entamoeba histolytica antibody, and Aspergillus fumigatus antibody. MELISA could be an excellent substitute for time-consuming conventional ELISA for rapid diagnosis of diseases in cases of medical urgency, outbreak of infectious diseases, and screening of samples in blood banks or emigration counters. PMID:22033289

  6. Smartphone instrument for portable enzyme-linked immunosorbent assays

    PubMed Central

    Long, Kenneth D.; Yu, Hojeong; Cunningham, Brian T.

    2014-01-01

    We demonstrate the utilization of a smartphone camera as a spectrometer that is capable of measuring Enzyme Linked Immunosorbent Assays (ELISA) at biologically-relevant concentrations with the aid of a custom cradle that aligns a diffraction grating and a collimating lens between a light source and the imaging sensor. Two example biomarkers are assayed using conventional ELISA protocols: IL-6, a protein used diagnostically for several types of cancer, and Ara h 1, one of the principle peanut allergens. In addition to the demonstration of limits of detection at medically-relevant concentrations, a screening of various cookies was completed to measure levels of peanut cross-contamination in local bakeries. The results demonstrate the utility of the instrument for quantitatively performing broad classes of homogeneous colorimetric assays, in which the endpoint readout is the color change of a liquid sample. PMID:25426311

  7. Smartphone instrument for portable enzyme-linked immunosorbent assays.

    PubMed

    Long, Kenneth D; Yu, Hojeong; Cunningham, Brian T

    2014-11-01

    We demonstrate the utilization of a smartphone camera as a spectrometer that is capable of measuring Enzyme Linked Immunosorbent Assays (ELISA) at biologically-relevant concentrations with the aid of a custom cradle that aligns a diffraction grating and a collimating lens between a light source and the imaging sensor. Two example biomarkers are assayed using conventional ELISA protocols: IL-6, a protein used diagnostically for several types of cancer, and Ara h 1, one of the principle peanut allergens. In addition to the demonstration of limits of detection at medically-relevant concentrations, a screening of various cookies was completed to measure levels of peanut cross-contamination in local bakeries. The results demonstrate the utility of the instrument for quantitatively performing broad classes of homogeneous colorimetric assays, in which the endpoint readout is the color change of a liquid sample. PMID:25426311

  8. Enzyme-linked immunosorbent assay for triclocarban in aquatic environments.

    PubMed

    Zeng, Kun; Zou, Yanmin; Liu, Jianxia; Wei, Wei; Zhang, Meng; Zhou, Jun; Zhang, Zhen; Gai, Zikuan

    2015-01-01

    A sensitive, competitive indirect enzyme-linked immunosorbent assay (ELISA) was developed for the detection of triclocarban (TCC) in waters and sediments. Haptens were synthesized by derivatizing the paraposition of a phenyl moiety of TCC. The synthesized hapten was then coupled to bovine thyroglobulin to be used as an immunogen, based on which, a high affinity monoclonal antibody 4D5 was produced with the hybridoma technique. Under the optimized conditions, using the monoclonal antibody, excellent performances of the assay were obtained: satisfactory sensitivity (IC50 (50% inhibition concentration) value, 0.43 ng/mL; limit of detection, 0.05 ng/mL); good linear range (0.05-10 ng/mL); and satisfactory accuracy (recoveries 70.7-107% in waters; 74.8-98.3% in sediments). Furthermore, TCC was found with the concentration ranging from not detected to 422.12 ng/L in waters and from 6.68 ng/g to 78.67 ng/g in sediments in Yunliang River, Ancient Canal and Hongqiao Port in Zhenjiang City. In conclusion ELISA could be applied for monitoring TCC in aquatic environments. PMID:26540528

  9. Simple standardized enzyme-linked immunosorbent assay for human antibodies to Entamoeba histolytica.

    PubMed Central

    Lin, T M; Halbert, S P; Chiu, C T; Zarco, R

    1981-01-01

    A simple solid-phase enzyme-linked immunosorbent assay procedure for the detection of human antibodies to Entamoeba histolytica was developed which showed a high degree of correlation with the agar gel diffusion, counterelectrophoresis, and indirect hemagglutination methods, as well as with clinical data. The enzyme-linked immunosorbent assay is rapid (1 h 15 min, total incubation time), and the reported values are referenced to a positive control so that they correlate with levels of antibody sufficient to be detected by the gel diffusion methods. The enzyme-linked immunosorbent assay is highly reproducible, specific, and sensitive; it can be used qualitatively or quantitatively. PMID:6262370

  10. Construction of a Simple, Inexpensive Multiple Enzyme-Linked Immunosorbent Assay Microdilution Plate Washer

    PubMed Central

    Stobbs, L. W.

    1990-01-01

    In this paper, plans are given for the construction of an inexpensive enzyme-linked immunosorbent assay plate washer from readily available materials. The wash unit uses an intermittent wash cycle based on a wash manifold cycling over the microdilution plates for a predetermined time. Laboratory tests showed that the unit provided reliable, rapid washing of plates with tap water, with no detectable contamination between wells. Substrate absorbance values for test samples from machine-washed plates were equal to or greater than absorbance values for corresponding samples from plates washed manually by an accepted protocol, by using either enzyme-linked immunosorbent assay wash buffer or tap water. Images PMID:16348216

  11. Detection of sugarcane yellow leaf virus by direct antigen coated enzyme-linked immunosorbent assay

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The L9 (34) orthogonal diagram was applied to optimize detection conditions of direct antigen coated enzyme-linked immunosorbent assay (DAC-ELISA) for Sugarcane yellow leaf virus (SCYLV) in sugarcane. Statistic analyses indicated that 150 µL of SCYLV in juice and leaf crude extract antigens was the ...

  12. Detection by Enzyme-Linked Immunosorbent Assay of Antibodies to West Nile virus in Birds

    PubMed Central

    Dupuis, Alan P.; Nicholas, David; Young, Donna; Maffei, Joseph; Kramer, Laura D.

    2002-01-01

    We adapted an indirect immunoglobulin G enzyme-linked immunosorbent assay to facilitate studies of West Nile virus (WNV) and evaluated its application to taxonomically diverse avian species. Anti-WNV antibodies were detected in 23 bird species, including many exotic species, demonstrating its value in studies of WNV epizootiology. PMID:12194778

  13. DEVELOPMENT OF ENZYME-LINKED IMMUNOSORBENT ASSAYS FOR RESIDUE ANALYSIS OF DIFLUBENZURON AND BAY SIR 8514

    EPA Science Inventory

    Three enzyme-linked immunosorbent assays (ELISA's) were developed for the benzoylphenylurea insect growth regulators (IGRs) diflubenzuron, BAY SIR 8514, and some of their analogues. All three ELISA's were based on antibodies raised against an N-carboxypropyl hapten of diflubenzur...

  14. Development of an Enzyme Linked Immunosorbent Assay to Detect Chicken Parvovirus Specific Antibodies

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Here we report the development and application of an enzyme linked immunosorbent assay to detect parvovirus-specific antibodies in chicken sera. We used an approach previously described for other parvoviruses to clone and express viral structural proteins in insect cells from recombinant baculovirus...

  15. AN ENZYME LINKED IMMUNOSORBENT ASSAY FOR THE HO-1 ISOFORM OF HEME OXYGENASE

    EPA Science Inventory

    AN ENZYME LINKED IMMUNOSORBENT ASSAY FOR THE HO-1 ISOFORM OF HEME OXYGENASE

    Heme oxygenase (HO) occurs in biological tissues as two major isoforms HO-1 and HO-2. HO-1 is inducible by many treatments, particularly oxidative stress-related conditions such as depletion of gl...

  16. Biological Monitoring of 3-Phenoxybenzoic Acid in Urine by an Enzyme -Linked Immunosorbent Assay

    EPA Science Inventory

    An enzyme-linked immunosorbent assay (ELISA) method was employed for determination of the pyrethroid biomarker, 3-phenoxybenzoic acid (3-PBA) in human urine samples. The optimized coating antigen concentration was 0.5 ng/mL with a dilution of 1:4000 for the 3-PBA antibody and 1:6...

  17. A Direct, Competitive Enzyme-Linked Immunosorbent Assay (ELISA) as a Quantitative Technique for Small Molecules

    ERIC Educational Resources Information Center

    Powers, Jennifer L.; Rippe, Karen Duda; Imarhia, Kelly; Swift, Aileen; Scholten, Melanie; Islam, Naina

    2012-01-01

    ELISA (enzyme-linked immunosorbent assay) is a widely used technique with applications in disease diagnosis, detection of contaminated foods, and screening for drugs of abuse or environmental contaminants. However, published protocols with a focus on quantitative detection of small molecules designed for teaching laboratories are limited. A…

  18. An enzyme-linked immunosorbent assay for determination of dicyclanil in animal tissue

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Dicyclanil is a pyrimidine-derived insect growth regulator used in veterinary medicine for the prevention of myiasis or fly-strike. It is toxic to animals and humans. In this paper, for the first time, a competitive indirect enzyme-linked immunosorbent assay was developed for the determination of ...

  19. USE OF ENZYME-LINKED IMMUNOSORBENT ASSAYS (ELISA) FOR THE DETERMINATION OF TRITON X NONIONIC DETERGENTS

    EPA Science Inventory

    An enzyme-linked immunosorbent assay (ELISA) for 4-t-octylphenyl ethoxylates such as Triton X-100 was developed. Both the 4-t-octylphenyl and the ethoxylate moiety were required for antibody recognition since members of the Triton N series showed low cross-reactivity, and polyeth...

  20. Enzyme-linked immunosorbent assay detection and bioactivity of Cry1Ab protein fragments

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Enzyme-linked immunosorbent assay (ELISA) has emerged as the preferred detection method for Cry proteins in environmental matrices. Concerns exist that ELISAs are capable of detecting fragments of Cry proteins, which may lead to an over-estimation of the concentration of these proteins in the enviro...

  1. Quantification of Dehalospirillum multivorans in Mixed-Culture Biofilms with an Enzyme-Linked Immunosorbent Assay

    PubMed Central

    Bauer-Kreisel, P.; Eisenbeis, M.; Scholz-Muramatsu, H.

    1996-01-01

    A fast, highly selective and sensitive method to quantify specific biomasses in mixed-culture biofilms is described. It consists of detachment of a biofilm from its support material, resolution of the detached biofilm flocs in order to separate the enclosed cells and antigens, and quantification of specific biomass by an enzyme-linked immunosorbent assay. PMID:16535389

  2. Quantification of Dehalospirillum multivorans in Mixed-Culture Biofilms with an Enzyme-Linked Immunosorbent Assay.

    PubMed

    Bauer-Kreisel, P; Eisenbeis, M; Scholz-Muramatsu, H

    1996-08-01

    A fast, highly selective and sensitive method to quantify specific biomasses in mixed-culture biofilms is described. It consists of detachment of a biofilm from its support material, resolution of the detached biofilm flocs in order to separate the enclosed cells and antigens, and quantification of specific biomass by an enzyme-linked immunosorbent assay. PMID:16535389

  3. Evaluation of solubilized herpes simplex virus membrane antigen by enzyme-linked immunosorbent assay.

    PubMed Central

    Jeansson, S; Forsgren, M; Svennerholm, B

    1983-01-01

    An antigen prepared by solubilization of membranes from herpes simplex virus (HSV)-infected cells with deoxycholate was evaluated by enzyme-linked immunosorbent assay. The deoxycholate-solubilized antigen, previously shown to contain all major HSV glycoproteins, was noninfectious and adsorbed easily and reproducibly to a polystyrene surface at pH 9.6. The deoxycholate-solubilized antigen provided an enzyme-linked immunosorbent assay of high sensitivity and reproducibility with complete correlation with complement fixation for the diagnosis of acute HSV infection. The correlation with neutralization and immunofluorescence for the presence or absence of anti-HSV activity was very good. Comparison with an HSV envelope preparation yielded results slightly in favor of the deoxycholate-solubilized antigen. The assay seems to be useful for demonstration of intrathecal production of antibody activity in HSV encephalitis. PMID:6315767

  4. Glycoprotein-based enzyme-linked immunosorbent assays for serodiagnosis of infectious laryngotracheitis.

    PubMed

    Kanabagatte Basavarajappa, Mallikarjuna; Song, Haichen; Lamichhane, Chinta; Samal, Siba K

    2015-05-01

    For detection of infectious laryngotracheitis virus (ILTV) antibody, glycoprotein B-, C-, and D-based enzyme-linked immunosorbent assays (B-, C-, and D-ELISAs, respectively) were developed. The B- and D-ELISAs showed enhanced detection of anti-ILTV antibodies in infected chickens compared to that of the commercial ELISA. Furthermore, the D-ELISA was efficient in detecting seroconversion with vectored vaccine, using recombinant Newcastle disease virus (rNDV) expressing glycoprotein D (gD) as the vaccine vector. PMID:25694519

  5. Glycoprotein-Based Enzyme-Linked Immunosorbent Assays for Serodiagnosis of Infectious Laryngotracheitis

    PubMed Central

    Kanabagatte Basavarajappa, Mallikarjuna; Song, Haichen; Lamichhane, Chinta

    2015-01-01

    For detection of infectious laryngotracheitis virus (ILTV) antibody, glycoprotein B-, C-, and D-based enzyme-linked immunosorbent assays (B-, C-, and D-ELISAs, respectively) were developed. The B- and D-ELISAs showed enhanced detection of anti-ILTV antibodies in infected chickens compared to that of the commercial ELISA. Furthermore, the D-ELISA was efficient in detecting seroconversion with vectored vaccine, using recombinant Newcastle disease virus (rNDV) expressing glycoprotein D (gD) as the vaccine vector. PMID:25694519

  6. Development of heterologous enzyme linked immunosorbent assay for dehydroepiandrosterone in serum.

    PubMed

    Shrivastav, Tulsidas G; Chaube, Shail K; Kariya, Kiran P; Bhanot, Sana; Singh, Rita; Kumar, Dinesh

    2010-01-01

    Homologous and heterologous combinations of enzyme conjugate with immunogen in steroid enzyme immunoassay (EIA) influence labeled steroid recognition by an antibody that affects the sensitivity of the assay. To develop dehydroepiandrosterone (DHEA) enzyme linked immunosorbent assay (ELISA), antibodies were generated against dehydroepiandrosterone-17-carboxymethyloxime-bovine serum albumin (DHEA-17-CMO-BSA) and dehydroepiandrosterone-7-carboxymethyloxime- bovine serum albumin (DHEA-7-CMO-BSA). Two dehydroepiandrosterone (DHEA) horse radish peroxidise (HRP) enzyme conjugates were prepared using two dehydroepiandrosterone derivatives (DHEA-17-CMO and DHEA-7-CMO). Four combinations of homologous and heterologous assays were evaluated. The use of heterologous combination improved the sensitivity of the assay. PMID:21113840

  7. Porcine respiratory coronavirus in Quebec: Serological studies using a competitive inhibition enzyme-linked immunosorbent assay

    PubMed Central

    Jabrane, Ahmed; Elazhary, Youssef; Talbot, Brian G.; Ethier, Raymond; Dubuc, Claude; Assaf, Robert

    1992-01-01

    Porcine respiratory coronavirus (PRCV) was identified for the first time in Quebec, using a blocking enzyme-linked immunosorbent assay (ELISA). Unlike the virus neutralization test (VNT), this ELISA was able to distinguish transmissible gastroenteritis virus (TGEV) from PRCV. Among the 15 seropositive fattening herds from group A, sera containing PRCV antibodies represented 74.8%, whereas those with TGEV antibodies represented only 7.2%. In group B, which consisted of 15 sow herds, nine herds expressed only PRCV-specific antibodies while the other herds had animals positive for TGEV-specific antibodies. PMID:17424115

  8. Cross-Reactivity of Fusarium spp. in the Aspergillus Galactomannan Enzyme-Linked Immunosorbent Assay

    PubMed Central

    Esposto, Maria Carmela; Prigitano, Anna; Grancini, Anna; Ossi, Cristina; Cavanna, Caterina; Cascio, Giuliana Lo

    2012-01-01

    Nine of 11 hematological patients with disseminated/deep-seated Fusarium infection tested at least twice for Aspergillus galactomannan (GM) had repeated positive results in the absence of Aspergillus isolation in culture. The centrifuged supernatants of 12 Fusarium isolates were tested by a GM enzyme-linked immunosorbent assay (EIA). All the isolates produced positive reactions when tested undiluted. These results show cross-reactivity of Fusarium spp. with Aspergillus GM that may constitute a drawback with respect to the specificity of the Platelia EIA. PMID:22205818

  9. Efficacy of enzyme-linked immunosorbent assay for rapid diagnosis of Bordetella pertussis infection.

    PubMed Central

    Lawrence, A J; Paton, J C

    1987-01-01

    We examined the diagnostic efficacy of an enzyme-linked immunosorbent assay (ELISA) for class-specific antibodies to Bordetella pertussis in acute-phase sera collected from 1,240 patients with suspected pertussis. A total of 833 serum specimens (67%) yielded positive results. The proportion of positive results increased to 77% if a second (convalescent-phase) serum was also tested. By comparison, a bacterial agglutination test for B. pertussis antibodies was positive in only 21% of acute-phase specimens and 50% of paired specimens. The high proportion of acute-phase sera which were ELISA positive indicates that a measurable serologic response has usually occurred by the time the diagnosis is suspected. Thus, the ELISA is potentially the most rapid means of laboratory confirmation of B. pertussis infection. PMID:2891724

  10. Sandwich enzyme-linked immunosorbent assay for detection of excretory secretory antigens in humans with fascioliasis.

    PubMed Central

    Espino, A M; Finlay, C M

    1994-01-01

    A sandwich enzyme-linked immunosorbent assay has been developed for the detection of Fasciola hepatica excretory secretory (ES) antigens in stool specimens of infected humans. The assay uses antibodies against F. hepatica ES antigens. A monoclonal antibody (ES78, mouse immunoglobulin G2a) was used to capture ES antigens, and a rabbit polyclonal antibody, peroxidase conjugate, was used to identify ES antigens. Thirteen of 14 patients with parasitological evidence of fascioliasis had a detectable concentration of ES antigens (more than 15 ng/ml). None of the stool specimens from controls and from patients with parasites other than F. hepatica showed a positive reaction, suggesting the absence of cross-reactions in this assay. When the 14 patients were retested 2 months after treatment, all of the specimens from the 11 parasitologically cured patients were negative by the antigen detection assay while the specimens from the 3 patients with persisting F. hepatica eggs in their stools remained positive. PMID:8126178

  11. An enzyme-linked immunosorbent assay for bromodeoxyuridine incorporation using fixed microcultures

    SciTech Connect

    Muir, D.; Varon, S.; Manthorpe, M. )

    1990-03-01

    We report a quantitative method by which a single microculture can be examined for cell morphology; cell number; DNA synthesis; and expression of cell antigens. This method first involves measuring by enzyme-linked immunosorbent assay (ELISA) the total bromodeoxyuridine (BrdU) incorporation into DNA by monolayer microcultures. The BrdU-ELISA measurement was followed by simultaneous immunostaining for BrdU-positive nuclei and for a cytoplasmic antigen. The method was applied to the measurement of mitogen-induced proliferation of rat sciatic nerve Schwann cell and cerebral astroglia microcultures. The ELISA measurement of BrdU incorporation compares favorably with measurements of tritiated thymidine incorporation and offers the additional advantages that the same microculture can subsequently be examined for cell number, for cell morphology, and for the percentage of cells having BrdU-labeled nuclei and other antigens.

  12. A First Application of Enzyme-Linked Immunosorbent Assay for Screening Cyclodiene Insecticides in Ground Water

    USGS Publications Warehouse

    Dombrowski, T.R.; Thurman, E.M.; Mohrman, G.B.

    1996-01-01

    A commercially available enzyme-linked immunosorbent assay (ELISA) plate kit for screening of cyclodiene insecticides (aldrin, chlordane, dieldrin, endosulfan, endrin, and heptachlor) was evaluated for sensitivity, cross reactivity, and overall performance using groundwater samples from a contaminated site. Ground-water contaminants included several pesticide compounds and their manufacturing byproducts, as well as many other organic and inorganic compounds. Cross-reactivity studies were carried out for the cyclodiene compounds, and results were compared to those listed by the manufacturer. Data obtained were used to evaluate the sensitivity of the ELISA kit to the cyclodiene compounds in ground water samples with a contaminated matrix. The method quantitation limit for the ELISA kit was 15 ??g/L (as chlordane). Of the 56 ground-water samples analyzed using the ELISA plate kits, more than 85% showed cyclodiene insecticide contamination. The ELISA kit showed excellent potential as a screening tool for sites with suspected groundwater contamination by insecticides.

  13. Use of an enzyme-linked immunosorbent assay to measure antigenaemia during acute plague*

    PubMed Central

    Williams, James E.; Gentry, Mary K.; Braden, Carol A.; Leister, Flora; Yolken, Robert H.

    1984-01-01

    An enzyme-linked immunosorbent assay (ELISA) was developed to measure concentrations of the specific F1 antigen of the plague bacillus in biological fluids. The assay employed a monoclonal antibody to capture the antigen. Sensitivity of the assay was 0.4 ng of F1 antigen. ELISA-inhibition was used to confirm the specificity of the reactions. This assay detected F1 antigen in two of ten sera from patients with acute bubonic plague and indicated that antigenaemia in man during plague may reach levels of 4-8 μg of F1 antigen per ml of serum. The probability for a correct serodiagnosis of plague was improved when the patients' sera were tested for both antibody and antigen. Two patients with antigenaemia did not have antibody, while two patients with antibody lacked antigenaemia. PMID:6380787

  14. Determination of PCBs in fish using enzyme-linked immunosorbent assay (ELISA)

    USGS Publications Warehouse

    Lasrado, J.A.; Santerre, C.R.; Zajicek, J.L.; Stahl, J.R.; Tillitt, D.E.; Deardorff, D.

    2003-01-01

    Polychlorinated biphenyls (PCBs) were determined in fish tissue using an enzyme-linked immunosorbent assay (ELISA). Standard curves for Aroclor 1248, 1254, and 1260 in catfish tissue were developed with ranges from 0.05 to 0.5 ppm and 0.5 to 5.0 ppm. Wild fish were initially analyzed using gas chromatography/electron-capture detection (GC/ECD) and those having residues within the standard curve ranges were analyzed with ELISA. Results obtained using ELISA and GC/ECD were not significantly different (p < 0.05) from 0.05 to 0.5 ppm. From 0.5 to 5.0 ppm, the standard curve for Aroclor 1254 was the best predictor of total PCB in wild fish samples.

  15. An enzyme-linked immunosorbant assay using polyclonal antibodies against bacopaside I.

    PubMed

    Phrompittayarat, Watoo; Putalun, Waraporn; Tanaka, Hiroyuki; Wittaya-Areekul, Sakchai; Jetiyanon, Kanchalee; Ingkaninan, Kornkanok

    2007-02-12

    Bacopa monnieri (L.) Wettst. (Brahmi) is a medicinal plant used as a memory enhancer in Ayurvedic medicines. Its active components are triterpenoid glycosides namely pseudojujubogenin and jujubogenin glycosides. In order to analyze these saponin glycosides, an enzyme-linked immunosorbant assay (ELISA) was developed using polyclonal antibodies against bacopaside I, one of the pseudojujubogenin glycosides found in the plant. Bacopaside I was conjugated with a bovine albumin serum (BSA) to prepare an immunogen. The bacopaside I-BSA conjugate was immunized to a rabbit for producing polyclonal antibodies (PAbs). The results showed that the antibodies were raised specifically against pseudojujubogenin glycosides. An ELISA using anti-bacopaside I PAbs was performed in the range of 1.95-62.5 ng mL(-1) of bacopaside I and the limit of detection was 0.1 ng mL(-1). The method was validated and the applicability of the ELISA for analyzing saponin glycosides from Brahmi was demonstrated. PMID:17386577

  16. Magnetic enzyme-linked immunosorbent assay (MELISA) for determination of specific IgG in paracoccidioidomycosis.

    PubMed

    de Camargo, Z P; Guesdon, J L; Drouhet, E; Improvisi, L

    1984-01-01

    A magnetic solid phase enzyme-linked immunosorbent assay (MELISA) for quantification of IgG antibodies to somatic and metabolic antigens of Paracoccidioides brasiliensis was developed. Activation of magnetic polyacrylamide agarose beads with concanavalin A was superior to glutaraldehyde activation, and test sensitivity was higher for somatic than for metabolic antigens. Comparative MELISA, counterimmunoelectrophoresis and erythroimmunoassay tests with sera from 33 proven cases of paracoccidioidomycosis, 14 cases of histoplasmosis and 20 normal human sera showed the MELISA could distinguish antibody levels in paracoccidioidomycosis from those in normal sera; however two sera from histoplasmosis cases cross-reacted in the MELISA. MELISA is a rapid test (5-6 h) and the results suggest it has considerable potential value for assay of anti-P. brasiliensis antibodies. PMID:6438813

  17. Detection of Giardia duodenalis antigen in coprolites using a commercially available enzyme-linked immunosorbent assay.

    PubMed

    Gonçalves, Marcelo Luiz Carvalho; Araújo, Adauto; Duarte, Rosemere; da Silva, Joaquim Pereira; Reinhard, Karl; Bouchet, Françoise; Ferreira, Luiz Fernando

    2002-01-01

    The objective of this experiment was to assess the utility of a commercially available enzyme-linked immunosorbent assay (ELISA) kit for diagnosis of giardiasis in archaeological human remains. The kit, a monoclonal antibody assay, is used to detect the presence of Giardia-specific antigen 65 (GSA65) in human faeces. We utilized the assay in ancient faecal material. The material included desiccated faeces found in mummies or in archaeological sites, and sediments from latrines. A total of 83 specimens, previously examined microscopically for parasites, were examined. The ELISA detected 3 positive samples, dated to about 1200 AD, 1600 AD and 1700 AD. The ELISA was superior to direct observation. It was possible to identify G. duodenalis cysts by direct microscopy in only one of these samples. The results did not show cross-reactivity between this protozoan and helminths. The use of ELISA to detect G. duodenalis coproantigen could help the diagnosis of giardiasis in ancient human remains. PMID:12625140

  18. An enzyme-linked immunosorbent assay using detergent-soluble Plasmodium vivax antigen for seroepidemiological surveys.

    PubMed

    González-Cerón, L; Rodríguez, M H

    1991-01-01

    An enzyme-linked immunosorbent assay (ELISA) to detect antibodies to Plasmodium vivax parasites in human sera was developed using P. vivax-infected erythrocytes from local malarious patients in southern Mexico. Infected cells were concentrated using a discontinuous Percoll gradient and detergent-soluble antigens obtained using Triton X100. The use of detergent and the addition of protease inhibitors to the antigen preparation ensured high sensitivity and reproducibility of the assay. No cross reactions were observed in sera immune to other protozoan, helmintic and bacterial infections, although some cross reactivity was seen in P. falciparum immune sera. A strong correlation between antibody titre values obtained by the ELISA and those obtained using an IFAT was observed. In a small field trial, carried out in a village where malaria transmission occurs, both ELISA and IFAT produced similar seroepidemiological profiles with regard to frequency of positive antibody titres and their distribution among the different age groups of the population. PMID:1949138

  19. Substitution of carbonate buffer by water for IgG immobilization in enzyme linked immunosorbent assay.

    PubMed

    Shrivastav, Tulsidas G; Basu, Anupam; Kariya, Kiran P

    2003-01-01

    The first step of enzyme linked immunosorbent assay (ELISA), namely, adsorption of antigen or antibody to the plastic microtiter well plate, was studied as a function of insolubility of IgG in water. Immobilization efficiency was assessed in terms of number of wells coated per milliliter of primary antiserum. We have compared different coating/immobilization protocols, i.e., direct and indirect immobilization of primary antibody to the plastic microtiter well plate using carbonate buffer and phosphate buffer with glutaraldehyde. We have observed efficient coating when the immobilization of primary antibody through an immunobridge technique was performed, where water was used as a coating medium. It gave a higher number of wells coated per milliliter of anti-serum (primary or secondary) than other compared coating protocols and it allowed the use of serum (non-immune) and anti-serum (primary and secondary antibody) dilutions, avoiding the need for gamma-globulin purification from normal and immunized serum. PMID:12778971

  20. Evaluation of enzyme-linked immunosorbent assay for diagnosis of Clostridium perfringens enterotoxemias.

    PubMed

    el Idrissi, A H; Ward, G E

    1992-06-15

    Two double sandwich enzyme-linked immunosorbent assays (ELISA) for Clostridium perfringens beta and epsilon toxins were assessed for routine diagnosis of enterotoxemias on intestinal contents of 151 sheep that died suddenly. Conventional tests (mouse assay and culture of organism) showed that 21 specimens were positive for Clostridium perfringens type C (beta toxin) and 39 were positive for Clostridium perfringens type D (epsilon toxin) enterotoxemias. Comparison of the ELISA results with conventional assays gave sensitivity and specificity rates respectively of 90.5% and 89.2% for beta toxin assay and 97.4% and 94.6% for epsilon toxin assay. With further refinement to improve the performance of the assay for beta toxin these tests could serve as a substitute for conventional tests in the laboratory diagnosis of Clostridium perfringens types B, C and D enterotoxemias. PMID:1496812

  1. Microchip-based enzyme-linked immunosorbent assay (microELISA) system with thermal lens detection.

    PubMed

    Sato, Kiichi; Yamanaka, Maho; Hagino, Tomokazu; Tokeshi, Manabu; Kimura, Hiroko; Kitamori, Takehiko

    2004-12-01

    A microchip-based enzyme-linked immunosorbent assay (microELISA) system was developed and interferon-gamma was successfully determined. The system was composed of a microchip with a Y-shaped microchannel and a dam structure, polystyrene microbeads, and a thermal lens microscope (TLM). All reactions required for the immunoassay were done in the microchannel by successive introduction of a sample and regents. The enzyme reaction product, in a liquid phase, was detected downstream in the channel using the TLM as substrate solution was injected. The antigen-antibody reaction time was shortened by the microchip integration. The limit of the determination was improved by adopting the enzyme label. Moreover, detection procedures were greatly simplified and required time for the detection was significantly cut. The system has good potential to be developed as a small and automated high throughput analyzer. PMID:15570367

  2. Comparison of immunodiffusion and enzyme linked immunosorbent assay for antibodies to four Aspergillus species.

    PubMed Central

    Froudist, J H; Harnett, G B; McAleer, R

    1989-01-01

    Antigenic extracts were prepared from Aspergillus fumigatus, A niger, A flavus and A terreus for use in enzyme linked immunosorbent assay (ELISA) and immunodiffusion (ID) tests for Aspergillus antibodies to determine whether the use of antigenic extracts from species other than A fumigatus increased the sensitivity of the ELISA. ELISA titres correlated well with positive ID tests. Patient titres by ELISA were significantly higher than control titres for all species. Patient titres to A niger were also significantly higher than titres to the other species. Total number of ID bands to A fumigatus correlated significantly with anti-A fumigatus ELISA titres. It is concluded that the use of antigenic extracts from species other than A fumigatus improves the sensitivity of the ELISA. PMID:2511230

  3. Serological diagnosis of typhoid fever by enzyme-linked immunosorbent assay (ELISA).

    PubMed

    Srivastava, L; Srivastava, V K

    1986-09-01

    Serum sample from 22 bacteriologically proved cases of typhoid fever, 41 febrile cases who were culture negative and 70 sick and healthy age-matched controls were tested for enzyme-linked immunosorbent assay (ELISA) IgG and IgM antibodies using Salmonella typhi LPS antigen. IgG and IgM antibodies were present in 72.7% and 81.8% respectively as against Widal test which was positive in 40.9% in proved cases. In febrile controls IgG and IgM ELISA antibodies were present in 80.4% and 60.9% respectively as against 53.6% by Widal test. This difference between the two tests was statistically significant P less than 0.001. ELISA test was more sensitive than the Widal test and hence it may be useful in rapid serodiagnosis of typhoid fever and also in circumstances where bacteriological techniques are not available. PMID:2430509

  4. Rapid enzyme-linked immunosorbent assay (ELISA) for Aspergillus fumigatus antibodies.

    PubMed Central

    Richardson, M D; Stubbins, J M; Warnock, D W

    1982-01-01

    A rapid enzyme-linked immunosorbent assay (ELISA) where component incubation periods were shortened to one hour, was compared with agar gel double diffusion (AGDD) and a standard ELISA procedure for detecting antibodies to Aspergillus fumigatus in 28 asthmatic patients with suspected allergic aspergillosis. Using two A fumigatus antigens the rapid ELISA compared well with AGDD and the standard ELISA method. Eleven sera that reacted with both antigens in AGDD were all positive against antigen 1 in both forms of ELISA, but two failed to react with antigen 2 in the standard ELISA and three failed to react with this antigen in the rapid method. Thirteen AGDD-negative sera were also negative in both forms of ELISA. The rapid ELISA provides a sensitive and reproducible test for routine serological investigation of allergic aspergillosis. PMID:6813358

  5. Determination of xanthine oxidase in human serum by a competitive enzyme-linked immunosorbent assay (ELISA).

    PubMed

    Battelli, M G; Abbondanza, A; Musiani, S; Buonamici, L; Strocchi, P; Tazzari, P L; Gramantieri, L; Stirpe, F

    1999-03-01

    Xanthine oxidase was purified from human milk and used to immunise rabbits. A competitive immunoenzymatic assay with purified enzyme and rabbit antiserum was optimised to measure xanthine oxidase in human serum, the lowest detectable amount being 0.03 pmol of enzymatic protein. Thus, the test (i) is sensitive enough to determine xanthine oxidase in human serum, being more sensitive than the spectrophotometric method, (ii) it is more convenient for clinical laboratories than other sensitive tests and (iii) it has the advantage over the enzyme activity-based assays of also detecting inactive enzyme molecules. A competitive enzyme-linked immunosorbent assay (ELISA) was used to measure the serum xanthine oxidase level in healthy donors and in patients with liver diseases, and it was found that any concentration below 1 mg/L is in the normal range. PMID:10217635

  6. Diagnosis of loxoscelism in a child confirmed with an enzyme-linked immunosorbent assay and noninvasive tissue sampling

    PubMed Central

    Stoecker, William V.; Green, Jonathan A.; Gomez, Hernan F.

    2011-01-01

    Background Confirmation of mild bites caused by Loxosceles reclusa with swab testing has not been previously documented, to our knowledge. Methods We report a case using an enzyme-linked immunosorbent assay (ELISA) test. Results A lesion lacking necrosis or other specific signs of loxoscelism was confirmed by identification of the Loxosceles venom and further confirmed by identification of a spider found in the patient’s bed. Limitations This is a pilot single-case report for this enzyme-linked immunosorbent assay test. Conclusions A sensitive and specific enzyme-linked immunosorbent assay designed to detect Loxosceles venom, using a specimen obtained by swabbing the lesion, can aid in diagnosis of loxoscelism. PMID:17052500

  7. Investigation of cross-reactions against Trichinella spiralis antigens by enzyme-linked immunosorbent assay and enzyme-linked immunoelectrotransfer blot assay in patients with various diseases.

    PubMed Central

    De-la-Rosa, J L; Alcantara, P; Correa, D

    1995-01-01

    Data regarding cross-reactions against Trichinella spiralis in humans are scarce and controversial. For this reason, we tested serum samples from patients with typhoid fever, brucellosis, toxoplasmosis, amoebiasis, cysticercosis, trichocephaliasis, ascariasis, and onchocerciasis against an antigenic extract of T. spiralis infective larvae in an enzyme-linked immunosorbent assay (ELISA) and an enzyme-linked immunoelectrotransfer blot (EITB) assay. All except one serum sample from the group of patients with onchocerciasis were negative in the ELISA; in the EITB assay, only faint bands were observed with the samples from patients with onchocerciasis and ascariasis and negative results were obtained with the samples from patients with other diseases. In conclusion, cross-reactions were found only in the groups of patients with other nematode infections and were of very low magnitude, most of them virtually negative. PMID:7719905

  8. QUANTITATIVE ENZYME-LINKED IMMUNOSORBENT ASSAY FOR DETERMINATION OF POLYCHLORINATED BIPHENYLS IN ENVIRONMENTAL SOIL AND SEDIMENT SAMPLES

    EPA Science Inventory

    An enzyme-linked immunosorbent assay (ELISA) for the quantitative determination of Aroclors 1242, 1248, 1254, and 1260 in soil and sediments was developed and its performance compared with that of gas chromatography (GC). The detection limits for Aroclors 1242 and 1248 in soil ar...

  9. DEVELOPMENT OF AN ENZYME-LINKED IMMUNOSORBENT ASSAY FOR THE DETECTION OF ANTIBODY TO EPIZOOTIC HEMORRHAGIC DISEASE OF DEER VIRUS

    Technology Transfer Automated Retrieval System (TEKTRAN)

    An enzyme linked immunosorbent assay has been developed to detect antibodies to epizootic hemorrhagic disease of deer virus (EHDV). The assay incorporates a monoclonal antibody to EHDV serotype 2 (EHDV-2) that demonstrates specificity for the viral structural protein, VP7. The assay was evaluated ...

  10. A sensitive monoclonal antibody-based enzyme-linked immunosorbent assay for chlorpyrifos residue determination in Chinese agricultural smaples

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A monoclonal antibody-based competitive antibody-coated enzyme-linked immunosorbent assay (ELISA) was developed and optimized for determining chlorpyrifos residue in agricultural products. The IC50 and IC10 of this ELISA were 3.3 ng/mL and 0.1 ng/mL respectively. The average recoveries recovery rate...

  11. COMPARISON OF BIOASSAY AND ENZYME-LINKED IMMUNOSORBENT ASSAY FOR QUANTIFICATION OF 'SPODOPTERA FRUGIPERDA' NUCLEAR POLYHEDROSIS VIRUS IN SOIL

    EPA Science Inventory

    Standard curves with known amounts of Spodoptera frugiperda nuclear polyhedrosis virus (NPV) in soil were established with a bioassay and with an enzyme-linked immunosorbent assay (ELISA). The bioassay detected as few as 4 x 10 to the 4th power polyhedral inclusion bodies (PIB)/g...

  12. Immunological Tools: Engaging Students in the Use and Analysis of Flow Cytometry and Enzyme-linked Immunosorbent Assay (ELISA)

    ERIC Educational Resources Information Center

    Ott, Laura E.; Carson, Susan

    2014-01-01

    Flow cytometry and enzyme-linked immunosorbent assay (ELISA) are commonly used techniques associated with clinical and research applications within the immunology and medical fields. The use of these techniques is becoming increasingly valuable in many life science and engineering disciplines as well. Herein, we report the development and…

  13. Droplet-Free Digital Enzyme-Linked Immunosorbent Assay Based on a Tyramide Signal Amplification System.

    PubMed

    Akama, Kenji; Shirai, Kentaro; Suzuki, Seigo

    2016-07-19

    Digital enzyme-linked immunosorbent assay (ELISA) is a single molecule counting technology and is one of the most sensitive immunoassay methods. The key aspect of this technology is to concentrate enzyme reaction products from a single target molecule in femtoliter droplets. This study presents a novel Digital ELISA that does not require droplets; instead, enzyme reaction products are concentrated using a tyramide signal amplification system. In our method, tyramide substrate reacts with horseradish peroxidase (HRP) labeled with an immunocomplex on beads, and the substrate is converted into short-lived radical intermediates. By adjusting the bead concentration in the HRP-tyramide reaction and conducting the reaction using freely moving beads, tyramide radicals are deposited only on beads labeled with HRP and there is no diffusion to other beads. Consequently, the fluorescence signal is localized on a portion of the beads, making it possible to count the number of labeled beads digitally. The performance of our method was demonstrated by detecting hepatitis B surface antigen with a limit of detection of 0.09 mIU/mL (139 aM) and a dynamic range of over 4 orders of magnitude. The obtained limit of detection represents a >20-fold higher sensitivity than conventional ELISA. Our method has potential applications in simple in vitro diagnostic systems for detecting ultralow concentrations of protein biomarkers. PMID:27322525

  14. One step enzyme linked immunosorbent assay for direct estimation of serum testosterone.

    PubMed

    Shrivastav, Tulsidas G; Basu, Anupam; Kariya, Kiran P

    2003-01-01

    One step competitive enzyme linked immunosorbent assay (ELISA) for direct estimation of testosterone in human serum is described. Testosterone-3-O-carboxymethyl-oxime-bovine serum albumin (testosterone-3-O-CMO-BSA), was used as immunogen and testosterone-3-O-carboxymethyl-oxime-adipic-acid dihydrazide-horseradish peroxidase (testosterone-3-O-CMO-ADH-HRP) was used as tracer. To the testosterone antibody coated microtiter wells, standard or serum samples (100 microL), along with testosterone-3-O-CMO-ADH-HRP conjugate (100 microL) were incubated for 1 h at 37 degrees C. Bound enzyme activity was measured by using tetra methyl benzidine/hydrogen peroxide (TMB/H2O2) as a substrate. In this new strategy, charcoal stripped pooled human serum spiked with non-cross reactive C18, C19, C21, and C27 steroids, used for preparing the standards and blocking the sex hormone binding globulin (SHBG)/and other steroid binding globulins (SBG). The sensitivity of the assay was 0.015 ng/mL. The intra-assay and inter-assay coefficients of variation (CVs) were ranged from 7.8 to 11.8 and 4.8 to 10.4, respectively. The serum testosterone values, obtained by this method, were correlated well with those obtained by radioimmunoassay r = .98 (n = 100). PMID:12778972

  15. Determination of diosgenin content in medicinal plants with enzyme-linked immunosorbent assay.

    PubMed

    Li, Jiaru; Yang, Dingyu; Yu, Kun; He, Ji; Zhang, Yingjun

    2010-11-01

    Many medicinal plants contain diosgenin, which has a significant medicinal value. However, there is currently no effective and rapid analytical method to determine the diosgenin content of plants or products. In the present work we have developed an indirect competitive enzyme-linked immunosorbent assay (ELISA) for the quantitative determination of diosgenin in herbal medicines. Diosgenin was conjugated with bovine serum albumin (BSA) for immunization. A polyclonal antibody developed in rabbits against a diosgenin-BSA conjugate was shown to be specific for diosgenin. The developed ELISA assay was highly sensitive, specific, and easy to perform. In addition, it gave more precise results with less variation than other methods that have been used in the past, including gravimetric and spectrophotometric assays, and correlated well with high-performance liquid chromatography. The diosgenin content determined by ELISA varied widely, with the highest and lowest values in rhizomes or tubers of Paris polyphylla and Dioscorea opposita Thunb. "Jiao-ban Yam", respectively, differing by more than 9000-fold. These results suggest that the ELISA method can be used as a rapid, simple, sensitive, and accurate tool for quantitative analysis of samples containing diosgenin, and may provide an important criterion for quality evaluation and a valuable tool for quality control of diosgenin-containing medicinal plants. PMID:20549594

  16. [Development of direct competitive enzyme-linked immunosorbent assay for the determination of domoic acid].

    PubMed

    Wang, Qian; Cheng, Jin-Ping; Gao, Li-Li; Dong, Yu; Xi, Lei

    2012-02-01

    To develop a direct competitive enzyme-linked immunosorbent assay (ELISA) for rapid detection of domoic acid concentrations, HRP (horse radish peroxidase) was successfully linked to DA using EDC. The concentration of DA was quantitatively analyzed on the basic of the specific immune responses between the DA- HRP and the monoclonal antibodies made in advance. Calibration curve were established after the optimization of reaction conditions such as the type of blocking solution, the blocking time and the incubation temperature. The results show that, the best reaction condition of the direct competitive ELISA is 1% gelatin, blocking 1 h at 37 degrees C, incubating 1 h at 37 degrees C after the monoclonal antibodies added. The detect limit is 3.58 ng x mL(-1), the coefficient of variation between the holes is below 15%, and the recovery is 80% - 120%. The whole analysis process could be completed within 1.5 h. It meets the requirements of rapid and batch detection of domoic acid. The method will have broad development prospects. PMID:22509610

  17. Biotin-Streptavidin Competition Mediates Sensitive Detection of Biomolecules in Enzyme Linked Immunosorbent Assay.

    PubMed

    Lakshmipriya, Thangavel; Gopinath, Subash C B; Tang, Thean-Hock

    2016-01-01

    Enzyme Linked Immunosorbent Assay (ELISA) is the gold standard assay for detecting and identifying biomolecules using antibodies as the probe. Improving ELISA is crucial for detecting disease-causing agents and facilitating diagnosis at the early stages of disease. Biotinylated antibody and streptavidin-conjugated horse radish peroxide (streptavidin-HRP) often are used with ELISA to enhance the detection of various kinds of targets. In the present study, we used a competition-based strategy in which we pre-mixed free biotin with streptavidin-HRP to generate high-performance system, as free biotin occupies some of the biotin binding sites on streptavidin, thereby providing more chances for streptavidin-HRP to bind with biotinylated antibody. ESAT-6, which is a protein secreted early during tuberculosis infection, was used as the model target. We found that 8 fM of free biotin mixed with streptavidin-HRP anchored the higher detection level of ESAT-6 by four-fold compared with detection without free biotin (only streptavidin-HRP), and the limit of detection of the new method was 250 pM. These results suggest that biotin-streptavidin competition can be used to improve the diagnosis of analytes in other types of sensors. PMID:26954237

  18. Milk matrix effects on antibody binding analyzed by enzyme-linked immunosorbent assay and biolayer interferometry.

    PubMed

    Brandon, David L; Adams, Lisa M

    2015-04-01

    Biolayer interferometry (BLI) was employed to study the impact of the milk matrix on the binding of ricin to asialofetuin (ASF) and to antibodies. This optical sensing platform used ligands immobilized covalently or via biotin-streptavidin linkage, and the results were compared to those obtained by enzyme-linked immunosorbent assay (ELISA). In sandwich ELISA, the binding of ricin to ASF was dramatically decreased when galactose was present during the analyte or detection antibody binding step. Low concentrations of milk (1%, v/v) produced a similar reduction in ricin binding to ASF but not to a high-affinity monoclonal antibody (mAb), increasing the dissociation rate of ASF-ricin complexes up to 100-fold. The effect of milk on the binding of ricin to ASF was ascribable to dialyzable factors, and milk sugar can account for these effects. The use of high-affinity mAbs in ELISA effectively limits the milk matrix effect on ricin analysis. PMID:25822824

  19. Development of an indirect enzyme-linked immunosorbent assay for the organophosphorus pesticide paraoxon-methyl.

    PubMed

    Li, Zhuo-Kun; Zhu, Ying-Yue; Yin, Xiang-Gang; Peng, Chi-Fang; Chen, Wei; Liu, Li-Qiang; Yin, Li-Mei; Xu, Chuan-Lai

    2009-01-01

    In the present study, the synthesis of hapten for the organophosphorus (OP) pesticide paraoxon-methyl was developed, with a spacer arm (aminocarboxylic acid) attached at the aromatic ring. It was conjugated to bovine serum albumin (BSA) for use as an immunogen and to ovalbumin (OVA) for coating antigen for ELISA testing. Rabbits were immunized with the immunogen and two polyclonal antisera were produced and screened against the coating antigen using competitive indirect enzyme-linked immunosorbent assay (ELISA). For application to textile samples, the influence of several factors such as organic solvent, ionic strength, and pH on the ELISA results were studied. Under optimized conditions, the quantitative working range was 0.012-1.158 microg/mL with a limit of detection (LOD) of 0.005 microg/mL and the IC(50) was 0.115 microg/mL.There was negligible cross reactivity (CR) with other OP pesticides. The recoveries obtained by standard paraoxon-methyl addition to the different textile samples such as cotton, wool and muslin delaine were all from 86.0% to 108.0%. Therefore, the optimized ELISA may become a new convenient and economical analytical tool for monitoring paraoxon-methyl residues in textile samples. PMID:19811409

  20. A Review of Cry Protein Detection with Enzyme-Linked Immunosorbent Assays.

    PubMed

    Albright, Vurtice C; Hellmich, Richard L; Coats, Joel R

    2016-03-23

    The widespread use of Cry proteins in insecticide formulations and transgenic crops for insect control has led to an increased interest in the environmental fate of these proteins. Although several detection methods are available to monitor the fate of Cry proteins in the environment, enzyme-linked immunosorbent assays (ELISAs) have emerged as the preferred detection method, due to their cost-effectiveness, ease of use, and rapid results. Validation of ELISAs is necessary to ensure accurate measurements of Cry protein concentrations in the environment. Validation methodology has been extensively researched and published for the areas of sensitivity, specificity, accuracy, and precision; however, cross validation of ELISA results has been studied to a lesser extent. This review discusses the use of ELISAs for detection of Cry proteins in environmental samples and validation of ELISAs and introduces cross validation. The state of Cry protein environmental fate research is considered through a critical review of published literature to identify areas where the use of validation protocols can be improved. PMID:26949828

  1. Development of an enzyme-linked immunosorbent assay for bisphenol a using chicken immunoglobulins.

    PubMed

    De Meulenaer, Bruno; Baert, Katleen; Lanckriet, Heikki; Van Hoed, Vera; Huyghebaert, Andre

    2002-09-11

    Bisphenol A was coupled, after derivatization into a suitable hapten, to bovine serum albumin and ovalbumin in order to produce immunizing and coating antigens. The immunizing antigens were injected into chickens, which allowed the isolation of specific bisphenol A immunoglobulins from the egg yolk. These antibodies were used in an indirect competitive enzyme-linked immunosorbent assay for the determination of bisphenol A in aqueous solutions. Various parameters, influencing the assay sensitivity, were evaluated. The applicability of the assay for the determination of bisphenol A in milk was also studied. The assay was not as sensitive as other analytical techniques used in bisphenol A analysis, since typical I(50) levels of 2.5 microM were reached in aqueous solutions. This study nevertheless illustrates the usefulness and the potency of chicken antibodies in the analysis of migration residues from packaging materials using immunochemical techniques. In addition, the assay showed to be quite specific for bisphenol A as well. Only for bisphenol A analogues, cross reactivities of about 40% were reached, enabling the use of the antibodies for the screening of bisphenol A and alike compounds. PMID:12207461

  2. Single-dilution enzyme-linked immunosorbent assay for quantification of antigen-specific salmonid antibody

    USGS Publications Warehouse

    Alcorn, S.W.; Pascho, R.J.

    2000-01-01

    An enzyme-linked immunosorbent assay (ELISA) was developed on the basis of testing a single dilution of serum to quantify the level of antibody to the p57 protein of Renibaclerium salmoninarum in sockeye salmon (Oncorhynchus nerka). The levels of antibody were interpolated from a standard curve constructed by relating the optical densities (OD) produced by several dilutions of a high-titer rainbow trout (O. mykiss) antiserum to the p57 protein. The ELISA OD values produced by as many as 36 test sera on each microplate were compared with the standard curve to calculate the antigen-specific antibody activity. Repeated measurements of 36 samples on 3 microplates on each of 6 assay dates indicated that the mean intraassay coefficient of variation (CV) was 6.68% (range, 0-23%) and the mean interassay CV was 8.29% (range, 4-16%). The antibody levels determined for the serum sample from 24 sockeye salmon vaccinated with a recombinant p57 protein generally were correlated with the levels determined by endpoint titration (r2 = 0.936) and with results from another ELISA that was based on extrapolation of antibody levels from a standard curve (r2 = 0.956). The single-dilution antibody ELISA described here increases the number of samples that can be tested on each microplate compared with immunoassays based on analysis of several dilutions of each test serum. It includes controls for interassay standardization and can be used to test fish weighing <3 g.

  3. Characterization by enzyme-linked immunosorbent assay of monoclonal antibodies to Pisum and Avena phytochrome

    SciTech Connect

    Cordonnier, M.M.; Greppin, H.; Pratt, L.H.

    1984-01-01

    Nine monoclonal antibodies to pea (Pisum sativum L.) and 16 to oat (Avena sativa L.) phytochrome are characterized by enzyme-linked immunosorbent assay against phytochrome from six different sources: pea, zucchini (Cucurbita pepo L.), lettuce (Lactuca sativa L.), oat, rye (Secale cereale L.), and barley (Hordeum vulgare L.). All antibodies were raised against phytochrome with a monomer size near 120,000 daltons. Nevertheless, none of them discriminated qualitatively between 118/114-kilodalton oat phytochrome and a photoreversible, 60-kilodalton proteolytic degradation product derived from it. In addition, none of the 23 antibodies tested discriminated substantially between phytochrome - red-absorbing form and phytochrome - far red-absorbing form. Two antibodies to pea and six to oat phytochrome also bound strongly to phytochrome from the other species, even though these two plants are evolutionarily widely divergent. Of these eight antibodies, two bound significantly to all of the six phytochrome preparations tested, indicating that these two may recognize highly conserved regions of the chromoprotein. Since the molecular function of phytochrome is unknown, these two antibodies may serve as unique probes for regions of this pigment that are important to its mode of action. 27 references, 3 figures, 1 table.

  4. Detection of antibodies and antigens of human parvovirus B19 by enzyme-linked immunosorbent assay.

    PubMed Central

    Anderson, L J; Tsou, C; Parker, R A; Chorba, T L; Wulff, H; Tattersall, P; Mortimer, P P

    1986-01-01

    Acute-phase serum from a patient with aplastic crisis provided sufficient human parvovirus B19 to make a monoclonal antibody against B19 and to develop antigen and immunoglobulin M (IgM) and IgG antibody detection enzyme-linked immunosorbent assays (ELISAs). The indirect capture antibody method was used for all three assays. Antigen was detected in 8 of 29 sera drawn within 2 days of onset of illness from patients with aplastic crisis. These sera had high titers of virus by electron microscopy and DNA hybridization and had no detectable B19 antibody. Antigen was not detected in serum specimens that had low titers of B19 DNA and had B19 antibody. With the IgM ELISA, we detected B19 IgM in over 85% of clinical cases of aplastic crisis and fifth disease and less than 2% of controls. The prevalence of B19 IgG antibodies increased with age. Approximately 2% of children less than 5 years of age and 49% of adults greater than 20 years of age had B19 IgG antibodies. The B19 antibody ELISAs are sensitive and specific tests to detect B19 infections. PMID:3021807

  5. Development of an enzyme-linked immunosorbent assay for atrazine monitoring in water samples.

    PubMed

    Lima, Diana L D; Schneider, Rudolf J; Esteves, Valdemar I

    2013-05-01

    The implementation of the Water Framework Directive (2000/60/EC) requires the establishment of monitoring programs. However, conventional procedures for sample preparation prior to chromatographic analysis are rather expensive and time consuming, being the development of cost-effective and easy tool a necessity. The aim of this work was to develop an enzyme-linked immunosorbent assay (ELISA) able to determine atrazine in water samples. Matrix effects evaluation showed that the increase of humic acid (HA) concentration leads to flattened calibration curves and to the loss of the sigmoidal shape. However, such interference was overcome, by the presence of an environmental sample buffer, incubated together with the samples. Recoveries from 88.5 to 119.2 % were obtained in the presence of HA concentrations up to 20 mg L(-1). An analytical range from 0.003 to 1 μg L(-1) was obtained, and atrazine was detected in a sewage treatment plant with concentrations ranging from 14 to 52 ng L(-1). PMID:23054792

  6. Mixed enzyme-linked immunosorbent assay (MELISA) for HLA class I antigen: a plasma membrane marker.

    PubMed

    Bjerrum, O W; Borregaard, N

    1990-03-01

    This study introduces a simple, reproducible assay for HLA class I antigen using antibodies against beta 2-microglobulin and the heavy chain on HLA. The sandwich technique was named mixed enzyme-linked immunosorbent assay (MELISA), and was designed for identification of plasma membranes in neutrophil subcellular fractions. The subcellular localization of HLA was identical to that of other plasma membrane markers, [3H]concanavalin A and detergent-independent alkaline phosphatase, and was unchanged by stimulation of cells by weak and strong secretagogues. In addition to the presence as part of the HLA complex in the plasma membrane uncomplexed beta 2-microglobulin is present in the specific granules of neutrophils. However, the release of beta 2-microglobulin from intact neutrophils stimulated with formyl-methionylleucylphenylalanine was much higher than could be explained by exocytosis of specific granules. Subcellular fractionation studies demonstrated that beta 2-microglobulin is localized in fractions characterized by latent alkaline phosphatase and released from this novel secretory compartment in response to stimulation with formyl-methionylleucylphenylalanine. PMID:2181625

  7. Performance of a Pneumolysin Enzyme-Linked Immunosorbent Assay for Diagnosis of Pneumococcal Infections▿

    PubMed Central

    del Mar García-Suárez, María; Cima-Cabal, María Dolores; Villaverde, Roberto; Espinosa, Emma; Falguera, Miquel; de Los Toyos, Juan R.; Vázquez, Fernando; Méndez, Francisco J.

    2007-01-01

    A pneumolysin-specific enzyme-linked immunosorbent assay (PLY-ELISA) for the detection of pneumolysin in urine was developed and evaluated in comparison with the commercially available Binax Now Streptococcus pneumoniae test (Binax, Portland, ME) for the diagnosis of pneumococcal infections. Assay sensitivity was evaluated using urine from 108 patients with culture-confirmed pneumococcal infections. In adults, the sensitivity and specificity of the PLY-ELISA were 56.6% and 92.2%, respectively. In children with nasopharyngeal pneumococcal carriage, PLY-ELISA and Binax Now S. pneumoniae test sensitivities were 62.5% and 87.5%, respectively, while specificities were 94.4% and 27.8%, respectively. In children with nonnasopharyngeal pneumococcal carriage, PLY-ELISA and Binax Now S. pneumoniae test sensitivities were 68.7% and 93.7%, respectively, and test specificities were 94.1% and 41.2%, respectively. The persistence of pneumolysin in urine of pneumococcal pneumonia patients decreased significantly after 4 to 6 days of treatment. Our data suggest that combining the high specificity of the PLY-ELISA with the high sensitivity of the Binax Now S. pneumoniae test would enable pneumococcal infections to be accurately diagnosed in children. PMID:17728474

  8. Enzyme-linked immunosorbent assay for detection and identification of coxsackieviruses A.

    PubMed Central

    Yolken, R H; Torsch, V M

    1981-01-01

    Coxsackieviruses A are known to cause a wide range of human disease processes. However, because many coxsackieviruses A present in clinical specimens do not produce a recognizable cytopathic effect in readily available tissue culture systems, infections with coxsackieviruses A are often difficult to diagnose. We have thus developed enzyme-linked immunosorbent assay (ELISA) systems for the detection and serotyping of coxsackievirus A antigens. The assays consist of a double-antibody ELISA which utilizes type-specific monkey and mouse coxsackievirus antisera. Although some cross-reactivity was noted, the ELISA systems correctly identified the serotypes of 22 to 23 coxsackievirus A complement fixation antigens available for testing. Testing of tissue culture fluids revealed that antigen could often be detected by ELISA before the appearance of a cytopathic effect. In addition, the infecting coxsackievirus A antigen could be unequivocally identified in 8 of 11 stool specimens obtained from patients with coxsackievirus A infections. The ELISA system might thus represent an important tool in the diagnosis and study of coxsackievirus A infections. PMID:6260675

  9. Sandwich Enzyme-Linked Immunosorbent Assay for Detecting Sesame Seed in Foods.

    PubMed

    Koppelman, Stef J; Söylemez, Gülsen; Niemann, Lynn; Gaskin, Ferdelie E; Baumert, Joseph L; Taylor, Steve L

    2015-01-01

    Small amounts of sesame can trigger allergic reactions in sesame-allergic patients. Because sesame is a widely used food ingredient, analytical methods are needed to support quality control and food safety programs in the food industry. In this study, polyclonal antibodies against sesame seed proteins were raised, and an enzyme-linked immunosorbent assay (ELISA) was developed for the detection and quantification of sesame seed residue in food. A comparison was made between this ELISA and other assays, particularly focusing on recovery of sesame seed residue from different food matrices. The developed ELISA is sensitive with a lower limit of quantification of 0.5 ppm and shows essentially no cross-reactivity with other foods or food ingredients (92 tested). The ELISA has a good recovery for analyzing sesame-based tahini in peanut butter, outperforming one other test. In a baked bread matrix, the ELISA has a low recovery, while two other assays perform better. We conclude that a sensitive and specific ELISA can be constructed based on polyclonal antibodies, which is suitable for detection of small amounts of sesame seed relevant for highly allergic patients. Furthermore, we conclude that different food products may require different assays to ensure adequate quantification of sesame. PMID:26783532

  10. Sandwich Enzyme-Linked Immunosorbent Assay for Detecting Sesame Seed in Foods

    PubMed Central

    Koppelman, Stef J.; Söylemez, Gülsen; Niemann, Lynn; Gaskin, Ferdelie E.; Baumert, Joseph L.; Taylor, Steve L.

    2015-01-01

    Small amounts of sesame can trigger allergic reactions in sesame-allergic patients. Because sesame is a widely used food ingredient, analytical methods are needed to support quality control and food safety programs in the food industry. In this study, polyclonal antibodies against sesame seed proteins were raised, and an enzyme-linked immunosorbent assay (ELISA) was developed for the detection and quantification of sesame seed residue in food. A comparison was made between this ELISA and other assays, particularly focusing on recovery of sesame seed residue from different food matrices. The developed ELISA is sensitive with a lower limit of quantification of 0.5 ppm and shows essentially no cross-reactivity with other foods or food ingredients (92 tested). The ELISA has a good recovery for analyzing sesame-based tahini in peanut butter, outperforming one other test. In a baked bread matrix, the ELISA has a low recovery, while two other assays perform better. We conclude that a sensitive and specific ELISA can be constructed based on polyclonal antibodies, which is suitable for detection of small amounts of sesame seed relevant for highly allergic patients. Furthermore, we conclude that different food products may require different assays to ensure adequate quantification of sesame. PMID:26783532

  11. Monoclonal-based enzyme-linked immunosorbent assay and immunochromatographic assay for enrofloxacin in biological matrices.

    PubMed

    Watanabe, Hiroo; Satake, Atsuko; Kido, Yasumasa; Tsuji, Akio

    2002-01-01

    Enrofloxacin has been increasingly used in veterinary medicine to treat microbial infections. A simple and reliable analytical method for this drug is required. The current determination by high performance liquid chromatography (HPLC) is sensitive but labor-intensive. This paper reports an enzyme-linked immunosorbent assay (ELISA) using a monoclonal antibody (MAb) and the development of a rapid test kit based on immunochromatography. The detection limits using the ELISA were 10 ppb for chicken liver and muscle, and 1 ppb for cattle milk, respectively. The mean recovery values were 77.3-96.0% for chicken liver, 72.4-92.0% for chicken muscle and 84.0-99.0% for cattle milk. The detection limits using the kit were ca. 100 ppb for chicken muscle and ca. 10 ppb for cattle milk, respectively. All ELISA results for assay of chicken liver, chicken muscle and cattle milk were confirmed using HPLC which is used as the routine assay. The HPLC (x) and ELISA (y) results showed close correlation for chicken liver (y = 8.7 + 0.85x, r2 = 0.99, n = 25), chicken muscle (y = -3.9 + 0.94x, r2 = 0.98, n = 25) and cattle milk (y = 18.4 + 0.92x, r2 = 0.99, n = 25). PMID:11827405

  12. Serodiagnosis of human and animal pythiosis using an enzyme-linked immunosorbent assay.

    PubMed Central

    Mendoza, L; Kaufman, L; Mandy, W; Glass, R

    1997-01-01

    Conventional serodiagnosis of Pythium insidiosum infections involves the use of the immunodiffusion (ID) test. This test specifically diagnoses human and animal pythiosis. The test, however, has limited sensitivity and does not detect some culturally proven cases of the disease. Because of the increased recognition of pythiosis among humans and animals, we developed and evaluated an enzyme-linked immunosorbent assay (ELISA) using a soluble antigen from broken hyphae of P. insidiosum. Studies were carried out with sera from five humans and eight animals with culturally and/or histologically proven pythiosis. Some of these sera were negative in the ID test for pythiosis. Heterologous case sera from thirteen humans and two horses, plus 5 sera from healthy humans and 17 from healthy animals, were tested. Of the pythiosis case sera tested, the ID test detected only 8 of 13 (61.5%), whereas the ELISA detected all of them (100%). The ID and ELISA tests were entirely specific and gave negative results or low titers respectively, with sera from humans and animals with heterologous fungal infections or with no apparent illness. No correlation was found between the height of the ELISA titers and negative or positive sera in the ID test. Our results indicate that the ELISA is a reliable serodiagnostic test for pythiosis. It is as specific as the ID test but more sensitive. PMID:9384295

  13. Identification of Ancient Silk Using an Enzyme-linked Immunosorbent Assay and Immuno-fluorescence Microscopy.

    PubMed

    Liu, Miaomiao; Xie, Jun; Zheng, Hailing; Zhou, Yang; Wang, Bing; Hu, Zhiwen

    2015-01-01

    The identification of ancient silk is of great importance in both archaeology and academia. In the present work, a specific antibody having the characteristics of low cost, easy operation and extensive applicability was developed directly through immunizing rabbits with complete antigen (silk fibroin, SF). Then, antibody-based immunoassays, i.e. enzyme-linked immunosorbent assay (ELISA) and immuno-fluorescence microscopy (IFM), were established and conducted in tandem to identify the corresponding protein in ancient silks. The anti-SF antibody exhibits high sensitivity and specificity for the identification of modern and ancient silks. The detection limit of the ELISA method is about 0.1 ng/mL, and no cross-reactions with other possible interference antigens have been noted. IFM makes it possible to localize target proteins in archaeological samples, and also ensure the reliability of the ELISA results. Based on these advantages, immunological techniques have the potential to become powerful analytical tools at archaeological sites and conservation science laboratories. PMID:26656824

  14. Detection of Borrelia burgdorferi in urine of Peromyscus leucopus by inhibition enzyme-linked immunosorbent assay.

    PubMed

    Magnarelli, L A; Anderson, J F; Stafford, K C

    1994-03-01

    An inhibition enzyme-linked immunosorbent assay was developed to detect Borrelia burgdorferi, the etiologic agent of Lyme borreliosis, in urine from white-footed mice (Peromyscus leucopus). Of the 87 urine specimens tested from 87 mice collected in widely separated tick-infested sites in Connecticut, 57 (65.5%) contained detectable concentrations of spirochetal antigens. Forty-seven (62.7%) of 75 serum samples analyzed contained antibodies to B. burgdorferi. In culture work with tissues from bladders, kidneys, spleens, or ears, 50 of 87 mice (57.5%) were infected with B. burgdorferi. Thirty-eight (76%) of 50 infected mice had antigens of this spirochete in urine, while 36 (72%) individuals had infected bladders. Of those with infected bladders, 24 (66.7%) mice excreted subunits or whole cells of B. burgdorferi into urine. Successful culturing of B. burgdorferi from mouse tissues, the presence of serum antibodies to this bacterium, and detection of antigens to this spirochete in urine provide further evidence that multiple assays can be performed to verify the presence of B. burgdorferi in P. leucopus. PMID:8195393

  15. Magnetic nanoparticle based purification and enzyme-linked immunosorbent assay using monoclonal antibody against enrofloxacin.

    PubMed

    Kim, Nam-Gun; Kim, Myeong-Ae; Park, Young-Il; Jung, Tae-Sung; Son, Seong-Wan; So, ByungJae; Kang, Hwan-Goo

    2015-01-01

    Monoclonal anti-enrofloxacin antibody was prepared for a direct competitive enzyme-linked immunosorbent assay (ELISA) and purification system using monoclonal antibody (mAb) coupled magnetic nanoparticles (MNPs). The IC50 values of the developed mAb for enrofloxacin (ENR), ciprofloxacin, difloxacin, sarafloxacin, pefloxacin, and norfloxacin were 5.0, 8.3, 9.7, 21.7, 36.0, and 63.7 ng/mL, respectively. The lowest detectable level of ENR was 0.7 ng/mL in the prepared ELISA system. To validate the developed ELISA in the food matrix, known amounts of ENR were spiked in meat and egg samples at 10, 20 and 30 ng/mL. Recoveries for ENR ranged from 72.9 to 113.16% with a coefficient of variation (CV) of 2.42 to 10.11%. The applicability of the mAb-MNP system was verified by testing the recoveries for ENR residue in three different matrices. Recoveries for ENR ranged from 75.16 to 86.36%, while the CV ranged from 5.08 to 11.53%. Overall, ENR-specific monoclonal antibody was prepared and developed for use in competitive to ELISAs for the detection of ENR in animal meat samples. Furthermore, we suggest that a purification system for ENR using mAb-coupled MNPs could be useful for determination of ENR residue in food. PMID:26040610

  16. Detection of tetracosactide in plasma by enzyme-linked immunosorbent assay (ELISA).

    PubMed

    Martin, Laurent; Chaabo, Ayman; Lasne, Françoise

    2015-06-01

    As a synthetic analogue of adrenocorticotropic hormone (ACTH), tetracosactide is prohibited in sport by the World Anti-Doping Agency (WADA). An enzyme-linked immunosorbent assay (ELISA) method is proposed for detection of this drug in plasma. Since its structure corresponds to the 24 N-terminal of the 39 amino acids of the natural endogenous peptide ACTH, tetracosactide can be detected with a commercial ELISA kit for ACTH that uses antibodies, the epitopes of which are located in the 1-24 part of ACTH. However, an essential condition for detection specificity is the preliminary total clearance of endogenous ACTH in the plasma samples. This is achieved by a preparative step based on cation-exchange chromatography before ELISA. The method is specific and sensitive (LOD: 30 pg/mL) and may be used as a screening analysis in anti-doping control. The pre-analytical conditions are shown to be of the upmost importance and recommendations for blood collection (EDTA tubes), sample transport (4 °C) and plasma sample storage (-20 °C) are presented. PMID:25219545

  17. Synthetic peptide-based enzyme-linked immunosorbent assay for serodiagnosis of visceral leishmaniasis.

    PubMed Central

    Fargeas, C; Hommel, M; Maingon, R; Dourado, C; Monsigny, M; Mayer, R

    1996-01-01

    Synthetic peptides, derived from the amino acid sequence of a Leishmania donovani clone, were used to develop an enzyme-linked immunosorbent assay (ELISA) for detecting antibodies against L. donovani. For this purpose, five peptides were conjugated to a protein carrier, human serum albumin (HSA), by using a heterobifunctional reagent, epsilon-maleimidocaproic acid N-hydroxysuccinimide ester, to obtain a well-defined product. The sensitivity and the specificity of the peptide-specific ELISA were determined with a panel of 106 serum samples from individuals living in areas where visceral leishmaniasis is endemic; sera from post-kala azar dermal leishmaniasis-infected patients and from individuals suffering from other infectious diseases were also included. ELISAs were performed with either a single peptide-HSA conjugate or a mixture of two peptide-HSA conjugates. Ninety-seven percent of the serum samples from patients with visceral leishmaniasis had detectable antibodies to one or more of the single synthetic peptides. ELISA with a single peptide-HSA conjugate proved to be less sensitive (less than 71%) but more specific (up to 93%) than ELISA with crude promastigote antigens (80% sensitivity and 79% specificity); when a combination of two different peptide-HSA conjugates was used, the test increased both in sensitivity and in specificity. Chemically defined peptide-protein conjugates improve the reproducibility and reliability of ELISA for the serodiagnosis of L. donovani infection. PMID:8788994

  18. Click chemistry armed enzyme-linked immunosorbent assay to measure palmitoylation by hedgehog acyltransferase

    PubMed Central

    Lanyon-Hogg, Thomas; Masumoto, Naoko; Bodakh, George; Konitsiotis, Antonio D.; Thinon, Emmanuelle; Rodgers, Ursula R.; Owens, Raymond J.; Magee, Anthony I.; Tate, Edward W.

    2015-01-01

    Hedgehog signaling is critical for correct embryogenesis and tissue development. However, on maturation, signaling is also found to be aberrantly activated in many cancers. Palmitoylation of the secreted signaling protein sonic hedgehog (Shh) by the enzyme hedgehog acyltransferase (Hhat) is required for functional signaling. To quantify this important posttranslational modification, many in vitro Shh palmitoylation assays employ radiolabeled fatty acids, which have limitations in terms of cost and safety. Here we present a click chemistry armed enzyme-linked immunosorbent assay (click–ELISA) for assessment of Hhat activity through acylation of biotinylated Shh peptide with an alkyne-tagged palmitoyl-CoA (coenzyme A) analogue. Click chemistry functionalization of the alkyne tag with azido-FLAG peptide allows analysis through an ELISA protocol and colorimetric readout. This assay format identified the detergent n-dodecyl β-d-maltopyranoside as an improved solubilizing agent for Hhat activity. Quantification of the potency of RU-SKI small molecule Hhat inhibitors by click–ELISA indicated IC50 values in the low- or sub-micromolar range. A stopped assay format was also employed that allows measurement of Hhat kinetic parameters where saturating substrate concentrations exceed the binding capacity of the streptavidin-coated plate. Therefore, click–ELISA represents a nonradioactive method for assessing protein palmitoylation in vitro that is readily expandable to other classes of protein lipidation. PMID:26334609

  19. An enzyme-linked immunosorbent assay for diagnosis of Fasciola gigantica infection in cattle and buffaloes.

    PubMed

    Krishna Murthy, C M; Souza, Placid E D

    2015-12-01

    The enzyme-linked immunosorbent assay (ELISA) was evaluated for the diagnosis of Fasciola gigantica infection in cattle and buffaloes. The excretory-secretory (E-S Ag) antigen of F. gigantica adult flukes obtained after invitro incubation was used as an antigen. The test was conducted with 276 sera collected from cattle and buffaloes which included 22 sera each from naturally infected cattle and buffaloes (known positive serum) and with similar number of samples with healthy cattle and buffaloes (known negative serum). The positive results were observed in 18 and 19 of the sera from naturally infected cattle and buffaloes with sensitivity of 81.8 and 86.3 % respectively. Out of 188 serum samples which were found negative on faecal examination 32 (34 %) sera of cattle and 40 (42.5 %) sera of buffaloes were found positive by ELISA respectively. The sensitivity of the test was found to be 91.6 and 95.6 % in cattle and buffaloes respectively. PMID:26688653

  20. Biotin-Streptavidin Competition Mediates Sensitive Detection of Biomolecules in Enzyme Linked Immunosorbent Assay

    PubMed Central

    Lakshmipriya, Thangavel; Gopinath, Subash C. B.; Tang, Thean-Hock

    2016-01-01

    Enzyme Linked Immunosorbent Assay (ELISA) is the gold standard assay for detecting and identifying biomolecules using antibodies as the probe. Improving ELISA is crucial for detecting disease-causing agents and facilitating diagnosis at the early stages of disease. Biotinylated antibody and streptavidin-conjugated horse radish peroxide (streptavidin-HRP) often are used with ELISA to enhance the detection of various kinds of targets. In the present study, we used a competition-based strategy in which we pre-mixed free biotin with streptavidin-HRP to generate high-performance system, as free biotin occupies some of the biotin binding sites on streptavidin, thereby providing more chances for streptavidin-HRP to bind with biotinylated antibody. ESAT-6, which is a protein secreted early during tuberculosis infection, was used as the model target. We found that 8 fM of free biotin mixed with streptavidin-HRP anchored the higher detection level of ESAT-6 by four-fold compared with detection without free biotin (only streptavidin-HRP), and the limit of detection of the new method was 250 pM. These results suggest that biotin-streptavidin competition can be used to improve the diagnosis of analytes in other types of sensors. PMID:26954237

  1. Alemtuzumab: validation of a sensitive and simple enzyme-linked immunosorbent assay.

    PubMed

    Jilani, Iman; Keating, Michael; Giles, Francis J; O'Brien, Susan; Kantarjian, Hagop M; Albitar, Maher

    2004-12-01

    Alemtuzumab (MabCampath) is a humanized rat monoclonal antibody that targets the CD52 antigen. It has been approved for the treatment of patients with resistant chronic lymphocytic leukaemia (CLL). Measuring plasma/serum levels of alemtuzumab is important for optimizing the dosing and scheduling of therapy; however, current assays in serum or plasma, based on the capture of alemtuzumab using CD52, are complicated and difficult to adapt for high throughput testing. We developed a simple sandwich enzyme-linked immunosorbent assay (ELISA) to measure alemtuzumab that takes advantage of the remaining rat sequence in alemtuzumab. Using specific anti-rat immunoglobulin (Ig) antibodies (absorbed against human Ig), alemtuzumab levels were measured in the serum and plasma of patients treated with alemtuzumab. Levels were similar between plasma and serum samples, in fresh samples and samples stored at 4 degrees C for 24 h, but were significantly lower in samples stored at room temperature for 24h. The assay was successfully used to determine serum alemtuzumab pre- and post-treatment. This assay is simple and adaptable for high throughput testing, with a limit of detection of 0.05 microg/ml and a coefficient of variation of +/-12.5%. No false positivity was observed in >200 samples tested. This validated assay should help optimize the dosing and scheduling of alemtuzumab therapy. The underlying principles are also applicable to the measurement of other humanized antibodies using an appropriate anti-Ig. PMID:15475065

  2. Recombinant antigen-based enzyme-linked immunosorbent assay for diagnosis of Baylisascaris procyonis larva migrans.

    PubMed

    Dangoudoubiyam, Sriveny; Vemulapalli, Ramesh; Ndao, Momar; Kazacos, Kevin R

    2011-10-01

    Baylisascaris larva migrans is an important zoonotic disease caused by Baylisascaris procyonis, the raccoon roundworm, and is being increasingly considered in the differential diagnosis of eosinophilic meningoencephalitis in children and young adults. Although a B. procyonis excretory-secretory (BPES) antigen-based enzyme-linked immunosorbent assay (ELISA) and a Western blot assay are useful in the immunodiagnosis of this infection, cross-reactivity remains a major problem. Recently, a recombinant B. procyonis antigen, BpRAG1, was reported for use in the development of improved serological assays for the diagnosis of Baylisascaris larva migrans. In this study, we tested a total of 384 human patient serum samples in a BpRAG1 ELISA, including samples from 20 patients with clinical Baylisascaris larva migrans, 137 patients with other parasitic infections (8 helminth and 4 protozoan), and 227 individuals with unknown/suspected parasitic infections. A sensitivity of 85% and a specificity of 86.9% were observed with the BpRAG1 ELISA, compared to only 39.4% specificity with the BPES ELISA. In addition, the BpRAG1 ELISA had a low degree of cross-reactivity with antibodies to Toxocara infection (25%), while the BPES antigen showed 90.6% cross-reactivity. Based on these results, the BpRAG1 antigen has a high degree of sensitivity and specificity and should be very useful and reliable in the diagnosis and seroepidemiology of Baylisascaris larva migrans by ELISA. PMID:21832102

  3. [Shellfish monitoring system for paralytic shellfish toxins using enzyme-linked immunosorbent assay].

    PubMed

    Shinozaki, Takashi; Watanabe, Ryuichi; Kawatsu, Kentaro; Sakurada, Kiyonari; Takahi, Shinya; Ueno, Ken-ichi; Matsushima, Ryoji; Suzuki, Toshiyuki

    2013-01-01

    We investigated the applicability of enzyme-linked immunosorbent assay (PSP-ELISA) using a monoclonal antibody against paralytic shellfish toxins (PST) for screening oysters collected at several coastal areas in Kumamoto prefecture, Japan. Oysters collected between 2007 and 2010 were analyzed by PSP-ELISA. As an alternative calibrant, a naturally contaminated oyster extract was used to quantify toxins in the oyster samples. The toxicity of the calibrant oyster extract determined by the official testing method, mouse bioassay (MBA), was 4 MU/g. Oyster samples collected over 3 years showed a similar toxin profile to the alternative standard, resulting in good agreement between the PSP-ELISA and the MBA. The PSP-ELISA method was better than the MBA in terms of sensitivity, indicating that it may be useful for earlier warning of contamination of oysters by PST in the distinct coastal areas. To use the PSP-ELISA as a screening method prior to MBA, we finally set a screening level at 2 MU/g PSP-ELISA for oyster monitoring in Kumamoto prefecture. We confirmed that there were on samples exceeding the quarantine level (4 MU/g) in MBA among samples quantified as below the screening level by the PSP-ELISA. It was concluded that the use of PSP-ELISA could reduce the numbers of animals needed for MBA testing. PMID:24389470

  4. Development of an enzyme-linked immunosorbent assay specific to Sudan red I.

    PubMed

    Xu, Ting; Wei, Ke Yi; Wang, Jia; Eremin, Sergei A; Liu, Shang Zhong; Li, Qing X; Li, Ji

    2010-10-01

    To obtain antibodies to develop an enzyme-linked immunosorbent assay (ELISA) for the analysis of Sudan red I, haptens were designed and synthesized via four different strategies: (i) attachment of a spacer at the para position of the benzene ring, (ii) attachment of a spacer at the naphthol part, (iii) attachment of a spacer at the hydroxyl group of the Sudan red I molecule, and (iv) use of a fragment of the target molecule. A total of 10 haptens were used to generate immunogens, coating antigens, and polyclonal antibodies. One of the heterologous ELISAs developed exhibited an IC(50) of 1.6 ng/ml, a limit of detection (LOD) of 0.03 ng/ml, and a dynamic range between 0.1 and 14 ng/ml. The assay had 13% cross-reactivity with Para red and negligible cross-reactivity with other structure-related compounds. This ELISA was much more specific than those published previously. This assay was used to determine Sudan red I residues in tomato sauce and chili powder samples after simple pretreatment. The results were validated by comparison with high-performance liquid chromatography (HPLC). The average recoveries of Sudan red I by ELISA and HPLC were in ranges of 70-97% and 82-114%, respectively, indicating suitability of the developed ELISA for screening of Sudan red I in foods. PMID:20522332

  5. Development and Evaluation of a PCR-Enzyme-Linked Immunosorbent Assay for Diagnosis of Human Brucellosis

    PubMed Central

    Morata, Pilar; Queipo-Ortuño, María I.; Reguera, Jose M.; García-Ordoñez, Miguel A.; Cárdenas, Ana; Colmenero, Juan D.

    2003-01-01

    In order to overcome some of the limitations of conventional microbiological techniques in the diagnosis of human brucellosis, a simple PCR-enzyme-linked immunosorbent assay (PCR-ELISA) was developed. After amplification of a 223-bp sequence of a gene that codes for the synthesis of an immunogenetic membrane protein specific for the Brucella genus (BCSP31), the digoxigenin-labeled amplified product was hybridized with a biotinylated capture probe which was complementary to the inner part of the amplicon. The hybrid was captured on streptavidin-coated microtiter plates and detected by using an antidigoxigenin Fab-peroxidase conjugate. The detection limit of the PCR-ELISA in a background of 3.5 μg of human genomic DNA was 10 fg (two bacterial cells). The PCR-ELISA showed an analytical sensitivity higher than that of ethidium bromide staining and equal to that obtained by conventional PCR followed by dot blot hybridization. In 59 peripheral blood samples from 57 consecutive patients with active brucellosis and 113 control samples, the PCR-ELISA was found to be 94.9% sensitive and 96.5% specific, whereas the sensitivity of the blood culture was only 70.1%. Since the assay can be performed in 1 day, is very reproducible, is easily standardized, and avoids the risk of infection in laboratory workers, this PCR-ELISA seems to be a practical and reliable tool for the diagnosis of human brucellosis. PMID:12517839

  6. Quantification of Sorafenib in Human Serum by Competitive Enzyme-Linked Immunosorbent Assay.

    PubMed

    Saita, Tetsuya; Yamamoto, Yuta; Noda, Satoshi; Shioya, Makoto; Hira, Daiki; Andoh, Akira; Morita, Shin-Ya; Terada, Tomohiro; Shin, Masashi

    2015-01-01

    The multikinase inhibitor sorafenib has been used in the treatment of hepatocellular carcinoma, renal cell carcinoma, and differentiated thyroid carcinoma. Here we have demonstrated the production of the first specific antibody against sorafenib. Anti-sorafenib serum was obtained by immunizing mice with an antigen conjugated with bovine serum albumin and carboxylic modified 4-(4-aminophenoxy)-N-methyl-2-pyridinecarboxamide (AMPC) using the N-succinimidyl ester method. Enzyme labeling of sorafenib with horseradish peroxidase was similarly performed using carboxylic modified AMPC. A simple competitive enzyme-linked immunosorbent assay (ELISA) for sorafenib was developed using the principle of direct competition between sorafenib and the enzyme marker for anti-sorafenib antibody, which had been adsorbed by the plastic surface of a microtiter plate. Serum sorafenib concentrations lower than 0.04 µg/mL were reproducibly measurable using the ELISA. This ELISA was specific to sorafenib and showed very slight cross-reactivity (2.5%) with a major metabolite, sorafenib N-oxide. The values of serum sorafenib levels from 32 patients measured by this ELISA were comparable with those measured by HPLC, and there was a strong correlation between the values determined by the two methods (Y=1.016X-0.137, r=0.979). The specificity and sensitivity of the ELISA for sorafenib should provide a valuable new tool for use in therapeutic drug monitoring and pharmacokinetic studies of sorafenib. PMID:26521829

  7. [An urease enzyme linked immunosorbent assay for detection of Helicobacter pylori infection].

    PubMed

    Ding, S Z; Jia, B Q; Liu, X G

    1993-05-01

    A sensitive and specific serological diagnostic test for Helicobacter pylori infection has been developed and validated in 120 patients with dyspeptic symptoms undergoing endoscopy. This test is to use urease, a protein unique to H. pylori, as the basis for the enzyme linked immunosorbent assay (ELISA) that detects serum H. pylori urease antibodies. The ELISA mean optical density (OD) in H. pylori-positive group is higher than that in H. pylori-negative group (0.57 +/- 0.23 vs 0.24 +/- 0.15, P < 0.001), a cut-off 0.3 OD yields a sensitivity of 95% and a specificity of 93%. Serum absorption test showed that Escherichia coli, Klebsiella pneumonia, Proteus mirabilis, Yersinia enterocolotica, Pseudomonas aeruginosa cell lysate do not influence serum H. pylori urease antibody level, though they all have urease except E. coli. The result implied that H. pylori urease can be a good antigen to detect serum H. pylori antibody and it would be useful for epidemiological survey and routine diagnostic approach. Nearly half of the blood donors showed positive result with H. pylori urease antibody. It is suggested that H. pylori infection is quite common in the asymptomatic population. PMID:8269756

  8. Biotin-streptavidin enzyme-linked immunosorbent assay for detecting Tetrabromobisphenol A in electronic waste.

    PubMed

    Bu, Dan; Zhuang, Huisheng; Zhou, Xinchu; Yang, Guangxin

    2014-03-01

    Tetrabromobisphenol A (TBBPA) is a widely used brominated flame retardant. A sensitive and selective indirect competitive biotin-streptavidin-amplified enzyme-linked immunosorbent assay (BA-ELISA) was developed for detecting TBBPA. The optimal hapten of TBBPA was 2-(2,6-dibromo-4-(2-(3,5-dibromo-4-hydroxyphenly)propan-2-yl)) acetic acid. Several physiochemical factors that influence assay performance, such as optimal coupling concentration of immunogen and antibody, organic solvent, ionic strength, and pH, were studied and optimized. The limit of detection (IC10) was 0.027 ng/mL and the median inhibitory concentration (IC50) was 0.58 ng/mL. The BA-ELISA was highly selective, with low cross-reactivity with TBBPA analogs. Finally, the assay was used to detect TBBPA in electronic waste samples. The results are consistent with those using liquid chromatography, which proves that the proposed immunoassay is accurate and receptive. This BA-ELISA method is suitable for the rapid and sensitive screening of TBBPA in environmental monitoring. PMID:24468340

  9. Enzyme-linked immunosorbent assay by image analysis using a charge-coupled device array detector.

    PubMed

    Sánchez, F G; Díaz, A N; Lovillo, J

    1996-07-15

    This paper describes a fluorescence enzyme-linked immunosorbent assay (ELISA) for the quantification of (+/-)-2-(2, 4-dichlorophen-oxy)propionic methyl ester (dichlorprop methyl ester). Antibodies for dichlorprop methyl ester were produced by immunizing rabbits with a conjugate of dichlorprop methyl ester with bovine serum albumin. Data acquisition on microtiter wells is performed by a spectrofluorometer through a fiber optic and by a charge-coupled device camera. A correlation was obtained between the image analysis data on ELISA and the data acquired by the spectrofluorometer. The results demonstrate that the fluorescence image analysis performed by the charge-coupled device detector is applicable to ELISA, and the analysis time, sensitivity, and precision of the ELISA procedure are compared to conventional fluorescence ELISA performed by the spectrofluorometer. The ELISA procedure was selective for structurally similar compounds or usually found in formulation pesticides. Concentrations for 50% displacement curves were dichlorprop, 83.59 microg/ml, and 2,4,5-T, 388.23 microg/ml; triclopyr, ioxynil, bentazone, and MCPA had no response. PMID:8660618

  10. Comparison of Mycoplasma arthritidis strains by enzyme-linked immunosorbent assay, immunoblotting, and DNA restriction analysis.

    PubMed Central

    Washburn, L R; Voelker, L L; Ehle, L J; Hirsch, S; Dutenhofer, C; Olson, K; Beck, B

    1995-01-01

    Twenty Mycoplasma arthritidis strains or isolates were compared by a combination of enzyme-linked immunosorbent assay by an antiserum adsorption technique, Western immunoblotting, and restriction analysis of chromosomal DNA. Antigenic markers that defined strains related to strains 158p10p9, PG6, and H606 were identified. In addition, restriction analysis allowed all 20 strains to be divided into six groups. Results of restriction analysis corresponded generally with antigenic similarities, although the former did not allow grouping with as fine a precision as the latter. However, intrastrain antigenic variability, which is common among many Mycoplasma species, including M. arthritidis, introduced a complicating factor into our attempts at antigenic analysis. While serologic and antigenic analyses remain useful, we recommend that they be used with caution and in combination with other techniques for identifying and characterizing new isolates and newly acquired strains. Combinations of these techniques have proven to be useful in our laboratory for quality control and for uncovering interesting relationships among strains subjected to animal passage and their less virulent antecedents and among strains originally classified as the same but obtained from different sources and maintained, sometimes for decades, in different laboratories. PMID:7494014

  11. Development of two enzyme-linked immunosorbent assays for detection of endosulfan residues in agricultural products.

    PubMed

    Wang, Shuo; Zhang, Jun; Yang, Zhiyan; Wang, Junping; Zhang, Yan

    2005-09-21

    Two competitive immunoassays, a laboratory assay based on microwell plates and a field test based on the use of polystyrene tubes, have been developed for the detection of endosulfan in agricultural products. The limit of detection for the microwell plate format was 0.8 +/- 0.1 microg/kg, and the limit of detection for the tube format was 1.6 +/- 0.2 microg/kg. A simple, rapid, and efficient extraction method was employed, and 76-112% recoveries of spiked samples were obtained. Methanol extracts of some agricultural product samples such as grape, carrot, spinach, and tobacco could be analyzed directly by immunoassay after dilution in 0.5% fish skin gelatin-phosphate buffered saline. In contrast, extracts of green tea caused significant interference in the assay, and a number of simple cleanup methods were ineffective in removing interference. However, use of the coagulating reagent polyvinyl pyrrolidone removed the matrix effect effectively. For the validation of the enzyme-linked immunosorbent assay (ELISA) tests, samples were analyzed by ELISA and gas chromatography (GC) after solid phase extraction. The relationship between data obtained using the tube assay and microwell assay was good (the lowest r(2) value was 0.94), and also, the immunoassay assay data correlated well with data obtained from GC analysis (the lowest r(2) value was 0.93). The developed immunoassay methods are the suitable methods for the rapid quantitative and reliable determination of endosulfan residues in agricultural products. PMID:16159161

  12. Direct immunofluorescence and enzyme-linked immunosorbent assays for evaluating chlorinated hydrocarbon degrading bacteria

    SciTech Connect

    Brigmon, R.L.; Franck, M.M.; Brey, J.; Fliermans, C.B.; Scott, D.; Lanclos, K.

    1997-06-01

    Immunological procedures were developed to enumerate chlorinated hydrocarbon degrading bacteria. Polyclonal antibodies (Pabs) were produced by immunizing New Zealand white rabbits against 18 contaminant-degrading bacteria. These included methanotrophic and chlorobenzene (CB) degrading species. An enzyme-linked immunosorbent assay (ELISA) was used to test for specificity and sensitivity of the Pabs. Direct fluorescent antibodies (DFAs) were developed with these Pabs against select methanotrophic bacteria isolated from a trichloroethylene (TCE) contaminated landfill at the Savannah River Site (SRS) and cultures from the American Type Culture Collection (ATCC). Analysis of cross reactivity testing data showed some of the Pabs to be group specific while others were species specific. The threshold of sensitivity for the ELISA is 105 bacteria cells/ml. The DFA can detect as few as one bacterium per ml after concentration. Results from the DFA and ELISA techniques for enumeration of methanotrophic bacteria in groundwater were higher but not significantly different (P < 0.05) compared to indirect microbiological techniques such as MPN. These methods provide useful information on in situ community structure and function for bioremediation applications within 1--4 hours of sampling.

  13. Sandwich-dot enzyme-linked immunosorbent assay for the detection of canine distemper virus

    PubMed Central

    Li, Zhi; Zhang, Yanlong; Wang, Huiguo; Jin, Jinhua; Li, Wenzhe

    2013-01-01

    A sandwich-dot enzyme-linked immunosorbent assay (dot ELISA) was developed for the detection of canine distemper virus (CDV). In 56 dogs suspected to have CD the rates of detection of CDV antigen in samples of blood lymphocytes and palpebral conjunctiva by dot ELISA and ELISA were, respectively, 91% (49/54) and 81% (44/54) for the lymphocyte samples and 88% (28/32) and 75% (24/32) for the conjunctival samples. The CDV detection limits were 10 ng/50 μL for dot ELISA and 40 ng/50 μL for ELISA. The reliability of dot ELISA relative to electron microscopy was 96% with 22 samples: all 21 samples in which CDV particles were observed by electron microscopy yielded positive results with dot ELISA; the single sample in which particles were not observed yielded false-positive results with dot ELISA. The results indicate that the dot ELISA developed can serve as a reliable rapid diagnostic test in suspected cases of CD and also be useful for epidemiologic surveillance of the disease. PMID:24124274

  14. Development a monoclonal antibody-based enzyme-linked immunosorbent assay for screening carotenoids in eggs.

    PubMed

    Peng, Dapeng; Liao, Feng; Pan, Yuanhu; Chen, Dongmei; Liu, Zhenli; Wang, Yulian; Yuan, Zonghui

    2016-07-01

    In this study, a monoclonal antibody (mAb) with broad-specificity against several carotenoid analogs with equal or similar efficacy was prepared. The obtained mAb C11, with the IgG1 isotype, showed cross-reactivity (CR) with canthaxanthin (100%), β-ionone acid (140.4%), β-carotene (92.9%), capsanthin (90.1%), β-apo-8'-carotenal (92.7%), and xanthophyll (95.8%). Using the mAb C11, a highly sensitive and inexpensive indirect competitive enzyme linked immunosorbent assay (ic-ELISA) was developed with a simple sample preparation procedure for the simultaneous detection of these carotenoid compounds in eggs. The limit of detection of the various carotenoids ranged from 1.31mgkg(-1) to 1.48mgkg(-1). Recoveries from egg yolks spiked with the above carotenoids ranged from 91.8% to 113.3%, with coefficients of variation (CVs) of less than 14.8%. These results suggest that the developed ic-ELISA is a sensitive, specific, accurate, and inexpensive method that is suitable for the screening of carotenoid residues in routine monitoring. PMID:26920278

  15. Paper-based colorimetric enzyme linked immunosorbent assay fabricated by laser induced forward transfer

    PubMed Central

    Katis, Ioannis N.; Holloway, Judith A.; Madsen, Jens; Faust, Saul N.; Garbis, Spiros D.; Smith, Peter J. S.; Voegeli, David; Bader, Dan L.; Eason, Robert W.; Sones, Collin L.

    2014-01-01

    We report the Laser Induced Forward Transfer (LIFT) of antibodies from a liquid donor film onto paper receivers for application as point-of-care diagnostic sensors. To minimise the loss of functionality of the active biomolecules during transfer, a dynamic release layer was employed to shield the biomaterial from direct exposure to the pulsed laser source. Cellulose paper was chosen as the ideal receiver because of its inherent bio-compatibility, liquid transport properties, wide availability and low cost, all of which make it an efficient and suitable platform for point-of-care diagnostic sensors. Both enzyme-tagged and untagged IgG antibodies were LIFT-printed and their functionality was confirmed via a colorimetric enzyme-linked immunosorbent assay. Localisation of the printed antibodies was exhibited, which can allow the creation of complex 2-d patterns such as QR codes or letters for use in a final working device. Finally, a calibration curve was determined that related the intensity of the colour obtained to the concentration of active antibodies to enable quantitative assessment of the device performance. The motivation for this work was to implement a laser-based procedure for manufacturing low-cost, point-of-care diagnostic devices on paper. PMID:24926392

  16. Single-dilution enzyme-linked immunosorbent assay for quantification of antigen-specific salmonid antibody.

    PubMed

    Alcorn, S W; Pascho, R J

    2000-05-01

    An enzyme-linked immunosorbent assay (ELISA) was developed on the basis of testing a single dilution of serum to quantify the level of antibody to the p57 protein of Renibacterium salmoninarum in sockeye salmon (Oncorhynchus nerka). The levels of antibody were interpolated from a standard curve constructed by relating the optical densities (OD) produced by several dilutions of a high-titer rainbow trout (O. mykiss) antiserum to the p57 protein. The ELISA OD values produced by as many as 36 test sera on each microplate were compared with the standard curve to calculate the antigen-specific antibody activity. Repeated measurements of 36 samples on 3 microplates on each of 6 assay dates indicated that the mean intraassay coefficient of variation (CV) was 6.68% (range, 0-23%) and the mean interassay CV was 8.29% (range, 4-16%). The antibody levels determined for the serum sample from 24 sockeye salmon vaccinated with a recombinant p57 protein generally were correlated with the levels determined by endpoint titration (r2 = 0.936) and with results from another ELISA that was based on extrapolation of antibody levels from a standard curve (r2 = 0.956). The single-dilution antibody ELISA described here increases the number of samples that can be tested on each microplate compared with immunoassays based on analysis of several dilutions of each test serum. It includes controls for interassay standardization and can be used to test fish weighing <3 g. PMID:10826838

  17. An enzyme-linked immunosorbent assay for hypoxia marker binding in tumours.

    PubMed Central

    Raleigh, J. A.; La Dine, J. K.; Cline, J. M.; Thrall, D. E.

    1994-01-01

    An enzyme-linked immunosorbent assay (ELISA) has been developed for measuring the in vivo binding of a hexafluorinated 2-nitroimidazole (CCI-103F) in tumour tissue biopsies. The binding of CCI-103F is believed to reflect the presence of hypoxia in tumours. The ELISA provides a sensitive and convenient method of measuring CCI-103F binding which does not require the injection of radioactive reagents. The ELISA is based on reagents prepared from synthetic antigens formed by the reductive activation and binding of CCI-103F to proteins in novel test tube experiments. Calibration of the ELISA involved comparing the ELISA with the radioactivity contained either in protein-CCI-103F adducts formed in vitro with tritiated CCI-103F or in tissues isolated from a tumour-bearing dog which had been injected with tritium-labelled CCI-103F. The two approaches to calibration are compared. The scope and limitation of the ELISA for measuring the binding of CCI-103F is discussed and an example of the application of the ELISA to measuring changes in tumour hypoxia in canine patients undergoing fractionated radiation therapy is presented. Images Figure 3 PMID:8286212

  18. Gluten determination by gliadin enzyme-linked immunosorbent assay kit: interlaboratory study.

    PubMed

    Gabrovská, Dana; Rysová, Jana; Filová, Vanda; Plicka, Jan; Cuhra, Petr; Kubík, Martin; Barsová, Sona

    2006-01-01

    An interlaboratory study with 10 participants was performed to obtain validation and performance data for an enzyme-linked immunosorbent assay (ELISA) kit developed for quantitative gluten determination in foods. The ELISA kit used for this study is based on 2 monoclonal and 1 polyclonal antibody developed by Immunotech, a Beckman Coulter Co. This kit did not show any false positive results or cross-reactivity with oat, rice, maize, and buckwheat. The gliadin standard from the Working Group on Prolamin Analysis and Toxicity was included in the kit as reference material for calibration. All participants obtained a gliadin ELISA kit with Standard Operational Procedure and a form for recording test results. The study included 13 samples labeled as "gluten-free" and 2 samples spiked by wheat flour. Seven samples had gliadin content below the limit of quantitation (LOQ) of the method, and 1 sample exceeded the highest calibration level. Gliadin content in the range from 10 to 157 mg/kg (1st day) and from 11 to 183 mg/kg (2nd day) was found in 7 samples (including 2 spiked samples). Results of these samples were used for further statistical analysis and evaluation. The Cochran, Dixon, and Mandel statistical tests were applied for detection of outliers. The LOQ of the kit was estimated. PMID:16512241

  19. Enzyme-linked immunosorbent assay for ultratrace determination of antibiotics in aqueous samples.

    PubMed

    Kumar, Kuldip; Thompson, Anita; Singh, Ashok K; Chander, Yogesh; Gupta, Satish C

    2004-01-01

    Two commercially available enzyme-linked immunosorbent assay (ELISA) kits that are commonly used for tylosin or tetracycline residues in meat and milk were adapted for ultratrace analysis of these antibiotics in surface and ground waters. These two antibiotics are commonly fed to swine, turkeys, and cattle at subtherapeutic doses for growth promotion purposes. Both ELISA techniques were found to be highly sensitive and selective for the respective antibiotics with detection limits of 0.10 and 0.05 microg L(-1) for tylosin and tetracycline, respectively. The recovery of both tylosin and tetracycline from spiked samples of lake waters, runoff samples, soil saturation extracts, and nanopure water was close to 100%. Tetracycline ELISA was highly specific for tetracycline and chlortetracycline but not for other forms of tetracycline (oxytetracycline, demeclocycline, and doxycycline). Analysis of a few liquid swine manure samples by liquid chromatography-mass spectrometry (LC-MS) showed lower concentrations for chlortetracycline as compared with concentrations obtained using ELISA. However, the concentrations of tylosin from ELISA were comparable with that of LC-MS. The lower concentrations of chlortetracycline obtained by LC-MS in manure samples indicate the presence of other similar or transformed compounds that were detected by ELISA but not determined by LC-MS. These results indicate that both ELISA kits can be useful tools for low-cost screening of tylosin, tetracycline, and chlortetracycline in environmental waters. Furthermore, both ELISA procedures are rapid, portable, and easily adaptable for testing of multiple samples simultaneously. PMID:14964379

  20. Magnetic nanoparticle based purification and enzyme-linked immunosorbent assay using monoclonal antibody against enrofloxacin

    PubMed Central

    Kim, Nam-Gun; Kim, Myeong-Ae; Park, Young-Il; Jung, Tae-Sung; Son, Seong-Wan; So, ByungJae

    2015-01-01

    Monoclonal anti-enrofloxacin antibody was prepared for a direct competitive enzyme-linked immunosorbent assay (ELISA) and purification system using monoclonal antibody (mAb) coupled magnetic nanoparticles (MNPs). The IC50 values of the developed mAb for enrofloxacin (ENR), ciprofloxacin, difloxacin, sarafloxacin, pefloxacin, and norfloxacin were 5.0, 8.3, 9.7, 21.7, 36.0, and 63.7 ng/mL, respectively. The lowest detectable level of ENR was 0.7 ng/mL in the prepared ELISA system. To validate the developed ELISA in the food matrix, known amounts of ENR were spiked in meat and egg samples at 10, 20 and 30 ng/mL. Recoveries for ENR ranged from 72.9 to 113.16% with a coefficient of variation (CV) of 2.42 to 10.11%. The applicability of the mAb-MNP system was verified by testing the recoveries for ENR residue in three different matrices. Recoveries for ENR ranged from 75.16 to 86.36%, while the CV ranged from 5.08 to 11.53%. Overall, ENR-specific monoclonal antibody was prepared and developed for use in competitive to ELISAs for the detection of ENR in animal meat samples. Furthermore, we suggest that a purification system for ENR using mAb-coupled MNPs could be useful for determination of ENR residue in food. PMID:26040610

  1. Comparison of enzyme-linked immunosorbent assay with enzyme-linked fluorescence assay with automated readers for detection of rubella virus antibody and herpes simplex virus.

    PubMed

    Shekarchi, I C; Sever, J L; Nerurkar, L; Fuccillo, D

    1985-01-01

    The enzyme-linked immunosorbent assay (ELISA) was compared with the enzyme-linked fluorescence assay (ELFA) for the detection of rubella antibody and herpes simplex virus antigen. Test parameters, specimens, antigen or antibody, and conjugates for the two types of assays were identical except that p-nitrophenyl phosphate was used as the substrate for the ELISA and 4-methylumbelliferyl phosphate was used as the substrate for ELFA. Automated readers were used for both assays. Antibody titers and sensitivity of antigen detection were quite similar for ELISA and ELFA. ELFA for rubella antibody, however, could be conducted with less antigen or shorter substrate incubation time (5 min for ELFA versus 30 min for ELISA). For herpes simplex virus antigen detection, ELFA could also be read after a shorter substrate incubation time (15 min for ELFA versus 30 min for ELISA). Clear polystyrene microtiter plates routinely used for ELISA could be used for ELFA, but clear polyvinyl chloride plates had high background fluorescence. Black polystyrene and polyvinyl chloride plates gave lower background fluorescence than did clear plates. ELFA is of particular value as a substitute for ELISAs in which long substrate incubations are required or antigens of only low titer are available. PMID:2981902

  2. A sandwich enzyme-linked immunosorbent assay for the detection of almonds in foods.

    PubMed

    Hlywka, J J; Hefle, S L; Taylor, S L

    2000-02-01

    An enzyme-linked immunosorbent assay was developed to detect almonds as potential allergenic contaminants in food. Polyclonal antibodies directed against roasted almonds were partially purified from immunized sheep and rabbits and used as capture and secondary antibodies, respectively, in a sandwich-type, 96-well plate format. Food samples and almond-spiked samples were extracted 1:10 in phosphate-buffered saline at 60 degrees C for 2 h, centrifuged, and applied to wells coated with sheep anti-almond antibody. After incubation, washing, and the addition of rabbit anti-almond antibody, the amount of almond present was detected with the subsequent addition of goat anti-rabbit immunoglobulin G-alkaline phosphatase conjugate and p-nitrophenyl phosphate substrate. Plate absorbances were read at 410 nm, and standard curves were developed in all matrices to quantify unknowns. Antibodies developed were specific for almond; however, some cross-reactivity was observed with extracts of some tree nuts and sesame seeds. Sodium dodecylsulfate-polyacrylamide gel electrophoresis and Western immunoblotting indicated that sheep anti-almond antibody recognized proteins extracted from black walnuts, Brazil nuts, cashews, hazelnuts, macadamia nuts, pistachios, and sesame seeds in addition to those from almond. The assay was optimized to detect less than 1 ppm of almond and was used successfully to determine almond residues in cereal and chocolate without cross-reacting interferences. A retail survey of 20 brands of cereal demonstrated that the assay produced statistically consistent results. This assay provides a useful quality control tool for the food industry for the protection of consumers allergic to almonds. PMID:10678432

  3. Correlation between centromere protein-F autoantibodies and cancer analyzed by enzyme-linked immunosorbent assay

    PubMed Central

    2013-01-01

    Background Centromere protein-F (CENP-F) is a large nuclear protein of 367 kDa, which is involved in multiple mitosis-related events such as proper assembly of the kinetochores, stabilization of heterochromatin, chromosome alignment and mitotic checkpoint signaling. Several studies have shown a correlation between CENP-F and cancer, e.g. the expression of CENP-F has been described to be upregulated in cancer cells. Furthermore, several studies have described a significant correlation between the expression of autoantibodies to CENP-F and cancer. Methods Autoantibodies to CENP-F were detected in a small number of samples during routine indirect immunofluorescence (IIF) analysis for anti-nuclear antibodies (ANA) using HEp-2 cells as substrate. Using overlapping synthetic peptides covering a predicted structural maintenance of chromosomes (SMC) domain, we developed an enzyme-linked immunosorbent assay (ELISA) for detection of CENP-F antibodies. Results Analyzing the reactivity of the sera positive in IIF for CENP-F antibodies to overlapping CENP-F peptides, we showed that autoantibodies to several peptides correlate with the presence of antibodies to CENP-F and a diagnosis of cancer, as increased CENP-F antibody expression specific for malignant cancer patients to five peptides was found (A9, A12, A14, A16, A27). These antibodies to CENP-F in clinical samples submitted for ANA analysis were found to have a positive predictive value for cancer of 50%. Furthermore, the expression of cancer-correlated CENP-F antibodies seemed to increase as a function of time from diagnosis. Conclusion These results conform to previous findings that approximately 50% of those patients clinically tested for ANA analyses who express CENP-F antibodies are diagnosed with cancer, confirming that these antibodies may function as circulating tumor markers. Thus, a peptide-based CENP-F ELISA focused on the SMC domain may aid in identifying individuals with a potential cancer. PMID:23978088

  4. Development of Two Antibody Detection Enzyme-Linked Immunosorbent Assays for Serodiagnosis of Human Chronic Fascioliasis

    PubMed Central

    Cabán-Hernández, Kimberly; Gaudier, José F.; Ruiz-Jiménez, Caleb

    2014-01-01

    Coprological examination based on egg detection in stool samples is currently used as the gold standard for the diagnosis of human fascioliasis. However, this method is not effective during the acute phase of the disease and has poor sensitivity during the chronic phase. Serodiagnosis has become an excellent alternative to coprological examination in efforts to combat the effects of fascioliasis on human and animal health. Two novel recombinant Fasciola hepatica proteins, i.e., a ferritin (FhFtn-1) and a tegument-associated protein (FhTP16.5), were used as antigens to develop in-house enzyme-linked immunosorbent assay (ELISA) methods. The assays were optimized and validated using 152 serum samples from humans with a known infection status, including healthy subjects, patients with chronic fascioliasis, and patients with other parasitic diseases. The FhFtn-1 ELISA was shown to be 96.6% sensitive and 95.7% specific; the respective parameters for the FhTP16.5 ELISA were 91.4% and 92.4%. The performances of the FhFtn-1 and FhTP16.5 ELISAs were compared with that of an available commercial test (the DRG test) using a subset of serum samples. Our in-house tests were slightly more sensitive than the DRG test in detecting antibodies against F. hepatica, but the differences were not statistically significant. In conclusion, the present study provides evidence for the potential of the FhFtn-1 and FhTP16.5 ELISAs as diagnostic tools for human fascioliasis, as might be implemented in conjunction with standard assays for large-scale screenings in areas where the disease is endemic and for the detection of occasional cases in clinical laboratories. PMID:24353000

  5. Characterization of enterotoxigenic Bacteroides fragilis by a toxin-specific enzyme-linked immunosorbent assay.

    PubMed Central

    Van Tassell, R L; Lyerly, D M; Wilkins, T D

    1994-01-01

    Within the past decade, certain strains of Bacteroides fragilis have been associated with diarrhea in humans and cytotoxic activity on certain colon carcinoma cell lines. An enzyme-linked immunosorbent assay (ELISA) for detecting the enterotoxin of B. fragilis in cultures and stools was developed by using high-titer monospecific goat and rabbit antitoxins in an indirect format. The lower limit of detection for purified toxin was approximately 0.05 micrograms/ml; the linear range was from 0.05 to 10 microgram/ml. Using the ELISA to screen cultures of toxigenic and nontoxigenic strains of B. fragilis, we observed 100% correlation with 16 known toxigenic strains which had various cytotoxic activities on HT-29 cells. In addition, we found 6 of 62 previously untested strains also to be positive in both assays. Stability studies revealed that although the cytotoxic activities of crude and purified toxin preparations incubated at elevated temperatures were rapidly lost, the ELISA responses were not significantly reduced. Sodium dodecyl sulfate(SDS)-polyacrylamide gel electrophoresis and SDS-capillary electrophoresis showed that the purified toxin autodigested to several stable peptides. Studies on partially purified membranes from the toxigenic strains revealed the presence of several membrane-associated components which were noncytotoxic but strongly immunoreactive in the ELISA. Preliminary studies with spiked feces indicated that the ELISA may be useful for screening not only cultures for the enterotoxigenic B. fragilis but also stool specimens. Ongoing studies are focusing on determining the nature of the toxin's apparent proteolytic capabilities and investigating the feasibility of using the ELISA on stool specimens from healthy and diarrheic humans. Images PMID:8556504

  6. Development of Rapid Enzyme-Linked Immunosorbent Assays for Detection of Antibodies to Burkholderia pseudomallei

    PubMed Central

    Suttisunhakul, Vichaya; Wuthiekanun, Vanaporn; Brett, Paul J.; Khusmith, Srisin; Day, Nicholas P. J.; Burtnick, Mary N.; Limmathurotsakul, Direk

    2016-01-01

    Burkholderia pseudomallei, the causative agent of melioidosis, is an environmental bacillus found in northeast Thailand. The mortality rate of melioidosis is ∼40%. An indirect hemagglutination assay (IHA) is used as a reference serodiagnostic test; however, it has low specificity in areas where the background seropositivity of healthy people is high. To improve assay specificity and reduce the time for diagnosis, four rapid enzyme-linked immunosorbent assays (ELISAs) were developed using two purified polysaccharide antigens (O-polysaccharide [OPS] and 6-deoxyheptan capsular polysaccharide [CPS]) and two crude antigens (whole-cell [WC] antigen and culture filtrate [CF] antigen) of B. pseudomallei. The ELISAs were evaluated using serum samples from 141 culture-confirmed melioidosis patients from Thailand along with 188 healthy donors from Thailand and 90 healthy donors from the United States as controls. The areas under receiver operator characteristic curves (AUROCC) using Thai controls were high for the OPS-ELISA (0.91), CF-ELISA (0.91), and WC-ELISA (0.90), while those of CPS-ELISA (0.84) and IHA (0.72) were lower. AUROCC values using U.S. controls were comparable to those of the Thai controls for all ELISAs except IHA (0.93). Using a cutoff optical density (OD) of 0.87, the OPS-ELISA had a sensitivity of 71.6% and a specificity of 95.7% for Thai controls; for U.S. controls, specificity was 96.7%. An additional 120 serum samples from tuberculosis, scrub typhus, or leptospirosis patients were evaluated in all ELISAs and resulted in comparable or higher specificities than using Thai healthy donors. Our findings suggest that antigen-specific ELISAs, particularly the OPS-ELISA, may be useful for serodiagnosis of melioidosis in areas where it is endemic and nonendemic. PMID:26912754

  7. Enzyme-linked immunosorbent assay for coproantigen detection of Trichuris vulpis in dogs.

    PubMed

    Elsemore, David A; Geng, Jinming; Flynn, Laurie; Cruthers, Larry; Lucio-Forster, Araceli; Bowman, Dwight D

    2014-03-26

    Infections with Trichuris vulpis, the canine whipworm, may be challenging to diagnose even though characteristic bipolar eggs are shed by mature worms and may be recovered from feces. Decreased detection sensitivities because of using flotation solutions with specific gravities <1.3 and a lengthy prepatent period can lessen the diagnostician's ability to detect infection. Coproantigen detection in feces is becoming an accepted form of diagnosing parasitic infections and can circumvent some of the factors that affect egg recovery. The development of an enzyme-linked immunosorbent assay (ELISA) for the detection of whipworm-specific coproantigens in the feces of dogs with experimental and natural T. vulpis infections is reported herein. Whipworm-specific coproantigens were evidenced in feces from experimentally infected dogs using the newly developed ELISA starting as early as day 23 postinfection, while eggs were not detected in feces until day 69. In addition, 1,156 field fecal samples were tested using fecal flotation methods and the newly developed whipworm ELISA. Of these, 27 samples were found by flotation to be whipworm egg positive, while 35 had detectable antigen on the ELISA. Discrepant results were obtained in 12 samples; 2 egg-positive samples tested ELISA negative, and 10 ELISA-positive samples did not contain detectable egg levels. Using the fecal ELISA for the detection of whipworms in dogs should allow for earlier detection of infection, aid the identification of cases in the face of low egg shedding, and increase detection sensitivity as most commercial laboratories are using flotation solutions not optimal for T. vulpis egg detection. PMID:24670954

  8. Enzyme-linked immunosorbent assay (ELISA) for the anthropogenic marker isolithocholic acid in water.

    PubMed

    Baldofski, Stefanie; Hoffmann, Holger; Lehmann, Andreas; Breitfeld, Stefan; Garbe, Leif-Alexander; Schneider, Rudolf J

    2016-11-01

    Bile acids are promising chemical markers to assess the pollution of water samples with fecal material. This study describes the optimization and validation of a direct competitive enzyme-linked immunosorbent assay for the bile acid isolithocholic acid (ILA). The quantification range of the optimized assay was between 0.09 and 15 μg/L. The assay was applied to environmental water samples. Most studies until now were focused on bile acid fractions in the particulate phase of water samples. In order to avoid tedious sample preparation, we undertook to evaluate the dynamics and significance of ILA levels in the aqueous phase. Very low concentrations in tap and surface water samples made a pre-concentration step necessary for this matrix as well as for wastewater treatment plant (WWTP) effluent. Mean recoveries for spiked water samples were between 97% and 109% for tap water and WWTP influent samples and between 102% and 136% for WWTP effluent samples. 90th percentiles of intra-plate and inter-plate coefficients of variation were below 10% for influents and below 20% for effluents and surface water. ILA concentrations were quantified in the range of 33-72 μg/L in influent, 21-49 ng/L in effluent and 18-48 ng/L in surface water samples. During wastewater treatment the ILA levels were reduced by more than 99%. ILA concentrations of influents determined by ELISA and LC-MS/MS were in good agreement. However, findings in LC-ELISA experiments suggest that the true ILA levels in concentrated samples are lower due to interfering effects of matrix compounds and/or cross-reactants. Yet, the ELISA will be a valuable tool for the performance check and comparison of WWTPs and the localization of fecal matter input into surface waters. PMID:27544648

  9. An enzyme linked immunosorbent assay (ELISA) for the determination of the human haptoglobin phenotype

    PubMed Central

    Levy, Nina S.; Vardi, Moshe; Blum, Shany; Miller-Lotan, Rachel; Afinbinder, Yefim; Cleary, Patricia A.; Paterson, Andrew D.; Bharaj, Bhupinder; Snell-Bergeon, Janet K.; Rewers, Marian J.; Lache, Orit; Levy, Andrew P.

    2013-01-01

    Background Haptoglobin (Hp) is an abundant serum protein which binds extracorpuscular hemoglobin (Hb). Two alleles exist in humans for the Hp gene, denoted 1 and 2. Diabetic individuals with the Hp 2-2 genotype are at increased risk of developing vascular complications including heart attack, stroke, and kidney disease. Recent evidence shows that treatment with vitamin E can reduce the risk of diabetic vascular complications by as much as 50% in Hp 2-2 individuals. We sought to develop a rapid and accurate test for Hp phenotype (which is 100% concordant with the three major Hp genotypes) to facilitate widespread diagnostic testing as well as prospective clinical trials. Methods A monoclonal antibody raised against human Hp was shown to distinguish between the three Hp phenotypes in an enzyme linked immunosorbent assay (ELISA). Hp phenotypes obtained in over 8000 patient samples using this ELISA method were compared with those obtained by polyacrylamide gel electrophoresis or the TaqMan PCR method. Results Our analysis showed that the sensitivity and specificity of the ELISA test for Hp 2-2 phenotype is 99.0% and 98.1%, respectively. The positive predictive value and the negative predictive value for Hp 2-2 phenotype is 97.5% and 99.3%, respectively. Similar results were obtained for Hp 2-1 and Hp 1-1 phenotypes. In addition, the ELISA was determined to be more sensitive and specific than the TaqMan method. Conclusions The Hp ELISA represents a user-friendly, rapid and highly accurate diagnostic tool for determining Hp phenotypes. This test will greatly facilitate the typing of thousands of samples in ongoing clinical studies. PMID:23492570

  10. Development of an enzyme-linked immunosorbent assay for vitellogenin of Morelet's crocodile (Crocodylus moreletii).

    PubMed

    Selcer, Kyle W; Nespoli, Lisa M; Rainwater, Thomas R; Finger, Adam G; Ray, David A; Platt, Steven G; Smith, Philip N; Densmore, Llewellyn D; McMurry, Scott T

    2006-05-01

    The purpose of this study was to develop an immunoassay for vitellogenin in Morelet's crocodile (Crocodylus moreletii). Blood was collected from wild-caught crocodiles in Belize. Plasma samples from adult females taken during the breeding season were used for vitellogenin purification and samples from adult males were used for comparison. No differences were detected between males and females for plasma total protein concentration, as measured by Coomassie assay. However, denaturing polyacrylamide gel electrophoresis (SDS-PAGE) revealed that female plasma contained a 210-kDa protein, presumably the vitellogenin monomer, that was absent in adult male plasma. The identity of the putative vitellogenin was confirmed by its cross-reactivity in Western blots with a vitellogenin antiserum that was generated against a conserved vitellogenin peptide sequence. Crocodile vitellogenin was purified by two successive rounds of DEAE chromatography. The purified protein had an apparent molecular mass of 450 kDa, as determined by gel filtration chromatography, and 210 kDa on SDS-PAGE. An indirect enzyme-linked immunosorbent assay (ELISA) was then developed for C. moreletii vitellogenin. The detection limit of the assay was 20.0 ng/mL. The intra- and inter-assay coefficients of variation were 5.3% and 9.8%, respectively. The recovery of vitellogenin diluted into male plasma was 94.7%. The ELISA assay revealed that vitellogenin levels of adult female plasma during the breeding season ranged from 1.8 to 3.1 mg/mL with a mean of 2.5+/-0.25 mg/mL. No vitellogenin was detected in adult male plasma. Induction of vitellogenin in Morelet's crocodile may be a useful model system for field studies of crocodile reproduction and for investigations of endocrine disruption in this species. PMID:16448857

  11. Evaluation of Three Commercial Enzyme-Linked Immunosorbent Assays for Diagnosis of Chagas’ Disease

    PubMed Central

    Oelemann, Walter M. R.; Teixeira, Maria Da Glória M.; Veríssimo Da Costa, Giovani C.; Borges-Pereira, José; De Castro, José Adail F.; Coura, José Rodrigues; Peralta, José Mauro

    1998-01-01

    Chagas’ disease is a common cause of morbidity in Latin American countries. In Brazil, naturally occurring transmission of its etiologic agent, Trypanosoma cruzi, has been almost completely abolished through effective control programs aimed at the triatomid insect vector. Thus, transfusion of blood from infected donors has become the major route for contracting Chagas’ disease due to the socioeconomically motivated migration of residents from areas where the disease is endemic to the larger urban centers. Therefore, the employment of screening tests is mandatory for all blood banks throughout the country. We compared the diagnostic performances of three commercially available screening assays used in routine testing in Brazilian blood banks: the Abbott Chagas antibody enzyme immunoassay (Abbott Laboratórios do Brasil, São Paulo), the BIOELISACRUZI kit (Biolab-Mérieux, Rio de Janeiro, Brazil), and the BIOZIMA Chagas kit (Polychaco S.A.I.C., Buenos Aires, Argentina). The evaluation was performed with sera obtained from chagasic patients and healthy residents of four different areas in Brazil where Chagas’ disease is either endemic or emergent and where clinical manifestations of the disease and circulating parasite strains vary. The results obtained with each kit were compared to matched in-house enzyme-linked immunosorbent assay and immunofluorescence assay data obtained for each sample. Depending on the area under investigation, the three commercial kits produced specificity values between 93.3 and 100.0%, sensitivity values between 97.7 and 100%, and accuracies ranging from 93.6 to 100.0%. PMID:9705367

  12. Assessment of Dextran Antigenicity of Intravenous Iron Preparations with Enzyme-Linked Immunosorbent Assay (ELISA)

    PubMed Central

    Neiser, Susann; Koskenkorva, Taija S.; Schwarz, Katrin; Wilhelm, Maria; Burckhardt, Susanna

    2016-01-01

    Intravenous iron preparations are typically classified as non-dextran-based or dextran/dextran-based complexes. The carbohydrate shell for each of these preparations is unique and is key in determining the various physicochemical properties, the metabolic pathway, and the immunogenicity of the iron-carbohydrate complex. As intravenous dextran can cause severe, antibody-mediated dextran-induced anaphylactic reactions (DIAR), the purpose of this study was to explore the potential of various intravenous iron preparations, non-dextran-based or dextran/dextran-based, to induce these reactions. An IgG-isotype mouse monoclonal anti-dextran antibody (5E7H3) and an enzyme-linked immunosorbent assay (ELISA) were developed to investigate the dextran antigenicity of low molecular weight iron dextran, ferumoxytol, iron isomaltoside 1000, ferric gluconate, iron sucrose and ferric carboxymaltose, as well as isomaltoside 1000, the isolated carbohydrate component of iron isomaltoside 1000. Low molecular weight iron dextran, as well as dextran-based ferumoxytol and iron isomaltoside 1000, reacted with 5E7H3, whereas ferric carboxymaltose, iron sucrose, sodium ferric gluconate, and isolated isomaltoside 1000 did not. Consistent results were obtained with reverse single radial immunodiffusion assay. The results strongly support the hypothesis that, while the carbohydrate alone (isomaltoside 1000) does not form immune complexes with anti-dextran antibodies, iron isomaltoside 1000 complex reacts with anti-dextran antibodies by forming multivalent immune complexes. Moreover, non-dextran based preparations, such as iron sucrose and ferric carboxymaltose, do not react with anti-dextran antibodies. This assay allows to assess the theoretical possibility of a substance to induce antibody-mediated DIARs. Nevertheless, as this is only one possible mechanism that may cause a hypersensitivity reaction, a broader set of assays will be required to get an understanding of the mechanisms that may

  13. An analytical model applied to a multicenter pneumococcal enzyme-linked immunosorbent assay study.

    PubMed

    Plikaytis, B D; Goldblatt, D; Frasch, C E; Blondeau, C; Bybel, M J; Giebink, G S; Jonsdottir, I; Käyhty, H; Konradsen, H B; Madore, D V; Nahm, M H; Schulman, C A; Holder, P F; Lezhava, T; Elie, C M; Carlone, G M

    2000-06-01

    Pneumococcal conjugate vaccines will eventually be licensed after favorable results from phase III efficacy trials. After licensure of a conjugate vaccine for invasive pneumococcal disease in infants, new conjugate vaccines will likely be licensed primarily on the basis of immunogenicity data rather than clinical efficacy. Analytical methods must therefore be developed, evaluated, and validated to compare immunogenicity results accurately within and between laboratories for different vaccines. At present no analytical technique is uniformly accepted and used in vaccine evaluation studies to determine the acceptable level of agreement between a laboratory result and the assigned value for a given serum sample. This multicenter study describes the magnitude of agreement among 12 laboratories quantifying an identical series of 48 pneumococcal serum specimens from 24 individuals (quality-control sera) by a consensus immunoglobulin G (IgG) enzyme-linked immunosorbent assay (ELISA) developed for this study. After provisional or trial antibody concentrations were assigned to the quality-control serum samples for this study, four methods for comparison of a series of laboratory-determined values with the assigned concentrations were evaluated. The percent error between assigned values and laboratory-determined concentrations proved to be the most informative of the four methods. We present guidelines that a laboratory may follow to analyze a series of quality-control sera to determine if it can reproduce the assigned antibody concentrations within an acceptable level of tolerance. While this study focused on a pneumococcal IgG ELISA, the methods that we describe are easily generalizable to other immunological assays. PMID:10834951

  14. Development of Rapid Enzyme-Linked Immunosorbent Assays for Detection of Antibodies to Burkholderia pseudomallei.

    PubMed

    Suttisunhakul, Vichaya; Wuthiekanun, Vanaporn; Brett, Paul J; Khusmith, Srisin; Day, Nicholas P J; Burtnick, Mary N; Limmathurotsakul, Direk; Chantratita, Narisara

    2016-05-01

    Burkholderia pseudomallei, the causative agent of melioidosis, is an environmental bacillus found in northeast Thailand. The mortality rate of melioidosis is ∼40%. An indirect hemagglutination assay (IHA) is used as a reference serodiagnostic test; however, it has low specificity in areas where the background seropositivity of healthy people is high. To improve assay specificity and reduce the time for diagnosis, four rapid enzyme-linked immunosorbent assays (ELISAs) were developed using two purified polysaccharide antigens (O-polysaccharide [OPS] and 6-deoxyheptan capsular polysaccharide [CPS]) and two crude antigens (whole-cell [WC] antigen and culture filtrate [CF] antigen) of B. pseudomallei The ELISAs were evaluated using serum samples from 141 culture-confirmed melioidosis patients from Thailand along with 188 healthy donors from Thailand and 90 healthy donors from the United States as controls. The areas under receiver operator characteristic curves (AUROCC) using Thai controls were high for the OPS-ELISA (0.91), CF-ELISA (0.91), and WC-ELISA (0.90), while those of CPS-ELISA (0.84) and IHA (0.72) were lower. AUROCC values using U.S. controls were comparable to those of the Thai controls for all ELISAs except IHA (0.93). Using a cutoff optical density (OD) of 0.87, the OPS-ELISA had a sensitivity of 71.6% and a specificity of 95.7% for Thai controls; for U.S. controls, specificity was 96.7%. An additional 120 serum samples from tuberculosis, scrub typhus, or leptospirosis patients were evaluated in all ELISAs and resulted in comparable or higher specificities than using Thai healthy donors. Our findings suggest that antigen-specific ELISAs, particularly the OPS-ELISA, may be useful for serodiagnosis of melioidosis in areas where it is endemic and nonendemic. PMID:26912754

  15. Detection of walnut residues in foods using an enzyme-linked immunosorbent assay.

    PubMed

    Niemann, Lynn; Taylor, Steve L; Hefle, Susan L

    2009-08-01

    Tree nuts, including walnuts, can be responsible for allergic reactions. Food manufacturers have the responsibility to declare the presence of walnuts on packaged foods even when trace residues may be present from the use of shared equipment or the adventitious contamination of ingredients. The aim of this study was to develop a rapid, sensitive, and specific enzyme-linked immunosorbent assay (ELISA) method for the detection of walnut protein residues. Mixtures of raw and roasted English walnuts of several varieties were defatted, powdered, and used as separate antigens in sheep and New Zealand white rabbits. An ELISA was developed using the sheep antiroasted walnut serum as the capture reagent and rabbit antiroasted walnut serum as the detector reagent followed by addition of commercial goat anti-rabbit IgG antibody labeled with alkaline phosphatase and subsequent substrate addition. The performance of the ELISA was validated by testing known amounts of walnut (0 to 100 ppm) either spiked into or manufactured into milk chocolate, cookies, muffins, or ice cream. Recoveries of 1 to 100 ppm walnut-in-chocolate ranged from 71.6% to 119%+/- 7% to 16.5%. The walnut ELISA has a detection limit of 1 ppm (1 microg/g) walnut in several food matrices. Substantial cross-reactivity was observed with pecan while minimal cross-reactivity was noted for hazelnut, mustard, mace, and poppy seed among almost 100 foods and food ingredients tested. This walnut ELISA can be used to detect undeclared walnut residues in foods and ingredients and as a tool to validate the effectiveness of allergen control programs for walnuts. PMID:19723237

  16. Sandwich enzyme-linked immunosorbent assay (ELISA) for detection of cashew nut in foods.

    PubMed

    Gaskin, Ferdelie E; Taylor, Steve L

    2011-01-01

    The presence of undeclared cashew can pose a health risk to cashew-allergic consumers. The food industry has the responsibility to declare the presence of cashews on packaged foods even when trace residues are or might be present. The objective of this study was to develop a rapid, sensitive, and specific enzyme-linked immunosorbent assay (ELISA) for the detection of cashew residues. Raw and roasted cashews were defatted and used separately to immunize sheep, goats, and rabbits. The cashew ELISA was developed using sheep and rabbit polyclonal anti-roasted cashew sera as capture and detector reagents, respectively, with visualization through an alkaline phosphatase-mediated substrate reaction. The cashew ELISA was shown to have a limit of quantification of 1 ppm (1 μg cashew/g). The ELISA was highly specific except that substantial cross-reactivity was noted with pistachio and a lesser degree of cross-reactivity was noted with hazelnut. The performance of the ELISA was assessed by manufacturing cookies, ice cream, and milk chocolate with added known amounts (0 to 1000 ppm) of cashew. The mean percent recoveries for ice cream, cookies, and milk chocolate were 118%± 2.9%, 84.3%± 4.0%, and 104%± 3.0%, respectively. In a limited retail survey, 4/5 retail samples with cashew declared on ingredient labels tested positive for cashew compared to 5/36 samples of foods with precautionary labels indicating the possible presence of one or more tree nuts and 0/18 samples without cashew declared on the label in any manner. The cashew ELISA can be used to detect undeclared cashew residue in foods and as a potential tool for the food industry to assess the effectiveness of allergen control strategies and to guarantee compliance with food labeling regulatory requirements. PMID:22416731

  17. Evaluation of various plastic microtiter plates with measles, toxoplasma, and gamma globulin antigens in enzyme-linked immunosorbent assays.

    PubMed Central

    Shekarchi, I C; Sever, J L; Lee, Y J; Castellano, G; Madden, D L

    1984-01-01

    Seventeen lots of microtiter plates which differed in lot, batch, plastic type, or manufacturer were evaluated as solid-phase carriers in enzyme-linked immunosorbent assays for antibodies to measles, toxoplasma, and human gamma globulin. Most plates of polystyrene or polyvinyl chloride were found to give acceptable binding. The final choice depended on the antigen to be attached. Variations in binding between lots, batches, and types of plastic were found. Well-to-well variation was found to be of greater statistical significance than edge effect and should be a consideration in selection of a plate lot for enzyme-linked immunosorbent assays. Lots of plates should be pretested by the investigator to determine whether there is good binding of the antigen to be used and whether there is low plate-to-plate and well-to-well variation. PMID:6199371

  18. Immunodiagnosis of Human Fascioliasis by an Enzyme-Linked Immunosorbent Assay (ELISA) and a Micro-ELISA

    PubMed Central

    Carnevale, Silvana; Rodríguez, Mónica I.; Santillán, Graciela; Labbé, Jorge H.; Cabrera, Marta G.; Bellegarde, Enrique J.; Velásquez, Jorge N.; Trgovcic, Jorge E.; Guarnera, Eduardo A.

    2001-01-01

    Enzyme-linked immunosorbent assay (ELISA) and micro-ELISA were evaluated for their ability to detect anti-Fasciola hepatica antibodies in humans by using excretory-secretory antigen. The sensitivity of each method was 100%, but the specificity was 100% for ELISA and 97% for micro-ELISA. The micro-ELISA could be used as a screening assay and ELISA could be used as a confirmatory method for the serodiagnosis of human fascioliasis. PMID:11139214

  19. PCR–Enzyme-Linked Immunosorbent Assay for Detection and Identification of Campylobacter Species: Application to Isolates and Stool Samples

    PubMed Central

    Metherell, L. A.; Logan, J. M. J.; Stanley, J.

    1999-01-01

    We report a PCR–enzyme-linked immunosorbent assay which identifies Campylobacter species by capture hybridization of a single-stranded 16S rRNA gene amplicon with species-specific probes in a microtiter plate format. Specificities were confirmed for both reference and field strains, but the type strain of Campylobacter coli was atypical. The assay was rapid, informative, and usable with stool-extracted DNA. PMID:9889235

  20. Should South Africa Be Performing Nucleic Acid Testing on HIV Enzyme-Linked Immunosorbent Assay-Negative Samples?▿

    PubMed Central

    Gous, Natasha; Scott, Lesley; Perovic, Olga; Venter, Francios; Stevens, Wendy

    2010-01-01

    The frequency of acute HIV infection (AHI) among HIV-1 enzyme-linked immunosorbent assay (ELISA)-negative samples received from general hospital patient admissions was assessed. Of 3,005 samples pooled for nucleic acid testing, a prevalence of 0.13% was found. Pooled nucleic acid testing may be feasible for low-cost identification of AHI in high-prevalence settings. PMID:20610671

  1. Environmental Technology Verification Report for Abraxis Ecologenia® 17β-Estradiol (E2) Microplate Enzyme-Linked Immunosorbent Assay (ELISA) Test Kits

    EPA Science Inventory

    This verification test was conducted according to procedures specifiedin the Test/QA Planfor Verification of Enzyme-Linked Immunosorbent Assay (ELISA) Test Kis for the Quantitative Determination of Endocrine Disrupting Compounds (EDCs) in Aqueous Phase Samples. Deviations to the...

  2. Application of an enzyme-linked immunosorbent assay (ELISA) to determine paraquat residues in milk, beef, and potatoes

    SciTech Connect

    Van Emon, J.; Seiber, J.; Hammock, B.

    1987-09-01

    The USDA Food Safety and Inspection Service (FSIS) has included paraquat on its list of compounds to be considered for monitoring in foods. However, present methods do not easily accommodate the processing of large numbers of samples, thus limiting routine monitoring of the compound. The conventional method, based on spectrophotometry of reduced paraquat solutions, requires time-consuming sample preparation. Although the advantages of immunoassays for pesticide residue analysis have been pointed out, the reported immunoassays for paraquat have only been applied to cases of clinical poisoning or human exposure assessment. In this study, spiked milk, potato, and beef were analyzed directly, without prior cleanup, by an enzyme-linked immunosorbent assay (ELISA).

  3. Development of a monoclonal antibody-based enzyme-linked immunosorbent assay for luteoloside detection in Flos Lonicerae Japonicae.

    PubMed

    Zhang, Bo; Nan, Tiegui; Zhan, Zhilai; Kang, Liping; Yang, Jian; Yuan, Yuan; Wang, Baomin; Huang, Luqi

    2016-09-01

    Flos Lonicerae Japonicae (FLJ), the flower bud of Lonicera japonica Thunb. (Caprifoliaceae), is a widely used traditional Chinese medicine with various pharmacological activities. Luteoloside is a major active compound and a quality control marker of FLJ. Luteolin-7-O-glucuronide (LG), an analog of luteoloside, was conjugated with bovine serum albumin (BSA) and ovalbumin (OVA) to create the immunogen and coating antigen, respectively. A sensitive and specific monoclonal antibody (mAb), designated as mAb3A4, was generated with LG-BSA. To screen the authenticity and quality of FLJ, an indirect competitive enzyme-linked immunosorbent assay (icELISA) was established. The concentration of luteoloside producing 50 % inhibition and the working range of the icELISA were 42.3 and 9.1-258.1 μg L(-1), respectively. The icELISA showed cross-reactivity values of 2414, 402, 230, and <1 % for LG, baicalin, scutellarin, and other analogs of luteoloside, respectively. The average recovery of luteoloside in the FLJ samples as determined by icELISA ranged from 83.0 to 112.5 %. The luteoloside content was determined for different Lonicera herbal samples with icELISA, and the results were confirmed by high-performance liquid chromatography analysis. Thus, this icELISA is suitable for the quality assurance of FLJ samples. Graphical abstract Specific monoclonal antibody-based enzyme-linked immunosorbent assay for luteoloside. PMID:26892641

  4. Utilization of the enzyme linked immunosorbent assay technique (ELISA) for the diagnosis of typhoid fever.

    PubMed

    Hernández-Velarde, R; Sánchez-Castillo, J; Díaz-Godinez, M C; Muñóz-Hernández, O

    1980-01-01

    50 sera from patients with a bacteriological diagnosis of typhoid fever and 325 sera from healthy individuals were studied to quantify antibodies against S. typhi somatic antigen by means of Widal's technique and the enzyme linked inmmunosorbent assay ELISA and by means of the latter, determine the minimum diagnostic titer. Only 2.3 per cent of the healthy population had titers up to 1:300 while sera from patients varied from 1:150 (2 per cent) to 1:2500. A titer was established by ELISA--1:3000 as suggestive of typhoid fever. Widal's agglutination reaction was positive in 1:160 titers in 50 per cent of patients while ELISA technique was positive in 98 per cent of cases (p < 0.001). ELISA technique was more efficient, rapid and sensitive than Widal's test and its utilization is proposed for the clincial laboratory as the method of choice in the serological diagnosis of typhoid fever. PMID:7000022

  5. Enzyme-Linked Immunosorbent Assay for Quantitating the Humoral Immune Response to the Colonization Factor Antigen of Enterotoxigenic Escherichia coli

    PubMed Central

    Clegg, Steven; Evans, Dolores G.; Evans, Doyle J.

    1980-01-01

    The enzyme-linked immunosorbent assay technique was used to quantitate, in milligrams per milliliter, anti-colonization factor antigen/I (CFA/I) immunoglobulin G (IgG) in acute- and convalescent-phase sera of individuals who experienced diarrhea associated with CFA/I-positive enterotoxigenic Escherichia coli. Purified CFA/I was used as antigen to coat polystyrene Microtiter plate wells for the determination of anti-CFA/I antibody. A reference anti-CFA/I IgG preparation was obtained by affinity chromatography of a high-titered serum with a CFA/I-Sepharose 4B column; IgG was the only class of immunoglobulin detectable in this serum as anti-CFA/I. Goat anti-human IgG conjugated to alkaline phosphatase was used in the enzyme-linked immunosorbent assay. Quantitation of IgG in the reference anti-CFA/I serum was achieved by comparison with a known sample of pure human IgG. Anti-CFA/I in test sera was quantitated by titration with CFA/I-coated Microtiter plate wells in the enzyme-linked immunosorbent assay, using a standard curve obtained with the reference anti-CFA/I serum. Anti-CFA/I IgG in paired sera was determined as percentage of total IgG by using the radial immunodiffusion technique to quantitate total IgG for each test serum. Diarrhea with isolation of CFA/I-positive enterotoxigenic E. coli was associated with a significant rise in serum anti-CFA/I IgG when these values were expressed as either milligrams of IgG per milliliter or as percentage of total IgG, although the response varied quantitatively and nonresponders were detected. None of the matched controls showed an anti-CFA/I IgG response. Further elucidation of the immune response to enterotoxigenic E. coli can now be accomplished by applying these methods to determine the class and specificity of immunoglobulins in external secretions such as saliva and intestinal contents. PMID:6103870

  6. Serological evaluation of thin-layer immunoassay-enzyme-linked immunosorbent assay for antibody detection in human trichinellosis.

    PubMed

    Gómez-Priego, A; Crecencio-Rosales, L; de-La-Rosa, J L

    2000-09-01

    A new immunoenzymatic test, named the thin-layer immunoassay-enzyme-linked immunosorbent assay (TIA-ELISA), was evaluated for antibody detection in human trichinellosis using excretion and secretion products prepared from Trichinella spiralis muscle larvae. Serum samples from people with positive muscle biopsies or symptoms compatible with the disease (n = 8 or 26, respectively), all reactive in enzyme-linked immunoelectrotransfer blot assay (EITB), as well as 67 serum samples from healthy, EITB-negative people, were tested in an ELISA and TIA-ELISA. TIA-ELISA was performed in polystyrene plastic petri dishes by adding dots of 10 microl each of antigen (7 microg/ml) followed by adding diluted serum and the conjugate. Finally, the substrate mixed with agar was added to develop the reaction. Enzymatic by-products were easily detected by the naked eye as defined dots. Sensitivity and specificity were 76 and 94% for ELISA, and both parameters were 91% for TIA-ELISA. The kappa correlation indices for both tests in relation to EITB were 0.73 and 0.80, respectively. The TIA-ELISA can be carried out with common laboratory equipment in 3 h and uses lower quantities of antigen than EITB and ELISA. Since TIA-ELISA is easy to perform, cheap, sensitive, and specific, the test could be an acceptable alternative to use in clinical laboratories lacking specialized equipment needed for ELISA and EITB and in field studies for antibody detection in human trichinellosis. PMID:10973459

  7. Purification and characterisation of a fimbrial haemagglutinin from Bordetella pertussis for use in an enzyme-linked immunosorbent assay.

    PubMed

    Askelöf, P; Granström, M; Gillenius, P; Lindberg, A A

    1982-02-01

    The fimbrial haemagglutinin (F-HA) of Bordetella pertussis grown on solid medium was extracted with 1M sodium acetate for 72 h at 20 degree C, and partially purified by Sephacryl S-300 gel chromatography. A pooled fraction with fimbrial haemmagglutinating activity was shown to contain fimbriae haemagglutinating activity was shown to contain fimbriae of the expected morphology by electron microscopy. Chemical and biological assays showed that the F-HA fraction contained some heat-labile agglutinogen and lipopolysaccharide but no measureable lymphocytosis-promoting factor or heat-labile toxin. The F-HA fraction used as antigen in an enzyme-linked immunosorbent assay (ELISA) permitted the detection of antibodies in convalescent serum from a patient with whooping cough. The impurities, heat-labile agglutinogens and lipopolysaccharide, did not contribute to the ELISA activity. The method for preparation of the F-HA antigen is simple, reproducible and gives a high yield. PMID:6292428

  8. Evaluation of a commercially available enzyme-linked immunosorbent assay for the diagnosis of canine sarcoptic mange.

    PubMed

    Curtis, C F

    2001-02-24

    This study was designed to assess the accuracy of a commercial enzyme-linked immunosorbent assay for the diagnosis of canine scabies. Serum samples from 37 dogs were examined blind; 12 had sarcoptic mange confirmed by the identification of mites in skin scrapings, 12 were atopic (with positive intradermal reactions to one or more aeroallergens, including Dermatophagoides farinae), and 13 were healthy dogs with no history of skin disease. Optical density values of more than 0.16 were considered positive, 0.145 to 0.16 were considered questionable and less than 0.145 were considered negative. Ten of the 12 dogs with scabies were positive, all 12 atopic dogs were negative, and 11 of the 13 healthy dogs were negative and two were questionable. PMID:11289551

  9. Evaluation of an enzyme-linked immunosorbent assay (ELISA) for the serological diagnosis of sarcoptic mange in dogs.

    PubMed

    Lower, K S; Medleau, L M; Hnilica, K; Bigler, B

    2001-12-01

    Canine scabies is a challenging disease to diagnose because sarcoptic mites are hard to find on skin scrapings. The purpose of this study was to evaluate a serologic enzyme-linked immunosorbent assay (ELISA) as an aid in the diagnosis of canine scabies. In addition, serum samples were obtained post treatment to determine the duration and persistence of circulating scabies antibodies after resolution of natural infection. Nineteen dogs diagnosed with sarcoptic mange and 38 control dogs were tested. Sixteen scabies-infested dogs showed positive pretreatment ELISA results (84.2% sensitivity). Thirty-four control dogs showed negative ELISA results (89.5% specificity). In the 11 scabies dogs from which multiple post treatment serum samples were obtained, detectable antibodies were not present 1 month after treatment in four cases, but were present for 1-4.5 months post treatment in seven dogs. Our results suggest that this scabies ELISA test is useful in the diagnosis of canine scabies. PMID:11844220

  10. Use of an indirect enzyme-linked immunosorbent assay for detection of antibodies in sheep naturally infected with Salmonella Abortusovis.

    PubMed

    Wirz-Dittus, Sophie; Belloy, Luc; Doherr, Marcus G; Hüssy, Daniela; Sting, Reinhard; Gabioud, Patricia; Waldvogel, Andreas S

    2010-07-01

    An indirect enzyme-linked immunosorbent assay (ELISA) was modified and validated to detect antibodies against Salmonella Abortusovis in naturally infected sheep. The ELISA was validated with 44 positive and 45 negative control serum samples. Compared with the immunoblot, the sensitivity and specificity of the assay were 98% and 100%, respectively. To follow antibody levels over time, samples from 12 infected ewes were collected at 1, 3, and 10 months after abortion. All animals showed antibody levels above the cutoff value throughout the observation period. One and 3 months after abortion, high antibody levels could be detected in all but one animal, whereas after 10 months, 9 animals had markedly lower but still positive antibody levels. The test characteristics and evidence for the persistence of detectable antibody levels in all infected animals for up to 10 months indicates that the ELISA can be used for herd surveillance testing. PMID:20622222

  11. Application of immunomagnetic particles to enzyme-linked immunosorbent assay (ELISA) for improvement of detection sensitivity of HCG.

    PubMed

    Kuo, Hsiao-Ting; Yeh, Jay Z; Wu, Po-Hua; Jiang, Chii-Ming; Wu, Ming-Chang

    2012-01-01

    This investigation was aimed at using superparamagnetic particles to enzyme-linked immunosorbent assay (SPIO-ELISA) of human chorionic gonadotropin (hCG) to enhance detection sensitivity of hCG. We found that N-(3-dimethyl aminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC) was the best cross-linking reagent to link anti hCG α antibody to superparamagnetic particle (SPIO-anti hCG α antibody immunomagnetic particle). To improve the specificity of the assay, a horse radish peroxidase (HRP)-labeled anti-hCG beta monoclonal antibody was used to detect captured hCG using double antibody sandwich ELISA assay. SPIO-ELISA application to determine hCG increased the sensitivity to 1 mIU/mL, which is a level of sensitivity enabling the diagnosis of pregnancy during the early gestational period. PMID:22963487

  12. A serosurvey using enzyme-linked immunosorbent assay for antibodies against poultry pathogens in ostriches (Struthio camelus) from Zimbabwe.

    PubMed

    Cadman, H F; Kelly, P J; Zhou, R; Davelaar, F; Mason, P R

    1994-01-01

    Horseradish peroxidase-conjugated goat anti-ostrich IgG was raised and used in commercial enzyme-linked immunosorbent assay kits to detect antibodies reactive with 11 poultry pathogens in sera from 149 ostriches from nine farms around Zimbabwe. Antibodies were detected to turkey rhinotracheitis virus (99%), Newcastle disease virus (23%), avian reovirus (19%), infectious bursal disease virus (15%), avian encephalomyelitis virus (15%), Mycoplasma gallisepticum and/or M. synoviae (11%), reticuloendotheliosis virus (10%), Salmonella enteritidis (8%), avian leukosis virus (3%), infectious bronchitis virus (2%), and Pasteurella multocida (< 1%). Although evidence of prior infection with turkey rhinotracheitis and newcastle disease virus was present on all farms tested, there was marked variation between farms in the prevalence of exposure to other poultry pathogens. PMID:7832718

  13. Modified enzyme-linked immunosorbent assay strategy using graphene oxide sheets and gold nanoparticles functionalized with different antibody types.

    PubMed

    Lin, Hongjun; Liu, Yingfu; Huo, Jingrui; Zhang, Aihong; Pan, Yiting; Bai, Haihong; Jiao, Zhang; Fang, Tian; Wang, Xin; Cai, Yun; Wang, Qingming; Zhang, Yangjun; Qian, Xiaohong

    2013-07-01

    Gold nanoparticles (GNPs) and graphene oxide (GO) sheets are excellent nano carriers in many analytical methods. In this study, a modified enzyme-linked immunosorbent assay (ELISA) strategy was developed using antibody-functionalized GO sheets and GNPs. This modification significantly reduced the limit of detection (LOD) and cost greatly of this assay. The applicability of the method was demonstrated by detecting HSP70 in a human serum sample. This result suggests that the 3G-ELISA method is feasible to detect an antigen in a complex mixture, and the LOD is up to 64-fold and the cost is as low as one-tenth of the conventional ELISA method. PMID:23713797

  14. Enzyme-linked immunosorbent assay for detection of serum antibodies to Pasteurella haemolytica cytotoxin (leukotoxin) in cattle.

    PubMed Central

    Mosier, D A; Confer, A W; Hall, S M; Gentry, M J; Panciera, R J

    1986-01-01

    An enzyme-linked immunosorbent assay (ELISA) was developed for detection of bovine serum antibodies to the cytotoxin (leukotoxin) of Pasteurella haemolytica. A partially purified, cytotoxic, and immunogenic protein obtained from supernatants of logarithmic-phase P. haemolytica was used as the ELISA antigen. Preadsorption of sera with various cytotoxic, somatic, and capsular antigen preparations demonstrated that the assay was specific for anticytotoxin antibodies. ELISA anticytotoxin titers had a strong, significant correlation to cytotoxin-neutralizing-antibody titers. The ELISA, however, was more rapid and allowed for greater numbers of samples to be run than did the neutralization technique. ELISA anticytotoxin titers were high in cattle vaccinated with a live P. haemolytica vaccine, whereas unvaccinated cattle and cattle receiving a P. haemolytica bacterin had low ELISA anticytotoxin titers. A significant positive correlation between ELISA titers and resistance to experimental bovine pneumonic pasteurellosis was present. PMID:3745419

  15. Immunodiagnosis of fascioliasis using sandwich enzyme-linked immunosorbent assay for detection of Fasciola gigantica paramyosin antigen

    PubMed Central

    Abou-Elhakam, Hany Mohamed Adel; Bauomy, Ibraheem Rabia; El Deeb, Somaya Osman; El Amir, Azza Mohamed

    2013-01-01

    Background: Many immunological techniques have been developed over years using the different Fasciola antigens for diagnosis of parasitic infection and to replace the parasitological techniques, which are time consuming and usually lack sensitivity and reproducibility. Materials and Methods: In this study, Fasciola gigantica paramyosin (Pmy) antigen was early detected in cattle sera using sandwich enzyme-linked immunosorbent assay (ELISA), to evaluate the Pmy antigen performance in diagnosis. This work was conducted on 135 cattle blood samples, which were classified according to parasitological investigation into, healthy control (30), fascioliasis (75), and other parasites (30) groups. Results: The sensitivity of Sandwich ELISA was 97.33%, and the specificity was 95%, in comparison with parasitological examination, which recorded 66.66% sensitivity and 100% specificity, respectively. Conclusions: It was clear that the native F. gigantica Pmy is considered as a powerful antigen in early immunodiagnosis of fascioliasis, using a highly sensitive and specific sandwich ELISA technique. PMID:23961441

  16. A competitive enzyme-linked immunosorbent assay for quantification of tetrastatin in body fluids and tumor extracts.

    PubMed

    Dupont-Deshorgue, A; Oudart, J B; Brassart, B; Deslee, G; Perotin, J M; Diebold, M D; Monboisse, J C; Ramont, L; Brassart-Pasco, S

    2015-08-01

    Basement membrane collagens or derived fragments are measured in biological fluids such as blood and urine of patients and appear to be useful for diagnosis, prognostication, or treatment monitoring as proposed for endostatin, a fragment of collagen XVIII, or tumstatin, a fragment of collagen IV. Tetrastatin, the NC1 alpha 4 collagen IV domain, was previously reported to inhibit tumor growth and angiogenesis. The aim of this study was to develop and validate a method to measure tetrastatin concentrations in human fluids. We developed a competitive enzyme-linked immunosorbent assay (ELISA). It allowed measuring tetrastatin levels in human serum, bronchial aspiration and bronchoalveolar lavage fluids, and lung tissue extracts. The tetrastatin level was significantly higher in tumor tissues than in healthy lung tissues. Tetrastatin competitive ELISA could be useful to quantify tetrastatin in tissues and biological fluids for the diagnosis or prognostication of diseases in which basement membrane metabolism may be altered, especially tumor progression. PMID:25935259

  17. Serodiagnosis of syphilis by enzyme-linked immunosorbent assay with purified recombinant Treponema pallidum antigen 4D.

    PubMed

    Radolf, J D; Lernhardt, E B; Fehniger, T E; Lovett, M A

    1986-06-01

    An enzyme-linked immunosorbent assay (ELISA) for syphilis has been developed that detects IgG antibody to purified recombinant Treponema pallidum surface antigen 4D. The 4D ELISA was capable of detecting 25 ng of 4D antigen-specific antibody. Neither 172 nonsyphilitic sera nor 20 false-positive sera in the Venereal Disease Research Laboratory test reacted in the 4D ELISA. The sensitivity of the 4D ELISA was comparable to that of the adsorbed fluorescent treponemal antibody test in primary, secondary, and latent disease. Most sera from patients with yaws or pinta were also reactive, a result indicating that a 4D antigen-like molecule also exists in the closely related pathogenic treponemes Treponema pertenue and Treponema carateum. PMID:3517186

  18. Enzyme-linked immunosorbent assay for detection of type A streptococcal exotoxin: kinetics and regulation during growth of Streptococcus pyogenes.

    PubMed Central

    Houston, C W; Ferretti, J J

    1981-01-01

    We describe the detection and quantitation of type A streptococcal exotoxin (erythrogenic toxin, streptococcal pyrogenic exotoxin) by an enzyme-linked immunosorbent assay. This sensitive and specific technique detected microgram amounts of type A exotoxin and was useful for studying the kinetics and regulation of type A exotoxin production during the growth of Streptococcus pyogenes NY5. Maximum production of type A exotoxin was observed during the mid-log phase of growth, similar to the production of other streptococcal extracellular products. When S. pyogenes NY5 was grown at 42 degrees C, decreases in both growth and type A exotoxin production were observed. The results obtained when we studied the influence of nutrient additives and metal ions on the production of type A exotoxin led to the conclusion that none of these factors significantly affected type A exotoxin synthesis and that regulation was constitutive. Images PMID:7026447

  19. Evaluation of enzyme-linked immunosorbent and alternative assays for detection of HIV antibodies using panels of Brazilian sera.

    PubMed

    Ivo-Dos-Santos, J; Mello, D L; Couto-Fernandez, J C; Passos, R M; Dias-Carneiro, L A; Castilho, E A; Galvão-Castro, B

    1990-01-01

    Sera from 472 Brazilian subjects, confirmed to be either positive or negative for HIV antibodies and comprising the total clinical spectrum of HIV infection, were utilized in the evaluation of six commercially available enzyme-linked immunosorbent assays (ELISA), as well as of four alternative assays, namely indirect immunofluorescence (IIF), passive hemagglutination (PHA), dot blot and Karpas AIDS cell test. The sensitivities ranged from 100% (Abbott and Roche ELISA) to 84.2% (PHA) and the specificities ranged from 99.3% (IIF) to 80.2% (PHA). The sensitivity and specificity of the PHA and the sensitivity of the Karpas cell test were significantly lower than those of the other tests. Although the IFF and dot blot had good sensitivities and specificities, the six ELISA were more attractive than those tests when other parameters such as ease of reading and duration of assay were considered. PMID:2095632

  20. A CMOS image sensor with stacked photodiodes for lensless observation system of digital enzyme-linked immunosorbent assay

    NASA Astrophysics Data System (ADS)

    Takehara, Hironari; Miyazawa, Kazuya; Noda, Toshihiko; Sasagawa, Kiyotaka; Tokuda, Takashi; Kim, Soo Hyeon; Iino, Ryota; Noji, Hiroyuki; Ohta, Jun

    2014-01-01

    A CMOS image sensor with stacked photodiodes was fabricated using 0.18 µm mixed signal CMOS process technology. Two photodiodes were stacked at the same position of each pixel of the CMOS image sensor. The stacked photodiodes consist of shallow high-concentration N-type layer (N+), P-type well (PW), deep N-type well (DNW), and P-type substrate (P-sub). PW and P-sub were shorted to ground. By monitoring the voltage of N+ and DNW individually, we can observe two monochromatic colors simultaneously without using any color filters. The CMOS image sensor is suitable for fluorescence imaging, especially contact imaging such as a lensless observation system of digital enzyme-linked immunosorbent assay (ELISA). Since the fluorescence increases with time in digital ELISA, it is possible to observe fluorescence accurately by calculating the difference from the initial relation between the pixel values for both photodiodes.

  1. Micro-light-pipe array with an excitation attenuation filter for lensless digital enzyme-linked immunosorbent assay

    NASA Astrophysics Data System (ADS)

    Takehara, Hironari; Nagasaki, Mizuki; Sasagawa, Kiyotaka; Takehara, Hiroaki; Noda, Toshihiko; Tokuda, Takashi; Ohta, Jun

    2016-03-01

    Digital enzyme-linked immunosorbent assay (ELISA) is used for detecting various biomarkers with hypersensitivity. We have been developing compact systems by replacing the fluorescence microscope with a CMOS image sensor. Here, we propose a micro-light-pipe array structure made of metal filled with dye-doped resin, which can be used as a fabrication substrate of the micro-reaction-chamber array of digital ELISA. The possibility that this structure enhances the coupling efficiency for fluorescence was simulated using a simple model. To realize the structure, we fabricated a 30-µm-thick micropipe array by copper electroplating around a thick photoresist pattern. The typical diameter of each fabricated micropipe was 10 µm. The pipes were filled with yellow-dye-doped epoxy resin. The transmittance ratio of fluorescence and excitation light could be controlled by adjusting the doping concentration. We confirmed that an angled excitation light incidence suppressed the leakage of excitation light.

  2. Enzyme-Linked Immunosorbent Assay for Specific Identification and Enumeration of Azospirillum brasilense Cd. in Cereal Roots †

    PubMed Central

    Levanony, Hanna; Bashan, Yoav; Kahana, Zvi E.

    1987-01-01

    The enzyme-linked immunosorbent assay is suggested as a reliable, sensitive, and highly specific method for the identification and enumeration of Azospirillum brasilense Cd. As few as 105 CFU/ml can be practically identified by this method. At higher bacterial numbers, sensitivity increased linearly up to 5 × 108 CFU/ml, yielding useful standard curves. No cross-reaction was found either with different closely related Azospirillum strains or with other rhizosphere bacteria. The method allows for a specific identification of A. brasilense Cd. both in pure cultures and in mixtures with other bacterial species, even when the colony morphology is variable. The method was successfully applied to assess the degree of root colonization on various cereals by A. brasilense Cd. PMID:16347284

  3. An enzyme-linked immunosorbent assay (ELISA) for the identification of Naegleria fowleri in environmental water samples.

    PubMed

    Reveiller, Fabienne L; Varenne, Marie-Pierre; Pougnard, Claire; Cabanes, Pierre-Andre; Pringuez, Emmanuelle; Pourima, Benedicte; Legastelois, Stephane; Pernin, Pierre

    2003-01-01

    Naegleria fowleri, a free-living amoeba, is the causative agent of primary amoebic meningoencephalitis, a fatal human disease of the central nervous system often contracted after swimming in fresh water. Identifying sites contaminated by N. fowleri is important in order to prevent the disease. An Enzyme-Linked ImmunoSorbent Assay (ELISA) has been developed for the specific identification of N. fawleri in primary cultures of environmental water samples. Of 939 samples isolated from artificially heated river water and screened by ELISA, 283 were positive. These results were subsequently confirmed by isoelectric focusing, the established reference method. A sensitivity of 97.4% and a specificity of 97% were obtained. These results indicate that this ELISA method is reliable and can be considered as a powerful tool for the detection of N. fowleri in environmental water samples. PMID:12744523

  4. Enzyme-Linked Immunosorbent Assay Using Vertical Micro Reactor Stack for the Detection of Biomolecules

    NASA Astrophysics Data System (ADS)

    Matsui, Katsuhiro; Morimoto, Syohei; Asano, Toshifumi; Ukita, Yoshiaki; Kato, Dai-Ichiro; Takeo, Masahiro; Utsumi, Yuichi; Negoro, Seiji

    Microreactors and micro total analysis system (μTAS) are recognized as powerful tools for genomics, proteomics, clinical diagnostics, and environmental testing. In this paper, we describe enzyme linked immunosorvent assay (ELISA) using a new microreactor with a vertical fluid flow operation. This microreactor is composed of two reaction vessels stacked on the vertical lines through PMMA fluid filters (φ3mm). The fluid filters constructed by deep X-ray lithography possess 2,100 pores (φ 40 μm), and have valve functions, which maintain liquid layer in each reaction vessel. In addition, the liquid can be selectively transferred by air pressure from upper vessel to lower, and vice versa. As a model of ELISA using the microreactor, we planed to detect mouse immunoglobulin (IgG). We bound the goat anti-IgG antibody to the surface of the PMMA filters, and assayed the IgG by ELISA using anti-IgG antibody/ peroxidase conjugate. We found that the mouse IgG (100 ng/ml) was quantitatively detected within 45 min of analytical period, which was ca. 1/3 of the period required for the conventional method using micro titer plate.

  5. Protein G-based enzyme-linked immunosorbent assay for anti-MPB70 antibodies in bovine tuberculosis.

    PubMed Central

    Harboe, M; Wiker, H G; Duncan, J R; Garcia, M M; Dukes, T W; Brooks, B W; Turcotte, C; Nagai, S

    1990-01-01

    MPB70 is a highly species specific protein which is secreted from Mycobacterium bovis during culture. To investigate whether antibodies against MPB70 can be used as an indicator of infection with M. bovis, an enzyme-linked immunosorbent assay was developed, based on the use of biotinylated protein G, to provide a common indicator for antibody formation in different species. During experimental infection with M. bovis in cattle, a characteristic pattern of anti-MPB70 antibody production was observed with an initial flat plateau followed by a marked rise 18 to 20 weeks after infection. Skin testing with bovine tuberculin purified protein derivative (PPD), which was shown to contain antibody-reactive MPB70, was a potent stimulator of antibody production in infected animals. In experimentally infected cattle, we observed an inverse relationship between antibody activity and delayed-type hypersensitivity skin test reactions. In natural M. bovis infections, skin testing with PPD was also a potent stimulator of anti-MPB70 formation. Comparison between the enzyme-linked immunosorbent assay for antibodies to MPB70 and that for antibodies to the widely cross-reacting M. bovis BCG antigen 85B in animals with M. bovis, Mycobacterium avium, Mycobacterium paratuberculosis, and Corynebacterium pseudotuberculosis infections showed that formation of antibody to MPB70 was highly specific for infection with M. bovis. The use of an MPB70-containing PPD preparation for skin testing followed by this anti-MPB70 assay is a highly specific indicator of M. bovis infection. Adjustment of the test conditions is expected to provide an increased sensitivity of the procedure for the diagnosis of natural M. bovis infections. PMID:2191012

  6. Evaluation of microtiter-plate enzyme-linked immunosorbent assay for the analysis of triazine and chloroacetanilide herbicides in rainfall

    USGS Publications Warehouse

    Pomes, M.L.; Thurman, E.M.; Aga, D.S.; Goolsby, D.A.

    1998-01-01

    Triazine and chloroacetanilide concentrations in rainfall samples collected from a 23-state region of the United States were analyzed with microtiter-plate enzyme-linked immunosorbent assay (ELISA). Thirty-six percent of rainfall samples (2072 out of 5691) were confirmed using gas chromatography/mass spectrometry (GC/MS) to evaluate the operating performance of ELISA as a screening test. Comparison of ELISA to GC/MS results showed that the two ELISA methods accurately reported GC/MS results (m = 1), but with more variability evident with the triazine than with the chloroacetanilide ELISA. Bayes's rule, a standardized method to report the results of screening tests, indicated that the two ELISA methods yielded comparable predictive values (80%), but the triazine ELISA yielded a false- positive rate of 11.8% and the chloroacetanilide ELISA yielded a false- negative rate of 23.1%. The false-positive rate for the triazine ELISA may arise from cross reactivity with an unknown triazine or metabolite. The false-negative rate of the chloroacetanilide ELISA probably resulted from a combination of low sensitivity at the reporting limit of 0.15 ??g/L and a distribution characterized by 75% of the samples at or below the reporting limit of 0.15 ??g/L.Triazine and chloroacetanilide concentrations in rainfall samples collected from a 23-state region of the United States were analyzed with microtiter-plate enzyme-linked immunosorbent assay (ELISA). Thirty-six percent of rainfall samples (2072 out of 5691) were confirmed using gas chromatography/mass spectrometry (GC/MS) to evaluate the operating performance of ELISA as a screening test. Comparison of ELISA to GC/MS results showed that the two ELISA methods accurately reported GC/MS results (m = 1), but with more variability evident with the triazine than with the chloroacetanilide ELISA. Bayes's rule, a standardized method to report the results of screening tests, indicated that the two ELISA methods yielded comparable predictive

  7. Evaluation of a commercial enzyme-linked immunosorbent assay for detection of antibodies against the H5 subtype of Influenza A virus in waterfowl

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Serologic tools for rapid testing of subtype-specific influenza A (IA) virus antibody in wild birds and poultry are limited. In the current study, the ID Screen Influenza H5 Antibody Competition enzyme-linked immunosorbent assay (ELISA) was tested for the detection of antibodies to the H5 subtype o...

  8. AN ENZYME LINKED IMMUNOSORBENT ASSAY (ELISA) METHOD FOR THE URINARY BIOMONITORING OF 2,4-DICHLOROPHRENOCYACETIC ACID (2,4-D)

    EPA Science Inventory

    An enzyme-linked immunosorbent assay (ELISA) method was developed to quantitatively measure 2,4-dichlorophenoyacetic acid (2,4-D) in human urine. Samples were diluted (1:5) with phosphate-buffered saline, 0.05% Tween 20, with 0.02% sodium azide, and analyzed by a 96-microwekk pl...

  9. Development of an enzyme-linked immunosorbent assay for determination of the furaltadone etabolite, 3-amino-5-morpholinomethyl-2-oxazolidinone (AMOZ) in animal tissues

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A rapid, sensitive, and specific competitive direct enzyme-linked immunosorbent assay (cdELISA) for determination of protein bound 3-amino-5-morpholinomethyl-2-oxazolidinone (AMOZ) residues is described to monitor the illegal use of furaltadone. Polyclonal and monoclonal antibodies were produced in...

  10. Validation of an enzyme-linked immunosorbent assay that detects Histoplasma capsulatum antigenuria in Colombian patients with AIDS for diagnosis and follow-up during therapy.

    PubMed

    Caceres, Diego H; Scheel, Christina M; Tobón, Angela M; Ahlquist Cleveland, Angela; Restrepo, Angela; Brandt, Mary E; Chiller, Tom; Gómez, Beatriz L

    2014-09-01

    We validated an antigen capture enzyme-linked immunosorbent assay (ELISA) in Colombian persons with AIDS and proven histoplasmosis and evaluated the correlation between antigenuria and clinical improvement during follow-up. The sensitivity of the Histoplasma capsulatum ELISA was 86%, and the overall specificity was 94%. The antigen test successfully monitored the response to therapy. PMID:25008902

  11. Development of a Multianalyte Enzyme-Linked Immunosorbent Assay for Permethrin and Aroclors and Its Implementation for Analysis of Soil/Sediment and House Dust ExtractsExtracts

    EPA Science Inventory

    Development of a multianalyte enzyme-linked immunosorbent assay (ELISA) for detection of permethrin and aroclors 1248 or 1254, and implementation of the assay for analysis of soil/sediment samples are described. The feasibility of using the multianalyte ELISA to monitor aroclors ...

  12. Analysis of Mycobacterium avium subsp. paratuberculosis Antigens Used in an In-house Enzyme-linked Immunosorbent Assay for Johne's Disease

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We developed a novel enzyme-linked immunosorbent assay (ELISA), called EVELISA, for the detection of MAP infection in cattle which showed a higher sensitivity than current ELISA test. We previously reported that the use of heat-killed M. flavascens for pre-absorption of cross-reactive antibodies im...

  13. Comparison of enzyme-linked immunosorbent assay, surface plasmon resonance and biolayer interferometry for screening of deoxynivalenol in wheat and wheat dust

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A sample preparation method was developed for the screening of deoxynivalenol (DON) in wheat and wheat dust. Extraction was carried out with water and was successful due to the polar character of DON. For detection, an enzyme-linked immunosorbent assay (ELISA) was compared to the sensor-based techni...

  14. Highly broad-specific and sensitive enzyme-linked immunosorbent assay for screening sulfonamides: Assay optimization and application to milk samples

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A broad-specific and sensitive immunoassay for the detection of sulfonamides was developed by optimizing the conditions of an enzyme-linked immunosorbent assay (ELISA) in regard to different monoclonal antibodies (MAbs), assay format, immunoreagents, and several physicochemical factors (pH, salt, de...

  15. Enzyme-linked immunosorbent assay detection of trichothecenes produced by the Bioherbicide Myrothecium verrucaria in cell cultures, extracts, and plant tissues

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Commercially available enzyme linked immunosorbent assay (ELISA) plates for trichothecene detection, possessing cross-reactivity with several trichothecene mycotoxins (e.g., verrucarin A, and J, roridin A, L-2, E, and H), were tested for their ability to detect trichothecenes produced by a strain of...

  16. Skin test and Gamma Interferon enzyme-linked Immunosorbent assay results in Sheep exposed to dead Mycobacterium avium subspecies paratuberculosis Organisms

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Cell mediated immunity (CMI) diagnostic tests, such as the gamma interferon enzyme-linked immunosorbent assay (IFN-gamma ELISA) and the johnin skin test, have the potential to detect animals infected with Mycobacterium avium subspecies paratuberculosis (MAP) early in the course of the disease. While...

  17. Simultaneous determination of 13 fluoroquinolone and 22 sulfonamide residues in milk by a dual-colorimetric enzyme-linked immunosorbent assay

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Enzyme-linked immunosorbent assays (ELISAs) usually focus on the detection of a single analyte or a single group of analytes, e.g., fluoroquinolones or sulfonamides. However, it is often necessary to simultaneously monitor the two classes of antimicrobial residues in different food matrices. In th...

  18. Enzyme-linked immunosorbent assay for a soluble antigen of Renibacterium salmoninarum, the causative agent for salmonid bacterial kidney disease

    USGS Publications Warehouse

    Pascho, R.J.; Mulcahy, D.

    1987-01-01

    A double-antibody enzyme-linked immunosorbent assay (ELISA) for detection of a soluble fraction of Renibacterium salmoninarum was developed from components extracted from the supernatant of an R. salmoninarum broth culture. The Costar® Serocluster™ EIA microplate gave the highest absorbance and signal-to-noise ratios among seven types tested. Including Tween 80 in the wash buffer resulted in higher absorbances than Tween 20 when antigen was present. Background absorbance did not increase when Tween 80 was added to the wash buffer, but did when Tween 80 replaced Tween 20 in antigen and conjugate diluents. Adsorption of coating antibody peaked within 4 h at 37 °C and 16 h at 4 °C. Antigen attachment to antibody-coated microplate wells depended more on incubation temperature than duration; we adopted a 3-h incubation at 25 °C. Conjugate incubation for longer than 1 h at 37 °C or 3 h at 25 °C resulted in unacceptable background levels. No cross-reactions resulted from heat-extracted antigens of 10 other species of bacteria. The optimized ELISA is a 6-h test that enables detection of levels of soluble antigen as low as 2–20 ng.

  19. Detection of group C rotavirus antigens and antibodies in animals and humans by enzyme-linked immunosorbent assays.

    PubMed Central

    Tsunemitsu, H; Jiang, B; Saif, L J

    1992-01-01

    Enzyme-linked immunosorbent assays (ELISAs) were developed to detect group (gp) C rotavirus antigens and antibodies. Both assays were confirmed to be specific for gp C rotavirus by using serogroup A, B, and C rotaviruses; hyperimmune antisera to these serogroups of rotaviruses; and paired serum specimens from animals infected with gp C rotaviruses. The ELISA for antigen detection reacted not only with porcine gp C rotaviruses but also with human and bovine gp C rotaviruses. Following experimental challenge of gnotobiotic pigs with porcine gp C rotavirus, the virus was found by ELISA in all diarrheic feces. A high prevalence of antibodies to gp C rotaviruses was detected in sera from adult pigs (93 to 97%) and cattle (47 to 56%) in the United States and Japan. However, no antibody to gp C rotavirus was detected in the sera (n = 20) of adult horses in the United States. In human sera from Hokkaido, Japan, 3% of children and 13% of adults possessed antibody to gp C rotaviruses. These results suggest that the ELISA that we developed may be useful for surveying gp C rotavirus infections in animals and humans. On the basis of serology, gp C rotavirus infections are common in pigs and cattle in the United States and Japan, but they occur at lower levels in humans from the Hokkaido area of Japan. PMID:1323577

  20. Serodiagnosis of Neospora caninum infection in cattle by enzyme-linked immunosorbent assay with recombinant truncated NcSAG1.

    PubMed

    Chahan, Bayin; Gaturaga, Irungu; Huang, Xiaohong; Liao, Min; Fukumoto, Shinya; Hirata, Haruyuki; Nishikawa, Yoshihumi; Suzuki, Hiroshi; Sugimoto, Chihiro; Nagasawa, Hideyuki; Fujisaki, Kozo; Igarashi, Ikuo; Mikami, Takeshi; Xuan, Xuenan

    2003-12-30

    Neospora caninum is a veterinary medically important pathogen capable of causing abortion in cattle and neuromuscular paralysis in dogs. The surface antigen 1 of N. caninum (NcSAG1) is an important candidate for the development of a diagnostic reagent for neosporosis. In order to establish an effective diagnostic method, the gene encoding truncated NcSAG1 (NcSAG1t) lacking a signal peptide and C-terminal hydrophobic regions was cloned and expressed in Escherichia coli as a fusion protein with glutathione S-transferase (GST). The purified GST-NcSAG1t was tested in an enzyme-linked immunosorbent assay (ELISA) for the detection of N. caninum antibodies in cattle. The ELISA with GST-NcSAG1t clearly differentiated between immunofluorescent antibody test (IFAT)-positive and -negative sera from cattle. In addition, the ELISA detected no cross-reactivity with sera from mice experimentally infected with the closely related parasite Toxoplasma gondii. Field serum samples collected from cattle in Brazil were examined for the diagnosis of neosporosis by using the ELISA. Of the 197 samples analyzed, 66 (33.5%) samples were positive for antibodies to N. caninum. Of the 66 ELISA-positive samples, 60 (90%) samples were confirmed as positive by Western blot analysis with whole parasite antigens. These results suggest that the recombinant NcSAG1t could be a reliable reagent for use as an antigen in ELISA for the serodiagnosis of N. caninum infection in cattle. PMID:14729165

  1. Use of hydrophilic extra-viral domain of canine distemper virus H protein for enzyme-linked immunosorbent assay development

    PubMed Central

    Cho, Ki-hyun; Kim, Jeongmi; Yoo, Hyun-ah; Kim, Dae-hee; Park, Seung-yong; Song, Chang-seon; Choi, In-soo

    2014-01-01

    Simple methods for measuring the levels of serum antibody against canine distemper virus (CDV) would assist in the effective vaccination of dogs. To develop an enzyme-linked immunosorbent assay (ELISA) specific for CDV, we expressed hydrophilic extra-viral domain (HEVD) protein of the A75/17-CDV H gene in a pET 28a plasmid-based Escherichia (E.) coli vector system. Expression was confirmed by dot and Western blotting. We proposed that detection of E. coli-expressed H protein might be conformation-dependent because intensities of the reactions observed with these two methods varied. The H gene HEVD protein was further purified and used as an antigen for an ELISA. Samples from dogs with undetectable to high anti-CDV antibody titers were analyzed using this HEVD-specific ELISA and a commercial CDV antibody detection kit (ImmunoComb). Levels of HEVD antigenicity measured with the assays and immunochromatography correlated. These data indicated that the HEDV protein may be used as antigen to develop techniques for detecting antibodies against CDV. PMID:25234325

  2. Dual-color plasmonic enzyme-linked immunosorbent assay based on enzyme-mediated etching of Au nanoparticles.

    PubMed

    Guo, Longhua; Xu, Shaohua; Ma, Xiaoming; Qiu, Bin; Lin, Zhenyu; Chen, Guonan

    2016-01-01

    Colorimetric enzyme-linked immunosorbent assay utilizing 3'-3-5'-5-tetramethylbenzidine(TMB) as the chromogenic substrate has been widely used in the hospital for the detection of all kinds of disease biomarkers. Herein, we demonstrate a strategy to change this single-color display into dual-color responses to improve the accuracy of visual inspection. Our investigation firstly reveals that oxidation state of 3'-3-5'-5-tetramethylbenzidine (TMB(2+)) can quantitatively etch gold nanoparticles. Therefore, the incorporation of gold nanoparticles into a commercial TMB-based ELISA kit could generate dual-color responses: the solution color varied gradually from wine red (absorption peak located at ~530 nm) to colorless, and then from colorless to yellow (absorption peak located at ~450 nm) with the increase amount of targets. These dual-color responses effectively improved the sensitivity as well as the accuracy of visual inspection. For example, the proposed dual-color plasmonic ELISA is demonstrated for the detection of prostate-specific antigen (PSA) in human serum with a visual limit of detection (LOD) as low as 0.0093 ng/mL. PMID:27599832

  3. An enzyme-linked immunosorbent assay for detecting 3-phenoxybenzoic acid in plasma and its application in farmers and consumers

    PubMed Central

    Thiphom, Sarunya; Prapamontol, Tippawan; Chantara, Somporn; Mangklabruks, Ampica; Suphavilai, Chaisuree; Ahn, Ki Chang; Gee, Shirley J.; Hammock, Bruce D.

    2013-01-01

    The aim of this study was to identify a plasma biomarker of exposure to pyrethroid insecticides. A major metabolite, 3-phenoxybenzoic acid (3-PBA), can be detected in urine but urinary 3-PBA cannot be used to assess the active dose. The 3-PBA-adduct represents a much more persistent class of biomarkers than metabolites excreted into urine, having half lives up to several weeks or months. We developed an enzyme-linked immunosorbent assay (ELISA) for total 3-PBA including adduct formed after alkaline hydrolysis, liquid-liquid extraction (LLE) and solid phase extraction (SPE) of the sample. The developed ELISA had an IC50 value of 26.7 ng/mL. The intra- and inter-assay coefficients of variation (%CV) were lower than 5% and were within the optimum condition variance (OCV) range. The LLE cleanup technique satisfactorily eliminated the matrix effect from plasma samples before SPE and ELISA analysis yielding good recoveries (85.9–99.4%) with a limit of quantitation (LOQ, 5 ng/mL) that was 30- to 47-fold more sensitive than previous studies. Moreover, the developed method could separate more than 80% of 3-PBA from adduct form. The method was successfully applied to the detection of the target in real samples obtained from consumers (n=50) and farmers (n=50). To our knowledge, this is the first ELISA method for detecting 3-PBA in human plasma and applied to a field study. PMID:23667388

  4. Comparison of four different enzyme-linked immunosorbent assays for serological diagnosis of Salmonella enteritidis infections in experimentally infected chickens.

    PubMed Central

    van Zijderveld, F G; van Zijderveld-van Bemmel, A M; Anakotta, J

    1992-01-01

    The program for the eradication of Salmonella enteritidis from chickens in The Netherlands is based on bacteriological examination of breeding flocks. There is a great need for a specific and sensitive serological screening test. For that purpose, we developed four different enzyme-linked immunosorbent assays (ELISAs), i.e., an indirect ELISA with S. enteritidis flagellin, an indirect ELISA with S. enteritidis lipopolysaccharide, a double-antibody sandwich blocking ELISA that uses monoclonal antibodies against S. enteritidis flagellin (GM-DAS blocking ELISA), and a double-antibody sandwich ELISA that uses monoclonal antibodies against S. enteritidis lipopolysaccharide. In the present study, we compare the results of those ELISAs with sera from experimentally infected 1-day-old chickens and with sera and eggs from experimentally infected laying hens. Experimental infections were induced with strains of S. enteritidis phage types 1 and 2, S. typhimurium, and S. panama. Sera were collected up to days 44 and 39 after infection from 1-day-old chickens and laying hens, respectively. Only the GM-DAS blocking ELISA was able to discriminate between S. enteritidis infections and infections with the other serotypes. This ELISA had both a sensitivity and a specificity of 100% for all serum samples from experimentally infected chickens. A field study is in progress to evaluate whether this test can be implemented in the Dutch S. enteritidis eradication program. PMID:1400954

  5. Electrochemical enzyme-linked immunosorbent assay (ELISA) for α-fetoprotein based on glucose detection with multienzyme-nanoparticle amplification.

    PubMed

    Liu, Qin-Lan; Yan, Xiao-Hui; Yin, Xiao-Mao; Situ, Bo; Zhou, Han-Kun; Lin, Li; Li, Bo; Gan, Ning; Zheng, Lei

    2013-01-01

    Since glucose biosensors are one of the most popular and widely used point-of-care testing devices, a novel electrochemical enzyme-linked immunosorbent assay (ELISA) for protein biomarkers has been developed based on a glucose detection strategy. In this study, α-fetoprotein (AFP) was used as the target protein. An electrochemical ELISA system was constructed using anti-AFP antibodies immobilized on microwell plates as the capture antibody (Ab1) and multi-label bioconjugates as signal tracer. The bioconjugates were synthesized by attaching glucoamylase and the secondary anti-AFP antibodies (Ab2) to gold nanoparticles (AuNPs). After formation of the sandwich complex, the Ab2-glucoamylase-AuNPs conjugates converted starch into glucose in the presence of AFP. The concentration of AFP can be calculated based on the linear relation between AFP and glucose, the concentration of which can be detected by the glucose biosensor. When the AFP concentration ranged from 0.05 to 100 ng/mL, a linear calibration plot (i (µA) = 13.62033 - 2.86252 logCAFP (ng/mL), r = 0.99886) with a detection limit of 0.02 ng/mL was obtained under optimal conditions. The electrochemical ELISA developed in this work shows acceptable stability and reproducibility, and the assay for AFP spiked in human serum also shows good recovery (97.0%-104%). This new method could be applied for detecting any protein biomarker with the corresponding antibodies. PMID:24129276

  6. Coupling solid-phase extraction and enzyme-linked immunosorbent assay for ultratrace determination of herbicides in pristine water

    USGS Publications Warehouse

    Aga, D.S.; Thurman, E.M.

    1993-01-01

    Solid-phase extraction (SPE) and enzyme-linked immunosorbent assay (ELISA) were coupled for automated trace analysis of pristine water samples containing 2-chloro-4-ethylamino-6-isopropylamine-s-triazine (atrazine) and 2-chloro-2???,6???-diethyl-N-(methoxymethyl)acetanilide (alachlor). The isolation of the two herbicides on a C18-resin involved the selection of an elution solvent that both removes interfering substances and is compatible with ELISA. Ethyl acetate was selected as the elution solvent followed by a solvent exchange with methanol/water (20/80, % v/v). The SPE-ELISA method has a detection limit of 5.0 ng/L (5 ppt), >90% recovery, and a relative standard deviation of ??10%. The performance of a microtiter plate-based ELISA and a magnetic particle-based ELISA coupled to SPE was also evaluated. Although the sensitivity of the two ELISA methods was comparable, the precision using magnetic particles was improved considerably (??10% versus ??20%) because of the faster reaction kinetics provided by the magnetic particles. Finally, SPE-ELISA and isotope dilution gas chromatography/ mass spectrometry correlated well (correlation coefficient of 0.96) for lake-water samples. The SPE-ELISA method is simple and may have broader applications for the inexpensive automated analysis of other contaminants in water at trace levels.

  7. Dual-color plasmonic enzyme-linked immunosorbent assay based on enzyme-mediated etching of Au nanoparticles

    PubMed Central

    Guo, Longhua; Xu, Shaohua; Ma, Xiaoming; Qiu, Bin; Lin, Zhenyu; Chen, Guonan

    2016-01-01

    Colorimetric enzyme-linked immunosorbent assay utilizing 3′-3-5′-5-tetramethylbenzidine(TMB) as the chromogenic substrate has been widely used in the hospital for the detection of all kinds of disease biomarkers. Herein, we demonstrate a strategy to change this single-color display into dual-color responses to improve the accuracy of visual inspection. Our investigation firstly reveals that oxidation state of 3′-3-5′-5-tetramethylbenzidine (TMB2+) can quantitatively etch gold nanoparticles. Therefore, the incorporation of gold nanoparticles into a commercial TMB-based ELISA kit could generate dual-color responses: the solution color varied gradually from wine red (absorption peak located at ~530 nm) to colorless, and then from colorless to yellow (absorption peak located at ~450 nm) with the increase amount of targets. These dual-color responses effectively improved the sensitivity as well as the accuracy of visual inspection. For example, the proposed dual-color plasmonic ELISA is demonstrated for the detection of prostate-specific antigen (PSA) in human serum with a visual limit of detection (LOD) as low as 0.0093 ng/mL. PMID:27599832

  8. Detection of Francisella tularensis-specific antibodies in patients with tularemia by a novel competitive enzyme-linked immunosorbent assay.

    PubMed

    Sharma, Neekun; Hotta, Akitoyo; Yamamoto, Yoshie; Fujita, Osamu; Uda, Akihiko; Morikawa, Shigeru; Yamada, Akio; Tanabayashi, Kiyoshi

    2013-01-01

    A novel competitive enzyme-linked immunosorbent assay (cELISA) was developed and evaluated for detection of antibodies against Francisella tularensis in humans. The assay is based on the ability of serum antibodies to inhibit the binding of monoclonal antibodies (MAbs) directed against F. tularensis lipopolysaccharide antigens. The assay was evaluated using serum samples of tularemia patients, inactivated F. tularensis-immunized rabbits, and F. tularensis-infected mice. Antibodies against F. tularensis were successfully detected in serum samples of tularemia patients as well as the immunized and infected animals. The cELISA method was compared to indirect ELISA (iELISA) and the commonly used microagglutination test (MA) using serum samples of 19 tularemia patients and 50 healthy individuals. The sensitivity and specificity of cELISA were 93.9 and 96.1%, respectively, in comparison to the iELISA. MA was less sensitive than cELISA with a sensitivity and specificity of only 81.8 and 98.0%, respectively. A high degree of correlation (R(2) = 0.8226) was observed between cELISA and iELISA results. The novel cELISA developed in this study appears to be highly sensitive and specific for serodiagnosis of human tularemia. The potential of the MAb-based cELISA to be used in both human and animal samples emphasizes its usefulness for serological survey of tularemia among multiple animal species. PMID:23114700

  9. Detection of Francisella tularensis-Specific Antibodies in Patients with Tularemia by a Novel Competitive Enzyme-Linked Immunosorbent Assay

    PubMed Central

    Sharma, Neekun; Hotta, Akitoyo; Yamamoto, Yoshie; Fujita, Osamu; Uda, Akihiko; Morikawa, Shigeru; Yamada, Akio

    2013-01-01

    A novel competitive enzyme-linked immunosorbent assay (cELISA) was developed and evaluated for detection of antibodies against Francisella tularensis in humans. The assay is based on the ability of serum antibodies to inhibit the binding of monoclonal antibodies (MAbs) directed against F. tularensis lipopolysaccharide antigens. The assay was evaluated using serum samples of tularemia patients, inactivated F. tularensis-immunized rabbits, and F. tularensis-infected mice. Antibodies against F. tularensis were successfully detected in serum samples of tularemia patients as well as the immunized and infected animals. The cELISA method was compared to indirect ELISA (iELISA) and the commonly used microagglutination test (MA) using serum samples of 19 tularemia patients and 50 healthy individuals. The sensitivity and specificity of cELISA were 93.9 and 96.1%, respectively, in comparison to the iELISA. MA was less sensitive than cELISA with a sensitivity and specificity of only 81.8 and 98.0%, respectively. A high degree of correlation (R2 = 0.8226) was observed between cELISA and iELISA results. The novel cELISA developed in this study appears to be highly sensitive and specific for serodiagnosis of human tularemia. The potential of the MAb-based cELISA to be used in both human and animal samples emphasizes its usefulness for serological survey of tularemia among multiple animal species. PMID:23114700

  10. Lipopolysaccharide-Based Enzyme-Linked Immunosorbent Assay for Experimental Use in Detection of Antibodies to Lawsonia intracellularis in Pigs

    PubMed Central

    Kroll, J. J.; Eichmeyer, M. A.; Schaeffer, M. L.; McOrist, S.; Harris, D. L.; Roof, M. B.

    2005-01-01

    An enzyme-linked immunosorbent assay (ELISA) for Lawsonia intracellularis was developed and compared with a whole-cell antigen-based immunofluorescence antibody test (IFAT). The antigen-containing lipopolysaccharide (LPS) was derived from Percoll gradient purified cultures of L. intracellularis by using a modification of the Westphal hot phenol procedure. The antigen was bound directly to polystyrene 96-well microtiter plates, and the assay was performed in an indirect ELISA format. Specificity and sensitivity values based on 80 known positive and 80 known negative serum samples from controlled experimental trials were 93.7% and 88.7%, respectively. Serological results from a controlled L. intracellularis challenge exposure study confirmed the high specificity and sensitivity of this assay (100% and 99.5%, respectively). Comparisons between the LPS ELISA and the IFAT in detecting anti-Lawsonia antibodies in this controlled study revealed significantly more LPS ELISA-positive pigs than IFAT-positive pigs on days 21, 28, 35, and 42 (P = 0.003, 0.030, 0.002, and 0.006, respectively). This indirect ELISA (LPS ELISA) test is an improved method of detecting antibodies in pigs soon after exposure to L. intracellularis, regardless of isolate type (vaccine or wild type) in experimental studies. The LPS ELISA may be used as a tool to support future research trials on vaccine efficacy and to further understand the immune response induced by L. intracellularis. PMID:15939742

  11. Presumptive diagnosis of subclinical infections utilizing computer-assisted analysis of sequential enzyme-linked immunosorbent assays against multiple antigens.

    PubMed

    Mallinson, E T; Snyder, D B; Marquardt, W W; Russek-Cohen, E; Savage, P K; Allen, D C; Yancey, F S

    1985-09-01

    One-hundred-seventy-two serum samples, collected sequentially from four flocks of egg- and meat-type chickens, were evaluated for antibodies to multiple infectious agents by enzyme-linked immunosorbent assay (MELISA). The MELISA system used provided simultaneous measurement of antibody titers against avian infectious bronchitis (IB), infectious bursal disease (BD), Newcastle disease, avian encephalomyelitis and reovirus infections, and Mycoplasma gallisepticum. The use of computer-generated graphic print outs of relative MELISA titers provided immediate visulization of over 740 data points and convenient detection of any temporal changes in median titer class (MTC). The temporally changing MTC, or flock profiles obtained, indicated that negligible or waning IB immunity may be a common occurrence in previously vaccinated commercial chickens. These profiles further suggested that, despite no IB revaccination, these same flocks experienced episodes of reexposure to IB which otherwise may have been difficult to detect by conventional clinical or diagnostic laboratory protocols. MELISA profiles and sequential histologic examinations of bursas of Fabricius also provided evidence of a possible BD vaccination problem in young chickens that also experienced excessive losses from coccidiosis, ulcerative enteritis, and Marek's disease. Short sampling intervals were found to foster the detection and definition of fluctuations in MTC which otherwise may have been missed. PMID:4048057

  12. Evaluation of a Commercial Enzyme Linked Immunosorbent Assay (ELISA) for the Determination of the Neurotoxin BMAA in Surface Waters

    PubMed Central

    Faassen, Elisabeth J.; Beekman, Wendy; Lürling, Miquel

    2013-01-01

    The neurotoxin β-N-methylamino-L-alanine (BMAA) is suspected to play a role in Alzheimer’s disease, Parkinson’s disease and amyotrophic lateral sclerosis. Because BMAA seems to be produced by cyanobacteria, surface waters are screened for BMAA. However, reliable analysis of BMAA requires specialized and expensive equipment. In 2012, a commercial enzyme-linked immunosorbent assay (ELISA) for determination of BMAA in surface waters was released. This kit could enable fast and relatively cheap screening of surface waters for BMAA. The objective of this study was to determine whether the BMAA ELISA kit was suitable for the determination of BMAA concentrations in surface waters. We hypothesised that the recovery of spiked samples was close to 100% and that the results of unspiked sample analysis were comparable between ELISA and liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis. However, we found that recovery was higher than 100% in most spiked samples, highest determined recovery was over 400%. Furthermore, the ELISA gave a positive signal for nearly each tested sample while no BMAA could be detected by LC-MS/MS. We therefore conclude that in its current state, the kit is not suitable for screening surface waters for BMAA. PMID:23762331

  13. Evaluation of a commercial enzyme linked immunosorbent assay (ELISA) for the determination of the neurotoxin BMAA in surface waters.

    PubMed

    Faassen, Elisabeth J; Beekman, Wendy; Lürling, Miquel

    2013-01-01

    The neurotoxin β-N-methylamino-L-alanine (BMAA) is suspected to play a role in Alzheimer's disease, Parkinson's disease and amyotrophic lateral sclerosis. Because BMAA seems to be produced by cyanobacteria, surface waters are screened for BMAA. However, reliable analysis of BMAA requires specialized and expensive equipment. In 2012, a commercial enzyme-linked immunosorbent assay (ELISA) for determination of BMAA in surface waters was released. This kit could enable fast and relatively cheap screening of surface waters for BMAA. The objective of this study was to determine whether the BMAA ELISA kit was suitable for the determination of BMAA concentrations in surface waters. We hypothesised that the recovery of spiked samples was close to 100% and that the results of unspiked sample analysis were comparable between ELISA and liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis. However, we found that recovery was higher than 100% in most spiked samples, highest determined recovery was over 400%. Furthermore, the ELISA gave a positive signal for nearly each tested sample while no BMAA could be detected by LC-MS/MS. We therefore conclude that in its current state, the kit is not suitable for screening surface waters for BMAA. PMID:23762331

  14. Enzyme-linked immunosorbent assay for serological diagnosis of Nocardia brasiliensis and clinical correlation with mycetoma infections.

    PubMed Central

    Salinas-Carmona, M C; Welsh, O; Casillas, S M

    1993-01-01

    We previously identified three immunodominant antigens obtained from a Nocardia brasiliensis cell extract and recognized by sera from mycetoma patients (M. C. Salinas-Carmona, L. Vera, O. Welsh, and M. Rodríguez, Zentralbl. Bakteriol. 276:390-397, 1992). In the present work, we obtained a crude extract from a mass culture of N. brasiliensis HUJEG-1 and purified two immunodominant antigens, the 26- and 24-kDa proteins, by using simple physiochemical techniques. With these antigens, we developed a conventional solid-phase enzyme-linked immunosorbent assay and tested 30 serum samples from mycetoma patients, 29 from tuberculosis patients, 24 from a leprosy group, and 31 from healthy individuals. Our results show for the first time statistically significant differences in serology among these groups. All mycetoma patients with a positive culture for N. brasiliensis had absorbance values higher than 0.3. On the other hand, the mycobacterium-infected patients as well as the healthy individuals all had absorbance values below that level. Moreover, we found a close correlation between the clinical condition of the mycetoma patients and the anti-26- and anti-24-kDa protein antibody concentrations. We therefore propose the use of this assay in routine clinical laboratories to confirm the diagnosis of N. brasiliensis infection in human mycetoma cases. In addition, the possible application of this assay in the serodiagnosis of Nocardia asteroides infection is also discussed. Images PMID:8263174

  15. Performance comparison of immunodiffusion, enzyme-linked immunosorbent assay, immunochromatography and hemagglutination for serodiagnosis of human pythiosis.

    PubMed

    Chareonsirisuthigul, Takol; Khositnithikul, Rommanee; Intaramat, Akarin; Inkomlue, Ruchuros; Sriwanichrak, Kanchana; Piromsontikorn, Savittree; Kitiwanwanich, Sureewan; Lowhnoo, Tassanee; Yingyong, Wanta; Chaiprasert, Angkana; Banyong, Ramrada; Ratanabanangkoon, Kavi; Brandhorst, Tristan T; Krajaejun, Theerapong

    2013-05-01

    Pythiosis is a life-threatening infectious disease caused by the fungus-like organism Pythium insidiosum. Morbidity and mortality rates of pythiosis are high. The treatment of choice for pythiosis is surgical debridement of infected tissue. Early and accurate diagnosis is critical for effective treatment. In-house serodiagnostic tests, including immunodiffusion (ID), enzyme-linked immunosorbent assay (ELISA), immunochromatography (ICT) and hemagglutination (HA) have been developed to detect antibodies against P. insidiosum in sera. This study compares the diagnostic performance of ID, ELISA, ICT, and HA, using sera from 37 pythiosis patients and 248 control subjects. ICT and ELISA showed optimal diagnostic performance (100% sensitivity, specificity, positive predictive value and negative predictive value). ICT was both rapid and user-friendly. ELISA results were readily quantitated. ID is relatively insensitive. HA was rapid, but diagnostic performance was poor. Understanding the advantages offered by each assay facilitates selection of an assay that is circumstance-appropriate. This will promote earlier diagnoses and improved outcomes for patients with pythiosis. PMID:23537786

  16. Evaluation of the bead enzyme-linked immunosorbent assay for detection of cholera toxin directly from stool specimens.

    PubMed Central

    Ramamurthy, T; Bhattacharya, S K; Uesaka, Y; Horigome, K; Paul, M; Sen, D; Pal, S C; Takeda, T; Takeda, Y; Nair, G B

    1992-01-01

    A highly sensitive bead enzyme-linked immunosorbent assay (bead ELISA) for detection of cholera toxin (CT) was evaluated for direct detection of CT from stool specimens of patients with acute secretory diarrhea. Of the 75 stool samples examined, 59 yielded biochemically, and serologically confirmed strains of Vibrio cholerae O1. The bead ELISA was positive for CT in stool supernatants in 50 (84.7%) of the 59 samples from which V. cholerae O1 was isolated. In addition, the bead ELISA was positive for three stool specimens which were negative by culture. The free CT present in 48 of the 50 stool samples positive by culture for V. cholerae O1 and for CT by bead ELISA was completely absorbed by anti-CT immunoglobulin G. All of the 59 strains of V. cholerae O1 biotype eltor isolated in this study produced in vitro CT. The concentration of CT present in the bead ELISA-positive stool samples ranged between 26 pg/ml and greater than 100 ng/ml. This evaluation study demonstrates that the bead ELISA is a sensitive and simple method for direct detection of CT in nonsterile stool samples, and we recommend routine use of this assay for detection of CT in stool samples and culture supernatants in clinical and reference laboratories. PMID:1629335

  17. Detection of Cronobacter Genus in Powdered Infant Formula by Enzyme-linked Immunosorbent Assay Using Anti-Cronobacter Antibody

    PubMed Central

    Song, Xinjie; Shukla, Shruti; Lee, Gibaek; Park, Sunhyun; Kim, Myunghee

    2016-01-01

    Cronobacter species (Cronobacter spp.) are hazardous foodborne pathogens associated with baby food, powdered infant formula (PIF). To develop a rapid and sensitive method for simultaneous detection of seven Cronobacter spp. in PIF, an indirect non-competitive enzyme-linked immunosorbent assay (INC-ELISA) was developed based on a novel immunoglobulin G (IgG), anti-Cronobacter IgG. The developed INC-ELISA was able to detect seven Cronobacter spp. at concentrations ranging from (5.6 ± 0.30) × 103 to (2.1 ± 0.01) × 105 colony forming unit (CFU)/mL in pure culture. Further, INC-ELISA employing anti-Cronobacter IgG was applicable for analysis of PIF samples contaminated with less than <10 cells of Cronobacter spp. per 25 g of PIF in 36 h. The developed antibody showed slight cross-reactivity with Franconibacter pulveris (LMG 24057) at high concentration (108 CFU/mL). The INC-ELISA method displayed excellent specificity without compromising cross-reactivity with other foodborne pathogens. The INC-ELISA assay method developed in this study using a novel anti-Cronobacter IgG facilitated highly sensitive, efficient, and rapid detection of Cronobacter spp. in baby food. PMID:27493642

  18. Quality Evaluation of Five Commercial Enzyme Linked Immunosorbent Assay Kits for Detecting Aflatoxin B1 in Feedstuffs

    PubMed Central

    Sun, Dan-dan; Gu, Xu; Li, Jun-guo; Yao, Ting; Dong, Ying-chao

    2015-01-01

    The objective of this study was to evaluate the quality of five commercial enzyme linked immunosorbent assay (ELISA) kits (A, B, C, D, and E) from different suppliers for detecting aflatoxin B1 (AFB1). AFB1-free corn samples supplemented with different levels of AFB1 (5, 10, and 20 μg/kg) were used as positive controls and 6 replicates of each control sample were tested to evaluate the accuracy and precision of these kits. In addition, we also evaluated the performance of these ELISA kits for AFB1 in 30 feed samples, including corn, distillers dried grains with soluble, wheat samples, soybean meal, and poultry feed, which were verified by high performance liquid chromatography. Results showed that the coefficients of variation ranged from 1.18% to 16.22% in intra-plate and 2.85% to 18.04% in inter-plate for the determination of AFB1. The half maximal inhibitory concentration for five kits ranged from 3.72 to 7.22 μg/kg. The quantitation limits of AFB1 were all under the legal limit in China but somewhat inconsistent with kit instructions. Although the recovery rate of four of the five kits were either less than 90% or more than 110%, all these values were acceptable in practice. Two kits had high false positive rates (C and E). In conclusion, our results revealed that the qualities of five tested ELISA kits were significantly different. PMID:25924961

  19. Antigen-capture enzyme-linked immunosorbent assays for detection of different H5 avian Influenza A virus.

    PubMed

    Chiu, Yi-Chung; Chu, Wen-Yu; Tsao, Zak; Wang, Ching-Ho

    2012-07-01

    Avian Influenza A virus (AIV) subtype H5 is divided into American and Eurasian lineages, according to hemagglutinin gene sequences. Although methods for detecting H5 AIVs have been described, no H5 strain-specific detection method has been reported. The purpose of the present study was to develop an antigen-capture enzyme-linked immunosorbent assay (ACE) to detect and differentiate between the American and the Eurasian H5 AIVs. Monoclonal antibodies (mAbs) against the HA fragment of a Eurasian H5N2 AIV were used as the capture antibodies as well as the detector antibodies after labeling with horseradish peroxidase to develop an ACE. One mAb was selected for detecting the American as well as the Eurasian H5 AIVs. The other mAb was used for detecting only the Eurasian H5N2 but not the American H5N2 AIVs, H6N1 AIVs, or Newcastle disease virus. The ACEs developed would be useful for detection and differentiation of H5 AIVs from the Eurasian and the American H5 AIVs. PMID:22621946

  20. A Magnetic Nanoparticle Based Enzyme-Linked Immunosorbent Assay for Sensitive Quantification of Zearalenone in Cereal and Feed Samples

    PubMed Central

    Zhang, Xian; Wang, Xin; Sun, Mengjiao; Zhang, Xiaofeng; Song, Houhui; Yan, Yaxian; Sun, Jianhe; Li, Xiaoliang; Fang, Weihuan

    2015-01-01

    A novel enzyme-linked immunosorbent assay based on magnetic nanoparticles and biotin/streptavidin-HRP (MNP-bsELISA) was developed for rapid and sensitive detection of zearalenone (ZEN). The detection signal was enhanced and the sensitivity of the assay was improved by combined use of antibody-conjugated magnetic nanoparticles and biotin-streptavidin system. Under the optimized conditions, the regression equation for quantification of ZEN was y = −0.4287x + 0.3132 (R2 = 0.9904). The working range was 0.07–2.41 ng/mL. The detection limit was 0.04 ng/mL and IC50 was 0.37 ng/mL. The recovery rates of intra-assay and inter-assay ranged from 92.8%–111.9% and 91.7%–114.5%, respectively, in spiked corn samples. Coefficients of variation were less than 10% in both cases. Parallel analysis of cereal and feed samples showed good correlation between MNP-bsELISA and liquid chromatograph-tandem mass spectrometry (R2 = 0.9283). We conclude that this method is suitable for rapid detection of zearalenone in cereal and feed samples in relevant laboratories. PMID:26492271

  1. Enzyme-linked immunosorbent assay for rubella immunoglobulin G: new method for attachment of antigens to microtiter plates.

    PubMed Central

    Skurrie, I J; Gilbert, G L

    1983-01-01

    Many of the enzyme-linked immunosorbent assay (ELISA) techniques previously described for detection of rubella-specific antibodies employ complex technology not available in routine diagnostic laboratories. The method described allows the use of commercially available rubella hemagglutination inhibition (HI) antigen. Passive adsorption of these antigens to plastic is variable, but with the use of albumin as a bridge, it is possible to attach the antigen reliably to the plastic wells. Over 1,500 sera were tested by both HI and ELISA techniques to detect the presence of rubella antibodies. These sera were selected with a bias towards those with low levels of rubella-specific antibody, since it has been demonstrated that it is in this range that discrepancies are more likely to occur between HI and ELISA techniques. In 99% of the sera tested, the results of both techniques were in agreement. On the basis of these results, the technique offers a useful alternative to the routine rubella HI test and other ELISA techniques which need sophisticated antigen preparations. PMID:6863497

  2. Development of enzyme-linked immunosorbent assays for two forms of vitellogenin in Japanese common goby (Acanthogobius flavimanus).

    PubMed

    Ohkubo, Nobuyuki; Mochida, Kazuhiko; Adachi, Shinji; Hara, Akihiko; Hotta, Komei; Nakamura, Yukio; Matsubara, Takahiro

    2003-05-01

    Two vitellogenins (Vgs) were detected in serum from estradiol-17beta (E(2))-injected Japanese common goby (Acanthogobius flavimanus). Vitellogenins with molecular masses of 530 kDa (Vg-530) and 320 kDa (Vg-320) were purified, and used to raise specific antisera in rabbits. Sandwich enzyme-linked immunosorbent assays (ELISAs) for Vg-530 and Vg-320 were developed using the antisera and the isolated Vgs. The sensitivity ranges of these ELISAs were 1.25-160 ng/ml for Vg-530 and 0.26-66 ng/ml for Vg-320, and very low cross-reactivity was found with the alternate Vg in each assay. Treatment of male gobys with E(2) by injection and immersion induced both Vgs in sera in a dose-dependent manner. The mean concentrations of the Vgs increased from 10 ng/L E(2) exposure for three weeks. Serum concentrations of the two Vgs in field-collected maturing females increased in accordance with increment of E(2) level and ovarian development, and the mean concentrations of Vg-530 were higher than those of Vg-320 in maturing female. These results indicate that the sandwich ELISAs for Vg-530 and Vg-320 developed in the present study is useful as an assay system for surveys of estrogenic activity in coastal areas of Japan. PMID:12714018

  3. An enzyme-linked immunosorbent assay for detection of Theileria parva antibodies in cattle using a recombinant polymorphic immunodominant molecule.

    PubMed

    Katende, J; Morzaria, S; Toye, P; Skilton, R; Nene, V; Nkonge, C; Musoke, A

    1998-05-01

    Field and experimental bovine infection sera were used in immunoblots of sporozoite and schizont lysates of Theileria parva to identify candidate diagnostic antigens. Four parasite antigens of Mr 67,000 (p67), 85,000 (the polymorphic immunodominant molecule, PIM), 104,000 (p104), and 150,000 (p150) were selected for a more detailed analysis. The p67 and p104 antigens were present only in the sporozoite lysates, whereas PIM and p150 were found in both sporozoite and schizont lysates. The four antigens were expressed as recombinant fusion proteins and were compared with each other in an enzyme-linked immunosorbent assay (ELISA) and in the whole-schizont-based indirect fluorescent antibody test (IFAT) in terms of their ability to detect antibodies in sera of experimentally infected cattle. The PIM-based ELISA provided a higher degree of sensitivity and specificity than did the ELISA using the other three recombinant antigens or the IFAT. Further evaluation of the PIM-ELISA using experimental sera derived from cattle infected with different hemoparasites and field sera from endemic and nonendemic T. parva areas showed that the assay had a sensitivity of > 99% and a specificity of between 94% and 98%. PMID:9610640

  4. Evaluation of different enzyme-linked immunosorbent assays for the diagnosis of brucellosis due to Brucella melitensis in sheep.

    PubMed

    García-Bocanegra, Ignacio; Allepuz, Alberto; Pérez, Julio José; Alba, Anna; Giovannini, Armando; Arenas, Antonio; Candeloro, Luca; Pacios, Alberto; Saez, José Luís; González, Miguel Ángel

    2014-03-01

    Six serological assays for the diagnosis of ovine brucellosis, due to Brucella melitensis were evaluated. Reference serum samples from sheep of known B. melitensis infection status (n=118) were assessed using the Rose Bengal test (RBT), complement fixation test (CFT) and four commercial enzyme-linked immunosorbent assays (ELISAs), including two indirect ELISAs (iELISAs), one competitive ELISA (cELISA) and one blocking ELISA (bELISA). The highest differential positive rates (DPR) were obtained with the cELISA and bELISA, while the lowest DPR was estimated using iELISAs. A latent class analysis was performed to estimate the accuracy of the CFT, RBT and bELISA using 1827 sera from sheep undergoing testing as part of a surveillance and control programme. Lower sensitivity and specificity were obtained for the three serological tests when the field samples were used. A higher DPR was achieved by the CFT, compared to bELISA and RBT. The results suggest that ELISAs, and particularly the bELISA, might be suitable for inclusion in the European Union legislation on intra-community trade for diagnosing B. melitensis infection in sheep, as it has a similar test performance compared to the RBT. PMID:24456797

  5. Development of an indirect enzyme-linked immunosorbent assay for the detection of leptospiral antibodies in dogs.

    PubMed Central

    Ribotta, M J; Higgins, R; Gottschalk, M; Lallier, R

    2000-01-01

    Serology plays an important role in the diagnosis of leptospirosis. Few laboratories have the resources, expertise, or facilities to perform the microscopic agglutination test (MAT). Thus, there is a need for a rapid and simple serological test that could be used in any diagnostic laboratory. In this study, a genus-specific, heat-stable antigenic preparation from Leptospira interrogans serovar pomona was used in an enzyme-linked immunosorbent assay (ELISA) for the detection of leptospiral antibodies in dog sera. This antigenic preparation reacted with rabbit antisera against L. interrogans serovars bratislava, autumnalis, icterohaemorrhagiae and pomona and with rabbit antiserum against L. kirschneri serovar grippotyphosa. The ELISA showed a relative specificity of 95.6% with 158 dog sera which were negative at a dilution of 1:100 in the MAT for serovars pomona, bratislava, icterohaemorrhagiae, autumnalis, hardjo, and grippotyphosa. The relative sensitivity of this assay with 21 dog sera that revealed serovars MAT titres of > or =100 to different serovars was 100%. This assay is easily standardized, technically more advantageous than MAT, and uses an antigenic preparation that can be routinely prepared in large amounts. It was concluded that this ELISA is sufficiently sensitive test to be used as an initial screening test for the detection of leptospiral antibodies in canine sera, with subsequent confirmation of positive test results with the MAT. PMID:10680654

  6. Analyses of mammalian sera in enzyme-linked immunosorbent assays with different strains of Borrelia burgdorferi sensu lato.

    PubMed

    Magnarelli, L A; Anderson, J F; Johnson, R C

    1995-04-01

    Blood samples were collected from cottontail rabbits (Sylvilagus floridanus), raccoons (Procyon lotor), white-footed mice (Peromyscus leucopus), and white-tailed deer (Odocoileus virginianus) between 1977 and 1991 in southern Connecticut and New York State (USA) and were tested for antibodies against eight strains of Borrelia burgdorferi sensu lato in enzyme-linked immunosorbent assays. Among these spirochetes were six strains of B. burgdorferi sensu stricto, one strain of B. garinii (=IP90) and a strain (IPF) in group VS461. Sera from each study group reacted positively to all strains having origins in North America and Eurasia. Assay sensitivities normally ranged between 85% and 100% for all study groups. The lowest sensitivity (66%) was noted when mouse sera were tested with B. garinii, an isolate from Ixodes persulcatus in the former Soviet Union. Differences in serum reactivity to various strains were noted for all study groups, but because of multiple shared antigens among the closely related spirochetes tested, the selection of a particular North American strain of B. burgdorferi sensu stricto did not appear to be a critical factor for optimal assay performance. Locally obtained strains of this bacterium are preferred as coating antigens for serologic testing because of their availability. PMID:8583632

  7. Sensitive radioimmunoassay and enzyme-linked immunosorbent assay for the simultaneous determination of chloroquine and its metabolites in biological fluids

    SciTech Connect

    Escande, C.; Chevalier, P.; Verdier, F.; Bourdon, R. )

    1990-01-01

    Two new methods for the simultaneous determination of chloroquine and its two main metabolites (monodesethylchloroquine and bisdesethylchloroquine) in biological samples, radioimmunoassay (RIA) and enzyme-linked immunosorbent assay (ELISA), are described. Antiserum is produced in rabbits immunized with N-(2-carboxyethyl)desethylchloroquine:protein conjugate. Besides chloroquine, this antiserum recognizes with good affinity the two main metabolites, monodesethylchloroquine and bisdesethylchloroquine (70 and 40% of crossreaction, respectively). Amodiaquine cross reacts by 4.5%; cross reactions with monodesethylamodiaquine, bisdesethylamodiaquine, and other antimalarial drugs are less than 1%. No extraction step or sample preparation is required for either system. Sensitivity limits are, respectively, 0.70 nM (3 pg of chloroquine sulfate measured in 10 microL of plasma sample) for RIA, and 10 nM (22 pg of chloroquine sulfate measured in 5 microL of plasma sample) for ELISA. The interassay coefficients of variation are, respectively, less than 10 and less than 16% for RIA and ELISA in the range 14-410 nM (6-180 ng/mL). The results of both methods are well correlated (r = 0.97) and correlate with spectrophotometry (r = 0.98) and HPLC results (r = 0.93). Because of their high sensitivity, both methods can be used in the case of chloroquine poisoning and in the control of malaria prophylaxis and treatment.

  8. Detection of enterotoxigenic Escherichia coli colonization factor antigen I in stool specimens by an enzyme-linked immunosorbent assay.

    PubMed Central

    Evans, D G; Evans, D J; Clegg, S

    1980-01-01

    An enzyme-linked immunosorbent assay (ELISA) was employed to detect and quantitate the fimbrial colonization factor antigen (CFA/I) of enterotoxigenic Escherichia coli in stool specimens obtained from adult cases of diarrhea in which CFA/I-positive E. coli was the known causative agent. The inhibition method, or blocking technique, was used. In this method, a standardized dilution of human anti-CFA/I serum was preincubated with dilutions of stool extract before transfer to CFA/I-coated microtiter plate wells, and then ELISA was performed with alkaline phosphatase-conjugated anti-human immunoglobulin. CFA/I purified from E. coli strain H-10407 (O78:H11) was used. Acute-phase diarrheal stool specimens were found to contain approximately 3.0 mg of antigen (mean value) per g stool, whereas control (CFA/I-negative) specimens contained insignificant amounts (less than 0.03 mg/g) of antigen. Also, CFA/I was detected in culture fluids of CFA/I positive enterotoxigenic E. coli belonging to a variety of serotypes and was undetectable in similar preparations from P-strains (spontaneous CFA/I-negative derivatives) of the same test cultures. Equivalent results were obtained in ELISA tests by using bacterial cells taken from isolated colonies grown on CFA agar. These results indicate that the ELISA technique will be useful for the diagnosis of diarrhea caused by CFA/I-positive enterotoxigenic E. coli. PMID:7031075

  9. Comparison of methods of immobilization to enzyme-linked immunosorbent assay plates for the detection of sugar chains.

    PubMed

    Satoh, A; Fukui, E; Yoshino, S; Shinoda, M; Kojima, K; Matsumoto, I

    1999-11-15

    The immobilization of carbohydrates for solid-phase assays, including enzyme-linked immunosorbent assay (ELISA), is difficult because they are hydrophilic. We developed four new methods for the immobilization of oligosaccharides. ELISA plates were first coated with methyl vinyl ether-maleic anhydride copolymer (MMAC) and an excess of active anhydride groups was introduced. They were subsequently reacted, in four different ways, to bind oligosaccharides. In method 1, the anhydride groups were reacted with hydrazide groups, in the presence of adipic acid dihydrazide, and then coupled to the reducing ends of sugar chains by reductive amination. In method 2, the anhydride groups were reacted with p-aminophenyl glycoside obtained by reduction with p-nitrophenyl glycoside. In method 3, the anhydride groups were reacted with 1, 6-hexamethylenediamine. Aminooxy groups were coupled to the amino groups introduced and then aminooxyacetic acid with carbodiimide and ligated to oligosaccharides by oxime formation. In method 4, stereospecifically aminated oligosaccharides reacted with the anhydride groups. We compared, in solid-phase assays systems, the ability of lectins to detect oligosaccharides immobilized with either one of these four new methods or one of the two methods previously described. Detection of sugars with lectins is useful because, in most cases, they recognize sugars stereospecifically. The immobilization method should therefore be carefully selected to avoid changing the configuration and substitution in C-1. PMID:10552909

  10. Enhanced binding of capsular polysaccharides of Cryptococcus neoformans to polystyrene microtitration plates for enzyme-linked immunosorbent assay.

    PubMed

    Cherniak, R; Cheeseman, M M; Reyes, G H; Reiss, E; Todaro, F

    1988-01-01

    A sensitive enzyme-linked immunosorbent assay (ELISA) to measure antibodies against capsular polysaccharide was developed, based on the enhanced binding of polysaccharide to polystyrene microtitration plates. The wells of the microtitration plate were primed with an adipic acid dihydrazide derivative of bovine serum albumin (AH-BSA) (100 micrograms/mL, 0.01 M NaPO4-0.14 M NaCl, pH 7.2 (PBS]. Capsular polysaccharide, the glucuronoxylomannan of Cryptococcus neoformans serotype A, was oxidized with NaIO4 for 5 min; the reaction was then quenched with ethylene glycol. The partially oxidized polysaccharide was dialyzed vs. PBS, and its concentration was adjusted to 50 micrograms/mL with PBS. This solution (100 microL/well) was covalently bound to the AH-BSA primed microtitration plates through formation of a Schiff base between the hydrazide group on the AH-BSA and the aldehyde groups on the polysaccharide. Antimouse IgG-alkaline phosphatase conjugate was used in an indirect ELISA to measure captured murine monoclonal antibodies directed against glucuronoxylomannan. Mean absorbances, after 15 min, were 0.13 in negative control wells, and greater than 0.7 in test wells. No intermediate steps were required to block nonspecific binding of antibody. PMID:3064947

  11. Smartphone-interfaced lab-on-a-chip devices for field-deployable enzyme-linked immunosorbent assay

    PubMed Central

    Chen, Arnold; Wang, Royal; Bever, Candace R. S.; Xing, Siyuan; Pan, Tingrui

    2014-01-01

    The emerging technologies on mobile-based diagnosis and bioanalytical detection have enabled powerful laboratory assays such as enzyme-linked immunosorbent assay (ELISA) to be conducted in field-use lab-on-a-chip devices. In this paper, we present a low-cost universal serial bus (USB)-interfaced mobile platform to perform microfluidic ELISA operations in detecting the presence and concentrations of BDE-47 (2,2′,4,4′-tetrabromodiphenyl ether), an environmental contaminant found in our food supply with adverse health impact. Our point-of-care diagnostic device utilizes flexible interdigitated carbon black electrodes to convert electric current into a microfluidic pump via gas bubble expansion during electrolytic reaction. The micropump receives power from a mobile phone and transports BDE-47 analytes through the microfluidic device conducting competitive ELISA. Using variable domain of heavy chain antibodies (commonly referred to as single domain antibodies or Nanobodies), the proposed device is sensitive for a BDE-47 concentration range of 10−3–104 μg/l, with a comparable performance to that uses a standard competitive ELISA protocol. It is anticipated that the potential impact in mobile detection of health and environmental contaminants will prove beneficial to our community and low-resource environments. PMID:25553178

  12. Recombinant Antigen-Based Enzyme-Linked Immunosorbent Assay for Diagnosis of Baylisascaris procyonis Larva Migrans ▿

    PubMed Central

    Dangoudoubiyam, Sriveny; Vemulapalli, Ramesh; Ndao, Momar; Kazacos, Kevin R.

    2011-01-01

    Baylisascaris larva migrans is an important zoonotic disease caused by Baylisascaris procyonis, the raccoon roundworm, and is being increasingly considered in the differential diagnosis of eosinophilic meningoencephalitis in children and young adults. Although a B. procyonis excretory-secretory (BPES) antigen-based enzyme-linked immunosorbent assay (ELISA) and a Western blot assay are useful in the immunodiagnosis of this infection, cross-reactivity remains a major problem. Recently, a recombinant B. procyonis antigen, BpRAG1, was reported for use in the development of improved serological assays for the diagnosis of Baylisascaris larva migrans. In this study, we tested a total of 384 human patient serum samples in a BpRAG1 ELISA, including samples from 20 patients with clinical Baylisascaris larva migrans, 137 patients with other parasitic infections (8 helminth and 4 protozoan), and 227 individuals with unknown/suspected parasitic infections. A sensitivity of 85% and a specificity of 86.9% were observed with the BpRAG1 ELISA, compared to only 39.4% specificity with the BPES ELISA. In addition, the BpRAG1 ELISA had a low degree of cross-reactivity with antibodies to Toxocara infection (25%), while the BPES antigen showed 90.6% cross-reactivity. Based on these results, the BpRAG1 antigen has a high degree of sensitivity and specificity and should be very useful and reliable in the diagnosis and seroepidemiology of Baylisascaris larva migrans by ELISA. PMID:21832102

  13. Ultrasensitive enzyme-linked immunosorbent assay (ELISA) of proteins by combination with the thio-NAD cycling method

    PubMed Central

    Watabe, Satoshi; Kodama, Hiromi; Kaneda, Mugiho; Morikawa, Mika; Nakaishi, Kazunari; Yoshimura, Teruki; Iwai, Atsushi; Miura, Toshiaki; Ito, Etsuro

    2014-01-01

    An ultrasensitive method for the determination of proteins is described that combines an enzyme-linked immunosorbent assay (ELISA) and a thionicotinamide-adenine dinucleotide (thio-NAD) cycling method. A sandwich method using a primary and a secondary antibody for antigens is employed in an ELISA. An androsterone derivative, 3α-hydroxysteroid, is produced by the hydrolysis of 3α-hydroxysteroid 3-phosphate with alkaline phosphatase linked to the secondary antibody. This 3α-hydroxysteroid is oxidized to a 3-ketosteroid by 3α- hydroxysteroid dehydrogenase (3α-HSD) with a cofactor thio-NAD. By the opposite reaction, the 3-ketosteroid is reduced to a 3α-hydroxysteroid by 3α-HSD with a cofactor NADH. During this cycling reaction, thio-NADH accumulates in a quadratic function-like fashion. Accumulated thio-NADH can be measured directly at an absorbance of 400 nm without any interference from other cofactors. These features enable us to detect a target protein with ultrasensitivity (10−19 mol/assay) by measuring the cumulative quantity of thio-NADH. Our ultrasensitive determination of proteins thus allows for the detection of small amounts of proteins only by the application of thio-NAD cycling reagents to the usual ELISA system. PMID:27493498

  14. An enzyme-linked immunosorbent assay for detection of avian influenza virus subtypes H5 and H7 antibodies

    PubMed Central

    2013-01-01

    Background Avian influenza virus (AIV) subtypes H5 and H7 attracts particular attention because of the risk of their potential pathogenicity in poultry. The haemagglutination inhibition (HI) test is widely used as subtype specific test for serological diagnostics despite the laborious nature of this method. However, enzyme-linked immunosorbent assays (ELISAs) are being explored as an alternative test method. H5 and H7 specific monoclonal antibodies were experimentally raised and used in the development of inhibition ELISAs for detection of serological response specifically directed against AIV subtypes H5 and H7. The ELISAs were evaluated with polyclonal chicken anti-AIV antibodies against AIV subtypes: H1N2, H5N2, H5N7, H7N1, H7N7, H9N9, H10N4 and H16N3. Results Both the H5 and H7 ELISA proved to have a high sensitivity and specificity and the ELISAs detected H5 and H7 antibodies earlier during experimental infection than the HI test did. The reproducibility of the ELISA’s performed at different times was high with Pearson correlation coefficients of 0.96-0.98. Conclusions The ELISAs are a potential alternative to the HI test for screening of large amounts of avian sera, although only experimental sera were tested in this study. PMID:24256721

  15. Quantification of osteoclastic resorption of the bovine otic capsule in vitro by an enzyme-linked immunosorbent assay.

    PubMed

    Frisch, T; Foged, N T; Sørensen, M S; Bretlau, P

    2000-01-01

    The bony shell surrounding the inner ear is known to have a very pronounced centripetal inhibition of remodelling in vivo, with almost no bone turnover immediately adjacent to the perilymphatic spaces and a gradually increasing turnover rate towards outer parts of the bony otic capsule. By the use of in vitro markers of bone resorption, including an enzyme-linked immunosorbent assay for quantification of type I collagen degradation and a colorimetric enzyme assay for quantification of osteoclast tartrate-resistant acid phosphatase activity, this study demonstrates that there are no ex vivo differences in bone matrix resorption between the inner and outer parts of the otic capsule when exposed to seeded osteoclasts from rabbits. Thus, the unique spatial distribution of perilabyrinthine bone turnover is not caused by a shift in resorbability from inner to outer capsular bone that is due to inherent bone quality differences particular to these bone compartments. More likely, the sustained action of some intravital 'field force', originating from the inner ear spaces, is responsible for the unique spatial distribution of the otic capsular bone turnover found in vivo. Though the character of this force is not yet defined, it is appealing to relate it to the large electromagnetic potential gradient present in the inner ear. PMID:10965257

  16. ENZYME-LINKED IMMUNOSORBENT ASSAY FOR SCREENING DIOXIN SOIL CONTAMINATION BY UNCONTROLLED COMBUSTION DURING INFORMAL RECYCLING IN SLUMS

    PubMed Central

    Trindade, Mirta; Nording, Malin; Nichkova, Mikaela; Spinnel, Erik; Haglund, Peter; Last, Michael S.; Gee, Shirley; Hammock, Bruce; Last, Jerold A.; González-Sapienza, Gualberto; Brena, Beatriz M.

    2010-01-01

    Uncontrolled combustion due to garbage recycling is a widespread activity among slum dwellers in distressed economy countries and has been indicated as a major source of dioxin contamination. However, because of the high cost and complexity of gas chromatography/high-resolution mass spectrometry (GC-HRMS) analysis, the magnitude of the problem remains largely unknown. The present study describes a first approach toward the use of a dioxin antibody-based enzyme-linked immunosorbent assay (ELISA) as the basis for a sustainable, simple, and low-cost monitoring program to assess the toxicological impact of uncontrolled combustion in slums. A panel of 16 samples was analyzed by GC-HRMS and ELISA on split extracts. Close to 20% of the analyzed samples showed dioxin concentrations up to almost twice the guidance level for residential soil in several countries, pointing out the need for performing a large-scale monitoring program. Despite the potential for variations in dioxin congener distribution due to the mixed nature of the incinerated material, there was a good correlation between the toxic equivalents as determined by GC-HRMS and ELISA. Furthermore, an interlaboratory ELISA validation showed that the capacity to perform the dioxin ELISA was successfully transferred between laboratories. It was concluded that the ELISA method performed very well as a screening tool to prioritize samples for instrumental analysis, which allows cutting down costs significantly. PMID:18522475

  17. Development of monoclonal antibodies and quantitative sandwich enzyme linked immunosorbent assay for the characteristic sialoglycoprotein of edible bird's nest.

    PubMed

    Zhang, Shiwei; Lai, Xintian; Liu, Xiaoqing; Li, Yun; Li, Bifang; Huang, Xiuli; Zhang, Qinlei; Chen, Wei; Lin, Lin; Yang, Guowu

    2013-01-01

    The article presents a sandwich enzyme linked immunosorbent assay (ELISA) for identification of edible bird's nest. The characteristic sialoglycoproteins were found by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and purified by liquid-phase isoelectric focusing (LIEF). According to the analysis, the molecular weight was 106-128 kDa and the isoelectric point was ≤pH 3.0. Two anti-characteristic sialoglycoprotein monoclonal antibodies were produced. The monoclonal antibodies were examined by western-blot assay. One of the monoclonal antibody was used as coating and the other as the enzyme-labeled antibody after being coupled to horseradish peroxidase (HRP). Based on the optimized ELISA condition, the method was established with IC(50) of 1.5 ng/mL, and low cross-reactivity with various fake materials (<0.01%). ELISA provided a suitable means for screening of a large number of samples. The coefficients of variation were between 2.9% and 5.8%. PMID:23323981

  18. Development of enzyme-linked immunosorbent assays for Sudan dyes in chilli powder, ketchup and egg yolk.

    PubMed

    Anfossi, Laura; Baggiani, Claudio; Giovannoli, Cristina; Giraudi, Gianfranco

    2009-06-01

    This study aimed at developing sensitive competitive enzyme-linked immunosorbent assays (ELISAs) for the banned Sudan dyes using polyclonal antibodies. Three different formats were developed and characterized in terms of sensitivity, selectivity and rapidity. A competitive indirect ELISA was developed, which showed an IC(50) of 3.8 microg l(-1). Two competitive direct ELISAs were also developed, in which the antibody was added before or simultaneously with the other reagents; the first showed an IC(50) of 8.3 microg l(-1) and the latter showed an IC(50) of 4.9 microg l(-1). Nevertheless, considering dilution of extracts which is needed to offset matrix interference, the limits of detection of the three formats were substantially the same (10 microg kg(-1)). The antibodies in all three test formats were able to recognize Sudan I and partially Sudan II, III and IV; no cross-reactivity was observed with the five edible dyes. Twenty food samples, including chilli powder, paprika, ketchup, and egg, were extracted by a simple sample preparation and very limited dilution. Extracts were analyzed by the developed competitive direct ELISA with the simultaneous addition of reagents. A good correlation was observed (y = 1.19 x-10.0, r(2) = 0.991, n = 20) when the data was compared with that obtained through a conventional HPLC method. PMID:19680953

  19. The use of enzyme-linked immunosorbent assays (ELISA) for the determination of pollutants in environmental and industrial wastes.

    PubMed

    Hirobe, M; Goda, Y; Okayasu, Y; Tomita, J; Takigami, H; Ike, M; Tanaka, H

    2006-01-01

    Twelve enzyme-linked immunosorbent assays (ELISA), for the determination of surfactants [linear alkylbenzene sulfonates (LAS), alkyl ethoxylates (AE), and alkylphenol ethoxylates (APE)], endocrine disruptors [alkylphenol (AP), AP + APE, and bisphenol A (BPA)], estrogens [17beta-estradiol (E2), estrone (El), estrogen (ES: El + E2 + estriol (E3)), 1 7alfa-ethynylestradiol (EE2)], dioxins and polychlorinated biphenyls (PCBs), were validated on environmental water and industrial wastes. The lowest quantification limits of these ELISAs were 0.05 microg/L (BPA, E2, El, ES and EE2), 2 microg/L (AE), 3 microg/L (dioxins and PCBs), 5 microg/L (AP, AP + APE) and 20 microg/L (LAS and APE). To apply these ELISAs to environmental or industrial waste samples, simple and appropriate pre-treatment methods were also developed for each ELISA. With optimized pre-treatments, the values of ELISAs were well co-related, in all cases, to those of instrumental analytical methods such as liquid chromatography (HPLC), liquid chromatography-tandem mass spectrometry (LC-MS/MS), and high-resolution gas chromatography mass spectrometry (HR-GC-MS), etc. PMID:17302299

  20. The role of iron in neurodegeneration—Mössbauer spectroscopy, electron microscopy, enzyme-linked immunosorbent assay and neuroimaging studies

    NASA Astrophysics Data System (ADS)

    Galazka-Friedman, Jolanta; Bauminger, Erika R.; Szlachta, Karol; Friedman, Andrzej

    2012-06-01

    The possible role of iron in neurodegeneration was studied by various techniques: electron microscopy, enzyme-linked immunosorbent assay, Mössbauer spectroscopy, atomic absorption, ultrasonography and magnetic resonance imaging. The measurements were made on human tissues extracted from liver and from brain structures involved in diseases of the human brain: substantia nigra (Parkinson’s, PD), hippocampal cortex (Alzheimer’s, AD) and globus pallidus (progressive supranuclear palsy, PSP). The sizes of the iron cores of ferritin, the main iron storage compound in tissues, were found to be smaller in brain than in liver. Brain ferritin has a higher proportion of H to L chains compared to liver. A significant decrease of the concentration of L chains in PD compared to control was found. No increase in the concentration of iron in PD versus control was detected; however, there was an increase of labile iron, which constitutes only 2‰ of brain iron. In AD an increase in the concentration of ferritin was noticed, without a significant increase in iron concentration. In PSP an increase of total iron was observed. Our findings suggest that the mechanisms leading to the death of nerve cells in these three diseases may be different, although all may be related to iron mediated oxidative stress.

  1. Development of an equine coronavirus-specific enzyme-linked immunosorbent assay to determine serologic responses in naturally infected horses.

    PubMed

    Kooijman, Lotte J; Mapes, Samantha M; Pusterla, Nicola

    2016-07-01

    Equine coronavirus (EqCoV) infection has been documented in most reports through quantitative qPCR analysis of feces and viral genome sequencing. Although qPCR is used to detect antigen during the acute disease phase, there is no equine-specific antibody test available to study EqCoV seroprevalence in various horse populations. We developed an enzyme-linked immunosorbent assay (ELISA) targeting antibodies to the spike (S) protein of EqCoV and validated its use, using acute and convalescent sera from 83 adult horses involved in 6 outbreaks. The EqCoV S protein-based ELISA was able to reliably detect antibodies to EqCoV in naturally infected horses. The greatest seroconversion rate was observed in horses with clinical signs compatible with EqCoV infection and EqCoV qPCR detection in feces. The EqCoV S protein-based ELISA could be used effectively for seroepidemiologic studies in order to better characterize the overall infection rate of EqCoV in various horse populations. PMID:27216723

  2. Development of enzyme linked immunosorbent assay (ELISA) for the detection of root-knot nematode Meloidogyne incognita.

    PubMed

    Kapur-Ghai, J; Kaur, M; Goel, P

    2014-09-01

    Root-knot nematodes (Meloidogyne incognita) are obligate, sedentary plant endoparasites that are extremely polyphagous in nature and cause severe economic losses in agriculture. Hence, it is essential to control the parasite at an early stage. For any control strategy to be effective, an early and accurate diagnosis is of paramount importance. Immunoassays have the inherent advantages of sensitivity and specificity; have the potential to identify and quantify these plant-parasitic nematodes. Hence, in the present studies, enzyme-linked immunosorbent assay (ELISA) has been developed for the detection of M.incognita antigens. First an indirect ELISA was developed for detection and titration of anti-M.incognita antibodies. Results indicated as high as 320 K titre of the antisera. Finally competitive inhibition ELISA was developed employing these anti-M.incognita antibodies for detection of M.incognita antigens. Sensitivity of ELISA was 10 fg. Competitive inhibition ELISA developed in the present studies has the potential of being used as an easy, rapid, specific and sensitive diagnostic tool for the detection of M.incognita infection. PMID:25035590

  3. A recombinant nucleocapsid protein-based indirect enzyme-linked immunosorbent assay to detect antibodies against porcine deltacoronavirus

    PubMed Central

    SU, Mingjun; LI, Chunqiu; GUO, Donghua; WEI, Shan; WANG, Xinyu; GENG, Yufei; YAO, Shuang; GAO, Jing; WANG, Enyu; ZHAO, Xiwen; WANG, Zhihui; WANG, Jianfa; WU, Rui; FENG, Li; SUN, Dongbo

    2015-01-01

    Recently, porcine deltacoronavirus (PDCoV) has been proven to be associated with enteric disease in piglets. Diagnostic tools for serological surveys of PDCoV remain in the developmental stage when compared with those for other porcine coronaviruses. In our study, an indirect enzyme-linked immunosorbent assay (ELISA) (rPDCoV-N-ELISA) was developed to detect antibodies against PDCoV using a histidine-tagged recombinant nucleocapsid (N) protein as an antigen. The rPDCoV-N-ELISA did not cross-react with antisera against porcine epidemic diarrhea virus, swine transmissible gastroenteritis virus, porcine group A rotavirus, classical swine fever virus, porcine circovirus-2, porcine pseudorabies virus, and porcine reproductive and respiratory syndrome virus; the receiver operating characteristic (ROC) curve analysis revealed 100% sensitivity and 90.4% specificity of the rPDCoV-N-ELISA based on samples of known status (n=62). Analyses of field samples (n=319) using the rPDCoV-N-ELISA indicated that 11.59% of samples were positive for antibodies against PDCoV. These data demonstrated that the rPDCoV-N-ELISA can be used for epidemiological investigations of PDCoV and that PDCoV had a low serum prevalence in pig population in Heilongjiang province, northeast China. PMID:26668175

  4. Preparation of Antibodies and Development of an Enzyme-Linked Immunosorbent Assay for the Tyrosine Kinase Inhibitors Lapatinib and Nilotinib.

    PubMed

    Saita, Tetsuya; Yamamoto, Yuta; Shin, Masashi; Nakano, Yukitaka

    2015-01-01

    In this paper, we describe the production of the first specific antibodies against the tyrosine kinase inhibitors lapatinib and nilotinib. Anti-lapatinib antibody was obtained by immunizing rabbits with an antigen conjugated with bovine serum albumin using 3-chloro-4-((3-fluorobenzyl)oxy)aniline. Anti-nilotinib antibody was produced by immunizing mice with an antigen conjugated with bovine serum albumin using 2-(5-amino-2-methylanilino)-4-(3-pyridyl)pyrimidine. The generated antibodies were used to develop highly sensitive and specific enzyme-linked immunosorbent assays (ELISAs) for lapatinib and nilotinib in human serum. The assays were capable of detecting lapatinib and nilotinib at serum concentrations as low as 40 and 8 ng/mL, respectively. Using the two ELISAs, drugs levels were easily measured in the serum of rats after a single dose oral administration of lapatinib or nilotinib. The assays are therefore expected be valuable tools for therapeutic drug monitoring in the clinical setting and pharmacokinetic studies of lapatinib and nilotinib. PMID:26424026

  5. Commercial Milk Enzyme-Linked Immunosorbent Assay (ELISA) Kit Reactivities to Purified Milk Proteins and Milk-Derived Ingredients.

    PubMed

    Ivens, Katherine O; Baumert, Joseph L; Taylor, Steve L

    2016-07-01

    Numerous commercial enzyme-linked immunosorbent assay (ELISA) kits exist to quantitatively detect bovine milk residues in foods. Milk contains many proteins that can serve as ELISA targets including caseins (α-, β-, or κ-casein) and whey proteins (α-lactalbumin or β-lactoglobulin). Nine commercially-available milk ELISA kits were selected to compare the specificity and sensitivity with 5 purified milk proteins and 3 milk-derived ingredients. All of the milk kits were capable of quantifying nonfat dry milk (NFDM), but did not necessarily detect all individual protein fractions. While milk-derived ingredients were detected by the kits, their quantitation may be inaccurate due to the use of different calibrators, reference materials, and antibodies in kit development. The establishment of a standard reference material for the calibration of milk ELISA kits is increasingly important. The appropriate selection and understanding of milk ELISA kits for food analysis is critical to accurate quantification of milk residues and informed risk management decisions. PMID:27272960

  6. A novel enzyme-linked immunosorbent assay for ethynylestradiol using a long-chain biotinylated EE2 derivative.

    PubMed

    Schneider, Christian; Schöler, Heinz F; Schneider, Rudolf J

    2004-04-01

    Ethynylestradiol (EE2) is one of the most potent endocrine disrupting compounds capable to induce estrogenic effects even at trace level concentrations in the aquatic environment. Methods for detecting EE2 in such concentrations are generally based on GC or HPLC coupled to at least one mass spectrometer. Another approach are immunoassays and sensor systems but for most designs, derivatives of EE2 are required (e.g. for coupling to carrier proteins, enzyme or fluorescent labels, etc.). Here we present the straightforward synthesis and complete characterization of a new long-chain biotinylated EE2 derivative. The new EE2 derivative is used as tracer in a direct competitive enzyme-linked immunosorbent assay (ELISA) for the determination of EE2. With pure water, the limit of detection (LOD, signal-to-noise ratio, S/N = 3) and the test midpoint were found to be 14 and 136 ng l(-1), respectively. Cross reactivity (CR) was tested for 10 endogenous steroids and the BSA-conjugate used for immunization, as well as a synthetic precursor of the conjugate. Among the naturally occurring compounds, CR was determined to be maximum for metabolites of EE2 conjugated at ring-position 3 (17% and 37% for 3-glucuronide and 3-sulphate, respectively). Assay stability was tested against humic substances and organic solvents. Increasing amounts of organic solvents in the sample caused a clear decrease in sensitivity, presence of humic substances lead to an overestimation of EE2. PMID:15183690

  7. A modified enzyme-linked immunosorbent assay adapted for immunodetection of low amounts of water-insoluble proteins.

    PubMed

    Godfrin, Dominique; Sénéchal, Hélène; Sutra, Jean-Pierre; Busnel, Jean-Marc; Desvaux, François-Xavier; Peltre, Gabriel

    2007-09-30

    A mixture of thiourea, urea and CHAPS (TUC) is an excellent solvent compatible with isoelectrofocusing (IEF) separation of water-insoluble protein extracts, and their subsequent two-dimensional gel electrophoresis is an important step in proteomic studies. The main aim of this work was to quantify extremely low amounts of water-insoluble proteins contained, for instance, in samples collected in bio-aerosol samplers. High CHAPS concentrations solubilize many proteins. However, enzyme-linked immunosorbent assay (ELISA), which is the most popular immunodetection method of quantifying antigens, is unfortunately not compatible with these high CHAPS concentrations and with the low protein concentrations of TUC extracts. The most common mixture used to solubilize these proteins contains 2 mol l(-1) thiourea, 7 mol l(-1) urea and 5% w/v CHAPS. This paper shows that these components inhibit the adsorption and/or recognition of proteins on microtitration plates, preventing antigen quantification under classic ELISA conditions. We have tried several solvents (ethanol, isopropanol, acetonitrile and trichloroacetic acid) to make the TUC-soluble proteins stick to the ELISA plates, and ethanol was shown to be the most appropriate. In this study, we have defined a new ELISA protocol allowing rapid and sensitive detection of low concentrations (60-500 ng ml(-1)) of water-insoluble proteins extracted with high concentrations of TUC. PMID:17706662

  8. A recombinant nucleocapsid protein-based indirect enzyme-linked immunosorbent assay to detect antibodies against porcine deltacoronavirus.

    PubMed

    Su, Mingjun; Li, Chunqiu; Guo, Donghua; Wei, Shan; Wang, Xinyu; Geng, Yufei; Yao, Shuang; Gao, Jing; Wang, Enyu; Zhao, Xiwen; Wang, Zhihui; Wang, Jianfa; Wu, Rui; Feng, Li; Sun, Dongbo

    2016-05-01

    Recently, porcine deltacoronavirus (PDCoV) has been proven to be associated with enteric disease in piglets. Diagnostic tools for serological surveys of PDCoV remain in the developmental stage when compared with those for other porcine coronaviruses. In our study, an indirect enzyme-linked immunosorbent assay (ELISA) (rPDCoV-N-ELISA) was developed to detect antibodies against PDCoV using a histidine-tagged recombinant nucleocapsid (N) protein as an antigen. The rPDCoV-N-ELISA did not cross-react with antisera against porcine epidemic diarrhea virus, swine transmissible gastroenteritis virus, porcine group A rotavirus, classical swine fever virus, porcine circovirus-2, porcine pseudorabies virus, and porcine reproductive and respiratory syndrome virus; the receiver operating characteristic (ROC) curve analysis revealed 100% sensitivity and 90.4% specificity of the rPDCoV-N-ELISA based on samples of known status (n=62). Analyses of field samples (n=319) using the rPDCoV-N-ELISA indicated that 11.59% of samples were positive for antibodies against PDCoV. These data demonstrated that the rPDCoV-N-ELISA can be used for epidemiological investigations of PDCoV and that PDCoV had a low serum prevalence in pig population in Heilongjiang province, northeast China. PMID:26668175

  9. Hapten synthesis, monoclonal antibody production and development of a competitive indirect enzyme-linked immunosorbent assay for erythromycin in milk.

    PubMed

    Wang, Zhanhui; Mi, Tiejun; Beier, Ross C; Zhang, Huiyan; Sheng, Yajie; Shi, Weimin; Zhang, Suxia; Shen, Jianzhong

    2015-03-15

    Erythromycin is an antibiotic used extensively in veterinary practice worldwide for treatment, prevention and growth promotion. In this work, monoclonal antibodies (Mabs) against erythromycin were produced and used to develop a competitive indirect enzyme-linked immunosorbent assay (ciELISA) for the determination of erythromycin in milk. A novel carboxyphenyl derivative of erythromycin (ERO-CMO) was synthesized and conjugated with bovine serum (BSA) for use as the immunogen or ovalbumin (OVA) as the coating antigen. Four hybridoma cell lines were isolated, which produced Mabs that competed with erythromycin. The 6C1 and 5B2 Mabs had IC50 values for erythromycin of 14.40 and 0.94 μg L(-)(1), respectively. These Mabs demonstrated high cross-reactivity to the macrolides containing 14-membered rings, but not to oleandomycin. No cross-reactivity was observed for 12 macrolides that contained 15 or 16-membered lactone rings or for 2 pleuromutilins. The ciELISA developed using the 5B2 Mab afforded recovery values that ranged from 76.9% to 85.7% with only a 10-fold sample dilution prior to analysis. PMID:25308648

  10. Recombinase polymerase and enzyme-linked immunosorbent assay as a DNA amplification-detection strategy for food analysis.

    PubMed

    Santiago-Felipe, S; Tortajada-Genaro, L A; Puchades, R; Maquieira, A

    2014-02-01

    Polymerase chain reaction in conjunction with enzyme-linked immunosorbent assay (PCR-ELISA) is a well-established technique that provides a suitable rapid, sensitive, and selective method for a broad range of applications. However, the need for precise rapid temperature cycling of PCR is an important drawback that can be overcome by employing isothermal amplification reactions such as recombinase polymerase amplification (RPA). The RPA-ELISA combination is proposed for amplification at a low, constant temperature (40°C) in a short time (40 min), for the hybridisation of labelled products to specific 5'-biotinylated probes/streptavidin in coated microtiter plates at room temperature, and for detection by colorimetric immunoassay. RPA-ELISA was applied to screen common safety threats in foodstuffs, such as allergens (hazelnut, peanut, soybean, tomato, and maize), genetically modified organisms (P35S and TNOS), pathogenic bacteria (Salmonella sp. and Cronobacter sp.), and fungi (Fusarium sp.). Satisfactory sensitivity and reproducibility results were achieved for all the targets. The RPA-ELISA technique does away with thermocycling and provides a suitable sensitive, specific, and cost-effective method for routine applications, and proves particularly useful for resource-limited settings. PMID:24456598

  11. Demonstration of immunoglobulin M class antibodies to Toxoplasma gondii antigenic component p35000 by enzyme-linked antigen immunosorbent assay.

    PubMed Central

    Lindenschmidt, E G

    1986-01-01

    On the basis that 89% of 48 acute-phase toxoplasmosis patients showed immunoglobulin M (IgM) class antibodies to the 35,000-molecular-weight antigenic component (p35000) of Toxoplasma gondii, as demonstrated by IgM immunoblotting, the antigen was purified by sucrose gradient centrifugation and enzyme labeled for use in an enzyme-linked antigen immunosorbent assay (ELA) for the demonstration of IgM class antibodies to the p35000 component. The ELA showed a specificity of 96% with 139 serum specimens at a serum dilution of only 1:5. The test serologically detected 73 symptomatic acute-phase toxoplasmosis patients; 64 were positive in the 19S IgM indirect immunofluorescent-antibody test, and 9 were negative, although they showed IgM antibodies to p35000, as demonstrated by IgM immunoblotting. Also, the ELA turned out to be independent of IgM rheumatoid factors in six acute-phase toxoplasmosis serum specimens. PMID:3536996

  12. Rapid diagnosis of typhoid fever by enzyme-linked immunosorbent assay detection of Salmonella serotype typhi antigens in urine.

    PubMed

    Fadeel, Moustafa Abdel; Crump, John A; Mahoney, Frank J; Nakhla, Isabelle A; Mansour, Adel M; Reyad, Baheia; El Melegi, Dawlat; Sultan, Yehia; Mintz, Eric D; Bibb, William F

    2004-03-01

    We developed and evaluated an enzyme-linked immunosorbent assay (ELISA) using monoclonal antibodies to capture somatic antigen 9 (O9), flagellar antigen d (Hd), and the Vi capsular polysaccharide antigen (Vi) from the urine of persons with and without typhoid fever. Sequential urine samples were collected from 44 patients with blood culture-confirmed typhoid fever and from two control groups. The first control group included patients with brucellosis (n = 12) and those with clinically diagnosed, non-typhoid, acute, febrile illness (n = 27). The second control group was a sample of healthy volunteer laboratory workers (n = 11). When assessed relative to date of fever onset, sensitivity was highest during the first week for all three antigens: Vi was detected in the urine of nine (100%) patients, O9 in 4 (44%) patients, and Hd in 4 (44%) patients. Sequential testing of two urine samples from the same patient improved test sensitivity. Combined testing for Vi with O9 and Hd produced a trend towards increased sensitivity without compromising specificity. The specificity for Vi exceeded 90% when assessed among both febrile and healthy control subjects, but was only 25% when assessed among patients with brucellosis. Detection of urinary Vi antigen with this ELISA shows promise for the diagnosis of typhoid fever, particularly when used within the first week after fever onset. However, positive reactions for Vi antigen in patients with brucellosis must be understood before urinary Vi antigen detection can be developed further as a useful rapid diagnostic test. PMID:15031525

  13. Enzyme-linked immunosorbent assay for detection of Salmonella typhi Vi antigen in urine from typhoid patients.

    PubMed

    Barrett, T J; Snyder, J D; Blake, P A; Feeley, J C

    1982-02-01

    Because typhoid fever continues to be a major cause of illness in many developing countries, there is a clear need for a sensitive and specific test that will permit rapid laboratory diagnosis of the disease. An enzyme-linked immunosorbent assay (ELISA) has recently been developed and tested, both in the laboratory and in a clinical situation, for its ability to detect Vi antigen in urine. The ELISA was capable of detecting as little as 1 ng of purified Vi antigen per ml in urine, compared with 100 ng/ml detectable by a previously tested coagglutination method. It could also detect antigen in urine diluted as much as 1:1,024 in normal urine. In tests of urine specimens from six stool culture-positive persons in a small typhoid outbreak in the United States, the ELISA detected antigen in specimens from four of the six patients. The ELISA also proved to be specific, giving no false-positive results for specimens from 50 persons who did not have typhoid fever. The apparent high sensitivity and specificity of this ELISA make it a promising test for rapid diagnosis of typhoid fever. PMID:7040446

  14. Restricted access supramolecular solvents for sample treatment in enzyme-linked immuno-sorbent assay of mycotoxins in food.

    PubMed

    García-Fonseca, Sergio; Ballesteros-Gómez, Ana; Rubio, Soledad

    2016-09-01

    A restricted access supramolecular solvent (SUPRAS-RAM) made up of tetradecanoic acid reverse micelles is proposed as a wide-scope and low-cost strategy for the treatment of agrifood samples prior to enzyme-linked immunosorbent assays (ELISA). The approach was assessed for the determination of ochratoxin A (OTA) in wines and spices and aflatoxin B1 (AFB1) in cereals, two ubiquitous mycotoxins that were selected as representative contaminants for this study. The samples were selected to cover a variety of matrices in terms diverse composition and high complexity. Macromolecules such as proteins and carbohydrates were not-co-extracted due to the restricted access properties of the SUPRAS that are provided by chemical and physical mechanisms. In this sense, analyte extraction and clean-up were carried out in a single step. Parameters determining the extraction efficiency were studied and optimized. Certified reference materials were used for method validation. Recoveries of OTA ranged between 83% and 96% in wines (with relative standard deviation, RSD, of about 10%) and between 81% and 93% in spices (RSD 7%). Recoveries for AFB1 in wheat ranged from 75% to 85% (RSD 8%). The detection limits were all below the maximum levels established for OTA and for AFB1 by EU directives. This method offers a green and low-cost alternative to the organic solvent-based extraction and/or immunoaffinity columns-based cleanup of complex samples prior to ELISA. PMID:27543022

  15. Rapid detection of Nocardia amarae in the activated sludge process using enzyme-linked immunosorbent assay (ELISA).

    PubMed

    Iwahori, K; Miyata, N; Morisada, S; Suzuki, N

    2000-01-01

    Nocardia amarae, a mycolic acid-containing bacterium, has often been reported to cause foaming of activated sludge in wastewater treatment plants. In this study, the number of N. amarae cells in the activated sludge process was estimated by enzyme-linked immunosorbent assay (ELISA) with anti-N. amarae polyclonal antibody. Use of the antibody enabled N. amarae to be detected at levels of 10(4) to 10(7) colony forming units. On the other hand, the antibody reacted with only a small portion of activated sludge, in which no N. amarae cells were detected by the plate count method. Competitive ELISA was employed to estimate the N. amarae cells in samples taken from a municipal wastewater treatment plant, including raw wastewater and activated sludge foam. The cell numbers estimated by competitive ELISA corresponded well with those obtained by plate counts. Hence, the antibody produced in this study was shown to be effective for the rapid monitoring of N. amarae in the activated sludge process. PMID:16232779

  16. Development of a heterologous enzyme-linked immunosorbent assay for organophosphorus pesticides with phage-borne peptide

    PubMed Central

    Hua, Xiude; Liu, Xiaofeng; Shi, Haiyan; Wang, Yanru; Kim, Hee Joo; Gee, Shirley J.; Wang, Minghua; Liu, Fengquan; Hammock, Bruce D.

    2015-01-01

    An enzyme-linked immunosorbent assay (ELISA) was developed to detect organophosphorus pesticides using a phage-borne peptide that was isolated from a cyclic 8-residue peptide phage library. The IC50 values of the phage ELISA ranged from 1.4 to 92.1 μg L−1 for eight organophosphorus pesticides (parathion-methyl, parathion, fenitrothion, cyanophos, EPN, paraoxon-methyl, paraoxon, fenitrooxon). The sensitivity was improved 120- and 2-fold compared to conventional homologous and heterologous ELISA, respectively. The selectivity of the phage ELISA was evaluated by measuring its cross-reactivity with 23 organophosphorus pesticides, among which eight were the main cross-reactants. The spike recoveries were between 66.1% and 101.6% for the detection of single pesticide residues of parathion-methyl, parathion and fenitrothion in Chinese cabbage, apple and greengrocery, and all of the coefficient of variation were less than or equal to 15.9%. Moreover, the phage ELISA results were validated by gas chromatography. The results indicate that isolating phage-borne peptides from phage display libraries is an alternative method for the development of a heterologous immunoassay and that the developed assay has a lower limit of detection than the chemically synthesized competitor assay. PMID:26290688

  17. A toxin-free enzyme-linked immunosorbent assay for the analysis of aflatoxins based on a VHH surrogate standard.

    PubMed

    Wang, Yanru; Li, Peiwu; Zhang, Qi; Hu, Xiaofeng; Zhang, Wen

    2016-09-01

    A toxin-free enzyme-linked immunosorbent assay (ELISA) for aflatoxins was developed using an anti-idiotype nanobody VHH 2-5 as surrogate standard. Anti-idiotype nanobody VHH 2-5 was generated by immunizing an alpaca with anti-aflatoxin monoclonal antibody 1C11. This assay was used to detect aflatoxins in agro-products after a simple extraction with 75 % methanol/H2O. Aflatoxin concentration was calculated by a two-step approach: the concentration of VHH 2-5 was first obtained by a four-parameter logistic regression from the detected absorbance value at 450 nm, and then converted to aflatoxin concentration by a linear equation. The assay exhibits a limit of detection (LOD) of 0.015 ng mL(-1), which is better than or comparable with conventional immunoassays. The performance of our VHH surrogate-based ELISA was further validated with a high-performance liquid chromatography (HPLC) method for total aflatoxins determination in 20 naturally contaminated peanut samples, displaying a good correlation (R (2) = 0.988). In conclusion, the proposed assay represents a first example applying an anti-idiotype VHH antibody as a standard surrogate in ELISA. With the advantages of high stability and ease of production, the VHH antibody-based standard surrogate can be extended in the future to immunoassays for other highly toxic compounds. Graphical Abstract ᅟ. PMID:27002610

  18. Detection of Cronobacter Genus in Powdered Infant Formula by Enzyme-linked Immunosorbent Assay Using Anti-Cronobacter Antibody.

    PubMed

    Song, Xinjie; Shukla, Shruti; Lee, Gibaek; Park, Sunhyun; Kim, Myunghee

    2016-01-01

    Cronobacter species (Cronobacter spp.) are hazardous foodborne pathogens associated with baby food, powdered infant formula (PIF). To develop a rapid and sensitive method for simultaneous detection of seven Cronobacter spp. in PIF, an indirect non-competitive enzyme-linked immunosorbent assay (INC-ELISA) was developed based on a novel immunoglobulin G (IgG), anti-Cronobacter IgG. The developed INC-ELISA was able to detect seven Cronobacter spp. at concentrations ranging from (5.6 ± 0.30) × 10(3) to (2.1 ± 0.01) × 10(5) colony forming unit (CFU)/mL in pure culture. Further, INC-ELISA employing anti-Cronobacter IgG was applicable for analysis of PIF samples contaminated with less than <10 cells of Cronobacter spp. per 25 g of PIF in 36 h. The developed antibody showed slight cross-reactivity with Franconibacter pulveris (LMG 24057) at high concentration (10(8) CFU/mL). The INC-ELISA method displayed excellent specificity without compromising cross-reactivity with other foodborne pathogens. The INC-ELISA assay method developed in this study using a novel anti-Cronobacter IgG facilitated highly sensitive, efficient, and rapid detection of Cronobacter spp. in baby food. PMID:27493642

  19. Use of hydrophilic extra-viral domain of canine distemper virus H protein for enzyme-linked immunosorbent assay development.

    PubMed

    Cho, Ki-hyun; Kim, Jeongmi; Yoo, Hyun-ah; Kim, Dae-hee; Park, Seung-yong; Song, Chang-seon; Choi, In-soo; Lee, Joong-bok

    2014-12-01

    Simple methods for measuring the levels of serum antibody against canine distemper virus (CDV) would assist in the effective vaccination of dogs. To develop an enzyme-linked immunosorbent assay (ELISA) specific for CDV, we expressed hydrophilic extra-viral domain (HEVD) protein of the A75/17-CDV H gene in a pET 28a plasmid-based Escherichia (E.) coli vector system. Expression was confirmed by dot and Western blotting. We proposed that detection of E. coli-expressed H protein might be conformation- dependent because intensities of the reactions observed with these two methods varied. The H gene HEVD protein was further purified and used as an antigen for an ELISA. Samples from dogs with undetectable to high anti-CDV antibody titers were analyzed using this HEVD-specific ELISA and a commercial CDV antibody detection kit (ImmunoComb). Levels of HEVD antigenicity measured with the assays and immunochromatography correlated. These data indicated that the HEDV protein may be used as antigen to develop techniques for detecting antibodies against CDV. PMID:25234325

  20. Comparison of an Enzyme-Linked Immunosorbent Assay with an Immunochromatographic Assay for Detection of Lincomycin in Milk and Honey.

    PubMed

    Cao, Shanshan; Song, Shanshan; Liu, Liqiang; Kong, Na; Kuang, Hua; Xu, Chuanlai

    2015-01-01

    An enzyme-linked immunosorbent assay (ELISA) and an immunochromatographic assay were constructed for the detection of lincomycin (LIN) in both milk and honey samples based on the monoclonal antibody named 5F6. The half-maximum inhibition of ELISA was 0.3 ng/mL after optimizing pH and ionic strength conditions; the limit of detection was 0.07 ng/mL. The cross-reactivity with clindamycin was 0.6%. LIN recovery in spiked milk and honey samples ranged from 84.6% to 115.6% with intra-assay coefficient variations of 1.7-25.4% and inter-assay coefficient variations of 2.7-8.9%. The detection limits were estimated as 2.1 µg/L for milk and 2.1 µg/kg for honey samples. The immunochromatographic assay revealed a LIN cut-off value of 10 ng/mL in PBS, 5 ng/mL in milk, and 120 ng/g in honey, and a visual lower detection limit of 2.5 ng/mL, 1 ng/mL and 30 ng/g in PBS, milk and honey, respectively. The immunochromatographic assay is preferred for large-scale practical application for its simpler pretreatment and satisfied sensitivity compared with ELISA assay. PMID:26107744

  1. Evaluation of Enzyme-Linked Immunosorbent Assays for Detection of Mycoplasma bovis-Specific Antibody in Bison Sera

    PubMed Central

    Sacco, Randy E.; Olsen, Steven C.

    2013-01-01

    Mycoplasma bovis has recently emerged as a significant and costly infectious disease problem in bison. A method for the detection of M. bovis-specific serum antibodies is needed in order to establish prevalence and transmission patterns. Enzyme-linked immunosorbent assays (ELISAs) validated for the detection of M. bovis-specific serum IgG in cattle are commercially available, but their suitability for bison sera has not been determined. A collection of bison sera, most from animals with a known history of infection or vaccination with M. bovis, was tested for M. bovis-specific IgG using commercially available kits as well as an in-house ELISA in which either cattle or bison M. bovis isolates were used as a source of antigen. Comparison of the results demonstrates that ELISAs optimized for cattle sera may not be optimal for the identification of bison seropositive for M. bovis, particularly those with low to moderate antibody levels. The reagent used for the detection of bison IgG and the source of the antigen affect the sensitivity of the assay. Optimal performance was obtained when the capture antigen was derived from bison isolates rather than cattle isolates and when a protein G conjugate rather than an anti-bovine IgG conjugate was used for the detection of bison IgG. PMID:23843427

  2. Development of a flatfish-specific enzyme-linked immunosorbent assay for Fsh using a recombinant chimeric gonadotropin.

    PubMed

    Chauvigné, François; Verdura, Sara; Mazón, María José; Boj, Mónica; Zanuy, Silvia; Gómez, Ana; Cerdà, Joan

    2015-09-15

    In flatfishes with asynchronous and semicystic spermatogenesis, such as the Senegalese sole (Solea senegalensis), the specific roles of the pituitary gonadotropins during germ cell development, particularly of the follicle-stimulating hormone (Fsh), are still largely unknown in part due to the lack of homologous immunoassays for this hormone. In this study, an enzyme-linked immunosorbent assay (ELISA) for Senegalese sole Fsh was developed by generating a rabbit antiserum against a recombinant chimeric single-chain Fsh molecule (rFsh-C) produced by the yeast Pichia pastoris. The rFsh-C N- and C-termini were formed by the mature sole Fsh β subunit (Fshβ) and the chicken glycoprotein hormone common α subunit (CGA), respectively. Depletion of the antiserum to remove anti-CGA antibodies further enriched the sole Fshβ-specific antibodies, which were used to develop the ELISA using the rFsh-C for the standard curve. The sensitivity of the assay was 10 and 50 pg/ml for Fsh measurement in plasma and pituitary, respectively, and the cross-reactivity with a homologous recombinant single-chain luteinizing hormone was 1%. The standard curve for rFsh-C paralleled those of serially diluted plasma and pituitary extracts of other flatfishes, such as the Atlantic halibut, common sole and turbot. In Senegalese sole males, the highest plasma Fsh levels were found during early spermatogenesis but declined during enhanced spermiation, as found in teleosts with cystic spermatogenesis. In pubertal males, however, the circulating Fsh levels were as high as in adult spermiating fish, but interestingly the Fsh receptor in the developing testis containing only spermatogonia was expressed in Leydig cells but not in the primordial Sertoli cells. These results indicate that a recombinant chimeric Fsh can be used to generate specific antibodies against the Fshβ subunit and to develop a highly sensitive ELISA for Fsh measurements in diverse flatfishes. PMID:25449660

  3. Serological diagnosis of bovine neosporosis by Neospora caninum monoclonal antibody-based competitive inhibition enzyme-linked immunosorbent assay.

    PubMed

    Baszler, T V; Knowles, D P; Dubey, J P; Gay, J M; Mathison, B A; McElwain, T F

    1996-06-01

    Neospora caninum, a protozoan parasite closely related to Toxoplasma gondii, causes abortion and congenital infection in cattle. To investigate specific methods of antemortem diagnosis, the antibody responses of infected cows were evaluated by immunoblot assay and competitive inhibition enzyme-linked immunosorbent assay (CI-ELISA) by using a monoclonal antibody (MAb), MAb 4A4-2, against N. caninum tachyzoites. MAb 4A4-2 bound diffusely to the exterior surface of N. caninum tachyzoites and recognized a single 65-kDa band in immunoblots. MAb 4A4-2 was unreactive to antigens of two closely related apicomplexan protozoa, Toxoplasma gondii and Sarcocystis cruzi. Binding of MAb 4A4-2 was inhibited by mild periodate treatment of N. caninum antigen, demonstrating the carbohydrate nature of the epitope. Immunoblot analysis of N. caninum tachyzoite antigens with sera from cows with confirmed Neospora-induced abortion revealed at minimum 14 major antigens ranging from 11 to 175 kDa. Although the recognized antigens varied from cow to cow, antigens of 116, 65, and 25 kDa were detected in all cows with abortion confirmed to be caused by N. caninum. The binding of MAb 4A4-2 to N. caninum tachyzoite antigen was consistently inhibited by sera from Neospora-infected cows in a CI-ELISA format and was not inhibited by sera from Neospora antibody-negative cows. Furthermore, sera from cattle experimentally infected with T. gondii, S. cruzi, Sarcocystis hominis, or Sarcocystis hirsuta, which had cross-reactive antibodies recognizing multiple N. caninum antigens by immunoblot assay, did not inhibit binding of MAb 4A4-2 in the CI-ELISA. Thus, MAb 4A4-2 binds a carbohydrate epitope on a single N. caninum tachyzoite surface antigen that is recognized consistently and specifically by Neospora-infected cattle. PMID:8735092

  4. The Diagnosis of Human Fascioliasis by Enzyme-Linked Immunosorbent Assay (ELISA) Using Recombinant Cathepsin L Protease

    PubMed Central

    Gonzales Santana, Bibiana; Vasquez Camargo, Fabio; Parkinson, Michael

    2013-01-01

    Background Fascioliasis is a worldwide parasitic disease of domestic animals caused by helminths of the genus Fasciola. In many parts of the world, particularly in poor rural areas where animal disease is endemic, the parasite also infects humans. Adult parasites reside in the bile ducts of the host and therefore diagnosis of human fascioliasis is usually achieved by coprological examinations that search for parasite eggs that are carried into the intestine with the bile juices. However, these methods are insensitive due to the fact that eggs are released sporadically and may be missed in low-level infections, and fasciola eggs may be misclassified as other parasites, leading to problems with specificity. Furthermore, acute clinical symptoms as a result of parasites migrating to the bile ducts appear before the parasite matures and begins egg laying. A human immune response to Fasciola antigens occurs early in infection. Therefore, an immunological method such as ELISA may be a more reliable, easy and cheap means to diagnose human fascioliasis than coprological analysis. Methodology/Principal findings Using a panel of serum from Fasciola hepatica-infected patients and from uninfected controls we have optimized an enzyme-linked immunosorbent assay (ELISA) which employs a recombinant form of the major F. hepatica cathepsin L1 as the antigen for the diagnosis of human fascioliasis. We examined the ability of the ELISA test to discern fascioliasis from various other helminth and non-helminth parasitic diseases. Conclusions/Significance A sensitive and specific fascioliasis ELISA test has been developed. This test is rapid and easy to use and can discriminate fasciola-infected individuals from patients harbouring other parasites with at least 99.9% sensitivity and 99.9% specificity. This test will be a useful standardized method not only for testing individual samples but also in mass screening programs to assess the extent of human fascioliasis in regions where this

  5. Evaluation by enzyme-linked immunosorbent assay (ELISA) of Renibacterium salmoninarum bacterins affected by persistence of bacterial antigens

    USGS Publications Warehouse

    Pascho, R.J.; Goodrich, T.D.; McKibben, C.L.

    1997-01-01

    Rainbow trout Oncorhynchus mykiss were injected intraperitoneally with a bacterin containing killed Renibacterium salmoninarum cells delivered alone or in an oil-based adjuvant. We evaluated the relative abilities of the batterins to prevent the initiation or progression of infection in fish challenged by waterborne exposure to R. salmoninarum. Sixty-one days after vaccination, fish were held for 24 h in water containing either no bacteria or approximately 1.7 x 103, 1.7 x 105, or 5.3 x 106 live R. salmoninarum cells/mL. An enzyme-linked immunosorbent assay (ELISA) was used to monitor changes in the levels of R. salmoninarum antigen in live fish before and after the immersion challenges. High levels of R. salmoninarum antigens were detected by ELISA in kidney-spleen tissue homogenates from vaccinated fish immediately before the challenges. Levels of those antigens remained high in the tissues of unchallenged fish throughout the study. We found that the ELISA used in this study may be unsuitable for evaluating the efficacy of batterins because it did not distinguish antigens produced by the challenge bacteria during an infection from those of the bacterins. Groups of control and vaccinated fish also were injected with either 1.7 x 104 or 1.7 x 106 R. salmoninarum cells and served as R. salmoninarum virulence controls. Relative survival among the various subgroups in the injection challenge suggests that adverse effects might have been associated with the adjuvant used in this study. The lowest survival at both injection challenge levels was among fish vaccinated with bacteria in adjuvant.

  6. Comparison of Seven Commercial Antigen and Antibody Enzyme-Linked Immunosorbent Assays for Detection of Acute Dengue Infection

    PubMed Central

    Jarman, Richard G.; Gibbons, Robert V.; Tanganuchitcharnchai, Ampai; Mammen, Mammen P.; Nisalak, Ananda; Kalayanarooj, Siripen; Bailey, Mark S.; Premaratna, Ranjan; de Silva, H. Janaka; Day, Nicholas P. J.; Lalloo, David G.

    2012-01-01

    Seven commercial assays were evaluated to determine their suitability for the diagnosis of acute dengue infection: (i) the Panbio dengue virus Pan-E NS1 early enzyme-linked immunosorbent assay (ELISA), second generation (Alere, Australia); (ii) the Panbio dengue virus IgM capture ELISA (Alere, Australia); (iii) the Panbio dengue virus IgG capture ELISA (Alere, Australia); (iv) the Standard Diagnostics dengue virus NS1 antigen ELISA (Standard Diagnostics, South Korea); (v) the Standard Diagnostics dengue virus IgM ELISA (Standard Diagnostics, South Korea); (vi) the Standard Diagnostics dengue virus IgG ELISA (Standard Diagnostics, South Korea); and (vii) the Platelia NS1 antigen ELISA (Bio-Rad, France). Samples from 239 Thai patients confirmed to be dengue virus positive and 98 Sri Lankan patients negative for dengue virus infection were tested. The sensitivities and specificities of the NS1 antigen ELISAs ranged from 45 to 57% and 93 to 100% and those of the IgM antibody ELISAs ranged from 85 to 89% and 88 to 100%, respectively. Combining the NS1 antigen and IgM antibody results from the Standard Diagnostics ELISAs gave the best compromise between sensitivity and specificity (87 and 96%, respectively), as well as providing the best sensitivity for patients presenting at different times after fever onset. The Panbio IgG capture ELISA correctly classified 67% of secondary dengue infection cases. This study provides strong evidence of the value of combining dengue virus antigen- and antibody-based test results in the ELISA format for the diagnosis of acute dengue infection. PMID:22441389

  7. Serosurveillance for Francisella tularensis Among Wild Animals in Japan Using a Newly Developed Competitive Enzyme-Linked Immunosorbent Assay

    PubMed Central

    Sharma, Neekun; Hotta, Akitoyo; Yamamoto, Yoshie; Uda, Akihiko; Fujita, Osamu; Mizoguchi, Toshio; Shindo, Junji; Park, Chun-Ho; Kudo, Noboru; Hatai, Hitoshi; Oyamada, Toshifumi; Yamada, Akio; Morikawa, Shigeru

    2014-01-01

    Abstract Tularemia, a highly infectious zoonotic disease caused by Francisella tularensis, occurs sporadically in Japan. However, little is known about the prevalence of the disease in wild animals. A total of 632 samples obtained from 150 Japanese black bears, 142 Japanese hares, 120 small rodents, 97 rats, 53 raptors, 26 Japanese monkeys, 21 Japanese raccoon dogs, 20 masked palm civets, and three Japanese red foxes between 2002 and 2010 were investigated for the presence of antibodies to F. tularensis by competitive enzyme-linked immunosorbent assay (cELISA) and the commonly used microagglutination (MA) test. Seropositive cELISA and MA results were obtained in 23 and 18 Japanese black bears, three and two Japanese raccoon dogs, and two and one small rodents, respectively. All MA-positive samples (n=21) were also positive by cELISA. Six of seven samples that were only positive by cELISA were confirmed to be antibody-positive by western blot analysis. These findings suggest that cELISA is a highly sensitive and useful test for serosurveillance of tularemia among various species of wild animals. Because this is the first study to detect F. tularensis–seropositive Japanese raccoon dogs, these could join Japanese black bears as sentinel animals for tularemia in the wild in Japan. Further continuous serosurveillance for F. tularensis in various species of wild animals using appropriate methods such as cELISA is important to assess the risks of human exposure and to improve our understanding of the ecology of F. tularensis in the wild. PMID:24689989

  8. Serosurveillance for Francisella tularensis among wild animals in Japan using a newly developed competitive enzyme-linked immunosorbent assay.

    PubMed

    Sharma, Neekun; Hotta, Akitoyo; Yamamoto, Yoshie; Uda, Akihiko; Fujita, Osamu; Mizoguchi, Toshio; Shindo, Junji; Park, Chun-Ho; Kudo, Noboru; Hatai, Hitoshi; Oyamada, Toshifumi; Yamada, Akio; Morikawa, Shigeru; Tanabayashi, Kiyoshi

    2014-04-01

    Tularemia, a highly infectious zoonotic disease caused by Francisella tularensis, occurs sporadically in Japan. However, little is known about the prevalence of the disease in wild animals. A total of 632 samples obtained from 150 Japanese black bears, 142 Japanese hares, 120 small rodents, 97 rats, 53 raptors, 26 Japanese monkeys, 21 Japanese raccoon dogs, 20 masked palm civets, and three Japanese red foxes between 2002 and 2010 were investigated for the presence of antibodies to F. tularensis by competitive enzyme-linked immunosorbent assay (cELISA) and the commonly used microagglutination (MA) test. Seropositive cELISA and MA results were obtained in 23 and 18 Japanese black bears, three and two Japanese raccoon dogs, and two and one small rodents, respectively. All MA-positive samples (n=21) were also positive by cELISA. Six of seven samples that were only positive by cELISA were confirmed to be antibody-positive by western blot analysis. These findings suggest that cELISA is a highly sensitive and useful test for serosurveillance of tularemia among various species of wild animals. Because this is the first study to detect F. tularensis-seropositive Japanese raccoon dogs, these could join Japanese black bears as sentinel animals for tularemia in the wild in Japan. Further continuous serosurveillance for F. tularensis in various species of wild animals using appropriate methods such as cELISA is important to assess the risks of human exposure and to improve our understanding of the ecology of F. tularensis in the wild. PMID:24689989

  9. Assessment of two enzyme-linked immunosorbent assay tests marketed for detection of ruminant proteins in finished feed.

    PubMed

    Myers, Michael J; Yancy, Haile F; Farrell, Dorothy E; Washington, Jewell D; Deaver, Christine M; Frobish, Russell A

    2007-03-01

    The performance characteristics of two enzyme-linked immunosorbent assay (ELISA) test kits, ELISA Technologies' MELISA-Tek test and Tepnel BioSystems' BioKit for (Cooked) Species Identification test, designed to detect ruminant proteins in animal feed, were evaluated. The test kits were evaluated by using acceptance criteria developed by the U.S. Food and Drug Administration's Center for Veterinary Medicine Office of Research for evaluating selectivity, sensitivity, ruggedness, and specificity. The acceptance criteria for determining success used a statistical approach requiring a 90% probability of achieving the correct response within a 95% confidence interval. In practice, this measure requires the test to achieve the correct response 58 times for every 60 samples evaluated, or a 96.7% accuracy rate. A minimum detection level of 0.1% bovine meat and bone meal (BMBM) was required, consistent with the sensitivity of the analytical methods presently used by the U.S. Food and Drug Administration. Selectivity was assessed by testing 60 dairy feed samples that contained no added animal proteins; sensitivity was determined by evaluating 60 samples (per level of fortification) of this same feed that contained 0.025, 0.05, 0.1, 0.25, 0.5, 1, or 2% BMBM. The MELISA-Tek test passed the acceptance set-point criteria for selectivity assessment but failed the sensitivity assessment at all levels except at the 2% level. The MELISA-Tek test came close to passing at the 1% level, detecting true-positive findings at a rate of 93%, but failed at lower levels, in spite of the label claim of 0.5% sensitivity. The BioKit for (Cooked) Species Identification test detected only 2 of 17 samples fortified at the 2% BMBM level and failed to detect any other BMBM-fortified samples. The results of this evaluation indicate that neither test is adequate for regulatory use. PMID:17388061

  10. Development of a monoclonal antibody-based enzyme-linked immunosorbent assay for the herbicide chlorimuron-ethyl.

    PubMed

    Zhao, Jing; Yi, Guo-Xiang; He, Su-Ping; Wang, Bao-Min; Yu, Cai-Xia; Li, Gang; Zhai, Zhi-Xi; Li, Zhao-Hu; Li, Qing X

    2006-07-12

    Hybridomas secreting a monoclonal antibody (mAb) against the herbicide chlorimuron-ethyl (CE) were produced by fusing the mouse myeloma cell line (SP2/0) with splenocytes from a mouse immunized against the conjugate of the sulfonamide moiety of CE and bovine serum albumin (BSA). The mAb, designated 1F5C5A10, had very weak affinity with metsulfuron, ethametsulfuron, pyrazosulfuron, bensulfuron, and chlorsulfuron. Two mAb-based indirect competitive enzyme-linked immunosorbent assays (icELISA) were developed. A conventional icELISA (icELISA-I) showed a concentration of half-maximum inhibition (IC(50)) of 11.6 ng/mL with a dynamic range of 1.6-84 ng/mL. A simplified icELISA (icELISA-II) had an IC(50) of 28.7 ng/mL and a dynamic range of 2.2-372 ng/mL. The two assays were tested on spiked water and soil samples. CE (1-500 ng/mL) fortified in water samples could be analyzed directly without any sample preparation by both immunoassays with an average recovery between 74 and 114%. icELISA-II, but not icELISA-I, was able to accurately analyze the herbicide residues in the crude soil extracts with recoveries between 99 and 129% without obvious matrix effects due to its lesser amount of sample used. In contrast to icELISA-I, icELISA-II is more convenient, whereas it consumes more reagents of coating antigen and goat anti-mouse IgG-peroxidase. PMID:16819901

  11. Rapid competitive enzyme-linked immunosorbent assay for detection of antibodies to peste des petits ruminants virus.

    PubMed

    Choi, Kang-Seuk; Nah, Jin-Ju; Ko, Young-Joon; Kang, Shien-Young; Jo, Nam-In

    2005-04-01

    Peste des petits ruminants (PPR) is a contagious viral disease of small ruminants that is of economic importance in Africa, the Middle East, and Asia. We developed a rapid competitive enzyme-linked immunosorbent assay (rapid c-ELISA) for the diagnosis and surveillance of PPR. This assay detects PPR virus (PPRV) antibodies in serum samples by quantifying the amount of monoclonal antibody (MAb) P-3H12 after 30 min of incubation of a serum-MAb conjugate mixture on plates coated with a PPRV recombinant nucleocapsid protein (rPPRV-N). We tested 249 PPRV-positive serum samples and 733 PPRV-negative serum samples from field ruminants. The threshold of percent inhibition (PI) was determined to be <50 on the basis of the mean PI plus 3 standard deviations for sera from PPRV-negative ruminants. The relative specificity and sensitivity of the rapid c-ELISA were 98.5% (722 of 733 serum samples) and 93.4% (234 of 249 serum samples), respectively. The rapid c-ELISA sensitively detected PPRV antibodies in hyperimmune sera (virus neutralization test [VNT] titer, >512), even at dilutions > or = 512 in normal goat serum, and as early as 6 to 13 days postinfection from 12 goats, each of which was infected with one of the four PPRV lineages. Hyperimmune sera from animals experimentally vaccinated with rinderpest virus gave positive results by the rapid c-ELISA when the rinderpest virus VNT titers were >512, although the rapid c-ELISA titers were very low (2 to 16). However, the rapid c-ELISA was negative when the rinderpest virus VNT titer was < or = 128. The rapid c-ELISA developed in the present work provides a short turnaround time and could be a useful tool for the diagnosis of PPR and screening for PPRV in the field. PMID:15817764

  12. Validation of a von Willebrand factor antigen enzyme-linked immunosorbent assay and newly developed collagen-binding assay

    PubMed Central

    Burgess, Hilary; Wood, Darren

    2008-01-01

    No single test is comprehensive enough to detect all of the variants of von Willebrand Disease (VWD), making determination of both concentration and function of von Willebrand Factor (VWF) important for an accurate diagnosis. The objective of the study was to validate a newly developed VWF collagen binding assay (VWF:CB) and VWF antigen enzyme-linked immunosorbent assay (ELISA) developed at the Ontario Veterinary College (OVC VWF:Ag). Linearity, sensitivity, and coefficients of variation were determined. The Asserachrom VWF:Ag ELISA was used as the reference assay for this study. Concordance correlation and Bland-Altman plots were used to evaluate agreement between both VWF:Ag assays. The VWF:CB accuracy was assessed by degree of association with the VWF:Ag assays, and the VWF:Ag to VWF:CB ratio. All assays were assessed for their ability to distinguish between VWD negative and VWD positive patients. Linearity, intra-assay coefficients of variation, and inter-assay coefficients of variation were acceptable for both the newly developed VWF:CB (R2 = 0.97, average CV = 4.4, and 15, respectively) and OVC VWF:Ag assays (R2 = 0.96, average CV = 7.9, and 5.9, respectively). Agreement between the OVC VWF:Ag assay and reference assay was excellent (ρc = 0.89), and although differences between assay results precluded interchangeable use of the assays, both successfully distinguished VWD positive and VWD negative dogs (P < 0.0001). The VWF:CB showed a strong association with both VWF:Ag assays (R2 = 0.86, 0.82) and VWF:Ag to VWF:CB ratios (≤ 1) were as expected. The excellent performance of both assays in this validation study confirm their reliability and potential for clinical application. PMID:19086374

  13. Application of an Improved Enzyme-Linked Immunosorbent Assay Method for Serological Diagnosis of Canine Leishmaniasis ▿

    PubMed Central

    Santarém, Nuno; Silvestre, Ricardo; Cardoso, Luís; Schallig, Henk; Reed, Steven G.; Cordeiro-da-Silva, Anabela

    2010-01-01

    Accurate diagnosis of canine leishmaniasis (CanL) is essential toward a more efficient control of this zoonosis, but it remains problematic due to the high incidence of asymptomatic infections. In this study, we present data on the development of enzyme-linked immunosorbent assay (ELISA)-based techniques for the detection of antibodies against the recombinant protein Leishmania infantum cytosolic tryparedoxin peroxidase (LicTXNPx) and a comparison of the results with those employing soluble Leishmania antigens from promastigote or amastigote forms and the homologue recombinant protein L. infantum mitochondrial TXNPx (LimTXNPx). Moreover, we offer an evaluation of the diagnostic potential of rK39 for CanL in the Portuguese canine population and propose an improvement to the existing ELISA-based serological techniques by combining the LicTXNPx and rK39 antigens as a Leishmania antigen mixture (LAM). The data demonstrated that ELISAs based on soluble promastigote or amastigote antigens had generally higher levels of sensitivity for detection of antibodies in symptomatic or asymptomatic dogs than for detection of those against isolated recombinant proteins. Nevertheless, the specificities were found to be similar for all target antigens used. Importantly, the LAM-ELISA methodology improved the overall sensitivity, maintaining a high overall level of specificity. In addition, it was demonstrated that the detection of anti-LAM IgG2 can increase the accuracy of the serological diagnosis. Overall, the obtained results showed that the strategy of combining two well-defined Leishmania antigens, LicTXNPx and rK39, proved to be a sensitive and specific improvement to current serological diagnosis of CanL, being a useful tool for the detection of both clinical and subclinical forms of canine Leishmania infection. PMID:20164286

  14. Optimization and standardization of an enzyme-linked immunosorbent assay protocol for serodiagnosis of Actinobacillus pleuropneumoniae serotype 5.

    PubMed Central

    Trottier, Y L; Wright, P F; Larivière, S

    1992-01-01

    An indirect enzyme-linked immunosorbent assay protocol has been optimized with special emphasis given to assay standardization and quality control. Technical aspects such as choice of a microplate, antigen immobilization, buffer composition, optimal screening dilution of sera, and kinetics of the enzymatic reaction were studied and evaluated in order to design a standard protocol offering maximal analytical sensitivity and specificity, as well as to obtain minimal within- and between-plate variability. Among the 27 plates tested, the Nunc 475-094 and 269-620 immunoplates were found to be the best in terms of high positive-to-negative ratio and low variability. No significant differences in antigen immobilization were found by using buffers of various compositions or pHs; however, the presence of magnesium ions (Mg2+; 0.02 M) resulted in a twofold increase in nonspecific background. An optimal screening dilution of sera was established at 1:200. A 1-h incubation period for test serum was found to be optimal. Maximum enzymatic activity for peroxidase was obtained by adjusting both substrate (H2O2) and hydrogen donor [2,2' -azinobis(3-ethylbenz-thiazoline sulfonic acid)] concentrations to 4 and 1 mM, respectively. To control between-plate variability, a timing protocol was adopted. Within-plate variability was also controlled by using a sample placement configuration pattern. Sliding scales were determined by repeated testing of a cross section of samples to set acceptance limits for both within- and between-plate variability. These limits were used in a quality control program to monitor assay performance. The results obtained suggest that this standardized protocol might be useful in the serodiagnosis of Actinobacillus pleuropneumoniae serotype 5. PMID:1734068

  15. Evaluation of Recombinant Leptospira Antigen-Based Enzyme-Linked Immunosorbent Assays for the Serodiagnosis of Leptospirosis

    PubMed Central

    Flannery, Brendan; Costa, Dirceu; Carvalho, Fernanda Pinheiro; Guerreiro, Hygia; Matsunaga, James; Da Silva, Emilson Domingos; Ferreira, Antonio Gomes Pinto; Riley, Lee W.; Reis, Mitermayer G.; Haake, David A.; Ko, Albert I.

    2001-01-01

    There is an urgent need for development of new serodiagnostic strategies for leptospirosis, an emerging zoonosis with worldwide distribution. We have evaluated the diagnostic utility of five recombinant antigens in enzyme-linked immunosorbent assays (ELISAs) for serodiagnosis of leptospirosis. Sera from 50 healthy residents of a high-incidence region were used to determine cutoff values for 96% specificity. In paired sera from 50 cases of leptospirosis confirmed by the microscopic agglutination test, immunoglobulin G (IgG) but not IgM reacted with the recombinant leptospiral proteins. The recombinant LipL32 IgG ELISA had the highest sensitivities in the acute (56%) and convalescent (94%) phases of leptospirosis. ELISAs based on recombinant OmpL1, LipL41, and Hsp58 had sensitivities of 16, 24, and 18% during the acute phase and 72, 44, and 32% during convalescence, respectively. Compared to sera from healthy individuals, patient sera did not react significantly with recombinant LipL36 (P > 0.05). Recombinant LipL32 IgG ELISA demonstrated 95% specificity among 100 healthy individuals, and specificities ranging from 90 to 97% among 30 dengue patients, 30 hepatitis patients, and 16 patients with diseases initially thought to be leptospirosis. Among 39 Venereal Disease Research Laboratory test-positive individuals and 30 Lyme disease patients, 13 and 23% of sera, respectively, reacted positively with the rLipL32 antigen. These findings indicate that rLipL32 may be an useful antigen for the serodiagnosis of leptospirosis. PMID:11526167

  16. Optimization and validation of enzyme-linked immunosorbent assay for the determination of endosulfan residues in food samples.

    PubMed

    Zhang, Yan; Liu, Jun W; Zheng, Wen J; Wang, Lei; Zhang, Hong Y; Fang, Guo Z; Wang, Shuo

    2008-02-01

    In this study, an enzyme-linked immunosorbent assay (ELISA) was optimized and applied to the determination of endosulfan residues in 20 different kinds of food commodities including vegetables, dry fruits, tea and meat. The limit of detection (IC(15)) was 0.8 microg kg(-1) and the sensitivity (IC(50)) was 5.3 microg kg(-1). Three simple extraction methods were developed, including shaking on the rotary shaker at 250 r min(-1) overnight, shaking on the rotary shaker for 1 h and thoroughly mixing for 2 min. Methanol was used as the extraction solvent in this study. The extracts were diluted in 0.5% fish skin gelatin (FG) in phosphate-buffered saline (PBS) at various dilutions in order to remove the matrix interference. For cabbage (purple and green), asparagus, Japanese green, Chinese cabbage, scallion, garland chrysanthemum, spinach and garlic, the extracts were diluted 10-fold; for carrots and tea, the extracts were diluted 15-fold and 900-fold, respectively. The extracts of celery, adzuki beans and chestnuts, were diluted 20-fold to avoid the matrix interference; ginger, vegetable soybean and peanut extracts were diluted 100-fold; mutton and chicken extracts were diluted 10-fold and for eel, the dilution was 40-fold. Average recoveries were 63.13-125.61%. Validation was conducted by gas chromatography (GC) and gas chromatography-mass spectrometry (GC-MS). The results of this study will be useful to the wide application of an ELISA for the rapid determination of pesticides in food samples. PMID:18246504

  17. Development of an Enzyme-Linked Immunosorbent Assay Method Specific for the Detection of G-Group Aflatoxins.

    PubMed

    Li, Peiwu; Zhou, Qian; Wang, Ting; Zhou, Haiyan; Zhang, Wen; Ding, Xiaoxia; Zhang, Zhaowei; Chang, Perng-Kuang; Zhang, Qi

    2016-01-01

    To detect and monitor G-group aflatoxins in agricultural products, we generated class-specific monoclonal antibodies that specifically recognized aflatoxins G₁ and G₂. Of the final three positive and stable hybridomas obtained, clone 2G6 produced a monoclonal antibody that had equal sensitivity to aflatoxins G₁ and G₂, and did not cross-react with aflatoxins B₁, B₂, or M₁. Its IC50 values for aflatoxins G₁ and G₂ were 17.18 ng·mL(-1) and 19.75 ng·mL(-1), respectively. Using this new monoclonal antibody, we developed a competitive indirect enzyme-linked immunosorbent assay (CI-ELISA); the method had a limit of detection of 0.06 ng·mL(-1). To validate this CI-ELISA, we spiked uncontaminated peanut samples with various amounts of aflatoxins G₁ and G₂ and compared recovery rates with those determined by a standard HPLC method. The recovery rates of the CI-ELISA ranging from 94% to 103% were comparable to those of the HPLC (92% to 102%). We also used both methods to determine the amounts of G-group aflatoxins in five peanut samples contaminated by aflatoxin B₁-positive, and their relative standard deviations ranged from 8.4% to 17.7% (under 20%), which demonstrates a good correlation between the two methods. We further used this CI-ELISA to assess the ability of 126 fungal strains isolated from peanuts or field soils to produce G-group aflatoxins. Among these, seven stains producing different amounts of G-group aflatoxins were identified. Our results showed that the monoclonal antibody 2 G6-based CI-ELISA was suitable for the detection of G-group aflatoxins present in peanuts and also those produced by fungi. PMID:26729164

  18. Cellphone-Based Hand-Held Microplate Reader for Point-of-Care Testing of Enzyme-Linked Immunosorbent Assays.

    PubMed

    Berg, Brandon; Cortazar, Bingen; Tseng, Derek; Ozkan, Haydar; Feng, Steve; Wei, Qingshan; Chan, Raymond Yan-Lok; Burbano, Jordi; Farooqui, Qamar; Lewinski, Michael; Di Carlo, Dino; Garner, Omai B; Ozcan, Aydogan

    2015-08-25

    Standard microplate based enzyme-linked immunosorbent assays (ELISA) are widely utilized for various nanomedicine, molecular sensing, and disease screening applications, and this multiwell plate batched analysis dramatically reduces diagnosis costs per patient compared to nonbatched or nonstandard tests. However, their use in resource-limited and field-settings is inhibited by the necessity for relatively large and expensive readout instruments. To mitigate this problem, we created a hand-held and cost-effective cellphone-based colorimetric microplate reader, which uses a 3D-printed opto-mechanical attachment to hold and illuminate a 96-well plate using a light-emitting-diode (LED) array. This LED light is transmitted through each well, and is then collected via 96 individual optical fibers. Captured images of this fiber-bundle are transmitted to our servers through a custom-designed app for processing using a machine learning algorithm, yielding diagnostic results, which are delivered to the user within ∼1 min per 96-well plate, and are visualized using the same app. We successfully tested this mobile platform in a clinical microbiology laboratory using FDA-approved mumps IgG, measles IgG, and herpes simplex virus IgG (HSV-1 and HSV-2) ELISA tests using a total of 567 and 571 patient samples for training and blind testing, respectively, and achieved an accuracy of 99.6%, 98.6%, 99.4%, and 99.4% for mumps, measles, HSV-1, and HSV-2 tests, respectively. This cost-effective and hand-held platform could assist health-care professionals to perform high-throughput disease screening or tracking of vaccination campaigns at the point-of-care, even in resource-poor and field-settings. Also, its intrinsic wireless connectivity can serve epidemiological studies, generating spatiotemporal maps of disease prevalence and immunity. PMID:26159546

  19. Enzyme-linked immunosorbent assay for detection of organophosphorylated butyrylcholinesterase: A biomarker of exposure to organophosphate agents

    SciTech Connect

    Wang, Liming; Du, Dan; Lu, Donglai; Lin, Chiann Tso; Smith, Jordan N.; Timchalk, Charles; Liu, Fengquan; Wang, Jun; Lin, Yuehe

    2011-05-05

    A sandwich enzyme-linked immunosorbent assay (sELISA) is developed for detection of organophosphorylated butyrylcholinesterase (OP-BChE), a potential biomarker for human exposure to organophosphate insecticides and nerve agents. A pair of antibodies specific to OP-BChE adduct were identified through systematic screening of several anti BChE antibodies (anti-BChE) and anti-phosphoserine antibodies (anti-Pser) from different sources. The selected anti-BChE (set as capture antibody) antibodies recognize both phosphorylated and nonphosphorylated BChE. These antibodies can therefore be used to capture both BChE and OP-BChE from the sample matrices. The anti- Pser (set as detecting antibody) was used to recognize the OP moiety of OP-BChE adducts. With the combination of the selected antibody pair, several key parameters (such as the concentration of anti-BChE and anti-Pser, and the blocking agent) were optimized to enhance the sensitivity and selectivity of the sELISA. Under the optimal conditions, the sELISA has shown a wide linear range from 0.03 nM to 30 nM, with a detection limit of 0.03 nM. Furthermore, the sELISA was successfully applied to detect OP-BChE using in-vitro biological samples such as rat plasma spiked with OP-BChE with excellent adduct recovery (z>99 %). These results demonstrate that this novel approach holds great promise to develop an ELISA kit and offers a simple and cost-effective tool for screening/evaluating exposure to organophosphate insecticides and nerve agents.

  20. Development of an Enzyme-Linked Immunosorbent Assay Method Specific for the Detection of G-Group Aflatoxins

    PubMed Central

    Li, Peiwu; Zhou, Qian; Wang, Ting; Zhou, Haiyan; Zhang, Wen; Ding, Xiaoxia; Zhang, Zhaowei; Chang, Perng-Kuang; Zhang, Qi

    2015-01-01

    To detect and monitor G-group aflatoxins in agricultural products, we generated class-specific monoclonal antibodies that specifically recognized aflatoxins G1 and G2. Of the final three positive and stable hybridomas obtained, clone 2G6 produced a monoclonal antibody that had equal sensitivity to aflatoxins G1 and G2, and did not cross-react with aflatoxins B1, B2, or M1. Its IC50 values for aflatoxins G1 and G2 were 17.18 ng·mL−1 and 19.75 ng·mL−1, respectively. Using this new monoclonal antibody, we developed a competitive indirect enzyme-linked immunosorbent assay (CI-ELISA); the method had a limit of detection of 0.06 ng·mL−1. To validate this CI-ELISA, we spiked uncontaminated peanut samples with various amounts of aflatoxins G1 and G2 and compared recovery rates with those determined by a standard HPLC method. The recovery rates of the CI-ELISA ranging from 94% to 103% were comparable to those of the HPLC (92% to 102%). We also used both methods to determine the amounts of G-group aflatoxins in five peanut samples contaminated by aflatoxin B1-positive, and their relative standard deviations ranged from 8.4% to 17.7% (under 20%), which demonstrates a good correlation between the two methods. We further used this CI-ELISA to assess the ability of 126 fungal strains isolated from peanuts or field soils to produce G-group aflatoxins. Among these, seven stains producing different amounts of G-group aflatoxins were identified. Our results showed that the monoclonal antibody 2 G6-based CI-ELISA was suitable for the detection of G-group aflatoxins present in peanuts and also those produced by fungi. PMID:26729164

  1. Enzyme-linked immunosorbant assay (ELISA) of size-selected crotalid venom antigens by Wyeth's polyvalent antivenom.

    PubMed

    Schaeffer, R C; Randall, H; Resk, J; Carlson, R W

    1988-01-01

    The binding of Antivenom (Crotalidae) Polyvalent to fractions from crude venoms of eight crotalid and one viperid snake, obtained by high performance size-exclusion chromatography, was determined with an indirect enzyme-linked immunosorbent assay (ELISA). Most of the large (greater than 30,000 mol. wt) molecular mass crotalid venom fractions were associated with high (greater than 0.7 absorbance units) ELISA values. Similarly, the medium (13,000-30,000 mol. wt) and small (less than 14,000 mol. wt) molecular mass crotalid venom fractions were coincident with moderate (0.3-0.7 absorbance units) and low (less than 0.3 absorbance units) ELISA levels. Some variability in this pattern was seen with individual venom fractions. A distinctly different pattern of ELISA values were observed with two rattlesnake venoms: the South American (Crotalus durissus terrificus) and Mojave desert (Crotalus scutulatus scutulatus) rattlesnakes. The elution profile from these venoms showed a progression of low to moderate ELISA values within the large molecular mass fractions. This pattern was followed by a decline to low ELISA values throughout the remainder of the elution profile. When saw scaled viper (Echis carinatus leucogaster) venom fractions were tested, only background ELISA values were detected with antivenom. Similarly, background ELISA values were associated with the small molecular mass fractions of all venoms tested. In addition, the elution position for the basic peptides of southern Pacific (Crotalus viridis helleri) and timber (Crotalus h. horridus) rattlesnake venoms showed minimal ELISA values. These data support the view that except for the venom of C. durissus terrificus and C. s. scutulatus, most antivenom antibodies bind large (greater than 30,000 mol. wt) venom fractions. Thus, antivenom contains minimal levels of antibodies to the basic peptides in these venoms. PMID:3347932

  2. Development of a highly sensitive and specific enzyme-linked immunosorbent assay for detection of Sudan I in food samples.

    PubMed

    Han, Dan; Yu, Meng; Knopp, Dietmar; Niessner, Reinhard; Wu, Mei; Deng, Anping

    2007-08-01

    A highly selective and sensitive indirect competitive enzyme-linked immunosorbent assay (ELISA) for Sudan I was developed. Two hapten derivatives with different lengths of carboxylic spacer at the azo-bound para-position were synthesized and coupled to carrier proteins. The hapten-bovine serum albumin (BSA) conjugates were used as immunogens, while the hapten-ovalbumin (OA) conjugates were applied as coating antigens. The antisera which were obtained from four immunized rabbits were characterized in terms of sensitivity and specificity. At optimal experimental conditions it was found that IC50 and LOD values of seven pairs based on four antisera and two coating antigens were in the range of 0.3-2 ng/mL and 0.02-0.1 ng/mL, respectively. The most sensitive ELISA could be established with Sudan I-propionic acid-OA coating antigen and the antiserum which was obtained with the corresponding immunogen. The cross-reactivity values of the four antisera with Sudan II, III, and IV was estimated with 0.1-14.3%. No cross-reactivity was found with six edible colorants Sunset yellow, Amarant, Kermes, Indigotin, Bright blue and Lemon yellow, indicating high specificity for Sudan I. Six food samples were fortified with Sudan I and extracted by simple sample preparation. The methanolic extracts after dilution with methanol:water (5:95, v/v) were analyzed by the developed ELISA. Assay precision and accuracy was estimated by determination of three replicates. Acceptable recovery rates of 92.5-114% and intra-assay coefficients of variation of 5.9-24.8% were obtained. The data were validated by conventional HPLC method. As revealed, both methods were highly correlated (r = 0.9851, n = 7), demonstrating the applicability of the developed ELISA for Sudan I analysis in food samples. PMID:17622156

  3. Immunoglobulin G1 Enzyme-Linked Immunosorbent Assay for Diagnosis of Johne's Disease in Red Deer (Cervus elaphus)

    PubMed Central

    Griffin, J. Frank T.; Spittle, Evelyn; Rodgers, Christie R.; Liggett, Simon; Cooper, Marc; Bakker, Douwe; Bannantine, John P.

    2005-01-01

    This study was designed to develop a customized enzyme-linked immunosorbent assay (ELISA) for the serodiagnosis of Johne's disease (JD) in farmed deer. Two antigens were selected on the basis of their superior diagnostic readouts: denatured purified protein derivative (PPDj) and undenatured protoplasmic antigen (PpAg). ELISA development was based on the antigen reactivity of the immunoglobulin G1 (IgG1) isotype, which is a highly specific marker for mycobacterial disease seroreactivity in deer. Sensitivity estimates and test parameters were established using 102 Mycobacterium paratuberculosis-infected animals from more than 10 deer herds, and specificity estimates were determined using 508 uninfected animals from 5 known disease-free herds. A receiver-operated characteristic analysis determined that at a cut point of 50 ELISA units, there was a specificity of 99.5% and sensitivities of 84.0% with PPDj antigen, 88.0% with PpAg, and 91.0% when the antigens were used serially in a composite test. Estimated sensitivity was further improved using recombinant protein antigens unique for M. paratuberculosis, which identified infected animals that were unreactive to PPDj or PpAg. While 80% of animals that were seropositive in the IgG1 ELISA had detectable histopathology, the assay could also detect animals with subclinical disease. The test was significantly less sensitive (75%) for animals that were culture positive for M. paratuberculosis but with no detectable pathology than for those with pathological evidence of JD (>90%). When the IgG1 ELISA was used annually over a 4-year period in a deer herd with high levels of clinical JD, it eliminated clinical disease, increased production levels, and reduced JD-related mortality. PMID:16339063

  4. Specific serum immunoglobulin D, detected by antibody capture enzyme-linked immunosorbent assay (ELISA), in cytomegalovirus infection.

    PubMed Central

    Mortensen, J; Nielsen, S L; Sørensen, I; Andersen, H K

    1989-01-01

    An antibody capture enzyme-linked immunosorbent assay (ELISA) was developed for the detection of immunoglobulin D (IgD) antibodies to cytomegalovirus (CMV) in sera from blood donors and various groups of patients infected with CMV. This method has previously been found especially valuable in detecting specific antibodies of the IgM, IgE, IgA and IgG class in patients with CMV infection. Specific CMV IgD antibodies were found in 37% of CMV seropositive blood donors and in 47 (88%) of the 53 patients investigated, including bone marrow transplant and renal allograft transplant patients, patients with CMV mononucleosis, neonates with CMV infection and AIDS patients with CMV infection. The highest IgD reactivity was found in patients having either a primary post-transplant CMV infection or CMV mononucleosis. The IgD reactivity in patients with AIDS and in neonates was low. It was also found that in the acute phase of CMV infection the development of CMV antibodies of the IgD class was similar to the development of antibodies of the other classes. The maintenance of IgD activity in some patients together with the presence of CMV IgD antibodies in a great proportion of the blood donors indicates that the development of CMV IgD antibodies resembles that of the IgG class. Determination of specific IgD antibodies offered no advantage over determination of specific antibodies of the IgM, IgE and IgA classes in the diagnosis of CMV infection. PMID:2539278

  5. Development of an Enzyme-linked Immunosorbent Assay Using Vitellin for Vitellogenin Measurement in the Pale Chub, Zacco platypus

    PubMed Central

    Lim, Eun-Suk; Lee, Eun Hee; Kim, Myung Hee; Han, Chang-Hee; Lee, Sung-Kyu

    2013-01-01

    Objectives Fish vitellogenin (VTG) is produced in the female liver during oogenesis through the estradiol cycle and produced in the male liver by endocrine disrupting chemicals (EDCs) such as alkylphenols. In this study, we propose that the VTG concentration in the pale chub could be detected using monoclonal antibodies and polyclonal antibodies against vitellin (Vn) in a VTG enzyme-linked immunosorbent assay (ELISA) system. Methods Monoclonal antibodies and polyclonal antibodies were produced using the Vn extracted from the matured ovum of the ovary. The VTG was extracted from the plasma of the male pale chub. The Vn and VTG were confirmed by measuring the molecular weight of their proteins using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and the specificity of the antibodies was checked through western blotting methods. The assay system was validated with respect to optimal assay concentrations, specificity, recovery, and intra- and inter-assay variations. Results The Vn consisted of two protein bands with apparent molecular weights of 64 and 37 kDa. The SDS-PAGE indicated protein weights of 146 and 77 kDa in the VTG. The assay range was 15.6 ng/mL to 2,000 ng/mL, and the value of the intra- and inter-assay variations were within 10.0% and 14.7%, respectively. The recovery rate was 99.5±5.5%. Conclusions A sandwich ELISA was developed that could be used to qualify the VTG of pale chub in screening for EDCs. Pale chub is an ideal species for observing estrogen activity in the environment because of its extensive habitat and extensive food chain. The ELISA developed here would be more favorable than those for other species for determining the effect of long-term food chain accumulation of EDCs in aquatic environments. PMID:24498593

  6. Development of monoclonal antibodies to pre-haptoglobin 2 and their use in an enzyme-linked immunosorbent assay (ELISA).

    PubMed

    Flanagan, J J; Arjomandi, A; Delanoy, M L; Du Paty, E; Galea, P; Laune, D; Rieunier, F; Walker, R P; Binder, S R

    2014-04-01

    Haptoglobins (HPs) are alpha 2-globulin proteins that bind free hemoglobin in plasma to prevent oxidative damage. HPs are produced as preproteins that are proteolytically cleaved in the ER into alpha and beta chains prior to forming mature, functional tetramers. Two alleles exist in humans (HP1 and HP2), therefore three genotypes are present in the population, i.e., HP1-1, HP2-1, and HP2-2. A biochemical role for nascent haptoglobin 2 (pre-haptoglobin 2 or pre-HP2) as the only known modulator of intestinal permeability has been established. In addition, elevated levels of serum pre-HP2 have been detected in multiple conditions including celiac disease and type I diabetes, which are believed to result in part through dysregulation of the intestinal barrier. In this study, we report the development of a monoclonal antibody that is specific for pre-HP2 with a binding affinity in the nanomolar range. Additional antibodies with specificities for preHP but not mature haptoglobin were also characterized. A sandwich enzyme-linked immunosorbent assay (ELISA) was established and validated. The ELISA showed high specificity for pre-HP2 even in the presence of excess pre-HP1 or mature haptoglobins, and has excellent linearity and inter- and intra-assay reproducibility with a working range from 3.1ng/mL to 200ng/mL. Testing of sera from 76 healthy patients revealed a non-Gaussian distribution of pre-HP2 levels with a mean concentration of 221.2ng/mL (95% CI: 106.5-335.9ng/mL) and a median value of 23.9ng/mL. Compared to current approaches, this ELISA offers a validated, monoclonal-based method with high sensitivity and specificity for measuring pre-HP2 in human serum. PMID:24583194

  7. Limitations of the BP26 Protein-Based Indirect Enzyme-Linked Immunosorbent Assay for Diagnosis of Brucellosis

    PubMed Central

    Xin, Ting; Yang, Hongjun; Wang, Nan; Wang, Fang; Zhao, Peng; Wang, Haiguang; Mao, Kairong; Zhu, Hongfei

    2013-01-01

    Brucellosis is a serious zoonosis that occurs worldwide, and its diagnosis is typically based on the detection of antibodies against Brucella lipopolysaccharide (LPS). However, the specificity of the LPS-based test is compromised by cross-reactivity with Escherichia coli O157:H7 and Yersinia enterocolitica O:9. Also, diagnosis based on the LPS test cannot differentiate between vaccinated and infected individuals. The detection of the 26-kDa cytosoluble protein (BP26) antibody is considered an alternative that circumvents these drawbacks because it is exclusively expressed by infectious Brucella. A BP26-based enzyme-linked immunosorbent assay (ELISA) has been tried for the diagnosis of Brucella-infected animals and humans, but a few results showed that BP26 couldn't react with all Brucella-positive sera. In order to explore whether different animals could produce antibodies against BP26 after being infected with various Brucella species, we infected sheep, goats, and beef cattle with common virulent reference Brucella species. All sera were collected from the experimental animals and tested using both LPS-based ELISAs and BP26-based ELISAs. The results showed that all Brucella-infected individuals could produce high levels of antibodies against LPS, but only B. melitensis 16M- and B. melitensis M28-infected sheep and B. melitensis 16M- and B. abortus 2308-infected goats could produce antibodies against BP26. Therefore, we concluded that the BP26-based indirect ELISA (i-ELISA) showed both Brucella species and host specificity, which obviously limits its reliability as a substitute for the traditional LPS-based ELISA for the detection of brucellosis. PMID:23863503

  8. An enzyme-linked immunosorbent assay for lipovitellin quantification in copepods: a screening tool for endocrine toxicity.

    PubMed

    Volz, David C; Chandler, G Thomas

    2004-02-01

    Vitellogenin (VTG) has been widely used as a biomarker of estrogenic exposure in fish, leading to the development of standardized assays for VTG quantification. However, standardized quantitative assays for invertebrate, particularly crustacean, lipovitellin (also known as vitellin [VTN]) are lacking. In this study, a fluorescence-based VTN enzyme-linked immunosorbent assay (ELISA) was developed to quantify microquantities of VTN in the estuarine, sediment-dwelling copepod Amphiascus tenuiremis. This ELISA utilizes a VTN-specific polyclonal antibody developed against amphipod (Leptocheirus plumulosus) embryo VTN and exhibits specificity toward female copepod proteins. In routine assays, the working range of the ELISA was 31.25 to 1,000 ng/ml (75-25% specific binding/maximum antibody binding [B/B0]) with a 50% B/B0 intra- and interassay variation of 3.9% (n = 9) and 12.5% (n = 26), respectively. This ELISA is capable of detecting VTN as low as 2 ng/ml, and can accurately detect VTN in as few as four copepods. The ELISA significantly discriminated positive (gravid female) and negative (male) samples, and was suitable for screening endocrine toxicity in copepods. Stage-I juvenile copepods were individually reared to adults in aqueous microvolumes of the phenylpyrazole insecticide, fipronil, and whole-body homogenate extracts were assayed for VTN levels. Fipronil-exposed virgin adult females, but not males, exhibited significantly higher levels of VTN relative to control males and females. This crustacean VTN ELISA is likely useful for evaluating endocrine activity of environmental toxicants in copepods and other crustacean species. PMID:14982375

  9. Development of an enzyme-linked immunosorbent assay for detection of cellular and in vivo LRRK2 S935 phosphorylation.

    PubMed

    Delbroek, Lore; Van Kolen, Kristof; Steegmans, Liesbeth; da Cunha, Raquel; Mandemakers, Wim; Daneels, Guy; De Bock, Pieter-Jan; Zhang, Jinwei; Gevaert, Kris; De Strooper, Bart; Alessi, Dario R; Verstreken, Patrik; Moechars, Diederik W

    2013-03-25

    After the discovery of kinase activating mutations in leucine-rich repeat kinase 2 (LRRK2) as associated with autosomal dominant forms of Parkinson's disease, inhibition of the kinase is being extensively explored as a disease modifying strategy. As signaling properties and substrate(s) of LRRK2 are poorly documented, autophosphorylation has been an important readout for the enzyme's activity. Western blotting using anti-phospho-S910 or S935 LRRK2 antibodies showed effectiveness in demonstrating inhibitory effects of compounds. In this communication we describe two types of enzyme-linked immunosorbent assays (ELISA) to determine LRRK2 protein levels and kinase activity. Both assays take advantage of the sensitivity of the earlier described total and pS935 antibodies for detection (Nichols et al., Biochem. J. 2010) [10]. The first assay is based on anti-GFP-based capturing of overexpressed LRRK2 and is highly suitable to show cellular effects of kinase inhibitors in a 96-well format. In the other platform anti-LRRK2-based capturing allows detection of endogenously expressed LRRK2 in rat tissue with no significant signal in tissue from LRRK2 knockout rats. Furthermore, both assays showed a significant reduction in pS935 levels on cellular and transgenic R1441C/G LRRK2. With the anti-LRRK2 ELISA we were able to detect LRRK2 phosphorylation in human peripheral blood mononuclear cells (PBMC). To conclude, we report two sensitive assays to monitor LRRK2 expression and kinase activity in samples coming from cellular and in vivo experimental settings. Both can show their value in drug screening and biomarker development but will also be useful in the elucidation of LRRK2-mediated signaling pathways. PMID:23313773

  10. Multiserotype Enzyme-Linked Immunosorbent Assay as a Diagnostic Aid for Periodontitis in Large-Scale Studies

    PubMed Central

    Pussinen, P. J.; Vilkuna-Rautiainen, T.; Alfthan, G.; Mattila, K.; Asikainen, S.

    2002-01-01

    Periodontitis is a common chronic oral infection caused by gram-negative bacteria, including Actinobacillus actinomycetemcomitans and Porphyromonas gingivalis. Periodontitis evokes inflammatory host response locally in the periodontium but also systemically. The systemic humoral antibody response against oral pathogens can conveniently be measured by an immunoassay. The aim of the study was to measure serum immunoglobulin G class antibodies against A. actinomycetemcomitans and P. gingivalis by an enzyme-linked immunosorbent assay (ELISA) in which mixtures of several serotypes of the pathogens were used as antigens to avoid biasing of the results in favor of a particular strain. For A. actinomycetemcomitans the antigen consisted of six strains representing serotypes a, b, c, d, and e and one nonserotypeable strain. In the P. gingivalis ELISA, antigens representing serotypes a, b, and c were used. Serum samples from 90 subjects, including 35 samples from patients with diagnosed periodontitis, 10 samples from periodontally healthy controls, and 45 samples from randomly selected apparently healthy volunteers (referred to as “healthy subjects”), were tested. For both pathogens the antibody levels (means ± standard deviations) of the patients—expressed as area under the dilution curve—were significantly higher than those for healthy controls or healthy subjects, with values for A. actinomycetemcomitans and P. gingivalis, respectively, as follows: patients, 22.60 ± 9.94 mm2 and 26.72 ± 11.13 mm2; healthy controls, 9.99 ± 3.92 mm2 and 6.90 ± 3.38 mm2; and healthy subjects, 16.85 ± 6.67 mm2 and 8.51 ± 4.23 mm2. The serotype mixture ELISA is suitable for measuring antibodies against periodontal pathogens in large epidemiological studies in order to evaluate the role of periodontitis as a risk factor for other diseases. PMID:11825965

  11. Development of an enzyme-linked immunosorbant assay (ELISA) for the serodiagnosis of canine dermatophytosis caused by Microsporum canis.

    PubMed

    Peano, Andrea; Rambozzi, Luisa; Gallo, Maria G

    2005-04-01

    Abstract In dogs, dermatophytosis should be considered in any case of alopecic, papular or pustular lesion. The aim of this study was to develop an enzyme-linked immunosorbant assay (ELISA) as an aid in the diagnosis of canine dermatophytosis. The antigen used was a whole fungal extract obtained from an isolate of Microsporum canis cultured on a liquid medium from the parasitized hair of a cat with patches of alopecia. To assess the ELISA performances, sera from 18 dogs with dermatophytosis caused by M. canis (group A, n = 18), 20 dogs with skin diseases other than dermatophytosis and 22 healthy dogs (group B, n = 42) were tested. Four further animals were tested: three with dermatophytosis caused by M. gypseum and one by T. mentagrophytes. A significant difference (P < 0.01, Wilcoxon's test, w = 364) was found between IgG-specific levels of sera of recently M. canis-infected dogs (infection < 15 days) and controls (although three dogs had negative titres at this stage). A highly significant difference (P < 0.001, w = 462) was noted between controls and dogs with infection of longer duration (> 30 days). All dogs had positive titres at this stage. A highly significant correlation (P < 0.001, Spearman's test, rho = 0.86) between duration of infection and IgG concentration was noted. The test has good sensitivity (83.3%) and high specificity (95.2%) but some dogs retained positive titres after elimination of infection. The sensitivity is higher than that of direct microscopic hair examination and similar to that of fungal culture with DTM (dermatophyte test medium). PMID:15842540

  12. Multicenter Evaluation of a Commercial PCR-Enzyme-Linked Immunosorbent Assay Diagnostic Kit (Onychodiag) for Diagnosis of Dermatophytic Onychomycosis▿

    PubMed Central

    Savin, C.; Huck, S.; Rolland, C.; Benderdouche, M.; Faure, O.; Noacco, G.; Menotti, J.; Candolfi, E.; Pelloux, H.; Grillot, R.; Coupe, S.; Derouin, F.

    2007-01-01

    We prospectively evaluated a new PCR-enzyme-linked immunosorbent assay kit (Onychodiag; BioAdvance, France) for the diagnosis of dermatophytic onychomycosis by testing nail samples from 438 patients with suspected onychomycosis and from 108 healthy controls in three independent laboratories. In two laboratories, samples were collected by trained mycologists as close as possible to the lesions (proximal samples). In one laboratory, samples were collected by other physicians. All samples were processed by conventional mycological techniques and by Onychodiag, blindly to the mycological results. An additional distal sample, collected by clipping the nail plate, was obtained from 75 patients and tested with Onychodiag alone. In patients with culture-proven dermatophytic onychomycosis, the sensitivity of Onychodiag was 83.6% (87.9% including the gray zone) and ranged from 75 to 100% according to the laboratory and the sampling conditions. The specificity was 100% when healthy subjects were considered true negative controls. Onychodiag was positive on 68 patient samples that were sterile or yielded nondermatophyte species in culture. Based on the results of Onychodiag for mycologically proven positive samples and true-negative samples, these results were considered true positives, and the poor performance of mycology on these samples was attributed to inconvenient sampling conditions or to contaminants. When tested on distal samples, Onychodiag was positive in 49/53 (92%) cases of proven dermatophytic onychomycosis. Finally, with either proximal or distal samples, Onychodiag provided a diagnosis of dermatophytic onychomycosis within 24 to 48 h after sampling, and its sensitivity was close to that of mycological techniques applied to proximal samples. PMID:17287330

  13. Recombinant nucleocapsid protein-based enzyme-linked immunosorbent assay for detection of antibody to turkey coronavirus.

    PubMed

    Abdelwahab, Mohamed; Loa, Chien Chang; Wu, Ching Ching; Lin, Tsang Long

    2015-06-01

    Nucleocapsid (N) protein gene of turkey coronavirus (TCoV) was expressed in a prokaryotic system and used to develop an enzyme-linked immunosorbent assay (ELISA) for detection of antibody to TCoV. Anti-TCoV hyperimmune turkey serum and normal turkey serum were used as positive or negative controls for optimization of the ELISA. Goat anti-turkey IgG (H+L) conjugated with horseradish peroxidase was used as detector antibody. Three hundred and twenty two turkey sera from the field were used to evaluate the performance of ELISA and determine the cut-off point of ELISA. The established ELISA was also examined with serum samples obtained from turkeys experimentally infected with TCoV. Those serum samples were collected at various time intervals from 1 to 63 days post-infection. The optimum conditions for differentiation between anti-TCoV hyperimmune serum and normal turkey serum were recombinant TCoV N protein concentration at 20 μg/ml, serum dilution at 1:800, and conjugate dilution at 1:10,000. Of the 322 sera from the field, 101 were positive for TCoV by immunofluorescent antibody assay (IFA). The sensitivity and specificity of the ELISA relative to IFA test were 86.0% and 96.8%, respectively, using the optimum cut-off point of 0.2 as determined by logistic regression method. Reactivity of anti-rotavirus, anti-reovirus, anti-adenovirus, or anti-enterovirus antibodies with the recombinant N protein coated on the ELISA plates was not detected. These results indicated that the established antibody-capture ELISA in conjunction with recombinant TCoV N protein as the coating protein can be utilized for detection of antibodies to TCoV in turkey flocks. PMID:25745958

  14. Enzyme-linked immunosorbent assay with major outer membrane proteins of Brucella melitensis to measure immune response to Brucella species.

    PubMed Central

    Hunter, S B; Bibb, W F; Shih, C N; Kaufmann, A F; Mitchell, J R; McKinney, R M

    1986-01-01

    We developed an enzyme-linked immunosorbent assay (ELISA) system to measure human immunoglobulin G (IgG) and IgM response to the major outer membrane proteins of Brucella melitensis. The ELISA was more sensitive in detecting antibody than a standard microagglutination (MA) test with B. abortus antigen. Of 101 sera from persons with suspected brucellosis, 79 (78.2%) gave ELISA IgM titers greater than or equal to the B. abortus MA titer without 2-mercaptoethanol (2ME), which measures both IgM and IgG. Of the 101 sera, 97% gave ELISA IgG titers greater than or equal to the MA with 2ME titer. A total of 58 sera, drawn from 11 human patients from 1 to 29 weeks after onset of brucellosis, gave higher geometric mean titers for the ELISA IgG test than for the MA with 2ME test. These 58 sera also gave ELISA IgM geometric mean titers that were greater than or within one doubling dilution of the geometric mean titers of MA without 2ME. In addition to detecting antibody response to B. abortus, B. melitensis, and B. suis, the ELISA was sensitive to antibody response to human and canine infections with B. canis. The B. canis antibody response is not detected by the MA test with B. abortus antigen. The ELISA, with a standard preparation of major outer membrane proteins of B. melitensis as antigen, appears to be useful in measuring antibody response in humans to infections by all species of Brucella known to infect humans. PMID:3095364

  15. Sensitive and specific enzyme-linked immunosorbent assay for detecting serum antibodies against Mycobacterium avium subsp. paratuberculosis in fallow deer.

    PubMed

    Prieto, José M; Balseiro, Ana; Casais, Rosa; Abendaño, Naiara; Fitzgerald, Liam E; Garrido, Joseba M; Juste, Ramon A; Alonso-Hearn, Marta

    2014-08-01

    The enzyme-linked immunosorbent assay (ELISA) is the diagnostic test most commonly used in efforts to control paratuberculosis in domestic ruminants. However, commercial ELISAs have not been validated for detecting antibodies against Mycobacterium avium subsp. paratuberculosis in wild animals. In this study, we compared the sensitivities and specificities of five ELISAs using individual serum samples collected from 41 fallow deer with or without histopathological lesions consistent with paratuberculosis. Two target antigenic preparations were selected, an ethanol-treated protoplasmic preparation obtained from a fallow deer M. avium subsp. paratuberculosis isolate (ELISAs A and B) and a paratuberculosis protoplasmic antigen (PPA3) (ELISAs C and D). Fallow deer antibodies bound to the immobilized antigens were detected by using a horseradish peroxidase (HRP)-conjugated anti-fallow deer IgG antibody (ELISAs A and C) or HRP-conjugated protein G (ELISAs B and D). A commercially available assay, ELISA-E, which was designed to detect M. avium subsp. paratuberculosis antibodies in cattle, sheep, and goats, was also tested. Although ELISAs A, C, and E had the same sensitivity (72%), ELISAs A and C were more specific (100%) for detecting fallow deer with lesions consistent with paratuberculosis at necropsy than was the ELISA-E (87.5%). In addition, the ELISA-A was particularly sensitive for detecting fallow deer in the latent stages of infection (62.5%). The antibody responses detected with the ELISA-A correlated with both the severity of enteric lesions and the presence of acid-fast bacteria in gut tissue samples. In summary, our study shows that the ELISA-A can be a cost-effective diagnostic tool for preventing the spread of paratuberculosis among fallow deer populations. PMID:24872517

  16. Sensitive and Specific Enzyme-Linked Immunosorbent Assay for Detecting Serum Antibodies against Mycobacterium avium subsp. paratuberculosis in Fallow Deer

    PubMed Central

    Prieto, José M.; Balseiro, Ana; Casais, Rosa; Abendaño, Naiara; Fitzgerald, Liam E.; Garrido, Joseba M.; Juste, Ramon A.

    2014-01-01

    The enzyme-linked immunosorbent assay (ELISA) is the diagnostic test most commonly used in efforts to control paratuberculosis in domestic ruminants. However, commercial ELISAs have not been validated for detecting antibodies against Mycobacterium avium subsp. paratuberculosis in wild animals. In this study, we compared the sensitivities and specificities of five ELISAs using individual serum samples collected from 41 fallow deer with or without histopathological lesions consistent with paratuberculosis. Two target antigenic preparations were selected, an ethanol-treated protoplasmic preparation obtained from a fallow deer M. avium subsp. paratuberculosis isolate (ELISAs A and B) and a paratuberculosis protoplasmic antigen (PPA3) (ELISAs C and D). Fallow deer antibodies bound to the immobilized antigens were detected by using a horseradish peroxidase (HRP)-conjugated anti-fallow deer IgG antibody (ELISAs A and C) or HRP-conjugated protein G (ELISAs B and D). A commercially available assay, ELISA-E, which was designed to detect M. avium subsp. paratuberculosis antibodies in cattle, sheep, and goats, was also tested. Although ELISAs A, C, and E had the same sensitivity (72%), ELISAs A and C were more specific (100%) for detecting fallow deer with lesions consistent with paratuberculosis at necropsy than was the ELISA-E (87.5%). In addition, the ELISA-A was particularly sensitive for detecting fallow deer in the latent stages of infection (62.5%). The antibody responses detected with the ELISA-A correlated with both the severity of enteric lesions and the presence of acid-fast bacteria in gut tissue samples. In summary, our study shows that the ELISA-A can be a cost-effective diagnostic tool for preventing the spread of paratuberculosis among fallow deer populations. PMID:24872517

  17. Comparison of monoclonal antibody-based sandwich enzyme-linked immunosorbent assay and virus isolation for detection of peste des petits ruminants virus in goat tissues and secretions.

    PubMed Central

    Saliki, J T; House, J A; Mebus, C A; Dubovi, E J

    1994-01-01

    A monoclonal antibody-based sandwich enzyme-linked immunosorbent assay (S-ELISA) was developed for specific detection of peste des petits ruminants virus. Compared with virus isolation in Vero cell cultures using 89 paired tissue and secretion samples from six experimentally infected goats, S-ELISA was significantly more sensitive (71.9% versus 65.2%; P < 0.05). The S-ELISA is a suitable alternative to virus isolation. PMID:8051266

  18. Evaluation of the PANBIO Brucella Immunoglobulin G (IgG) and IgM Enzyme-Linked Immunosorbent Assays for Diagnosis of Human Brucellosis

    PubMed Central

    Araj, George F.; Kattar, Mireille M.; Fattouh, Layla G.; Bajakian, Kayane O.; Kobeissi, Sara A.

    2005-01-01

    PANBIO Brucella immunoglobulin G (IgG) and IgM enzyme-linked immunosorbent assays (ELISAs) were assessed against Brucella standard agglutination tube and Coombs tests. The sensitivities of ELISA IgG and IgM were 91% and 100%, respectively, while the specificity was 100% for both. These ELISAs are simple, rapid, and reliable for the diagnosis of human brucellosis. PMID:16275951

  19. Evaluation of the PANBIO Brucella immunoglobulin G (IgG) and IgM enzyme-linked immunosorbent assays for diagnosis of human brucellosis.

    PubMed

    Araj, George F; Kattar, Mireille M; Fattouh, Layla G; Bajakian, Kayane O; Kobeissi, Sara A

    2005-11-01

    PANBIO Brucella immunoglobulin G (IgG) and IgM enzyme-linked immunosorbent assays (ELISAs) were assessed against Brucella standard agglutination tube and Coombs tests. The sensitivities of ELISA IgG and IgM were 91% and 100%, respectively, while the specificity was 100% for both. These ELISAs are simple, rapid, and reliable for the diagnosis of human brucellosis. PMID:16275951

  20. Development of an enzyme-linked immunosorbent assay to detect chicken serum antibody to glycoprotein G of infectious laryngotracheitis virus.

    PubMed

    Shil, Niraj K; Markham, Philip F; Noormohammadi, Amir H; O'Rourke, Denise; Devlin, Joanne M

    2012-09-01

    Infectious laryngotracheitis (ILT) is a significant upper respiratory tract disease of chickens and has a worldwide distribution. Diagnostic enzyme-linked immunosorbent assays (ELISAs) are commonly used in ILT disease control programs. These ELISAs generally detect serum antibody to infectious laryngotracheitis virus (ILTV) and frequently utilize whole virus as the ELISA antigen. This study investigated the use of recombinant glycoprotein G (gG) of ILTV as an alterative to the use of whole virus antigen. Codon-optimized ILTV gG was expressed in Escherichia coli as a fusion protein with a maltose binding protein tag (gG-MBP). Another gG fusion protein with a 6-histidine tag (gG-His) was expressed in a baculovirus expression system. Following purification, the proteins were assessed for their suitability to be used as an antigen in an ELISA to detect ILTV-specific antibodies in sera from commercial and specific-pathogen-free (SPF) birds. The gG-MBP antigen showed some nonspecific reactions with chicken sera, but the gG-HIS antigen was found to be suitable for differentiating between sera collected from ILTV-vaccinated and unvaccinated chickens. The highest levels of agreement between the results from the gG-HIS ELISA and the commercial Trop-ILT ELISA were achieved using a cut-off value for positivity equal to the geometric mean antibody concentration of the sera from the unvaccinated birds plus 1 SD. This produced a very good level of agreement (kappa [kappa] value of 0.821) using sera from commercial birds and a moderate level of agreement (kappa value of 0.506) using sera from SPF birds. Importantly, this ELISA was also tested for its ability to discriminate between sera collected from SPF chickens vaccinated with a gG deletion mutant candidate vaccine strain of ILTV (gG-ve ILTV) and sera collected from SPF chickens vaccinated with other ILTV strains. The results showed that the gG-His ELISA has the potential to serve as a companion diagnostic tool in conjunction

  1. Multicountry prospective clinical evaluation of two enzyme-linked immunosorbent assays and two rapid diagnostic tests for diagnosing dengue fever.

    PubMed

    Pal, Subhamoy; Dauner, Allison L; Valks, Andrea; Forshey, Brett M; Long, Kanya C; Thaisomboonsuk, Butsaya; Sierra, Gloria; Picos, Victor; Talmage, Sara; Morrison, Amy C; Halsey, Eric S; Comach, Guillermo; Yasuda, Chadwick; Loeffelholz, Michael; Jarman, Richard G; Fernandez, Stefan; An, Ung Sam; Kochel, Tadeusz J; Jasper, Louis E; Wu, Shuenn-Jue L

    2015-04-01

    We evaluated four dengue diagnostic devices from Alere, including the SD Bioline Dengue Duo (nonstructural [NS] 1 Ag and IgG/IgM), the Panbio Dengue Duo Cassette (IgM/IgG) rapid diagnostic tests (RDTs), and the Panbio dengue IgM and IgG capture enzyme-linked immunosorbent assays (ELISAs) in a prospective, controlled, multicenter study in Peru, Venezuela, Cambodia, and the United States, using samples from 1,021 febrile individuals. Archived, well-characterized samples from an additional 135 febrile individuals from Thailand were also used. Reference testing was performed on all samples using an algorithm involving virus isolation, in-house IgM and IgG capture ELISAs, and plaque reduction neutralization tests (PRNT) to determine the infection status of the individual. The primary endpoints were the clinical sensitivities and specificities of these devices. The SD Bioline Dengue Duo had an overall sensitivity of 87.3% (95% confidence interval [CI], 84.1 to 90.2%) and specificity of 86.8% (95% CI, 83.9 to 89.3%) during the first 14 days post-symptom onset (p.s.o.). The Panbio Dengue Duo Cassette demonstrated a sensitivity of 92.1% (87.8 to 95.2%) and specificity of 62.2% (54.5 to 69.5%) during days 4 to 14 p.s.o. The Panbio IgM capture ELISA had a sensitivity of 87.6% (82.7 to 91.4%) and specificity of 88.1% (82.2 to 92.6%) during days 4 to 14 p.s.o. Finally, the Panbio IgG capture ELISA had a sensitivity of 69.6% (62.1 to 76.4%) and a specificity of 88.4% (82.6 to 92.8%) during days 4 to 14 p.s.o. for identification of secondary dengue infections. This multicountry prospective study resulted in reliable real-world performance data that will facilitate data-driven laboratory test choices for managing patient care during dengue outbreaks. PMID:25588659

  2. Multicountry Prospective Clinical Evaluation of Two Enzyme-Linked Immunosorbent Assays and Two Rapid Diagnostic Tests for Diagnosing Dengue Fever

    PubMed Central

    Dauner, Allison L.; Valks, Andrea; Forshey, Brett M.; Long, Kanya C.; Thaisomboonsuk, Butsaya; Sierra, Gloria; Picos, Victor; Talmage, Sara; Morrison, Amy C.; Halsey, Eric S.; Comach, Guillermo; Yasuda, Chadwick; Loeffelholz, Michael; Jarman, Richard G.; Fernandez, Stefan; An, Ung Sam; Kochel, Tadeusz J.; Jasper, Louis E.; Wu, Shuenn-Jue L.

    2015-01-01

    We evaluated four dengue diagnostic devices from Alere, including the SD Bioline Dengue Duo (nonstructural [NS] 1 Ag and IgG/IgM), the Panbio Dengue Duo Cassette (IgM/IgG) rapid diagnostic tests (RDTs), and the Panbio dengue IgM and IgG capture enzyme-linked immunosorbent assays (ELISAs) in a prospective, controlled, multicenter study in Peru, Venezuela, Cambodia, and the United States, using samples from 1,021 febrile individuals. Archived, well-characterized samples from an additional 135 febrile individuals from Thailand were also used. Reference testing was performed on all samples using an algorithm involving virus isolation, in-house IgM and IgG capture ELISAs, and plaque reduction neutralization tests (PRNT) to determine the infection status of the individual. The primary endpoints were the clinical sensitivities and specificities of these devices. The SD Bioline Dengue Duo had an overall sensitivity of 87.3% (95% confidence interval [CI], 84.1 to 90.2%) and specificity of 86.8% (95% CI, 83.9 to 89.3%) during the first 14 days post-symptom onset (p.s.o.). The Panbio Dengue Duo Cassette demonstrated a sensitivity of 92.1% (87.8 to 95.2%) and specificity of 62.2% (54.5 to 69.5%) during days 4 to 14 p.s.o. The Panbio IgM capture ELISA had a sensitivity of 87.6% (82.7 to 91.4%) and specificity of 88.1% (82.2 to 92.6%) during days 4 to 14 p.s.o. Finally, the Panbio IgG capture ELISA had a sensitivity of 69.6% (62.1 to 76.4%) and a specificity of 88.4% (82.6 to 92.8%) during days 4 to 14 p.s.o. for identification of secondary dengue infections. This multicountry prospective study resulted in reliable real-world performance data that will facilitate data-driven laboratory test choices for managing patient care during dengue outbreaks. PMID:25588659

  3. Clinical Utility of an Enzyme-Linked Immunosorbent Assay for Detecting Anti-Melanoma Differentiation-Associated Gene 5 Autoantibodies

    PubMed Central

    Sato, Shinji; Murakami, Akihiro; Kuwajima, Akiko; Takehara, Kazuhiko; Mimori, Tsuneyo; Kawakami, Atsushi; Mishima, Michiaki; Suda, Takafumi; Seishima, Mariko; Fujimoto, Manabu; Kuwana, Masataka

    2016-01-01

    Objective Autoantibodies to melanoma differentiation-associated gene 5 (MDA5) are specifically expressed in patients with dermatomyositis (DM) and are associated with a subset of DM patients with rapidly progressive interstitial lung disease (RP-ILD). Here, we examined the clinical utility of a newly developed enzyme-linked immunosorbent assay (ELISA) system for detecting these antibodies. Methods Here we developed an improved ELISA for detecting anti-MDA5 antibodies. We then performed a multicenter clinical study involving 8 medical centers and enrolled 242 adult patients with polymyositis (PM)/DM, 190 with non-PM/DM connective tissue disease (CTD), 154 with idiopathic interstitial pneumonia (IIP), and 123 healthy controls. Anti-MDA5 antibodies in the patients’ serum samples were quantified using our newly developed ELISA, and the results were compared to those obtained using the gold-standard immunoprecipitation (IP) assay. In addition, correlations between the ELISA-quantified anti-MDA5 antibodies and clinical characteristics were evaluated. Results In patients with PM/DM, the anti-MDA5 antibody measurements obtained from the ELISA and IP assay were highly concordant; the ELISA exhibited an analytical sensitivity of 98.2%, specificity of 100%, positive predictive value of 100%, and negative predictive value of 99.5% (compared to the IP assay). Anti-MDA5 antibodies were detected in 22.7% of the DM patients, but not in any of the patients with PM, non-PM/DM CTD, or IIP. Clinically amyopathic DM, RP-ILD, arthritis, and fever were more prevalent in DM patients who were anti-MDA5 antibody-positive than in those who were antibody-negative (P ≤ 0.0002 for all comparisons). In addition, anti-MDA5 antibody-positive patients with RP-ILD exhibited higher antibody levels than those without RP-ILD (P = 0.006). Conclusion Our newly developed ELISA can detect anti-MDA5 antibodies as efficiently as the gold standard IP assay and has the potential to facilitate the routine

  4. Development of a sandwich enzyme-linked immunosorbent assay (ELISA) for detection of buckwheat residues in food.

    PubMed

    Panda, Rakhi; Taylor, Steve L; Goodman, Richard E

    2010-08-01

    Buckwheat is a pseudocereal (an eudicot with seed qualities and uses similar to those of monocot cereals, family Poaceae) that is consumed in some Asian countries as a staple, and in some western countries as a health food. Allergic reactions to buckwheat are common in some countries. The objective was to develop a specific and sensitive sandwich enzyme-linked immunosorbent assay (ELISA) to detect traces of buckwheat that might inadvertently contaminate other foods in order to assure accurate labeling and consumer protection. Buckwheat-specific antibodies produced in 3 species of animals were tested for specificity and titer by direct ELISA and immunoblot. A sandwich ELISA was developed utilizing pooled rabbit antibuckwheat sera to capture buckwheat proteins and pooled goat antibuckwheat sera, followed by enzyme-labeled rabbit antigoat immunoglobulin G (IgG), to detect bound buckwheat proteins. The lower limit of quantification (LOQ) of the sandwich ELISA was 2 parts per million (ppm) of buckwheat in the presence of complex food matrices. The ELISA is highly specific with no cross-reactivity to any of 80 food ingredients and matrices tested. Validation studies conducted with buckwheat processed into noodles and muffins showed greater than 90% and 60% recovery, respectively. The percent recovery of buckwheat from noodles was similar to that achieved with a commercial buckwheat ELISA kit (ELISA Systems Pty. Ltd., Windsor, Queensland, Australia) at high buckwheat concentrations. However, the sensitivity of this ELISA was greater than the commercial ELISA. This newly developed ELISA is sufficiently specific and sensitive to detect buckwheat residues in processed foods to protect buckwheat-allergic subjects from potential harm. Practical Application: Buckwheat is becoming a common food ingredient in a number of processed foods due to potentially beneficial nutritional properties, without the celiac disease inducing glutenin proteins of wheat and related cereals. However

  5. Evaluation of Two Enzyme-Linked Immunosorbent Assays for Detecting Salmonella enterica subsp. enterica Serovar Dublin Antibodies in Bulk Milk

    PubMed Central

    Veling, J.; van Zijderveld, F. G.; van Zijderveld-van Bemmel, A. M.; Schukken, Y. H.; Barkema, H. W.

    2001-01-01

    Two enzyme-linked immunosorbent assays (ELISAs) for the detecting Salmonella enterica subsp. enterica serovar Dublin antibodies in bulk milk were developed and evaluated for potential use in control programs. The ELISAs were based on either lipopolysacharide (LPS ELISA) or flagellar antigen (GP ELISA). Sensitivity was determined with 79 case herds with a wide range of clinical signs. Specificity was determined with 125 Dutch and 200 Swedish control herds. The relation between antibodies in bulk milk, antibodies in serum, and the level of milk production of individual cows was studied with 61 case herds. The optimal optical density (OD) values of the LPS ELISA and the GP ELISA were determined to be 0.2 and 0.5, respectively. The sensitivities of the LPS ELISA and the GP ELISA were 54 and 63%, respectively, with a specificity of 98% for both ELISAs with samples from the Dutch control herds. The specificities for samples from the Swedish herds were 100% for the LPS ELISA and 95% for the GP ELISA. The sensitivity of the combination of tests was 65% when samples were run in parallel, and the specificity was 100% when samples were run in series, irrespective of whether the samples came from Dutch or Swedish control herds. The variance (R2) in the OD value for bulk milk samples could be explained by the percentage of seropositive lactating cows in a herd with the LPS ELISA for 51% of the samples and with the GP ELISA for 72%. The variance in the OD value was best explained by the combination of the percentage of seropositive lactating cows in the herd and the mean log10 serum antibody titer for that herd (R2 = 62% for the LPS ELISA and R2 = 75% for the GP ELISA). Case herds more often tested negative by the ELISA with bulk milk when the percentage of seropositive lactating cows was less than 5%. It is concluded that both ELISAs with bulk milk can be used in control programs to distinguish between infected and noninfected herds. Specificity can be increased by using the

  6. Determination of Cry9C protein in corn-based foods by enzyme-linked immunosorbent assay: interlaboratory study.

    PubMed

    Trucksess, N W

    2001-01-01

    The performance of a commercially available enzyme-linked immunosorbent assay kit (Enviro-Logix) was assessed for the determination of Cry9C protein, which is produced by the genetically modified corn StarLink, in 8 types of corn-based foods (starch, refined oil, soft tortillas, tortilla chips, corn flakes, corn puffs, corn muffins, and corn bread) in an interlaboratory study involving 7 laboratories in the United States. The assay kit is a double antibody sandwich and is based on the specific interaction between antibody and antigen. The Cry9C protein analyte is sandwiched between 2 antibodies, one to capture the analyte and the other is conjugated to the enzyme, horseradish peroxidase. The enzyme uses tetramethylbenzidine/peroxide for color development. A strong acid stopping reagent is then used to change the color from blue to a stable yellow. The intensity of the color is proportional to the concentration of the Cry9C protein. In this study blind duplicates of control samples (blank material prepared from non- StarLink corn), spiked samples (blank material with the addition of Cry9C protein), and samples containing incurred analyte (products prepared with StarLink corn) were analyzed. Cry9C protein from 2 different sources was used to spike the food products. Cry9C protein produced and purified from a bacterial host was used to prepare spiked test samples at 2.72 and 6.8 ng/g. Cry9C protein from StarLink corn flour was used to prepare spiked samples at 1.97 ng/g. Average recoveries for samples spiked with corn flour Cry9C protein at 1.97 ng/g ranged from 73 to 122%, within-laboratory relative standard deviations (RSDr) ranged from 6 to 22%, and between-laboratories relative standard deviations (RSDR) ranged from 16 to 56%. Average recoveries for samples spiked with bacterial Cry9C protein at 2.72 and 6.8 ng/g ranged from 27 to 96% and from 32 to 113%, respectively; RSDr values ranged from 10 to 35% and from 7 to 38%, respectively; and the RSDR ranged from 28

  7. Comparison of an enzyme-linked immunosorbent assay with indirect hemagglutination and hemagglutination inhibition for determination of rubella virus antibody: evaluation of immune status with commercial reagents in a clinical laboratory.

    PubMed Central

    Truant, A L; Barksdale, B L; Huber, T W; Elliott, L B

    1983-01-01

    Comparative evaluations of immune status for rubella virus are described for enzyme-linked immunosorbent assay, hemagglutination inhibition, and indirect hemagglutination. A 92.1% agreement between enzyme-linked immunosorbent assay and indirect hemagglutination assay was demonstrated for rubella immune status. Enzyme-linked immunosorbent assay and hemagglutination inhibition demonstrated a 92.6% agreement and were compared in an attempt to define the quantitative usefulness of comparisons of single sera for determining immune status. These data support the relative lack of correlation between single enzyme-linked immunosorbent assay and hemagglutination inhibition quantitative values. Enzyme immunoassay was, however, an acceptable alternative to hemagglutination inhibition for the determination of immune status to rubella virus. PMID:6338030

  8. Development of an Enzyme-Linked Immunosorbent Assay and Immunoaffinity Column Chromatography for Saikosaponin d Using an Anti-Saikosaponin d Monoclonal Antibody.

    PubMed

    Sai, Jiayang; Zhao, Yan; Shan, Wenchao; Qu, Baoping; Zhang, Yue; Cheng, Jinjun; Qu, Huihua; Wang, Qingguo

    2016-03-01

    This work developed a novel immunochemical approach for the quality control of saikosaponin d using an enzyme-linked immunosorbent assay. Splenocytes from mice immunized with the saikosaponin d-bovine serum albumin conjugate were fused with the hypoxanthine-aminopterin-thymidine-sensitive mouse myeloma SP2/0 cell line, and a hybridoma secreting monoclonal antibody against saikosaponin d was successfully obtained. The prepared anti-saikosaponin d monoclonal antibody 1E7F3 has a novel characteristic, showing weak reactivity with compounds that are structurally related to saikosaponin d. Using monoclonal antibody 1E7F3, a specific and reliable enzyme-linked immunosorbent assay was developed to detect saikosaponin d. The system shows a full measurement range from 156.25 to 5000.00 ng × mL(-1). Both intra-assay and inter-assay repeatability and precision were achieved, with relative standard deviations lower than 10.00 %. The recovery rates ranged from 92.36 % to 101.00 %, meeting the requirements for biological samples. There was a good correlation between the enzyme-linked immunosorbent assay and high-performance liquid chromatography analyses of saikosaponin d, and the saikosaponin d levels in formulated Chinese medicines were successfully determined. Furthermore, immunoaffinity column chromatography was established using this anti-saikosaponin d monoclonal antibody, and the elution profile of saikosaponin d was detected by a Bio-Rad QuadTec UV/Vis detector at 203 nm. The results demonstrate that we generated a reliable and more efficient assay system for measuring saikosaponin d and provide a potential approach for purifying and separating saikosaponin d. PMID:26824622

  9. Detection of Specific Antibodies against Tembusu Virus in Ducks by Use of an E Protein-Based Enzyme-Linked Immunosorbent Assay

    PubMed Central

    Yin, Xiuchen; Lv, Rang; Chen, Xiaodan; Hua, Ronghong

    2013-01-01

    We developed an enzyme-linked immunosorbent assay (ELISA) using eukaryotically expressed E protein as the antigen (termed E-ELISA) to detect antibodies to tembusu virus (TMUV) in ducks. The E-ELISA did not react with antisera to other known pathogens, indicating the E protein is specific for recognizing anti-TMUV antibodies. Compared to the serum neutralization test, the specificity and sensitivity of the E-ELISA was 93.2 and 97.8%, respectively. Therefore, this E-ELISA is a sensitive and rapid method for detecting antibodies against TMUV in ducks. PMID:23616462

  10. Comparison of different antigen preparations as substrates for use in passive hemagglutination and enzyme-linked immunosorbent assays for detection of antibody against bovine enteric coronavirus.

    PubMed Central

    Crouch, C F; Raybould, T J

    1983-01-01

    Purified coronavirus, detergent extracts of purified coronavirus, and virus-infected Madin-Darby bovine kidney cells were evaluated as antigen substrates in enzyme-linked immunosorbent assay (ELISA) and passive hemagglutination systems. Only detergent-extracted and -unextracted, purified viruses were reactive as antigen substrates in ELISA, whereas all three antigen preparations could be used for sensitization of erythrocytes in the passive hemagglutination assay. The passive hemagglutination system with infected cell extracts exhibited a similar level of sensitivity and specificity to the ELISA system employing purified coronavirus but enabled 300 times more tests to be performed per volume of virus-infected cell culture. PMID:6309897

  11. Development of an Enzyme-Linked Immunosorbent Assay Based on Fusion VP2332-452 Antigen for Detecting Antibodies against Aleutian Mink Disease Virus

    PubMed Central

    Chen, Xiaowei; Song, Cailing; Liu, Yun; Qu, Liandong; Liu, Dafei

    2015-01-01

    For detection of Aleutian mink disease virus (AMDV) antibodies, an enzyme-linked immunosorbent assay (ELISA) was developed using the recombinant VP2332-452 protein as an antigen. Counterimmunoelectrophoresis (CIEP) was used as a reference test to compare the results of the ELISA and Western blotting (WB); the specificity and sensitivity of the VP2332-452 ELISA were 97.9% and 97.3%, respectively, which were higher than those of WB. Therefore, this VP2332-452 ELISA may be a preferable method for detecting antibodies against AMDV. PMID:26582828

  12. Comparative study of colony hybridization with synthetic oligonucleotide probes and enzyme-linked immunosorbent assay for identification of enterotoxigenic Escherichia coli.

    PubMed Central

    Sommerfelt, H; Svennerholm, A M; Kalland, K H; Haukanes, B I; Bjorvatn, B

    1988-01-01

    On the basis of the published nucleotide sequences of the genes that code for the heat-labile toxin LTh and the heat-stable toxins STaI and STaII of human enterotoxigenic Escherichia coli, a 34-mer and two 33-mer oligonucleotide probes were synthesized. To compare their relative efficacies in the detection and differentiation of enterotoxigenic E. coli, a colony hybridization technique using these probes and a GM1 ganglioside enzyme-linked immunosorbent assay using monoclonal anti-LT and anti-ST antibodies were used with 76 strains of E. coli with known enterotoxin profiles. For further evaluation of probe specificity, the enterotoxigenic bacteria Vibrio cholerae O1 and non-O1 and Yersinia enterocolitica were examined with the colony hybridization technique. The sensitivity of colony hybridization compared favorably with that of GM1 ganglioside enzyme-linked immunosorbent assay, and the two assays showed a high level of concordance in specific detection and differentiation of E. coli with various enterotoxin profiles (kappa = 0.906, P less than 0.00001). The probes did not hybridize with DNAs from strains of V. cholerae O1 or non-O1 or Y. enterocolitica. PMID:3281978

  13. A competitive enzyme-linked immunosorbent assay specific for murine hepcidin-1: correlation with hepatic mRNA expression in established and novel models of dysregulated iron homeostasis

    PubMed Central

    Gutschow, Patrick; Schmidt, Paul J.; Han, Huiling; Ostland, Vaughn; Bartnikas, Thomas B.; Pettiglio, Michael A.; Herrera, Carolina; Butler, James S.; Nemeth, Elizabeta; Ganz, Tomas; Fleming, Mark D.; Westerman, Mark

    2015-01-01

    Mice have been essential for distinguishing the role of hepcidin in iron homeostasis. Currently, investigators monitor levels of murine hepatic hepcidin-1 mRNA as a surrogate marker for the bioactive hepcidin protein itself. Here, we describe and validate a competitive, enzyme-linked immunosorbent assay that quantifies hepcidin-1 in mouse serum and urine. The assay exhibits a biologically relevant lower limit of detection, high precision, and excellent linearity and recovery. We also demonstrate correlation between serum and urine hepcidin-1 values and validate the competitive enzyme-linked immunosorbent assay by analyzing plasma hepcidin response of mice to physiological challenges, including iron deficiency, iron overload, acute blood loss, and inflammation. Furthermore, we analyze multiple murine genetic models of iron dysregulation, including β-thalassemia intermedia (Hbbth3/+), hereditary hemochromatosis (Hfe−/−, Hjv−/−, and Tfr2Y245X/Y245X), hypotransferrinemia (Trfhpx/hpx), heterozygous transferrin receptor 1 deficiency (Tfrc+/−) and iron refractory iron deficiency anemia (Tmprss6−/− and Tmprss6hem8/hem8). Novel compound iron metabolism mutants were also phenotypically characterized here for the first time. We demonstrate that serum hepcidin concentrations correlate with liver hepcidin mRNA expression, transferrin saturation and non-heme liver iron. In some circumstances, serum hepcidin-1 more accurately predicts iron parameters than hepcidin mRNA, and distinguishes smaller, statistically significant differences between experimental groups. PMID:25425686

  14. A novel mu-capture enzyme-linked immunosorbent assay based on recombinant proteins for sensitive and specific diagnosis of hemorrhagic fever with renal syndrome.

    PubMed Central

    Zöller, L G; Yang, S; Gött, P; Bautz, E K; Darai, G

    1993-01-01

    Hantavirus nucleocapsid protein has recently been identified as a major antigen inducing an early and long-lasting humoral immune response in patients with hemorrhagic fever with renal syndrome. A mu-capture enzyme-linked immunosorbent assay utilizing recombinant nucleocapsid proteins of Hantavirus strains Hantaan 76-118 (Hantaan serotype) and CG 18-20 (Puumala serotype) as diagnostic antigens and specific monoclonal antibodies as the detection system has been developed. Histidine-tailed recombinant proteins were expressed in Escherichia coli and purified in a single step by affinity chromatography on a nickel-chelate resin. The assay was evaluated with a panel of sera from patients with hemorrhagic fever with renal syndrome originating from various geographic regions. The overall sensitivity of the mu-capture enzyme-linked immunosorbent assay (both recombinant antigens) was 100%, and its specificity was also found to be 100%. Immunoglobulin M antibodies were detected as early as on day 3, and maximum titers were obtained between days 8 and 25 after onset of the disease. The assay was regularly found to be positive within 3 to 4 months but in some cases up to 2 years after the acute phase of the disease. Images PMID:8099085

  15. Antibody responses of cows during an outbreak of neosporosis evaluated by indirect fluorescent antibody test and different enzyme-linked immunosorbent assays.

    PubMed

    Dubey, J P; Jenkins, M C; Adams, D S; McAllister, M M; Anderson-Sprecher, R; Baszler, T V; Kwok, O C; Lally, N C; Björkman, C; Uggla, A

    1997-12-01

    Serum samples from 70 (33 aborting and 37 non-aborting) dairy cows from a herd in California were analyzed for Neospora caninum antibodies in different laboratories by various serologic assays including enzyme-linked immunosorbent assay (ELISA) with recombinant antigens (Nc4.1 and Nc14.1), kinetic ELISA, whole tachyzoite lysate ELISA, immunostimulating complex (iscom) ELISA, antigen capture competitive inhibition ELISA, and by the indirect fluorescent antibody test. Eighteen percent of pregnant cows in this herd had aborted within 2 mo of the index case. All 70 cows had antibodies to N. caninum by at least 1 of the tests. Antibody levels to N. caninum in aborting cows as a group were higher than in nonaborting cows. However, it was concluded that no serological test could be used to establish definitively that N. caninum caused the abortion in an individual cow. PMID:9406780

  16. A solid phase enzyme-linked immunosorbent assay for the antigenic detection of Legionella pneumophila (serogroup 1): A compliment for the space station diagnostic capability

    NASA Technical Reports Server (NTRS)

    Hejtmancik, Kelly E.

    1987-01-01

    It is necessary that an adequate microbiology capability be provided as part of the Health Maintenance Facility (HMF) to support expected microbial disease events and environmental monitoring during long periods of space flight. The application of morphological and biochemical studies to confirm the presence of certain bacterial and fungal disease agents are currently available and under consideration. This confirmation would be facilitated through employment of serological methods to aid in the identification of bacterial, fungal, and viral agents. A number of serological approaches are currently being considered, including the use of Enzyme Linked Immunosorbent Assay (ELISA) technology, which could be utilized during microgravity conditions. A solid phase, membrane supported ELISA for the detection of Legionella pneumophila, an expected disease agent, was developed to show a potential model system that would meet the HMF requirements and specifications for the future space station. These studies demonstrate the capability of membrane supported ELISA systems for identification of expected microbial disease agents as part of the HMF.

  17. Development and evaluation of a blocking enzyme-linked immunosorbent assay and virus neutralization assay to detect antibodies to viral hemorrhagic septicemia virus

    USGS Publications Warehouse

    Wilson, Anna; Goldberg, Tony; Marcquenski, Susan; Olson, Wendy; Goetz, Frederick; Hershberger, Paul; Hart, Lucas M.; Toohey-Kurth, Kathy

    2014-01-01

    Viral hemorrhagic septicemia virus (VHSV) is a target of surveillance by many state and federal agencies in the United States. Currently, the detection of VHSV relies on virus isolation, which is lethal to fish and indicates only the current infection status. A serological method is required to ascertain prior exposure. Here, we report two serologic tests for VHSV that are nonlethal, rapid, and species independent, a virus neutralization (VN) assay and a blocking enzyme-linked immunosorbent assay (ELISA). The results show that the VN assay had a specificity of 100% and sensitivity of 42.9%; the anti-nucleocapsid-blocking ELISA detected nonneutralizing VHSV antibodies at a specificity of 88.2% and a sensitivity of 96.4%. The VN assay and ELISA are valuable tools for assessing exposure to VHSV.

  18. Development and Evaluation of a Blocking Enzyme-Linked Immunosorbent Assay and Virus Neutralization Assay To Detect Antibodies to Viral Hemorrhagic Septicemia Virus

    PubMed Central

    Wilson, Anna; Goldberg, Tony; Marcquenski, Susan; Olson, Wendy; Goetz, Frederick; Hershberger, Paul; Hart, Lucas

    2014-01-01

    Viral hemorrhagic septicemia virus (VHSV) is a target of surveillance by many state and federal agencies in the United States. Currently, the detection of VHSV relies on virus isolation, which is lethal to fish and indicates only the current infection status. A serological method is required to ascertain prior exposure. Here, we report two serologic tests for VHSV that are nonlethal, rapid, and species independent, a virus neutralization (VN) assay and a blocking enzyme-linked immunosorbent assay (ELISA). The results show that the VN assay had a specificity of 100% and sensitivity of 42.9%; the anti-nucleocapsid-blocking ELISA detected nonneutralizing VHSV antibodies at a specificity of 88.2% and a sensitivity of 96.4%. The VN assay and ELISA are valuable tools for assessing exposure to VHSV. PMID:24429071

  19. Application of a monoclonal antibody-based enzyme-linked immunosorbent assay for detection of an inflammatory response antigen in subclinical mastitic milk samples.

    PubMed Central

    Ball, H J; Finlay, D; Mackie, D P; Greer, D; Pollock, D; McNair, J

    1991-01-01

    A monoclonal antibody to a 23.5-kDa bovine inflammatory antigen present in high levels in mastitic milk has been used in an antigen-capture enzyme-linked immunosorbent assay (ELISA) to screen milk samples from herds of cattle for subclinical mastitis. The results from 20 herds with a total of 2,612 quarter samples are presented. Good correlation was observed between the ELISA level and the milk cell count (MCC). The results demonstrated an average of 5% false negatives (1.8% associated with isolates of Staphylococcus aureus and/or Streptococcus spp.) and 7.7% false positives for each herd in relation to mastitic (greater than 400,000 cells per ml) or nonmastitic (less than 400,000 cells per ml) MCCs. Images PMID:1761683

  20. Development and application of an enzyme-linked immunosorbent assay (ELISA) for the quantification of amygdalin, a cyanogenic glycoside, in food.

    PubMed

    Bolarinwa, Islamiyat F; Orfila, Caroline; Morgan, Michael R A

    2014-07-01

    Amygdalin is a member of the cyanogenic glycoside group of plant secondary metabolites capable of generating hydrogen cyanide under certain conditions. As a consequence, the cyanogenic glycosides have been associated with incidents of acute and subacute food poisoning. Specific antibodies were raised against an amygdalin-bovine serum albumin immunogen synthesized using a novel approach. The antibodies were used in a microtitration plate enzyme-linked immunosorbent assay (ELISA) for the quantification, for the first time, of amygdalin in commercially available foods. Correlation of results with high-performance liquid chromatography was very high (r = 0.983). The limit of detection of the immunoassay was 200 ± 0.05 pg mL(-1), and the 50% inhibitory concentration of amygdalin was 50 ± 0.02 ng mL(-1), making the ELISA particularly sensitive. PMID:24905893

  1. Use of circumsporozoite protein enzyme-linked immunosorbent assay compared with microscopic examination of salivary glands for calculation of malaria infectivity rates in mosquitoes (Diptera: Culicidae) from Cameroon.

    PubMed

    Fontenille, D; Meunier, J Y; Nkondjio, C A; Tchuinkam, T

    2001-05-01

    A survey in Cameroon compared the usefulness of the circumsporozoite protein enzyme-linked immunosorbent assay (CSP ELISA) to dissection and microscopic examination of anopheline salivary glands for measuring infectivity rates in anopheline mosquitoes. The salivary glands of 375 females, belonging to four species were examined for sporozoites. After microscopic examination, the glands as well as all the remaining heads and thoraces were tested by ELISA. The sensitivity of ELISA was 100% (18/18), confidence interval (CI) (78.1-100) and the specificity was 99.7% (357/358), CI (98.2 100). The Kappa value, agreement between examination of the glands and salivary gland ELISA, was 0.97. The head thorax CSP ELISA overestimated the true salivary gland infection rate by 12.0%. The results obtained in Central Africa in a village with perennial transmission highly justified the use of the ELISA for measuring the entomological inoculation rate. PMID:11372973

  2. Development of an enzyme-linked immunosorbent assay to determine the numbers of chemolithotrophic bacteria at acid-mine-drainage sites. Technical report (Final)

    SciTech Connect

    Blake, R.C.; Revis, N.W.; Holdsworth, G.

    1990-09-01

    Thiobacillus ferrooxidans is a prominent member of a group of chemo-lithotrophic bacteria that bear principal responsibility for the formation of acid mine drainage. A prototype enzyme-linked immunosorbent assay (ELISA) for enumerating and qualifying T. ferrooxidans was assembled and characterized. The immunoassay protocol consisted of sequential incubations of the sample with (i) the primary antibody, (ii) the enzyme-labeled secondary antibody, and (iii) a chromogenic substrate specific for the enzyme lable. The necessary reagents comprised primary polyclonal rabbit antibodies directed against T. ferrooxidans ATCC 23270, alkaline phosphatase-copled goat anti-rabbit polyclonal antibodies, and phenolphrhalein monophosphate. The ELISA developed herein correctly identified whether iron-oxidizing bacteria were present in each of 4 samples supplied and analyzed by an independent laboratory. Sufficient preliminary data was obtained to warrant further research and development activities.

  3. Development of a novel monoclonal antibody to human inducible co-stimulator ligand (ICOSL): Biological characteristics and application for enzyme-linked immunosorbent assay.

    PubMed

    Hu, Xiaohan; Wu, Jian; An, Jingnan; Hu, Yumin; Shen, Yu; Liu, Cuiping; Zhang, Xueguang

    2016-07-01

    ICOSL (B7-H2, CD275), a co-stimulatory molecule of the B7 superfamily, functions as a positive signal in immune response. To investigate whether ICOSL could be released into sera and the possible biological function of soluble ICOS (sICOSL), we generated and characterized a functional anti-human ICOSL monoclonal antibody (mAb), 20B10, and developed a novel enzyme-linked immunosorbent assay (ELISA) based on two anti-human ICOSL antibodies with different epitope specificities. Using the ELISA system, we found that sICOSL in the serum of healthy donors increases in an age-dependent manner and that the matrix metalloproteinase inhibitor (MMPI) could suppress sICOSL production. Together, these data demonstrate that the existence of circulating sICOSL in human serum might play an important role in immunoregulation. PMID:27138044

  4. A Dot enzyme linked immunosorbent assay (Dot ELISA): comparison with standard fluorescent antibody test (FAT) for the diagnosis of rabies in animals.

    PubMed

    Jayakumar, R; Nachimuthu, K; Padmanaban, V D

    1995-09-01

    A modified enzyme linked immunosorbent assay (Dot ELISA) is described for visual detection of rabies antigen in animals. The test materials were dotted onto the nitrocellulose paper and allowed to react with rabies antiserum. The bound antigen--anti-body were reacted with a peroxidase conjugated antirabbit immunoglobulin. Positive reactions were easily visualized as brown dots after enzyme degradation of the substrate. A total of 400 specimens from various geographical locations were tested with the dot ELISA technique, and also with the fluorescent antibody test (FAT), which was used as a reference method. The concordance between the two tests was 95.25%. The dot ELISA may have potential applications as a rapid, simple and economical field test in the diagnosis of rabies. PMID:8549116

  5. Bovine viral diarrhea virus antigen detection across whole cattle hides using two antigen-capture enzyme-linked immunosorbent assays.

    PubMed

    Vander Ley, Brian L; Ridpath, Julia F; Sweiger, Shaun H

    2012-05-01

    Bovine viral diarrhea virus is a costly disease of cattle that can be controlled by vaccination, biosecurity, and removal of persistently infected cattle. Development and proficiency testing of assays to identify persistently infected cattle requires substantial quantities of known positive- and negative-sample material. The objective of this study was to determine what sections of bovine skin contained Bovine viral diarrhea virus antigen. Two commercially available antigen-capture enzyme-linked immunoassays were used to test subsamples representing the entire skin of 3 persistently infected calves. Both assays detected Bovine viral diarrhea virus antigen in the samples indicated for use by assay protocol. However, one assay identified all subsamples as positive, while the second assay identified 64.4% of subsamples as positive. These results show that use of samples other than those specified by the assay protocol must be validated for each individual assay. In this study, alternative sample sites and use of the entire hide for proficiency testing would be acceptable for only one of the assays tested. PMID:22529122

  6. Identification of seven surface-exposed Brucella outer membrane proteins by use of monoclonal antibodies: immunogold labeling for electron microscopy and enzyme-linked immunosorbent assay.

    PubMed Central

    Cloeckaert, A; de Wergifosse, P; Dubray, G; Limet, J N

    1990-01-01

    A panel of monoclonal antibodies (MAbs) to seven Brucella outer membrane proteins were characterized. These antibodies were obtained by immunizing mice with sodium dodecyl sulfate-insoluble (SDS-I) fractions, cell walls, or whole bacterial cells of Brucella abortus or B. melitensis. Enzyme-linked immunosorbent assays were used to screen the hybridoma supernatants and to determine their binding at the surface of rough and smooth B. abortus and B. melitensis cells. The outer membrane proteins (OMPs) recognized by these antibodies were the proteins with molecular masses of 25 to 27 kDa and 36 to 38 kDa (porin) (major proteins) and the proteins with molecular masses of 10, 16.5, 19, 31 to 34, and 89 kDa (minor proteins). Surface exposure of these OMPs was visualized by electron microscopy by using the MAbs and immunogold labeling. Binding of the MAbs on whole rough bacterial cells indicates that the 10-, 16.5-, 19-, 25- to 27-, 31- to 34-, 36- to 38-, and 89-kDa OMPs are exposed at the cell surface. However, enzyme-linked immunosorbent assay results indicate a much better binding of the anti-OMP MAbs on rough strains than on the corresponding smooth strains except for the anti-19-kDa MAb. Immunoelectron microscopy showed that on smooth B. abortus cells only the 89- and 31- to 34-kDa OMPs were not accessible to the MAbs tested. Binding of the anti-31- to 34-kDa MAb at the cell surface was observed for the rough B. abortus cells and for the rough and smooth B. melitensis cells. These results indicate the importance of steric hindrance due to the presence of the long lipopolysaccharide O side chains in the accessibility of OMPs on smooth Brucella strains and should be considered when undertaking vaccine development. Images PMID:1701417

  7. Development of an Enzyme-Linked Immunosorbent Spot Assay To Measure Serum-Neutralizing Antibodies against Coxsackievirus B3

    PubMed Central

    Yang, Lisheng; He, Delei; Tang, Min; Li, Zhiqun; Liu, Che; Xu, Longfa; Chen, Yixin; Du, Hailian; Zhao, Qinjian; Zhang, Jun; Xia, Ningshao

    2014-01-01

    Coxsackievirus B3 (CVB3) is the most common pathogen that induces acute and chronic viral myocarditis in children. The cytopathic effect (CPE)-based neutralization test (Nt-CPE) and the plaque reduction neutralization test (PRNT) are the most common methods for measuring neutralizing antibody titers against CVB3 in blood serum samples. However, these two methods are inefficient for CVB3 vaccine clinical trials, which require the testing of a large number of serum specimens. In this study, we developed an efficient neutralization test based on the enzyme-linked immunospot (Nt-ELISPOT) assay for measuring CVB3-neutralizing antibodies. This modified ELISPOT assay was based on the use of a monoclonal antibody against the viral capsid protein VP1 to detect the cells that are infected with CVB3, which, after immunoperoxidase staining, are counted as spots using an automated ELISPOT analyzer. Using the modified ELISPOT assay, we characterized the infection kinetics of CVB3 and divided the infection process of CVB3 on a cluster of cells into four phases. The stability of the Nt-ELISPOT was then evaluated. We found that over a wide range of infectious doses (102 to 106.5× 50% tissue culture infectious dose [TCID50] per well), the neutralizing titers of the sera were steady as long as they were tested during the log phase or the first half of the stationary phase of growth of the spots. We successfully shortened the testing period from 7 days to approximately 20 h. We also found that there was a good correlation (R2 = 0.9462) between the Nt-ELISPOT and the Nt-CPE assays. Overall, the Nt-ELISPOT assay is a reliable and efficient method for measuring neutralizing antibodies in serum. PMID:24391137

  8. Quantification of Ponceau 4R in Foods by Indirect Competitive Enzyme-Linked Immunosorbent Assay (icELISA).

    PubMed

    Dong, Yaqing; Zhang, Jie; Xing, Yue; Song, Zhaorui; Wang, Yufen; Meng, Meng; Deng, Chuan; Tong, Zhongsheng; Yin, Yongmei; Xi, Rimo

    2015-07-22

    As one of the rarely allowable azo dyes, ponceau 4R can be added in some foods in some countries. However, it is necessary to develop a credible and rapid analytical method for its monitoring, because of its potentially harmful risk. The hapten of ponceau 4R was first designed and synthesized by introducing a primary amine group into the structure of ponceau 4R. Based on the well-prepared hapten, the immunogen of ponceau 4R was prepared using glutaraldehyde to link ponceau 4R to the carrier protein. The triggered polyclonal antibody was obtained and tested by ELISA to optimize the proper dilution. An icELISA was developed for ponceau 4R, and the IC50 of the method is 36.82 ng/mL. The limit of detection is 0.80 ng/mL, and the linear range is 1-10000 ng/mL. Five selected structural analogues have no cross-reactivity with the anti-ponceau 4R polyclonal antibody (<0.3). In three food samples (grape juice, carbonated beverage, and RIO cocktail), the assay exhibits good stability and reproducibility with a recovery range of 93.87-103.77%, and the intra- and interassay coefficients of variation were <11.73%. The results indicate that the proposed icELISA is sensitive, accurate, specific, and simple, which provides an alternative for the detection of ponceau 4R in foods. PMID:26138666

  9. Double-antibody sandwich enzyme-linked immunosorbent assay for quantitation of endoglucanase I of Trichoderma reesei.

    PubMed Central

    Bühler, R

    1991-01-01

    A sensitive and specific enzyme-liked immunosorbent assay for endoglucanase I (EG-I) has been developed. The monoclonal antibody a-EG-I 2, directed against an epitope on the core part of the enzyme, was used to capture the antigen in microtiter plate wells. A second, polyclonal antibody against the enzyme was then used to detect and quantitate the bound antigen. The test was specific for EG-I; neither endoglucanase II nor cellobiohydrolase I or II interfered. As little as 20 pg of EG-I protein could be detected. The coefficients of variation were 3.8% within plates and 6% between plates for a diluted Trichoderma reesei culture supernatant that contained 31 ng of EG-I per ml. Binding of the antigen to the monoclonal antibody was pH dependent and restricted to values between pH 6.5 and 10.5 with a maximum around pH 9. Standard solutions of EG-I were very stable at concentrations as low as 5 ng/ml when prepared in buffer that contained 1% bovine serum albumin and that was stored at -20 degrees C. After 37 weeks the antigenicity was still 97%. With this test it was possible to monitor the production of EG-I in a cellulase-producing strain of T. reesei and to demonstrate the apparent absence of the enzyme in a strain with the eglI gene deleted. PMID:1781689

  10. Enzyme-linked immunosorbent assay for detection of serum or mucosal isotype specific IgG and IgA whole virus antibody to influenza A virus in swine

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Enzyme-linked immunosorbent assays (ELISA) can be used to detect isotype specific anti-influenza antibodies in biological samples to characterize the porcine immune response to influenza A virus. The isotype antibody assay is based on an indirect ELISA using whole influenza virus as antigen and dete...

  11. Absorption of p,p'-dichlorodiphenyldichloroethylene and dieldrin in largemouth bass from a 60-D slow-release pellet and detection using a novel enzyme-linked immunosorbent assay method for blood plasma

    USGS Publications Warehouse

    Muller, Jennifer K.; Sepulveda, Maria S.; Borgert, Christopher J.; Gross, Timothy S.

    2005-01-01

    This work describes the uptake of two organochlorine pesticides from slow-release pellets by largemouth bass and the utility of a blood plasma enzyme-linked immunosorbent assay (ELISA) method for exposure verification. We measured blood and tissue levels by gas chromatography/mass spectrometry and by a novel ELISA method, and present a critical comparison of the results.

  12. Low-Cost Nanoribbon Sensors for Protein Analysis in Human Serum Using a Miniature Bead-Based Enzyme-Linked Immunosorbent Assay.

    PubMed

    Hu, Chunxiao; Zeimpekis, Ioannis; Sun, Kai; Anderson, Sally; Ashburn, Peter; Morgan, Hywel

    2016-05-01

    We describe a low cost thin-film transistor (TFT) nanoribbon sensor for detection of the inflammatory biomarker C-reactive protein (CRP) in human serum via a miniature bead-based enzyme-linked immunosorbent assay (ELISA). The TFT nanoribbon sensor measures the reaction products from the ELISA via pH changes. The bead-based ELISA decouples the protein functionalization steps from the sensor surface, increasing the signal and simplifying the assay. The ability to directly sense proteins in human serum in this way overcomes the Debye length limitation associated with nanowire and nanoribbon biosensors. Compared to classically fabricated nanowires, the TFT nanoribbon sensors are simple, extremely easy to fabricate, and should therefore be much cheaper to manufacture. TFT nanoribbon sensors, configured to measure pH, were used for quantitative detection of CRP spiked into human serum at concentrations as low as 0.2 ng/mL, which is 10 000 times lower than needed for diagnostic purposes, providing the potential for applications that require very high sensitivity. PMID:27035411

  13. Evaluation of two Neospora caninum recombinant antigens for use in an enzyme-linked immunosorbent assay for the diagnosis of bovine neosporosis.

    PubMed

    Lally, N C; Jenkins, M C; Dubey, J P

    1996-05-01

    Neospora caninum is a recently described apicomplexan parasite which causes paralysis and death in dogs. Neospora parasites also cause abortion and neonatal morbidity in cattle, sheep, goats, and horses, and neosporosis is emerging as an important cause of bovine abortion worldwide. The purpose of this study was to identify N. caninum cDNA clones encoding antigens that would be useful for the immunodiagnosis of bovine neosporosis. Two N. caninum tachyzoite cDNA clones expressing antigens that were recognized by serum from naturally and experimentally infected cattle were identified. The DNA sequences of these clones were determined, and the inserts were subcloned into the plasmid expression vector pTrcHisB. Both recombinant antigens, expressed as fusion proteins with a His6 tag, were purified on a nickel-chelating affinity column and evaluated in separate enzyme-linked immunosorbent assays (ELISAs). Both recombinant antigen ELISAs were capable of distinguishing between sera from Neospora-infected cows and sera from uninfected control cows. Furthermore, both assays were able to detect an antibody response in animals that were experimentally inoculated with N. caninum. Neither antigen showed evidence of cross-reactivity with serum from animals inoculated with the closely related parasites Toxoplasma gondii, Sarcocystis cruzi, Sarcocystis hominis, and Sarcocystis hirsuta. PMID:8705668

  14. Performance and reliability of five commercial enzyme-linked immunosorbent assay kits in screening for anti-human immunodeficiency virus antibody in high-risk subjects.

    PubMed Central

    Ozanne, G; Fauvel, M

    1988-01-01

    Anti-human immunodeficiency virus enzyme-linked immunosorbent assay kits marketed by Electro-Nucleonics Inc. (ENI), Genetic Systems Corp. (GSC), Organon Teknika Inc. (OTI), Ortho Diagnostic Systems Inc. (ODSI), and Wellcome Diagnostics (WD) were evaluated by using 289 randomly selected serum samples from a high-risk population and 53 serum samples likely to produce false-positive results. The radioimmunoprecipitation assay was used as the reference test. Sensitivities ranged from 96.51% (ODSI, WD) to 97.67% (ENI, GSC, OTI). Sera showing antibodies to viral glycoproteins only produced the false-negative results. Specificities ranged from 99.6% (ENI, GSC, ODSI, OTI) to 100% (WD). False-positive results were obtained with sera from patients with autoimmune disease or Epstein-Barr virus infection. Only results from GSC and OTI kits were distributed in two compact clusters well segregated on either side of the cutoff point. ODSI and GSC kits had the best intralot reproducibility. The GSC kit had the best interlot reproducibility. Cutoff values for ODSI and GSC kits were the least variable. Intraplate repeatability was good for all kits. Sample localization was not an important source of variability. Our results do not point out one outstanding kit among the five evaluated. However, the GSC kit showed the best overall results. PMID:3170712

  15. Enzyme-linked immunosorbent assay (ELISA) for the detection of use of the synthetic cannabinoid agonists UR-144 and XLR-11 in human urine.

    PubMed

    Mohr, Amanda L A; Ofsa, Bill; Keil, Alyssa Marie; Simon, John R; McMullin, Matthew; Logan, Barry K

    2014-09-01

    Ongoing changes in the synthetic cannabinoid drug market create the need for relevant targeted immunoassays for rapid screening of biological samples. We describe the validation and performance characteristics of an enzyme-linked immunosorbent assay designed to detect use of one of the most prevalent synthetic cannabinoids in urine, UR-144, by targeting its pentanoic acid metabolite. Fluorinated UR-144 (XLR-11) has been demonstrated to metabolize to this common product. The assay has significant cross-reactivity with UR-144-5-OH, UR-144-4-OH and XLR-11-4-OH metabolites, but <10% cross-reactivity with the parent compounds, and no measurable cross-reactivity with other synthetic cannabinoids and their metabolites at concentrations of <1,000 ng/mL. The assay's cutoff is 5 ng/mL relative to the pentanoic acid metabolite of UR-144, which is used as the calibrator. The method was validated with 90 positive and negative control urine samples for UR-144, XLR-11 and its metabolites tested versus liquid chromatography-tandem mass spectrometry. The accuracy, sensitivity and specificity were determined to be 100% for the assay at the specified cutoff. PMID:24908262

  16. Pistachio (Pistacia vera L.) Detection and Quantification Using a Murine Monoclonal Antibody-Based Direct Sandwich Enzyme-Linked Immunosorbent Assay.

    PubMed

    Liu, Changqi; Chhabra, Guneet S; Sathe, Shridhar K

    2015-10-21

    A commercially available direct sandwich enzyme-linked immunosorbent assay (ELISA) (BioFront Technologies, Tallahassee, FL, USA) using murine anti-pistachio monoclonal antibodies (mAbs) as capture and detection antibodies was evaluated. The assay was sensitive (limit of detection = 0.09 ± 0.02 ppm full fat pistachio, linear detection range = 0.5-36 ppm, 50% maximum signal concentration = 7.9 ± 0.7 ppm), reproducible (intra- and inter-assay variability < 24% CV), and rapid (post-extraction testing time ∼ 1.5 h). The target antigen was stable and detectable in whole pistachio seeds subjected to autoclaving (121 °C, 15 psi, 15, 30 min), blanching (100 °C, 5, 10 min), frying (191 °C, 1 min), microwaving (500, 1000 W, 3 min), and dry roasting (140 °C, 30 min; 168 °C, 12 min). No cross-reactivity was observed in 156 food matrices, each tested at 100,000 ppm, suggesting the ELISA to be pistachio specific. The pistachio recovery ranges for spiked (10 ppm) and incurred (10-50000 ppm) food matrices were 93.1-125.6% and 35.7-112.2%, respectively. The assay did not register any false-positive or -negative results among the tested commercial and laboratory prepared samples. PMID:26416205

  17. Monoclonal antibody production and indirect competitive enzyme-linked immunosorbent assay development of 3-methyl-quinoxaline-2-carboxylic acid based on novel haptens.

    PubMed

    Li, Guopeng; Zhao, Liang; Zhou, Feng; Li, Jiaying; Xing, Yuan; Wang, Tiangang; Zhou, Xilong; Ji, Baoping; Ren, Wanpeng

    2016-10-15

    Two novel immunizing haptens of 3-methyl-quinoxaline-2-carboxylic acid (MQCA) were synthesized and conjugated with cationized bovine serum albumin. Female BALB/c mice were immunized with above conjugates, splenocytes were fused with Sp2/0 cells to produce monoclonal antibody. Compared with previous studies, antibodies raised in this work showed higher sensitivity. Meantime, a novel heterologous coating hapten was also prepared. The indirect competitive enzyme-linked immunosorbent assay (icELISA) based on the optimum condition showed an IC50 of 3.1μg/kg (ppb), and the linear range of 0.46-10.5ppb for MQCA. The limit of detect (LOD) of MQCA in swine muscle, swine liver and chicken was 0.32, 0.54, and 0.28ppb, respectively. The LOD of this assay can satisfy the minimum required performance levels (4ppb) for MQCA. These results indicated that the proposed ELISA, with high sensitivity and specificity, as well as good reproducibility and accuracy, is suitable for determination of MQCA residues in food samples. PMID:27173564

  18. Selective and sensitive determination of cypermethrin in fish via enzyme-linked immunosorbent assay-like method based on molecularly imprinted artificial antibody-quantum dot optosensing materials.

    PubMed

    Xiao, Ting-Ting; Shi, Xi-Zhi; Jiao, Hai-Feng; Sun, Ai-Li; Ding, Hao; Zhang, Rong-Rrong; Pan, Dao-Dong; Li, De-Xiang; Chen, Jiong

    2016-01-15

    Molecularly imprinted silica layers appended to quantum dots (MIP-QDs) with customized selective artificial recognition sites were fabricated in this study by optimizing the ratio of the functional monomer to the template. Scanning electron microscopy, transmission electron microscopy, Brunauer–emmett–teller, Fourier transform infrared spectroscopy, and selectivity assay analyses were also performed. Results demonstrated that the selective fluorescence quenching properties of MIP-QDs toward cypermethrin (CYP) are due to strong interactions between these molecules. An enzyme-linked immunosorbent assay (ELISA)-like method based on the MIP-QDs was established under optimal conditions. The fluorescence quenching observed from this method showed a linear relationship with CYP concentration over the range of 0.05–60.0 mg/kg with a correlation coefficient of 0.9838. Good recovery (82.7–92.4%) and a relative standard deviation of less than 10.1% were obtained from fish samples spiked with three levels of CYP. This method also demonstrated a low detection limit of 1.2 μg/kg. The ELISA-like method based on MIP-QDs can be successfully employed to detect residual of CYP in fish samples. PMID:26283587

  19. Serodiagnostic comparison of enzyme-linked immunosorbent assay and surface plasmon resonance for the detection of antibody to Porcine circovirus type 2

    PubMed Central

    Cho, Ho-Seong; Kim, Tae-Jung; Lee, Jae-Il; Park, Nam-Yong

    2006-01-01

    This paper describes the cloning and expression of the capsid protein of Porcine circovirus type 2 (PCV2) in an Escherichia coli expression system that was used to produce a fusion protein for subsequent immunologic studies: enzyme-linked immunosorbent assay (ELISA) and surface plasmon resonance (SPR). Polymerase chain reaction was used to amplify the gene encoding the capsid protein from the DNA of PCV2. The protein was then cloned into a pRSET prokaryotic expression vector. Western blot analysis revealed that the recombinant protein gave strong signals on a polyvinylidene difluoride membrane when exposed to the serum from a pig infected with PCV2. The expressed protein was purified and used as an antigen for the ELISA and SPR study. A protein chip based on SPR was developed, and the diagnostic potential of SPR was compared with that of ELISA with the use of 70 serum samples obtained from 6 pig farms. There was a strong positive correlation between the ELISA and SPR titers (r = 0.877, P < 0.01). Therefore, this recombinant capsid protein can be used as an antigen for serologic studies, and the SPR, a label-free method, appears to be a valuable and reproducible tool in the serodiagnosis of a PCV2 infection. PMID:17042378

  20. Development of an enzyme-linked immunosorbent assay for thiacloprid in soil and agro-products with phage-displayed peptide.

    PubMed

    Yin, Wei; Hua, Xiude; Liu, Xiaofeng; Shi, Haiyan; Gee, Shirley J; Wang, Minghua; Hammock, Bruce D

    2015-07-15

    A monoclonal antibody (3A5) that can recognize thiacloprid was produced, and a linear 8-residue peptide phage library was constructed. Six phage-displayed peptides were isolated from the linear 8-residue peptide phage library and a cyclic 8-residue peptide phage library. A phage enzyme-linked immunosorbent assay (ELISA) was developed to detect thiacloprid using a phage-displayed peptide. Under the optimal conditions, the half-maximal inhibition concentration (IC50) and the limit of detection (IC10) of the developed phage ELISA were 8.3 and 0.7 μg/L, respectively. Compared with the conventional ELISA, the sensitivity was improved more than 3-fold. The cross-reactivity (CR) was less than 0.08% for the tested structural analogues and was regarded as negligible. The recoveries of thiacloprid ranged from 80.3% to 116.3% in environmental and agricultural samples, which conformed to the requirements for residue detection. The amount of thiacloprid detected by phage ELISA in the samples was significantly correlated with that detected by high-performance liquid chromatography. The current study indicates that isolating phage-displayed peptides from phage display libraries is an alternative method for the development of a sensitive immunoassay and that the developed assay is a potentially useful tool for detecting thiacloprid in environmental and agricultural samples. PMID:25908560

  1. Enzyme-linked immunosorbent assays for the detection of equine antibodies specific to a recombinant Fasciola hepatica surface antigen in an endemic area.

    PubMed

    Arias, María Sol; Piñeiro, Pablo; Hillyer, George V; Francisco, Iván; Cazapal-Monteiro, Cristiana Filipa; Suárez, José Luis; Morrondo, Patrocinio; Sánchez-Andrade, Rita; Paz-Silva, Adolfo

    2012-02-01

    The utility of an enzyme-linked immunosorbent assay to determine the sensitization against the trematode Fasciola hepatica in horses from an endemic area (NW Spain) was assessed. Blood samples were collected from 536 horses and tested against a 2.9-kDa recombinant surface protein (FhrAPS) to estimate the presence of IgG antibodies. Data were analysed regarding several intrinsic (age, gender and breed) and extrinsic factors (aptitude and housing). The farm size (number of horses/farm) was also considered. Sixty percent (95% CI 56, 64) of the horses were positive to the FhrAPS-ELISA, with a significantly higher seroprevalence in the mares (67%). Foals reached the lowest percentage of sensitization against the trematode (12%), and a significant positive correlation between the seroprevalence of fasciolosis and the age of the horses was established. When considering all the factors together, the seroprevalence of fasciolosis was initially classified into two groups (nodes) regarding the age of the horses. The node composed of the horses older than 1 year was then divided into two other clusters according to their gender. The mares were finally classified and grouped into two nodes regarding their breed. We concluded that the FhrAPS-ELISA is very useful for the demonstration of specific equine IgG antibodies against F. hepatica. An elevated risk of exposition to this trematode in horses maintained in endemic areas was proven. The possible role of horses as reservoirs for F. hepatica infections is discussed. PMID:21847600

  2. Development and application of an indirect enzyme-linked immunosorbent assay for serological survey of Japanese encephalitis virus infection in dogs.

    PubMed

    Shimoda, Hiroshi; Inthong, Natnaree; Noguchi, Keita; Terada, Yutaka; Nagao, Yumiko; Shimojima, Masayuki; Takasaki, Tomohiko; Rerkamnuaychoke, Worawut; Maeda, Ken

    2013-01-01

    Japanese encephalitis virus (JEV) causes serious acute encephalitis in humans and horses. Although dogs are good sentinels for assessing the risk of JEV infection to humans, a virus neutralization test has been the only method available for measuring the levels of JEV antibody in dogs. In this study, an indirect enzyme-linked immunosorbent assay (ELISA) using purified viral particles as an antigen, was developed for serological survey of JEV infection in dogs. In dogs inoculated experimentally with JEV, the ELISA detected anti-JEV IgM 3 days after infection, with IgM levels peaking 7 days after infection. Anti-JEV IgG was detected 14 days after infection and peaked on 21-28 days after infection. Virus neutralization titers correlated with anti-JEV immunoglobulins measured by the ELISA. To test the utility of the new assay, the seroprevalence of JEV infection among 102 dogs in Kyushu, Japan, was examined by IgG ELISA and by virus neutralization. The correlation coefficient between the IgG ELISA and virus neutralization was 0.813 (p<0.001); comparison of the IgG ELISA and virus neutralization showed a sensitivity and specificity of 82% and 98%, respectively. The IgG ELISA was used to survey dogs in Bangkok, Thailand and 51% of these dogs were found seropositive for JEV. These data suggest that in the capital city of Thailand, the risk of infection with JEV remains high. PMID:23046992

  3. Standardization of an enzyme linked immunosorbent assay (ELISA) for detecting circulating toxic venom antigens in patients stung by the scorpion Tityus serrulatus.

    PubMed

    de Rezende, N A; Dias, M B; Campolina, D; Chavéz-Olortegui, C; Amaral, C F

    1995-01-01

    The sensitivity and specificity of an enzyme-linked immunosorbent assay (ELISA) for the detection of circulating antigens from toxic components of Tityus serrulatus scorpion venom was determined in patients stung by T. serrulatus before antivenom administration. Thirty-seven patients were classified as mild cases and 19 as moderate or severe cases. The control absorbance in the venom assay was provided by serum samples from 100 individuals of same socioeconomic group and geographical area who had never been stung by scorpions or treated with horse antisera. The negative cutoff value (mean + 2 SD) corresponded to a venom concentration of 4.8 ng/ml. Three out of the 100 normal sera were positive, resulting in a specificity of 97%. The sensitivity of the ELISA when all cases of scorpion sting were included was 39.3%. When mild cases were excluded, the sensitivity increased to 94.7%. This study showed that this ELISA can be used for the detection of circulating venom toxic antigens in patients with systemic manifestations following. T. serrulatus sting but cannot be used for clinical studies in mild cases of envenoming since the test does not discriminate mild cases from control patients. PMID:7569644

  4. Analysis of the Fungicide Boscalid in Horticultural Crops Using an Enzyme-Linked Immunosorbent Assay and an Immunosensor Based on Surface Plasmon Resonance.

    PubMed

    Hirakawa, Yuki; Yamasaki, Tomomi; Harada, Ayako; Ohtake, Toshiya; Adachi, Kayo; Iwasa, Seiji; Narita, Hiroshi; Miyake, Shiro

    2015-09-16

    A direct competitive enzyme-linked immunosorbent assay (dc-ELISA) and an immunosensor based on surface plasmon resonance (SPR-sensor) were developed for fungicide boscalid determination in horticultural crops. To produce antiboscalid monoclonal antibodies (MoAb BSC7 and MoAb BSC72) for these assays, a hapten of boscalid was synthesized and conjugated to keyhole limpet hemocyanin for Balb/c mouse immunization. The working range of the dc-ELISA was 0.8-16 ng/mL with MoAb BSC7 and 2.5-120 ng/mL with MoAb BSC72, and that of the SPR-sensor was 17-80 ng/mL with MoAb BSC7. The dc-ELISA and SPR-sensor were compared for their sensitivity in determining boscalid residues at the maximum residue limit of 1-40 mg/kg for horticultural crops in Japan. Recovery of the spiked boscalid was 85-109% by the SPR-sensor and 100-124% by the dc-ELISA. On real tomato samples, the results obtained by both of these immunoassays correlated well with the results obtained by high-performance liquid chromatography. PMID:26340386

  5. Determination of alachlor and its sulfonic acid metabolite in water by solid-phase extraction and enzyme-linked immunosorbent assay

    USGS Publications Warehouse

    Aga, D.S.; Thurman, E.M.; Pomes, M.L.

    1994-01-01

    Solid-phase extraction (SPE) and enzyme-linked immunosorbent assay (ELISA) were combined for the trace analysis of the herbicide alachlor and its major soil metabolite, ethanesulfonic acid (ESA). The anti-alachlor antibody cross-reacted with ESA, which produced false-positive detections of alachlor in water samples by immunoassay screens. Alachlor and ESA were isolated from water by SPE on a C18 resin and eluted sequentially with ethyl acetate and methanol. Alachlor is soluble in ethyl acetate while the anionic ESA is not. Thus ESA remained adsorbed on the C18 resin and was eluted later with methanol. The combination of SPE with ELISA effectivety separated and quantified both alachlor and ESA using the same antibody for two ELISA methods. The general method may have applicability for the separation of other herbicides and their ionic metabolites. The SPE-ELISA method has a, detection limit of 0.01 ??g/L for alachlor and 0.05 ??g/L for ESA, with a precision of ?? 10%. Analyses of surface and ground water samples were confirmed by gas chromatography/mass spectrometry and high-performance liquid chromatography with photodiode-array detection. Results showed widespread occurrence of ESA in surface and ground water of the midwestern United States, with concentrations ranging from 10 ??g/L.

  6. Comparison of the membrane-filtration fluorescent antibody test, the enzyme-linked immunosorbent assay, and the polymerase chain reaction to detect Renibacterium salmoninarum in salmon ovarian fluid

    USGS Publications Warehouse

    Pascho, R.J.; Chase, D.; McKibben, C.L.

    1998-01-01

    Ovarian fluid samples from naturally infected chinook salmon (Oncorhynchus tshawytscha) were examined for the presence of Renibacterium salmoninarum by the membrane-filtration fluorescent antibody test (MF-FAT), an antigen capture enzyme-linked immunosorbent assay (ELISA), and a nested polymerase chain reaction (PCR). On the basis of the MF-FAT, 64% (66/103) samples contained detectable levels of R. salmoninarum cells. Among the positive fish, the R. salmoninarum concentrations ranged from 25 cells/ml to 4.3 3 109 cells/ml. A soluble antigenic fraction of R. salmoninarum was detected in 39% of the fish (40/103) by the ELISA. The ELISA is considered one of the most sensitive detection methods for bacterial kidney disease in tissues, yet it did not detect R. salmoninarum antigen consistently at bacterial cell concentrations below about 1.3 3 104 cells/ml according to the MF-FAT counts. When total DNA was extracted and tested in a nested PCR designed to amplify a 320-base-pair region of the gene encoding a soluble 57-kD protein of R. salmoninarum, 100% of the 100 samples tested were positive. The results provided strong evidence that R. salmoninarum may be present in ovarian fluids thought to be free of the bacterium on the basis of standard diagnostic methods.

  7. Mutations in the rpoB Gene of Rifampin-Resistant Mycobacterium tuberculosis Isolates in Spain and Their Rapid Detection by PCR–Enzyme-Linked Immunosorbent Assay

    PubMed Central

    Garcia, Lucia; Alonso-Sanz, Mercedes; Rebollo, Maria J.; Tercero, Juan C.; Chaves, Fernando

    2001-01-01

    Genetic alterations in the rpoB gene were characterized in 50 rifampin-resistant (Rifr) clinical isolates of Mycobacterium tuberculosis complex from Spain. A rapid PCR–enzyme-linked immunosorbent assay (ELISA) technique for the identification of rpoB mutations was evaluated with isolates of the M. tuberculosis complex and clinical specimens from tuberculosis patients that were positive for acid-fast bacilli (AFB). Sequence analysis demonstrated 11 different rpoB mutations among the Rifr isolates in the study. The most frequent mutations were those associated with codon 531 (24 of 50; 48%) and codon 526 (11 of 50; 22%). Although the PCR-ELISA does not permit characterization of the specific Rifr allele within each strain, 10 of the 11 Rifr genotypes were correctly identified by this method. We used the PCR-ELISA to predict the rifampin susceptibility of M. tuberculosis complex organisms from 30 AFB-positive sputum specimens. For 28 samples, of which 9 contained Rifr organisms and 19 contained susceptible strains, results were concordant with those based on culture-based drug susceptibility testing and sequencing. Results from the remaining two samples could not be interpreted because of low bacillary load (microscopy score of 1+ for 1 to 9 microorganisms/100 fields). Our results suggest that the PCR-ELISA is an easy technique to implement and could be used as a rapid procedure for detecting rifampin resistance to complement conventional culture-based methods. PMID:11325996

  8. Analysis of benzo[a]pyrene in vegetable oils using molecularly imprinted solid phase extraction (MISPE) coupled with enzyme-linked immunosorbent assay (ELISA).

    PubMed

    Pschenitza, Michael; Hackenberg, Rudolf; Niessner, Reinhard; Knopp, Dietmar

    2014-01-01

    This paper describes the development of a molecularly imprinted polymer-based solid phase extraction (MISPE) method coupled with enzyme-linked immunosorbent assay (ELISA) for determination of the PAH benzo[a]pyrene (B[a]P) in vegetable oils. Different molecularly imprinted polymers (MIPs) were prepared using non-covalent 4-vinylpyridine/divinylbenzene co-polymerization at different ratios and dichloromethane as porogen. Imprinting was done with a template mixture of phenanthrene and pyrene yielding a broad-specific polymer for PAHs with a maximum binding capacity (Q) of ~32 μg B[a]P per 50 mg of polymer. The vegetable oil/n-hexane mixture (1:1, (v/v)) was pre-extracted with acetonitrile, the solvent evaporated, the residue reconstituted in n-hexane and subjected to MISPE. The successive washing with n-hexane and isopropanol revealed most suitable to remove lipid matrix constituents. After elution of bound PAHs from MISPE column with dichloromethane, the solvent was evaporated, the residue reconstituted with dimethyl sulfoxide and diluted 100-fold with methanol/water (10:90, (v/v)) for analysis of B[a]P equivalents with an ELISA. The B[a]P recovery rates in spiked vegetable oil samples of different fatty acid composition were determined between 63% and 114%. The presence of multiple PAHs in the oil sample, because of MIP selectivity and cross-reactivity of the ELISA, could yield overestimated B[a]P values. PMID:24887045

  9. Competitive-inhibition enzyme-linked immunosorbent assay for detection of serum antibodies to caprine arthritis-encephalitis virus: diagnostic tool for successful eradication.

    PubMed

    Herrmann, Lynn M; Cheevers, William P; McGuire, Travis C; Adams, D Scott; Hutton, Melinda M; Gavin, William G; Knowles, Donald P

    2003-03-01

    A competitive-inhibition enzyme-linked immunosorbent assay (cELISA) was evaluated for the detection of serum antibodies to the surface envelope (SU) of caprine arthritis-encephalitis virus (CAEV) in goats. This assay utilized 96-well microtiter plates containing CAEV-63 SU captured by monoclonal antibody (MAb) F7-299 and measured the competitive displacement of horseradish peroxidase-conjugated MAb GPB 74A binding by undiluted goat sera (F. Ozyörük, W. P. Cheevers, G. A. Hullinger, T. C. McGuire, M. Hutton, and D. P. Knowles, Clin. Diagn. Lab. Immunol. 8:44-51, 2001). Two hundred serum samples from goats in the United States were used to determine the sensitivity and specificity of cELISA based on the immunoprecipitation (IP) of [(35)S]methionine-labeled viral antigens as a standard of comparison. A positive cELISA was defined as >33.2% inhibition of MAb 74A binding based on 2 standard deviations above the mean percent inhibition of 140 IP-negative serum samples. At this cutoff value, there were 0 of 60 false-negative sera (100% sensitivity) and 5 of 140 false-positive sera (96.4% specificity). Additional studies utilized IP-monitored cELISA to establish a CAEV-free herd of 1,640 dairy goats. PMID:12626453

  10. Serological analysis by enzyme-linked immunosorbent assay using recombinant antigen LipL32 for the diagnosis of swine leptospirosis.

    PubMed

    Hartleben, Cláudia P; Leal, Fernanda M A; Monte, Leonardo G; Hartwig, Daiane D; Seixas, Fabiana K; Vasconcellos, Sílvio A; Brihuega, Bibiana; Dellagostin, Odir A

    2013-02-01

    Leptospirosis is an important global zoonotic disease caused by pathogenic Leptospira spp. species. Swine leptospirosis has a major economic impact because pigs are sources of animal protein and by-products. The signs of swine leptospirosis are abortion, stillbirth, birth of weak or ill piglets, appearing 14-60 days after infection. The reference method for diagnosis of leptospirosis is the microscopic agglutination test (MAT), in which serum samples are reacted with live antigen suspensions of leptospiral serovars. However, MAT is laborious and time consuming as a diagnostic procedure when dealing with a large number of samples; therefore, efforts are being made to develop novel, sensitive, and specific diagnostic tests for leptospirosis. In this study, a recombinant LipL32 based on enzyme-linked immunosorbent assay (rLipL32/ELISA) was evaluated as a screening test for the detection of pathogenic leptospiral-specific antibodies. A total of 86 swine serum samples tested by MAT were used to develop rLipL32/ELISA. Compared to positive and negative sera tested by MAT, rLipL32/ELISA showed 100 % sensitivity, 85.1 % specificity, and 91.86 % accuracy. No positive reaction for other bacterial diseases (enzootic pneumonia and brucellosis) was observed. The rLipL32/ELISA reported in this study is a specific, sensitive, and convenient test for the detection of antibodies against swine leptospiral infection and can be used as a rapid screening test in epidemiological surveys. PMID:23064970

  11. Validation and use of an indirect enzyme-linked immunosorbent assay for detection of antibodies to West Nile virus in American Alligators (Alligator mississippiensis) in Florida.

    PubMed

    Jacobson, Elliott R; Johnson, April J; Hernandez, Jorge A; Tucker, Sylvia J; Dupuis, Alan P; Stevens, Robert; Carbonneau, Dwayne; Stark, Lillian

    2005-01-01

    In October 2002, West Nile virus (WNV) was identified in farmed American alligators (Alligator mississippiensis) in Florida showing clinical signs and having microscopic lesions indicative of central nervous system disease. To perform seroepidemiologic studies, an indirect enzyme-linked immunosorbent assay (ELISA) was developed to determine exposure of captive and wild alligators to WNV. To validate the test, a group of WNV-seropositive and -seronegative alligators were identified at the affected farm using hemagglutination inhibition (HAI) and the plaque reduction neutralization test (PRNT). The indirect ELISA utilized a rabbit anti-alligator immunoglobulins polyclonal antibody as the secondary antibody, and inactivated WNV-infected Vero cells were used as the coating antigen. For all samples (n=58), the results of the ELISA were consistent with the HAI and PRNT findings. Plasma was collected from 669 free-ranging alligators from 21 sites across Florida in April and October 2003. Four samples collected in April and six in October were positive for WNV antibodies using HAI, PRNT, and the indirect ELISA. This indicated that wild alligators in Florida have been exposed to WNV. These findings can be used as a baseline for future surveys. PMID:15827216

  12. A Colorimetric Enzyme-Linked Immunosorbent Assay (ELISA) Detection Platform for a Point-of-Care Dengue Detection System on a Lab-on-Compact-Disc

    PubMed Central

    Thiha, Aung; Ibrahim, Fatimah

    2015-01-01

    The enzyme-linked Immunosorbent Assay (ELISA) is the gold standard clinical diagnostic tool for the detection and quantification of protein biomarkers. However, conventional ELISA tests have drawbacks in their requirement of time, expensive equipment and expertise for operation. Hence, for the purpose of rapid, high throughput screening and point-of-care diagnosis, researchers are miniaturizing sandwich ELISA procedures on Lab-on-a-Chip and Lab-on-Compact Disc (LOCD) platforms. This paper presents a novel integrated device to detect and interpret the ELISA test results on a LOCD platform. The system applies absorption spectrophotometry to measure the absorbance (optical density) of the sample using a monochromatic light source and optical sensor. The device performs automated analysis of the results and presents absorbance values and diagnostic test results via a graphical display or via Bluetooth to a smartphone platform which also acts as controller of the device. The efficacy of the device was evaluated by performing dengue antibody IgG ELISA on 64 hospitalized patients suspected of dengue. The results demonstrate high accuracy of the device, with 95% sensitivity and 100% specificity in detection when compared with gold standard commercial ELISA microplate readers. This sensor platform represents a significant step towards establishing ELISA as a rapid, inexpensive and automatic testing method for the purpose of point-of-care-testing (POCT) in resource-limited settings. PMID:25993517

  13. Development and Evaluation of an In-House Enzyme-Linked Immunosorbent Assay for Early Diagnosis and Monitoring of Human Pythiosis

    PubMed Central

    Krajaejun, Theerapong; Kunakorn, Mongkol; Niemhom, Sopaporn; Chongtrakool, Piriyaporn; Pracharktam, Roongnapa

    2002-01-01

    Human pythiosis is an emerging, fatal, infectious disease caused by Pythium insidiosum and occurs in both tropical and subtropical countries. Thalassemic patients, farmers, and aquatic-habitat residents are predisposed to this disease. Delayed treatment due to the long time required for isolation and identification of the causative organism, as well as the difficulty in obtaining internal organ specimens, results in high morbidity and mortality. To facilitate rapid diagnosis, an in-house enzyme-linked immunosorbent assay (ELISA) for the detection of immunoglobulin G antibodies against P. insidiosum was developed and evaluated for the diagnosis and monitoring of human pythiosis. Sixteen sera were collected from seven culture-proven human pythiosis cases. A total of 142 sera from thalassemic patients, from patients with other infectious diseases, and from healthy blood donors served as controls. All sera were tested in duplicate. By choosing a suitable cutoff point to maximize sensitivity and specificity, sera from pythiosis cases were all determined to be positive, whereas sera from control groups were all determined to be negative. ELISA signals from serial samples of sera taken from treated patients showed gradually declining levels of antibodies to P. insidiosum. The ELISA test was highly sensitive (100%) and specific (100%) and was useful for early diagnosis and for monitoring the treatment for pythiosis. PMID:11874882

  14. Development and evaluation of an enzyme-linked immunosorbent assay for serum Vi antibodies for detection of chronic Salmonella typhi carriers.

    PubMed Central

    Losonsky, G A; Ferreccio, C; Kotloff, K L; Kaintuck, S; Robbins, J B; Levine, M M

    1987-01-01

    An enzyme-linked immunosorbent assay (ELISA) measuring, in serum, immunoglobulin G (IgG), IgM, and IgA to Vi capsular polysaccharide antigen that was tyraminated (Vi-Tyr) to increase its binding efficiency to microtiter plates was compared with the standard passive hemagglutination assay (PHA) as a screening test for chronic Salmonella typhi carriers. Initially, three populations were evaluated: 22 healthy U.S. adults, 17 young Chilean adults with acute typhoid fever, and 51 Chileans who had bacteriologically confirmed S. typhi chronic carriage. IgG-specific Vi-Tyr antibodies were preferentially present in the S. typhi chronic carrier state. A total of 44 of 51 (81%) chronic carriers, 0 of 22 (0%) healthy U.S. adults, and 2 of 17 (12%) Chileans with acute typhoid fever had reciprocal IgG Vi-Tyr ELISA antibody titers in serum of greater than or equal to 200. The IgG Vi-Tyr ELISA was then compared with the PHA as a screening test for chronic carriers in 141 Chilean female food handlers. One woman was serologically incriminated as a carrier by both the IgG ELISA and PHA; her coprocultures were positive for S. typhi. One other woman, identified as a carrier by PHA, was negative by culture and IgG ELISA. The IgG Vi-Tyr ELISA is as sensitive as the PHA (86 versus 76%) and as specific (95 versus 95%) in screening for chronic carriers. PMID:3429619

  15. A liquid-phase-blocking concanavalin A enzyme-linked immunosorbent assay for the detection of antibodies against Newcastle disease virus in serum of free-ranging pigeons.

    PubMed

    de Oliveira, Elisabete Schirato; Silva, Ketherson Rodrigues; Fernando, Filipe Santos; Gonçalves, Mariana Costa Mello; Fernandes, Camila Cesário; Borzi, Mariana Monezi; dos Santos, Romeu Moreira; Tamanini, Maria de Lourdes Feres; Montassier, Maria de Fátima da Silva; Montassier, Helio José

    2013-11-01

    A competitive liquid-phase-blocking concanavalin A enzyme-linked immunosorbent assay (LPB-ConA-ELISA) was developed in the current study. The assay used ConA as a capture reagent, and the sera of specific pathogen-free chickens immunized with nonpurified Newcastle disease virus (NDV) suspension as detector antibodies, to detect and quantify specific antiviral antibodies in serum samples from free-ranging pigeons. The comparison between the LPB-ConA-ELISA and the hemagglutination inhibition (HI) test for the detection of antibodies in serum samples from 107 pigeons showed significant correlation between the assays (r = 0.875), a high sensitivity (100%), specificity (95.8%), accuracy (96.3%) for the ELISA, and good agreement (κ = 0.83) between the 2 assays. The results of this study suggest that the LPB-ConA-ELISA could be a useful alternative to HI test in the serodiagnosis of NDV in pigeons, or other species of birds. PMID:24100439

  16. Development and field application of a competitive enzyme-linked immunosorbent assay for detection of Newcastle disease virus antibodies in chickens and ducks.

    PubMed

    Phan, L V; Park, M-J; Kye, S-J; Kim, J-Y; Lee, H-S; Choi, K-S

    2013-08-01

    A competitive enzyme-linked immunosorbent assay (C-ELISA) using a baculovirus-expressed recombinant nucleocapsid protein antigen (rNDV-N) and an rNDV-N-specific monoclonal antibody (5B3) was developed for the detection of Newcastle disease virus (NDV) antibodies, and its diagnostic performance was evaluated. The specificity and sensitivity of the C-ELISA was found to be 98.4 and 98.9%, respectively, for chickens, and 98.2 and 97.9% for ducks. However, the C-ELISA showed weak cross-reaction with hyperimmune antisera to some other avian paramyxovirus serotypes. In all experimentally vaccinated chickens, seroconversion rates at 7 d postinoculation were 100 and 40% when measured by C-ELISA and hemagglutination inhibition (HI), respectively. In field trials, the C-ELISA showed positive results in 98.9% of HI-positive sera and 40.8% of HI-negative sera from NDV-vaccinated chickens (n = 705). In domestic ducks (n = 158) from NDV-positive duck farms (n = 8), the positive rates according to C-ELISA were significantly higher than those according to the HI test. At the same time, 98.1% of ducks (n = 209) from NDV-negative duck farms (n = 11) were also negative by C-ELISA. Our results indicate that C-ELISA could be a useful alternative to HI testing for detecting NDV antibodies in different avian species such as chickens and ducks. PMID:23873550

  17. Use of a commercial enzyme-linked immunosorbent assay for rapid detection of Giardia duodenalis in dog stools in the environment: a Bayesian evaluation.

    PubMed

    Papini, Roberto; Carreras, Giulia; Marangi, Marianna; Mancianti, Francesca; Giangaspero, Annunziata

    2013-05-01

    Giardia duodenalis is considered a potentially zoonotic protozoan. Some animal species, including infected dogs, may play an important role in the spread of Giardia cysts through environmental contamination with their feces. In the present study, a commercial enzyme-linked immunosorbent assay (ELISA) was used to examine 143 samples of dog feces collected in urban areas as an indicator of the risk of field contamination. Using a Bayesian statistical approach, the ELISA showed a sensitivity of 88.9% and a specificity of 95.8% with positive and negative predictive values of 89.6% and 95.4%, respectively. The test affords the advantage of rapid processing of fecal samples without a complex technical structure and extensive costly labor. Moreover, the present results show that the assay provides public health veterinarians with a practical tool that can be used in screening programs, as a valid alternative or as an adjunct to other tests, in order to assess the biological risk of exposure to G. duodenalis cysts from dogs in human settlements. However, the test may not be completely accurate for human health risk evaluation, as it does not discriminate between zoonotic and non-zoonotic isolates. PMID:23604261

  18. Development of a monoclonal antibody-based enzyme-linked immunosorbent assay for the analysis of 6-benzylaminopurine and its ribose adduct in bean sprouts.

    PubMed

    Zhang, Wei; He, Lishan; Zhang, Rui; Guo, Suqin; Yue, Huanfang; Ning, Xiangxue; Tan, Guiyu; Li, Qing X; Wang, Baomin

    2016-09-15

    6-Benzylaminopurine (6-BA), a cytokinin plant growth regulator, has been banned for use in bean sprout production in China. An indirect competitive enzyme-linked immunosorbent assay (icELISA) was developed with a specific monoclonal antibody (mAb 3E5). The assay showed a half-maximum inhibition concentration (IC50) and detection range of 18.9 ng/mL and 3.6-106 ng/mL, respectively. Recoveries of 6-BA spiked in home cultured bean sprout samples averaged from 75% to 89% with a correlation coefficient (R(2)) of 0.998 between the results determined by icELISA and those by liquid chromatography-electrospray ionization quadrupole Orbitrap mass spectrometry (LC-ESI-MS). LC-ESI-MS showed that 6-BA had been partially metabolized to 6-benzylaminopurine riboside (6-BAR) in the positive samples. The content of 6-BA determined by icELISA was about 5-70 times higher than that of LC-ESI-MS because mAb 3E5 had 315% cross-reactivity with 6-BAR. Such icELISA being ultra-sensitive to 6-BAR would allow quick monitoring of 6-BA by detecting 6-BAR as a potential biomarker. PMID:27080901

  19. Recombinant protein-based enzyme-linked immunosorbent assay and immunochromatographic tests for detection of immunoglobulin G antibodies to severe acute respiratory syndrome (SARS) coronavirus in SARS patients.

    PubMed

    Guan, Ming; Chen, Hsiao Ying; Foo, Shen Yun; Tan, Yee-Joo; Goh, Phuay-Yee; Wee, Shock Hwa

    2004-03-01

    An enzyme-linked immunosorbent assay (ELISA) and a rapid immunochromatographic test for detection of immunoglobulin G (IgG) antibodies in severe acute respiratory syndrome (SARS) patients were developed by utilizing the well-characterized recombinant proteins Gst-N and Gst-U274. The ELISA detected IgG antibodies to SARS-CoV in all 74 convalescent-phase samples from SARS patients while weakly cross-reacting to only 1 of the 210 control sera from healthy donors. This finding thus led to a kit sensitivity, specificity, and accuracy of 100, 99.5, and 99.6%, respectively. The test thus provided a positive predictive value (PPV) of 98.7% and a negative predictive value (NPV) of 100%. In addition, the ELISA gave a positive delta of 5.4 and a negative delta of 3.6, indicating an excellent differentiation between positives and negatives. The same recombinant proteins were also applied to a newly developed platform for the development of a 15-min rapid test. The resulting rapid test has an excellent agreement of 99.6%, with a kappa value of 1.00, with the ELISA. Again, this rapid test was able to detect 100% of the samples tested (n = 42) while maintaining a specificity of 99.0% (n = 210). The PPV and NPV for the rapid test thus reached 95.3 and 100%, respectively. PMID:15013977

  20. Evaluation of an O antigen enzyme-linked immunosorbent assay for screening of milk samples for Salmonella dublin infection in dairy herds.

    PubMed Central

    Hoorfar, J; Lind, P; Bitsch, V

    1995-01-01

    Levels of antibodies to the O antigens (O:1,9,12) of Salmonella dublin were tested in 1355 serum, 1143 cow milk and 160 bulk milk samples from dairy herds using an enzyme-linked immunosorbent assay (ELISA). In order to define the background reaction, milk samples from all lactating cows and serum samples from 9 animals were collected in each of 20 salmonellosis-free herds located on the island of Bornholm, where cattle salmonellosis has not been reported. Similar samples were collected from all stalled animals in 10 herds with recent (< 6 months) outbreaks of salmonellosis located in Jutland, where salmonella infection is enzootic. Using herd history of salmonellosis, herd location and clinical status of the herds as criteria, the optimal cutoff in the milk ELISA was determined as being at least 5% of the samples having optical density > 0.5, resulting in herd sensitivity of 1.0 and herd specificity of 0.95. While none of the sera in the herds from Bornholm was ELISA positive, 2 herds had a few reactors in the milk ELISA. Using the same cutoff, all but 1 bulk milk sample from 150 herds on Bornholm was ELISA-negative, and all 10 salmonellosis-positive herds from Jutland were ELISA-positive. A significant correlation was found between ELISA reactions in milk and in serum of cows (34% and 32% respectively, rs = 0.69, P < 0.001).(ABSTRACT TRUNCATED AT 250 WORDS) PMID:7648527

  1. Aggregated silver nanoparticles based surface-enhanced Raman scattering enzyme-linked immunosorbent assay for ultrasensitive detection of protein biomarkers and small molecules.

    PubMed

    Liang, Jiajie; Liu, Hongwu; Huang, Caihong; Yao, Cuize; Fu, Qiangqiang; Li, Xiuqing; Cao, Donglin; Luo, Zhi; Tang, Yong

    2015-06-01

    Lowering the detection limit is critical to the design of bioassays required for medical diagnostics, environmental monitoring, and food safety regulations. The current sensitivity of standard color-based analyte detection limits the further use of enzyme-linked immunosorbent assays (ELISAs) in research and clinical diagnoses. Here, we demonstrate a novel method that uses the Raman signal as the signal-generating system of an ELISA and combines surface-enhanced Raman scattering (SERS) with silver nanoparticles aggregation for ultrasensitive analyte detection. The enzyme label of the ELISA controls the dissolution of Raman reporter-labeled silver nanoparticles through hydrogen peroxide and generates a strong Raman signal when the analyte is present. Using this assay, prostate-specific antigen (PSA) and the adrenal stimulant ractopamine (Rac) were detected in whole serum and urine at the ultralow concentrations of 10(-9) and 10(-6) ng/mL, respectively. The methodology proposed here could potentially be applied to other molecules detection as well as PSA and Rac. PMID:25928837

  2. Rapid Determination of Ractopamine Residues in Edible Animal Products by Enzyme-Linked Immunosorbent Assay: Development and Investigation of Matrix Effects

    PubMed Central

    Zhang, Yan; Wang, FengXia; Fang, Li; Wang, Shuo; Fang, GuoZhen

    2009-01-01

    To determine ractopamine residues in animal food products (chicken muscle, pettitoes, pig muscle, and pig liver), we established a rapid direct competitive enzyme-linked immunosorbent assay (ELISA) using a polyclonal antibody generated from ractopamine-linker-BSA. The antibody showed high sensitivity and specificity in phosphate buffer, with an IC50 of 0.6 ng/mL, and the limit of detection was 0.04 ng/mL. The matrix effect of the samples was easily eliminated by one-step extraction with PBS, without any organic solution or clean-up procedure such as SPE or liquid-liquid extraction, making it a much more simple and rapid method than previously reported ones. The detection limit in blank samples was 0.2 μg/kg. To validate this new RAC (ractopamine hydrochloride) ELISA, a RAC-free pig liver sample spiked at three different concentrations was prepared and analyzed by HPLC and ELISA. The results showed a good correlation between the data of ELISA and HPLC (R2 > 0.95), which proves that the established ELISA is accurate enough to quantify the residue of RAC in the animal derived foods. PMID:19826637

  3. Avidity of the Immunoglobulin G Response to a Neisseria meningitidis Group C Polysaccharide Conjugate Vaccine as Measured by Inhibition and Chaotropic Enzyme-Linked Immunosorbent Assays▿

    PubMed Central

    Harris, Shannon L.; Tsao, How; Ashton, Lindsey; Goldblatt, David; Fernsten, Philip

    2007-01-01

    Antibody avidity, the strength of the multivalent interaction between antibodies and their antigens, is an important characteristic of protective immune responses. We have developed an inhibition enzyme-linked immunosorbent assay (ELISA) to measure antibody avidity for the capsular polysaccharide (PS) of Neisseria meningitidis group C (MnC) and determined the avidity constants (KDs) for 100 sera from children immunized with an MnC PS conjugate vaccine. The avidity constants were compared to the avidity indices (AI) obtained for the same sera using a chaotropic ELISA protocol. After the primary immunization series, the geometric mean (GM) KD was 674 nM and did not change in the months following immunization. However, the GM avidity did increase after the booster dose (GM KD, 414 nM 1 month after booster immunization). In contrast, the GM AI increased from an initial value of 118 after the primary immunization series to 147 6 months after the completion of the primary immunization series and then further increased to 178 after booster immunization. At the individual subject level, the avidity constant and AI correlated after the primary immunization series and after booster immunization but not prior to boosting. This work suggests that the AI, as measured by the chaotropic ELISA, in contrast to the KD, reflects changes that render antibody populations less susceptible to disruption by chaotropic agents without directly affecting the strength of the binding interactions. PMID:17287312

  4. Characterization of monoclonal antibodies to human group B rotavirus and their use in an antigen detection enzyme-linked immunosorbent assay.

    PubMed Central

    Burns, J W; Welch, S K; Nakata, S; Estes, M K

    1989-01-01

    Three monoclonal antibodies (MAbs)--B5C9, B5E4, and B10G10--to human group B rotavirus, an agent implicated in epidemic outbreaks of diarrhea in the People's Republic of China, primarily in adults, were prepared. MAb reactivity was decreased when virus preparations were treated with EDTA, suggesting reactivity with the outer-capsid protein(s). Competition experiments suggested that these MAbs recognize overlapping epitopes within a single antigenic site. A simple antigen detection enzyme-linked immunosorbent assay (ELISA) specific for the human group B rotavirus was established by using these MAbs as capture antibodies. Fifteen clinical samples obtained from three epidemic areas in the People's Republic of China and previously shown by Chinese scientists to contain group B virus were all positive in the MAb capture antigen detection ELISA, whereas none of the 57 samples lacking the group B virus reacted in the test. The results suggest that this MAb capture antigen detection ELISA will be useful to identify outbreaks caused by the human group B rotavirus and to monitor possible spread of the virus. Images PMID:2536755

  5. Enzyme-linked immunosorbent assay with worm vomit and cercarial secretions of Schistosoma mansoni to detect infections in an endemic focus of Burkina Faso.

    PubMed

    Bahgat, M; Sorgho, H; Ouédraogo, J B; Poda, J N; Sawadogo, L; Ruppel, A

    2006-03-01

    Cercariae and adult Schistosoma mansoni were used to prepare, respectively, cercarial secretions (CS) and worm vomit (WoV). These were used as antigens in an enzyme-linked immunosorbent assay (ELISA) to test the IgG-reactivity of sera obtained in an S. mansoni-endemic area of Burkina Faso. Among the egg-excreting individuals (n = 240), 94.6% reacted positively with WoV, but only 62.9% with CS, thus suggesting a high diagnostic sensitivity of WoV, but not of CS. Among those individuals without detectable eggs in two Kato-Katz thick smears from different stool specimens (n = 215), the respective percentages of positive IgG reactivity were 78.1% and 63.3%. These positive reactions in the absence of detectable eggs are interpreted in terms of limited sensitivity of parasitological stool examinations. Optical density values in ELISA with CS, but not with WoV, correlated negatively with age, which may reflect decreasing exposure to cercariae in older individuals. PMID:16469168

  6. Cross-reactivity of antibodies with phenolic compounds in pistachios during quantification of ochratoxin A by commercial enzyme-linked immunosorbent assay kits.

    PubMed

    Lee, Hyun Jung; Meldrum, Alexander D; Rivera, Nicholas; Ryu, Dojin

    2014-10-01

    Ochratoxin A (OTA), a nephrotoxic mycotoxin, naturally occurs in wide range of agricultural commodities. Typical screening of OTA involves various enzyme-linked immunosorbent assay (ELISA) methods. Pistachio (Pistacia vera L.) is a rich source of phenolic compounds that may result in a false positive due to structural similarities to OTA. The present study investigated the cross-reactivity profiles of phenolic compounds using two commercial ELISA test kits. High-performance liquid chromatography was used to confirm the concentration of OTA in the pistachio samples and compared with the results obtained from ELISA. When the degree of interaction and 50 % inhibitory concentration of phenolic compounds were determined, the cross-reactivity showed a pattern similar to that observed with the commercial ELSIA kits, although quantitatively different. In addition, the degree of interaction increased with the increasing concentration of phenolic compounds. The ELISA value had stronger correlations with the content of total phenolic compound, gallic acid, and catechin (R(2) = 0.757, 0.732, and 0.729, respectively) compared with epicatechin (R(2) = 0.590). These results suggest that phenolic compounds in pistachio skins may cross-react with the OTA antibody and lead to a false positive or to an overestimation of OTA concentration in ELISA-based tests. PMID:25285493

  7. Simple Identification of Human Taenia Species by Multiplex Loop-Mediated Isothermal Amplification in Combination with Dot Enzyme-Linked Immunosorbent Assay.

    PubMed

    Nkouawa, Agathe; Sako, Yasuhito; Okamoto, Munehiro; Ito, Akira

    2016-06-01

    For differential detection of Taenia solium, Taenia saginata, and Taenia asiatica, loop-mediated isothermal amplification (LAMP) assay targeting the cytochrome c oxidase subunit 1 gene has been recently developed and shown to be sensitive, specific, and effective. However, to achieve differential identification, one specimen requires three reaction mixtures containing a primer set of each Taenia species separately, which is complex and time consuming and increases the risk of cross-contamination. In this study, we developed a simple differential identification of human Taenia species using multiplex LAMP (mLAMP) in combination with dot enzyme-linked immunosorbent assay (dot-ELISA). Forward inner primers of T. solium, T. saginata, and T. asiatica labeled with fluorescein isothiocyanate (FITC), digoxigenin (DIG), and tetramethylrhodamine (TAMRA), respectively, and biotin-labeled backward inner primers were used in mLAMP. The mLAMP assay succeeded in specific amplification of each respective target gene in a single tube. Furthermore, the mLAMP product from each species was easily distinguished by dot-ELISA with an antibody specific for FITC, DIG, or TAMRA. The mLAMP assay in combination with dot-ELISA will make identification of human Taenia species simpler, easier, and more practical. PMID:27044566

  8. Development and Application of a Double-Antigen Sandwich Enzyme-Linked Immunosorbent Assay for Detection of Antibodies to Porcine Circovirus 2

    PubMed Central

    Ge, Meng; Luo, Wei; Jiang, Daliang; Li, Runcheng; Zhao, Wenwei; Chen, Guoliang; Yang, Xingdong

    2012-01-01

    A double-antigen sandwich enzyme-linked immunosorbent assay (ELISA) is described for detection of porcine circovirus 2 (PCV2) antibodies using the well-characterized recombinant PCV2 capsid protein. In a comparative test of 394 pig sera against an indirect immunofluorescence (IIF) test and a commercial ELISA kit (also based on the recombinant PCV2 capsid protein), the results showed that the diagnostic sensitivity, specificity, and accuracy of the assay were, respectively, 90.61, 94.02, and 91.62% compared with IIF and 94.38, 95.28, and 94.67% compared with the commercial ELISA kit. Assay of 12 PCV-free pigs over a 5-week period produced only PCV2-negative titers by all 3 methods. These results and the seroprofiles of 4 pig farms obtained by both the commercial ELISA kit and the double-antigen sandwich ELISA indicate that the sandwich ELISA is a reliable method for detection of antibodies to PCV2. Additionally, the method described here permits the use of undiluted test serum samples simultaneously loaded with horseradish peroxidase (HRP)-conjugated antigen into the test well, and the complete test procedure can be performed in less than 90 min. This double-antigen sandwich ELISA should be a useful tool to aid swine industry professionals in deciding the intervention strategies for the control of PCV2-associated diseases. PMID:22815145

  9. Antigen-capture blocking enzyme-linked immunosorbent assay based on a baculovirus recombinant antigen to differentiate Transmissible gastroenteritis virus from Porcine respiratory coronavirus antibodies.

    PubMed

    López, Lissett; Venteo, Angel; García, Marga; Camuñas, Ana; Ranz, Ana; García, Julia; Sarraseca, Javier; Anaya, Carmen; Rueda, Paloma

    2009-09-01

    A new commercially available antigen-capture, blocking enzyme-linked immunosorbent assay (antigen-capture b-ELISA), based on baculovirus truncated-S recombinant protein of Transmissible gastroenteritis virus (TGEV) and 3 specific monoclonal antibodies, was developed and evaluated by examining a panel of 453 positive Porcine respiratory coronavirus (PRCoV), 31 positive TGEV, and 126 negative field sera by using another commercially available differential coronavirus b-ELISA as the reference technique to differentiate TGEV- from PRCoV-induced antibodies. The recombinant S protein-based ELISA appeared to be 100% sensitive for TGEV and PRCoV detection and highly specific for TGEV and PRCoV detection (100% and 92.06%, respectively), when qualitative results (positive or negative) were compared with those of the reference technique. In variability experiments, the ELISA gave consistent results when the same serum was evaluated on different wells and different plates. These results indicated that truncated recombinant S protein is a suitable alternative to the complete virus as antigen in ELISA assays. The use of recombinant S protein as antigen offers great advantages because it is an easy-to-produce, easy-to-standardize, noninfectious antigen that does not require further purification or concentration. Those advantages represent an important improvement for antigen preparation, in comparison with other assays in which an inactivated virus from mammalian cell cultures is used. PMID:19737754

  10. Preparation and application of monoclonal antibodies for a sandwich enzyme-linked immunosorbent assay of the major soybean allergen, Gly m Bd 30K.

    PubMed

    Tsuji, H; Bando, N; Kimoto, M; Okada, N; Ogawa, T

    1993-08-01

    In order to obtain probes suitable for the determination of the major soybean allergen, Gly m Bd 30 K, in soybean-related processed foods, we have prepared two monoclonal antibodies, F5 of IgG2a and H6 of IgM, by the fusion of P3U1 myeloma cells with the spleen cells of BALB/c mice immunized with the reductively carboxymethylated allergen (RCM-allergen). The two monoclonal antibodies were shown to be specific to the intact allergen as well as the RCM-allergen and to recognize distinct epitopes on the allergen. Of the monoclonal antibodies, F5 was conjugated with peroxidase. H6 and the labeled F5 were applied as the fixing antibody and the labeled first antibody, respectively, for a direct sandwich enzyme-linked immunosorbent assay of the allergen. When the allergen was extracted from the soybean-related foods with Tris-HCl buffer containing sodium dodecylsulfate and mercaptoethanol, the allergen, Gly m Bd 30 K, was shown to be measured in a range of 5-500 ng in this assay. PMID:7506767

  11. Use of Peptide-Based Enzyme-Linked Immunosorbent Assay followed by Immunofluorescence Assay To Document Ehrlichia chaffeensis as a Cause of Febrile Illness in Nicaragua.

    PubMed

    Chikeka, Ijeuru; Matute, Armando J; Dumler, J Stephen; Woods, Christopher W; Mayorga, Orlando; Reller, Megan E

    2016-06-01

    Ehrlichia chaffeensis, the etiologic agent of human monocytic ehrlichiosis (HME), has been extensively studied as a cause of acute febrile illness and an emerging tick-borne zoonosis in the United States. Limited data suggest its presence in other regions, including Central and South America but not Nicaragua to date. Diagnosis of E. chaffeensis infection by indirect immunofluorescence assay (IFA) is the reference standard due to its presumed high sensitivity and specificity, but IFA is impractical, variably reproducible, and cumbersome for large epidemiologic studies and for clinical diagnosis in resource-poor regions. We evaluated a high-throughput, objective peptide-based enzyme-linked immunosorbent assay (ELISA) for use alone or in combination with IFA. We found that it performed best as a screening test (sensitivity, 100%; specificity, 84%) to reduce the proportion of serum samples that were required by the more cumbersome and subjective IFA testing to <20%. Using a two-step diagnostic approach (IFA is performed if the ELISA is positive), we identified E. chaffeensis or a serologically and antigenically similar organism as a heretofore unrecognized cause of acute febrile illness in humans in Nicaragua and demonstrated the utility of the peptide ELISA as a screening tool for large-scale clinical studies. PMID:27053675

  12. Diagnostic accuracy of an IgM enzyme-linked immunosorbent assay and comparison with 2 polymerase chain reactions for early diagnosis of human leptospirosis.

    PubMed

    Vanasco, N B; Jacob, P; Landolt, N; Chiani, Y; Schmeling, M F; Cudos, C; Tarabla, H; Lottersberger, J

    2016-04-01

    Enzyme-linked immunosorbent assay (ELISA) tests and polymerase chain reaction (PCR) may play a key role for early detection and treatment of human leptospirosis in developing countries. The aims of this study were to develop and validate an IgM ELISA under field conditions and to compare the diagnostic accuracy among IgG, IgM ELISAs, conventional PCR (cPCR), and real-time PCR (rtPCR) for early detection of human leptospirosis. Overall accuracy of IgM ELISA was sensitivity of 87.9%, specificity of 97.0%, and area under the curve of 0.940. When the 4 methods were compared, IgM ELISA showed the greatest diagnostic accuracy (J=0.6) followed by rtPCR (J=0.4), cPCR (J=0.2) and IgG ELISA (J=0.1). Our results support the use of IgM ELISA and rtPCR for early diagnosis of the disease. Moreover, due to their high specificity, they could be also useful to replace or supplement microscopic agglutination test as a confirmatory test, allowing more confirmations. PMID:26867967

  13. Evaluation and Validation of the Detection of soluble Triggering Receptor Expressed on Myeloid Cells 1 by Enzyme-linked immunosorbent Assay

    PubMed Central

    Hasibeder, Astrid; Stein, Pamela; Brandwijk, Ricardo; Schild, Hansjörg; Radsak, Markus P.

    2015-01-01

    Triggering receptor expressed on myeloid cells (TREM)-1 plays an important role in innate immune responses and is upregulated under infectious as well as non-infectious conditions. In addition, a soluble TREM-1 variant (sTREM-1) is detectable in sera or bronchoalveolar-lavage fluids from patients. Currently, various studies are difficult to compare, since the methods of detection by enzyme-linked immunosorbent assays (ELISA) vary among different research groups. In this study, we compared three different s-TREM-1 specific ELISAs and identified individual assay characteristics finding notable differences in sTREM-1 concentrations in part depending on the employed buffers. Investigating potential confounding factors for sTREM-1 detection, serum heat-inactivation (HI) showed improved recovery compared to non-HI (NHI) serum, reproducible by addition of complement and re-heat-inactivation. Hence we identified complement as a heat-sensitive confounder in some sTREM-1 ELISAs. We conclude that it is difficult to directly compare data of several studies, in particular if different ELISAs are engaged. Immunoassays for research use only are in general hampered by lack of standardization. Further standardization is needed until sTREM-1 ELISA is capable for better reproducibility of studies and clinical application. PMID:26480887

  14. Evaluation of enzyme-linked immunosorbent assays performed on milk and serum samples for detection of neosporosis and leukosis in lactating dairy cows

    PubMed Central

    Walsh, Robert B.; Kelton, David F.; Hietala, Sharon K.; Duffield, Todd F.

    2013-01-01

    Serum and milk samples from 1229 cows on 22 Ontario dairy farms were individually tested for antibodies specific for bovine leukosis virus (BLV) and Neospora caninum by enzyme-linked immunosorbent assay (ELISA). Antibodies against BLV were present in 361 serum samples (29.4%) and 369 milk samples (30.0%). Comparing the 2 tests, agreement was almost perfect (k = 0.86; 95% CI = 0.83 to 0.90) and the proportions of samples positive were not significantly different (P = 0.56). Both tests identified the same 3 herds free of bovine leukosis virus. Antibodies against N. caninum were detected in 138 serum samples (11.2%), and 111 milk samples (9.0%). Agreement between the 2 tests was moderate (k = 0.52; 95% CI = 0.43 to 0.59). Four herds were free of neosporosis by the serum test, while 10 herds were negative by the milk test. The ELISA on milk samples facilitates sample collection to classify herds free of BLV; the milk N. caninum ELISA was less reliable in predicting herd-level infection. PMID:24082160

  15. Development of an enzyme-linked immunosorbent assay system for detecting β'-component (Onk k 5), a major IgE-binding protein in salmon roe.

    PubMed

    Shimizu, Yutaka; Oda, Hiroshi; Seiki, Kohsuke; Saeki, Hiroki

    2015-08-15

    A novel enzyme-linked immunosorbent assay (ELISA) system has been established for selective detection of chum salmon (Oncorhynchus keta) yolk protein (SYP). Rabbit and rat polyclonal Immunoglobulin G antibodies to β'-component (the major allergic protein in fish roe; anti-β) were applied for designing the ELISA system. The sandwich ELISA using rabbit anti-β for the capture antibody and horseradish peroxidase-labeled F(ab')2 fragment of rat anti-β for the detection antibody obtained high sensitivity and narrow specificity for SYP. Protein extraction using sodium dodecyl sulfate and 2-mercaptoethanol ensured strict specificity of the ELISA, and components of three popular processed foods had no effect on the ELISA response. The limits of determination and quantification of SYP were estimated to be 0.78 μg/g and 2.60 μg/g of food sample, respectively. In conclusion, the developed ELISA system has a probability to be applied for the detection of contaminated chum salmon roe in processed food. PMID:25794755

  16. Determination of the folate content in cladodes of nopal (Opuntia ficus indica) by microbiological assay utilizing Lactobacillus casei (ATCC 7469) and enzyme-linked immunosorbent assay.

    PubMed

    Ortiz-Escobar, Tania Breshkovskaya; Valverde-González, Maria Elena; Paredes-López, Octavio

    2010-05-26

    Prickly pear cactus has been an important food source in Mexico since ancient times due to its economical and ecological benefits and potential nutraceutical value. Nevertheless, studies on the nutritional aspects and health benefits have been scarce. The purpose of this study was to assess, apparently for the first time, the folate contents of cladodes of nopal by a microbiological assay, using Lactobacillus casei (ATCC 7469) in extracts that were enzymatically treated to release the bound vitamin, employing single, dual, and trienzymatic procedures, and using the enzyme-linked immunosorbent assay (ELISA). We used Opuntia cladodes of different length sizes. The microbiological assay showed some differences among enzyme treatments and sizes of nopal; the trienzyme treatment (alpha-amylase-protease-conjugase) was more efficient in determining the folate content in nopal, giving 5.0 ng/g in the small size cladodes at 54 h of testing time, while ELISA showed no significant differences in the folate content among sizes of cladodes (5.5-5.62 ng/g at 0 min testing time). Both techniques may be used for the assessment of folate content in cladodes, but ELISA is more rapid and reliable. PMID:20441169

  17. Application of 3D Printing Technology in Increasing the Diagnostic Performance of Enzyme-Linked Immunosorbent Assay (ELISA) for Infectious Diseases

    PubMed Central

    Singh, Harpal; Shimojima, Masayuki; Shiratori, Tomomi; An, Le Van; Sugamata, Masami; Yang, Ming

    2015-01-01

    Enzyme-linked Immunosorbent Assay (ELISA)-based diagnosis is the mainstay for measuring antibody response in infectious diseases and to support pathogen identification of potential use in infectious disease outbreaks and clinical care of individual patients. The development of laboratory diagnostics using readily available 3D printing technologies provides a timely opportunity for further expansion of this technology into immunodetection systems. Utilizing available 3D printing platforms, a ‘3D well’ was designed and developed to have an increased surface area compared to those of 96-well plates. The ease and rapidity of the development of the 3D well prototype provided an opportunity for its rapid validation through the diagnostic performance of ELISA in infectious disease without modifying current laboratory practices for ELISA. The improved sensitivity of the 3D well of up to 2.25-fold higher compared to the 96-well ELISA provides a potential for the expansion of this technology towards miniaturization and Lab-On-a-Chip platforms to reduce time, volume of reagents and samples needed for such assays in the laboratory diagnosis of infectious and other diseases including applications in other disciplines. PMID:26184194

  18. Increased sensitivity of 3D-Well enzyme-linked immunosorbent assay (ELISA) for infectious disease detection using 3D-printing fabrication technology.

    PubMed

    Singh, Harpal; Shimojima, Masayuki; Fukushi, Shuetsu; Le Van, An; Sugamata, Masami; Yang, Ming

    2015-01-01

    Enzyme-linked Immunosorbent Assay or ELISA -based diagnostics are considered the gold standard in the demonstration of various immunological reaction including in the measurement of antibody response to infectious diseases and to support pathogen identification with application potential in infectious disease outbreaks and individual patients' treatment and clinical care. The rapid prototyping of ELISA-based diagnostics using available 3D printing technologies provides an opportunity for a further exploration of this platform into immunodetection systems. In this study, a '3D-Well' was designed and fabricated using available 3D printing platforms to have an increased surface area of more than 4 times for protein-surface adsorption compared to those of 96-well plates. The ease and rapidity in designing-product development-feedback cycle offered through 3D printing platforms provided an opportunity for its rapid assessment, in which a chemical etching process was used to make the surface hydrophilic followed by validation through the diagnostic performance of ELISA for infectious disease without modifying current laboratory practices for ELISA. The higher sensitivity of the 3D-Well (3-folds higher) compared to the 96-well ELISA provides a potential for the expansion of this technology towards miniaturization platforms to reduce time, volume of reagents and samples needed for laboratory or field diagnosis of infectious diseases including applications in other disciplines. PMID:26406036

  19. Commercial enzyme-linked immunosorbent assay versus polymerase chain reaction for the diagnosis of chronic Chagas disease: a systematic review and meta-analysis

    PubMed Central

    do Brasil, Pedro Emmanuel Alvarenga Americano; Castro, Rodolfo; de Castro, Liane

    2016-01-01

    Chronic Chagas disease diagnosis relies on laboratory tests due to its clinical characteristics. The aim of this research was to review commercial enzyme-linked immunosorbent assay (ELISA) and polymerase chain reaction (PCR) diagnostic test performance. Performance of commercial ELISA or PCR for the diagnosis of chronic Chagas disease were systematically searched in PubMed, Scopus, Embase, ISI Web, and LILACS through the bibliography from 1980-2014 and by contact with the manufacturers. The risk of bias was assessed with QUADAS-2. Heterogeneity was estimated with the I2 statistic. Accuracies provided by the manufacturers usually overestimate the accuracy provided by academia. The risk of bias is high in most tests and in most QUADAS dimensions. Heterogeneity is high in either sensitivity, specificity, or both. The evidence regarding commercial ELISA and ELISA-rec sensitivity and specificity indicates that there is overestimation. The current recommendation to use two simultaneous serological tests can be supported by the risk of bias analysis and the amount of heterogeneity but not by the observed accuracies. The usefulness of PCR tests are debatable and health care providers should not order them on a routine basis. PCR may be used in selected cases due to its potential to detect seronegative subjects. PMID:26814640

  20. Determination of ochratoxin A in food: comparison of a stable isotope dilution assay, liquid chromatography-fluorescence detection and an enzyme-linked immunosorbent assay.

    PubMed

    Lindenmeier, Michael; Schieberle, Peter; Rychlik, Michael

    2011-05-01

    Quantitative results for the mycotoxin ochratoxin A (OTA), obtained by a stable isotope dilution assay (SIDA) were compared with two commonly used analytical methods for OTA quantitation. For this, different types of food, such as wheat, coffee, sultanas, and blood sausages, were analyzed. Because results obtained by the SIDA method were closest to the certified contents of an OTA reference material, data obtained by this method were considered as reference data. For liquid chromatography-fluorescence detection, a clean-up by solid phase extraction on silica was found to be necessary, and a correction for recovery had to be performed to match the data from the SIDA experiments. The enzyme-linked immunosorbent assay (ELISA) strongly overestimated the OTA content in coffee and nutmeg therefore an extract clean-up by immunoaffinity chromatography had to be used to match the SIDA results. Following this sample preparation, ELISA gave correct qualitative and semiquantitative results, and proved to be a suitable screening method. SIDA was also established as a valuable tool to quantify OTA in meat products, when using a clean-up procedure developed recently for blood samples. PMID:23605702

  1. Preparation of antibodies and development of an enzyme-linked immunosorbent assay (ELISA) for the determination of doxycycline antibiotic in milk samples.

    PubMed

    Adrian, Javier; Fernández, Fátima; Sánchez-Baeza, Francisco; Marco, M-Pilar

    2012-04-18

    This paper reports the development of an immunoassay for the specific analysis of doxycycline (DC), a congener of the tetracycline antibiotic family (TCs), in milk samples. This is the first time that DC antibody production is reported, based on a rationally designed and well-characterized immunizing hapten. The chemical structure of the immunizing hapten (13-[(2-carboxyethyl)thiol]-5-hydroxy-6-α-deoxytetracycline, TC1) was designed to maximize recognition of the tetracycline characteristic moiety defined as lower periphery of the TCs plus the region of the upper periphery composed by the hydroxyl group at position C(5) (B ring) and the dimethylamino group in ring A. Polyclonal antibodies raised against TC1 coupled to horseshoe crab hemocianyn (HCH) were used to develop a homologous indirect competitive enzyme-linked immunosorbent assay (ELISA). The microplate ELISA can detect DC in buffer down to 0.1 μg L(-1). The ELISA has been proven to tolerate a wide range of ionic strengths and pH values. The assay is very selective for DC with a minor recognition of methacycline (32% of cross-reactivity). Experiments performed with whole milk samples demonstrate that samples can be directly analyzed after a simple treatment method, reaching detectability values below 5 μg L(-1). PMID:22486559

  2. Development of a monoclonal antibody against domoic acid and its application in enzyme-linked immunosorbent assay and colloidal gold immunostrip.

    PubMed

    Tsao, Zih-Jay; Liao, Yi-Chun; Liu, Biing-Hui; Su, Ching-Chyuan; Yu, Feng-Yih

    2007-06-27

    A monoclonal antibody (mAb) specific to domoic acid was produced from a stable hybridoma cell line, 9F1F11, generated by the fusion of P3/NS1/1-AG4-1 myeloma cells with spleen cells isolated from a Balb/c mouse immunized with domoic acid--keyhole limpet hemocyanin. The 9F1F11 mAb belongs to the immunoglobulin G1 (kappa-chain) isotype. A competitive direct enzyme-linked immunosorbent assay (cdELISA) and a competitive indirect ELISA were established for antibody characterization. In the cdELISA, the concentration causing 50% inhibition (IC50) of binding of domoic acid-horseradish peroxidase to the antibody by domoic acid was found to be 0.58 ng/mL. A sensitive and rapid mAb-based colloidal gold immunostrip was also developed. The immunostrip assay, which has a detection limit of 5 ng/mL for domoic acid, can be completed in 10 min. Analysis of domoic acid in blue mussel samples revealed that data obtained from immunostrip were in a good agreement with those obtained from cdELISA. The mAb-based cdELISA and immunostrip assay established in this study were sensitive and accurate for rapid screening of domoic acid in shellfish samples. PMID:17542614

  3. Dengue virus serotyping based on envelope and membrane and nonstructural protein NS1 serotype-specific capture immunoglobulin M enzyme-linked immunosorbent assays.

    PubMed

    Shu, Pei-Yun; Chen, Li-Kuang; Chang, Shu-Fen; Su, Chien-Ling; Chien, Li-Jung; Chin, Chuan; Lin, Ting-Hsiang; Huang, Jyh-Hsiung

    2004-06-01

    Envelope and membrane (E/M) and nonstructural protein NS1 serotype-specific capture Immunoglobulin M (IgM) enzyme-linked immunosorbent assays (ELISAs) were developed to differentiate four dengue virus serotypes. A total of 93 anti-dengue virus IgM-positive serum samples collected between days 5 and 45 of illness from 59 confirmed dengue patients were analyzed. The results showed that positive serotype specificity could be identified for 86.1 and 47.6% of serum samples tested for E/M-specific IgM antibodies versus 83.3 and 42.9% of serum samples tested for NS1-specific IgM antibodies from patients with primary and secondary dengue virus infections, respectively. Dual analyses with both E/M and NS1 serotype-specific capture IgM ELISAs showed that positive serotype specificity could be correctly identified for 98.6 and 61.9% of all of the primary and secondary serum samples tested, respectively. These findings suggested that E/M and NS1 serotype-specific capture IgM ELISAs have the potential to be of use in dengue virus serotyping. PMID:15184425

  4. Development of a monoclonal antibody against ochratoxin A and its application in enzyme-linked immunosorbent assay and gold nanoparticle immunochromatographic strip.

    PubMed

    Liu, Biing-Hui; Tsao, Zih-Jay; Wang, Jing-Jhih; Yu, Feng-Yih

    2008-09-15

    A monoclonal antibody (mAb) specific to ochratoxin A (OTA) was produced from a stable hybridoma cell line, 9C9H9, generated by the fusion of P3/NS1/1-AG4-1 myeloma cells with spleen cells isolated from a BALB/c mouse immunized with OTA-keyhole limpet hemocyanin. The 9C9H9 mAb belongs to the immunoglobulin G1 (kappa chain) isotype. A competitive direct enzyme-linked immunosorbent assay (cdELISA) and a competitive indirect ELISA were established for antibody characterization. The concentrations causing 50% inhibition of binding of OTA-horseradish peroxidase to the antibody by OTA, OTB, and OTC were found to be 0.32, 0.17, and 0.28 ng/mL, respectively, in the cdELISA. A sensitive and rapid mAb-based gold nanoparticle immunochromatographic strip was also developed using this mAb. This strip has a detection limit of 5 ng/mL for OTA and can be completed in 10 min. Analysis of OTA in coffee samples revealed that data obtained from immunochromatographic strip were in a good agreement with those obtained from cdELISA. The mAb-based cdELISA and immunochromatographic strip assay established in this study were sensitive and accurate for rapid screening of OTA in coffee samples. PMID:18698802

  5. Comparative studies of a real-time PCR method and three enzyme-linked immunosorbent assays for the detection of central nervous system tissues in meat products.

    PubMed

    Schönenbrücher, Holger; Göbel, Katrin Annette; Abdulmawjood, Amir; Richt, Jürgen A; Bülte, Michael

    2008-10-01

    The removal of certain central nervous system (CNS) tissues (part of the bovine spongiform encephalopathy risk material) from the food chain is one of the highest priority tasks associated with avoiding contamination of the human food chain with the agent of bovine spongiform encephalopathy. A recently developed real-time PCR assay and three commercially available enzyme-linked immunosorbent assays (ELISAs) for the detection of CNS tissues in minced meat and three types of heat-treated sausages were evaluated. Bovine brain was used for spiking of internal reference material, and its detectability was examined during storage times of 12 months (for frozen minced meat and liver sausage) and 24 months (for sausages treated with medium and high heat). The real-time PCR method and both ELISA kits detected 0.1% CNS tissue in frozen minced meat and 0.1 or 1% CNS tissue in heat-treated meat products. The detectability of the amplified mRNA target region with the PCR assay was similar to the detectability of antigen by the ELISAs. Because the real-time PCR method also can be used to distinguish cattle, ovine, and caprine CNS tissues from porcine CNS tissues, it seems to be suitable as a routine diagnostic test for the sensitive and specific detection of CNS tissues in meat and meat products. PMID:18939753

  6. A Colorimetric Enzyme-Linked Immunosorbent Assay (ELISA) Detection Platform for a Point-of-Care Dengue Detection System on a Lab-on-Compact-Disc.

    PubMed

    Thiha, Aung; Ibrahim, Fatimah

    2015-01-01

    The enzyme-linked Immunosorbent Assay (ELISA) is the gold standard clinical diagnostic tool for the detection and quantification of protein biomarkers. However, conventional ELISA tests have drawbacks in their requirement of time, expensive equipment and expertise for operation. Hence, for the purpose of rapid, high throughput screening and point-of-care diagnosis, researchers are miniaturizing sandwich ELISA procedures on Lab-on-a-Chip and Lab-on-Compact Disc (LOCD) platforms. This paper presents a novel integrated device to detect and interpret the ELISA test results on a LOCD platform. The system applies absorption spectrophotometry to measure the absorbance (optical density) of the sample using a monochromatic light source and optical sensor. The device performs automated analysis of the results and presents absorbance values and diagnostic test results via a graphical display or via Bluetooth to a smartphone platform which also acts as controller of the device. The efficacy of the device was evaluated by performing dengue antibody IgG ELISA on 64 hospitalized patients suspected of dengue. The results demonstrate high accuracy of the device, with 95% sensitivity and 100% specificity in detection when compared with gold standard commercial ELISA microplate readers. This sensor platform represents a significant step towards establishing ELISA as a rapid, inexpensive and automatic testing method for the purpose of point-of-care-testing (POCT) in resource-limited settings. PMID:25993517

  7. Comparison of the membrane-filtration fluorescent antibody test, the enzyme-linked immunosorbent assay, and the polymerase chain reaction to detect Renibacterium salmoninarum in salmonid ovarian fluid.

    PubMed

    Pascho, R J; Chase, D; McKibben, C L

    1998-01-01

    Ovarian fluid samples from naturally infected chinook salmon (Oncorhynchus tshawytscha) were examined for the presence of Renibacterium salmoninarum by the membrane-filtration fluorescent antibody test (MF-FAT), an antigen capture enzyme-linked immunosorbent assay (ELISA), and a nested polymerase chain reaction (PCR). On the basis of the MF-FAT, 64% (66/103) samples contained detectable levels of R. salmoninarum cells. Among the positive fish, the R. salmoninarum concentrations ranged from 25 cells/ml to 4.3 x 10(9) cells/ml. A soluble antigenic fraction of R. salmoninarum was detected in 39% of the fish (40/103) by the ELISA. The ELISA is considered one of the most sensitive detection methods for bacterial kidney disease in tissues, yet it did not detect R. salmoninarum antigen consistently at bacterial cell concentrations below about 1.3 x 10(4) cells/ml according to the MF-FAT counts. When total DNA was extracted and tested in a nested PCR designed to amplify a 320-base-pair region of the gene encoding a soluble 57-kD protein of R. salmoninarum, 100% of the 100 samples tested were positive. The results provided strong evidence that R. salmoninarum may be present in ovarian fluids thought to be free of the bacterium on the basis of standard diagnostic methods. PMID:9526862

  8. Development of a sensitive and specific indirect enzyme-linked immunosorbent assay based on a baculovirus recombinant antigen for detection of specific antibodies against Ehrlichia canis.

    PubMed

    López, Lissett; Venteo, Angel; Aguirre, Enara; García, Marga; Rodríguez, Majosé; Amusátegui, Inmaculada; Tesouro, Miguel A; Vela, Carmen; Sainz, Angel; Rueda, Paloma

    2007-11-01

    An indirect enzyme-linked immunosorbent assay (ELISA) based on baculovirus recombinant P30 protein of Ehrlichia canis and the 1BH4 anticanine IgG monoclonal antibody was developed and evaluated by examining a panel of 98 positive and 157 negative sera using the indirect fluorescent antibody (IFA) test as the reference technique. The P30-based ELISA appeared to be sensitive and specific (77.55% and 95.54%, respectively) when qualitative results (positive/negative) were compared with those of the IFA test; the coefficient of correlation (R) between the 2 tests was 0.833. Furthermore, it was possible to establish a mathematical formula for use in comparing the results of both techniques. These results indicate that recombinant P30 antigen-based ELISA is a suitable alternative of the IFA test for simple, consistent, and rapid serodiagnosis of canine ehrlichiosis. Moreover, the use of this recombinant protein as antigen offers a great advantage for antigen preparation in comparison with other techniques in which the whole E. canis organism is used as antigen. PMID:17998551

  9. A novel double recognition enzyme-linked immunosorbent assay based on the nucleocapsid protein for early detection of European porcine reproductive and respiratory syndrome virus infection.

    PubMed

    Venteo, A; Rebollo, B; Sarraseca, J; Rodriguez, M J; Sanz, A

    2012-04-01

    Precise and rapid detection of porcine reproductive respiratory syndrome virus (PRRSV) infection in swine farms is critical. Improvement of control procedures, such as testing incoming gilt and surveillance of seronegative herds requires more rapid and sensitive methods. However, standard serological techniques detect mainly IgG antibodies. A double recognition enzyme-linked immunosorbent assay (DR-ELISA) was developed for detection of antibodies specific to European PRRSV. This new assay can recognize both IgM and IgG antibodies to PRSSV which might be useful for detecting in routine surveillance assays pigs that are in the very early stages of infection and missed by conventional assays detecting only IgG antibodies. DR-ELISA is based on the double recognition of antigen by antibody. In this study, the recombinant nucleocapsid protein (N) of PRRSV was used both as the coating and the enzyme-conjugated antigen. To evaluate the sensitivity of the assay at early stages of the infection, sera from 69 pigs infected with PRRSV were collected during successive days post infection (pi) and tested. While standard methods showed low sensitivity rates before day 14 pi, DR-ELISA detected 88.4% seropositive samples at day 7 showing greater sensitivity at early stages of the infection. Further studies were carried out to assess the efficiency of the new assay, and the results showed DR-ELISA to be a sensitive and accurate method for early diagnosis of EU-PRRSV infection. PMID:22342444

  10. Indirect enzyme-antibody sandwich enzyme-linked immunosorbent assay for quantification of TAXI and XIP type xylanase inhibitors in wheat and other cereals.

    PubMed

    Beaugrand, Johnny; Gebruers, Kurt; Ververken, Cedric; Fierens, Ellen; Dornez, Emmie; Goddeeris, Bruno M; Delcour, Jan A; Courtin, Christophe M

    2007-09-19

    To quantify Triticum aestivum xylanase inhibitor (TAXI) and xylanase inhibiting protein (XIP) type proteins in cereals in general and wheat ( T. aestivum) in particular, a robust enzyme-linked immunosorbent assay (ELISA) using an uncommon enzyme-antibody sandwich format was developed. Bacillus subtilis glycoside hydrolase family (GH) 11 and Aspergillus oryzae GH 10 xylanases were selected for coating ELISA plate wells to capture TAXI and XIP, respectively, prior to probing with antibodies. The detection threshold of the developed ELISA was much lower than that of the currently used xylanase inhibitor assay and the recently described Western blot approach. Because of its broad dynamic range (TAXI, 30-600 ng/mL, and XIP, 3-60 ng/mL), one proper standard extract dilution can be used for analyzing different wheat varieties, whereas for the currently used colorimetric assay, often different dilutions need to be analyzed. The TAXI ELISA for wheat was successfully adapted for barley ( Hordeum vulgare) and could also be used for other cereals. PMID:17715986

  11. An Application of Outer Membrane Protein P6-Specific Enzyme-Linked Immunosorbent Assay for Detection of Haemophilus influenzae in Middle Ear Fluids and Nasopharyngeal Secretions

    PubMed Central

    Hotomi, Muneki; Togawa, Akihisa; Kono, Masamitsu; Sugita, Gen; Sugita, Rinya; Fujimaki, Yutaka; Kamide, Yosuke; Uchizono, Akihiro; Kanesada, Keiko; Sawada, Shoichi; Okitsu, Naohiro; Masuda, Hisayo; Tanaka, Hideaki; Tanaka, Yumi; Yamanaka, Noboru

    2013-01-01

    An enzyme-linked immunosorbent assay specific to outer membrane protein P6 (P6-ELISA) was applied for detecting Haemophilus influenzae in middle ear fluids (MEFs) from acute otitis media (AOM) patients and in nasopharyngeal secretions (NPSs) from acute rhinosinusitis patients. P6-ELISA had a sensitivity of 83.3% for MEFs and 71.5% for NPSs and a specificity of 85.6% for MEFs and 92.5% for NPSs, respectively. Real-time PCR exhibited significant differences in the number of ompP1 gene copies among samples determined by P6-ELISA to be positive and negative for H. influenzae. However, because the P6-ELISA test has the reactivity in Haemophilus species include two commensals H. haemolyticus and H. parainfluenzae, it is thus a weak method in order to detect only NTHi correctly. Consequently, diagnosis using the P6-ELISA should be based on an overall evaluation, including the results of other related examinations and clinical symptoms to prevent misleading conclusions in clinical setting. PMID:24015192

  12. Enzyme-linked immunosorbent assay and polymerase chain reaction performance using Mexican and Guatemalan discrete typing unit I strains of Trypanosoma cruzi.

    PubMed

    Ballinas-Verdugo, Martha; Reyes, Pedro Antonio; Mejia-Dominguez, Ana; López, Ruth; Matta, Vivian; Monteón, Victor M

    2011-12-01

    Thirteen Trypanosoma cruzi isolates from different geographic regions of Mexico and Guatemala belonging to discrete typing unit (DTU) I and a reference CL-Brener (DTU VI) strain were used to perform enzyme-linked immunosorbent assay (ELISA) and polymerase chain reaction (PCR). A panel of 57 Mexican serum samples of patients with chronic chagasic cardiopathy and asymptomatic infected subjects (blood bank donors) were used in this study. DNA from the above 14 T. cruzi strains were extracted and analyzed by PCR using different sets of primers designed from minicircle and satellite T. cruzi DNA. The chronic chagasic cardiopathy serum samples were easily recognized with ELISA regardless of the source of antigenic extract used, even with the CL-Brener TcVI, but positive serum samples from blood bank donors in some cases were not recognized by some Mexican antigenic extracts. On the other hand, PCR showed an excellent performance despite the set of primers used, since all Mexican and Guatemalan T. cruzi strains were correctly amplified. In general terms, Mexican, Guatemalan, and CL-Brener T. cruzi strains are equally good sources of antigen when using the ELISA test to detect Mexican serum samples. However, there are some strains with poor performance. The DTU I strains are easily detected using either kinetoplast or satellite DNA target designed from DTU VI strains. PMID:21867413

  13. A sensitive monoclonal antibody-based enzyme-linked immunosorbent assay for the detection of bifenthrin in a chemical soil barrier.

    PubMed

    Hua, Xiude; Liu, Xiaofeng; Yin, Wei; Xia, Yazhong; Zhou, Qiujun; Lu, Yiwen; Li, Wei; Shi, Haiyan; Liu, Fengquan; Wang, Minghua

    2015-01-01

    A sensitive monoclonal antibody-based enzyme monoclonal antibody-based enzyme-linked immunosorbent assay (ELISA) was developed to detect bifenthrin in a chemical soil barrier for termite control. LBc ((2-methyl[1,1-biphenyl]-3-methoxy) carbonyl propionic acid) was conjugated to bovine serum albumin (BSA) for producing monoclonal antibody. After optimization, the IC50 and limit of detection (LOD, IC10) were 0.05 mg L(-1) and 0.004 mg L(-1), respectively. The sensitivity was improved 40-fold compared to polyclonal antibody-based ELISA reported earlier. No cross-reactivity was measured for the other analogues such as cyhalothrin, cyhalothric acid, cypermethrin, deltamethrin, fenpropathrin, fenvalerate, ethofenprox and tetramethrin except 2-methyl-3-biphenylmethyanol. Spiked recoveries were between 83.5% and 104.7% for the detection of bifenthrin in loess, red soil and black soil. All the relative standard deviations (RSDs) were less than or equal to 15.0%. Moreover, the ELISA for authentic samples showed reliability and high correlation with gas chromatography. The developed ELISA is an alternative tool for simple, sensitive and accurate monitoring of bifenthrin in chemical soil barriers. PMID:25261814

  14. Development of cathepsin-L cysteine proteinase based Dot-enzyme-linked immunosorbent assay for the diagnosis of Fasciola gigantica infection in buffaloes.

    PubMed

    Varghese, Anju; Raina, O K; Nagar, Gaurav; Garg, Rajat; Banerjee, P S; Maharana, B R; Kollannur, Justin D

    2012-02-10

    Native cathepsin-L cysteine proteinase (28 kDa) was purified from the excretory secretory products of Fasciola gigantica and was used for sero-diagnosis of F. gigantica infection in buffaloes by Dot-enzyme-linked immunosorbent assay (Dot-ELISA). The test detected F. gigantica field infection in these animals with a sensitivity of ∼ 90%. No specific IgG antibody binding was displayed by sera obtained from 76 buffaloes considered to be Fasciola and other parasite-free by microscopic examination of faeces and necropsy examination of liver, rumen and intestine. Additionally, sera from 156 Fasciola-free buffaloes, yet infected with Gigantocotyle explanatum, Paramphistomum epiclitum, Gastrothylax spp., Strongyloides papillosus and hydatid cyst were all negative, indicating that F. gigantica cathepsin-L cysteine proteinase does not cross-react with these helminth parasites in natural infection of the host. The data indicated that cathepsin-L cysteine proteinase based Dot-ELISA reached ∼ 90% sensitivity and 100% specificity with relation to above parasites in the detection of bubaline fasciolosis. The present Dot-ELISA diagnostic assay is relevant to the field diagnosis of F. gigantica infection in buffaloes. PMID:22055612

  15. Comparison of Fecal Antigen Detection Using Enzyme Linked Immunosorbent Assay With the Auramine Phenol Staining Method for Diagnosis of Human Cryptosporidiosis

    PubMed Central

    Jafari, Rasool; Maghsood, Amir Hossein; Safari, Marzieh; Latifi, Milad; Fallah, Mohammad

    2015-01-01

    Background: Fecal antigen detection using the enzyme linked immunosorbent assay (ELISA) and oocyst detection using auramine phenol (AP) staining methods, are told to be more sensitive compared to other conventional methods, for diagnosis of cryptosporidiosis. Objectives: The aim of the present study was to evaluate the antigen-detection capacity in the stool specimens using ELISA and oocyst detection by AP staining methods, for the diagnosis of human cryptosporidiosis. Materials and Methods: A total of 228 fecal samples were collected from residents of rural areas of Hamadan, West of Iran. Each fecal sample was divided into two parts, one kept frozen at -20˚C for Ag-capture ELISA and the other in 10% formalin for the AP staining method. Cryptosporidium Ag-detection ELISA procedure was performed according to the manual of the manufacturer. The preserved samples concentrated using the formalin-ether concentration technique were stained with AP and then investigated under florescent microscopy. Results: Eight (3.5%) and three (1.3%) out of 228 fecal samples were positive for Cryptosporidium infection by ELISA and AP staining methods, respectively. Cryptosporidium Ag-detection using ELISA showed an increased frequency of the infection, compared to the AP staining method (P = 0.062). Conclusions: For epidemiological studies and diagnostic purposes of the Cryptosporidium infection, especially in asymptomatic individuals, Ag-detection ELISA is an easy to perform and accurate method, compared to other conventional microscopic methods. PMID:25825642

  16. Field estimation of the flock-level diagnostic specificity of an enzyme-linked immunosorbent assay for Avian metapneumovirus antibodies in turkeys.

    PubMed

    Muñoz-Zanzi, Claudia; Trampel, Darrell; Hanson, Tim; Harrison, Kristen; Goyal, Sagar; Cortinas, Roberto; Lauer, Dale

    2009-03-01

    Routine serologic testing for Avian metapneumovirus (AMPV) infection of turkey flocks at slaughter is currently being used to monitor changes in the occurrence of AMPV infection in endemic areas and can also be used to detect the emergence of infection in currently unaffected areas. Because of the costs associated with false-positive results, particularly in areas that are free of AMPV infection, there is a need to obtain improved estimates of flock-level specificity (SP). The objective of this study was to estimate flock-level SP of a program to monitor AMPV infection in turkey flocks at processing using a standard enzyme-linked immunosorbent assay (ELISA). A study was carried out in which 37 AMPV-free flocks from 7 Midwest operations were followed serologically. Six percent, 3%, and 0.2% of total samples tested AMPV positive at 8 weeks, 12 weeks, and at processing, respectively. Overall, flock-level SP increased as the cutoff increased and as age increased. Flock-level SP at processing was 97%, if a cutoff of 1 was used (the flock was classified as positive if at least 1 sample tested positive), and 100%, if any other cutoff was used. Administration of antibiotics (P = 0.02) and vaccination for Bordetella avium (P = 0.08) were positively associated with the probability of (false) positive test results. These findings suggest possible cross-reactions with other infections and highlight the need to consider variable diagnostic performance depending on farm conditions. PMID:19286505

  17. Development and Evaluation of an Enzyme-Linked Immunosorbent Assay for Use in the Detection of Bovine Tuberculosis in Cattle ▿ †

    PubMed Central

    Waters, W. R.; Buddle, B. M.; Vordermeier, H. M.; Gormley, E.; Palmer, M. V.; Thacker, T. C.; Bannantine, J. P.; Stabel, J. R.; Linscott, R.; Martel, E.; Milian, F.; Foshaug, W.; Lawrence, J. C.

    2011-01-01

    As a consequence of continued spillover of Mycobacterium bovis into cattle from wildlife reservoirs and increased globalization of cattle trade with associated transmission risks, new approaches such as vaccination and novel testing algorithms are seriously being considered by regulatory agencies for the control of bovine tuberculosis. Serologic tests offer opportunities for identification of M. bovis-infected animals not afforded by current diagnostic techniques. The present study describes assay development and field assessment of a new commercial enzyme-linked immunosorbent assay (ELISA) that detects antibody to M. bovis antigens MPB83 and MPB70 in infected cattle. Pertinent findings include the following: specific antibody responses were detected at ∼90 to 100 days after experimental M. bovis challenge, minimal cross-reactive responses were elicited by infection/sensitization with nontuberculous Mycobacterium spp., and the apparent sensitivity and specificity of the ELISA with naturally infected cattle were 63% and 98%, respectively, with sensitivity improving as disease severity increased. The ELISA also detected infected animals missed by the routine tuberculin skin test, and antibody was detectable in bulk tank milk samples from M. bovis-infected dairy herds. A high-throughput ELISA could be adapted as a movement, border, or slaughter surveillance test, as well as a supplemental test to tuberculin skin testing. PMID:21918115

  18. A polymerase chain reaction and enzyme linked immunosorbent assay based approach for diagnosis and differentiation between vaccinated and infected cattle with Mycobacterium bovis

    PubMed Central

    Sabry, Mohamed; Elkerdasy, Ahmed

    2014-01-01

    Background: In most African and Arabic countries tuberculosis (TB) causes great economic losses in bovine species and constitutes serious zoonotic problem. As the traditional diagnostic method delay the research because of low sensitivity and specificity, a rapid method of diagnosis is of outmost importance. Aim: The study was designed to evaluate the two rapid diagnostic methods of TB in cattle, further to differentiate between infected and bacillus Calmette-Guerin (BCG) vaccinated animals. Materials and Methods: Intradermal tuberculin test was applied to 300 cattle. Of these cattle, 15 cattle were vaccinated from cattle negative to tuberculin test with BCG. Blood samples were taken for lymphocyte separation to apply polymerase chain reaction (PCR) upon and for serum preparation for the enzyme-linked immunosorbent assay (ELISA) application, this blood collected from 65 cattle classified into three groups, viz. positive tuberculin test (35 animals), negative tuberculin test (15 animals), and vaccinated cow with BCG (15 animals). From blood samples lymphocytes were separated and the isolated lymphocytes were subjected to PCR and serum for ELISA application. Blood samples, specimens from lymph nodes and specific tissues were taken for PCR and for cultivation and isolation of Mycobacterium bovis. Results and Conclusions: The results of this study revealed that PCR can be used as rapid efficient and accurate diagnostic test in detection of ruminant TB. Moreover, cattle's ELISA reading showed higher sensitivity in positive tuberculin animals. However, the differentiations between vaccinated and infected animals not clear by using a single antigen only. PMID:24741280

  19. Comparison of Enzyme-Linked Immunosorbent Assay, Surface Plasmon Resonance and Biolayer Interferometry for Screening of Deoxynivalenol in Wheat and Wheat Dust.

    PubMed

    Sanders, Melanie; McPartlin, Daniel; Moran, Kara; Guo, Yirong; Eeckhout, Mia; O'Kennedy, Richard; De Saeger, Sarah; Maragos, Chris

    2016-04-01

    A sample preparation method was developed for the screening of deoxynivalenol (DON) in wheat and wheat dust. Extraction was carried out with water and was successful due to the polar character of DON. For detection, an enzyme-linked immunosorbent assay (ELISA) was compared to the sensor-based techniques of surface plasmon resonance (SPR) and biolayer interferometry (BLI) in terms of sensitivity, affinity and matrix effect. The matrix effects from wheat and wheat dust using SPR were too high to further use this screenings method. The preferred ELISA and BLI methods were validated according to the criteria established in Commission Regulation 519/2014/EC and Commission Decision 2002/657/EC. A small survey was executed on 16 wheat lots and their corresponding dust samples using the validated ELISA method. A linear correlation (r = 0.889) was found for the DON concentration in dust versus the DON concentration in wheat (LOD wheat: 233 μg/kg, LOD wheat dust: 458 μg/kg). PMID:27077883

  20. Comparison of Enzyme-Linked Immunosorbent Assay, Surface Plasmon Resonance and Biolayer Interferometry for Screening of Deoxynivalenol in Wheat and Wheat Dust

    PubMed Central

    Sanders, Melanie; McPartlin, Daniel; Moran, Kara; Guo, Yirong; Eeckhout, Mia; O’Kennedy, Richard; De Saeger, Sarah; Maragos, Chris

    2016-01-01

    A sample preparation method was developed for the screening of deoxynivalenol (DON) in wheat and wheat dust. Extraction was carried out with water and was successful due to the polar character of DON. For detection, an enzyme-linked immunosorbent assay (ELISA) was compared to the sensor-based techniques of surface plasmon resonance (SPR) and biolayer interferometry (BLI) in terms of sensitivity, affinity and matrix effect. The matrix effects from wheat and wheat dust using SPR were too high to further use this screenings method. The preferred ELISA and BLI methods were validated according to the criteria established in Commission Regulation 519/2014/EC and Commission Decision 2002/657/EC. A small survey was executed on 16 wheat lots and their corresponding dust samples using the validated ELISA method. A linear correlation (r = 0.889) was found for the DON concentration in dust versus the DON concentration in wheat (LOD wheat: 233 μg/kg, LOD wheat dust: 458 μg/kg). PMID:27077883

  1. A modified agglutination test for Neospora caninum: development, optimization, and comparison to the indirect fluorescent-antibody test and enzyme-linked immunosorbent assay.

    PubMed

    Packham, A E; Sverlow, K W; Conrad, P A; Loomis, E F; Rowe, J D; Anderson, M L; Marsh, A E; Cray, C; Barr, B C

    1998-07-01

    Current serologic tests used to detect antibodies to Neospora caninum require species-specific secondary antibodies, limiting the number of species that can be tested. In order to examine a wide variety of animal species that may be infected with N. caninum, a modified direct agglutination test (N-MAT) similar to the Toxoplasma gondii modified direct agglutination test (T-MAT) was developed. This test measures the direct agglutination of parasites by N. caninum-specific antibodies in serum, thus eliminating the need for secondary host-specific anti-isotype sera. The N-MAT was compared to the indirect fluorescent-antibody test (IFAT) and the enzyme-linked immunosorbent assay (ELISA) with a "gold standard" serum panel from species for which secondary antibodies were available (n = 547). All positive samples tested were from animals with histologically confirmed infections. Up to 16 different species were tested. The N-MAT gave a higher sensitivity (100%) and specificity (97%) than the ELISA (74 and 94%, respectively) and had a higher sensitivity but a lower specificity than the IFAT (98 and 99%, respectively). The reduced specificity of the N-MAT was due to false-positive reactions in testing fetal fluids with particulate matter or severely hemolyzed serum. Overall, the N-MAT proved to be highly sensitive and specific for both naturally and experimentally infected animals, highly reproducible between and within readers, easy to use on large sample sizes without requiring special equipment, and useful in testing serum from any species without modification. PMID:9665950

  2. Evaluation of an IgG Enzyme-Linked Immunosorbent Assay as a Serological Assay for Detection of Mycoplasma bovis Infection in Feedlot Cattle.

    PubMed

    Wawegama, Nadeeka K; Markham, Philip F; Kanci, Anna; Schibrowski, Meghan; Oswin, Sally; Barnes, Tamsin S; Firestone, Simon M; Mahony, Timothy J; Browning, Glenn F

    2016-05-01

    Mycoplasma bovis is a pathogen of emerging significance in cattle throughout the world that is causing a range of diseases, including mastitis, arthritis, and pneumonia. The limited availability and efficacy of current diagnostic and prophylactic tools for its control and its increasing antimicrobial resistance are contributing to its increasing importance in beef and dairy cattle. We have developed an indirect IgG enzyme-linked immunosorbent assay (ELISA) based on a recombinant fragment of the MilA protein and have shown its potential as an effective diagnostic tool. To more comprehensively estimate the diagnostic sensitivity and specificity of this IgG ELISA for detection of infection with M. bovis in cattle and to define a suitable cutoff for use in the field, we further assessed its performance in experimentally infected calves in a closed beef herd and by applying Bayesian latent class modeling to laboratory testing results from 7,448 cattle entering Australian feedlots. The most effective cutoff points were estimated to be 68.6 antibody units (AU) for experimentally infected calves and to be 58.7 AU for a closed adult herd. Under field conditions, in feedlot cattle the globally optimal cutoff was estimated to be 105 AU. At this cutoff, the diagnostic sensitivity was 94.3% (95% probability interval [PI], 89.9% to 99.6%) with a diagnostic specificity of 94.4% (95% PI, 90.3% to 99.6%). Applying this 105 AU cutoff, 13.1% of cattle were seropositive for infection with M. bovis on entry into feedlots, and 73.5% were seropositive when followed up approximately 6 weeks later suggesting a high risk of infection shortly after entry into feedlots. PMID:26912757

  3. Differentiation between Human Coronaviruses NL63 and 229E Using a Novel Double-Antibody Sandwich Enzyme-Linked Immunosorbent Assay Based on Specific Monoclonal Antibodies ▿

    PubMed Central

    Sastre, Patricia; Dijkman, Ronald; Camuñas, Ana; Ruiz, Tamara; Jebbink, Maarten F.; van der Hoek, Lia; Vela, Carmen; Rueda, Paloma

    2011-01-01

    Human coronaviruses (HCoVs) are responsible for respiratory tract infections ranging from common colds to severe acute respiratory syndrome. HCoV-NL63 and HCoV-229E are two of the four HCoVs that circulate worldwide and are close phylogenetic relatives. HCoV infections can lead to hospitalization of children, elderly individuals, and immunocompromised patients. Globally, approximately 5% of all upper and lower respiratory tract infections in hospitalized children are caused by HCoV-229E and HCoV-NL63. The latter virus has recently been associated with the childhood disease croup. Thus, differentiation between the two viruses is relevant for epidemiology studies. The aim of this study was to develop a double-antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) as a potential tool for identification and differentiation between HCoV-NL63 and HCoV-229E. The nucleocapsid (N) proteins of HCoV-NL63 and HCoV-229E were expressed in an Escherichia coli system and used to immunize mice in order to obtain monoclonal antibodies (MAbs) specific for each virus. Three specific MAbs to HCoV-NL63, one MAb specific to HCoV-229E, and four MAbs that recognized both viruses were obtained. After their characterization, three MAbs were selected in order to develop a differential DAS-ELISA. The described assay could detect up to 3 ng/ml of N protein and 50 50% tissue culture infective doses/ml of virus stock. No cross-reactivity with other human coronaviruses or closely related animal coronaviruses was found. The newly developed DAS-ELISA was species specific, and therefore, it could be considered a potential tool for detection and differentiation of HCoV-NL63 and HCoV-229E infections. PMID:21084464

  4. An enzyme-linked immunosorbent assay to detect PCR products of the rfbS gene from serogroup D salmonellae: a rapid screening prototype.

    PubMed Central

    Luk, J M; Kongmuang, U; Tsang, R S; Lindberg, A A

    1997-01-01

    We describe a digoxigenin-based enzyme-linked immunosorbent assay (DIG-ELISA) following a PCR to detect the amplified lipopolysaccharide rfbS gene as a means for rapid screening of serogroup D salmonellae in stool specimens. For pure bacterial cultures, the sensitivity of the PCR DIG-ELISA was approximately 10 bacteria. In the presence of stool materials, the salmonellae were first isolated by an immunomagnetic separation technique with an O9-specific monoclonal antibody. MATy-O9, followed by PCR and DIG-ELISA. The corresponding sensitivity was about 10 to 100 bacteria. To evaluate the assay performance clinically, 203 stool samples from patients with diarrhea were subjected to the routine culture techniques and the PCR ELISA method with overnight enrichment. The conventional culture method identified 145 salmonellae (31 serogroup B, 27 serogroup C, 83 serogroup D, and 5 serogroup E isolates) and 58 non-salmonella bacteria. The PCR ELISA method correctly identified all 82 serogroup D salmonellae (A405 by ELISA, 2.54 +/- 0.74) but was negative for the other Salmonella serogroups (A405, 0.26 +/- 0.08; n = 63) and non-Salmonella isolates (A405, 0.16 +/- 0.04; n = 58). In order to obtain a visible result, the assay takes approximately 6 h (PCR, 4 h; ELISA, 2 h), along with brief enrichment cultivation of the samples (from 4 to 16 h). Thus, the PCR DIG-ELISA offers a fast, accurate, semiquantitative means of detecting infectious agents such as salmonellae, and future robotic automation is possible. PMID:9041418

  5. Comparison of enzyme-linked immunosorbent assay and gas chromatography procedures for the detection of cyanazine and metolachlor in surface water samples

    USGS Publications Warehouse

    Schraer, S.M.; Shaw, D.R.; Boyette, M.; Coupe, R.H.; Thurman, E.M.

    2000-01-01

    Enzyme-linked immunosorbent assay (ELISA) data from surface water reconnaissance were compared to data from samples analyzed by gas chromatography for the pesticide residues cyanazine (2-[[4-chloro-6-(ethylamino)-l,3,5-triazin-2-yl]amino]-2-methylpropanenitrile ) and metolachlor (2-chloro-N-(2-ethyl-6-methylphenyl)-N-(2-methoxy-1-methylethyl)acetamide). When ELISA analyses were duplicated, cyanazine and metolachlor detection was found to have highly reproducible results; adjusted R2s were 0.97 and 0.94, respectively. When ELISA results for cyanazine were regressed against gas chromatography results, the models effectively predicted cyanazine concentrations from ELISA analyses (adjusted R2s ranging from 0.76 to 0.81). The intercepts and slopes for these models were not different from 0 and 1, respectively. This indicates that cyanazine analysis by ELISA is expected to give the same results as analysis by gas chromatography. However, regressing ELISA analyses for metolachlor against gas chromatography data provided more variable results (adjusted R2s ranged from 0.67 to 0.94). Regression models for metolachlor analyses had two of three intercepts that were not different from 0. Slopes for all metolachlor regression models were significantly different from 1. This indicates that as metolachlor concentrations increase, ELISA will over- or under-estimate metolachlor concentration, depending on the method of comparison. ELISA can be effectively used to detect cyanazine and metolachlor in surface water samples. However, when detections of metolachlor have significant consequences or implications it may be necessary to use other analytical methods.

  6. Detection of Type A, B, E, and F Clostridium botulinum Neurotoxins in Foods by Using an Amplified Enzyme-Linked Immunosorbent Assay with Digoxigenin-Labeled Antibodies

    PubMed Central

    Sharma, Shashi K.; Ferreira, Joseph. L.; Eblen, Brian S.; Whiting, Richard C.

    2006-01-01

    An amplified enzyme-linked immunosorbent assay (ELISA) for the detection of Clostridium botulinum complex neurotoxins was evaluated for its ability to detect these toxins in food. The assay was found to be suitable for detecting type A, B, E, and F botulinum neurotoxins in a variety of food matrices representing liquids, solid, and semisolid food. Specific foods included broccoli, orange juice, bottled water, cola soft drinks, vanilla extract, oregano, potato salad, apple juice, meat products, and dairy foods. The detection sensitivity of the test for these botulinum complex serotypes was found to be 60 pg/ml (1.9 50% lethal dose [LD50]) for botulinum neurotoxin type A (BoNT/A), 176 pg/ml (1.58 LD50) for BoNT/B, 163 pg/ml for BoNT/E (4.5 LD50), and 117 pg/ml for BoNT/F (less than 1 LD50) in casein buffer. The test could also readily detect 2 ng/ml of neurotoxins type A, B, E, and F in a variety of food samples. For specificity studies, the assay was also used to test a large panel of type A C. botulinum, a smaller panel of proteolytic and nonproteolytic type B, E, and F neurotoxin-producing Clostridia, and nontoxigenic organisms using an overnight incubation of toxin production medium. The assay appears to be an effective tool for large-scale screening of the food supply in the event of a botulinum neurotoxin contamination event. PMID:16461671

  7. Evaluation of a monoclonal antibody-based enzyme-linked immunosorbent assay for detection of Campylobacter fetus in bovine preputial washing and vaginal mucus samples.

    PubMed

    Brooks, B W; Devenish, J; Lutze-Wallace, C L; Milnes, D; Robertson, R H; Berlie-Surujballi, G

    2004-10-01

    A monoclonal antibody-based enzyme-linked immunosorbent assay (ELISA) was described and evaluated for use as a presumptive screening test for detection of Campylobacter fetus in bovine preputial washing and vaginal mucus samples. A total of 725 diagnostic samples collected in the field and submitted in Clark's transport enrichment medium (TEM) were analyzed. Cultural isolation of C. fetus was used as the standard for comparison. After incubation of the TEM vials for 4-5 days, fluid was removed for culture and ELISA testing. A sandwich ELISA format was used and the target antigen was C. fetus lipopolysaccharides (LPS). A rabbit anti-C. fetus polyclonal antiserum was used as the capture antibody. Murine monoclonal antibodies (MAbs) to C. fetus serotype A and B LPS core and O-polysaccharides and a goat anti-mouse horseradish peroxidase conjugate were used as detection antibodies. ELISA and culture results for the diagnostic samples were in complete agreement. Seven hundred and eight samples were negative by both tests. All 17 culture positive samples were positive by ELISA with a MAb to LPS core. The ELISA with MAbs to LPS O-polysaccharides detected all culture positive samples with the homologous C. fetus serotype. Sixty-six preputial wash samples from three known C. fetus culture positive bulls were also analyzed. Forty-nine of these samples were positive by both ELISA and culture, 16 were positive by ELISA only, and one was negative by both ELISA and culture. The results indicate that this ELISA is useful as a screening test for the detection of C. fetus in diagnostic samples. PMID:15381269

  8. Development of a sensitive monoclonal-based enzyme-linked immunosorbent assay for monitoring T-2 toxin in food and feed.

    PubMed

    Peng, Dapeng; Chang, Fangfang; Wang, Yulian; Chen, Dongmei; Liu, Zhenli; Zhou, Xiaodong; Feng, Liang; Yuan, Zonghui

    2016-04-01

    The consumption of food or feed contaminated with high levels of T-2 toxin may cause adverse health effects in humans and other animals. In this study, to monitor T-2 toxin rapidly in food and feed, a sensitive and specific monoclonal antibody (mAb) against T-2 toxin was generated and a simple and rapid indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) developed. T-2 toxin was first converted to T-2-hemisuccinate (T-2HS) and T-2-hemiglutarate (T-2HG), which were then conjugated to bovine serum albumin (BSA) and ovalbumin (OVA) to prepare an immunogen and coating antigen, respectively. After the inoculation of female Balb/c mice and cell fusions, one cell line, 4D8, with the IgG1 isotype was obtained. The 4D8 antibody exhibited the ability specifically to recognise T-2 toxin with IC50 1.46 µg l(-1). Based on this 4D8 mAb, an optimised ic-ELISA protocol was developed using only methanol-water (7:3, v/v) in feed and cereal samples and ethyl acetate in muscle samples. The limits of detection of T-2 toxin in various sample matrices varied from 0.07 to 15.8 µg kg(-1); the recoveries ranged from 50.3% to 113.6%; and the CVs were less than 19.0%. These results suggest that the prepared mAb and the developed ic-ELISA method will be a useful tool for detecting T-2 toxin in foods and feeds. PMID:26933973

  9. Development of an indirect competitive enzyme-linked immunosorbent assay based on the multiepitope peptide for the synchronous detection of staphylococcal enterotoxin A and G proteins in milk.

    PubMed

    Liang, Mingyan; Zhang, Tingting; Liu, Xuelan; Fan, Yanan; Xia, Shenglin; Xiang, Yiqing; Liu, Ziqi; Jinnian, Li

    2015-02-01

    Staphylococcal food poisoning (SFP), one of the most common foodborne diseases, results from ingestion of staphylococcal enterotoxins (SEs) in foods. In our previous studies, we found that SEA and SEG were two predominant SE proteins produced by milkacquired S. aureus isolates. Here, a tandemly arranged multiepitope peptide (named SEAGepis) was designed with six linear B-cell epitopes derived from SEA or SEG and was heterologously expressed. The SEAGepis-specific antibody was prepared by immunizing rabbit with rSEAGepis. Then, an indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) based on rSEAGepis and the corresponding antibody was developed to simultaneously detect SEA and SEG. Under the optimized conditions, the ic-ELISA standard curve for rSEAGepis was constructed in the concentration range of 0.5 to 512 ng/ml, and the average coefficients of variation of intra- and interassay were 4.28 and 5.61% during six standard concentrations. The average half-maximal inhibitory concentration was 5.07 ng/ml, and the limit of detection at a signal-to-noise ratio of 3 was 0.52 ng/ml. The anti-rSEAGepis antibody displayed over 90% cross-reactivity with SEA and SEG but less than 0.5% cross-reactivity with other enterotoxins. Artificially contaminated milk with different concentrations of rSEAGepis, SEA, and SEG was detected by the established ic-ELISA; the recoveries of rSEAGepis, SEA, and SEG were 91.1 to 157.5%, 90.3 to 134.5%, and 89.1 to 117.5%, respectively, with a coefficient of variation below 12%. These results demonstrated that the newly established ic-ELISA possessed high sensitivity, specificity, stability, and accuracy and could potentially be a useful analytical method for synchronous detection of SEA and SEG in milk. PMID:25710152

  10. Evaluation of the sensitivity and specificity of an enzyme-linked immunosorbent assay for diagnosing brucellosis in African buffalo (Syncerus caffer).

    PubMed

    Gorsich, Erin E; Bengis, Roy G; Ezenwa, Vanessa O; Jolles, Anna E

    2015-01-01

    Brucellosis is a disease of veterinary and public health importance worldwide. In sub-Saharan Africa, where the bacterium Brucella abortus has been identified in several free-ranging wildlife species, successful disease control may be dependent on accurate detection in wildlife reservoirs, including African buffalo (Syncerus caffer). We estimated the sensitivity and specificity of a commercial enzyme-linked immunosorbent assay (ELISA) (IDEXX Brucellosis Serum Ab test, IDEXX Laboratories, Westbrook, Maine, USA) for B. abortus based on a data set of 571 serum samples from 258 buffalo in the Kruger National Park, South Africa. We defined a pseudogold standard test result as those buffalo that were consistently positive or negative on two additional serologic tests, namely, the rose bengal test (RBT) and the complement fixation test (CFT). The ELISA's cutoff value was selected using receiver operating characteristics analysis, the pseudogold standard, and a threshold criterion that maximizes the total sensitivity and specificity. Then, we estimated the sensitivity and specificity of all three tests using Bayesian inference and latent class analysis. The ELISA had an estimated sensitivity of 0.928 (95% Bayesian posterior credibility interval [95% BCI] = 0.869-0.974) and specificity of 0.870 (95% BCI = 0.836-0.900). Compared with the ELISA, the RBT had a higher estimated sensitivity of 0.986 (95% BCI = 0.928-0.999), and both the RBT and CFT had higher specificities, estimated to be 0.992 (95% BCI = 0.971-0.996) and 0.998 (95% BCI = 0.992-0.999), respectively. Therefore, no single serologic test perfectly detected the antibody. However, after adjustment of cutoff values for South African conditions, the IDEXX Brucellosis Serum Ab Test may be a valuable additional screening test for brucellosis in Kruger National Park's African buffalo. PMID:25397998

  11. Simple Indirect Enzyme-Linked Immunosorbent Assay to Detect Antibodies Against Bovine Viral Diarrhea Virus, Based on Prokaryotically Expressed Recombinant MBP-NS3 Protein

    PubMed Central

    Mahmoodi, Pezhman; Seyfi Abad Shapouri, Masoud Reza; Ghorbanpour, Masoud; Haji Hajikolaei, Mohammad Rahim; Lotfi, Mohsen; Pourmahdi Boroujeni, Mahdi; Daghari, Maryam

    2015-01-01

    Background: Bovine viral diarrhea (BVD) is an economically important disease of cattle distributed worldwide. Diagnosis of BVD relies on laboratory-based detection of its viral causing agent or virus specific antibodies and the most common laboratory method for this purpose is Enzyme-Linked Immunosorbent Assay (ELISA). Objectives: The current study was aimed to develop a simple indirect ELISA to detect antibodies against Bovine Viral Diarrhea Virus (BVDV) in the sera of infected cattle. Materials and Methods: A new simple indirect ELISA method was developed to detect BVDV infection by prokaryotically (Escherichia coli, BL21 strain) expressed recombinant whole nonstructural protein 3 (NS3) of BVDV (NADL strain). Four hundred bovine serum samples were evaluated by the newly developed NS3-ELISA and virus neutralization test (VNT) as the gold standard method to diagnose BVD. Among these samples, 289 sera had been previously tested by a commercial ELISA kit. Results: Statistical analyses showed a very high correlation between the results of the developed NS3-ELISA and VNT (kappa coefficient = 0.935, P < 0.001), with the relative sensitivity and specificity of 94% and 98.8%, respectively. There was also a high correlation between the results of NS3-ELISA and the commercial ELISA kit (kappa coefficient = 0.802, P < 0.001) with the relative sensitivity and specificity of 90.72% and 91.15%, respectively. Conclusions: The newly developed simple indirect ELISA showed high sensitivity and specificity with respect to VNT. Developing such a simple, sensitive, and specific ELISA which is much less expensive than the available commercial ELISA kits can improve the detection of BVDV infections, help to eliminate the disease from herds, and decrease economic losses caused by this disease. PMID:25964844

  12. Impact of grey zone sample testing by enzyme-linked immunosorbent assay in enhancing blood safety: Experience at a tertiary care hospital in North India

    PubMed Central

    Solanki, Archana; Singh, Abhay; Chaudhary, Rajendra

    2016-01-01

    Background: Enzyme-linked immunosorbent assay (ELISA) used for screening blood donors for transfusion transmitted infections (TTIs) can sometimes fail to detect blood donors who are recently infected or possessing the low strength of pathogen. Estimation of a grey zone in ELISA testing and repeat testing of grey zone samples can further help in reducing the risks of TTI in countries where nucleic acid amplification testing for TTIs is not feasible. Materials and Methods: Grey zone samples with optical density (OD) lying between cut-off OD and 10% below the cut-off OD (cut-off OD × 0.9) were identified during routine ELISA testing. On performing repeat ELISA testing on grey zone samples in duplicate, the samples showing both OD value below grey zone were marked nonreactive, and samples showing one or both OD value in the grey zone were marked indeterminate. The samples on repeat testing showing one or both OD above cut-off value were marked positive. Results: About 119 samples (77 for hepatitis B virus [HBV], 23 for human immunodeficiency virus [HIV], and 19 for hepatitis C virus [HCV]) were found to be in grey zone. On repeat testing of these samples in duplicate, 70 (58.8%) samples (45 for HBV, 12 for HIV, and 13 for HCV) were found to be reactive. Six (5%) samples (four for HBV, one for HIV, and one for HCV) were found to be indeterminate. Conclusion: Seventy donors initially screened negative, were found out to be potentially infectious on repeat grey zone testing. Thus, estimation of grey zone samples with repeat testing can further enhance the safety of blood transfusion. PMID:27011675

  13. Field evaluation of an immunoglobulin G anti-F1 enzyme-linked immunosorbent assay for serodiagnosis of human plague in Madagascar.

    PubMed Central

    Rasoamanana, B; Leroy, F; Boisier, P; Rasolomaharo, M; Buchy, P; Carniel, E; Chanteau, S

    1997-01-01

    Bacteriological isolation of Yersinia pestis is the reference test for confirming plague infection, but recovery of the pathogen from human samples is usually very poor. When the etiology of the disease cannot be bacteriologically confirmed, it may be useful to possess alternative tests such as detection of specific circulating antibodies to help guide the diagnosis. In the present study, the immunoglobulin G (IgG) anti-F1 enzyme-linked immunosorbent assay (ELISA) has been applied to various human sera to evaluate its large-scale applicability in the high-endemicity plague foci of Madagascar. The sensitivity of the test was found to be 91.4%, and its specificity was 98.5%. The positive and negative predictive values were 96 and 96.6%, respectively. Seroconversion was observed on day 7 after onset of the disease. Patients with a positive ELISA result could be separated into high (82%) and low (18%) IgG anti-F1 responders. Cross-reactions with eight other infectious diseases prevalent in Madagascar were scarce and were found in 1 of 27 Mycobacterium tuberculosis-, 3 of 34 Schistosoma haematobium-, and 1 of 41 Salmonella-infected patients. Finally, the efficiency of the IgG anti-F1 ELISA was evaluated during the Mahajanga, Madagascar, plague outbreak of 1995. When the number of ELISA-positive patients was added to the number of bacteriologically confirmed and probable cases, the number of positive patients was increased by 35%. In conclusion, although it does not replace bacteriology, IgG anti-F1 ELISA is a useful and powerful tool for retrospective diagnosis and epidemiological surveillance of plague outbreaks. PMID:9302210

  14. An indirect competitive biotin-streptavidin enzyme-linked immunosorbent assay for the determination of dimethyl phthalate (DMP) in milk and milk products.

    PubMed

    Sun, Rui Y; Zhuang, Hui S

    2015-01-01

    After the "plasticizer event" in Taiwan, phthalic acid esters (PAEs) have been listed in "Inedible materials possibly added into food illegally" and "Commonly abused food additives." As one of the PAEs family, DMP has long been a problem of great concern due to its potential impacts on human health. In order to detect DMP with high sensitivity and specificity, a sensitive indirect competitive biotin-streptavidin enzyme-linked immunosorbent assay (BA-ELISA) has been established in this study. A high-titer rabbit polyclonal antibody (pAb-DMP) targeting DMP was obtained, and the procedures of BA-ELISA were optimized for the determination of DMP in milk and milk products. Under optimal conditions, good linearity was achieved within a range of 0.024 to 6.027 μg L(-1), with low cross-reactivity values for DMP structural analogues (lower than 10%). The median inhibitory concentration (IC50) was 0.356 μg L(-1) and the limit of detection (LOD) was 0.0082 μg L(-1). Finally, the concentrations of DMP in milk and milk products ranged from 1.03 μg kg(-1) to 7.23 μg kg(-1) by BA-ELISA. Satisfactory recoveries (90.26-112.38%) and coefficient of variation (CV) values (5.08-8.46%) were obtained. These results were consistent with those using gas chromatography-mass spectrometry (GC-MS), which further confirmed that the proposed BA-ELISA was accurate, specific, reliable and rapid for routine monitoring trace DMP residues in foodstuff, especially milk and milk products. PMID:25714459

  15. Development of a competitive enzyme-linked immunosorbent assay for detection of antibodies against the 3B protein of foot-and-mouth disease virus.

    PubMed

    Yang, Ming; Parida, Satya; Salo, Tim; Hole, Kate; Velazquez-Salinas, Lauro; Clavijo, Alfonso

    2015-04-01

    Foot-and-mouth disease (FMD) is one of the most highly contagious and economically devastating diseases, and it severely constrains the international trade of animals. Vaccination against FMD is a key element in the control of FMD. However, vaccination of susceptible animals raises critical issues, such as the differentiation of infected animals from vaccinated animals. The current study developed a reliable and rapid test to detect antibodies against the conserved, nonstructural proteins (NSPs) of the FMD virus (FMDV) to distinguish infected animals from vaccinated animals. A monoclonal antibody (MAb) against the FMDV NSP 3B was produced. A competitive enzyme-linked immunosorbent assay (cELISA) for FMDV/NSP antibody detection was developed using a recombinant 3ABC protein as the antigen and the 3B-specific MAb. Sera collected from naive, FMDV experimentally infected, vaccinated carrier, and noncarrier animals were tested using the 3B cELISA. The diagnostic specificity was 99.4% for naive animals (cattle, pigs, and sheep) and 99.7% for vaccinated noncarrier animals. The diagnostic sensitivity was 100% for experimentally inoculated animals and 64% for vaccinated carrier animals. The performance of this 3B cELISA was compared to that of four commercial ELISA kits using a panel of serum samples established by the World Reference Laboratory for FMD at The Pirbright Institute, Pirbright, United Kingdom. The diagnostic sensitivity of the 3B cELISA for the panel of FMDV/NSP-positive bovine serum samples was 94%, which was comparable to or better than that of the commercially available NSP antibody detection kits. This 3B cELISA is a simple, reliable test to detect antibodies against FMDV nonstructural proteins. PMID:25651918

  16. Serological evidence of infectious salmon anaemia virus (ISAV) infection in farmed fishes, using an indirect enzyme-linked immunosorbent assay (ELISA).

    PubMed

    Kibenge, Molly T; Opazo, Beatriz; Rojas, Alejandro H; Kibenge, Frederick S B

    2002-08-15

    Antibody detection tests are rarely used for diagnostic purposes in fish diseases. Infectious salmon anaemia (ISA) caused by ISA virus (ISAV) is an emerging disease of Atlantic salmon Salmo salar L. The virus has also been isolated from diseased coho salmon Oncorhynchus kisutch in Chile. An indirect enzyme-linked immunosorbent assay (ELISA) that should facilitate serodiagnosis of ISAV infection, the study of epidemiology, and the control of ISA in farmed fishes has been developed using purified ISAV as the coating antigen, and monoclonal antibodies that detect fish immunoglobulins bound to the antigen on the plate. Application of the test to a random sample of farmed Atlantic salmon from the Bay of Fundy, New Brunswick, Canada, positively identified 5 of the 7 ISAV RT-PCR-positive fish, and all 10 RT-PCR-negative fish were also negative in the ELISA. Some RT-PCR-negative fish had an elevated non-specific antibody reactivity suggestive of chronic infection or resistance to ISAV. This test was also able to detect 11 of the 14 coho salmon pooled serum samples from a clinically affected farm in Chile that were positive by the virus neutralization (VN) test, and 2 of the 4 VN-negative samples. We conclude that this ELISA would be suitable as a routine test for ISAV infection or for assessing ISAV vaccine efficacy before placing smolts in sea cages, and for testing fishes in sea cages to detect level of resistance to ISA. The assay enables vaccination in combination with depopulation control methods. PMID:12240966

  17. Development and Validation of Digital Enzyme-Linked Immunosorbent Assays for Ultrasensitive Detection and Quantification of Clostridium difficile Toxins in Stool.

    PubMed

    Song, Linan; Zhao, Mingwei; Duffy, David C; Hansen, Joshua; Shields, Kelsey; Wungjiranirun, Manida; Chen, Xinhua; Xu, Hua; Leffler, Daniel A; Sambol, Susan P; Gerding, Dale N; Kelly, Ciarán P; Pollock, Nira R

    2015-10-01

    The currently available diagnostics for Clostridium difficile infection (CDI) have major limitations. Despite mounting evidence that toxin detection is paramount for diagnosis, conventional toxin immunoassays are insufficiently sensitive and cytotoxicity assays too complex; assays that detect toxigenic organisms (toxigenic culture [TC] and nucleic acid amplification testing [NAAT]) are confounded by asymptomatic colonization by toxigenic C. difficile. We developed ultrasensitive digital enzyme-linked immunosorbent assays (ELISAs) for toxins A and B using single-molecule array technology and validated the assays using (i) culture filtrates from a panel of clinical C. difficile isolates and (ii) 149 adult stool specimens already tested routinely by NAAT. The digital ELISAs detected toxins A and B in stool with limits of detection of 0.45 and 1.5 pg/ml, respectively, quantified toxins across a 4-log range, and detected toxins from all clinical strains studied. Using specimens that were negative by cytotoxicity assay/TC/NAAT, clinical cutoffs were set at 29.4 pg/ml (toxin A) and 23.3 pg/ml (toxin B); the resulting clinical specificities were 96% and 98%, respectively. The toxin B digital ELISA was 100% sensitive versus cytotoxicity assay. Twenty-five percent and 22% of the samples positive by NAAT and TC, respectively, were negative by the toxin B digital ELISA, consistent with the presence of organism but minimal or no toxin. The mean toxin levels by digital ELISA were 1.5- to 1.7-fold higher in five patients with CDI-attributable severe outcomes, versus 68 patients without, but this difference was not statistically significant. Ultrasensitive digital ELISAs for the detection and quantification of toxins A and B in stool can provide a rapid and simple tool for the diagnosis of CDI with both high analytical sensitivity and high clinical specificity. PMID:26202120

  18. Serodiagnosis of pleuropneumonia using enzyme-linked immunosorbent assay with capsular polysaccharide antigens of Actinobacillus pleuropneumoniae serotypes 1, 2, 5 and 7.

    PubMed Central

    Bossé, J T; Johnson, R P; Rosendal, S

    1990-01-01

    Capsular polysaccharide antigens of serotypes 1, 2, 5 and 7 of Actinobacillus pleuropneumoniae were used in enzyme-linked immunosorbent assays (ELISAs) to test sera from experimentally infected and field pigs. Specific reactions were found in sera of experimental pigs with antigens of serotypes 1, 5 and 7 whereas the serotype 2 antigen was cross-reactive. A 1:200 serum dilution was used for testing of 300 sera from 21 swine herds in southern Ontario. Cases of pleuropneumonia had occurred in 11 of these herds, but not in the others. The negative cut-off value was the mean optical density at 405 nm (OD405) + three standard deviations (SD) for 16 negative reference sera. Sera from four pigs naturally infected with Actinobacillus suis were tested and found to react to varying degrees with each of the antigens. Therefore a second cut-off value was determined as the mean OD405 + 2 SD for the A. suis sera. Sera which, in the ELISA produced OD readings above the latter cut-off were considered positive for antibodies to A. pleuropneumoniae; those which were lower than the former cut-off were considered negative. Readings between the two cut-off values may have been due to low positive titers or cross-reactivity, possibly with A. suis, and could not be used to predict pleuropneumonia. Of the pleuropneumonia-free herds, none had positive reactors to serotypes 5 or 7, whereas one and two herds had positive reactors to serotypes 1 and 2, respectively. Of the pleuropneumonia positive herds, six had positive reactors to serotype 1, one to serotype 2, four to serotype 5, and eight to serotype 7. PMID:2249177

  19. Development and Characterization of Monoclonal Antibodies to Yellow Fever Virus and Application in Antigen Detection and IgM Capture Enzyme-Linked Immunosorbent Assay.

    PubMed

    Adungo, Ferdinard; Yu, Fuxun; Kamau, David; Inoue, Shingo; Hayasaka, Daisuke; Posadas-Herrera, Guillermo; Sang, Rosemary; Mwau, Matilu; Morita, Kouichi

    2016-08-01

    Yellow fever (YF) is an acute hemorrhagic viral infection transmitted by mosquitoes in Africa and South America. The major challenge in YF disease detection and confirmation of outbreaks in Africa is the limited availability of reference laboratories and the persistent lack of access to diagnostic tests. We used wild-type YF virus sequences to generate recombinant envelope protein in an Escherichia coli expression system. Both the recombinant protein and sucrose gradient-purified YF vaccine virus 17D (YF-17D) were used to immunize BALB/c mice to generate monoclonal antibodies (MAbs). Eight MAbs were established and systematically characterized by indirect enzyme-linked immunosorbent assay (ELISA), Western blot analysis, and immunofluorescence assay (IFA). The established MAbs showed strong reactivity with wild-type YF virus and recombinant protein with no detectable cross-reactivity to dengue virus or Japanese encephalitis virus. Epitope mapping showed strong binding of three MAbs to amino acid positions 1 to 51, while two MAbs mapped to amino acid positions 52 to 135 of the envelope protein. The remaining three MAbs did not show reactivity to envelope fragments. The established MAbs exert no neutralization against wild-type YF and 17D viruses (titer of <10 for both strains). The applicability of MAbs 8H3 and 3F4 was further evaluated using IgM capture ELISA. A total of 49 serum samples were analyzed, among which 12 positive patient and vaccinee samples were correctly identified. Using serum samples that were 2-fold serially diluted, the IgM capture ELISA was able to detect all YF-positive samples. Furthermore, MAb-based antigen detection ELISA enabled the detection of virus in culture supernatants containing titers of about 1,000 focus-forming units. PMID:27307452

  20. Reactivity of heat-stable Leptospira antigenic preparation used in enzyme-linked immunosorbent assay for detection of antibodies in swine serum.

    PubMed

    Wasiński, B; Pejsak, Z

    2012-01-01

    Serology plays an important role in laboratory diagnosis of leptospirosis. Apart from the most often used microscopic agglutination test (MAT), enzyme-linked immunosorbent assay (ELISA) seems to be useful especially in screenings of animal herds. The ELISA used for detection of antibodies against selected Leptospira serogroups in swine serum samples was investigated during the study. An essential element of this test is heat-stable antigenic preparation from cultures of Leptospira interrogans serovars Icterohaemorrhagiae, Pomona and L. borgpetersenii serovar Sejroe. The aim of the present study was to identify and analyze ELISA heat-stable antigen fractions playing a role in the reaction with leptospiral antibodies indicated in swine serum. Reactivity of the three-component antigenic preparation was compared in immunoblotting with reactivity of six heat-stable antigenic preparations made from the following single serovars: L. interrogans serovars Icterohaemorrhagiae, Pomona, Canicola, L. borgpetersenii serovars Sejroe, Tarassovi and L. kirshneri serovar Grippotyphosa. All antigenic preparations were submitted to SDS-PAGE and transferred to a nitrocellulose membrane using a semidry system. After the transfer, the membrane was incubated with diluted swine serum containing antibodies specific for one of the six above mentioned Leptospira serovars. For the three-component antigenic preparation and antigens prepared from single serovars the immunoblot revealed reaction of sera with fractions of the 20-26 kDa region and around the 14.5 kDa region. The investigated heat-stable Leptospira antigenic preparation contains fractions demonstrating serogroup- and species-specificity. Fraction 20-26 kDa showed serogroup-specific activity, whereas the fraction around 14.5 kDa showed species-specific activity. PMID:22708354

  1. Chicken single-chain antibody fused to alkaline phosphatase detects Aspergillus pathogens and their presence in natural samples by direct sandwich enzyme-linked immunosorbent assay.

    PubMed

    Xue, Sheng; Li, He-Ping; Zhang, Jing-Bo; Liu, Jin-Long; Hu, Zu-Quan; Gong, An-Dong; Huang, Tao; Liao, Yu-Cai

    2013-11-19

    A sensitive and specific analytical method to detect ubiquitous aflatoxigenic Aspergillus pathogens is essential for monitoring and controlling aflatoxins. Four highly reactive chicken single-chain variable fragments (scFvs) against soluble cell wall proteins (SCWPs) from Aspergillus flavus were isolated by phage display. The scFv antibody AfSA4 displayed the highest activity toward both A. flavus and A. parasiticus and specifically recognized a surface target of their cell walls as revealed by immunofluorescence localization. Molecular modeling revealed a unique compact motif on the antibody surface mainly involving L-CDR2 and H-CDR3. As measured by surface plasmon resonance, AfSA4 fused to alkaline phosphatase had a higher binding capability and 6-fold higher affinity compared with AfSA4 alone. Immunoblot analyses showed that the fusion had good binding capacity to SCWP components from the two fungal species. Direct sandwich enzyme-linked immunosorbent assays with mouse antiaspergillus monoclonal antibody mAb2A8 generated in parallel as a capture antibody revealed that the detection limit of the two fungi was as low as 10(-3) μg/mL, 1000-fold more sensitive than that reported previously (1 μg/mL). The fusion protein was able to detect fungal concentrations below 1 μg/g of maize and peanut grains in both artificially and naturally contaminated samples, with at least 10-fold more sensitivity than that reported (10 μg/g) thus far. Thus, the fusion can be applied in rapid, simple, and specific diagnosis of Aspergillus contamination in field and stored food/feed commodities. PMID:24128348

  2. Influence of pigments and protein aging on protein identification in historically representative casein-based paints using enzyme-linked immunosorbent assay.

    PubMed

    Ren, Fang; Atlasevich, Natalya; Baade, Brian; Loike, John; Arslanoglu, Julie

    2016-01-01

    A systematic study on the influence of pigments and sample aging on casein identification was performed on 30 reconstructed paints. The protein in all the paints was extracted into solution for analysis. The amount of protein that can be retrieved for solution-based analysis in each of the reconstructed paints was studied with a well-developed NanoOrange method before and after artificial aging. The results showed that in the paints with calcium phosphate (in bone black) and copper carbonate, hydroxide, or acetate (in verdigris and azurite), the amount of protein that can be retrieved for liquid-phase analysis is much smaller than the other paints, indicating that the protein degradation was accelerated significantly in those paints. Carbon (in vine black), calcium carbonate (in natural chalk), and calcium sulfate (terra alba gypsum and ground alabaster) did not affect much the amount of protein that can be retrieved in the paints compared to non-pigmented binder, meaning that the protein degradation rate was not affected much by those pigments. Artificial aging was observed to decrease the amount of retrievable protein in all the reconstructed paints that were studied. The enzyme-linked immunosorbent assay (ELISA) method was applied to the 28 reconstructed paints (except two verdigris paints) to assess the protein identification. The ELISA responses from the different paints were compared at fixed protein concentrations. Natural chalk, bone black, raw sienna, stack lead white, and cochineal red-violet lake had the lowest ELISA signal in this study, which indicated that the binding sites (epitopes) on the target protein in these paints are likely to deteriorate more than those in the other paints. Artificial aging did not influence the ELISA response as much as the pigments when the protein concentration was kept the same for the paints that were studied. PMID:26472321

  3. A protein A/G indirect enzyme-linked immunosorbent assay for the detection of anti-Brucella antibodies in Arctic wildlife.

    PubMed

    Nymo, Ingebjørg H; Godfroid, Jacques; Åsbakk, Kjetil; Larsen, Anett K; das Neves, Carlos G; Rødven, Rolf; Tryland, Morten

    2013-05-01

    A species-independent indirect enzyme-linked immunosorbent assay (iELISA) based on chimeric protein A/G was established for the detection of anti-Brucella antibodies in Arctic wildlife species and compared to previously established brucellosis serological tests for hooded seals (Cystophora cristata), minke whales (Balaenoptera acutorostrata), sei whales (Balaenoptera borealis), fin whales (Balaenoptera physalus), and polar bears (Ursus maritimus), as well as bacteriology results for reindeer and caribou (Rangifer tarandus sp.). The protein A/G iELISA results were consistent with the other serological tests with Cohen kappa values between 0.47 and 0.92, and the protein A/G iELISA can thus offer a technically simple method for these species yielding results consistent with established brucellosis serological tests. Receiver operator characteristics analysis proved that the reindeer and caribou protein A/G iELISA results were consistent with the bacteriological gold standard with an area under the curve of 0.99, and the protein A/G iELISA was thus validated as a sensitive and specific serological method for the detection of anti-Brucella antibodies in reindeer and caribou. The binding of the antibodies from the respective species to protein A and G were also evaluated in the iELISA. The antibodies from hooded seals and polar bears reacted stronger to protein A than to G. The sei whale, fin whale, reindeer, and caribou antibodies reacted stronger to protein G than to A. The minke whale antibodies reacted to both protein A and G. There was a strong correlation (r s = 0.88-0.98) between the optical density results obtained with the iELISA with protein A/G and protein A or G, showing that protein A/G is as well suited as protein A or G for the detection of anti-Brucella antibodies in these species with the iELISA. PMID:23572454

  4. Identification and expression of Babesia ovis secreted antigen 1 and evaluation of its diagnostic potential in an enzyme-linked immunosorbent assay.

    PubMed

    Sevinc, Ferda; Cao, Shinuo; Xuan, Xuenan; Sevinc, Mutlu; Ceylan, Onur

    2015-05-01

    In order to identify immunoreactive proteins that are usable for the immunological diagnosis of Babesia ovis infections, a phage lambda cDNA expression library was constructed and screened using parasite-specific immune serum. Immunoscreening resulted in the identification of a full-length cDNA clone encoding a secreted protein designated Babesia ovis secreted antigen 1 (BoSA1). The full-length BoSA1 cDNA contained a 1,137-bp open reading frame that encoded a protein of 378 amino acids, with a signal peptide and 2 internal repeat domains. The theoretical molecular mass of the mature protein was 42.5 kDa. Recombinant BoSA1 (rBoSA1) protein was expressed in Escherichia coli strain DH5α cells as a glutathione S-transferase (GST) fusion protein and was purified by affinity chromatography. Purified rBoSA1 was tested for reactivity with sera from animals experimentally or naturally infected with B. ovis, in an indirect enzyme-linked immunosorbent assay (ELISA). The results showed that specific antibodies against rBoSA1 were detectable on days 7 and 8 of the experimental infection and were maintained during the sampling period. Additionally, 38 field sera taken from sheep naturally infected with B. ovis gave strong positive reactions in the ELISA between day 20 and day 30 of treatment. As a result, the identified recombinant BoSA1 protein seems to be a promising diagnostic antigen that is usable for the development of serological assays for the diagnosis of ovine babesiosis. This is the first report on the molecular cloning, expression, and potential use of a recombinant antigen for the diagnosis of ovine babesiosis. PMID:25694531

  5. Evaluation of a commercial enzyme-linked immunosorbent assay for detection of immunoglobulin M antibody in diagnosis of human leptospiral infection.

    PubMed Central

    Winslow, W E; Merry, D J; Pirc, M L; Devine, P L

    1997-01-01

    The PanBio Leptospira immunoglobulin M (IgM) enzyme-linked immunosorbent assay (ELISA) is a commercially available screening test for the diagnosis of acute leptospiral infection. The ability of the test to diagnose early or recent Leptospira interrogans infection was assessed by testing sera with known microagglutination test (MAT) titers to serovars pomona, hardjo, copenhageni, and australis. The IgM ELISA detected all 41 cases of early or recent leptospiral infection (sensitivity, 100%), with a positive ELISA result seen in many cases before MAT antibody titers reached 1:50. Thirty-eight of 41 patients showed seroconversion (fourfold or greater increase in titer by MAT, 2 of 41 patients had a single sample with elevated titer, and 1 patient from whom leptospires were isolated from a blood sample failed to show MAT titers, despite a seroconversion (negative to positive result) in the ELISA. Follow-up sera obtained from 8 of 12 patients (67%) for 3 to 48 months after the acute stage of illness showed persisting IgM antibody. However, the range of levels detected in these samples (maximum ELISA ratio, 2.0) was lower than the range seen when infection was recent. Reactivity in the IgM ELISA was observed for only 1 of 59 serum samples from asymptomatic donors (specificity, 98%) and 16 of 233 serum samples from patients with Ross River virus, brucella, Epstein-Barr virus, cytomegalovirus, mycoplasma, Q-fever, toxoplasma, hepatitis A virus, Treponema pallidum, or Borrelia burgdorferi infection (specificity, 93%), with the majority of these patients showing lower levels of IgM in comparison to those in patients with leptospiral infection. We conclude that this ELISA is sufficiently sensitive for use as an initial screen for leptospiral infections, with subsequent confirmation of positive test results by MAT. PMID:9230359

  6. Detection and serogroup differentiation of Salmonella spp. in food within 30 hours by enrichment-immunoassay with a T6 monoclonal antibody capture enzyme-linked immunosorbent assay.

    PubMed Central

    Ng, S P; Tsui, C O; Roberts, D; Chau, P Y; Ng, M H

    1996-01-01

    We previously described an antigen capture enzyme-linked immunosorbent assay which makes use of monoclonal antibody T6, which recognizes an epitope on the outer core polysaccharide of Salmonella lipopolysaccharide molecules that is common to almost all Salmonella serovars. In this paper, we show that this assay can detect between 10(5) and 10(7) Salmonella cells per ml even in the presence of excess Escherichia coli. A total of 153 of 154 (99%) serogroup A to E strains and 51 of 78 (71%) serogroup F to 67 strains were reactive as determined by this assay. This corresponds to a detection rate of approximately 98% of all salmonellae known to affect humans. None of the 65 strains of non-Salmonella bacteria tested positive. Taking advantage of the O-factor polysaccharides also present on the antigen captured by the immobilized T6 antibody, we showed that 136 of 154 Salmonella serogroup A to E strains (88%) were correctly differentiated according to their serogroups by use of enzyme conjugates of a panel of O-factor-specific monoclonal antibodies. We evaluated this assay for the detection and serogroup differentiation of salmonellae directly from enrichment cultures of simulated food, eggs, pork, and infant formula milk. All 26 samples which had been contaminated with Salmonella spp. were detected by T6 (100% sensitivity), with only one false-positive result from 101 samples not contaminated by Salmonella spp. (99% specificity). The detection time was substantially reduced to between 17 and 29 h, depending on the enrichment methods used. Since there were no false-negative results, we concluded that this enrichment-immunoassay method can afford rapid screening for Salmonella spp. in food samples. PMID:8779567

  7. Performance Assessment and Comparability of a Commercial Enzyme-Linked Immunosorbent Assay Kit with Liquid Chromatography-Tandem Mass Spectrometry for Chloramphenicol Residues in Crab and Shrimp.

    PubMed

    Jester, Edward L E; Loader, Jared I; El Said, Kathleen R; Abraham, Ann; Flores Quintana, Harold A; Plakas, Steven M

    2016-01-01

    Monitoring for chloramphenicol (CAP) in aquaculture products is primarily performed by liquid chromatography-tandem mass spectrometry (LC-MS/MS), which requires expensive equipment and specialized training. Many laboratories prefer to screen samples with facile and high-throughput enzyme-linked immunosorbent assay (ELISA) kits for CAP residues before submitting samples for LC-MS/MS quantification and confirmation. We evaluated the performance of a Ridascreen (R-Biopharm) ELISA kit for CAP in spiked and incurred crab and shrimp muscle at levels bracketing the minimum required performance level for analysis (0.3 ng/g). The Ridascreen ELISA kit incorporates antibody directed against CAP. Incurred CAP levels in crab and shrimp muscle were verified using LC-MS/MS. We found good repeatability (relative standard deviation) of the ELISA in spiked and incurred crab and shrimp muscle samples, with values ranging from 6.8 to 21.7%. Recoveries of CAP from tissues spiked at 0.15 to 0.60 ng/g ranged from 102 to 107%. Minimal cross-reactivity with blank crab and shrimp muscle matrix components was observed. ELISA data were highly correlated with those of LC-MS/MS for CAP in incurred muscle tissue. We believe this study to be the first evaluation of the performance and comparability of a CAP ELISA kit and LC-MS/MS for determination of CAP residues, as well as their elimination, in crab muscle. Our findings support the use of this ELISA kit for screening purposes and, when used in conjunction with validated instrumental methods, for regulatory monitoring of CAP in these species. PMID:26735037

  8. Modified Indirect Porcine Circovirus (PCV) Type 2-Based and Recombinant Capsid Protein (ORF2)-Based Enzyme-Linked Immunosorbent Assays for Detection of Antibodies to PCV

    PubMed Central

    Nawagitgul, Porntippa; Harms, Perry A.; Morozov, Igor; Thacker, Brad J.; Sorden, Steven D.; Lekcharoensuk, Chalermpol; Paul, Prem S.

    2002-01-01

    Postweaning multisystemic wasting syndrome of swine associated with porcine circovirus (PCV) is a recently reported and economically important disease. Simple and reliable diagnostic methods are needed for detecting antibodies to PCV type 2 (PCV2) for monitoring of PCV infection. Here, we report the development of two modified indirect enzyme-linked immunosorbent assays (ELISAs): a PCV2 ELISA based on cell-culture-propagated PCV2 and an ORF2 ELISA based on recombinant major capsid protein. PCV2 and ORF2 ELISA detected antibodies to PCV2 and the capsid protein, respectively, in sera from pigs experimentally infected with PCV2 as early as 14 and 21 days postinoculation (dpi). The kinetics of the antibody response to PCV2 and the major capsid protein were similar. Repeatability tests revealed that the coefficients of variation of positive sera within and between runs for both assays were less than 30%. To validate the assays, PCV2 and ORF2 ELISAs were performed with 783 serum samples of young and adult pigs collected from different herds in the Midwestern United States and compared with an indirect immunofluorescent assay (IIF). Six out of 60 samples collected from nursery and growing pigs in 1987 were positive by both ELISA and IIF. Compared with IIF, the diagnostic sensitivity, specificity, and accuracy of PCV2 and ORF2 ELISAs were similar (>90%). The tests showed no cross-reactivity with antibodies to porcine parvovirus and porcine reproductive and respiratory syndrome virus. There was good agreement between the two ELISAs and between the ELISAs and IIF. The availability of the two ELISAs should accelerate our understanding of the host immune response to PCV2 and facilitate the development of prevention and control strategies by elucidating the ecology of PCV2 within swine populations. PMID:11777826

  9. Evaluation of a Commercial Enzyme-Linked Immunosorbent Assay for the Diagnosis of Bovine Tuberculosis from Milk Samples from Dairy Cows

    PubMed Central

    Wilson, Tania; Luo, Dongwen; Voges, Hinrich; Linscott, Richard; Martel, Edmond; Lawrence, John C.; Neill, Mark A.

    2013-01-01

    Milk samples from dairy cows provide a ready source of material for measuring antibody responses to Mycobacterium bovis antigens. In this study, we evaluated the IDEXX enzyme-linked immunosorbent assay (ELISA) for the measurement of antibody responses to M. bovis antigens MPB70 and MPB83 in milk samples from New Zealand cattle. Test sensitivities for individual milk and serum samples were assessed in samples collected from 44 M. bovis-infected cows, and test specificities were assessed in milk samples collected from 356 cows from tuberculosis (TB)-free herds. Milk vat samples were collected from 505 herds from regions with relatively high or low prevalences of infection. The ELISA had a sensitivity of 50% and a specificity of 97.5% for milk samples, and the test sensitivities for milk and serum samples were the same. Dilution of the positive test milk samples in milk from noninfected cows at 1/10, 1/20, and 1/50 dilutions reduced the proportions of positive responses to 13/21, 9/21, and 4/21, respectively. Small differences were observed in the ELISA responses of milk samples from individual TB-free cows collected at different times during lactation. No significant differences were detected in the ELISA responses of milk vat samples collected from infected and noninfected herds. This study shows that milk samples can be substituted for serum samples for screening individual cows for M. bovis infection, and pooling of milk samples from 10 to 20 animals can result in a reduction in the sensitivity by approximately 50%. However, screening of milk vat samples is unlikely to be useful in countries with low prevalences of M. bovis in cattle and large herd sizes. PMID:24132605

  10. Identification and Expression of Babesia ovis Secreted Antigen 1 and Evaluation of Its Diagnostic Potential in an Enzyme-Linked Immunosorbent Assay

    PubMed Central

    Cao, Shinuo; Xuan, Xuenan; Sevinc, Mutlu; Ceylan, Onur

    2015-01-01

    In order to identify immunoreactive proteins that are usable for the immunological diagnosis of Babesia ovis infections, a phage lambda cDNA expression library was constructed and screened using parasite-specific immune serum. Immunoscreening resulted in the identification of a full-length cDNA clone encoding a secreted protein designated Babesia ovis secreted antigen 1 (BoSA1). The full-length BoSA1 cDNA contained a 1,137-bp open reading frame that encoded a protein of 378 amino acids, with a signal peptide and 2 internal repeat domains. The theoretical molecular mass of the mature protein was 42.5 kDa. Recombinant BoSA1 (rBoSA1) protein was expressed in Escherichia coli strain DH5α cells as a glutathione S-transferase (GST) fusion protein and was purified by affinity chromatography. Purified rBoSA1 was tested for reactivity with sera from animals experimentally or naturally infected with B. ovis, in an indirect enzyme-linked immunosorbent assay (ELISA). The results showed that specific antibodies against rBoSA1 were detectable on days 7 and 8 of the experimental infection and were maintained during the sampling period. Additionally, 38 field sera taken from sheep naturally infected with B. ovis gave strong positive reactions in the ELISA between day 20 and day 30 of treatment. As a result, the identified recombinant BoSA1 protein seems to be a promising diagnostic antigen that is usable for the development of serological assays for the diagnosis of ovine babesiosis. This is the first report on the molecular cloning, expression, and potential use of a recombinant antigen for the diagnosis of ovine babesiosis. PMID:25694531

  11. Enzyme-linked immunosorbent assay and colloidal gold immunoassay for ochratoxin A: investigation of analytical conditions and sample matrix on assay performance.

    PubMed

    Wang, Xiang-Hong; Liu, Tao; Xu, Na; Zhang, Yan; Wang, Shuo

    2007-10-01

    A polyclonal antibody against ochratoxin A (OTA) was produced from rabbits immunized with the OTA-BSA conjugate. A competitive direct enzyme-linked immunosorbent assay (cdELISA) and a membrane-base colloidal gold immunoassay in flow-through format were developed for the rapid detection of OTA in various food matrices. In the cdELISA, the concentration causing 50% inhibition was 0.07 ng mL(-1), and the effects of different chemical conditions (ionic strength, pH value, and organic solvent) were studied. The sensitivity of the assay was higher than those previously reported. A simple, rapid, and efficient extraction method was developed and 74-110% recoveries of spiked samples were obtained. Fifty percent methanol extracts of some food samples such as barley, wheat, oat, corn, rice, and raisins could be analyzed directly by immunoassay after dilution in PBS; grape juice and beer samples could be analyzed directly after dilution with PBS; for coffee samples, a more complex method was used to remove the matrix effect effectively. Membrane-based colloidal gold immunoassays had a visual detection limit of 1.0 ng mL(-1) for OTA with a detection time of less than 10 min. For the validation of the cdELISA and membrane-based colloidal gold immunoassay, samples were analyzed by high-performance liquid chromatography. The correlation between data obtained using the microwell assay and HPLC was good (R2 = 0.984). The developed immunoassay methods are suitable for the rapid quantitative or qualitative determination of OTA in food samples. PMID:17668189

  12. Use of the Normalized Absorbance Ratio as an Internal Standardization Approach To Minimize Measurement Error in Enzyme-Linked Immunosorbent Assays for Diagnosis of Human Papillomavirus Infection▿

    PubMed Central

    Ramanakumar, Agnihotram V.; Thomann, Patricia; Candeias, Joao M.; Ferreira, Silvaneide; Villa, Luisa L.; Franco, Eduardo L.

    2010-01-01

    The serological detection of antibodies against human papillomavirus (HPV) antigens is a useful tool to determine exposure to genital HPV infection and in predicting the risk of infection persistence and associated lesions. Enzyme-linked immunosorbent assays (ELISAs) are commonly used for seroepidemiological studies of HPV infection but are not standardized. Intra- and interassay performance variation is difficult to control, especially in cohort studies that require the testing of specimens over extended periods. We propose the use of normalized absorbance ratios (NARs) as a standardization procedure to control for such variations and minimize measurement error. We compared NAR and ELISA optical density (OD) values for the strength of the correlation between serological results for paired visits 4 months apart and HPV-16 DNA positivity in cervical specimens from a cohort investigation of 2,048 women tested with an ELISA using HPV-16 virus-like particles. NARs were calculated by dividing the mean blank-subtracted (net) ODs by the equivalent values of a control serum pool included in the same plate in triplicate, using different dilutions. Stronger correlations were observed with NAR values than with net ODs at every dilution, with an overall reduction in nonexplained regression variability of 39%. Using logistic regression, the ranges of odds ratios of HPV-16 DNA positivity contrasting upper and lower quintiles at different dilutions and their averages were 4.73 to 5.47 for NARs and 2.78 to 3.28 for net ODs, with corresponding significant improvements in seroreactivity-risk trends across quintiles when NARs were used. The NAR standardization is a simple procedure to reduce measurement error in seroepidemiological studies of HPV infection. PMID:20053861

  13. Evaluation of different modifications of acid-fast staining techniques and stool enzyme-linked immunosorbent assay in detecting fecal Cryptosporidium in diarrheic HIV seropositive and seronegative patients

    PubMed Central

    Parghi, Ekta; Dash, Lona; Shastri, Jayanthi

    2014-01-01

    Rational: The role of Cryptosporidium as an agent of human diarrhea has been redefined over the past decade following recognition of the strong association between cases of cryptosporidiosis and immune deficient individuals (such as those with AIDS). Purpose: The purpose of this study is to determine the prevalence of enteric parasites and to compare the diagnostic utility of stool enzyme-linked immunosorbent assay (ELISA) with various modifications of acid-fast (AF) staining in detection of Cryptosporidium in stool samples of diarrheic patients. Materials and Methods: Stool samples from 186 cases comprising of 93 HIV seropositive and 93 seronegative patients were included. These were subjected to routine and microscopic examination as well as various modifications of AF staining for detection of coccidian parasites and ELISA for the detection of Cryptosporidium. Results: The prevalence of enteric parasites was 54.8% and of Cryptosporidium was 17.2% in HIV seropositive patients while it was 29.0% and 5.4%, respectively in seronegative patients. Of the 186 cases, 33 cases (17.7%) were positive for Cryptosporidium by stool ELISA as compared to 21 (11.3%) by modified AF staining (gold standard) showing sensitivity and specificity of 100% and 92.7%, respectively. The maximum cases of Cryptosporidium (21; 11.3%) were detected by AF staining using 3% acid alcohol. Conclusion: ELISA is a simple, useful, and rapid tool for detection of Cryptosporidium in stool, especially for large scale population studies. However, the role of modified AF staining in detection of Cryptosporidium and other coccidian parasites is important. Based on the results of various modifications of AF staining, the present study recommends the use of 3% acid alcohol along with 10% H2SO4. PMID:25250230

  14. Interlaboratory standardization of the sandwich enzyme-linked immunosorbent assay designed for MATS, a rapid, reproducible method for estimating the strain coverage of investigational vaccines.

    PubMed

    Plikaytis, Brian D; Stella, Maria; Boccadifuoco, Giuseppe; DeTora, Lisa M; Agnusdei, Mauro; Santini, Laura; Brunelli, Brunella; Orlandi, Luca; Simmini, Isabella; Giuliani, Marzia; Ledroit, Morgan; Hong, Eva; Taha, Muhamed-Kheir; Ellie, Kim; Rajam, Gowrisankar; Carlone, George M; Claus, Heike; Vogel, Ulrich; Borrow, Ray; Findlow, Jamie; Gilchrist, Stefanie; Stefanelli, Paola; Fazio, Cecilia; Carannante, Anna; Oksnes, Jan; Fritzsønn, Elisabeth; Klem, Anne-Marie; Caugant, Dominique A; Abad, Raquel; Vázquez, Julio A; Rappuoli, Rino; Pizza, Mariagrazia; Donnelly, John J; Medini, Duccio

    2012-10-01

    The meningococcal antigen typing system (MATS) sandwich enzyme-linked immunosorbent assay (ELISA) was designed to measure the immunologic cross-reactivity and quantity of antigens in target strains of a pathogen. It was first used to measure the factor H-binding protein (fHbp), neisserial adhesin A (NadA), and neisserial heparin-binding antigen (NHBA) content of serogroup B meningococcal (MenB) isolates relative to a reference strain, or "relative potency" (RP). With the PorA genotype, the RPs were then used to assess strain coverage by 4CMenB, a multicomponent MenB vaccine. In preliminary studies, MATS accurately predicted killing in the serum bactericidal assay using human complement, an accepted correlate of protection for meningococcal vaccines. A study across seven laboratories assessed the reproducibility of RPs for fHbp, NadA, and NHBA and established qualification parameters for new laboratories. RPs were determined in replicate for 17 MenB reference strains at laboratories A to G. The reproducibility of RPs among laboratories and against consensus values across laboratories was evaluated using a mixed-model analysis of variance. Interlaboratory agreement was very good; the Pearson correlation coefficients, coefficients of accuracy, and concordance correlation coefficients exceeded 99%. The summary measures of reproducibility, expressed as between-laboratory coefficients of variation, were 7.85% (fHbp), 16.51% (NadA), and 12.60% (NHBA). The overall within-laboratory measures of variation adjusted for strain and laboratory were 19.8% (fHbp), 28.8% (NHBA), and 38.3% (NadA). The MATS ELISA was successfully transferred to six laboratories, and a further laboratory was successfully qualified. PMID:22875603

  15. Development and Comparative Evaluation of a Plate Enzyme-Linked Immunosorbent Assay Based on Recombinant Outer Membrane Antigens Omp28 and Omp31 for Diagnosis of Human Brucellosis

    PubMed Central

    Tiwari, Sapana; Kumar, Ashu; Mangalgi, Smita; Rathod, Vedika; Prakash, Archana; Barua, Anita; Arora, Sonia; Sathyaseelan, Kannusamy

    2013-01-01

    Brucellosis is an important zoonotic infectious disease of humans and livestock with worldwide distribution and is caused by bacteria of the genus Brucella. The diagnosis of brucellosis always requires laboratory confirmation by either isolation of pathogens or detection of specific antibodies. The conventional serological tests available for the diagnosis of brucellosis are less specific and show cross-reactivity with other closely related organisms. These tests also necessitate the handling of Brucella species for antigen preparation. Therefore, there is a need to develop reliable, rapid, and user-friendly systems for disease diagnosis and alternatives to vaccine approaches. Keeping in mind the importance of brucellosis as an emerging infection and the prevalence in India, we carried out the present study to compare the recombinant antigens with the native antigens (cell envelope and sonicated antigen) of Brucella for diagnosis of human brucellosis by an indirect plate enzyme-linked immunosorbent assay (ELISA). Recombinant outer membrane protein 28 (rOmp28) and rOmp31 antigens were cloned, expressed, and purified in the bacterial expression system, and the purified proteins were used as antigens. Indirect plate ELISAs were then performed and standardized for comparison of the reactivities of recombinant and native antigens against the 433 clinical samples submitted for brucellosis testing, 15 culture-positive samples, and 20 healthy donor samples. The samples were separated into four groups based on their positivity to rose bengal plate agglutination tests (RBPTs), standard tube agglutination tests (STATs), and 2-mercaptoethanol (2ME) tests. The sensitivities and specificities of all the antigens were calculated, and the rOmp28 antigen was found to be more suitable for the clinical diagnosis of brucellosis than the rOmp31 antigen and native antigens. The rOmp28-based ELISA showed a very high degree of agreement with the conventional agglutination tests and

  16. Production of monoclonal antibody for okadaic acid and its utilization in an ultrasensitive enzyme-linked immunosorbent assay and one-step immunochromatographic strip.

    PubMed

    Liu, Biing-Hui; Hung, Chun-Tse; Lu, Chuan-Chen; Chou, Hong-Non; Yu, Feng-Yih

    2014-02-12

    Okadaic acid (OA) is a common marine biotoxin that accumulates in bivalves and causes diarrhetic shellfish poisoning (DSP). This study generated a monoclonal antibody (mAb) specific to OA from a hybridoma cell line, 6B1A3, which was obtained by fusion of myeloma cells (P3/NS1/1-AG4-1) with spleen cells isolated from a BALB/c mouse immunized with OA-γ-globulin. The 6B1A3 mAb belongs to the immunoglobulin G1 (κ chain) isotype. Both competitive direct and indirect enzyme-linked immunosorbent assays (ELISAs) were established for characterization of the antibody. The concentrations causing 50% inhibition of binding of OA-horseradish peroxidase to the antibody by OA were calculated to be 0.077 ng/mL in the cdELISA. A rapid and sensitive mAb-based gold nanoparticle immunochromatographic strip was also established. This proposed strip has a detection limit of 5 ng/mL for OA and can be finished in 10 min. Extensive analyses of 20 seafood samples with ELISA revealed that 10 were slightly contaminated with OA, with a mean concentration of 0.892 ng/g. Analysis of OA in shellfish samples showed that data acquired by the immunochromatographic strip agreed well with those acquired by the ELISA. The mAb-based ELISA and immunochromatographic strip assay developed in this study have adequate sensitivity and accuracy for rapid screening of OA in shellfish samples. PMID:24446876

  17. Appropriate coating methods and other conditions for enzyme-linked immunosorbent assay of smooth, rough, and neutral lipopolysaccharides of Pseudomonas aeruginosa.

    PubMed

    Bantroch, S; Bühler, T; Lam, J S

    1994-01-01

    Smooth, rough, and neutral forms of lipopolysaccharide (LPS) from Pseudomonas aeruginosa were used to assess the appropriate conditions for effective enzyme-linked immunosorbent assay (ELISA) of LPS. Each of these forms of well-defined LPS was tested for the efficiency of antigen coating by various methods as well as to identify an appropriate type of microtiter plate to use. For smooth LPS, the standard carbonate-bicarbonate buffer method was as efficient as the other sensitivity-enhancing plate-coating methods compared. The rough LPS, which has an overall hydrophobic characteristic, was shown to adhere effectively, regardless of the coating method used, to only one type of microtiter plate, CovaLink. This type of plate has secondary amine groups attached on its polystyrene surface by carbon chain spacers, which likely favors hydrophobic interactions between the rough LPS and the well surfaces. Dehydration methods were effective for coating microtiter plates with the neutral LPS examined, which is composed predominantly of a D-rhamnan. For the two dehydration procedures, LPS suspended in water or the organic solvent chloroform-ethanol was added directly to the wells, and the solvent was allowed to dehydrate or evaporate overnight. Precoating of plates with either polymyxin or poly-L-lysine did not give any major improvement in coating with the various forms of LPS. The possibility of using proteinase K- and sodium dodecyl sulfate-treated LPS preparations for ELISAs was also investigated. Smooth LPS prepared by this method was as effective in ELISA as LPS prepared by the hot water-phenol method, while the rough and neutral LPSs prepared this way were not satisfactory for ELISA. PMID:7496923

  18. Seroprevalence of human respiratory syncytial virus and human metapneumovirus in healthy population analyzed by recombinant fusion protein-based enzyme linked immunosorbent assay

    PubMed Central

    2012-01-01

    Background Human respiratory syncytial virus (hRSV) and human metapneumovirus (hMPV) are two of the most frequent respiratory pathogens that circulate worldwide. Infection with either virus can lead to hospitalization of young children, immunocompromised people and the elderly. A better understanding of the epidemiological aspects, such as prevalence of these viruses in the population will be of significant importance to the scientific community. The aim of this study was to gain some detailed knowledge on the humoral immune response to both viruses in different populations of individuals. Findings The fusion protein (F) of hRSV and hMPV was expressed in the baculovirus and Escherichia coli systems, respectively, and used as antigen in two independent enzyme-linked immunosorbent assays (ELISAs) for detection of specific antibodies in human sera. The seroprevalence of each virus in a large cohort of individuals with ages ranging from 0 to 89 years old was determined. Although the general distribution of the antibody response to each virus in the different age group was similar, the prevalence of hRSV appeared to be higher than that of hMPV in most of them. The group of children with ages between 0 and 2 showed the highest seronegative rates. After this age, an increase in the antibody response was observed, most likely as the result of new infections or even due to reinfections. Conclusions The use of these specific F-ELISAs in seroepidemiological studies might be helpful for a better understanding of the human antibody response to these viruses. PMID:22748150

  19. Differentiation between human coronaviruses NL63 and 229E using a novel double-antibody sandwich enzyme-linked immunosorbent assay based on specific monoclonal antibodies.

    PubMed

    Sastre, Patricia; Dijkman, Ronald; Camuñas, Ana; Ruiz, Tamara; Jebbink, Maarten F; van der Hoek, Lia; Vela, Carmen; Rueda, Paloma

    2011-01-01

    Human coronaviruses (HCoVs) are responsible for respiratory tract infections ranging from common colds to severe acute respiratory syndrome. HCoV-NL63 and HCoV-229E are two of the four HCoVs that circulate worldwide and are close phylogenetic relatives. HCoV infections can lead to hospitalization of children, elderly individuals, and immunocompromised patients. Globally, approximately 5% of all upper and lower respiratory tract infections in hospitalized children are caused by HCoV-229E and HCoV-NL63. The latter virus has recently been associated with the childhood disease croup. Thus, differentiation between the two viruses is relevant for epidemiology studies. The aim of this study was to develop a double-antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) as a potential tool for identification and differentiation between HCoV-NL63 and HCoV-229E. The nucleocapsid (N) proteins of HCoV-NL63 and HCoV-229E were expressed in an Escherichia coli system and used to immunize mice in order to obtain monoclonal antibodies (MAbs) specific for each virus. Three specific MAbs to HCoV-NL63, one MAb specific to HCoV-229E, and four MAbs that recognized both viruses were obtained. After their characterization, three MAbs were selected in order to develop a differential DAS-ELISA. The described assay could detect up to 3 ng/ml of N protein and 50 50% tissue culture infective doses/ml of virus stock. No cross-reactivity with other human coronaviruses or closely related animal coronaviruses was found. The newly developed DAS-ELISA was species specific, and therefore, it could be considered a potential tool for detection and differentiation of HCoV-NL63 and HCoV-229E infections. PMID:21084464

  20. A Modified Agglutination Test for Neospora caninum: Development, Optimization, and Comparison to the Indirect Fluorescent-Antibody Test and Enzyme-Linked Immunosorbent Assay

    PubMed Central

    Packham, Andrea E.; Sverlow, Karen W.; Conrad, Patricia A.; Loomis, Emily F.; Rowe, Joan D.; Anderson, Mark L.; Marsh, Antoinette E.; Cray, Carolyn; Barr, Bradd C.

    1998-01-01

    Current serologic tests used to detect antibodies to Neospora caninum require species-specific secondary antibodies, limiting the number of species that can be tested. In order to examine a wide variety of animal species that may be infected with N. caninum, a modified direct agglutination test (N-MAT) similar to the Toxoplasma gondii modified direct agglutination test (T-MAT) was developed. This test measures the direct agglutination of parasites by N. caninum-specific antibodies in serum, thus eliminating the need for secondary host-specific anti-isotype sera. The N-MAT was compared to the indirect fluorescent-antibody test (IFAT) and the enzyme-linked immunosorbent assay (ELISA) with a “gold standard” serum panel from species for which secondary antibodies were available (n = 547). All positive samples tested were from animals with histologically confirmed infections. Up to 16 different species were tested. The N-MAT gave a higher sensitivity (100%) and specificity (97%) than the ELISA (74 and 94%, respectively) and had a higher sensitivity but a lower specificity than the IFAT (98 and 99%, respectively). The reduced specificity of the N-MAT was due to false-positive reactions in testing fetal fluids with particulate matter or severely hemolyzed serum. Overall, the N-MAT proved to be highly sensitive and specific for both naturally and experimentally infected animals, highly reproducible between and within readers, easy to use on large sample sizes without requiring special equipment, and useful in testing serum from any species without modification. PMID:9665950

  1. Mycoplasma agassizii strain variation and distinct host antibody responses explain differences between enzyme-linked immunosorbent assays and Western blot assays.

    PubMed

    Wendland, Lori D; Klein, Paul A; Jacobson, Elliott R; Brown, Mary B

    2010-11-01

    The precarious status of desert (Gopherus agassizii) and gopher (G. polyphemus) tortoises has resulted in conservation efforts that now include health assessment as an important component of management decision-making. Mycoplasmal upper respiratory tract disease (URTD) is one of very few diseases in chelonians for which comprehensive and rigorously validated diagnostic tests exist. In this study, serum samples obtained from eight Gopherus tortoises documented at necropsy to (i) be enzyme-linked immunosorbent assay (ELISA) seropositive using the PS6 antigen, (ii) be infected with Mycoplasma agassizii as indicated by direct isolation of the pathogen from the respiratory surfaces, and (iii) have histological lesions of mycoplasmal URTD were used to evaluate four distinct clinical isolates of M. agassizii as antigens for ELISA and Western blot analyses. Each animal sample reacted in the Western blot with its homologous M. agassizii strain, but recognition of heterologous M. agassizii strains was variable. Further, individual animals varied significantly with respect to the specific proteins recognized by the humoral immune response. An additional 114 Gopherus serum samples were evaluated using ELISA antigens prepared from the four distinct M. agassizii strains; A₄₀₅ values were significantly correlated (r² goodness of fit range, 0.708 to 0.771; P < 0.0001) for all antigens tested. The results confirm that strain variation is responsible for the observed differences between Western blot binding patterns. Thus, reliance on a single M. agassizii strain as an antigen in Western blot assays may provide false-negative results. This could have adverse consequences for the well-being of these environmentally sensitive hosts if false-negative animals were relocated to sites consisting of true-negative populations. PMID:20810678

  2. Improved Enzyme-Linked Immunosorbent Assay To Reveal Mycoplasma agassizii Exposure: a Valuable Tool in the Management of Environmentally Sensitive Tortoise Populations▿

    PubMed Central

    Wendland, Lori D.; Zacher, Laurie A.; Klein, Paul A.; Brown, Daniel R.; Demcovitz, Dina; Littell, Ramon; Brown, Mary B.

    2007-01-01

    The precarious status of desert (Gopherus agassizii) and gopher (Gopherus polyphemus) tortoises has resulted in research and conservation efforts that include health assessments as a substantial component of management decision-making. Therefore, it is critical that available diagnostic tests for diseases impacting these species undergo rigorous standardization and validation. Since 1992, analysis of exposure of tortoises to Mycoplasma agassizii, an etiological agent of upper respiratory tract disease, has relied on the detection of specific M. agassizii antibody by enzyme-linked immunosorbent assay (ELISA). We report here substantive refinements in the diagnostic assay and discuss the implications of its use in wildlife conservation and management. The ELISA has been refined to include more stringent quality control measures and has been converted to a clinically more meaningful titer reporting system, consistent with other diagnostic serologic tests. The ELISA results for 5,954 desert and gopher tortoises were plotted, and a subset of these serum samples (n = 90) was used to determine end-point titers, to establish an optimum serum dilution for analyzing samples, and to construct a standard curve. The relationship between titer and A405 was validated using 77 serum samples from known positive (n = 48) and negative (n = 29) control tortoises from prior transmission studies. The Youden index, J, and the optimal cut point, c, were estimated using ELISA results from the 77 control sera. Based on this evaluation, the refinement has substantially improved the performance of the assay (sensitivity of 0.98, specificity of 0.99, and J of 0.98), thus providing a clinically more reliable diagnostic test for this important infection of tortoises. PMID:17626160

  3. Improved enzyme-linked immunosorbent assay to reveal Mycoplasma agassizii exposure: a valuable tool in the management of environmentally sensitive tortoise populations.

    PubMed

    Wendland, Lori D; Zacher, Laurie A; Klein, Paul A; Brown, Daniel R; Demcovitz, Dina; Littell, Ramon; Brown, Mary B

    2007-09-01

    The precarious status of desert (Gopherus agassizii) and gopher (Gopherus polyphemus) tortoises has resulted in research and conservation efforts that include health assessments as a substantial component of management decision-making. Therefore, it is critical that available diagnostic tests for diseases impacting these species undergo rigorous standardization and validation. Since 1992, analysis of exposure of tortoises to Mycoplasma agassizii, an etiological agent of upper respiratory tract disease, has relied on the detection of specific M. agassizii antibody by enzyme-linked immunosorbent assay (ELISA). We report here substantive refinements in the diagnostic assay and discuss the implications of its use in wildlife conservation and management. The ELISA has been refined to include more stringent quality control measures and has been converted to a clinically more meaningful titer reporting system, consistent with other diagnostic serologic tests. The ELISA results for 5,954 desert and gopher tortoises were plotted, and a subset of these serum samples (n = 90) was used to determine end-point titers, to establish an optimum serum dilution for analyzing samples, and to construct a standard curve. The relationship between titer and A405 was validated using 77 serum samples from known positive (n = 48) and negative (n = 29) control tortoises from prior transmission studies. The Youden index, J, and the optimal cut point, c, were estimated using ELISA results from the 77 control sera. Based on this evaluation, the refinement has substantially improved the performance of the assay (sensitivity of 0.98, specificity of 0.99, and J of 0.98), thus providing a clinically more reliable diagnostic test for this important infection of tortoises. PMID:17626160

  4. Mycoplasma agassizii Strain Variation and Distinct Host Antibody Responses Explain Differences between Enzyme-Linked Immunosorbent Assays and Western Blot Assays ▿

    PubMed Central

    Wendland, Lori D.; Klein, Paul A.; Jacobson, Elliott R.; Brown, Mary B.

    2010-01-01

    The precarious status of desert (Gopherus agassizii) and gopher (G. polyphemus) tortoises has resulted in conservation efforts that now include health assessment as an important component of management decision-making. Mycoplasmal upper respiratory tract disease (URTD) is one of very few diseases in chelonians for which comprehensive and rigorously validated diagnostic tests exist. In this study, serum samples obtained from eight Gopherus tortoises documented at necropsy to (i) be enzyme-linked immunosorbent assay (ELISA) seropositive using the PS6 antigen, (ii) be infected with Mycoplasma agassizii as indicated by direct isolation of the pathogen from the respiratory surfaces, and (iii) have histological lesions of mycoplasmal URTD were used to evaluate four distinct clinical isolates of M. agassizii as antigens for ELISA and Western blot analyses. Each animal sample reacted in the Western blot with its homologous M. agassizii strain, but recognition of heterologous M. agassizii strains was variable. Further, individual animals varied significantly with respect to the specific proteins recognized by the humoral immune response. An additional 114 Gopherus serum samples were evaluated using ELISA antigens prepared from the four distinct M. agassizii strains; A405 values were significantly correlated (r2 goodness of fit range, 0.708 to 0.771; P < 0.0001) for all antigens tested. The results confirm that strain variation is responsible for the observed differences between Western blot binding patterns. Thus, reliance on a single M. agassizii strain as an antigen in Western blot assays may provide false-negative results. This could have adverse consequences for the well-being of these environmentally sensitive hosts if false-negative animals were relocated to sites consisting of true-negative populations. PMID:20810678

  5. Development and Characterization of Monoclonal Antibodies to Yellow Fever Virus and Application in Antigen Detection and IgM Capture Enzyme-Linked Immunosorbent Assay

    PubMed Central

    Adungo, Ferdinard; Kamau, David; Inoue, Shingo; Hayasaka, Daisuke; Posadas-Herrera, Guillermo; Sang, Rosemary; Mwau, Matilu

    2016-01-01

    Yellow fever (YF) is an acute hemorrhagic viral infection transmitted by mosquitoes in Africa and South America. The major challenge in YF disease detection and confirmation of outbreaks in Africa is the limited availability of reference laboratories and the persistent lack of access to diagnostic tests. We used wild-type YF virus sequences to generate recombinant envelope protein in an Escherichia coli expression system. Both the recombinant protein and sucrose gradient-purified YF vaccine virus 17D (YF-17D) were used to immunize BALB/c mice to generate monoclonal antibodies (MAbs). Eight MAbs were established and systematically characterized by indirect enzyme-linked immunosorbent assay (ELISA), Western blot analysis, and immunofluorescence assay (IFA). The established MAbs showed strong reactivity with wild-type YF virus and recombinant protein with no detectable cross-reactivity to dengue virus or Japanese encephalitis virus. Epitope mapping showed strong binding of three MAbs to amino acid positions 1 to 51, while two MAbs mapped to amino acid positions 52 to 135 of the envelope protein. The remaining three MAbs did not show reactivity to envelope fragments. The established MAbs exert no neutralization against wild-type YF and 17D viruses (titer of <10 for both strains). The applicability of MAbs 8H3 and 3F4 was further evaluated using IgM capture ELISA. A total of 49 serum samples were analyzed, among which 12 positive patient and vaccinee samples were correctly identified. Using serum samples that were 2-fold serially diluted, the IgM capture ELISA was able to detect all YF-positive samples. Furthermore, MAb-based antigen detection ELISA enabled the detection of virus in culture supernatants containing titers of about 1,000 focus-forming units. PMID:27307452

  6. Development and use of an indirect enzyme-linked immunosorbent assay for detection of iridovirus exposure in gopher tortoises (Gopherus polyphemus) and eastern box turtles (Terrapene carolina carolina).

    PubMed

    Johnson, April J; Wendland, Lori; Norton, Terry M; Belzer, Bill; Jacobson, Elliott R

    2010-05-19

    Iridoviruses, pathogens typically associated with fish and amphibians, have recently been shown to cause acute respiratory disease in chelonians including box turtles, red-eared sliders, gopher tortoises, and Burmese star tortoises. Case reports of natural infections in several chelonian species in the United States have been reported, however the prevalence remains unknown in susceptible populations of free-ranging chelonians. To determine the prevalence of iridovirus exposure in free-ranging gopher tortoises (Gopherus polyphemus) in the southeast United States, an indirect enzyme-linked immunosorbent assay (ELISA) was developed and used to evaluate plasma samples from wild gopher tortoises (G. polyphemus) from: Alabama (n=9); Florida (n=658); Georgia (n=225); Louisiana (n=12); Mississippi (n=28); and unknown locations (68) collected between 2001 and 2006. Eight (1.2%) seropositive tortoises were identified from Florida and seven (3.1%) from Georgia for an overall prevalence of 1.5%. Additionally, a population of eastern box turtles was sampled from a private nature sanctuary in Pennsylvania that experienced an outbreak of iridovirus the previous year, which killed 16 turtles. Only 1 turtle out of 55 survivors tested positive (1.8%). Results suggest a low exposure rate in chelonians to this pathogen; however, it is suspected that this is an underestimate of the true prevalence. Since experimental transmission studies and past outbreaks have shown a high rate of mortality in infected turtles, turtles may die before they develop an antibody response. Further, the duration of the antibody response is unknown and may also cause an underestimate of the true prevalence. PMID:19931321

  7. Highly scalable, uniform, and sensitive biosensors based on top-down indium oxide nanoribbons and electronic enzyme-linked immunosorbent assay.

    PubMed

    Aroonyadet, Noppadol; Wang, Xiaoli; Song, Yan; Chen, Haitian; Cote, Richard J; Thompson, Mark E; Datar, Ram H; Zhou, Chongwu

    2015-03-11

    Nanostructure field-effect transistor (FET) biosensors have shown great promise for ultra sensitive biomolecular detection. Top-down assembly of these sensors increases scalability and device uniformity but faces fabrication challenges in achieving the small dimensions needed for sensitivity. We report top-down fabricated indium oxide (In2O3) nanoribbon FET biosensors using highly scalable radio frequency (RF) sputtering to create uniform channel thicknesses ranging from 50 to 10 nm. We combine this scalable sensing platform with amplification from electronic enzyme-linked immunosorbent assay (ELISA) to achieve high sensitivity to target analytes such as streptavidin and human immunodeficiency virus type 1 (HIV-1) p24 proteins. Our approach circumvents Debye screening in ionic solutions and detects p24 protein at 20 fg/mL (about 250 viruses/mL or about 3 orders of magnitude lower than commercial ELISA) with a 35% conduction change in human serum. The In2O3 nanoribbon biosensors have 100% device yield and use a simple 2 mask photolithography process. The electrical properties of 50 In2O3 nanoribbon FETs showed good uniformity in on-state current, on/off current ratio, mobility, and threshold voltage. In addition, the sensors show excellent pH sensitivity over a broad range (pH 4 to 9) as well as over the physiological-related pH range (pH 6.8 to 8.2). With the demonstrated sensitivity, scalability, and uniformity, the In2O3 nanoribbon sensor platform makes great progress toward clinical testing, such as for early diagnosis of acquired immunodeficiency syndrome (AIDS). PMID:25636984

  8. Development of a sandwich enzyme-linked immunosorbent assay for the detection of CD44v3 using exon v3- and v6-specific monoclonal antibody pairs.

    PubMed

    Jeoung, Mee Hyun; Kim, Taek-Keun; Shim, Hyunbo; Lee, Sukmook

    2016-09-01

    It has been suggested that soluble CD44 levels in cancer patient sera may be closely associated with tumor progression and metastasis. However, to date, there has been limited methodology for detecting the soluble CD44 variant 3 isoform (CD44v3). Herein, using phage display technology, we isolated monoclonal antibodies specific to exon v3 or v6 of CD44 (CD44-exonv3 or CD44-exonv6) from a human synthetic antibody library. We also confirmed the specificity of antibody binding to CD44-exonv3 or -exonv6. Label-free kinetic analysis using the Octet biolayer interferometry system showed that the Kd values of the anti-CD44-exonv3 and anti-CD44-exonv6 antibodies for CD44v3-10 are approximately 1.1nM and 1.5nM, respectively. Finally, we developed a sandwich enzyme-linked immunosorbent assay (ELISA) using the anti-CD44-exonv3 and anti-CD44-exonv6 antibody pairs. The minimum detection limit of the assay was 6.2ng/ml CD44v3-10 and the linear range was up to 125ng/ml. Intra- and inter-assay coefficients of variation were 2.2% and 2.9%, respectively. The intra- and inter-assay recoveries were 99.3% and 105.3%, respectively. Taken together, these results suggest that this novel sandwich ELISA using the anti-CD44-exonv3 and anti-CD44-exonv6 antibody pairs will be useful for the detection of soluble CD44v3 in cancer patient sera. PMID:27288967

  9. Development and validation of an indirect competitive enzyme-linked immunosorbent assay for the screening of tylosin and tilmicosin in muscle, liver, milk, honey and eggs.

    PubMed

    Peng, Dapeng; Ye, Shengqiang; Wang, Yulian; Chen, Dongmei; Tao, Yanfei; Huang, Lingli; Liu, Zhenli; Dai, Menghong; Wang, Xiaoqing; Yuan, Zonghui

    2012-01-11

    Incorrect use of tylosin and tilmicosin could result in allergy and select resistance. To monitor the illegal use of these antibiotics in animals, a monoclonal-based indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) has been established. Several haptens were synthesized and conjugated to carrier protein. Female Balb/c mice were inoculated with the four different conjugates to produce monoclonal antibodies according to the schemes of immunization. Aftercell fusion and culture several times, nine hybridoma cell lines were isolated. Only one, 3C4 that has isotype IgG2a, was selected for detailed study. The cross-reactivity of the monoclonal antibody 3C4 to tylosin and tilmicosin was 100% and 51% respectively. The standard curves based on the tylosin and tilmicosin matrix calibration ranged from 2.5 to 40 μg L(-1), with an IC(50) value of 6.1 μg L(-1) and 12.1 μg L(-1), respectively. The limits of detection of the ic-ELISA ranged from 5.1 μg kg(-1) to 13.8 μg kg(-1) in edible animal tissues. The recoveries were 74.1% to 120.7% with less than 18.6% of the coefficient of variation when tylosin and tilmicosin were spiked in various biological matrices with the concentrations of 25.0-200.0 μg kg(-1). Good correlations between the results of the ic-ELISA and high performance liquid chromatography were observed in the incurred tissues. These results suggest that the ic-ELISA is a sensitive, accurate and low-cost method that would be a useful tool for the screening of the residues of tylosin and tilmicosin in muscle, liver, milk, honey and eggs. PMID:22136611

  10. Chromatographic purification and characterization of antigens A and D from Mycobacterium paratuberculosis and their use in enzyme-linked immunosorbent assays for diagnosis of paratuberculosis in sheep.

    PubMed Central

    Sugden, E A; Brooks, B W; Young, N M; Watson, D C; Nielsen, K H; Corner, A H; Turcotte, C; Michaelides, A; Stewart, R B

    1991-01-01

    The protein antigens A and D were purified from culture filtrates and sonic extracts of laboratory strains of Mycobacterium paratuberculosis by salt precipitation and chromatography. The characterization of antigen A is shown here, and both antigens were evaluated along with lipoarabinomannan antigen in indirect enzyme-linked immunosorbent assays (ELISA) for the serodiagnosis of ovine paratuberculosis. After anion-exchange (DEAE-5PW) and hydrophobic (phenyl-5PW) chromatography using high-performance liquid chromatography, antigen A showed a prominant band in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) at 31 kDa with small amounts of low-molecular-mass proteins but with no evidence of antigen D. A single precipitin arc was evident with purified antigen A in crossed immunoelectrophoresis. The determination of the N-terminal amino acid sequence showed a high degree of homology between the 31-kDa component of antigen A and antigens of the BCG85 complex of Mycobacterium bovis BCG, a total of 24 of 26 residues being identical to those of BCG85C. A prominant SDS-PAGE band at 400 kDa and a single crossed-immunoelectrophoresis arc was also evident for antigen D after gel filtration (Sephacryl S-200), anion-exchange (DEAE-Sephacel), and concanavalin A-Sepharose affinity chromatography. By ELISA, purified antigen A detected antibody in the sera of 18 of 22 paratuberculosis-infected sheep (82% sensitivity), whereas the purified antigen D detected antibody in all 22 infected animals (100% sensitivity). Combined ELISA results showed increased specificity with some loss in sensitivity. Images PMID:1761688

  11. Humoral immune responses of Brucella-infected cattle, sheep, and goats to eight purified recombinant Brucella proteins in an indirect enzyme-linked immunosorbent assay.

    PubMed Central

    Letesson, J J; Tibor, A; van Eynde, G; Wansard, V; Weynants, V; Denoel, P; Saman, E

    1997-01-01

    Brucellosis research is currently focused on the identification of nonlipopolysaccharide (LPS) antigens which could potentially be useful for the specific serologic diagnosis of brucellosis as well as for vaccinal prophylaxis. On the basis of previous reports, we selected eight Brucella proteins (OMP36, OMP25, OMP19, OMP16, OMP10, p17, p15, and p39) as candidate antigens to be further evaluated. The genes encoding these proteins were cloned, sequenced, and overexpressed in Escherichia coli. The recombinant proteins were purified with a polyhistidine tag and metal chelate affinity chromatography and evaluated in an indirect enzyme-linked immunosorbent assay (iELISA). The specificity of the iELISA was determined with sera from healthy cattle, sheep, and goats and ranged from 95 to 99%, depending on the recombinant antigen and the species tested. Sera from experimentally infected, and from naturally infected, animals were used to evaluate the sensitivity of the iELISA. The antiprotein antibody response was often delayed when compared to the anti-smooth LPS (S-LPS) response and was limited to animals which developed an active brucellosis infection (experimentally infected pregnant animals and sheep and goats from areas where brucellosis is still endemic). Among the recombinant antigens, the three cytoplasmic proteins (p17, p15, and p39) gave the most useful results. More than 80% of the animals positive in S-LPS serology were also positive with one of these cytoplasmic proteins alone or a combination of two of them. None of the recombinant antigens detected experimentally infected nonpregnant cows and sheep or naturally infected cattle. This study is a first step towards the development of a multiprotein diagnostic reagent for brucellosis. PMID:9302205

  12. Diagnosis and Clinical Virology of Lassa Fever as Evaluated by Enzyme-Linked Immunosorbent Assay, Indirect Fluorescent-Antibody Test, and Virus Isolation

    PubMed Central

    Bausch, D. G.; Rollin, P. E.; Demby, A. H.; Coulibaly, M.; Kanu, J.; Conteh, A. S.; Wagoner, K. D.; McMullan, L. K.; Bowen, M. D.; Peters, C. J.; Ksiazek, T. G.

    2000-01-01

    The Lassa virus (an arenavirus) is found in West Africa, where it sometimes causes a severe hemorrhagic illness called Lassa fever. Laboratory diagnosis has traditionally been by the indirect fluorescent-antibody (IFA) test. However, enzyme-linked immunosorbent assays (ELISAs) for Lassa virus antigen and immunoglobulin M (IgM) and G (IgG) antibodies have been developed that are thought to be more sensitive and specific. We compared ELISA and IFA testing on sera from 305 suspected cases of Lassa fever by using virus isolation with a positive reverse transcription-PCR (RT-PCR) test as the “gold standard.” Virus isolation and RT-PCR were positive on 50 (16%) of the 305 suspected cases. Taken together, Lassa virus antigen and IgM ELISAs were 88% (95% confidence interval [CI], 77 to 95%) sensitive and 90% (95% CI, 88 to 91%) specific for acute infection. Due to the stringent gold standard used, these likely represent underestimates. Diagnosis could often be made on a single serum specimen. Antigen detection was particularly useful in providing early diagnosis as well as prognostic information. Level of antigenemia varied inversely with survival. Detection by ELISA of IgG antibody early in the course of illness helped rule out acute Lassa virus infection. The presence of IFA during both acute and convalescent stages of infection, as well as significant interobserver variation in reading the slides, made interpretation difficult. However, the assay provided useful prognostic information, the presence of IFA early in the course of illness correlating with death. The high sensitivity and specificity, capability for early diagnosis, and prognostic value of the ELISAs make them the diagnostic tests of choice for the detection of Lassa fever. PMID:10878062

  13. Accurate Detection of Adenylation Domain Functions in Nonribosomal Peptide Synthetases by an Enzyme-linked Immunosorbent Assay System Using Active Site-directed Probes for Adenylation Domains.

    PubMed

    Ishikawa, Fumihiro; Miyamoto, Kengo; Konno, Sho; Kasai, Shota; Kakeya, Hideaki

    2015-12-18

    A significant gap exists between protein engineering and enzymes used for the biosynthesis of natural products, largely because there is a paucity of strategies that rapidly detect active-site phenotypes of the enzymes with desired activities. Herein, we describe a proof-of-concept study of an enzyme-linked immunosorbent assay (ELISA) system for the adenylation (A) domains in nonribosomal peptide synthetases (NRPSs) using a combination of active site-directed probes coupled to a 5'-O-N-(aminoacyl)sulfamoyladenosine scaffold with a biotin functionality that immobilizes probe molecules onto a streptavidin-coated solid support. The recombinant NRPSs have a C-terminal His-tag motif that is targeted by an anti-6×His mouse antibody as the primary antibody and a horseradish peroxidase-linked goat antimouse antibody as the secondary antibody. These probes can selectively capture the cognate A domains by ligand-directed targeting. In addition, the ELISA technique detected A domains in the crude cell-free homogenates from the Escherichia coli expression systems. When coupled with a chromogenic substrate, the antibody-based ELISA technique can visualize probe-protein binding interactions, which provides accurate readouts of the A-domain functions in NRPS enzymes. To assess the ELISA-based engineering of the A domains of NRPSs, we reprogramed 2,3-dihydroxybenzoic acid (DHB)-activating enzyme EntE toward salicylic acid (Sal)-activating enzymes and investigated a correlation between binding properties for probe molecules and enzyme catalysts. We generated a mutant of EntE that displayed negligible loss in the kcat/Km value with the noncognate substrate Sal and a corresponding 48-fold decrease in the kcat/Km value with the cognate substrate DHB. The resulting 26-fold switch in substrate specificity was achieved by the replacement of a Ser residue in the active site of EntE with a Cys toward the nonribosomal codes of Sal-activating enzymes. Bringing a laboratory ELISA technique

  14. Interpretations of Antibody Responses to Salmonella enterica Serotype Enteritidis gm Flagellin in Poultry Flocks Are Enhanced by a Kinetics-Based Enzyme-Linked Immunosorbent Assay

    PubMed Central

    McDonough, Patrick L.; Jacobson, Richard H.; Timoney, John F.; Mutalib, Ahmed; Kradel, David C.; Chang, Yung-fu; Shin, Sang J.; Lein, Donald H.; Trock, Susan; Wheeler, Kaye

    1998-01-01

    Many regulatory and diagnostic programs for the detection of Salmonella enterica serotype Enteritidis infection in commercial poultry flocks have relied on rapid Pullorum agglutination tests to screen birds because of the shared antigens of S. enterica Enteritidis and S. enterica Pullorum and Gallinarum; however, the use of the enzyme-linked immunosorbent assay (ELISA) format affords better analytical sensitivity than crude agglutination tests. In this study, we adapted our earlier conventional indirect ELISA, using gm flagellin as the antigen, to a kinetics-based, computer-controlled ELISA (KELA). The KELA was used to screen for flagellin antibody from three commercial flocks: (i) a large flock involved in a U.S. Department of Agriculture trace back from a human S. enterica Enteritidis foodborne outbreak (n = 3,209), (ii) a flock infected with the endemic S. enterica Enteritidis serotype but which also had multiple other salmonella serotypes (n = 65), and (iii) an S. enterica Pullorum-infected flock (n = 12). The first flock (S. enterica Enteritidis prevalence of 2.45% based on culture) provided a field test of the KELA and allowed the calculation of diagnostic sensitivity (D-Sn) and diagnostic specificity (D-Sp). With a cutoff of 10 (used for screening flocks [i.e., high sensitivity]), the KELA has a D-Sn of 95.2% and a D-Sp of 18.5%; with a cutoff of 140 (used in confirmatory flock testing [i.e., high specificity]), the KELA has a D-Sn of 28.0% and a D-Sp of 99.1%. We found that with a cutoff of 60 (D-Sn = 63.1%; D-Sp = 91.6%), we could eliminate reactions in the KELA caused by other non-S. enterica Enteritidis salmonellae. The KELA was also compared to two commercial rapid Pullorum tests, the Solvay (D-Sn = 94.9%; D-Sp = 55.5%) and the Vineland (D-Sn = 62.0%; D-Sp = 75.3%). PMID:9665965

  15. A novel 57-kDa merozoite protein of Babesia gibsoni is a prospective antigen for diagnosis and serosurvey of canine babesiosis by enzyme-linked immunosorbent assay.

    PubMed

    Aboge, Gabriel Oluga; Jia, Honglin; Terkawi, Mohamad Alaa; Goo, Younkyoung; Kuriki, Ken; Nishikawa, Yoshifumi; Igarashi, Ikuo; Suzuki, Hiroshi; Xuan, Xuenan

    2007-10-21

    We isolated a novel single copy gene encoding a 57-kDa merozoite protein of Babesia gibsoni (BgP57). The nucleotide sequence of the cDNA was 2387 bp with an open reading frame (ORF) of 1644 bp encoding a 57-kDa predicted polypeptide having 547 amino acid residues. The recombinant BgP57 (rBgP57) without a predicted signal peptide was expressed in Escherichia coli as a soluble glutathione S-transferase (GST) fusion protein. Western blotting showed that the corresponding native protein was 57-kDa, consistent with molecular weight of predicted mature polypeptide. An indirect enzyme-linked immunosorbent assay (ELISA) using the rBgP57 detected specific antibodies in the sequential sera from a dog experimentally infected with B. gibsoni. Moreover, the antigen did not cross-react with antibodies to B. canis sub-species and closely related apicomplexan parasites indicating that the rBgP57 was a specific antigen for B. gibsoni antibodies. The diagnostic performance of ELISA based on rBgP57 using 107 sera from B. gibsoni-naturally infected dogs was the same as the previously identified rBgP32 but performed better than the previously studied rBgP50. Although, seminested PCR detected higher proportions (82%) of positive samples than the ELISAs, the Mcnemar's chi-square test showed that there was no significant difference in relative effectiveness of rBgP57-ELISA and seminested PCR (chi(2)=2.70; P=0.1003) in identifying positive samples. The rBgP57-ELISA when used in combination with rBgP32-ELISA and rBgP50-ELISA appeared to improve sensitivity of the rBgP57-ELISA for detection of B. gibsoni antibodies. Overall, the rBgP57-ELISA and seminested PCR when used in combination, could improve epidemiological surveys and clinical diagnosis of B. gibsoni infection. PMID:17706873

  16. Detection of serum antibodies to ovine progressive pneumonia virus in sheep by using a caprine arthritis-encephalitis virus competitive-inhibition enzyme-linked immunosorbent assay.

    PubMed

    Herrmann, Lynn M; Cheevers, William P; Marshall, Katherine L; McGuire, Travis C; Hutton, Melinda M; Lewis, Gregory S; Knowles, Donald P

    2003-09-01

    A competitive-inhibition enzyme-linked immunosorbent assay (cELISA) for detection of antibodies to the surface envelope (SU) of caprine arthritis-encephalitis virus (CAEV) was recently reported (L. M. Herrmann, W. P. Cheevers, T. C. McGuire, D. Scott Adams, M. M. Hutton, W. G. Gavin, and D. P. Knowles, Clin. Diagn. Lab. Immunol. 10:267-271, 2003). The cELISA utilizes CAEV-63 SU captured on microtiter plates using the monoclonal antibody (MAb) F7-299 and measures competitive displacement of binding of the anti-CAEV MAb GPB 74A by goat serum. The present study evaluated the CAEV cELISA for detection of antibodies to ovine progressive pneumonia virus (OPPV) in sheep. Three hundred thirty-two sera were randomly selected from 21,373 sheep sera collected throughout the United States to determine the sensitivity and specificity of cELISA and agar gel immunodiffusion (AGID) based on immunoprecipitation (IP) of [35S]methionine-labeled OPPV antigens as a standard of comparison. A positive cELISA test was defined as >20.9 percent inhibition (% I) of MAb 74A binding based on two standard deviations above the mean % I of 191 IP-negative sheep sera. At this cutoff, there were 2 of 141 false-negative sera (98.6% sensitivity) and 6 of 191 false-positive sera (96.9% specificity). Sensitivity and specificity values for IP-monitored AGID were comparable to those for cELISA for 314 of 332 sera with unambiguous AGID results. Concordant results by cELISA and IP resolved 16 of the 18 sera that were indeterminate by AGID. Additional studies evaluated cELISA by using 539 sera from a single OPPV-positive flock. Based on IP of 36 of these sera, there was one false-negative by cELISA among 21 IP-positive sera (95.5% sensitivity) and 0 of 15 false-positives (100% specificity). We conclude that the CAEV cELISA can be applied to detection of OPPV antibodies in sheep with high sensitivity and specificity. PMID:12965917

  17. Alpha2-plasmin inhibitor and alpha2-macroglobulin-plasmin complexes in plasma. Quantitation by an enzyme-linked differential antibody immunosorbent assay.

    PubMed Central

    Harpel, P C

    1981-01-01

    An enzyme-linked differential antibody immunosorbent assay has been developed for the quantification of alpha2-plasmin inhibitor-plasmin and alpha2-macroglobulin-plasmin complexes. In this method the inhibitor-plasmin complex is bound to a surface by an inhibitor-specific antibody, and the plasmin bound to the inhibitor is quantified by a second antibody, rabbit antiplasminogen F(ab')2, labeled with alkaline phosphatase. The hydrolysis of p-nitrophenyl phosphate by the alkaline phosphatase is expressed in femtomoles of plasminogen per milliliter, by reference to a standard plasminogen curve. Inhibitor-enzyme complexes were generated in plasma by the addition of plasmin or of urokinase. The concentration of plasmin added was well below the plasma concentration of alpha2-plasmin inhibitor (1 microM) or of alpha2-macroglobulin (3.5 microM), so that neither inhibitor would be fully saturated with enzyme. Under these conditions increasing amounts of plasmin generated an increase in both alpha2-plasmin inhibitor-plasmin and alpha2-macroglobulin-plasmin complexes. Varying amounts of plasmin were incubated with each of the purified inhibitors in the concentration found in plasma, and the complexes. Varying amounts of plasmin were incubated with each of the purified inhibitors in the concentration found in plasma, and the complexes that formed were quantified by immunoassay. These studies made it possible to quantify the distribution of plasmin between the two inhibitors in plasmin or urokinase-treated plasma. In plasmin-treated plasma, 10% or less of the plasmin bound to both inhibitors was in complex with alpha2-macroglobulin. In contrast, between 19 and 51% of the plasmin generated in urokinase-activated plasma was bound to alpha2-macroglobulin. Thus, major changes in the distribution of plasma were observed, according to whether plasmin was added to plasma or whether plasminogen was activated endogenously. The pattern of inhibitor plasmin complexes generated in vivo by

  18. Double-Antigen Enzyme-Linked Immunosorbent Assay for Detection of Hepatitis E Virus-Specific Antibodies in Human or Swine Sera ▿

    PubMed Central

    Hu, Wei Ping; Lu, Yang; Precioso, Nestor Amadeo; Chen, Hsiao Ying; Howard, Teresa; Anderson, David; Guan, Ming

    2008-01-01

    A new double-antigen sandwich-based enzyme-linked immunosorbent assay (ELISA) for the detection of total antibodies (immunoglobulin G [IgG] and IgM) specific for hepatitis E virus (HEV) was developed by utilizing well-characterized recombinant protein ET2.1 and its peroxidase-labeled counterpart. Our study showed that the ELISA detected all the positive patient samples (n = 265) regardless of whether they contained IgM or IgG antibodies, or both, while it maintained an excellent specificity of 98.8% with samples from various patient or healthy control groups (total number of samples, 424). The test had a detection limit for anti-HEV IgG antibodies that was equivalent to 62 mIU/ml of the international reference. Compared with the serological status of the specimens determined on the basis of tests performed at the individual collection sites, the testing outcome generated by the new ELISA had a good agreement of 99.3%, with a kappa value of 0.985. The positive predictive value and the negative predictive value for the new test reached 98.1% and 100%, respectively. This ELISA had a positive delta value of 4.836 and a negative delta value of 3.314 (where delta is a measure of the number of standard deviations by which the cutoff is separated from the mean of the sample groups) (N. Crofts, W. Maskill, and I. D. Gust, J. Virol. Methods 22:51-59, 1988), indicating that it had an excellent ability to differentiate the infected and noninfected cohorts. Furthermore, the new design enables the detection of antibodies not only in human samples but also in pig samples. Our preliminary data showed that the ELISA could detect seroconversion in samples from pigs at as early as 14 days postinoculation. The potential utility of detecting specific antibodies in pigs will be an added advantage for managing the disease, with suggested zoonotic implications. PMID:18495846

  19. Serodiagnosis of Toxoplasma gondii infection in farm animals (horses, swine, and sheep) by enzyme-linked immunosorbent assay using chimeric antigens.

    PubMed

    Ferra, Bartłomiej; Holec-Gąsior, Lucyna; Kur, Józef

    2015-10-01

    Toxoplasma gondii infects all warm-blooded animals including humans, causing serious public health problems and great economic loss in the animal husbandry. Commonly used serological tests for diagnosis of toxoplasmosis involve preparation of whole Toxoplasma lysate antigen (TLA) from tachyzoites. The production of this antigen is associated with high costs and lengthy preparation and the possibility of staff infection. There are also some difficulties in the standardization of such tests. One approach in order to improve the diagnosis of T. gondii infection is to use recombinant chimeric antigens in place of the TLA, which was confirmed by studies in the serodiagnosis of toxoplasmosis in humans. In this paper, we assess, for the first time, the diagnostic utility of five T. gondii recombinant chimeric antigens (MIC1-MAG1-SAG1S, SAG1L-MIC1-MAG1, SAG2-GRA1-ROP1S, SAG2-GRA1-ROP1L, and GRA1-GRA2-GRA6) in immunoglobulin G (IgG) enzyme-linked immunosorbent assays (IgG ELISAs) with sera from three different groups of livestock animals (horses, pigs, and sheep). The reactivity of individual chimeric antigens was analyzed in relation to the results obtained in IgG ELISAs based on a mixture of three antigens (M1: rSAG1+rMIC1+rMAG1, M2: rSAG2+rGRA1+rROP1, and M3: rGRA1+rGRA2+rGRA6) and referenced to TLA. All chimeric antigens were characterized by high specificity (100%), and the sensitivity of the IgG ELISAs based on chimeric antigens was variable (between 28.4% and 100%) and mainly dependent on the animal species. The chimeric antigens were generally more reactive than mixtures of three antigens. The most effective for the diagnosis of toxoplasmosis was SAG2-GRA1-ROP1L, which can detect specific anti-T. gondii antibodies in 100%, 93.8%, and 100% of positive serum samples from horses, pigs, and sheep, respectively. The present study shows that recombinant chimeric antigens can be successfully used to diagnose T. gondii infection in farm animals, and can replace the commonly

  20. Development of an enzyme-linked immunosorbent assay for the detection of human calretinin in plasma and serum of mesothelioma patients

    PubMed Central

    2010-01-01

    Background Calretinin is one of the well-established immunohistochemical markers in the diagnostics of malignant mesothelioma (MM). Its utility as a diagnostic tool in human blood, however, is scarcely investigated. The aim of this study was to develop an enzyme-linked immunosorbent assay (ELISA) for human calretinin in blood and to assess its usefulness as a potential minimally invasive diagnostic marker for MM. Methods Initially, attempts were made to establish an assay using commercially available antibodies and to optimize it by including a biotin-streptavidin complex into the assay protocol. Subsequently, a novel ELISA based on polyclonal antibodies raised in rabbit immunized with human recombinant calretinin was developed. The assay performance in human serum and plasma (EDTA/heparin) and the influence of calcium concentrations on antibody recognition were studied. Stability of spiked-in calretinin in EDTA plasma under different storage conditions was also examined. In preliminary studies serum and plasma samples from 97 healthy volunteers, 35 asbestos-exposed workers, and 42 MM patients were analyzed. Results The mean detection range of the new ELISA was 0.12 to 8.97 ng/ml calretinin. The assay demonstrated markedly lower background and significantly higher sensitivity compared to the initially contrived assay that used commercial antibodies. Recovery rate experiments confirmed dependence of calretinin antibody recognition on calcium concentration. Calcium adjustment is necessary for calretinin measurement in EDTA plasma. Spiked-in calretinin revealed high stability in EDTA plasma when stored at room temperature, 4°C, or after repeated freeze/thaw cycles. Median calretinin values in healthy volunteers, asbestos workers, and MM patients were 0.20, 0.33, and 0.84 ng/ml, respectively (p < 0.0001 for healthy vs. MM, p = 0.0036 for healthy vs. asbestos-exposed, p < 0.0001 for asbestos-exposed vs. MM). Median values in patients with epithelioid and biphasic MM

  1. Development of a highly sensitive and specific monoclonal antibody-based enzyme-linked immunosorbent assay (ELISA) for detection of Sudan I in food samples.

    PubMed

    Wang, Yuzhen; Wei, Dapeng; Yang, Hong; Yang, Yuan; Xing, Weiwei; Li, Yuan; Deng, Anping

    2009-03-15

    The use of Sudan I as an additive in food products has been prohibited in the European Union and many other countries. In this study, a highly sensitive and specific monoclonal antibody (mAb)-based indirect competitive enzyme-linked immunosorbent assay (ELISA) for the detection of Sudan I in food samples was developed. The hapten derivative with a three-carbon-atom length of carboxylic spacer at the azobound para-position was synthesized and coupled to carrier proteins. The hapten-bovine serum albumin (BSA) conjugate was used as an immunogen, while the hapten-ovalbumin (OVA) conjugate was applied as a coating antigen. The mAb against Sudan I was produced by hybridoma technique and the corresponding ELISA was characterized in terms of sensitivity, specificity, precision and accuracy. At optimal experimental conditions, the standard curve was constructed in concentrations of 0.1-100 ngmL(-1). The values of IC(50) for nine standard curves were in the range of 1.1-2.0 ngmL(-1) and the LOD at a signal-to-noise ratio of 3 (S/N=3) was 0.07-0.14 ngmL(-1). The cross-reactivity values of the mAb with Sudan II, III and IV were 9.5%, 33.9% and 0.95%; no cross-reactivity was found with other six edible colorants: Lemon yellow, Bright blue, Indigotin, Kermes, Amarant and Sunset yellow, indicating the assay displays not only high sensitivity but also high specificity as well. The organic solvent effect on the assay was tested. It was observed that the ELISA was tolerated to 30% of methanol and 10% of acetonitrile without significant loss of IC(50) value. Six food samples were spiked with Sudan I and the methanolic extracts after appropriate dilution were analyzed by ELISA. Acceptable recovery rates of 88.2-110.5% and coefficients of variation of 2.5-17.4% were obtained. The ELISA for nine spiked samples was confirmed by high-performance liquid chromatography (HPLC) with a high correlation coefficient of 0.9840 (n=9). The mAb-based ELISA proven to be a feasible quantitative

  2. Assessment of colostral transfer and systemic availability of immunoglobulin G in new-born foals using a newly developed enzyme-linked immunosorbent assay (ELISA) system.

    PubMed

    Erhard, M H; Luft, C; Remler, H P; Stangassinger, M

    2001-06-01

    To measure the immunoglobulin G (IgG) concentration in colostrum, milk and serum samples, a sandwich enzyme-linked immunosorbent assay (ELISA) detection system was developed. The system provided high reproducibility and sensitivity for routine diagnostic purposes. The period of fluctuating serum concentrations of IgG was monitored in new-born foals and their mares for a period of 6 weeks postnatum and postpartum, respectively. All foals received colostrum from their mares. The mean IgG concentration in the precolostral mare serum was approximately 19.0 mg/ml and decreased significantly to 13.8 mg/ml within the first 24 h postpartum. The IgG value fell to a minimum of 11.2 mg/ml by day 21 and increased to 21.6 mg/ml by day 42 postpartum. Within the first 4 h postpartum, mean IgG concentrations of 54.5 mg/ml were measured in the colostrum. A significant decrease to 10.1 mg IgG/ml colostrum was then noted 9-12 h postpartum. The mean IgG concentrations in foal serum increased from 0.3 mg/ml (precolostral value) to 9.6 mg/ml within 5-8 h postnatum. After 13-16 h postnatum, the highest IgG value of 15.7 mg/ml was reached. Over time the mean IgG concentration decreased significantly to 7.9 mg/ml at day 35. At the end of the observation period (day 42 postnatum) the mean IgG concentration once again increased to 11.2 mg/ml serum. In addition, the possible influence of various parameters on IgG concentration were examined. No significant influences could be shown by the breed, mare age, number of pregnancies, days of gestation, month foaled, foal sex, or the different farms. Finally, the cumulative incidence of failure of passive transfer (FPT) defined as IgG levels < 4 mg/ml foal serum, and partial FPT (PFPT) at levels ranging from 4 to 8 mg/ml foal serum was determined. From a total of 70 foals, 10.0% showed FPT and 18.6% showed PFPT. PMID:11686785

  3. Comparison of cytomegalovirus (CMV) enzyme-linked immunosorbent spot and CMV quantiferon gamma interferon-releasing assays in assessing risk of CMV infection in kidney transplant recipients.

    PubMed

    Abate, Davide; Saldan, Alda; Mengoli, Carlo; Fiscon, Marta; Silvestre, Cristina; Fallico, Loredana; Peracchi, Marta; Furian, Lucrezia; Cusinato, Riccardo; Bonfante, Luciana; Rossi, Barbara; Marchini, Francesco; Sgarabotto, Dino; Rigotti, Paolo; Palù, Giorgio

    2013-08-01

    Assessing cytomegalovirus (CMV)-specific cell-mediated immunity (CMI) represents an appealing strategy for identifying transplant recipients at risk of infection. In this study, we compared two gamma interferon-releasing assays (IGRAs), Quantiferon-CMV and CMV enzyme-linked immunosorbent spot (ELISPOT), to determine the ability of each test to predict protective CMV-specific T-cell responses. Two hundred twenty-one Quantiferon-CMV and ELISPOT tests were conducted on 120 adult kidney transplant recipients (KTRs), including 100 CMV-seropositive transplant recipients (R+) and 20 CMV-seronegative transplant recipients of a CMV-positive donor (D+/R-). As a control cohort, 39 healthy adult subjects (including 33 CMV-seropositive and 6 CMV-seronegative subjects) were enrolled. CMV IgG serology was used as a reference for both tests. In the CMV-seropositive individuals, the ELISPOT and Quantiferon-CMV assays provided 46% concordance with the serology, 12% discordance, 18% disagreement between ELISPOT or Quantiferon-CMV and the serology, and 24% gray areas when one or both tests resulted in weak positives. None of the CMV-seronegative subjects showed detectable responses in the ELISPOT or the Quantiferon-CMV test. In transplant recipients, both the ELISPOT and Quantiferon-CMV assays positively correlated with each other and negatively correlated with CMV DNAemia in a significant way (P<0.05). During the antiviral prophylaxis, all 20 D+/R- KTRs we examined displayed undetectable Quantiferon-CMV and ELISPOT results, and there was no evidence of CMV seroconversion. The receiving operator curve (ROC) statistical analysis revealed similar specificities and sensitivities in predicting detectable viremia (areas under the curve [AUC], 0.66 and 0.62 for Quantiferon-CMV and ELISPOT, respectively). ELISPOT and Quantiferon-CMV values of >150 spots/200,000 peripheral blood mononuclear cells (PBMCs) and >1 to 6 IU gamma interferon (IFN-γ) were associated with protection from CMV

  4. Adaptations of a competitive enzyme-linked immunosorbent assays for the detection of antibodies to influenza a virus in horse sera for use in wild aquatic birds.

    PubMed

    Hoque, M A; Skerratt, L F; Garland, S; Burgess, G W; Selleck, P

    2012-12-01

    We applied a competitive enzyme-linked immunosorbent assay for the detection of antibodies for influenza A in equine sera to their detection in sera from wild aquatic birds. Suboptimal results were obtained for the optical density (OD) of the monoclonal antibody (MAb) control and reproducibility between duplicate analyses in the initial assessment. It was therefore necessary to modify the assay to deliver increased reliability and reproducibility while maintaining adequate sensitivity. We optimized reagent concentrations to obtain optimal OD values (close to 2) for the monoclonal antibody control and used 2, 2'-Azino-bis: 3-Benzthiazoline-6-Sulphonic Acid as an alternative chromogen to potentially reduce variability in duplicate analyses. The original assay was compared with the optimized versions, with and without post coating, for the detection of avian influenza viral antibodies in 240 sera obtained from wild plumed whistling ducks. A separate analytical sensitivity study on diluted positive field sera of plumed whistling ducks and a test of antigen stability after post coating were also performed. Some quantitative differences were detected between the original and modified assays. The original assay recorded higher percentage inhibition results which were potentially indicative of increased sensitivity. However, when reagent concentrations were increased in the original assay to the same levels as used in the modified versions, there were no quantitative differences for practical purposes. The original assay produced a median (OD) value of 0.81 for the (MAb) controls that is at the limit of acceptability. By contrast, the modified assays always produced acceptable optical density values for MAb controls. Our overall results indicated the modified assays were potentially more reliable (OD values close to 2), and of adequate sensitivity compared to the original assay in the detection of avian influenza viral antibodies in wild bird sera. Although further

  5. Nonstructural Protein 1-Specific Immunoglobulin M and G Antibody Capture Enzyme-Linked Immunosorbent Assays in Diagnosis of Flaviviral Infections in Humans

    PubMed Central

    Galula, Jedhan Ucat; Shen, Wen-Fan; Davis, Brent S.

    2014-01-01

    IgM antibody- and IgG antibody-capture enzyme-linked immunosorbent assays (MAC/GAC-ELISAs) targeted at envelope protein (E) of dengue viruses (DENV), West Nile virus, and Japanese encephalitis virus (JEV) are widely used as serodiagnostic tests for presumptive confirmation of viral infection. Antibodies directed against the flavivirus nonstructural protein 1 (NS1) have been proposed as serological markers of natural infections among vaccinated populations. The aim of the current study is to optimize an IgM and IgG antibody-capture ELISA (MAC/GAC-ELISA) to detect anti-NS1 antibodies and compare it with anti-E MAC/GAC-ELISA. Plasmids to express premembrane/envelope (prM/E) or NS1 proteins of six medically important flaviviruses, including dengue viruses (DENV-1 to DENV-4), West Nile virus (WNV), and Japanese encephalitis virus (JEV), were constructed. These plasmids were used for the production of prM/E-containing virus-like particles (VLPs) and secreted NS1 (sNS1) from COS-1 cells. Archived clinical specimens from patients with confirmed DENV, JEV, and WNV infections, along with naive sera, were subjected to NS1-MAC/GAC-ELISAs before or after depletion of anti-prM/E antibodies by preabsorption with or without VLPs. Human serum specimens from previously confirmed DENV infections showed significantly enhanced positive-to-negative (P/N) ratios for NS1-MAC/GAC-ELISAs after the depletion of anti-prM/E antibodies. No statistical differences in sensitivities and specificities were found between the newly developed NS1- and VLP-MAC/GAC-ELISAs. Further application of the assays to WNV- and JEV-infected serum panels showed similar results. A novel approach to perform MAC/GAC-ELISAs for NS1 antibody detection was successfully developed with great potential to differentiate antibodies elicited by the tetravalent chimeric yellow fever-17D/dengue vaccine or DENV infection. PMID:25502522

  6. Nonstructural protein 1-specific immunoglobulin M and G antibody capture enzyme-linked immunosorbent assays in diagnosis of flaviviral infections in humans.

    PubMed

    Chao, Day-Yu; Galula, Jedhan Ucat; Shen, Wen-Fan; Davis, Brent S; Chang, Gwong-Jen J

    2015-02-01

    IgM antibody- and IgG antibody-capture enzyme-linked immunosorbent assays (MAC/GAC-ELISAs) targeted at envelope protein (E) of dengue viruses (DENV), West Nile virus, and Japanese encephalitis virus (JEV) are widely used as serodiagnostic tests for presumptive confirmation of viral infection. Antibodies directed against the flavivirus nonstructural protein 1 (NS1) have been proposed as serological markers of natural infections among vaccinated populations. The aim of the current study is to optimize an IgM and IgG antibody-capture ELISA (MAC/GAC-ELISA) to detect anti-NS1 antibodies and compare it with anti-E MAC/GAC-ELISA. Plasmids to express premembrane/envelope (prM/E) or NS1 proteins of six medically important flaviviruses, including dengue viruses (DENV-1 to DENV-4), West Nile virus (WNV), and Japanese encephalitis virus (JEV), were constructed. These plasmids were used for the production of prM/E-containing virus-like particles (VLPs) and secreted NS1 (sNS1) from COS-1 cells. Archived clinical specimens from patients with confirmed DENV, JEV, and WNV infections, along with naive sera, were subjected to NS1-MAC/GAC-ELISAs before or after depletion of anti-prM/E antibodies by preabsorption with or without VLPs. Human serum specimens from previously confirmed DENV infections showed significantly enhanced positive-to-negative (P/N) ratios for NS1-MAC/GAC-ELISAs after the depletion of anti-prM/E antibodies. No statistical differences in sensitivities and specificities were found between the newly developed NS1- and VLP-MAC/GAC-ELISAs. Further application of the assays to WNV- and JEV-infected serum panels showed similar results. A novel approach to perform MAC/GAC-ELISAs for NS1 antibody detection was successfully developed with great potential to differentiate antibodies elicited by the tetravalent chimeric yellow fever-17D/dengue vaccine or DENV infection. PMID:25502522

  7. Analysis by enzyme-linked immunosorbent assay and 2-dimensional electrophoresis of haptoglobin in the high-density lipoprotein fraction in cows.

    PubMed

    Kanno, H; Katoh, N

    2001-01-01

    Haptoglobin (Hp) is a hemoglobin (Hb)-binding acute-phase protein. Besides its relevance in inflammation, Hp is involved in the regulation of lipid metabolism. In cattle, in addition to the lipoprotein-deficient fraction, Hp is distributed in high-density lipoprotein (HDL) and very high-density lipoprotein (VHDL) fractions. The purpose of this study was to determine Hp concentrations in the lipoprotein fractions using an enzyme-linked immunosorbent assay (ELISA) based on the affinity with Hb, and also to detect structural differences of HDL Hp from that in the lipoprotein-deficient fraction using 2-dimensional electrophoresis. When purified Hp was used as the antigen for the ELISA, the detection limit was 7.4 ng/ml and linearity was obtained from 14.8 to 475 ng/ml. The correlation coefficient between the ELISA and single radial immunodiffusion was 0.884. The ELISA was shown to be applicable to evaluate Hp concentrations in the lipoprotein fractions. Hp concentrations in the lipoprotein fractions were in the range of 0.94 to 8.77 microg of Hp/ml (n = 4), and concentration ratios were 0.2 to 0.3% of whole serum Hp. Of the lipoprotein fractions, Hp was most abundant in HDL, moderate in VHDL and faint in chylomicrons, the very low-density lipoprotein fraction and low-density lipoprotein fraction. By 2-dimensional electrophoresis, alpha- and beta-chains of serum Hp were each separated into 5 spots, and their isoelectric point (pI) values were from 5.05 to 6.28 in the alpha-chain and from 5.92 to 6.95 in the beta-chain. The pI values of HDL Hp were indistinguishable from those of serum Hp. These results indicate that the ELISA based on the affinity with Hb is useful for evaluating Hp concentrations in lipoprotein fractions, and also suggest that HDL Hp is structurally similar to that in the lipoprotein-deficient fraction. PMID:11217066

  8. Rapid Mycobacterial Liquid Culture-Screening Method for Mycobacterium avium Complex Based on Secreted Antigen-Capture Enzyme-Linked Immunosorbent Assay▿

    PubMed Central

    Shin, Sung Jae; Anklam, Kelly; Manning, Elizabeth J. B.; Collins, Michael T.

    2009-01-01

    Sensors in automated liquid culture systems for mycobacteria, such as MGIT, BacT/Alert 3D, and Trek ESP II, flag growth of any type of bacteria; a positive signal does not mean that the target mycobacteria are present. All signal-positive cultures thus require additional and often laborious testing. An immunoassay was developed to screen liquid mycobacterial cultures for evidence of Mycobacterium avium complex (MAC). The method, called the MAC-enzyme-linked immunosorbent assay (ELISA), relies on detection of MAC-specific secreted antigens in liquid culture. Secreted MAC antigens were captured by the MAC-ELISA with polyclonal anti- Mycobacterium avium subsp. paratuberculosis chicken immunoglobulin Y (IgY), detected using rabbit anti-MAC IgG, and then revealed using horseradish peroxidase-conjugated goat anti-rabbit IgG. When the MAC-ELISA was evaluated using pure cultures of known mycobacterial (n = 75) and nonmycobacterial (n = 17) organisms, no false-positive or false-negative MAC-ELISA results were found. By receiver operator characteristic (ROC) analysis of 1,275 previously identified clinical isolates, at the assay optimal cutoff the diagnostic sensitivity and specificity of the MAC-ELISA were 92.6% (95% confidence interval [95% CI], 90.3 to 94.5) and 99.9% (95% CI, 99.2 to 100), respectively, with an area under the ROC curve of 0.992. Prospective evaluation of the MAC-ELISA with an additional 652 clinical samples inoculated into MGIT ParaTB medium and signaling positive per the manufacturer's instructions found that the MAC-ELISA was effective in determining those cultures that actually contained MAC species and warranting the resources required to identify the organism by PCR. Of these 652 MGIT-positive cultures, the MAC-ELISA correctly identified 96.8% (of 219 MAC-ELISA-positive cultures) as truly containing MAC mycobacteria, based on PCR or high-performance liquid chromatography (HPLC) as reference tests. Only 6 of 433 MGIT signal-positive cultures (1

  9. Occurrence and Distribution of Pesticides in the St. Lucie River Watershed, South-Central Florida, 2000-01, Based on Enzyme-Linked Immunosorbent Assay (ELISA) Screening

    USGS Publications Warehouse

    Lietz, A.C.

    2003-01-01

    The St. Lucie River watershed is a valuable estuarine ecosystem and resource in south-central Florida. The watershed has undergone extensive changes over the last century because of anthropogenic activities. These activities have resulted in a complex urban and agricultural drainage network that facilitates the transport of contaminants, including pesticides, to the primary canals and then to the estuary. Historical data indicate that aquatic life criteria for selected pesticides have been exceeded. To address this concern, a reconnaissance was conducted to assess the occurrence and distribution of selected pesticides within the St. Lucie River watershed. Numerous water samples were collected from 37 sites among various land-use categories (urban/built-up, citrus, cropland/pastureland, and inte-grated). Samples were collected at inflow points to primary canals (C-23, C-24, and C-44) and at control structures along these canals from October 2000 to September 2001. Samples were screened for four pesticide classes (triazines, chloroacetanilides, chlorophenoxy compounds, and organophosphates) by using Enzyme-Linked Immunosorbent Assay (ELISA) screening. A temporal distribution of pesticides within the watershed was made based on samples collected at the integrated sites during different rainfall events between October 2000 and September 2001. Triazines were detected in 32 percent of the samples collected at the integrated sites. Chloroacetanilides were detected in 60 percent of the samples collected at the integrated sites, with most detections occurring at one site. Chlorophenoxy compounds were detected in 17 percent of the samples collected at the integrated sites. Organophosphates were detected in only one sample. A spatial distribution and range of concentration of pesticides at the 37 sampling sites in the watershed were determined among land-use categories. Triazine concentrations ranged from highest to lowest in the citrus, urban/built-up, and integrated areas

  10. Quality assurance/quality control of foot and mouth disease solid phase competition enzyme-linked immunosorbent assay--Part II. Quality control: comparison of two charting methods to monitor assay performance.

    PubMed

    Goris, N; De Clercq, K

    2005-12-01

    Diagnostic laboratories are increasingly required to meet stringent quality standards, and validated assays are needed to achieve formal accreditation. Validation of test methods is often considered to be finalised when the assay parameters and characteristics have been established. However, like any process, diagnostic assays are subject to random variation resulting in shifts in the mean test values. Continuous monitoring of assays using control charts will alert the interpreter of changes in performance. For this purpose, several charting methods have been developed and implemented. This paper compares the Shewhart and the exponentially weighted moving average (EWMA) control charts with respect to the day to day monitoring of internal quality control samples for the foot and mouth disease solid phase competition enzyme-linked immunosorbent assay. Both chart types are equally sensitive to shifts, but the EWMA method seems to provide the best balance between false rejection and false acceptance. PMID:16642771

  11. Potential application of nonstructural protein NS1 serotype-specific immunoglobulin G enzyme-linked immunosorbent assay in the seroepidemiologic study of dengue virus infection: correlation of results with those of the plaque reduction neutralization test.

    PubMed

    Shu, Pei-Yun; Chen, Li-Kuang; Chang, Shu-Fen; Yueh, Yi-Yun; Chow, Ling; Chien, Li-Jung; Chin, Chuan; Yang, Hui-Hua; Lin, Ting-Hsiang; Huang, Jyh-Hsiung

    2002-05-01

    An NS1 serotype-specific indirect enzyme-linked immunosorbent assay (ELISA) was developed to differentiate primary and secondary dengue virus infections and serotypes of primary dengue virus infection. For this report, we carried out retrospective seroepidemiologic studies on serum samples collected from residents of Liuchiu Hsiang, Pingtung County, an isolated island in southern Taiwan during 1997-1998. The results demonstrated that good correlation existed between dengue virus NS1 serotype-specific immunoglobulin G (IgG) ELISA and dengue virus plaque reduction neutralization test (PRNT). Our data suggested that NS1 serotype-specific IgG ELISA could replace PRNT for seroepidemiologic studies to differentiate Japanese encephalitis and dengue virus infections and for dengue virus serotyping. PMID:11980973

  12. Evaluation of the performance of a rapid enzyme-linked immunosorbent assay in the detection of Anaplasma phagocytophilum antibodies in horses.

    PubMed

    Veronesi, Fabrizia; Passamonti, Fabrizio; Moretti, Annabella; Morganti, Giulia; Vardi, Doron Moshe; Laus, Fulvio; Marenzoni, Maria Luisa; Spaterna, Andrea; Coletti, Mauro; Fioretti, Daniela Piergili

    2014-05-01

    The aim of this study was to evaluate the performance of a commercially available rapid enzyme-linked immonosorbent assay, the Snap® 4Dx test, in the detection of Anaplasma phagocytophilum antibodies in horses. Two hundred apparently healthy horses (asymptomatic) and 244 animals showing clinical symptoms (symptomatic), were tested for A. phagocytophilum immunoglobulin G (IgG) antibodies using both the Snap® 4Dx kit and an indirect fluorescence antibody test (IFAT), with the latter serving as a comparative test. Horses belonging to the symptomatic group were also tested for evidence of active infection with A. phagocytophilum by analysis of IFAT IgM titers and PCR assay amplifying a specific fragment of the 16S rRNA gene. The overall agreement between the results obtained using the two tests, as well as the relative performance exhibited by the Snap® 4Dx test in the two groups, was assessed. Forty of the 45 animals (89%) testing positive for IgG antibodies using IFAT were correctly identified using Snap® 4Dx testing. The agreement between the results of the two tests was very high (k>0.9), with almost identical performances in both symptomatic and asymptomatic animals. Conversely, within the symptomatic group, only 44% (no. 11/25) of Snap® 4Dx positives appeared to be associated with a state of active infection, whereas the remaining 56% (no. 14/25) were related both to not infected animals (no. 1) and to horses whose status of infection needed further evaluations to be confirmed (no. 13/25). This study suggests that the Snap® 4Dx test could represent a valid screening method for use during epidemiological surveys of equine populations. Nevertheless, in-clinic application of the test does not appear to be merited. PMID:24745728

  13. Applications of PCR (real-time and MassTag) and enzyme-linked immunosorbent assay in diagnosis of respiratory infections and diarrheal illness among deployed U.S. military personnel during exercise Balikatan 2009, Philippines.

    PubMed

    Velasco, John Mark S; Yoon, In-Kyu; Mason, Carl J; Jarman, Richard G; Bodhidatta, Ladaporn; Klungthong, Chonticha; Silapong, Sasikorn; Valderama, Maria Theresa G; Wongstitwilairoong, Tippa; Torres, Arturo G; De Cecchis, Daniel P; Pavlin, Julie A

    2011-10-01

    Laboratory-based surveillance for diarrheal and respiratory illness was conducted at the 2009 Republic of the Philippines-United States Balikatan exercise to determine the presence of specific pathogens endemic in the locations where the military exercises were conducted. Ten stool and 6 respiratory specimens were obtained from individuals meeting case definitions for diarrhea or respiratory illness. Stool specimens were frozen in dry ice and remotely tested using enzyme-linked immunosorbent assay for Rotavirus, Astrovirus, Adenovirus, Entamoeba histolytica, Giardia, and Cryptosporidium and polymerase chain reaction for enterotoxigenic Escherichia coli, Campylobacter, Shigella, Vibrio, Salmonella, and Norovirus. Eight (4 for Campylobacter jejuni, 2 for Campylobacter coli, 1 for Norovirus genogroup II, and 1 for both Campylobacter coli and enterotoxigenic Escherichia coli) of 10 samples were positive for at least 1 enteric pathogen. MassTag polymerase chain reaction for influenza A and B, respiratory syncytial virus groups A and B, human coronavirus-229E and human coronavirus-OC43, human metapneumovirus, enterovirus, human parainfluenza viruses 2,3, and 4a, human adenovirus, Haemophilus influenzae, Neisseria meningitidis, Streptococcus pneumoniae, Legionella pneumonia, and Mycoplasma pneumonia was done on respiratory specimens. Out of 6 samples, 3 tested positive for H. influenzae; 1 tested positive for both H. influenzae and human parainfluenza virus 3; and 2 tested negative. Laboratory-based surveillance can be useful in determining etiologies of diarrheal and respiratory illness of deployed military personnel. PMID:22128641

  14. Specific and sensitive enzyme-linked immunosorbent assays for analysis of residual allergenic food proteins in commercial bottled wine fined with egg white, milk, and nongrape-derived tannins.

    PubMed

    Rolland, Jennifer M; Apostolou, Effie; de Leon, Maria P; Stockley, Creina S; O'Hehir, Robyn E

    2008-01-23

    Regulations introduced by the Food Standards Australia New Zealand in December 2002 require all wine and wine product labels in Australia to identify the presence of a processing aid, additive or other ingredient, which is known to be a potential allergen. The objective of this study was to establish sensitive assays to detect and measure allergenic proteins from commonly used processing aids in final bottled wine. Sensitive and specific enzyme-linked immunosorbent assays (ELISA) were developed and established for the proteins casein, ovalbumin, and peanut. Lower limits of detection of these proteins were 8, 1, and 8 ng/mL, respectively. A panel of 153 commercially available bottled Australian wines were tested by these ELISA, and except for two red wines known to contain added whole eggs, residuals of these food allergens were not detected in any wine. These findings are consistent with a lack of residual potentially allergenic egg-, milk-, or nut-derived processing aids in final bottled wine produced in Australia according to good manufacturing practice at a concentration that could cause an adverse reaction in egg, milk, or peanut/tree-nut allergic adult consumers. PMID:18163561

  15. Comparison of Dissociation-Enhanced Lanthanide Fluorescent Immunoassays to Enzyme-Linked Immunosorbent Assays for Detection of Staphylococcal Enterotoxin B, Yersinia pestis-Specific F1 Antigen, and Venezuelan Equine Encephalitis Virus

    PubMed Central

    Smith, Darci R.; Rossi, Cynthia A.; Kijek, Todd M.; Henchal, Erik A.; Ludwig, George V.

    2001-01-01

    The dissociation-enhanced lanthanide fluorescent immunoassays (DELFIA) were developed for the detection of staphylococcal enterotoxin B, Yersinia pestis-specific F1 antigen, and Venezuelan equine encephalitis virus. These assays were compared to previously developed enzyme-linked immunosorbent assays (ELISAs) by determining the sensitivity or limit of detection (LOD), the dynamic range, and the reproducibility of each assay in a number of different sample matrices. The sensitivity and specificity of each assay were then determined by using a small panel of blinded spiked and nonspiked samples. All three DELFIAs demonstrated at least 1 log greater sensitivity than corresponding ELISAs utilizing the same reagents and showed an increase in dynamic range of at least 2 log10 concentrations. This increased LOD resulted in higher sensitivity rates for the DELFIA. The specificity of all of the assays evaluated was 100%, and no sample matrix effects were observed in either format. However, the reproducibility of the DELFIA was poor due to randomly distributed wells exhibiting excessive background signal (hot wells), which occurred throughout the evaluation. As this technology matures, the reproducibility of these assays should improve, as will the ability to identify hot wells. Despite its sensitivity, the logistical burden associated with the DELFIA and the technical expertise required to complete assays and interpret the data limit the application of this technology to reference or large clinical laboratories. PMID:11687442

  16. Enhanced immunoassay for porcine circovirus type 2 antibody using enzyme-loaded and quantum dots-embedded shell-core silica nanospheres based on enzyme-linked immunosorbent assay.

    PubMed

    Wu, Long; Li, Xuepu; Shao, Kang; Ye, Shiyi; Liu, Chen; Zhang, Chenjun; Han, Heyou

    2015-08-01

    Boosting the detection sensitivity of enzyme-linked immunosorbent assay (ELISA) is significant to the early clinical diagnosis of various diseases. Here, we developed a versatile immunosensor using silica nanospheres as carriers for sensitive detection of porcine circovirus type 2 (PCV2) antibody. With HRP enzyme covalently immobilized on the silica nanospheres and CdSe nanocrystals embedded inside, these signal probes were successfully utilized in the sensitive detection of PCV2 antibody by ELISA, fluorometry and square-wave voltammetry (SWV). To further demonstrate the performance of the immunosensor, Human IgG (HIgG) was used as a model analyte. Since more HRP and CdSe QDs were loaded, 5-, 200- and 400-fold enhancements in amplified ELISA, fluorometry and voltammetry responses for HIgG could be achieved compared to conventional ELISA. The respective detection limits of theses methods for HIgG were 3.9, 0.1 and 0.05 ng mL(-1) with a RSD below 5% for amplified ELISA, fluorescence and SWV measurements. Additionally, a 100-fold improvement was obtained in the detection sensitivity for PCV2 antibody immunoassay. The versatile immunosensor exhibits good sensitivity, stability and reproducibility, suggesting its potential applications in clinical diagnostics. PMID:26320802

  17. Sequence analysis of Malaysian infectious bursal disease virus isolate and the use of reverse transcriptase nested polymerase chain reaction enzyme-linked immunosorbent assay for the detection of VP2 hypervariable region.

    PubMed

    Phong, S F; Hair-Bejo, M; Omar, A R; Aini, I

    2003-01-01

    The VP2 hypervariable region of P97/302 local infectious bursal disease virus (IBDV) isolate was amplified by the reverse transcriptase (RT) nested polymerase chain reaction (PCR) and cloned. This region of P97/302 local isolate was sequenced and compared with eight other reported IBDV sequences. The result showed that P97/302 IBDV was most identical to the reported very virulent IBDV strains because it has amino acid substitutions at positions 222, 256, 294, and 299, which encode alanine, isoleucine, isoleucine, and serine, respectively. This region can be digested with restriction enzymes of Taq1, Sty1, Ssp1 but not with Sac1. The P97/302 isolate was then used for the optimization of RT nested PCR enzyme-linked immunosorbent assay (ELISA). The RT nested PCR ELISA was able to detect 10(-4) dilution of the infected bursa homogenates and was 10 times more sensitive when compared with the agarose gel detection method. The RT nested PCR ELISA can detect up to 0.48 ng of the PCR product. The specificity of this nested PCR ELISA was also high (100%). PMID:12713171

  18. Sensitivity and specificity enhanced enzyme-linked immunosorbent assay by rational hapten modification and heterogeneous antibody/coating antigen combinations for the detection of melamine in milk, milk powder and feed samples.

    PubMed

    Cao, Biyun; Yang, Hong; Song, Juan; Chang, Huafang; Li, Shuqun; Deng, Anping

    2013-11-15

    The adulteration of food products with melamine has led to an urgent requirement for sensitive, specific, rapid and reliable quantitative/screening methods. To enhance the sensitivity and specificity of the enzyme-linked immunosorbent assay (ELISA) for the detection of melamine in milk, milk powder and feed samples, rational hapten modification and heterogeneous antibody/coating antigen combinations were adopted. Three melamine derivatives with different length of carboxylic spacer at the end were synthesized and linked to carrier proteins for the production of immunogens and coating antigens. Monoclonal antibody against melamine was produced by hybridoma technology. Under optimal experimental conditions, the standard curves of the ELISAs for melamine were constructed in range of 0.1-100 ng mL(-1). The sensitivity was 10-300 times enhanced compared to those in the published literatures. The cross-reactivity values of the ELISAs also demonstrated the assays exhibited high specificity. Five samples were spiked with melamine at different concentrations and detected by the ELISA. The recovery rates of 72.8-123.0% and intra-assay coefficients of variation of 0.8-18.9% (n=3) were obtained. The ELISA for milk sample was confirmed by high-performance liquid chromatography with a high correlation coefficient of 0.9902 (n=6). The proposed ELISA was proven to be a feasible quantitative/screening method for melamine analysis. PMID:24148389

  19. Production of Monoclonal Antibodies against the Major Capsid Protein of the Lactococcus Bacteriophage ul36 and Development of an Enzyme-Linked Immunosorbent Assay for Direct Phage Detection in Whey and Milk

    PubMed Central

    Moineau, Sylvain; Bernier, Denis; Jobin, Marie; Hébert, Jacques; Klaenhammer, Todd R.; Pandian, Sithian

    1993-01-01

    The only major structural protein (35 kDa) of the lactococcal small isometric-headed bacteriophage ul36, a member of the P335 species, was isolated from a preparative sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. Monoclonal antibodies (MAbs) were raised against the denatured 35-kDa protein. Six MAbs were selected and characterized. Western blots (immunoblots) showed that all MAbs recognized the 35 kDa but also a 45 kDa that is in lower concentration in the phage structure. Binding inhibition assays identified five families of MAbs that recognized nonoverlapping epitopes of the 35- and 45-kDa proteins. Immunoelectron microscopy showed that these two proteins are localized within the phage head, therefore indicating that the 35 kDa is a major capsid protein of ul36 and that the 45 kDa is a minor capsid protein. With two MAbs, a sandwich enzyme-linked immunosorbent assay (ELISA) was developed for direct detection of lactococcal phages in whey and milk samples. Whey and milk components, however, interfered with the conduct of the assay. Partial denaturation of milk samples by heat treatment in the presence of SDS and β-mercaptoethanol removed the masking effect and increased the sensitivity of the assay by 100-fold. With the method used here, 107 PFU/ml were detected by the ELISA within 2 h without any steps to enrich or isolate bacteriophages. Images PMID:16348980

  20. Evaluation of Chicken IgY Generated Against Canine Parvovirus Viral-Like Particles and Development of Enzyme-Linked Immunosorbent Assay and Immunochromatographic Assay for Canine Parvovirus Detection.

    PubMed

    He, Jinxin; Wang, Yuan; Sun, Shiqi; Zhang, Xiaoying

    2015-11-01

    Immunoglobulin Y (IgY) antibodies were generated against canine parvovirus virus-like particles (CPV-VLPs) antigen using chickens. Anti-CPV-VLPs-IgY was extracted from hen egg yolk and used for developing enzyme-linked immunosorbent assay (ELISA) and immunochromatographic assay (ICA) for the detection of CPV in dog feces. The cutoff negative values for anti-CPV-VLPs-IgY were determined using negative fecal samples (already confirmed by polymerase chain reaction [PCR]). In both ELISA and ICA, there was no cross-reaction with other diarrheal pathogens. Thirty-four fecal samples were collected from dogs with diarrhea, of which 26.47% were confirmed as CPV-positive samples by PCR, while 29.41% and 32.35% of the samples were found to be positive by ELISA and ICA, respectively. The developed ELISA and ICA exhibited 97.06% and 94.12% conformity with PCR. Higher sensitivity and specificity were observed for IgY-based ELISA and ICA. Thus, they could be suitable for routine use in the diagnosis of CPV in dogs. PMID:26469376

  1. A study of cross-reactivity in serum samples from dogs positive for Leishmania sp., Babesia canis and Ehrlichia canis in enzyme-linked immunosorbent assay and indirect fluorescent antibody test.

    PubMed

    Oliveira, Trícia Maria F de Sousa; Furuta, Patrícia I; de Carvalho, Débora; Machado, Rosangela Z

    2008-01-01

    To verify the presence of cross-reaction among leishmaniosis, ehrlichiosis and babesiosis in serological diagnostics used in human visceral leishmaniasis control programs, serum samples from leishmaniasis endemic and non-endemic areas were collected and tested by Indirect Fluorescent Antibody (IFAT) and Enzyme-linked immunosorbent assay (ELISA). All serum samples from endemic areas were positive for Leishmania sp., by ELISA and IFAT, 51% positive for Babesia canis and 43% for Ehrlichia canis by IFAT. None of the serum samples from non-endemic areas were positive for Leishmania sp., by IFAT, but 67% were positive for B. canis and 78% for E. canis using the same test. When tested by ELISA for Leishmania sp., four samples from non-endemic area were positive. These dogs were then located and no clinical signs, parasites or antibody was detected in new tests for a six month period. Only one of these 4 samples was positive for B. canis by IFAT and ELISA and three for E. canis by IFAT. The results of the work suggest a co-infection in the endemic area and no serological cross-reaction among these parasites by IFAT and ELISA. PMID:18554433

  2. Comparison of capture immunoglobulin M (IgM) and IgG enzyme-linked immunosorbent assay (ELISA) and nonstructural protein NS1 serotype-specific IgG ELISA for differentiation of primary and secondary dengue virus infections.

    PubMed

    Shu, Pei-Yun; Chen, Li-Kuang; Chang, Shu-Fen; Yueh, Yi-Yun; Chow, Ling; Chien, Li-Jung; Chin, Chuan; Lin, Ting-Hsiang; Huang, Jyh-Hsiung

    2003-07-01

    We have found that NS1 serotype-specific immunoglobulin G (IgG) enzyme-linked immunosorbent assay (ELISA) can be used to differentiate primary and secondary dengue virus infections. This is due to the fact that the NS1-specific IgG antibody cannot be detected before day 9 of illness for primary infection, so the NS1-specific IgG antibodies measured in acute-phase sera must come from previous infection. Comparison of NS1 serotype-specific IgG ELISA with envelope- and membrane-specific capture IgM and IgG ELISA in the differentiation of primary and secondary dengue virus infections showed good correlation (95.90% agreement). Most important, we have found that the serotype of the dengue virus from the majority of patients with primary infection could be correctly identified when convalescent-phase or postinfection sera were analyzed by NS1 serotype-specific IgG ELISA. These findings suggested that NS1 serotype-specific IgG ELISA could be reliably applied for serodiagnosis and seroepidemiological study of dengue virus infection. PMID:12853395

  3. Clinical and Immunological Studies of 332 Japanese Patients Tentatively Diagnosed as Anti-BP180-type Mucous Membrane Pemphigoid: A Novel BP180 C-terminal Domain Enzyme-linked Immunosorbent Assay.

    PubMed

    Yasukochi, Atsushi; Teye, Kwesi; Ishii, Norito; Hashimoto, Takashi

    2016-08-23

    Diagnosis of anti-BP180-type mucous membrane pemphigoid (BP180-MMP) is frustrated by the difficulty of detecting BP180 reactivity. A total of 721 patients with suspected MMP, selected from a cohort of 4,698 patients with autoimmune bullous disease (AIBD), were included in this study. Of these, 332 patients were tentatively diagnosed as BP180-MMP if they showed IgG/IgA reactivity with the epidermal side of 1M NaCl-split-skin and/or positive reactivity with BP180 in at least one of our antigen detection methods. Clinically, a predominance of female patients was found. Oral mucosal and cutaneous lesions were found in 85.5% and 41.0% of patients, respectively, and frequent treatments were systemic steroids, tetracycline/minocycline and diaminodiphenyl sulfone. Various immunological methods, including a newly developed BP180 C-terminal domain enzyme-linked immunosorbent assay (ELISA), revealed frequent reactivity with BP180 C-terminal and NC16a domains. Some patients reacted with BP180 and other antigens, indicating that BP180-MMP tends to concur with other AIBDs. This large study of patients with suspected BP180-MMP indicates the difficulty of diagnosis of BP180-MMP and the diagnostic usefulness of BP180 C-terminal domain ELISA. PMID:26984589

  4. Efficient silkworm expression of single-chain variable fragment antibody against ginsenoside Re using Bombyx mori nucleopolyhedrovirus bacmid DNA system and its application in enzyme-linked immunosorbent assay for quality control of total ginsenosides.

    PubMed

    Sakamoto, Seiichi; Pongkitwitoon, Benyakan; Nakamura, Seiko; Maenaka, Katsumi; Tanaka, Hiroyuki; Morimoto, Satoshi

    2010-09-01

    A single-chain variable fragment (scFv) antibody against ginsenoside Re (G-Re) have been successfully expressed in the silkworm larvae using Bombyx mori nucleopolyhedrovirus (BmNPV) bacmid DNA system. The baculovirus donor vector for expression of scFv against G-Re (GRe-scFv) was constructed to contain honeybee melittin signal sequence to accelerate secretion of the recombinant GRe-scFv into the haemolymph of silkworm larvae. Functional recombinant GRe-scFv was purified by cation exchange chromatography followed by immobilized metal ion affinity chromatography. The yield of purified GRe-scFv was 6.5 mg per 13 silkworm larvae, which is equivalent to 650 mg/l of the haemolymph, exhibiting extremely higher yield than that expressed in Escherichia coli (1.7 mg/l of culture medium). It was revealed from characterization that GRe-scFv retained similar characteristic of the parental monoclonal antibody (MAb) against G-Re (MAb-4G10), making it possible to develop indirect competitive enzyme-linked immunosorbent assay (icELISA) for quality control of total ginsenosides in various ginsengs. The detectable range for calibration of G-Re by developed icELISA shows 0.05-10 microg/ml. These results clearly suggested that the silkworm expression system is quite useful for the expression of functional scFv that frequently required time- and cost-consuming re-folding when it expressed in E. coli. PMID:20592135

  5. Faunal distribution of fleas and their blood-feeding preferences using enzyme-linked immunosorbent assays from farm animals and human shelters in a new rural region of southern Iran.

    PubMed

    Moemenbellah-Fard, Mohammad Djaefar; Shahriari, Bahador; Azizi, Kourosh; Fakoorziba, Mohammad Reza; Mohammadi, Jalal; Amin, Masoume

    2016-03-01

    Blood sucking insects, such as fleas, are responsible for the transmission of many infectious disease-causing agents which impose an intolerable burden on the health of people living particularly in endemic parts of the world. Fleas (Insecta: Siphonaptera) are found in many parts of the world including Iran. Both adult male and female fleas are obligatory ectoparasites. They are one of the main public health concerns as a result of their nuisance or the potential to act as vectors of a number of medically-important pathogens. The current study was conducted to examine the geographical distribution and fauna of fleas and their anthropophagic index in part of Fars province, southern Iran. This study was the first to be done in Iran. A total of 20 villages were randomly selected. From October 2011 to May 2012, adult fleas were collected by direct hand catch from human to animal shelters. Overall 848 fleas, most of which were blood-fed, were captured from the floor or the body of farm animal hosts (cattle, sheep, goat and hens). Only two different genera of fleas were identified, the main species (99.76 %) was human flea, Pulex irritans. The village of Shamsabad was the most heavily infested area. P. irritans had an anthropophagic index of 15 % using indirect enzyme-linked immunosorbent assays (ELISA). It could be concluded that P. irritans is widely distributed in this area. Based on their blood feeding activity, fleas thus posed a serious health threat to residents and their economically important livestock in this part of Iran. PMID:27065620

  6. Also with a restrictive transfusion policy, screening with second-generation anti-hepatitis C virus enzyme-linked immunosorbent assay would have reduced post-transfusion hepatitis C after open-heart surgery.

    PubMed

    Mathiesen, U L; Karlsson, E; Foberg, U; Frydén, A; Franzén, L; Widell, A; Bodemar, G

    1993-07-01

    The incidence of post-transfusion hepatitis non-A, non-B (PTH-NANB) was prospectively assessed among open-heart surgery patients from the southeast region of Sweden before the introduction of antihepatitis C virus (HCV) blood donor screening. Blood samples for alanine aminotransferase analysis were drawn before and 2, 3, and 4 months after transfusion. Surgery was performed in four centres. Of 190 transfused and followed-up patients 2 (1.1%) contracted PTH-NANB, both operated on at the centre with significantly fewer transfusions than the other centres. One patient had antibodies to HCV detected by first-generation (C100-3) and later by second-generation anti-HCV enzyme-linked immunosorbent assay (ELISA-2) and by positive second-generation recombinant immunoblot assay (4-RIBA). The other patient, although negative by first-generation anti-HCV ELISA, was positive by second-generation ELISA and by 4-RIBA. Both patients were hepatitis C-viremic by polymerase chain reaction (PCR). All the six donors implicated in the two hepatitis cases were first-generation anti-HCV-negative, but two, one for each patient, were positive by second-generation anti-HCV ELISA. This finding was confirmed by positive 4-RIBA in only 1 donor, the other being 'indeterminate'. However, in both donors hepatitis C viremia was found by PCR. This study shows that the second-generation anti-HCV ELISA will further reduce the risk for PTH-NANB/C and draws attention to the problem of evaluation of confirmatory tests. PMID:7689744

  7. Enumeration of Gut-Homing β7-Positive, Pathogen-Specific Antibody-Secreting Cells in Whole Blood from Enterotoxigenic Escherichia coli- and Vibrio cholerae-Infected Patients, Determined Using an Enzyme-Linked Immunosorbent Spot Assay Technique.

    PubMed

    Bhuiyan, Taufiqur Rahman; Hoq, Mohammad Rubel; Nishat, Naoshin Sharmin; Al Mahbuba, Deena; Rashu, Rasheduzzaman; Islam, Kamrul; Hossain, Lazina; Dey, Ayan; Harris, Jason B; Ryan, Edward T; Calderwood, Stephen B; Svennerholm, Ann-Mari; Qadri, Firdausi

    2016-01-01

    Vibrio cholerae and enterotoxigenic Escherichia coli (ETEC) are noninvasive mucosal pathogens that cause acute watery diarrhea in people in developing countries. Direct assessment of the mucosal immune responses to these pathogens is problematic. Surrogate markers of local mucosal responses in blood are increasingly being studied to determine the mucosal immune responses after infection. However, the volume of blood available in children and infants has limited this approach. We assessed whether an approach that first isolates β7-positive cells from a small volume of blood would allow measurement of the antigen-specific immune responses in patients with cholera and ETEC infection. β7 is a cell surface marker associated with mucosal homing. We isolated β7-expressing cells from blood on days 2, 7, and 30 and used an enzyme-linked immunosorbent spot (ELISPOT) assay to assess the gut-homing antibody-secreting cells (ASCs) specific to pathogen antigens. Patients with ETEC diarrhea showed a significant increase in toxin-specific gut-homing ASCs at day 7 compared to the levels at days 2 and 30 after onset of illness and to the levels in healthy controls. Similar elevations of responses to the ETEC colonization factors (CFs) CS6 and CFA/I were observed in patients infected with CS6- and CFA/I-positive ETEC strains. Antigen-specific gut-homing ASCs to the B subunit of cholera toxin and cholera-specific lipopolysaccharides (LPS) were also observed on day 7 after the onset of cholera using this approach. This study demonstrates that a simple ELISPOT assay can be used to study the mucosal immunity to specific antigens using a cell-sorting protocol to isolate mucosal homing cells, facilitating measurement of mucosal responses in children following infection or vaccination. PMID:26512047

  8. Validation of an enzyme-linked immunosorbent assay for the quantification of human IgG directed against the repeat region of the circumsporozoite protein of the parasite Plasmodium falciparum

    PubMed Central

    2012-01-01

    Background Several pre-erythrocytic malaria vaccines based on the circumsporozoite protein (CSP) antigen of Plasmodium falciparum are in clinical development. Vaccine immunogenicity is commonly evaluated by the determination of anti-CSP antibody levels using IgG-based assays, but no standard assay is available to allow comparison of the different vaccines. Methods The validation of an anti-CSP repeat region enzyme-linked immunosorbent assay (ELISA) is described. This assay is based on the binding of serum antibodies to R32LR, a recombinant protein composed of the repeat region of P. falciparum CSP. In addition to the original recombinant R32LR, an easy to purify recombinant His-tagged R32LR protein has been constructed to be used as solid phase antigen in the assay. Also, hybridoma cell lines have been generated producing human anti-R32LR monoclonal antibodies to be used as a potential inexhaustible source of anti-CSP repeats standard, instead of a reference serum. Results The anti-CSP repeats ELISA was shown to be robust, specific and linear within the analytical range, and adequately fulfilled all validation criteria as defined in the ICH guidelines. Furthermore, the coefficient of variation for repeatability and intermediate precision did not exceed 23%. Non-interference was demonstrated for R32LR-binding sera, and the assay was shown to be stable over time. Conclusions This ELISA, specific for antibodies directed against the CSP repeat region, can be used as a standard assay for the determination of humoral immunogenicity in the development of any CSP-based P. falciparum malaria vaccine. PMID:23173602

  9. Development of 2 types of competitive enzyme-linked immunosorbent assay for detecting antibodies to the rinderpest virus using a monoclonal antibody for a specific region of the hemagglutinin protein.

    PubMed

    Khamehchian, S; Madani, R; Rasaee, M J; Golchinfar, F; Kargar, R

    2007-06-01

    A competitive enzyme-linked immunosorbent assay (C-ELISA) has been developed and standardized for the detection of antibodies to the rinderpest virus (RPV) in sera from cattle, sheep, and goats. The test is specific for rinderpest because it does not detect antibodies to peste-des-petits-ruminants virus (PPRV). The test depends on the ability of the monoclonal antibody (MAb) directed against the hemagglutinin (H) protein of RPV to compete with the binding of RPV antibodies in the positive serum to the H protein of this virus. This MAb recognized a region from amino acids 575 to 583 on the H protein of RPV that is unique to the RPV H protein and is not present on the hemagglutinin-neuraminidase protein of PPRV. Another C-ELISA (peptide C-ELISA) was set up using this specific region as an antigen. A threshold value of 64.4% inhibition was established for the RPV C-ELISA, with 90 known RPV-negative and 30 RPV-positive serum samples. Using common serum samples, a cutoff value of 43.0% inhibition for the peptide C-ELISA was established. Based on statistical analysis, the overall sensitivity and specificity of the RPV C-ELISA, relative to those of a commercial kit, were found to be 90.00% and 103.33%, respectively. However, the sensitivity and specificity of the peptide C-ELISA were found to be 180.00% and 73.33%, respectively. Although a common MAb in 2 new C-ELISA systems was used, variation in their percent inhibition, due to the use of different antigens, was observed. Taking into consideration the difference in percent inhibition of the 2 described assays and the commercial kit (50%), it was found that the RPV C-ELISA and the peptide C-ELISA are more specific and sensitive tools than the commercial kit for assessing herd immune status and for epidemiologic surveillance. PMID:17668032

  10. Significance of quantitative enzyme-linked immunosorbent assay (ELISA) results in evaluation of three ELISAs and Western blot tests for detection of antibodies to human immunodeficiency virus in a high-risk population.

    PubMed Central

    Nishanian, P; Taylor, J M; Korns, E; Detels, R; Saah, A; Fahey, J L

    1987-01-01

    The characteristics of primary (first) tests with three enzyme-linked immunosorbent assay (ELISA) kits for human immunodeficiency virus (HIV) antibody were determined. The three ELISAs were performed on 3,229, 3,130, and 685 specimens from high-risk individuals using the Litton (LT; Litton Bionetics Laboratory Products, Charleston, S.C.), Dupont (DP; E. I. du Pont de Nemours & Co., Inc., Wilmington, Del.), and Genetic Systems (GS; Genetic Systems, Seattle, Wash.) kits, respectively. Evaluation was based on the distribution of quantitative test results (such as optical densities), a comparison with Western blot (WB) results, reproducibility of the tests, and identification of seroconverters. The performances of the GS and the DP kits were good by all four criteria and exceeded that of the LT kit. Primary ELISA-negative results were not always confirmed with repeat ELISA and by WB testing. The largest percentage of these unconfirmed negative test results came from samples with quantitative results in the fifth percentile nearest the cutoff. Thus, supplementary testing was indicated for samples with test results in this borderline negative range. Similarly, borderline positive primary ELISA results that were quantitatively nearest (fifth percentile) the cutoff value were more likely to be antibody negative on supplementary testing than samples with high antibody values. In this study, results of repeated tests by GS ELISA showed the least change from first test results. DP ELISA showed more unconfirmed primary positive test results, and LT ELISA showed more unconfirmed primary negative test results. Designation of a specimen with a single ELISA quantitative level near the cutoff value as positive or negative should be viewed with skepticism. A higher than normal proportion of specimens with high negative optical densities by GS ELISA (fifth percentile nearest the cutoff) and also negative by WB were found to be from individuals in the process of seroconversion. PMID

  11. Serum Antibodies from a Subset of Horses Positive for Babesia caballi by Competitive Enzyme-Linked Immunosorbent Assay Demonstrate a Protein Recognition Pattern That Is Not Consistent with Infection

    PubMed Central

    Awinda, Peter O.; Mealey, Robert H.; Williams, Laura B. A.; Conrad, Patricia A.; Packham, Andrea E.; Reif, Kathryn E.; Grause, Juanita F.; Pelzel-McCluskey, Angela M.; Chung, Chungwon; Bastos, Reginaldo G.; Kappmeyer, Lowell S.; Howe, Daniel K.; Ness, SallyAnne L.; Knowles, Donald P.

    2013-01-01

    Tick-borne pathogens that cause persistent infection are of major concern to the livestock industry because of transmission risk from persistently infected animals and the potential economic losses they pose. The recent reemergence of Theileria equi in the United States prompted a widespread national survey resulting in identification of limited distribution of equine piroplasmosis (EP) in the U.S. horse population. This program identified Babesia caballi-seropositive horses using rhoptry-associated protein 1 (RAP-1)–competitive enzyme-linked immunosorbent assay (cELISA), despite B. caballi being considered nonendemic on the U.S. mainland. The purpose of the present study was to evaluate the suitability of RAP-1–cELISA as a single serological test to determine the infection status of B. caballi in U.S. horses. Immunoblotting indicated that sera from U.S. horses reacted with B. caballi lysate and purified B. caballi RAP-1 protein. Antibody reactivity to B. caballi lysate was exclusively directed against a single ∼50-kDa band corresponding to a native B. caballi RAP-1 protein. In contrast, sera from experimentally and naturally infected horses from regions where B. caballi is endemic bound multiple proteins ranging from 30 to 50 kDa. Dilutions of sera from U.S. horses positive by cELISA revealed low levels of antibodies, while sera from horses experimentally infected with B. caballi and from areas where B. caballi is endemic had comparatively high antibody levels. Finally, blood transfer from seropositive U.S. horses into naive horses demonstrated no evidence of B. caballi transmission, confirming that antibody reactivity in cELISA-positive U.S. horses was not consistent with infection. Therefore, we conclude that a combination of cELISA and immunoblotting is required for the accurate serodiagnosis of B. caballi. PMID:24049108

  12. Development of a Highly Specific IgM Enzyme-Linked Immunosorbent Assay for Bartonella henselae Using Refined N-Lauroyl-Sarcosine-Insoluble Proteins for Serodiagnosis of Cat Scratch Disease.

    PubMed

    Otsuyama, Ken-Ichiro; Tsuneoka, Hidehiro; Kondou, Kaori; Yanagihara, Masashi; Tokuda, Nobuko; Shirasawa, Bungo; Ichihara, Kiyoshi

    2016-04-01

    The conventional anti-Bartonella henselaeIgM enzyme-linked immunosorbent assay (IgM-ELISA) methods for diagnosing cat scratch disease (CSD) remain poor in both sensitivity and specificity. We sought to develop an IgM-ELISA with improved accuracy in the serodiagnosis of CSD by exploring the antigens that are most suitable for an ELISA. We prepared 5 different protein antigens: antigen I (sonicatedB. henselaewhole-cell antigen), antigen II (N-lauroyl-sarcosine-insoluble antigen), antigen III (processed sarcosine-soluble antigen), and antigen IV and antigen V (sarcosine-insoluble and sarcosine-soluble antigens refined by DEAE-Sepharose Fast Flow ion-exchange chromatography). The IgM antibodies in the sera of 47 patients with clinically suspected CSD (24 definite, 23 suspected) and of 85 healthy individuals were examined by ELISAs using the 5 antigens, and the results were compared with those of an IgM indirect fluorescent antibody assay (IgM-IFA). In a reference panel, which consisted of 5 positive and 5 negative sera, antigen I and antigen III failed to distinguish between the two statuses, whereas the other three antigens succeeded in distinguishing between them. When the cutoff value was set at the 98th percentile of the ELISA index for healthy individuals, the sensitivity of IgM-IFA for the 24 cases of definite CSD was 54%, whereas the sensitivities of the IgM-ELISAs with antigen II, IV, and V were 75%, 83%, and 75%, respectively. The sensitivities of these three IgM-ELISAs for all 47 of the clinically suspected cases were 49%, 64%, and 51%, respectively. In contrast, the sensitivity of IgM-IFA was 28%. These results indicate that the refined sarcosine-insoluble proteins (antigen IV), which possessed the highest specificity among the 5 antigens, are the most appropriate for developing an IgM-ELISA for the highly specific serodiagnosis of CSD. PMID:26865692

  13. Combination of reverse transcription real-time polymerase chain reaction and antigen capture enzyme-linked immunosorbent assay for the detection of animals persistently infected with Bovine viral diarrhea virus.

    PubMed

    Yan, Lifang; Zhang, Shuping; Pace, Lanny; Wilson, Floyd; Wan, Henry; Zhang, Michael

    2011-01-01

    Bovine viral diarrhea virus (BVDV) is an economically important pathogen of cattle. A successful control program requires early detection and removal of persistently infected (PI) animals. The objective of the current study was to develop, validate, and apply a cost-effective testing scheme for the detection of BVDV PI animals in exposed herds. Pooled samples were screened by using a real-time reverse transcription polymerase chain reaction (real-time RT-PCR), and individual positives were identified with an antigen capture enzyme-linked immunosorbent assay (ACE). The detection limits of the optimized real-time RT-PCR were 10 and 100 RNA copies per reaction for BVDV-1 and BVDV-2, respectively. The semiquantitative results of real-time RT-PCR and ACE or real-time RT-PCR and immunohistochemistry were moderately correlated. The threshold cycle of real-time RT-PCR performed on pooled samples was significantly correlated with the pool size (R(2)  =  0.993). The least-cost pool sizes were 50 at a prevalence of 0.25-0.5% and 25 at a prevalence of 0.75-2.0%. By using the combined real-time RT-PCR and ACE procedure, 111 of 27,932 samples (0.4%) tested positive for BVDV. At this prevalence, cost reduction associated with the application of real-time RT-PCR and ACE ranged from 61% to 94%, compared with testing individual samples by ACE, immunohistochemistry, or real-time RT-PCR. Real-time RT-PCR screening also indicated that 92.94% of PI animals were infected with BVDV-1, 3.53% with BVDV-2, and 3.53% with both BVDV-1 and BVDV-2. Analysis of the 5'-untranslated region of 22 isolates revealed the predominance of BVDV-1b followed by BVDV-2a. PMID:21217023

  14. Erysipelothrix rhusiopathiae and Mycoplasma hyopneumoniae: the sensitivities of enzyme-linked immunosorbent assays for detecting vaccinated sows of unknown disease status using serum and colostrum, and the correlation of the results for sow serum, colostrum, and piglet serum.

    PubMed

    Jenvey, Caitlin J; Reichel, Michael P; Cockcroft, Peter D

    2015-03-01

    Due to relatively high concentrations of immunoglobulins, colostrum has the potential to improve the sensitivity of diagnostic tests for diseases in pigs when compared with serum. It is possible that colostrum could improve the sensitivity of the antibody enzyme-linked immunosorbent assay (ELISA) compared with serum. Colostrum is also essential for piglets, providing protection against infections in the first few weeks and months of life. The sensitivity of 2 commercially available ELISAs, one for the detection of Erysipelothrix rhusiopathiae and the second for Mycoplasma hyopneumoniae antibodies, when used with sow colostrum in comparison with serum was investigated. The correlation of maternal E. rhusiopathiae- and M. hyopneumoniae-specific antibody levels with specific-antibody serum levels in the piglet was also determined. The sensitivity was defined as the proportion of vaccinated sows that were correctly identified as vaccinated at a given cutoff point. The true disease status of the sows with regard to the 2 infections was unknown. Blood and colostrum samples were collected from 20 sows, 10 primiparous and 10 multiparous, and blood samples were also collected from the piglets of each sow, 48-72 hr post-farrowing. The sensitivities of both ELISAs were significantly improved when using colostrum compared with serum. Sow serum and colostrum optical density (OD) values were significantly correlated. The mean sow OD values for serum for E. rhusiopathiae and M. hyopneumoniae and colostrum for E. rhusiopathiae were significantly correlated with piglet serum OD levels. If the improved sensitivity of colostrum can be demonstrated in infected animals, this will increase the ability of the test to identify infected animals using both individual and pooled colostrum. Testing serum and/or colostrum using ELISA can be useful predictors of piglet disease-specific OD values. PMID:25613041

  15. Generation of E. coli-derived virus-like particles of porcine circovirus type 2 and their use in an indirect IgG enzyme-linked immunosorbent assay.

    PubMed

    Zhang, Yan; Wang, Zhanfeng; Zhan, Yang; Gong, Qian; Yu, Wanting; Deng, Zhibang; Wang, Aibing; Yang, Yi; Wang, Naidong

    2016-06-01

    Porcine circovirus type 2 (PCV2) causes increased mortality and poor growth or weight loss in apparently healthy swine. Therefore, methods to detect PCV2-specific antibodies in swine serum are important for prevention, diagnosis, and control of PCV2-associated diseases (PCVAD). In this study, PCV2 virus-like particles (VLPs) were used to develop a rapid, simple and economical indirect enzyme-linked immunosorbent assay to detect (with high sensitivity) PCV2-specific antibodies in swine serum. The PCV2 capsid protein (Cap) was overexpressed in E. coli after optimizing the cap gene. Subsequently, the soluble Cap was rapidly purified in one step by automated fast protein liquid chromatography (FPLC). The purified PCV2 Cap was shown by transmission electron microscopy and gel filtration chromatography to be capable of self-assembling into VLPs in vitro. Using the purified VLPs as antigens, optimal operating conditions for the VLP ELISA were determined. The concentration of PCV2 VLPs was 1 µg/ml per well, and the dilution factors for swine serum and horseradish peroxidase (HRP)-labeled goat anti-pig antibody were 1:150 and 1:4000, respectively. Out of 241 serum samples tested with this assay, 83.4 % were found to be positive. Importantly, the VLP ELISA had a total coincidence rate of 97.4 % (74/76) compared to an Ingezim PCV2 ELISA IgG assay. In summary, this rapid, inexpensive VLP ELISA has the potential to greatly facilitate large-scale investigations of PCV2-associated serotypes. PMID:26973229

  16. Optimal Cutoff and Accuracy of an IgM Enzyme-Linked Immunosorbent Assay for Diagnosis of Acute Scrub Typhus in Northern Thailand: an Alternative Reference Method to the IgM Immunofluorescence Assay.

    PubMed

    Blacksell, Stuart D; Lim, Cherry; Tanganuchitcharnchai, Ampai; Jintaworn, Suthatip; Kantipong, Pacharee; Richards, Allen L; Paris, Daniel H; Limmathurotsakul, Direk; Day, Nicholas P J

    2016-06-01

    The enzyme-linked immunosorbent assay (ELISA) has been proposed as an alternative serologic diagnostic test to the indirect immunofluorescence assay (IFA) for scrub typhus. Here, we systematically determine the optimal sample dilution and cutoff optical density (OD) and estimate the accuracy of IgM ELISA using Bayesian latent class models (LCMs). Data from 135 patients with undifferentiated fever were reevaluated using Bayesian LCMs. Every patient was evaluated for the presence of an eschar and tested with a blood culture for Orientia tsutsugamushi, three different PCR assays, and an IgM IFA. The IgM ELISA was performed for every sample at sample dilutions from 1:100 to 1:102,400 using crude whole-cell antigens of the Karp, Kato, and Gilliam strains of O. tsutsugamushi developed by the Naval Medical Research Center. We used Bayesian LCMs to generate unbiased receiver operating characteristic curves and found that the sample dilution of 1:400 was optimal for the IgM ELISA. With the optimal cutoff OD of 1.474 at a sample dilution of 1:400, the IgM ELISA had a sensitivity of 85.7% (95% credible interval [CrI], 77.4% to 86.7%) and a specificity of 98.1% (95% CrI, 97.2% to 100%) using paired samples. For the ELISA, the OD could be determined objectively and quickly, in contrast to the reading of IFA slides, which was both subjective and labor-intensive. The IgM ELISA for scrub typhus has high diagnostic accuracy and is less subjective than the IgM IFA. We suggest that the IgM ELISA may be used as an alternative reference test to the IgM IFA for the serological diagnosis of scrub typhus. PMID:27008880

  17. Optimal Cutoff and Accuracy of an IgM Enzyme-Linked Immunosorbent Assay for Diagnosis of Acute Scrub Typhus in Northern Thailand: an Alternative Reference Method to the IgM Immunofluorescence Assay

    PubMed Central

    Blacksell, Stuart D.; Tanganuchitcharnchai, Ampai; Jintaworn, Suthatip; Kantipong, Pacharee; Richards, Allen L.; Day, Nicholas P. J.

    2016-01-01

    The enzyme-linked immunosorbent assay (ELISA) has been proposed as an alternative serologic diagnostic test to the indirect immunofluorescence assay (IFA) for scrub typhus. Here, we systematically determine the optimal sample dilution and cutoff optical density (OD) and estimate the accuracy of IgM ELISA using Bayesian latent class models (LCMs). Data from 135 patients with undifferentiated fever were reevaluated using Bayesian LCMs. Every patient was evaluated for the presence of an eschar and tested with a blood culture for Orientia tsutsugamushi, three different PCR assays, and an IgM IFA. The IgM ELISA was performed for every sample at sample dilutions from 1:100 to 1:102,400 using crude whole-cell antigens of the Karp, Kato, and Gilliam strains of O. tsutsugamushi developed by the Naval Medical Research Center. We used Bayesian LCMs to generate unbiased receiver operating characteristic curves and found that the sample dilution of 1:400 was optimal for the IgM ELISA. With the optimal cutoff OD of 1.474 at a sample dilution of 1:400, the IgM ELISA had a sensitivity of 85.7% (95% credible interval [CrI], 77.4% to 86.7%) and a specificity of 98.1% (95% CrI, 97.2% to 100%) using paired samples. For the ELISA, the OD could be determined objectively and quickly, in contrast to the reading of IFA slides, which was both subjective and labor-intensive. The IgM ELISA for scrub typhus has high diagnostic accuracy and is less subjective than the IgM IFA. We suggest that the IgM ELISA may be used as an alternative reference test to the IgM IFA for the serological diagnosis of scrub typhus. PMID:27008880

  18. Bayesian Validation of the Indirect Immunofluorescence Assay and Its Superiority to the Enzyme-Linked Immunosorbent Assay and the Complement Fixation Test for Detecting Antibodies against Coxiella burnetii in Goat Serum.

    PubMed

    Muleme, Michael; Stenos, John; Vincent, Gemma; Campbell, Angus; Graves, Stephen; Warner, Simone; Devlin, Joanne M; Nguyen, Chelsea; Stevenson, Mark A; Wilks, Colin R; Firestone, Simon M

    2016-06-01

    Although many studies have reported the indirect immunofluorescence assay (IFA) to be more sensitive in detection of antibodies to Coxiella burnetii than the complement fixation test (CFT), the diagnostic sensitivity (DSe) and diagnostic specificity (DSp) of the assay have not been previously established for use in ruminants. This study aimed to validate the IFA by describing the optimization, selection of cutoff titers, repeatability, and reliability as well as the DSe and DSp of the assay. Bayesian latent class analysis was used to estimate diagnostic specifications in comparison with the CFT and the enzyme-linked immunosorbent assay (ELISA). The optimal cutoff dilution for screening for IgG and IgM antibodies in goat serum using the IFA was estimated to be 1:160. The IFA had good repeatability (>96.9% for IgG, >78.0% for IgM), and there was almost perfect agreement (Cohen's kappa > 0.80 for IgG) between the readings reported by two technicians for samples tested for IgG antibodies. The IFA had a higher DSe (94.8%; 95% confidence interval [CI], 80.3, 99.6) for the detection of IgG antibodies against C. burnetii than the ELISA (70.1%; 95% CI, 52.7, 91.0) and the CFT (29.8%; 95% CI, 17.0, 44.8). All three tests were highly specific for goat IgG antibodies. The IFA also had a higher DSe (88.8%; 95% CI, 58.2, 99.5) for detection of IgM antibodies than the ELISA (71.7%; 95% CI, 46.3, 92.8). These results underscore the better suitability of the IFA than of the CFT and ELISA for detection of IgG and IgM antibodies in goat serum and possibly in serum from other ruminants. PMID:27122484

  19. Development and validation of a competitive enzyme-linked immunosorbent assay for the measurement of total plasma immunoglobulins in healthy loggerhead sea (Caretta caretta) and green turtles (Chelonia mydas).

    PubMed

    Kaplan, Amy J; Stacy, Nicole I; Jacobson, Elliott; Le-Bert, Carolina R; Nollens, Hendrik H; Origgi, Francesco C; Green, Linda G; Bootorabi, Shadi; Bolten, Alan; Hernandez, Jorge A

    2016-01-01

    The quantification of circulating plasma immunoglobulins represents a valuable diagnostic tool in human and veterinary immunology, although its application is very limited in reptile medicine to date. The objectives of our study were the development and standardization of a competitive enzyme-linked immunosorbent assay (cELISA) for the measurement of total plasma immunoglobulins (Igs; both IgM and IgY) in loggerhead sea turtles (LST; Caretta caretta; n = 254) and green turtles (GT; Chelonia mydas; n = 111), the establishment of reference intervals for Ig for both species, and the examination of associations between Ig and total protein (TP), condition index, and water temperature. The cELISA for Ig was successfully developed and optimized. Reference intervals for Ig were 0.38-0.94 g/dL in LST (median: 0.59 g/dL; range: 0.16-2.15 g/dL) and 0.40-0.85 g/dL in GT (median: 0.58 g/dL; range: 0.18-1.80 g/dL). In LST, there were positive linear relationships of Ig with TP, and TP with Ig and condition index, and a negative relationship of Ig with condition index. The positive linear relationships of Ig with TP, and TP with Ig were also identified in GT. These positive associations of Ig and TP were expected, as Ig represents fractions of TP, and TP reportedly increases with straight carapace length and weight. The negative association of Ig with condition index may indicate potential biological variations. The cELISA and reference intervals for total Ig of LST and GT presented herein have the potential to be useful as a diagnostic and research tool for sea turtle immunology. PMID:26699523

  20. Suitability of real-time quantitative polymerase chain reaction and enzyme-linked immunosorbent assay for cry9C detection in Mexican corn tortillas: fate of DNA and protein after alkaline cooking.

    PubMed

    Quirasco, Maricarmen; Schoel, Bernd; Plasencia, Javier; Fagan, John; Galvez, Amanda

    2004-01-01

    Alkaline-cooked corn, called nixtamal, is the basis for many traditional corn products such as tortillas, chips, and taco shells that are used widely in Mexico and Central America and in the preparation of snack foods that are consumed globally. To assess the effects of alkaline and thermal treatments on the detectability of DNA and protein for the presence of genetically modified sequences, various nixtamalized products were prepared from blends of conventional white corn containing 0.1, 1.0, and 10% transgenic corn (event CBH 351, StarLink). Real-time quantitative polymerase chain reactions (RTQ-PCR) and immunoassays were used to determine the cry9C gene and protein, respectively, in unprocessed corn kernels, freshly prepared alkaline-cooked and ground corn (masa), masa flour, tortillas prepared from masa by heat treatment, chips prepared from damp masa dough by deep frying, and from tortillas processed at high (200 degrees C) and low temperatures (70 degrees C). In spite of progressive degradation of genomic DNA during processing, RTQ-PCR genetic analysis allowed detection and quantification of the cry9C gene in all products prepared from 10, 1, and 0.1% StarLink corn, except deep-fried chips containing 0.1% StarLink. Enzyme-linked immunosorbent assays readily detected <1 ppm cry9C protein in all blends of unprocessed corn (10, 1, and 0.1% StarLink) as well as in nonfried tortilla and masa products. This technique was not suitable for thermally treated nixtamalized products containing <1% transgenic corn. PMID:15287662

  1. Detection of endocervical anti-Chlamydia trachomatis immunoglobulin A in pregnant women by a rapid, 6-minute enzyme-linked immunosorbent assay: comparison with PCR and chlamydial antigen detection methods.

    PubMed Central

    Witkin, S S; Bongiovanni, A M; Inglis, S R

    1997-01-01

    There is a need for a rapid, uncomplicated, and inexpensive test for Chlamydia trachomatis infection in women. We evaluated the ability of a 6-min enzyme-linked immunosorbent assay (ELISA) that requires no laboratory equipment (IgA Rapid SeroTest; Savyon Diagnostics) to detect C. trachomatis immunoglobulin A (IgA) in the endocervices of 167 inner-city pregnant women and compared the results with DNA amplification (Amplicor PCR; Roche Diagnostics) and antigen detection (Chlamydiazyme; Abbott Laboratories) performed on the same women. Anti-C. trachomatis IgA was detected in the cervices of 32 women (19.2%). Samples from 23 women (13.8%) were PCR positive, while chlamydial antigen was present in 20 women (12.0%). There was only 1 sample (4.3%) that was positive by PCR but negative by ELISA; 10 samples were ELISA positive and PCR negative. In contrast, seven samples (30.4%) were PCR positive but Chlamydiazyme negative and four were Chlamydiazyme positive and PCR negative. Compared to PCR, the IgA ELISA had a sensitivity of 95.7%, a specificity of 93.1%, a positive predictive value of 68.8%, and a negative predictive value of 99.3%. The antigen assay had a sensitivity of only 69.6%, a specificity of 97.2%, a positive predictive value of 80.0%, and a negative predictive value of 95.2%. In high-risk groups where laboratory testing is not available, or where the patient might not return to obtain her testing result and be treated, the Rapid IgA SeroTest is a viable alternative for detection of cervical C. trachomatis in pregnant women. PMID:9196193

  2. Serological diagnosis of hantavirus infections by an enzyme-linked immunosorbent assay based on detection of immunoglobulin G and M responses to recombinant nucleocapsid proteins of five viral serotypes.

    PubMed

    Elgh, F; Lundkvist, A; Alexeyev, O A; Stenlund, H; Avsic-Zupanc, T; Hjelle, B; Lee, H W; Smith, K J; Vainionpää, R; Wiger, D; Wadell, G; Juto, P

    1997-05-01

    Worldwide, hantaviruses cause more than 100,000 human infections annually. Rapid and accurate methods are important both in monitoring acute infections and for epidemiological studies. We and others have shown that the amino termini of hantavirus nucleocapsid proteins (Ns) are sensitive tools for the detection of specific antibodies in hantavirus disease. Accordingly, we expressed truncated Ns (amino acids 1 to 117) in Escherichia coli from the five hantaviruses known to be pathogenic to man; Hantaan (HTN), Seoul (SEO), Dobrava (DOB), Sin Nombre (SN), and Puumala (PUU) viruses. In order to obtain pure antigens for use in an enzyme-linked immunosorbent assay (ELISA), the recombinant proteins were purified by polyhistidine-metal chelate affinity chromatography. Polyclonal animal antisera and a panel of serum specimens from hantavirus-infected individuals from Scandinavia, Slovenia, Russia, Korea, China, and the United States were used to evaluate the usefulness of the method. With both human and animal sera, it was possible to designate the antibody response into two groups: those with HTN, SEO, and DOB virus reactivity on the one hand and those with SN and PUU virus reactivity on the other. In sera from Scandinavia, European Russia, and the United States, the antibody response was directed mainly to the PUU and SN virus group. The sera from Asia reacted almost exclusively with the HTN, SEO, and DOB types of viruses. This was true for both the immunoglobulin M (IgM) and IgG antibody responses, indicating that this type of discrimination can be done during the acute phase of hantavirus infections. Both the HTN, SEO, and DOB virus and the PUU and SN virus types of antibody response patterns were found in patients from the Balkan region (Solvenia). PMID:9114393

  3. Use of Seroconversion Panels To Estimate Delay in Detection of Anti-Human Immunodeficiency Virus Antibodies by Enzyme-Linked Immunosorbent Assay of Pooled Compared to Singleton Serum Samples

    PubMed Central

    Novack, Lena; Galai, Noya; Yaari, Arieh; Orgel, Mordechai; Shinar, Eilat; Sarov, Batia

    2006-01-01

    The transfusion of unsafe blood worldwide accounts for 5 to 15% of new human immunodeficiency virus (HIV) infections, most of which occur in sub-Saharan Africa. While developed countries now apply PCR testing of pooled samples, some developing countries still do not have universal screening policies. More efficient low-cost procedures for the screening of pooled samples have the potential to encourage mass screening efforts in resource-poor settings. The aim of this study was to estimate the delay in the detection of HIV antibodies in pooled serum samples compared to that in singleton serum samples by enzyme-linked immunosorbent assay (ELISA) and to evaluate the risk of transfusion-transmitted HIV infection during the window period. Serial blood samples obtained from five HIV seroconversion panels were mixed with HIV-seronegative blood samples to create pools of 6, 12, 16, 24, 32, and 48 samples. The delay in detection of the first anti-HIV antibody-positive sample in tests with pooled samples was calculated for each pool size and compared to that obtained by testing of singleton samples and statistically evaluated by a robust log-linear regression analysis. The risk of a false-negative (FN) result caused by dilution was estimated by use of the incidence risk/window period model. The additional risk of transmission related to ELISA screening of pooled samples for HIV did not exceed 9% of the current risk of an FN result (estimated to be 1/1,067,000). The countries with virus prevalence rates in donors of less than 15% are expected to save up to 30% in the number of tests. ELISA screening of pooled samples could be considered in settings where the testing of blood supplies for HIV is not routinely done. PMID:16891511

  4. Use of seroconversion panels to estimate delay in detection of anti-human immunodeficiency virus antibodies by enzyme-linked immunosorbent assay of pooled compared to singleton serum samples.

    PubMed

    Novack, Lena; Galai, Noya; Yaari, Arieh; Orgel, Mordechai; Shinar, Eilat; Sarov, Batia

    2006-08-01

    The transfusion of unsafe blood worldwide accounts for 5 to 15% of new human immunodeficiency virus (HIV) infections, most of which occur in sub-Saharan Africa. While developed countries now apply PCR testing of pooled samples, some developing countries still do not have universal screening policies. More efficient low-cost procedures for the screening of pooled samples have the potential to encourage mass screening efforts in resource-poor settings. The aim of this study was to estimate the delay in the detection of HIV antibodies in pooled serum samples compared to that in singleton serum samples by enzyme-linked immunosorbent assay (ELISA) and to evaluate the risk of transfusion-transmitted HIV infection during the window period. Serial blood samples obtained from five HIV seroconversion panels were mixed with HIV-seronegative blood samples to create pools of 6, 12, 16, 24, 32, and 48 samples. The delay in detection of the first anti-HIV antibody-positive sample in tests with pooled samples was calculated for each pool size and compared to that obtained by testing of singleton samples and statistically evaluated by a robust log-linear regression analysis. The risk of a false-negative (FN) result caused by dilution was estimated by use of the incidence risk/window period model. The additional risk of transmission related to ELISA screening of pooled samples for HIV did not exceed 9% of the current risk of an FN result (estimated to be 1/1,067,000). The countries with virus prevalence rates in donors of less than 15% are expected to save up to 30% in the number of tests. ELISA screening of pooled samples could be considered in settings where the testing of blood supplies for HIV is not routinely done. PMID:16891511

  5. Enhanced expression of the Erns protein of classical swine fever virus in yeast and its application in an indirect enzyme-linked immunosorbent assay for antibody differentiation of infected from vaccinated animals.

    PubMed

    Luo, Yuzi; Li, Lin; Austermann-Busch, Sophia; Dong, Mei; Xu, Jingjing; Shao, Lina; Lei, Jianlin; Li, Na; He, Wen-Rui; Zhao, Bibo; Li, Su; Li, Yongfeng; Liu, Lihong; Becher, Paul; Sun, Yuan; Qiu, Hua-Ji

    2015-09-15

    Classical swine fever (CSF), caused by classical swine fever virus (CSFV), is a devastating disease of swine worldwide. Although a mandatory vaccination with the modified live vaccine C-strain has been implemented in China for decades, CSF remains a serious threat to the swine industry. To facilitate the control and eradication of CSF in China, the E2-based marker vaccine rAdV-SFV-E2, an adenovirus-delivered, alphavirus replicon-vectored vaccine, has been developed. Accordingly, an accompanying discriminatory test that allows differentiating infected from vaccinated animals (DIVA) is required. Here, the enhanced expression of E(rns) protein of CSFV was achieved in the methyltropic yeast Pichia pastoris by codon-optimization of the E(rns) gene, and an indirect enzyme-linked immunosorbent assay (iELISA) based on the yeast-expressed E(rns) (yE(rns)) was developed and evaluated. The optimized iELISA was able to detect CSFV-specific antibodies in the serum samples from the CSFV-infected pigs as early as 6 days post-infection, and discriminate the CSFV-infected pigs from those vaccinated with rAdV-SFV-E2. The iELISA was evaluated using a panel of swine sera, and showed comparable sensitivity (94.6%) and specificity (97.1%), and the consistence rates with the virus neutralization test were 96.8% for CSFV-infected swine sera, 83.3% for C-strain-vaccinated swine sera, and 95.0% for field swine sera. In addition, the iELISA showed higher sensitivity (90.4%) compared with PrioCHECK CSFV E(rns) (59.6%). Taken together, the yE(rns)-based iELISA is specific and sensitive, representing a promising DIVA test for E2-based marker vaccines against CSF. PMID:26005003

  6. Evaluation of an Erns-based enzyme-linked immunosorbent assay to distinguish Classical swine fever virus-infected pigs from pigs vaccinated with CP7_E2alf.

    PubMed

    Pannhorst, Katrin; Fröhlich, Andreas; Staubach, Christoph; Meyer, Denise; Blome, Sandra; Becher, Paul

    2015-07-01

    Infections with Classical swine fever virus (CSFV) are a major economic threat to pig production. To combat CSF outbreaks and to maintain trade, new marker vaccines were developed that allow differentiation of infected from vaccinated animals (DIVA principle). The chimeric pestivirus CP7_E2alf was shown to be safe and efficacious. Its DIVA strategy is based on the detection of CSFV E(rns)-specific antibodies that are only developed on infection. However, for the new marker vaccine to be considered a valuable control tool, a validated discriminatory assay is needed. One promising candidate is the already commercially available enzyme-linked immunosorbent assay, PrioCHECK CSFV E(rns) ELISA (Prionics BV, Lelystad, The Netherlands). Four laboratories of different European Union member states tested 530 serum samples and country-specific field sera from domestic pigs and wild boar. The ELISA displayed a good robustness. However, based on its reproducibility and repeatability, ranges rather than single values for diagnostic sensitivity and specificity were defined. The ELISA displayed a sensitivity of 90-98% with sera from CSFV-infected domestic pigs. A specificity of 89-96% was calculated with sera from domestic pigs vaccinated once with CP7_E2alf. The ELISA detected CSFV infections in vaccinated domestic pigs with a sensitivity of 82-94%. The sensitivity was lower with sera taken ≤21 days post-challenge indicating that the stage of CSFV infection had a considerable influence on testing. Taken together, the PrioCHECK CSFV E(rns) ELISA can be used for detection of CSFV infections in CP7_E2alf-vaccinated and nonvaccinated domestic pig populations, but should only be applied on a herd basis by testing a defined number of animals. PMID:26179095

  7. Identification of high-affinity anti-IL-1. alpha. autoantibodies in normal human serum as an interfering substance in a sensitive enzyme-linked immunosorbent assay for IL-1. alpha

    SciTech Connect

    Mae, N.; Liberato, D.J.; Chizzonite, R.; Satoh, H. )

    1991-04-01

    A highly reproducible, sensitive, and specific sandwich enzyme-linked immunosorbent assay (ELISA) for recombinant human IL-1 {alpha} (rhIL-1 alpha) has been developed. Results from this ELISA have demonstrated that the concentration of rhIL-1 {alpha} added to normal human serum (NHS) decreased by 16.3% after 3 h and 24.9% after 6 h at room temperature. Molecular exclusion column chromatography with Sephacryl S-300 HR revealed that 125I-labeled IL-1 {alpha} added to normal human serum rapidly formed higher molecular weight complexes without indication of proteolytic degradation. The observed reduction in immunoreactivity was correlated with this protein complex formation and accounted for the apparent instability of rhIL-1 {alpha} in NHS. Immunoblot analysis indicated that the molecular weight of the binding protein was 150-160K, and the IL-1 {alpha} binding activity was removed and recovered from NHS by Protein-G affinity chromatography; indicating that the binding protein was IL-1 {alpha}-specific IgG. The binding of 125I-labeled IL-1 {alpha} to the serum binding proteins could be inhibited by unlabeled IL-1 alpha (IC50 = 7.4 {times} 10(-11) M) but not by unlabeled IL-1 {beta}. Kinetic analysis with 125I-labeled IL-1 alpha revealed that the average binding affinity of these IL-1 {alpha}-specific IgGs was 4.7 {times} 10(10) M-1. These results suggest that these autoantibodies may interfere with the detection of IL-1 {alpha} in human serum by various assay systems and also could be a regulator of circulating IL-1 {alpha}.

  8. Comparison of Sorbitol MacConkey Agar and a Two-Step Method Which Utilizes Enzyme-Linked Immunosorbent Assay Toxin Testing and a Chromogenic Agar To Detect and Isolate Enterohemorrhagic Escherichia coli

    PubMed Central

    Novicki, Thomas J.; Daly, Judy A.; Mottice, Susan L.; Carroll, Karen C.

    2000-01-01

    Enterohemorrhagic Escherichia coli (EHEC) and specifically serotype O157:H7 are a significant cause of hemorrhagic gastrointestinal disease and the hemolytic uremic syndrome. Methods currently used in clinical microbiology labs, such as sorbitol-MacConkey (SMAC) agar, reliably detect only O157:H7. We have evaluated a two-step method that has the potential to identify and isolate all EHEC serotypes, including serotype O157:H7. This method utilizes a chromogenic selective-differential medium for the isolation of E. coli together with an enzyme-linked immunosorbent assay (ELISA) that detects the Shiga-like toxins Stx1 and Stx2. Both are commercially available and usable in a wide range of clinical microbiology laboratories. Compared to a Vero cell cytotoxic assay, SMAC had sensitivities of 23.5% for the identification of all EHEC serotypes and of 50.0% for the identification of O157:H7 alone. The two-step method had sensitivities of 76.5 and 100%, respectively. The ELISA alone had a sensitivity of 82.4% in the detection of Stx1 and Stx2. The specificity was 100% in all cases. Overall, 14 EHEC isolates were obtained: 8 (58%) O157:H7, 2 (14%) O26, 2 (14%) O111:NM, 1 (7%) O103:H2, and 1 (7%) O121:H19. All but one were isolated during the months of May to September. The two-step method was found to be considerably more expensive than SMAC for both positive and negative samples. PMID:10655343

  9. AhpC, AhpD, and a secreted 14-kilodalton antigen from Mycobacterium avium subsp. paratuberculosis distinguish between paratuberculosis and bovine tuberculosis in an enzyme-linked immunosorbent assay.

    PubMed

    Olsen, I; Tryland, M; Wiker, H G; Reitan, L J

    2001-07-01

    Sera from cattle naturally infected with Mycobacterium avium subsp. paratuberculosis (n = 56) and naturally (n = 4) and experimentally (n = 8) infected with Mycobacterium bovis were tested for the presence of antibodies against paratuberculosis antigens. An enzyme-linked immunosorbent assay (ELISA) was established based on absorption of M. avium subsp. paratuberculosis antigens on a hyperimmune antiserum against M. avium subsp. avium proteins in order to remove cross-reacting antigens. This absorbed-antigen ELISA recognized 66% of animals with paratuberculosis (37 of 56), while none of the animals with naturally occurring bovine tuberculosis (TB) had detectable antibodies. However, the animals with experimental bovine TB also responded in this ELISA. Similar results were found in a commercial ELISA, showing that neither of these tests was able to distinguish between paratuberculosis and bovine TB. The sera were further tested for antibody activities against purified AhpC and AhpD, which are proteins constitutively expressed by M. avium subsp. paratuberculosis, and against a secreted 14-kDa protein present in culture filtrates from the M. avium complex. Elevated antibody levels to AhpC, AhpD, and the 14-kDa antigen were found in 27% (13 of 48), 15% (7 of 48), and 27% (13 of 48), respectively, of the cattle with paratuberculosis. Together these ELISAs were positive with 35% (17 of 48) of the animals. None of the animals with bovine TB had detectable antibodies against any of the purified proteins despite their high levels of cross-reacting antibodies. These results show that purified specific antigens are needed to differentiate between paratuberculosis and bovine TB in ELISA. PMID:11427429

  10. A comparison of titers of anti-Brucella antibodies of naturally infected and healthy vaccinated cattle by standard tube agglutination test, microtiter plate agglutination test, indirect hemagglutination assay, and indirect enzyme-linked immunosorbent assay

    PubMed Central

    Mohan, Anju; Saxena, Hari Mohan; Malhotra, Puneet

    2016-01-01

    Aim: We determined the antibody response in cattle naturally infected with brucellosis and normal healthy adult cattle vaccinated during calf hood with strain 19. Materials and Methods: The antibody titers were measured by standard tube agglutination test (STAT), microtiter plate agglutination test (MAT), indirect hemagglutination assay (IHA), and indirect enzyme-linked immunosorbent assay (iELISA) as per standard protocols. Results: The mean STAT titers were 1.963±0.345 in infected cattle and 1.200±0.155 in healthy vaccinated cattle. The difference was extremely significant (p<0.0001). The mean MAT titers were 2.244±0.727 in infected cattle and 1.200±0.155 in healthy vaccinated cattle. The difference was very significant (p<0.005). The mean IHA titers in infected cattle were 2.284±0.574, and those in healthy vaccinated cattle were 1.200±0.155. The difference was extremely significant (p=0.0002). However, the difference in mean iELISA titers of infected cattle (1.3678±0.014) and healthy vaccinated cattle (1.367±0.014) was non-significant. The infected animals showed very high titers of agglutinating antibodies compared to the vaccinated animals. However, it cannot be ascertained whether these antibodies are due to vaccine or response to infection. Since the infected animals had been vaccinated earlier, the current infection may suggest that vaccination was unable to induce protective levels of antibody. The heightened antibody response after infection may also indicate a secondary immune response to the antigens common to the vaccine strain and wild Brucella organisms. Conclusion: The brucellosis infected animals showed very high titers of agglutinating antibodies compared to the vaccinated animals. PMID:27536032

  11. Rapid and sensitive detection of immunoglobulin M (IgM) and IgG antibodies against canine distemper virus by a new recombinant nucleocapsid protein-based enzyme-linked immunosorbent assay.

    PubMed

    von Messling, V; Harder, T C; Moennig, V; Rautenberg, P; Nolte, I; Haas, L

    1999-04-01

    Canine distemper morbillivirus (CDV) infection causes a frequently fatal systemic disease in a broad range of carnivore species, including domestic dogs. In CDV infection, classical serology provides data of diagnostic and prognostic values (kinetics of seroconversion) and is also used to predict the optimal vaccination age of pups. Routine CDV serology is still based on time- and cost-intensive virus neutralization assays (V-NA). Here, we describe a new capture-sandwich enzyme-linked immunosorbent assay (ELISA) that uses recombinant baculovirus-expressed nucleocapsid (N) protein of a recent CDV wild-type isolate (2544/Han95) for the detection of CDV-specific antibodies in canine sera. Recombinant antigen was produced with high efficacy in Heliothis virescens larvae. The capture-sandwich ELISA enabled a clear-cut qualitative evaluation of the CDV-specific immunoglobulin G (IgG) and IgM serostatuses of 196 and 35 dog sera, respectively. Inter-rater agreement analysis (kappa = 0.988) indicated that the ELISA can be used unrestrictedly as a substitute for the V-NA for the qualitative determination of CDV-specific IgG serostatus. In an attempt to semiquantify N-specific antibodies, a one-step-dilution (alpha method) IgG-specific ELISA was implemented. Alpha values of >/=50% showed very good inter-rater agreement (kappa = 0.968) with V-NA titers of >/=1/100 50% neutralizing dose (ND50) as measured against the central European CDV wild-type isolate 2544/Han95 in canine sera originating from northern Germany. An ND50 titer of 1/100 is considered a threshold, and titers of >/=1/100 indicate a resilient, protective immunity. CDV N-specific antibodies of the IgM class were detected by the newly developed ELISA in 9 of 15 sera obtained from dogs with symptoms of acute distemper. In leucocytes of 5 of the 15 dogs (all of which were also IgM positive) CDV RNA was detected by reverse transcription (RT)-PCR. The recombinant capture-sandwich ELISA detecting N-specific antibodies

  12. Rapid and Sensitive Detection of Immunoglobulin M (IgM) and IgG Antibodies against Canine Distemper Virus by a New Recombinant Nucleocapsid Protein-Based Enzyme-Linked Immunosorbent Assay

    PubMed Central

    von Messling, Veronika; Harder, Timm C.; Moennig, Volker; Rautenberg, Peter; Nolte, Ingo; Haas, Ludwig

    1999-01-01

    Canine distemper morbillivirus (CDV) infection causes a frequently fatal systemic disease in a broad range of carnivore species, including domestic dogs. In CDV infection, classical serology provides data of diagnostic and prognostic values (kinetics of seroconversion) and is also used to predict the optimal vaccination age of pups. Routine CDV serology is still based on time- and cost-intensive virus neutralization assays (V-NA). Here, we describe a new capture-sandwich enzyme-linked immunosorbent assay (ELISA) that uses recombinant baculovirus-expressed nucleocapsid (N) protein of a recent CDV wild-type isolate (2544/Han95) for the detection of CDV-specific antibodies in canine sera. Recombinant antigen was produced with high efficacy in Heliothis virescens larvae. The capture-sandwich ELISA enabled a clear-cut qualitative evaluation of the CDV-specific immunoglobulin G (IgG) and IgM serostatuses of 196 and 35 dog sera, respectively. Inter-rater agreement analysis (κ = 0.988) indicated that the ELISA can be used unrestrictedly as a substitute for the V-NA for the qualitative determination of CDV-specific IgG serostatus. In an attempt to semiquantify N-specific antibodies, a one-step-dilution (alpha method) IgG-specific ELISA was implemented. Alpha values of ≥50% showed very good inter-rater agreement (κ = 0.968) with V-NA titers of ≥1/100 50% neutralizing dose (ND50) as measured against the central European CDV wild-type isolate 2544/Han95 in canine sera originating from northern Germany. An ND50 titer of 1/100 is considered a threshold, and titers of ≥1/100 indicate a resilient, protective immunity. CDV N-specific antibodies of the IgM class were detected by the newly developed ELISA in 9 of 15 sera obtained from dogs with symptoms of acute distemper. In leucocytes of 5 of the 15 dogs (all of which were also IgM positive) CDV RNA was detected by reverse transcription (RT)-PCR. The recombinant capture-sandwich ELISA detecting N-specific antibodies of the

  13. An increased level of the Concanavalin A-positive IgG in the serum of patients with gastric cancer as evaluated by a lectin enzyme-linked immunosorbent assay (LELISA).

    PubMed

    Klaamas, K; Kodar, K; Kurtenkov, O

    2008-01-01

    All human immunoglobulins are glycosylated. The changes in IgG glycosylation are associated with autoimmune disorders and pregnancy. Little is known about IgG glycosylation in patients with cancer. A lectin enzyme-linked immunosorbent assay (LELISA) based method was developed for measuring the Concanavalin A - positive IgG in the serum. Its rationale is as follows: PtA was used as a capture agent for binding IgG via the Fc fragment. Then IgG and the ConA-positive glycans on the IgG were detected using an anti-human IgG-F(ab)2 alkaline phosphatase conjugate or biotinylated ConA, respectively. The index ConA binding/total IgG was calculated. Serum samples from patients with gastric carcinoma (n=53) and healthy blood transfusion donors (n=24) were analysed. The protein A-agarose and ConA-sepharose affinity chromatography was applied to the purification of IgG, ConA-positive IgG, and Fab fragments. The LELISA, SDS-PAGE and Western blot methods were used to analyse the purified IgG and Fab fragments. A significantly higher ConA binding to IgG was found in patients with cancer compared to that of blood donors (ConA index = 1.07+/-0.08 (95% CI) and 0.81+/-0.08, respectively; P=0.0002). In donors, a significant correlation between the level of IgG bound to PtA and the ConA binding (r=0.85; p<0.001) was observed. Patients with gastric cancer showed a less pronounced, though significant correlation (r=0.33; P=0.02). Only the Fd fragment of the Fabs derived from both total serum IgG and ConA-positive fraction of IgG contained the ConA-positive glycans. The comparison of the purified IgG and Fab fragments derived from healthy blood donors and patient with gastric cancer showed no difference in either SDS-PAGE, immunoblotting or LELISA pattern. The LELISA is simple, reproducible and suitable for the evaluation of IgG glycosylation changes. The level of ConA positive serum IgG was found to be increased in patients with cancer. No convincing evidence of the presence of

  14. Monoclonal antibodies to conformational epitopes of the surface glycoprotein of caprine arthritis-encephalitis virus: potential application to competitive-inhibition enzyme-linked immunosorbent assay for detecting antibodies in goat sera.

    PubMed

    Ozyörük, F; Cheevers, W P; Hullinger, G A; McGuire, T C; Hutton, M; Knowles, D P

    2001-01-01

    Four immunoglobulin G1 monoclonal antibodies (MAbs) to the gp135 surface envelope glycoprotein (SU) of the 79-63 isolate of caprine arthritis-encephalitis virus (CAEV), referred to as CAEV-63, were characterized and evaluated for their ability to compete with antibody from CAEV-infected goats. Three murine MAbs (MAbs GPB16A, 29A, and 74A) and one caprine MAb (MAb F7-299) were examined. All MAbs reacted in nitrocellulose dot blots with native CAEV-63 SU purified by MAb F7-299 affinity chromatography, whereas none reacted with denatured and reduced SU. All MAbs reacted in Western blots with purified CAEV-63 SU or the SU component of whole-virus lysate following denaturation in the absence of reducing agent, indicating that intramolecular disulfide bonding was essential for epitope integrity. Peptide-N-glycosidase F digestion of SU abolished the reactivities of MAbs 74A and F7-299, whereas treatment of SU with N-acetylneuraminate glycohydrolase (sialidase A) under nonreducing conditions enhanced the reactivities of all MAbs as well as polyclonal goat sera. MAbs 29A and F7-299 were cross-reactive with the SU of an independent strain of CAEV (CAEV-Co). By enzyme-linked immunosorbent assay (ELISA), the reactivities of horseradish peroxidase (HRP)-conjugated MAbs 16A and 29A with homologous CAEV-63 SU were <10% of that of HRP-conjugated MAb 74A. The reactivity of HRP-conjugated MAb 74A was blocked by sera from goats immunized with CAEV-63 SU or infected with CAEV-63. The reactivity of MAb 74A was also blocked by sera from goats infected with a CAEV-Co molecular clone, although MAb 74A did not react with CAEV-Co SU in Western blots. Thus, goats infected with either CAEV-63 or CAEV-Co make antibodies that inhibit binding of MAb 74A to CAEV-63 SU. A competitive-inhibition ELISA based on displacement of MAb 74A reactivity has potential applicability for the serologic diagnosis of CAEV infection. PMID:11139194

  15. Evaluation of the Double Agar Gel Immunodiffusion Test and of the Enzyme-Linked Immunosorbent Assay in the Diagnosis and Follow-Up of Patients with Chronic Pulmonary Aspergillosis

    PubMed Central

    de Azevedo, Priscila Zacarias; Sylvestre, Tatiane Fernanda; Cavalcante, Ricardo de Souza; de Carvalho, Lídia Raquel; Moris, Daniela Vanessa; de Oliveira, Maria Luiza Cotrim Sartor; Mendes, Rinaldo Poncio

    2015-01-01

    The diagnosis of chronic pulmonary aspergillosis (CPA) depends on the radiologic image and the identification of specific antibodies. The present study aimed to evaluate accuracy parameters of enzyme-linked immunosorbent assay (ELISA) and of the determination of serum galactomannan level in the diagnosis of patients with CPA, comparing these results with the double agar gel immunodiffusion (DID) test. In addition, the prevalence of cross-reactivity and the serological progression after treatment were evaluated by comparing DID and ELISA. Six study groups were formed: G1: 22 patients with CPA, 17 of whom had Aspergillus fungus ball, one chronic cavitary pulmonary aspergillosis (CCPA) and four chronic fibrosing pulmonary aspergillosis (CFPA); G2: 28 patients with pulmonary tuberculosis (TB); G3: 23 patients with histoplasmosis (HST); G4: 50 patients with paracoccidioidomycosis (PCM); G5: 20 patients with cryptococcosis (CRC); and G6: 200 healthy controls. Serum antibodies were measured by DID and ELISA, with two antigen preparations—Aspergillus fumigatus (DID1, ELISA1) and a pool of A. fumigatus, A. flavus and A. niger antigens (DID2, ELISA2). The Platélia Aspergillus Enzyme Immunoassay (EIA) kit was used to measure galactomannan. The cut-off points of ELISA were determined for each antigen preparation and for the 95% and 99% confidence intervals. Despite the low sensitivity, DID was the technique of choice due to its specificity, positive and negative predictive values and positive likelihood ratio–especially with the antigen pool and due to the low frequency of cross-reactivity. ELISA1 and a 0.090 cut-off showed high sensitivity, specificity and negative predictive value, but a high frequency of cross-reactivity with CRC. The best degree of agreement was observed between ELISA1 and ELISA2. The detection of serum galactomannan showed high sensitivity, comparable to ELISA2. The immunodiffusion test showed an excellent relationship with the progression after

  16. Evaluation of Indirect Enzyme-Linked Immunosorbent Assays and IgG Avidity Assays Using a Protein A-Peroxidase Conjugate for Serological Distinction between Brucella abortus S19-Vaccinated and -Infected Cows ▿

    PubMed Central

    Pajuaba, Ana C. A. M.; Silva, Deise A. O.; Mineo, José R.

    2010-01-01

    This study aimed to evaluate the use of protein A-peroxidase (horseradish peroxidase [HRPO]) in indirect enzyme-linked immunosorbent assays (iELISAs) and IgG avidity assays for serological distinction between Brucella abortus S19-vaccinated and -infected cows. Four groups were analyzed: GI, 41 nonvaccinated seropositive cows; GII, 79 S19-vaccinated heifers analyzed at 3 months postvaccination; GIII, 105 S19-vaccinated cows analyzed after 24 months of age; and GIV, 278 nonvaccinated seronegative cows. IgG levels and avidity to B. abortus smooth lipopolysaccharide (S-LPS) were determined using anti-bovine IgG-HRPO or protein A-HRPO conjugates. Similar levels of IgG anti-S-LPS were found with GI using both conjugates. Lower IgG levels were detected with GII, GIII, and GIV using protein A-HRPO. Both conjugates showed high performance in discriminating GI from GIII, with high sensitivity (Se; 97.6%) and specificity (Sp; 97.1%). Protein A-HRPO was better in distinguishing GI from GIV (Se, 97.6%; Sp, 94.6%) and GI from GII (Se, 80.5%; Sp, 94.9%). Protein A-HRPO excluded a higher number of positive samples with GII and GIV. IgG avidity showed that protein A-HRPO, but not anti-IgG-HRPO, was able to distinguish nonvaccinated from vaccinated cattle, showing a higher avidity index (AI) with GI than with GII, with 78% of serum samples in GII showing an AI of <50%. Therefore, the iELISA using B. abortus S-LPS antigen and protein A-HRPO conjugate for preferential detection of the IgG2 subclass was shown to be suitable for serological distinction between S19-vaccinated and -infected cows. Also, antibodies generated after vaccination showed lower avidity, suggesting a role for the IgG2 subclass as an antibody of higher-affinity maturation after infection, constituting an additional tool for differentiating vaccinated from infected cattle. PMID:20147498

  17. Evaluation of indirect enzyme-linked immunosorbent assays and IgG avidity assays using a protein A-peroxidase conjugate for serological distinction between Brucella abortus S19-vaccinated and -infected cows.

    PubMed

    Pajuaba, Ana C A M; Silva, Deise A O; Mineo, José R

    2010-04-01

    This study aimed to evaluate the use of protein A-peroxidase (horseradish peroxidase [HRPO]) in indirect enzyme-linked immunosorbent assays (iELISAs) and IgG avidity assays for serological distinction between Brucella abortus S19-vaccinated and -infected cows. Four groups were analyzed: GI, 41 nonvaccinated seropositive cows; GII, 79 S19-vaccinated heifers analyzed at 3 months postvaccination; GIII, 105 S19-vaccinated cows analyzed after 24 months of age; and GIV, 278 nonvaccinated seronegative cows. IgG levels and avidity to B. abortus smooth lipopolysaccharide (S-LPS) were determined using anti-bovine IgG-HRPO or protein A-HRPO conjugates. Similar levels of IgG anti-S-LPS were found with GI using both conjugates. Lower IgG levels were detected with GII, GIII, and GIV using protein A-HRPO. Both conjugates showed high performance in discriminating GI from GIII, with high sensitivity (Se; 97.6%) and specificity (Sp; 97.1%). Protein A-HRPO was better in distinguishing GI from GIV (Se, 97.6%; Sp, 94.6%) and GI from GII (Se, 80.5%; Sp, 94.9%). Protein A-HRPO excluded a higher number of positive samples with GII and GIV. IgG avidity showed that protein A-HRPO, but not anti-IgG-HRPO, was able to distinguish nonvaccinated from vaccinated cattle, showing a higher avidity index (AI) with GI than with GII, with 78% of serum samples in GII showing an AI of <50%. Therefore, the iELISA using B. abortus S-LPS antigen and protein A-HRPO conjugate for preferential detection of the IgG2 subclass was shown to be suitable for serological distinction between S19-vaccinated and -infected cows. Also, antibodies generated after vaccination showed lower avidity, suggesting a role for the IgG2 subclass as an antibody of higher-affinity maturation after infection, constituting an additional tool for differentiating vaccinated from infected cattle. PMID:20147498

  18. Establishment of an Algorithm Using prM/E- and NS1-Specific IgM Antibody-Capture Enzyme-Linked Immunosorbent Assays in Diagnosis of Japanese Encephalitis Virus and West Nile Virus Infections in Humans.

    PubMed

    Galula, Jedhan U; Chang, Gwong-Jen J; Chuang, Shih-Te; Chao, Day-Yu

    2016-02-01

    The front-line assay for the presumptive serodiagnosis of acute Japanese encephalitis virus (JEV) and West Nile virus (WNV) infections is the premembrane/envelope (prM/E)-specific IgM antibody-capture enzyme-linked immunosorbent assay (MAC-ELISA). Due to antibody cross-reactivity, MAC-ELISA-positive samples may be confirmed with a time-consuming plaque reduction neutralization test (PRNT). In the present study, we applied a previously developed anti-nonstructural protein 1 (NS1)-specific MAC-ELISA (NS1-MAC-ELISA) on archived acute-phase serum specimens from patients with confirmed JEV and WNV infections and compared the results with prM/E containing virus-like particle-specific MAC-ELISA (VLP-MAC-ELISA). Paired-receiver operating characteristic (ROC) curve analyses revealed no statistical differences in the overall assay performances of the VLP- and NS1-MAC-ELISAs. The two methods had high sensitivities of 100% but slightly lower specificities that ranged between 80% and 100%. When the NS1-MAC-ELISA was used to confirm positive results in the VLP-MAC-ELISA, the specificity of serodiagnosis, especially for JEV infection, was increased to 90% when applied in areas where JEV cocirculates with WNV, or to 100% when applied in areas that were endemic for JEV. The results also showed that using multiple antigens could resolve the cross-reactivity in the assays. Significantly higher positive-to-negative (P/N) values were consistently obtained with the homologous antigens than those with the heterologous antigens. JEV or WNV was reliably identified as the currently infecting flavivirus by a higher ratio of JEV-to-WNV P/N values or vice versa. In summary of the above-described results, the diagnostic algorithm combining the use of multiantigen VLP- and NS1-MAC-ELISAs was developed and can be practically applied to obtain a more specific and reliable result for the serodiagnosis of JEV and WNV infections without the need for PRNT. The developed algorithm should provide great

  19. Establishment of an Algorithm Using prM/E- and NS1-Specific IgM Antibody-Capture Enzyme-Linked Immunosorbent Assays in Diagnosis of Japanese Encephalitis Virus and West Nile Virus Infections in Humans

    PubMed Central

    Galula, Jedhan U.; Chang, Gwong-Jen J.

    2015-01-01

    The front-line assay for the presumptive serodiagnosis of acute Japanese encephalitis virus (JEV) and West Nile virus (WNV) infections is the premembrane/envelope (prM/E)-specific IgM antibody-capture enzyme-linked immunosorbent assay (MAC-ELISA). Due to antibody cross-reactivity, MAC-ELISA-positive samples may be confirmed with a time-consuming plaque reduction neutralization test (PRNT). In the present study, we applied a previously developed anti-nonstructural protein 1 (NS1)-specific MAC-ELISA (NS1-MAC-ELISA) on archived acute-phase serum specimens from patients with confirmed JEV and WNV infections and compared the results with prM/E containing virus-like particle-specific MAC-ELISA (VLP-MAC-ELISA). Paired-receiver operating characteristic (ROC) curve analyses revealed no statistical differences in the overall assay performances of the VLP- and NS1-MAC-ELISAs. The two methods had high sensitivities of 100% but slightly lower specificities that ranged between 80% and 100%. When the NS1-MAC-ELISA was used to confirm positive results in the VLP-MAC-ELISA, the specificity of serodiagnosis, especially for JEV infection, was increased to 90% when applied in areas where JEV cocirculates with WNV, or to 100% when applied in areas that were endemic for JEV. The results also showed that using multiple antigens could resolve the cross-reactivity in the assays. Significantly higher positive-to-negative (P/N) values were consistently obtained with the homologous antigens than those with the heterologous antigens. JEV or WNV was reliably identified as the currently infecting flavivirus by a higher ratio of JEV-to-WNV P/N values or vice versa. In summary of the above-described results, the diagnostic algorithm combining the use of multiantigen VLP- and NS1-MAC-ELISAs was developed and can be practically applied to obtain a more specific and reliable result for the serodiagnosis of JEV and WNV infections without the need for PRNT. The developed algorithm should provide great

  20. Evaluation of a Caprine Arthritis-Encephalitis Virus/Maedi-Visna Virus Indirect Enzyme-Linked Immunosorbent Assay in the Serological Diagnosis of Ovine Progressive Pneumonia Virus in U.S. Sheep

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Serological diagnostic testing of sheep and goats using enzyme immunosorbent assays (ELISAs) is the most common method of determining small ruminant lentivirus (SRLV) infection. A caprine arthritis-encephalitis virus (CAEV)/maedi-visna virus (MVV) indirect (i) ELISA, which utilizes MVV EV1 capsid a...