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Sample records for elisa enzyme-linked immunosorbent

  1. The enzyme-linked immunosorbent assay (ELISA).

    PubMed

    1976-01-01

    The enzyme-linked immunosorbent assay "ELISA" developed in recent years represents a significant addition to existing serological tools. Encouraging preliminary results obtained through its application to a number of parasitic diseases during the last two years indicate the value of further investigations and trials which will permit a true evaluation.Although the technique is easy to perform and quite sensitive, there are certain problems to be solved before it becomes widely usable. In the present Memorandum the technical details are given and the advantages and shortcomings of the procedure are discussed. Present applications and future prospects are reviewed.

  2. 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…

  3. 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…

  4. Predicting detection limits of enzyme-linked immunosorbent assay (ELISA) and bioanalytical techniques in general.

    PubMed

    Zhang, Shiyun; Garcia-D'Angeli, Alexa; Brennan, Joseph P; Huo, Qun

    2014-01-21

    The detection limit is one of the most important performance parameters for bioanalytical techniques. Here we present a generic method to estimate the detection limit of biomolecular assays based on a step-by-step analysis of the assay procedure. Enzyme-linked immunosorbent assay (ELISA) is used here as an example; however, much of the information presented in this article may be applied to other types of biomolecular assays and analytical techniques. A clear understanding of what affects the detection limit can help researchers to evaluate different bio-analytical techniques properly, and to design better strategies to optimize and achieve the best analytical performance.

  5. Timed ELISA: an alternative approach to quantitative enzyme-linked immunosorbent assay.

    PubMed

    Muller, R

    1997-10-01

    As the uses for ELISA (enzyme-linked immunosorbent assay) increase, so does the need for a quantitative procedure that does not require a spectrophotometer or other expensive equipment. 'Timed ELISA' employs an 'iodine clock' as the final step such that quantitative measurements may be made using a stopwatch. Catalase, coupled to the primary antibody, reduces the concentration of H2O2 available to generate iodine in the clock reaction. Iodine stains the starch component blue, but catalase prolongs the time taken for the change in colour to be observed. After the time delay occurs the transition to full colour development is extremely rapid (< 1 s) at all analyte concentrations, allowing clear definition of the end point. The performance of Timed ELISA is similar to that obtained using a horseradish peroxidase-conjugated system employing the customary spectrophotometric determination.

  6. 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.

  7. The effects of detergent on the enzyme-linked immunosorbent assay (ELISA) of blood group substances.

    PubMed

    McCabe, J P; Fletcher, S M; Jones, M N

    1988-04-06

    The detergents 1-0-n-octyl-beta-D-glucopyranoside (OBG) and sodium n-dodecyl sulphate (SDS) have been used to extract blood group substances from human erythrocyte membranes for detection by enzyme-linked immunosorbent assay (ELISA). The effect of detergent concentration on the extraction process and detection by ELISA have been investigated. Detergent extraction increased the ELISA response relative to response from membrane suspensions approximately 1000-fold. Optimum responses occurred using detergent concentrations near the critical micelle concentration (cmc) for OBG and below the cmc for SDS. High detergent concentrations interfered with the ELISA but this effect was reduced by dilution of the extracts before adsorption of antigen on the microtitre wells. The interference effects of detergent on ELISA were also investigated using ovarian cyst glycoproteins as antigen. It was found that detergents inhibit the assay at the initial stage by competing with antigens for adsorption sites on the microtitre well surface and that subsequent detergent can displace pre-bound antigen. The results are discussed in terms of detergent binding to proteins (and glycoproteins) in relation to free (unbound) detergent concentration.

  8. Detection of platelet antibodies by enzyme-linked immunosorbent assay (ELISA). Comparative studies with the indirect immunofluorescence assay.

    PubMed

    Horai, S; Claas, F H; van Rood, J J

    1981-08-01

    A newly developed enzyme-linked immunosorbent assay (ELISA) for the detection of platelet antibodies was compared with the platelet immunofluorescence test (PIF). A good correlation was found between both assays. However, ELISA seems to be more sensitive than PIF. Some sera reacted only in ELISA whereas no sera were found that were negative in ELISA and positive in PIF. When comparing the antibody titres, ELISA is at least 8 times more sensitive than PIF.

  9. Validation of a KHV antibody enzyme-linked immunosorbent assay (ELISA).

    PubMed

    Bergmann, S M; Wang, Q; Zeng, W; Li, Y; Wang, Y; Matras, M; Reichert, M; Fichtner, D; Lenk, M; Morin, T; Olesen, N J; Skall, H F; Lee, P-Y; Zheng, S; Monaghan, S; Reiche, S; Fuchs, W; Kotler, M; Way, K; Bräuer, G; Böttcher, K; Kappe, A; Kielpinska, J

    2017-05-04

    Koi herpesvirus (KHV) causes KHV disease (KHVD). The virus is highly contagious in carp or koi and can induce a high mortality. Latency and, in some cases, a lack of signs presents a challenge for virus detection. Appropriate immunological detection methods for anti-KHV antibodies have not yet been fully validated for KHV. Therefore, it was developed and validated an enzyme-linked immunosorbent assay (ELISA) to detect KHV antibodies. The assay was optimized with respect to plates, buffers, antigens and assay conditions. It demonstrated high diagnostic and analytical sensitivity and specificity and was particularly useful at the pond or farm levels. Considering the scale of the carp and koi industry worldwide, this assay represents an important practical tool for the indirect detection of KHV, also in the absence of clinical signs. © 2017 John Wiley & Sons Ltd.

  10. Enzyme-linked immunosorbent assay (ELISA) for antibodies against Campylobacter jejuni, and its clinical application.

    PubMed

    Walder, M; Forsgren, A

    1982-12-01

    Antibody response to Campylobacter jejuni/coli (CJC) was investigated, using an enzyme-linked immunosorbent assay, ELISA. With a mixture of lipopolysaccharide from two CJC strains as antigen in ELISA, all 24 tested rabbit anti-CJC sera showed high antibody levels. However, only 70% of sera from patients with Campylobacter enteritis demonstrated an antibody response against the combined LPS antigen, using paired sera. In addition, the results obtained suggested non-specific binding of human immunoglobulin. When 24 formalinized whole CJC bacteria were used as antigen in ELISA, all corresponding rabbit antisera reacted with one strain (M 14). Essentially no unspecific binding of human immunoglobulin was obtained. Antibodies were detected in sera from healthy blood donors and at a lower level in sera from children, suggesting early immunization. In 67 enteritis patients with positive stool cultures for CJC, a significantly increased level of IgG antibodies could be detected in single or paired serum samples from 82% of the patients. An IgG titre increase occurred early in the course of infection, suggesting a boosting of an earlier immunization. IgM antibodies could be detected in the same sera in 77% of the patients. Considering both IgG and IgM analyses of the enteritis sera, 94% of the patients were positive in Campylobacter ELISA serology compared with only 5% of healthy controls.

  11. 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.

  12. 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.

  13. 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

  14. 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…

  15. 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…

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

    PubMed

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

    2016-07-21

    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

  17. 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

  18. Evaluation of the enzyme-linked immunosorbent assay (ELISA) and other serological tests for the diagnosis of toxoplasmosis

    PubMed Central

    Carlier, Y.; Bout, D.; Dessaint, J. P.; Capron, A.; Van Knapen, F.; Ruitenberg, E. J.; Bergquist, R.; Huldt, G.

    1980-01-01

    The enzyme-linked immunosorbent assay (ELISA) was evaluated in human toxoplasmosis in three laboratories using their own procedures. The same batch of serum samples was investigated in the three laboratories. ELISA results were compared by statistical analysis both with one another and with those of the dye test (DT), immunofluorescence (IF), complement fixation test (CFT), and indirect haemagglutination (IHA). Highly significant correlations were obtained between the three laboratories with ELISA using two different antigens and enzyme conjugates. The correlations between ELISA and the other serological tests showed the following sequence: CFT>IF>IHA>DT. Highly significant correlations were obtained between ELISA using anti-γ-chain and anti-total immunoglobulin conjugates. The agreement in discrimination between sera with low and high antibody levels was good for all the different ELISA techniques but discrimination between positive and negative sera depended rather on the ELISA procedure used. PMID:6991147

  19. An enzyme-linked immunosorbent assay (ELISA) for methylphenidate (Ritalin ) in urine.

    PubMed

    Lewis, Mark G; Lewis, John G; Elder, Peter A; Moore, Grant A

    2003-09-01

    A direct enyzme-linked immunosorbent assay (ELISA) for urinary immunoreactive methylphenidate (Ritalin), in which a standard 96-well microtiter plate is used, is described. For this ELISA, a methylphenidate-thyroglobulin conjugate is immobilized to the microtiter plate and competes with methylphenidate in the standard or urine sample for antibody-binding sites. After washing, the sheep methylphenidate antibody bound to immobilized methylphenidate is detected with peroxidase-labelled goat antisheep IgG. Following a further wash, tetramethylbenzidine is added, color is developed, and the plate is read at 450 nm on an ELISA plate reader. This method is unaffected by drugs of abuse and is suitable for routine use in the toxicology laboratory.

  20. 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...

  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. An enzyme-linked immunosorbent assay (ELISA) for PBS Z1, a defective phage of Bacillus subtilis.

    PubMed

    Steensma, H Y; Duermeyer, W

    1979-09-01

    The sensitivity, reproducibility and specificity of an enzyme-linked immunosorbent assay (ELISA) for the defective phage PBS Z1 of Bacillus subtilis have been investigated. It was shown that phages in concentrations between 10(8) and 2.5 X 10(10) particles/ml could be assayed with this method. The coefficient of variation for concentrations between 5 X 10(8) and 5 X 10(9) particles/ml was approx. 10%. From some other Bacillus phages tested, only the defective phages resembling PBS Z1 in morphology were detected efficiently with the ELISA for PBS Z1. A comparison is made between ELISA and other assays for PBS Z1.

  4. Enzyme-linked immunosorbent assay (ELISA) and blocking with bovine serum albumin (BSA)--not all BSAs are alike.

    PubMed

    Xiao, Yuhong; Isaacs, Stuart N

    2012-10-31

    The enzyme-linked immunosorbent assay (ELISA) is an extremely common and powerful laboratory technique for detecting proteins by antibodies. Researchers frequently use bovine serum albumin (BSA) as a blocking agent to prevent non-specific binding of antigens and antibodies to the microtiter well. While studying the interactions of the vaccinia virus complement control protein (VCP) with complement, we found non-specific binding of VCP to BSA and identify a BSA preparation that did not result in non-specific binding. This work draws attention to the fact that not all BSA preparations are alike. It also highlights the need to perform critical controls to ensure that ELISA reactants do not inappropriately bind to the blocking agent.

  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.

  6. 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.

  7. 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

  8. An enzyme-linked immunosorbent assay (ELISA) for quantification of human collectin 11 (CL-11, CL-K1).

    PubMed

    Selman, L; Henriksen, M L; Brandt, J; Palarasah, Y; Waters, A; Beales, P L; Holmskov, U; Jørgensen, T J D; Nielsen, C; Skjodt, K; Hansen, S

    2012-01-31

    Collectin 11 (CL-11), also referred to as collectin kidney 1 (CL-K1), is a pattern recognition molecule that belongs to the collectin group of proteins involved in innate immunity. It interacts with glycoconjugates on pathogen surfaces and has been found in complex with mannose-binding lectin-associated serine protease 1 (MASP-1) and/or MASP-3 in circulation. Mutation in the CL-11 gene was recently associated with the developmental syndrome 3MC. In the present study, we established and thoroughly validated a sandwich enzyme-linked immunosorbent assay (ELISA) based on two different monoclonal antibodies. The assay is highly sensitive, specific and shows excellent quantitative characteristics such as reproducibility, dilution linearity and recovery (97.7-104%). The working range is 0.15-34 ng/ml. The CL-11 concentration in two CL-11-deficient individuals affected by the 3MC syndrome was determined to be below 2.1 ng/ml. We measured the mean serum CL-11 concentration to 284 ng/ml in 100 Danish blood donors, with a 95% confidence interval of 269-299 ng/ml. There was no significant difference in the CL-11 concentration measured in matched serum and plasma samples. Storage of samples and repeated freezing and thawing to a certain extent did not influence the ELISA. This ELISA offers a convenient and reliable method for studying CL-11 levels in relation to a variety of human diseases and syndromes.

  9. An antigen-capture enzyme-linked immunosorbent assay (ELISA) to detect isometamidium chloride in Oncorhynchus spp.

    PubMed

    Ardelli, B F; Woo, P T

    2000-02-09

    An antigen-capture enzyme-linked immunosorbent assay (ELISA) was developed to detect and measure isometamidium chloride in the plasma of Oncorhynchus tshawytscha and O. mykiss. Isometamidium-ovalbumin conjugate and anti-isometamidium antibodies were used to coat polystyrene plates. The peroxidase saturation technique was used to optimize the coating antigen concentration; it demonstrated low affinity of the isometamidium-ovalbumin conjugate but high affinity of the anti-isometamidium antibodies for polystyrene surface sites. The optimal conditions of antiisometamidium antibodies to coat plates was at pH 7.3 and a 1:1000 dilution (0.0012 mg ml(-1) protein). The ELISA was sensitive as it detected 0.0006 mg ml(-1) of isometamidium in fish plasma. Isometamidium diluted with saline could not be detected at concentrations less than 0.05 mg ml(-1). The results indicate that this ELISA is much more sensitive when isometamidium is bound to plasma than unbound isometamidium in saline.

  10. Immunological tools: engaging students in the use and analysis of flow cytometry and enzyme-linked immunosorbent assay (ELISA).

    PubMed

    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 evaluation of a novel half-semester course that focused on introducing undergraduate and graduate students to advance conceptual and technical skills associated with flow cytometry and ELISA, with emphasis on applications, experimental design, and data analysis. This course was offered in the North Carolina State University Biotechnology Program over three semesters and consisted of weekly lectures and laboratories. Students performed and/or analyzed flow cytometry and ELISA in three separate laboratory exercises: (1) identification of transgenic zebrafish hematopoietic cells, (2) analysis of transfection efficiency, and (3) analysis of cytokine production upon lipopolysaccharide stimulation. Student learning outcomes were achieved as demonstrated by multiple means of assessment, including three laboratory reports, a data analysis laboratory practicum, and a cumulative final exam. Further, anonymous student self-assessment revealed increased student confidence in the knowledge and skill sets defined in the learning outcomes. Copyright © 2014 The International Union of Biochemistry and Molecular Biology.

  11. Comparison between conventional indirect competitive enzyme-linked immunosorbent assay (icELISA) and simplified icELISA for small molecules.

    PubMed

    Zhao, Jing; Li, Gang; Yi, Guo-Xiang; Wang, Bao-Min; Deng, Ai-Xing; Nan, Tie-Gui; Li, Zhao-Hu; Li, Qing X

    2006-06-30

    A simplified indirect competitive enzyme-linked immunosorbent assay (icELISA) for small molecules was established by modifying the procedure of conventional icELISA. The key change was that the analyte, antibody, and enzyme-labeled second antibody in the simplified icELISA were added in one step, whereas in conventional icELISA these reagents were added in two separate steps. Three small chemicals, namely zeatin riboside, glycyrrhetinic acid, and chlorimuron-ethyl, were used to verify the new assay format and compare the results obtained from conventional icELISA and simplified icELISA. The results indicated that, under optimized conditions, the new assay offered several advantages over the conventional icELISA, which are simpler, less time consuming and higher sensitive although it requires more amount of reagents. The assay sensitivity (IC50) was improved for 1.2-1.4-fold. Four licorice roots samples were analyzed by conventional icELISA and simplified icELISA, as well as liquid chromatography (LC). There was no significant difference among the content obtained from the three methods for each sample. The correlation between data obtained from conventional icELISA and simplified icELISA analyses was 0.9888. The results suggest that the simplified icELISA be useful for high throughput screening of small molecules.

  12. 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

  13. Detection of mustard, egg, milk, and gluten in salad dressing using enzyme-linked immunosorbent assays (ELISAs).

    PubMed

    Lee, Poi-Wah; Niemann, Lynn M; Lambrecht, Debra M; Nordlee, Julie A; Taylor, Steve L

    2009-06-01

    Enzyme-linked immunosorbent assay (ELISA) is a commonly used method for the detection of trace amounts of potentially allergenic protein residues in foods. However, food matrices and processing conditions can affect the detection of protein residues. The effects of acidity on the detectability of several allergenic proteins commonly found in salad dressing using ELISAs was investigated. First, recovery experiments were performed on salad dressing formulated with 0 to 1000 ppm mustard flour (mustard). The mean percent recovery for mustard from the salad dressing was only 7.7%+/- 1.6%. When the pH of the salad dressing was adjusted to pH 7 prior to spiking with mustard, recovery improved to 94.1%+/- 7.6%. However, if the pH was adjusted to pH 7 after spiking and extraction, the recovery was only 11.1%+/- 1.7%. When vinegar was spiked with mustard flour at pH 3, 3.5, and 4, detectability of mustard was lowest at pH 3. Basic extraction of mustard proteins from salad dressing did not improve the mustard detection. Acidic salad dressing matrices reduced the detectability of mustard by the mustard ELISA probably because of acid precipitation of mustard proteins that renders them insoluble and nonextractable. Commercial salad dressings containing 100 ppm (mg/kg) of egg, milk, or gluten were analyzed every 2 to 4 d for 90 d using 3 commercially available ELISAs. A decrease in the detection of the egg, milk, and gluten in the salad dressing upon storage was observed. Our study highlighted the importance of evaluating the utility of various ELISAs for specific food matrices and the recovery as a function of product storage.

  14. Application of enzyme-linked immunosorbent assay (ELISA) in diagnosis of allergic bronchopulmonary aspergillosis.

    PubMed

    Greenberger, P A; Patterson, R

    1982-02-01

    IgE and IgG antibodies against Af were measured by ELISA in sera from 15 patients with ABPA and compared with antibody levels in six patients with extrinsic asthma and immediate-type skin reactivity to Af (prick skin test 3+ or 4+) but no other evidence for ABPA. The assay used polyvinyl microhemagglutination plates as a solid phase to adsorb antigens of Af and a double-antibody system of class-specific anti-human globulin and alkaline phosphatase-conjugated goat anti-rabbit globulin. IgE-Af and IgG-Af were significantly greater among patients with ABPA than in patients with asthma, suggesting that this assay may be used as an important diagnostic aid. The advantages of ELISA over radioimmunoassay are discussed in regard to early detection of an indolent pulmonary disease.

  15. Application of enzyme-linked immunosorbent assay (ELISA) in diagnosis of allergic bronchopulmonary aspergillosis

    SciTech Connect

    Greenberger, P.A.; Patterson, R.

    1982-02-01

    IgE and IgG antibodies against Af were measured by ELISA in sera from 15 patients with ABPA and compared with antibody levels in six patients with extrinsic asthma and immediate-type skin reactivity to Af (prick skin test 3+ or 4+) but no other evidence for ABPA. The assay used polyvinyl microhemagglutination plates as a solid phase to absorb antigens of Af and a double-anti-rabbit globulin. IgE-Af and IgG-Af were significantly greater among patients with ABPA than in patients with asthma, suggesting that this assay may be used as an important diagnostic aid. The advantages of ELISA over radioimmunoassay are discussed in regard to early detection of an indolent pulmonary disease.

  16. Sandwich enzyme-linked immunosorbent assay (ELISA) for the detection of lupine residues in foods.

    PubMed

    Kaw, C H; Hefle, S L; Taylor, S L

    2008-10-01

    Lupine has been increasingly used in food applications due to its high nutritional value and excellent functional properties. However, lupine provokes allergic reactions in susceptible individuals. The presence of undeclared lupine residues in foods can pose a serious health risk to lupine-allergic individuals. Therefore, the objective of this research was to develop a sandwich-type ELISA for the detection of lupine residues in foods. Lupine flour derived from Lupinus albus was used to immunize 3 rabbits and a sheep. Pooled lupine-specific antibodies were partially purified from the sera by ammonium sulfate precipitation. A sandwich lupine ELISA with a limit of quantification (LOQ) of 1 ppm was developed by utilizing the rabbit antisera as the capture reagent and the sheep antiserum as the detector reagent. The binding of the antigen-antibody complex was visualized by the addition of commercial rabbit antisheep IgG antibody labeled with alkaline phosphatase with subsequent addition of p-nitrophenyl phosphate substrate to produce a colored product for quantification. Minor cross-reactivity was observed with soy (Glycine max) and black bean (Castanospermum australe). The performance of the lupine ELISA was evaluated in reference food standards (beef frankfurter and apple cinnamon muffin) and laboratory-prepared cooked frankfurters and corn muffins. The mean percent recovery for lupine spiked-frankfurters and corn muffins were 108.4%+/- 8.8% and 103.1%+/- 11.5%, respectively. The sandwich-type lupine ELISA developed in this study provides food manufacturers and regulatory agencies with an effective analytical tool to detect and quantify lupine residues in processed foods.

  17. A recombinant antigen-based enzyme-linked immunosorbent assay (ELISA) for lungworm detection in seals.

    PubMed

    Ulrich, Sophia Arlena; Lehnert, Kristina; Siebert, Ursula; Strube, Christina

    2015-09-02

    Pinnipeds are frequently infected by the lungworms Otostrongylus circumlitus and Parafilaroides gymnurus (Metastrongyloidea). Infections are frequently associated with secondary bacterial bronchopneumonia and are often lethal. To date, a reliable lungworm diagnosis in individual seals is only possible during necropsy as examination of faeces collected from resting places does not allow assignment to individuals. Therefore, a diagnostic tool for lungworm detection in living seals is desirable for monitoring health of seals in the wild and in captivity. Previously, an ELISA based on recombinant bovine lungworm major sperm protein (MSP) as diagnostic antigen was developed for lungworm diagnosis in cattle. In the present study, this test was adapted for detection of antibodies against lungworms in harbour (Phoca vitulina) and grey seals (Halichoerus grypus). Furthermore, sera of northern elephant seals (Mirounga angustirostris) were tested to evaluate whether the harbour/grey seal ELISA is suitable for this seal species as well. For ELISA evaluation, lungworm-positive and -negative sera of harbour and grey seals were analysed using horseradish peroxidase (HRP)-conjugated Protein A as secondary antibody. Optical density was measured and a receiver operating characteristic (ROC) analysis was performed to determine a cut-off value. Potential cross-reactions were examined by testing serum of seals positive for gastrointestinal and heart nematodes, but negative for lungworm infections. In addition, sera of northern elephant seals were analysed. Harbour and grey seal serum samples showed significant differences in optical density (OD) between serum of infected and uninfected animals resulting in a cut-off value of 0.422 OD with a specificity of 100% (95% CI: 87.23-100%) and a sensitivity of 97.83% (95% CI: 88.47-99.94%). Cross-reactions with heart or gastrointestinal nematodes were not observed. Analysis of northern elephant seal samples resulted in detection of antibodies

  18. Development of an enzyme-linked immunosorbent assay (ELISA) for identification of venoms from snakes in the Agkistrodon genus.

    PubMed

    Li, Q; Ownby, C L

    1994-11-01

    An enzyme-linked immunosorbent assay (ELISA) using a purified myotoxin from the venom of Agkistrodon contortrix laticintus (broad-banded copperhead) as immunogen was developed for potential use in the identification of envenomation by snakes belonging to the genus Agkistrodon native to North America. The specificity of the assay was tested using a total of 43 venom samples from snakes of diverse geographic locations. Venom samples used for cross-reactivity determination represent eight snake families including 14 species from the genus Crotalus. The assay detected venom from all Agkistrodon species tested without significant cross-reactivity with other venoms except for samples from two species of Bothrops which do not occur naturally north of Southern Mexico. The detection limit of the assay was 2 ng/ml for homologous crude venom dissolved in normal human serum. The assay was highly accurate in correlating optical densities with venom concentrations (r = 0.997). The presence of the antigen in experimental envenomations was readily detected by the assay at an i.m. injection dosage of 0.1 microgram/g. This ELISA is a promising test for identification of envenomations by species of Agkistrodon found in most of North America. It can also be used to study the kinetics of the myotoxin in experimental envenomations.

  19. Comparison of four functionalization methods of gold nanoparticles for enhancing the enzyme-linked immunosorbent assay (ELISA)

    PubMed Central

    Ciaurriz, Paula; Fernández, Fátima; Tellechea, Edurne; Moran, Jose F

    2017-01-01

    The enzyme-linked immunosorbent assay (ELISA) technique is based on the specific recognition ability of the molecular structure of an antigen (epitope) by an antibody and is likely the most important diagnostic technique used today in bioscience. With this methodology, it is possible to diagnose illness, allergies, alimentary fraud, and even to detect small molecules such as toxins, pesticides, heavy metals, etc. For this reason, any procedures that improve the detection limit, sensitivity or reduce the analysis time could have an important impact in several fields. In this respect, many methods have been developed for improving the technique, ranging from fluorescence substrates to methods for increasing the number of enzyme molecules involved in the detection such as the biotin–streptavidin method. In this context, nanotechnology has offered a significant number of proposed solutions, mainly based on the functionalization of nanoparticles from gold to carbon which could be used as antibody carriers as well as reporter enzymes like peroxidase. However, few works have focused on the study of best practices for nanoparticle functionalization for ELISA enhancement. In this work, we use 20 nm gold nanoparticles (AuNPs) as a vehicle for secondary antibodies and peroxidase (HRP). The design of experiments technique (DOE) and four different methods for biomolecule loading were compared using a rabbit IgG/goat anti-rabbit IgG ELISA model (adsorption, directional, covalent and a combination thereof). As a result, AuNP probes prepared by direct adsorption were the most effective method. AuNPs probes were then used to detect gliadin, one of the main components of wheat gluten, the protein composite that causes celiac disease. With this optimized approach, our data showed a sensitivity increase of at least five times and a lower detection limit with respect to a standard ELISA of at least three times. Additionally, the assay time was remarkably decreased. PMID:28243563

  20. 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

  1. An enzyme-linked immunosorbent assay (ELISA) for quantification of human collectin 11 (CL-11, CL-K1)

    PubMed Central

    Selman, L.; Henriksen, M.L.; Brandt, J.; Palarasah, Y.; Waters, A.; Beales, P.L.; Holmskov, U.; Jørgensen, T.J.D.; Nielsen, C.; Skjodt, K.; Hansen, S.

    2012-01-01

    Collectin 11 (CL-11), also referred to as collectin kidney 1 (CL-K1), is a pattern recognition molecule that belongs to the collectin group of proteins involved in innate immunity. It interacts with glycoconjugates on pathogen surfaces and has been found in complex with mannose-binding lectin-associated serine protease 1 (MASP-1) and/or MASP-3 in circulation. Mutation in the CL-11 gene was recently associated with the developmental syndrome 3MC. In the present study, we established and thoroughly validated a sandwich enzyme-linked immunosorbent assay (ELISA) based on two different monoclonal antibodies. The assay is highly sensitive, specific and shows excellent quantitative characteristics such as reproducibility, dilution linearity and recovery (97.7–104%). The working range is 0.15–34 ng/ml. The CL-11 concentration in two CL-11-deficient individuals affected by the 3MC syndrome was determined to be below 2.1 ng/ml. We measured the mean serum CL-11 concentration to 284 ng/ml in 100 Danish blood donors, with a 95% confidence interval of 269–299 ng/ml. There was no significant difference in the CL-11 concentration measured in matched serum and plasma samples. Storage of samples and repeated freezing and thawing to a certain extent did not influence the ELISA. This ELISA offers a convenient and reliable method for studying CL-11 levels in relation to a variety of human diseases and syndromes. PMID:22301270

  2. High seroprevalence of Mycoplasma pneumoniae IgM in acute Q fever by enzyme-linked immunosorbent assay (ELISA).

    PubMed

    Lai, Chung-Hsu; Chang, Lin-Li; Lin, Jiun-Nong; Chen, Wei-Fang; Kuo, Li-Li; Lin, Hsi-Hsun; Chen, Yen-Hsu

    2013-01-01

    Q fever is serologically cross-reactive with other intracellular microorganisms. However, studies of the serological status of Mycoplasma pneumoniae and Chlamydophila pneumoniae during Q fever are rare. We conducted a retrospective serological study of M. pneumoniae and C. pneumoniae by enzyme-linked immunosorbent assay (ELISA), a method widely used in clinical practice, in 102 cases of acute Q fever, 39 cases of scrub typhus, and 14 cases of murine typhus. The seropositive (57.8%, 7.7%, and 0%, p<0.001) and seroconversion rates (50.6%, 8.8%, and 0%, p<0.001) of M. pneumoniae IgM, but not M. pneumoniae IgG and C. pneumoniae IgG/IgM, in acute Q fever were significantly higher than in scrub typhus and murine typhus. Another ELISA kit also revealed a high seropositivity (49.5%) and seroconversion rate (33.3%) of M. pneumoniae IgM in acute Q fever. The temporal and age distributions of patients with positive M. pneumoniae IgM were not typical of M. pneumoniae pneumonia. Comparing acute Q fever patients who were positive for M. pneumoniae IgM (59 cases) with those who were negative (43 cases), the demographic characteristics and underlying diseases were not different. In addition, the clinical manifestations associated with atypical pneumonia, including headache (71.2% vs. 81.4%, p=0.255), sore throat (8.5% vs. 16.3%, p=0.351), cough (35.6% vs. 23.3%, p=0.199), and chest x-ray suggesting pneumonia (19.3% vs. 9.5%, p=0.258), were unchanged between the two groups. Clinicians should be aware of the high seroprevalence of M. pneumoniae IgM in acute Q fever, particularly with ELISA kits, which can lead to misdiagnosis, overestimations of the prevalence of M. pneumoniae pneumonia, and underestimations of the true prevalence of Q fever pneumonia.

  3. 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.

  4. 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...

  5. 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...

  6. Diagnosis of loxoscelism in two Turkish patients confirmed with an enzyme-linked immunosorbent assay (ELISA) and non-invasive tissue sampling.

    PubMed

    Akdeniz, Sedat; Green, Jonathan A; Stoecker, William V; Gomez, Hernan F; Keklikçi, S Ugur

    2007-05-01

    Confirmed envenomations due to Loxosceles reclusa have not been previously documented in Turkey, to our knowledge. This brief report describes two Turkish patients with suspected envenomation by Loxosceles spider bites on the eyelids. Material obtained by swabbing the lesions with gauze was tested using a venom-specific enzyme-linked immunosorbent assay (ELISA). Both patients tested positive for the presence of Loxosceles venom.

  7. Chemiluminometric enzyme-linked immunosorbent assays (ELISA)-on-a-chip biosensor based on cross-flow chromatography.

    PubMed

    Cho, Il-Hoon; Paek, Eui-Hwan; Kim, Young-Kee; Kim, Joo-Ho; Paek, Se-Hwan

    2009-01-26

    A chemiluminometric biosensor system for point-of-care testing has been developed using an immuno-chromatographic assay combined with an enzyme (e.g., horseradish peroxidase) tracer that produces a light signal measurable on a simple detector. Cross-flow chromatography, a method previously investigated by our laboratory, was utilized in order to accomplish sequential antigen-antibody binding and signal generation. This enzyme-linked immunosorbent assay (ELISA) was effectively carried out on a plastic chip that was redesigned to simplify the fabrication process. To enhance the sensitivity, biotin-streptavidin capture technology was employed in preparing an immuno-strip that was then incorporated onto the chip in order to generate the ELISA-on-a-chip (EOC) biosensor. Samples containing cardiac troponin I (cTnI) were analyzed using the EOC. A chemiluminescent signal proportional to the analyte concentration was produced by adding a luminogenic substrate to the tracer enzyme complexed with the analyte on the chip. The luminescent signal was detected in a dark chamber mounted with a cooled charge-coupled device and the signal was converted to optical density for quantification. This EOC biosensor system was capable of detecting cTnI present in serum at concentrations as low as 0.027 ng mL(-1), 30 times lower than those measured using the conventional rapid test kit with colloidal gold as the tracer. In addition, the final data was acquired within 30s after the addition of the enzyme substrate, which was faster than the detection time required when using a colorimetric substrate with the same tracer enzyme.

  8. 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).

  9. Novel fluorescent probe for highly sensitive bioassay using sequential enzyme-linked immunosorbent assay-capillary isoelectric focusing (ELISA-cIEF).

    PubMed

    Henares, Terence G; Uenoyama, Yuta; Nogawa, Yuto; Ikegami, Ken; Citterio, Daniel; Suzuki, Koji; Funano, Shun-ichi; Sueyoshi, Kenji; Endo, Tatsuro; Hisamoto, Hideaki

    2013-06-07

    This paper presents a novel rhodamine diphosphate molecule that allows highly sensitive detection of proteins by employing sequential enzyme-linked immunosorbent assay and capillary isoelectric focusing (ELISA-cIEF). Seven-fold improvement in the immunoassay sensitivity and a 1-2 order of magnitude lower detection limit has been demonstrated by taking advantage of the combination of the enzyme-based signal amplification of ELISA and the concentration of enzyme reaction products by cIEF.

  10. Evaluation of guppy (Poecilia reticulata Peters) immunization against Tetrahymena sp. by enzyme-linked immunosorbent assay (ELISA).

    PubMed

    Sharon, Galit; Nath, Pulak R; Isakov, Noah; Zilberg, Dina

    2014-09-15

    Analysis of the effectiveness of guppy (Poecilia reticulata Peters) immunization based on measurements of antibody (Ab) titers suffers from a shortage of reagents that can detect guppy antibodies (Abs). To overcome this problem, we immunized mice with different preparations of guppy immunoglobulins (Igs) and used the mouse antisera to develop a quantitative enzyme-linked immunosorbent assay (ELISA). The most efficient immunogen for mouse immunization was guppy Igs adsorbed on protein A/G beads. Antisera from mice boosted with this immunoglobulin (Ig) preparation were highly specific and contained high Ab titers. They immunoreacted in a Western blot with Ig heavy and light chains from guppy serum, and Ig heavy chain from guppy whole-body homogenate. The mouse anti-guppy Ig was applied in an ELISA aimed at comparing the efficiency of different routes of guppy immunization against Tetrahymena: (i) anal intubation with sonicated Tetrahymena (40,000 Tetrahymena/fish in a total volume of 10 μL) mixed with domperidon, deoxycholic acid and free amino acids (valine, leucine, isoleucine, phenylalanine and tryptophan), or (ii) intraperitoneal (i.p.) injection of sonicated Tetrahymena in complete Freund's adjuvant (15,000 Tetrahymena/fish in total a volume of 20 μL). Negative control fish were anally intubated with the intubation mixture without Tetrahymena, or untreated. ELISA measurement of anti-Tetrahymena Ab titer revealed a significantly higher level of Abs in i.p.-immunized guppies, compared to the anally intubated and control fish. In addition, the efficiency of immunization was tested by monitoring guppy mortality following (i) i.p. challenge with Tetrahymena (900 Tetrahymena/fish) or (ii) cold stress followed by immersion in water containing 10,000 Tetrahymena/mL. Fish mortality on day 14 post-Tetrahymena infection by i.p. injection exceeded 50% in the control and anally intubated fish, compared to 31% in i.p.-immunized fish. Immunization did not protect from

  11. Detection of Klebsiella pneumoniae antibodies in Aotus l. lemurinus (Panamanian owl monkey) using an enzyme linked immunosorbent assay (ELISA) test.

    PubMed

    Obaldia, N

    1991-04-01

    An enzyme linked immunosorbent assay (ELISA), was adapted to detect antibodies against Klebsiella pneumoniae in Aotus l. lemurinus monkeys. It was used to define the prevalence of infection and the immunogenicity of an Al(OH)3 bacterin in a population of laboratory born A. l. lemurinus monkeys. This represents a preliminary step to reduce K. pneumoniae produced mortality. A striking finding during a cross-sectional prevalence study was that none of the babies of less than 2 months old had detectable levels of antibody. The antibody prevalence gradually increased in all other age groups reaching 87.5% in the 8-10-month-old group. These results indicate that infection with K. pneumoniae occurred sometime between 2 and 6 months of age, probably as a result of oral-faecal contamination and a change in the feeding and grooming behaviour. To determine whether infants had maternal antibodies or if they were asymptomatic carriers of the bacterium, a cross-sectional study was done in 15 infants less than 4 months old and their mothers. K. pneumoniae antibodies were detected in 11/15 mothers with serum titers ranging from 1:4 to greater than 1:256 and the bacterium was isolated from 3 babies and one mother and her baby. Results showed that no maternal antibodies remained in babies older than 3 weeks old. A prospective study indicated a reduction in mortality from 20% for the previous 3 years to 3.7% (3/79) in AL(OH)3 K. pneumoniae bacterin vaccinated infants born during 1988-89.

  12. Experimental conditions affecting the sensitivity of enzyme-linked immunosorbent assay (ELISA) for detection of hepatitis B surface antigen (HBsAg)*

    PubMed Central

    Fields, Howard A.; Davis, Candace L.; Bradley, Daniel W.; Maynard, James E.

    1983-01-01

    The sensitivity of an enzyme-linked immunosorbent assay (ELISA) for the detection of hepatitis B surface antigen (HBsAg) was improved 16 to 32 times after examination of various solid-phase supports, different antibody preparations as capture antibody, and different conditions for adsorbing capture antibody to the solid-phase. Comparisons were made by checkerboard titration analysis and by sensitivity studies, both of which demonstrated essentially equivalent results. Endpoints were determined by visual inspection and by spectrophotometry using o-phenylenediamine as substrate. The assay was as sensitive as commercially available radioimmunoassays without the requirement of affinity chromatography purified reagents, expensive instrumentation, or radioisotopes. PMID:6340847

  13. Diagnostic tests for amoebic liver abscess: comparison of enzyme-linked immunosorbent assay (ELISA) and counterimmunoelectrophoresis (CIE).

    PubMed

    Restrepo, M I; Restrepo, Z; Elsa Villareal, C L; Aguirre, A; Restrepo, M

    1996-01-01

    The liver abscess is the most frequent extraintestinal complication of intestinal amoebiasis: its diagnosis is suggested by the clinical picture but it must be confirmed by paraclinic tests. Themost stringent diagnosis requires identification of E. histolytica. But this is possible only in a few cases. Serological tests greatly improve the diagnosis of this severe complication of amoebiasis. We compared the Enzyme Linfed Immunosorbent Assay and the Counterimmunoelectrophoresis techniques. Both techniques were used to detect amoebic antibodies in 50 control patients, 30 patients with liver abscess and 30 patients with intestinal amoebiasis. All the sera from control patients gave negative results in both techniques. When analysing the sera from patients with intestinal amoebiasis, 10% of them were positive by ELISA but non by CIE. The sera of patients with liver abscess, we found that 90% were positive by the ELISA method and 66.6% by the CIE technique. In patients with amoebic liver abscess, the results showed that the ELISA was more sensitive than the CIE, as it presented a higher sensitivity (100%) than that of the CIE technique (66%).

  14. Cascade enzyme-linked immunosorbent assay (CELISA).

    PubMed

    Lee, Young-mi; Jeong, Yujin; Kang, Hyo Jin; Chung, Sang J; Chung, Bong Hyun

    2009-10-15

    Immunoassays are representative biochemical detection methods. Among them, sandwich-type immunoassays, typified by sandwich ELISA, have used in disease diagnosis or biochemical detection with high target selectivity. Horseradish peroxidase and alkaline phosphatase have been typically used for signal amplification in ELISA. Recently developed sandwich-type immunoassays such as biobarcode immunoassays, immuno-PCR, and immuno-RCA have improved sensitivity by changing mainly the signal amplification method. To develop a novel amplification method in ELISA, an enzyme-cascading system was incorporated into an ELISA, and the new assay is termed a cascading enzyme-linked immunosorbent assay (CELISA). This CELISA includes a trypsinogen-enterokinase combination as the cascading enzyme system, and was used to detect alpha-fetoprotein (AFP), which is a liver cancer marker, and prostate-specific antigen (PSA). Using a colorimetric reagent for signal generation, CELISA had 0.1-10pM limits-of-detection for AFP and PSA in whole human serum and assay buffers, depending on the platform, well plate, or microbead type used. This study represents the first example that incorporated an enzyme cascading step in an ELISA system, resulting in successful signal amplification with sensitive detection of pathogenic antigens in serum.

  15. 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

    2015-05-18

    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.

  16. 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

    2014-07-09

    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.

  17. Serodiagnosis of pulmonary tuberculosis in Argentina by enzyme-linked immunosorbent assay (ELISA) of IgG antibody to Mycobacterium tuberculosis antigen 5 and tuberculin purified protein derivative.

    PubMed

    Balestrino, E A; Daniel, T M; de Latini, M D; Latini, O A; Ma, Y; Scocozza, J B

    1984-01-01

    IgG antibody to Mycobacterium tuberculosis antigen 5 and tuberculin purified protein derivative (PPD) was measured, by enzyme-linked immunosorbent assay (ELISA), in serum samples from 86 patients with active pulmonary tuberculosis and 91 non-tuberculous control subjects from Santa Fé, Argentina. The geometric mean titre for the tuberculosis patients was 74.6 with antigen 5 and 99.5 with PPD. In 91 control subjects the geometric mean titres were 3.6 and 15.6 respectively. Titres were not related to tuberculin reactor status or prior BCG vaccination. At a serum dilution end-point of 1:40, ELISA with antigen 5 had a sensitivity of 81.4% and a specificity of 93.4% for tuberculosis. At 1:40, ELISA with PPD showed a sensitivity of 82.6% and a specificity of 54.9% for tuberculosis. Applied at a serum dilution of 1:40 to a hypothetical model population with a tuberculosis prevalence of 2%, ELISA using antigen 5 would correctly classify 93.2% of persons and ELISA with PPD, 55.5%. At a dilution of 1:80, accuracy is increased to 99.3% with antigen 5 and 83.3% with PPD, but sensitivity decreases to 64.0% with antigen 5 and 72.1% with PPD. Thus, antigen 5 is more accurate than PPD for the diagnosis of tuberculosis using ELISA.

  18. Serological diagnosis of pertussis: IgM, IgA and IgG antibodies against Bordetella pertussis measured by enzyme-linked immunosorbent assay (ELISA).

    PubMed

    Viljanen, M K; Ruuskanen, O; Granberg, C; Salmi, T T

    1982-01-01

    Igm, IgA and IgG antibodies against Bordetella pertussis were measured by enzyme-linked immunosorbent assay (ELISA) with an ultrasonicate of formalin-killed bacteria (a mixture of strains 1, 2 and 1, 2, 3) as antigen and disposable polystyrene 9-cuvette blocks as the solid phase. The specificity properties of the assay were assessed by an inhibition technique. Of the microbes tested, only B. parapertussis was able to cause a significant inhibition. In addition, IgM and IgA antibodies against B. pertussis were only found in some sporadic cases of respiratory infections caused by other microbes. Sera, nasal swabs and cough plates were received from 198 patients with suspected whooping-cough. ELISA determinations were mostly made from only one serum sample of each patient. Paired sera were studied only from the culture-positive infants under 3 months of age. The number of positive cultures was highest in group under 3 months of age (41%), where the frequency of positive ELISA was lowest (20%). The use of paired sera strikingly increased the number of ELISA-positive individuals in this youngest patient group. In later life, the relationship between these tests changed: isolation was positive in only about 10% of the patients, whereas 29-64% yielded positive titres in ELISA. This study shows that pertussis ELISA is a valuable aid in the rapid diagnosis of pertussis, particularly of the atypical forms of the disease which mostly are culture-negative.

  19. Serodiagnosis of pulmonary tuberculosis in Argentina by enzyme-linked immunosorbent assay (ELISA) of IgG antibody to Mycobacterium tuberculosis antigen 5 and tuberculin purified protein derivative

    PubMed Central

    Balestrino, E. A.; Daniel, T. M.; de Latini, M. D. S.; Latini, O. A.; Ma, Y.; Scocozza, J. B.

    1984-01-01

    IgG antibody to Mycobacterium tuberculosis antigen 5 and tuberculin purified protein derivative (PPD) was measured, by enzyme-linked immunosorbent assay (ELISA), in serum samples from 86 patients with active pulmonary tuberculosis and 91 non-tuberculous control subjects from Santa Fé, Argentina. The geometric mean titre for the tuberculosis patients was 74.6 with antigen 5 and 99.5 with PPD. In 91 control subjects the geometric mean titres were 3.6 and 15.6 respectively. Titres were not related to tuberculin reactor status or prior BCG vaccination. At a serum dilution end-point of 1:40, ELISA with antigen 5 had a sensitivity of 81.4% and a specificity of 93.4% for tuberculosis. At 1:40, ELISA with PPD showed a sensitivity of 82.6% and a specificity of 54.9% for tuberculosis. Applied at a serum dilution of 1:40 to a hypothetical model population with a tuberculosis prevalence of 2%, ELISA using antigen 5 would correctly classify 93.2% of persons and ELISA with PPD, 55.5%. At a dilution of 1:80, accuracy is increased to 99.3% with antigen 5 and 83.3% with PPD, but sensitivity decreases to 64.0% with antigen 5 and 72.1% with PPD. Thus, antigen 5 is more accurate than PPD for the diagnosis of tuberculosis using ELISA. PMID:6439426

  20. Use of the Falcon assay screening test--enzyme-linked immunosorbent assay (FAST-ELISA) and the enzyme-linked immunoelectrotransfer blot (EITB) to determine the prevalence of human fascioliasis in the Bolivian Altiplano.

    PubMed

    Hillyer, G V; Soler de Galanes, M; Rodriguez-Perez, J; Bjorland, J; Silva de Lagrava, M; Ramirez Guzman, S; Bryan, R T

    1992-05-01

    A collaborative study between the University of Puerto Rico School of Medicine, the Centers for Disease Control, the Bolivian Ministry of Health, and private voluntary organizations (Foster Parents Plan International and Danchurchaid) working in Bolivia has identified a region in the northwestern Altiplano of Bolivia near Lake Titicaca as harboring the highest prevalence of human fascioliasis in the world reported to date. Two serologic techniques (the Falcon assay screening test-enzyme-linked immunosorbent assay [FAST-ELISA] and the enzyme-linked immunoelectrotransfer blot [EITB]) were used in the determination of its prevalence. One hundred serum samples and 73 stool samples were obtained from Aymara Indians from Corapata, Bolivia. Antibody absorbance levels to Fasciola hepatica excretion-secretion antigens were compared with EITB banding patterns using the same antigen preparation. A positive FAST-ELISA result was defined as an absorbance value greater than the mean plus three standard deviations of two sets of normal negative controls (Puerto Rican and Bolivian). Using this criterion, 53 of 100 sera tested were found positive by this technique. Within this group, 19 (95%) of 20 individuals who were parasite positive were also positive by FAST-ELISA. An additional 24 individuals who were negative for F. hepatica eggs and 10 individuals for whom no specimens were received were also positive by FAST-ELISA. Among the 53 individuals negative for F. hepatica eggs, 29 were also negative by FAST-ELISA. The EITB analysis of the sera from confirmed infected individuals revealed at least three F. hepatica (Fh) bands with molecular weights of 12, 17, and 63 kD, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

  1. Nucleoprotein-based indirect enzyme-linked immunosorbent assay (indirect ELISA) for detecting antibodies specific to Ebola virus and Marbug virus.

    PubMed

    Huang, Yi; Zhu, Youjie; Yang, Mengshi; Zhang, Zhenqing; Song, Donglin; Yuan, Zhiming

    2014-12-01

    Full-length nucleoproteins from Ebola and Marburg viruses were expressed as His-tagged recombinant proteins in Escherichia coli and nucleoprotein-based enzyme-linked immunosorbent assays (ELISAs) were established for the detection of antibodies specific to Ebola and Marburg viruses. The ELISAs were evaluated by testing antisera collected from rabbit immunized with Ebola and Marburg virus nucleoproteins. Although little cross-reactivity of antibodies was observed in anti-Ebola virus nucleoprotein rabbit antisera, the highest reactions to immunoglobulin G (IgG) were uniformly detected against the nucleoprotein antigens of homologous viruses. We further evaluated the ELISA's ability to detect antibodies to Ebola and Marburg viruses using human sera samples collected from individuals passing through the Guangdong port of entry. With a threshold set at the mean plus three standard deviations of average optical densities of sera tested, the ELISA systems using these two recombinant nucleoproteins have good sensitivity and specificity. These results demonstrate the usefulness of ELISA for diagnostics as well as ecological and serosurvey studies of Ebola and Marburg virus infection.

  2. 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.

  3. Evaluation of an enzyme-linked immunosorbent assay (ELISA) for the detection of Campylobacter jejuni antibodies, and comparison with a complement fixation test (CFT).

    PubMed

    Oosterom, J; den Uyl, C H; Bänffer, J R; Lauwers, S; Huisman, J; Busschbach, A E; Poelma, F G; Bellemans, R

    1985-01-01

    An enzyme-linked immunosorbent assay (ELISA) was developed for the detection of total anti-Campylobacter immunoglobulins in human sera. In this assay disintegrated Campylobacter bacteria were used as the antigen. Absorption tests including other possibly enteropathogenic bacterial species showed that the ELISA system displayed a high immunological specificity for Campylobacter. Using this ELISA it was found that in about 80% of Campylobacter patients these Campylobacter antibodies are produced to almost maximal levels within 8 days after onset of disease, and that they may persist for at least 4 months. Indeed, Campylobacter antibodies were demonstrated at low levels in a large number of control sera. However, accepting an antibody titre of 1:640 as indicative of Campylobacter infection, the statistical sensitivity of the ELISA system was 77% and the specificity 95%. In an epidemiological survey a high association was demonstrated between the severity of Campylobacter-related symptoms and antibody titre values. Assessment of Campylobacter antibody titres by means of this ELISA and by a complement fixation test in 92 sera from index patients and contacts with and without symptoms showed a high association of results.

  4. Evaluation of sensitivity and specificity of enzyme-linked immunosorbent assay (ELISA) for detecting antidesmoglein 1 and 3 in Thai patients with pemphigus vulgaris and foliaceus.

    PubMed

    Kulkollakarn, Suthinee; Wattanakrai, Penpun; Vachiramon, Vasanop; Chalidapongse, Parichart

    2008-11-01

    Pemphigus is an acquired autoimmune blistering skin diseases, of which pemphigus vulgaris (PV) and pemphigus foliaceus (PF) are two major subtypes. A novel commercial enzyme-linked immunosorbent assay (ELISA) against Dsg1 and Dsg3 has been well established for diagnosis and prediction of disease activity in PF and PV. At present, the benefit of anti-Dsg 1 and anti-Dsg 3 IgG by ELISA in the diagnosis of pemphigus in Thai patients has never been reported. The objective of the present study is to evaluate the sensitivity and specificity of ELISA for detecting antidesmoglein 1 and 3 in Thai patients with pemphigus. Retrospective review of anti-Dsg1 and anti-Dsg3 antibody ELISA test results from 48 serum samples collected from 27 patients with PV seven patients with PF and 14 controls. The sensitivity of Dsg1 and Dsg3 ELISA for all patients with PV was 64% and 77.8% respectively. When subgrouped into only PV patients with new diagnosis, the sensitivity of Dsg 1 and Dsg 3 ELISA increased to 85.7% and 100%. In all PF patients, the sensitivity of anti-Dsg 1 ELISA was 71.4% and 100% for newly diagnosed PF cases. Anti-Dsg 3 was not detected in the PF group. The specificity of ELISA for anti-Dsg 1 and anti-Dsg 3 in both types of pemphigus was 85.7% and 92.3% respectively. Dsg 1 and Dsg 3 ELISA is a simple, highly sensitive and specific test in Thai pemphigus patients with 100% sensitivity in the diagnosis of both new pemphigus vulgaris and foliaceus patients.

  5. Effects of thermal processing on the enzyme-linked immunosorbent assay (ELISA) detection of milk residues in a model food matrix.

    PubMed

    Downs, Melanie L; Taylor, Steve L

    2010-09-22

    Food products and ingredients are frequently tested for the presence of undeclared allergenic food residues (including milk) using commercial enzyme-linked immunosorbent assays (ELISAs). However, little is understood about the efficacy of these kits with thermally processed foods. This study evaluated the performance of three milk ELISA kits with a model food processed by several methods. A model food (pastry dough squares) was spiked with nonfat dry milk at several concentrations. The pastry squares were processed by boiling (100 °C for 2 min), baking (190 °C for 30 min), frying (190 °C for 2 min), and retorting (121 °C for 20 min with 17 psi overpressure). Samples were analyzed with three commercial ELISA kits: Neogen Veratox Total Milk, ELISA Systems β-lactoglobulin, and ELISA Systems casein. The detection of milk residues depended upon the type of processing applied to the food and the specific milk analyte targeted by the ELISA kit. Poor recoveries were obtained in all processed samples (2-10% of expected values) using the β-lactoglobulin kit. Better recoveries were obtained in boiled samples (44 and 59%, respectively) using the total milk and casein kits. However, these kits performed poorly with baked (9 and 21%) and fried (7 and 18%) samples. Moderate recoveries were observed in retorted samples (23 and 28%). The decreased detection in processed samples is likely due to protein modifications, including aggregation and Maillard reactions, which affect the solubility and immunoreactivity of the antigens detected by the ELISA methods. The observed decreases in ELISA detection of milk are dramatic enough to affect risk-assessment decisions. However, a lower detection of milk residues does not necessarily indicate decreased allergenicity. These ELISA kits are not acceptable for all applications, and users should understand the strengths and limitations of each method.

  6. Enzyme-Linked Immunosorbent Assays.

    PubMed

    Hornbeck, Peter V

    2015-08-03

    This unit describes six different ELISA systems for the detection of specific antibodies, soluble antigens, or cell-surface antigens. In all six systems, soluble reactants are removed from solution after specifically binding to solid-phase reactants. In the first four protocols, solid-phase reactants are prepared by adsorbing an antigen or antibody onto plastic microtiter plates; in the next two protocols, the solid-phase reactants are cell-associated molecules. In all protocols, the solid-phase reagents are incubated with secondary or tertiary reactants covalently coupled to an enzyme. Unbound conjugates are washed out and a chromogenic or fluorogenic substrate is added. As the substrate is hydrolyzed by the bound enzyme conjugate, a colored or fluorescent product is generated. Finally, the product is detected visually or with a microtiter plate reader. The amount of product generated is proportional to the amount of analysate in the test mixture. One of the support protocols can be used to optimize the different ELISAs. A second support protocol presents a method for preparing alkaline phosphatase conjugates. Copyright © 2015 John Wiley & Sons, Inc.

  7. Application of 3D Printing Technology in Increasing the Diagnostic Performance of Enzyme-Linked Immunosorbent Assay (ELISA) for Infectious Diseases.

    PubMed

    Singh, Harpal; Shimojima, Masayuki; Shiratori, Tomomi; An, Le Van; Sugamata, Masami; Yang, Ming

    2015-07-08

    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.

  8. The use of the enzyme-linked immunosorbent assay (ELISA) for the detection and quantification of specific antibody from cell cultures.

    PubMed Central

    Kelly, B S; Levy, J G; Sikora, L

    1979-01-01

    The solid phase enzyme linked immunosorbent assay (ELISA) has been used to quantify anti-keyhole limpet haemocyanin (anti-KLH) antibody in the serum of KLH-immune C57Bl/6 mice. When spleen cells from immune mice were cultured overnight in ELISA microtitre wells to which KLH had been adsorbed it was found that easily quantifiable amounts of anti-KLH antibody were synthesized and were detectable. It was found further that spleen cells from KLH-primed mice, when cultured in vitro in the presence of KLH, transferred to KLH-labelled ELISA plates, and cultured overnight, also produced detectable levels of antibody. Levels of antibody were detectable only after 4 and 5 days of in vitro stimulation. A comparison was made between detectable numbers of plaque forming cells to sheep red blood cells (SRBC) in SRBC primed CBA mice and levels of antibody detected by the ELISA procedure. It was found that the sensitivities of the two tests were comparable. The applications of this technique to the study of in vitro antibody synthesis using soluble antigens are discussed. PMID:381177

  9. 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

  10. AN ENZYME LINKED IMMUNOSORBENT ASSAY (ELISA) METHOD FOR MONITORING 2,4 DICHLOROPHENOXYACETIC ACID (2,4-D) EXPOSURES

    EPA Science Inventory

    Abstract describes a streamlined ELISA method developed to quantitatively measure 2,4-D in human urine samples. Method development steps and comparison with gas chromatography/mass spectrometry are presented. Results indicated that the ELISA method could be used as a high throu...

  11. Analysis of Benzo[a]pyrene in Vegetable Oils Using Molecularly Imprinted Solid Phase Extraction (MISPE) Coupled with Enzyme-Linked Immunosorbent Assay (ELISA)

    PubMed Central

    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

  12. Standardisation and comparison of serial dilution and single dilution enzyme linked immunosorbent assay (ELISA) using different antigenic preparations of the Babesia (Theileria) equi parasite.

    PubMed

    Kumar, Sanjay; Kumar, Yogesh; Malhotra, Dharam V; Dhar, Shruti; Nichani, Anil K

    2003-01-01

    Serial dilution and single dilution enzyme linked immunosorbent assays (ELISA) were standardised and their sensitivity and specificity were compared for serodiagnosis of Babesia equi infection. The antibody titres of 24 donkey sera of known identity were determined separately by serial dilution ELISA using three different B. equi antigens namely whole merozoite (WM), cell membrane (CM) and high speed supernatant (HSS). The ratios of the optical density (OD) of known positive and known negative sera at different serum dilutions were calculated and termed as the positive/negative (P/N) ratio. The coefficients of correlation (r) were calculated between the P/N ratios at different dilutions of sera and the log10 antibody titres of the same sera were ascertained by serial dilution ELISA. The highest value of 'r' was obtained at a serum dilution of 1:200. From log10 antibody titre of sera (y) and their P/N ratio at a dilution of 1:200 (x), regression equations (y = a + bx) were calculated separately for the three antigens. Test sera were diluted to 1:200, their OD were read in duplicate wells and were converted to the P/N ratio. Antibody titres were predicted from the P/N ratio using a regression equation separately for the three antigens. Titres obtained by both ELISAs were not significantly different from each other, thus confirming that single dilution ELISA could be successfully used to replace conventional serial dilution ELISA. The sensitivity, specificity and predictive value of single dilution ELISA was validated statistically using 42 B. equi disease-positive sera and 106 B. equi disease-negative sera. The WM antigen was found to be the most sensitive with a higher predictive value for negative test sera as compared to the CM or HSS antigens. Sera positive for other equine infections including Babesia caballi showed no cross-reaction with the three B. equi antigens in ELISA, thus the test was immunologically specific. Antibody titres of 109 unknown field donkey

  13. Development of an analytical protocol for a fast, sensitive and specific protein recognition in paintings by enzyme-linked immunosorbent assay (ELISA).

    PubMed

    Palmieri, M; Vagnini, Manuela; Pitzurra, L; Rocchi, P; Brunetti, B G; Sgamellotti, A; Cartechini, L

    2011-03-01

    Enzyme-linked immunosorbent assay (ELISA) analysis of proteins offers a particularly promising approach for investigations in cultural heritage on account of its appreciated properties of being highly specific, sensitive, relatively fast, and cost-affordable with respect to other conventional techniques. In spite of that, it has never been fully exploited for routine analyses of painting materials in consideration of several analytical issues that inhibited its diffusion in conservation science: limited sample dimensions, decrease of binder solubility and reduced availability of antibody bonding sites occurring with protein degradation. In this study, an ELISA analytical protocol suited for the identification of aged denatured proteins in ancient painting micro-samples has been developed. We focused on the detection of bovine β-casein and chicken ovalbumin as markers of bovine milk (or casein) and chicken albumen, respectively. A systematic experimentation of the ELISA protocol has been carried out on mock-ups of mural and easel painting prepared with 13 different pigments to assess limits and strengths of the method when applied for the identification of proteins in presence of a predominant inorganic matrix. The analytical procedure has been optimized with respect to protein extraction, antibodies' concentrations, incubation time and temperature; it allows the detection of the investigated proteins with sensitivity down to nanograms. The optimized protocol was then tested on artificially aged painting models. Analytical results were very encouraging and demonstrated that ELISA allows for protein analysis also in degraded painting samples. To address the feasibility of the developed ELISA methodology, we positively investigated real painting samples and results have been cross-validated by gas chromatography-mass spectrometry.

  14. Validation of an indirect enzyme-linked immunosorbent assay for the detection of antibody against Brucella abortus in cattle sera using an automated ELISA workstation.

    PubMed

    Paweska, J T; Potts, A D; Harris, H J; Smith, S J; Viljoen, G J; Dungu, B; Brett, O L; Bubb, M; Prozesky, L

    2002-03-01

    An automated indirect enzyme-linked immunosorbent assay (I-ELISA) for the serological diagnosis of bovine brucellosis was developed and validated in-house. A total of 4,803 cattle sera from South Africa (n = 3,643), Canada (n = 652), Germany (n = 240), France (n = 73) and the USA (n = 195) was used. The South African panel of sera represented 834 sera known to be positive by the Rose Bengal test (RBT), serum agglutination test (SAT) and complement fixation test (CFT), 2709 sera that were negative by CFT, and 100 sera from animals vaccinated with a standard dose of Brucella abortus strain 19. Overseas sera were obtained from reference non-vaccinated brucella-free cattle (n = 834), naturally infected (n = 72), experimentally infected (n = 71), and vaccinated animals (n = 83). Also 100 sera collected from cattle in Canada and known to be positive by competitive ELISA (C-ELISA) were used. The intermediate ranges ("borderline" range for the interpretation of test results) were derived from two-graph receiver operating characteristics analysis. The lowest values of the misclassification cost-term analysis obtained from testing overseas panels, covered lower I-ELISA cut-off PP values (0.02-3.0) than those from local panels (1.5-5.0). The relatively low cut-off PP values selected for I-ELISA were due to the fact that the positive control used represents a very strong standard compared to other reference positive sera. The greater overlap found between negative and positive cattle sera from South Africa than that between reference overseas panels was probably due to the different criteria used in classifying these panels as negative (sera from true non-diseased/non-infected animals) or positive (sera from true diseased/infected animals). The diagnostic sensitivity of the I-ELISA (at the optimum cut-off value) was 100% and of the CFT 83.3%. The diagnostic specificity of I-ELISA was 99.8% and of the CFT 100%. Estimate of Youden's index was higher for the I-ELISA (0.998) than

  15. Development of a polyclonal antibody-based enzyme-linked immunosorbent assay (ELISA) for detection of Sunset Yellow FCF in food samples.

    PubMed

    Xing, Yue; Meng, Meng; Xue, Huyin; Zhang, Taichang; Yin, Yongmei; Xi, Rimo

    2012-09-15

    Sunset Yellow FCF is widely used as food additives to make foods more attractive. Due to its abuse and potential risk to human health, Sunset Yellow FCF is precisely limited to use in food. To monitor the illegal use of Sunset Yellow FCF, a polyclonal antibody-based indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) with satisfactory sensitivity and specificity was developed. A carboxyl group was introduced to Sunset Yellow FCF, then the modified hapten was coupled with carrier proteins to synthesize the immunogen and coating antigen. The IC(50) value of 0.52 ng mL(-1) and detection limit of 25 pg mL(-1) (in buffer) were achieved by this method. The cross-reactivity values of the antibodies with six structurally related colorants were less than 1.5%, indicating the high selectivity. Three kinds of food samples (beverage, dried beancurd, braised pork) and serum were chosen to evaluate the application of the immunoassay in real systems. The limits of detection (LOD) in the above three food samples were 0.12, 0.04 and 1.11, respectively (mean+3SD). The recovery (94%-106%), intra-assay (<5%) and inter-assay (<12%) coefficients of variation in foods and serum samples were also acceptable. The results suggest that this ELISA method is a specific, sensitive and simple method for the determination of Sunset Yellow FCF additives.

  16. A simple, specific high-throughput enzyme-linked immunosorbent assay (ELISA) for quantitative determination of melatonin in cell culture medium.

    PubMed

    Li, Ye; Cassone, Vincent M

    2015-09-01

    A simple, specific, high-throughput enzyme-linked immunosorbent assay (ELISA) for quantitative determination of melatonin was developed for directly measuring melatonin in cell culture medium with 10% FBS. This assay adopts a commercial monoclonal melatonin antibody and melatonin-HRP conjugate, so it can be applied in multiple labs rapidly with low cost compared with commercial RIA and ELISA kits. In addition, the procedure is much simpler with only four steps: 1) sample/conjugate incubation, 2) plate washing, 3) TMB color reaction and 4) reading of results. The standards of the assay cover a wide working range from 100 pg/mL to 10 ng/mL. The sensitivity was 68 pg/mL in cell culture medium with 10% FBS and 26 pg/mL in PBS with as little as 25 μL sample volume. The recovery of melatonin from cell culture medium was 101.0%. The principal cross-reacting compound was 5-methoxytryptophol (0.1%). The variation coefficients of the assay, within and between runs, ranged between 6.68% and 15.76% in cell culture medium. The mean linearity of a series diluted cell culture medium sample was 105% (CV=5%), ranging between 98% and 111%, y=5.5263x+0.0646, R(2)=0.99. The assay enables small research and teaching labs to reliably measure this important neurohormone.

  17. Development of enzyme-linked immunosorbent assay (ELISA) for the detection of neomycin residues in pig muscle, chicken muscle, egg, fish, milk and kidney.

    PubMed

    Wang, S; Xu, B; Zhang, Y; He, J X

    2009-05-01

    A colorimetric competitive direct enzyme-linked immunosorbent assay (ELISA) method was developed using polyclonal antibody to determine neomycin residues in food of animal origin. No cross-reactivity of the antibody was observed with other aminoglycosides. The limit of detection of the method was 0.1μg/kg. A simple and efficient sample extraction method was established with recoveries of neomycin ranged from 75% to 105%. The detection limits were 5μg/kg(l) in pig muscle, chicken muscle, fish and milk, 10μg/kg in kidney and 20μg/kg in egg, respectively. Chemiluminescence assay was developed for detecting neomycin residues in pig muscle and chicken muscle. The limit of detection of the method was 0.015μg/kg, and the detection limits were 1.5μg/kg in pig muscle and 6μg/kg in chicken muscle. The ELISA tests were validated by HPLC, and the results showed a good correlation (r(2)) which was greater than 0.9.

  18. Enzyme-linked immunosorbent assay (ELISA) for brown trout vitellogenin for use in the detection of environmental estrogens

    SciTech Connect

    Gamble, A.; Solomon, K.; Sherry, J.; Fielden, M.; Hodson, P.

    1995-12-31

    Vitellogenin (Vg) is a phospholipoprotein egg yolk precursor found in females of most oviparous vertebrates. Vitellogenin induction in male teleosts is known to result from exposure to xenoestrogens. Using antisera for brown trout Vg, the authors adapted an (ELISA) for in vivo Vg in brown trout (Salmo trutta). The authors will use the Vg bioassay to detect the estrogenic effects of effluents, contaminated sediments and suspect chemicals. Vg production in male fish was induced by intraperitoneal injection of 11-B estradiol. Harvested and purified Vg was used in an assay based on competition between soluble Vg and Vg adsorbed in microtitre wells for binding sites on rabbit anti-Vg antibody. Adsorbed Vg-antibody complexes were subsequently revealed by an alkaline phosphatase technique. The reaction intensity was measured as absorbance and used to quantify the amount of antibody bound to adsorbed Vg. The amount of bound antibody was inversely proportional to the amount of vitellogenin in the fish plasma. Preliminary results show that the assay`s working range, corresponding to 80--20% binding, was 14--355 ng/ml, with 50% binding (IC{sub 50}) around 71 ng/mi. Calculated at 50% binding, the intra-assay variation was less than 8% (n = 9) and the inter-assay variation was also less than 8% (n =4). The assay`s sensitivity was 0.067 ng/mi and the limit of detection was 0.134 ng/ml. The authors will present the results of Vg bioassays for the presence of estrogen mimics in industrial effluent.

  19. Development of a sandwich enzyme linked immunosorbent assay (ELISA) for the quantification of iduronate-2-sulfate sulfatase.

    PubMed

    Sosa, Angela Catalina; Espejo, Angela Johana; Rodriguez, Edwin Alexander; Lizaraso, Lina Maria; Rojas, Andrea; Guevara, Johana; Echeverri, Olga Yaneth; Barrera, Luis Alejandro

    2011-05-31

    Iduronate-2-sulfate sulfatase (IDS; EC 3.1.6.13) is an enzyme that belongs to human sulfatases. IDS deficiency causes the Hunter syndrome or mucopolysaccharidosis type II (MPS II; OMIM 309900). We have been developing an expression system for human recombinant IDS (hrIDS) in Pichia pastoris, therefore a method was required for its detection during production and purification processes, which could be used also to measure the enzyme in human fluids. In this study, an immunoquantification assay for human and recombinant IDS was developed with the combination of two antibodies. Rabbit IgG and chicken IgY were used as IDS capture and detection antibodies, respectively. Chicken IgY antibodies were developed against specific amino acid sequences present in IDS but absent in other human sulfatases. hrIDS produced in P. pastoris, commercial hrIDS, and normal human plasma samples were used as antigens and immunoquantification results were compared to enzyme activity. The technique was linear over the range 8 to 500 ng mL(-1) using commercial hrIDS. The concentration range detected for IDS in normal human plasma was 14.43 to 287.88 ng mL(-1). The hrIDS was detected in P. pastoris cultures even when the enzyme was inactive, which is convenient for monitoring the production of recombinant proteins. These results show that chicken site-specific antibodies provide a good alternative, as a substitute of monoclonal antibodies, for the detection of human proteins. This is the first report on the development of an ELISA system to detect and quantify IDS with IgY antibodies.

  20. Monoclonal antibodies suitable for incorporation into a competitive enzyme-linked immunosorbent assay (ELISA) for detection of specific antibodies to Leptospira interrogans serovar pomona.

    PubMed

    Surujballi, O; Elmgren, C

    2000-01-01

    Monoclonal antibodies (mAb) were produced by fusing Sp2/0-Ag14 myeloma cells with spleen cells from BALB/c and ND4 mice that were immunized with killed Leptospira interrogans serovar pomona whole cells. Thirty hybridomas which produced antibodies (of the IgG1, IgG2a, IgG2b, or IgG3 isotype) that bound to epitopes on the serovar pomona whole cell antigen were identified by an indirect enzyme-linked immunosorbent assay (ELISA). Twenty-eight of these 30 mAbs cross-reacted in the indirect ELISA with at least one whole cell antigen prepared from 12 other pathogenic Leptospira serovars, and/or with whole cell antigen from the non-pathogenic Leptospira biflexa serovar patoc. The two serovar pomona-specific mAbs, which were designated M897 and M898, were obtained from the ND4 mouse and were both of the IgG1 isotype. In competitive ELISAs, M897 and M898 were inhibited from binding to the pomona antigen by bovine sera with anti-serovar pomona microscopic agglutination test (MAT) titres ranging from 100 to 6400. No significant inhibition was observed with pomona MAT-negative sera or with sera from animals experimentally infected with serovars canicola, copenhageni, grippotyphosa, hardjo type hardjobovis or sejroe. The epitopes recognized by M897 and M898 were both highly susceptible to sodium meta-periodate oxidation, indicating a carbohydrate composition. Neither of these mAbs reacted in immunoblots with the separated components of the serovar pomona whole cell antigen.

  1. Development of sensitive direct and indirect enzyme-linked immunosorbent assays (ELISAs) for monitoring bisphenol-A in canned foods and beverages.

    PubMed

    Lu, Yang; Peterson, Joshua Richard; Gooding, John Justin; Lee, Nanju Alice

    2012-06-01

    Enzyme-linked immunosorbent assays (ELISAs) are investigated in this work for the detection of bisphenol-A (BPA), a plastic monomer and a critical contaminant in food and environment. A series of polyclonal antibodies generated in vivo using BPA-butyrate-protein conjugate and BPA-valerate-protein conjugate were evaluated on direct and indirect competitive assay formats with five competing haptens (BPA-butyrate, BPA-valerate, BPA-crotonate, BPA-acetate, and BPA-2-valerate). Two indirect ELISAs and one direct ELISA exhibiting high sensitivity and specificity for BPA were developed. The 50 % inhibition of antibody binding (IC(50)) values were 0.78 ± 0.01-1.20 ± 0.26 μg L(-1), and the limits of detection as measured by the IC(20) values were 0.10 ± 0.03-0.20 ± 0.04 μg L(-1). The assays were highly specific to BPA, only displaying low cross-reactivity (3-8 % for the indirect assays and 26 % for the direct assay) for 4-cumylphenol (4-CP), at pH 7.2. The degree of cross-reaction of 4-CP was influenced by the antibody/hapten conjugate combination, assay conditions, and the assay format. The assays were optimized for the analysis of BPA in canned vegetables, bottled water and carbonated drinks. The limits of quantification for these three evaluated sample types, based on the spike and recovery data, were 0.5, 2.5, and 100 μg L(-1), respectively.

  2. Enzyme-linked immunosorbent assay (ELISA) using a specific monoclonal antibody as a new tool to detect Sudan dyes and Para red.

    PubMed

    Ju, Chunmei; Tang, Yong; Fan, Huiying; Chen, Jinding

    2008-07-28

    To set up an immunoassay-based method to detect Sudan dyes and Para red, we generated a monoclonal antibody (Mab) using a specially designed carboxyl derivative of Sudan I (CSD I) as the immunogen. CSD I was synthesized by azocoupling reaction using 2-naphthol and diazotised 4-aminobenzoic acid. The antibody was obtained from a hybridoma, which was derived from the fusion of the mouse myeloma SP2/0 cells and the splenocytes from the mice immunized with the CSD I-bovine serum albumin (BSA) conjugate. In addition, we showed that the Mab was highly specific for Sudan I, III and Para red. The limit of detection was approximately 0.01ngmL(-1) in phosphate-buffered saline (PBS) buffer and 0.5ngg(-1) in chilli tomato sauce. The recoveries of Sudan I, III and Para red for the chilli tomato sauce were from 84% to 99% and coefficients of variation were from 14.9% to 33.3%. Thus, the enzyme-linked immunosorbent assay (ELISA) method is a rapid and high throughput screening tool to detect Sudan dyes and Para red in food products.

  3. 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

  4. Detection of flagellar antigen of Campylobacter jejuni and Campylobacter coli in canine faeces with an enzyme-linked immunosorbent assay (ELISA)--new prospects for diagnosis.

    PubMed

    Monfort, J D; Bech-Nielsen, S; Stills, H F

    1994-01-01

    A new diagnostic procedure was developed to detect the flagellar antigen of Campylobacter jejuni and Campylobacter coli in canine faecal specimens and was tested on faecal samples from random-source dogs obtained from the local dog pound. Extraction of acid-soluble proteins was performed on faecal specimens and the extracted material was evaluated using species-specific monoclonal antibodies in an enzyme-linked immunosorbent assay. The assay detected all C. jejuni or C. coli infected specimens compared with direct selective faecal culture. One of 18 faecal specimens culture-negative for C. jejuni was identified as positive by the assay, i.e. a false positive rate of 1 of 18 (5.6%) and a corresponding specificity of 94.4%. These results suggest that the screening procedure developed to detect flagellar antigens of C. jejuni and C. coli in canine faecal samples should be further investigated as a diagnostic alternative to culture.

  5. The enzyme-linked immunosorbent assay (ELISA) as a serological test for detecting antibodies against Leptospira interrogans serovar hardjo in sheep.

    PubMed

    Adler, B; Faine, S; Gordon, L M

    1981-09-01

    The enzyme-liked immunosorbent assay (ELISA) was compared with the standard microscopic agglutination test (MAT) as a method for detecting antibodies against Leptospira interrogans serovar hardjo in sheep. Peak antibody levels detected by the 2 tests occurred at different times following experimental infection of sheep. In serums from flocks of sheep with naturally acquired infection there was a 95% correlation between MAT and ELISA with respect to the presence or absence of antibody to serovar hardjo, although the levels of correlation of the titres of the 2 tests was low. The 2 tests appeared to measure different antigen-antibody systems. The ELISA would be a useful test for screening large numbers of serums for antibodies to L. interrogans serovar hardjo.

  6. 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. © The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  7. Immunometric Antibody Sandwich Enzyme-Linked Immunosorbent Assay.

    PubMed

    Kohl, Thomas O; Ascoli, Carl A

    2017-06-01

    The antibody sandwich enzyme-linked immunosorbent assay (ELISA) is the most commonly used assay for rapid and accurate detection of antigens. It displays greater sensitivity compared with the indirect ELISA and can be used to determine absolute antigen concentrations in unknown samples provided purified antigen standards are available, although it requires the use of two different antibodies. Briefly, wells are coated with antigen-specific capture antibody then incubated with samples containing unknown antigen. Washing removes unbound antigen and exogenous sample protein before incubation with a second antigen-specific detection antibody, washing, and reincubation with a reporter-labeled tertiary antibody. After tertiary antibody is washed off, substrate is added and hydrolysis is measured spectrophotometrically. The signal intensity is directly proportional to the concentration of the antigen in the test sample. © 2017 Cold Spring Harbor Laboratory Press.

  8. Risk factors for herds to test positive for Mycobacterium avium ssp. paratuberculosis-antibodies with a commercial milk enzyme-linked immunosorbent assay (ELISA) in Ontario and western Canada.

    PubMed

    Sorge, Ulrike S; Lissemore, Kerry; Godkin, Ann; Jansen, Jocelyn; Hendrick, Steven; Wells, Scott; Kelton, David F

    2012-09-01

    The objectives of this study were to identify risk factors associated with i) a Mycobacterium avium subsp. paratuberculosis (MAP)-antibody milk enzyme-linked immunosorbent assay (MAP milk ELISA)-positive herd status, and ii) the within-herd MAP milk ELISA-positive prevalence in Canadian dairy herds. This prospective cohort study was conducted between 2005 and 2009 on 226 herds in Ontario and western Canada, which participated in a voluntary risk assessment (RA)-based Johne's disease control program. Two MAP milk ELISA and risk assessments and a previsit survey were available per herd. The overall farm RA scores alone could not be used to predict whether a herd would test positive for MAP antibodies. However, the results of this study indicated that increasing the likelihood of exposing calves to MAP through certain management practices, as assessed with the RA, increased the likelihood of a herd being test-positive for MAP antibodies.

  9. Enzyme-linked immunosorbent assay for Potomac horse fever disease.

    PubMed Central

    Pretzman, C I; Rikihisa, Y; Ralph, D; Gordon, J C; Bech-Nielsen, S

    1987-01-01

    An enzyme-linked immunosorbent assay (ELISA) for immunoglobulin G (IgG) and IgM in natural and experimental infections of equids with Ehrlichia risticii was developed. Ehrlichial organisms purified from an infected mouse macrophage cell line were used as the antigen. IgM was separated from serum IgG by the expedient of spun-column chromatography, allowing the use of an indirect ELISA for quantitation of both IgG and IgM in the test sera. Among 16 paired sera from horses exhibiting clinical signs of Potomac horse fever, 8 were positive by the indirect fluorescent-antibody test (IFA), 11 were positive by the IgG ELISA, and 8 were positive by the IgM ELISA. All IFA-positive specimens were positive by the IgG ELISA, which appeared to be more sensitive than the IFA. In all cases, the IgG ELISA alone would have sufficed for diagnosis when acute- and convalescent-phase sera were available. When 26 single acute- or convalescent-phase serum samples were tested, the IFA detected 8, the IgG ELISA detected 10, and the IgM ELISA detected 6 positive serum specimens. The kinetics of IgG and IgM responses as determined by ELISA in two experimentally infected ponies which survived infection and challenges revealed that specific IgM was short-lived, falling to undetectable levels by day 60 postinoculation, whereas specific IgG persisted for more than 1 year. IgM and IgG were detected as early as days 1 and 10, respectively, postinoculation. The results suggest that the ELISA is more sensitive than the IFA and that the IgM ELISA may provide a means for early diagnosis of Potomac horse fever at or before the onset of clinical signs. PMID:3539995

  10. Enzyme-linked immunosorbent assay (ELISA) measurement of vitellogenin in plasma and liver histopathology in barfin plaice Liopsetta pinnifasciata from Amursky Bay, Sea of Japan.

    PubMed

    Shved, Nikita; Kumeiko, Vadim; Syasina, Iraida

    2011-12-01

    Vitellogenin (Vg) of the barfin plaice Liopsetta pinnifasciata was isolated and purified. In native polyacrylamide gel electrophoresis, Vg appeared as one band. After being subjected to sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), Vg fraction produced several polypeptides with molecular masses of 180, 98, 70, 52, 41 and 37 kDa. MALDI-TOF mass spectrometry (MS) of the 180- and 98-kDa Vg polypeptides from the SDS-PAGE gel and de novo sequencing of their four peptide fragments based on MS/MS analysis confirmed that the purified proteins were vitellogenins, which shared high similarity with the Vgs of the barfin flounder Verasper moseri and Atlantic halibut Hippoglossus hippoglossus. The most part of the predicted sequences obtained from the L. pinnifasciata 180-kDa polypeptide has previously been found in the V. moseri vitellogenin type B, the sequences obtained from the 98-kDa polypeptide were found in V. moseri vitellogenin type A, so these findings allow us to propose that L. pinnifasciata has at least two different forms of Vg. Rabbit polyclonal antibodies against Vg were produced, and a quantitative enzyme-linked immunosorbent assay was developed. The concentration of Vg in barfin plaice from the moderately contaminated area of Amursky Bay in the Sea of Japan was detected based on the maturity stage of their gonads. In November 2008, the Vg concentration in the plasma of females with advanced oogenesis varied from 5.295 to 28.367 mg/ml (mean 16.38 ± 6.73 mg/ml, CV = 41.1%); in the plasma of males, the concentration ranged from non-detectable to 0.957 mg/ml (0.29 ± 0.42 mg/ml, CV = 127.9%). In October 2009, the Vg concentration in female plasma was lower than in November 2008 (2.21-13.87 mg/ml). High individual variability of plasma Vg was characteristic for maturing males (CV = 200.3%) and immature females (CV = 255.5%), and there was no significant difference between plasma Vg concentrations in males captured in November 2008 and

  11. Development of enzyme-linked immunosorbent assay (ELISA) for glutathione S-transferase (GST-S) protein in the intertidal copepod Tigriopus japonicus and its application for environmental monitoring.

    PubMed

    Rhee, Jae-Sung; Kim, Bo-Mi; Jeong, Chang-Bum; Leung, Kenneth Mei Yee; Park, Gyung Soo; Lee, Jae-Seong

    2013-11-01

    To utilize the GST-S protein as a useful biomarker for environmental contamination, we developed a polyclonal antibody-based enzyme-linked immunosorbent assay (ELISA) in the intertidal copepod Tigriopus japonicus. Two polyclonal antibodies, TJ-GST-S1 and TJ-GST-S2, were raised against two TJ-GST-S synthetic peptides. Also a recombinant TJ-GST-S protein was purified as a standard for ELISA development. Each polyclonal antibody was tested by Western blot analysis and indirect ELISA. Of two polyclonal antibodies, TJ-GST-S2 ELISA was further employed due to its wide range of detection and the limit of specificity compared to those of TJ-GST-S1 ELISA system. After exposure to 4 metals (Ag, As, Cd, and Cu) to T. japonicus, the amount of TJ-GST-S protein was significantly elevated in a concentration-dependent manner. Also, TJ-GST-S protein was upregulated at relative high concentrations of B[α]P, PCB, and TBT. In this paper, we suggest that T. japonicas ELISA for TJ-GST-S2 is useful as a potential indicator system for marine contaminants. Copyright © 2013 Elsevier Ltd. All rights reserved.

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

    USDA-ARS?s Scientific Manuscript database

    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...

  13. A review of Cry protein detection with enzyme-linked immunosorbent assays

    USDA-ARS?s Scientific Manuscript database

    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 acc...

  14. Diagnosis of intestinal acariasis with avidin-biotin system enzyme-linked immunosorbent assay

    PubMed Central

    Zhang, Rong-Bo; Huang, Yong; Li, Chao-Pin; Cui, Yu-Bao

    2004-01-01

    AIM: To explore the value of avidin-biotin system enzyme-linked immunosorbent assay (ABC-ELISA) in diagnosis of intestinal acariasis. METHODS: Mite-specific IgG levels in serum of 48 patients with intestinal acariasis were measured with ABC-ELISA. The sensitivity of this method was compared with that of staphylococcal protein A enzyme-linked immunosorbent assay (SPA-ELISA). RESULTS: The positive rate of mite-specific IgG detected with ABC-ELISA and SPA-ELISA was 89.58% (43/48) and 56.25% (27/48), respectively. The positive rate with ABC-ELISA was statistically higher than that with SPA-ELISA (χ2 = 13.50, P < 0.01). CONCLUSION: ABC-ELISA is an effective method for the diagnosis of intestinal acariasis. PMID:15112362

  15. Indirect enzyme-linked immunosorbent assay for the mycotoxin zearalenone.

    PubMed Central

    Liu, M T; Ram, B P; Hart, L P; Pestka, J J

    1985-01-01

    A competitive indirect enzyme-linked immunosorbent assay (ELISA) was developed for the detection of zearalenone, an estrogenic mycotoxin. Zearalenone was converted to zearalenone-6'-carboxymethyloxime and conjugated to bovine serum albumin and poly-L-lysine for use as immunogen and solid-phase marker, respectively. Immunization of rabbits with the bovine serum albumin conjugate resulted in zearalenone antibody titers of 20,480 in 11 weeks. A competitive indirect ELISA was conducted by simultaneously incubating zearalenone with zearalenone antiserum over zearalenone-6'-carboxymethyloxime poly-L-lysine solid phase and then determining the bound rabbit immunoglobulin with goat anti-rabbit peroxidase conjugate. Response range for zearalenone in the resulting competition curve was between 1 and 50 ng/ml. Reactivities of this antiserum for alpha-zearalenol, beta-zearalenol, alpha-zearalanol, and beta-zearalanol were, respectively, 50, 12, 6, and 3% of that found for zearalenone. By using the competitive indirect ELISA, zearalenone was detectable in methanol-water extracts of corn, wheat, and pig feed samples. PMID:2932054

  16. Evaluation of musk by enzyme-linked immunosorbent assay.

    PubMed

    Ahn, Kwang Seok; Hahn, Bum-Soo; Lee, Jong Pill; Chang, Seung Yeup; Lee, Hyung Kyu; Jeong, Choon Sik; Kim, Yeong Shik

    2002-04-01

    We report the development of enzyme-linked immunosorbent assay (ELISA) for the quantitative analysis of a unique musk protein (MP-1) in musk samples. Musk defatted with ethyl acetate/methanol (9:1, v/v) was dipped in cold water and ammonium sulfate was added to the supernatant up to 85% saturation. The resulting precipitate was applied to a Bio-Gel P-100 chromatography. The fraction eluted at the void region was collected and it was consecutively purified by affinity chromatography on a DEAE Affi-Gel Blue and on anion-exchange columns containing DEAE-Sepharose CL-6B. This protein was determined to be homogeneous by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under denaturing conditions with an apparent molecular weight of 35000 Da and was called as musk protein-1 (MP-1). Polyclonal antibodies of MP-1 were produced by injecting it into a rabbit. These antibodies were reactive to the aqueous extract of musk and the pure antigen. The ELISA could be applied to detect nano gram quantities of the antigen in musk samples. This method made it possible to distinguish musk samples from different origins.

  17. Enzyme-Linked Immunosorbent-Assay for Deoxynivalenol (DON)

    PubMed Central

    Ji, Fang; Li, Hua; Xu, Jianhong; Shi, Jianrong

    2011-01-01

    Deoxynivalenol (DON), one of the trichothecene mycotoxins, is a worldwide contaminant of wheat and barley, especially when infected by Fusarium graminearum, the causative agent of an epidemic wheat disease called Fusarium Head Blight. Because of the high risk of DON ingestion and the possibility of frequent exposure, it is important to develop a rapid and highly sensitive method for easy identification and quantification of DON in grain samples. In this study, we have developed an indirect competitive enzyme-linked immunosorbent assay (ELISA) to detect DON in wheat. We conjugated 3-O-Hemisuccinyl-DON (3HS-DON) to Bovine serum albumin (BSA) and Ovalbumin (OVA), and obtained DON-specific mice antisera. The indirect competitive ELISA revealed that the optimal concentration of mice serum and the coated antigen was 1/1600 and 1/1500, respectively. The antiserum cross-reacted with the trichothecenes 3-acetyl-DON and T-2 toxin, reaching about 55.2% and 6.3%, respectively, as compared with DON. Results showed that the assay could be performed satisfactorily using an extraction buffer containing less than 15% methanol. Recovery from DON was 82–93% in grains. The linear detection range of DON in grains was between 0.01 and 100 μg/mL. PMID:22069751

  18. Immunometric Double-Antibody Sandwich Enzyme-Linked Immunosorbent Assay.

    PubMed

    Kohl, Thomas O; Ascoli, Carl A

    2017-06-01

    The double-antibody sandwich enzyme-linked immunosorbent assay (ELISA) is preferentially used to determine the concentration of unknown antibody in a sample. Pure antigen is not required in this assay; however, the use of a reporter-labeled detection antibody is essential. The double-antibody sandwich ELISA is suitable for epitope mapping of different monoclonal antibodies that have been generated against a single antigen. First, plates are coated with a capture antibody specific for immunoglobulins generated by immunization of a host species. Next, the test antibody solution (e.g., serum) is incubated with the capture antibody to facilitate binding. The plates are washed to remove unbound antibody, and then antigen is added. The plates are washed again followed by the addition of an antigen-specific reporter-labeled antibody. Following incubation, unbound reporter antibody is washed off, and reporter-specific substrate is added. Reporter-mediated substrate hydrolysis is visualized and measured. The signal is proportional to the number of test antibodies present in the serum. © 2017 Cold Spring Harbor Laboratory Press.

  19. Development and application of dot-enzyme-linked immunosorbent (dot-ELISA) assay for detection of Brucella melitensis and evaluation of the shedding pattern in infected goats.

    PubMed

    Onilude, Opeyemi Mayowa; Mohd Yusoff, Sabri; Emikpe, Benjamin Obukowho; Tanko, Polycarp; Shahrom, Salisi M; Effendy, Mohammed

    2017-01-01

    Early and accurate diagnosis of Brucella melitensis is essential for the treatment and control of brucellosis both in animals and humans. The thrust for the development of a rapid diagnostic technique to overcome the limitations of conventional microbiological and serological tests brought about this investigation on the development and application of dot-ELISA for antigen and antibody detection in infected goats. Fifteen apparently healthy Boer aged 2-3 years which tested negative for brucellosis using PCR and ELISA, were grouped into A (10 goats infected intraocularly with 10(7) CFU of B. melitensis) and B (5 goats) as control. Discharges (ocular, nasal, and vaginal) and blood were collected at days 3, 7, 10, 14, weekly until 42 post-infection (pi) for dot-ELISA, PCR, and RBPT. Dot-ELISA detected B. melitensis antigen and antibody in group A at day 3 and 7 pi, respectively with adequate sensitivity and specificity relative to PCR and RBPT. The bacteria shedding detected from discharges at day 3 pi in the nasal and ocular route with dot-ELISA. Group B were consistently negative. Values such as speed, simplicity, field adaptability, high sensitivity, and specificity make dot-ELISA a rapid and adequate technique for diagnosis of brucellosis in B. melitensis infected goats within few hours.

  20. Evaluation of an enzyme-linked immunosorbent assay (ELISA) kit for the detection of botulinum neurotoxins A, B, E, and F in selected food matrices.

    PubMed

    Singh, Ajay; Datta, Shomik; Sachdeva, Amita; Maslanka, Susan; Dykes, Janet; Skinner, Guy; Burr, Donald; Whiting, Richard C; Sharma, Shashi K

    2015-01-01

    The mouse bioassay (MBA) is the only accepted standard method for detection of botulinum neurotoxins (BoNTs) in foods. The ELISA method has several advantages over the MBA and is therefore widely used for in vitro detection of BoNTs. The US Food and Drug Administration (FDA) and the Centers for Disease Control and Prevention (CDC) conducted a precollaborative study to evaluate the applicability of Botulinum Toxin ELISA kits for the detection of BoNT serotypes A, B, E, and F in a variety of food matrices. In this study, food samples (e.g., broccoli, salami, smoked salmon, green beans, orange juice, tomato juice, low-fat plain yogurt, whole milk, liquid infant formula milk, and liquid eggs) were spiked with high, medium, and low concentration BoNT serotypes A, B, E, and F. Samples (unspiked and spiked) were tested at both laboratories by the ELISA kits. All food samples were positive for BoNTs by ELISA in both laboratories at medium and high spiking levels; a positive ELISA result in low spiked samples was both serotype and laboratory dependent. Overall, the ELISA method appears to be an effective preliminary screening method for BoNT detection in food matrices.

  1. A survey for antibodies to equine arteritis virus in donkeys, mules and zebra using virus neutralisation (VN) and enzyme linked immunosorbent assay (ELISA).

    PubMed

    Paweska, J T; Binns, M M; Woods, P S; Chirnside, E D

    1997-01-01

    A seroepidemiological survey of donkeys in South Africa (n = 4300) indicated a wide distribution and increasing prevalence of antibodies to equine arteritis virus (EAV). Donkey sera inhibited equine arteritis virus infection in virus neutralisation (VN) tests and in ELISA specifically bound to a recombinant antigen derived from the Bucyrus isolate of EAV. These results suggest that donkeys have been exposed to the same serotype of this virus as circulates among horses. A good correlation existed between EAV neutralising antibody titres and ELISA absorbance values (0.8631); the ELISA was sensitive and specific (99.2% and 80.3% respectively) for donkey sera when compared to the VN test and the recombinant ELISA antigen did not cross-react with sera positive for common African equine pathogens. VN+ ELISA+ donkeys were also found in Morocco and Zimbabwe and seropositive mules in both South Africa and Morocco. No seropositive zebra (n = 266) were detected from game reserves or zoos in 9 countries. The results confirm that in addition to horses and donkeys, mules are naturally infected with EAV.

  2. Seroprevalence of equine granulocytic anaplasmosis and lyme borreliosis in Canada as determined by a point-of-care enzyme-linked immunosorbent assay (ELISA)

    PubMed Central

    Schvartz, Gili; Epp, Tasha; Burgess, Hilary J.; Chilton, Neil B.; Pearl, David L.; Lohmann, Katharina L.

    2015-01-01

    Equine granulocytic anaplasmosis (EGA) and Lyme borreliosis (LB) are an emerging concern in Canada. We estimated the seroprevalence of EGA and equine LB by testing 376 convenience serum samples from 3 provinces using a point-of-care SNAP® 4Dx® ELISA (IDEXX Laboratories, Westbrook, Maine, USA), and investigated the agreement between the point-of-care ELISA and laboratory-based serologic tests. The estimated seroprevalence for EGA was 0.53% overall (0.49% in Saskatchewan, 0.71% in Manitoba), while the estimated seroprevalence for LB was 1.6% overall (0.49% in Saskatchewan, 2.86% in Manitoba). There was limited agreement between the point-of-care ELISA and an indirect fluorescent antibody test for EGA (kappa 0.1, PABAK 0.47) and an ELISA/Western blot combination for LB (kappa 0.23, PABAK 0.71). While the SNAP® 4Dx® ELISA yielded expected seroprevalence estimates, further evaluation of serologic tests for the purposes of disease exposure recognition may be needed. PMID:26028677

  3. Quantitation of Acidothermus cellulolyticus E1 endoglucanase and Thermomonospora fusca E{sub 3} exoglucanase using enzyme-linked immunosorbent assay (ELISA)

    SciTech Connect

    Nieves, R.A.; Chou, Y.C.; Himmel, M.E.

    1995-12-31

    Two distinct quantitative indirect ELISAs were developed to determine the concentration of recombinant cellulose enzymes in culture filtrates. A monoclonal antibody (E1P7) was used as the primary antibody in developing an ELISA specific for Acidothermus cellulolyticus E1 endoglucanase. Likewise, a polyclonal rabbit serum (Ab684) was used to develop an ELISA specific for Thermomonospora fusca E{sub 3} exoglucanase. Dose-response curves indicated a dynamic range for both assays between 0.01 and 0.08 {mu}g/mL (1-8 ng/assay) when purified enzymes were used as standards. These assays have been used to estimate concentrations of secreted recombinant E1 and/or E{sub 3} in culture supernatants of Streptomyces lividans strain TK24 in which the corresponding genes have been cloned and expressed.

  4. Diagnostic performance of an indirect enzyme-linked immunosorbent assay (ELISA) to detect bovine leukemia virus antibodies in bulk-tank milk samples

    PubMed Central

    Nekouei, Omid; Durocher, Jean; Keefe, Greg

    2016-01-01

    This study assessed the diagnostic performance of a commercial ELISA for detecting bovine leukemia virus antibodies in bulk-tank milk samples from eastern Canada. Sensitivity and specificity of the test were estimated at 97.2% and 100%, respectively. The test was recommended as a cost-efficient tool for large-scale screening programs. PMID:27429469

  5. [Enzyme-linked immunosorbent assay (ELISA) for detection of antibodies to Salmonella Typhi lipopolysaccharide O and capsular polysaccharide Vi antigens in persons from outbreak of typhoid fever].

    PubMed

    Rastawicki, Waldemar; Kałużewski, Stanisław

    2015-01-01

    The laboratory diagnosis of typhoid fever is dependent upon either isolation of S. Typhi from a clinical sample or the detection of raised titers of serum antibodies in the Widal test or the passive hemagglutination assay (PHA). In this study we evaluated the usefulness of ELISA for detection of antibodies to S. Typhi lipopolysaccharide O and capsular polysaccharide Vi antigens in the sera of persons from outbreak of typhoid fever. Fifteen serum samples from patients with laboratory confirmed typhoid fever and 140 sera from persons suspected for contact with typhoid fever patients from outbreak in 1974/75 in Poland were tested by ELISA. Additionally, as the control group, we tested 115 sera from blood donors for the presence of S. Typhi anti-LPS and anti-Vi antibodies. Anti-LPS and anti-Vi antibodies were detected in 80% and 53.3% of sera obtained from patients with laboratory confirmed typhoid fever, respectively. The high percentages of positive results in ELISA were also noted in the group of persons suspected for contact with typhoid fever patients (51.4% and 45%) but not in the group of blood donors (7.8% and 6.1%, respectively). The ELISA could be a useful tool for the serological diagnosis of typhoid fever in patients who have clinical symptoms but are culture negative, especially during massive outbreaks of typhoid fever.

  6. Comparison of an enzyme-linked immunosorbent assay (ELISA) to gas chromatography (GC) - measurement of polychlorinated biphenyls (PCBs) in selected US fish extracts

    USGS Publications Warehouse

    Zajicek, J.L.; Tillitt, D.E.; Schwartz, T.R.; Schmitt, C.J.; Harrison, R.O.

    2000-01-01

    The analysis of PCBs in fish tissues by immunoassay methods was evaluated using fish collected from a US monitoring program, the National Contaminant Biomonitoring Program of the US Department of Interior, Fish and Wildlife Service. Selected composite whole fish samples, which represented widely varying concentrations and sources of PCBs, were extracted and subjected to congener PCB analysis by gas chromatography (GC) and total PCB analysis using an ELISA (ePCBs) calibrated against technical Aroclor 1248. PCB congener patterns in these fishes were different from the patterns found in commercial Aroclors or their combinations as demonstrated by principal component analysis of normalized GC congener data. The sum of the PCB congeners measured by GC (total-PCBs) ranged from 37 to 4600 ng/g (wet weight). Concentrations of PCBs as determined by the ELISA method were positively correlated with total-PCBs and the ePCBs/total-PCBs ratios for individual samples ranged from 1 to 6. Ratios of ePCBs/total-PCBs for dilutions of Aroclors 1242, 1254, and 1260 and for matrix spikes range from 0.6 for 1242 to 2.5 for 1254 and 1260. These results suggest that higher chlorinated PCB congeners have higher affinity for the anti-PCB antibodies. Partial least squares with latent variable analysis of GC and ELISA data of selected Aroclors and fish samples also support the conclusion that ELISA derived PCB concentrations are dependent on the degree on chlorination.

  7. Development of a highly sensitive and specific enzyme-linked immunosorbent assay (ELISA) for the detection of phenylethanolamine A in tissue and feed samples and confirmed by liquid chromatography tandem mass spectrometry (LC-MS/MS).

    PubMed

    Cao, Biyun; He, Guangzhao; Yang, Hong; Chang, Huafang; Li, Shuqun; Deng, Anping

    2013-10-15

    Phenylethanolamine A (PA) is a new emerged β-adrenergic agonist illegally used as feed additives for growth promotion. In this study, a highly sensitive and specific indirect competitive enzyme-linked immunosorbent assay (ELISA) for the detection of PA in tissue and feed samples was developed and confirmed by liquid chromatography tandem mass spectrometry (LC-MS/MS). By reduction of nitryl group to amino group, the PA derivative was synthesized and coupled to carrier proteins with diazobenzidine method. The antisera obtained from four immunized rabbits were characterized in terms of sensitivity and specificity. All antisera displayed high sensitivity with IC50 values lower than 0.48 ng mL(-1). The most sensitive ELISA was established with IC50 and limit of detection (LOD) values of 0.049 ng mL(-1) and 0.003 ng mL(-1), respectively. The cross-reactivity (CR) values of the antisera with three frequently used β-adrenergic agonists (clenbuterol, salbutamol and ractopamine) were lesser than 0.39%; there was no CR of the antisera with other six compounds including two structurally related substances (isoproterenol, phenylephrine). To investigate the accuracy and precision of the assay, swine kidney, liver, meat and feed samples were fortified with PA at different content and analyzed by ELISA. Acceptable recovery rates of 92.2-113.7% and intra-assay coefficients of variation of 3.8-10.9% (n=3) were achieved. Seven spiked samples were simultaneously analyzed by ELISA and LC-MS/MS. There was a high correlation coefficient of 0.9956 (n=7) between the two methods. The proposed ELISA proven to be a feasible quantitative/screening method for PA analysis in tissue and feed samples with the properties of high sensitivity and specificity, high sample throughput and low expensive.

  8. 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...

  9. 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...

  10. Utilisation of an enzyme-linked immunosorbent assay (ELISA) for determination of alkylphenols in various environmental matrices. Comparison with LC-MS/MS method.

    PubMed

    Pasquet, Camille; Vulliet, Emmanuelle

    2011-10-15

    Among the wide range of substances discharged continuously in the environment, alkylphenols became a major focus of environmental research in the last decades, as it was found that they possess endocrine disrupting properties. Knowledge about the occurrence and levels of alkylphenols in environment is critical for the risk assessment of these compounds on both ecosystem and human health. However, the analysis of traces of alkylphenols in environmental matrices is a very difficult task, and the suitable methods involve generally an extraction followed by an extensive sample clean-up before detection, steps often time-consuming and costly. In order to reduce the analysis time, obtain a high throughput of analysis and thus improve work efficiency, the objective of the present study is to investigate the use of immunochemical technique (ELISA) for the determination of nonylphenol and octylphenol in soils and various kinds of water. To our knowledge, this is the first time that the determination of alkylphenols in soil using immunoassay technique is described. A methodology is developed, based on the combination of a single preparation step and the use of a simply ELISA kit. The performances of the method are compared with LC-MS/MS, considered as reference. The developed procedure offers the sensitivity and selectivity necessary for the detection of the target alkylphenols in the ng/g or ng/L range, and is successfully applied to the analysis of several samples. Results indicate that alkylphenols are quantified with concentrations in the same order than LC-MS/MS, meaning that ELISA may be useful not only in screening the samples and get a positive/negative response, but also it allows a good approximation of the concentrations.

  11. Establishing an indirect sandwich enzyme-linked-immunosorbent-assay (ELISA) for the detection of antibodies against Histomonas meleagridis from experimentally infected specific pathogen-free chickens and turkeys

    PubMed Central

    Windisch, M.; Hess, M.

    2010-01-01

    No serological method suitable for large screening of antibodies against Histomonas meleagridis in poultry is available so far, the objective targeted in the present investigation. Consequently, an ELISA was developed as a suitable tool for this purpose. Investigating serum samples from non-infected specific pathogen-free (spf) chickens and commercial turkeys a high-background signal was noticed when ELISA plates were directly coated with purified parasitic cells. This signal was significantly reduced by coating the plates with a polyclonal rabbit antibody, raised against histomonads, prior to the addition of the antigen. Adopting this approach five antigen preparations were compared and a high reproducibility could be demonstrated reflected by a very low coefficient of variation of 5.3% and 1.7% for the chicken and turkey sera, respectively. After this initial development all further experiments were carried out with one set of plates and the same antigen preparation. Investigating chicken sera obtained from birds infected at 14 days of life, OD values above a predetermined cut-off value were observed 2 weeks post-infection and a rise of IgG antibodies was noticed until 6 weeks post-infection, when the experiment was terminated. Non-protected turkeys infected at 6 weeks of age displayed an increasing IgG response until 14 days post-infection, prior to the death of animals due to histomonosis. In comparison, the majority of turkeys vaccinated with attenuated histomonads, obtained through prolonged passaging and challenged 4 weeks later with virulent parasites, displayed a demonstrable antibody response after the challenge only. Antibody titres increased until 4 weeks post-challenge when the birds were killed and the study was terminated. Altogether, the developed indirect sandwich ELISA proved to be a quick and efficient method to detect IgG antibodies against H. meleagridis in sera of experimentally infected chickens and turkeys and will be a helpful tool to

  12. Establishing an indirect sandwich enzyme-linked-immunosorbent-assay (ELISA) for the detection of antibodies against Histomonas meleagridis from experimentally infected specific pathogen-free chickens and turkeys.

    PubMed

    Windisch, M; Hess, M

    2009-04-06

    No serological method suitable for large screening of antibodies against Histomonas meleagridis in poultry is available so far, the objective targeted in the present investigation. Consequently, an ELISA was developed as a suitable tool for this purpose. Investigating serum samples from non-infected specific pathogen-free (spf) chickens and commercial turkeys a high-background signal was noticed when ELISA plates were directly coated with purified parasitic cells. This signal was significantly reduced by coating the plates with a polyclonal rabbit antibody, raised against histomonads, prior to the addition of the antigen. Adopting this approach five antigen preparations were compared and a high reproducibility could be demonstrated reflected by a very low coefficient of variation of 5.3% and 1.7% for the chicken and turkey sera, respectively. After this initial development all further experiments were carried out with one set of plates and the same antigen preparation. Investigating chicken sera obtained from birds infected at 14 days of life, OD values above a predetermined cut-off value were observed 2 weeks post-infection and a rise of IgG antibodies was noticed until 6 weeks post-infection, when the experiment was terminated. Non-protected turkeys infected at 6 weeks of age displayed an increasing IgG response until 14 days post-infection, prior to the death of animals due to histomonosis. In comparison, the majority of turkeys vaccinated with attenuated histomonads, obtained through prolonged passaging and challenged 4 weeks later with virulent parasites, displayed a demonstrable antibody response after the challenge only. Antibody titres increased until 4 weeks post-challenge when the birds were killed and the study was terminated. Altogether, the developed indirect sandwich ELISA proved to be a quick and efficient method to detect IgG antibodies against H. meleagridis in sera of experimentally infected chickens and turkeys and will be a helpful tool to

  13. Evaluation of a commercial enzyme-linked immunosorbent assay (ELISA) kit for serological diagnosis of Helicobacter pylori infection in a group of non-ulcer dyspepsia sufferers.

    PubMed Central

    Ching, C. K.; Thompson, S.; Buxton, C.; Holgate, C.; Holmes, G. K.

    1993-01-01

    Helicobacter pylori infection of the stomach is associated with gastritis and peptic ulceration and may be causative. A noninvasive test for this organism might be useful in managing some patients with dyspepsia without the need for further investigation. We have evaluated a new commercially available serological test (Helico-G ELISA, Porton Cambridge, UK) for this infection to assess its diagnostic accuracy in a retrospective study of 115 patients with non-ulcer dyspepsia. Sixty-three of these patients (55%) were found to have H. pylori infection and gastritis on histology. A sensitivity of 81% and specificity of 90% were obtained. No significant fall in the antibody titres was found in a subgroup of 15 patients who were selected to complete a course of triple therapy despite significant improvement in their dyspepsia score and confirmed eradication of H. pylori organism in 80% of these patients. We conclude that the test has limited value in aiding clinical decision of managing patients with dyspepsia. PMID:8208642

  14. Is a Plasmodium lactate dehydrogenase (pLDH) enzyme-linked immunosorbent (ELISA)-based assay a valid tool for detecting risky malaria blood donations in Africa?

    PubMed

    Atchade, Pascal S; Doderer-Lang, Cécile; Chabi, Nicodème; Perrotey, Sylvie; Abdelrahman, Tamer; Akpovi, Casimir D; Anani, Ludovic; Bigot, André; Sanni, Ambaliou; Candolfi, Ermanno

    2013-08-08

    Malaria is a leading cause of mortality in southern Benin. The main causative agent, Plasmodium falciparum, poses a threat on critical transfusions in pregnant women and children. This study's objective was to compare the performance of different malaria screening methods in blood donors in southern Benin, a malaria-endemic country. Blood from 2,515 voluntary blood donors in Benin was collected over a period of 10 months in ethylenediaminetetraacetic acid (EDTA) tubes, which were then classified according to extraction time: long rainy season, short dry season, short rainy season, and long dry season. Microscopic examination was used to count parasites. Parasite density (PD) was expressed as the number of parasites per μL of blood. Pan Plasmodium pLDH detection was assessed by an ELISA-malaria antigen test. Using crude soluble P. falciparum antigens, an ELISA-malaria antibody test detected anti-Plasmodium antibodies. Among the 2,515 blood donors (2,025 males and 488 females) screened, the rate of asymptomatic Plasmodium carriage was 295/2,515 (11.72%, 95% CI: 10.5-13.1%). Males had a higher infection rate (12.4%) than did females (8.8%). Parasite density was very low: between seven and100 parasites per μL of blood was reported in 80% of donors with parasitaemia. Three Plasmodium species were diagnosed: P. falciparum in 280/295 patients (95.0%), Plasmodium malariae in 14/295 (5.0%), and Plasmodium ovale in 1/295 (0.34%). Malaria prevalence in donors was higher during the rainy seasons (13.7%) compared with the dry seasons (9.9%). The use of a highly sensitive assay enabled pan Plasmodium pLDH detection in 966/2,515 (38.4%, 95% CI: 36.5%-40.3%). Malaria antibody prevalence was 1,859/2,515 (73.9%, 95% CI: 72.16-75.6%). Donors' antigenaemia and antibody levels varied significantly (P <0.05) over the course of the four seasons. The highest antigenaemia rate 323/630 (51.3%), was observed during the short rainy season, while the highest antibody prevalence, 751/886 (84

  15. Aspergillus Galactomannan Enzyme-Linked Immunosorbent Assay Cross-Reactivity Caused by Invasive Geotrichum capitatum

    PubMed Central

    Giacchino, Mareva; Chiapello, Nadia; Bezzio, Stefania; Fagioli, Franca; Saracco, Paola; Alfarano, Alda; Martini, Vincenza; Cimino, Giuseppe; Martino, Pietro; Girmenia, Corrado

    2006-01-01

    We report three cases of invasive Geotrichum capitatum infection in patients with acute leukemia for which an enzyme-linked immunosorbent assay (ELISA) for Aspergillus galactomannan was positive, with no evidence of aspergillosis. Supernatants obtained from suspensions of 17 G. capitatum strains gave positive reactions with the Aspergillus galactomannan ELISA. These clinical and laboratory data seem to suggest that G. capitatum produces a soluble antigen that is cross-reactive with Aspergillus galactomannan. PMID:16954294

  16. Enzyme-linked immunosorbent assay for leucine and methionine enkephalins

    SciTech Connect

    Zamboni, G.; Jones, C.A.; Hughes, J.

    1983-04-01

    An enzyme-linked immunosorbent assay for enkephalins was developed by coupling the peptides to a carrier molecule (bovine serum albumin) in order to allow the antibody-antigen reaction to take place in the solid phase. The assay was shown to be highly reproducible. Its sensitivity was 14 nmol/liter for leucine enkephalin and 27 nmol/liter for methionine enkephalin, which is similar to that obtained when the same antibodies were used in radioimmunoassay.

  17. Validation and evaluation of VapA-specific IgG and IgG subclass enzyme-linked immunosorbent assays (ELISAs) to identify foals with Rhodococcus equi pneumonia.

    PubMed

    Sanz, M G; Oliveira, A F; Loynachan, A; Page, A; Svansson, V; Giguère, S; Horohov, D W

    2016-01-01

    Rhodococcus equi (Rhodococcus hoagii/Prescottella equi) is a common cause of foal pneumonia, but its diagnosis remains a challenge for equine veterinarians. While the VapA-specific (virulence-associated protein A) immunoglobulin G (IgG) enzyme-linked immunosorbent assay (ELISA) has low sensitivity and specificity for detecting pneumonic foals, little is known about VapA-specific IgG subclasses. To evaluate the performance of VapA-specific ELISA for IgG and its subclasses IgGa, IgGb and IgG(T) in the early diagnosis of pneumonia caused by R. equi. Assay validation followed by assessment of diagnostic performance using archived samples from animals of known status. Serum samples from exposed (n = 125) and nonexposed adult horses (n = 10) and from experimentally challenged and naturally infected foals were used for ELISA validation. Post mortem and tissue culture records of the last 24 years from the Institute for Experimental Pathology at the University of Iceland in Keldur, Iceland laboratory were evaluated to confirm the absence of R. equi cases in Iceland. The diagnostic performance of VapA-specific IgG and its subclasses was evaluated using banked serum samples from pneumonic (n = 21) and healthy foals (n = 80). To evaluate each IgG assay, a cut-off value was selected based on receiver operating characteristic curve analysis and used to calculate sensitivity and specificity. The intra- and interassay coefficients of variation were calculated for each ELISA. Using sera from Iceland, where R. equi infection has not been reported, the VapA-specific IgG ELISA differentiated exposed from nonexposed horses. When used to identify infected foals, VapA-specific IgG, IgGa and IgGb had no diagnostic value. In contrast, IgG(T) had high sensitivity and specificity. Horses from Iceland are not exposed to VapA(+) R. equi and can serve as negative controls. VapA-specific IgG subclasses, with the exception of IgG(T), are poor predictors of disease. Further

  18. Brood stock segregation of spring chinook salmon Oncorhynchus tshawytscha by use of the enzyme-linked immunosorbent assay (ELISA) and the fluorescent antibody technique (FAT) affects the prevalence and levels of Renibacterium salmoninarum infection in progeny

    USGS Publications Warehouse

    Pascho, Ronald J.; Elliott, Diane G.; Streufert, Jonathan M.

    1991-01-01

    A study of the effect of maternal Renibacterium salmoninarum infection levels on the prevalence and levels of bacterial kidney disease (BKD) in progeny fish was conducted at a production salmon hatchery. A total of 302 mating pairs of spring chinook salmon Oncorhynchus tshawytscha was screened in August 1988 for R. salmoninarum by an enzyme-linked immunosorbent assay (ELISA). On the basis of ELISA testing of kidney tissues from all fish and the testing of ovarian fluid samples from a subsample of the females by a direct membrane filtration fluorescent antibody technique (MF-FAT), selected egg lots were segregated into 2 groups of 30 egg lots or about 135 000 eggs each. One group contained egg lots from male and female parents that had low R. salmoninarum infection levels or tested negative for R. salmoninarum (low-BKD group), and the other group contained egg lots from female parents with relatively high R. salmoninarum infection levels and male parents with various infection levels (high-BKD group). The progeny groups were maintained in separate rearing units supplied with untreated river water, and were monitored for R. salmoninarum by the ELISA until they were released from the hatchery in April 1990. Total mortality of the juvenile fish was higher (p = 0.0001) in the high-BKD group (20%) than in the low-BKD group (10 %). Mortality in the high-BKD group was highest after the fish were moved from nursery tanks to raceways, and clinical BKD became evident in this group. During the 11 mo of raceway rearing, mortality in the high-BKD group was 17 % compared with 5 % for the low-BKD group. An ELISA analysis of smolts just before release showed an R. salmoninarum infection rate of 85 % in the high-BKD group and 62 % in the low-BKD group. Of the positive fish, 98 % in the low-BKD group and 55 % in the high-BKD group had low infection levels, whereas 36 % in the high-BKD group and only 1 % in the low-BKD group had high infection levels. The results of this research

  19. Development of a simple gel permeation clean-up procedure coupled to a rapid disequilibrium enzyme-linked immunosorbent assay (ELISA) for the detection of Sudan I dye in spices and sauces.

    PubMed

    Oplatowska, Michalina; Stevenson, Paul J; Schulz, Claudia; Hartig, Lutz; Elliott, Christopher T

    2011-09-01

    Sudan dyes have been found to be added to chilli and chilli products for illegal colour enhancement purposes. Due to the possible carcinogenic effect, they are not authorized to be used in food in the European Union or the USA. However, over the last few years, many products imported from Asian and African countries have been reported via the Rapid Alert System for Food and Feed in the European Union to be contaminated with these dyes. In order to provide fast screening method for the detection of Sudan I (SI), which is the most widely abused member of Sudan dyes family, a unique (20 min without sample preparation) direct disequilibrium enzyme-linked immunosorbent assay (ELISA) was developed. The assay was based on polyclonal antibodies highly specific to SI. A novel, simple gel permeation chromatography clean-up method was developed to purify extracts from matrices containing high amounts of fat and natural pigments, without the need for a large dilution of the sample. The assay was validated according to the Commission Decision 2002/657/EC criteria. The detection capability was determined to be 15 ng g(-1) in sauces and 50 ng g(-1) in spices. The recoveries found ranged from 81% to 116% and inter- and intra-assay coefficients of variation from 6% to 20%. The assay was used to screen a range of products (85 samples) collected from different retail sources within and outside the European Union. Three samples were found to contain high amounts (1,649, 722 and 1,461 ng g(-1)) of SI by ELISA. These results were confirmed by liquid chromatography-tandem mass spectrometry method. The innovative procedure allows for the fast, sensitive and high throughput screening of different foodstuffs for the presence of the illegal colorant SI.

  20. 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

  1. EVALUATION OF AN ENZYME-LINKED IMMUNOSORBENT ASSAY FOR BIOLOGICAL MONITORING OF 3-PHENOXYBENZOIC ACID IN URINE

    EPA Science Inventory

    Abstract describes the development of an enzyme-linked immunosorbent assay (ELISA) method for monitoring 2,4-dichlorophenoxyacetic acid (2,4-D exposures). The ELISA is compared with a gas chromatograhy/mass spectrometry procedure. ELISA method development steps and comparative ...

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

    PubMed

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

    2006-11-01

    Confirmation of mild bites caused by Loxosceles reclusa with swab testing has not been previously documented, to our knowledge. We report a case using an enzyme-linked immunosorbent assay (ELISA) test. 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. This is a pilot single-case report for this enzyme-linked immunosorbent assay test. 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.

  3. Direct comparison of the histidine-rich protein-2 enzyme-linked immunosorbent assay (HRP-2 ELISA) and malaria SYBR green I fluorescence (MSF) drug sensitivity tests in Plasmodium falciparum reference clones and fresh ex vivo field isolates from Cambodia

    PubMed Central

    2013-01-01

    Background Performance of the histidine-rich protein-2 enzyme-linked immunosorbent assay (HRP-2 ELISA) and malaria SYBR Green I fluorescence (MSF) drug sensitivity tests were directly compared using Plasmodium falciparum reference strains and fresh ex vivo isolates from Cambodia against a panel of standard anti-malarials. The objective was to determine which of these two common assays is more appropriate for studying drug susceptibility of “immediate ex vivo” (IEV) isolates, analysed without culture adaption, in a region of relatively low malaria transmission. Methods Using the HRP-2 and MSF methods, the 50% inhibitory concentration (IC50) values against a panel of malaria drugs were determined for P. falciparum reference clones (W2, D6, 3D7 and K1) and 41 IEV clinical isolates from an area of multidrug resistance in Cambodia. Comparison of the IC50 values from the two methods was made using Wilcoxon matched pair tests and Pearson’s correlation. The lower limit of parasitaemia detection for both methods was determined for reference clones and IEV isolates. Since human white blood cell (WBC) DNA in clinical samples is known to reduce MSF assay sensitivity, SYBR Green I fluorescence linearity of P. falciparum samples spiked with WBCs was evaluated to assess the relative degree to which MSF sensitivity is reduced in clinical samples. Results IC50 values correlated well between the HRP-2 and MSF methods when testing either P. falciparum reference clones or IEV isolates against 4-aminoquinolines (chloroquine, piperaquine and quinine) and the quinoline methanol mefloquine (Pearson r = 0.85-0.99 for reference clones and 0.56-0.84 for IEV isolates), whereas a weaker IC50 value correlation between methods was noted when testing artemisinins against reference clones and lack of correlation when testing IEV isolates. The HRP-2 ELISA produced a higher overall success rate (90% for producing IC50 best-fit sigmoidal curves), relative to only a 40% success rate for the

  4. Specificity of a sandwich enzyme-linked immunosorbent assay for detecting Aspergillus galactomannan.

    PubMed Central

    Swanink, C M; Meis, J F; Rijs, A J; Donnelly, J P; Verweij, P E

    1997-01-01

    The specificity of a sandwich enzyme-linked immunosorbent assay (ELISA) for detecting Aspergillus galactomannan was tested with exoantigens of 29 fungi cultured from clinical specimens. Cross-reactivity was observed with Penicillium chrysogenum, Penicillium digitatum, and Paecilomyces variotii. Furthermore, 40 serum samples obtained from bacteremic patients with hematologic malignancies were retrospectively tested by sandwich ELISA. False-positive reactions with the serum were reproducible but did not correspond with the results of culture of specific microorganisms. Moreover, the microorganisms cultured from the blood showed no reactivity by the sandwich ELISA. PMID:8968919

  5. Enzyme-linked immunosorbent assay for the detection and identification of plant pathogenic bacteria (in particular for Erwinia amylovora and Clavibacter michiganensis subsp. sepedonicus).

    PubMed

    Kokoskova, Blanka; Janse, Jaap D

    2009-01-01

    Enzyme-linked immunosorbent assay (ELISA) is the most commonly used serological diagnostic technique. A number of different ELISA formats can be used for the detection of bacterial plant pathogens and in particular Erwinia amylovora and Clavibacter michiganensis subsp. sepedonicus.

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

    USDA-ARS?s Scientific Manuscript database

    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...

  7. 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

  8. Enzyme-linked immunosorbent assay for detection of respiratory syncytial virus infection: application to clinical samples.

    PubMed Central

    McIntosh, K; Hendry, R M; Fahnestock, M L; Pierik, L T

    1982-01-01

    An enzyme-linked immunosorbent assay (ELISA) for respiratory syncytial virus antigens was applied to the rapid diagnosis of acute infections in children and was compared with viral culture and immunofluorescence tests. The ELISA test employed commercially available reagents and was run on a day-to-day basis as specimens were received in the laboratory. Sensitivity and specificity by ELISA were 82 and 95%, respectively, compared with culture. In the same specimens, the sensitivity and specificity by immunofluorescence were 86 and 96%, respectively. Nasopharyngeal aspirates were proven to be a better source of viral antigen than were nasopharyngeal swabs. ELISA-positive samples remained positive even when left unrefrigerated for a week or mailed to the laboratory in plastic containers. Respiratory syncytial virus ELISA, like culture, became negative as the disease progressed and showed no superiority over culture for diagnosis late in the illness. PMID:6749895

  9. 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.

  10. Detection of immunoglobulin M in cerebrospinal fluid from syphilis patients by enzyme-linked immunosorbent assay.

    PubMed Central

    Lee, J B; Farshy, C E; Hunter, E F; Hambie, E A; Wobig, G H; Larsen, S A

    1986-01-01

    Cerebrospinal fluid (CSF) samples were evaluated in an immunoglobulin M enzyme-linked immunosorbent assay (IgM ELISA) for syphilis with sonic extracts of Treponema pallidum coated on polystyrene plates. The ELISA procedure was reproducible, and T. pallidum antigens were stable., A total of 15 CSF samples from patients with neurosyphilis, 18 CSF samples from patients with syphilis, 12 CSF samples from patients treated for syphilis, and 494 CSF samples from patients with neurologic or other systemic diseases were tested. The IgM ELISA gave reactive results in all of six symptomatic and congenital neurosyphilitic patients and none of nine asymptomatic neurosyphilitic patients. Of 524 CSF samples from nonneurosyphilitic individuals, 513 were nonreactive, resulting in 98% test specificity. The IgM ELISA in CSF should prove to be useful for confirmation of symptomatic neurosyphilis. PMID:3533984

  11. 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...

  12. 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...

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

    PubMed

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

    2013-10-01

    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.

  14. 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.

  15. Effect of treatment on serum antibody to Hymenolepis nana detected by enzyme-linked immunosorbent assay.

    PubMed

    Castillo, R M; Grados, P; Carcamo, C; Miranda, E; Montenegro, T; Guevara, A; Gilman, R H

    1991-02-01

    An enzyme-linked immunosorbent assay (ELISA) was developed to measure serum immunoglobulin G antibodies in 65 patients infected with Hymenolepis nana and 30 noninfected patients. Antibody was detected in 51 of 65 (sensitivity, 79%) and 5 of 30 H. nana-negative patients (specificity, 83%). Nine patients infected with H. nana were treated with praziquantel (20 to 25 mg/kg of body weight). Antibody disappeared from the sera at 90 days in six patients, five of whom had eliminated H. nana. Antibody persisted in three patients in whom H. nana infection did not clear after treatment. The H. nana ELISA had a high rate of cross-reactions with sera from patients with cysticercosis (8 of 29 [28%]) and hydatidosis (8 of 23 [35%]). The ELISA for H. nana may be useful for defining the epidemiology of H. nana infections, especially in areas free from cysticercosis and hydatidosis.

  16. Effect of treatment on serum antibody to Hymenolepis nana detected by enzyme-linked immunosorbent assay.

    PubMed Central

    Castillo, R M; Grados, P; Carcamo, C; Miranda, E; Montenegro, T; Guevara, A; Gilman, R H

    1991-01-01

    An enzyme-linked immunosorbent assay (ELISA) was developed to measure serum immunoglobulin G antibodies in 65 patients infected with Hymenolepis nana and 30 noninfected patients. Antibody was detected in 51 of 65 (sensitivity, 79%) and 5 of 30 H. nana-negative patients (specificity, 83%). Nine patients infected with H. nana were treated with praziquantel (20 to 25 mg/kg of body weight). Antibody disappeared from the sera at 90 days in six patients, five of whom had eliminated H. nana. Antibody persisted in three patients in whom H. nana infection did not clear after treatment. The H. nana ELISA had a high rate of cross-reactions with sera from patients with cysticercosis (8 of 29 [28%]) and hydatidosis (8 of 23 [35%]). The ELISA for H. nana may be useful for defining the epidemiology of H. nana infections, especially in areas free from cysticercosis and hydatidosis. PMID:2007652

  17. Evaluation of Babesia bigemina 200 kDa recombinant antigen in enzyme-linked immunosorbent assay.

    PubMed

    Altangerel, Khukhuu; Alhassan, Andy; Iseki, Hiroshi; Sivakumar, Thillaiampalam; Boldbaatar, Damdinsuren; Yokoyama, Naoaki; Igarashi, Ikuo

    2009-07-01

    A truncated fragment of the gene encoding the 200-kDa protein (P200) of Babesia bigemina was cloned into a plasmid vector, pGEX-4 T-1 and expressed in Escherichia coli as a glutathione-S-transferase fused protein. An indirect enzyme-linked immunosorbent assay (ELISA) using the rp200/CT detected specific antibodies in cattle experimentally infected with B. bigemina. Furthermore, the antigen did not cross-react with antibodies to Babesia bovis, a closely related Babesia parasite indicating that rp200/CT is a specific antigen for the diagnosis of B. bigemina infection. Additionally, ELISA using p200/CT and polymerase chain reaction were conducted on serum and corresponding DNA samples obtained from field cattle to evaluate the diagnostic utility of the p200/CT antigen. Results from the current study suggest that p200/CT ELISA is a sensitive and specific method for improved serodiagnosis of B. bigemina infection.

  18. Inhibition Enzyme-Linked Immunosorbent Assay for Serotyping of Group B Streptococcal Isolates

    PubMed Central

    Arakere, Gayathri; Flores, Aurea E.; Ferrieri, Patricia; Frasch, Carl E.

    1999-01-01

    Group B Streptococcus (GBS) is one of the most common organisms causing neonatal sepsis as well as serious infections in adults. Serotyping the organism is important in studying the epidemiology of the disease as well as deciding a course of treatment. There are several methods available for serotyping. Most of them need high-titered sera and are not quantitative. We are reporting a new inhibition enzyme-linked immunosorbent assay (ELISA) for serotyping which is sensitive and specific compared to the conventional methods but does not need high-titered serotype-specific antisera, as the specificity is controlled by the polysaccharide coating on the ELISA plates. The method can also be quantitative, and we have measured polysaccharide elaborated by different serotype V strains. Thus, the inhibition ELISA method will be useful in serotyping for epidemiological studies, assessing virulence, and performing strain selection for vaccine production. PMID:10405402

  19. Development of an enzyme-linked immunosorbent assay for studying Vibrio cholerae cell surface antigens.

    PubMed Central

    Cryz, S J; Fürer, E; Germanier, R

    1982-01-01

    An enzyme-linked immunosorbent assay for the measurement of antibodies directed against cell surface antigens of Vibrio cholerae (CSA ELISA) was developed. NaN3-killed whole cells of V. cholerae, adsorbed to polystyrene tubes, were used as immobilized antigens. The assay was capable of detecting antibodies directed against lipopolysaccharide and non-lipopolysaccharide surface antigens. In addition, the CSA ELISA was capable of detecting non-vibriocidal antibody. An antiserum raised in rabbits by immunization with live V. cholerae 1418 (Ogawa, El Tor) was capable of reacting with various heterologous strains of V. cholerae used as immobilized antigens. Therefore, common antigens shared by V. cholerae strains could be detected by using the CSA ELISA. PMID:7107860

  20. 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

  1. Comparison of enzyme-linked immunosorbent assay, DNA hybridization, hemagglutination, and electron microscopy for detection of canine parvovirus infections.

    PubMed Central

    Teramoto, Y A; Mildbrand, M M; Carlson, J; Collins, J K; Winston, S

    1984-01-01

    Canine fecal samples were analyzed by enzyme-linked immunosorbent assays (ELISA) by using monoclonal antibodies to the canine parvovirus hemagglutinating protein. These data were compared with results obtained with DNA hybridization assays, hemagglutination assays, and electron microscopy. The highest correlation was observed between the ELISA and the hemagglutination tests, with 94.4% of samples showing agreement. Lower correlation was obtained between ELISA and DNA hybridization tests (73.3%). Correlation between ELISA and electron microscopy was 60.9%. The studies indicated that the ELISA can be used as a sensitive and specific diagnostic assay for canine parvovirus infections. Images PMID:6092425

  2. 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.

  3. Enzyme-linked immunosorbent assay for immunoglobulin M specific antibody for the diagnosis of melioidosis.

    PubMed Central

    Kunakorn, M; Boonma, P; Khupulsup, K; Petchclai, B

    1990-01-01

    Indirect hemagglutination (IHA) is commonly used for serodiagnosis of melioidosis. However, in endemic areas, high background titers in normal populations and occasional low titers in patients with septicemic melioidosis prompted a search for a more sensitive and more specific method of serodiagnosis. An indirect fluorescent-antibody test for immunoglobulin M (IgM) specific antibody to Pseudomonas pseudomallei was more sensitive and more specific, but fluorescence microscopes are rarely available in the endemic areas. An enzyme-linked immunosorbent assay (ELISA) for IgM antibody is an attractive alternative. An indirect ELISA for IgM antibody (IgM ELISA) and an IgM antibody capture ELISA for melioidosis were developed. Both tests, together with IHA, were evaluated for 153 serum specimens from blood donors and 16 serum specimens from 16 melioidosis patients. It was found that IHA, the IgM ELISA, and the IgM antibody capture ELISA had sensitivities of 88, 88, and 75%, respectively, with specificities of 97.4, 92.2, and 91.5%, respectively. When IHA was combined with IgM ELISA, a sensitivity of 100% and a specificity of 95.4% were obtained. The IgM ELISA and IHA should be used in combination for serodiagnosis of melioidosis. PMID:2199494

  4. Evaluation of a commercially available enzyme-linked immunosorbent assay for Giardia lamblia antigen in stool.

    PubMed Central

    Addiss, D G; Mathews, H M; Stewart, J M; Wahlquist, S P; Williams, R M; Finton, R J; Spencer, H C; Juranek, D D

    1991-01-01

    The lack of a quick, simple, and inexpensive diagnostic test has limited the ability of public health officials to rapidly assess and control outbreaks of Giardia lamblia in child day-care centers. We evaluated the performance of a commercially available enzyme-linked immunosorbent assay (ELISA) for the detection of a G. lamblia-associated antigen in stool. Stool specimens were collected from the diapers of 426 children attending 20 day-care centers, fixed in 10% Formalin and polyvinyl alcohol, and examined by microscopy by Formalin concentration and trichrome staining techniques. Specimens were also tested visually and spectrophotometrically by ELISA. Of 99 tests positive by microscopy, 93 were visually positive by ELISA (sensitivity, 93.9%). Of 534 tests negative for G. lamblia by microscopy, 32 (6.0%) were ELISA positive. However, on the basis of examination of multiple specimens from the same child, none of these could be considered false-positive ELISAs; the specificity of the ELISA was therefore 100%. The sensitivity of both microscopy and ELISA improved as the number of specimens per child increased. An optical density value of greater than 0.040 was 98.0% sensitive and 100% specific for G. lamblia. This ELISA, which appeared to be more sensitive for G. lamblia than did microscopic examination of stool, should be useful as an epidemiologic tool, particularly in day-care settings, and may also have a role in confirming clinical diagnoses of giardiasis. PMID:1864930

  5. 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

  6. Use of an Inhibition Enzyme-Linked Immunosorbent Assay for Quantification of Capsular Polysaccharide or Proteins in Vaccines▿

    PubMed Central

    Inzana, Thomas J.; Champion, Anna

    2007-01-01

    An inhibition enzyme-linked immunosorbent assay (ELISA) is described for quantification of capsular polysaccharide or proteins in vaccines and other samples containing whole cells or extracts of Actinobacillus pleuropneumoniae. The assay can be used to quantify any antigen that can be purified and for which highly specific antibodies are not available. The assay can be carried out by any laboratory capable of performing an ELISA. PMID:17267591

  7. Enzyme-linked immunosorbent assay screening then indirect immunofluorescence confirmation of antinuclear antibodies: a statistical analysis.

    PubMed

    Copple, Susan S; Sawitzke, Allen D; Wilson, Andrew M; Tebo, Anne E; Hill, Harry R

    2011-05-01

    The purpose of this study was to analyze antinuclear antibody (ANA) screening by enzyme-linked immunosorbent assay (ELISA) followed by indirect fluorescent antibody (IFA) testing to confirm and characterize the pattern and titer of the antibody. We evaluated 4 ANA ELISAs and 1 HEp-2 IFA substrate in 224 clinically defined serum samples consisting of 30 from systemic lupus erythematosus (SLE) cases, 94 from rheumatoid arthritis cases, and 100 from healthy donors plus 495 serum samples submitted for routine ANA testing and 12 reference serum samples distributed by the Centers for Disease Control and Prevention. IFA tests were read independently by 2 certified medical technologists. ELISA sensitivities ranged from 90% to 97% compared with 80% by IFA in the SLE serum samples. The ELISAs had specificities of 36% to 94%, whereas the IFA had 99% specificity. Overall, ELISAs for ANA assays demonstrated better sensitivity and good specificity, suggesting ELISA is a more cost-effective alternative to IFA testing for initial ANA screening. Samples positive by ANA ELISA should be tested on HEp-2 to determine the titer and pattern.

  8. Comparison of five commercial enzyme-linked immunosorbent assays for detection of antibodies to Bordetella pertussis.

    PubMed

    Kösters, K; Riffelmann, M; Dohrn, B; von König, C H

    2000-05-01

    Measuring antibodies to Bordetella pertussis antigens is mostly done by enzyme-linked immunosorbent assays (ELISAs). We compared the performance of five commercially available ELISA kits with the help of 65 serum specimens which were repetitively tested for evaluation of the kits. The specimens contained 20 paired serum samples from patients with clinical pertussis, 15 samples were from children vaccinated with a diphtheria-tetanus-acellular pertussis vaccine, seven specimens were taken from an interlaboratory comparison of ELISAs, and there were three reference preparations from the Food and Drug Administration's (FDA's) Laboratory of Pertussis and from our laboratory. Reference values were obtained from the FDA or from results obtained with an in-house ELISA. Commercial ELISAs were compared with respect to their reproducibility and variability, their ability to detect significant titer rises in paired serum samples, their ability to detect an immune response after vaccination, and the comparability of semiquantitative and quantitative results. Reproducibility was generally good (>89%), intra-assay variation ranged from 2.4 to 28.7%, and indeterminate results were recorded in up to 18.5% of all specimens. Most kits correctly identified the antibody response to an acellular pertussis vaccine. None of the commercial kits identified all cases of pertussis correctly, and the sensitivity ranged between 60 and 95%. All five commercial ELISAs showed great discrepancies when comparing semiquantitative results and contained obviously different antigen preparations. Our data suggest that the five commercial ELISAs tested here need further improvement and standardization.

  9. Enzyme-Linked Immunosorbent Assays for Detection of Equine Antibodies Specific to Sarcocystis neurona Surface Antigens†

    PubMed Central

    Hoane, Jessica S.; Morrow, Jennifer K.; Saville, William J.; Dubey, J. P.; Granstrom, David E.; Howe, Daniel K.

    2005-01-01

    Sarcocystis neurona is the primary causative agent of equine protozoal myeloencephalitis (EPM), a common neurologic disease of horses in the Americas. We have developed a set of enzyme-linked immunosorbent assays (ELISAs) based on the four major surface antigens of S. neurona (SnSAGs) to analyze the equine antibody response to S. neurona. The SnSAG ELISAs were optimized and standardized with a sample set of 36 equine sera that had been characterized by Western blotting against total S. neurona parasite antigen, the current gold standard for S. neurona serology. The recombinant SnSAG2 (rSnSAG2) ELISA showed the highest sensitivity and specificity at 95.5% and 92.9%, respectively. In contrast, only 68.2% sensitivity and 71.4% specificity were achieved with the rSnSAG1 ELISA, indicating that this antigen may not be a reliable serological marker for analyzing antibodies against S. neurona in horses. Importantly, the ELISA antigens did not show cross-reactivity with antisera to Sarcocystis fayeri or Neospora hughesi, two other equine parasites. The accuracy and reliability exhibited by the SnSAG ELISAs suggest that these assays will be valuable tools for examining the equine immune response against S. neurona infection, which may help in understanding the pathobiology of this accidental parasite-host interaction. Moreover, with modification and further investigation, the SnSAG ELISAs have potential for use as immunodiagnostic tests to aid in the identification of horses affected by EPM. PMID:16148170

  10. Enzyme-linked immunosorbent assays for detection of equine antibodies specific to Sarcocystis neurona surface antigens.

    PubMed

    Hoane, Jessica S; Morrow, Jennifer K; Saville, William J; Dubey, J P; Granstrom, David E; Howe, Daniel K

    2005-09-01

    Sarcocystis neurona is the primary causative agent of equine protozoal myeloencephalitis (EPM), a common neurologic disease of horses in the Americas. We have developed a set of enzyme-linked immunosorbent assays (ELISAs) based on the four major surface antigens of S. neurona (SnSAGs) to analyze the equine antibody response to S. neurona. The SnSAG ELISAs were optimized and standardized with a sample set of 36 equine sera that had been characterized by Western blotting against total S. neurona parasite antigen, the current gold standard for S. neurona serology. The recombinant SnSAG2 (rSnSAG2) ELISA showed the highest sensitivity and specificity at 95.5% and 92.9%, respectively. In contrast, only 68.2% sensitivity and 71.4% specificity were achieved with the rSnSAG1 ELISA, indicating that this antigen may not be a reliable serological marker for analyzing antibodies against S. neurona in horses. Importantly, the ELISA antigens did not show cross-reactivity with antisera to Sarcocystis fayeri or Neospora hughesi, two other equine parasites. The accuracy and reliability exhibited by the SnSAG ELISAs suggest that these assays will be valuable tools for examining the equine immune response against S. neurona infection, which may help in understanding the pathobiology of this accidental parasite-host interaction. Moreover, with modification and further investigation, the SnSAG ELISAs have potential for use as immunodiagnostic tests to aid in the identification of horses affected by EPM.

  11. Simple assay for staphylococcal enterotoxins A, B, and C: modification of enzyme-linked immunosorbent assay.

    PubMed Central

    Stiffler-Rosenberg, G; Fey, H

    1978-01-01

    The enzyme-linked immunosorbent assay (ELISA) introduced for the detection of staphylococcal enterotoxins by Saunders et al., Simon and Terplan, and ourselves has proved to be a simple, reliable, and sensitive test. A new modification is described that uses polystyrene balls (diameter, 6 mm) coated individually with antibody against one of the toxins A, B, or C. In a single tube, 20 ml of the food extract was incubated with the three balls differently stained, which were then each tested for the uptake of enterotoxin by a competitive ELISA. A concentration of 0.1 ng or less of enterotoxin per ml can be measured, making tedious concentration procedures of the extracts superfluous. Culture supernatants and extracts from foods artificially or naturally contaminated with toxin were successfully examined. Cross-reactions did not occur, and nonspecific interfering substances did not create serious problems. PMID:365877

  12. Development of an enzyme-linked immunosorbent assay for the insecticide imidacloprid.

    PubMed

    Li, K; Li, Q X

    2000-08-01

    Enzyme-linked immunosorbent assays (ELISAs) were developed for imidacloprid, a neonicotinoid insecticide. Haptens were designed in such ways that spacer arms were introduced on either the pyridinyl or the imidazolidinyl ring of imidacloprid. Two sets of polyclonal antibodies were raised from rabbits immunized with two different immunogens and were characterized with an indirect ELISA format. Cross-reactivities and effects of organic solvents on the assays were evaluated. One set of antibodies shows approximately equal cross-reactivities to imidacloprid and its major metabolites with half-maximum inhibition concentrations (I(50)) of 73-88 ppb. Another is specific to imidacloprid with an I(50) of 35 ppb. The assay was initially applied to the analysis of imidacloprid in fortified water, coffee cherry, and bean extracts.

  13. Optimization of a human papillomavirus-specific enzyme-linked immunosorbent assay.

    PubMed

    Karem, Kevin L; Poon, Alysia C; Bierl, Cynthia; Nisenbaum, Rosane; Unger, Elizabeth

    2002-05-01

    A strategy was developed for the control, standardization, and critical evaluation of an enzyme-linked immunosorbent assay (ELISA) for the detection of human papillomavirus-specific immunoglobulin G in human sera. Control human sera, polyclonal animal sera, and monoclonal antibodies were used to establish optimal assay parameters, including antigen coating, serum dilutions, and criteria for daily reproducibility, monitoring, and rejection of assays. Three evaluation techniques were used in parallel to define an optimal cutoff absorbance value that yields greater than 93% sensitivity and 98.5% specificity in the assay's ability to discriminate positive and negative control sera. This strategy provides an optimal method by which to determine cutoff absorbance values for ELISA.

  14. Optimization of a Human Papillomavirus-Specific Enzyme-Linked Immunosorbent Assay

    PubMed Central

    Karem, Kevin L.; Poon, Alysia C.; Bierl, Cynthia; Nisenbaum, Rosane; Unger, Elizabeth

    2002-01-01

    A strategy was developed for the control, standardization, and critical evaluation of an enzyme-linked immunosorbent assay (ELISA) for the detection of human papillomavirus-specific immunoglobulin G in human sera. Control human sera, polyclonal animal sera, and monoclonal antibodies were used to establish optimal assay parameters, including antigen coating, serum dilutions, and criteria for daily reproducibility, monitoring, and rejection of assays. Three evaluation techniques were used in parallel to define an optimal cutoff absorbance value that yields greater than 93% sensitivity and 98.5% specificity in the assay's ability to discriminate positive and negative control sera. This strategy provides an optimal method by which to determine cutoff absorbance values for ELISA. PMID:11986263

  15. Enzyme-linked immunosorbent assay for efficient detection of antibody to bluetongue virus in pronghorn (Antilocapra americana).

    PubMed

    Drolet, B S; Mills, K W; Belden, E L; Mecham, J O

    1990-01-01

    An indirect enzyme-linked immunosorbent assay (ELISA), using cell-associated viral antigen, was developed for detection of antibody to bluetongue virus (BTV) in field-collected pronghorn (Antilocapra americana) sera. To test the applicability of the ELISA to seroepizootiologic studies, pronghorn serum samples from three Wyoming counties (USA) were tested. Bluetongue virus ELISA results were compared to those of the bluetongue immunodiffusion assay. Discrepant serum samples were retested for reaction to either BTV or epizootic hemorrhagic disease virus. The pronghorn BTV ELISA gave rapid, quantitative, objective results and should facilitate testing large numbers of sera for BT diagnostic and seroepizootiologic studies.

  16. 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

  17. Enzyme-linked immunosorbent assay for the determination of five organophosphorus pesticides in camellia oil.

    PubMed

    Liu, Yihua; Guo, Yirong; Zhu, Guonian; Tang, Fubin

    2014-07-01

    A matrix solid-phase dispersion and direct competitive enzyme-linked immunosorbent assay (MSPD-ELISA) was developed for five organophosphorus pesticides (OPs) in camellia oil. Seven haptens with different substituents in the aromatic ring were used to prepare different competitors; the ELISA showed highest sensitivity and specificity to OPs when the competitor had moderate heterology to the immunizing hapten. Several assay conditions were optimized to increase the ELISA sensitivity. The optimized ELISA for five OPs had 50% inhibitory concentrations of 6.3 ng/ml (parathion), 18.9 ng/ml (methyl parathion), 120.7 ng/ml (fenitrothion), 110.4 ng/ml (fenthion), and 20.7 ng/ml (phoxim). The average recoveries of five OPs in camellia oil ranged from 75.7 to 105.3%, with the interassay coefficient of variations ranging from 6.0 to 13.4%. Compared with the results previously reported, the ELISA that was developed in the present study showed a much higher sensitivity. Additionally, MSPD was used in the sample preparation to minimize the matrix effect. Recoveries from the method developed here were in agreement with those obtained by gas chromatography, which indicated that the detection performance of the MSPD-ELISA could meet the regulatory requirements of different governments and international organizations.

  18. 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.

  19. 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.

  20. 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

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

    PubMed

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

    2016-12-01

    The continuing use of transgenic crops has led to an increased interest in the fate of insecticidal crystalline (Cry) proteins in the environment. Enzyme-linked immunosorbent assays (ELISAs) have 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 overestimation of the concentration of these proteins in the environment. Five model systems were used to generate fragments of the Cry1Ab protein, which were then analyzed by ELISAs and bioassays. Fragments from 4 of the model systems were not detectable by ELISA and did not retain bioactivity. Fragments from the proteinase K model system were detectable by ELISA and retained bioactivity. In most cases, ELISAs appear to provide an accurate estimation of the amount of Cry proteins in the environment, as detectable fragments retained bioactivity and nondetectable fragments did not retain bioactivity. Environ Toxicol Chem 2016;35:3101-3112. © 2016 SETAC. © 2016 SETAC.

  2. Enzyme-linked immunosorbent assays using immune complexes for the diagnosis of tuberculosis.

    PubMed

    Pereira Arias-Bouda, Lenka M; Kuijper, Sjoukje; van Deutekom, Henk; van Gijlswijk, Rob; Pekel, Inge; Jansen, Henk M; Kolk, Arend H J

    2003-12-01

    The serodiagnosis of tuberculosis has long been the subject of investigation, but we still lack a test with widespread clinical utility. The poor sensitivity and specificity of commercial assays precludes their use as the sole means of diagnosis. All of these assays use mycobacterial antigens adsorbed onto a surface. Little attention has been paid to changes in antigen conformation that may occur as a result of passive coating of these antigens to solid supports like polystyrene. Such changes may cause technical artifacts resulting in false-positive (FP) and false-negative (FN) reactions. We have developed two different enzyme-linked immunosorbent assay (ELISA) systems, in which human serum antibodies and target antigens of Mycobacterium tuberculosis are able to associate and dissociate freely in solution to form immune complexes. In one ELISA, rabbit antibodies against M. tuberculosis, passively coated in the ELISA wells, capture the immune complexes (ICs). In the other ELISA, the ICs are detected by these same rabbit antibodies but are first captured by passively coated goat anti-rabbit IgG. We have compared these two ELISA systems with an ELISA using M. tuberculosis antigens passively adsorbed to the solid polystyrene surface of the plate. We studied sera from 81 patients with tuberculosis and 47 healthy subjects. The differences between tuberculosis (TB) patients and healthy subjects were statistically significant in all three of our ELISA systems. However, the ELISA systems using soluble M. tuberculosis antigens distinguished better between TB patients and healthy subjects than the ELISA using surface-adsorbed M. tuberculosis antigens. We suggest that in the latter ELISA, passive adsorption of the target antigens induces conformational change, generating altered epitopes that are recognized by antibodies present in the serum from even healthy people. These altered conformational epitopes are recognized by antibodies that were originally evoked by antigens

  3. Accuracy of enzyme-linked immunosorbent assays for quantification of antibodies against Aleutian mink disease virus.

    PubMed

    Farid, A H; Rupasinghe, P P

    2016-09-01

    There is a growing interest among mink ranchers to select their stock for tolerance to the Aleutian mink disease virus (AMDV). Enzyme-linked immunosorbent assays (ELISA) are used to identify mink which have low anti-AMDV antibody titres and are expected to tolerate the AMDV infection. The objective of this study was to calculate the accuracy of three ELISA systems which were performed on blood or serum of AMDV-inoculated American mink (Neovison vison) at five laboratories in Canada, USA, Finland, the Netherlands and Denmark. The accuracy was determined by comparing the ELISA results with antibody titres measured by the counter-immunoelectrophoresis (CIEP) using 10 two-fold serial dilutions of the plasma. Antibody titres of 880 black mink which were inoculated with a spleen homogenate from a naturally infected mink were measured between 16 and 176 weeks post-inoculation. Each ELISA result from every laboratory covered a wide range of antibody titres and the Spearman's rank correlation coefficients between CIEP and ELISA results from different laboratories varied between 0.41 and 0.83, indicating a low to moderate accuracy of ELISA systems for ranking mink by antibody titre. The recombinant VP2-based ELISA used in the Netherlands and Finland ranked the mink by antibody titres more accurately than did the AMDV-G-based ELISA platforms developed in Denmark and the USA, suggesting that the source of antigen was one of the factors affecting the accuracy of ELISA results. It was concluded that the ELISA systems, particularly those based on AMDV-G antigen, require further refinement to improve their accuracy for ranking mink by antibody titre. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

  4. Double-conjugate enzyme-linked immunosorbent assay for immunoglobulins G and M against Treponema pallidum.

    PubMed Central

    Farshy, C E; Hunter, E F; Larsen, S A; Cerny, E H

    1984-01-01

    An enzyme-linked immunosorbent assay (ELISA) for the simultaneous measurement of immunoglobulin G (IgG) and IgM was developed to detect antibodies to Treponema pallidum. Wells of polystyrene microtiter plates were coated with T. pallidum antigen, diluted patient serum was added, and IgG and IgM which bound to the T. pallidum antigen were measured by the simultaneous addition of alkaline phosphatase-labeled anti-human IgG and horseradish peroxidase-labeled anti-human IgM. Bound IgG was detected first, followed by bound IgM. After development of the procedure, 145 categorized sera were evaluated: 60 from individuals without syphilis; 62 from patients with syphilis, including 22 with primary, 20 with secondary, and 20 with latent phases of syphilis; and 23 from patients with rheumatoid arthritis. Of the 60 sera from individuals without syphilis, 100% were nonreactive for IgG antibody and 16% were reactive for IgM. Of the 23 sera from patients with rheumatoid arthritis, 3 were reactive for IgG and 3 were nonreactive for IgM. Of the 62 sera from patients with syphilis, 61 (98%) were reactive for IgG antibody with increased titers as the stage of syphilis increased, whereas IgM reactivity decreased. This enzyme-linked immunosorbent assay appears to be a simple method for the simultaneous measurement of antibodies under equal assay conditions. PMID:6394613

  5. Vitellogenin assay by enzyme-linked immunosorbant assay as a biomarker of endocrine disruptor chemicals pollution.

    PubMed

    Ebrahimi, Mansour

    2007-09-15

    Research is ongoing to develop screening and testing programmes for endocrine disrupting effects of new chemicals and in the focus of this development are the fish test species common carp (Cyprinus carpio). In this study we have developed quantitative enzyme linked immunosorbent assays (ELISA) for VTS in common carp. The working range of the ELISA was 11.25 to 2000 ng mL(-1) (75-25% specific binding/maximum antibody binding [B/B0]) with a 50% B/B0 intra- and interassay variation of 3.9% (n=10) and 12.5% (n=30), respectively. This ELISA is capable of detecting VTG as low as 6 ng mL(-1) and can accurately detect VTG in even 10 microL of plasma. The ELISA was applied to measurement of VTG production by male carp (Cyprinous carpio, Cyprinidae) fish exposure to ethynylestradiol. The results showed that the amount of VTG produced in plasma of exposed fish increased in logaritmic order comparing to the control group and the ELISA described here could be used as an indicator of water pollution to estrogenic pollutants.

  6. Enzyme-linked immunosorbent assay for detection of filovirus species-specific antibodies.

    PubMed

    Nakayama, Eri; Yokoyama, Ayaka; Miyamoto, Hiroko; Igarashi, Manabu; Kishida, Noriko; Matsuno, Keita; Marzi, Andrea; Feldmann, Heinz; Ito, Kimihito; Saijo, Masayuki; Takada, Ayato

    2010-11-01

    Several enzyme-linked immunosorbent assays (ELISAs) for the detection of filovirus-specific antibodies have been developed. However, diagnostic methods to distinguish antibodies specific to the respective species of filoviruses, which provide the basis for serological classification, are not readily available. We established an ELISA using His-tagged secreted forms of the transmembrane glycoproteins (GPs) of five different Ebola virus (EBOV) species and one Marburg virus (MARV) strain as antigens for the detection of filovirus species-specific antibodies. The GP-based ELISA was evaluated by testing antisera collected from mice immunized with virus-like particles as well as from humans and nonhuman primates infected with EBOV or MARV. In our ELISA, little cross-reactivity of IgG antibodies was observed in most of the mouse antisera. Although sera and plasma from some patients and monkeys showed notable cross-reactivity with the GPs from multiple filovirus species, the highest reactions of IgG were uniformly detected against the GP antigen homologous to the virus species that infected individuals. We further confirmed that MARV-specific IgM antibodies were specifically detected in specimens collected from patients during the acute phase of infection. These results demonstrate the usefulness of our ELISA for diagnostics as well as ecological and serosurvey studies.

  7. Enzyme-linked immunosorbent assay using monoclonal antibodies for identification of mycobacteria from early cultures.

    PubMed Central

    Verstijnen, C P; Ly, H M; Polman, K; Richter, C; Smits, S P; Maselle, S Y; Peerbooms, P; Rienthong, D; Montreewasuwat, N; Koanjanart, S

    1991-01-01

    A simple enzyme-linked immunosorbent assay (ELISA) for the identification of cultured mycobacteria belonging to the Mycobacterium tuberculosis complex, the Mycobacterium avium complex, and Mycobacterium kansasii has been developed (R. Schöningh, C. P. H. J. Verstijnen, S. Kuijper, and A. H. J. Kolk. J. Clin. Microbiol. 28:708-713, 1990). The test for the routine identification of cultured mycobacteria was introduced in five clinical laboratories located in Tanzania, Thailand, Vietnam, and The Netherlands. The ELISA can be conducted without an ELISA reader since the test can be read visually. The results of identification of 255 strains of the M. tuberculosis complex by microbiological means and by ELISA were compared; the specificity and the sensitivity were 100%. For the M. avium complex, the specificity was 100% and the sensitivity was 64%. All 26 M. kansasii strains tested could be identified as M. kansasii. The ELISA described here proved to be useful in both well- and modestly equipped laboratories and may replace the microbiological method of identification of M. tuberculosis and M. kansasii. PMID:1909344

  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. 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

  10. Serodiagnosis of Chagas' disease by enzyme-linked immunosorbent assay using two synthetic peptides as antigens.

    PubMed Central

    Peralta, J M; Teixeira, M G; Shreffler, W G; Pereira, J B; Burns, J M; Sleath, P R; Reed, S G

    1994-01-01

    An enzyme-linked immunosorbent assay (ELISA) was developed for detecting antibodies against Trypanosoma cruzi. Two synthetic T. cruzi peptides, TcD and PEP2, were used. The specificity and sensitivity of the peptide ELISA were determined with 260 serum samples from individuals living in an area in which Chagas' disease is endemic. ELISAs were performed with the peptides singly or in combination. The evaluation of these tests showed that 168 (93.8%) of 179 serum samples from T. cruzi-infected patients were positive when TcD peptide was used as antigen; 164 (91.6%) samples were positive with PEP2, and 178 (99.4%) samples were positive when the two peptides were combined. Thus, the sensitivity of the ELISA using the two peptides exceeded 99%. The specificity was evaluated by using a panel of 118 serum samples that included samples from 81 individuals living in an area of endemicity with negative serology for Chagas' disease and from 37 patients from areas in which T. cruzi was not endemic but with other pathologies, such as leishmaniasis, tuberculosis, and leprosy. Only two false-positive serum samples were found in this group of individuals, giving a test specificity of more than 98%. Because these peptides can be synthesized and are very stable at room temperature, the use of such reagents can improve the standardization and reproducibility of ELISAs for the serodiagnosis of T. cruzi infection. PMID:8027352

  11. Validation of 2 commercial Neospora caninum antibody enzyme linked immunosorbent assays

    PubMed Central

    Wu, John T.Y.; Dreger, Sally; Chow, Eva Y.W.; Bowlby, Evelyn E.

    2002-01-01

    Abstract This is a validation study of 2 commercially available enzyme linked immunosorbent assays (ELISA) for the detection of antibodies against Neospora caninum in bovine serum. The results of the reference sera (n = 30) and field sera from an infected beef herd (n = 150) were tested by both ELISAs and the results were compared statistically. When the immunoblotting results of the reference bovine sera were compared to the ELISA results, the same identity score (96.67%) and kappa values (K) (0.93) were obtained for both ELISAs. The sensitivity and specificity values for the IDEXX test were 100% and 93.33% respectively. For the Biovet test 93.33% and 100% were obtained. The corresponding positive (PV+) and negative predictive (PV−) values for the 2 assays were 93.75% and 100% (IDEXX), and 100% and 93.75% (Biovet). In the 2nd study, competitive inhibition ELISA (c-ELISA) results on bovine sera from an infected herd were compared to the 2 sets of ELISA results. The identity scores of the 2 ELISAs were 98% (IDEXX) and 97.33% (Biovet). The K values calculated were 0.96 (IDEXX) and 0.95 (Biovet). For the IDEXX test the sensitivity and specificity were 97.56% and 98.53%, whereas for the Biovet assay 95.12% and 100% were recorded, respectively. The corresponding PV+ and PV− values were 98.77% and 97.1% (IDEXX), and 100% and 94.44% (Biovet). Our validation results showed that the 2 ELISAs worked equally well and there was no statistically significant difference between the performance of the 2 tests. Both tests showed high reproducibility, repeatability and substantial agreement with results from 2 other laboratories. A quality assurance based on the requirement of the ISO/IEC 17025 standards has been adopted throughout this project for test validation procedures. PMID:12418782

  12. Neonatal tetanus in Peru: risk assessment with modified enzyme-linked immunosorbent assay and toxoid skin test.

    PubMed Central

    Vernacchio, L; Madico, G; Verastegui, M; Diaz, F; Collins, T S; Gilman, R H

    1993-01-01

    We used a modified enzyme-linked immunosorbent assay (ELISA) to investigate tetanus immunity in 232 pregnant Peruvian women. One hundred forty-two (61.2%) had protective antitoxin titers (> or = 0.01 IU/mL). Protective titers correlated positively with the number of toxoid doses reported during the current pregnancy. A majority of women reporting no toxoid doses during the current pregnancy had at least one prenatal health care visit. We evaluated a toxoid skin test in 44 of the subjects, but it correlated poorly with the ELISA. The modified ELISA is a useful in vitro method for studying tetanus immunity in the developing world. PMID:8259811

  13. Evaluation of two commercial enzyme-linked immunosorbent assay kits for the detection of Mycoplasma gallisepticum antibodies.

    PubMed

    Kempf, I; Gesbert, F; Guittet, M; Bennejean, G; Stipkovits, L

    1994-06-01

    Sensitivity and specificity of two commercial Mycoplasma gallisepticum (MG) enzyme-linked immunosorbent assay (ELISA) kits, rapid slide agglutination (SA) and haemagglutination inhibition (HI) tests were compared using sera from specific pathogen free chickens, turkeys or ducks which had been inoculated with various avian mycoplasmas, bacteria or with a reovirus. Results show that sensitivity of SA was superior to ELISA and HI tests in the ability to detect antibodies formed in early response to MG infection. However, both ELISA kits and HI tests had a higher degree of specificity.

  14. Consequences of Interference of Milk with Chemoattractants for Enzyme-Linked Immunosorbent Assay Quantifications▿

    PubMed Central

    Rainard, P.

    2010-01-01

    Concentrations of the chemoattractants CXCL1, CXCL2, CXCL3, CXCL8, and C5a in milk were reduced by the preparation of milk whey by high-speed centrifugation or with rennet. About half of the chemoattractants (35 to 65%) were associated with the casein micelle sediment, except when whey was prepared by acidification. Consequently, quantification of chemoattractants should be carried out preferentially with skimmed milk samples or, whenever whey is needed, with acidic whey samples. The interference of milk or milk whey with the enzyme-linked immunosorbent assays (ELISAs) used to quantify the chemoattractants was moderate, as long as tetramethylbenzidine (TMB), not ABTS [2,2′-azino-bis-(3-ethylbenzthiazoline-sulfonate)], was used as the substrate of peroxidase. These considerations will help to assess more precisely a component of the immune response of the mammary gland to infection. PMID:20237202

  15. A murine monoclonal antibody based enzyme-linked immunosorbent assay for almond (Prunus dulcis L.) detection.

    PubMed

    Su, Mengna; Venkatachalam, Mahesh; Liu, Changqi; Zhang, Ying; Roux, Kenneth H; Sathe, Shridhar K

    2013-11-13

    A sandwich enzyme-linked immunosorbent assay (ELISA) using anti-almond soluble protein rabbit polyclonal antibodies as capture antibodies and murine monoclonal antibody 4C10 as the detection antibodies was developed. The assay is specific and sensitive (3-200 ng almond protein/mL) for almond detection. The standardized assay is accurate (<15% CV) and reproducible (intra- and inter assay variability <15% CV). The assay did not register any cross-reactivity with the tested food matrices, suggesting the assay to be almond amandin specific. The assay could detect the presence of declared almond in the tested matched commercial samples. Further, the assay reliably detected the presence of almonds in the laboratory prepared food samples spiked with almond flour.

  16. Evaluation of specificity of indirect enzyme-linked immunosorbent assay for diagnosis of human Q fever.

    PubMed Central

    Uhaa, I J; Fishbein, D B; Olson, J G; Rives, C C; Waag, D M; Williams, J C

    1994-01-01

    Ninety-five acute- and convalescent-phase serum specimens from 48 patients suspected of having rickettsial or Legionella infections were assayed for antibodies to Coxiella burnetii, the causative agent of Q fever. To evaluate the specificity of the indirect enzyme-linked immunosorbent assay (ELISA) for human Q fever, we compared the ELISA results with those of the indirect immunofluorescence antibody (IFA) test. The ELISA data were analyzed by two different criteria for a positive test. The first criterion for positive results by ELISA was based upon diagnostic titers established in a study of 150 subjects who had no demonstrable cellular or humoral immune responses to C. burnetii phase I or phase II whole cells or phase I lipopolysaccharide. The second criterion was based upon diagnostic antibody titers in a study of 51 subjects who had been diagnosed as having clinical Q fever and had fourfold or greater rises in humoral immune responses to C. burnetii phase I and phase II whole-cell antigens. A comparison of the ELISA and IFA test results of the 95 serum specimens indicated excellent agreement between the tests (Kappa = 92.9%; P < 0.05). None of the 38 patients whose etiologies were confirmed serologically as Legionnaires' disease or rickettsial diseases other than Q fever were classified as positive for C. burnetii by the ELISA. Only one patient identified by the IFA test as having Q fever was not scored positive by the ELISA. These results suggest that the ELISA is useful for epidemiologic screening and as a diagnostic test for human Q fever. PMID:8077404

  17. Reliable enzyme-linked immunosorbent assay for the determination of soybean proteins in processed foods.

    PubMed

    Morishita, Naoki; Kamiya, Kumiko; Matsumoto, Takashi; Sakai, Shinobu; Teshima, Reiko; Urisu, Atsuo; Moriyama, Tatsuya; Ogawa, Tadashi; Akiyama, Hiroshi; Morimatsu, Fumiki

    2008-08-27

    Among allergenic foods, soybean is known as a food causing adverse reactions in allergenic patients. To clarify the validity of labeling, the specific and sensitive detection method for the analysis of the soybean protein would be necessary. The p34 protein, originally characterized to be p34 as an oil-body associated protein in soybean, has been identified as one of the major allergenic proteins and named Gly m Bd 30K. A novel sandwich enzyme-linked immunosorbent assay (ELISA) for the detection and quantification of the soybean protein in processed foods was developed using polyclonal antibodies raised against p34 as a soybean marker protein and the specific extraction buffer for extract. The developed sandwich ELISA method was highly specific for the soybean protein. The limit of detection (LOD) and the limit of quantification (LOQ) of the developed ELISA were 0.47 ng/mL (equivalent to 0.19 microg/g in foods) and 0.94 ng/mL (equivalent to 0.38 microg/g in foods), respectively. The recovery ranged from 87.7 to 98.7%, whereas the intra- and interassay coefficients of variation were less than 4.2 and 7.5%, respectively. This study showed that the developed ELISA method is a specific, precise, and reliable tool for the quantitative analysis of the soybean protein in processed foods.

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

    PubMed

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

    2015-12-01

    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.

  19. 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.

  20. 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

  1. 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).

  2. 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.

  3. 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

  4. Development and application of an enzyme linked immunosorbent assay for Clostridium perfringens type A enterotoxin.

    PubMed Central

    Bartholomew, B A; Stringer, M F; Watson, G N; Gilbert, R J

    1985-01-01

    An enzyme linked immunosorbent assay (ELISA) has been developed to quantitate faecal Clostridium perfringens enterotoxin in the investigation of C perfringens food poisoning. The sandwich ELISA could be carried out in 24 h and was sensitive enough to detect as little as 5 ng/g of enterotoxin in faeces. Specificity of the assay was shown by comparing results with those obtained from other standard toxin assays, such as double gel diffusion and counterimmunoelectrophoresis, and by the assay of faecal material from control groups. By means of the ELISA method, 515 faecal samples from 50 separate outbreaks of C perfringens food poisoning were examined, together with 21 food samples from 12 of the outbreaks. A clear distinction was noted between faecal samples collected on the first two days of an outbreak, where 77% were enterotoxin positive, and those specimens collected later than the second day, when only 33% had detectable enterotoxin. The ELISA is recommended as a valuable tool in the investigation of C perfringens foodborne illness. PMID:2857184

  5. A sandwich enzyme-linked immunosorbent assay for the quantification of insoluble membrane and scaffold proteins.

    PubMed

    Geumann, Constanze; Grønborg, Mads; Hellwig, Michaela; Martens, Henrik; Jahn, Reinhard

    2010-07-15

    Enzyme-linked immunosorbent assays (ELISAs) are applied for the quantification of a vast diversity of small molecules. However, ELISAs require that the antigen is present in a soluble form in the sample. Accordingly, the few ELISAs described so far targeting insoluble proteins such as integral membrane and scaffold proteins have been restricted by limited extraction efficiencies and the need to establish an individual solubilization protocol for each protein. Here we describe a sandwich ELISA that allows the quantification of a diverse array of synaptic membrane and scaffold proteins such as munc13-1, gephyrin, NMDA R1 (N-methyl-d-aspartate receptor subunit 1), synaptic vesicle membrane proteins, and SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors). The assay is based on initial solubilization by the denaturing detergent sodium dodecyl sulfate (SDS), followed by partial SDS removal using the detergent Triton X-100, which restores antigenicity while keeping the proteins in solution. Using recombinant standard proteins, we determined assay sensitivities of 78ng/ml to 77pg/ml (or 74-0.1fmol). Calibration of the assay using both immunoblotting and mass spectroscopy revealed that in some cases correction factors need to be included for absolute quantification. The assay is versatile, allows parallel processing and automation, and should be applicable to a wide range of hitherto inaccessible proteins.

  6. Penicillinase-based enzyme-linked immunosorbent assay for the detection of plant viruses.

    PubMed

    Sudarshana, M R; Reddy, D V

    1989-10-01

    A penicillinase (PNC)-based, enzyme-linked immunosorbent assay (ELISA) was standardized to detect maize mosaic virus (MMV) in sorghum leaf extracts, peanut mottle virus (PMV) in pea leaf extracts, and tomato spotted wilt virus (TSWV) in peanut leaf extracts. Rabbit Fc-specific antibodies were conjugated with PNC by a single step glutaraldehyde bridge. Among several indicators tested, bromothymol blue (BTB) was found suitable for measuring PNC activity under simulated conditions. Two reagents, starch-iodine complex (SIC) and a mixed pH indicator, containing bromocresol purple and BTB (2:1) used earlier for the PNC-based ELISA, were compared with BTB for utilization in the PNC-based ELISA. SIC gave a slightly higher virus titre than BTB or the mixed pH indicator, but it often gave nonspecific reactions. Sodium or potassium salts of penicillin-G at 0.5-1.0 mg/ml and BTB at 0.2 mg/ml were found to be suitable as substrate-indicator mixture for PNC-based ELISA. The sensitivity of the PNC system was comparable to those of the alkaline phosphatase (ALP) and horseradish peroxidase (HRP) systems in detecting MMV, PMV, and TSWV. The PNC conjugate could be used at a greater dilution than those of the ALP and HRP conjugates and the BTB substrate mixture was stable for at least 3 weeks at 4 degrees C. Penicillin is readily available in developing countries, and at a substantially lower cost than p-nitrophenyl phosphate, the commonly used substrate for ALP in the plate ELISA. Thus the PNC-based ELISA provides a less expensive means for assaying plant viruses by ELISA.

  7. Competitive and indirect enzyme-linked immunosorbent assays for Mycobacterium bovis infections based on MPB70 and lipoarabinomannan antigens.

    PubMed Central

    Sugden, E A; Stilwell, K; Rohonczy, E B; Martineau, P

    1997-01-01

    A competitive enzyme-linked immunosorbent assay (C-ELISA) using M. bovis BCG Tokyo culture filtrate as antigen and anti-MPB70 4C3/17 monoclonal antibody was developed for use in multiple animal species. An analysis of the C-ELISA data for cattle and bison serum panels revealed specificities of 68% to 85% and sensitivities of 85% to 89%. Receiver operator characteristics (ROC) of this data revealed areas of 81% to 92% for C-ELISA and demonstrated that C-ELISA as well as the indirect ELISA protocols, MPB70-ELISA and LAM-ELISA, discriminate M. bovis infected animals from non-infected animals for these particular panels. The kappa statistic values for agreement beyond chance between C-ELISA and MPB70-ELISA were determined after ELISA cutoffs were adjusted to minimize false positives. There were poor to excellent agreements between C-ELISA and MPB70-ELISA in all species tested (Bovidae, Cervidae, and Camelidae) that were consistently higher than the kappa statistic between C-ELISA and LAM-ELISA. The humoral response to one antigen and little or no response to the other in many animals argued for a parallel interpretation of C-ELISA and LAM-ELISA to increase sensitivity. PMID:9008794

  8. Serological Evaluation of Thin-Layer Immunoassay–Enzyme-Linked Immunosorbent Assay for Antibody Detection in Human Trichinellosis

    PubMed Central

    Gómez-Priego, Alberto; Crecencio-Rosales, Lidia; de-la-Rosa, Jorge-Luis

    2000-01-01

    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 μl each of antigen (7 μg/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

  9. 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 ...

  10. 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 ...

  11. 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 Central

    Caceres, Diego H.; Scheel, Christina M.; Tobón, Ángela M.; Ahlquist Cleveland, Angela; Restrepo, Ángela; Brandt, Mary E.; Chiller, Tom

    2014-01-01

    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

  12. A double antibody sandwich enzyme-linked immunosorbent assay for detection of soft-shelled turtle iridovirus antigens.

    PubMed

    Zhang, M; Yang, J X; Lin, X M; Zhu, C H; He, J Q; Liu, H; Lin, T L

    2010-08-01

    A double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) for detection of the soft-shelled turtle iridovirus (STIV) was developed using a specific monoclonal antibody (mAb) against STIV and anti-STIV rabbit serum. Using DAS-ELISA, the detection limit of STIV was found to be 10(3)PFU/ml. The positive rate of 15 STIV samples was 100%, while the positive rate of 100 other aquatic virus samples was 0%. These data show that DAS-ELISA is highly specific and sensitive for the detection of STIV. In clinical tests, 128 samples isolated from pond-reared turtles were subjected to DAS-ELISA and PCR. The overall agreement between the results obtained by DAS-ELISA and PCR was 98.4%. The results indicate that the DAS-ELISA method could be used for diagnosing diseases caused by STIV. Copyright (c) 2010 Elsevier B.V. All rights reserved.

  13. [Enzyme-linked immunosorbent assay in the diagnosis of campylobacteriosis].

    PubMed

    Jakubczak, Aleksandra; Rastawicki, Waldemar; Jagielski, Marek

    2007-01-01

    Campylobacter jejuni and Campylobacter coli are the bacterial cause of human gastroenteritis commonly reported worldwide. The serodiagnosis of Campylobacter infections is not routinely done in Poland so the aim of this study was to evaluation of ELISA in the diagnosis ofcampylobacteriosis. Serum samples obtained from 145 patients with gastroenteritis were tested by ELISA with 7 different heat-stable antigens of C. jejuni and one of C. coli and by the commercial Virion/Serion ELISA with purified 45 kDa outer membrane protein of C. jejuni. Antibodies for heat-stable antigens of C. jejuni were detected statistically more often than antibodies for heat-stable antigens of C. coli and for purifled protein of C. jejuni. We found significant differences in the frequency of detection of antibodies to different heat-stable antigens, ranged from 18.6% to 68.9% of positive results, what indicate for serological heterogenicity of C. jejuni strains isolated in Poland. The results of our study showed usefulness of ELISA in serological diagnosis of campylobacteriosis. However it is necessary to serotype the C. jejuni strains isolated in Poland to find the appropriate C. jejuni serotype for using in ELISA.

  14. Evaluation of an antigen capture enzyme-linked immunosorbent assay for detection of Cryptosporidium oocysts.

    PubMed Central

    Newman, R D; Jaeger, K L; Wuhib, T; Lima, A A; Guerrant, R L; Sears, C L

    1993-01-01

    The diagnosis of the small (4- to 6-microns) Cryptosporidium oocysts is labor intensive and relies on stool concentration, with subsequent staining and microscopy. The primary purpose of this study was to evaluate the clinical utility of an antigen capture enzyme-linked immunosorbent assay (ELISA) (LMD Laboratories, Carlsbad, Calif.) in detecting Cryptosporidium oocysts in human stools. A total of 591 specimens (76 diarrheal, 515 control) obtained from 213 inhabitants of an urban slum in northeastern Brazil were examined by both ELISA and conventional microscopic examination (CME) of formalin-ethyl acetate-concentrated stool samples stained with modified acid-fast and auramine stains. Forty-eight diarrheal stools (63.2%) were positive for Cryptosporidium oocysts by CME, with 40 of these positive by ELISA. Thirty-five control stools (6.8%) had Cryptosporidium oocysts detected by CME, with 15 of these also positive by ELISA. All of the 480 nondiarrheal stools and all but one of the diarrheal stools negative by CME were negative by ELISA. The test had an overall sensitivity of 66.3% and a specificity of 99.8% (positive predictive value, 98.2%; negative predictive value, 94.8%). In the evaluation of human diarrheal stool samples, the test sensitivity increased to 83.3%, with a specificity of 96.4%, and, in analysis of samples from individual patients with diarrhea, the sensitivity was 87.9%, with a specificity of 100%. These results indicate that this stool ELISA is sensitive and specific for the detection of Cryptosporidium oocysts in human diarrheal stool specimens but has limited use in epidemiologic studies for the diagnosis of asymptomatic Cryptosporidium infection. PMID:8370732

  15. Development of an enzyme-linked immunosorbent assay for serodiagnosis of ringworm infection in cattle.

    PubMed

    Bagut, Elena Tatiana; Cambier, Ludivine; Heinen, Marie-Pierre; Cozma, Vasile; Monod, Michel; Mignon, Bernard

    2013-08-01

    The aim of this study was to develop an in-house enzyme-linked immunosorbent assay (ELISA) for the serological diagnosis of ringworm infection in cattle. We used available recombinant forms of Trichophyton rubrum dipeptidyl peptidase V (TruDppV) and T. rubrum leucin aminopeptidase 2 (TruLap2), which are 98% identical to Trichophyton verrucosum orthologues. Field serum samples from 135 cattle with ringworm infection, as confirmed by direct microscopy, fluorescence microscopy, and PCR, and from 55 cattle without any apparent skin lesions or history of ringworm infection that served as negative controls were used. Sensitivities, specificities, and positive and negative predictive values were determined to evaluate the diagnostic value of our ELISA. Overall, the ELISAs based on recombinant TruDppV and TruLap2 discriminated well between infected animals and healthy controls. Highly significant differences (P < 0.0001, Mann-Whitney U test) were noted between optical density values obtained when sera from infected versus control cattle were tested. The ELISA developed for the detection of specific antibodies against DppV gave 89.6% sensitivity, 92.7% specificity, a 96.8% positive predictive value, and a 78.4% negative predictive value. The recombinant TruLap2-based ELISA displayed 88.1% sensitivity, 90.9% specificity, a 95.9% positive predictive value, and a 75.7% negative predictive value. To the best of our knowledge, this is the first ELISA based on recombinant antigens for assessing immune responses to ringworm infection in cattle; it is particularly suitable for epidemiological studies and also for the evaluation of vaccines and/or vaccination procedures.

  16. Performance of commercial enzyme-linked immunosorbent assays for detection of antibodies to Bordetella pertussis.

    PubMed

    Riffelmann, M; Thiel, K; Schmetz, J; Wirsing von Koenig, C H

    2010-12-01

    Measuring antibodies to Bordetella pertussis antigens is mostly done by enzyme-linked immunosorbent assays (ELISAs). We compared the performance of ELISA kits that were commercially available in Germany. Eleven measured IgG antibodies, and nine measured IgA antibodies. An in-house ELISA with purified antigens served as a reference method. Samples included two WHO reference preparations, the former Food and Drug Administration (FDA)/Center for Biologics Evaluation and Research (CBER) reference preparations, serum samples from patients with clinically suspected pertussis, and serum samples from patients having received a combined tetanus, diphtheria, and pertussis (Tdap) vaccination. Kits using pertussis toxin (PT) as an antigen showed linearity compared to the WHO Reference preparation (r2 between 0.82 and 0.99), and these kits could quantify antibodies according to the reference preparation. ELISA kits using mixed antigens showed no linear correlation to the reference preparations. Patient results were compared to results of in-house ELISAs using a dual cutoff of either ≥100 IU/ml anti-PT IgG or ≥40 IU/ml anti-PT IgG together with ≥12 IU/ml anti-PT IgA. The sensitivities of kits measuring IgG antibodies ranged between 0.84 and 1.00. The specificities of kits using PT as an antigen were between 0.81 and 0.93. The specificities of kits using mixed antigens were between 0.51 and 0.59 and were thus not acceptable. The sensitivities of kits measuring IgA antibodies ranged between 0.53 and 0.73, and the specificities were between 0.67 and 0.94, indicating that IgA antibodies may be of limited diagnostic value. Our data suggest that ELISAs should use purified PT as an antigen and be standardized to the 1st International Reference preparation.

  17. Enzyme-linked immunosorbent assay for detection of antibody to human herpesvirus 6.

    PubMed Central

    Chokephaibulkit, K; Brunell, P A; Vimal, V; Long, C; Schnabel, K; Hall, C B

    1997-01-01

    The results obtained with an enzyme-linked immunosorbent assay (ELISA) for detection of human herpesvirus 6 (HHV-6) immunoglobulin G using a single 1:100 dilution of serum correlated well with those found by an indirect fluorescence microscopic assay (IFA) (r = 0.71). Concordant results were found in all 7 paired serum samples obtained from patients with acute primary infections and in 37 of 41 (90.24%) single serum samples. Fourteen serum samples (25%) which yielded nonspecific results by IFA were evaluable by ELISA. In a serologic survey using the ELISA, a disproportionate number of 12-month-old infants had low difference-of-optical-density values, suggesting that maternal antibody might persist beyond a year of age. This finding and the rises in antibody to HHV-6 found in patients with primary cytomegalovirus infections might lead to overestimation of HHV-6 infection rates in young children in seroprevalence studies. Other herpesvirus infections produced lesser effects on anti-HHV-6. PMID:9384290

  18. Chromium functionalized diglyme plasma polymer coating enhances enzyme-linked immunosorbent assay performance.

    PubMed

    Welch, Nicholas G; Madiona, Robert M T; Easton, Christopher D; Scoble, Judith A; Jones, Robert T; Muir, Benjamin W; Pigram, Paul J

    2016-11-10

    Ensuring the optimum orientation, conformation, and density of substrate-bound antibodies is critical for the success of sandwich enzyme-linked immunosorbent assays (ELISAs). In this work, the authors utilize a diethylene glycol dimethyl ether plasma polymer (DGpp) coating, functionalized with chromium within a 96 well plate for the enhanced immobilization of a capture antibody. For an equivalent amount of bound antibody, a tenfold improvement in the ELISA signal intensity is obtained on the DGpp after incubation with chromium, indicative of improved orientation on this surface. Time-of-flight secondary-ion-mass-spectrometry (ToF-SIMS) and principal component analysis were used to probe the molecular species at the surface and showed ion fragments related to lysine, methionine, histidine, and arginine coupled to chromium indicating candidate antibody binding sites. A combined x-ray photoelectron spectroscopy and ToF-SIMS analysis provided a surface molecular characterization that demonstrates antibody binding via the chromium complex. The DGpp+Cr surface treatment holds great promise for improving the efficacy of ELISAs.

  19. Broad-spectrum enzyme-linked immunosorbent assay for detection of Legionella soluble antigens.

    PubMed Central

    Tang, P W; Toma, S

    1986-01-01

    An enzyme-linked immunosorbent assay (ELISA) was developed which detected soluble antigens from culture extracts of Legionella pneumophila serogroups 1 to 8, L. micdadei, L. bozemanii serogroups 1 and 2, L. dumoffii, L. gormanii, L. longbeachae serogroups 1 and 2, L. wadsworthii, L. oakridgensis, L. anisa, L. feeleii serogroup 1, and L. jordanis. The assay was approximately 10-fold more sensitive for the eight L. pneumophila serogroups than for the other Legionella species tested. The ELISA detected Legionella antigens in the urine specimens of 25 of 35 patients with L. pneumophila serogroup 1, 3, 4, 6, and 8; L. micdadei; and L. longbeachae serogroup 1 infections. None of the 334 urine specimens from patients with either non-Legionella pneumonia or urinary tract infections was positive. For 10 patients from whom sequential urine specimens were available, Legionella antigens were not detectable from 7 to 19 days after laboratory diagnosis. Test sensitivity was not affected by heavy bacterial contamination. This ELISA offers the detection of a broad spectrum of Legionella antigens by a single test. PMID:3771744

  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. 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.

  2. Standardization of Immunoglobulin M Capture Enzyme-Linked Immunosorbent Assays for Routine Diagnosis of Arboviral Infections

    PubMed Central

    Martin, Denise A.; Muth, David A.; Brown, Teresa; Johnson, Alison J.; Karabatsos, Nick; Roehrig, John T.

    2000-01-01

    Immunoglobulin M antibody-capture enzyme-linked immunosorbent assay (MAC-ELISA) is a rapid and versatile diagnostic method that readily permits the combination of multiple assays. Test consolidation is especially important for arthropod-borne viruses (arboviruses) which belong to at least three virus families: the Togaviridae, Flaviviridae, and Bunyaviridae. Using prototype viruses from each of these families and a panel of well-characterized human sera, we have evaluated and standardized a combined MAC-ELISA capable of identifying virus infections caused by members of each virus family. Furthermore, by grouping antigens geographically and utilizing known serological cross-reactivities, we have reduced the number of antigens necessary for testing, while maintaining adequate detection sensitivity. We have determined that a 1:400 serum dilution is most appropriate for screening antiviral antibody, using a positive-to-negative ratio of ≥2.0 as a positive cutoff value. With a blind-coded human serum panel, this combined MAC-ELISA was shown to have test sensitivity and specificity that correlated well with those of other serological techniques. PMID:10790107

  3. Serodiagnosis of bovine leptospirosis by IgG-enzyme-linked immunosorbent assay and latex agglutination test.

    PubMed

    Senthilkumar, T M A; Subathra, M; Ramadass, P; Ramaswamy, V

    2010-02-01

    The efficacy of a recombinant leptospiral outer membrane protein LipL41 as an antigen for conducting IgG-Enzyme linked immunosorbent assay (ELISA) and latex agglutination test (LAT) for serodiagnosis of bovine leptospirosis was evaluated. The recombinant LipL41 antigen developed and used for detecting the antibodies was specific in detection of the pathogenic serovars of Leptospira, as the expression of the LipL41 antigen is restricted only to pathogenic leptospires. A total of 430 bovine serum samples were subjected to IgG-ELISA and LAT, and the sensitivity and specificity were assessed in comparison with microscopic agglutination test (MAT). The sensitivity and specificity of IgG-ELISA and LAT were 86.84% and 93.16%, and 95.42% and 98.33% respectively. Both the tests are found to be sensitive, specific and concurred with the standard MAT. The study concluded that the rLipL41 protein could be used as a potential diagnostic antigen in different assay formats for bovine leptospirosis.

  4. 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.

  5. Detection of lymphoproliferative disease virus by an enzyme-linked immunosorbent assay.

    PubMed Central

    Patel, J. R.; Shilleto, R. W.

    1987-01-01

    Hitherto, detection of lymphoproliferative disease virus (LPDV), a C-type retrovirus of turkeys, has proved difficult since no tissue culture or serological assay has been available. Development of serological tests has been hampered by the problems of raising virus-specific antisera. An indirect enzyme-linked immunosorbent assay (ELISA) is reported, using a viral antiserum raised with bromelain-digested virus. The assay specifically detected purified virus at a concentration of 250 ng/ml or greater. In an experiment to detect virus in plasma from turkeys over a period of 4 weeks following LPDV infection, ELISA results correlated closely with the viral reverse transcriptase activity. Both assays were of similar sensitivity and detected small amounts of virus in high-speed pellets of turkey plasma. Evidence is presented indicating that LPDV-infected or hyperimmunized turkeys do not produce readily detectable circulating viral antibodies. In reciprocal ELISA tests, using antibodies to group-specific antigens of other avian retrovirus groups (avian sarcoma-leukosis (ASLV) and reticuloendotheliosis (REV] no antigenic cross-reaction was found between LPDV, ASLV and REV. PMID:2448156

  6. Enzyme-linked immunosorbent assay for Helicobacter pylori needs adjustment for the population investigated.

    PubMed

    Hoang, Thi Thu Ha; Wheeldon, Thuc-Uyen; Bengtsson, Carina; Phung, Dac Cam; Sörberg, Mikael; Granström, Marta

    2004-02-01

    Helicobacter pylori infection and peptic ulcer disease are common in developing countries, e.g., Vietnam. An enzyme-linked immunosorbent assay (ELISA) for screening of patients and for seroepidemiology is a useful tool but needs to be validated in the population studied. We used in-house ELISA with sonicated Swedish and Vietnamese strains as antigens to measure immunoglobulin G antibodies after absorption with sonicated Campylobacter jejuni in sera from 270 H. pylori culture-confirmed peptic ulcer patients, 128 Swedish urea-breath test and immunoblot-positive healthy controls, and 432 Vietnamese immunoblot-positive population controls. Sonicated whole-cell antigen based on the local strains showed a significantly better performance. Immunoblot-positive peptic ulcer patients had significantly higher antibody concentrations than immunoblot-positive population controls, necessitating a lower cutoff level if serology is used for screening or epidemiological purposes. The study shows that the parameters of ELISA for H. pylori need to be adjusted for the population being investigated.

  7. Enzyme-Linked Immunosorbent Assay for Helicobacter pylori Needs Adjustment for the Population Investigated

    PubMed Central

    Hoang, Thi Thu Ha; Wheeldon, Thuc-Uyen; Bengtsson, Carina; Phung, Dac Cam; Sörberg, Mikael; Granström, Marta

    2004-01-01

    Helicobacter pylori infection and peptic ulcer disease are common in developing countries, e.g., Vietnam. An enzyme-linked immunosorbent assay (ELISA) for screening of patients and for seroepidemiology is a useful tool but needs to be validated in the population studied. We used in-house ELISA with sonicated Swedish and Vietnamese strains as antigens to measure immunoglobulin G antibodies after absorption with sonicated Campylobacter jejuni in sera from 270 H. pylori culture-confirmed peptic ulcer patients, 128 Swedish urea-breath test and immunoblot-positive healthy controls, and 432 Vietnamese immunoblot-positive population controls. Sonicated whole-cell antigen based on the local strains showed a significantly better performance. Immunoblot-positive peptic ulcer patients had significantly higher antibody concentrations than immunoblot-positive population controls, necessitating a lower cutoff level if serology is used for screening or epidemiological purposes. The study shows that the parameters of ELISA for H. pylori need to be adjusted for the population being investigated. PMID:14766827

  8. Enzyme linked immunosorbent assay for detecting benzodiazepines in urine.

    PubMed

    Laurie, D; Mason, A J; Piggott, N H; Rowell, F J; Seviour, J; Strachan, D; Tyson, J D

    1996-07-01

    A relatively simple ELISA technique was developed for the detection of a range of benzodiazepines (BZs) in urine. The assay employs a mouse anti-oxazepam antibody that is highly specific for the BZs. The limit of detection using 10 microliters samples of urine was 0.3 microgram ml-1 oxazepam. N-Desmethyldiazepam showed equal cross-reactivity to oxazepam, 11 BZs cross-reacted weakly and flurazepam and chlordiazepoxide did not cross-react at levels reported to be found in urine. No cross-reactivity was observed with drugs of abuse and a range of therapeutic drugs commonly found in urine. The assay was used as a screen to detect the presence of BZs in urine from 88 addicts that had been screened by the EMIT technique and a radioreceptor assay (RRA) for BZs. The ELISA produced two false negatives that were EMIT and RRA positive whereas the EMIT produced four different false negatives that were positive by both ELISA and RRA. Thirty-three positives were common to all three assays. The ELISA was also used to monitor nitrazepam-like activity in the urine of a greyhound receiving 5 mg oral medication and the results were compared with those obtained by RRA. Both assays were able to detect nitrazepam-like activity for up to 10 h post-administration.

  9. Development of a specific monoclonal antibody for grouper (Epinephelus guaza) identification by an indirect enzyme-linked immunosorbent assay.

    PubMed

    Asensio, Luis; González, Isabel; Rodríguez, Miguel A; Mayoral, Belén; Lopez-Calleja, Inés; Hernández, Pablo E; García, Teresa; Martín, Rosario

    2003-05-01

    Identification of fish species adulteration is important for consumer protection and the enforcement of food-labeling laws. A monoclonal antibody (MAb) generated against soluble muscle proteins from grouper (Epinephelus guaza) has been used in two indirect enzyme-linked immunosorbent assay (ELISA) formats (microtiter plates and immunostick tubes) for the rapid authentication of grouper fillets. The 3D12 MAb was produced with the use of the hybridoma technique and tested against several commonly consumed fish species by ELISA. The 3D12 MAb specifically reacted with grouper samples and could be useful for the discrimination of grouper among other, less-valued, fish species sold in the marketplace.

  10. 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. Copyright © 2013 Elsevier Ltd. All rights reserved.

  11. 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.

  12. Quantitative analysis of phosphinothricin-N-acetyltransferase in genetically modified herbicide tolerant pepper by an enzyme-linked immunosorbent assay.

    PubMed

    Shim, Youn-Young; Shin, Weon-Sun; Moon, Gi-Seong; Kim, Kyung-Hwan

    2007-04-01

    An immunoassay method was developed to quantitatively detect phosphinothricin-N-acetyltransferase (PAT) encoded by the Bialaphos resistance (bar) gene in genetically modified (GM) pepper. The histidine-tagged PAT was overexpressed in Escherichia coli M15 (pQE31-bar) and efficiently purified by Ni2+ affinity chromatography. A developed sandwich enzyme-linked immunosorbent assay (S-ELISA) method (detection limit: 0.01 microg/ml) was 100-fold more sensitive than a competitive indirect ELISA (CI-ELISA) method or Western blot analysis in detecting the recombinant PAT. In real sample tests, PAT in genetically modified herbicide-tolerant (GMHT) peppers was successfully quantified [4.9 +/- 0.4 microg/g of sample (n = 6)] by the S-ELISA method. The S-ELISA method developed here could be applied to other GMHT crops and vegetables producing PAT.

  13. Comparison of measles virus-specific antibody titres as measured by enzyme-linked immunosorbent assay and virus neutralisation assay.

    PubMed

    van den Hof, Susan; van Gageldonk-Lafeber, Arianne B; van Binnendijk, Robert S; van Gageldonk, Pieter G M; Berbers, Guy A M

    2003-10-01

    We assessed whether measles virus-specific antibody levels in the Dutch population as estimated by an enzyme-linked immunosorbent assay (ELISA) were comparable with estimates by virus neutralisation assay (NT), prompted by a relatively low ELISA seroprevalence in the 10-21-year-old group. We tested 791 sera from individuals aged 2-49 years both in ELISA and NT. Seroprevalence in the 10-21-year-old group was 93.4% (95% confidence interval (CI) 89.5-97.2%) in ELISA versus 97.2% (CI 94.7-99.6%) in NT. There was good agreement between NT and ELISA seroprevalences in the vaccinated 2-9-year-olds and the unvaccinated 22-49-year-olds.

  14. Evaluation of an enzyme-linked immunosorbent assay using purified, deglycosylated histoplasmin for different clinical manifestations of histoplasmosis

    PubMed Central

    Guimarães, Allan Jefferson; Pizzini, Claudia Vera; De Abreu Almeida, Marcos; Peralta, José Mauro; Nosanchuk, Joshua Daniel; Zancopé-Oliveira, Rosely Maria

    2010-01-01

    Diagnosis of invasive fungal diseases remains problematic, especially in undeveloped countries. We have developed an enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies to Histoplasma capsulatum using metaperiodate treated purified histoplasmin (ptHMIN). Our ELISA was validated comparing sera from patients with histoplasmosis, related mycoses, and healthy individuals. The overall test specificity was 96%, with sensitivities of 100% (8/8) in acute disease, 90% (9/10) in chronic disease, 89% (8/9) in disseminated infection in individuals without HIV infection, 86% (12/14) in disseminated disease in the setting of HIV infection and 100% (3/3) in mediastinal histoplasmosis. These parameters are superior to the use of untreated histoplasmin in diagnostic ELISAs. The high specificities, sensitivities, and simplicity of our ELISA support further development of a deglycosylated HMIN ELISA for clinical use and for monitoring the humoral immune response during therapy in patients with chronic and disseminated histoplasmosis. PMID:21691458

  15. Normalized enzyme-linked immunosorbent assay for determining immunoglobulin G antibodies to cytomegalovirus.

    PubMed Central

    Kiefer, D J; Phelps, D A; Halbert, S P

    1983-01-01

    A normalized enzyme-linked immunosorbent assay for the determination of immunoglobulin G antibodies to cytomegalovirus is described. The rapid assay involves three 30-min incubations and permits the quantitation of antibody levels with a single-specimen dilution in conjunction with a reference antibody preparation. The results obtained with the normalized enzyme-linked immunosorbent assay correlated closely with the results of complement fixation titrations and another commercially available enzyme-linked immunosorbent assay. The specificity of the procedure was further demonstrated by viral absorption, using cytomegalovirus from two different sources and other viral antigen preparations, including rubella and influenza. The reproducibility of the normalized test results is good and allows for greater uniformity of reporting on a day-to-day basis, as well as between laboratories. PMID:6309900

  16. Serological identification of oral Bacteroides spp. by enzyme-linked immunosorbent assay.

    PubMed Central

    Ebersole, J L; Frey, D E; Taubman, M A; Smith, D J; Socransky, S S; Tanner, A C

    1984-01-01

    A rapid method for identifying black-pigmented oral Bacteroides spp. is described. Species-specific rabbit antisera to Bacteroides gingivalis, B. intermedius, and B. melaninogenicus were used in an enzyme-linked immunosorbent assay to identify clinical isolates of black-pigmented Bacteroides spp. from humans. The results showed excellent agreement with biochemical identification of B. gingivalis and B. intermedius. Only 36% of the B. melaninogenicus isolates were identified with the enzyme-linked immunosorbent assay, suggesting that this group of black-pigmented Bacteroides spp. is made up of more than one serotype. The serological enzyme-linked immunosorbent assay should enable rapid identification of black-pigmented Bacteroides spp. isolated from sites of oral diseases and may also be used to identify the presence of these organisms in complex bacterial mixtures from oral sites. PMID:6736225

  17. Comparative evaluation of the enzyme-linked immunosorbent assay in the laboratory diagnosis of brucellosis.

    PubMed Central

    De Klerk, E; Anderson, R

    1985-01-01

    An enzyme-linked immunosorbent assay was adapted to measure total and Brucella abortus-specific immunoglobulin M antibodies. The results were compared with those of conventional serological tests for B. abortus antibody on the sera of a number of normal controls, apparently healthy occupationally exposed workers, and patients with suspected acute brucellosis. Relative to other tests, the B. abortus enzyme-linked immunosorbent assay was found to be both highly sensitive and highly specific. The serological results obtained in occupationally exposed workers indicate a higher "normal range" for this group and therefore a possibility of false-positive results and overdiagnosis. It is therefore important to establish a separate "normal range" for occupationally exposed workers. Investigation of patients with acute brucellosis showed that the enzyme-linked immunosorbent assay for immunoglobulin M was the most sensitive serodiagnostic test and was likely to be of value in the serodiagnosis of acute brucellosis in occupationally exposed workers. PMID:3920241

  18. Enzyme-linked immunosorbent assay for detection of respiratory syncytial virus infection: development and description.

    PubMed Central

    Hendry, R M; McIntosh, K

    1982-01-01

    An indirect enzyme-linked immunosorbent assay for detection of respiratory syncytial virus (RSV) antigens was developed, using commercially available antisera. Horse anti-RSV and calf antiserum to bovine RSV were used as capture and detector antibodies, respectively. The assay could detect as few as 50 PFU of unpurified RSV per ml in infected cell culture supernatant fluids and as little as 10 ng of affinity-purified RSV antigen per ml. No cross-reactions were observed with heterologous virus types. Freeze-thaw treatment had no effect on RSV enzyme-linked immunosorbent assay titers, but viral transport medium inhibited RSV enzyme-linked immunosorbent assay titers from 10- to 100-fold. The assay can be easily performed in 24 h and is a sensitive and specific method for the detection of RSV antigens. PMID:6749894

  19. 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

  20. An enzyme-linked immunosorbent assay for detection of pyrene and related polycyclic aromatic hydrocarbons.

    PubMed

    Meng, X Y; Li, Y S; Zhou, Y; Zhang, Y Y; Yang, L; Qiao, B; Wang, N N; Hu, P; Lu, S Y; Ren, H L; Liu, Z S; Zhang, J H; Wang, X R

    2015-03-15

    Polycyclic aromatic hydrocarbons (PAHs) can form DNA-binding compounds that show genotoxicity and carcinogenicity. Pyrene, as a PAH, was covalently linked to carrier protein bovine serum albumin and ovalbumin. A monoclonal antibody (McAb) was produced that showed high cross-reactivity values with chrysene (169.73%), benzo[a]pyrene (693.34%), benzo[a]anthracene (16.36%), and indeno[1,2,3-cd]pyrene (40.96%) and showed no significant cross-reactivity values with other homologues (<0.1%). A competitive enzyme-linked immunosorbent assay (ELISA) was developed for detection of pyrene and some homologues in water samples. The detection limit of the assay was 65.08 pg ml(-1). The average recoveries of PAHs from tap water, lake water, and mineral water were 99.13, 99.74, and 99.19%, respectively, indicating that matrices of water samples do not interfere with the assay. The results demonstrated that the developed ELISA seems to be a potential method for monitoring of pyrene and some homologous PAHs in water samples.

  1. Development of a competitive enzyme-linked immunosorbent assay for diethylcarbamazine.

    PubMed

    Mitsui, Y; Takamura, N; Fujimaki, Y; Yamaguchi, T; Kitagawa, T; Aoki, Y

    1996-08-01

    A sensitive and reproducible competitive enzyme-linked immunosorbent assay (ELISA) for the determination of the concentration of diethylcarbamazine (DEC) in biological fluids was developed. Since DEC has no functional group to conjugate with bovine serum albumin (BSA), N-(2-aminoethyl)-N-ethyl-4-methyl-1-piperazinecarboxamide (DEC-NH2) was first synthesized. This compound was then converted to carboxyl DEC (DEC-COOH) and conjugated to BSA and to poly-L-lysine for use as immunogen and solid-phase marker, respectively. The competitive ELISA was conducted by simultaneously incubating DEC with mouse anti-DEC antiserum over DEC-poly-L-lysine solid phase. Subsequently, the binding of anti-DEC antibody was detected by using sheep anti-mouse IgG peroxidase conjugate as a tracer. The reliability, determined by the coefficient of variation for inter and intra-assay, was satisfactory. The cross-reactivities of anti-DEC antibodies with DEC metabolites, related compounds and ivermectin were negligible. Using this assay, DEC levels were easily determined in serum of Mongolian jirds (Meriones unguiculatus) up to 4 hours following a single dose of DEC citrate base (100 mg/kg of body weight) via intraperitoneal route.

  2. Detection of flunixin in greyhound urine by a kinetic enzyme-linked immunosorbent assay.

    PubMed

    Brady, T C; Yang, T J; Hyde, W G; Kind, A J; Hill, D W

    1997-01-01

    A two-step kinetic enzyme-linked immunosorbent assay was developed to detect the presence of flunixin in the urine of greyhound dogs. The assay system was developed using polyclonal antiflunixin antisera, a rabbit albumin-flunixin conjugate adsorbed onto polystyrene microtiter strips, and flunixin reference standards for calibration. The assay parameters were optimized and the performance characteristics were determined. The quantitative intra- and inter-run precisions (%CV) of the analysis of replicate (n = 10) flunixin-spiked urine samples were 9.9-12.5% and 10.2-13.6%, respectively. The linear dynamic range was 1-100 ng/mL, and the quantitative accuracy, as determined by calculation of percent error of measured flunixin in flunixin-spiked drug-free greyhound urine, was -16% to +14% over this range. The I50 of the ELISA was 17.3 ng/mL. The limit of detection was 25 ng/mL in greyhound urine. The reactivity in the assay system relative to flunixin (100%) was 147% for flunixin glucuronide, 25% for clonixin, and 5% for niflumic acid. The ELISA was capable of detecting total flunixin for up to 72 h in dogs administered flunixin at 0.55 mg/kg orally and up to 96 h in a dog that was administered flunixin at 1.0 mg/kg orally.

  3. 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. Copyright © 2016. Published by Elsevier Ltd.

  4. Measurement of inflammatory biomarkers in synovial tissue extracts by enzyme-linked immunosorbent assay.

    PubMed

    Rosengren, Sanna; Firestein, Gary S; Boyle, David L

    2003-11-01

    We developed methods for measuring inflammatory biomarkers (cytokines, chemokines, and metalloproteinases) in synovial biopsy specimens from patients with rheumatoid arthritis (RA) and osteoarthritis (OA). Soluble extracts of synovial fragments were prepared with mild detergent and analyzed by enzyme-linked immunosorbent assay (ELISA) for interleukin 1beta (IL-1beta), IL-6, IL-8, tumor necrosis factor alpha (TNF-alpha), and matrix metalloproteinase 3. The optimal detergent was 0.1% Igepal CA-630, which interfered minimally with ELISA detection but extracted 80% of IL-6 from synovial tissue. Upon spiking, 81 to 107% of added biomarkers could be recovered. To determine within-tissue variability, multiple biopsy specimens from each RA synovial extract were analyzed individually. A resulting coefficient of variation of 35 to 62% indicated that six biopsy specimens per synovial extract would result in a sampling error of < or = 25%. Preliminary power analysis suggested that 8 to 15 patients per group would suffice to observe a threefold difference before and after treatment in a serial biopsy clinical study. The previously described significant differences in IL-1beta, IL-6, IL-8, and TNF-alpha levels between RA and OA could be detected, thereby validating the use of synovial extracts for biomarker analysis in arthritis. These methods allow monitoring of biomarker protein levels in synovial tissue and could potentially be applied to early-phase clinical trials to provide a preliminary estimate of drug efficacy.

  5. [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.

  6. Direct Comparison of the Histidine-rich Protein-2 Enzyme-linked Immunosorbent Assay (HRP-2 ELISA) and Malaria SYBR Green I Fluorescence (MSF) Drug Sensitivity Tests in Plasmodium falciparum Reference Clones and Fresh ex vivo Field Isolates from Cambodia

    DTIC Science & Technology

    2013-07-12

    assay (HRP-2 ELISA) and malaria SYBR Green I fluorescence (MSF) drug sensitivity tests were directly compared using Plasmodium falciparum reference...Southeast Asia. Keywords: Immediate ex vivo Plasmodium falciparum drug susceptibility testing, HRP-2 ELISA, Malaria SYBR green fluorescence assay, Cambodia... malaria drug susceptibility assay using a reference clone to improve comparisons of Plasmodium falciparum field isolates. Malar J 2012, 11:325. 22. Le

  7. Enzyme-linked immunosorbent assay typing of California serogroup viruses isolated in Canada.

    PubMed

    Artsob, H; Spence, L P; Th'ng, C

    1984-08-01

    A procedure was developed to type California serogroup viruses by an antibody-capture, enzyme-linked immunosorbent assay. Seven California serogroup members from North America were distinguished, including snowshoe hare, La Crosse, California encephalitis, San Angelo, Jamestown Canyon, Keystone, and trivittatus. Extensive cross-reactions were observed between Jamestown Canyon and the closely related South River strain. The enzyme-linked immunosorbent assay method was successfully applied to the typing of 77 California serogroup viruses isolated in Canada, including 61 snowshoe hare, 13 Jamestown Canyon, and 3 trivittatus topotypes.

  8. Enzyme-linked immunosorbent assay for diagnosis of Amphimerus spp. liver fluke infection in Humans.

    PubMed

    Cevallos, William; Calvopiña, Manuel; Nipáz, Victoria; Vicente-Santiago, Belén; López-Albán, Julio; Fernández-Soto, Pedro; Guevara, Ángel; Muro, Antonio

    2017-05-01

    Amphimerus spp. is a liver fluke that infects humans and domestic animals. It is highly prevalent in some Ecuadorian communities. Currently, diagnosis is based on the microscopic observation of eggs in faeces, but this has variable sensitivity. More sensitive methods are needed for diagnostic testing. The main objective of this work was to develop an enzyme-linked immunosorbent assay (ELISA) using crude antigens from Amphimerus spp. adult worms to detect anti-Amphimerus IgG in human sera. Crude somatic antigens were obtained from adult Amphimerus spp. worms. Human sera from 119 patients were tested: 48 from individuals with a confirmed Amphimerus spp. infection, 78 from non-infected Ecuadorians living in the endemic region, 60 from persons living in non-endemic areas (20 Ecuadorians, 20 Europeans, and 20 Africans), and 33 who had other parasitic and non-parasitic infections. Results were analysed using the receiver-operator characteristic (ROC) curve analysis with an area under curve (AUC) value of 0.967. The accuracy of the ELISA was high. The sensitivity was 85.0% [95% confidence interval (CI): 80.3-89.7%] and the specificity was 71.0% (95% CI: 65.2-76.8%). Some cross reactivity was detected against Paragonimus mexicanus, Fasciola hepatica, Schistosomiasis, Taenia solium, Strongyloides stercoralis, Mansonella spp., and Vampirolepis nana. We have developed the first ELISA technique that detects anti-Amphimerus IgG in human sera with good sensitivity, repeatability and reproducibility. However, more specific antigens are needed to further enhance performance of this assay. Regardless, this ELISA test could be useful for early diagnosis and prompt treatment of human Amphimerus spp. infections.

  9. Enzyme-linked immunosorbent assay for diagnosis of Amphimerus spp. liver fluke infection in Humans

    PubMed Central

    Cevallos, William; Calvopiña, Manuel; Nipáz, Victoria; Vicente-Santiago, Belén; López-Albán, Julio; Fernández-Soto, Pedro; Guevara, Ángel; Muro, Antonio

    2017-01-01

    BACKGROUND Amphimerus spp. is a liver fluke that infects humans and domestic animals. It is highly prevalent in some Ecuadorian communities. Currently, diagnosis is based on the microscopic observation of eggs in faeces, but this has variable sensitivity. More sensitive methods are needed for diagnostic testing. OBJECTIVE The main objective of this work was to develop an enzyme-linked immunosorbent assay (ELISA) using crude antigens from Amphimerus spp. adult worms to detect anti-Amphimerus IgG in human sera. METHODS Crude somatic antigens were obtained from adult Amphimerus spp. worms. Human sera from 119 patients were tested: 48 from individuals with a confirmed Amphimerus spp. infection, 78 from non-infected Ecuadorians living in the endemic region, 60 from persons living in non-endemic areas (20 Ecuadorians, 20 Europeans, and 20 Africans), and 33 who had other parasitic and non-parasitic infections. PRINCIPAL FINDINGS Results were analysed using the receiver-operator characteristic (ROC) curve analysis with an area under curve (AUC) value of 0.967. The accuracy of the ELISA was high. The sensitivity was 85.0% [95% confidence interval (CI): 80.3-89.7%] and the specificity was 71.0% (95% CI: 65.2-76.8%). Some cross reactivity was detected against Paragonimus mexicanus, Fasciola hepatica, Schistosomiasis, Taenia solium, Strongyloides stercoralis, Mansonella spp., and Vampirolepis nana. MAIN CONCLUSIONS We have developed the first ELISA technique that detects anti-Amphimerus IgG in human sera with good sensitivity, repeatability and reproducibility. However, more specific antigens are needed to further enhance performance of this assay. Regardless, this ELISA test could be useful for early diagnosis and prompt treatment of human Amphimerus spp. infections. PMID:28443982

  10. Development and Application of a Saccharomyces cerevisiae-Expressed Nucleocapsid Protein-Based Enzyme-Linked Immunosorbent Assay for Detection of Antibodies against Infectious Bronchitis Virus

    PubMed Central

    Gibertoni, Aliandra M.; Montassier, Maria de Fátima S.; Sena, Janete A. D.; Givisiez, Patrícia E. N.; Furuyama, Cibele R. A. G.; Montassier, Hélio J.

    2005-01-01

    A Saccharomyces cerevisiae-expressed nucleocapsid (N) polypeptide of the M41 strain of infectious bronchitis virus (IBV) was used as antigen in a recombinant yeast-expressed N protein-based enzyme-linked immunosorbent assay (Y-N-ELISA). The Y-N-ELISA was rapid, sensitive, and specific for detecting chicken serum antibodies to IBV, and it compared favorably with a commercial ELISA. PMID:15815038

  11. Comparison of in-house biotin-avidin tetanus IgG enzyme-linked-immunosorbent assay (ELISA) with gold standard in vivo mouse neutralization test for the detection of low level antibodies.

    PubMed

    Sonmez, Cemile; Coplu, Nilay; Gozalan, Aysegul; Akin, Lutfu; Esen, Berrin

    2017-06-01

    Detection of anti-tetanus antibody levels is necessary for both determination of the immune status of individuals and also for planning preventive measures. ELISA is the preferred test among in vitro tests however it can be affected by the cross reacting antibodies. A previously developed in-house ELISA test was found not reliable for the antibody levels ≤1.0IU/ml. A new method was developed to detect low antibody levels correctly. The aim of the present study was to compare the results of the newly developed in-house biotin-avidin tetanus IgG ELISA test with the in vivo mouse neutralization test, for the antibody levels ≤1.0IU/ml. A total of 54 serum samples with the antibody levels of three different levels, =0.01IU/ml, 0.01-0.1IU/ml, 0.1-1IU/ml, which were detected by in vivo mouse neutralization test were studied by the newly developed in-house biotin-avidin tetanus IgG ELISA test. Test was validated by using five different concentrations (0.01IU/ml, 0.06IU/ml, 0.2IU/ml, 0.5IU/ml, 1.0IU/ml). A statistically significant correlation (r(2)=0.9967 p=0,001) between in vivo mouse neutralization test and in-house biotin-avidin tetanus IgG ELISA test, was observed. For the tested concentrations intra-assay, inter-assay, accuracy, sensitivity, specificity and coefficients of variations were determined as ≤15%. In-house biotin-avidin tetanus IgG ELISA test can be an alternative method to in vivo mouse neutralization method for the detection of levels ≤1.0IU/ml. By using in-house biotin-avidin tetanus IgG ELISA test, individuals with non protective levels, will be reliably detected. Copyright © 2017. Published by Elsevier B.V.

  12. Application of enzyme-linked immunosorbent assay for quantification of the insecticides imidacloprid and thiamethoxam in honey samples.

    PubMed

    Ma, Huixin; Xu, Yanjun; Li, Qing X; Xu, Ting; Wang, Xintong; Li, Ji

    2009-05-01

    An enzyme-linked immunosorbent assay (ELISA) was used for the determination of residues of imidacloprid and thiamethoxam insecticides in honey after simple dilution of the samples without either extraction or cleanup. The ELISA enabled accurate determination of imidacloprid and thiamethoxam down to limits of 20 and 5 ng g(-1) in honey, respectively. Average recoveries of imidacloprid and thiamethoxam from the fortified honey samples were 90-120 and 96-122%, and coefficients of variation ranged 5-12 and 3-15%, respectively. The results from the ELISA agreed well with those by liquid chromatography-mass spectrometry (LC-MS) for the insecticide-spiked samples, with a correlation coefficient (r(2)) of 0.96 and a regression coefficient (slope) of 1.03. The results indicate that ELISA is a suitable tool for the quantification of imidacloprid and thiamethoxam in honey.

  13. Detection of antibodies to legionnaires disease organism by microagglutination and micro-enzyme-linked immunosorbent assay tests.

    PubMed Central

    Farshy, C E; Klein, G C; Feeley, J C

    1978-01-01

    Microagglutination and micro-enzyme-linked immunosorbent assay (ELISA) tests with easily prepared, safe, heat-killed antigens for detecting antibodies to the legionnaires disease organism have been developed. A safranin-stained whole-cell antigen is used in the microagglutination test, and a simply prepared soluble antigen is used in the micro-ELISA tests. The microagglutination test detected elevated titers in 97.2% of the sera from patients with legionnaires disease. Three variations of the micro-ELISA test with anti-human immunoglobulin G, immunoglobulin M, and Fab peroxidase-labeled conjugates revealed elevated titers with 74.3, 82.9, and 88.6% of the sera, respectively. The microagglutination and the micro-ELISA tests used in combination detected 100% of the elevated titers. PMID:357440

  14. Detection of proline-rich proteins for the identification of saliva by enzyme-linked immunosorbent assay.

    PubMed

    Igoh, Akihisa; Tomotake, Sho; Doi, Yusuke

    2015-05-01

    Saliva is one of the most common body fluids found at a crime scene. Therefore, identifying saliva is important in forensic science. However, the current protein marker assays used to identify saliva are not sufficiently specific. Although proline-rich proteins (PRPs) are highly specific for saliva, their forensic potential has not yet been investigated. In this study, we developed enzyme-linked immunosorbent assays (ELISAs) to detect acidic salivary PRP HaeIII subfamily 1/2 (PRH1/2) and basic salivary PRP 2 (PRB2). The specificity, sensitivity, and efficiency of the ELISAs for PRH1/2 and PRB2 were compared with those of the ELISA for statherin (STATH), a known protein marker for saliva. The levels of PRH1/2 were significantly higher in saliva and saliva stains than in other body fluids (nasal secretions, urine, semen, vaginal fluid, blood, and sweat). PRB2 and STATH were detected in both nasal secretions and saliva. The PRH1/2 ELISA showed sensitivity similar to that of STATH ELISA. The detection rate of PRH1/2 ELISA was almost similar to that of STATH ELISA, followed by the ELISA for PRB2. The PRH1/2 ELISA had higher specificity for saliva than STATH ELISA. Therefore, the PRH1/2 ELISA has potential as a method to identify saliva for forensic investigation.

  15. 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

  16. Application of dispersive liquid-liquid microextraction for estrogens' quantification by enzyme-linked immunosorbent assay.

    PubMed

    Lima, Diana L D; Silva, Carla Patrícia; Schneider, Rudolf J; Otero, Marta; Esteves, Valdemar I

    2014-07-01

    Estrogens, such as 17β-estradiol (E2) and 17α-ethinylestradiol (EE2), are the major responsible for endocrine-disrupting effects observed in aquatic environments due to their high estrogenic potency, even at concentrations ranging from pgL(-1) to ng L(-1). Thus, it is essential to develop analytical methodologies suitable for monitoring their presence in water samples. Dispersive liquid-liquid microextraction (DLLME) was used as a pre-concentration step prior to the quantification of E2 and EE2 by enzyme-linked immunosorbent assay (ELISA). First, an evaluation of the effect of DDLME on the E2 and EE2 ELISA calibration curves was performed. Since the extraction procedure itself had an influence on the ELISA optical density (OD), it became necessary to subject, not only the samples, but also all the standards to the DLLME process. Working ranges were determined, being between 1.2 and 8000 ng L(-1), for E2, and between 0.22 and 1500 ng L(-1), for EE2. The influence of organic matter, both in the extraction and quantification, was evaluated and it was observed that its presence in the solution did not affect considerably the calibration curve. Recovery rates were also determined, ranging from 77% to 106% for ultrapure water and from 104% to 115% for waste water samples, the most complex ones in what concerns matrix effects. Results obtained when applying the proposed method to real water samples can be considered quite satisfying. Moreover, the obtained working ranges encompass values generally reported in literature, confirming the practical use of the method for environmental samples.

  17. Accuracy of a rapid enzyme-linked immunosorbent assay to measure progesterone in mares

    PubMed Central

    Relave, Fabien; Lefebvre, Réjean C.; Beaudoin, Sandra; Price, Christopher

    2007-01-01

    The aim of this study was to validate an enzyme-linked immunosorbent assay (ELISA) for the measurement of progesterone (P4) in mares. Specifically, the objectives were as follows: 1) to determine the specificity and sensitivity of the ELISA test for determination of P4, 2) to measure the potential agreement between the 2 people performing the test, and 3) to evaluate the effect of time on the outcome. Ten mares were sampled on the day before ovulation (D-1), and on days 1 (D1), 3 (D3), and 5 (D5) following ovulation, during the reproductive season. While mares were cycling regularly, estrus was induced by the injection of 5 mg of prostaglandin (PGF2) and monitored starting on the 4th day by daily transrectal palpation and ultrasonography to determine the time of ovulation. Blood was collected and all samples (n = 96) were assayed for P4 by a semiquantitative ELISA, by chemiluminescent immunoassay, and by radioimmunoassay (RIA). Based on the RIA, values of P4 on D-1, D1, D3, and D5 were significantly different (P < 0.0001) with mean and standard deviation(s) of 0.004, s = 0.52; 2.05, s = 2.58; 8.37, s = 4.17; and 12.76, s = 4.00 ng/mL respectively. The sensitivity and specificity of the semiquantitative assay were 94% and 95%, respectively for the lowest values of P4 (< 1.0 ng/mL). The value of kappa was 0.90 between 2 individuals performing the test. In conclusion, these results suggest that the semi-quantitative test may be used reliably and economically to evaluate P4 levels in equine plasma in the clinical situation. PMID:17824324

  18. Interlaboratory agreement among results of human papillomavirus type 16 enzyme-linked immunosorbent assays.

    PubMed Central

    Strickler, H D; Hildesheim, A; Viscidi, R P; Shah, K V; Goebel, B; Drummond, J; Waters, D; Sun, Y; Hubbert, N L; Wacholder, S; Brinton, L A; Han, C L; Nasca, P C; McClimens, R; Turk, K; Devairakkam, V; Leitman, S; Martin, C; Schiller, J T

    1997-01-01

    Serological assays for measuring antibodies to human papillomavirus type 16 (HPV-16) virus-like particles (VLPs) have become important epidemiologic tools in recent years. However, the interlaboratory replicability of these assays has not been assessed. In this investigation, three laboratories tested a panel of specimens obtained from two different groups: 265 subjects in a vulvar cancer case-control study and 107 healthy volunteer blood donors. Each laboratory used an enzyme-linked immunosorbent assay (ELISA), but no attempt was made to standardize assay procedures among the three laboratories. The data showed good day-to-day intralaboratory replicability in laboratory 1 (correlation coefficient, > or = 0.88) and good intra-assay variability in laboratory 3 (correlation coefficient, > or = 0.93). Interlaboratory correlations, likewise, ranged between 0.61 and 0.80 in both case-control study subjects and healthy blood donors, indicating that ELISA optical density (OD) values between laboratories were linearly related regardless of the population. Kappa coefficients (kappa), based on each laboratory's categorical interpretation of its results (as positive or negative), showed good agreement (kappa, > 0.6) in case-control study subjects and moderate agreement (kappa, > or = 0.4) in blood donors, a population that had few strongly positive sera. When OD values near seropositive cutoffs were treated as indeterminates, there was little discordance between laboratories in either population. The data suggest that each laboratory measured the same humoral immune response and that their HPV-16 VLP ELISAs performed similarly (Pearson correlations). Interlaboratory differences, however, probably due to reagents and procedures, were considerably greater than intralaboratory day-to-day variability. Interlaboratory agreement in determining seropositivity (kappa) could be improved by sharing positive and negative serum controls and by treating marginal results as indeterminate

  19. Enzyme-linked immunosorbent assay for Salmonella serology using lipopolysaccharide antigen.

    PubMed

    Smith, B P; Dilling, G W; House, J K; Konrad, H; Moore, N

    1995-10-01

    Enzyme-linked immunosorbent assay (ELISA) using Salmonella lipopolysaccharide (LPS) to measure specific IgG titers in cattle has proven useful. Serology can be used to assess vaccine responses and infection rates, to detect carriers, and to aid in epidemiologic studies. The objective of this study was to assess cross-reactions using sera from cattle vaccinated with different Salmonella serogroups. ELISA plates using lipopolysaccharide from serogroup B, C1, C3, D1 or E1 as the plate antigens were tested. LPS was extracted from Salmonella typhimurium (Serogroup B; somatic antigens 01, 4, 12), S. montevideo (C1; 06, 7), S. kentucky (C3; 08, 20), S. dublin (D1; 01, 9, 12) and S. anatum (E1; 03, 10) using the Westphal method. Fifteen cows were found to be seronegative for all 5 of these serogroup antigens. Each cow was then vaccinated 3 times at 2-week intervals with a killed Salmonella bacterin. The 15 different serotypes used for vaccination were chosen to represent a wide array of Salmonella serogroups with a wide array of somatic "O" antigens expressed, including somatic antigens 1, 2, 3, 4, 6, 7, 8, 9, 10, 11, 12, 13, 14, 18, 19, 20, 22, 23, 25, and 27. With each antigen tested, the highest ELISA titers were seen with sera from cattle vaccinated with homologous O antigens, indicating that reactions were highly O antigen-specific. Some cross reactions between subgroups sharing one O factor antigen were found; these titers were lower than those found with homologous serogroups sharing 2 or more antigens. Only serum from the cow vaccinated from S. anatum (group E; antigens 03, 10) cross-reacted at a low titer with group C1 (O somatic antigens 6, 7) and D1 (O somatic antigens 1, 9, 12) plate antigens, with which no somatic antigens were shared. We conclude from these results that Salmonella serology using LPS antigens is highly O antigen-specific and predictable.

  20. 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

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

    USDA-ARS?s Scientific Manuscript database

    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 ...

  2. Enzyme-linked immunosorbent assay antibody responses to a temperature-sensitive mutant of Pseudomonas aeruginosa.

    PubMed Central

    Sordelli, D O; Rojas, R A; Cerquetti, M C; Hooke, A M; Degnan, P J; Bellanti, J A

    1985-01-01

    The serum immunoglobulin G and M responses induced by immunization of mice with temperature-sensitive mutant A/10/25 of Pseudomonas aeruginosa were evaluated by enzyme-linked immunosorbent assay. These antibody responses were immunotype specific, and the immunoglobulin G antibody level, although low, was still significant by day 52 after vaccination. PMID:3930404

  3. 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...

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

    USDA-ARS?s Scientific Manuscript database

    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...

  5. 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...

  6. Inhibition enzyme-linked immunosorbent assay for detection of Pseudomonas fluorescens on meat surfaces.

    PubMed Central

    Eriksson, P V; di Paola, G N; Pasetti, M F; Manghi, M A

    1995-01-01

    An inhibition enzyme-linked immunosorbent assay was developed for Pseudomonas fluorescens enumeration of meat surfaces. The assay detected contamination levels as low as 3 x 10(5) bacteria per ml and could be completed within 4 h. It could be used as a framework for a test system for quantifying P. fluorescens spoilage in meat products. PMID:7887624

  7. Comparison of five commercial anti-tetanus toxoid immunoglobulin G enzyme-linked immunosorbent assays.

    PubMed

    Perry, A L; Hayes, A J; Cox, H A; Alcock, F; Parker, A R

    2009-12-01

    Five commercially available enzyme-linked immunosorbent assays for the measurement of anti-tetanus toxoid immunoglobulin G (IgG) antibodies were evaluated for performance. The data suggest that there are manufacturer-dependent differences in sensitivity and accuracy for the determination of tetanus toxoid IgG antibodies that could result in different diagnostic interpretations.

  8. A survey of 17α-ethinylestradiol and mestranol residues in Hawkesbury River, Australia, using a highly specific enzyme-linked immunosorbent assay (ELISA) demonstrates the levels of potential biological significance.

    PubMed

    Uraipong, Chatchaporn; Allan, Robin D; Li, Chunhua; Kennedy, Ivan R; Wong, Victor; Lee, Nanju Alice

    2017-10-01

    This study reports on the potential status of 17α-ethinylestradiol (EE2) and mestranol (MeEE2) residues in aquatic environments in New South Wales (NSW), Australia, based on the analysis by a specific ELISA we developed. Polyclonal antibodies were raised against the EE2 hapten with a linker attached at the C3-position to direct the antibody binding towards the ring D of EE2/MeEE2. Using this approach, an ELISA highly specific to EE2 and MeEE2 was successfully developed, showing less than 3.1% cross-reactivity (% CR) with other major steroidal sex hormones and their derivatives. The assay performed with the limit of detection (LOD) of 0.04 ± 0.01µg/L for both EE2 and MeEE2, and the limit of quantitation (LOQ) of 0.05 ± 0.01ng/L when it was coupled with the SM2-Biobeads solid phase extraction. Prior to conducting the survey study, it was validated against the gas chromatography-mass spectrophotometry (GC-MS) method, which showed high correlation with R(2) of 0.934. Fresh surface water samples collected at different sites along Hawkesbury River in New South Wales (NSW) were analyzed for the EE2/ MeEE2 residues using the developed ELISA. The EE2/MeEE2 levels were found to range between 4.1 and 8.3ng/L in Emigrant Creek, NSW, where the primary activity was macadamia plantation, and higher levels between 15 and 29ng/L in South Creek, NSW, Greater Western Sydney at sites upstream and downstream of the municipal sewage treatment plants. Copyright © 2017 Elsevier Inc. All rights reserved.

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

    USDA-ARS?s Scientific Manuscript database

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

    USDA-ARS?s Scientific Manuscript database

    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...

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

    USDA-ARS?s Scientific Manuscript database

    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...

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

    USDA-ARS?s Scientific Manuscript database

    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...

  13. Microfluidic tectonics platform: A colorimetric, disposable botulinum toxin enzyme-linked immunosorbent assay system.

    PubMed

    Moorthy, Jaisree; Mensing, Glennys A; Kim, Dongshin; Mohanty, Swomitra; Eddington, David T; Tepp, William H; Johnson, Eric A; Beebe, David J

    2004-06-01

    A fabrication platform for realizing integrated microfluidic devices is discussed. The platform allows for creating specific microsystems for multistep assays in an ad hoc manner as the components that perform the assay steps can be created at any location inside the device via in situ fabrication. The platform was utilized to create a prototype microsystem for detecting botulinum neurotoxin directly from whole blood. Process steps such as sample preparation by filtration, mixing and incubation with reagents was carried out on the device. Various microfluidic components such as channel network, valves and porous filter were fabricated from prepolymer mixture consisting of monomer, cross-linker and a photoinitiator. For detection of the toxoid, biotinylated antibodies were immobilized on streptavidin-functionalized agarose gel beads. The gel beads were introduced into the device and were used as readouts. Enzymatic reaction between alkaline phosphatase (on secondary antibody) and substrate produced an insoluble, colored precipitate that coated the beads thus making the readout visible to the naked eye. Clinically relevant amounts of the toxin can be detected from whole blood using the portable enzyme-linked immunosorbent assay (ELISA) system. Multiple layers can be realized for effective space utilization and creating a three-dimensional (3-D) chaotic mixer. In addition, external materials such as membranes can be incorporated into the device as components. Individual components that were necessary to perform these steps were characterized, and their mutual compatibility is also discussed.

  14. 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.

  15. 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

  16. Evaluation of an enzyme-linked immunosorbent assay for quantitation of antibodies to Junin virus in human sera.

    PubMed

    García Franco, S; Ambrosio, A M; Feuillade, M R; Maiztegui, J I

    1988-01-01

    An enzyme-linked immunosorbent assay (ELISA) was evaluated for the quantitation of anti-Junin virus (JV) antibodies, in 83 selected cases of Argentine haemorrhagic fever (AHF). Serum samples were studied in two groups to facilitate comparative analysis; the first group was ELISA with indirect immunofluorescence (IF) test, in the second ELISA with plaque reduction neutralization test (PRINT). From the results obtained by using ELISA and IF on the same serum samples, a clear tendency of ELISA to demonstrate seroconversion for JV earlier and at higher frequency than IF test was noted. Simultaneous titration of specific antibodies by ELISA and PRNT tests rendered significantly correlated titers (r = 0.81), both methods being equivalently specific (100%). The demonstration of specific antibodies by ELISA in two cases that were undetected by the PRNT test resulted in a higher sensitivity index for ELISA than for PRNT (100% vs 97%). It is concluded that ELISA could efficiently replace IF and PRNT tests for the diagnosis of AHF.

  17. 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.

  18. Sandwich enzyme-linked immunosorbent assay for the quantification of human serum albumin fragment 408-423 in bodily fluids.

    PubMed

    Mohr, Katharina B; Zirafi, Onofrio; Hennies, Mark; Wiese, Sebastian; Kirchhoff, Frank; Münch, Jan

    2015-05-01

    Urinary levels of human serum albumin (hSA) fragment 408-423 have been proposed to represent an early marker for graft-versus-host disease (GvHD) and chronic kidney diseases. Here, we developed an enzyme-linked immunosorbent assay (ELISA) for the quantification of hSA(408-423). The sandwich ELISA has a detection limit of 0.5ng/ml and is highly specific for hSA(408-423) because it does not cross-react with other albumin fragments or the full-length precursor. This ELISA allows rapid and convenient quantification of hSA(408-423) in bodily fluids, further clarifying the prognostic and diagnostic value of this peptide in GvHD, kidney disease, and other disorders. Copyright © 2015 Elsevier Inc. All rights reserved.

  19. Seroprevalence of human immunodeficiency virus in north Indian blood donors using third and fourth generation Enzyme linked immunosorbent assay.

    PubMed

    Malhotra, Sheetal; Marwaha, Neelam; Saluja, Karan

    2013-07-01

    The percentage of HIV cases attributable to blood transfusion has decreased significantly in the last decade. The newer 4(th) generation Enzyme linked immunosorbent assay (ELISA) has been shown to have increased sensitivity compared to 3(rd) generation ELISA. To estimate the seroprevalence of HIV among blood donors using 4(th) generation ELISA assay and to compare it with the 3(rd) generation ELISA. This prospective study involved 10,200 blood donors- 6,800 were voluntary donors (3400-students and 3400-non students) and 3400 were replacement donors. All blood units were tested with 3(rd) as well as 4(th) generation ELISA. All samples found reactive or in grey zone with either 3(rd) or 4(th) generation ELISA were retested by Western blot (WB). The seroprevalence of HIV was estimated to be 1.37/1000 donations (0.14%) with 3(rd) generation ELISA compared to 3.62/1000 donations (0.36%) with 4(th) generation ELISA (p>0.05). The seroprevalence of HIV among voluntary donors was estimated to be 1.32/1000 donations (0.13%) with 3(rd) generation ELISA and 3.67/1000 donations (0.36%) with 4(th) generation ELISA. The prevalence of HIV among replacement donors was 1.47/1000 donations (0.15%) with 3(rd) generation ELISA and 3.52/1000 donations (0.35%) with 4(th) generation ELISA. 4(th) generation HIV ELISA detects a higher number of seroreactive donors compared to 3(rd) generation ELISA. However, larger studies are required with confirmatory tests for both 3(rd) and 4(th) generation ELISA for making any policy changes.

  20. 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

  1. Detection, quantification, and glycotyping of prion protein in specifically activated enzyme-linked immunosorbent assay plates.

    PubMed

    Triantaphyllidou, I E; Sklaviadis, T; Vynios, D H

    2006-12-15

    The conversion of a normal glycoprotein, prion protein (PrP(C)), to its abnormal protease-resistant isoform (PrP(Sc)) seems to be one of the main factors underlying the pathogenesis of spongiform encephalopathies. There are many studies indicating that PrP interacts with glycosaminoglycans, and we exploited this interaction to develop a sensitive solid phase assay for detection of both PrP forms. Glycosaminoglycans, such as chondroitin sulfate and heparin, were immobilized by their negative charge to enzyme-linked immunosorbent assay (ELISA) plate wells activated by glutaraldehyde and spermine. PrP in the samples examined (recombinant PrP or tissue homogenate) was allowed to interact with glycans. The interaction of recombinant PrP was more efficient against immobilized chondroitin sulfate of type A, and a linear correlation with concentration was demonstrated. From this curve, the concentration of each one of the PrP isoforms in biological samples can be determined. In addition, and taking into account that glycosylation of prion protein is species specific, we used similarly activated ELISA plate wells to determine different PrP glycoforms. A monoclonal antibody against PrP was immobilized, and PrP present in the samples (brain homogenates) was bound and visualized by various lectins. The most interesting outcome of the study is the differential binding of ricinus communis agglutinin I to the normal and scrapie brain homogenates. Dattura stramonium lectin and wheat germ agglutinin seem to bind almost equally to both samples, and all three have an increased sensitivity to PrP(Sc) after proteinase K digestion.

  2. Development of an enzyme-linked immunosorbent assay for the veratrum plant teratogens: cyclopamine and jervine.

    PubMed

    Lee, Stephen T; Panter, Kip E; Gaffield, William; Stegelmeier, Bryan L

    2003-01-29

    Veratrum californicum was responsible for large losses of sheep grazing high mountain ranges in central Idaho in the 1950s. Veratrum induces various birth defects including the cyclopic-type craniofacial defect (monkey-faced lambs) that is specifically induced in lambs after pregnant ewes grazed the plant on the 14th day of gestation. The steroidal alkaloids cyclopamine (1) and jervine (2) were isolated from Veratrum and shown to be primarily responsible for the malformations. Cyclopamine (1) and jervine (2) are potent teratogens that inhibit Sonic hedgehog (Shh) signaling during gastrulation-stage embryonic development, producing cyclopia and holoprosencephaly. Although losses to the sheep industry from Veratrum are now relatively infrequent, occasional incidents of toxicoses and craniofacial malformations are still reported in sheep and other species. However, the benefits to biomedical research using cyclopamine (1) as a tool to study human diseases have greatly expanded. A competitive inhibition enzyme-linked immunosorbent assay (ELISA) to detect and measure cyclopamine (1) and jervine (2) was developed using polyclonal antibodies produced in ewes. The limits of detection of the assay were 90.0 and 22.7 pg for cyclopamine (1) and jervine (2), respectively. This assay was used for the detection and measurement of cyclopamine (1) spiked into sheep blood. The simple extraction-ELISA methods developed in this study demonstrate the potential of using these techniques for the rapid screening of biological samples to detect the presence and concentration of cyclopamine (1) and jervine (2) and will be beneficial to pharmacological studies and livestock diagnostics.

  3. Antigenic Variation in Bacteroides forsythus Detected by a Checkerboard Enzyme-Linked Immunosorbent Assay

    PubMed Central

    Sims, Tom J.; Mancl, Lloyd A.; Braham, Pamela H.; Page, Roy C.

    1998-01-01

    Evidence indicating that multiple serotypes of Bacteroides forsythus participate in rapidly progressing periodontal infections has not been reported previously. Our aim was to develop an assay for detecting subsets of B. forsythus clinical isolates which differ in serogroup membership and subsets of patients with immunoglobulin G (IgG) responses which differ in serogroup recognition. A checkerboard enzyme-linked immunosorbent assay (ELISA) was used to assess variation in the IgG binding profiles of 22 clinical isolates in sera from 28 patients with early-onset rapidly progressive periodontitis. To accommodate the maximum number of isolates and sera in a given assay run, a multiplate assay grid with standard 96-well microtest plates was established. Single dilutions of individual sera were placed in rows crossing columns of isolate-coated wells, and antigen-specific IgG immobilized in the wells was measured as ELISA absorbance. Pooled sera and isolates were assayed in parallel to serve as negative controls for variation in IgG binding profiles. Correlation and hierarchical cluster analysis of the absorbance data matrix showed that the isolates could be sorted into at least four clusters based on variations in their IgG binding profiles across different sera. Furthermore, at least two patient clusters were defined by variations in their serum IgG antigen recognition profiles across different isolates. We conclude that multiple serogroups of B. forsythus exist and that different serogroups are dominant in the antibody response of different patients. The method applied here could be used to serologically classify clinical isolates of other species which evoke a serum antibody response in patients. PMID:9729543

  4. An Analytical Model Applied to a Multicenter Pneumococcal Enzyme-Linked Immunosorbent Assay Study

    PubMed Central

    Plikaytis, Brian D.; Goldblatt, David; Frasch, Carl E.; Blondeau, Christine; Bybel, Michael J.; Giebink, G. Scott; Jonsdottir, Ingileif; Käyhty, Helena; Konradsen, Helle Bossen; Madore, Dace V.; Nahm, Moon H.; Schulman, Cheryl A.; Holder, Patricia F.; Lezhava, Tamar; Elie, Cheryl M.; Carlone, George M.

    2000-01-01

    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

  5. Serologic testing for avian influenza viruses in wild birds: comparison of two commercial competition enzyme-linked immunosorbent assays.

    PubMed

    Pérez-Ramírez, Elisa; Rodríguez, Vanessa; Sommer, Dagmar; Blanco, Juan Manuel; Acevedo, Pelayo; Heffels-Redmann, Ursula; Höfle, Ursula

    2010-03-01

    Serologic testing of wild birds for avian influenza virus (AIV) surveillance poses problems due to species differences and nonspecific inhibitors that may be present in sera of wild birds. Recently available competitive enzyme-linked immunosorbent assay (cELISA) kits offer a new species-independent approach. In this study we compare two commercial competitive cELISAs, using a total of 184 serum and plasma samples from 23 species of wild birds belonging to 10 orders. Thirteen samples were from experimentally high pathogenicity AI and low pathogenicity AI infected red-legged partridges (Alectoris rufa), 77 samples were from a flock of sentinel hybrid ducks confirmed infected by AI by real-time PCR, and 94 samples were from wild birds admitted to a rehabilitation center. Both ELISAs detected AI antibodies in the experimentally infected partridges, whereas hemagglutination inhibition (HI) was negative. Concordance in results between the two ELISAs was 51.5%. When specific subtype-H5/H7 HI-positive samples were considered for comparison, ELISA 1 appeared to perform better on ducks, whereas ELISA 2 appeared to perform better in other wild bird species. Overall, 68.2% of H5/H7 positive samples tested positive by ELISA 1 and 36% by ELISA 2. Both ELISAs detected AIV-antibody-positive samples negative by specific HI against 9 of the 16 existing hemagglutinin (HA) subtypes. Presumably this reflects either higher sensitivity of cELISA when compared to HI, presence of antibodies against HA subtypes not tested, or unspecific reactions. Performance of ELISA 1 on ducks appears to be comparable to in-house cELISA previously used by other authors in wild birds, but requires a relatively large sample volume. Alternatively, although ELISA 2 required a smaller sample volume, it was less effective at identifying HI-positive samples. The results reflect the necessity of validation of cELISA tests for individual species or at least families, as required by the OIE.

  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. Comparison of an enzyme-linked immunosorbent assay with serum neutralization test for serodiagnosis of porcine epidemic diarrhea virus infection.

    PubMed

    Oh, Jin Sik; Song, Dae Sub; Yang, Jeong Sun; Song, Ju Young; Moon, Hyoung Joon; Kim, Tae Yung; Park, Bong Kyun

    2005-12-01

    An indirect porcine epidemic diarrhea (PED) virus (PEDV) enzyme-linked immunosorbent assay (ELISA) was compared with the serum neutralization (SN) test by testing 46 samples from experimentally infected sows, 73 samples from naive sows, and 1,024 field sow samples from 48 commercial swine farms of undefined PED status. The SN test and the ELISA were performed using PEDV, KPEDV-9 strain. Viral proteins as a coating antigen of PEDV ELISA were extracted from the cytoplasm of PEDV-infected Vero cells using a non-ionic detergent, Triton X-100, and a simple protocol of PEDV ELISA was followed. The presence of antibodies in these experimental samples was confirmed by SN and ELISA in which the sensitivity of the ELISA was 89.1%, and the corresponding specificity was 94.5%. On testing 1,024 field samples, an overall agreement of 84.2% was generated between the SN and ELISA. This study demonstrates that the PEDV ELISA is a useful serodiagnostic screening test at herd level for detecting swine antibodies against PEDV.

  8. Establishment of a miniaturized enzyme-linked immunosorbent assay for human transferrin quantification using an intelligent multifunctional analytical plate.

    PubMed

    Spies, Peter; Chen, Guo Jun; Gygax, Daniel

    2008-11-01

    A miniaturized enzyme-linked immunosorbent assay (ELISA) with a reaction volume of 5 microl for human transferrin quantification has successfully been developed using an intelligent multifunctional analytical plate (IMAPlate 5RC96), the first miniature analytical platform capable of manually performing parallel liquid transfer, reaction, and analysis. This is the first article to validate the platform for the ELISA application. The data obtained from the standards in this miniaturized ELISA can well be fitted by a one-site binding reaction mode, the coefficient of variation (CV) of the whole plate for an artificial sample (spiking a known concentration of human transferrin into the assay diluent) is 7.0%, and the mean recovery is between 94 and 114% (n=96), comparable to the values from conventional ELISA in a 96-well format plate. The IMAPlate 5RC96-based miniaturized ELISA not only can reduce sample and reagent consumption to 5% of the conventional ELISA but also can shorten the reaction time. Combined with the advantages brought by miniaturization, the easy-to-handle, parallel, and simultaneous liquid transfer features of the IMAPlate 5RC96 provide a completely new lab tool for manually performing high-throughput ELISA. Our results demonstrate that the IMAPlate 5RC96 is a convenient, robust, high-throughput lab device feasible for miniaturized ELISA in an ordinary laboratory.

  9. Development of an enzyme-linked immunosorbent assay for Bartonella henselae infection detection.

    PubMed

    Ferrara, F; Di Niro, R; D'Angelo, S; Busetti, M; Marzari, R; Not, T; Sblattero, D

    2014-09-01

    Several serological diagnostics rely on enzyme-linked immunosorbent assay (ELISA) to detect bacterial infections. However, for some pathogens, including Bartonella henselae, diagnosis still depends on manually intensive, time-consuming assays including micro-immunofluorescence, Western blotting or indirect immunofluorescence. For such pathogens, there is obviously still a need to identify antigens to establish a reliable, fast and high-throughput assay (Dupon et al. ). We evaluated two B. henselae proteins to develop a novel serological ELISA: a well-known antigen, the 17-kDa protein, and GroEL, identified during this study by a proteomic approach. When serum IgG were tested, the specificity and sensitivity were 76 and 65·7% for 17-kDa, respectively, and 82 and 42·9% for GroEL, respectively. IgM were found to be more sensitive and specific for both proteins: 17-kDa protein, specificity 86·2% and sensitivity 75%; GroEL, specificity 97·7% and sensitivity 45·3%. IgM antibodies were also measured in lymphoma patients and patients with Mycobacterium tuberculosis infection to assess the usefulness of our ELISA to distinguish them from B. henselae infected patients. The resulting specificities were 89·1 and 93·5% for 17-kDa protein and GroEL, respectively. Combining the results from the two tests, we obtained a sensitivity of 82·8% and a specificity of 83·9%. Our work described and validated a proteomic approach suitable to identify immunogenic proteins useful for developing a serological test of B. henselae infection. A reliable serological assay for the diagnosis of Cat Scratch Disease (CSD) - a pathological condition caused by Bartonella henselae infection - has not yet been developed. Such an assay would be extremely useful to discriminate between CSD and other pathologies with similar symptoms but different aetiologies, for example lymphoma or tuberculosis. We investigate the use of two B. henselae proteins - GroEL and 17-kDa - to develop a serological

  10. Advantages of soybean peroxidase over horseradish peroxidase as the enzyme label in chemiluminescent enzyme-linked immunosorbent assay of sulfamethoxypyridazine.

    PubMed

    Sakharov, Ivan Yu; Berlina, Anna N; Zherdev, Anatoly V; Dzantiev, Boris B

    2010-03-24

    An indirect competitive chemiluminescent enzyme-linked immunosorbent assay (CL-ELISA) of sulfamethoxypyridazine (SMP) was developed. The conjugates of streptavidin with cationic horseradish peroxidase (HRP) and anionic soybean peroxidase (SbP) were used in CL-ELISA for the detection of biotinylated anti-SMP antibodies. For streptavidin-HRP conjugate-catalyzed chemiluminescence measured 20 s after the initiation of the enhanced chemiluminescence reaction (ECR), the limit of detection (IC(10)), the IC(50) value, and the working range in CL-ELISA of SMP are 0.3, 12.4, and 1.2-85.0 ng/mL, respectively. An increase in the time interval between the ECR initiation and the luminescence measurement results in the loss in the quality of analytical measurements because of the time-dependent quenching of chemiluminescence typical of the HRP-catalyzed ECR. In the case of SbP-based CL-ELISA of SMP, the limit of detection, the IC(50) value, and the working range (0.025, 0.17, and 0.045-0.63 ng/mL, respectively) are better than those for HRP-based CL-ELISA. Furthermore, the analytical parameters of SbP-based CL-ELISA remain unchanged during a long period of time (for at least 30 min). The recovery values from four spiked milk samples with different concentrations of SMP in SbP-based CL-ELISA vary from 70 to 130%.

  11. Quantitative enzyme-linked immunosorbent assay for determination of polychlorinated biphenyls in environmental soil and sediment samples.

    PubMed

    Johnson, J C; Van Emon, J M

    1996-01-01

    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 are 10.5 and 9 ng/g, respectively. The assay linear dynamic range is 50-1333 ng/g. Cross-reactivity of the assay with 37 structurally related potential cocontaminants in environmental soil samples was examined; none of the chlorinated anisoles, benzenes, or phenols exhibited >3% cross-reactivity, with <0.1% cross-reactivity being the norm. Soil spike recoveries of 107% and 104% were obtained for Aroclors 1242 and 1248, respectively, for a spike level of 5 mg/kg, with corresponding relative standard deviations of 14% and 17%. One hundred forty-eight environmental soil, sediment, and paper pulp samples, obtained from two EPA listed Superfund sites, were analyzed by ELISA and standard GC methods. Samples were extracted for ELISA analysis by shaking with methanol. Additional extractions of the same samples were performed either with supercritical carbon dioxide or by Soxhlet extraction with methanol. ELISA results for both the supercritical fluid and the Soxhlet extracts were in close agreement with the GC results, while the ELISA results for the methanol shake extracts were not. The data for the environmental samples demonstrated the capability of the ELISA to provide accurate results and reinforced the dependence of any detection method, including ELISA, on appropriate extraction procedures.

  12. Evaluation of commercial enzyme-linked immunosorbent assays to identify psychedelic phenethylamines.

    PubMed

    Kerrigan, Sarah; Mellon, Monica Brady; Banuelos, Stephanie; Arndt, Crystal

    2011-09-01

    The 2C, 2C-T, and DO series of designer drugs pose a number of challenges to forensic toxicology laboratories. Although these drugs are seized by law enforcement agencies throughout the United States, they are not readily detected in forensic toxicology laboratories. A systematic evaluation of the cross-reactivity of 9 commercial enzyme-linked immunosorbent assays (ELISAs) was conducted using 11 designer drugs. Cross-reactivity was measured towards 2,5-dimethoxy-4-bromophenethylamine (2C-B), 2,5-dimethoxyphenethylamine (2C-H), 2,5-dimethoxy4-iodophenethylamine (2C-I), 2,5-dimethoxy-4ethylthiophenethylamine (2C-T-2), 2,5-dimethoxy-4isopropylthiophenethylamine (2C-T-4), 2,5-dimethoxy-4propylthiophenethylamine (2C-T-7), 2,5-dimethoxy-4bromoamphetamine (DOB), 2,5-dimethoxy-4-ethylamphetamine (DOET), 2,5-dimethoxy-4-iodoamphetamine (DOI), 2,5-dimethoxy-4-methylamphetamine (DOM), and 4methylthioamphetamine (4-MTA). Cross-reactivity towards the 2C, 2C-T, and DO series of psychedelic amphetamines was < 0.4%. Concentrations as high as 50,000 ng/mL in urine, which greatly exceed those expected in forensic case samples, were not sufficient to produce a positive result. The only substance to produce any measurable cross-reactivity was 4-MTA. Cross-reactivities of 5 and 7% were obtained using four methamphetamine/MDMA directed assays, 25 and 200% using two amphetamine-directed assays. The absence of any measurable cross-reactivity towards the 10 2C, 2C-T, and DO psychedelic phenethylamines makes it harder to detect these drugs using routine screening. As a consequence, laboratories that rely upon immunoassay rather than more broad spectrum chromatographic screening techniques, may fail to detect these powerful psychedelic substances.

  13. Serum biotin in Japanese children: Enzyme-linked immunosorbent assay measurement.

    PubMed

    Wakabayashi, Kenji; Kodama, Hiroko; Ogawa, Eishin; Sato, Yasuhiro; Motoyama, Kahoko; Suzuki, Mitsuyoshi

    2016-09-01

    Biotin deficiency has been reported in Japanese infants fed special formulas for medical reasons, including those with milk allergy and congenital metabolic diseases, because these formulas contain little biotin. Serum biotin measurement is useful for diagnosing biotin deficiency. We applied a simple and rapid method to analyze serum biotin, and established normal ranges for children and adults. Serum biotin in 188 healthy Japanese children aged 0-4 years and in 25 healthy adults was analyzed using a Biotin ELISA Kit (immundiagnostik). The effects of various conditions on the measurement of serum biotin were also examined. Median biotin in children aged 0-4 years was 10.4 ng/dL (IQR, 7.9-13.4 ng/dL), and that in adults was 12.9 ng/dL (IQR, 10.8-15.8 ng/dL). Normal range was 4.7-22.0 ng/dL in children and 8.4-20.5 ng/dL in adults (calculated using two-sided 95%CI). Measurements obtained with this method were not affected by frozen storage, freeze-thaw, or hemolysis, indicating that serum biotin can be analyzed accurately under these conditions, with a possible application to plasma samples. Serum biotin was significantly lower in children than in adults, with the normal range being 4.7-22.0 ng/dL in children and 8.4-20.5 ng/dL in adults. This simple and accurate enzyme-linked immunosorbent assay method is useful for diagnosing biotin deficiency. © 2016 The Authors. Pediatrics International published by John Wiley & Sons Australia, Ltd on behalf of Japan Pediatric Society.

  14. Detection of Leptospira-Specific Antibodies Using a Recombinant Antigen-Based Enzyme-Linked Immunosorbent Assay

    PubMed Central

    Chen, Hua-Wei; Zhang, Zhiwen; Halsey, Eric S.; Guevara, Carolina; Canal, Enrique; Hall, Eric; Maves, Ryan; Tilley, Drake H.; Kochel, Tadeusz J.; Ching, Wei-Mei

    2013-01-01

    We produced three highly purified recombinant antigens rLipL32, rLipL41, and rLigA-Rep (leptospiral immunoglobulin-like A repeat region) for the detection of Leptospira-specific antibodies in an enzyme-linked immunosorbent assay (ELISA). The performance of these recombinant antigens was evaluated using 121 human sera. Among them, 63 sera were microscopic agglutination test (MAT)-confirmed positive sera from febrile patients in Peru, 22 sera were indigenous MAT-negative febrile patient sera, and 36 sera were from patients with other febrile diseases from Southeast Asia, where leptospirosis is also endemic. Combining the results of immunoglobulin M (IgM) and IgG detection from these three antigens, the overall sensitivity is close to 90% based on the MAT. These results suggest that an ELISA using multiple recombinant antigens may be used as an alternative method for the detection of Leptospira-specific antibodies. PMID:24166046

  15. 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.

  16. Gelatin particle indirect agglutination and enzyme-linked immunosorbent assay for diagnosis of strongyloidiasis using Strongyloides venezuelensis antigen.

    PubMed

    Huaman, Maria Cecilia; Sato, Yoshiya; Aguilar, Jose Luis; Terashima, Angelica; Guerra, Humberto; Gotuzzo, Eduardo; Kanbara, Hiroji

    2003-01-01

    Routine microscopical examination of stool specimens for diagnosis of strongyloidiasis is insensitive and serological methods using Strongyloides stercoralis antigen are at present not available for field studies. We evaluated 2 techniques, enzyme-linked immunosorbent assay (ELISA) and gelatin particle indirect agglutination (GPIA), using an antigen obtained from the rodent parasite, S. venezuelensis. Fifty-four Peruvian patients with different clinical forms of strongyloidiasis were studied: 12 asymptomatic, 31 symptomatic, and 11 hyperinfection cases. Our results demonstrate that both ELISA and GPIA using S. venezuelensis antigen are useful for diagnosis of strongyloidiasis, with sensitivities of 74.1% and 98.2%, respectively and a specificity of 100% for both techniques. We found that GPIA is a highly sensitive test for patients with suspected chronic infection and/or hyperinfection. In the hyperinfection cases, significantly lower concentrations of specific immunoglobulin antibodies and eosinophils (P < 0.001) were found compared with the asymptomatic and symptomatic cases.

  17. 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

  18. Serologic diagnosis of canine and equine borreliosis: use of recombinant antigens in enzyme-linked immunosorbent assays.

    PubMed Central

    Magnarelli, L A; Flavell, R A; Padula, S J; Anderson, J F; Fikrig, E

    1997-01-01

    Serum samples from dogs and equids suspected of having canine or equine borreliosis, respectively, were analyzed in polyvalent enzyme-linked immunosorbent assays (ELISAs) with whole-cell or recombinant antigens of Borrelia burgdorferi sensu stricto. Purified preparations of recombinant antigens included outer surface protein A (OspA), OspB, OspC, OspE, OspF, and p41-G (a fragment of flagellin). Of the 36 dog sera that reacted positively to whole-cell antigen, 32 (88.9%) contained antibodies to one or more recombinant antigens. Reactivities to OspF (88.9% positive) and p41-G (75% positive) were most prevalent. In analyses of 30 equid sera positive in an ELISA with whole cells, 24 (80%) contained antibodies to one or more recombinant antigens. Seropositivities in ELISAs with p41-G (50% positive) and OspF (46.7% positive) were more than twofold greater than in ELISAs with OspA, OspB, or OspC (10 to 20% positive). In parallel tests of eight canine and three equine sera, there was good agreement in results of Western blot (immunoblot) analyses and ELISAs. Although dog and equid sera with antibodies to whole-cell B. burgdorferi frequently reacted positively to one or more recombinant antigens, the inclusion of OspF and p41-G antigens in ELISAs was most useful in the serologic diagnosis of canine and equine borreliosis. PMID:8968901

  19. Development of a cellular tau enzyme-linked immunosorbent assay method for screening GSK-3β inhibitors.

    PubMed

    Cho, Goang-Won; Noh, Min-Young; Kang, Byung Yong; Ku, Il-Whea; Park, Jiseon; Hong, Yoon-Ho; Kim, Myung-Hwa; Kim, Seung Hyun

    2011-10-01

    Glycogen synthase kinase-3β (GSK-3β), a serine/threonine kinase also known as tau protein kinase I, has been implicated in the pathogenic conditions of Alzheimer's disease. Many investigators have focused on GSK-3 inhibitor as a therapeutic drug. In this study, we established a cell-based assay for the screening of novel GSK-3β inhibitors. For this purpose, four-repeat tau cDNAs were stably expressed in human embryonic kidney 293 (HEK293) cells (HEK293-Tau). The proliferation of HEK293-Tau cells was no different from that of HEK293 cells, as measured by the bromodeoxyuridine enzyme-linked immunosorbent assay (BrdU ELISA). The concentration-dependent reduction of tau phosphorylation by GSK-3 inhibitors, LiCl, Chir98023, and SB415286, was examined by immunoblot analysis and Tau ELISA (in situ ELISA). Highly consistent data were obtained, suggesting that this novel ELISA method is highly reproducible. Using this ELISA strategy, we isolated a few candidate compounds, including compounds 114 and 149, from several hundreds of synthetic agents and demonstrated that such candidates protect nerve growth factor-differentiated PC12 cells against amyloid-β-induced cell death. These data indicate that this Tau ELISA method in HEK293-Tau cells may be a suitable cell-based assay system to screen for GSK-3β inhibitors.

  20. 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

  1. 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

  2. Comparison of commercial enzyme-linked immunosorbent assays and agar gel immunodiffusion tests for the serodiagnosis of equine infectious anemia

    PubMed Central

    2004-01-01

    Abstract The purpose of this study was to estimate the performance characteristics (accuracy, detection limit, and precision) of commercially available enzyme-linked immunosorbent assay (ELISA) and agar gel immunodiffusion (AGID) kits in comparison with a reference AGID kit for the detection of equine infectious anemia (EIA) antibodies in horses for regulatory use in Canada. A total of 285 positive and 315 negative samples by the reference AGID were tested blindly on 2 other AGID and 4 ELISA kits. Commercially available AGID kits for the serodiagnosis of EIA were found equivalent. The 3 ELISAs directed against antibodies to the p26 core protein also performed relatively well in comparison with the reference AGID, with excellent relative accuracy and acceptable precision. The single ELISA directed against antibodies to the gp45 trans-membrane viral protein yielded a lower relative sensitivity. The performance characteristics of the ELISAs directed against antibodies to p26 are, therefore, adequate to support the implementation of ELISA for regulatory purposes in Canada. PMID:15581219

  3. Comparison of commercial enzyme-linked immunosorbent assays and agar gel immunodiffusion tests for the serodiagnosis of equine infectious anemia.

    PubMed

    Paré, Julie; Simard, Carole

    2004-10-01

    The purpose of this study was to estimate the performance characteristics (accuracy, detection limit, and precision) of commercially available enzyme-linked immunosorbent assay (ELISA) and agar gel immunodiffusion (AGID) kits in comparison with a reference AGID kit for the detection of equine infectious anemia (EIA) antibodies in horses for regulatory use in Canada. A total of 285 positive and 315 negative samples by the reference AGID were tested blindly on 2 other AGID and 4 ELISA kits. Commercially available AGID kits for the serodiagnosis of EIA were found equivalent. The 3 ELISAs directed against antibodies to the p26 core protein also performed relatively well in comparison with the reference AGID, with excellent relative accuracy and acceptable precision. The single ELISA directed against antibodies to the gp45 trans-membrane viral protein yielded a lower relative sensitivity. The performance characteristics of the ELISAs directed against antibodies to p26 are, therefore, adequate to support the implementation of ELISA for regulatory purposes in Canada.

  4. Enzyme-linked immunosorbent assay for screening dioxin soil contamination by uncontrolled combustion during informal recycling in slums.

    PubMed

    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

    2008-11-01

    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.

  5. Evaluation of the recombinant LipL32 in enzyme-linked immunosorbent assay for the serodiagnosis of bovine leptospirosis.

    PubMed

    Bomfim, Maria Rosa Quaresma; Ko, Albert; Koury, Matilde Cota

    2005-08-10

    The recombinant leptospiral protein LipL32 was evaluated for use in the diagnosis of bovine leptospirosis by enzyme-linked immunosorbent assay (rLipL32 IgG ELISA). The microscopic agglutination test (MAT) of 150 serum samples from cattle suspected of leptospirosis showed that 125 (83.3%) samples had positive reciprocal agglutination titres, which ranged from 100 to 1600. The highest titres were observed for the serovars Hardjoprajitno and Bratislava. In the rLipL32 IgG ELISA, 83.3% of the samples were positive. The sensitivity of IgG ELISA for 125 bovine sera, which had MAT titres of greater than or equal to 100, was 100%. ELISA showed a specificity of 100% with 58 bovine sera, which were negative at a 1:50 dilution in the MAT for Leptospira interrogans serovars. When analytical specificity of the IgG ELISA was evaluted using 60 bovine serum samples from animals showing serum antibodies to other pathogens that cause abortion in cattle, such as Babesia sp., Anaplasma sp. and Brucella sp. and no cross-reaction was observed. The recombinant LipL32 IgG ELISA can be an alternative to the MAT for diagnosis of leptospiral infection in cattle.

  6. Evaluation of a dengue IgG indirect enzyme-linked immunosorbent assay and a Japanese encephalitis IgG indirect enzyme-linked immunosorbent assay for diagnosis of secondary dengue virus infection.

    PubMed

    Inoue, Shingo; Alonzo, Maria T G; Kurosawa, Yae; Mapua, Cynthia A; Reyes, Joyce D; Dimaano, Efren M; Alera, Maria Theresa P; Saito, Mariko; Oishi, Kazunori; Hasebe, Futoshi; Matias, Ronald R; Natividad, Filipinas F; Morita, Kouichi

    2010-03-01

    To establish a new method for the diagnosis of dengue secondary infection, 187 serum samples from the patients with dengue secondary infection, 40 serum samples from the patients with dengue primary infection, and 44 serum samples from the healthy volunteers were tested using the dengue IgG indirect enzyme-linked immunosorbent assay (DEN IgG ELISA). The results of the test were compared with those from the dengue hemagglutination inhibition (DEN HI) test, which has been recommended as the gold standard by the World Health Organization (WHO, 1997). Japanese encephalitis IgG indirect ELISA (JE IgG ELISA) was also performed to measure anti-flavivirus IgG, which cross-reacts with the Japanese encephalitis virus, to test the possibility of an alternative to DEN IgG ELISA. The results of DEN IgG and JE IgG ELISAs were highly correlated with those of the DEN HI test. In the DEN IgG ELISA, a titer of 1:29,000 was the cut-off value for the diagnosis of dengue secondary infection (91.5% accuracy [95% confidence interval, CI], 90.9% sensitivity [95%CI], and 92.9% specificity [95%CI]). A titer of 1:52,000 was the cut-off value for dengue secondary infection using JE IgG ELISA (95.6% accuracy [95%CI], 98.9% sensitivity [95%CI], and 88.1% specificity [95%CI]). In conclusion, this study confirmed that the results of both DEN IgG and JE IgG ELISAs were highly correlated with the results of DEN HI test. Thus, these ELISAs are simple, rapid, sensitive, and quantitative tests that can be used in the determination of dengue secondary infection.

  7. Babesia gibsoni: Serodiagnosis of infection in dogs by an enzyme-linked immunosorbent assay with recombinant BgTRAP.

    PubMed

    Goo, Youn-Kyoung; Jia, Honglin; Aboge, G Oluga; Terkawi, M Alaa; Kuriki, Ken; Nakamura, Chinatsu; Kumagai, Akiko; Zhou, Jinlin; Lee, Eung-goo; Nishikawa, Yoshifumi; Igarashi, Ikuo; Fujisaki, Kozo; Xuan, Xuenan

    2008-04-01

    The thrombospondin-related adhesive protein of Babesia gibsoni (BgTRAP) is known as an immunodominant antigen and is, therefore, considered as a candidate for the development of a diagnostic reagent for canine babesiosis. The recombinant BgTRAP (rBgTRAP) expressed in Escherichia coli was tested in an enzyme-linked immunosorbent assay (ELISA) for detecting antibodies to B. gibsoni in dogs. The ELISA with rBgTRAP clearly differentiated between B. gibsoni-infected dog sera and specific pathogen-free (SPF) dog sera. The sera collected from dogs experimentally infected with closely related parasites, B. canis canis, B. canis vogeli, B. canis rossi, and Neospora caninum, showed no cross-reactivity by the ELISA with rBgTRAP. A total of 107 blood samples collected from dogs that had been diagnosed as having babesiosis at veterinary hospitals in Japan were examined for the diagnosis of B. gibsoni infection by the ELISA and PCR. Ninety-six (89.7%) and 89 (83.2%) of the tested samples were positive by the ELISA and PCR, respectively, while 11 (10.3%) and 4 (3.7%) were ELISA+/PCR- and ELISA-/PCR+, respectively. In addition, the sensitivity of the ELISA with rBgTRAP was much higher than that of previously established ELISAs with rBgP50, rBgSA1, and rBgP32. These results indicate that the rBgTRAP is the most promising diagnostic antigen for the detection of an antibody to B. gibsoni in dogs and that the combined ELISA/PCR approach could provide the most reliable diagnosis for clinical sites.

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

    PubMed

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

    2014-01-01

    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.

  9. 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

  10. Development of an enzyme-linked immunosorbent assay for residue analysis of the insecticide emamectin benzoate in agricultural products.

    PubMed

    Kondo, Mika; Yamashita, Hiroshi; Uchigashima, Mikiko; Kono, Takeshi; Takemoto, Toshihide; Fujita, Masahiro; Saka, Machiko; Iwasa, Seiji; Ito, Shigekazu; Miyake, Shiro

    2009-01-28

    A direct competitive enzyme-linked immunosorbent assay (dc-ELISA) for the analysis of emamectin residues in agricultural products was developed using a prepared mouse monoclonal antibody. The working range was 0.3-3.0 ng/mL, and the 50% inhibition concentration (IC(50)) was 1.0 ng/mL. The assay was sufficiently sensitive for analysis of the maximum residue limits in agricultural products in Japan (>0.1 microg/g). Emamectin residues contain the following metabolites: the 4''-epi-amino analogue, the 4''-epi-(N-formyl)amino analogue, the 4''-epi-(N-formyl-N-methyl)amino analogue, and the 8,9-Z isomer. The dc-ELISA reacted with these compounds at ratios of 113, 55, 38, and 9.1% of the IC(50) value of emamectin benzoate. Seven kinds of vegetables were spiked with emamectin benzoate at concentrations of 15-300 ng/g, and the recoveries were 91-117% in the dc-ELISA. The dc-ELISA results agreed reasonably well with results obtained by liquid chromatography-tandem mass spectrometry (LC-MS/MS) using spiked samples and actual (incurred) samples. The results indicate that the dc-ELISA was useful for the analysis of emamectin benzoate residues in agricultural products.

  11. Monoclonal antibody-based competitive enzyme-linked immunosorbent assay for detection of morbillivirus antibody in marine mammal sera.

    PubMed

    Saliki, J T; Lehenbauer, T W

    2001-05-01

    A competitive enzyme-linked immunosorbent assay (cELISA), using two monoclonal antibodies (MAbs), was developed and compared with the standard virus neutralization test (VNT) for detecting antibodies against canine distemper virus (CDV) and phocine distemper virus (PDV) in sera from dogs and various species of marine mammals. The test depends on the blocking of MAb binding to solid-phase antigen in the presence of positive serum. Test conditions were optimized by using control VNT-negative and -positive sera specific for CDV and PDV. A positive cutoff value of 30% inhibition, which represents the mean cutoff of a VNT-negative population (n = 623) plus 2 standard deviations, was adopted for the test. A total of 736 serum samples were tested by the new cELISA and by the VNT as the "gold standard." An unexpected but useful finding was the ability of this CDV- and PDV-specific cELISA to also detect antibodies against the related pair dolphin morbillivirus and porpoise morbillivirus. Based on a subpopulation of 625 sera used in statistical analyses, the overall sensitivity and specificity of cELISA relative to those of the VNT were 94.9 and 97.7%, respectively. Because the cELISA proved to be nearly as sensitive and specific as the VNT while being simpler and more rapid, it would be an adequate screening test for suspect CDV or PDV cases and would also be useful for epidemiological surveillance of morbilliviral infections in marine mammal populations.

  12. Evaluation of an enzyme-linked immunosorbent assay for detection of antibodies to Junin virus in rodents.

    PubMed

    Morales, María A; Calderón, Gladys E; Riera, Laura M; Ambrosio, Ana M; Enría, Delia A; Sabattini, Marta S

    2002-05-01

    Junin virus is the etiological agent of Argentine hemorrhagic fever, a serious rodent-borne disease. An enzyme-linked immunosorbent assay (ELISA) to detect Junin virus IgG antibodies in rodents was evaluated using sera from 27 Calomys musculinus and five Calomys laucha, inoculated experimentally with a live attenuated strain of this arenavirus. The test performance was compared against an indirect immunofluorescence assay (IFA). The ELISA had a sensitivity and specificity of 100% and a reproducibility of 87.9% for samples with titers above the selected cut-off value. IFA had lower sensitivity (53%) with the same specificity. The ELISA results were similar, whether carried out on whole blood or serum samples, thus eliminating the need for serum separation. A high correlation (K=0.86) between ELISA and IFA results was obtained from 1011 wild sigmodontine and murine rodents collected within and outside of the Argentine hemorrhagic fever endemic area. These results indicate that Junin virus IgG ELISA is the most suitable assay for detection of Junin virus antibodies in rodent samples.

  13. Synthesis of novel haptens and development of an enzyme-linked immunosorbent assay for quantification of histamine in foods.

    PubMed

    Luo, Lin; Xu, Zhen-Lin; Yang, Jin-Yi; Xiao, Zhi-Li; Li, Yong-Jun; Beier, Ross C; Sun, Yuan-Ming; Lei, Hong-Tao; Wang, Hong; Shen, Yu-Dong

    2014-12-24

    Novel haptens were designed and synthesized to prepare antibodies against free histamine, but none resulted in producing suitable antibodies for developing an enzyme-linked immunosorbent assay (ELISA). However, an antiserum was obtained having high specificity and affinity to p-nitrobenzoylated histamine (NPHA), which can be easily formed from reaction between histamine and p-nitrobenzoic acid N-hydroxysuccinimide ester (PNBA-OSu) under mild conditions. Based on rabbit polyclonal antibodies, a competitive indirect ELISA (ciELISA) for histamine determination in foods was developed. After ciELISA and derivatization optimization, the assay showed good sensitivity, with limits of detection of 1.8 mg/kg, 93.6 μg/L, and 93.6 μg/kg in fish, red wine, and yoghurt, respectively, with negligible cross-reactivity with related biogenic amines and amino acids. Average recovery of histamine in fortified food samples ranged from 80.9% to 110.1% with coefficients of variation below 16.3%. Good correlation between the ciELISA and liquid chromatography-tandem mass spectrometry was obtained for spiked food samples.

  14. Quantitative evaluation of pork adulteration in raw ground beef by radial immunodiffusion and enzyme-linked immunosorbent assay.

    PubMed

    Martin, D R; Chan, J; Chiu, J Y

    1998-12-01

    Quantitative estimates are important to establish whether pork adulteration in ground beef is accidental or intentional. A standard agar gel radial immunodiffusion (RID) test using forensic-grade antiserum to porcine albumin and an enzyme-linked immunosorbent assay (ELISA) using forensic-grade anti-porcine glycoprotein immunoglobulin were used to determine from 1 to 75% raw pork in raw ground beef. The RID test, which incorporated 1.5% anti-pork serum in 1% immunodiffusion agar, formed precipitin rings with pork albumin in agar wells. A linear standard curve was obtained by plotting the diffusion area against standard pork concentrations ranging from 0 to 80%. For the ELISA the endpoint optical density increased linearly versus log % pork between 0.0625% and 2% pork. In spiked samples, the RID test had a detection limit of 3 to 5%, a coefficient of variation (CV) of 22%, and a recovery of 105%. The ELISA had a detection limit of 1%, a CV of 18%, and a recovery of 114%. The mean recovery from the spiked samples by the ELISA and RID test was not significantly different (P > 0.05) from the known sample amounts. Quantitation by RID of 28 ground beef samples (27 of which were DTEK ELISA-positive for pork adulteration) revealed a wide range of pork content, with values as high as 48%.

  15. Detection of Campylobacter jejuni and Campylobacter coli in environmental waters by PCR enzyme-linked immunosorbent assay.

    PubMed

    Sails, A D; Bolton, F J; Fox, A J; Wareing, D R A; Greenway, D L A

    2002-03-01

    A PCR enzyme-linked immunosorbent assay (ELISA) assay was applied to the detection of Campylobacter jejuni and Campylobacter coli in environmental water samples after enrichment culture. Bacterial cells were concentrated from 69 environmental water samples by using filtration, and the filtrates were cultured in Campylobacter blood-free broth. After enrichment culture, DNA was extracted from the samples by using a rapid-boiling method, and the DNA extracts were used as a template in a PCR ELISA assay. A total of 51 samples were positive by either PCR ELISA or culture; of these, 43 were found to be positive by PCR ELISA and 43 were found to be positive by culture. Overall, including positive and negative results, 59 samples were concordant in both methods. Several samples were positive in the PCR ELISA assay but were culture negative; therefore, this assay may be able to detect sublethally damaged or viable nonculturable forms of campylobacters. The method is rapid and sensitive, and it significantly reduces the time needed for the detection of these important pathogens by 2 to 3 days.

  16. Detection of Campylobacter jejuni and Campylobacter coli in Environmental Waters by PCR Enzyme-Linked Immunosorbent Assay

    PubMed Central

    Sails, A. D.; Bolton, F. J.; Fox, A. J.; Wareing, D. R. A.; Greenway, D. L. A.

    2002-01-01

    A PCR enzyme-linked immunosorbent assay (ELISA) assay was applied to the detection of Campylobacter jejuni and Campylobacter coli in environmental water samples after enrichment culture. Bacterial cells were concentrated from 69 environmental water samples by using filtration, and the filtrates were cultured in Campylobacter blood-free broth. After enrichment culture, DNA was extracted from the samples by using a rapid-boiling method, and the DNA extracts were used as a template in a PCR ELISA assay. A total of 51 samples were positive by either PCR ELISA or culture; of these, 43 were found to be positive by PCR ELISA and 43 were found to be positive by culture. Overall, including positive and negative results, 59 samples were concordant in both methods. Several samples were positive in the PCR ELISA assay but were culture negative; therefore, this assay may be able to detect sublethally damaged or viable nonculturable forms of campylobacters. The method is rapid and sensitive, and it significantly reduces the time needed for the detection of these important pathogens by 2 to 3 days. PMID:11872483

  17. Detecting clothianidin residues in environmental and agricultural samples using rapid, sensitive enzyme-linked immunosorbent assay and gold immunochromatographic assay.

    PubMed

    Li, Ming; Hua, Xiude; Ma, Ming; Liu, Jisong; Zhou, Liangliang; Wang, Minghua

    2014-11-15

    Two rapid, sensitive immunoassays based on monoclonal antibody for detecting clothianidin were developed and applied in agricultural samples: a quantitative enzyme-linked immunosorbent assay (ELISA) and a semiquantitative gold immunochromatographic assay (GICA). Under optimal conditions, the half-maximal inhibition concentration (IC50) and the limit of detection (LOD, IC10) of clothianidin were 25.6 and 3.8 ng mL(-1) for ELISA. GICA using colloidal gold-MAb probe had a visual detection limit of 8 ng mL(-1), and the results can be judged by the naked eye within 10 min. The cross-reactivities of the immunoassays with its analogues were negligible except for that with dinotefuran. For the spiked agricultural samples, recoveries of 78.0 to 114.5% with relative standard deviations (RSDs) of 3.2 to 12.8% were achieved for ELISA and further evaluated by GICA. Furthermore, the results of ELISA and GICA for the authentic samples correlated well with those obtained by HPLC. Overall, the proposed ELISA and GICA are satisfactory for rapid, sensitive, and quantitative/semiquantitative detection of clothianidin residues in agricultural samples.

  18. Development of an enzyme-linked immunosorbent assay for detecting antibodies in sera of Brucella suis-infected swine.

    PubMed Central

    Thoen, C O; Hopkins, M P; Armbrust, A L; Angus, R D; Pietz, D E

    1980-01-01

    An enzyme-linked immunosorbent assay was developed using a heat-killed Brucella suis antigen for detecting antibodies in the sera of swine from which B. suis was isolated. Optimal enzyme-linked immunosorbent assay reactions were obtained using heat-killed B. suis antigen at a concentration comparable to McFarland Standard No. 1. Statistically significant differences were observed in the enzyme-linked immunosorbent assay results of 40 animals from which B. suis was isolated and the results for 48 noninfected swine at serum dilutions of 1:25 and 1:50 (P < 0.0001). The enzyme-linked immunosorbent assay is a rapid reproducible test which can be readily automated that appears to have practical value for screening large numbers of breeding and slaughter swine for brucellosis. PMID:7000319

  19. 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

  20. Performance of Competitive and Indirect Enzyme-Linked Immunosorbent Assays, Gel Immunoprecipitation with Native Hapten Polysaccharide, and Standard Serological Tests in Diagnosis of Sheep Brucellosis

    PubMed Central

    Marín, C. M.; Moreno, E.; Moriyón, I.; Díaz, R.; Blasco, J. M.

    1999-01-01

    Competitive and standard enzyme-linked immunosorbent assays (ELISAs), rose bengal (RB), complement fixation, and agar gel immunoprecipitation with native hapten (AGID-NH) were compared by using sera from Brucella-free, Brucella melitensis-infected, and B. melitensis Rev1-vaccinated sheep. The most sensitive tests were indirect ELISA and RB, and the most specific tests were AGID-NH and competitive ELISA. We show that RB followed by AGID-NH is a simple and effective system for diagnosing sheep brucellosis. PMID:10066666

  1. Evaluation of Vircell Enzyme-Linked Immunosorbent Assay and Indirect Immunofluorescence Assay for Detection of Antibodies against Legionella pneumophila

    PubMed Central

    Diederen, Bram M. W.; Kluytmans, Jan A. J. W.; Peeters, Marcel F.

    2006-01-01

    We evaluated the abilities of the Vircell immunoglobulin G (IgG) and IgM indirect immunofluorescence assay (IFA) for Legionella pneumophila serogroup 1, the IgM and IgG enzyme-linked immunosorbent assay (ELISA) for Legionella pneumophila serogroup 1, and the IgM-plus-IgG ELISA for Legionella pneumophila serogroups 1 to 6 to diagnose Legionnaires' disease (LD) in a well-described sample of patients with and without LD. Also, we determined the agreements, sensitivities, and specificities of the different Vircell assays in comparison to a validated ELISA (Serion classic ELISA). Clinical sensitivity and specificity were 74.6% and 96.6%, respectively, for the IgM IFA, 65.1% and 88.0% for the IgG IFA, 92.3% and 100% for the IgM ELISA, 43.3% and 96.6% for the IgG ELISA, and 90.8% and 100% for the IgM-plus-IgG ELISA. Compared to Serion classic ELISA, agreement, sensitivity, and specificity were 80.0%, 83.1%, and 78.4%, respectively, for the IgM IFA, 75.2%, 66.0%, and 79.5% for the IgG IFA, 89.5%, 82.0%, and 97.6% for the IgM ELISA, 81.9%, 88.9%, and 78.0% for the IgG ELISA, and 93.5%, 90.0%, and 96.6% for the IgM-plus-IgG ELISA. The value of a positive diagnostic result obtained by the Vircell IgM IFA, the Vircell IgG IFA, and the Vircell IgG ELISA might not be acceptable for a diagnostic assay. Both the high specificities and sensitivities of the Vircell IgM ELISA and the IgM-plus-IgG ELISA and the high correlation with the Serion classic ELISA indicate that they are useful in the diagnosis of LD. PMID:16522778

  2. A monoclonal antibody based enzyme-linked immunosorbent assay for detection of phenylethanolamine A in tissue of swine.

    PubMed

    Li, Yansong; Lu, Shiying; Liu, Zengshan; Sun, Lihui; Guo, Junfei; Hu, Pan; Zhang, Junhui; Zhang, Yuanyuan; Wang, Yang; Ren, Honglin; Meng, Xingyu; Zhou, Yu

    2015-01-15

    Phenylethanolamine A (PEA) is a phenethanolamine member of the family of β-adrenergic agonists compounds illegally used as feed additives for growth promotion. In this study, PEA was covalently linked to carrier protein cationized bovine serum albumin (cBSA) and egg albumin (OVA). A monoclonal antibody specific for PEA was produced and characteristics of monoclonal antibody (McAb) were studied. Based on the McAb, an enzyme-linked immunosorbent assay (ELISA) for detection of PEA was developed, which showed an IC50 value of 6.25 ng mL(-1) and a detection limit of 0.19 ng mL(-1). The average recovery of PEA from spiked samples was 103.4%, which demonstrated that the matrices of meat where PEA may be found do not interfere with the assay. Copyright © 2014 Elsevier Ltd. All rights reserved.

  3. Modification of Clearview Tuberculosis (TB) Enzyme-Linked Immunosorbent Assay for TB Patients Not Infected with HIV

    PubMed Central

    Kantele, Anu; Sandboge, Bettina; Sirén, Marita; Valleala, Heikki; Tuompo, Riitta; Pusa, Liana; Erkinjuntti-Pekkanen, Riitta; Knuuttila, Aija; Ku, Cheng-Lung; Chi, Chih-Yu; Vasankari, Tuula; Tuuminen, Tamara

    2013-01-01

    Diagnosis of active tuberculosis by detection of urinary lipoarabinomannan (uLAM) from Mycobacterium tuberculosis is an attractive approach. Concentrating urine 100-fold allowed quantitation of uLAM at levels equal to picograms/ml of nonconcentrated urine. The approach of concentrating urine 100-fold improved the clinical sensitivity of the Clearview TB enzyme-linked immunosorbent assay (ELISA) from 7% to 57% yet impaired its specificity from 97% to 89%. (This study has been registered at University Hospital of Turku under registration no. 47/180/2009, Helsinki University Central Hospital under 149/2010, University Hospital of Kuopio under 105/2010, and China Medical University Hospital, Taichung, under DMR-99-IRB-075-2.) PMID:23825194

  4. Salivary Desmoglein Enzyme-Linked Immunosorbent Assay for Diagnosis of Pemphigus Vulgaris: A Noninvasive Alternative Test to Serum Assessment

    PubMed Central

    Khatami, Alireza; Seyedin, Zahra; Daneshpazhooh, Maryam

    2015-01-01

    Background. Serum desmoglein enzyme-linked immunosorbent assay (ELISA) is used for the diagnosis and monitoring of pemphigus diseases. Objectives. To compare the diagnostic accuracy of salivary antidesmoglein (Dsg) 1 and 3 ELISA in the diagnosis of pemphigus vulgaris (PV) patients with that of serum desmogleins ELISA. Methods. Eighty-six untreated PV patients and 180 age- and sex-matched PV-free controls were recruited in this case-control study. PV was diagnosed based on clinical, histopathological, and direct immunofluorescence findings. After processing, serum and salivary anti-Dsg 1 and 3 were measured by the ELISA method using Euroimmun kit (Lübeck, Germany). Results. Using the cut-off point of 20 relative units (RU)/mL, the serum anti-Dsg 1 and 3 ELISA were positive in 62 (72.1%) and 83 (96.5%) patients, respectively, and the salivary anti-Dsg 1 and 3 ELISA were positive in 31 (36.1%) and 63 (73.3%) patients, respectively. The specificity of salivary anti-Dsg 1 and anti-Dsg 3 were both 98.9%. Optimal cut-off values of 7.7 and 13.4 RU/mL were determined for the salivary anti-Dsg 1 and anti-Dsg 3 ELISA, respectively. Conclusion. Salivary anti-Dsg 1 and 3 ELISA with high specificities (98.9%) could be suggested as safe and noninvasive methods for the diagnosis of PV when obtaining a blood sample is difficult. PMID:25688364

  5. A novel fusion protein-based indirect enzyme-linked immunosorbent assay for the detection of bovine tuberculosis.

    PubMed

    Liu, Siguo; Guo, Sheping; Wang, Chunlai; Shao, Meili; Zhang, Xiuhua; Guo, Yang; Gong, Qiang

    2007-05-01

    Enzyme-linked immunosorbent assay (ELISA) for diagnosis of bovine tuberculosis has been widely explored over the years. Three Mycobacterium bovis-specific antigen genes, namely, mpb70, mpb83, and esat-6 were recombined in tandem by spliced overlap extension technology and expressed in Escherichia coli to obtain the fusion protein (rM70-83-E6). Western blot analysis showed that rM70-83-E6 can specifically react with bovine tuberculosis-positive sera but not those from cattle infected with other bovine diseases such as bovine paratuberculosis. An indirect ELISA (iELISA) method was established with rM70-83-E6 as the diagnostic antigen. The diagnostic criteria were determined using 150 serum samples from healthy cattle. Analyses of 85 serum samples from cattle with bovine tuberculosis and 100 serum samples from healthy cattle demonstrated that the sensitivity of the iELISA was 69.4% (59/85) and the specificity was 96.0% (96/100). Moreover, 46 out of 67 purified protein derivative (PPD) skin test-positive samples were also positive by iELISA, giving a positive coincidence of 68.7%, while all 50 PPD skin test-negative samples were negative by iELISA, giving a negative coincidence of 100%. The total coincidence between iELISA and the PPD skin test was 82.1%. This study demonstrated that iELISA using rM70-83-E6 antigen is simple, sensitive and easy to perform and can be used to analysis of a large number of samples for serodiagnosis of bovine tubercuiosis.

  6. 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

  7. Development and validation of an enzyme-linked immunosorbent assay for the diagnosis of porcine proliferative enteropathy.

    PubMed

    Wattanaphansak, Suphot; Asawakarn, Tanong; Gebhart, Connie J; Deen, John

    2008-03-01

    The objective of this study was to develop an indirect enzyme-linked immunosorbent assay (ELISA) using a sonicated pure culture of Lawsonia intracellularis as the antigen (So-ELISA). A total of 332 serum samples, consisting of 232 experimentally infected animals and 100 animals naturally infected with L. intracellularis, were used to assess the diagnostic sensitivity. Three hundred and fifty-five sera from uninfected animals were used to determine the diagnostic specificity. The receiver operating characteristic and mean +3 standard deviation of optical density (OD) values from uninfected animals were used for selecting cut-off points. The diagnostic accuracy of So-ELISA was considered to be high as the area under the curve index was 0.991 with 0.0029 standard error. The optimal cut-off for So-ELISA was set at 0.45 OD with 89.8% sensitivity and 99.4% specificity based on a combination of good sensitivity and high specificity. No cross-reactivity was found in sera from pigs exposed to Brachyspira pilosicoli, B. hyodysenteriae, Campylobacter mucosalis, C. jejuni, or C. coli. Inter- and intracoefficient of variation of all control sera tested with So-ELISA was less than 10%. The observed agreements between So-ELISA and the immunoperoxidase monolayer assay tested with experimental challenge animals and field samples were 95.08% with 0.88 kappa and 90.65% with 0.74 kappa value, respectively. So-ELISA was able to detect the seroconversion of infected animals at 2 to 4 weeks after exposure to L. intracellularis. Based on the validation results, So-ELISA could be used as an alternative serology for proliferative enteropathy diagnosis.

  8. 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

  9. Bluetongue in Bosnia: comparisons of competitive enzyme-linked immunosorbent assay and standard agar gel immunodiffusion tests.

    PubMed

    Velić, L; Velić, R; Bajrović, T; Dukić, B; Camo, D

    2004-01-01

    At the end of August 2002, clinical symptoms of bluetongue (BT) (fever between 39 degrees C and 41 degrees C, muco-purulent or bloody nasal discharge, oedema of the lips and the intramandibular space, foot lesions including laminitis and coronitis in some cases, diarrhoea and dysentery) were recorded in Pramenka sheep flocks in north-east Bosnia in August 2002. A total of 9 599 serum samples (ovine: 8 967; bovine: 632) from 40 communities of Bosnia and Herzegovina were tested for the presence of anti-bluetongue virus (BTV) antibodies using competitive enzyme-linked immunosorbent assay (c-ELISA) and the standard agar gel immunodiffusion (AGID) test. The c-ELISA revealed BTV-seropositive reactions in 187 (1.94%) samples and the AGID test detected 141 (1.53%) cases. Complete agreement was recorded between the c-ELISA and AGID test results for bovine sera. These results indicate that the ability of c-ELISA to detect anti-bluetongue virus antibodies in ovine sera was superior to that of the AGID. All positive sera were collected from animals in the river areas of Bosnia and Herzegovina.

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

    PubMed

    Chen, Arnold; Wang, Royal; Bever, Candace R S; Xing, Siyuan; Hammock, Bruce D; Pan, Tingrui

    2014-11-01

    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)-10(4 ) 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.

  11. 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

    2014-02-06

    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.

  12. Detection of group D salmonellae including Salmonella Enteritidis in eggs by polymyxin-based enzyme-linked immunosorbent assay.

    PubMed

    Blais, Burton W; Martinez-Perez, Amalia

    2008-02-01

    A high-throughput, rapid method was devised for the detection of Salmonella Enteritidis in egg products. For each target organism, preenrichment in nutrient broth was followed by selective enrichment in Rappaport-Vassiliadis soya peptone and tetrathionate brilliant green broths or by plating on modified semisolid Rappaport Vassiliadis (MSRV) agar medium. The presence of Salmonella Enteritidis was determined by subjecting portions of the selective broth cultures or swarming growth on MSRV medium to an enzyme-linked immunosorbent assay (ELISA) procedure using polymyxin immobilized in the wells of a microtiter plate as a high-affinity adsorbent for lipopolysaccharide (LPS) antigens. Sample extracts were reacted with polymyxin-coated microwells, and captured LPS antigens were detected immunoenzymatically with a commercially available Salmonella factor O9-specific antibody. The polymyxin-ELISA was 100% sensitive and 100% specific for Salmonella strains bearing the O9 antigen. When the ELISA was combined with enrichment using either the selective broths or plating on MSRV medium, the system was an effective means for detection of Salmonella Enteritidis in artificially inoculated egg products. The polymyxin-ELISA is a simple and inexpensive assay for group D salmonellae (including Salmonella Enteritidis) in a convenient 96-well microtiter plate format, making this system ideally suited for processing large numbers of samples.

  13. Development of an enzyme-linked immunosorbent assay for equine infectious anemia virus detection using recombinant Pr55gag.

    PubMed

    Archambault, D; Wang, Z M; Lacal, J C; Gazit, A; Yaniv, A; Dahlberg, J E; Tronick, S R

    1989-06-01

    To provide more sensitive and convenient methods for the detection of equine infectious anemia virus (EIAV), we developed an enzyme-linked immunosorbent assay (ELISA) employing the EIAV gag precursor (Pr55gag) produced by using recombinant DNA techniques. The antigenic reactivity of the recombinant EIAV Pr55gag was found to be equivalent to that of the virion p24gag and elicited high-titered antiserum in rabbits. When a large number of horse sera were analyzed for the presence of antibodies to EIAV by this ELISA, a radioimmunoassay for EIAV p15gag, or the standard agar gel immunodiffusion test, there was 98.7% concordance among the assays. By using the ELISA it was possible to specifically detect antibodies earlier after experimental infection of horses with EIAV than with the other two tests. A competition ELISA developed in order to detect EIAV gag antigens was found to be approximately 15 times more sensitive than the radioimmunoassay for EIAV p15gag. Antigens of other animal lentiviruses as well as those of the prototype oncovirus failed to compete in this assay.

  14. 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.

  15. Development of a Recombinant Protein-Based Enzyme-Linked Immunosorbent Assay for Diagnosis of Mycoplasma bovis Infection in Cattle

    PubMed Central

    Wawegama, Nadeeka K.; Kanci, Anna; Marenda, Marc S.; Markham, Philip F.

    2014-01-01

    Mycoplasma bovis causes a range of diseases in cattle, including mastitis, arthritis, and pneumonia. However, accurate serological diagnosis of infection remains problematic. The studies described here aimed to identify an antigen that might be used to develop a more specific and sensitive diagnostic assay. A 226-kDa immunogenic protein was consistently detected in Western blots by antibodies in sera from calves experimentally infected with M. bovis. This protein was shown to be a membrane protein with lipase activity and was named mycoplasma immunogenic lipase A (MilA). Different regions of MilA were expressed in Escherichia coli as glutathione S-transferase (GST) fusion proteins and recombinant products from the amino-terminal end shown to have strong immunoreactivity with M. bovis-specific bovine sera. The most immunoreactive fusion protein, GST-MilA-ab, was used to develop indirect IgM and IgG enzyme-linked immunosorbent assays (ELISAs). The IgM ELISA detected M. bovis-specific IgM antibody 2 weeks after infection with 97.1% sensitivity and had a specificity of 63.3%, while the IgG ELISA detected M. bovis-specific IgG 3 weeks after infection with 92.86% sensitivity and had a specificity of 98.7%, demonstrating that the IgG ELISA has potential for use as a sensitive and specific assay for detecting infection in cattle. PMID:24334686

  16. A highly rapid and simple competitive enzyme-linked immunosorbent assay for monitoring paralytic shellfish poisoning toxins in shellfish.

    PubMed

    Kawatsu, Kentaro; Kanki, Masashi; Harada, Tetsuya; Kumeda, Yuko

    2014-11-01

    Using a streptavidin-coated well plate, a biotin-labelled anti-gonyautoxin 2/3 monoclonal antibody GT-13A, and a decarbamoyl saxitoxin-peroxidase conjugate, a direct competitive enzyme-linked immunosorbent assay (PSP-ELISA) was developed for monitoring paralytic shellfish poisoning (PSP) toxins in shellfish. This assay is simple to perform and can be completed in approximately 20 min. The PSP-ELISA was compared to the mouse bioassay (MBA) for the detection of PSP toxins in shellfish samples (n=83) collected from the coast of Osaka Prefecture, Japan. When positive and negative results were indicated based on the regulatory limit for PSP toxins (4 mouse unit(MU)/g of shellfish meat), the PSP-ELISA results showed a sensitivity of 100% (25 of 25) and a specificity of 89.7% (52 of 58 samples) compared to the MBA results. These results suggest that the PSP-ELISA could be used as a rapid and simple screening method prior to the MBA. Copyright © 2014 Elsevier Ltd. All rights reserved.

  17. 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

  18. An indirect competitive enzyme-linked immunosorbent assay for determination of norfloxacin in waters using a specific polyclonal antibody.

    PubMed

    Cui, Jianlan; Zhang, Kun; Huang, Qiuxin; Yu, Yiyi; Peng, Xianzhi

    2011-02-28

    A specific polyclonal anti-norfloxacin antibody was obtained, and a sensitive indirect competitive enzyme-linked immunosorbent assay (icELISA) was developed for determining trace amounts of norfloxacin in various waters. Good linearity was achieved in the range from 0.1 to 10 μg L(-1). The average IC(50) value was determined to be 2.2 μg L(-1) and the limit of detection was 0.016 μg L(-1) at a signal-to-noise ratio of 3 in phosphate-buffered saline buffer. Recoveries of norfloxacin at various spiking levels ranged from 74 to 105% in groundwater, surface water, treated and untreated wastewater samples, with relative standard deviations of 3-5%. The assay was applied for determining norfloxacin in municipal wastewater, surface water, and groundwater collected in a metropolis of China. Raw wastewater samples were only submitted to filtration and pH adjustment while the other water samples were pre-concentrated by solid phase extraction prior to the icELISA assay. Good agreement of the results obtained by the icELISA and liquid chromatography tandem mass spectrometry further confirmed the reliability and accuracy of the icELISA for rapid detection of norfloxacin in waters. Copyright © 2010 Elsevier B.V. All rights reserved.

  19. The development of a noncompetitive enzyme-linked immunosorbent assay for oncorhynchid growth hormone using monoclonal antibodies.

    PubMed

    Farbridge, K J; Leatherland, J F

    1991-07-01

    The development of a sensitive and specific two-site, or sandwich, noncompetitive enzyme-linked immunosorbent assay (ELISA) for oncorhynchid growth hormone (GH) using monoclonal antibodies (MCAs) is reported. The MCAs were generated by the fusion of myeloma cells with spleen cells from mice that had been immunized with chum salmon (Oncorhynchus keta) recombinant GH. The MCAs specifically recognized the GH-secreting acidophils in the proximal pars distalis of immature male rainbow trout (Oncorhynchus mykiss) pituitaries. Affinity chromatography using one of the MCAs isolated a single protein with a molecular weight of 22,500 from a rainbow trout pituitary extract. The ELISA recognized recombinant chum salmon GH and the affinity-purified protein but did not recognize chum salmon prolactin, gonadotropin I or II, nor several mammalian hormone preparations. The ELISA recognized GH in rainbow trout, coho salmon (Oncorhynchus kisutch), and chinook salmon (Oncorhynchus tshawytscha) pituitary extracts, but not in goldfish (Carassius auratus) extracts, and recognized GH in rainbow trout, coho salmon, lake sturgeon (Acipenser fulvescens), and bowfin (Amia calva) plasma, but not in goldfish, yellow bullhead (Ictalurus natalis), or lamprey (Petromyzon marinus) plasma. The sensitivity of the ELISA was less than 1.56 ng/ml and circulating levels of GH in the plasma of coho salmon and rainbow trout plasma were measured as 75 and 35 ng equivalents/ml, respectively.

  20. Enzyme-linked immunosorbent assay for quantitation of attachment and ingestion stages of bacterial phagocytosis.

    PubMed Central

    Athamna, A; Ofek, I

    1988-01-01

    Research on phagocytosis of bacteria is often hampered by the inability to distinguish quantitatively between bacteria that have been ingested by phagocytic cells and those which are attached to the surface of the cells. A method using the enzyme-linked immunosorbent assay technique to simply and accurately measure the rate of bacterial ingestion by phagocytic cells is described. The method is based on the ability of antibacterial antibodies to bind to bacteria attached to but not internalized by phagocytic cells. The attached bacteria were quantitated by enzyme-linked immunosorbent assay. Compared with the number of bacteria at zero time (17 bacteria attached per phagocyte) only 10 to 20% of the bacteria remained attached to phagocytic cells after incubation for 30 min at 37 degrees C. The decrease in detected attached bacteria at 37 degrees C was due to internalization of the bacteria by phagocytic cells, since upon disruption of the monolayer, most of the ingested bacteria were recovered, and at 4 degrees C, most of the bacteria remained extracellularly attached. The proposed attachment and ingestion assay is easy to perform, allows the detection of specific attachment of test bacteria, and provides objective quantitation of attached and ingested bacteria. Most importantly, the assay allows testing of ingestion rates of bacteria under many variables on the same day. PMID:2893805

  1. Indirect immunofluorescence test and enzyme-linked immunosorbent assay for detection of Campylobacter pylori.

    PubMed Central

    Schaber, E; Umlauft, F; Stöffler, G; Aigner, F; Paulweber, B; Sandhofer, F

    1989-01-01

    An indirect immunofluorescence test (IIF) has been developed for detecting Campylobacter pylori in gastroduodenal biopsies. This test was compared with standard methods of C. pylori diagnosis, namely Gram staining and urease test, in a study population of 226 patients; 121 of the biopsy specimens were cultured for C. pylori as well. C. pylori colonization was detected in 154 of 226 patients (68%) by at least one of these methods (IIF, 96%; Gram staining, 78%; urease test, 60%; cultivation, 55%). Serum samples from 191 patients of the study population were screened for circulating antibodies to C. pylori by an indirect enzyme-linked immunosorbent assay with whole, untreated bacteria as antigen. Of these serum specimens, 140 (73%) revealed absorbance readings above the limit of positivity, which was determined as an optical density of greater than 0.35 at 405/620 nm. Of 132 serum specimens, 128 (97%) from patients with C. pylori detected in biopsies, but only 12 (20%) of 59 specimens from those without C. pylori detection showed elevated specific antibody levels. Our data revealed that IIF proved to be the superior rapid, sensitive, and specific diagnostic method. The correlation between microbiological findings and the immune response favors our enzyme-linked immunosorbent assay as an additional tool in C. pylori diagnosis. PMID:2644295

  2. Development of a monoclonal antibody against deoxynivalenol for magnetic nanoparticle-based extraction and an enzyme-linked immunosorbent assay.

    PubMed

    Lee, Hyuk-Mi; Song, Sung-Ok; Cha, Sang-Ho; Wee, Sung-Bok; Bischoff, Karyn; Park, Sung-Won; Son, Seong-Wan; Kang, Hwan-Goo; Cho, Myung-Haing

    2013-01-01

    Monoclonal antibody (mAb, NVRQS-DON) against deoxynivalenol (DON) was prepared. DON-Ag coated enzyme linked immunosorbent assay (ELISA) and DON-Ab coated ELISA were prepared by coating the DON-BSA and DON mAb. Quantitative DON calculation ranged from 50 to 4,000 ng/mL for DON-Ab coated ELISA and from 25 to 500 ng/mL for DON-Ag coated ELISA. 50% of inhibitory concentration values of DON, HT-2, 15-acetyl-DON, and nivalenol were 23.44, 22,545, 5,518 and 5,976 ng/mL based on the DON-Ab coated ELISA. Cross-reactivity levels of the mAb to HT-2, 15-acetyl-DON, and nivalenol were 0.1, 0.42, and 0.40%. The intra- and interassay precision coefficient variation (CV) were both <10%. In the mAb-coated ELISA, mean DON recovery rates in animal feed (0 to 1,000 mg/kg) ranged from 68.34 to 95.49% (CV; 4.10 to 13.38%). DON in a buffer solution (250, 500 and 1,000 ng/mL) was isolated using 300 mg of NVRQS-DON and 3 mg of magnetic nanoparticles (MNPs). The mean recovery rates of DON using this mAb-MNP system were 75.2, 96.9, and 88.1% in a buffer solution spiked with DON (250, 500, and 1,000 ng/mL). Conclusively we developed competitive ELISAs for detecting DON in animal feed and created a new tool for DON extraction using mAb-coupled MNPs.

  3. 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.

  4. 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.

    PubMed

    Muller, Jennifer K; Sepúlveda, María S; Borgert, Christopher J; Gross, Timothy S

    2005-08-01

    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.

  5. Validation of a Monoclonal Antibody-Based Capture Enzyme-Linked Immunosorbent Assay for Detection of Campylobacter fetus

    PubMed Central

    Devenish, J.; Brooks, B.; Perry, K.; Milnes, D.; Burke, T.; McCabe, D.; Duff, S.; Lutze-Wallace, C. L.

    2005-01-01

    A monoclonal antibody (MAb)-based antigen capture enzyme-linked immunosorbent assay (ELISA) was compared with the routine culture methodology for the detection of Campylobacter fetus subspecies from bovine and ovine field samples inoculated into Clark's transport enrichment medium (TEM). The work was a collaboration between two different diagnostic laboratories, one in Canada and the other in England. In both labs, TEM samples were incubated for 4 days at 35°C and then tested by culture and ELISA. The ELISA consisted of initial screening with MAb M1825 against C. fetus subspecies core lipopolysaccharide (LPS). All samples positive on ELISA screening were then retested by ELISA with MAb M1825 and MAbs M1177, M1183, and M1194, which recognize serotype A- and/or serotype B-specific C. fetus subspecies LPS epitopes. The Canadian samples consisted of 1,060 preputial washings from 529 bulls, of which 18 were positive by both culture and ELISA and 1,042 were negative by both methods. The English samples consisted of 321 tissue specimens, mostly stomach contents and placentas, from 190 aborted ovine and bovine fetuses. A total of 262 samples were negative by culture and ELISA, 52 samples were positive by culture and ELISA, and 7 samples were culture negative but ELISA positive. The results for all 70 culture-positive isolates were confirmed by conventional biochemical methods as C. fetus subsp. fetus, with 39 presumptively identified by the ELISA as serotype A and 30 presumptively identified as serotype B and with one sample containing isolates presumptively identified as serotype A and serotype B. A receiver operating characteristic analysis of the combined ELISA data from both countries resulted in an area under the curve of 0.997, with a sensitivity of 100% and a specificity of 99.5% relative to the results of culture. The data confirm that this ELISA method can be used as an excellent test for the screening of field samples in TEM for the presence of C. fetus

  6. Validation of a monoclonal antibody-based capture enzyme-linked immunosorbent assay for detection of Campylobacter fetus.

    PubMed

    Devenish, J; Brooks, B; Perry, K; Milnes, D; Burke, T; McCabe, D; Duff, S; Lutze-Wallace, C L

    2005-11-01

    A monoclonal antibody (MAb)-based antigen capture enzyme-linked immunosorbent assay (ELISA) was compared with the routine culture methodology for the detection of Campylobacter fetus subspecies from bovine and ovine field samples inoculated into Clark's transport enrichment medium (TEM). The work was a collaboration between two different diagnostic laboratories, one in Canada and the other in England. In both labs, TEM samples were incubated for 4 days at 35 degrees C and then tested by culture and ELISA. The ELISA consisted of initial screening with MAb M1825 against C. fetus subspecies core lipopolysaccharide (LPS). All samples positive on ELISA screening were then retested by ELISA with MAb M1825 and MAbs M1177, M1183, and M1194, which recognize serotype A- and/or serotype B-specific C. fetus subspecies LPS epitopes. The Canadian samples consisted of 1,060 preputial washings from 529 bulls, of which 18 were positive by both culture and ELISA and 1,042 were negative by both methods. The English samples consisted of 321 tissue specimens, mostly stomach contents and placentas, from 190 aborted ovine and bovine fetuses. A total of 262 samples were negative by culture and ELISA, 52 samples were positive by culture and ELISA, and 7 samples were culture negative but ELISA positive. The results for all 70 culture-positive isolates were confirmed by conventional biochemical methods as C. fetus subsp. fetus, with 39 presumptively identified by the ELISA as serotype A and 30 presumptively identified as serotype B and with one sample containing isolates presumptively identified as serotype A and serotype B. A receiver operating characteristic analysis of the combined ELISA data from both countries resulted in an area under the curve of 0.997, with a sensitivity of 100% and a specificity of 99.5% relative to the results of culture. The data confirm that this ELISA method can be used as an excellent test for the screening of field samples in TEM for the presence of C. fetus

  7. Specificity of Cryptococcus neoformans factor sera determined by enzyme-linked immunosorbent assay and dot enzyme assay.

    PubMed Central

    Belay, T; Cherniak, R; Shinoda, T

    1993-01-01

    An indirect enzyme-linked immunosorbent assay (ELISA) and a dot enzyme assay (DEA) were used to determine the specificities of Cryptococcus neoformans factor sera to serotype type-specific capsular polysaccharides, glucuronoxylomannans (GXMs). Pure and chemically characterized GXMs were obtained from representative isolates of C. neoformans serotypes A, B, C, and D. Distinctive specificity patterns and quantitative differences were observed for each factor serum when the selected GXMs were studied by ELISA. The specificity patterns for each factor serum determined by DEA almost completely paralleled the ELISA results. The serotype specificities demonstrated by ELISA and DEA were similar to previously reported results that were obtained by slide agglutination studies of whole cells. On the basis of the ELISA and DEA activity patterns, factor sera 5, 6, and 8 were specific for serotypes B, C, and D, respectively; factor serum 1 was strongly reactive to all serotypes; factor serum 2 was specific for serotypes A, B, and D; factor serum 3 was specific for serotypes A and D; and factor serum 4 was specific for serotypes B and C. The specificity of factor serum 7 for serotype A was demonstrated by DEA only. Structural variation was indicated among the serotype C isolates studied because a unique activity pattern versus factor serum 6 was observed for each isolate. The quantitative differences in the activity of the GXMs from five serotype C isolates suggest that mannopyranoside residues substituted O-2 and O-4 with xylose are essential elements of the determinant responsible for the observed activity of factor 6. No significant differences in activity patterns and specificities of factor serum 6 were observed when O-deacetylated GXMs were substituted for the native GXMs. Our results show that ELISA and DEA are valuable techniques for the serological analysis of cryptococcal factor sera and GXMs. Images PMID:7685739

  8. Limitations of the BP26 protein-based indirect enzyme-linked immunosorbent assay for diagnosis of Brucellosis.

    PubMed

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

    2013-09-01

    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.

  9. Development of an Enzyme Linked Immunosorbent Assay and an Immunochromatographic Assay for Detection of Organophosphorus Pesticides in Different Agricultural Products

    PubMed Central

    Hua, Xiude; Yang, Jifei; Wang, Limin; Fang, Qingkui; Zhang, Gaiping; Liu, Fengquan

    2012-01-01

    Objective Organophosphorus (OP) pesticides are considered hazardous substances because of their high toxicity to nontarget species and their persistence in the environment and agricultural products. Therefore, it is important to develop a rapid, sensitive, and economical method for detecting OP pesticides and their residues in food and the environment. Methods A broad, selective monoclonal antibody (MAb) for organophosphorus pesticides was produced. Based on the MAb, an enzyme linked immunosorbent assay (ELISA) and an immunochromatography assay (ICA) for detecting OP pesticides in different agricultural products were developed using a binding inhibition format on microtiter plates and a membrane strip, respectively. Results Under the optimized conditions, the IC50 values of the ELISA ranged from 3.7 to 162.2 ng mL–1 for the 8 OP pesticides. The matrix interferences of Apple, Chinese cabbage, and greengrocery were removed by 40-fold dilution, the recoveries from spiked samples ranged from 79.1% to 118.1%. The IC50 values of ICA for the 8 OP pesticides ranged from 11.8 to 470.4 ng mL−1. The matrix interference was removed from the Chinese cabbage and Apple samples with 5-fold dilution, and the interference was removed from the greengrocery samples with 20-fold dilution. The recoveries from the spiked samples ranged between 70.6 and 131.9%. The established ELISA and ICA were specific selectivity for the 8 OP pesticides. Conclusions The established ELISA is a sensitive screening method for the detection of OP pesticides, but the ELISA detection method depends on a laboratory platform and requires a relative long assay time and several steps operation. The established ICA is very useful as a screening method for the quantitative, semi-quantitative or qualitative detection of OP pesticides in agricultural products, and it has advantages over ELISA methods with regard to factors such as the testing procedure, testing time, and matrix interferences, among others. PMID

  10. 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

  11. 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.

  12. Use of the protective antigen of Erysipelothrix rhusiopathiae in the enzyme-linked immunosorbent assay and latex agglutination.

    PubMed

    Sato, H; Yamazaki, Y; Tsuchiya, K; Aoyama, T; Akaba, N; Suzuki, T; Yokoyama, A; Saito, H; Maehara, N

    1998-09-01

    To establish a safe and convenient serodiagnostic method for swine erysipelas, a purified protective protein antigen of Erysipelothrix rhusiopathiae, which included a large amount of protective protein (64 kDa protein), was used for enzyme-linked immunosorbent assay (ELISA) and the latex agglutination (LA) test. In the ELISA, the antisera to four different serovars (1a, 2, 5 and 20) of E. rhusiopathiae exhibit a positive reaction, while antisera to other species of bacteria (Listeria monocytogenes, Staphylococcus aureus, Streptococcus suis, Rhodococcus equi and Corynebacterium pseudotuberculosis) exhibit a negative reaction. In the LA test, the antisera to three different serovars (1a, 2 and 5) of E. rhusiopathiae reacted with P64-sensitized latex beads, while the antiserum to serovar 20 (2553 strain) did not. Moreover, the antisera to other species of bacteria (Listeria monocytogenes, Staphylococcus aureus, Streptococcus suis, Rhodococcus equi and Corynebacterium pseudotuberculosis) did not in this test. Comparing the results of the growth agglutination (GA), ELISA and LA tests of 284 swine sera, there was a high degree of correlation among the results. The detection of anti-E. rhusiopathiae antibodies in the GA, ELISA and LA tests were compared using sera from pigs immunized with P64, alkaline extract (AE) and live-cell vaccine (LV). In all three tests, anti-E. rhusiopathiae antibodies could be detected 1 week after immunization. The serum antibody titre as determined by the LA test increased moderately, as did that by the GA test, while that determined by ELISA increased rapidly. These results suggested that ELISA could be used to monitor changes in anti-E. rhusiopathiae antibody titre and the LA test could be used in the screening test for swine erysipelas.

  13. Evaluation of three enzyme-linked immunosorbent assays for sarcoptic mange diagnosis and assessment in the Iberian ibex, Capra pyrenaica.

    PubMed

    Ráez-Bravo, Arián; Granados, José Enrique; Serrano, Emmanuel; Dellamaria, Debora; Casais, Rosa; Rossi, Luca; Puigdemont, Anna; Cano-Manuel, Francisco Javier; Fandos, Paulino; Pérez, Jesús María; Espinosa, José; Soriguer, Ramón Casimiro; Citterio, Carlo; López-Olvera, Jorge Ramón

    2016-10-21

    Sarcoptic mange is a contagious skin disease caused by the mite Sarcoptes scabiei, affecting different mammalian species worldwide including the Iberian ibex (Capra pyrenaica), in which mortalities over 90 % of the population have been reported. No efficient diagnostic methods are available for this disease, particularly when there are low mite numbers and mild or no clinical signs. In this study, three enzyme-linked immunosorbent assays (ELISA) developed for dog (ELISA A), Cantabrian chamois (Rupicapra pyrenaica parva) (ELISA B) and Alpine chamois (Rupicapra rupicapra) (ELISA C), were evaluated to detect specific antibodies (IgG) to sarcoptic mange in Iberian ibex sera. Serum samples from 131 Iberian ibexes (86 healthy and 45 scabietic) were collected from 2005 to 2012 in the Sierra Nevada Natural and National Parks (southern Spain). Based on visual inspection, ibexes were classified into one of three categories, namely healthy (without scabietic compatible lesions), mildly affected (skin lesions over less than 50 % of the body surface) and severely affected (skin lesions over more than 50 % of the body surface). The optimal cut-off point, specificity, sensitivity and the area under the curve (AUC) were calculated, and the agreement between tests was determined. Moreover, differences in the optical density (OD) related to scabies severity have been evaluated for the best test. ELISA C showed better performance than the two other tests, reaching higher values of sensitivity (93.0 %) and specificity (93.5 %) against the visual estimation of the percentage of affected skin, chosen as the gold standard. Significantly higher concentrations of specific antibodies were observed with this test in the mildly and severely infested ibexes than in healthy ones. Our results revealed that ELISA C was an optimal test to diagnose sarcoptic mange in the Iberian ibex. Further studies characterizing immune response during the course of the disease, including spontaneous or drug

  14. Comparison of performance of serum and plasma in panbio dengue and Japanese encephalitis virus enzyme-linked immunosorbent assays.

    PubMed

    Blacksell, Stuart D; Lee, Sue J; Chanthongthip, Anisone; Taojaikong, Thaksinaporn; Thongpaseuth, Soulignasack; Hübscher, Tanja; Newton, Paul N

    2012-09-01

    We examined the comparative performance of serum and plasma (in dipotassium EDTA) in Panbio Dengue enzyme-linked immunosorbent assays (ELISAs) for detection of non-structural protein 1 (NS1), IgM, and IgG, and a dengue/Japanese encephalitis virus (JEV) combination IgM ELISA in a prospective series of 201 patients with suspected dengue in Laos. Paired comparisons of medians from serum and plasma samples were not significantly different for Dengue IgM, and NS1 which had the highest number of discordant pairs (both 2%; P = 0.13 and P = 0.25, respectively). Comparison of qualitative final diagnostic interpretations for serum and plasma samples were not significantly different: only 1.5% (3 of 201 for Dengue/JEV IgM and Dengue IgG) and 2.0% (4 of 201; IgM and NS1) showed discordant pairs. These results demonstrate that plasma containing EDTA is suitable for use in these ELISAs.

  15. 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

  16. Borrelia burgdorferi enzyme-linked immunosorbent assay for discrimination of OspA vaccination from spirochete infection.

    PubMed Central

    Zhang, Y Q; Mathiesen, D; Kolbert, C P; Anderson, J; Schoen, R T; Fikrig, E; Persing, D H

    1997-01-01

    Recombinant Lyme disease vaccines based on purified preparations of outer surface protein A (OspA) have been shown to be effective in preventing transmission of Borrelia burgdorferi in experimental animal models and are now being tested in humans. Since the most widely used screening tests for Lyme disease are based on a whole-cell sonicate of B. burgdorferi, serologic false positivity in vaccinated persons could result from reactivity to OspA within the antigen preparation. In order to avoid serologic false positivity in vaccinated subjects, we developed an immunoassay based on a low-passage-number, naturally occurring variant of B. burgdorferi which lacks the plasmid encoding OspA and OspB. The use of an antigen preparation derived from this organism provided sensitive and specific detection of B. burgdorferi seropositivity in experimental animals and in human Lyme disease cases. The OspA-B-negative enzyme-linked immunosorbent assay (ELISA) also appeared to be capable of discriminating the vaccinated state from vaccine failure and natural infection in experimental animals. Sera from human subjects participating in a vaccine trial gave false-positive results with an ELISA based on an OspA-containing strain, but no such reactivity was observed when the OspA-negative ELISA was used. We conclude that low-passage-number OspA-B-negative isolates in immunoassays may become useful for the immunologic discrimination of the vaccinated state, natural infection, and vaccine failure. PMID:8968914

  17. Screening survey of deoxynivalenol in beer from the European market by an enzyme-linked immunosorbent assay.

    PubMed

    Papadopoulou-Bouraoui, A; Vrabcheva, T; Valzacchi, S; Stroka, J; Anklam, E

    2004-06-01

    Deoxynivalenol (DON) was analysed in 313 beer samples collected from the European retail market using a commercially available immunoassay kit (enzyme-linked immunosorbent assay, ELISA). The incidence rate was about 87%, while most samples (73%) had contamination levels lower than 20 ng m(-1). The contamination ranged between 4.0 and 56.7 ng ml(-1), with an average of 13.5 ng ml(-1). A statistically significant correlation between alcohol levels and DON contamination was found, as well as a significant difference between bottom, top and spontaneous fermenting beers. Twenty-seven beer samples were compared using a second ELISA kit and a good correlation was obtained between the two kits (r = 0.93). Although when compared with gas chromatography-mass spectrometry the ELISA tended to overestimate the results, a good correlation (r=0.94) between the two methods was observed. Monitoring of DON in beer is important considering that DON production is dependent on the weather and that it can contribute significantly to the tolerable daily intake of DON, especially for frequent beer consumers.

  18. 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.

  19. A novel hapten and monoclonal-based enzyme-linked immunosorbent assay for sulfonamides in edible animal tissues.

    PubMed

    Zhou, Qi; Peng, Dapeng; Wang, Yulian; Pan, Yuanhu; Wan, Dan; Zhang, Xiya; Yuan, Zonghui

    2014-07-01

    For high-throughput monitoring of the residues of sulfonamides (SAs) in edible animal tissues, a novel hapten and monoclonal-based indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) was developed. The novel hapten was synthesized and conjugated to carrier protein as immunogen. The spleen cells of the inoculated mice expressing group-specificity against SAs were fused. The obtained monoclonal antibody 4E5 showed the cross-reactivity (CR) to 16 structurally different SAs. Based on this antibody, an optimised icELISA protocol was carried out with only phosphate-buffered saline for the fast extraction of SAs in the tissues. The limits of detection of SAs in chicken ranged from 1.5 to 22.3μgkg(-1). The recoveries were 70.6-121% with less than 24.1% relative standard deviation. The developed ic-ELISA showed a good correlation with high performance liquid chromatography. It would be a useful tool for the screening of residues of SAs in edible animal tissues. Copyright © 2014 Elsevier Ltd. All rights reserved.

  20. Nationwide survey of leptospira antibodies in dogs in Japan: results from microscopic agglutination test and enzyme-linked immunosorbent assay.

    PubMed

    Iwamoto, Emiko; Wada, Yuko; Fujisaki, Yuka; Umeki, Saori; Jones, Miyuki Y; Mizuno, Takuya; Itamoto, Kazuhito; Maeda, Ken; Iwata, Hiroyuki; Okuda, Masaru

    2009-09-01

    Leptospirosis is an infectious disease caused by Leptospira interrogans sensu lato and is common in both humans and animals. In the present study, serum samples were collected from 801 dogs across all 47 prefectures in Japan, and evaluated with a microscopic agglutination test (MAT), using 5 major L. interrogans serovars (Icterohaemorrhagiae, Canicola, Autumnalis, Hebdomadis, and Australis) as antigens, and an enzyme-linked immunosorbent assay (ELISA) using recombinant OmpL1 protein as the antigen. Across all dogs tested, 217 (27.0%) and 29 (3.6%) were MAT- and ELISA-positive, respectively. However, evidence strongly suggests that MAT also detected antibodies produced by vaccination. Of 243 dogs never inoculated with any canine vaccine, 41 (16.9%) from 23 prefectures were MAT and/or ELISA positive. The most commonly detected serovar was Icterohaemorrhagiae (22 dogs, 19 prefectures). Our results suggest that there are dogs with subclinical Leptospira infection throughout Japan. To the best of our knowledge, the present study is the first nationwide survey of Leptospira infection in dogs, and the findings are relevant not only for clinical veterinary medicine but also for public health.

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

    PubMed

    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.

  2. Anti-Protective Antigen IgG Enzyme-Linked Immunosorbent Assay for Diagnosis of Cutaneous Anthrax in India

    PubMed Central

    Ghosh, N.

    2012-01-01

    Anthrax caused by Bacillus anthracis is a public health problem in several developing countries whose main source of income is farming. Anthrax is a disease of herbivorous animals, and humans can be infected by handling infected animals or contaminated animal products. Specific diagnostic tests are unavailable in India for the detection and confirmation of cutaneous anthrax in humans. Here, we describe the development of an enzyme-linked immunosorbent assay (ELISA) for detection of serum antibodies against Bacillus anthracis protective antigen in the Indian population. A total of 405 serum samples collected from different groups were tested by the developed ELISA. The assay provided a specificity of 99.41% (95% confidence interval [CI], 97.89 to 99.93) and a sensitivity of 100% (CI, 94.4 to 100) using a cutoff value of 0.29 ELISA unit (EU). The positive predictive value (PPV) and negative predictive value (NPV) of the assay were 97% and 100%, respectively. The efficiency and J index for the reliability of the assay were 99.5% and 0.994, respectively. The assay can be a very useful tool for surveillance as well as for diagnosis of cutaneous anthrax cases in India. PMID:22718130

  3. PCR enzyme-linked immunosorbent assay for diagnosis of leishmaniasis in human immunodeficiency virus-infected patients.

    PubMed Central

    Costa, J M; Durand, R; Deniau, M; Rivollet, D; Izri, M; Houin, R; Vidaud, M; Bretagne, S

    1996-01-01

    A PCR enzyme-linked immunosorbent assay (ELISA) involving the use of bone marrow aspirates (BMA) and blood samples (BS) for the diagnosis of visceral leishmaniasis (VL) in human immunodeficiency virus-infected patients was developed with primers selected from the sequence of the small-subunit rRNA gene and compared with direct examination and in vitro cultivation. The PCR was optimized for routine diagnosis: processing of samples with lysis of erythrocytes without isolation of leukocytes, enzymatic prevention of contamination, internal control of the reaction, and ELISA testing in a microtitration plate hybridization. Of 79 samples (33 BMA and 46 BS) from 77 patients without VL, all the results were negative. Fifty-three samples (9 BMA and 44 BS) were obtained from 13 patients with VL: 6 samples drawn during anti-Leishmania treatment were negative whatever the technique used, and 47 samples (9 BMA and 38 BS) were positive with at least one technique. The sensitivities were 51% (24 of 47), 81% (38 of 47), and 98% (46 of 47) for direct examination, culture, and PCR, respectively. Thus, PCR ELISA is reliable for diagnosing VL in human immunodeficiency virus-infected patients, and blood sampling should be sufficient for the follow-up. PMID:8784604

  4. Determination of urinary and salivary cotinine using gas and liquid chromatography and enzyme-linked immunosorbent assay.

    PubMed

    Kuo, H W; Yang, J S; Chiu, M C

    2002-03-05

    The objective of this study was to compare cotinine concentrations in urine and saliva using gas chromatography (GC), high-performance liquid chromatography (HPLC) and enzyme-linked immunosorbent assay (ELISA). Ninety-four subjects were selected (27 smokers and 67 non-smokers) and interviewed using questionnaire. Of the non-smokers, 39 had been exposed to environmental tobacco smoke (ETS) and 28 had not been exposed to ETS. Cotinine levels among smokers were highest using all three measurements, followed by ETS exposed subjects and non-smokers. Cotinine levels in urine, using HPLC, correlated significantly with levels measured using ELISA (r=0.92) and GC-nitrogen-phosphorus detection (NPD) (r=0.92). Salivary cotinine levels measured using ELISA did not correlate significantly with either HPLC (r=0.37) or GC-NPD (r=0.33) measurements. Multiple regression models were used to adjust for age, gender, drug use and health status, and it was found that cotinine levels in urine and saliva were significantly correlated with smoking pack-year. The authors conclude that urinary cotinine concentration is a more accurate biomarker for ETS than salivary cotinine concentration.

  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. Monoclonal antibody production and the development of an indirect competitive enzyme-linked immunosorbent assay for screening spiramycin in milk.

    PubMed

    Jiang, Wenxiao; Zhang, Huiyan; Li, Xiangmei; Liu, Xinxin; Zhang, Suxia; Shi, Weimin; Shen, Jianzhong; Wang, Zhanhui

    2013-11-20

    To monitor spiramycin (SP) residue in milk, a monoclonal antibody (mAb)-based indirect competitive enzyme-linked immunosorbent assay (icELISA) was developed. This study described the preparation of three immunogens and the production of a high-affinity mAb. After optimization, the 50% inhibition concentration (IC50) for the developed icELISA was estimated as 0.97 ng/mL in the assay buffer, and the limit of detection and limit of quantitation were 2.51 and 4.40 μg/L in the milk matrix. The newly developed assay demonstrated negligible cross-reactivity with 15 other macrolide antibiotics, but not with kitasamycin (23.4%). The mean recoveries ranged from 81 to 103% for the spiked samples (5, 10, and 50 μg/L), and the coefficient of variation ranged from 5.4 to 9.6%. The icELISA was validated by LC-MS/MS method, and all results demonstrated that it was a suitable screening method for detecting SP residue in milk without requiring a cleanup process.

  7. Immunologic diversity among serogroup 1 Legionella pneumophila urinary antigens demonstrated by monoclonal antibody enzyme-linked immunosorbent assays.

    PubMed Central

    Kohler, R B; Wilde, C; Johnson, W; Joly, J; Wheat, L J; Baker, R; Misfeldt, M

    1988-01-01

    We tested urine specimens from 222 patients with serogroup 1 Legionella pneumophila pneumonia in two enzyme-linked immunosorbent assays (ELISAs) which used different monoclonal antibodies (A and B) as detector antibodies. Of 171 specimens which contained enough antigen to be detected in the ELISAs, 169 reacted in only one of the two assays. A total of 25 patients whose infections were acquired in any of three Indianapolis hospitals excreted antigen reactive with monoclonal antibody B, but 18 patients who were treated for infections acquired elsewhere reacted with monoclonal antibody A. The urinary antigen ELISA reactivity patterns correlated with the reactivity patterns of L. pneumophila isolates when a separate panel of seven monoclonal antibodies was used. The isolate patterns, in turn, correlated well with environmental isolate patterns from two of the hospitals with nosocomial cases. We conclude that at least two different epitopes exist on the antigen molecules in urine from patients with serogroup 1 L. pneumophila pneumonia and that the subtyping of urinary antigens can be useful epidemiologically. PMID:2460492

  8. 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.

  9. Effect of heat inactivation of serum on Bordetella pertussis antibody determination by enzyme-linked immunosorbent assay.

    PubMed

    Lopez, A L; Pineda, E; Garakian, A; Cherry, J D

    1998-01-01

    The effect of heat inactivation on Bordetella pertussis antibodies determined by enzyme-linked immunosorbent assay (ELISA) was studied. Sera were heated at increasing temperatures (from 30 to 50 degrees C at 5 degrees C increments and from 52 to 70 degrees C at 2 degrees C increments). Between 30 and 50 degrees C, no significant differences were observed in immunoglobin G (IgG) antibodies to pertussis toxin (PT). From 50 to 56 degrees C the antibody values were twofold higher than those of uninactivated sera; at 64 degrees C the values were 3.6- to 9.1-fold higher. The increase in PT IgG antibody values was more pronounced in sera with low antibody values. ELISA antibody values of sera from a vaccine trial were determined in unheated and heat inactivated sera. The geometric mean value (GMV) of the heat inactivated samples was 3.2 times the geometric mean value of the uninactivated sera. ELISA IgG antibodies to filamentous hemagglutinin, fimbriae-2, and pertactin were studied, and the values of heat inactivated sera did not differ significantly from the values of the uninactivated sera. Our findings indicate that heat inactivation of sera leads to higher, variable, and false-positive PT and IgG values.

  10. Detection of residual toxin in tissues of ricin-poisoned mice by sandwich enzyme-linked immunosorbent assay and immunoprecipitation.

    PubMed

    Men, Jinshuan; Lang, Liwei; Wang, Chenyu; Wu, Junhua; Zhao, Yu; Jia, Pei-Yuan; Wei, Wenqing; Wang, Yuxia

    2010-06-15

    This work aimed to evaluate a method to detect the residual ricin in animal tissues. Immunoprecipitation and sandwich enzyme-linked immunosorbent assay (ELISA) were used to detect ricin in the tissues of intoxicated mice. The monoclonal antibodies (Mabs) 4C13 and 3D74 were used to assay the whole ricin molecules via sandwich ELISA. Mab 4C13 was conjugated with Sepharose 4B to capture ricin or ricin A chain by immunoprecipitation. Mice injected intravenously with ricin at the dosage of 5 microg/mouse were killed at different time points after intoxication. The serum, liver, kidney, lung, and intestine were harvested. High levels of ricin were found in serum and liver samples at each poisoning time point by sandwich ELISA, suggesting the possibility of determining ricin intoxication by detecting residual ricin in the serum. However, this method turned out to be ineffective for examining ricin in the kidney, lung, and intestine of poisoned mice. Although the same tissue samples of intoxicated mice were analyzed by immunoprecipitation, positive bands were found. This indicated that some components in the kidney, lung, and intestine could bind with ricin and interfere in its binding activity with the coated antibody. Immunoprecipitation could be used to measure the existence of ricin in these samples. Copyright (c) 2010 Elsevier Inc. All rights reserved.

  11. Enzyme-linked immunosorbent assay for measuring ileal symbiont intracellularis-specific immunoglobulin G response in sera of pigs.

    PubMed Central

    Holyoake, P K; Cutler, R S; Caple, I W; Monckton, R P

    1994-01-01

    Proliferative enteritis (PE) is a common intestinal disease on pig farms. The disease is caused by ileal symbiont (IS) intracellularis (Campylobacter-like organisms) bacteria. An enzyme-linked immunosorbent assay (ELISA) was developed to measure IS intracellularis-specific immunoglobulin G (IgG) response in the sera of pigs. The antigen used in the ELISA was filtered, percoll gradient-purified IS intracellularis extracted from the intestines of pigs affected with proliferative hemorrhagic enteropathy. The antibody responses of pigs challenged with intestinal homogenates from pigs affected with proliferative hemorrhagic enteropathy containing IS intracellularis or percoll-gradient purified IS intracellularis were low and variable. The low IgG titers measured in challenged pigs support previous findings that IgG plays a minor role in the immune response of pigs to IS intracellularis. On a farm in which infection was endemic, pigs seroconverted at between 7 and 24 weeks of age. High IgG titers, indicative of maternally acquired antibody, were present in 3-week-old pigs. The IgG titers in piglets were lowest at 6 weeks of age, which approximates the age of onset of clinical disease. These results suggest that IgG plays a role in determining the susceptibilities of pigs to natural infection. Measurements of seroconversion by the ELISA might aid in epidemiological investigations of PE in naturally infected herds. However, the variable antibody responses in experimentally challenged pigs would seem to limit its usefulness as an antemortem diagnostic test for PE. PMID:7989553

  12. 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. © 2016 The Author(s).

  13. 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.

  14. Evaluation of enzyme-linked immunosorbent assays for detection of Mycoplasma bovis-specific antibody in bison sera.

    PubMed

    Register, Karen B; Sacco, Randy E; Olsen, Steven C

    2013-09-01

    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.

  15. A sensitive and selective enzyme-linked immunosorbent assay for the analysis of Para red in foods.

    PubMed

    Wang, Jia; Wei, Keyi; Li, Hao; Li, Qing X; Li, Ji; Xu, Ting

    2012-05-07

    Para red is a synthetic dye and a potential genotoxic carcinogen. A hapten mimicking Para red structure was synthesized by introducing a carboxyl to the naphthol part of Para red and coupled to carrier protein to form an immunogen for the production of specific antibodies. A sensitive and selective enzyme-linked immunosorbent assay (ELISA) was developed for the detection of Para red in food samples. The limit of detection and inhibition half-maximum concentrations of Para red in phosphate buffered saline with 10% methanol were 0.06 and 2.2 ng mL(-1), respectively. Cross-reactivity values of the ELISA with the tested compounds including Sudan red I, II, III, IV, and G, sunset yellow, 2-naphthol, and 4-nitroaniline were ≤0.2%. This assay was used to determine Para red in tomato sauce, chilli sauce, chilli powder and sausage samples after ultrasonic extraction, cleanup and concentration steps. The average recoveries, repeatability (intraday extractions and analysis), and intra-laboratory reproducibility (interday extractions and analysis) were in the range 90-108%, 4-12% and 8-17%, respectively. This assay was compared to a high-performance liquid chromatographic method for 28 samples, displaying a good correlation (R(2) = 0.95). Para red residues in 53 real world samples determined by ELISA were below the limit of detection.

  16. Monoclonal antibody capture enzyme-linked immunosorbent assay for detection of bovine enteric coronavirus.

    PubMed Central

    Crouch, C F; Raybould, T J; Acres, S D

    1984-01-01

    Monoclonal antibodies reactive with three different viral polypeptides were evaluated singly and in combination as the capture antibody(s) in an enzyme-linked immunosorbent assay system for the detection of bovine enteric coronavirus. Similar levels of sensitivity were found for all combinations tested. A sensitive, highly specific, and reproducible assay for the detection of bovine enteric coronavirus was developed, using a mixture of two of these monoclonal antibodies reactive with antigenic components either external or internal to the virion. These monoclonal antibodies were bound indirectly to 96-well plates via rabbit anti-mouse immunoglobulin. After sample application and incubation, virus was detected by using rabbit anti-coronavirus peroxidase conjugate followed by enzyme substrate and chromagen. Fecal samples from a single herd of cows were screened for the presence of coronavirus by this assay. Five percent of clinically normal cows were found to be shedding coronavirus. Images PMID:6325490

  17. Preparation of Mycoplasma hyopneumoniae antigen for the enzyme-linked immunosorbent assay.

    PubMed Central

    Kazama, S; Yagihashi, T; Seto, K

    1989-01-01

    Methods of preparation of Mycoplasma hyopneumoniae antigens for the enzyme-linked immunosorbent assay to detect specific antibody, and properties of the antigens, are described. The reactivity and specificity of antigen prepared by Sephacryl S-300 column chromatography after treatment of M. hyopneumoniae cells with Tween 20 (S-300 antigen) were superior to those of antigen prepared by Sephadex G-25 column chromatography after treatment with Tween 20, or to lipid antigen. There were no differences among strains MI-3, J and VPP11 of M. hyopneumoniae. The S-300 antigen did not show cross-reactivity against porcine hyperimmune sera produced by M. hyorhinis, M. hyosynoviae, M. hyopharyngis, M. flocculare and Acholeplasma granularum. Antibody was first detected in sera of pigs inoculated intranasally with M. hyopneumoniae at two to four weeks after inoculation and seven to eight weeks after pigs were contact-exposed to the same mycoplasma. PMID:2523756

  18. 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

  19. Establishment of indirect enzyme-linked immunosorbent assay for artificial musk.

    PubMed

    Shen, Fang; Bai, Jin-Ye; Meng, Yu; Xiao, Xuan; Zhang, Shu; Zhu, Xiu-Yuan; Cheng, Gui-Fang

    2014-12-01

    Anti-inflammatory component (AIC) known as an important constituent of artificial musk exhibits bioactive effects on many pharmacological models. This study describes an immunochemical assay for the quality control of artificial musk in traditional Chinese medicine using an enzyme-linked immunosorbent assay. A polyclonal antibody against AIC was produced by rabbit. The method, at an effective measuring range of 3.13-200 ng/ml of AIC, 3 ng/ml limit of detection, and no cross-reaction with natural musk, successfully detected artificial musk in many traditional Chinese prescriptions containing artificial musk. The results demonstrated that a novel and reliable assay system for detection of artificial musk was generated.

  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. Validation and Field Evaluation of a Competitive Enzyme-Linked Immunosorbent Assay for Diagnosis of Babesia bovis Infections in Argentina

    PubMed Central

    Dominguez, Mariana; Echaide, Ignacio; de Echaide, Susana Torioni; Wilkowsky, Silvina; Zabal, Osvaldo; Mosqueda, Juan J.; Schnittger, Leonhard

    2012-01-01

    Infections by Babesia bovis limit cattle production and cause important economic losses in tropical and subtropical areas around the world. Monitoring of calf sera can be used to detect unprotected cattle herds and to decide on strategic control measures, as well as for epidemiological studies. Merozoite surface antigen 2c (MSA-2c) is an immunodominant surface protein expressed in B. bovis merozoites and sporozoites and contains B-cell epitopes that are conserved among geographic isolates. A monoclonal antibody against recombinant MSA-2c (rMSA-2c) was previously shown to inhibit the binding of anti-B. bovis antibodies to a parasite B-cell epitope in a competitive enzyme-linked immunosorbent assay (cELISA) format. In the work at hand, the parameters of this cELISA were reevaluated and adjusted when necessary, and a cutoff value was determined by receiver operator characteristic (ROC) curve analysis of a total of 357 bovine sera of known reactivity, as assessed by indirect immunofluorescence assay (IFAT). The established rMSA-2c cELISA demonstrated a specificity of 98% and a sensitivity of 96.2%. An additional set of 303 field bovine sera from regions where ticks are endemic and tick-free regions of Argentina was tested by both rMSA-2c cELISA and IFAT, and the results were shown to be in very good agreement (kappa index, 0.8325). The performance shown by rMSA-2c cELISA in the detection of B. bovis-specific antibodies and its suitability for standardization and large-scale production, as well as the possibility of its application in most veterinary diagnostic laboratories, make the assay a powerful tool for the surveillance of herd immunity as a strategic measure for the control of bovine babesiosis. PMID:22492742

  2. Monoclonal Antibody-Based Competitive Enzyme-Linked Immunosorbent Assay for Detection of Morbillivirus Antibody in Marine Mammal Sera

    PubMed Central

    Saliki, Jeremiah T.; Lehenbauer, Terry W.

    2001-01-01

    A competitive enzyme-linked immunosorbent assay (cELISA), using two monoclonal antibodies (MAbs), was developed and compared with the standard virus neutralization test (VNT) for detecting antibodies against canine distemper virus (CDV) and phocine distemper virus (PDV) in sera from dogs and various species of marine mammals. The test depends on the blocking of MAb binding to solid-phase antigen in the presence of positive serum. Test conditions were optimized by using control VNT-negative and -positive sera specific for CDV and PDV. A positive cutoff value of 30% inhibition, which represents the mean cutoff of a VNT-negative population (n = 623) plus 2 standard deviations, was adopted for the test. A total of 736 serum samples were tested by the new cELISA and by the VNT as the “gold standard.” An unexpected but useful finding was the ability of this CDV- and PDV-specific cELISA to also detect antibodies against the related pair dolphin morbillivirus and porpoise morbillivirus. Based on a subpopulation of 625 sera used in statistical analyses, the overall sensitivity and specificity of cELISA relative to those of the VNT were 94.9 and 97.7%, respectively. Because the cELISA proved to be nearly as sensitive and specific as the VNT while being simpler and more rapid, it would be an adequate screening test for suspect CDV or PDV cases and would also be useful for epidemiological surveillance of morbilliviral infections in marine mammal populations. PMID:11326007

  3. 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.

  4. Postabsorptive hyperglucagonemia in patients with type 2 diabetes mellitus analyzed with a novel enzyme-linked immunosorbent assay.

    PubMed

    Matsuo, Toshihiro; Miyagawa, Jun-Ichiro; Kusunoki, Yoshiki; Miuchi, Masayuki; Ikawa, Takashi; Akagami, Takafumi; Tokuda, Masaru; Katsuno, Tomoyuki; Kushida, Akira; Inagaki, Takashi; Namba, Mitsuyoshi

    2016-05-01

    The aims of the present study were to investigate the performance of a novel sandwich enzyme-linked immunosorbent assay (ELISA) for measuring glucagon (1-29) with monoclonal antibodies against both the C- and N-terminal regions of glucagon (1-29), and to analyze the differences in plasma levels and responses of glucagon (1-29) to oral glucose loading in normal glucose tolerance (NGT) subjects and patients with type 2 diabetes mellitus. The cross-reactivity against proglucagon fragments using the ELISA kit and two types of conventional radioimmunoassay (RIA) kits was evaluated. A 75-g oral glucose tolerance test was carried out with NGT subjects and patients with type 2 diabetes mellitus, and the glucagon (1-29) concentration was measured using three types of kit. The ELISA kit clearly had the lowest cross-reactivity against miniglucagon (19-29) and glicentin (1-61). The oral glucose tolerance test was carried out with 30 NGT and 17 patients with type 2 diabetes mellitus. The glucagon (1-29) levels measured by the ELISA kit after glucose loading were significantly higher at all time-points in the type 2 diabetes mellitus group than in the NGT group. However, the glucagon (1-29) levels measured by one RIA kit were significantly higher in the NGT group, and those measured with the other RIA kit were approximately the same among the groups. The novel sandwich ELISA accurately determines plasma glucagon (1-29) concentrations with much less cross-reactivity against other proglucagon fragments than conventional RIA kits.

  5. Pneumococcal Type 22F Polysaccharide Absorption Improves the Specificity of a Pneumococcal-Polysaccharide Enzyme-Linked Immunosorbent Assay

    PubMed Central

    Concepcion, Nelydia F.; Frasch, Carl E.

    2001-01-01

    The specificity of the immune response to the 23-valent pneumococcal-polysaccharide (PS) vaccine in healthy adults and to a pneumococcal conjugate vaccine in infants was examined by measuring immunoglobulin G (IgG) antibody titers by enzyme-linked immunosorbent assay (ELISA) and the opsonophagocytosis assay. ELISA measures total antipneumococcal IgG titers including the titers of functional and nonfunctional antibodies, while the opsonophagocytosis assay measures only functional-antibody titers. Twenty-four pairs of pre- and post-pneumococcal vaccination sera from adults were evaluated (ELISA) for levels of IgG antibodies against serotypes 4, 6B, 9V, 14, 18C, 19F, and 23F. Twelve of the pairs were also examined (opsonophagocytosis assay) for their functional activities. The correlation coefficients between assay results for most types ranged from 0.75 to 0.90, but the correlation coefficient was only about 0.6 for serotypes 4 and 19F. The specificities of these antibodies were further examined by the use of competitive ELISA inhibition. A number of heterologous polysaccharides (types 11A, 12F, 15B, 22F, and 33A) were used as inhibitors. Most of the sera tested showed cross-reacting antibodies, in addition to those removed by pneumococcal C PS absorption. Our data suggest the presence of a common epitope that is found on most pneumococcal PS but that is not absorbed by purified C PS. Use of a heterologous pneumococcal PS (22F) to adsorb the antibodies to the common epitope increased the correlation between the IgG ELISA results and the opsonophagocytosis assay results. The correlation coefficient improve from 0.66 to 0.92 for type 4 and from 0.63 to 0.80 for type 19F. These common-epitope antibodies were largely absent in infants at 7 months of age, suggesting the carbohydrate nature of the epitope. PMID:11238206

  6. Enzyme-linked immunosorbent assay employing a recombinant antigen for detection of protective antibody against swine erysipelas.

    PubMed

    Imada, Yumiko; Mori, Yasuyuki; Daizoh, Masaji; Kudoh, Kazuma; Sakano, Tetsuya

    2003-11-01

    The specificities and sensitivities of five recombinant proteins of the surface protective antigen (SpaA) of Erysipelothrix rhusiopathiae were examined by indirect enzyme-linked immunosorbent assay (ELISA) with the aim of developing a reliable serological test for the detection of protective antibody against E. rhusiopathiae. Fully mature protein and the N-terminal 416 amino acids (SpaA416) showed sufficient antigenicities, and further examination was done with SpaA416 because of its higher yield. The antibody titers of pigs experimentally immunized with commercial live vaccine and two types of inactivated vaccines clearly increased after immunization, and all pigs were completely protected against challenge with virulent strains. On the other hand, the antibody titers of nonimmunized control pigs remained very low until they were challenged, and all showed severe symptoms or subsequently died. Interference with the production of antibody against live vaccine by maternal antibody or porcine respiratory and reproductive syndrome virus infection 1 week after vaccination was also clearly detected. Because the ELISA titer correlated well with the protection results, the specificity and sensitivity of the ELISA were further evaluated with sera collected from pigs reared on 1 farm on which animals had acute septicemia, 2 farms on which the animals were infected or free from infection, and 10 farms on which the animals were vaccinated with live vaccine, among others. The ELISA titers clearly revealed the conditions of the herds. These results indicate that the SpaA416 ELISA is an effective method not only for evaluating pigs for the presence of protective antibody levels resulting from vaccination or maternal antibody but also for detecting antibody produced by natural infection. This test has important potential for the effective control of swine erysipelas.

  7. 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

    2015-12-28

    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.

  8. Development of dioxin toxicity evaluation method in human milk by enzyme-linked immunosorbent assay--assay validation for human milk.

    PubMed

    Sugawara, Yukio; Saito, Koichi; Ogawa, Masahiko; Kobayashi, Susumu; Shan, Guomin; Sanborn, James R; Hammock, Bruce D; Nakazawa, Hiroyuki; Matsuki, Yasuhiko

    2002-03-01

    In this study, the development of a toxicity evaluation method for dioxins in human milk by enzyme-linked immunosorbent assay (ELISA) was reported. A total of 17 human milk samples were tested by ELISA and by gas chromatography/mass spectrometry (GC/MS) to assess whether the ELISA performed on samples obtained from primiparas could be considered as reliable enough for identifying a dioxins contamination in human milk. The concept of toxicity equivalent quantity (TEQ) screening was validated by comparing TEQ values for a set of human milk samples to the ELISA responses predicted for those samples. A fairly good correlation (r = 0.920) between immunoassay and GC/MS was achieved for human milk. This ELISA should be useful for biological samples monitoring.

  9. Herpes simplex virus type-selective enzyme-linked immunosorbent assay with Helix pomatia lectin-purified antigens.

    PubMed Central

    Svennerholm, B; Olofsson, S; Jeansson, S; Vahlne, A; Lycke, E

    1984-01-01

    Helix pomatia lectin-purified antigens with specific reactivity to herpes simplex virus type 1 (HSV-1) and HSV-2 antibodies in human sera were used in an enzyme-linked immunosorbent assay. The type specificity of the antigens was assessed by double immunodiffusion precipitation in gel against rabbit HSV-1 and HSV-2 hyperimmune sera, and by enzyme-linked immunosorbent assay with human reference sera containing antibodies to either type of HSV. Fifty-two sera from patients with documented infection with either HSV-1 or HSV-2 were assayed for HSV type-specific immunoglobulin G antibodies. The reactivity of the sera against lectin-purified antigens correlated completely with the results of virus typing. We conclude that HSV type-specific immunoglobulin G antibodies can easily be measured by enzyme-linked immunosorbent assay with the use of Helix pomatia lectin-purified HSV-1 and HSV-2 antigens. Images PMID:6321548

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

    NASA Astrophysics Data System (ADS)

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

    2016-09-01

    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.

  11. 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

  12. Effect of hapten structures on specific and sensitive enzyme-linked immunosorbent assays for N-methylcarbamate insecticide metolcarb.

    PubMed

    Zhang, Qi; Wu, Yunru; Wang, Limin; Hu, Baishi; Li, Peiwu; Liu, Fengquan

    2008-09-05

    Five different haptens of the N-methylcarbamate insecticide metolcarb were designed and synthesized. All of the haptens were conjugated with ovalbumin (OVA) for the coating antigen, and one hapten containing all of the structure of metolcarb was conjugated with bovine serum albumin (BSA) for the immunogen. Two polyclonal antisera were raised against the BSA conjugate, and ten antibody/coating conjugate combinations were selected for studies of assay sensitivity and specificity for metolcarb. A class-specific combination was found, with the I50 of the assay ranged from 0.64 to 20.98 microg mL(-1) for seven tested N-methylcarbamate insecticides except for pirimicarb. Considering titer, I50 and cross-reactivity of all combinations of antibody/coating conjugate, a competitive indirect enzyme-linked immunosorbent assay (ELISA) in a homologous system, whose limit of detection (LoD) reached 1.4 ng mL(-1), was presented. The results of competitive ELISAs indicated that coating hapten structure can significantly affect not only assay sensitivity but also its specificity.

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

    PubMed

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

    2015-10-20

    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.

  14. 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

  15. 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. Copyright © 2015 Elsevier Inc. All rights reserved.

  16. Development and validation of an enzyme-linked immunosorbent assay for antibodies against Mycobacterium bovis in European wild boar.

    PubMed

    Aurtenetxe, Olaia; Barral, Marta; Vicente, Joaquín; de la Fuente, José; Gortázar, Christian; Juste, Ramón A

    2008-11-01

    Bovine tuberculosis (bTB) remains a significant problem in some parts of Spain largely because of contacts between cattle and wildlife reservoirs in extensive grazing systems. European Wild boar (Sus scrofa) is one of the species involved in the transmission of the disease to other species. Fast and simple detection methods would be critical for assessing infection prevalence, study the mechanisms of pathogen transmission and monitoring the effects of TB control measures. An enzyme-linked immunosorbent assay (ELISA) to detect antibodies against Mycobacterium bovis in wild boar serum was developed and validated on 185 sera from TB positive and negative wild boar. Based on antigen inoculation of captive animals as well as tuberculosis compatible lesions, culture results and molecular analysis of hunted individuals, animals were allocated into two groups: tuberculosis positive group and tuberculosis negative group. After optimization of the positive to negative ratio using different combinations of serum dilutions and conjugate concentrations, the test yielded a sensitivity of 72.60% and a specificity of 96.43% for the best cut-off. Although some negative group animals showed an ELISA positive reaction (< 3%), this assay showed a high potential for accurate diagnosis of TB in wild boar, as its large dynamic range supported a good discriminatory power and a satisfactory balance between sensitivity and specificity.

  17. Seroprevalence of contagious ecthyma in goats of Assam: An analysis by indirect enzyme-linked immunosorbent assay

    PubMed Central

    Bora, Mousumi; Bora, Durlav Prasad; Barman, Nagendra Nath; Borah, Biswajyoti; Das, Sutopa

    2016-01-01

    Aim: The objective of this study was to screen the prevalence of contagious ecthyma (CE) among the goat population of Assam owing to its high prevalence rate. Materials and Methods: In this study, a total of 231 serum samples were collected from 12 districts of Assam during September 2013 to July 2014. The serum samples were tested for the presence of antibodies against Orf virus (ORFV) by indirect enzyme-linked immunosorbent assay (ELISA). Indirect ELISA was standardized using purified Orf reference virus produced in bulk in primary lamb testes cells. Results: Studies on seroprevalence showed 76.62% of goats were seropositive. The total number of animals were divided into different age groups starting from 0-2 months, 2-4 months, 4-6 months, and above 8 months and accordingly highest prevalence of antibodies against ORFV was recorded in the age-group above 8 months of age. Significantly, lower rates of infection were observed in goats of age group 2-4 months. This study recorded that seropositivity from naturally infected animals and in contact apparently healthy animals to be 53.67% and 46.32%, respectively. Conclusion: The results indicated that CE is a prevalent infection in goats of Assam, and the healthy population is at increased risk of infection. PMID:27733808

  18. Development of species-specific enzyme-linked immunosorbent assay for diagnosis of Johne's disease in cattle.

    PubMed Central

    Vannuffel, P; Gilot, P; Limbourg, B; Naerhuyzen, B; Dieterich, C; Coene, M; Machtelinckx, L; Cocito, C

    1994-01-01

    The previously described (M. De Kesel, P. Gilot, M.-C. Misonne, M. Coene, and C. Cocito, J. Clin. Microbiol., 31:947-954, 1993) a362 recombinant polypeptide of Mycobacterium paratuberculosis was used as reagent for an enzyme-linked immunosorbent assay (ELISA). This ELISA, which is endowed with species specificity with respect to the other mycobacteria, was applied to the analysis of bovine paratuberculosis (Johne's disease), an endemic mycobacteriosis of cattle caused by M. paratuberculosis. The distribution of anti-a362 antibodies in the cattle population was analyzed by a computer program (mixture population model) to determine a cutoff value for the test. The prevalence of a362 seropositivity in the Belgian bovine population was estimated to be 12%. The sensitivity of the a362 assay was 70%, as determined with reference sera from the U.S. National Repository of Paratuberculosis Specimens. Some 40% of the animals in the herds with paratuberculosis analyzed were found to be positive by the a362 assay. The latter proved to be 95% specific with respect to both healthy and tuberculous cattle. PMID:8051246

  19. 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.

  20. Development and evaluation of an enzyme-linked immunosorbent assay to detect Histoplasma capsulatum antigenuria in immunocompromised patients.

    PubMed

    Scheel, Christina M; Samayoa, Blanca; Herrera, Alejandro; Lindsley, Mark D; Benjamin, Lynette; Reed, Yvonne; Hart, John; Lima, Sandra; Rivera, Blanca E; Raxcaco, Gabriella; Chiller, Tom; Arathoon, Eduardo; Gómez, Beatriz L

    2009-06-01

    Histoplasma capsulatum infection causes significant morbidity and mortality in human immunodeficiency virus-infected individuals, particularly those in countries with limited access to rapid diagnostics or antiretroviral therapies. The fungus easily disseminates in persons with AIDS, resulting in progressive disseminated histoplasmosis (PDH), which can progress rapidly to death if undiagnosed. The availability of a simple, rapid method to detect H. capsulatum infection in less developed countries where the infection is endemic would dramatically decrease the time to diagnosis and treatment of PDH. We have developed an antigen-capture enzyme-linked immunosorbent assay (ELISA) to detect PDH antigenuria in infected patients. The assay uses polyclonal antibodies against H. capsulatum as both capture and detection reagents, and a standard reference curve is included to quantify antigenuria and ensure reproducibility. We evaluated this assay using specimens collected from patients with AIDS and culture-proven histoplasmosis in a Guatemalan clinic (n = 48), from healthy persons (n = 83), and from patients with other, nonhistoplasmosis diseases (n = 114). The ELISA demonstrated a sensitivity of 81% and a specificity of 95% in detecting H. capsulatum antigen in urine. This assay relies on simple technology that can be performed in institutions with limited resources. Use of this test will facilitate rapid diagnosis of PDH in countries where mortality is high, expediting treatment and likely reducing PDH-related mortality.

  1. Development and Evaluation of an Enzyme-Linked Immunosorbent Assay To Detect Histoplasma capsulatum Antigenuria in Immunocompromised Patients▿

    PubMed Central

    Scheel, Christina M.; Samayoa, Blanca; Herrera, Alejandro; Lindsley, Mark D.; Benjamin, Lynette; Reed, Yvonne; Hart, John; Lima, Sandra; Rivera, Blanca E.; Raxcaco, Gabriella; Chiller, Tom; Arathoon, Eduardo; Gómez, Beatriz L.

    2009-01-01

    Histoplasma capsulatum infection causes significant morbidity and mortality in human immunodeficiency virus-infected individuals, particularly those in countries with limited access to rapid diagnostics or antiretroviral therapies. The fungus easily disseminates in persons with AIDS, resulting in progressive disseminated histoplasmosis (PDH), which can progress rapidly to death if undiagnosed. The availability of a simple, rapid method to detect H. capsulatum infection in less developed countries where the infection is endemic would dramatically decrease the time to diagnosis and treatment of PDH. We have developed an antigen-capture enzyme-linked immunosorbent assay (ELISA) to detect PDH antigenuria in infected patients. The assay uses polyclonal antibodies against H. capsulatum as both capture and detection reagents, and a standard reference curve is included to quantify antigenuria and ensure reproducibility. We evaluated this assay using specimens collected from patients with AIDS and culture-proven histoplasmosis in a Guatemalan clinic (n = 48), from healthy persons (n = 83), and from patients with other, nonhistoplasmosis diseases (n = 114). The ELISA demonstrated a sensitivity of 81% and a specificity of 95% in detecting H. capsulatum antigen in urine. This assay relies on simple technology that can be performed in institutions with limited resources. Use of this test will facilitate rapid diagnosis of PDH in countries where mortality is high, expediting treatment and likely reducing PDH-related mortality. PMID:19357311

  2. Comparison of immunodiffusion and enzyme linked immunosorbent assay in the detection of abnormal antibodies in pigeon breeder's disease.

    PubMed Central

    Simpson, C.; Shirodaria, P. V.; Evans, J. P.; Simpson, D. I.; Stanford, C. F.

    1992-01-01

    AIMS: To compare the sensitivity of two methods for the detection of serum antibodies to pigeon faecal antigens in patients with pigeon breeder's disease. METHODS: Serum samples stored at -20 degrees C from 50 patients with pigeon breeder's disease, 50 control samples from patients with other respiratory diseases, and 50 healthy blood donors were examined for the precipitating antibodies and IgG antibodies to antigens present in extract of pigeon droppings by immunodiffusion and enzyme linked immunosorbent assay (ELISA), respectively. RESULTS: Both antigen preparations of pigeon dropping extract were equally effective. A positive immunodiffusion reaction gave one or more precipitin lines and these antibodies were detected only in undiluted sera from 80% of the patients with pigeon breeder's disease. In the ELISA the sera were tested at a starting dilution of 1 in 100 because positive reactions were observed with sera from healthy blood donors at lower dilutions. All sera which gave optical density readings above 3 SD of the control value were considered to have IgG antibodies. These antibodies were detected in sera from all the patients with pigeon breeder's disease. The antibody titres were much higher in those patients who had precipitating antibodies (range 800-51,200) than those without (range 100-800). The antibodies were not detected in the sera of patients with respiratory diseases or healthy blood donors by either method. CONCLUSIONS: Antibodies to pigeon dropping antigens were detected by immunodiffusion and ELISA in sera from patients with pigeon breeder's disease but not in control sera. ELISA was a more sensitive method for detecting antibodies and therefore seems to have considerable potential as a routine technique in the serological diagnosis of pigeon breeder's disease. PMID:1624596

  3. Evaluation of four enzyme-linked immunosorbent assays for the serologic survey of avian influenza in wild bird species.

    PubMed

    Claes, Gerwin; Vangeluwe, Didier; Van der Stede, Yves; van den Berg, Thierry; Lambrecht, Bénédicte; Marché, Sylvie

    2012-12-01

    Wild birds that reside in aquatic environments are the major reservoir of avian influenza viruses (AIVs). Since this reservoir of AIVs forms a constant threat for poultry, many countries have engaged in AIV surveillance. More and more commercial enzyme-linked immunosorbent assays (ELISA) are available for serologic surveillance, but these tests are often developed and validated for use in domestic poultry. However, for a correct interpretation of ELISA test results from wild bird sera, more information is needed. In the present study, four ELISA test kits (ID-Vet IDScreen, IDEXX FlockChek AI MultiS-Screen Ab Test Kit, Synbiotics FluDETECTBE, and BioChek AIMSp) were compared for the serologic analysis of 172 serum samples from mallard, mute swan, and Canada goose. Samples were selected based on ID-Vet IDScreen results to obtain an approximately equal number of positive and negative samples. In addition, 92 serum samples from experimentally infected specific-pathogen-free (SPF) chickens and Pekin ducks were included in the tests for validation purposes. Cohen's kappa statistics and Spearman correlation coefficients were calculated for each combination of two tests and for each bird species. Test agreement for mallard sera varied from poor to moderate, while test results for Canada goose and swan sera agreed from fair to almost perfect. The best agreement was obtained with sera from experimentally infected SPF chickens and Pekin ducks. This study shows that some care must be taken before using nucleoprotein ELISAs for the testing of sera from wild birds and that more reliable validation studies should be considered before their use in the serologic surveillance of wild birds.

  4. Development of a quantitative enzyme-linked immunosorbent assay for vitellin in the mysid Neomysis integer (Crustacea: Mysidacea).

    PubMed

    Ghekiere, An; Fenske, Martina; Verslycke, Tim; Tyler, Charles; Janssen, Colin

    2005-09-01

    Mysid crustaceans have been put forward by several regulatory bodies as suitable test organisms to screen and test the potential effects of environmental endocrine disruptors. Despite the well-established use of mysid reproductive endpoints such as fecundity, egg development time, and time to first brood release in standard toxicity testing, little information exists on the hormonal regulation of these processes. Control of vitellogenesis is being studied intensively because yolk is an excellent model for studying mechanisms of hormonal control, and vitellogenesis can be chemically disrupted. Yolk protein or vitellin is a major source of nourishment during embryonic development of ovigorous egg-laying invertebrates. The accumulation of vitellin during oocyte development is vital for the production of viable offspring. In this context, we developed a competitive enzyme-linked immunosorbent assay (ELISA) for vitellin of the estuarine mysid Neomysis integer. Mysid vitellin was isolated using gel filtration, and the purified vitellin was used to raise polyclonal antibodies. The ELISA was sensitive within a working range of 4 to 500 ng vitellin/mL. Serial dilutions of whole body homogenates from female N. integer and the vitellin standard showed parallel binding curves, validating the specificity of the ELISA. The intra- and interassay coefficients of variation were 8.2% and 13.8%, respectively. Mysid vitellin concentrations were determined from ovigorous females and eggs at different developmental stages. The availability of a quantitative mysid vitellin ELISA should stimulate further studies on the basic biology of this process in mysids. Furthermore, it could provide a means to better understand and predict chemically induced reproductive effects in mysids.

  5. A novel monoclonal antibody-based enzyme-linked immunosorbent assay to determine luteinizing hormone in bovine plasma.

    PubMed

    Borromeo, V; Berrini, A; De Grandi, F; Cremonesi, F; Fiandanese, N; Pocar, P; Secchi, C

    2014-07-01

    The development of a novel enzyme-linked immunosorbent assay (ELISA) for determining luteinizing hormone (LH) in bovine plasma is described. Anti-bovine LH (bLH) monoclonal antibodies (mAbs) were produced and characterized. One mAb recognizing the bLH β subunit was used for immunoaffinity purification of substantial amounts of biologically active bLH from pituitary glands. The purified bLH in combination with 2 anti-bLH β subunit mAbs was used to develop a sandwich ELISA, which satisfied all the criteria required to investigate LH secretory patterns in the bovine species. The ELISA standard curve was linear over the range 0.05 to 2.5 ng/mL, and the assay proved suitable for measuring bLH in plasma without any prior treatment of samples. Cross-reactivity and recovery tests confirmed the specificity of the method. The intra- and inter-assay coefficients of variation ranged between 3.41% and 9.40%, and 9.29% and 15.84%, respectively. The analytical specificity of the method was validated in vivo by provocative tests for LH in heifers, using the LH releasing peptide gonadotropin-releasing hormone. In conclusion, the adoption of mAbs for this ELISA for coating the wells and labeling, combined with the easy one-step production of reference bLH, ensures long-term continuity in large-scale measurements of LH in the bovine species. Copyright © 2014 Elsevier Inc. All rights reserved.

  6. A direct competitive enzyme-linked immunosorbent assay for rapid detection of anilofos residues in agricultural products and environmental samples.

    PubMed

    Zhang, Yan; Gao, Ai H; Liu, Bing; Sheng, Wei; Tan, Chao; Yuan, Meng; Wang, Shuo

    2013-01-01

    A direct competitive enzyme-linked immunosorbent assay (dc-ELISA) was developed to measure anilofos levels in agricultural and environmental samples. The ELISA was developed using rabbit polyclonal antibodies against a hapten-protein conjugate of anilofos-bovine serum albumin. The limit of detection was 0.1 μg L(-1), and there was no cross-reactivity with other related pesticides or structurally similar compounds. The matrix effects of rice (aromatic rice, white rice, brown rice), corn, barley, wheat and soil were measured and removed by extraction and dilution with phosphate buffered saline with 0.05% Tween-20. For water samples (tap water and river water), the matrix effects were also removed by dilution with phosphate buffered saline with Tween-20. The detection limits for anilofos in authentic samples (aromatic rice, white rice, brown rice, corn, barley, wheat, soil, tap water and river water) were 2, 2, 2, 3, 2, 2, and 2 μg kg(-1), and 0.5 and 1 μg L(-1), respectively . The anilofos recovery ranged from 81.0-116.0% with a coefficient of variation of 1.7-9.0%. The method was validated using GC, and the results showed good correlation with the dc-ELISA data (r(2) = 0.9795). Forty-two cereal samples were randomly collected from different supermarkets and analyzed using the developed dc-ELISA. No anilofos was found in these products. The developed immunoassay is suitable for rapid quantitation of anilofos residues.

  7. Evaluation of a commercial enzyme-linked immunosorbent assay for detection of serum immunoglobulin G response to human herpesvirus 6.

    PubMed Central

    Sloots, T P; Kapeleris, J P; Mackay, I M; Batham, M; Devine, P L

    1996-01-01

    A rapid (60-min) commercially available enzyme-linked immunosorbent assay (ELISA) for the detection of immunoglobulin G (IgG) class antibodies to human herpesvirus 6 (HHV-6) was evaluated. The specificity of the ELISA for HHV-6 was confirmed by absorption studies, with the reactivities of HHV-6-positive sera being unaffected by other herpesviruses (cytomegalovirus, herpes simplex virus, and varicella-zoster virus) or the HSB2 cell line used to culture HHV-6. HHV-6 IgG antibody levels in a panel of 502 serum samples were determined by ELISA and an indirect immunofluorescence assay (IFA). Results obtained by the two methods were in close agreement, suggesting that the ELISA provides a suitable test method for the determination of HHV-6 IgG antibodies in a routine clinical laboratory. Both tests were positive in 398 cases (79%), and both were negative in 71 cases (14%), with a different result obtained by IFA and ELISA in only 33 cases (7%). Furthermore, absorption of sera with HHV-6 prior to assay revealed that the majority of these results were false positive (n = 8) or false negative (n = 23) in the IFA (true positives or negatives in the ELISA). Subsequently, the ELISA showed a sensitivity of 99.76% and a specificity of 98.75%. HHV-6-specific IgG levels were also determined in paired serum samples collected from 49 donors--14 with exanthem subitum (ES), 15 with ES which was complicated with central nervous system involvement, and 20 undergoing bone marrow transplantation--in whom HHV-6 infection had been demonstrated by virus isolation and/or PCR. All patients with ES or central nervous system complications showed an increase in HHV-6-specific IgG, indicating that this ELISA may be a useful aid in the diagnosis of these conditions. Furthermore, 14 of 20 patients undergoing bone marrow transplantation showed an increase in HHV-6-specific IgG levels, possibly reflecting a reactivation of HHV-6 in these patients. PMID:8904436

  8. 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

  9. Cryptococcal phospholipase B antigen is not detected in serum of patients infected with Cryptococcus neoformans using a sandwich enzyme-linked immunosorbent assay.

    PubMed

    Wu, Qi Xuan; Chen, Sharon C A; Santangelo, Rosemary T; Martin, Patricia; Malik, Richard; Sorrell, Tania C

    2007-05-01

    Extracellular phospholipase B (PLB) is a virulence determinant of Cryptococcus neoformans and Cryptococcus gattii. In this study, we developed a sensitive enzyme-linked immunosorbent assay (ELISA) for PLB antigen with a detection limit of 3.9 ng mL(-1). PLB was detected in culture supernatants of C. neoformans and C. gattii. PLB, however, was not detected in sera of seven human patients and 10 feline patients with active cryptococcosis. Furthermore, none of five rats with extensive pulmonary C. gattii infection had a positive ELISA test result. In conclusion, cryptococcal PLB could not be detected in serum using a PLB antigen-based ELISA. Despite its sensitivity, this ELISA is of limited diagnostic value. Exploration of further extracellular molecules suitable for serodiagnosis of active cryptococcal infection is warranted.

  10. Detection of aflatoxin B1 in imported food products into Japan by enzyme-linked immunosorbent assay and high performance liquid chromatography.

    PubMed

    Adachi, Y; Hara, M; Kumazawa, N H; Hirano, K; Ueno, I; Egawa, K

    1991-02-01

    In order to detect the presence of aflatoxin B1 (AFB1), the use of the enzyme-linked immunosorbent assay (ELISA) and recovery test was evaluated. The detection limit of ELISA for AFB1 was 1 pg/assay and the recovery from maize spiked with AFB1 exceeded 80%. AFB1 was detected by ELISA in seven out of twelve samples of imported food products including peanut, almond, red pepper, cocoa bean, black pepper, buckwheat, walnut, adlay, soybean, popcorn, and pistachio nut, and by high performance liquid chromatography (HPLC) in four of the samples. However, the content of AFB1 in these samples was less than 10 ng/g of the minimum value authorized by the Japanese sanitation law. These results demonstrate that ELISA is more sensitive than HPLC and imported food products are broadly contaminated with AFB1.

  11. 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

  12. Development and Evaluation of a Blocking Enzyme-Linked Immunosorbent Assay for Detection of Avian Metapneumovirus Type C-Specific Antibodies in Multiple Domestic Avian Species

    PubMed Central

    Turpin, Elizabeth A.; Lauer, Dale C.; Swayne, David E.

    2003-01-01

    The first cases of infection caused by avian metapneumoviruses (aMPVs) were described in turkeys with respiratory disease in South Africa during 1978. The causative agent was isolated and identified as a pneumovirus in 1986. aMPVs have been detected in domestic nonpoultry species in Europe, but tests for the detection of these viruses are not available in the United States. To begin to understand the potential role of domestic ducks and geese and wild waterfowl in the epidemiology of aMPV, we have developed and evaluated a blocking enzyme-linked immunosorbent assay (bELISA) for the detection of aMPV type C (aMPV-C)-specific antibodies. This assay method overcomes the species-specific platform of indirect ELISAs to allow detection of aMPV-C-specific antibodies from potentially any avian species. The bELISA was initially tested with experimental turkey serum samples, and the results were found to correlate with those of virus neutralization assays and indirect enzyme-linked immunosorbent assay (iELISA). One thousand serum samples from turkey flocks in Minnesota were evaluated by our bELISA, and the level of agreement of the results of the bELISA and those of the iELISA was 94.9%. In addition, we were able to show that the bELISA could detect aMPV-C-specific antibodies from experimentally infected ducks, indicating its usefulness for the screening of serum samples from multiple avian species. This is the first diagnostic assay for the detection of aMPV-C-specific antibodies from multiple avian species in the United States. PMID:12904358

  13. High performance enzyme-linked immunosorbent assay for determination of miroestrol, a potent phytoestrogen from Pueraria candollei.

    PubMed

    Yusakul, Gorawit; Udomsin, Orapin; Juengwatanatrakul, Thaweesak; Tanaka, Hiroyuki; Chaichantipyuth, Chaiyo; Putalun, Waraporn

    2013-06-27

    Pueraria candollei associated preparation is widely applied in folk Thai medicine for rejuvenating purpose in aged people, which correlated with its pharmacological activities reported by pre-clinical and clinical trials. Therefore, standardized products of this plant are needed by consumers and health care personnel. Miroestrol, a potent and stable phytoestrogen in P. candollei, exhibited potential to be biomarker for quality control of P. candollei samples in research or industrial levels. Indirect competitive enzyme-linked immunosorbant assay (ELISA) for miroestrol determination was developed and validated by using polyclonal antibody from rabbit immunization. The polyclonal antibody recognized specifically to miroestrol, which exhibited cross-reactivity to deoxymiroestrol and isomiroestrol with 6.68% and 1.05%, respectively. The linearity range of measurement was 0.73-3000 ng mL(-1), which coefficient of variation (CV) of both intra- and inter-plate determination was less than 5%. With spiked samples of known amount miroestrol, the percentages of recovery were 98.80-104.37% and 98.31-106.69% in P. candollei and its involved product samples, respectively. Validated ELISA was comparable with published HPLC method (R(2)=0.9996) (Yusakul et al.) in samples with various miroestrol contents. For application, the P. candollei involved preparations contained miroestrol 0.695±0.037-12.108±0.285 μg g(-1) dry wt. The developed ELISA was high performance for miroestrol determination, which could be applied for P. candollei quality control in research fields and industrial productions. Copyright © 2013 Elsevier B.V. All rights reserved.

  14. Indirect enzyme-linked immunosorbent assay for detection of Brucella melitensis-specific antibodies in goat milk.

    PubMed

    Funk, N D; Tabatabai, L B; Elzer, P H; Hagius, S D; Martin, B M; Hoffman, L J

    2005-02-01

    Brucella melitensis is the cause of brucellosis in sheep and goats, which often results in abortion. Few cases of B. melitensis infection in goats have occurred in the United States over the last 25 years. However, vigilance must be maintained, as it is for the bovine milk industry, to ensure that brucellosis is not introduced into the U.S. goat population. The objective of this study was to develop a sensitive and specific indirect enzyme-linked immunosorbent assay (iELISA) for the detection of B. melitensis-specific antibodies in goat milk. Brucella salt-extractable protein extract was employed as an antigen, and a horseradish peroxidase-labeled polyclonal anti-goat antibody was used as an anti-species conjugate. Thirteen of 13 (100%) individual infected goat milk samples tested positive and 134 of 134 (100%) uninfected bulk milk samples tested negative by the developed iELISA. Three positive milk samples with high, medium, and low absorbance values were used to simulate one positive animal in an otherwise negative herd. By this estimation, one high-titer animal could be detected in a herd of >1,600 animals. Detection estimates for medium- and low-titer animals were one positive animal per herd of <200 and 50 animals, respectively. Based on this estimation, it is recommended that herds be sampled in groups of 50 animals or less for bulk milk testing. The iELISA developed for this study was found to be sensitive and specific and shows potential for use as a bulk milk test for the detection of B. melitensis-specific antibodies in goat milk.

  15. Detection of egg drop syndrome virus antigen or genome by enzyme-linked immunosorbent assay or polymerase chain reaction.

    PubMed

    Dhinakar Raj, G; Sivakumar, S; Matheswaran, K; Chandrasekhar, M; Thiagarajan, V; Nachimuthu, K

    2003-10-01

    Mouse monoclonal antibodies (mAbs) were produced against an Indian isolate of egg drop syndrome (EDS) virus and characterized. Four hybridoma clones were secreting mAbs that bound to a 100 kDa protein, presumably the hexon protein. These mAbs were found to cross-react with two other Indian isolates of EDS virus and to the reference UK 127 strain. Three of these mAbs were mapped to the same epitope compared with the other mAb (F8), which bound to a different epitope. An antigen-capture enzyme-linked immunosorbent assay (AC-ELISA) was developed using the F8 mAbs as capture antibody and polyclonal chicken serum against EDS virus as detection antibody. A polymerase chain reaction (PCR) was used to detect the EDS viral genome. Following experimental infection of oestrogen-treated chickens with EDS virus, cloacal swabs, oviduct, uterus and spleen were collected at different days post-infection and used in both AC-ELISA and PCR, directly and after a single passage in embryonated duck eggs. The sensitivity and specificity of antigen detection by AC-ELISA or PCR was 95% and 98%, respectively. For diagnosis of EDS viral infections, PCR is recommended due to its ease and the lack of requirement of prepared reagents such as mAbs or conjugates. We recommend that PCR be performed directly on boiled tissue homogenates. Any negative samples may be passaged in embryonated duck eggs and the allantoic fluids tested by PCR before a conclusive negative diagnosis is given.

  16. Development of a sandwich enzyme-linked immunosorbent assay for dTMP-GH fusion protein by rational immunogen selection.

    PubMed

    Wang, Song; Shen, Mingqiang; Chen, Shilei; Wang, Cheng; Chen, Fang; Chen, Mo; Zhao, Gaomei; Ran, Xinze; Cheng, Tianmin; Su, Yongping; Xu, Yang; Wang, Junping

    2017-12-01

    dTMP-GH is a chimeric protein containing a tandem dimer of thrombopoietin mimetic peptide (dTMP) fused to human growth hormone (hGH) prepared previously by our team. It shows significant bioactivity in promoting thrombocytopoiesis, but detection of intact dTMP-GH in plasma is still a challenge due to the presence of endogenous hGH. In this study, a rabbit polyclonal antibody with high affinity to dTMP was obtained with a BSA-conjugated immunogen composed of 20 amino acids sequence spanning two TMP and the linker. A monoclonal antibody termed as 3B2 was screened out by using immunizing mice with whole dTMP-GH, which was proved to simultaneously interact with rhGH, TMP-GH, and dTMP-GH, respectively. In this study, we developed a specific and sensitive sandwich enzyme-linked immunosorbent assay (ELISA) with two antibodies (one polyclonal and one HRP-conjugated monoclonal) to quantify dTMP-GH. The polyclonal antibody and HRP-conjugated monoclonal antibody 3B2 were applied as the capture antibody and detection antibody, respectively. A good correlation between ELISA and bicinchoninic acid (BCA) assay in the quantification of diluted dTMP-GH was observed (r(2) = 0.996). Meanwhile, the standard curve of this ELISA method was found in a linear relationship between 0.2 and 10 ng/mL in the presence of rabbit plasma. In vivo experiments demonstrate that the newly developed method is effective to detect dTMP-GH in rabbits, which paves the way for further pharmacokinetic evaluation.

  17. 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.

  18. Enzyme-linked immunosorbent assay for detection and quantitation of capsular antigen of Haemophilus influenzae type b.

    PubMed Central

    Crosson, F J; Winkelstein, J A; Moxon, E R

    1978-01-01

    An enzyme-linked immunosorbent assay was developed to detect the presence of the ribose-ribitol phosphate capsular antigen of Haemophilus influenzae type b in laboratory and clinical specimens. The assay is simple, sensitive, specific, and quantitative and should prove to be of value in the diagnosis and management of H. influenzae infections. PMID:310425

  19. An enzyme-linked immunosorbent assay-based isolation procedure for verotoxigenic Escherichia coli.

    PubMed Central

    Milley, D G; Sekla, L H

    1993-01-01

    A colony enzyme-linked immunosorbent assay using the hydrophobic grid membrane filter format was developed for the isolation of verotoxigenic Escherichia coli from human and food samples. The method utilizes monoclonal antibodies directed against the verotoxins and is sensitive to all verotoxin 1- and/or 2-producing serotypes. E. coli that produced a minimum of 2 x 10(2) and 2 x 10(3) 50% cytotoxic doses per ml of verotoxins 1 and 2, respectively, were detectable. In a method comparison using human stool specimens, this procedure isolated 29% more E. coli O157 than did the standard sorbitol-MacConkey agar procedure, with no false-positive reactions. When applied to meat, 11 of 20 samples positive for verotoxin by polymyxin extraction yielded verotoxigenic E. coli of a variety of serotypes including O157:H7. Four false positives were noted. This procedure provides a sensitive means for the isolation of verotoxigenic E. coli and should facilitate recovery of those serotypes that are otherwise indistinguishable from nonpathogenic strains. PMID:8285713

  20. 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

  1. Reproducible enzyme-linked immunosorbent assay with a magnetic processing system for diagnosis of toxoplasmosis.

    PubMed Central

    Konishi, E; Takahashi, J

    1983-01-01

    An enzyme-linked immunosorbent assay with polycarbonate-coated iron beads as the solid phase and with magnetic processing devices was evaluated for the quantitation of antibodies to Toxoplasma gondii in human serum samples. Under the parameters and other basic conditions determined in this study, the assay was highly reproducible: coefficients of variation for the absorbance values obtained with the positive serum were 2.42% in same-day tests and 3.75% in day-to-day tests. Significant correlations were observed between the present assay system and other conventional serological tests: correlation coefficients were 0.960 with the dye test and 0.929 with the latex agglutination test. Statistical analysis based on the frequency distribution of absorbance values for dye-test-positive and dye-test-negative serum samples gave feasible border lines for distinguishing between positive and doubtful samples (0.357) and between doubtful and negative samples (0.266). Under this diagnostic criterion, the results of our assay system agreed remarkably well with those obtained by the dye test and the latex agglutination test, with consistencies of 94.9 and 93.9%, respectively. Images PMID:6833477

  2. An indirect enzyme linked immunosorbent assay for the detection of bovine antibodies to multiple Leptospira serovars

    PubMed Central

    2004-01-01

    Abstract An indirect enzyme linked immunosorbent assay was developed for the detection of bovine antibodies to multiple pathogenic Leptospira serovars, including canicola, copenhageni (represents icterohaemorrhagiae), grippotyphosa, hardjobovis, pomona, and sejroe. The antigen utilized in this assay was a sonicated mixture of equal parts of killed whole cells of each of the 6 serovars named above. A mouse monoclonal antibody against bovine immunoglobulin (Ig)G1 that was conjugated with horseradish peroxidase was used for detection of bound antibodies. This assay was evaluated with sera (n = 3107) that were microscopic agglutination test (MAT)-negative (at a 1:100 dilution) for each of the 6 serovars listed above and sera (n = 601) that were MAT-positive (at a 1:100 dilution) for 1, or any combination of the 6 listed serovars. In addition, sera from serial weekly bleedings of cows, which were individually experimentally infected with serovars hardjobovis, copenhageni, grippotyphosa, or canicola, were also tested in this assay. At an optimal cut-off point determined by receiver operating characteristic (ROC) curve analysis, the relative sensitivity and specificity of the assay were 93.5% (95% confidence interval = 91.2% to 95.3%) and 94.7% (95% confidence interval = 93.9% to 95.5%), respectively. This assay was able to detect antibody in the sera of animals experimentally infected with serovar hardjobovis as early as 1 week postinoculation PMID:14979428

  3. An indirect enzyme linked immunosorbent assay for the detection of bovine antibodies to multiple Leptospira serovars.

    PubMed

    Surujballi, Om; Mallory, Maria

    2004-01-01

    An indirect enzyme linked immunosorbent assay was developed for the detection of bovine antibodies to multiple pathogenic Leptospira serovars, including canicola, copenhageni (represents icterohaemorrhagiae), grippotyphosa, hardjobovis, pomona, and sejroe. The antigen utilized in this assay was a sonicated mixture of equal parts of killed whole cells of each of the 6 serovars named above. A mouse monoclonal antibody against bovine immunoglobulin (Ig)G1 that was conjugated with horseradish peroxidase was used for detection of bound antibodies. This assay was evaluated with sera (n = 3107) that were microscopic agglutination test (MAT)-negative (at a 1:100 dilution) for each of the 6 serovars listed above and sera (n = 601) that were MAT-positive (at a 1:100 dilution) for 1, or any combination of the 6 listed serovars. In addition, sera from serial weekly bleedings of cows, which were individually experimentally infected with serovars hardjobovis, copenhageni, grippotyphosa, or canicola, were also tested in this assay. At an optimal cut-off point determined by receiver operating characteristic (ROC) curve analysis, the relative sensitivity and specificity of the assay were 93.5% (95% confidence interval = 91.2% to 95.3%) and 94.7% (95% confidence interval = 93.9% to 95.5%), respectively. This assay was able to detect antibody in the sera of animals experimentally infected with serovar hardjobovis as early as 1 week postinoculation.

  4. 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

  5. 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.

  6. Development of a fibrinogen-specific sandwich enzyme-linked immunosorbent assay microarray assay for distinguishing between blood plasma and serum samples.

    PubMed

    Gonzalez, Rachel M; Zhang, Qibin; Zangar, Richard C; Smith, Richard D; Metz, Thomas O

    2011-07-01

    We have developed a fibrinogen-specific sandwich enzyme-linked immunosorbent assay (ELISA) microarray assay for use in qualitatively distinguishing between blood plasma and serum samples. Three capture antibodies (49D2, HPA001900, and F8512) were evaluated in conjunction with 1D6 as the detection antibody. The data show that 49D2 and (to a lesser extent) F8512 successfully identify previously unknown plasma and serum samples based on approximately a 28-fold difference in signal intensity between the sample types. This assay has utility in rapidly identifying previously archived clinical samples with incomplete annotation in a high-throughput manner prior to proteomic analyses.

  7. Evaluation of the antibody response in pigs vaccinated against Streptococcus suis capsular type 2 using a double-antibody sandwich enzyme-linked immunosorbent assay.

    PubMed Central

    Blouin, C; Higgins, R; Gottschalk, M; Simard, J

    1994-01-01

    A double-antibody sandwich enzyme-linked immunosorbent assay (ELISA) was standardized for the detection of specific antibodies following vaccination with Streptococcus suis capsular type 2 bacterins. No statistically significant increase of antibody titers was detected in vaccinated piglets compared to the nonvaccinated control group, even if a minority of piglets demonstrated an important postvaccinal response. Three of four vaccinated sows showed a low antibody response to vaccine and specific immunity was detected in piglets of only one litter of these three sows. Passive protection studies showed that none of the sera from vaccinated piglets were protective for mice whereas serum obtained from hyperimmunized pigs gave protection. PMID:8143253

  8. Quantitative determination of recombinant bovine somatotropin in commercial shrimp feed using a competitive enzyme-linked immunosorbent assay.

    PubMed

    Munro, James L; Boon, Virginia A

    2010-02-10

    Recombinant bovine somatotropin (rbST), also known as growth hormone, is used to enhance production and development of animals within the agriculture and aquaculture industries. Its use is controversial because of its potential effects on human and animal health. To screen for rbST in shrimp feed, a competitive enzyme-linked immunosorbent assay (ELISA) with an inhibition step was developed. Sample and rbST antibody (rabbit anti-rbST) were incubated at room temperature for 30 min. Subsequently, this competitive reaction was transferred to a microplate coated with rbST, using goat antirabbit IgG linked with horseradish peroxidise as the secondary antibody. Substrates for peroxidise were added, and the absorbance at 410 nm was determined. The applicability of the method was assessed using rbST extracted from "spiked" shrimp feed samples. The assay was reproducible and linear with R(2) values greater than 0.98 over the standard curve range of 20-500 microg/g. The intra- and interday precisions expressed as relative standard deviations were 3.4 and 5.3%, respectively. The mean recovery from 15 spiked feed samples was 105%. This assay will be a valuable tool for quantitative detection of rbST by both governments and commercial companies and can be modified for other types of feed.

  9. 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.

  10. Production of monoclonal antibody and development of enzyme-linked immunosorbent assay for kanamycin in biological matrices.

    PubMed

    Watanabe, H; Satake, A; Kido, Y; Tsuji, A

    1999-11-01

    Monoclonal antibodies (MAbs) against kanamycin were prepared by using a kanamycin-bovine gamma-globulin conjugate for the immunization of mice. Splenocytes from BALB/c immunized mice were fused with P3X63Ag8U.1 myeloma cells. This resulted in two hybridoma cell lines. Fifty per cent inhibition concentrations (IC50) for the MAbs were 2 and 5 ng ml-1. One MAb (IC50 = 2 ng ml-1) was named #22 and was used to develop quantitative assays for kanamycin by means of an enzyme-linked immunosorbent assay (ELISA). The detection limit was 0.2 ng ml-1 and the standard deviations were 0.2-4.4% for intra-assay and 0.6-4.7% for inter-assay, respectively. The detection limits using peroxidase were 4 ppb in cattle milk, cattle plasma, cattle urine, swine plasma, swine urine and chicken plasma. Using the MAb #22 produced, a rapid test kit based on an immunochromatographic method was developed. The detection limits using the kit were 50 ppb in cattle milk, cattle plasma, cattle urine and chicken plasma.

  11. Surface modification for enhancing antibody binding on polymer-based microfluidic device for enzyme-linked immunosorbent assay.

    PubMed

    Bai, Yunling; Koh, Chee Guan; Boreman, Megan; Juang, Yi-Je; Tang, I-Ching; Lee, L James; Yang, Shang-Tian

    2006-10-24

    A novel surface treatment method using poly(ethyleneimine) (PEI), an amine-bearing polymer, was developed to enhance antibody binding on the poly(methyl methacrylate) (PMMA) microfluidic immunoassay device. By treating the PMMA surface of the microchannel on the microfluidic device with PEI, 10 times more active antibodies can be bound to the microchannel surface as compared to those without treatment or treated with the small amine-bearing molecule, hexamethylenediamine (HMD). Consequently, PEI surface modification greatly improved the immunoassay performance of the microfluidic device, making it more sensitive and reliable in the detection of IgG. The improvement can be attributed to the spacer effect as well as the functional amine groups provided by the polymeric PEI molecules. Due to the smaller dimensions (140x125 microm) of the microchannel, the time required for antibody diffusion and adsorption onto the microchannel surface was reduced to only several minutes, which was 10 times faster than the similar process carried out in 96-well plates. The microchip also had a wider detection dynamic range, from 5 to 1000 ng/mL, as compared to that of the microtiter plate (from 2 to 100 ng/mL). With the PEI surface modification, PMMA-based microchips can be effectively used for enzyme linked immunosorbent assays (ELISA) with a similar detection limit, but much less reagent consumption and shorter assay time as compared to the conventional 96-well plate.

  12. 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.

  13. Simultaneous determination of soy isoflavone glycosides, daidzin and genistin by monoclonal antibody-based highly sensitive indirect competitive enzyme-linked immunosorbent assay.

    PubMed

    Sakamoto, Seiichi; Yusakul, Gorawit; Pongkitwitoon, Benyakan; Paudel, Madan Kumar; Tanaka, Hiroyuki; Morimoto, Satoshi

    2015-02-15

    Soy isoflavones are known as major bioactive compounds in soybean (Glycine max), which is an indispensable food. Despite their utility, the consumption of isoflavones has recently been limited because they exhibit oestrogenic and topoisomerase II inhibitory effects. To assess their intake limitation, accurate, sensitive, and effective quantitative analyses are necessary. In this study, we produced the monoclonal antibody (MAb) against daidzin (DZ) and applied it to an indirect competitive enzyme-linked immunosorbent assay (icELISA) for the simultaneous determination of DZ and genistin (GEN), which are known as two major soy isoflavone glycosides in soy products. Using the DZ-MAb, we developed a sensitive icELISA method, where the limit of detection for DZ and GEN was 1.95ng/ml. Several validation analyses revealed that the icELISA is sufficiently accurate and sensitive to be used to assess the overconsumption of soy isoflavones, which would lead to the safe dietary intake of soy products.

  14. Enzyme-linked immunosorbent assay for total isoflavonoids in Pueraria candollei using anti-puerarin and anti-daidzin polyclonal antibodies.

    PubMed

    Pongkitwitoon, Benyakan; Sakamoto, Seiichi; Tanaka, Hiroyuki; Tsuchihashi, Ryota; Kinjo, Junei; Morimoto, Satoshi; Putalun, Waraporn

    2010-05-01

    Pueraria candollei (White Kwao Khuer) is a medicinal plant containing puerarin, daidzin, genistin, daidzein, and genistein as major isoflavonoids used for its rejuvenating and estrogenic effects. In order to analyze these compounds, a single enzyme-linked immunosorbent assay (ELISA) for total isoflavonoids was developed using anti-puerarin and anti-daidzin polyclonal antibodies (PAbs). The range for calibration of isoflavonoids by ELISA was 0.05-6.25 microg/mL. Total isoflavonoid concentrations in P. candollei samples determined by the newly developed assay system showed good agreement with those analyzed by HPLC. Based on validation analysis, this analytical method by ELISA is a precise, accurate, and sensitive method for the determination of total isoflavonoids in P. candollei.

  15. 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.

  16. An enzyme-linked immunosorbent assay for monitoring of Aspergillus ochraceus growth in coffee powder, chilli powder and poultry feed.

    PubMed

    Anand, S; Rati, E R

    2006-01-01

    The work was carried out to develop an immunoassay for estimation of Aspergillus ochraceus biomass on solid substrate. An indirect noncompetitive enzyme-linked immunosorbent assay (ELISA) was developed for determination of fungal biomass in food commodities using antibody raised against A. ochraceus mycelial antigen. The sensitivity of the assay was linear in the range of 10-160 microg fungal biomass per millilitre extract of coffee (R(2)=0.989), poultry feed (R(2)=0.987) and chilli (R(2)=0.989). The growth of A. ochraceus in the food commodities like chilli, coffee beans and poultry feed, under the influence of two levels of moisture (20% and 30%) were monitored by the ELISA. The maximum fungal colonization was observed in poultry feed (9.8 and 11.8 mg g(-1)) followed by coffee beans (6.8 and 11.3 mg g(-1)) and chilli (5.1 and 6.3 mg g(-1)) at 20% and 30% moisture after 20 days of incubation. Similarly the fungus produced maximum ochratoxin A in poultry feed (25 and 120 microg g(-1)) followed by coffee beans (8 and 24 microg g(-1)) and chilli (0.2 and 0.45 microg g(-1)) at 20% and 30% moisture after 20 days of incubation. The method can be used for quantitative estimation of fungal biomass and comparison of fungal colonization in food substrates varying in composition. The method can be adapted for studying the fungal colonization in different solid substrates under different culture condition. The method is sensitive to mould colonization of >or=0.02% (w/w) and can be used for early detection of specific fungal infestation in food commodities.

  17. Detection of HLA class I-specific antibodies by the QuikScreen enzyme-linked immunosorbent assay.

    PubMed Central

    Lucas, D P; Paparounis, M L; Myers, L; Hart, J M; Zachary, A A

    1997-01-01

    The GTI QuikScreen test is an enzyme-linked immunosorbent assay (ELISA) that uses soluble HLA class I antigens as targets. In tests of 5,893 human serum specimens, we evaluated the reliability, sensitivity, and utility of the GTI QuikScreen test for detecting HLA class I-specific antibody. We found that the test could reliably detect HLA-specific antibodies of the immunoglobulin G (IgG) but not the IgM class. The degree of correlation with lymphocytotoxicity testing varied among the different serum sources, with the best correlation achieved with sera from renal transplant candidates (r > 0.7) and the poorest with sera from patients with end-stage liver disease (r = 0.26), possibly because of elevated alkaline phosphatase levels in the liver patients. Test reproducibility was high (96%), and test failure rate was low (1.7%). The test sensitivity is comparable to that of the antiglobulin cytotoxicity and, possibly, even flow cytometric tests. There was a highly significant (P < 0.001) correlation between the optical densities obtained in the ELISA and the percent panel reactive antibody determined by cytotoxicity testing. Therefore, although designed only to determine the presence or absence of HLA-specific antibody, GTI QuikScreen test results also provided an indication of the extent of sensitization. The test is one of the most effective and efficient ways to determine if antibodies producing a positive result in crossmatch tests are specific for HLA class I antigens. As an adjunct to serum screening by cytotoxicity testing, the GTI QuikScreen test can produce a substantial savings of time and effort that reduces the cost to the laboratory and to the patient. PMID:9144358

  18. Detection of nonhemagglutinating influenza a(h3) viruses by enzyme-linked immunosorbent assay in quantitative influenza virus culture.

    PubMed

    van Baalen, C A; Els, C; Sprong, L; van Beek, R; van der Vries, E; Osterhaus, A D M E; Rimmelzwaan, G F

    2014-05-01

    To assess the efficacy of novel antiviral drugs against influenza virus in clinical trials, it is necessary to quantify infectious virus titers in respiratory tract samples from patients. Typically, this is achieved by inoculating virus-susceptible cells with serial dilutions of clinical specimens and detecting the production of progeny virus by hemagglutination, since influenza viruses generally have the capacity to bind and agglutinate erythrocytes of various species through their hemagglutinin (HA). This readout method is no longer adequate, since an increasing number of currently circulating influenza A virus H3 subtype (A[H3]) viruses display a reduced capacity to agglutinate erythrocytes. Here, we report the magnitude of this problem by analyzing the frequency of HA-deficient A(H3) viruses detected in The Netherlands from 1999 to 2012. Furthermore, we report the development and validation of an alternative method for monitoring the production of progeny influenza virus in quantitative virus cultures, which is independent of the capacity to agglutinate erythrocytes. This method is based on the detection of viral nucleoprotein (NP) in virus culture plates by enzyme-linked immunosorbent assay (ELISA), and it produced results similar to those of the hemagglutination assay using strains with good HA activity, including A/Brisbane/059/07 (H1N1), A/Victoria/210/09 (H3N2), other seasonal A(H1N1), A(H1N1)pdm09, and the majority of A(H3) virus strains isolated in 2009. In contrast, many A(H3) viruses that have circulated since 2010 failed to display HA activity, and infectious virus titers were determined only by detecting NP. The virus culture ELISA described here will enable efficacy testing of new antiviral compounds in clinical trials during seasons in which nonhemagglutinating influenza A viruses circulate.

  19. 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.

  20. 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

  1. Comparison of two methods (microscopy and enzyme-linked immunosorbent assay) for the diagnosis of amebiasis.

    PubMed

    Tanyuksel, Mehmet; Yilmaz, Hasan; Ulukanligil, Mustafa; Araz, Engin; Cicek, Mutalip; Koru, Ozgur; Tas, Zeynep; Petri, William A

    2005-07-01

    Diagnosis of amebiasis is usually performed on a clinical basis alone in most endemic countries having limited economic resources. This epidemiological study was conducted using modern diagnostic tests for amebiasis in the southeastern region of Turkey, an endemic area for amebiasis. The population of this study included patients with symptomatic diarrhea/dysentery attending both Yuzuncu Yil University, Van and Harran University, Sanliurfa, Turkey. A total of 380 stool specimens were collected and examined for Entamoeba by light microscopy (fresh, lugol, and trichrome staining) and stool antigen detection based- enzyme-linked immunosorbent assay (EIA) test (TechLab Entamoeba histolytica II). 24% (91/380) of stool specimens were positive for E. histolytica/Entamoeba dispar trophozoites/cysts microscopically using trichrome staining. 13% (51/380) of the stool specimens were found to be positive for E. histolytica by the EIA test, including 15% (14/91) of microscopy (+) stool specimens and 13% (37/289) of microscopy (-) stool specimens. Enteric parasites were common in these populations with 66% (251/380) of the study population harboring more than one parasite. In addition to the 13% (51/380) of patients determined to have E. histolytica by EIA, eighty-six patients (22.6%) had Blastocystis hominis, 54 (14.2%) Entamoeba coli, 44 (11.5%) Giardia lamblia, 16 (4.2%) Chilomastix mesnili, 15 (3.9%) Iodamoeba bütschlii, 12 (3.1%) Hymenolepis nana, 9 (2.3%) Endolimax nana, 9 (2.3%) Dientamoeba fragilis, and 8 (2.1%) had Ascaris lumbricoides. We concluded that E. histolytica infection was found in 13% of the patients presenting with diarrhea in Van and Sanliurfa Turkey.

  2. International reference standards: antibody standards for the indirect enzyme-linked immunosorbent assay.

    PubMed

    Wright, P F; Tounkara, K; Lelenta, M; Jeggo, M H

    1997-12-01

    Reference standards are used to calibrate similar assay systems against an international reference protocol and to provide a template for the preparation of secondary and/or working standards. Three reference standards are recommended for the indirect enzyme-linked immunosorbent assay: a strong positive standard, a weak positive standard and a negative serum standard. The negative standard should be derived from a single serum or from a serum pool which exhibits typical background activity in the reference protocol. The strong and weak positive standards should be derived from a single serum or from a serum pool which typifies the humoral response (antibody) to natural infection. Suitable candidates for the positive reference standards should exhibit dose/response curves in the mid-range of antibody activity. The strong and weak positive standards should each be prepared from a one-time dilution in the negative standard, to yield antibody activities which are defined by specific points on the linear portion of the dose/response curve. The strong positive standard should represent an antibody activity (absorbance value) midway between the upper and central points and the weak positive standard should represent an antibody activity midway between the central and lower points of the linear portion of the curve. Owing to inherent differences among assay systems, antibody activities should be expressed in relative rather than in absolute terms. It is recommended that the antibody activity of the strong positive standard should denote 100% positivity. The activities of the weak positive and negative standards should then be expressed as relative percentages. Every set of international reference standards should be accompanied by an information sheet which includes, among other things, a plot of the dose/response curve and an indication of the dilutions used to prepare the standards.

  3. 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.

  4. A serological study of severe fever with thrombocytopenia syndrome using a virus neutralization test and competitive enzyme-linked immunosorbent assay

    PubMed Central

    Lee, Hyojin; Kim, Eun-Ju; Cho, In-Soo; Song, Jae-Young; Choi, Jeong Soo; Lee, Ji Youn

    2017-01-01

    Severe fever with thrombocytopenia syndrome (SFTS) is caused by the SFTS virus (SFTSV). The SFTSV appears to have a wide host range, as SFTSV-positive ticks have been isolated from both farm animals and wild rodents. Therefore, it is important to monitor SFTSV-positive animals to prevent the transmission of SFTSV from animals to humans. Previously, we developed a competitive enzyme-linked immunosorbent assay (cELISA) to detect SFTSV-specific antibodies from field animals and compared the cELISA results to those from an indirect immunofluorescence assay (IFA). In this study, cELISA results were compared to and evaluated against the results from both an IFA and a virus neutralization (VN) test of 193 bovine serum samples (including two bovine positive control sera) and 70 horse serum samples. The consistency (98.9%) between cELISA and VN results was higher than that (97.4%) between cELISA and IFA for the bovine serum samples. Similarly, for the horse serum samples, the consistency (88.6%) between cELISA and VN results was higher than that (84.3%) between the cELISA and IFA. These findings indicate that our newly developed cELISA can be used for surveillance or epidemiological studies of SFTSV in animals. PMID:27297411

  5. 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

  6. 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.

  7. Comparison of Three Enzyme-Linked Immunosorbent Assays for Detection of Immunoglobulin G Antibodies to Tetanus Toxoid with Reference Standards and the Impact on Clinical Practice▿

    PubMed Central

    van Hoeven, Karen H.; Dale, Connie; Foster, Phil; Body, Barbara

    2008-01-01

    Accurate determination of the concentrations of immunoglobulin G (IgG) antibody to tetanus toxoid is important in order to evaluate the immunogenicity of tetanus toxoid vaccines, determine immune competence in individual patients, and measure the prevalence of immunity in populations. The performance of three commercially available enzyme-linked immunosorbent assays (ELISAs) for IgG antibodies to tetanus toxoid were evaluated. Serially diluted NIBSC 76/589 and TE-3 human tetanus IgG immunoglobulin international reference standards were analyzed in quadruplicate using ELISAs manufactured by The Binding Site, Inc. (VaccZyme); Scimedx; and Euroimmun. In addition, IgG antibodies to tetanus toxoid were measured in 83 deidentified serum specimens using each manufacturer's ELISA. Each ELISA provided linear results when evaluated with the reference preparations. The Binding Site ELISA provided results that closely corresponded to the reference preparations (y = 1.09x − 0.08), whereas the Scimedx ELISA gave results that were consistently lower (y = 0.21x − 0.07) and the Euroimmun ELISA gave results that were consistently higher (y = 1.5x + 0.30) than the reference preparation concentrations. Using the recommended cutoff for each ELISA (<0.10 IU/ml), the overall agreement of all of the ELISA methods was 78%. Three of eighty-three (3.6%) human serum samples demonstrated inadequate immunity with all three assays. The Binding Site ELISA yielded nonprotective antibody concentrations in only these 3 samples, whereas 19 samples (22.9%) according to the Scimedx ELISA and 6 samples (7.2%) according to the Euroimmun ELISA demonstrated nonprotective concentrations. The performance characteristics of ELISAs for tetanus immunoglobulin titers were manufacturer dependent, and the differences translated into important disparities in reported results. PMID:18845832

  8. Comparison of three enzyme-linked immunosorbent assays for detection of immunoglobulin g antibodies to tetanus toxoid with reference standards and the impact on clinical practice.

    PubMed

    van Hoeven, Karen H; Dale, Connie; Foster, Phil; Body, Barbara

    2008-12-01

    Accurate determination of the concentrations of immunoglobulin G (IgG) antibody to tetanus toxoid is important in order to evaluate the immunogenicity of tetanus toxoid vaccines, determine immune competence in individual patients, and measure the prevalence of immunity in populations. The performance of three commercially available enzyme-linked immunosorbent assays (ELISAs) for IgG antibodies to tetanus toxoid were evaluated. Serially diluted NIBSC 76/589 and TE-3 human tetanus IgG immunoglobulin international reference standards were analyzed in quadruplicate using ELISAs manufactured by The Binding Site, Inc. (VaccZyme); Scimedx; and Euroimmun. In addition, IgG antibodies to tetanus toxoid were measured in 83 deidentified serum specimens using each manufacturer's ELISA. Each ELISA provided linear results when evaluated with the reference preparations. The Binding Site ELISA provided results that closely corresponded to the reference preparations (y=1.09x-0.08), whereas the Scimedx ELISA gave results that were consistently lower (y=0.21x-0.07) and the Euroimmun ELISA gave results that were consistently higher (y=1.5x+0.30) than the reference preparation concentrations. Using the recommended cutoff for each ELISA (<0.10 IU/ml), the overall agreement of all of the ELISA methods was 78%. Three of eighty-three (3.6%) human serum samples demonstrated inadequate immunity with all three assays. The Binding Site ELISA yielded nonprotective antibody concentrations in only these 3 samples, whereas 19 samples (22.9%) according to the Scimedx ELISA and 6 samples (7.2%) according to the Euroimmun ELISA demonstrated nonprotective concentrations. The performance characteristics of ELISAs for tetanus immunoglobulin titers were manufacturer dependent, and the differences translated into important disparities in reported results.

  9. 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

  10. PCR-enzyme-linked immunosorbent assay for detection and identification of Campylobacter species: application to isolates and stool samples.

    PubMed

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

    1999-02-01

    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.

  11. 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

  12. Early and specific diagnosis of seropositivity to HIVs by an enzyme-linked immunosorbent assay using env-derived synthetic peptides.

    PubMed

    Fenouillet, E; Sorensen, A M; Lacroix, M; Coutellier, A; Herson, S; Fretz-Foucault, C; Gluckman, J C

    1990-11-01

    We describe and evaluate the sensitivity and specificity of an enzyme-linked immunosorbent assay (ELISA) using a 22-amino-acid peptide corresponding to the carboxy-terminal end of HIV-1 gp120 and two 30-amino-acid long cyclic peptides including the two vicinal cysteines present on HIV-1 gp41 and on HIV-2 gp36. This test was evaluated. Data obtained with the Western blot (WB) and the peptide-based ELISA on a first panel composed of sera from 547 patients attending a specialized outpatient clinic (high-risk population) are in perfect agreement; moreover, 39 samples that had falsely been found positive with a viral lysate-based ELISA were not detected by peptide-based ELISA. The second panel was composed of 309 sera which were difficult to resolve using both WB and viral lysate-based ELISA. Using the peptide-based ELISA, 134 were found clearly positive and 173 clearly negative; only two were falsely positive. Finally, sera from 16 individuals examined at the time of seroconversion gave high absorbancy readings even if they were weakly reactive by WB (weak gp160 band). This test is thus highly sensitive and specific, and capable of detecting early seroconversion. It is also instrumental in clearly defining samples that are found indeterminate in the WB, and consequently it avoids the unnecessary follow-up required when a false-positive result is obtained using viral lysate-based ELISA.

  13. Utility of Schistosoma bovis adult worm antigens for diagnosis of human schistosomiasis by enzyme-linked immunosorbent assay and electroimmunotransfer blot techniques.

    PubMed

    Pardo, J; Carranza, C; Turrientes, M C; Pérez Arellano, J L; López Vélez, R; Ramajo, V; Muro, A

    2004-11-01

    Immunodiagnostic methods based on the detection of antibodies continue to be the most effective and practical methods for the diagnosis of imported schistosomiasis. Schistosoma bovis is a species whose final natural hosts are bovines, ovines, caprines, and small wild ruminants. Different studies have demonstrated the analogies existing between S. bovis and other Schistosoma species which affect humans. The objective of this work was to evaluate the utility of S. bovis adult worm antigens (AWA) for the diagnosis of imported human schistosomiasis by enzyme-linked immunosorbent assay (ELISA) and electroimmunotransfer blotting (EITB) techniques. By detecting eggs, the ELISA for S. bovis AWA was able to definitively detect imported cases with a sensitivity of 94%. The specificity of the ELISA for S. bovis AWA was 97%. There were no differences between the results of the S. bovis AWA ELISA for patients infected with Schistosoma mansoni and those infected with Schistosoma haematobium. The EITB technique showed bands of 85, 37, and 20 kDa, which are characteristic of infections with Schistosoma spp. Specific bands to indicate infection by different species of Schistosoma have not been detected. The combined use of the ELISA for S. bovis AWA and EITB increased the global sensitivity of the study to 97%. Our findings suggest that the ELISA for S. bovis AWA is a useful test for the immunodiagnosis of imported schistosomiasis and that EITB for detecting S. bovis AWA permits the confirmation of diagnosis when the ELISA for S. bovis AWA is positive.

  14. Utility of Schistosoma bovis Adult Worm Antigens for Diagnosis of Human Schistosomiasis by Enzyme-Linked Immunosorbent Assay and Electroimmunotransfer Blot Techniques

    PubMed Central

    Pardo, J.; Carranza, C.; Turrientes, M. C.; Arellano, J. L. Pérez; Vélez, R. López; Ramajo, V.; Muro, A.

    2004-01-01

    Immunodiagnostic methods based on the detection of antibodies continue to be the most effective and practical methods for the diagnosis of imported schistosomiasis. Schistosoma bovis is a species whose final natural hosts are bovines, ovines, caprines, and small wild ruminants. Different studies have demonstrated the analogies existing between S. bovis and other Schistosoma species which affect humans. The objective of this work was to evaluate the utility of S. bovis adult worm antigens (AWA) for the diagnosis of imported human schistosomiasis by enzyme-linked immunosorbent assay (ELISA) and electroimmunotransfer blotting (EITB) techniques. By detecting eggs, the ELISA for S. bovis AWA was able to definitively detect imported cases with a sensitivity of 94%. The specificity of the ELISA for S. bovis AWA was 97%. There were no differences between the results of the S. bovis AWA ELISA for patients infected with Schistosoma mansoni and those infected with Schistosoma haematobium. The EITB technique showed bands of 85, 37, and 20 kDa, which are characteristic of infections with Schistosoma spp. Specific bands to indicate infection by different species of Schistosoma have not been detected. The combined use of the ELISA for S. bovis AWA and EITB increased the global sensitivity of the study to 97%. Our findings suggest that the ELISA for S. bovis AWA is a useful test for the immunodiagnosis of imported schistosomiasis and that EITB for detecting S. bovis AWA permits the confirmation of diagnosis when the ELISA for S. bovis AWA is positive. PMID:15539523

  15. 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

  16. Development of polyclonal antibody-based indirect enzyme-linked immunosorbent assay for the detection of Alicyclobacillus strains in apple juice.

    PubMed

    Wang, Zhouli; Yue, Tianli; Yuan, Yahong; Cai, Rui; Guo, Caixia; Wang, Xin; Niu, Chen

    2012-11-01

    A sort of specific polyclonal anti-Alicyclobacillus antibody was generated by immunizing New Zealand white rabbits, and a sensitive indirect enzyme-linked immunosorbent assay (ELISA) was developed for Alicyclobacillus detection in apple juice. A set of experimental parameters such as concentration of antigen, dilutions of the antibody and goat anti-rabbit IgG-horseradish peroxidase conjugate, selection of the blocking reagent, incubation time, and temperature was optimized. The cross-reactivity of the antibody was evaluated by ELISA and the result was consistent with Western blot analysis. The detection limit of the ELISA was about 10(5) colony forming units (CFU)/mL in apple juice samples. Samples were detected by ELISA and conventional culture method, and the ELISA results gave a good agreement with the results obtained by plating on Alicyclobacillus acidoterrestris medium agar. ELISA takes a total detection time of 6 to 7 h, which is less than the time of conventional techniques requiring more than 24 to 48 h. These results indicated that the established ELISA was a potential useful analytical method for detection of Alicyclobacillus in apple juice. © 2012 Institute of Food Technologists®

  17. Evaluation of a recombinant LigB protein of Leptospira interrogans serovar Canicola in an enzyme-linked immunosorbent assay for the serodiagnosis of bovine leptospirosis.

    PubMed

    Sankar, Surya; Harshan, Hiron M; Somarajan, S R; Srivastava, S K

    2010-06-01

    A recombinant leptospiral lipoprotein, LigB, was evaluated for use in the diagnosis of bovine leptospirosis by enzyme-linked immunosorbent assay (rLigB IgG ELISA). The standard reference test (Microscopic agglutination test, MAT) of 200 serum samples from cattle suspected of leptospirosis showed that 95 (47.5%) samples had positive agglutination titres, which ranged from 100 to 1600. In rLigB IgG ELISA, 49% of the samples were positive. Sensitivity of IgG ELISA for 95 bovine sera, which had MAT titres of greater than or equal to 100, were 100%. ELISA showed a specificity of 97.1% with 105 bovine sera, which were negative at a 1:50 dilution in MAT for Leptospira interrogans serovars. The results of ELISA and MAT correspond very good. When analytical specificity of IgG ELISA was evaluated using bovine serum samples from animals showing the serum antibodies to other pathogens, no cross-reaction was observed. Thus the recombinant LigB IgG ELISA can be used instead of the MAT as an aid to the diagnosis of bovine leptospirosis. Copyright 2009. Published by Elsevier India Pvt Ltd.

  18. Development of a Blocking Enzyme-Linked Immunosorbent Assay for Detection of Serum Antibodies to O157 Antigen of Escherichia coli

    PubMed Central

    Laegreid, William; Hoffman, Mark; Keen, James; Elder, Robert; Kwang, Jimmy

    1998-01-01

    The O157 antigen of Escherichia coli shares structural elements with lipopolysaccharide (LPS) antigens of other bacterial species, notably Brucella abortus and Yersinia enterocolitica O9, a fact that confounds the interpretation of assays for anti-O157 antibodies. To address this problem, a blocking enzyme-linked immunosorbent assay (bELISA) was designed with E. coli O157:H7 LPS as the antigen and a monoclonal antibody specific for E. coli O157, designated 13B3, as the competing antibody. The bELISA had equivalent sensitivity to, and significantly higher specificity than, the indirect ELISA (iELISA), detecting anti-O157 antibodies in sera from cattle experimentally inoculated with O157:H7. Only 13% of sera from naive heifers vaccinated for or experimentally infected with B. abortus had increased anti-O157 bELISA titers, while 61% of anti-O157 iELISA titers were increased. The bELISA is a sensitive and specific method for the detection of serum antibodies resulting from exposure to E. coli O157. PMID:9521150

  19. 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

  20. Comparison of two new tests for rapid diagnosis of respiratory syncytial virus infections by enzyme-linked immunosorbent assay and immunofluorescence techniques.

    PubMed Central

    Freymuth, F; Quibriac, M; Petitjean, J; Amiel, M L; Pothier, P; Denis, A; Duhamel, J F

    1986-01-01

    The sensitivity and the specificity of two new commercial reagent tests, an indirect fluorescent antibody test (FAT) with a mouse monoclonal antibody (MAb) against respiratory syncytial virus (RSV) and an enzyme-linked immunosorbent assay (ELISA) RSV antigen detection kit, were determined by a comparison of results from these tests with those of tissue culture isolation and an indirect FAT with bovine polyclonal antibody (BPA). Of 251 nasal aspirates from infants with suspected RSV infection, positive results were found for 99 (39%) by the FAT-MAb, 93 (37%) by the FAT-BPA, and 87 (35%) by the ELISA; 69 of 240 (29%) were positive by cultures. The FAT-MAb was a more sensitive technique than cultures, with 87% sensitivity for the FAT-MAb and 84% for the ELISA. It was also more sensitive than the FAT-BPA, with 97% sensitivity for the FAT-MAb and 85% for the ELISA. This could be caused only by the distinctive volume of suspended specimens used in these tests. Of 171 negative culture specimens, positive (but not false-positive) results were found for 18% by the FAT-MAb and for 12% by the ELISA. Inversely, 13% of 69 culture positive specimens were FAT-MAb negative and 16% were ELISA negative, emphasizing the importance of tissue cultures for the maximum recovery of RSV, as well as for detection of other respiratory viruses. The FAT-MAb and ELISA were easy to perform and interpret, thus facilitating wider use. PMID:3536993

  1. The diagnosis of high altitude illness by the determination of plasma dermcidin isoform 2 levels by enzyme linked immunosorbent assay.

    PubMed

    Bank, Sarbashri; Ghosh, Rajeshwary; Jana, Pradipta; Bhattacharya, Suman; Sinha, Asru K

    2014-01-01

    High altitude illness (HAI) is a cluster of syndromes which develops due to the injury of the central nervous system produced by the reduction of the partial pressure of O2 in the atmosphere which disappears on decent. The HAI also results in a prothrombotic condition leading to acute coronary syndrome (ACS), which cannot be controlled on descent to the ground level. There is no diagnosis in HAI to forewarn of the impending ACS. A protein identified to be dermcidin isoform 2 (dermcidin), produced in the system due to environmental stresses, has been reported to be a potent diabetogenic agent. Investigation was carried out to determine the systemic stimulation of dermcidin synthesis at different levels of altitudes in normal adult male volunteers to assess the feasibility of developing a diagnosis for ACS in HAI due to dermcidin synthesis. Normal, nondiabetic, normotensive male volunteers (25 - 35 years old, n = 16) participated in the study. The plasma dermcidin level was determined by enzyme linked immunosorbent assay (ELISA) and by in vitro translation of dermcidin mRNA. The plasma insulin level was determined by ELISA and blood glucose level was determined in a glucometer (Behringer). The plasma dermcidin level in the volunteers at ground level was 10 +/- 2.10 nM and increased to 80 +/- 4.62 nM at 15000 feet altitude. For each 1000 feet increase of altitude, the dermcidin level increased by 5.83 +/- 0.21 nM with a Coefficient of Correlation "r" = +0.9405. The increase of plasma dermcidin level was found to be inversely related to the decrease of plasma insulin level from 23 microunit/mL to 5 microunit/mL from sea level to 15000 feet height ("r" = -0.9951) with concomitant increase of blood sugar level from 80 +/- 3.6 mg/dL to 135 +/- 2.01 mg/dL. These results suggest the feasibility of a diagnosis of a prediabetic condition by determining the plasma dermcidin level in HAI by simple ELISA which may also be useful to forewarn of the possibility of developing an

  2. Analysis of antibody responses to Hymenolepis nana infection in mice by the enzyme-linked immunosorbent assay and immunoprecipitation.

    PubMed

    Ito, A; Honey, R D; Scanlon, T; Lightowlers, M W; Rickard, M D

    1988-05-01

    Serum antibody responses in two strains of mice infected with embryonated eggs of Hymenolepis nana were analysed by the enzyme-linked immunosorbent assay (ELISA) and immunoprecipitation (IP) using sodium deoxycholate (DOC)-solubilized antigens prepared from embryonated eggs (eggs), mouse-derived cysticercoids (cysts) and adult tapeworms with immature segments only (adults). Highly susceptible dd mice, which harbour mature tapeworms for a long period (greater than 70 days), produced high levels of antibodies to all three different stages of H. nana. BALB/c mice, almost all of which expel adult tapeworms by 30 days after infection, produced high levels of antibody against egg antigens only. The high antibody titres to cyst and adult antigens in dd mice did not lead to expulsion of the worms. However, worms are rejected early in BALB/c mice when there is little or no detectable serum antibody. The antibody responses to eggs seen in BALB/c mice which had long since shed their adult worms were probably due to ingestion of eggs from faeces of other infected mice. Antibodies to eggs were not detected in BALB/c mice which were initially inoculated with eggs (day 0) and then treated with praziquantel on day 6 after the tissue phase of infection only. The different antibody responses to egg antigens and the other two antigens (cyst and adult) in BALB/c mice suggest a difference in antigen specificity between eggs and both cysts and adults. A major antigen component with Mr 32,000 appears to be specific to the egg (or oncosphere) stage of H. nana. Antibody to this major component of eggs was absorbed only with intact eggs, but not with intact cysts nor adults with immature segments only, so that the antigen appears to be on the surface of the oncosphere.

  3. A sensitive enzyme-linked immunosorbent assay amplified by biotin-streptavidin system for detecting non-steroidal anti-inflammatory drug ketoprofen.

    PubMed

    Bu, Dan; Zhuang, Hui S; Yang, Guang X

    2014-01-01

    A sensitive biotin-streptavidin-amplified enzyme-linked immunosorbent assay (BA-ELISA) method was developed for detecting non-steroidal anti-inflammatory drug ketoprofen. Compared with traditional ELISA method, the sensitivity of proposed immunoassay was enhanced by the biotin-streptavidin system. Under the optimal condition, the median inhibitory concentration (IC50) was 0.25 ng mL(-1), with minor cross-reactivity to a number of structural analogs. This developed assay was successfully applied to detect the ketoprofen residues in different fish samples, and good recoveries (72.6-105.5%) were obtained. The results indicated that this immunoassay method could specifically detect trace ketoprofen residues and could be widely used for routine monitoring of food samples.

  4. 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.

  5. Indirect Enzyme-Linked Immunosorbent Assay for Detection of Immunoglobulin G Reactive with a Recombinant Protein Expressed from the Gene Encoding the 116-Kilodalton Protein of Mycoplasma pneumoniae

    PubMed Central

    Duffy, Michael F.; Whithear, Kevin G.; Noormohammadi, Amir H.; Markham, Philip F.; Catton, Michael; Leydon, Jennie; Browning, Glenn F.

    1999-01-01

    Serology remains the method of choice for laboratory diagnosis of Mycoplasma pneumoniae infection. Currently available serological tests employ complex cellular fractions of M. pneumoniae as antigen. To improve the specificity of M. pneumoniae diagnosis, a recombinant protein was assessed as a serodiagnostic reagent. A panel of recombinant proteins were expressed from a cloned M. pneumoniae gene that encodes a 116-kDa surface protein antigen. The recombinant proteins were assessed for reactivity with patient sera and the most antigenic was further assessed for its serodiagnostic potential by indirect enzyme-linked immunosorbent assay (ELISA). The ELISA based on the recombinant protein was equivalent in sensitivity to the commercial test (Serodia Myco II; Fujirebio Inc.) to which it was compared. Southern and Western blotting data suggested that the recombinant protein derived from the 116-kDa protein of M. pneumoniae could provide a species-specific diagnostic tool, although further assessment is required. PMID:10074521

  6. Monoclonal antibodies to surface antigens of Mycobacterium tuberculosis and their use in a modified enzyme-linked immunosorbent spot assay for detection of mycobacteria.

    PubMed

    Glatman-Freedman, A; Martin, J M; Riska, P F; Bloom, B R; Casadevall, A

    1996-11-01

    Three monoclonal antibodies (MAbs) were generated from splenocytes of a BALB/c mouse immunized with heat-killed Mycobacterium tuberculosis. All three MAbs bound to surface epitopes of M. tuberculosis as shown by whole-cell enzyme-linked immunosorbent assay (ELISA), indirect immunofluorescence, and immunoelectron microscopy. One immunoglobulin M (IgM) MAb bound to lipoarabinomannan, the second IgM MAb bound to mycolyl-arabinogalactan-peptidoglycan complex, and the third MAb, an IgG3, bound to a surface epitope of an uncertain nature. The MAbs demonstrated different cross-reactivity patterns with other mycobacteria. Two of the MAbs were used to develop a modified ELISA spot assay for the detection of mycobacteria.

  7. Seroprevalence study of Equine rhinitis B virus (ERBV) in Australian weanling horses using serotype-specific ERBV enzyme-linked immunosorbent assays.

    PubMed

    Horsington, Jacquelyn; Hartley, Carol A; Gilkerson, James R

    2013-09-01

    Respiratory infections are a major burden in the performance horse industry. Equine rhinitis B virus (ERBV) has been isolated from horses displaying clinical respiratory disease, and ERBV-neutralizing antibodies have been detected in 50-80% of horses in reported surveys. Current ERBV isolation and detection methods may underestimate the number of ERBV-positive animals and do not identify multiple serotype infections. The aim of the current study was to develop a serotyping ERBV antibody-detection enzyme-linked immunosorbent assay (ELISA) and examine the seroprevalence of ERBV in a group of Australian weanling horses. ELISAs with high sensitivity and specificity were developed. The seroprevalence of ERBV in the weanling horses was high (74-86%); ERBV-3 antibodies were most prevalent (58-62%) and ERBV-2 antibodies were least prevalent (10-16%). Many horses were seropositive to 2 or more serotypes. All 3 serotypes of ERBV were detected, and concurrent positivity to multiple serotypes was common.

  8. Indirect enzyme-linked immunosorbent assay for immunoglobulin G and four immunoassays for immunoglobulin M to Toxoplasma gondii in a series of heart transplant recipients.

    PubMed Central

    Sluiters, J F; Balk, A H; Essed, C E; Mochtar, B; Weimar, W; Simoons, M L; Ijzerman, E P

    1989-01-01

    Toxoplasma gondii infections in heart transplant recipients were monitored by indirect enzyme-linked immunosorbent assay for immunoglobulin G (ELISA-IgG), indirect ELISA-IgM in serum IgM fractions, antibody capture ELISA-IgM, IgM-immunosorbent agglutination assay (ISAGA), and IgM immunoblotting. Basic immunosuppression consisted of cyclosporine and low-dose steroids. Before transplantation, 26 of 43 recipients showed serological evidence of infection. In serum samples from 15 (35%) recipients, specific antibodies were not detected. Approximately 50% of the heart donors, were toxoplasma seropositive. Eight of the fifteen seronegative recipients received hearts from toxoplasma-seropositive donors. In four of the eight recipients, seroconversion could be demonstrated with all tests used. In three of these four patients, clinical disease developed. One patient with strong serological evidence of toxoplasmosis died, but toxoplasma parasites and antigens were not detected at autopsy. In two patients, toxoplasma cysts were found in cardiac biopsies. Seroconversion was not prevented by the use of spiramycin prophylaxis in two recipients. Reactivations of latent infections or reinfections were detected by indirect ELISA in six (23%) seropositive recipients, but symptoms and signs of active T. gondii infection were not seen. Seroconversion and reactivation of infection were readily found by a combined use of indirect ELISA-IgG and ELISA-IgM and antibody capture ELISA-IgM. Discrepancies in results could be examined by immunoblotting. IgM-ISAGA retained stable positive values longer than IgM-ELISAs did. Cyclosporine treatment did not hamper detection of seroconversion but could cause antibody levels to remain relatively low in primary infections. Seronegative recipients should receive antitoxoplasma treatment on seroconversion. PMID:2654182

  9. Evaluation of Three Commercial Varicella-Zoster Virus IgG Enzyme-Linked Immunosorbent Assays in Comparison to the Fluorescent-Antibody-to-Membrane-Antigen Test

    PubMed Central

    Schäfler, Anna; Hofmann, Jörg; Schacke, Michael; Gruhn, Bernd; Wutzler, Peter

    2012-01-01

    Commercial serologic assays for varicella-zoster virus (VZV), which enable reliable determination of VZV immune status and are amenable to automation, are needed. The present study compares the automated performance of the VZV whole-cell enzyme-linked immunosorbent assay (ELISA) Enzygnost anti-VZV/IgG, the Euroimmun anti-VZV ELISA (IgG) based on highly purified viral proteins, and the VZV glycoprotein (gp)-based Serion ELISA Classic VZV IgG. The fluorescent-antibody-to-membrane-antibody (FAMA) test was used as a reference. A total of 638 serum samples from VZV-negative children, blood donors, varicella vaccinees, and bone marrow transplant recipients were included. The Enzygnost anti-VZV/IgG and the Serion ELISA Classic VZV IgG showed sensitivities of 99.6% and 99.2%, respectively, and the Euroimmun anti-VZV ELISA (IgG) had a significantly lower sensitivity of 90.5%. Specificity was calculated as 100% for both the Euroimmun anti-VZV ELISA (IgG) and for the Enzygnost anti-VZV/IgG, and the Serion ELISA Classic VZV IgG had a significantly lower specificity of 89.4%. Quantitative results of all ELISAs correlated well, but there was a poor quantitative correlation between the ELISAs and FAMA. In conclusion, this study does not show any superiority of a gp- and a protein-based ELISA compared to a whole-cell ELISA for the automated detection of VZV-specific IgG. The automated performance of the Enzygnost anti-VZV/IgG assay correlated best with the FAMA reference assay. PMID:22718131

  10. Detection of Antiendothelial Cell Antibodies by an Enzyme-Linked Immunosorbent Assay Using Antigens from Cell Lysate: Minimal Interference with Antinuclear Antibodies and Rheumatoid Factors

    PubMed Central

    Drouet, Christian; Nissou, Marie-France; Ponard, Denise; Arvieux, Josiane; Dumestre-Pérard, Chantal; Gaudin, Philippe; Imbert, Bernard; Massot, Christian; Sarrot-Reynauld, Françoise

    2003-01-01

    The objective of the present work was to set up a routine test adapted to screening for antiendothelial cell antibodies (AECAs) in serum samples with minimal interference from antinuclear antibodies (ANAs) or rheumatoid factors (RFs). We compared the titers of AECAs titrated following two enzyme-linked immunosorbent assays (ELISAs): (i) an ELISA with ethanol-fixed EA.hy926 monolayers as the antigenic substrate and (ii) an ELISA with nucleus-depleted lysates prepared from EA.hy926 cells and normalized for protein (1.0 to 1.7 mg/ml) and DNA (≤0.1 μg/ml) contents as a surrogate substrate (postnuclear supernatant ELISA [PNS-ELISA]). The AECA titers in 51 serum samples, including 28 samples containing ANAs, were compared. A significantly positive correlation (r = 0.77; P < 0.001) between the two series was shown only for the ANA-negative serum samples. Conversely, ANAs or RFs in samples were shown not to interfere in tests for AECAs by the PNS-ELISA. AECAs recognize their antigenic targets in postnuclear supernatants, which is representative of the endothelial antigenic content, with improvement of the reliability of the assay, a prerequisite to application of the assay for their evaluation in clinical practice. PMID:12965929

  11. Comparison of Laboratory-Developed and Commercial Monoclonal Antibody-Based Sandwich Enzyme-Linked Immunosorbent Assays for Almond (Prunus dulcis) Detection and Quantification.

    PubMed

    Liu, Changqi; Chhabra, Guneet S; Zhao, Jing; Zaffran, Valerie D; Gupta, Sahil; Roux, Kenneth H; Gradziel, Thomas M; Sathe, Shridhar K

    2017-09-04

    A commercially available monoclonal antibody (mAb)-based direct sandwich enzyme-linked immunosorbent assay (ELISA) kit (BioFront Technologies, Tallahassee, Fla., U.S.A.) was compared with an in-house developed mAb 4C10-based ELISA for almond detection. The assays were comparable in sensitivity (limit of detection < 1 ppm full fat almond, limit of quantification < 5 ppm full fat almond), specificity (no cross-reactivity with 156 tested foods at a concentration of 100000 ppm whole sample), and reproducibility (intra- and interassay variability < 15% CV). The target antigens were stable and detectable in whole almond seeds subjected to autoclaving, blanching, frying, microwaving, and dry roasting. The almond recovery ranges for spiked food matrices were 84.3% to 124.6% for 4C10 ELISA and 81.2% to 127.4% for MonoTrace ELISA. The almond recovery ranges for commercial and laboratory prepared foods with declared/known almond amount were 30.9% to 161.2% for 4C10 ELISA and 38.1% to 207.6% for MonoTrace ELISA. Neither assay registered any false-positive or negative results among the tested commercial and laboratory prepared samples. © 2017 Institute of Food Technologists®.

  12. A novel enzyme-linked immunosorbent assay for detection of Escherichia coli O157:H7 using immunomagnetic and beacon gold nanoparticles.

    PubMed

    Shen, Zhiqiang; Hou, Nannan; Jin, Min; Qiu, Zhigang; Wang, Jingfeng; Zhang, Bin; Wang, Xinwei; Wang, Jie; Zhou, Dongsheng; Li, Junwen

    2014-01-01

    This paper presents a functional nanoparticle-enhanced enzyme-linked immunosorbent assay (FNP-ELISA) for detection of enterohemorrhagic Escherichia coli (EHEC) O157:H7. Immunomagnetic nanoparticles (IMMPs) conjugated with monoclonal anti-O157:H7 antibody were used to capture E. coli O157:H7. Beacon gold nanoparticles (B-GNPs) coated with polyclonal anti-O157:H7 and biotin single-stranded DNA (B-DNA) were then subjective to immunoreaction with E. coli O157:H7, which was followed by streptavidin-horseradish peroxidase (Strep-HRP) conjugated with B-GNPs based on a biotin-avidin system. The solutions containing E. coli O157:H7, IMMPs, B-GNPs, and Strep-HRP were collected for detecting color change. The signal was significantly amplified with detection limits of 68 CFU mL(-1) in PBS and 6.8 × 10(2) to 6.8 × 10(3) CFU mL(-1) in the food samples. The FNP-ELISA method developed in this study was two orders of magnitude more sensitive than immunomagnetic separation ELISA (IMS-ELISA) and four orders of magnitude more sensitive than C-ELISA. The entire detection process of E. coli O157:H7 lasted only 3 h, and thus FNP-ELISA is considered as a time-saving method.

  13. Evaluation of spore wall protein 1 as an alternative antigen for the diagnosis of Encephalitozoon cuniculi infection of farmed foxes using an enzyme-linked immunosorbent assay.

    PubMed

    Meng, Xiangchun; Zheng, Jun; Gao, Yang; Zhang, Yanlong; Jia, Honglin

    2014-07-14

    The sequence encoding SWP1 was cloned from the genome of Encephalitozoon cuniculi. Recombinant SWP1 (rSWP1) was expressed in Escherichia coli and used to detect E. cuniculi infections in farmed foxes and dogs with an indirect enzyme-linked immunosorbent assay (ELISA) in the present study. The sera of foxes infected with E. cuniculi could be distinguished from the sera of foxes infected with Toxoplasma gondii, Neospora caninum, and Cryptosporidium parvum using the ELISA. In total, 198 fox samples collected in Liaoning were used to determine the prevalence of antibodies against this disease. The results showed that 16.7% of the fox serum samples were positive according to the ELISA using rSWP1, which agreed with the ELISA results based on recombinant PTP2 (rPTP2). The sensitivity and specificity of the ELISA based on rSWP1 suggest that this could be an alternative method for the diagnosis of E. cuniculi infections in foxes. In addition, 298 dog samples collected in Beijing, Shanghai, and Hunan were also detected in this study, of which six dog samples (2%) were positive according to the ELISA using rSWP1. To the best of our knowledge, this is the first study to demonstrate the serological prevalence of E. cuniculi infections in dogs and foxes in China. Copyright © 2014 Elsevier B.V. All rights reserved.

  14. Comparison of a multiplex flow cytometric assay with enzyme-linked immunosorbent assay for auantitation of antibodies to tetanus, diphtheria, and Haemophilus influenzae Type b.

    PubMed

    Pickering, Jerry W; Martins, Thomas B; Schroder, M Carl; Hill, Harry R

    2002-07-01

    We developed a multiplexed indirect immunofluorescence assay for antibodies to Haemophilus influenza type b (Hib) polysaccharide and the toxoids of Clostridium tetani (Tet) and Corynebacterium diphtheriae (Dip) based on the Luminex multiple-analyte profiling system. A pooled serum standard was calibrated against World Health Organization standards for Dip and Tet and an international standard for Hib. The multiplexed Luminex assay was compared to individual enzyme-linked immunosorbent assays (ELISAs) for the same analytes. By both methods, 75 (92.6%) of 81 of random serum samples had protective levels of antibody to Tet (> or = 0.1 IU/ml). For Dip, 81.5% of the samples had protective antibody levels (> or = 0.1 IU/ml) by ELISA and 80.2% had protective antibody levels by Luminex. Protective levels (> or = 1.0 microg/ml) of antibody to Hib were found in 45.0% of the samples tested by ELISA and in 39.0% of the samples tested by Luminex. The correlations (R(2)) between ELISA and Luminex of the 81 samples were 0.96, 0.96, and 0.91 for Tet, Dip, and Hib, respectively. There was also similar agreement between Luminex and ELISA for sera collected before and 1 month after Tet, Dip, and Hib vaccine administration. Both methods detected strong postvaccination responses. The Luminex method is an attractive alternative to ELISA since it reduces labor and reagent costs, as well as assay time.

  15. A novel enzyme-linked immunosorbent assay for detection of Escherichia coli O157:H7 using immunomagnetic and beacon gold nanoparticles

    PubMed Central

    2014-01-01

    This paper presents a functional nanoparticle-enhanced enzyme-linked immunosorbent assay (FNP-ELISA) for detection of enterohemorrhagic Escherichia coli (EHEC) O157:H7. Immunomagnetic nanoparticles (IMMPs) conjugated with monoclonal anti-O157:H7 antibody were used to capture E. coli O157:H7. Beacon gold nanoparticles (B-GNPs) coated with polyclonal anti-O157:H7 and biotin single-stranded DNA (B-DNA) were then subjective to immunoreaction with E. coli O157:H7, which was followed by streptavidin-horseradish peroxidase (Strep-HRP) conjugated with B-GNPs based on a biotin-avidin system. The solutions containing E. coli O157:H7, IMMPs, B-GNPs, and Strep-HRP were collected for detecting color change. The signal was significantly amplified with detection limits of 68 CFU mL-1 in PBS and 6.8 × 102 to 6.8 × 103 CFU mL-1 in the food samples. The FNP-ELISA method developed in this study was two orders of magnitude more sensitive than immunomagnetic separation ELISA (IMS-ELISA) and four orders of magnitude more sensitive than C-ELISA. The entire detection process of E. coli O157:H7 lasted only 3 h, and thus FNP-ELISA is considered as a time-saving method. PMID:24864164

  16. Detection of antibodies against Bordetella avium in turkeys by avidin-biotin enhancement of the enzyme-linked immunosorbent assay and the dot-immunobinding assay.

    PubMed

    Tsai, H J; Saif, Y M

    1991-01-01

    An avidin-biotin-enhanced enzyme-linked immunosorbent assay (AB-ELISA) and an avidin-biotin-enhanced dot-immunobinding (AB-DIB) assay for detecting antibody to Bordetella avium in turkey sera were developed and compared with the microagglutination (MA) test. Whole-cell antigen, biotin-labeled goat anti-turkey IgG conjugate, and horseradish-peroxidase-labeled streptavidin were used in the AB-ELISA and AB-DIB assay. The AB-ELISA and AB-DIB assay were sensitive, specific, and reproducible. These assays were superior to the MA test for measuring acquired and maternal antibodies against B. avium. All MA-positive sera were positive by two assays, but some sera negative by MA test had titers in the AB-ELISA and AB-DIB assay. AB-ELISA and AB-DIB titers showed a positive correlation (r = 0.866), and AB-ELISA was more sensitive than the AB-DIB assay.

  17. 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

  18. Rapid detection of Campylobacter coli, C. jejuni, and Salmonella enterica on poultry carcasses by using PCR-enzyme-linked immunosorbent assay.

    PubMed

    Hong, Yang; Berrang, Mark E; Liu, Tongrui; Hofacre, Charles L; Sanchez, Susan; Wang, Lihua; Maurer, John J

    2003-06-01

    Contamination of retail poultry by Campylobacter spp. and Salmonella enterica is a significant source of human diarrheal disease. Isolation and identification of these microorganisms require a series of biochemical and serological tests. In this study, Campylobacter ceuE and Salmonella invA genes were used to design probes in PCR-enzyme-linked immunosorbent assay (ELISA), as an alternative to conventional bacteriological methodology, for the rapid detection of Campylobacter jejuni, Campylobacter coli, and S. enterica from poultry samples. With PCR-ELISA (40 cycles), the detection limits for Salmonella and Campylobacter were 2 x 10(2) and 4 x 10(1) CFU/ml, respectively. ELISA increased the sensitivity of the conventional PCR method by 100- to 1,000-fold. DNA was extracted from carcass rinses and tetrathionate enrichments and used in PCR-ELISA for the detection of Campylobacter and S. enterica, respectively. With PCR-ELISA, Salmonella was detected in 20 of 120 (17%) chicken carcass rinses examined, without the inclusion of an enrichment step. Significant correlation was observed between PCR-ELISA and cultural methods (kappa = 0.83; chi-square test, P < 0.001) with only one false negative (1.67%) and four false positives (6.67%) when PCR-ELISA was used to screen 60 tetrathionate enrichment cultures for Salmonella. With PCR-ELISA, we observed a positive correlation between the ELISA absorbance (optical density at 405 nm) and the campylobacter cell number in carcass rinse, as determined by standard culture methods. Overall, PCR-ELISA is a rapid and cost-effective approach for the detection and enumeration of Salmonella and Campylobacter bacteria on poultry.

  19. 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

    2004-10-05

    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.

  20. Evaluation of enzyme-linked immunosorbent assay using milk samples as a potential screening test of bovine tuberculosis of dairy cows in Korea.

    PubMed

    Jeon, Bo-Young; Kim, Seung-Cheol; Je, Sungmo; Kwak, Jeongyeon; Cho, Jang-Eun; Woo, Jong-Tae; Seo, Sangkyo; Shim, Hang-Sub; Park, Byoung-Ok; Lee, Sung-Sik; Cho, Sang-Nae

    2010-06-01

    An enzyme-linked immunosorbent assay (ELISA) using bulk tank milk samples was evaluated as a screening test for bovine tuberculosis (TB), a contagious chronic disease of cattle. An ELISA with MPB70, a major antigen of Mycobacterium bovis was performed using paired sets of milk and sera samples from 33 tuberculin-positive and 43 tuberculin-negative cattle. Anti-MPB70 antibodies were detected in milk samples and there was a significant correlation between seroreactivities of milk and sera samples (R(2)=0.83). Using the tuberculin skin test as the reference test, the sensitivities of ELISA using milk and sera samples were 87.8% and 81.8%, respectively, and the specificities were 97.7% and 100%, respectively. In the screening test using bulk tank milk samples from 931 dairy herds in Whasung, Gyeonggi-do, Korea, the positive rate for anti-MPB70 antibody was 4.5% (42/931) and the tuberculin-positive rate was 2.8% (26/931). Individual milk samples (n=253) were collected from randomly selected 8 problematic and 3 negative herds (positive and negative in the screening test by MPB70 ELISA using bulk tank milk samples, respectively) and tested by MPB70 milk ELISA. In the problematic herds, positive rates were 10.5% (20/190) for anti-MPB70 antibodies in milk ELISA and 2.1% (4/190) in the tuberculin skin test. More than one dairy cows were positive by milk ELISA among the problematic herds, and all tuberculin-positive dairy cows were positive in the milk ELISA. Further, no positive cows were detected in negative herds both by milk ELISA and tuberculin skin test. These results suggest that an ELISA, using bulk tank milk samples, might be a potential efficient screening test for bovine TB of dairy cows. Copyright 2009 Elsevier Ltd. All rights reserved.

  1. Enzyme-linked immunosorbent assay for detection of immunoglobulin G antibodies to Escherichia coli Vero cytotoxin 1.

    PubMed

    Karmali, M A; Petric, M; Winkler, M; Bielaszewska, M; Brunton, J; van de Kar, N; Morooka, T; Nair, G B; Richardson, S E; Arbus, G S

    1994-06-01

    The frequency of Vero cytotoxin 1 (VT1)-neutralizing antibody (NAb) in serum specimens from 790 age-stratified (0 to 70 years) control individuals from Toronto was 61 of 790 (7.7%), with a peak of 19% in the 20- to 30-year-old age group and a second peak of 16.7% in the 60- to 70-year-old age group. A total of 568 serum specimens, including 538 from the 790 Toronto control subjects, 21 from patients from three outbreaks of VT-producing Escherichia coli (VTEC) infection, and 9 known VT1-NAb-positive serum specimens from patients with hemolytic-uremic syndrome (HUS), were then tested for the presence of anti-VT1 immunoglobulin G (IgG) by an enzyme-linked immunosorbent assay (ELISA). The mean ELISA values of 522 VT1-NAb-negative serum specimens and 46 VT1-NAb-positive serum specimens were 0.09 +/- 0.06 (range, 0 to 0.56) and 0.78 +/- 0.66 (range, 0.16 to 2.91), respectively (P < 0.001; Student's t test). With a breakpoint of 0.21 (mean ELISA value of the VT1-NAb-negative sera + 2 standard deviations), the sensitivity, specificity, positive predictive value, and negative predictive value of the VT1 IgG ELISA compared with those of the VT1-NAb assay were, respectively, 95.7, 98.7, 86.3, and 99.6%. There were nine discrepant serum specimens, of which seven were anti-VT1 IgG positive and VT1-NAb negative and two were anti-VT1 IgG negative and VT1-NAb positive. The ELISA was also used for testing 238 control serum specimens from The Netherlands, Japan, and India and acute- and convalescent-phase serum specimens from 42 Toronto patients with HUS. The frequencies of anti-VT1 IgG (with VT1-NAb frequencies in parantheses) in control sera from the Netherlands, Japan, and India were 6% (3%), 1.1% (0%), and 12% (10%), respectively, with no age clustering. The frequencies of anti-VT1 IgG seropositivity in HUS patients were 5 of 14 (35.7%) in patients with unknown toxin exposure, 2 of 22 (9.1%) in individuals with known exposure to VT1 plus VT2 or VT1 alone, and 0 of 6 (0%) in

  2. Enzyme-linked immunosorbent assay for detection of immunoglobulin G antibodies to Escherichia coli Vero cytotoxin 1.

    PubMed Central

    Karmali, M A; Petric, M; Winkler, M; Bielaszewska, M; Brunton, J; van de Kar, N; Morooka, T; Nair, G B; Richardson, S E; Arbus, G S

    1994-01-01

    The frequency of Vero cytotoxin 1 (VT1)-neutralizing antibody (NAb) in serum specimens from 790 age-stratified (0 to 70 years) control individuals from Toronto was 61 of 790 (7.7%), with a peak of 19% in the 20- to 30-year-old age group and a second peak of 16.7% in the 60- to 70-year-old age group. A total of 568 serum specimens, including 538 from the 790 Toronto control subjects, 21 from patients from three outbreaks of VT-producing Escherichia coli (VTEC) infection, and 9 known VT1-NAb-positive serum specimens from patients with hemolytic-uremic syndrome (HUS), were then tested for the presence of anti-VT1 immunoglobulin G (IgG) by an enzyme-linked immunosorbent assay (ELISA). The mean ELISA values of 522 VT1-NAb-negative serum specimens and 46 VT1-NAb-positive serum specimens were 0.09 +/- 0.06 (range, 0 to 0.56) and 0.78 +/- 0.66 (range, 0.16 to 2.91), respectively (P < 0.001; Student's t test). With a breakpoint of 0.21 (mean ELISA value of the VT1-NAb-negative sera + 2 standard deviations), the sensitivity, specificity, positive predictive value, and negative predictive value of the VT1 IgG ELISA compared with those of the VT1-NAb assay were, respectively, 95.7, 98.7, 86.3, and 99.6%. There were nine discrepant serum specimens, of which seven were anti-VT1 IgG positive and VT1-NAb negative and two were anti-VT1 IgG negative and VT1-NAb positive. The ELISA was also used for testing 238 control serum specimens from The Netherlands, Japan, and India and acute- and convalescent-phase serum specimens from 42 Toronto patients with HUS. The frequencies of anti-VT1 IgG (with VT1-NAb frequencies in parantheses) in control sera from the Netherlands, Japan, and India were 6% (3%), 1.1% (0%), and 12% (10%), respectively, with no age clustering. The frequencies of anti-VT1 IgG seropositivity in HUS patients were 5 of 14 (35.7%) in patients with unknown toxin exposure, 2 of 22 (9.1%) in individuals with known exposure to VT1 plus VT2 or VT1 alone, and 0 of 6 (0%) in

  3. Comparison of Immunofluorescence and Desmoglein Enzyme-linked Immunosorbent