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Sample records for igm enzyme immunoassay

  1. Enzyme immunoassays for IgG and IgM antibodies to Toxoplasma gondii based on enhanced chemiluminescence.

    PubMed Central

    Crouch, C F

    1995-01-01

    AIMS--To evaluate the clinical performance of enzyme immunoassays for IgG and IgM antibodies to Toxoplasma gondii based on enhanced chemiluminescence. METHODS--Classification of routine clinical samples from the originating laboratories was compared with that obtained using the chemiluminescence based assays. Resolution of discordant results was achieved by testing in alternative enzyme immunoassays (IgM) or by an independent laboratory using the dye test (IgG). RESULTS--Compared with resolved data, the IgM assay was found to be highly specific (100%) with a cut off selected to give optimal performance with respect to both the early detection of specific IgM and the detection of persistent levels of specific IgM (sensitivity 98%). Compared with resolved data, the IgG assay was shown to have a sensitivity and a specificity of 99.4%. CONCLUSIONS--The Amerlite Toxo IgM assay possesses high levels of sensitivity and specificity. Assay interference due to rheumatoid factor like substances is not a problem. The Amerlite Toxo IgG assay possesses good sensitivity and specificity, but is less sensitive for the detection of seroconversion than methods detecting both IgG and IgM. PMID:7560174

  2. Evaluation of serological diagnostic indices for mucocutaneous leishmaniasis: immunofluorescence tests and enzyme-linked immunoassays for IgG, IgM and IgA antibodies.

    PubMed Central

    Guimarães, M. C.; Celeste, B. J.; Franco, E. L.; Cucé, L. C.; Belda, W.

    1989-01-01

    The sensitivity, specificity, positive predictive value, negative predictive value, and efficiency of immunofluorescence (IF) and enzyme-linked immunoassays (ELISA) for IgG, IgM and IgA antibodies were assessed on sera from mucocutaneous leishmaniasis patients and controls. The sensitivity of the IgG-ELISA test was 93.3% with 95% confidence interval higher than what could be due to a random test not associated with the disease. The specificity of all tests, except the IgM-ELISA, gave indices that could not have been due to chance. The IgG-ELISA and IgG-IF had the highest positive predictive value and the kappa statistic showed that the strength of agreement between the disease and the test was strongest for IgG-ELISA. The IgG-ELISA had a negative predictive value with 95% confidence limits that were not due to chance alone. Efficiency was highest for IgG-ELISA and IgG-IF. These results were obtained using sera from patients with severe or long-standing disease and from controls in whom the disease was ruled out by a negative Montenegro skin test. In field surveys where the differences between cases and controls are less easy to define the diagnostic indices of these tests may vary with the disease prevalence. PMID:2699277

  3. Enzyme immunoassay for detection of immunoglobulin G (IgG), IgM, and IgA antibodies against type 6B pneumococcal capsular polysaccharide and cell wall C polysaccharide in chinchilla serum.

    PubMed Central

    Koskela, M; Harris, M; Giebink, G S

    1992-01-01

    Conjugation of the capsular polysaccharides of Streptococcus pneumoniae to protein carriers has introduced a new generation of pneumococcal vaccines which may be efficacious in preventing pneumococcal otitis media during infancy. The chinchilla model has been used extensively for studying the pathogenesis of pneumococcal otitis media and for testing the efficacy of early pneumococcal capsular polysaccharide (PCP) vaccines, but immunologic studies in the chinchilla have been limited by the lack of antibodies against specific immunoglobulin isotypes. By using affinity-purified rabbit immunoglobulin G (IgG) anti-chinchilla IgG, IgM, and IgA, we developed a sensitive enzyme immunoassay that is highly specific for IgG, IgM, and IgA antibodies against type 6B PCP (anti-6B) and against C polysaccharide in chinchilla serum. Antibody titers increased in serum from five chinchillas immunized with a type 6B outer membrane protein complex vaccine. Increases of anti-6B IgG and IgM antibody titers were more striking than increases of anti-6B IgA or anti-C polysaccharide IgG, IgM, or IgA titers were. PMID:1624568

  4. Diagnostic Accuracy of the InBios Scrub Typhus Detect Enzyme-Linked Immunoassay for the Detection of IgM Antibodies in Northern Thailand

    PubMed Central

    Blacksell, Stuart D.; Tanganuchitcharnchai, Ampai; Nawtaisong, Pruksa; Kantipong, Pacharee; Laongnualpanich, Achara; Day, Nicholas P. J.

    2015-01-01

    In this study, we examined the diagnostic accuracy of the InBios Scrub Typhus Detect IgM enzyme-linked immunosorbent assay (ELISA) and determined the optimal diagnostic optical density (OD) cutoffs for screening and diagnostic applications based on prospectively collected, characterized samples from undifferentiated febrile illness patients in northern Thailand. Direct comparisons with the serological gold standard, indirect immunofluorescence assay (IFA), revealed strong statistical correlation of ELISA OD values and IFA IgM titers. Determination of the optimal ELISA cutoff for seroepidemiology or screening purposes compared to the corresponding IFA reciprocal titer of 400 as previously described for Thailand was 0.60 OD, which corresponded to a sensitivity (Sn) of 84% and a specificity (Sp) of 98%. The diagnostic performance against the improved and more-stringent scrub typhus infection criteria (STIC), correcting for low false-positive IFA titers, resulted in an Sn of 93% and an Sp of 91% at an ELISA cutoff of 0.5 OD. This diagnostic ELISA cutoff corresponds to IFA reciprocal titers of 1,600 to 3,200, which greatly reduces the false-positive rates associated with low-positive IFA titers. These data are in congruence with the recently improved serodiagnostic positivity criteria using the Bayesian latent class modeling approach. In summary, the InBios Scrub Typhus Detect IgM ELISA is affordable and easy-to-use, with adequate diagnostic accuracy for screening and diagnostic purposes, and should be considered an improved alternative to the gold standard IFA for acute diagnosis. For broader application, regional cutoff validation and antigenic composition for consistent diagnostic accuracy should be considered. PMID:26656118

  5. Diagnostic Accuracy of the InBios Scrub Typhus Detect Enzyme-Linked Immunoassay for the Detection of IgM Antibodies in Northern Thailand.

    PubMed

    Blacksell, Stuart D; Tanganuchitcharnchai, Ampai; Nawtaisong, Pruksa; Kantipong, Pacharee; Laongnualpanich, Achara; Day, Nicholas P J; Paris, Daniel H

    2015-01-01

    In this study, we examined the diagnostic accuracy of the InBios Scrub Typhus Detect IgM enzyme-linked immunosorbent assay (ELISA) and determined the optimal diagnostic optical density (OD) cutoffs for screening and diagnostic applications based on prospectively collected, characterized samples from undifferentiated febrile illness patients in northern Thailand. Direct comparisons with the serological gold standard, indirect immunofluorescence assay (IFA), revealed strong statistical correlation of ELISA OD values and IFA IgM titers. Determination of the optimal ELISA cutoff for seroepidemiology or screening purposes compared to the corresponding IFA reciprocal titer of 400 as previously described for Thailand was 0.60 OD, which corresponded to a sensitivity (Sn) of 84% and a specificity (Sp) of 98%. The diagnostic performance against the improved and more-stringent scrub typhus infection criteria (STIC), correcting for low false-positive IFA titers, resulted in an Sn of 93% and an Sp of 91% at an ELISA cutoff of 0.5 OD. This diagnostic ELISA cutoff corresponds to IFA reciprocal titers of 1,600 to 3,200, which greatly reduces the false-positive rates associated with low-positive IFA titers. These data are in congruence with the recently improved serodiagnostic positivity criteria using the Bayesian latent class modeling approach. In summary, the InBios Scrub Typhus Detect IgM ELISA is affordable and easy-to-use, with adequate diagnostic accuracy for screening and diagnostic purposes, and should be considered an improved alternative to the gold standard IFA for acute diagnosis. For broader application, regional cutoff validation and antigenic composition for consistent diagnostic accuracy should be considered. PMID:26656118

  6. Gold bands as a suitable surface for enzyme immunoassays.

    PubMed

    Abad-Villar, Eva M; Fernndez-Abedul, M Teresa; Costa-Garca, Agustn

    2002-09-01

    Gold bands sputtered over a polymeric material, Kapton, are employed for the development of enzyme immunoassays. The immunological interaction takes place between human IgM and alkaline phosphatase (AP) conjugated anti-IgM. The model analyte (IgM) could be determined following a non-competitive design in the range of 0.05-5 ppm, with a limit of detection of 50 ppb. After the interaction, gold bands are sequentially inserted in a flow system and the extension of the reaction is followed through the enzymatic hydrolysis of naphthylphosphate, AP substrate. The product, naphthol, is oxidised to naphtoquinone in the gold band of the flow cell that constitutes the detector. Parameters affecting the interaction are studied and calibration curves are performed. The reproducibility between different bands (RSD=4%, n=5) and possibilities of regeneration are also detailed. PMID:12191928

  7. Enzyme linked immunoassay with stabilized polymer saccharide enzyme conjugates

    DOEpatents

    Callstrom, M.R.; Bednarski, M.D.; Gruber, P.R.

    1997-11-25

    An improvement in enzyme linked immunoassays is disclosed wherein the enzyme is in the form of a water soluble polymer saccharide conjugate which is stable in hostile environments. The conjugate comprises the enzyme which is linked to the polymer at multiple points through saccharide linker groups. 19 figs.

  8. Enzyme linked immunoassay with stabilized polymer saccharide enzyme conjugates

    DOEpatents

    Callstrom, Matthew R.; Bednarski, Mark D.; Gruber, Patrick R.

    1997-01-01

    An improvement in enzyme linked immunoassays is disclosed wherein the enzyme is in the form of a water soluble polymer saccharide conjugate which is stable in hostile environments. The conjugate comprises the enzyme which is linked to the polymer at multiple points through saccharide linker groups.

  9. Comparison of chemiluminescent immunoassay and ELISA for measles IgG and IgM.

    PubMed

    de Ory, Fernando; Minguito, Teodora; Balfagón, Pilar; Sanz, Juan C

    2015-08-01

    In the context of measles elimination, the identification of recent infections is important for clinical laboratories. Serological diagnosis is achieved by detecting specific IgG and IgM. Recently an automated chemiluminescent immunoassay (CLIA) (Liaison, DiaSorin, Italy) has been used to quantify the measles antibody. The aim of this study was to compare this assay with Enzygnost ELISA (Siemens, Germany), with final classification of discrepancies by indirect immunofluorescence (Euroimmun, Germany). For measles IgM, 204 sera were analyzed: 50 IgM-positive, 104 IgM-negative/IgG-positive, and 50 from other viral infections (B19V, rubella, mumps, CMV, and EBV). For the measles IgG assay, 162 samples were tested: 106 were positive and 56 were negative. For measles IgM, the sensitivity and specificity of CLIA against ELISA were 94% (95% CI: 83.2-98.6) and 100% (95% CI: 97.1-100), respectively; the corrected figures after the final classification of discrepancies were 100% (95% CI: 91.0-100) and 99.4% (95% CI: 96.1-100), respectively. In relation to IgG, the sensitivity and specificity of CLIA against ELISA were, respectively, 97.2% (95% CI: 91.7-99.4) and 92.9% (95% CI: 82.5-97.7), and 95.5% (95% CI: 89.5-98.3) and 100% (95% CI: 91.8-100) after the final classification. CLIA showed excellent sensitivity and specificity in detecting measles IgG and IgM antibodies, eliminating the need to aliquot specimens before carrying out the assay. PMID:26140432

  10. Competitive enzyme immunoassay for bovine growth hormone.

    PubMed

    Roh, S G; Matsunaga, N; Miyamoto, A; Hidaka, S; Hidari, H

    1997-02-01

    We developed an enzyme immunoassay (EIA) for bovine GH (bGH) which is based on indirect competitive immunoassay in culture medium from a bovine pituitary cell culture. 40 microliters cell culture samples (or bGH standard) and bGH antibody (rabbit anti-bGH) were added to the 96 well microplate coated with secondary antibody (Goat anti-rabbit IgG), and incubated for 24 h at 37 degrees C. Biotin-label bGH was added and incubated further for 24 h at 37 degrees C, and biotinylated bGH was linked with streptoavidin-peroxidase. Substrates for peroxidase were added to the plate and incubated for 1 h at 4 degrees C. The enzyme reaction was stopped with 4N H2SO4, and the absorbency at 450 nm was measured with an ELISA Reader. The coefficients of intra-assay and inter-assay variations were 4.13 approximately 7.59% and 3.71 approximately 8.27%, respectively. The regression equation and correlation coefficients with the radioimmunoassay (RIA) were y(RIA) = 1.9986 x (EIA) - 1.3921 and 0.9701 (n = 27), respectively. Collectively, the present assay provides a reliable alternative to RIA and offers the major advantage of eliminating radioactive reagents and counting equipment. PMID:9152634

  11. Neutralization enzyme immunoassay for influenza virus.

    PubMed Central

    Benne, C A; Harmsen, M; De Jong, J C; Kraaijeveld, C A

    1994-01-01

    A neutralization enzyme immunoassay (N-EIA) was developed for the detection of antibody titer rises in sera of patients infected with influenza A (H3N2) virus. In this N-EIA, a selected strain of influenza A (H3N2) virus was added to monolayers of LLC-MK2 cells in microtiter plates. After 24 h, the replicated virus could be demonstrated with a virus-specific enzyme-labeled monoclonal antibody. Preincubation of the influenza virus with convalescent-phase sera of patients infected with influenza A (H3N2) virus resulted 1 day later in decreased absorbance values that could be used for calculation of neutralization titers. From use of paired serum samples from 10 patients with a history of flu-like symptoms, the results obtained with N-EIA correlated well (r = 0.83) with those of the standard hemagglutination inhibition test. PMID:8027355

  12. Enzyme immunoassays with special reference to ELISA techniques.

    PubMed Central

    Voller, A; Bartlett, A; Bidwell, D E

    1978-01-01

    In this review outlines are given on various types of enzyme immunoassay. The applications to such enzyme immunoassays, especially ELISA, are dealth with in detail. It is concluded that these techniques have high sensitivity and will be suitable in due course as routine laboratory tests. PMID:78929

  13. [Detection of food additives by enzyme immunoassay].

    PubMed

    Yoshida, A; Takagaki, Y

    1995-09-01

    The analysis of synthesized food additives is generally performed by chromatography or spectrophotometry. However, the analytical procedures for natural food additives have been little reported so far because they are difficult to analyse chemically. We have attempted to apply enzyme immunoassay (EIA) to the analysis of natural food additives. Hen egg white lysozyme, as a food preservative, was determined by the competitive EIA, using mouse anti-HEL ascites. Carminic acid (CA), which is the main component of cochineal color, was determined by the competitive EIA, using monoclonal anti-CA antibody. Phycocyanin, which is the main component of spirulina color, was determined by the avidin-biotin sandwich EIA, using double monoclonal anti-phycocyanin antibodies. PMID:7474399

  14. Enzyme immunoassay for carminic acid in foods.

    PubMed

    Yoshida, A; Takagaki, Y; Nishimune, T

    1995-01-01

    A competitive enzyme immunoassay (EIA) for carminic acid was investigated. Monoclonal anticarminic acid antibody was obtained from A/J mice immunized with carminic acid-human immunoglobulin G (IgG) conjugate. Carminic acid was extracted with distilled water from beverage, jelly, candy, pasta sauce, yogurt, or ice cream samples. Ham or fish paste samples were digested with pronase, then carminic acid was extracted from samples with sodium hydroxide solution. The extract was diluted more than 10-fold with 1% gelatin in borate buffer solution. Microtiter plates were coated with carminic acid-bovine serum albumin (BSA) conjugate or just BSA. Goat anti-mouse IgG(H+L)-peroxidase complex was used as a second antibody, and 3,3',5,5'-tetramethylbenzidine was used as a substrate for the peroxidase. The working range for quantitative analysis was 0.3-10 ng/mL, and the detection limit was 0.2 micrograms/g original sample. Recoveries of carminic acid by this assay were > 95% for milk beverage and jelly, and > 85% for yogurt and fish paste. Carminic acid was detected in 7 of 26 red-colored commercial food products and ranged from 3.5 to 356 micrograms/g. This EIA system also responded to the structural analogue of carminic acid, laccaic acid. PMID:7756895

  15. DETECTION OF ROTAVIRUS WITH A NEW POLYCLONAL ANTIBODY ENZYME IMMUNOASSAY (ROTAZYME 2) AND A COMMERCIAL LATEX AGGLUTINATION TEXT (ROTALEX): COMPARISON WITH A MONOCLONAL ANTIBODY ENZYME IMMUNOASSAY

    EPA Science Inventory

    A total of 176 human fecal specimens were examined for the presence of rotavirus using four different assays: a monoclonal antibody enzyme immunoassay; the original polyclonal antibody enzyme immunoassay marketed by Abbott Laboratories, Chicago, IL (Rotazyme I); a modification of...

  16. Ability of TESTPACK ROTAVIRUS enzyme immunoassay to diagnose rotavirus gastroenteritis.

    PubMed Central

    Chernesky, M; Castriciano, S; Mahony, J; Spiewak, M; Schaefer, L

    1988-01-01

    TESTPACK ROTAVIRUS, a simple 10-min enzyme immunoassay, was compared with electron microscopy and Pathfinder enzyme immunoassay on feces from 172 patients of various ages with gastroenteritis. The percent sensitivities and specificities before blocking with antiserum were as follows: TESTPACK, 100% sensitivity and 99% specificity; Pathfinder, 95% sensitivity and 98% specificity. After blocking, the sensitivity and specificity, respectively, were 100% and 100% for TESTPACK and 95% and 99% for Pathfinder. TESTPACK ROTAVIRUS was more sensitive, but not significantly, than Pathfinder (P greater than 0.1) and the direct electron microscopy technique (P greater than 0.1). PMID:3069866

  17. DETECTION OF NORWALK VIRUS IN STOOLS BY ENZYME IMMUNOASSAY

    EPA Science Inventory

    The development of a solid-phase microtiter enzyme immunoassay (EIA) for detection of Norwalk virus antigen in stool samples is described. The EIA was compared with a previously developed radioimmunoassay (RIA) for detection of Norwalk virus antigen in stools obtained from 30 vol...

  18. Enzyme immunoassays as screening tools for catalysts and reaction discovery.

    PubMed

    Créminon, Christophe; Taran, Frédéric

    2015-05-11

    Enzyme immunoassays are incredibly powerful analytical tools for the quantifiable detection of target molecules in complex media. These techniques, which exploit the fantastic specific binding properties of antibodies, are fast, precise, selective and highly sensitive and thus perfectly adapted to high-throughput detection of important analytes. Although immunoassays have been used routinely by biologists for more than 50 years, especially for diagnostic purposes, it is only recently that chemists have used them to address pure chemical problems. In this feature article, we provide an overview of progress in the development of immunoassays and their use in two main fields of organic chemistry: the identification of efficient catalysts in libraries and the discovery of new chemical reactions. PMID:25765583

  19. Rapid diagnosis of acute mumps infection by a direct immunoglobulin M antibody capture enzyme immunoassay with labeled antigen.

    PubMed Central

    Gut, J P; Spiess, C; Schmitt, S; Kirn, A

    1985-01-01

    A new immunoglobulin M (IgM) antibody capture enzyme immunoassay with peroxidase-labeled mumps antigen (dMACEIA) is described, and its suitability for practical diagnosis of acute mumps infection is evaluated. All 54 patients with proven mumps infection that were tested showed mumps-specific IgM antibodies. On the other hand, no specific IgM antibodies were present in 16 cases of suspected mumps that could not be confirmed by classical complement fixation serology, and IgM mumps virus antibodies could be detected neither in the sera of 100 healthy individuals nor in those of 16 patients positive for rheumatoid factor. In all, 22 children with acute respiratory illness caused by parainfluenza virus and 44 patients with infections due to other viruses showed no IgM response in mumps dMACEIA. The particular characteristic in which complement fixation antibodies against mumps nucleocapsids appear before and disappear earlier than antibodies to the enveloped mumps virus could not be demonstrated in the dMACEIA. In an extensive epidemic of mumps virus infection, the dMACEIA gave a clear diagnosis of mumps infection in 200 out of 371 suspected cases. By day 2 of the illness, 71% of the patients had detectable IgM, and by day 3, all of them had detectable IgM. In 99% of the cases, dMACEIA gave a positive result in the first available serum specimens, most of which were negative for complement fixation antibodies. A positive but only moderate correlation was thus observed between the two serological procedures. IgM antibodies persisted for at least 6 weeks. The dMACEIA, performed in 3 h, offers a reliable, simple, and rapid alternative to routine methods for detection of acute mumps infection. PMID:3884652

  20. Assessment of immunoglobulin M enzyme immunoassays for diagnosis of measles.

    PubMed

    Tipples, Graham A; Hamkar, Rasool; Mohktari-Azad, Talat; Gray, Michael; Parkyn, Geoff; Head, Carol; Ratnam, Samuel

    2003-10-01

    We evaluated the performance of three commercial measles immunoglobulin M enzyme immunoassays from Meddens, Denka Seiken, and Behring. The sensitivities were determined to be 96.7% for the Meddens and Denka Seiken assays and 87.9% for the Behring assay. The specificities of the assays were determined to be 94.6% for Meddens, 98.2% for Denka Seiken, and 98.7% for Behring. PMID:14532222

  1. Rapid determination of dioxin in water by enzyme immunoassay

    SciTech Connect

    Wang, H.; Wang, L.; George J.E. III; Ward, G.K.

    1996-10-01

    Dioxin in water, soil, sediments and other sample matrices is usually determined by the EPA method 1613 which was developed by the EPA Office of Science and Technology. This method however requires expensive instruments (high resolution gas chromatography/high resolution mass spectrometry) and a highly trained analyst. In order to reduce the cost and turn around time, a dioxin enzyme immunoassay (EIA) was developed to rapidly analyze trace levels of 2,3,7,8-tetra chlorinated dibenzodioxin (TCDD) in water samples. Water samples were extracted using a 47 mm, C18 Empore extraction disk (3M). Dioxin was eluted with dichloromethame. EnvironGard reagents and microwell strip reader (Millipore Corporation) were used to perform the dioxin enzyme immunoassay. The extraction efficiency was also tested by GC/MS with Varian`s large volume injector and Selected Ion Storage technique. The working range of the dioxin enzyme immunoassay was from 15 pg/L to 100 pg/L. The precision and accuracy of EIA was determined by performing five replicates of reagent water spiked at a concentration of 25 pg/L. The recovery of the dioxin assay ranged from 74% to 122%, and % CV for five replicates was less than 15%. In general, EIA provides a relatively easy and cost effective means for measuring trace levels of dioxin in water samples.

  2. Enzyme immunoassay for the detection of group A streptococcal antigen.

    PubMed Central

    Knigge, K M; Babb, J L; Firca, J R; Ancell, K; Bloomster, T G; Marchlewicz, B A

    1984-01-01

    A competitive inhibition enzyme immunoassay for the detection of Streptococcus pyogenes directly from throat specimens or from solid bacteriological medium is described. Group A-specific polysaccharide adsorbed onto treated polystyrene beads, in conjunction with rabbit antibody to S. pyogenes, was used to determine the presence of the polysaccharide antigen. Inhibition values in excess of 65% were observed with 10(4) or more CFU of S. pyogenes per test. An inhibition of 25% was demonstrated with as few as 10(3) CFU per test. Heterologous microorganisms tested at 10(6) CFU per test reacted at levels of inhibition less than 25%. Two types of bacterial transport medium and swabs of different fiber compositions did not alter the assay performance. Accurate identification of S. pyogenes was achieved by testing single colonies picked directly from blood agar plates which had been incubated for 18 to 24 h. In addition, the assay was performed on throat specimens from children and adults having pharyngitis. A single-swab, blind study was conducted in which enzyme immunoassay reactivity was compared with results of blood agar culture and bacitracin sensitivity. When there were discordant results, serological identification was used as the confirmatory test. At an optimal cutoff value of 40% inhibition, sensitivity and specificity by enzyme immunoassay were 97.0% and 97.9%, respectively, as compared with confirmed culture results. The assay has an incubation time of 3 h and is a sensitive and specific method for the detection of S. pyogenes antigen. PMID:6386878

  3. Diagnostic performance indices for immunofluorescent tests and enzyme immunoassays of leishmaniasis sera from northern and north-eastern Brazil.

    PubMed Central

    Guimarães, M. C.; Celeste, B. J.; Franco, E. L.

    1990-01-01

    A total of 341 sera were screened for anti-Leishmania IgA, IgG, and IgM antibodies by immunofluorescent (IF) tests and enzyme immunoassay (ELISA). Altogether, 292 of the sera originated from patients with clinically as well as parasitologically diagnosed (positive lesion imprint or the Montenegro skin test) cutaneous leishmaniasis; 49 of the sera were from controls from the same base population. In terms of diagnostic performance, the ELISAs for IgG and IgM yielded indices of diagnostic utility, and the positive predictive value for the IgG-ELISA was 94.6%. A remarkably high specificity (100%) was obtained with the IgA-IF test, but its sensitivity was very low. PMID:2189584

  4. Rapid enzyme immunoassay for measurement of bovine progesterone.

    PubMed

    Claycomb, R W; Delwiche, M J; Munro, C J; BonDurant, R H

    1998-11-15

    Reproductive management is a primary financial concern of the dairy industry with missed estrus detection one of the major causes of lost income. A rapid enzyme immunoassay (EIA) was developed for on-line measurement of progesterone in bovine milk with a biosensor for detection of estrus. The EIA was developed using covalent binding microtiter wells, monoclonal antibody, horseradish peroxidase, and 3,3',5,5'-tetramethylbenzidine (TMB). The EIA took 8 min and had a dynamic response for progesterone in buffer and milk between 0.2 and 20 ng/ml. PMID:9871971

  5. Chemiluminescence enzyme immunoassay using ProteinA-bacterial magnetite complex

    NASA Astrophysics Data System (ADS)

    Matsunaga, Tadashi; Sato, Rika; Kamiya, Shinji; Tanaka, Tsuyosi; Takeyama, Haruko

    1999-04-01

    Bacterial magnetic particles (BMPs) which have ProteinA expressed on their surface were constructed using magA which is a key gene in BMP biosynthesis in the magnetic bacterium Magnetospirillum sp. AMB-1. Homogenous chemiluminescence enzyme immunoassay using antibody bound ProteinA-BMP complexes was developed for detection of human IgG. A good correlation between the luminescence yield and the concentration of human IgG was obtained in the range of 1-10 3 ng/ml.

  6. Use of cerebrospinal fluid enzyme immunoassay for diagnosis of neurosyphilis.

    PubMed

    Chan, Yung; Yeung, Kwok-Hung; Ho, Hing-Fung; Ho, King-Man; Tin-Keung Lam, Edman; Leung, Wai-Lin; Kam, Kai-Man

    2014-07-01

    Neurosyphilis is a difficult clinical stage of syphilis as there is no ideal method for diagnosis and workup requires lumbar puncture which may sometimes provide ambiguous results especially in HIV co-infected patients. Enzyme immunoassay is a widely used screening test for syphilis in serum, but its test performance was not well studied in cerebrospinal fluid. To examine the diagnostic performance of cerebrospinal fluid-enzyme immunoassay (CSF-EIA) in neurosyphilis, we conducted a prospective study for two years. All consecutive patients admitted for workup of neurosyphilis under the Social Hygiene Service, in Hong Kong, were included. Laboratory tests on CSF included several serological tests, CSF cell count, and protein. Forty-five patients were prospectively recruited, of which 29 people were living with HIV / AIDS. Using diagnostic case definition standard stipulated in the IUSTI 2008 guidelines, 17 patients satisfied the diagnosis of neurosyphilis. The CSF-EIA test provided 100% in both sensitivity and negative predictive value; its specificity was 46.4% (13/28, 95% CI 31.8-61%). Specificity improved to 80.8% (95% CI: 68.4-93.2%) with optical density cut-off value at 1.4 for cases with CSF red cell counts <600/mm(3) This is the first study on use of CSF-EIA in neurosyphilis. CSF-EIA showed high sensitivity and high negative predictive value in the study population and the presence of CSF red cells < 600/mm(3)might not affect its accuracy. PMID:24334293

  7. Chemiluminescence enzyme immunoassay for the determination of sulfamethoxydiazine

    NASA Astrophysics Data System (ADS)

    Wu, Yongjun; Yu, Songcheng; Yu, Fei; Yan, Nali; Qu, Lingbo; Zhang, Hongquan

    2011-10-01

    Sulfamethoxydiazine (SMD), which is often used for animal disease treatment, is harmful to human health. No SMD residue should be detected in food in some countries, such as USA and Japan. Therefore, it is significant to develop a high-throughput, high-sensitivity and accurate method for the determination of the content of SMD in food. In this paper, chemiluminescence enzyme immunoassay (CLEIA) was developed for quantification of SMD. For this method, the limit of detection was 3.2 pg/ml, the linear range was from 10 to 2000 pg/ml, the within-day and inter-day precision were below 13% and below 18%, respectively, and the recovery was from 85% to 105%. Milk and egg were selected as samples to be examined with this method, and the result indicated that this CLEIA method was suitable for screening and quality control of food.

  8. Performance of a cytomegalovirus IgG enzyme immunoassay kit modified to measure avidity.

    PubMed

    Prince, Harry E; Lapé-Nixon, Mary; Novak-Weekley, Susan M

    2014-06-01

    The measurement of cytomegalovirus (CMV) IgG avidity accurately discriminates recent and past CMV infections. We sought to determine if the Wampole Laboratories CMV IgG enzyme immunoassay (EIA) could be modified to measure avidity. The evaluation panel consisted of 156 serum samples we used in 2002 to validate a laboratory-developed EIA, in which 78 serum samples exhibited low avidity, 7 exhibited intermediate avidity, and 71 exhibited high avidity. The qualitative agreement between the two avidity assays was 94% (147/156); all 9 sera with discordant results exhibited intermediate avidity in one of the assays. The avidity index (AI) values in the two assays showed excellent correlation (r = 0.96, P < 0.0001). The definition of high avidity was verified for the Wampole assay by demonstrating high avidity in 91/93 (98%) recently collected CMV IgG-positive/IgM-negative serum samples. The performance of the Wampole avidity assay in a reference laboratory setting was assessed using 470 consecutive serum samples submitted for CMV IgG avidity testing. Surprisingly, 101 serum samples were negative when screened for CMV IgG using the Wampole kit per the package insert; 98 of these 101 serum samples were tested using a CMV IgG chemiluminescent immunoassay, and only 5 were positive. Of the 369 CMV IgG-positive samples, 6% exhibited low IgG avidity, 6% exhibited intermediate avidity, and 88% exhibited high avidity; CMV IgM detection rates were inversely related to AI levels. These findings show that (i) the Wampole CMV IgG EIA can be modified to measure CMV IgG avidity, (ii) many samples are apparently submitted for avidity testing without knowledge of their CMV IgG status, and (iii) most CMV IgG-positive sera submitted for avidity testing exhibit high avidity. PMID:24671557

  9. A one-step solid phase immunoassay for simultaneous detection of serum IgG and IgM antibodies to Borrelia burgdorferi.

    PubMed

    Schønau, A; Stender, H; Grauballe, P C

    1998-09-01

    A one-step immunoassay for simultaneous detection of serum IgG and IgM antibodies to Borrelia burgdorferi has been developed. The assay is based on C1q, which binds to immune complexes containing IgG and/or IgM antibodies. Micro-beads pre-coated with antibodies to human C1q are mixed with human serum samples and fluorochrome-labelled B. burgdorferi flagellum antigen. In the presence of serum IgG and/or IgM antibodies to B. burgdorferi, fluorochrome-labelled antigen/antibody complexes are formed. These are then bound by serum C1q and are subsequently captured on the anti-C1q-coated beads. The sample is analysed on a flow cytometer and the presence of fluorescent beads is, thus, indicative of a positive test result. In the present study the sensitivity and specificity of the assay are compared to those of the indirect IDEIA B. burgdorferi IgG and the mu-chain capture IDEIA B. burgdorferi IgM ELISAs for separate determination of IgG and IgM. Detection using a flow cytometer can be performed without separation of the beads from the reaction mixture, which means that in practice, the method is carried out as a one-step assay and it is, thus, very suitable for automation. Other advantages of this kind of assay includes an antibody/antigen reaction which occurs in solution and the potential of using the method for the detection of antibodies against several antigens from the same or different infectious agents (multi-parameter screening). PMID:9819119

  10. Immunoassay using /sup 125/I- or enzyme-labeled protein A and antigen-coated tubes

    SciTech Connect

    Gee, A.P.; Langone, J.J.

    1981-09-15

    Antigen-coated plastic tubes were used with /sup 125/I- or enzyme-labeled stapylococcal protein A in a general immunoassay method for antigens and haptens. Protein A reacts with immunoglobulin G(IgG) regardless of antibody specificity at sites distal to the antigen combining site and does not inhibit the immune reaction. It therefore serves as a general tracer and its use eliminates the need to purify and to label individual components for each assay. Macromolecular antigens were bound to polystyrene or polypropylene tubes by direct passive absorption. Haptens with free carboxyl groups were bound covalently to poly-L-lysine and these conjugates passively absorbed to the tube surface. Optimal assay conditions were established for the quantitative determination of immunoglobulins and the folate derivatives, methotrexate and 5-methyltetrahydrofolate, using /sup 125/I-labeled protein A or protein A labeled with alkaline phosphatase. The method has been used to estimate levels of IgG, IgA, Igm, and IgE in serum in volumes up to 1 ml.

  11. Affinity patterns of enzyme tracers for triazine immunoassays

    NASA Astrophysics Data System (ADS)

    Weller, Michael G.; Niessner, Reinhard

    1997-05-01

    Cross-reactivities are one of the most important characteristics of immunoassays. Nevertheless most cross- reactivity studies are only performed with small molecules, which are similar to the target analyte. The complex mechanism of the binding event of an antibody makes it likely that the orientation of the hapten plays a critical role. Therefore cross-reactivities of hapten-derivatives with spacers may be quite different compared to the simple compound, especially when the spacer has been coupled to a large molecule, like a marker enzyme (e.g. peroxidase). We examined the relative affinity patterns of 17 enzyme tracers to 16 monoclonal and polyclonal antibodies for the analysis of triazine herbicides (atrazine, terbuthylazine, etc.). This allows interesting discussions about the structure of the antibody binding site and the comparison of antibodies generated with different immunogens. In addition, some general rules for the selection of immunogen structures could be derived from the data. The figures shown in this paper facilitate to find suitable tracer haptens for one of the tested antibodies and support for instance the optimization of immunosensor regeneration, as tracer affinity is closely correlated to the dissociation rate constant of an antibody-tracer complex.

  12. Enzyme immunoassay of thyroxin with a centrifugal analyzer

    SciTech Connect

    Izquierdo, J.M.; Sotorrio, P.; Quiros, A.

    1982-01-01

    We have applied a homogeneous enzyme immunoassay for determination of thyroxinin serum to the ''Cobas Bio'' centrifugal analyzer. To unbind thyroxin from its protein complex, serum is treated for 20 min with a solution of NaOH containing ''Lipex,'' an agent for sequestering free fatty acids. The immunoenzymic reaction is then automatically performed by the analyzer at 37/sup 0/C. To 20 ..mu..L of sample mixture is added 125 ..mu..L of reagent (thyroxin antibodies and NAD/sup +/) and this mixture is incubated for 10 s. Then 25 ..mu..L of start reagent (enzyme-thyroxin conjugate and malate substrate) is added and the change in absorbance is monitored at 340 nm. The standard curve is linear up to at least 200 ..mu..g of thyroxin per liter. Within-assay precision (CV) varied from 1.1 to 2.9%, between-assay precision from 3.1 to 7.8%. Analytical recovery of thyroxin was complete. The deviation of control samples from target values ranged from -2.1% to 7.0%. Interference by hemoglobin or bilirubin is negligible. Results compare favorably with those by radioimmunoassay.

  13. DEVELOPMENT OF A CLASS-SELECTIVE ENZYME IMMUNOASSAY FOR URINARY PHENOLIC GLUCURONIDES. (R825433)

    EPA Science Inventory

    Class-selective immunoassays for the measurement of glucuronides in human urine can aid evaluation of human exposure to complex mixtures of xenobiotics. Therefore, an enzyme immunoassay (EIA) for the group-selective detection of phenolic Immunoassay

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Immunoassays are analytical methods that employ antibodies or molecules derived from antibodies for the essential binding reactions. The choice of immunoassay system for food safety analysis depends on the analyte, the matrix, and the requirements of the analysis (speed, throughput, sensitivity, spe...

  14. Immunoassays

    NASA Astrophysics Data System (ADS)

    Hsieh, Y.-H. Peggy

    Immunochemistry is a relatively new science that has developed rapidly in the last few decades. One of the most useful analytical developments associated with this new science is immunoassay. Originally immunoassays were developed in medical settings to facilitate the study of immunology, particularly the antibody-antigen interaction. Immunoassays now are finding widespread applications outside the clinical field because they are appropriate for a wide range of analytes ranging from proteins to small organic molecules. In the food analysis area, immunoassays are widely used for chemical residue analysis, identification of bacteria and viruses, and detection of proteins in food and agricultural products. Protein detection is important for determination of allergens and meat species content, seafood species identification, and detection of genetically modified plant tissues. While immunoassays of all formats are too numerous to cover completely in this chapter, there are several procedures that have become standard for food analysis because of their specificity, sensitivity, and simplicity.

  15. Rapid dioxin screening of milk and water by enzyme immunoassay

    SciTech Connect

    Harrison, R.O.; Carlson, R.E.; Shirkhan, H.

    1995-12-01

    A simple and easy to use enzyme immunoassay (EIA) system has been developed for rapid screening of 2,3,7,8-tetrachlorodibenzo-p-dioxin (2378D). This EIA has been adapted to analysis of water and milk using an automated system for extraction of liquid samples. Water analysis can be performed directly following extraction and solvent exchange with no extract clean-up. The same automated system is used for milk extraction and the extract is then cleaned chromatographically using the automated FMS Dioxin-Prep{trademark} System. Sensitivity for 2378D in the EIA is approximately 100 pg per analysis. Thus sensitivity to 10 ppt 2378D (whole weight basis) in milk is possible using only 50 ml or less of sample and sensitivity to 0.1 ppt 2378D in water is possible using 1-2 liters of sample. Total time for sample preparation and analysis is about 3 hours for water and 4.5 hours for milk.

  16. Evaluation of an enzyme immunoassay kit for estrogen receptor measurements.

    PubMed

    Raam, S; Vrabel, D M

    1986-08-01

    Using an enzyme immunoassay (EIA) procedure, we evaluated the ability of the glass beads in the ER-EIA kit of Abbott Laboratories, which were coated with monoclonal anti-estrogen receptor (ER) antibodies, to bind hormone-free and hormone-filled Type I ER in cytosols, buffer washes, and 0.4 mol/L KCI extracts of ultracentrifugal pellets of breast cancer tissue homogenates. The unmodified ER-EIA technique yields higher values than does the dextran-coated charcoal (DCC) method for Type I ER. However, the antibody-coated beads fail to bind hormone-free ER and react with only a certain proportion of ER-[3H]estradiol complexes. The antigen saturation limit of the beads could not be determined because the quantity of antigen bound by the beads was disproportionate and unrelated to the total amount of the antigen available in the cytosols. In buffer extracts of tissue pellets that were ER-negative by the DCC assay, the EIA method detected high quantities of ER. We recommend checking the ability of the monoclonal antibodies to recognize proteins other than Type I ER in the extra-nuclear and nuclear compartments of target cells before using them for immunohistochemical detection of ER. PMID:2426007

  17. Enzyme immunoassay for determination of gluten in foods: collaborative study.

    PubMed

    Skerritt, J H; Hill, A S

    1991-01-01

    A collaborative study was performed in 15 laboratories to validate a monoclonal antibody-based enzyme immunoassay (EIA) for determination of gluten in foods. The study included 13 samples: maize starch, "gluten-free" baking mixes, wheat flours, cookies, cooked meats, and a soup. Gluten was present in these samples at either zero or 0.02 to 10% by weight, i.e., over almost 3 orders of magnitude. The mean assay values for the foods varied from 88 to 105% of the actual amounts. The assay was quantitative for cereal products and the soup with repeatability (RDS-r, relative standard deviation) and reproducibility (RSD-R) of 16-22% and 24-33%, respectively. The assay was semiquantitative for the processed meat products (RSD-r 14 and 26% and RSD-R 46 and 56%), probably because gluten was unevenly distributed in the small (1 g) samples that were analyzed. The ELISA method produced no false positive results, and false negatives obtained with tannin-containing foods could be avoided by use of a modified sample extractant. None of the collaborators reported problems in following the protocol. The method has been adopted official first action by AOAC for determination of wheat gluten in foods. PMID:2050607

  18. Bioluminescent enzyme immunoassay for the detection of norovirus capsid antigen.

    PubMed

    Sakamaki, Nozomi; Ohiro, Yoshiyuki; Ito, Mitsuki; Makinodan, Mitsuru; Ohta, Tsubasa; Suzuki, Wataru; Takayasu, Susumu; Tsuge, Harufumi

    2012-12-01

    An ultrasensitive and fully automated bioluminescent enzyme immunoassay (BLEIA) was developed for the detection of norovirus (NV) capsid antigen. In the evaluation tests with recombinant virus-like particles, the BLEIA demonstrated broad reactivity against several NV genotypes (genotypes 1, 3, 4, 7, 8, and 12 in genogroup I [GI] and genotypes 1, 2, 3, 4, 5, 6, 12, and 13 in GII), a wide dose-response range from 0.25 pg/ml to 10,000 pg/ml, and good reproducibility with low coefficients of variation (CVs) (within-run CVs of <2.8%, between-day CVs of <3.7%). In the evaluation tests with NV-positive fecal samples, a good correlation (y = 0.66x - 3.21, r = 0.84) between the BLEIA and real-time quantitative reverse transcription-PCR was obtained. Furthermore, in the dilution test with NV specimens, the analytical sensitivity of NV was estimated to be 10(5) to 10(6) copies/g of fecal sample, indicating that the analytical sensitivity of the BLEIA is comparable to that of commercially available molecular methods. All assay steps are fully automated, the turnaround time is 46 min, and the throughput of the assay is 120 tests/h. These results indicate that the BLEIA is potentially useful for the rapid diagnosis of NV in epidemic and sporadic gastroenteritis. PMID:23081816

  19. Sensitivity and Specificity of Histoplasma Antigen Detection by Enzyme Immunoassay.

    PubMed

    Cunningham, Lauren; Cook, Audrey; Hanzlicek, Andrew; Harkin, Kenneth; Wheat, Joseph; Goad, Carla; Kirsch, Emily

    2015-01-01

    The objective of this study was to evaluate the sensitivity and specificity of an antigen enzyme immunoassay (EIA) on urine samples for the diagnosis of histoplasmosis in dogs. This retrospective medical records review included canine cases with urine samples submitted for Histoplasma EIA antigen assay between 2007 and 2011 from three veterinary institutions. Cases for which urine samples were submitted for Histoplasma antigen testing were reviewed and compared to the gold standard of finding Histoplasma organisms or an alternative diagnosis on cytology or histopathology. Sensitivity, specificity, negative predictive value, positive predictive value, and the kappa coefficient and associated confidence interval were calculated for the EIA-based Histoplasma antigen assay. Sixty cases met the inclusion criteria. Seventeen cases were considered true positives based on identification of the organism, and 41 cases were considered true negatives with an alternative definitive diagnosis. Two cases were considered false negatives, and there were no false positives. Sensitivity was 89.47% and the negative predictive value was 95.35%. Specificity and the positive predictive value were both 100%. The kappa coefficient was 0.9207 (95% confidence interval, 0.8131-1). The Histoplasma antigen EIA test demonstrated high specificity and sensitivity for the diagnosis of histoplasmosis in dogs. PMID:26355580

  1. Laboratory evaluation of a dot-blot enzyme immunoassay for serologic confirmation of illness due to Rickettsia conorii.

    PubMed

    Broadhurst, L E; Kelly, D J; Chan, C T; Smoak, B L; Brundage, J F; McClain, J B; Miller, R N

    1998-06-01

    Of the 169 United States Army soldiers who deployed on a field training exercise to a remote area of Botswana for two weeks in January 1992, more than 30% developed a febrile illness within five days of their return. A diagnosis of South African tick typhus was suggested by soldiers' exposure to ticks, as well as the presence of eschars and vesicles at the site of tick bites, and tender regional lymphadenopathies. This high attack rate, experienced during such a short exposure period, emphasizes the hazard of illness due to Rickettsia conorii to persons visiting endemic areas. A rapid, diagnostic, semiquantitative enzyme immunoassay (DS) for detection of IgG and IgM antibodies to R. conorii was performed on 209 acute and convalescent sera from soldiers in the outbreak and on 75 control sera. For the acute sera from soldiers meeting the probable case definition of having both regional lymphadenopathy and tick bite eschar, as judged by an IgG indirect fluorescent antibody (IFA) test, the resulting sensitivity and specificity of the DS test were 100% and 48%, respectively. In the analysis of the acute sera, the DS test identified as reactive more of the probable cases (62%) than either the IgG (16%) or IgM (55%) IFAs. This simple and rapid diagnostic test could be useful in establishing a preliminary diagnosis of R. conorii rickettsiosis in remote settings when immediate confirmation by IFA is impossible. PMID:9660464

  2. Optimization of condition for conjugation of enrofloxacin to enzymes in chemiluminescence enzyme immunoassay

    NASA Astrophysics Data System (ADS)

    Yu, Songcheng; Yu, Fei; Zhang, Hongquan; Qu, Lingbo; Wu, Yongjun

    2014-06-01

    In this study, in order to find out a proper method for conjugation of enrofloxacin to label enzymes, two methods were compared and carbodiimide condensation was proved to be better. The results showed that the binding ratio of enrofloxacin and alkaline phosphatase (ALP) was 8:1 and that of enrofloxacin and horseradish peroxidase (HRP) was 5:1. This indicated that conjugate synthesized by carbodiimide condensation was fit for chemiluminescence enzyme immunoassay (CLEIA). Furthermore, data revealed that dialysis time was an important parameter for conjugation and 6 days was best. Buffer to dilute conjugate had little effect on CLEIA. The storage condition for conjugates was also studied and it was shown that the conjugate was stable at 4 °C with no additive up to 30 days. These data were valuable for establishing CLEIA to quantify enrofloxacin.

  3. Chemiluminescence enzyme immunoassay using magnetic nanoparticles for detection of neuron specific enolase in human serum.

    PubMed

    Fu, Xiaoling; Meng, Meng; Zhang, Yu; Yin, Yongmei; Zhang, Xiangsheng; Xi, Rimo

    2012-04-13

    To detect a biomarker for small cell lung carcinoma, neuron specific enolase (NSE), a sensitive and specific chemiluminescence enzyme immunoassay was developed. Fluorescein isothiocyanate (FITC) labeled NSE capture antibody connected with NSE and alkaline phosphatase (ALP) labeled NSE detection antibody in a sandwich-type detection manner. This immune complex was further reacted with anti-FITC coated magnetic beads. In a magnetic field, the complex was enriched, and the sensitivity was thus enhanced. The limit of detection (LOD) of this method was <0.2 ng mL(-1). The proposed immunoassay was highly selective, and not interfered by hook effect. The recovery was >83.0% and the coefficient of variation was <10.0%. Human sera from 120 patients were tested with the presented and traditional chemiluminescence enzyme immunoassay. An excellent linear relationship was obtained between two techniques. Overall, this immunoassay offers a promising alternative for NSE detection than traditional clinical examinations. PMID:22444542

  4. Autoantibody testing for connective tissue diseases. Comparison of immunodiffusion, immunoblot, and enzyme immunoassay.

    PubMed

    Bridges, A J; Lorden, T E; Havighurst, T C

    1997-10-01

    We evaluated 500 consecutive patient serum samples for the presence of six autoantibodies by three antibody detection methods: immunodiffusion, immunoblot, and enzyme immunoassay. Clinical data were reviewed for each patient with positive antibody test results. Serum samples from 60 patients revealed antibodies to Sm, ribonucleoprotein (RNP), SSA/Ro, SSB/La, Scl-70, or Jo-1. There were 7 false-positive test results (1%). All three methods detected autoantibodies in 36 (68%) of 53 patients with connective tissue disease. Immunoblot was the most sensitive method to detect autoantibodies (92%). Enzyme immunoassay and immunodiffusion were less sensitive (81% and 74%, respectively). Antiribonucleoprotein and anti-SSB/La antibodies were more often detected by immunoblotting, whereas anti-SSA/Ro antibodies were more often detected by enzyme immunoassay. Newer antibody detection methods (immunoblot and enzyme immunoassay) are less time consuming than immunodiffusion and show good interassay sensitivity without loss of specificity. A combination of immunoblot and enzyme immunoassay yielded excellent assay sensitivity (100%) and specificity (99%) for detection of autoantibodies. PMID:9322593

  5. Magnetic enzyme immunoassay of anti-grass pollen specific-IgE in human sera.

    PubMed Central

    Guesdon, J L; David, B; Lapeyre, J

    1978-01-01

    This paper reports a magnetic solid-phase sandwich enzyme immunoassay for specific IgE antibodies in human sera. Crude extracts of grass pollen bound to magnetic polyacrylamide agarose beads were mixed with human serum to be tested. After washing in a magnetic rack, the beads were incubated with the glucose-oxidase-labelled sheep anti-IgE. The enzyme activity associated with the beads was measured by colorimetric assay. Results obtained from sixty-one human sera, as measured by the magnetic enzyme immunoassay, gave a linear correlation coefficient of 0.98 with the values as determined by radio-immunoassay. This procedure, which allows the grass pollen specific IgE in human sera, to be measured, is easy to perform, reproducible and may avoid the use of radioactive compounds. PMID:367648

  6. Comparison of an enzyme immunoassay with electron microscopic procedures for detecting rotavirus.

    PubMed Central

    Rubenstein, A S; Miller, M F

    1982-01-01

    The sensitivity and specificity of an enzyme immunoassay (Rotazyme), an ongrid immunoelectron microscopy procedure, and conventional negative stain electron microscopic techniques were compared. By using partially purified human rotavirus and simian rotavirus (SA-11) of known particle concentration, the enzyme immunoassay was essentially equivalent to the immunoelectron microscopic procedure and significantly more sensitive than conventional electron microscopic techniques. The level of sensitivity was approximately 10(6) particles per ml for simian rotavirus SA-11 and 10(7) particles per ml for human rotavirus. In an evaluation of 455 clinical samples by these techniques, a sensitivity of 98% and specificity of 92% were demonstrated. Samples negative by the immunoelectron microscopic procedure and positive by enzyme immunoassay could be confirmed by a blocking assay. Images PMID:6284793

  7. Comparison of five enzyme immunoassays, electron microscopy, and latex agglutination for detection of rotavirus in fecal specimens.

    PubMed Central

    Kok, T W; Burrell, C J

    1989-01-01

    Five different enzyme immunoassays, electron microscopy, and latex agglutination (Slidex; bioMerieux) were compared for the rapid detection of human rotavirus in fecal specimens. The enzyme immunoassay using rotavirus polyclonal antiserum (Dakopatts) with simple in-house modifications was shown by the use of confirmatory tests to be the most sensitive and specific procedure. PMID:2536758

  8. Preparation of biotinylated cypridina luciferase and its use in bioluminescent enzyme immunoassay.

    PubMed

    Wu, Chun; Kawasaki, Kosei; Ogawa, Yoko; Yoshida, Yasukazu; Ohgiya, Satoru; Ohmiya, Yoshihiro

    2007-02-15

    Cypridina luciferase, a well-known secretory enzyme involved in the bioluminescence of marine ostracod, is used to monitor gene expression in mammalian cells. Here we report the preparation of biotinylated Cypridina luciferase and its use in bioluminescent enzyme immunoassay (BLEIA). Recombinant Cypridina luciferase was expressed in yeast and successfully purified to near homogeneity. The luciferase was biotinylated with conventional biotinylation reagents, and the biotinylated lysine sites were determined by liquid chromatography-tandem mass spectrometry. The biotinylated luciferase was stable when stored at 4 degrees C. The stability of synthetic S-Cypridina luciferin was significantly improved by adding antioxidants to Tris-HCl buffer. The biotinylated luciferase and S-Cypridina luciferin were used in a model study of the immunoassay for interferon-alpha. The linearity of this immunoassay extended from 7.8 to 500 pg/mL interferon-alpha. Our results show that Cypridina luciferase is a very sensitive and versatile bioluminescent reporter. PMID:17297966

  9. Optimal Cutoff and Accuracy of an IgM Enzyme-Linked Immunosorbent Assay for Diagnosis of Acute Scrub Typhus in Northern Thailand: an Alternative Reference Method to the IgM Immunofluorescence Assay.

    PubMed

    Blacksell, Stuart D; Lim, Cherry; Tanganuchitcharnchai, Ampai; Jintaworn, Suthatip; Kantipong, Pacharee; Richards, Allen L; Paris, Daniel H; Limmathurotsakul, Direk; Day, Nicholas P J

    2016-06-01

    The enzyme-linked immunosorbent assay (ELISA) has been proposed as an alternative serologic diagnostic test to the indirect immunofluorescence assay (IFA) for scrub typhus. Here, we systematically determine the optimal sample dilution and cutoff optical density (OD) and estimate the accuracy of IgM ELISA using Bayesian latent class models (LCMs). Data from 135 patients with undifferentiated fever were reevaluated using Bayesian LCMs. Every patient was evaluated for the presence of an eschar and tested with a blood culture for Orientia tsutsugamushi, three different PCR assays, and an IgM IFA. The IgM ELISA was performed for every sample at sample dilutions from 1:100 to 1:102,400 using crude whole-cell antigens of the Karp, Kato, and Gilliam strains of O. tsutsugamushi developed by the Naval Medical Research Center. We used Bayesian LCMs to generate unbiased receiver operating characteristic curves and found that the sample dilution of 1:400 was optimal for the IgM ELISA. With the optimal cutoff OD of 1.474 at a sample dilution of 1:400, the IgM ELISA had a sensitivity of 85.7% (95% credible interval [CrI], 77.4% to 86.7%) and a specificity of 98.1% (95% CrI, 97.2% to 100%) using paired samples. For the ELISA, the OD could be determined objectively and quickly, in contrast to the reading of IFA slides, which was both subjective and labor-intensive. The IgM ELISA for scrub typhus has high diagnostic accuracy and is less subjective than the IgM IFA. We suggest that the IgM ELISA may be used as an alternative reference test to the IgM IFA for the serological diagnosis of scrub typhus. PMID:27008880

  10. Buprenorphine detection in urine using liquid chromatography-high-resolution mass spectrometry: comparison with cloned enzyme donor immunoassay (ThermoFisher) and homogeneous enzyme immunoassay (immunalysis).

    PubMed

    Belsey, Sarah L; Couchman, Lewis; Flanagan, Robert J

    2014-09-01

    A sensitive liquid chromatographic-high-resolution mass spectrometric (LC-HR-MS) assay for buprenorphine and its urinary metabolites has been developed that requires minimal sample preparation. The results obtained have been compared with those given by (i) cloned enzyme donor immunoassay (CEDIA) and (ii) homogeneous enzyme immunoassay (HEIA) in the analysis of patient urines submitted for buprenorphine analysis. Centrifuged urine (100 L) was diluted with internal standard solution (25 L) + LC eluent (875 L), and 50 L of the prepared sample were analyzed (Accucore Phenyl-Hexyl column). MS detection was in alternating positive and negative mode using heated electrospray ionization (ThermoFisher Q Exactive). Intra- and inter-assay accuracy and precision were 104-128 and <11%, respectively, at 5 g/L. Limits of detection were 1.3 g/L (buprenorphine, norbuprenorphine and buprenorphine glucuronide) and 2.5 g/L (norbuprenorphine glucuronide). Immunoassay sensitivity and selectivity were 97 and 100% (HEIA) and 99 and 84% (CEDIA), respectively, compared with LC-HR-MS. In 120 patient urines, norbuprenorphine glucuronide was easily the most abundant analyte except when adulteration with buprenorphine had occurred. The median immunoreactive buprenorphine species present (unhydrolysed urine) were 7.5 and 13% for HEIA and CEDIA, respectively. However, codeine, dihydrocodeine, morphine and morphine-3-glucuronide did not interfere in the HEIA assay. PMID:24925983

  11. ENZYME-LINKED IMMUNOASSAYS FOR THE DETECTION OF MICROBIAL ANTIGENS AND THEIR ANTIBODIES

    EPA Science Inventory

    The advantages of enzyme-immunoassay (EIA) over radioactive assay techniques are mainly convenience in use, in that the labelled immunoreagents are stable for long periods, and the precautions and disposal procedures required for radioisotopes are unnecessary. In addition, the us...

  12. Evaluation of a recombinant rhoptry protein 2 enzyme-linked immunoassay for the diagnosis of toxoplasmosis acquired during pregnancy

    PubMed Central

    Capobiango, Jaqueline Dario; Pagliari, Sthefany; Pasquali, Aline Kuhn Sbruzzi; Nino, Beatriz; Ferreira, Fernanda Pinto; Monica, Thaís Cabral; Tschurtschenthaler, Nely Norder; Navarro, Italmar Teodorico; Garcia, João Luis; Mitsuka-Breganó, Regina; Reiche, Edna Maria Vissoci

    2015-01-01

    The aim of this study was to evaluate an enzyme-linked immunoassay with recombinant rhoptry protein 2 (ELISA-rROP2) for its ability to detectToxoplasma gondii ROP2-specific IgG in samples from pregnant women. The study included 236 samples that were divided into groups according to serological screening profiles for toxoplasmosis: unexposed (n = 65), probable acute infection (n = 48), possible acute infection (n = 58) and exposed to the parasite (n = 65). When an indirect immunofluorescence assay forT. gondii-specific IgG was considered as a reference test, the ELISA-rROP2 had a sensitivity of 61.8%, specificity of 62.8%, predictive positive value of 76.6% and predictive negative value of 45.4% (p = 0.0002). The ELISA-rROP2 reacted with 62.5% of the samples from pregnant women with probable acute infection and 40% of the samples from pregnant women with previous exposure (p = 0.0180). Seropositivity was observed in 50/57 (87.7%) pregnant women with possible infection. The results underscored that T. gondii rROP2 is recognised by specific IgG antibodies in both the acute and chronic phases of toxoplasmosis acquired during pregnancy. However, the sensitivity of the ELISA-rROP2 was higher in the pregnant women with probable and possible acute infections and IgM reactivity. PMID:26517651

  13. Evaluation of a recombinant rhoptry protein 2 enzyme-linked immunoassay for the diagnosis of toxoplasmosis acquired during pregnancy.

    PubMed

    Capobiango, Jaqueline Dario; Pagliari, Sthefany; Pasquali, Aline Kuhn Sbruzzi; Nino, Beatriz; Ferreira, Fernanda Pinto; Monica, Thaís Cabral; Tschurtschenthaler, Nely Norder; Navarro, Italmar Teodorico; Garcia, João Luis; Mitsuka-Breganó, Regina; Reiche, Edna Maria Vissoci

    2015-09-01

    The aim of this study was to evaluate an enzyme-linked immunoassay with recombinant rhoptry protein 2 (ELISA-rROP2) for its ability to detect Toxoplasma gondii ROP2-specific IgG in samples from pregnant women. The study included 236 samples that were divided into groups according to serological screening profiles for toxoplasmosis: unexposed (n = 65), probable acute infection (n = 48), possible acute infection (n = 58) and exposed to the parasite (n = 65). When an indirect immunofluorescence assay forT. gondii-specific IgG was considered as a reference test, the ELISA-rROP2 had a sensitivity of 61.8%, specificity of 62.8%, predictive positive value of 76.6% and predictive negative value of 45.4% (p = 0.0002). The ELISA-rROP2 reacted with 62.5% of the samples from pregnant women with probable acute infection and 40% of the samples from pregnant women with previous exposure (p = 0.0180). Seropositivity was observed in 50/57 (87.7%) pregnant women with possible infection. The results underscored that T. gondii rROP2 is recognised by specific IgG antibodies in both the acute and chronic phases of toxoplasmosis acquired during pregnancy. However, the sensitivity of the ELISA-rROP2 was higher in the pregnant women with probable and possible acute infections and IgM reactivity. PMID:26517651

  14. Identification of Past and Recent Parvovirus B19 Infection in Immunocompetent Individuals by Quantitative PCR and Enzyme Immunoassays: a Dual-Laboratory Study

    PubMed Central

    Hedman, Lea; Dhanilall, Pravesh; Kantola, Kalle; Nurmi, Visa; Söderlund-Venermo, Maria; Brown, Kevin E.; Hedman, Klaus

    2014-01-01

    Parvovirus B19 (B19V) is a member of the family Parvoviridae, genus Erythrovirus. B19V-specific IgG and IgM react differently against conformational and linear epitopes of VP1 and VP2 antigens, leading to the development of IgG avidity and epitope type specificity (ETS) enzyme immunoassays (EIAs) for distinguishing past from recent infection. Additionally, B19V viral load determination (by quantitative PCR [qPCR]) is increasingly used in the staging of B19V infection. In this study, the utility of these methods is compared. A panel of 78 sera was jointly tested by the Virus Reference Department (VRD), London, United Kingdom, and the Haartman Institute (HI), Helsinki, Finland, using a number of EIAs, e.g., B19V-specific IgG and IgM, IgG avidity, and ETS EIAs. At VRD, the sera were also tested by a B19V viral load PCR (qPCR). By consensus analysis, 43 (55.1%) sera represented past infection, 28 (35.9%) sera represented recent infection, and 7 (9.0%) sera were indeterminate. Both VRD B19V qPCR and HI B19V VP2 IgM EIA gave the highest agreement with consensus interpretation for past or recent infection, with an overall agreement of 99% (95% confidence interval [CI], 92 to 100) and positive predictive value (PPV) of 100% (95% CI, 87 to 100). Nine sera designated as representing past infection by consensus analysis were B19V IgM positive by a commercial VRD B19V IgM EIA and B19V IgM negative by a new HI in-house B19V VP2 IgM EIA. A new VRD B19V IgG avidity EIA showed good (>95%) agreement (excluding equivocal results) with consensus interpretations for past or recent infection. Correct discrimination of past from recent B19V infection was achieved through application of qPCR or by appropriate selection of EIAs. PMID:24403307

  15. Cytomegalovirus Antibody in Cerebrospinal Fluid of Schizophrenic Patients Detected by Enzyme Immunoassay

    NASA Astrophysics Data System (ADS)

    Fuller Torrey, E.; Yolken, Robert H.; Winfrey, C. Jack

    1982-05-01

    By means of enzyme immunoassay techniques to detect the presence of antibody to cytomegalovirus, the cerebrospinal fluid of 178 patients with schizophrenia, 17 patients with bipolar disorders, and 11 other psychiatric patients was compared with that of 79 neurological patients and 41 normal control subjects. The cerebrospinal fluid of 20 of the schizophrenic patients and 3 of the patients with bipolar disorders showed significant increases in immunoglobulin M antibody to cytomegalovirus; no difference was found in patients on or off psychotropic medications.

  16. Automation of the enzyme immunoassay for the serodiagnosis of infectious diseases in cattle

    SciTech Connect

    Seawright, G.L.; Sanders, W.M.; Bryson, M.

    1980-01-01

    The enzyme immunoassay (EIA) is a versatile and highly sensitive new tool that can be used to detect a wide variety of infectious diseases or toxic agents and other low molecular weight compound. The paper describes the present state of development of the EIA-modified Technicon Autoanalyzer II instrumentation, standardization of quality control criteria for the automated EIA and standardization of the diagnostic decision process. (ACR)

  17. Detection of La Crosse arbovirus antigen in mosquito pools: application of chromogenic and fluorogenic enzyme immunoassay systems.

    PubMed Central

    Hildreth, S W; Beaty, B J; Meegan, J M; Frazier, C L; Shope, R E

    1982-01-01

    An enzyme immunoassay producing either a chromogenic or fluorogenic end product was developed and evaluated for detecting La Crosse viral antigen within mosquito pools. The enzyme immunoassay was found to be sensitive, detecting one infected mosquito within a pool of 100 mosquitoes, and specific, distinguishing between closely related California group viruses. Assays were completed within 5 h after the addition of test samples. La Crosse viral antigen could be readily detected in mosquito pools after seven freeze-thaw cycles. PMID:7047555

  18. A sensitive surface-enhanced Raman scattering enzyme-catalyzed immunoassay of respiratory syncytial virus.

    PubMed

    Zhan, Lei; Zhen, Shu Jun; Wan, Xiao Yan; Gao, Peng Fei; Huang, Cheng Zhi

    2016-02-01

    Respiratory viruses have become a major global health challenge which would benefit from advances in screening methods for early diagnosis. Respiratory syncytial virus (RSV) is one of the most important pathogen causing severe lower respiratory tract infections. Here we present a novel surface-enhanced Raman scattering (SERS) enzyme-catalyzed immunoassay of RSV by employing peroxidase substrate 3, 3'-5, 5'-tetramethylbenzidine (TMB) as Raman molecule. Horseradish peroxidase (HRP) attached to the detection antibody in a novel sandwich immunoassay catalyzes the oxidation of TMB by H2O2 to give a radical cation (TMB(+)), which could be easily adsorbed on the negatively charged surface of silver nanoparticles (AgNPs) through electrostatic interaction, inducing the aggregation of AgNPs and thus giving a strong SERS signal. A linear relationship was obtained between the Raman intensity and the amount of RSV in the range from 0.5 to 20 pg/mL, and the minimum detectable concentration of this SERS-based enzyme immunoassay was 0.05 pg/mL, which was 20 times lower than that found in the colorimetric method. PMID:26653454

  19. Variability in the adsorption properties of microtitre plates used as solid supports in enzyme immunoassay.

    PubMed

    Kricka, L J; Carter, T J; Burt, S M; Kennedy, J H; Holder, R L; Halliday, M I; Telford, M E; Wisdom, G B

    1980-05-01

    We evaluated the variability in the amount of protein adsorbed onto the surface of individual wells of an assortment of microtitre plates by use of procedures involving enzyme-protein conjugates. Coefficients of variation in adsorbed protein ranged from 5.2 to 29.5%. Microtitre plates show a distinct "edge effect"; wells at the edges of a plate adsorb more protein than those in the interior, which markedly affects results from immunoassays involving such plates. In a sandwich enzyme immunoassay the results for an individual sample varied by +/- 18%; in a competitive assay, values read from different calibration curves in the same plate varied by +/- 11%. Scanning electron microscopy and birefringence studies demonstrated no marked physical differences between individual wells on a plate. The variability in the amount of protein adsorbed onto the surface of individual wells on the same microtitre plate seriously affects the reliability and interpretation of results of immunoassays in which such plates are used as solid supports. PMID:6154544

  20. A direct dot-enzyme immunoassay to detect human ovulation.

    PubMed

    de Lauzon, S; Desfosses, B; Christeff, N; Hanquez, C; Cittanova, N

    1992-04-01

    This paper describes an original dot-enzyme-linked immunosorbent assay (ELISA) for predicting ovulation in women, based on the detection of the pre-ovulatory estrogen peak in urine. A monoclonal anti-estrogen antibody is used which recognizes not only free estrogens but also some of their urinary metabolites (17-glucuro- and sulfo-conjugates) allowing a direct assay on early morning urines. Antigen is immobilized as a spot on a nitrocellulose membrane which is immersed in urine in the presence of this antibody. A peroxidase-labeled second antibody allows the detection of the first antibody bound to the membrane. Antigen and anti-estrogen antibody concentrations are chosen to obtain a maximal enzymatic coloration of spots corresponding to basal urinary estrogen levels and no coloration corresponding to the pre-ovulatory surge. Six menstrual cycles were studied, comparing dot-ELISA results with patterns of: (1) urinary estrogens measured by RIA either directly or after hydrolysis and extraction, and (2) basal body temperatures. The validity of the pre-ovulatory signal obtained and the requirements for an adaptation of this methodology to a reliable home kit are discussed. PMID:1567785

  1. Application of enzyme immunoassay for postchemotherapy evaluation of human strongyloidiasis.

    PubMed

    Kobayashi, J; Sato, Y; Toma, H; Takara, M; Shiroma, Y

    1994-01-01

    Posttherapy evaluation of strongyloidiasis is frequently difficult because coprologic examination is not sensitive enough for diagnosis of chronic infection. In the present study, anti-Strongyloides enzyme-linked immunosorbent assay antibodies were monitored before and after treatment with thiabendazole and pyrvinium pamoate in 199 patients with chronic strongyloidiasis in Okinawa, Japan. A significant decrease in antibody levels was observed in patients who became negative for fecal larvae after the treatment, whereas the antibody levels did not show a significant change after the treatment in patients who were still harboring the parasite. In the group coprologically negative in the follow-up examination, however, many individuals did not show a significant fall in antibody titers after treatment, which suggests that these cases were equivocal for complete cure. By the subsequent fecal reexamination performed on the equivocal cases, approximately 20% were additionally found to be still harboring the parasite. These results indicate that serologic testing is useful to check whether a real cure has been achieved among the patients in whose fecal samples the presence of larvae has not been demonstrated after treatment. PMID:8026153

  2. A competitive chemiluminescence enzyme immunoassay for rapid and sensitive determination of enrofloxacin

    NASA Astrophysics Data System (ADS)

    Yu, Fei; Wu, Yongjun; Yu, Songcheng; Zhang, Huili; Zhang, Hongquan; Qu, Lingbo; Harrington, Peter de B.

    With alkaline phosphatase (ALP)-adamantane (AMPPD) system as the chemiluminescence (CL) detection system, a highly sensitive, specific and simple competitive chemiluminescence enzyme immunoassay (CLEIA) was developed for the measurement of enrofloxacin (ENR). The physicochemical parameters, such as the chemiluminescent assay mediums, the dilution buffer of ENR-McAb, the volume of dilution buffer, the monoclonal antibody concentration, the incubation time, and other relevant variables of the immunoassay have been optimized. Under the optimal conditions, the detection linear range of 350-1000 pg/mL and the detection limit of 0.24 ng/mL were provided by the proposed method. The relative standard deviations were less than 15% for both intra and inter-assay precision. This method has been successfully applied to determine ENR in spiked samples with the recovery of 103%-96%. It showed that CLEIA was a good potential method in the analysis of residues of veterinary drugs after treatment of related diseases.

  3. Enzyme immunoassay and liquid chromatography-fluorescence detection for amikacin in raw milk.

    PubMed

    Chen, Yiqiang; Chen, Qian; He, Lidong; Shang, Bingru; Zhang, Liying

    2012-11-15

    An enzyme immunoassay and a liquid chromatography (LC) method for amikacin (AMK) in raw milk were developed in this study. Anti-AMK antibody was prepared by immunizing rabbits with AMK-BSA conjugate. The developed immunoassay exhibited an IC(50) value of 1.30 ng/mL and the spiked recoveries at 25-1000 ng/mL ranged from 69.8% to 93.3% with coefficients of variation (CVs) of 8.5-17.6%. For LC analysis, AMK was derivatized with 9-fluorenylmethyl chloroformate, which was followed by C8 column separation and fluorescence detection. By trichloroacetic acid extraction and MCX column purification, the recoveries at spiked concentrations of 500-5000 ng/mL were 80.7-91.3% with CVs less than 6.3%. The two methods can be selectively used for rapidly screening or quantitatively determining AMK in raw milk. PMID:22868103

  4. Sugar additives improve signal fidelity for implementing two-phase resorufin-based enzyme immunoassays.

    PubMed

    Sandoz, Patrick A; Chung, Aram J; Weaver, Westbrook M; Di Carlo, Dino

    2014-06-17

    Enzymatic signal amplification based on fluorogenic substrates is commonly used for immunoassays; however, when transitioning these assays to a digital format in water-in-mineral oil emulsions, such amplification methods have been limited by the leakage of small reporting fluorescent probes. In the present study, we used a microfluidic system to study leakage from aqueous droplets in a controlled manner and confirmed that the leakage of fluorescent resorufin derivatives is mostly due to the presence of the lipophilic surfactant Span80, which is commonly used to preserve emulsion stability. This leakage can be overcome by the addition of specific sugars that most strongly interfered with the surfactants ability to form micelles in water. The application of the microfluidic system to the quantitative analysis of droplets and the implementation of the described sugar additives would allow for alternatives to fluorinated surfactant-based platforms and improve the signal fidelity in enzyme immunoassays implemented through multiphase microfluidics. PMID:24870310

  5. A sensitive and specific enzyme immunoassay for the detection of methyl ether derivatives of cyclomaltoheptaose.

    PubMed

    Djedaïni-Pilard, Florence; Nevers, Marie-Claire; Weisse, Sandrine; Grassi, Jacques; Perly, Bruno; Créminon, Christophe

    2003-09-26

    We have raised antibodies against two methylated derivatives of beta-CD, heptakis(2,6-di-O-methyl)cyclomaltoheptaose (Dimeb) and heptakis(2,3,6-tri-O-methyl)cyclomaltoheptaose (Trimeb). These antibodies were used to develop two specific and sensitive enzyme immunoassays, presenting a detection limit close to 500 and 30 pg/mL for Trimeb and Dimeb, respectively. Cross reactivities of different linear and cyclic maltooligosaccharides were investigated, demonstrating a high specificity against the structural features of the secondary hydroxyls rim. Several commercial Dimeb samples, containing different mixtures of partially methylated beta-cyclodextrin derivatives including RAMEB, which contains only a few amount of pure Dimeb, could be easily evaluated by the Dimeb immunoassay. Both of these assays have been shown to allow accurate measurement in plasma and urine, thus appearing as useful tools for further applications in biological material. PMID:14505876

  6. Distribution of phosphoneuroprotein 14 (PNP 14) in vertebrates: its levels as determined by enzyme immunoassay.

    PubMed

    Nakajo, S; Tsukada, K; Kameyama, H; Furuyama, Y; Nakaya, K

    1996-11-25

    We have established an enzyme immunoassay for phosphoneuroprotein 14 (PNP 14) which is mainly localized in the cytoplasmic matrix in presynaptic axon terminals and which is phosphorylated in vivo, as well as in vitro. Fab' prepared from rabbit IgG antibodies against bovine PNP 14 was conjugated with maleimide-horseradish peroxidase. The enzyme-conjugated Fab' was used as a second antibody in a sandwich enzyme immunoassay. This assay was able accurately to quantify 0.5-100 ng of rat PNP 14, as well as bovine PNP 14, and it was used for the determination of concentrations of PNP 14 in various rat tissues, neuroblastoma cells, and brains of other vertebrates. The concentrations of PNP 14 in the rat cerebrum, cerebellum, and testis were 1.1, 1.0, and 0.28 micrograms/mg of protein, respectively, and those in other tissues examined were less than 0.1 microgram/mg of protein. PNP 14 was also found in cultured cells, such as rat pheochromocytoma PC12 cells, NG108-15 cells, which are a hybrid between a mouse neuroblastoma and a rat glioma, mouse neuroblastoma Neuro-2a cells, and human neuroblastoma IMR32 cells. Furthermore, PNP 14-specific immunoreactivity was evaluated in the brains of various vertebrates, such as fish, frog, snake and chicken by immunoblot and enzyme immunoassay. The results revealed the immunoreactivity in the brains of all vertebrates examined and the levels were determined to be 0.6-2.1 micrograms bovine PNP 14 equivalents per mg of protein, suggesting that PNP 14 might be an essential component of the central nervous systems of vertebrates. PMID:9001721

  7. Application of enzyme immunoassays for the confirmation of clinically suspect plague in Namibia, 1982*

    PubMed Central

    Williams, James E.; Arntzen, Lorraine; Tyndal, Guy L.; Isaäcson, Margaretha

    1986-01-01

    An outbreak of plague occurred in Ovamboland, northern Namibia, late in 1982. Blood cultures, sera and blood clots were tested to obtain laboratory confirmations for clinically suspect cases of the disease. Isolation of the bacillus (Yersinia pestis) was attempted from blood cultures; sera were tested for antibody by passive haemagglutination (PHA) and enzyme-linked immunosorbent assay (ELISA). Sera and clots also were tested by ELISA for the specific F1 plague antigen. All the ELISA procedures were based on a monoclonal antibody to F1 antigen to ensure specificity. Thirty-eight cases were confirmed as plague: 50% by isolation, 34% by antibody responses, and 16% by the detection of antigenaemia. All isolates of Y. pestis were capable of producing F1 antigen, and significant antibody responses were observed in bacteriologically confirmed cases with paired sera. Patients who experienced sero-conversion had a higher IgM titre than IgG titre during the first nine days of hospitalization, while patients hospitalized for 17 or more days had IgG titres that were higher than the IgM titres. The relationship between IgM and IgG antibody titres is discussed with reference to identifying very recent infections. PHA titres increased and declined with IgM titres but were lower and more transient. ELISA procedures increased laboratory confirmations of plague by 23% above the numbers achieved using blood cultures and PHA tests alone. The ELISA to detect F1 antigen accounted for 86% of this increase by confirming cases where bacteriological isolation was not done. This ELISA did not replace the requirement for bacteriological isolation, since seven bacteraemic patients did not demonstrate antigenaemia. PMID:3492308

  8. Characterization of Treponema pallidum Particle Agglutination Assay-Negative Sera following Screening by Treponemal Total Antibody Enzyme Immunoassays

    PubMed Central

    Maple, P. A. C.; Ratcliffe, D.; Smit, E.

    2010-01-01

    Following a laboratory audit, a significant number of Treponema pallidum particle agglutination assay (TPPA)-negative sera were identified when TPPA was used as a confirmatory assay of syphilis enzyme immunoassay (EIA) screening-reactive sera (SSRS). Sera giving such discrepant results were further characterized to assess their significance. A panel of 226 sera was tested by the Abbott Murex ICE Syphilis EIA and then by the Newmarket Syphilis EIA II. TPPA testing was performed on 223 sera. Further testing by the Venereal Disease Research Laboratory (VDRL) test, the Mercia Syphilis IgM EIA, the fluorescent treponemal antibody (FTA-ABS) assay, and INNO-LIA immunoblotting was undertaken in discrepant cases. One hundred eighty-seven of 223 (83.8%) SSRS were TPPA reactive, while 26 (11.6%) sera which were reactive in both the ICE and Newmarket EIAs were nonreactive by TPPA. The majority (68%) of the TPPA-discrepant sera were from HIV-positive patients and did not represent early acute cases, based on previous or follow-up samples, which were available for 22/26 samples. FTA-ABS testing was performed on 24 of these sera; 14 (58.3%) were FTA-ABS positive, and 10 (41.7%) were FTA-ABS negative. Twenty-one of these 26 sera were tested by INNO-LIA, and an additional 4 FTA-ABS-negative samples were positive. In this study, significant numbers (18/26) of SSRS- and TPPA-negative sera were shown by further FTA-ABS and LIA (line immunoblot assay) testing to be positive. The reason why certain sera are negative by TPPA but reactive by treponemal EIA and other syphilis confirmatory assays is not clear, and these initial findings should be further explored. PMID:20844087

  9. [Detection of IgE specific for egg yolk by enzyme immunoassay. Description of a case].

    PubMed

    Carrillo Díaz, T; Cuevas Agustín, M; Moneo Goiri, I; Ibáñez Sandín, M D; Ureña Vilardell, V

    1986-01-01

    Food allergy is a common disease in our country, especially affecting atopic children. Egg-white hypersensitivity is frequently found in these patients. However, egg-yolk hypersensitivity is not usually reported in patients with egg allergy. This article describes a young patient with egg-yolk hypersensitivity, a 12 year old female patient with a medical history of contact urticaria, angioedema and severe acute bronchospasm shortly after the intake of small amounts of egg-yolk. All these episodes required treatment in emergency care units because of the severity of the symptoms. The patient did not describe any other food hypersensitivity and remained symptom-free after the intake of boiled or fried egg-white. She had clinical symptoms of grass pollen hypersensitivity and was therefore on specific immunotherapy at the time of the study. The skin prick-tests were positive to grass pollen and egg-yolk and were negative to mites, moulds, animal dander and to the common food tested (milk, fish, peanut, almond and hazel-nut). Total serum IgE was 1.160 UL/ml. The patient had a positive RAST to egg-white (0.0 PRU/ml) as well as to egg-yolk (8.6 PRU/ml). Furthermore, an indirect enzyme immunoassay as well as a reverse enzyme immunoassay also revealed the presence of specific IgE antibodies. The reverse enzyme immunoassay uses microtiter plates as a solid surface. These plates are coated with a monospecific antihuman IgE antibody. Thereafter, the serum samples are incubated overnight in the wells. After several washings, the presence of specific antibodies is revealed by means of a peroxidase conjugated allergen.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:3515886

  10. Enzyme immunoassay for specific determination of the synthetic estrogen, ethynyl estradiol, in plasma.

    PubMed

    Turkes, A; Dyas, J; Read, G F; Riad-Fahmy, D

    1981-06-01

    In this enzyme immunoassay for ethynyl estradiol, a conjugate of the 3-(O-carboxymethyl)ether with horseradish peroxidase is used as the label and a conjugate of the 6-(O-carboxymethyl)oxime with bovine serum albumin as the immunogen. A solid-phase antibody procedure is used in separating antibody-bound and free steroid. The lower limit of sensitivity of the assay is 2 pg per assay tube. Interference by synthetic progestagens was minimized by extracting the sample with an anti-estrogen serum before assay. Results obtained with a comparison radioimmunoassay and this procedure agreed well (r greater than 0.98). PMID:7016369

  11. An enzyme immunoassay for rat growth hormone - Applications to the study of growth hormone variants

    NASA Technical Reports Server (NTRS)

    Farrington, Marianne A.; Hymer, W. C.

    1987-01-01

    A sensitive and specific competitive enzyme immunoassay for rat growth hormone (GH) is described and its use in the detection of GH variants is demonstrated. In the present assay, soluble GH and GH adsorbed to a solid-phase support compete for monkey anti-GH antibody binding sites. The immobilized antibody-GH complex is detected and quantified using goat antimonkey immunoglobin G covalently conjugated to horseradish peroxidase. It is noted that the assay can be performed in 27 hours and that sensitivities in the range of 0.19 to 25 ng can be obtained in the region of 10 to 90 percent binding.

  12. Examination of the Rotazyme II enzyme immunoassay for the diagnosis of rotavirus gastroenteritis.

    PubMed Central

    Chernesky, M; Castriciano, S; Mahony, J; DeLong, D

    1985-01-01

    Rotazyme II, which is a shorter version of Rotazyme (less than 3 h), was compared with electron microscopy and Rotazyme for sensitivity and specificity on 229 human stool specimens. Compared with electron microscopy, the newer assay was found to be 99.4% sensitive and 97.3% specific for an overall agreement of 98.7%. After resolution of discordant results by blocking tests, the Rotazyme II enzyme immunoassay was shown to be more sensitive than electron microscopy and, therefore, 100% specific. PMID:2995441

  13. Application of magnetic nanoparticles in full-automated chemiluminescent enzyme immunoassay

    NASA Astrophysics Data System (ADS)

    Xie, Xiaomao; Ohnishi, Noriyuki; Takahashi, Yuki; Kondo, Akihiko

    2009-05-01

    The magnetic nanoparticles (MNPs) Therma-Max™ were used as a carrier to develop an automated sandwich chemiluminescent enzyme immunoassay (CLEIA) to detect thyroid-stimulating hormone (TSH) in a sensitive and specific way. The Therma-Max™ particles allow for automation because, unlike magnetic microspheres, they are completely dispersed in aqueous solution and allow for accurate automatic handling. Signal intensities detected with MNPs were 8-fold higher than those found with conventional micron-sized magnetic particles. A reproducibility study suggests that these particles allow for a stable detection method, as the coefficient of variation (CV) is less than 6% ( n=10).

  14. Enzyme immunoassay versus latex agglutination cryptococcal antigen assays in adults with non-HIV-related cryptococcosis.

    PubMed

    Panackal, Anil A; Dekker, John P; Proschan, Michael; Beri, Andrea; Williamson, Peter R

    2014-12-01

    We compared paired enzyme immunoassay (EIA) and latex agglutination (LA) assay results with 185 blood and 164 cerebrospinal fluid (CSF) samples from 44 and 33 non-HIV cryptococcosis patients, respectively. The LA assay cutoff of 1:256 in the blood and 1:32 in the CSF was most highly predictive of a positive EIA result. The EIA missed 18.4% detected by the LA assay in the blood samples and 7.8% detected by the LA assay in the CSF samples. We note here the improved sensitivity of the LA assay over the EIA in non-HIV patients. PMID:25253799

  15. Evaluation of a rapid enzyme immunoassay for diagnosis of hepatic amoebiasis.

    PubMed Central

    Kraoul, L; Adjmi, H; Lavarde, V; Pays, J F; Tourte-Schaefer, C; Hennequin, C

    1997-01-01

    We compared the capability of rapid enzyme immunoassay (EIA) to detect antiamoebic antibodies during hepatic amoebiasis with those of indirect hemagglutination and latex agglutination. EIA is simple to perform and rapid (20 min) and does not require any special equipment (optical reading is sufficient). EIA of 143 sera (including 43 from patients with proven hepatic amoebic abscess, 33 from patients with other hepatic disorders and/or parasitic infections, and 67 from healthy individuals) yielded a specificity, a sensitivity, and positive and negative predictive values of 100, 93, 100, and 97.1, respectively. This test could thus be considered another valuable tool for the diagnosis of hepatic amoebiasis. PMID:9163475

  16. Sensitive and specific enzyme immunoassays for antigenic trisaccharide from Bacillus anthracis spores.

    PubMed

    Dhénin, Sandrine G Y; Moreau, Vincent; Nevers, Marie-Claire; Créminon, Christophe; Djedaïni-Pilard, Florence

    2009-12-21

    A straightforward synthesis of an anthrose-containing trisaccharide derived from Bacillus anthracis was achieved. Antibodies raised against this hapten provide a highly sensitive enzyme immunoassay with a detection limit of 8.5 pmol mL(-1). By investigating the specificity of the antibodies obtained using different mono-, di- and trisaccharide synthetic analogues, we demonstrated that the epitope was mainly made up of the methyl group at C-5, the butamido group at C-4 and the hydroxyl at C-3 of the anthrose unit, the other parts of the trisaccharide appearing little involved in the recognition. PMID:20024115

  17. Enhanced competitive chemiluminescent enzyme immunoassay for the trace detection of insecticide triazophos.

    PubMed

    Jin, Maojun; Shao, Hua; Jin, Fen; Gui, Wenjun; Shi, Xiaomei; Wang, Jing; Zhu, Guonian

    2012-05-01

    A direct competitive chemiluminescent enzyme immunoassay (CLEIA) for triazophos was developed, which was based on the anti-THHe IgG monoclonal antibody and a heterogeneous enzyme tracer (THHu-HRP). Several components of chemiluminescent enhanced solution (CES) were optimized. The results showed that 1 mM of p-iodo-phenol, 0.625 mM of luminol, and 4 mM of H(2)O(2) had the best performance. Based on the study of CES, the influence of several factors (assay buffer, blocking substance, and solvent) on the immunoassay was investigated. The sensitivity for detection, IC(50) value was 0.87 ng/mL at a practical working concentration range between 0.04 ng/mL and 5 ng/mL and the limit of detection for triazophos was 0.063 ng/mL. The average recovery of triazophos added to lettuce, carrot, apple, water, and soil were 78.71%, 67.52%, 118.3%, 117.2%, and 122.0%, respectively. Finally, comparison between the methods of CLEIA and high-performance liquid chromatography-tandem mass spectrum (HPLC-MS/MS) was performed. The results obtained from CLEIA were in agreement with those of HPLC-MS/MS. PMID:22490114

  18. A rapid and sensitive 384-microtiter wells format chemiluminescent enzyme immunoassay for clenbuterol.

    PubMed

    Roda, A; Manetta, A C; Piazza, F; Simoni, P; Lelli, R

    2000-06-21

    A fast and sensitive chemiluminescent (CL) enzyme immunoassay for clenbuterol (CLB) analysis in bovine urine has been developed. Clenbuterol (CLB) specific polyclonal antibodies were raised in rabbit using a CLB azo derivative conjugated with ovalbumin. Horseradish peroxidase (HRP) was used as label and conjugated with the same derivative. In the developed competitive method, antibodies were immobilized on 384-wells black polystyrene microtiter plates; the sample volume was 20 mul and HRP-labeled CLB activity was immediately measured, using different CL substrates, after 10 min incubation time. Emitted light was recorded using a sensitive back-illuminated, cooled CCD camera or a conventional, photomultiplier-based micrtotiter plate reader. The developed method fulfills all the requirements of precision (CV below 10%) and accuracy (mean recovery from 96 to 110%) with a detection limit of 0.08 ppb in urine matrix. The use of 384-wells microtiter plate allows a 5-fold reduction in reagent quantity and the CL detection improves the detectability of the HRP-labeled tracer, thus reducing analysis time. The developed method is therefore suitable for high-throughput screening of CLB in urine samples, with reduced costs as compared with conventional colorimetric enzyme immunoassays, thanks to the possibility to optimize the system in non-equilibrium immunological conditions and with a very fast chemiluminescence detection of the HRP-label activity. PMID:18967990

  19. A rapid and sensitive 384-well microtitre format chemiluminescent enzyme immunoassay for 19-nortestosterone.

    PubMed

    Roda, Aldo; Manetta, Anna Chiara; Portanti, Ottavio; Mirasoli, Mara; Guardigli, Massimo; Pasini, Patrizia; Lelli, Rossella

    2003-01-01

    We developed a competitive chemiluminescent (CL) enzyme immunoassay for rapid, sensitive analysis of 19-nortestosterone (19-NT) in bovine urine. Anti-19-NT polyclonal antibodies were raised in rabbits using a 19-NT-hemisuccinate derivative conjugated with ovalbumin; the derivative was also conjugated with horseradish peroxidase (HRP) as a label. Antibodies were immobilized on 384-well black polystyrene microtitre plates and HRP-labelled 19-NT activity was measured using an efficient chemiluminescent substrate (SuperSignal ELISA Femto) after 3 min incubation. Emitted light was recorded using a conventional, photomultiplier-tube-based microtitre plate reader or a sensitive back-illuminated, cooled CCD camera. The developed method fulfils all the requirements of precision (intra- and inter-assay CV < 10%) and accuracy (mean recovery 94-112%), with a detection limit of 0.03 ppb (1.1 x 10(-9) mol/L) in a urine matrix. Chemiluminescence enhances detectability of the HRP-labelled tracer (thus lowering the limit of detection with respect to colorimetry) and reduces analysis time. The 384-well microtitre plate cuts the sample/reagent volume (20 microL), a five-fold reduction with respect to the conventional 96-well microtitre plate. The developed method is suitable for high-throughput screening of 19-NT in urine samples, with reduced costs as compared with conventional colorimetric enzyme immunoassays. PMID:12687626

  20. Development of sensitive direct chemiluminescent enzyme immunoassay for the determination of dihydroartemisinin in plasma.

    PubMed

    Zehnacker, Laura; Nevers, Marie-Claire; Sinou, Véronique; Parzy, Dominique; Créminon, Christophe; Parzy, Daniel; Azoulay, Stéphane

    2015-10-01

    Despite significant progress in prevention and therapy, malaria is still one of the world's leading major diseases due to its high morbidity and mortality. Recommended treatments by the World Health Organization include the use of artemisinin and artemisinin derivative-based combination therapies. To allow efficient patient monitoring during antimalarial therapy without the use of expensive apparatus, we developed a sensitive direct chemiluminescent enzyme immunoassay for the determination of dihydroartemisinin in biological fluids. To produce specific antibodies against dihydroartemisinin (DHA), a synthetic DHA derivative was coupled to bovine serum albumin as the immunogen. In parallel, a new, rapid, and efficient procedure to covalently link glycoprotein to all amine-containing molecules has been established and the enzyme tracer was prepared by chemically coupling the DHA derivative in combination with SBP rather than the more commonly used HRP. It allowed us to develop, after optimization of the luminescent reagent, a sensitive and stable luminescent EIA, with a LLOQ of 90 pg mL(-1). This assay compares favorably with the most efficient HPLC methods previously reported with a LLOQ close to 1 ng mL(-1) and shows good precision and efficiency since recovery from human plasma spiked with DHA ranged between 91 and 103%, with coefficients of variation of <13%. To date, no immunoassay for DHA has been applied to plasma analysis and this EIA should be very useful in all clinical laboratories for rapid and cost-effective analysis. PMID:26280205

  1. Dipstick enzyme immunoassay to detect Fusarium T-2 toxin in wheat.

    PubMed

    De Saeger, S; Van Peteghem, C

    1996-06-01

    A dipstick enzyme immunoassay for the rapid detection of Fusarium T-2 toxin in wheat was developed. An Immunodyne ABC membrane was precoated with rabbit anti-mouse immunoglobulins. After the strips were immersed in a solution of monoclonal anti-T-2 toxin antibodies, a direct competitive enzyme immunoassay was performed. This assay included the incubation of the antibody-coated dipsticks in a mixture of sample and T-2 toxin-horseradish peroxidase conjugate. Afterwards, the strips were placed in a chromogen-containing substrate solution (H202-3,3',5,5'-tetramethylbenzidine) for color reaction. The dot color intensity of toxin-positive dipsticks was visually distinguishable from that of the negative control. A portable colorimeter was used to confirm and quantify the visual observations. With coated strips, the tests could be performed in 45 min. The visual detection limit for T-2 toxin in buffer solution was 0.25 ng/ml. Artificially infected wheat samples were extracted with 80% methanol-water. A dilution of the raw extract of 1:8 was sufficient to avoid matrix effects. It was possible to make visually a clear distinction between the negative control and a wheat extract spiked with 12 ng/g. PMID:8787386

  2. Comparative studies with penicillinase, horseradish peroxidase, and alkaline phosphatase as enzyme labels in developing enzyme immunoassay of cortisol.

    PubMed

    Kumari, G Lakshmi; Dhir, Ravindra N

    2003-01-01

    Relative merit of different enzyme labels for measuring cortisol directly in serum by competitive enzyme immunoassay (ELISA) was examined. Cortisol-21-hemisuccinate was labeled separately with penicillinase, horseradish peroxidase (HRP), and alkaline phosphatase (ALP) under identical reaction conditions. Antibody developed in rabbits against cortisol-3-0-(carboxymethyl)-oxime-bovine serum albumin was used to coat polystyrene tubes that were precoated with anti-rabbit gamma globulin (ARGG). Cortisol standards were prepared in steroid-free human serum in buffer (1:4) contaning 8-anilino-1-naphthalene sulfonic acid (8-ANS). Assay buffer also consisted 8-ANS. The assay involved adding standard cortisol or serum sample to antibody-coated tubes, followed by addition of enzyme label and buffer, and incubation for 2 h at 37 degrees C. The whole procedure took 3 h for completion. All three labels proved to be sensitive, with a slope around -2.0. Although penicillinase as an enzyme label was highly sensitive and stable compared with others, the assays were not always accurate and precise, especially at low concentrations of cortisol. This was mainly due to the color reagent used for measuring penicillinase activity. Serum samples that underwent 2-3 freeze-thaw cycles gave high values with HRP label compared with ALP. Therefore, utilizing ALP as an enzyme label, an ELISA was developed and its performance was comparable with some of the commercial kits already in the market. PMID:12778970

  3. Enzyme immuno assay for the detection of virus specific IgG and IgM antibody in patients with haemorrhagic fever with renal syndrome.

    PubMed

    Ivanov, A P; Tkachenko, E A; Petrov, V A; Pashkov, A J; Dzagurova, T K; Vladimirova, T P; Voronkova, G M; van der Groen, G

    1988-01-01

    Consecutive serum samples collected from 235 patients with Haemorrhagic Fever with Renal Syndrome (HFRS), between two days and two years after onset of disease, have been analysed for the presence of IgG and IgM type of antibodies specific for Hanta-viruses. The sera were screened in parallel by a newly developed indirect Immuno Enzyme Assay (EIA) in parallel with Indirect Immunofluorescent Antibody Assay (IFA). In both tests the Hantaan virus strain 76-118 was used as the antigen. The EIA was much more sensitive than the IFA test for the detection of IgM type antibodies. With the indirect EIA IgM type antibodies against Hantaan virus 76-118 have been detected in HFRS patient's sera from the second day of illness indicating the usefulness of this test for the early serological diagnosis of this disease. PMID:2898929

  4. Diagnostic accuracy of an IgM enzyme-linked immunosorbent assay and comparison with 2 polymerase chain reactions for early diagnosis of human leptospirosis.

    PubMed

    Vanasco, N B; Jacob, P; Landolt, N; Chiani, Y; Schmeling, M F; Cudos, C; Tarabla, H; Lottersberger, J

    2016-04-01

    Enzyme-linked immunosorbent assay (ELISA) tests and polymerase chain reaction (PCR) may play a key role for early detection and treatment of human leptospirosis in developing countries. The aims of this study were to develop and validate an IgM ELISA under field conditions and to compare the diagnostic accuracy among IgG, IgM ELISAs, conventional PCR (cPCR), and real-time PCR (rtPCR) for early detection of human leptospirosis. Overall accuracy of IgM ELISA was sensitivity of 87.9%, specificity of 97.0%, and area under the curve of 0.940. When the 4 methods were compared, IgM ELISA showed the greatest diagnostic accuracy (J=0.6) followed by rtPCR (J=0.4), cPCR (J=0.2) and IgG ELISA (J=0.1). Our results support the use of IgM ELISA and rtPCR for early diagnosis of the disease. Moreover, due to their high specificity, they could be also useful to replace or supplement microscopic agglutination test as a confirmatory test, allowing more confirmations. PMID:26867967

  5. Comparison of three enzyme immunoassays for measuring 17beta-estradiol in flushed dairy manure wastewater.

    PubMed

    Hanselman, Travis A; Graetz, Donald A; Wilkie, Ann C

    2004-01-01

    Natural steroidal estrogens are an environmental concern because low nanogram per liter concentrations in water can adversely affect aquatic vertebrate species by disrupting the normal function of their endocrine systems. There is a critical need to accurately measure estrogens in dairy wastes, a potential source of estrogens such as 17beta-estradiol, to assess the risk of estrogen contamination of agricultural drainage waters resulting from land application. Commercially available enzyme immunoassay (EIA) kits have been used for measuring 17beta-estradiol in livestock manure, but it is not known if different EIAs provide similar results. We compared three EIAs by measuring 17beta-estradiol in two samples of flushed dairy manure wastewater (FDMW). The measured concentrations of 17beta-estradiol in FDMW differed according to the immunoassay used. The differences were attributed to a matrix interference associated with coextracted humic substances. Future research should develop methods that enable routine measurement of 17beta-estradiol in livestock wastes by more conclusive analytical techniques such as gas chromatography-mass spectrometry. PMID:15356254

  6. Clinical and microbiological effects of subgingival antimicrobial irrigation with citric acid as evaluated by an enzyme immunoassay and culture analysis.

    PubMed

    Renvert, S; Dahlén, G; Snyder, B

    1997-04-01

    The purpose of the present study was to compare an enzyme immunoassay with culture samples from untreated and non-surgically treated periodontal pockets and to assess the clinical and microbiological effects of citric acid irrigation as a supplement to scaling and root planing. The enzyme immunoassay used in this study is a chairside diagnostic tool aimed at identifying the presence of P. gingivalis, P. intermedia, and A. actinomycetemcomitans. Six sites with pocket depths > or = 6 mm in each of 16 patients were monitored for 24 weeks using clinical and microbiological parameters. In two out of the six sites, scaling and root planing was supplemented with subgingival citric acid irrigation of the pocket after completion of the mechanical treatment. The sensitivity of the immunoassay in relation to culture was calculated to 85.5% and the specificity to 90.2%. The immunoassay corresponded to a detection level of 10(4) as estimated by culture. Sites treated with a combination of scaling and irrigation with citric acid demonstrated a similar healing pattern as sites treated with scaling and root planing alone. The profile of the marker bacteria was almost parallel for the two groups. The results of this investigation thus indicated that the immunoassay can be used as a screening tool for selected periodontal pathogens and that adjunctive irrigation with citric acid has no measurable clinical or microbiological effects. PMID:9150039

  7. A new enzyme immunoassay to detect antibodies to arboviruses in the blood of wild birds.

    PubMed

    Chiles, R E; Reisen, W K

    1998-12-01

    A new indirect enzyme immunoassay (EIA) was developed to screen wild bird sera for antibodies against western equine encephalomyelitis (WEE) and St. Louis encephalitis (SLE) viruses. The detector antibody was made by immunizing rabbits with serum proteins pooled from single species representatives of four bird orders and was conjugated with horseradish peroxidase to allow visualization with the ABTS substrate in an EIA plate reader set at 405 nm. The detector antibody recognized a wide range of bird species and was more accurate, sensitive, and specific than a hemaglutination inhibition test when compared to a plaque reduction neutralization test (PRNT). EIA positive sera frequently could not be confirmed by PRNT; however, practically all sera positive by PRNT also were positive by EIA. The new EIA has been incorporated into our field research program and has been used to economically screen over 10,000 wild bird sera from 124 species for antibodies against WEE and SLE. PMID:9879069

  8. False-Positive Results Obtained with the Alexon ProSpecT Cryptosporidium Enzyme Immunoassay

    PubMed Central

    Doing, Kirk M.; Hamm, Jill L.; Jellison, Jo Ann; Marquis, Jessica A.; Kingsbury, Cindy

    1999-01-01

    Cryptosporidium is known to cause diarrhea in immunocompromised patients and is also associated with outbreaks of disease due to food-borne and waterborne parasites. Traditional procedures, involving iodine staining of wet mounts of stool sediments and trichrome staining, lack the sensitivity to detect Cryptosporidium. Special staining procedures, such as the modified acid-fast and safranin stains, are generally employed. Less labor-intensive antigen detection assays have simplified detection; however, careful attention to local epidemiology is important because false-positive tests occur. Here, we report two incidents involving 62 false-positive results obtained with the Alexon ProSpecT Cryptosporidium enzyme immunoassay, which were deemed false-positive based on negative results obtained from extensive microscopic examinations. PMID:10203528

  9. Detection of Pesticides and Pesticide Metabolites Using the Cross Reactivity of Enzyme Immunoassays

    USGS Publications Warehouse

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

    2001-01-01

    Enzyme immunoassay is an important environmental analysis method that may be used to identify many pesticide analytes in water samples. Because of similarities in chemical structure between various members of a pesticide class, there often may be an unwanted response that is characterized by a percentage of cross reactivity. Also, there may be cross reactivity caused by degradation products of the target analyte that may be present in the sample. In this paper, the concept of cross reactivity caused by degradation products or by nontarget analytes is explored as a tool for identification of metabolites or structurally similar compounds not previously known to be present in water samples. Two examples are examined in this paper from various water quality studies. They are alachlor and its metabolite, alachlor ethane sulfonic acid, and atrazine and its class members, prometryn and propazine. A method for using cross reactivity for the detection of these compounds is explained in this paper.

  10. Species identification of blood and saliva stains by enzyme-linked immunoassay (ELISA) using monoclonal antibody.

    PubMed

    Fletcher, S M; Dolton, P; Harris-Smith, P W

    1984-01-01

    An indirect enzyme-linked immunoassay (ELISA) method for the identification of human blood and saliva stains is reported. The method uses a monoclonal antibody which reacts with human immunoglobulin G (IgG) in extracts of blood and saliva stains up to 16 months old. Semen stain extracts gave weak or negative results. For routine screening purposes dilutions of 1:1000 for bloodstain extracts and 1:100 for saliva stain extracts would be suitable. Of 32 other animal species tested, only chimpanzee, mouse, rat, and eel cross-reacted significantly, and the presence of the last three was clearly indicated by appropriate controls. The monoclonal antibody gave poor results in the crossover and gel diffusion techniques. PMID:6699607

  11. Improvement of an enzyme immunoassay for the determination of mercury (II)

    SciTech Connect

    Marx, A.; Kroetz, E.; Hock, B.

    1998-07-01

    Three systems were tested for the optimization of a heterogeneous noncompetitive enzyme immunoassay (EIA) for the determination of Hg (II). The sensitivity of the nonoptimized Hg-EIA with a detection limit of 2.1 {micro}g/L Hg (II) was improved by an avidin-biotin-complex (ABC) amplification system to a 2-fold lower detection limit (1.1 {micro}g/L Hg (II)). A conventional competitive EIA with the competition reaction between bound and free Hg (II) for antibody (ab) binding sites yielded a detection limit of 1.0 {micro}g/L Hg (II). Further improvement of sensitivity could be achieved by a competitive displacement EIA. In this case ab molecules bound to immobilized haptens are displaced in the next step by free Hg (II). The detection limit of the displacement approach is 0.4 {micro}g/L Hg (II).

  12. Determination of metallothionein in biological fluids using enzyme-linked immunoassay with commercial antibody.

    PubMed

    Milnerowicz, Halina; Bizoń, Anna

    2010-01-01

    Metallothionein (MT) is a low molecular weight cysteine-rich protein with a number of roles in the pro/antioxidant balance and homeostasis of essential metals, such as zinc and copper, and in the detoxification of heavy metals, such as cadmium and mercury. Until now, detection of metallothionein in biological fluids remained difficult because of a lack of a broadly reactive commercial test. Meaningful comparison of the values of metallothionein concentrations reported by different authors using their specific isolation procedures and different conditions of enzyme-linked immunoassay is difficult due to the absence of a reference material for metallothionein. Therefore in the present study, we describe a quantitative assay for metallothionein in biological fluids such as plasma and urine performed by a direct enzyme-linked immunoassay using a commercially available monoclonal mouse anti-metallothionein clone E9 antibody and commercial standards of metallothionein from rabbit liver and a custom preparation of metallothionein from human liver. The sensitivity of the assay for the standard containing two isoforms MT-I and MT-II from human liver was 140 pg/well. The reactivity of the commercial standards and standards containing two isoforms MT-I and MT-II isolated from human liver in our laboratory with a commercial monoclonal mouse anti-metallothionein clone E9 antibody were similar. This suggests that the described ELISA test can be useful for determination of metallothionein concentration in biological fluids. The concentrations of metallothionein in human plasma, erythrocyte lysate and in urine of smoking and non-smoking healthy volunteers are reported. Tobacco smoking increases the extracellular metallothionein concentration (plasma and urine) but does not affect the intracellular concentration (erythrocyte lysate). PMID:20349027

  13. Evaluation of the Specificity of Two Enzyme Immunoassays for Coccidioidomycosis by Using Sera from a Region of Endemicity and a Region of Nonendemicity.

    PubMed

    Lindsley, Mark D; Ahn, YoonJi; McCotter, Orion; Gade, Lalitha; Hurst, Steven F; Brandt, Mary E; Park, Benjamin J; Litvintseva, Anastasia P

    2015-10-01

    Coccidioidomycosis (CM), a serious life-threatening fungal infection endemic to arid regions of the western United States and Mexico, can be challenging to diagnose in a timely manner. Commercially developed enzyme immunoassays (EIAs) (from Meridian Biosciences and Immuno-Mycologics [IMMY]) have provided faster, simpler means for serodiagnosis; however, independent evaluations have questioned EIA specificity, particularly IgM-positive/IgG-negative results. This study was conducted to evaluate EIA specificity among persons residing in Puerto Rico (n = 534), where CM is not endemic (who were not likely to have been exposed to Coccidioides spp.), compared to blood bank donors residing in Arizona (n = 1,218), where CM is endemic. Upon comparing serum reactivity between Puerto Rico and Arizona, the Meridian EIA showed a significant difference in IgG reactivity (0.37% versus 3.6%; P < 0.001) but not IgM reactivity (3.4% versus 2.4%; P = 0.31). No IgM-/IgG-reactive sera were detected among sera from Puerto Rico, compared to 7 (0.57%) sera from Arizona. Similar results were observed using the IMMY EIA, although significantly (P = 0.03) fewer IgM-reactive sera from Arizona were observed, compared to the Meridian EIA. EIA-reactive sera were also evaluated by immunodiffusion before and after 3- to 4-fold concentration of the sera. These results demonstrate that elevated IgG EIA reactivity is present in sera from healthy individuals in regions of endemicity and that IgM EIA reactivity observed in sera from individuals residing outside regions of endemicity is most likely nonspecific. Other criteria, including clinical and microbiological evaluations, should be taken into account when interpreting results from surveillance studies and other reporting measures. PMID:26245352

  14. Evaluation of the Specificity of Two Enzyme Immunoassays for Coccidioidomycosis by Using Sera from a Region of Endemicity and a Region of Nonendemicity

    PubMed Central

    Ahn, YoonJi; McCotter, Orion; Gade, Lalitha; Hurst, Steven F.; Brandt, Mary E.; Park, Benjamin J.; Litvintseva, Anastasia P.

    2015-01-01

    Coccidioidomycosis (CM), a serious life-threatening fungal infection endemic to arid regions of the western United States and Mexico, can be challenging to diagnose in a timely manner. Commercially developed enzyme immunoassays (EIAs) (from Meridian Biosciences and Immuno-Mycologics [IMMY]) have provided faster, simpler means for serodiagnosis; however, independent evaluations have questioned EIA specificity, particularly IgM-positive/IgG-negative results. This study was conducted to evaluate EIA specificity among persons residing in Puerto Rico (n = 534), where CM is not endemic (who were not likely to have been exposed to Coccidioides spp.), compared to blood bank donors residing in Arizona (n = 1,218), where CM is endemic. Upon comparing serum reactivity between Puerto Rico and Arizona, the Meridian EIA showed a significant difference in IgG reactivity (0.37% versus 3.6%; P < 0.001) but not IgM reactivity (3.4% versus 2.4%; P = 0.31). No IgM-/IgG-reactive sera were detected among sera from Puerto Rico, compared to 7 (0.57%) sera from Arizona. Similar results were observed using the IMMY EIA, although significantly (P = 0.03) fewer IgM-reactive sera from Arizona were observed, compared to the Meridian EIA. EIA-reactive sera were also evaluated by immunodiffusion before and after 3- to 4-fold concentration of the sera. These results demonstrate that elevated IgG EIA reactivity is present in sera from healthy individuals in regions of endemicity and that IgM EIA reactivity observed in sera from individuals residing outside regions of endemicity is most likely nonspecific. Other criteria, including clinical and microbiological evaluations, should be taken into account when interpreting results from surveillance studies and other reporting measures. PMID:26245352

  15. Self-Assembly of Ferritin Nanoparticles into an Enzyme Nanocomposite with Tunable Size for Ultrasensitive Immunoassay.

    PubMed

    Men, Dong; Zhang, Ting-Ting; Hou, Li-Wei; Zhou, Juan; Zhang, Zhi-Ping; Shi, Yuan-Yuan; Zhang, Jin-Li; Cui, Zong-Qiang; Deng, Jiao-Yu; Wang, Dian-Bing; Zhang, Xian-En

    2015-11-24

    The self-assembly of nanoparticles into larger superstructures is a powerful strategy to develop novel functional nanomaterials, as these superstructures display collective properties that are different to those displayed by individual nanoparticles or bulk samples. However, there are increasing bottlenecks in terms of size control and multifunctionalization of nanoparticle assemblies. In this study, we developed a self-assembly strategy for construction of multifunctional nanoparticle assemblies of tunable size, through rational regulation of the number of self-assembling interaction sites on each nanoparticle. As proof-of-principle, a size-controlled enzyme nanocomposite (ENC) was constructed by self-assembly of streptavidin-labeled horseradish peroxidase (SA-HRP) and autobiotinylated ferritin nanoparticles (bFNP). Our ENC integrates a large number of enzyme molecules, together with a streptavidin-coated surface, allowing for a drastic increase in enzymatic signal when the SA is bound to a biotinylated target molecule. As result, a 10 000-fold increase in sensitivity over conventional enzyme-linked immunosorbent assays (ELISA) methods was achieved in a cardiac troponin immunoassay. Our method presented here should provide a feasible approach for constructing elaborate multifunctional superstructures of tunable size useful for a broad range of biomedical applications. PMID:26431499

  16. Potential impact of different cytomegalovirus (CMV) IgM assays on an algorithm requiring IgM reactivity as a criterion for measuring CMV IgG avidity.

    PubMed

    Prince, Harry E; Lapé-Nixon, Mary; Brenner, Andrew; Pitstick, Nancy; Couturier, Marc Roger

    2014-06-01

    The measurement of cytomegalovirus (CMV) IgG avidity is a powerful tool for identifying individuals with recent CMV infection. Because such patients are expected to be positive for CMV IgM, several investigators have suggested that CMV IgG-positive sera first be screened for CMV IgM and then only the IgM-reactive sera be tested for avidity. We investigated the impact of different CMV IgM assays on such a reflexing algorithm using a panel of 369 consecutive IgG-positive serum samples submitted for avidity testing. A bead-based immunofluorescent assay (BIFA) identified 105 IgM-positive serum samples, whereas an IgM-capture enzyme immunoassay (EIA) identified 48 IgM-positive serum samples; this marked difference led us to evaluate additional CMV IgM assays. An enzyme-linked immunofluorescent assay (ELFA) and a chemiluminescent immunoassay (CIA) were used to test all sera with discordant BIFA/EIA results, all sera with concordant positive results, and selected sera with concordant negative results. The findings indicated that the ELFA would identify 74 CMV IgM-positive samples and the CIA would identify 64. Of the 23 low-avidity serum samples, 2 were IgM negative by BIFA, 3 by ELFA and CIA, and 4 by EIA; of the 23 intermediate-avidity serum samples, 6 were IgM negative by BIFA, 10 by ELFA, and 15 by EIA and CIA. In both these avidity groups, BIFA IgM-negative sera were also negative by the other 3 assays. These findings demonstrate that an algorithm requiring CMV IgM reactivity as a criterion for CMV IgG avidity testing does not identify all low-avidity sera and thus misses some cases of acute CMV infection. PMID:24671558

  17. Development of a competitive enzyme immunoassay for 17 alpha-19-nortestosterone.

    PubMed

    Van Look, L J; Jansen, E H; van den Berg, R H; Zomer, G; Vanoosthuyze, K E; Van Peteghem, C H

    1991-04-01

    Because 17 beta-19-nortestosterone and its esters are frequently used anabolic steroids in cattle in Europe, there is a need for an assay that can also detect certain metabolites. The enzyme immunoassay described here involves the detection and quantitation of the major metabolite 17 alpha-19-nortestosterone in urine. The assay is based on the coating of an antibody raised in a rabbit against 17 alpha-19-nortestosterone-3-carboxy-methyloxime-bovine serum albumin (17 alpha-19-NT-3-CMO-BSA), the competitive incubation of 17 alpha-19-NT and the 17 alpha-19-nortestosterone-3-CMO-horseradish peroxidase label, followed by the detection of the blue colour developed by the action of the enzyme on tetramethylbenzidine. The 3-CMO conjugate of 17 alpha-19-nortestosterone was used to produce an antibody with selective affinity for the 17 alpha-epimer. For the optimization of the assay, different coatings and incubation conditions were tested. The standard curve ranged between 0.98 and 4000 pg per well, with a B/B0 50% of +/- 65 pg per well. Colour was measured with a microtitre plate reader. The method was validated by means of certified blank and spiked cattle urine samples. PMID:1874849

  18. Enzyme immunoassay for rat growth hormone: applications to the study of growth hormone variants

    SciTech Connect

    Farrington, M.A.; Hymer, W.C.

    1987-06-29

    A sensitive and specific competitive enzyme immunoassay (EIA) for rat growth hormone was developed. In this assay soluble growth hormone and growth hormone adsorbed to a solid-phase support compete for monkey anti-growth hormone antibody binding sites. The immobilized antibody-growth hormone complex is detected and quantified using goat anti-monkey immunoglobin G covalently conjugated to horse radish peroxidase. Therefore, a high concentration of soluble growth hormone in the sample will result in low absorbance detection from the colored products of the enzyme reaction. Assay parameters were optimized by investigating the concentration of reagents and the reaction kinetics in each of the assay steps. The assay can be performed in 27 hours. A sensitivity range of 0.19 ng to 25 ng in the region of 10 to 90% binding was obtained. Near 50% binding (3 ng) the intraassay coefficient of variation (CV) was 5.54% and the interassay CV was 5.33%. The correlation coefficient (r/sup 2/) between radioimmunoassay and EIA was 0.956 and followed the curve Y = 0.78X + 1.0. 9 references, 6 figures.

  19. Use of monoclonal antibodies in an enzyme immunoassay for rapid identification of group B Streptococcus types II and III.

    PubMed Central

    Polin, R A; Kennett, R

    1980-01-01

    Streptococci belonging to Lancefield group B are frequently recognized as the etiological agents of sepsis and meningitis in young children. Current methods of identifying these organisms have not been universally accepted because of the time and complexity in performing the studies and a lack of reference antisera. We have developed hybrid myeloma (hybridoma) cell lines which secrete large amounts of antibody against types II and III group B streptococci. Antibodies harvested from supernatants react only with the bacterial strain that was used initially to immunize the animals. We have used the hybridoma antibodies in an enzyme immunoassay and have shown it to be a sensitive and reliable technique for typing group B streptococci. The use of hybridoma antibodies in the enzyme immunoassay may permit early detection of group B streptococcal antigen before cultures are visibly positive. PMID:6989855

  20. The Rapid Screening of Triazophos Residues in Agricultural Products by Chemiluminescent Enzyme Immunoassay

    PubMed Central

    Chen, Ge; Yang, Lihua; Jin, Maojun; Du, Pengfei; Zhang, Chan; Wang, Jian; Shao, Hua; Jin, Fen; Zheng, Lufei; Wang, Shanshan; She, Yongxin; Wang, Jing

    2015-01-01

    A highly sensitive chemiluminescent enzyme immunoassay (CLEIA) method was developed in this study for efficient screening of triazophos residues in a large number of samples. Based on the maximum residue limits (MRLs) set by China and CAC for triazophos in different agro-products, the representative apple, orange, cabbage, zucchini, and rice samples were selected as spiked samples, and the triazophos at the concentrations of the MRL values were spiked to blank samples. Subsequently, the five samples with the spiked triazophos standard were measured by CLEIA 100 times, and the detection results indicated that the correction factors of the apple, orange, cabbage, zucchini, and rice were determined as 0.79, 0.66, 0.85, 0.76, and 0.91, respectively. In this experiment, 1500 real samples were detected by both the CLEIA and the GC-MS methods. With the GC-MS method, 1462 samples were identified as negative samples and 38 samples as positive samples. Based on the correction factors, the false positive rate of the CLEIA method was 0.13%, and false negative rate was 0. The results showed that the established CLEIA method could be used to screen a large number of real samples. PMID:26218576

  1. Laboratory and epidemiologic evaluation of an enzyme immunoassay for antibodies to HTLV-III

    SciTech Connect

    Ward, J.W.; Grindon, A.J.; Feorino, P.M.; Schable, C.; Parvin, M.; Allen, J.R.

    1986-07-18

    The enzyme immunoassays (EIAs) for antibody to human T-cell lymphotropic virus type III (HTLV-III) were rapidly adopted for screening donated blood and plasma. To evaluate the significance of a positive EIA reaction, test performance was examined in a blood bank screening program. Specimens were tested by EIA, Western blot assay, and HTLV-III/lymphadenopathy-associated virus (LAV) culture. The EIA was positive in 0.25% of 67 190 blood donations. Specimens were categorized and 57.3% had low (weak) reactivity, 12.7% had moderate reactivity, and 30.0% had high reactivity. Highly reactive specimens were strongly associated with a positive Western blot or culture (86.7%) in contrast to moderately and weekly reactive specimens (1.9%). Twenty-five of 29 donors interviewed with a highly reactive EIA had risk factors for HTLV-III/LAV infection. Risk factors were not identified for 74 of 75 interviewed donors with specimens of lower reactivity. The minimum calculated specificity was 99.82%. The use of the HTLV-III EIA has virtually eliminated the use of blood and plasma for HTLV-III/LAV infected donors.

  2. Detection of Snake Venom in Post-Antivenom Samples by Dissociation Treatment Followed by Enzyme Immunoassay.

    PubMed

    Maduwage, Kalana P; O'Leary, Margaret A; Silva, Anjana; Isbister, Geoffrey K

    2016-01-01

    Venom detection is crucial for confirmation of envenomation and snake type in snake-bite patients. Enzyme immunoassay (EIA) is used to detect venom, but antivenom in samples prevents venom detection. We aimed to detect snake venom in post-antivenom samples after dissociating venom-antivenom complexes with glycine-HCl (pH 2.2) and heating for 30 min at 950 °C. Serum samples underwent dissociation treatment and then Russell's viper venom or Australian elapid venom measured by EIA. In confirmed Russell's viper bites with venom detected pre-antivenom (positive controls), no venom was detected in untreated post-antivenom samples, but was after dissociation treatment. In 104 non-envenomed patients (negative controls), no venom was detected after dissociation treatment. In suspected Russell's viper bites, ten patients with no pre-antivenom samples had venom detected in post-antivenom samples after dissociation treatment. In 20 patients with no venom detected pre-antivenom, 13 had venom detected post-antivenom after dissociation treatment. In another 85 suspected Russell's viper bites with no venom detected pre-antivenom, 50 had venom detected after dissociation treatment. Dissociation treatment was also successful for Australian snake envenomation including taipan, mulga, tiger snake and brown snake. Snake venom can be detected by EIA in post-antivenom samples after dissociation treatment allowing confirmation of diagnosis of envenomation post-antivenom. PMID:27136587

  3. Enzyme immunoassay for identification of Vibrio vulnificus in seawater, sediment, and oysters.

    PubMed Central

    Tamplin, M L; Martin, A L; Ruple, A D; Cook, D W; Kaspar, C W

    1991-01-01

    Historically, methods used to identify Vibrio vulnificus in environmental samples have been inadequate because isolation and identification procedures are time-consuming and fail to separate V. vulnificus from other bacterial species. We describe an enzyme immunoassay (EIA) and culture techniques which identified V. vulnificus in seawater, sediment, and oysters. The EIA used monoclonal antibody FRBT37 to a species-specific epitope of V. vulnificus. No cross-reactions were observed among 72 non-V. vulnificus strains comprising 34 species and 15 genera. In field trials, the EIA identified correctly 99.7% of 348 biochemically confirmed V. vulnificus isolates. The epitope corresponding to FRBT37 was found in cells lysed by Triton X-100, deionized H2O, and ultrasonication but was not found in culture supernatants, indicating that its location was intracellular. In addition, electron micrographs of V. vulnificus labeled with FRBT37-biotin-avidin-gold showed that epitope FRBT37 reacted with fragments of lysed cells but not whole cells. FRBT37 was expressed when V. vulnificus was cultured in different growth media. The minimum level of detection of the EIA was approximately 2,000 V. vulnificus cells per EIA well. Epitope FRBT37 was labile at 70 degrees C for 30 min. Immunoblot and EIA plate formats reduced assay time and facilitated handling large numbers of test samples. Images PMID:2059045

  4. Competitive enzyme-linked immunoassay for sialoglycoprotein of edible bird's nest in food and cosmetics.

    PubMed

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

    2012-04-11

    The proliferation of fake and inferior edible bird's nest (EBN) products has recently become an increasingly serious concern. To identify and classify EBN products, a competitive enzyme-linked immunoassay (ELISA) was developed to quantitate sialoglycoprotein in EBN used in food and cosmetic applications. The characteristic sialoglycoprotein in EBN was found, extracted, purified, and analyzed. Sialoglycoprotein, considered the main carrier of sialic acid in EBN, consisted of 106 and 128 kDa proteins. A monoclonal antibody that could recognize both proteins was prepared. The heat-treated process did not change the affinity of sialoglycoprotein with the antibody. An optimized ELISA method was established with a cross-reactivity of less than 0.1% and an IC(50) of 3.3 μg/mL. On the basis of different food and cosmetic samples, the limits of detection (LOD) were 10-18 μg/g. Recoveries of fortified samples at levels of 20 and 80 μg/g ranged from 81.5 to 96.5%, respectively. The coefficients of variation were less than 8.0%. PMID:22439641

  5. Establishment of magnetic beads-based enzyme immunoassay for detection of chloramphenicol in milk.

    PubMed

    Xu, Jing; Yin, Weiwei; Zhang, Yuanyang; Yi, Jian; Meng, Meng; Wang, Yabin; Xue, Huyin; Zhang, Taichang; Xi, Rimo

    2012-10-15

    In this research, magnetic beads-based enzyme immunoassays were investigated for rapid analysis of chloramphenicol (CAP) in milk. To improve sensitivity of CAP determination, two kinds of immunomagnetic separation methods were designed and compared. Magnetic polystyrene microspheres were conjugated with anti-CAP antibody (Method I) or goat-anti-mouse IgG (Method II). The whole determination could be finished in 1.25 h. Both methods showed high sensitivity to CAP in buffer, and obtained an IC(50) value of 0.05 ng mL(-1) for Method I and 0.4 ng mL(-1) for Method II. The methods showed high specificity, only showing a little cross-reaction towards CAP succinate. The two methods were applied to detect CAP in milk. The recovery rates were 80-106% and the coefficients of variation (CVs) were 4.7-15%. The immunomagnetic assay showed promising potential in rapid screening field for CAP analysis. Between the two methods, Method I is more sensitive, and Method II is more suitable for producing a general assay by changing a primary antibody for another analyte. PMID:23442720

  6. A magnetic particles-based chemiluminescence enzyme immunoassay for rapid detection of ovalbumin.

    PubMed

    Feng, Xiao-Li; Ren, Hong-Lin; Li, Yan-Song; Hu, Pan; Zhou, Yu; Liu, Zeng-Shan; Yan, Dong-Ming; Hui, Qi; Liu, Dong; Lin, Chao; Liu, Nan-Nan; Liu, Yan-Yan; Lu, Shi-Ying

    2014-08-15

    Egg allergy is an important public health and safety concern, so quantification and administration of food or vaccines containing ovalbumin (OVA) are urgently needed. This study aimed to establish a rapid and sensitive magnetic particles-chemiluminescence enzyme immunoassay (MPs-CLEIA) for the determination of OVA. The proposed method was developed on the basis of a double antibodies sandwich immunoreaction and luminol-H2O2 chemiluminescence system. The MPs served as both the solid phase and separator, the anti-OVA MPs-coated polyclonal antibodies (pAbs) were used as capturing antibody, and the horseradish peroxidase (HRP)-labeled monoclonal antibody (mAb) was taken as detecting antibody. The parameters of the method were evaluated and optimized. The established MPs-CLEIA method had a linear range from 0.31 to 100ng/ml with a detection limit of 0.24ng/ml. The assays showed low reactivities and less than 5% of intraassay and interassay coefficients of variation (CVs), and the average recoveries were between 92 and 97%. Furthermore, the developed method was applied in real samples analysis successfully, and the correlation coefficient with the commercially available OVA kit was 0.9976. Moreover, it was more rapid and sensitive compared with the other methods for testing OVA. PMID:24769049

  7. Clinically practical seminested PCR for Burkholderia pseudomallei quantitated by enzyme immunoassay with and without solution hybridization.

    PubMed Central

    Kunakorn, M; Markham, R B

    1995-01-01

    Diagnosis of melioidosis, an infectious disease caused by Burkholderia pseudomallei (formerly Pseudomonas pseudomallei), is made initially by antibody testing, which is not always sensitive or specific. We have developed two seminested PCR protocols combined with enzyme immunoassay (EIA) to detect the conserved ribosomal regulatory region of B. pseudomallei. Both PCRs used one biotinylated primer for capturing PCR products on EIA plates. One system, termed solution hybridization EIA (SHEIA), hybridized PCR products with a digoxigenin-labeled probe in solution. Another system, termed primer-labeled EIA (PLEIA), used a digoxigenin-labeled nested primer to generate products that were directly detected without hybridization. To prevent amplicon contamination, pre-PCR uracil DNA glycosylase treatment or post-PCR UV irradiation was incorporated into each system. By a rapid method of blood sample preparation for PCR, these systems had sensitivities of 75 bacteria per ml for SHEIA and 300 bacteria per ml for PLEIA. No nonspecific amplification of other bacterial DNAs was detected. This seminested PCR coupled with SHEIA or PLEIA fulfills all the requirements for a diagnostic test to be used in developing countries where B. pseudomallei is endemic. PMID:7559961

  8. Fieldable, real-time enzyme immunoassay kits for drugs on surfaces

    NASA Astrophysics Data System (ADS)

    Chiappini, Michele W.; Wendel, Gregory J.; Duquette, Peter H.; Hamilton, Martha J.; Chudzik, Stephen J.; Chappa, Ralph A.

    1994-03-01

    Immunoassays (e.g., RIA, EIA) have been demonstrated to be useful for rapid, convenient detection and semiquantitative analysis of drugs. Thermedics Detection, Inc. manufactures a rapid, sensitive, self-contained, disposable, EIA device, developed by Bio-Metric Systems, Inc., designed to allow untrained personnel to perform in field situations. This format has been developed for drugs in biological fluids and on surfaces. The analyte in the test sample competes with an enzyme-analyte conjugate for a limited number of immobilized antibody sites. The AccuPRESS Test format can detect analytes at 10 ppb in biological fluids, water, and soil, and on surfaces, such as suitcases, vehicles, tables and hands, with positive results indicated by clearly visible color development within 5 minutes. This format is designed to have all dry components and to have an ambient shelf life of greater than one year. The format is available for cocaine and opiate derivatives, including heroin, and is readily adaptable for use with numerous other drugs, explosives, and environmental pollutants.

  9. Determination of mite allergens in house dust using the enzyme immunoassay.

    PubMed

    Prester, Ljerka; Brcić Karaconji, Irena; Macan, Jelena

    2007-12-01

    The aim of this study was to determine the level of two major mite allergens Dermatophagoides pteronyssinus (Der p 1) and Dermatophagoides farinae (Der f 1) in 30 urban homes in Zagreb, Croatia, using the enzyme immunoassay with two monoclonal antibodies which has been established as the reference method for indoor allergen analysis. Dust samples were taken by vacuuming a carpeted area and collected on cellulose filters. The ranges of Der p 1 and Der f 1 were (0.1-12.5) microg g-1 (median 0.32 microg g-1) and (0.1-31.2) microg g-1 (median 0.35 microg g-1), respectively. Der p 1 and Der f 1 (>2 microg g-1) associated with increased risk of sensitization to mite allergens were found in approximately 16% homes for each allergen. The sum of allergen (Der p 1 + Der f 1) exceeded the lower threshold in 27% of homes. Analytical evaluation of the ELISA assay showed satisfactory results for precision (intra-assay CV <6.9%, inter-assay CV<13.3%), accuracy (91% to 93%), and sensitivity (2 ng mL-1). The ELISA assay for the measurement of dust mite allergens demonstrated very good analytical characteristics for routine laboratory use, and will provide the essential basis for our future studies of various indoor allergens. PMID:18063526

  10. Detection of galactomannan antigenemia by enzyme immunoassay in experimental invasive aspergillosis.

    PubMed Central

    de Repentigny, L; Boushira, M; Ste-Marie, L; Bosisio, G

    1987-01-01

    A sensitive enzyme immunoassay (EIA) for galactomannan antigenemia that avoids the use of radioisotopes was devised. Three carbohydrate-rich antigenic fractions were purified from Aspergillus fumigatus 2085: a cold alkali extract (CA) from mycelium, an acetone-precipitated pyridine extract (APSK-66) from mycelium, and a methanol precipitate from culture filtrate. CA and APSK-66 were further purified by gel filtration and ion-exchange chromatography, respectively. An acid hydrolysate of CA contained only mannose and galactose, as determined by gas-liquid chromatography. Rabbit antisera were raised against conidia, mycelia, and cell walls of A. fumigatus. By indirect EIA, the best immunoglobulin G response (1/8,000) was obtained against CA in rabbits immunized intravenously with cell walls. Antigenemia was detected by indirect EIA inhibition in heat-dissociated sera of four immunosuppressed rabbits that were infected intravenously but was absent in two uninfected controls. The circulating antigen was resistant to pronase, was adsorbed onto concanavalin A, and had a molecular size of 50 to 100 kilodaltons. PMID:3294887

  11. Diagnosis of American cutaneous leishmaniasis by enzyme immunoassay using membrane antigens of Leishmania (Viannia) braziliensis.

    PubMed

    Skraba, Cissiara Manetti; Pedroso, Raíssa Bocchi; Fiorini, Adriana; Rosado, Fábio Rogério; Aristides, Sandra Mara Alessi; Lonardoni, Maria Valdrinez Campana; Teixeira, Jorge Juarez Vieira; Silveira, Thaís Gomes Verzignassi

    2014-04-01

    This study evaluated the reactivity of membrane antigens of Leishmania (Viannia) braziliensis for the diagnosis of ACL by enzyme immunoassay (EIA). Promastigotes of L. (V.) braziliensis were grown in medium 199 and lysed in a sonicator. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting showed that specific proteins of L. (V.) braziliensis (apparent molecular weights 36 kDa and 48-56 kDa) were recognized by sera from ACL patients. These proteins were eluted from the SDS-PAGE and tested in EIA-IgG with sera from ACL patients, healthy individuals, patients with toxoplasmosis, paracoccidioidomycosis, syphilis, tuberculosis, leprosy, and Chagas disease. The EIA-IgG with membrane antigens allowed us to distinguish patients with ACL from healthy individuals and patients with other diseases (P < 0.0001), and showed a sensitivity of 93.3% and specificity of 90.8%, not including Chagas disease patients. 2D-SDS-PAGE followed by Western blotting was performed to improve the characterization of the antigens, and showed a component with isoelectric points near the acid pH side and apparent molecular weights of 48-56 kDa. The results showed good sensitivity and specificity of EIA-IgG with membrane antigens, indicating their potential use for diagnosis of ACL, as well as seroepidemiological surveys and follow-up of clinically cured patients. PMID:24485589

  12. The Rapid Screening of Triazophos Residues in Agricultural Products by Chemiluminescent Enzyme Immunoassay.

    PubMed

    Chen, Ge; Yang, Lihua; Jin, Maojun; Du, Pengfei; Zhang, Chan; Wang, Jian; Shao, Hua; Jin, Fen; Zheng, Lufei; Wang, Shanshan; She, Yongxin; Wang, Jing

    2015-01-01

    A highly sensitive chemiluminescent enzyme immunoassay (CLEIA) method was developed in this study for efficient screening of triazophos residues in a large number of samples. Based on the maximum residue limits (MRLs) set by China and CAC for triazophos in different agro-products, the representative apple, orange, cabbage, zucchini, and rice samples were selected as spiked samples, and the triazophos at the concentrations of the MRL values were spiked to blank samples. Subsequently, the five samples with the spiked triazophos standard were measured by CLEIA 100 times, and the detection results indicated that the correction factors of the apple, orange, cabbage, zucchini, and rice were determined as 0.79, 0.66, 0.85, 0.76, and 0.91, respectively. In this experiment, 1500 real samples were detected by both the CLEIA and the GC-MS methods. With the GC-MS method, 1462 samples were identified as negative samples and 38 samples as positive samples. Based on the correction factors, the false positive rate of the CLEIA method was 0.13%, and false negative rate was 0. The results showed that the established CLEIA method could be used to screen a large number of real samples. PMID:26218576

  13. Sensitive enzyme-amplified electrical immunoassay for protein A-bearing Staphylococcus aureus in foods.

    PubMed Central

    Brooks, J L; Mirhabibollahi, B; Kroll, R G

    1990-01-01

    An amperometric electrochemical immunoassay specific for protein A-bearing Staphylococcus aureus was developed. The method was based on a sandwich immunosorbent assay and incorporated an enzyme amplification step, using a NAD-specific redox cycle generating NADH (C. H. Stanley, A. Johannsson, and C. H. Self, J. Immunol. Methods 83:89-95, 1985). Reduction of the mediator, ferricyanide, was dependent on the initial concentration of antigen. The final potential was measured by using a Pt disk electrode polarized at +0.8 V to the Ag/AgCl reference electrode. The assay was rapid (4 h) and generated protein A- and cell (S. aureus)-dependent signals. The system was highly sensitive and could detect 10 pg of protein A ml-1 and less than 100 CFU of S. aureus ml-1. Similar sensitivities were observed with S. aureus cultures inoculated into beef and milk, but the sensitivity was reduced slightly (ca. 10(3) g-1) with samples of Cheddar cheese. PMID:2268148

  14. Development and validation of a simple, sensitive enzyme immunoassay for quantification of androstenedione in bull plasma.

    PubMed

    Mallick, Smrutirekha; Kumar, Bs Bharath; Prakash, B S; Aggrawal, Anjali; Pandita, Sujata

    2015-01-01

    As an alternative to radioimmunoassay a simple and highly sensitive enzyme immunoassay (EIA) was developed and validated for androstenedione quantification in plasma of Karan Fries bulls using second antibody coating technique. The wells of the microtitreplate were coated with affinity-purified goat immunoglobulin (antirabbit IgG) that binds the hormone specific antibody. The EIA was performed to analyze androstenedione directly in 40 μl of bull plasma. The androstenedione standards ranged from 0.20 to 200 pg/40 μl /well and the sensitivity of the assay was 5 pg/ml plasma. Serially diluted bull plasma containing high endogenous androstenedione showed good parallelism with bovine androstenedione standard curve. Intra- and inter-assay coefficients of variation (CV) were found to be 8 and 9%, respectively. Peripheral plasma androstenedione concentrations determined in young and adult bull samples ranged between 104-990 pg/ml and 184-2040 pg/ml, respectively. PMID:26290733

  15. Fecal steroid evaluation to monitor reproductive status in wild ungulate females using enzyme immunoassay commercial kits.

    PubMed

    Borque, Conception; Pérez-Garnelo, Sonia S; Delclaux, Maria; Martínez, Eva; De la Fuente, Julio

    2011-12-01

    Analysis of reproductive hormones in fecal samples is necessary for the noninvasive monitoring of reproductive status in free-ranging species. The aim of the present study was to establish an easy noninvasive method to monitor reproductive status in wild ungulate females. Feces were collected daily, weekly, or three or four times a week directly from the soil for a period ranging from 1 to 9.8 mo. Fecal estradiol and progestagens were monitored in nine wild ungulate females (Barbary sheep, Ammotragus lervia [n = 3]; European bison, Bison bonasus [n = 1]; auroch, Bos taurus primigenius [n = 2]; sitatunga, Tragelaphus spekii gratus [n = 2]; and Indian rhinoceros, Rhinoceros unicornis [n = 1]) by using commercially available enzyme immunoassay kits prepared for human serum or plasma. In the species evaluated in this study, luteal phase, abortion, and gestation patterns corresponded closely with changes in fecal progestagens. Luteal phase and gestation values differed significantly (P < 0.001) from basal values, whereas progestagens values after abortion were not significantly different (P > 0.05) from basal values. For estradiol excretory patterns, follicular phase and pregnancy values differed significantly (P < 0.001) from basal values, but differences between values after abortion and basal values were not significant (P > 0.05); length of estrous cycles were clearly defined through estradiol data. This study demonstrates that ovarian function in the wild ungulate females studied can be monitored by enzyme-linked immunosorbent assay (ELISA). Therefore, ELISA methodologies used here could be a practical alternative to other ELISAs that require more complex procedures or whose commercial availability is difficult. PMID:22204046

  16. An ultrasensitive and universal photoelectrochemical immunoassay based on enzyme mimetics enhanced signal amplification.

    PubMed

    Wang, Guang-Li; Shu, Jun-Xian; Dong, Yu-Ming; Wu, Xiu-Ming; Li, Zai-Jun

    2015-04-15

    An ultrasensitive photoelectrochemical (PEC) immunoassay based on signal amplification by enzyme mimetics was fabricated for the detection of mouse IgG (as a model protein). The PEC immunosensor was constructed by a layer-by-layer assembly of poly (diallyldimethylammonium chloride) (PDDA), CdS quantum dots (QDs), primary antibody (Ab1, polyclonal goat antimouse IgG), and the antigen (Ag, mouse IgG) on an indium-tin oxide (ITO) electrode. Then, the secondary antibody (Ab2, polyclonal goat antimouse IgG) combined to a bio-bar-coded Pt nanoparticle(NP)-G-quadruplex/hemin probe was used for signal amplification. The bio-bar-coded Pt NP-G-quadruplex/hemin probe could catalyze the oxidation of hydroquinone (HQ) using H2O2 as an oxidant, demonstrating its intrinsic enzyme-like activity. High sensitivity for the target Ag was achieved by using the bio-bar-coded probe as signal amplifier due to its high catalytic activity, a competitive nonproductive absorption of hemin and the steric hindrance caused by the polymeric oxidation products of HQ. For most important, the oxidation product of HQ acted as an efficient electron acceptor of the illuminated CdS QDs. The target Ag could be detected from 0.01pg/mL to 1.0ng/mL with a low detection limit of 6.0fg/mL. The as-obtained immunosensor exhibited high sensitivity, good stability and acceptable reproducibility. This method might be attractive for clinical and biomedical applications. PMID:25437365

  17. Anti-TNP monoclonal antibodies as reagents for enzyme immunoassay (ELISA).

    PubMed

    Léo, P; Ucelli, P; Augusto, E F; Oliveira, M S; Tamashiro, W M

    2000-12-01

    The aim of this study was to produce anti-TNP monoclonal antibodies (MAbs) that could be conjugated and used for the detection of antigen-antibody reactions, in which the antigen specific-antibody had been previously bound to trinitrophenyl (TNP). For hybridoma production, SP2/0-Ag14 cells were fused with spleen cells from mice previously immunized with TNP-ovalbumin (TNP-OVA). After 10 days, enzyme-linked immunoadsorbent assay (ELISA) was used to detect anti-TNP antibodies in the supernatants, and five cultures were found to be strictly positive for TNP. Three of these were subsequently cloned by limiting dilution, and 15 clones were chosen for expansion based on the criterion of high reactivity against TNP. Anti-TNP MAbs produced by those clones were isotyped as IgG1, and purified by Sepharose-protein G affinity cromatography from ascites developed in BALB/c mice. Two purified MAbs (1B2.1B6 and 1B2.1E12) were coupled to horseradish peroxidase (HRPO). The resulting conjugates were evaluated in ELISA tests for interferon-gamma and interleukin-4 detection, in which the secondary anti-cytokine antibodies were coupled either to TNP or biotin. The performance of anti-TNP conjugates in these assays were compared with a biotin-streptavidin/peroxidase system. Both types of conjugates were similarly able to detect cytokines with r2 (linear correlation coefficient) close to unity value. Growth studies of one of those hybridomas (1B2.1B6) yielded a specific growth rate of 0.042 h(-1) and a doubling time of 16.5 h. Data discussed here show that at least two MAbs against TNP raised in this work can be used as a reagent for enzyme immunoassays. PMID:11152399

  18. Validation of an automated microplate enzyme immunoassay for screening of postmortem blood for drugs of abuse.

    PubMed

    Spiehler, V R; Collison, I B; Sedgwick, P R; Perez, S L; Le, S D; Farnin, D A

    1998-01-01

    The objective of this study was to compare the sensitivity and specificity of an enzyme immunoassay employing antibodies bound to a microtiter plate (MPEIA) with those of two radioimmunoassays for screening postmortem blood from selected coroner's cases for drugs of abuse. The radioimmunoassays were a coated-tube radioimmunoassay (CTRIA) and a double antibody radioimmunoassay (DARIA). Specimens consisted of 260 postmortem blood specimens from coroner's cases. Immunoassay results (positive or negative) were compared with confirmed results on those cases by gas chromatography-mass spectrometry, alone or in combination with gas-liquid chromatography using either a nitrogen-phosphorus or flame-ionization detector. Sensitivity was calculated as the true-positive rate using chromatographic confirmation as the reference standard. Specificity was calculated as the true-negative rate. Sensitivity and specificity were calculated for 5-7 potential cutoff concentrations for the drug classes opiates, amphetamines, cocaine and metabolites, and barbiturates. For opiates, the sensitivity and specificity were 99% and 93%, respectively, for the MPEIA at a cutoff of 20-ng/mL morphine, compared with 94% and 96% for the CTRIA at a cutoff of 5-ng/mL morphine and > 99% and 96% for the DARIA at 20-ng/mL morphine. For cocaine and metabolites, the sensitivity and specificity were 96% and 93%, respectively, for the MPEIA at 50-ng/mL benzoylecgonine, compared with 93% and 96% for CTRIA at 50-ng/mL benzoylecgonine and 98% and 97% for the DARIA at 50-ng/mL benzoylecgonine. For amphetamines, the sensitivity and specificity were >99% and 91%, respectively, for the MPEIA at 25-ng/mL methamphetamine, compared with 93% and 86% for the CTRIA at 25-ng/mL methamphetamine and 83% and 89% for the DARIA at 50-ng/mL methamphetamine. For barbiturates, the sensitivity and specificity were > 99% and 92%, respectively, for the MPEIA at 50-ng/mL secobarbital, compared with 91% and 87% for the CTRIA at 500-ng/mL secobarbital and 79% and 95% for the DARIA at a cutoff of 1000-ng/mL phenobarbital. PMID:9847007

  19. Development of enzyme linked immunoassay for the simultaneous detection of carbaryl and metolcarb in different agricultural products.

    PubMed

    Sun, Jingwei; Dong, Tingting; Zhang, Yan; Wang, Shuo

    2010-05-01

    A direct competitive enzyme linked immunosorbent assay in multi-enzyme tracers format for the simultaneous analysis of carbaryl and metolcarb in agricultural products is described in this study. The concentrations of coating antibodies and enzyme tracer were studied. Under the optimum conditions, the limits of detection of carbaryl and metolcarb were 0.15 microg L(-1) and 1.2 microg L(-1), respectively. Determination of carbaryl and metolcarb in fruit juices and vegetables was accomplished by simple, rapid and efficient extraction methods. Recoveries of spiked samples were great than 70%. Validation of the immunosorbent assay was conducted by comparison of results from high performance liquid chromatography (HPLC). The correlations between the data obtained using multi-enzyme tracers enzyme linked immunosorbent assay and high performance liquid chromatography were good. Results indicated that the new strategy for developing immunoassay for simultaneous quantitative determination of carbaryl and metolcarb residues was suitable in this study. PMID:20433968

  20. Power-free chip enzyme immunoassay for detection of prostate specific antigen (PSA) in serum.

    PubMed

    Adel Ahmed, Heba; Azzazy, Hassan M E

    2013-11-15

    A power-free, portable "Chip EIA" was designed to render the popular Enzyme Linked Immunosorbent Assay (ELISA) more suitable for point-of-care testing. A number of microfluidic platforms have enabled miniaturization of the conventional microtitre plate ELISA, however, they require external pumping systems, valves, and electric power supply. The Chip EIA platform has eliminated the need for pumps and valves through utilizing a simple permanent magnet and magnetic nanoparticles. The magnetic nanoparticles act as solid support to capture the target and are then moved through chambers harboring different reagents necessary to perform a sandwich ELISA. The use of magnetic nanoparticles increases the volume-to-surface ratio reducing the assay time to 30 min. Changing the color of horseradish peroxidase (HRP) substrate to green indicates a positive result. In addition, a quantitative read-out was obtained through the use of cellphone camera imaging and analyzing the images using Matlab®. Cell phones, including smart ones, are readily available almost everywhere. The Chip EIA device was used to assay total prostate specific antigen (tPSA) in 19 serum samples. The PSA Chip EIA was tested for accuracy, precision, repeatability, and the results were correlated to the commercial Beckman Colter, Hybritech immunoassay® for determination of tPSA in serum samples with a Pearson correlation coefficient (R(2)=0.96). The lower detection limit of the PSA Chip EIA was 3.2 ng/mL. The assay has 88.9% recovery and good reproducibility (% CV of 6.5). We conclude that the developed Chip EIA can be used for detection of protein biomarkers in biological specimens. PMID:23811482

  1. Enzyme immunoassay using a novel recombinant polypeptide to detect human immunodeficiency virus env antibody.

    PubMed Central

    Thorn, R M; Beltz, G A; Hung, C H; Fallis, B F; Winkle, S; Cheng, K L; Marciani, D J

    1987-01-01

    A unique antigen, CBre3, has been synthesized from a genetically engineered clone to detect human immunodeficiency virus (HIV) env antibodies with high sensitivity and specificity. The antigen contains sequences derived from both envelope proteins of HIV, i.e., gp120 and gp41, and was purified free of Escherichia coli proteins detectable by Coomassie stain or immunoblotting with E. coli antiserum. The purified recombinant polypeptides were used as antigen in an enzyme immunoassay (EIA) to screen serum samples from healthy and HIV-infected individuals. The same samples were also tested by radioimmunoprecipitation (RIP) for gp120 and gp160 HIV antibodies. All samples containing gp120 and gp160 antibodies by RIP had CBre3 EIA values greater than 0.35 (n, 122; range, 0.37 to 2.1+; median, 1.65). All RIP HIV antibody-negative samples had CBre3 EIA values less than 0.25 (n, 140; mean, 0.052; standard deviation, 0.045; range, 0.00 to 0.22). The endpoint titer of a standard positive control serum was 1:10,000 by RIP and by CBre3 EIA. The assay was 100% accurate in three proficiency panels. It easily detected six samples from individuals whose infections were confirmed by culture; these samples were reactive only with p24 by Western blot. The samples also were positive for gp120 and gp160 antibodies by RIP. These data suggest that the CBre3 EIA can detect env antibodies as sensitively and specifically as RIP and with more sensitivity than Western blot. Images PMID:3475281

  2. Enzyme-immunoassay for the determination of metallothionein in human urine: application to environmental monitoring.

    PubMed

    Swierzcek, Sabina; Abuknesha, Ramadan A; Chivers, Ian; Baranovska, Irena; Cunningham, Phillip; Price, Robert G

    2004-01-01

    The objectives of this study were to develop an enzyme immunoassay for metallothioneins in human urine using a polyclonal antiserum and to demonstrate a possible relationship between the level of this biomarker and heavy metal exposure. The antiserum was raised in sheep against horse metallothionein conjugated to carboxylated bovine serum albumin. The antibody was used to construct a two-step competitive ELISA procedure. Human urine was treated with activated charcoal powder to remove traces of metallothioneins and known amounts of pure metallothioneins were added to provide standards for a standard curve. Metallothionein levels were measured in two groups of children living in areas of mild and high environmental pollution due mainly to heavy metals. A comparison was made between the biomarker levels and the levels of cadmium and lead in urine samples in the two groups. A group of children from a non-polluted area acted as controls. The results show that the detected levels of metallothioneins appear to correspond to levels of the two heavy metals studied and that there was an apparent relationship to the environmental exposure. Thus according to results of this study the increase in the metallothionein excretion seems to provide an indication of previous of exposure to metals. The ELISA procedure is sensitive and robust and can be used to screen large numbers of samples and is more rapid than the physical procedures currently used for analysis of these proteins. The assay can therefore be used as an additional tool for screening at-risk populations where either environmental or occupational exposure to divalent heavy metals is suspected. PMID:15764296

  3. Evaluation of an enzyme immunoassay kit for detecting cryptosporidium in faeces and environmental samples.

    PubMed Central

    Siddons, C. A.; Chapman, P. A.; Rush, B. A.

    1992-01-01

    AIMS: To evaluate a commercially available enzyme immunoassay based on a monoclonal antibody to a genus specific Cryptosporidium (IDEIA Cryptosporidium; Dako) antigen for detecting Cryptosporidium oocysts in faecal and environmental samples. METHODS: 435 human faecal samples and post-filtration deposits from 10 reservoir samples, and from six tap water samples seeded with Cryptosporidium oocysts, were examined by EIA according to the manufacturer's instructions, and by microscopic examination of phenolauramine stained smears. Samples giving discrepant results were examined by specific immunofluorescence, before and after concentration of oocysts. RESULTS: Sixteen (3.6%) faecal samples were positive by both microscopy and EIA; five (1.1%) were positive by microscopy of auramine-phenol stained smears (but were not confirmed by specific immunofluorescence) and negative by EIA; one (0.2%) was positive by EIA alone, but confirmed by specific immunofluorescence; and 362 (83.2%) were negative by both microscopy and EIA. Compared with immunofluorescence positive faecal samples, the sensitivity of conventional microscopy and EIA were 94% and 100%, and specificity 76.4% and 100%, respectively. Fifty one (11.7%) were not examined by microscopy due to detection of other pathogens in a previous sample from that patient, but were found to be negative by EIA. Ten reservoir water samples (not suspected of being linked to cases of cryptosporidiosis) were negative by both microscopy and EIA. Of six samples of tap water seeded with varying concentrations of Cryptosporidium oocysts, two (10(2) and 10(3) oocysts/l) were positive by both microscopy and EIA, two (10 and 1/l) by EIA alone, and two (0.1/l and unseeded water) were negative by both microscopy and EIA. CONCLUSIONS: The kit is simple and rapid to use and offers a less subjective method than microscopy for detecting Cryptosporidium in faecal samples submitted to a busy diagnostic laboratory. PMID:1624594

  4. Magnetic-particle-based, ultrasensitive chemiluminescence enzyme immunoassay for free prostate-specific antigen.

    PubMed

    Liu, Ruping; Wang, Cheng; Jiang, Quan; Zhang, Wei; Yue, Zhao; Liu, Guohua

    2013-11-01

    We report a magnetic-particle (MMP)-based chemiluminescence enzyme immunoassay (CLEIA) for free prostate-specific antigen (f-PSA) in human serum. In this method, the f-PSA is sandwiched between the anti-PSA antibody coated MMPs and alkaline phosphatase (ALP)-labeled anti-f-PSA antibody. The signal produced by the emitted photons from the chemiluminescent substrate (4-methoxy-4-(3-phosphatephenyl)-spiro-(1,2-dioxetane-3,2'-adamantane)) is directly proportional to the amount of f-PSA in a sample. The present MMP-based assay can detect f-PSA in the range of 0.1-30 ng mL(-1) with the detection limit of 0.1 ng mL(-1). The linear detection range could match the concentration range within the "diagnostic gray zone" of serum f-PSA levels (4-10 ng mL(-1)). The detection limit was sufficient for measuring clinically relevant f-PSA levels (>4 ng mL(-1)). Furthermore, the method was highly selective; it was unaffected by cross-reaction with human glandular kallikrein-2, a kallikrein-like serine protease that is 80% similar to f-PSA. The proposed method was finally applied to determine f-PSA in 40 samples of human sera. Results obtained using the method showed high correlation with those obtained using a commercially available microplate CLEIA kit (correlation coefficient, 0.9821). This strategy shows great potential application in the fabrication of diagnostic kits for determining f-PSA in serum. PMID:24139579

  5. Preparation of a Monoclonal Antibody against Gintonin and Its Use in an Enzyme Immunoassay.

    PubMed

    Lee, Byung-Hwan; Choi, Sun-Hye; Kim, Hyeon-Joong; Jung, Seok-Won; Kim, Hyunsook; Shin, Ho-Chul; Lee, Joon-Hee; Hwang, Sung-Hee; Kim, Hyoung-Chun; Nah, Seung-Yeol

    2015-01-01

    Gintonin is a novel ginseng-derived G protein-coupled lysophosphatidic acid (LPA) receptor ligand. Gintonin elicits an [Ca(2+)]i transient in animal cells via activation of LPA receptors. In vitro studies have shown that gintonin regulates various calcium-dependent ion channels and receptors. In in vivo studies, gintonin elicits anti-Alzheimer's disease activity through the activation of the non-amyloidogenic pathway and anti-metastatic effects through the inhibition of autotaxin. However, a method for gintonin quantitation in ginseng has not been developed. In the present study, we developed an enzyme immunoassay (EIA) to measure gintonin. A monoclonal antibody was raised in a mouse using gintonin as the immunogen, and an indirect competitive EIA was used to measure gintonin. The working range was 0.01-10 µg per assay. The anti-gintonin monoclonal antibody did not cross-react with the ginsenosides Ra, Rb1, Rb2, Rc, Rd, Re, Rf, Rg1, and Rg3 or with LPAs such as LPA C16:0, LPA C18:0, LPA C18:1, and LPA C18:2. Using a standard curve, we measured the amount of gintonin in various ginseng extract fractions. Interestingly, we only detected a little amount of gintonin in conventional hot water extracts of Korean red ginseng. However, we can measure gintonin after ethanol extraction of Korean red ginseng marc. Thus, gintonin can be extracted from ginseng with ethanol but not water, and the remaining Korean red ginseng marc can be used to obtain gintonin. These results indicate that the EIA with the anti-gintonin monoclonal antibody can be used to quantify gintonin in various ginseng preparations, including commercial ginseng products. PMID:26424022

  6. Caffeine clearance by enzyme multiplied immunoassay technique: a simple, inexpensive, and useful indicator of liver function.

    PubMed

    McDonagh, J E; Nathan, V V; Bonavia, I C; Moyle, G R; Tanner, A R

    1991-06-01

    The clinical value and sensitivity of serum caffeine clearance measurement has been evaluated as an indicator of hepatic disease. After a 17 hour caffeine exclusion period, 300 mg of caffeine citrate was administered orally to the study subjects. Serum samples were taken four and 16 hours later. Serum caffeine concentrations were measured using an enzyme multiplied immunoassay technique (EMIT) and a clearance value derived. Conventional liver function tests were measured at the same time. A total of 103 subjects attending the medical unit in a district general hospital were studied. Twenty one had alcoholic liver disease, 11 non-alcoholic cirrhosis, nine non-cirrhotic liver disease, 21 suspected liver disease, six hepatic tumours, and 35 were hospital and normal control subjects. Caffeine clearance values were lowest in subjects with alcoholic liver disease (median 0.19 ml/min/kg, range 0.04-0.61 ml/min/kg) and significantly reduced in all subjects with liver disease (median 0.32 ml/min/kg, range 0.04-2.68 ml/min/kg) compared with control subjects (median 1.27 ml/min/kg, p less than 0.001). In subjects with suspected liver disease subsequently shown to have another explanation for abnormal liver function test results, caffeine clearance values were normal (median 1.31 ml/min/kg, range 0.23-2.64 ml/min/kg) and significantly different, p less than 0.001, from those of subjects with liver disease. Serum albumen values were not different for these latter two groups. Using a cut off value of 0.86 ml/min/kg, caffeine clearance measurement was 100% sensitive for alcoholic liver disease and 89% sensitive for all liver disease. The respective sensitivities for conventional liver function test measurement were 76% and 83%. In the suspected liver disease group, caffeine clearance was abnormal in only 24%, conventional liver function tests were abnormal in 95%. The respective specificities for caffeine clearance and liver function test measurement in control subjects were 93% and 100%. Caffeine clearance determined by EMIT is a simple inexpensive hepatic metabolic function test. This study indicates that it is a more sensitive indicator of structural liver disease than conventional liver function tests, especially for alcoholic liver disease. The test could be widely introduced as a useful, repeatable assessment of hepatic function. PMID:2060878

  7. Evaluation of a novel chemiluminescent microplate enzyme immunoassay for hepatitis B surface antigen detection.

    PubMed

    Yang, Lin; Song, Liu-Wei; Fang, Lin-Lin; Wu, Yong; Ge, Sheng-Xiang; Li, Hui; Yuan, Quan; Zhang, Jun; Xia, Ning-Shao

    2016-02-01

    Hepatitis B virus surface antigen (HBsAg) is an important biomarker used in the diagnosis of hepatitis B virus (HBV) infection, but false-negative results are still reported in the detection of HBsAg using commercial assays. In this study, we evaluated the qualitative properties of a novel HBsAg chemiluminescence enzyme immunoassay (CLEIA) assay--WTultra. WHO standard sample dilution series and samples from low-level HBsAg carriers (<1 ng/mL) were used to evaluate the sensitivity of the WTultra assay. Boston Biomedica, Inc. (BBI) hepatitis B seroconversion panels were used to assess the ability of the WTultra assay to detect the window period. In addition, dilution series of 22 serum samples with different genotypes, serotypes and HBsAg mutations were used to assess the WTultra assay, and these were compared with other commercial assays. The lower detection limit of the WTultra assay was 0.012 IU/mL, and it showed a high sensitivity (97.52%, 95% CI, 94.95-99.00) in the detection of 282 low-level HBsAg carriers (<1 ng/mL). In samples with various HBV genotypes, serotypes and HBsAg mutations, the WTultra assay yielded 117 positive results in 132 samples, which was significantly higher than the results with the other four commercial assays (89, 83, 65 and 45, respectively, p<0.01). In the assays of mutant strains, the WTultra assay detected 82 positive results in 90 samples, which was significantly better than the results for the Hepanostika HBsAg Ultra (58 positive) and Architect (55 positive) (p<0.01) assays, which in turn were significantly better than the Murex V.3 (41 positive, p=0.026) and AxSYM V2 (29 positive, p<0.01) assays. However, in the detection of 42 samples of wild-type strains with various genotypes and serotypes, no significant differences were observed among the WTultra (35 positive), Architect (28 positive) and Hepanostika HBsAg Ultra (31 positive) assays. However, the WTultra assay detected significantly more samples than the Murex V.3 (24 positive, p<0.01) and AxSYM V2 (16 positive, p<0.01) assays. In conclusion, the WTultra HBsAg assay has a high detection sensitivity and presents excellent results for the detection of mutants. PMID:26615806

  8. Development of a sensitive enzyme immunoassay for measuring taipan venom in serum.

    PubMed

    Kulawickrama, S; O'Leary, M A; Hodgson, W C; Brown, S G A; Jacoby, T; Davern, K; Isbister, G K

    2010-07-01

    The detection and measurement of snake venom in blood is important for confirming snake identification, determining when sufficient antivenom has been given, detecting recurrence of envenoming, and in forensic investigation. Venom enzyme immunoassays (EIA) have had persistent problems with poor sensitivity and high background absorbance leading to false positive results. This is particularly problematic with Australasian snakes where small amounts of highly potent venom are injected, resulting in low concentrations being associated with severe clinical effects. We aimed to develop a venom EIA with a limit of detection (LoD) sufficient to accurately distinguish mild envenoming from background absorbance at picogram concentrations of venom in blood. Serum samples were obtained from patients with taipan bites (Oxyuranus spp.) before and after antivenom, and from rats given known venom doses. A sandwich EIA was developed using biotinylated rabbit anti-snake venom antibodies for detection. For low venom concentrations (i.e. <1 ng/mL) the assay was done before and after addition of antivenom to the sample (antivenom difference method). The LoD was 0.15 ng/mL for the standard assay and 0.1 ng/mL for the antivenom difference method. In 11 pre-antivenom samples the median venom concentration was 10 ng/mL (Range: 0.3-3212 ng/mL). In four patients with incomplete venom-induced consumption coagulopathy the median venom concentration was 2.4 ng/mL compared to 30 ng/mL in seven patients with complete venom-induced consumption coagulopathy. No venom was detected in any post-antivenom sample and the median antivenom dose prior to this first post-antivenom sample was 1.5 vials (1-3 vials), including 7 patients administered only 1 vial. In rats the assay distinguished a 3-fold difference in venom dose administered and there was small inter-individual variability. There was small but measurable cross-reactivity with black snake (Pseudechis), tiger snake (Notechis) and rough-scale snake (Tropidechis carinatus) venoms with the assay for low venom concentrations (<1 ng/mL). The use of biotinylation and the antivenom difference method in venom EIA produces a highly sensitive assay that will be useful for determining antivenom dose, forensic and clinical diagnosis. PMID:20223258

  9. Evaluation of a homogenous enzyme immunoassay for the detection of synthetic cannabinoids in urine

    PubMed Central

    Barnes, Allan J.; Young, Sheena; Spinelli, Eliani; Martin, Thomas M.; Klette, Kevin L.; Huestis, Marilyn A.

    2014-01-01

    Introduction The recent emergence and widespread availability of many new synthetic cannabinoids support the need for an accurate and high-throughput urine screen for these new designer drugs. We evaluated performance of the immunalysis homogeneous enzyme immunoassay (HEIA) to sensitively, selectively, and rapidly identify urinary synthetic cannabinoids. Methods 2443 authentic urine samples were analyzed with the HEIA that targets JWH-018 N-pentanoic acid, and a validated LC-MS/MS method for 29 synthetic cannabinoids and metabolites. Semiquantitative HEIA results were obtained, permitting performance evaluation at and around three cutoffs (5, 10 and 20 μg/L), and diagnostic sensitivity, specificity and efficiency determination. Performance challenges at ±25 and ±50% of each cutoff level, cross-reactivity and interferences also were evaluated. Results Sensitivity, specificity, and efficiency of the immunalysis HEIA K2 Spice kit with the manufacturer's recommended 10 μg/L cutoff were 75.6%, 99.6% and 96.8%, respectively, as compared to the reference LC-MS/MS method with limits of detection of 0.1 -10 μg/L. Performance at 5 μg/L was 92.2%, 98.1% and 97.4%, and for the 20 μg/L cutoff were 62.9%, 99.7% and 95.4%. Semi-quantitative results for in-house prepared standards were obtained from 2.5-30 μg/L, and documented acceptable linearity from 5-25 μg/L, with inter-day imprecision <30% (n = 17). Thirteen of 74 synthetic cannabinoids evaluated were classified as highly cross-reactive (≥50% at 10 μg/L); 4 showed moderate cross-reactivity (10–50% at 10 μg/L), 30 low cross-reactivity (<10% at 500 μg/L), and 27 <1% cross-reactivity at 500 μg/L. There was no interference from 102 investigated compounds. Only a mixture containing 1000 μg/L each of buprenorphine/norbuprenorphine produced a positive result above our proposed cutoff (5 μg/L) but below the manufacturer's recommended cutoff concentration (10 μg/L). Conclusion The Immunalysis HEIA K2 Spice kit required no sample preparation, had a high-throughput, and acceptable sensitivity, specificity and efficiency, offering a viable method for screening synthetic cannabinoids in urine that cross-react with JWH-018 N-pentanoic acid antibodies. PMID:24845968

  10. The measurement of triclosan in water using a magnetic particle enzyme immunoassay

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A sensitive magnetic particle-based immunoassay to determine triclosan (5-chloro-2-(2,4-dichlorophenoxy)phenol) in drinking water and wastewater was developed. Rabbit antiserum was produced by immunizing the rabbit with 6-(5-chloro-2-(2,4-dichlorophenoxy)phenoxy)hexanoic acid-keyhole limpet hemocya...

  11. Dried venous blood samples for the detection and quantification of measles IgG using a commercial enzyme immunoassay.

    PubMed Central

    Riddell, Michaela A.; Byrnes, Graham B.; Leydon, Jennie A.; Kelly, Heath A.

    2003-01-01

    OBJECTIVES: To determine whether samples of dried venous blood (DVB) were an acceptable alternative to serum for detecting measles-specific IgG in a commercial enzyme immunoassay. METHODS: Paired samples of serum and DVB were collected from 98 suspected cases of measles and 1153 schoolchildren in Victoria, Australia. All samples were tested using the Dade Behring Enzygnost Anti-Measles-Virus/IgG immunoassay. DVB samples were eluted using either the sample buffer provided with the kit or 5% dry milk powder in phosphate-buffered saline-Tween 20. FINDINGS: DVB samples eluted by sample buffer showed significantly better linear correlation to the serum samples than did DVB samples eluted in 5% dry milk in phosphate-buffered saline-Tween 20. To improve the comparability of serum and DVB samples an adjustment factor of 1.28 was applied to the optical density (OD) values of DVB. This adjustment also enabled quantification of the titre of measles IgG in mIU/ml directly from the OD value using the alpha calculation as specified by the kit protocol. For DVB samples stored for less than six months at 4 degrees C, the assay showed an overall sensitivity of 98.4% and a specificity of 97.2% compared with the results of serum testing. CONCLUSION: These results illustrate the potential for DVB samples to be widely used with the Dade Behring enzyme immunoassay system for determining the immunity of the individual and the population to the measles virus. PMID:14758429

  12. Multiplexed Electrochemical Immunoassay of Phosphorylated Proteins Based on Enzyme-Functionalized Gold Nanorod Labels and Electric Field-Driven Acceleration

    SciTech Connect

    Du, Dan; Wang, Jun; Lu, Donglai; Dohnalkova, Alice; Lin, Yuehe

    2011-09-09

    A multiplexed electrochemical immunoassay integrating enzyme amplification and electric field-driven strategy was developed for fast and sensitive quantification of phosphorylated p53 at Ser392 (phospho-p53 392), Ser15 (phospho-p53 15), Ser46 (phospho-p53 46) and total p53 simultaneously. The disposable sensor array has four spatially separated working electrodes and each of them is modified with different capture antibody, which enables simultaneous immunoassay to be conducted without cross-talk between adjacent electrodes. The enhanced sensitivity was achieved by multi-enzymes amplification strategy using gold nanorods (AuNRs) as nanocarrier for co-immobilization of horseradish peroxidase (HRP) and detection antibody (Ab2) at high ratio of HRP/Ab2, which produced an amplified electrocatalytic response by the reduction of HRP oxidized thionine in the presence of hydrogen peroxide. The immunoreaction processes were accelerated by applying +0.4 V for 3 min and then -0.2 V for 1.5 min, thus the whole sandwich immunoreactions could be completed in less than 5 min. The disposable immunosensor array shows excellent promise for clinical screening of phosphorylated proteins and convenient point-of-care diagnostics.

  13. Naked-eye detection as a universal approach to lower the limit of detection of enzyme-linked immunoassays.

    PubMed

    O'Connor, Erin F; Paterson, Sureyya; de la Rica, Roberto

    2016-05-01

    Colorimetric biosensors for the detection of analytes with the naked eye are required in environmental monitoring, point-of-care diagnostics, and analyses in resources constrained settings, where detection instruments may not be available. However, instrument-based detection methods are usually more adequate for detecting small variations in the signal compared to naked-eye detection schemes, and consequently the limit of detection of the latter is usually higher than the former. Here, we demonstrate that the limit of detection of colorimetric enzyme-linked immunoassays can be decreased several orders of magnitude when using naked-eye detection instead of a spectrophotometer for detecting the signal. The key step to lower the limit of detection is adding a small volume of chromogenic substrate during the signal generation step. This generates highly colored solutions that can be easily visualized with the naked eye and recorded with the camera of a mobile phone. The proposed method does not require expensive equipment or complex protocols to enhance the signal, and therefore it is a universal approach to lower the limit of detection of colorimetric enzyme-linked immunoassays. PMID:26970749

  14. Enhanced Colorimetric Immunoassay Accompanying with Enzyme Cascade Amplification Strategy for Ultrasensitive Detection of Low-Abundance Protein

    PubMed Central

    Gao, Zhuangqiang; Hou, Li; Xu, Mingdi; Tang, Dianping

    2014-01-01

    Methods based on enzyme labels have been developed for colorimetric immunoassays, but most involve poor sensitivity and are unsuitable for routine use. Herein, we design an enhanced colorimetric immunoassay for prostate-specific antigen (PSA) coupling with an enzyme-cascade-amplification strategy (ECAS-CIA). In the presence of target PSA, the labeled alkaline phosphatase on secondary antibody catalyzes the formation of palladium nanostructures, which catalyze 3,3′,5,5′-tetramethylbenzidine-H2O2 system to produce the colored products, thus resulting in the signal cascade amplification. Results indicated that the ECAS-CIA presents good responses toward PSA, and allows detection of PSA at a concentration as low as 0.05 ng mL−1. Intra- and inter-assay coefficients of variation are below 9.5% and 10.7%, respectively. Additionally, the methodology is validated for analysis of clinical serum specimens with consistent results obtained by PSA ELISA kit. Importantly, the ECAS-CIA opens a new horizon for protein diagnostics and biosecurity. PMID:24509941

  15. A Semester-long Student-directed Research Project Involving Enzyme Immunoassay: Appropriate for Immunology, Endocrinology, or Neuroscience Courses

    PubMed Central

    DeLuca, Jane

    2007-01-01

    The following project aimed at promoting integrated and long-lasting learning is described for an Immunology course, but it may be adapted to other disciplines. Students were asked to develop and carry out a research project to examine the relationship between immune function and stress. The experiments were required to include the assessment of salivary cortisol and salivary IgA (sIgA) with enzyme immunoassays. All other aspects of the experiments were developed by student groups with appropriate guidance from the instructor. Data are presented for one group project that assessed the effect of music on cortisol and sIgA. Overall levels of sIgA and cortisol were consistent with reported values. Students found a significant decrease in cortisol over time. Additionally, there was a trend that supported the overall student hypothesis regarding the effect of stress and immune function. Compared with the same Immunology course that included an instructor-designed experiment using enzyme immunoassays for cortisol and sIgA, several assessments (e.g., final grades and comments on student evaluations) show that overall learning seemed to be much better in the course with the student-directed research project. PMID:18056304

  16. Voltammetric determination of alkaline phosphatase and horseradish peroxidase activity using 3-indoxyl phosphate as substrate Application to enzyme immunoassay.

    PubMed

    Fanjul-Bolado, Pablo; Gonzlez-Garca, Mara Begoa; Costa-Garca, Agustn

    2004-10-01

    The use of 3-indoxyl phosphate (3-IP) as an electrochemical substrate for ELISAs with voltammetric detection was investigated. Indirect measurements of alkaline phosphatase (AP) and horseradish peroxidase (HRP) activity in solution were carried out. Picomolar levels of both enzymes can be detected, which enables the design of electrochemical immunoassays using this substrate. The enzymatic turnover of the substrate gives indigo blue, insoluble in aqueous solutions. This product is easily converted into its soluble parent compound, indigo carmine (IC), by addition of fuming sulphuric acid to the reaction media. IC shows a reversible voltammetric peak at the formal potential of -0.15V (versus Ag pseudo-reference electrode) when a screen-printed carbon electrode (SPCE) is used. The peak current of this process constitutes the analytical signal. Using this approach an ELISA assay to quantify pneumolysin (PLY, a toxin related to respiratory infections) was carried out using AP or HRP as enzymatic label. Calibration plots obtained are reported. 3-IP is demonstrated to be the first suitable substrate for the two most common enzyme labels used in immunoassays. PMID:18969625

  17. Magnetic Affinity Enzyme-Linked Immunoassay for Diagnosis of Schistosomiasis Japonicum in Persons with Low-Intensity Infection

    PubMed Central

    Yu, Qin; Yang, Hai; Feng, Youmei; Zhu, Yanhong; Yang, Xiangliang

    2012-01-01

    Most schistosome-endemic areas in China are characterized by low-intensity infections that are independent of prevalence. To establish an effective diagnostic method, we developed a magnetic affinity enzyme-linked immunoassay based on soluble egg antigens (SEA-MEIA) for diagnosing schistosomiasis in persons with low-intensity infection with Schistosoma japonicum by comparing it with a conventional enzyme-linked immunosorbent assay (ELISA). Our results showed that the SEA-MEIA had a higher sensitivity and greater precision in the diagnosis of low-intensity S. japonicum infections than the ELISA. In addition, when we used Pearson's correlation in associating SEA-MEIA with ELISA, a significant correlation existed between the two assays (r = 0.845, P < 0.001). Our data indicated that SEA-MEIA, with a higher sensitivity and greater ease of performance, would be valuable for diagnosis of schistosomiasis japonicum in persons with low-intensity infections. PMID:22869635

  18. Magnetic affinity enzyme-linked immunoassay for diagnosis of Schistosomiasis japonicum in persons with low-intensity infection.

    PubMed

    Yu, Qin; Yang, Hai; Feng, Youmei; Zhu, Yanhong; Yang, Xiangliang

    2012-10-01

    Most schistosome-endemic areas in China are characterized by low-intensity infections that are independent of prevalence. To establish an effective diagnostic method, we developed a magnetic affinity enzyme-linked immunoassay based on soluble egg antigens (SEA-MEIA) for diagnosing schistosomiasis in persons with low-intensity infection with Schistosoma japonicum by comparing it with a conventional enzyme-linked immunosorbent assay (ELISA). Our results showed that the SEA-MEIA had a higher sensitivity and greater precision in the diagnosis of low-intensity S. japonicum infections than the ELISA. In addition, when we used Pearson's correlation in associating SEA-MEIA with ELISA, a significant correlation existed between the two assays (r = 0.845, P < 0.001). Our data indicated that SEA-MEIA, with a higher sensitivity and greater ease of performance, would be valuable for diagnosis of schistosomiasis japonicum in persons with low-intensity infections. PMID:22869635

  19. Evaluation of performances of three DNA enzyme immunoassays for detection of Helicobacter pylori PCR products from biopsy specimens.

    PubMed Central

    Monteiro, L; Cabrita, J; Mégraud, F

    1997-01-01

    PCR is recognized as a promising method for the detection of Helicobacter pylori in gastric biopsy specimens. However, detection of PCR products by gel electrophoresis is difficult to implement in routine clinical laboratories. The aim of this study was to compare three new DNA enzyme immunoassays with the standard method in their ability to detect PCR products. The three assays were based on the amplification of a fragment of the ureC gene of H. pylori and a colorimetric hybridization assay. The first assay (GEN-ETI-K DNA enzyme immunoassay; Sorin, Sallugia, Italy) was based on the hybridization of amplified DNA with a probe bound in microtiter wells and detected with labelled anti-DNA antibody. The second assay (Pylori-prob; Biocode, Sclessin, Belgium) comprised a solid-phase sandwich hybridization system with a specific biotinylated probe being used for detection. Finally, the third assay (PCR enzyme-linked immunosorbent assay; Boehringer, Mannheim, Germany) was based on the hybridization of amplified DNA labelled with digoxigenin as a probe (used as a coating in microtiter wells) and detected with antidigoxigenin-peroxidase as conjugate. The sensitivity of the colorimetric assay was evaluated by using amplification products from PCR assays performed on several 10-fold dilutions of DNA from H. pylori CIP 101260, and the specificity was assessed with different urease-positive bacteria. Biopsy specimens from 199 patients were tested; 106 were classified as H. pylori positive, and 93 were classified as H. pylori negative by culture and/or histological examination as the "gold standard." The receiving operating characteristic curve was used to determine the best cutoff point for each assay. The detection of PCR products by colorimetric hybridization increases the sensitivity up to 100-fold compared to that with gel electrophoresis. The results are rapid (4 h) and easy to interpret and can be automated. PMID:9350762

  20. Amplified inhibition of the electrochemical signal of ferrocene by enzyme-functionalized graphene oxide nanoprobe for ultrasensitive immunoassay.

    PubMed

    Lai, Guosong; Cheng, Hui; Xin, Dinghong; Zhang, Haili; Yu, Aimin

    2016-01-01

    A nanoprobe-induced signal inhibition mechanism was designed for ultrasensitive electrochemical immunoassay at a chitosan-ferrocene (CS-Fc) based immunosensor. The nanoprobe was prepared by covalently loading signal antibody and high-content horseradish peroxidase (HRP) on the graphene oxide (GO) nanocarrier. The immunosensor was prepared through the stepwise assembly of gold nanoparticles (Au NPs) and capture antibody at a CS-Fc modified electrode. After sandwich immunoreaction, the GO-HRP nanoprobes were quantitatively captured onto the immunosensor surface and thus induced the production of a layer of insoluble film through the enzymatically catalytic reaction of the HRP labels. Both the dielectric immunocomplex formed on the immunosensor surface and the enzymatic precipitate with low electroconductivity led to the electrochemical signal decease of the Fc indicator, which was greatly amplified by the multi-enzyme signal amplification of the nanoprobe. Based on this amplified signal inhibition mechanism, a new ultrasensitive electrochemical immunoassay method was developed. Using carcinoembryonic antigen as a model analyte, this method showed a wide linear range over 5 orders of magnitude with a detection limit down to 0.54 pg/mL. Besides, the immunosensor showed good specificity, acceptable reproducibility and stability as well as satisfactory reliability for the serum sample analysis. PMID:26703270

  1. TRICLOSAN AND METHYL-TRICLOSAN MONITORING STUDY IN THE NORTHEAST OF SPAIN USING A MAGNETIC PARTICLE ENZYME IMMUNOASSAY AND CONFIRMATORY ANALYSIS BY GAS CHROMATOGRAPHY-MASS SPECTROMETRY

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The occurrence of triclosan in the water environment around a Mediterranean region was investigated. Triclosan and methyl-triclosan content of ninety five environmental samples were screened using a magnetic particle enzyme immunoassay. Positive samples were confirmed by solid phase extraction (SPE...

  2. A prospective field evaluation of an enzyme immunoassay: Detection of eastern equine encephalomyelitis virus antigen in pools of Culiseta melanura

    USGS Publications Warehouse

    Scott, T.W.; Olson, J.G.; Lewis, T.E.; Carpenter, J.W.; Lorenz, L.H.; Lembeck, L.A.; Joseph, S.R.; Pagac, B.B.

    1987-01-01

    A prospective field study was conducted to determine the sensitivity and specificity of an enzyme immunoassay (EIA) compared to virus isolation in cell culture for the detection of eastern equine encephalomyelitis (EEE) virus in naturally infected mosquitoes. A total of 10,811 adult female Culiseta melanura were collected in light traps during 1985 from four locations in Maryland. Eastern equine encephalomyelitis virus was isolated from 5 of 495 mosquito pools in African green monkey kidney and baby hamster kidney cell cultures. All five virus-infected pools were detected by the EIA, and all 490 uninfected pools were correctly scored as not containing virus. The EIA did not produce false positive or false negative results. Results support the assertion of previous researchers that the antigen detection EIA is a rapid, sensitive, specific, and simple alternative to traditional bioassays for the detection of EEE virus in mosquitoes.

  3. Enzyme immunoassay using native envelope glycoprotein (gp160) for detection of human immunodeficiency virus type 1 antibodies.

    PubMed Central

    Nair, B C; Ford, G; Kalyanaraman, V S; Zafari, M; Fang, C; Sarngadharan, M G

    1994-01-01

    An enzyme immunoassay using the purified native gp160 for the detection of human immunodeficiency virus type 1 (HIV-1) antibody was developed. This assay was determined to be highly specific, since (i) 157 serum samples that were confirmed negative by Western blot (immunoblot) (WB) were negative, (ii) 41 serum samples from populations with medical conditions that might cause nonspecific assay reactivity were all negative, and (iii) all 15 serum samples that showed false-positive reactions in one or more commercial HIV-1 screening tests were negative. The assay gave 100% specificity with a randomly selected and unlinked panel of 1,000 serum samples from healthy blood donors. The sensitivity of the assay was assessed by testing 238 samples confirmed as HIV-1 antibody positive by a standardized WB assay. All 238 serum samples (100%) were reactive in the native gp160 assay. In a dilution panel of 14 weakly WB-positive serum samples, 7 samples reacted two-to fivefold more strongly in the gp160 assay than in a virus lysate-based assay; the remaining 7 samples gave comparable reactivities in the two tests. The reactivities of 13 of these 14 serum samples in the gp160 assay were higher than in a commercial enzyme immunoassay that uses a recombinant envelope protein as the antigen. The native gp160 assay was more sensitive to identify seroconversion. In a well-characterized panel of sequential blood samples from a seroconverter, the new assay detected antibodies at least one sample ahead of the other commercial assays tested. PMID:8077388

  4. Enzyme-triggered tyramine-enzyme repeats on prussian blue-gold hybrid nanostructures for highly sensitive electrochemical immunoassay of tissue polypeptide antigen.

    PubMed

    Xu, Tisen; Zhang, Haiying; Li, Xuegui; Xie, Zhaohui; Li, Xiangyong

    2015-11-15

    A novel sandwich-type electrochemical immunoassay with sensitivity enhancement was developed for quantitative detection of tissue polypeptide antigen (TPA) by coupling with target-induced tyramine signal amplification on prussian blue-gold hybrid nanostructures. The immunosensor was prepared through immobilizing anti-TPA capture antibody on a cleaned screen-printed carbon electrode (SPCE). Prussian blue-gold hybrid nanostructures (PBGNS) labeled with horseradish peroxidase (HRP) and detection antibody were utilized as the signal-transduction tags. Upon target TPA introduction, the sandwiched immunocomplex was formed between capture antibody and detection antibody on the electrode. The carried HRP could trigger the formation of tyramine-HRP repeats on the PBGNS in the presence of H2O2. Using the doped prussian blue as the electron mediator, the conjugated HRP could catalyze the reduction of H2O2. Under the optimal conditions, the catalytic currents increased with the increasing target TPA in the dynamic range from 1.0 pg mL(-1) to 100 ng mL(-1) with a detection limit of 0.3 pg mL(-1). The reproducibility and specificity of the electrochemical immunoassay were acceptable. In addition, the contents of target TPA in nine human serum specimens were evaluated by using the developed electrochemical immunosensor, and the obtained results correlated well with those from commercially enzyme-linked immunosorbent assay (ELISA) method with a correlation coefficient of 0.9975. PMID:26067328

  5. Concordance of four commercial enzyme immunoassay and three immunoblot formats for the detection of Lyme borreliosis antibodies in human serum: the two-tier approach remains.

    PubMed

    Dickeson, David J; Chen, Sharon C-A; Sintchenko, Vitali G

    2016-04-01

    Serological tests show considerable variation in their ability to correctly diagnose Lyme borreliosis (LB). This study compared four commercially available screening enzyme immunoassays (EIA) for the detection of LB IgG using either whole cell lysate (WCL) antigens, purified proteins or recombinant antigens with the second-tier whole cell sonicate (WCS) western immunoblots or recombinant antigen line blots. A consensus between three EIA results from 222 patient sera was designated as a point of comparison for each method which gave 66 positive and 156 negative results. The positive predictive values (PPV) of WCL EIA were 40% for the MarDx Diagnostics Borrelia burgdorferi EIA 'combined' IgG and IgM (Trinity Biotech) and 55% for the EUROIMMUN plus VlsE IgG. These were significantly lower PPVs than that produced by the recombinant antigen-based EIA NovaLisa Borrelia burgdorferi IgG-ELISA (NovaTec Immunodiagnostica) and the EUROIMMUN Anti-Borrelia Select ELISA IgG (90% and 100%, respectively; p = 0.02). The WCS western immunoblot using B. burgdorferi and B. afzelii separately showed a high PPV of 91% but its positive agreement with consensus EIA result was only 65%. Another WCL western immunoblot with purified extracts of Osp C and VlsE, the Trinity Biotech EU Lyme + VlsE IgG Western Blot had a PPV of 92% while the recombinant line blot from EUROIMMUN, the Anti-Borrelia (IgG) EUROLINE-RN-AT, demonstrated a significantly reduced PPV of 70% with some non-specific reactions in sera containing antibodies to Leptospira species, Helicobacter pylori and Treponema pallidum. The use of recombinant antigens in EIA for LB IgG screening significantly improves the predictive values of serological results above those of WCL antigen EIA. Second tier WCS western immunoblots offer high PPVs, especially with added specific purified proteins, more so than in one recombinant line blot. PMID:27020501

  6. Development of radioimmunoassay and enzyme immunoassays for luteinizing hormone in dromedaries (Camelus dromedarius).

    PubMed

    Anouassi, A; Combarnous, Y

    1991-11-01

    Polyclonal antibodies against luteinizing hormone in dromedary (camLH) were raised in a rabbit and enabled the development of homologous immunoassays (radioimmunoassay, competitive enzymeimmunoassay (EIA) and sandwich EIA) for the measurement of circulating camLH in plasma. These assays were highly specific for camLH since neither dromedary follicle-stimulating hormone, growth hormone nor prolactin cross-reacted significantly. The lowest detection limit (0.08 ng/ml) was obtained with the sandwich EIA. In addition to its high specificity and sensitivity, this method does not require radiolabelled molecules or expensive laboratory facilities. It can be performed in the field using a portable, battery-powered plate reader. PMID:1787460

  7. Stability of monoclonal IgM antibodies freeze-dried in the presence of trehalose.

    PubMed

    Dráber, P; Dráberová, E; Nováková, M

    1995-04-12

    We describe the use of the disaccharide trehalose for stabilization of mouse monoclonal IgM antibodies during freeze-drying and prolonged storage at elevated temperatures. Spent culture media, ascitic fluids and isolated immunoglobulins were freeze-dried in the presence of trehalose, stored at different temperatures, and tested after rehydration for their binding to their corresponding antigens. Antibodies, directed against various types of antigens, effectively recovered their binding efficiency as tested in enzyme-linked immunoassays, flow cytometry and immunofluorescence. Application of trehalose for freeze-drying of labile monoclonal IgM antibodies permits convenient long-term storage of large quantities of antibodies, facilitates their transport at ambient temperature and simplifies the construction of pre-aliquoted kits based on such antibodies. PMID:7730665

  8. Rapid determination of recent cocaine use with magnetic particles-based enzyme immunoassays in serum, saliva, and urine fluids.

    PubMed

    Vidal, Juan C; Bertolín, Juan R; Bonel, Laura; Asturias, Laura; Arcos-Martínez, M Julia; Castillo, Juan R

    2016-06-01

    Cocaine is one of the most worldwide used illicit drugs. We report a magnetic particles-based enzyme-linked immunoassay (mpEIA) method for the rapid and sensitive determination of cocaine (COC) in saliva, urine and serum samples. Under optimized conditions, the limits of detections were 0.09ngmL(-1) (urine), 0.15ngmL(-1) (saliva), and 0.06ngmL(-1) COC (human serum). Sensitivities were in the range EC50=0.6-2.5ngmL(-1) COC. The cross-reactivity with the principal metabolite benzoylecgonine (BZE) was only 1.6%. Recovering percentages of doped samples (0, 10, 50, and 100ngmL(-1) of COC) ranged from about 86-111%. Some advantages of the developed mpEIA over conventional ELISA kits are faster incubations, improved reproducibility, and consumption of lower amounts of antibody and enzyme conjugates due to the use of magnetic beads. The reported method was validated following the guidelines on bioanalytical methods of the European Medicines Agency (2011). Unmetabolized COC detection has a great interest in pharmacological, pharmacokinetics, and toxicokinetics studies, and can be used to detect a very recent COC use (1-6h). PMID:27003120

  9. Enzyme-linked immunoassay for detection of Listeria monocytogenes in dairy products, seafoods, and meats: collaborative study.

    PubMed

    Curiale, M S; Lepper, W; Robison, B

    1994-01-01

    A collaborative study was conducted to evaluate Listeria-Tek, an enzyme-linked immunosorbent assay (ELISA) for detection of Listeria monocytogenes and other Listeria spp. in foods. The present ELISA method was compared to the U.S. Food and Drug Administration culture method for detection of L. monocytogenes in dairy products and seafoods and to the U.S. Department of Agriculture Food Safety and Inspection Service method for detection of L. monocytogenes in meats. Replicate samples of 6 food types (frankfurters, roast beef, Brie cheese, 2% milk, raw shrimp, and crab meat) inoculated with L. monocytogenes and uninoculated control samples were analyzed by the collaborators. L. monocytogenes was identified in 593 samples by the ELISA method and in 574 samples using culture procedures. Identical results were obtained for 506 positive samples and 419 negative samples using the ELISA and culture methods for an overall agreement rate of 85.6%. The enzyme-linked immunoassay for detection of L. monocytogenes in dairy, seafood, and meat products has been adopted first action by AOAC INTERNATIONAL. PMID:7819756

  10. A paper-based surface-enhanced resonance Raman spectroscopic (SERRS) immunoassay using magnetic separation and enzyme-catalyzed reaction.

    PubMed

    Chen, Yuanyuan; Cheng, Hanwen; Tram, Kha; Zhang, Shengfeng; Zhao, Yanhua; Han, Liyang; Chen, Zengping; Huan, Shuangyan

    2013-05-01

    In this study, a novel paper-based SERRS immunoassay based on magnetic separation and alkaline phosphatase (ALP) enzyme catalyzed hydrolysis reaction was developed. By using modified antibodies conjugated to magnetic beads, capture of mouse IgG followed by addition of ALP-labeled antibodies would form a sandwich-like immunoconjugate. After magnetic separation, 5-bromo-4-chloro-3-indolyl phosphate (BCIP), a low SERRS active compound, was added as the substrate for ALP to generate a high SERRS response. Detection was conducted on a silver colloid/PVP/filter paper SERS substrate by spotting a pre-aggregated silver colloid sol onto polyvinyl pyrrolidone (PVP) modified filter paper using a semi-automatic TLC sample applicator. The optimization of the highly SERS active paper-based substrate, dynamic hydrolysis process of BCIP, quantitative detection of IgG, and selectivity of the assay was studied in detail. By taking advantage of magnetic separation in order to decrease the background interference, the selective enzyme reaction involved in producing a highly SERRS active product could detect mouse IgG from 1 to 500 ng mL(-1) with a LOD of 0.33 ng mL(-1). PMID:23486763

  11. Anti-idiotypic nanobody-alkaline phosphatase fusion proteins: Development of a one-step competitive enzyme immunoassay for fumonisin B1 detection in cereal.

    PubMed

    Shu, Mei; Xu, Yang; Liu, Xing; Li, Yanping; He, Qinghua; Tu, Zhui; Fu, Jinheng; Gee, Shirley J; Hammock, Bruce D

    2016-06-14

    A rapid and sensitive one-step competitive enzyme immunoassay for the detection of FB1 was developed. The anti-idiotypic nanobody-alkaline phosphatase (Ab2β-Nb-AP) was validated by the AP enzyme activity and the properties of bounding to anti-FB1-mAb (3F11) through colorimetric and chemiluminescence analyses. The 50% inhibitory concentration and the detection limit (LOD) of colorimetric enzyme-linked immunosorbent assay (ELISA) for FB1 were 2.69 and 0.35 ng mL(-1), respectively, with a linear range of 0.93-7.73 ng mL(-1). The LOD of the chemiluminescence ELISA (CLIA) was 0.12 ng mL(-1), and the IC50 was 0.89 ± 0.09 ng mL(-1) with a linear range of 0.29-2.68 ng mL(-1). Compared with LC-MS/MS, the results of this assay indicated the reliability of the Ab2β-Nb-AP fusion protein based one-step competitive immunoassay for monitoring FB1 contamination in cereals. The Ab2β-Nb-AP fusion proteins have the potential to replace chemically-coupled probes in competitive enzyme immunoassay systems. PMID:27181644

  12. Comparative evaluation of tests for detection of parvovirus B19 IgG and IgM.

    PubMed

    de Ory, Fernando; Minguito, Teodora; Echevarra, Juan Emilio; Del Mar Mosquera, Mara; Fuertes, Antonio

    2014-03-01

    The aim of this study was to evaluate enzyme immunoassays (EIA) (Euroimmun, Lbeck, Germany) and chemiluminiscent immunoassays (CLIA) (Diasorin, Saluggia, Italy) in their application to detect B19V-IgM and -IgG. For this purpose, one hundred and ninety samples were studied. Of them, 101 came from recent infection cases (B19V-specific IgM (86) and/or PCR (87), 42 from past infections, 18 from non-infected, and 29 from other viral recent infections (Epstein-Barr virus, measles, and rubella). Samples were characterized by capture (for IgM), or indirect (for IgG) EIA (Biotrin, Dublin, Ireland); indeterminate samples were classified by indirect immunofluorescence (IIF) (Biotrin). All the samples were used for testing IgM assays, and all but the cases from other viral infections were used for IgG tests. For IgM, CLIA, and EIA identified 76 and 62 of 86 IgM positives, respectively (sensitivity 88.4% and 72.1%). Considering B19V IgM negative samples, negative result was obtained in 95 and 92 of 104, being the specificity values of CLIA and EIA 91.3% and 88.5%, respectively. For IgG, CLIA and EIA identified correctly 114 and 115 of the 122 positive samples (sensitivity 93.4% and 94.3%, respectively), and 39 and 36 of 39 negative samples (specificity 100% and 92.3%). As conclusion, CLIA methods can be used in clinical laboratories as adequate alternatives to the well-established Biotrin EIAs. PMID:23763266

  13. Sensitive and high-fidelity electrochemical immunoassay using carbon nanotubes coated with enzymes and magnetic nanoparticles.

    PubMed

    Piao, Yunxian; Jin, Zongwen; Lee, Dohoon; Lee, Hye-Jin; Na, Hyon-Bin; Hyeon, Taeghwan; Oh, Min-Kyu; Kim, Jungbae; Kim, Hak-Sung

    2011-03-15

    We demonstrate a highly sensitive electrochemical immunosensor based on the combined use of substrate recycling and carbon nanotubes (CNTs) coated with tyrosinase (TYR) and magnetic nanoparticles (MNP). Both TYR and MNP were immobilized on the surface of CNTs by covalent attachment, followed by additional cross-linking via glutaraldehyde treatment to construct multi-layered cross-linked TYR-MNP aggregates (M-EC-CNT). Magnetically capturable, highly active and stable M-EC-CNT were further conjugated with primary antibody against a target analyte of hIgG, and used for a sandwich-type immunoassay with a secondary antibody conjugated with alkaline phosphatase (ALP). In the presence of a target analyte, a sensing assembly of M-EC-CNT and ALP-conjugated antibody was attracted onto a gold electrode using a magnet. On an electrode, ALP-catalyzed hydrolysis of phenyl phosphate generated phenol, and successive TYR-catalyzed oxidation of phenol produced electrochemically measurable o-quinone that was converted to catechol in a scheme of substrate recycling. Combination of highly active M-EC-CNT and substrate recycling for the detection of hIgG resulted in a sensitivity of 27.6 nA ng(-1) mL(-1) and a detection limit of 0.19 ng mL(-1) (1.2 pM), respectively, representing better performance than any other electrochemical immunosensors relying on the substrate recycling with the TYR-ALP combination. The present immunosensing system also displayed a long-term stability by showing a negligible loss of electrochemical detection signal even after reagents were stored in an aqueous buffer at 4°C for more than 6 months. PMID:21242086

  14. Competitive chemiluminescent enzyme immunoassay for vitamin B12 analysis in human milk

    Technology Transfer Automated Retrieval System (TEKTRAN)

    BACKGROUND Few accurate data exist on the concentration of vitamin B12 in human milk. Binding of the vitamin to haptocorrin (HC) can interfere with the assay if not removed by pretreatment, and very low values can occur in women with poor B12 status. This study evaluated two competitive enzyme bind...

  15. Enzyme immunoassay of antibody to Rochalimaea quintana: diagnosis of trench fever and serologic cross-reactions among other rickettsiae.

    PubMed

    Hollingdale, M R; Herrmann, J E; Vinson, J W

    1978-05-01

    Enzyme immunoassay (EIA) tests were used to diagnose trench fever and to determine cross-reactions of Rochalimaea quintana with other rickettsiae. The results were compared with those obtained by counterimmunoelectrophoresis (CIE). All sera from cases of primary or relapsed forms of trench fever were positive both in EIA, with serum antibody titers of 1:20-1:640, and in CIE, giving one to three precipitin lines. Sera from patients with other rickettsial infections were also tested for reactivity with R. quintana antigen: typhus group (Rickettsia prowazekii, Ricketsia mooseri), 15 sera; spotted fever group (Ricketsia ricketsii, Rickettsia akari), eight sera; and scrub typhus (Rickettsia tsutsugamushi), six sera. Strong reactions occurred with four sera from patients with scrub typhus, giving one or two lines in CIE and EIA titers of 1:40-1:160; these results were extended to guinea pig antisera to R. tsutsugamushi. About 50% of typhus group sera reacted with a single line in CIE and had antibody titers of 1:20-1:80 by EIA. The results show that EIA is accurate for the diagnosis of trench fever and, with the results obtained by CIE, suggest that R. quintana is antigenically related to R. tsutsugamushi and possibly to rickettsiae in the typhus group as well. PMID:351072

  16. Comparison of direct and indirect enzyme immunoassays with direct ultracentrifugation before electron microscopy for detection of rotaviruses.

    PubMed Central

    Hammond, G W; Ahluwalia, G S; Barker, F G; Horsman, G; Hazelton, P R

    1982-01-01

    A direct and an indirect enzyme immunoassay (EIA) were evaluated against a standard of electron microscopy after direct ultracentrifugation of the specimen for their performances in detecting rotaviruses. The indirect EIA had variable background activity which influenced test specificity. The indirect EIA control (test system without the detector antibody) plus a regression line (which reflected background noise) improved test specificity. However, the results of direct EIA (Rotazyme; Abbott Laboratories, North Chicago, Ill.) sensitivity (86%) and specificity (96%) were better than those of the indirect EIA in tests on 73 rotavirus-positive and 78 rotavirus-negative specimens. Endpoint titrations of purified SA-11 rotavirus showed greater sensitivity of the direct EIA test. Electron microscopy, performed after direct ultracentrifugation, and direct EIA were approximately 2 log10 more sensitive in the detection of purified SA-11 rotavirus than was electron microscopy with standard methods of unconcentrated specimen preparation. Direct EIA test are potentially sensitive, specific, and practical for the rapid detection of rotaviruses from human clinical specimens. Further studies are needed before EIA methods for detection of human rotaviruses can be equated with the level of reliability of results obtainable with sensitive electron microscopy techniques. PMID:6286720

  17. Detection of Tritrichomonas foetus by PCR and DNA Enzyme Immunoassay Based on rRNA Gene Unit Sequences

    PubMed Central

    Felleisen, Richard S. J.; Lambelet, Natacha; Bachmann, Philipp; Nicolet, Jacques; Müller, Norbert; Gottstein, Bruno

    1998-01-01

    Tritrichomonas foetus is the causative agent of bovine tritrichomonosis, a sexually transmitted disease leading to infertility and abortion. Diagnosis is hampered by putative contamination of samples with intestinal or coprophilic trichomonadid protozoa which might be mistaken for T. foetus. Therefore, we developed a PCR test optimized for applicability in routine diagnosis. Amplification is based upon primers TFR3 and TFR4 directed to the rRNA gene units of T. foetus. In order to avoid potential carryover contamination by products of previous amplification reactions, conditions were adapted to the use of the uracil DNA glycosylase system. Furthermore, documentation and interpretation of results were facilitated by including a DNA enzyme immunoassay for the detection of amplification products. Specificity was confirmed with genomic material from different related trichomonadid protozoa. The high sensitivity of the test allowed the detection of a single T. foetus organism in diagnostic culture medium or about 50 parasites per ml of preputial washing fluid. The present methods are thus proposed as (i) confirmatory tests for microscopic diagnosis following diagnostic in vitro cultivation and (ii) a direct T. foetus screening test with diagnostic samples. PMID:9466768

  18. Evaluation of two new enzyme immunoassay reagents for diagnosis of histoplasmosis in a cohort of clinically characterized patients.

    PubMed

    Zhang, Chen; Lei, Guang-Sheng; Lee, Chao-Hung; Hage, Chadi A

    2015-11-01

    The performance characteristics of the recently available analyte-specific reagent based enzyme immunoassay (ASR-EIA) and in vitro diagnostic (IVD) kit for urine Histoplasma antigen detection were evaluated in a cohort of 50 clinically characterized patients with histoplasmosis and 50 control patients. Overall sensitivity and specificity of the ASR-EIA were significantly improved compared with those of the IVD kit (sensitivity 72% vs. 22%, P<.001, specificity 98% vs. 84%, P = .014). Fourteen specimens from patients with clinically characterized histoplasmosis (five with pulmonary histoplasmosis and nine with progressive disseminated histoplasmosis) were falsely negative by ASR-EIA. All 10 specimens from patients with severe symptoms of progressive disseminated histoplasmosis were positive by ASR-EIA, although the average reading value of these 10 specimens was not significantly different from that of others with positive results. Compared to the MiraVista antigen assay, both the IVD kit and the ASR-EIA were significantly less sensitive in detecting Histoplasma antigen in the urine of patients with histoplasmosis. The ASR-EIA and MiraVista assay had comparable specificity. In conclusion, the ASR-EIA has improved performance compared with the IVD kit in the detection of Histoplasma antigen in the urine. However, users should be aware of the potential for false negative results using the currently recommended cutoff value. PMID:26337088

  19. Broad-Specificity Chemiluminescence Enzyme Immunoassay for (Fluoro)quinolones: Hapten Design and Molecular Modeling Study of Antibody Recognition.

    PubMed

    Zeng, Haopeng; Chen, Jiahong; Zhang, Chijian; Huang, Xin-An; Sun, Yuanming; Xu, Zhenlin; Lei, Hongtao

    2016-04-01

    On the basis of the structural features of (fluoro)quinolones (FQs), pazufloxacin was first used as a generic immunizing hapten to raise a broad-specificity antibody. The obtained polyclonal antibody exhibited broad cross-reactivity ranging from 5.19% to 478.77% with 21 FQs. Furthermore, the antibody was able to recognize these FQs below their maximum residue limits (MRLs) in an indirect competitive chemiluminescence enzyme immunoassay (ic-CLEIA), with the limit of detection (LOD) ranging from 0.10 to 33.83 ng/mL. For simply pretreated milk samples with spiked FQs, the ic-CLEIA exhibited an excellent recovery with a range of 84.6-106.9% and an acceptable coefficient of variation below 15%, suggesting its suitability and reliability for the use of a promising tool to detect FQs. Meanwhile, comparative molecular field analysis (CoMFA) and comparative molecular similarity indices analysis (CoMSIA) models, with statistically significant correlation coefficients (q(2)CoMFA = 0.559, r(2)CoMFA = 0.999; q(2)CoMSIA = 0.559, r(2)CoMSIA = 0.994), were established to investigate the antibody recognition mechanism. These two models revealed that in the antibody, the active cavity binding FQs' 7-position substituents worked together with another cavity (binding FQs' 1-position groups) to crucially endow the high cross-reactivity. This investigation will be significant for better exploring the recognition mechanism and for designing new haptens. PMID:26976361

  20. Enzyme immunoassay for the detection of porcine gelatine in edible bird's nests.

    PubMed

    Tukiran, Nur Azira; Ismail, Amin; Mustafa, Shuhaimi; Hamid, Muhajir

    2015-01-01

    Porcine gelatine is a common adulterant found in edible bird's nests (EBNs) used to increase the net weight prior to sale. This study aimed to develop indirect enzyme-linked immunosorbent assays (ELISAs) for porcine gelatine adulteration using anti-peptide polyclonal antibodies. Three indirect ELISAs were developed (PAB1, 2 and 3), which had limits of detection (LODs) of 0.12, 0.10 and 0.11 µg g(-1), respectively. When applied to standard solutions of porcine gelatine, the inter- and intra-assays showed coefficients of variation (CVs) less than 20% and were able to detect at least 0.5 ng µg(-1) (0.05%) porcine gelatine in spiked samples. The proposed ELISA offers attractions for quality control in the EBN industry. PMID:25861981

  1. Nanogold-penetrated poly(amidoamine) dendrimer for enzyme-free electrochemical immunoassay of cardiac biomarker using cathodic stripping voltammetric method.

    PubMed

    Zhang, Bo; Zhang, Yi; Liang, Wenbin; Cui, Bin; Li, Jiabei; Yu, Xuejun; Huang, Lan

    2016-01-21

    Methods based on immunoassays have been developed for cardiac biomarkers, but most involve the low sensitivity and are unsuitable for early disease diagnosis. Herein we design an electrochemical immunoassay for sensitive detection of myoglobin (a cardiac biomarker for acute myocardial infarction) by using nanogold-penetrated poly(amidoamine) dendrimer (AuNP-PAMAM) for signal amplification without the need of natural enzymes. The assay was carried out on the monoclonal mouse anti-myoglobin (capture) antibody-anchored glassy carbon electrode using polyclonal rabbit anti-myoglobin (detection) antibody-labeled AuNP-PAMAM as the signal tag. In the presence of target myoglobin, the sandwiched immunocomplex could be formed between capture antibody and detection antibody. Accompanying AuNP-PAMAM, the carried gold nanoparticles could be directly determined via stripping voltammetric method under acidic conditions. Under optimal conditions, the detectable electrochemical signal increased with the increasing target myoglobin in the sample within a dynamic working range from 0.01 to 500 ng mL(-1) with a detection limit of 3.8 pg mL(-1). The electrochemical immunoassay also exhibited high specificity and good precision toward target myoglobin. Importantly, our strategy could be applied for quantitative monitoring of myoglobin in human serum specimens, giving well matched results with those obtained from commercialized enzyme-linked immunosorbent assay (ELISA) method. PMID:26724762

  2. Use of enzyme immunoassay for large water-quality surveys of major herbicides

    SciTech Connect

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

    1996-10-01

    Commercially available enzyme-linked immunosorbent assay (ELISA) was used for the determination of major herbicides in several large water-quality surveys of surface water, rainwater, and ground water throughout the United States. The ELISA results were compared with gas chromatography/mass spectrometry (GC/MS) for accuracy and cross reactivity. In total, five compounds were analyzed: alachlor, atrazine, cyanazine, metolachlor, and (2,4-dichlorophenoxy) acetic acid (2,4-D). Results indicated that the ELISA and GC/MS results were comparable for cyanazine and metolachlor. The atrazine ELISA correlated well with GC/MS for surface- and ground-water samples from the central United States but did not correlate with samples from Texas where the cotton triazine, prometryn, is used. Results using the alachlor ELISA were poor because of cross reactivity with the metabolite, alachlor ethane-sulfonic acid. The ELISA for (2,4-dichlorophenoxy) acetic acid was insensitive at concentrations that occur in most surface water.

  3. Comparative evaluation of the WHO and DAKOPATTS enzyme-linked immunoassay kits for rotavirus detection.

    PubMed Central

    Flewett, T. H.; Arias, C. F.; Avendano, L. F.; Ghafoor, A.; Mathan, M. M.; Mendis, L.; Moe, K.; Bishop, R. F.

    1989-01-01

    Faeces obtained from 1,163 children (including 66 newborn babies) were analysed in parallel for the presence of rotavirus particles using two enzyme-linked immunosorbent assay kits. The kits had been formulated by the WHO Collaborating Centre for Reference and Research on Rotavirus (WHO-ELISA kit) and by DAKOPATTS (DAKO-ELISA kit) to be suitable for use in laboratories in developing countries. The kits were evaluated in laboratories in Burma, Chile, India, Mexico, Pakistan, Sri Lanka and the United Kingdom. Comparison of the results obtained with the two kits indicated that the DAKO-ELISA had an overall sensitivity of 97% and a specificity of 97% relative to the WHO-ELISA. In individual laboratories the DAKO-ELISA (K349) kit had a sensitivity in the range 90-100%, and a specificity of 85-100%. The kit showed a sensitivity of 100% and a specificity of 98% in assays on faeces obtained from newborn babies. We conclude that the DAKO-ELISA is as sensitive and specific as the WHO-ELISA for the detection of rotavirus antigen in faeces. PMID:2680139

  4. Determination of Aspergillus fumigatus allergen 1 in poultry farms using the enzyme immunoassay.

    PubMed

    Prester, Ljerka; Macan, Jelena; Matković, Kristina; Vucemilo, Marija

    2010-06-01

    Poultry farms contain high levels of allergenic fungi, and Aspergillus spp. is the most common genus of moulds. Aspergillus fumigatus antigens are responsible for the development of several respiratory diseases including asthma. The aim of this study was to measure the mass fraction of Asp f 1, a major allergen of Asperillus fumigatus in 37 indoor dust samples collected from four poultry farms in a rural area of the Zagreb County (Croatia) using the enzyme-linked immunosorbent assay. More than 62 % of dust samples had detectable Asp f 1 levels (limit of detection 3.6 ng g(-1)). The overall mean Asp f 1 level was 17.9 ng g(-1) [range (3.8 to 72.4) ng g(-1)]. Satisfactory results were obtained for analytical within-run imprecision (6.7 %), between-run imprecision (10.5 %), and accuracy (91 % to 115 %). Microclimate parameters (air temperature, relative humidity, and velocity) were within the recommended ranges in all poultry farms. This study has shown that Asp f 1 settles on dust at poultry farms and that occupational exposure to this allergen deserves monitoring in livestock buildings. PMID:20587390

  5. Development of an enzyme immunoassay using a monoclonal antibody against the psychoactive diterpenoid salvinorin A.

    PubMed

    Paudel, Madan Kumar; Shirota, Osamu; Sasaki-Tabata, Kaori; Tanaka, Hiroyuki; Sekita, Setsuko; Morimoto, Satoshi

    2013-09-27

    Salvinorin A (1), the main active constituent in Salvia divinorum, is a highly selective kappa-opioid receptor agonist with hallucinogenic effects, which is regulated in several countries. In the present study, a monoclonal antibody (mAb) against 1 was prepared, and an indirect competitive enzyme-linked immunosorbent assay (icELISA) system was developed for the detection of salvinorins. To raise mAbs against 1, salvinorin B (2) hemisuccinate was synthesized and used to prepare the immunogen 2-bovine serum albumin conjugate. This technique was used to prepare a hybridoma cell line, 3D5, which secreted a mAb that recognized 1. The mAb was shown to have specificity for 1 and other salvinorins in cross-reactivity tests. The intra-assay calibration range by icELISA using the mAb against 1 was 0.0195-0.625 μg/mL. After validating the icELISA using intra- and interassays, a recovery experiment and analysis of several plants in the family Lamiaceae, including S. divinorum, confirmed that the analytical method based on ELISA is not only simple but also precise, accurate, sensitive, and sufficiently reliable. The results indicate that icELISA is a useful tool in the identification of S. divinorum. PMID:23987562

  6. Chronic Chagas Disease Diagnosis: A Comparative Performance of Commercial Enzyme Immunoassay Tests.

    PubMed

    Santos, Fred Luciano Neves; de Souza, Wayner Vieira; Barros, Michelle da Silva; Nakazawa, Mineo; Krieger, Marco Aurélio; Gomes, Yara de Miranda

    2016-05-01

    There is a significant heterogeneity in reported performance of serological assays for Chagas disease diagnosis. The conventional serology testing in laboratory diagnosis and in blood banks is unsatisfactory because of a high number of inconclusive and misclassified results. We aimed to assess the quality of four commercially available enzyme-linked immunosorbent assay tests for their ability to detect Trypanosoma cruzi antibodies in 685 sera samples. Cross-reactivity was assessed by using 748 sera from patients with unrelated diseases. Initially, we found that the reactivity index against T. cruzi antigen was statistically higher in sera from Chagas disease patients compared with those from non-chagasic patients, supporting the notion that all evaluated tests have a good discriminatory ability toward the diagnosis of T. cruzi infection in patients in the chronic phase of the disease. Although all tests were similarly sensitive for diagnosing T. cruzi infection, there were significant variations in terms of specificity and cross-reactivity among them. Indeed, we obtained divergent results when testing sera from patient with unrelated diseases, particularly leishmaniasis, with the levels of cross-reactivity being higher in tests using whole T. cruzi extracts compared with those using recombinant proteins. Our data suggest that all four tests may be used for the laboratory diagnosis and routine blood screening diagnose for Chagas disease. We also emphasize that, despite their general good performance, caution is needed when analyzing the results when these tests are performed in areas where other diseases, particularly leishmaniasis, are endemic. PMID:26976886

  7. Rapid screening of flonicamid residues in environmental and agricultural samples by a sensitive enzyme immunoassay.

    PubMed

    Liu, Zhenjiang; Zhang, Zhen; Zhu, Gangbing; Sun, Jianfan; Zou, Bin; Li, Ming; Wang, Jiagao

    2016-05-01

    A fast and sensitive polyclonal antibody-based enzyme-linked immunosorbent assay (ELISA) was developed for the analysis of flonicamid in environmental and agricultural samples. Two haptens of flonicamid differing in spacer arm length were synthesized and conjugated to proteins to be used as immunogens for the production of polyclonal antibodies. To obtain most sensitive combination of antibody/coating antigen, two antibodies were separately screened by homologous and heterologous assays. After optimization, the flonicamid ELISA showed that the 50% inhibitory concentration (IC50 value) was 3.86mgL(-1), and the limit of detection (IC20 value) was 0.032mgL(-1). There was no cross-reactivity to similar tested compounds. The recoveries obtained after the addition of standard flonicamid to the samples, including water, soil, carrot, apple and tomato, ranged from 79.3% to 116.4%. Moreover, the results of the ELISA for the spiked samples were largely consistent with the gas chromatography (R(2)=0.9891). The data showed that the proposed ELISA is an alternative tool for rapid, sensitive and accurate monitoring of flonicamid in environmental and agricultural samples. PMID:26897400

  8. Development of ultrasensitive direct chemiluminescent enzyme immunoassay for determination of aflatoxin M1 in milk.

    PubMed

    Vdovenko, Marina M; Lu, Chuan-Chen; Yu, Feng-Yih; Sakharov, Ivan Yu

    2014-09-01

    A direct competitive chemiluminescent enzyme-linked immunosorbent assay (CL-ELISA) for detecting aflatoxin M1 (AFM1) was developed. To improve the sensitivity of the assay, a mixture of 3-(10'-phenothiazinyl)-propane-1-sulfonate (SPTZ) and 4-morpholinopyridine (MORPH) was used to enhance peroxidase-induced CL. The concentrations of the coating anti-AFM1 antibody and the conjugate of AFB1 with horseradish peroxidase the conditions of the chemiluminescent assay were varied to optimise the condition of the chemiluminescent assay. The lower detection limit values and dynamic working range of CL-ELISA of AFM1 were 0.001 ng mL(-1) and 0.002-0.0075 ng mL(-1), respectively. A 20-fold dilution of milk samples prevented a matrix effect of the milk and allowed measurement of AFM1 at concentrations below than the maximum acceptable limit. Values of recovery within and between assays were 81.5-117.6% and 86-110.6%, respectively. The results of using the developed CL-ELISA to analyse samples of six brands of milk that were purchased in Taiwan revealed that AFM1 was absent from all studied samples. PMID:24731347

  9. Prospective Evaluation of a New Aspergillus IgG Enzyme Immunoassay Kit for Diagnosis of Chronic and Allergic Pulmonary Aspergillosis.

    PubMed

    Dumollard, C; Bailly, S; Perriot, S; Brenier-Pinchart, M P; Saint-Raymond, C; Camara, B; Gangneux, J P; Persat, F; Valot, S; Grenouillet, F; Pelloux, H; Pinel, C; Cornet, M

    2016-05-01

    Anti-Aspergillus IgG antibodies are important biomarkers for the diagnosis of chronic pulmonary aspergillosis (CPA) and allergic bronchopulmonary aspergillosis (ABPA). We compared the performance of a new commercial enzyme immunoassay (EIA) (Bordier Affinity Products) with that of the Bio-Rad and Virion\\Serion EIAs. This assay is novel in its association of two recombinant antigens with somatic and metabolic antigens of Aspergillus fumigatus In a prospective multicenter study, 436 serum samples from 147 patients diagnosed with CPA (136 samples/104 patients) or ABPA (94 samples/43 patients) and from 205 controls (206 samples) were tested. We obtained sensitivities of 97%, 91.7%, and 86.1%, and specificities of 90.3%, 91.3%, and 81.5% for the Bordier, Bio-Rad, and Virion\\Serion tests, respectively. The Bordier kit was more sensitive than the Bio-Rad kit (P < 0.01), which was itself more sensitive than the Virion\\Serion kit (P = 0.04). The Bordier and Bio-Rad kits had similar specificity (P = 0.8), both higher than that of the Virion\\Serion kit (P = 0.02). The area under the receiver operating characteristic (ROC) curves confirmed the superiority of the Bordier kit over the Bio-Rad and the Virion\\Serion kits (0.977, 0.951, and 0.897, respectively; P < 0.01 for each comparison). In a subset analysis of 279 serum samples tested with the Bordier and Bio-Rad kits and an in-house immunoprecipitin assay (IPD), the Bordier kit had the highest sensitivity (97.7%), but the IPD tended to be more specific (71.2 and 84.7%, respectively; P = 0.10). The use of recombinant, somatic, and metabolic antigens in a single EIA improved the balance of sensitivity and specificity, resulting in an assay highly suitable for use in the diagnosis of chronic and allergic aspergillosis. PMID:26888904

  10. Prevalence of Human Calicivirus Infections in Kenya as Determined by Enzyme Immunoassays for Three Genogroups of the Virus

    PubMed Central

    Nakata, Shuji; Honma, Shinjiro; Numata, Kazuko; Kogawa, Keiko; Ukae, Susumu; Adachi, Noriaki; Jiang, Xi; Estes, Mary K.; Gatheru, Zippora; Tukei, Peter M.; Chiba, Shunzo

    1998-01-01

    An epidemiological survey on human calicivirus (HuCV) infections and associated gastroenteritis in infants was conducted to clarify the prevalence of HuCV infections in infants and adults in Kenya. Enzyme immunoassays (EIAs) for three genogroups of HuCVs, Norwalk virus (NV), Mexico virus (MXV), and Sapporo virus (SV), were used to detect antigen or antibody. We tested 1,431 stool samples obtained from children younger than 6 years old with acute gastroenteritis who visited outpatient clinics in three districts in Kenya from August 1991 to July 1994. Thirty-two (2.2%) of these stool samples were positive for SV antigen. Only one (0.1%) of 1,186 samples was positive for NV antigen and none of 246 samples was positive for MXV antigen. One hundred ninety-three serum samples were tested for antibodies to NV and MXV, and 64 of them were examined for antibody to SV. The pattern of the age-related prevalence of serum antibody to NV was different from that of antibodies to MXV and SV. The acquisition of serum antibodies to HuCVs in the three genogroups appeared in early childhood, at about 1 to 2 years of age. The prevalence of serum antibody to NV was low (about 60%) throughout adulthood compared with a high prevalence of antibody (∼80 to 90%) to MXV and SV. These data indicate that infections with viruses in the three genogroups of HuCVs are common in Kenya, and immunological responses to NV may be different from those to MXV and SV. The EIAs for the detection of NV and MXV antigens appear to be quite specific for prototype NV and MXV strains, respectively, so that they can detect only a few strains of HuCVs related to them. Alternatively, NV and MXV caused less severe infections that did not bring children to the outpatient clinics for gastroenteritis in Kenya. PMID:9774557

  11. Detection of Campylobacter in Stool and Determination of Significance by Culture, Enzyme Immunoassay, and PCR in Developing Countries

    PubMed Central

    Platts-Mills, James A.; Liu, Jie; Gratz, Jean; Mduma, Esto; Amour, Caroline; Swai, Ndealilia; Taniuchi, Mami; Begum, Sharmin; Peataro Yori, Pablo; Tilley, Drake H.; Lee, Gwenyth; Shen, Zeli; Whary, Mark T.; Fox, James G.; McGrath, Monica; Kosek, Margaret; Haque, Rashidul

    2014-01-01

    Campylobacter is a common bacterial enteropathogen that can be detected in stool by culture, enzyme immunoassay (EIA), or PCR. We compared culture for C. jejuni/C. coli, EIA (ProSpecT), and duplex PCR to distinguish Campylobacter jejuni/C. coli and non-jejuni/coli Campylobacter on 432 diarrheal and matched control stool samples from infants in a multisite longitudinal study of enteric infections in Tanzania, Bangladesh, and Peru. The sensitivity and specificity of culture were 8.5% and 97.6%, respectively, compared with the results of EIA and 8.7% and 98.0%, respectively, compared with the results of PCR for C. jejuni/C. coli. Most (71.6%) EIA-positive samples were positive by PCR for C. jejuni/C. coli, but 27.6% were positive for non-jejuni/coli Campylobacter species. Sequencing of 16S rRNA from 53 of these non-jejuni/coli Campylobacter samples showed that it most closely matched the 16S rRNA of C. hyointestinalis subsp. lawsonii (56%), C. troglodytis (33%), C. upsaliensis (7.7%), and C. jejuni/C. coli (2.6%). Campylobacter-negative stool spiked with each of the above-mentioned Campylobacter species revealed reactivity with EIA. PCR detection of Campylobacter species was strongly associated with diarrhea in Peru (odds ratio [OR] = 3.66, P < 0.001) but not in Tanzania (OR = 1.56, P = 0.24) or Bangladesh (OR = 1.13, P = 0.75). According to PCR, Campylobacter jejuni/C. coli infections represented less than half of all infections with Campylobacter species. In sum, in infants in developing country settings, the ProSpecT EIA and PCR for Campylobacter reveal extremely high rates of positivity. We propose the use of PCR because it retains high sensitivity, can ascertain burden, and can distinguish between Campylobacter infections at the species level. PMID:24452175

  12. Validation of an enzyme immunoassay for the measurement of faecal glucocorticoid metabolites in spotted hyenas (Crocuta crocuta).

    PubMed

    Benhaiem, Sarah; Dehnhard, Martin; Bonanni, Roberto; Hofer, Heribert; Goymann, Wolfgang; Eulenberger, Klaus; East, Marion L

    2012-09-01

    The use of enzyme immunoassays (EIAs) to measure faecal glucocorticoid metabolites (fGCM) is a useful non-invasive technique to monitor adrenocortical activity in vertebrates. The first objective of this study was to validate an 'in-house' EIA (cortisol-3-CMO) for the measurement of fGCM concentrations in spotted hyenas. High-performance liquid chromatography (HPLC) was used to characterise fGCM in samples from a captive hyena that received an i.v. injection of [(3)H] cortisol. All HPLC fractions were analysed with the EIA for the presence and quantities of radiolabelled fGCM. Radiolabelled fGCM consisted of substances with a higher polarity than cortisol and substances of lower polarity that eluted between cortisol and corticosterone. Authentic radiolabelled cortisol was not detected. The EIA measured substantial amounts of immunoreactivity corresponding to the radioactive peaks. It also detected a significant increase in fGCMs after an adrenocorticotropic hormone (ACTH) challenge in two other captive animals and a significant increase in fGCMs in a fourth captive animal after anaesthesia. The second objective was to investigate an age effect on fGCM: we conducted pairwise comparisons of fGCM concentrations in individual free-ranging juvenile spotted hyenas when less than 6 months of age and when between 6 and 24 months of age. We expected juveniles to experience a more unpredictable and therefore more stressful environment when younger than when older. When younger, juveniles had significantly higher fGCM concentrations than when they were older. Our results demonstrate that our assay can be used to assess adrenocortical activity in spotted hyenas. PMID:22634955

  13. Highly Sensitive Detection of Hepatitis B Virus Surface Antigen by Use of a Semiautomated Immune Complex Transfer Chemiluminescence Enzyme Immunoassay

    PubMed Central

    Maruki, Mari; Yamagaito, Takahiro; Muramatsu, Machiko; Sakai, Yasuhiro; Tobimatsu, Hiroaki; Kobayashi, Hironori; Mizuno, Yoshiteru; Hamaguchi, Yukio

    2013-01-01

    The performance of hepatitis B surface antigen (HBsAg) screening assays is continuously improved to reduce the risk of transfusion-associated hepatitis B. In this study, a semiautomated immune complex transfer chemiluminescence enzyme immunoassay (ICT-CLEIA) for the detection of HBsAg, which is as sensitive as hepatitis B virus (HBV) DNA PCR, was developed; the ICT-CLEIA assay performance was compared with the performance of the Architect HBsAg QT assay and HBV DNA PCR. The specificities in the initial assay and after retesting were 99.50% (1,988/1,998 samples) and 99.95% (1,997/1,998 samples), respectively. The analytical detection limit was determined to be 0.2 mIU/ml using the 2nd International WHO HBsAg standard, and the cutoff value (0.5 mIU/ml) of the ICT-CLEIA assay was 8.0 standard deviations (SD) above the mean of the HBsAg-negative specimens. The ICT-CLEIA assay could detect HBsAg even in the presence of anti-HBs antibodies and demonstrated a 23.6-day-shorter window period using commercially available HBsAg seroconversion panels than the Architect HBsAg QT assay. Furthermore, the monitoring of the viral kinetics by the ICT-CLEIA assay and the HBV DNA PCR produced very similarly shaped curves during both the HBsAg seroconversion and reverse seroconversion periods. Therefore, the ICT-CLEIA assay may be useful not only for an earlier detection of HBV reactivation but also for the monitoring of hepatitis B patients. PMID:23658266

  14. Comparison of a lateral flow milk progesterone test with enzyme immunoassay as an aid for reproductive status determination in cows.

    PubMed

    Waldmann, A; Raud, A

    2016-03-12

    The lateral flow test (LFT) is an immunochromatographic method that utilises an immunostrip for non-laboratory diagnostic purposes. The present study evaluated a milk progesterone LFT against the enzyme immunoassay (EIA) to confirm oestrus and a non-pregnancy diagnosis. In total, 277 milk samples from 70 cows were analysed, collected on the day of artificial insemination and at 19 days, 21 days and 24 days post insemination. The level of accuracy of the LFT compared with the EIA was 95.0 per cent for milk samples containing <2 ng/ml progesterone and 97.0 per cent for milk samples containing >10 ng/ml progesterone. The validation of oestrus by the LFT was 98.6 per cent accurate using 2 ng/ml progesterone as the EIA estimate for oestrus. The test performance for a non-pregnancy diagnosis was subject to the day of milk sampling, showing the highest accuracy on day 24 post insemination for both tests. When optimised for maximum specificity, and compared with rectal palpation, the LFT had a sensitivity and specificity for non-pregnancy diagnosis on day 24 post insemination of 75.0 per cent and 100.0 per cent, respectively, with an overall accuracy of 84.4 per cent. The corresponding characteristics for the quantitative EIA were 85.0 per cent, 100.0 per cent and 90.6 per cent, respectively. The LFT results compared favourably with the quantitative milk progesterone EIA. PMID:26873072

  15. Development of magnetic particle-based chemiluminescence enzyme immunoassay for the detection of 17beta-estradiol in environmental water.

    PubMed

    Xin, Tian-Bing; Wang, Xu; Jin, Hui; Liang, Shu-Xuan; Lin, Jin-Ming; Li, Zhen-Jia

    2009-09-01

    In the present work, a simple, fast, and highly sensitive chemiluminescence enzyme immunoassay for 17beta-estradiol (E2) in environmental water samples was developed, using magnetic particles (MPs) labeled with secondary antibody as both the immobilization matrix and the separation tools. The specific anti-E2 polyclonal antibody (PcAb) was produced against a conjugate of estradiol-bovine serum albumin. The specificity of the anti-E2 antibody was studied. The results showed that the antibody did not cross-react with the structurally related endocrine-disrupting compounds, including estrone, ethinyl E2, estriol, E2-17-glucuronide, E2-3-sulfate-17-glucuronide, androstenedione, and dihydrotestosterone. The water samples were pretreated with solid-phase extraction using C18 cartridges for the removal of matrix effects. Several physicochemical parameters including the dilution ratios of E2-6-horseradish peroxidase conjugate and anti-E2 PcAb, immunoreaction time, volume of chemiluminescent substrate and MPs, chemiluminescence reaction time, and pH of assay solution were studied and optimized. At optimal experimental conditions, it was found that the proposed method exhibited high performance with detection limit of 2.0 pg/mL, linear range of 20-1,200 pg/mL, and total assay time of 45 min. Both inter- and intra-assay coefficient of variation were less than 10%. The average recoveries of three different spiked concentration samples ranged from 86.3% to 108%. The method was successfully applied to the determination of E2 in river, waste, and tap water, and showed a good correlation with the commercially available radioimmunoassay kit. PMID:18841499

  16. Development and validation of an indirect enzyme immunoassay for detection of ovine antibody to Brucella ovis.

    PubMed

    Vigliocco, A M; Silva Paulo, P S; Mestre, J; Briones, G C; Draghi, G; Tossi, M; Nielsen, K

    1997-03-01

    An indirect enzyme-linked immunosorbent assay (ELISA) for the serodiagnosis of Brucella ovis infection was developed. The assay uses a mouse monoclonal antibody to bovine IgG1 horseradish peroxidase (HRPO) conjugate that cross-reacts with immunoglobulin from sheep and a purified antigen from Brucella ovis. The ELISA data were read and analyzed according to a targeting procedure. The ELISA results were compared with a cold complement fixation test (CFT). Sera from 675 rams from three uninfected flocks were used to determine the ELISA cut-off value (O.D. 405 nm: 0.095) and the diagnostic specificity of the ELISA (100%) and the CFT (99.69% +/- 0.42). The ELISA cut-off value was corroborated by receiver operating characteristic (ROC) analysis. Six hundred and forty semen and serum samples from 419 rams from two naturally infected flocks were collected before and after mating-time during two consecutive years. All semen samples were cultured and Brucella ovis was isolated from 28 samples. Sera from the 28 rams with positive semen were used to determine the diagnostic sensitivity of the ELISA (96.43% +/- 6.8) and of the CFT (including suspected positive samples with titers of 1:5; 88.89% +/- 11.85). Considering the CFT suspicious and the anti-complementary reactions as positive resulted in a diagnostic sensitivity value of 89.28% +/- 11.46. Six hundred and ten serum samples from the 640 sera were used to determine relative sensitivity (excluding sera with 1:5) at: ELISA/CFT 97.26% +/- 3.74 and CFT/ELISA was 71.72% +/- 8.87. The percent agreement, beyond chance measured by the Kappa index was 79.7. Relative sensitivity ELISA/CFT (including 1:5 titers in the CFT as positive) was 94.9% +/- 4.83 and CFT/ELISA was 72.84% +/- 8.59. The Kappa index was 79.4. PMID:9100335

  17. Universal phosphorescence immunoassay

    NASA Astrophysics Data System (ADS)

    Mantrova, Ekaterina Y.; Demcheva, Marina V.; Savitsky, Alexander P.; Ponomarev, Gely V.

    1993-05-01

    Pd-coproporphyrin I (Pd-CP) has optimum phosphorescence characteristics for application in immunoassay. The aim of this study is to work out a universal phosphorescence immunoassay method (UniPhIA) using monoclonal antibodies to Pd-CP and conjugates of various proteins with Pd-CP for detection of insulin. Pd-CP and monoclonal antibodies obtained allow a convenient method for determination of various antigens to be developed, which combines the universal character of the amplified enzyme-linked immunosorbent assay (a-ELISA) with a high sensitivity and the simplicity of the time-resolved phosphorescence immunoassay.

  18. Improved Standardization of the Bio-Rad Platelia Aspergillus Galactomannan Antigen Sandwich Enzyme Immunoassay Using the DS2 (Dynex) Enzyme-Linked Immunosorbent Assay (ELISA) Processing System.

    PubMed

    Gorton, R L; White, P L; Bagkeris, E; Cotterall, D; Desai, R; McHugh, T; Kibbler, C C

    2015-07-01

    The galactomannan enzyme immunoassay (GM-EIA) is widely utilized for the diagnosis of invasive aspergillosis (IA). There is inconsistent reproducibility of results between centers when the assay is processed manually. Automation of EIAs can reduce variation. This study investigated the semiautomation of the GM-EIA on the DS2 (Dynex) platform in the following three stages: (i) DS2 GM-EIA method validation with experimental samples, (ii) DS2 retesting of case-defined clinical samples, and (iii) a 12-month audit of DS2 GM-EIA performance. In stage i, Bland-Altman analysis demonstrated a reduced variance between optical density index (ODI) values for samples processed on two DS2 platforms (mean difference, -0.02; limits of agreement [LOA], -0.19 to 0.14) compared with the variance between samples processed manually and on a DS2 platform (mean difference, 0.02; LOA, -0.25 to 0.3). In stage ii, 100% (14/14 samples) qualitative agreement was observed for serum samples from patients with IA, with no significant change in the ODI values when samples were processed on the DS2 platform. A significant decrease in ODI values was observed for control serum samples on the DS2 platform (difference, 0.01; P = 0.042). In stage iii, a significant reduction in the frequency of equivocal results, from 5.56% (136/2,443 samples) to 1.56% (15/961 samples), was observed after DS2 automation (difference, 4.0%; 95% confidence interval [CI], 2.7 to 5.2%; P < 0.01), with an equivalent increase in negative results. This study demonstrates that GM-EIA automation may reduce intersite variability. Automation does not have an impact on the repeatability of truly positive results but contributes to a reduction in false-positive (equivocal) GM-EIA results, reducing the need to retest a significant proportion of samples. PMID:25878352

  19. A highly sensitive enzyme immunoassay for evaluation of 2'-deoxycytidine plasma level as a prognostic marker for breast cancer chemotherapy.

    PubMed

    Darwish, Ibrahim A; Mahmoud, Ashraf M; Aboul-Fadl, Tarek; Al-Majed, Abdul-Rahman A; Khalil, Nasr Y

    2009-01-26

    A highly sensitive competitive enzyme immunoassay (EIA) has been developed and validated for the determination of the plasma level of 2'-deoxycytidine (dCyd), the potential prognostic marker for breast cancer chemotherapy. This assay employed a monoclonal antibody that recognizes dCyd with a high specificity, and 5'-succinyl-dCyd (5'sdCyd) conjugate of bovine serum albumin (5'sdCyd-BSA) immobilized onto microplate wells as a solid phase. The assay involved a competitive binding reaction between dCyd, in plasma sample, and the immobilized 5'sdCyd-BSA for the binding sites of the anti-dCyd antibody. The bound antibody was quantified with horseradish peroxidase-labeled anti-immunoglobulin second antibody and 3,3',5,5'-tetramethylbenzidine as a peroxidase substrate. The concentration of dCyd in the sample was quantified by its ability to inhibit the binding of the antibody to the immobilized 5'sdCyd-BSA and subsequently the color formation in the assay. The assay limit of detection was 8 nM and the effective working range at relative standard deviations (R.S.D.s) of

  20. Capture-ELISA for serum IgM antibody to respiratory syncytial virus.

    PubMed Central

    Cevenini, R.; Donati, M.; Bertini, S.; Moroni, A.; Sambri, V.

    1986-01-01

    A four-component solid-phase capture enzyme immunoassay was set up to test for serum IgM antibody to respiratory syncytial (RS) virus and was compared with immunofluorescence assay (IFA). A total of 128 young children with acute respiratory infections were studied. Thirty-six were shown to be RS virus-positive by the detection of RS virus in nasopharyngeal secretions and 92 were RS virus-negative. A serum specimen was collected after admission to the hospital (days 0-4) and a further specimen was obtained during days 10-14. Out of 36 RS virus-positive patients, 28 (77.7%) were found to be positive for IgM by both capture-ELISA and IFA. Out of 92 RS virus-negative patients 5 (5.4%) were IgM-positive. Four false-positive results were obtained by IFA due to the presence of rheumatoid factor. The capture-ELISA was shown to be a reliable technique in detecting specific IgM antibody to RS virus. PMID:3540115

  1. Improved Standardization of the Bio-Rad Platelia Aspergillus Galactomannan Antigen Sandwich Enzyme Immunoassay Using the DS2 (Dynex) Enzyme-Linked Immunosorbent Assay (ELISA) Processing System

    PubMed Central

    White, P. L.; Bagkeris, E.; Cotterall, D.; Desai, R.; McHugh, T.; Kibbler, C. C.

    2015-01-01

    The galactomannan enzyme immunoassay (GM-EIA) is widely utilized for the diagnosis of invasive aspergillosis (IA). There is inconsistent reproducibility of results between centers when the assay is processed manually. Automation of EIAs can reduce variation. This study investigated the semiautomation of the GM-EIA on the DS2 (Dynex) platform in the following three stages: (i) DS2 GM-EIA method validation with experimental samples, (ii) DS2 retesting of case-defined clinical samples, and (iii) a 12-month audit of DS2 GM-EIA performance. In stage i, Bland-Altman analysis demonstrated a reduced variance between optical density index (ODI) values for samples processed on two DS2 platforms (mean difference, −0.02; limits of agreement [LOA], −0.19 to 0.14) compared with the variance between samples processed manually and on a DS2 platform (mean difference, 0.02; LOA, −0.25 to 0.3). In stage ii, 100% (14/14 samples) qualitative agreement was observed for serum samples from patients with IA, with no significant change in the ODI values when samples were processed on the DS2 platform. A significant decrease in ODI values was observed for control serum samples on the DS2 platform (difference, 0.01; P = 0.042). In stage iii, a significant reduction in the frequency of equivocal results, from 5.56% (136/2,443 samples) to 1.56% (15/961 samples), was observed after DS2 automation (difference, 4.0%; 95% confidence interval [CI], 2.7 to 5.2%; P < 0.01), with an equivalent increase in negative results. This study demonstrates that GM-EIA automation may reduce intersite variability. Automation does not have an impact on the repeatability of truly positive results but contributes to a reduction in false-positive (equivocal) GM-EIA results, reducing the need to retest a significant proportion of samples. PMID:25878352

  2. Measurements in international units of antibody to hepatitis B surface antigen(anti-HBs) after immunization with a yeast-derived, subtype adr hepatitis B vaccine are considerably different between chemiluminescent immunoassay (CLIA) and chemiluminescent enzyme immunoassay (CLEIA).

    PubMed

    Ogata, Norio

    2006-04-01

    The worldwide consensus of the minimum protective anti-HBs level against HBV infection is 10 mIU/mL on assays standardized by the World Health Organization (WHO) reference preparations. To investigate whether this value could be applied to recipients of yeast-derived recombinant HB vaccine containing the major surface protein of subtype adr (Bimmugen, Astellas Pharmaceutical, Tokyo), we compared anti-HBs measurements between chemiluminescent immunoassay (CLIA) (Architect Ausab, Abbott Japan, Tokyo) and chemiluminescent enzyme immunoassay (CLEIA) (Lumipulse Forte, Fujirebio, Tokyo) in given serum samples obtained from the vaccinees. The vaccine and the two assay methods are currently in a wide use in Japan. The study included 300 medical students who completed a standard vaccination course (0, 1 and 6 months). Serum samples obtained 1 month or 13 months after completing the vaccination were simultaneously tested for anti-HBs by CLIA and CLEIA. In 147 samples with quantifiable values on both CLIA and CLEIA (10 to 1000 mIU/mL) the geometric mean titer on CLEIA (225.0 mIU/mL) was significantly higher than that on CLIA (94.5 mIU/mL) (p < 0.0001). Of 26 subjects with CLIA measurements below 10 mIU/mL, 15 samples (57.7%) showed CLEIA measurements more than 10 mIU/mL. Thus, in the subtype adr-vaccinees CLEIA demonstrated considerably high serum anti-HBs measurements compared to CLIA and discordance in determining critical anti-HBs level of 10 mIU/mL was observed in more than half the samples. This suggests that the minimum HBV-protective anti HBs titer of 10 mIU/mL is difficult to be introduced to Japan where subtype adr-HB vaccines or -HBV infection are prevalent, unless characteristics of assay methods are carefully evaluated. PMID:16722452

  3. [Rheumatoid factor activity as a disturbing factor in the serological diagnosis of specific IgM antibodies].

    PubMed

    Lindenschmidt, E G

    1984-04-01

    Rheumatoid factors (RF) are autoantibodies mainly directed against autologous IgG. They belong at most to the IgM class antibodies. It is demonstrated at groups with unsolved hepatitis B, rubella, syphilis and toxoplasmose infection that RF do occur not rarely at these patients even without rheumatoid arthritis. This is probably due to stimulation by antigen-IgG-complexes. During serologic detection of specific IgM antibodies they present an antigen independent mu-specificity. So the test for specific IgM might even loose its diagnostic and possibly therapy indicating value. It is shown how the disturbance by RF can be calculated after adsorption with aggregated IgG. Also RF can be titrated by an enzyme immunoassay (ELISA). With IgG coated latex particles RF can be eliminated prior to the IgM-test. Solid phase techniques which are applied with enzyme-coupled antigen instead of marked anti-IgM cannot be disturbed by RF significantly. PMID:6398795

  4. A New Enzyme Immunoassay for the Quantitative Determination of Classical Autotaxins (ATXα, ATXβ, and ATXγ) and Novel Autotaxins (ATXδ and ATXε)

    PubMed Central

    Tokuhara, Yasunori; Kurano, Makoto; Shimamoto, Satoshi; Igarashi, Koji; Nojiri, Takahiro; Kobayashi, Tamaki; Masuda, Akiko; Ikeda, Hitoshi; Nagamatsu, Takeshi; Fujii, Tomoyuki; Aoki, Junken; Yatomi, Yutaka

    2015-01-01

    Background Autotaxin (ATX) is a secreted enzyme that converts lysophosphatidylcholine to lysophosphatidic acid, a potent bioactive lipid mediator, through its lysophospholipase D activity. Although five alternative splicing isoforms of ATX have been identified as ATXα, ATXβ, ATXγ, ATXδ, and ATXε and the expression patterns of each isoform differ among several tissues, the clinical significance of each isoform remains to be elucidated. Methods Anti-ATXβ and anti-ATXδ monoclonal antibodies were produced by immunization with recombinant human ATXβ and ATXδ expressed using a baculovirus system, respectively. We then developed enzyme immunoassays to measure the serum concentrations of “classical ATX” (ATXα, ATXβ, and ATXγ) and “novel ATX” (ATXδ and ATXε) antigens and evaluated the usefulness of these assays using human serum samples. Results The with-run and between-run precision, interference, detection limit, and linearity studies for the present assay were well validated. In healthy subjects, the serum concentrations of classical ATX and novel ATX were significantly (P < 0.01) higher in women than in men, while the ratios of classical ATX or novel ATX to total ATX were not different between women and men. The concentrations of both classical ATX and novel ATX in normal pregnant subjects and patients with chronic liver diseases or follicular lymphoma were significantly higher than those in healthy subjects, while the ratio of both ATX isoforms to total ATX did not vary among these groups. Conclusions We have developed a new enzyme immunoassay to determine the concentrations of classical ATX and novel ATX in human serum. These assays may be helpful for elucidating the distinct functional roles of each ATX isoform, which are largely unknown at present. PMID:26083365

  5. Magnetic bead-based enzyme-chromogenic substrate system for ultrasensitive colorimetric immunoassay accompanying cascade reaction for enzymatic formation of squaric acid-iron(III) chelate.

    PubMed

    Lai, Wenqiang; Tang, Dianping; Zhuang, Junyang; Chen, Guonan; Yang, Huanghao

    2014-05-20

    This work reports on a simple and feasible colorimetric immunoassay with signal amplification for sensitive determination of prostate-specific antigen (PSA, used as a model) at an ultralow concentration by using a new enzyme-chromogenic substrate system. We discovered that glucose oxidase (GOx), the enzyme broadly used in enzyme-linked immunosorbent assay (ELISA), has the ability to stimulate in situ formation of squaric acid (SQA)-iron(III) chelate. GOx-catalyzed oxidization of glucose leads to the formation of gluconic acid and hydrogen peroxide (H2O2). The latter can catalytically oxidize iron(II) to iron(III), which can rapidly (<1 min) coordinate with the SQA. Formation of the iron-squarate complex causes the color of the solution to change from bluish purple to bluish red accompanying the increasing absorbance with the increment of iron(III) concentration. On the basis of the SQA-iron(III) system, a new immunoassay protocol with GOx-labeled anti-PSA detection antibody can be designed for the detection of target PSA on capture antibody-functionalized magnetic immunosensing probe, monitored by recording the color or absorbance (λ = 468 nm) of the generated SQA-iron(III) chelate. The absorbance intensity shows to be dependent on the concentration of target PSA. A linear dependence between the absorbance and target PSA concentration is obtained under optimal conditions in the range from 1.0 pg mL(-1) to 30 ng mL(-1) with a detection limit (LOD) of 0.5 pg mL(-1) (0.5 ppt) estimated at the 3Sblank level. The sensitivity displays to be 3-5 orders of magnitude better than those of most commercialized human PSA ELISA kits. In addition, the developed colorimetric immunoassay was validated by assaying 12 human serum samples, receiving in good accordance with those obtained by the commercialized PSA ELISA kit. Importantly, the SQA-based immunosensing system can be further extended for the detection of other low-abundance proteins or biomarkers by controlling the target antibody. PMID:24785462

  6. IMMUNOASSAY HUMAN EXPOSURE STUDIES

    EPA Science Inventory

    The Human Exposure Research Branch has developed several enzyme-linked immunosorbent assay (ELISA) methods to support human exposure assessment studies. Immunoassays to detect low levels (<10 ng/mL) of chlorpyrifos in food, track-in dirt and house dust have been applied to sam...

  7. Enzyme immunoassay for measurement of antibodies to herpes simplex virus infection: comparison with complement fixation, immunofluorescent-antibody, and neutralization techniques.

    PubMed Central

    Denoyel, G A; Gaspar, A; Nouyrigat, C

    1980-01-01

    An enzyme immunoassay (EIA) was developed for detecting antibody to herpes simplex virus, and the results were compared with those of complement fixation, indirect immunofluorescent-antibody, and plaque reduction neutralization tests. Test sera showed very little nonspecific reactivity even at a starting dilution as low as 1:10. EIA results showed excellent correlation with results obtained by the neutralization test, with an average gain in sensitivity of 1.65. EIA proved very useful in detecting current herpes simplex virus infection, and antibody appeared in all cases soon after clinical onset. EIA appears to be a rapid, sensitive, and specific method for routine demonstration of herpes simplex virus antibody in a clinical setting. PMID:6244329

  8. Clinical evaluation of the sensitivity and specificity of a commercially available enzyme immunoassay for detection of rubella virus-specific immunoglobulin M.

    PubMed Central

    Chernesky, M A; Wyman, L; Mahony, J B; Castriciano, S; Unger, J T; Safford, J W; Metzel, P S

    1984-01-01

    A solid-phase capture antigen enzyme immunoassay (Rubazyme-M) was evaluated for sensitivity and specificity on sera from 1,200 blood donors, 51 patients with rubella, 2 infants with congenital rubella, 104 patients with other infections, and 126 patients with immunological abnormalities. The sensitivity was 100% for sera tested between days 3 and 40 after the onset of symptoms of rubella virus infection. Rubella virus-specific immunoglobulin M was detected at birth in sera from congenitally infected infants and persisted for several months. Positive Rubazyme-M responses were observed in some patients in the absence of rubella diagnosis (one blood donor, three other infections, and two immunological abnormalities), providing a test specificity of 99.6%. None of 67 patients with rubella virus-specific immunoglobulin G antibody and high levels of rheumatoid factor were positive in the test. PMID:6386857

  9. Enzyme-amplified protein micorarray and a fluidic renewable surface fluorescence immunoassay for botulinum neurotoxin detection using high-affinity recombinant antibodies

    SciTech Connect

    Varnum, Susan M.; Warner, Marvin G.; Dockendorff, Brian P.; Anheier, Norman C.; Lou, Jianlong; Marks, James D.; Smith, Leonard A.; Feldhaus, Michael J.; Grate, Jay W.; Bruckner-Lea, Cindy J.

    2006-06-16

    With the use of high-affinity recombinant monoclonal antibodies against the receptor binding domain of botulinum neurotoxin A (BoNT/A), two separate immunoassay platforms were developed for either the sensitive or the rapid detection of BoNT/A. An enzyme-linked immunosorbent assay (ELISA) microarray was developed for the specific and sensitive detection of BoNT in buffer and clinical fluids. This assay has the sensitivity to detect BoNT in diverse samples down to 14 fM (1.4 pg/mL). Using the recombinant monoclonal antibodies, a renewable surface microcolumn sensor was developed for the rapid detection of BoNT/A in an automated fluidic system. While the ELISA microarray assay, because of its sensitivity, offers an alternative to the mouse bioassay, the renewable surface assay has potential as a rapid screening assay for the analysis of complex environmental samples.

  10. Comparison of a frozen human foreskin fibroblast cell assay to an enzyme immunoassay and toxigenic culture for the detection of toxigenic Clostridium difficile.

    PubMed

    Strachan, Alastair J; Evans, Natalie E; Williams, O Martin; Spencer, Robert C; Greenwood, Rosemary; Probert, Chris J

    2013-01-01

    This study set out to validate the Hs27 ReadyCell assay (RCCNA) as an alternative CCNA method compared against a commonly used commercial enzyme immunoassay (EIA) method and toxigenic culture (TC) reference standard. A total of 860 samples were identified from those submitted to the Health Protection Agency microbiology laboratories over a 30-week period. RCCNA performed much better than EIA when using TC as a gold standard, with sensitivities of 90.8% versus 78.6% and positive predictive value of 87.3% to 81.9%, respectively. The Hs27 Human Foreskin Fibroblast ReadyCells are an easy-to-use and a sensitive CCNA method for the detection of toxigenic Clostridium difficile directly from stool. A turnaround time of up to 48 h for a negative result and possible need for repeat testing make it an unsuitable method to be used in most clinical laboratory setting. PMID:23107315

  11. Development of a flow-through enzyme immunoassay and application in screening green coffee samples for ochratoxin A with confirmation by high-performance liquid chromatography.

    PubMed

    Sibanda, L; De Saeger, S; Bauters, T G; Nelis, H J; Van Peteghem, C

    2001-10-01

    A flow-through enzyme immunoassay has been developed for the screening of green coffee bean samples for ochratoxin A (OA) and was later used in a survey on OA in green coffee from different countries. The test has a sensitivity of 8 ng/g, and calculated recoveries ranged from 70 to 89% and from 86 to 95% for spiked and naturally contaminated samples, respectively. There were no significant differences in within-day and between-day assay performance (P > 0.05). Green coffee samples (15 Arabica and 7 Robusta) received from an international coffee trader were analyzed for intrinsic fungal contamination, screened for OA, and subsequently confirmed by high-performance liquid chromatography (HPLC). All 22 samples were contaminated by fungal species of the genus Aspergillus, while Penicillium species were isolated from a mere 13.6% of the total number of samples. Isolates were tested for their ability to produce OA, and only 3.9% were positive. There was no correlation between occurrence of OA-producing isolates and levels of OA in contaminated samples. Results of the screening procedure showed that 4 of the 22 samples were contaminated with 8 ng/g or higher. The HPLC method confirmed that the OA levels ranged from 27 to 168 ng/g. A fifth sample, which was shown to be negative during screening, had an OA concentration of 4 ng/g. There were no false negatives or positives recorded, and the flow-through enzyme immunoassay results correlated with those obtained by HPLC. PMID:11601711

  12. Comparison of PremierTM Rotaclone®, ProSpecTTM, and RIDASCREEN® Rotavirus Enzyme Immunoassay Kits for Detection of Rotavirus Antigen in Stool Specimens

    PubMed Central

    Gautam, Rashi; Lyde, Freda; Esona, Mathew D.; Quaye, Osbourne; Bowen, Michael D.

    2015-01-01

    Background Rotaviruses are the major cause of severe dehydrating diarrhea in children throughout the world. Enzyme immunoassays (EIA) have been the standard method for detection of rotavirus in stool specimens since the 1980s. The World Health Organization (WHO) Rotavirus Surveillance Network has proposed including three EIA kits in the WHO-GSM (Global Management System/Système Mondial de Gestion ) catalogue for easy procurement of EIA kits by participating rotavirus surveillance network laboratories. Objectives In this study, we conducted a comparative analysis of 3 commercially available enzyme immunoassay kits: PremierTM Rotaclone® (Meridian Bioscience, Inc.), ProSpecTTM (Oxoid, Ltd.) and RIDASCREEN® (R-biopharm AG) for rotavirus diagnostics. Study design Using reverse-transcriptase-PCR (RT-PCR) as the gold standard, the 3 EIA kits were evaluated by testing a stool panel consisting of 56 rotavirus-positive and 54 rotavirus negative samples. Results The sensitivities of the PremierTM Rotaclone®, ProSpecTTM and RIDASCREEN® kits were 76.8%, 75% and 82.1% respectively, but did not differ significantly. The specificity of all the 3 kits was 100%. The use of RT-PCR as a gold standard lowered the observed sensitivity of all 3 EIA kits but helps to reduce equivocal results that can be seen when another EIA or other non-molecular methods are used as the reference assay in comparison studies. Conclusion Our study found that all three kits are suitable for use by rotavirus surveillance programs. PMID:23850415

  13. Development of a Highly Specific IgM Enzyme-Linked Immunosorbent Assay for Bartonella henselae Using Refined N-Lauroyl-Sarcosine-Insoluble Proteins for Serodiagnosis of Cat Scratch Disease.

    PubMed

    Otsuyama, Ken-Ichiro; Tsuneoka, Hidehiro; Kondou, Kaori; Yanagihara, Masashi; Tokuda, Nobuko; Shirasawa, Bungo; Ichihara, Kiyoshi

    2016-04-01

    The conventional anti-Bartonella henselaeIgM enzyme-linked immunosorbent assay (IgM-ELISA) methods for diagnosing cat scratch disease (CSD) remain poor in both sensitivity and specificity. We sought to develop an IgM-ELISA with improved accuracy in the serodiagnosis of CSD by exploring the antigens that are most suitable for an ELISA. We prepared 5 different protein antigens: antigen I (sonicatedB. henselaewhole-cell antigen), antigen II (N-lauroyl-sarcosine-insoluble antigen), antigen III (processed sarcosine-soluble antigen), and antigen IV and antigen V (sarcosine-insoluble and sarcosine-soluble antigens refined by DEAE-Sepharose Fast Flow ion-exchange chromatography). The IgM antibodies in the sera of 47 patients with clinically suspected CSD (24 definite, 23 suspected) and of 85 healthy individuals were examined by ELISAs using the 5 antigens, and the results were compared with those of an IgM indirect fluorescent antibody assay (IgM-IFA). In a reference panel, which consisted of 5 positive and 5 negative sera, antigen I and antigen III failed to distinguish between the two statuses, whereas the other three antigens succeeded in distinguishing between them. When the cutoff value was set at the 98th percentile of the ELISA index for healthy individuals, the sensitivity of IgM-IFA for the 24 cases of definite CSD was 54%, whereas the sensitivities of the IgM-ELISAs with antigen II, IV, and V were 75%, 83%, and 75%, respectively. The sensitivities of these three IgM-ELISAs for all 47 of the clinically suspected cases were 49%, 64%, and 51%, respectively. In contrast, the sensitivity of IgM-IFA was 28%. These results indicate that the refined sarcosine-insoluble proteins (antigen IV), which possessed the highest specificity among the 5 antigens, are the most appropriate for developing an IgM-ELISA for the highly specific serodiagnosis of CSD. PMID:26865692

  14. Development of an enzyme immunoassay for the antibiotic cefquinome and its application for residue determination in cow's milk after therapeutical mastitis treatment.

    PubMed

    Thal, Johannes; Steffen, Monika; Meier, Bianca; Schneider, Elisabeth; Adriany, Ansgar; Usleber, Ewald

    2011-01-01

    The aim of this study was to develop and evaluate an enzyme immunoassay (EIA) for the cephalosporin antibiotic in milk, in combination with a new microbiological test system (brilliant black reduction test, BRT-P). Polyclonal antibodies against cefquinome were produced in rabbits, using cefquinome-keyhole limpet hemocyanine as the immunogen. These antibodies and a cefquinome-glucose oxidase conjugate were used in a competitive indirect EIA. The detection limit for cefquinome in milk was 1.5 ng ml(-1), recoveries were 80-128% at 4-40 ng ml(-1). Cross-reactivities with other cephalosporins/penicillins were all <1%. The EIA was used to determine cefquinome in incurred raw milk, the BRT-P (detection limit ≈ 20 ng ml(-1)) and a receptor assay (ßeta-s.t.a.r., detection limit ≈ 15 ng ml(-1)) were used in parallel. Five lactating cows, suffering from clinical mastitis, were treated with cefquinome by simultaneous intramammary and intramuscular injection. Cefquinome residues (maximum 10-27 μg ml(-1)) were most exclusively found in the udder quarter which was treated intramammary, residue levels in the other three quarters were low (<20 ng ml(-1)). Even in milk from intramammary-dosed quarters, residue levels fell below European Union maximum residue level (MRL, 20 μg kg(-1)) 2 days before the end of the withdrawal period. EIA, BRT-P, and ßeta-s.t.a.r. results showed acceptable agreement for milk samples, but the newly developed EIA is superior in aspects of sensitivity. In conclusion, this is the first one description of immunoassay and microbiological tests capable to determine cefquinome in milk at the MRL in incurred sample material. PMID:21103866

  15. EQCM Immunoassay for Phosphorylated Acetylcholinesterase as a Biomarker for Organophosphate Exposures Based on Selective Zirconia Adsorption and Enzyme-Catalytic Precipitation

    SciTech Connect

    Wang, Hua; Wang, Jun; Choi, Daiwon; Tang, Zhiwen; Wu, Hong; Lin, Yuehe

    2009-03-01

    A zirconia (ZrO2) adsorption-based immunoassay by electrochemical quartz crystal microbalance (EQCM) has been initially developed, aiming at the detection of phosphorylated acetylcholinesterase (AChE) as a potential biomarker for bio-monitoring exposures to organophosphate (OP) pesticides and chemical warfare agents. Hydroxyl-derivatized monolayer was preferably chosen to modify the crystal serving as the template for directing the electro-deposition of ZrO2 film with uniform nanostructures. The resulting ZrO2 film was utilized to selectively capture phosphorylated AChE from the sample media. Horseradish peroxidase (HRP)-labeled anti-AChE antibodies were further employed to recognize the captured phosphorylated protein. Enzyme-catalytic oxidation of the benzidine substrate resulted in the accumulation of insoluble product on the functionalized crystal. Ultrasensitive EQCM quantification by mass-amplified frequency responses as well as rapid qualification by visual color changes of product could be thus achieved. Moreover, 4-chloro-1-naphthol (CN) was comparably studied as an ideal chromogenic substrate for the enzyme-catalytic precipitation. Experimental results show that the developed EQCM technique can allow for the detection of phosphorylated AChE in human plasma. Such an EQCM immunosensing format opens a new door towards the development of simple, sensitive, and field-applicable biosensor for biologically monitoring low-level OP exposures.

  16. Immunoassay techniques.

    PubMed

    Wheeler, Michael J

    2013-01-01

    No other development has had such a major impact on the measurement of hormones as immunoassay. Reagents and assay kits can now be bought commercially but not for the more esoteric or new hormones. This chapter explains the basics of the immunoassay reaction and gives simple methods for immunoassays and immunometric assays and for the production of reagents for both antigenic and hapten hormones. Alternative methods are given for the preparation of labeled hormones as well as several possible separation procedures. The methods described here have been previously used in a wide range of assays and have stood the test of time. They will allow the production of usable immunoassays in a relatively short period of time. PMID:23996355

  17. Detection of measles virus-specific immunoglobulin M in dried venous blood samples by using a commercial enzyme immunoassay.

    PubMed

    Riddell, Michaela A; Leydon, Jennie A; Catton, Mike G; Kelly, Heath A

    2002-01-01

    The optical densities (ODs) of 216 dried venous blood (DVB) samples submitted to the Victorian Infectious Diseases Reference Laboratory as part of enhanced measles surveillance were compared to the ODs of the corresponding serum samples collected at the same time. DVB samples, stored for up to 24 months at 4 degrees C, were tested by the Dade Behring Enzygnost Anti-Measles-Virus/IgM immunoassay. Elution and testing conditions were optimized with the use of spiked DVB samples. The assay showed an overall sensitivity of 90.2% and a specificity of 98.8% for DVB samples compared to the results for serum. When the results were analyzed according to the length of time that the DVB sample had been stored, the assay was 100% sensitive and 97% specific according to the ODs for those samples stored for less than 6 months compared to the results for the corresponding serum samples, with 97.7% agreement between the results for the two sample types. These results demonstrate the potential for the use of DVB samples for the diagnosis of measles in routine diagnostic laboratories. PMID:11773084

  18. [Detection of IgM against the dengue++ virus in whole blood absorbed on filter paper].

    PubMed

    Vázquez, S; Sáenz, E; Huelva, G; González, A; Kourí, G; Guzmán, M

    1998-03-01

    This paper presents a standardized procedure for the detection of IgM antibodies to dengue virus in blood samples taken from filter paper. The samples were obtained from 118 patients, of whom 91 had been clinically diagnosed with dengue and 27 with a viral infection unrelated to that disease. The first group of patients came from Costa Rica and Nicaragua and the second group from Cuba. All the samples were tested for IgM antibody against dengue virus by means of a capture enzyme immunoassay (EIA). Analysis of the results for patients from all three countries together yielded a sensitivity of 98.1% and a specificity of 98.5% for the test done on whole blood on filter paper stored at 4 degrees C; agreement between the results of that test and those of the EIA using serum samples was 96%. In a comparison of the results obtained with three samples from the same patient--whole blood on filter paper stored at room temperature, the same type of sample stored at 4 degrees C, and serum--the agreement was 86%. This study demonstrates the high diagnostic sensitivity and specificity achieved when whole blood absorbed on filter paper is processed in the manner described in detail in the article. The authors recommend the use of this method in the dengue surveillance programs in the Region. PMID:9567651

  19. Development of a Reverse Transcription-PCR–DNA Enzyme Immunoassay for Detection of “Norwalk-Like” Viruses and Hepatitis A Virus in Stool and Shellfish

    PubMed Central

    Schwab, Kellogg J.; Neill, Frederick H.; Le Guyader, Françoise; Estes, Mary K.; Atmar, Robert L.

    2001-01-01

    Outbreaks of food- and waterborne gastroenteritis are being increasingly reported throughout the world. The analysis of environmental samples by newer diagnostic techniques such as reverse transcription-PCR (RT-PCR) amplification of nucleic acid has begun to identify human enteric viruses (predominantly “Norwalk-like” viruses [NLVs]) as the cause of many of these outbreaks. To streamline NLV detection from environmental samples such as shellfish, we have developed an RT-PCR–oligoprobe amplification and detection method using several new procedures that enable confirmed RT-PCR amplification and product detection in 1 day. The new steps include replacing reverse transcriptase and Taq polymerase with rTth polymerase, a heat-stable enzyme that functions as both a reverse transcriptase and DNA polymerase, in a single-tube, single-buffer, elevated temperature reaction. An internal standard Norwalk virus (NV) RNA control is added to each RT-PCR to identify sample inhibition, and thermolabile uracil N-glycosylase is incorporated into the reaction to prevent PCR product carryover contamination. Finally, RT-PCR-generated amplicons are detected in microtiter wells using virus-specific biotinylated oligoprobes in an enzyme-linked immunosorbent assay-based format. The DNA enzyme immunoassay is based on the capture of PCR product by biotinylated probes fixed onto individual streptavidin-coated wells. Using this method, low levels of NV were detected in stool and both NLV and hepatitis A virus were detected in bivalve mollusks following bioaccumulation. The method also successfully detected NLV in oysters implicated in an outbreak of NLV gastroenteritis. This method dramatically decreases the time needed for analysis and is amenable to automation. PMID:11157239

  20. Detection of Aspergillus galactomannan: comparison of an enzyme-linked immunoassay and a europium-linked time-resolved fluoroimmunoassay.

    PubMed

    Paugam, A; Sarfati, J; Romieu, R; Viguier, M; Dupouy-Camet, J; Latgé, J P

    1998-10-01

    With a view to improving the sensitivity of serological detection of Aspergillus galactomannan (GM), a europium-linked time-resolved fluoroimmunoassay was developed. This method was compared to an enzyme-linked immunosorbent assay using a peroxidase-conjugated detector antibody. No increase in the sensitivity of the detection of GM standards was seen with the europium-based fluoroimmunoassay. PMID:9738075

  1. Detection of Aspergillus Galactomannan: Comparison of an Enzyme-Linked Immunoassay and a Europium-Linked Time-Resolved Fluoroimmunoassay

    PubMed Central

    Paugam, A.; Sarfati, J.; Romieu, R.; Viguier, M.; Dupouy-Camet, J.; Latgé, J. P.

    1998-01-01

    With a view to improving the sensitivity of serological detection of Aspergillus galactomannan (GM), a europium-linked time-resolved fluoroimmunoassay was developed. This method was compared to an enzyme-linked immunosorbent assay using a peroxidase-conjugated detector antibody. No increase in the sensitivity of the detection of GM standards was seen with the europium-based fluoroimmunoassay. PMID:9738075

  2. Comparison of immunoglobulin A (IgA), IgG, and IgM enzyme-linked immunosorbent assays, plaque reduction neutralization assay, and complement fixation in detecting seroresponses to rotavirus vaccine candidates.

    PubMed Central

    Midthun, K; Pang, L Z; Flores, J; Kapikian, A Z

    1989-01-01

    In a phase 1 study to evaluate human-rhesus rotavirus reassortant vaccines, 116 infants 1 to 5 months of age received one of the following five preparations: the serotype 1 reassortant, the serotype 2 reassortant, rhesus rotavirus (serotype 3), a bivalent preparation (serotypes 1 and 3), or a placebo. Seroresponses to the different vaccines were measured by plaque reduction neutralization assay (PRNA); rotavirus-specific immunoglobulin A (IgA), IgG, and IgM enzyme-linked immunosorbent assays (ELISAs); and complement fixation (CF). The seroresponse rate, calculated by using a fourfold or greater antibody rise by any assay, was similar in the four vaccine groups (83 to 96%). When the data from all the vaccinees were pooled, IgA ELISA, IgG ELISA, and PRNA were comparable in detecting seroresponses (67, 62, and 70%, respectively) and more efficient than IgM ELISA (53%) and CF (44%). When the vaccinees were analyzed by age, the overall seroresponse rates were the same for infants 1 to 2 and 3 to 5 months old (90%). The IgA ELISA and PRNA were the most efficient for detecting antibody rises in both age groups. IgG ELISA was among the least efficient methods for detecting antibody rises in the younger age group but among the most efficient in the older age group (44 versus 78%). CF was among the least efficient methods in both age groups but was significantly better in the older age group than in the younger age group (54 versus 21%). Our findings show that ELISA, in particular rotavirus-specific IgA ELISA, is a sensitive indicator of vaccine takes in 1- to 5 month-old infants, the target population for vaccination. ELISA should also be very useful in demonstrating natural rotavirus infections in field studies in which a stool specimen from a diarrheal episode is not always available. The ELISA has the advantages of being easier and quicker and requiring less serum than PRNA, but it does not give serotype-specific information about the immune response. PMID:2556433

  3. Plasma cortisol and 11-ketotestosterone enzyme immunoassay (EIA) kit validation for three fish species: the orange clownfish Amphiprion percula, the orangefin anemonefish Amphiprion chrysopterus and the blacktip reef shark Carcharhinus melanopterus.

    PubMed

    Mills, S C; Mourier, J; Galzin, R

    2010-08-01

    Commercially available enzyme immunoassay (EIA) kits were validated for measuring steroid hormone concentrations in blood plasma from three fish species: the orange clownfish Amphiprion percula, the orangefin anemonefish Amphiprion chrysopterus and the blacktip reef shark Carcharhinus melanopterus. A minimum of 5 microl plasma was required to estimate hormone concentrations with both kits. These EIA kits are a simple method requiring minimal equipment, for measuring hormone profiles under field conditions. PMID:20701653

  4. Determination of zeranol and its metabolites in bovine muscle and liver by a chemiluminescence enzyme immunoassay: compared to an ultraperformance liquid chromatography tandem mass spectroscopy method.

    PubMed

    Haiyang, Jiang; Wenjun, Wang; Jinghui, Zhu; Xiaoqi, Tao; Jiancheng, Li; Xi, Xia; Kai, Wen; Fei, Xu; Zhaopeng, Wang; Min, Chen; Xiangmei, Li; Xiaoping, Wu; Shien, Wang; Shuangyang, Ding

    2014-06-01

    A chemiluminescent enzyme immunoassay (CLEIA) was compared to an ultraperformance liquid chromatography tandem mass spectroscopy (UPLC-MS/MS) procedure for the analysis of zeranol and its metabolites in bovine tissue samples. Apparent recoveries from fortified samples by both methods were comparable at 0.5-4.0 µg/kg and a significant correlation was obtained. For CLEIA analysis, hapten mimicking the analyte was first synthesized and conjugated with the carrier protein bovine serum albumin as the immunogen to produce monoclonal antibody. The obtained antibody showed extensive cross-reactivity toward zeranol metabolites (zearalanone). The limit of detection of CLEIA and UPLC-MS/MS was 0.05 µg/kg and 0.5 µg/kg, respectively. Recoveries of both methods for fortified samples were higher than 75.0% with the coefficient of variation less than 15%. These results indicated that the combination of screening with CLEIA and confirmation with UPLC-MS/MS for zeranol and its metabolites would be a reliable method for a large number of bovine samples. PMID:23934682

  5. Development of a nanobody-alkaline phosphatase fusion protein and its application in a highly sensitive direct competitive fluorescence enzyme immunoassay for detection of ochratoxin A in cereal

    PubMed Central

    Liu, Xing; Xu, Yang; Wan, De-bin; Xiong, Yong-hua; He, Zhen-yun; Wang, Xian-xian; Gee, Shirley J.; Ryu, Dojin; Hammock, Bruce D.

    2015-01-01

    A rapid and sensitive direct competitive fluorescence enzyme immunoassay (dc-FEIA) for ochratoxin A (OTA) based on a nanobody (Nb)-alkaline phosphatase (AP) fusion protein was developed. The VHH gene of Nb28 was subcloned into the expression vector pecan45 containing the AP double mutant gene. The Nb28-AP construct was transformed into E. coli BL21(DE3)plysS and soluble expression in bacteria was confirmed by SDS-PAGE and Western blot. Both the Nb properties and AP enzymatic activity were validated by colorimetric and fluorometric analysis. The 50% inhibitory concentration and the detection limit of the dc-FEIA were 0.13 ng/mL and 0.04 ng/mL, respectively, with a linear range of 0.060.43 ng/mL. This assay was compared with LC-MS/MS, and the results indicated the reliability of Nb-AP fusion protein-based dc-FEIA for monitoring OTA contamination in cereal. PMID:25531426

  6. Validation of an enzyme-immunoassay for the non-invasive monitoring of faecal testosterone metabolites in male cheetahs (Acinonyx jubatus).

    PubMed

    Pribbenow, Susanne; Wachter, Bettina; Ludwig, Carsten; Weigold, Annika; Dehnhard, Martin

    2016-03-01

    In mammals, the sex hormone testosterone is the major endocrine variable to objectify testicular activity and thus reproductive function in males. Testosterone is involved in the development and function of male reproductive physiology and sex-related behaviour. The development of a reliable androgen enzyme-immunoassay (EIA) to monitor faecal testosterone metabolites (fTM) is a powerful tool to non-invasively assess the gonadal status of males. We validated an epiandrosterone EIA for male cheetahs by performing a testosterone radiometabolism study followed by high-performance liquid chromatography (HPLC) analyses and excluding possible cross-reactivities with androgenic metabolites not derived from testosterone metabolism. The physiological and biological relevance of the epiandrosterone EIA was validated by demonstrating (1) a significant increase in fTM concentrations within one day in response to a testosterone injection, (2) a significant increase in fTM concentrations within one day in response to a gonadotropin-releasing hormone (GnRH) injection, which failed following a placebo injection, and (3) significant differences in fTM concentrations between adult male and adult female cheetahs and between adult and juvenile male cheetahs of a free-ranging population. Finally, we demonstrated stability of fTM concentrations measured in faecal samples exposed to ambient temperatures up to 72h. Our results clearly demonstrate that the epiandrosterone EIA is a reliable non-invasive method to monitor testicular activity in male cheetahs. PMID:26828820

  7. Development of a nanobody-alkaline phosphatase fusion protein and its application in a highly sensitive direct competitive fluorescence enzyme immunoassay for detection of ochratoxin A in cereal.

    PubMed

    Liu, Xing; Xu, Yang; Wan, De-bin; Xiong, Yong-hua; He, Zhen-yun; Wang, Xian-xian; Gee, Shirley J; Ryu, Dojin; Hammock, Bruce D

    2015-01-20

    A rapid and sensitive direct competitive fluorescence enzyme immunoassay (dc-FEIA) for ochratoxin A (OTA) based on a nanobody (Nb)-alkaline phosphatase (AP) fusion protein was developed. The VHH (variable domain of heavy chain antibody) gene of Nb28 was subcloned into the expression vector pecan45 containing the AP double-mutant gene. The Nb28-AP construct was transformed into Escherichia coli BL21(DE3)plysS, and soluble expression in bacteria was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot. Both the Nb properties and AP enzymatic activity were validated by colorimetric and fluorometric analysis. The 50% inhibitory concentration and the detection limit of the dc-FEIA were 0.13 and 0.04 ng/mL, respectively, with a linear range of 0.06-0.43 ng/mL. This assay was compared with LC-MS/MS, and the results indicated the reliability of Nb-AP fusion protein-based dc-FEIA for monitoring OTA contamination in cereal. PMID:25531426

  8. Evaluation of recombinant outer membrane protein C based indirect enzyme-linked immunoassay for the detection of Salmonella antibodies in poultry

    PubMed Central

    Manoj, Jinu; Agarwal, Rajesh K.; Sailo, Blessa; Wani, Mudasir Ahmed; Singh, Manoj Kumar

    2015-01-01

    Aim: To evaluate the efficacy of recombinant outer membrane proteinC (rOmpC) based enzyme-linked immunoassay (ELISA) for the diagnosis of salmonellosis in poultry. Materials and Methods: Three antigens were prepared, and the indirect ELISA was standardized using the antigens and the antiserum raised in chicken against Omp and rOmpC. Sera were collected from a total of 255 apparently healthy field chickens and screened for the presence of Salmonella antibodies by this ELISA. Results: The sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of Omp revealed major polypeptides at 36, 42 and 52 kDa, and the rOmpC was evident by a single protein band of 43 kDa. The Omp and rOmpC antigen revealed an optimum concentration of 78 and 156 ng, respectively, in the assay, while the whole cell antigen gave an optimum reaction at a concentration of 106 organisms/ml. The test was found to be specific as it did not react with any of the antisera of seven other organisms. The developed ELISA detected Salmonella antibodies from 22 (8.62%) samples with rOmpC antigen, while 24 (9.41%) samples gave a positive reaction with both Omp and whole cell antigens. Conclusion: We suggest rOmpC based indirect ELISA as a suitable screening tool for serological monitoring of poultry flocks.

  9. Magnetic affinity enzyme-linked immunoassay based on recombinant 26 kDa glutathione-S-transferase for serological diagnosis of schistosomiasis japonica.

    PubMed

    Yu, Qin; Yang, Hai; Feng, Youmei; Yang, Xiangliang; Zhu, Yanhong

    2012-12-01

    Schistosomiasis remains a serious worldwide public health problem. Improving the diagnostic assay for surveillance and monitoring will contribute to hastening the possible elimination of the disease in endemic regions. Therefore, this study aims to develop magnetic affinity enzyme-linked immunoassay (MEIA) for serological diagnosis of schistosomiasis based on recombinant 26kDa glutathione-S-transferase of Schistosoma japonicum (rSj26GST). BALB/c mice infected with S. japonicum cercariae (40 per mouse) were used. After infecting for 6 weeks, the antibody was detected by MEIA. All of the infected mouse sera were effectively determined by MEIA. Compared with the enzyme-linked immunosorbent assay (ELISA), MEIA has a higher ratio of the mean positive value to the mean negative value (P/N) at the same dilution ratio (3.92 versus 2.66). MEIA was further applied for diagnosis of human schistosomiasis. Sera from 28 schistosomiasis-confirmed patients with low-intensity infection, 15 treated patients, and 20 non-endemic negative controls, were used to assess the assay. The results showed that MEIA and ELISA had similarity in positive detection rates. However, the higher P/N of MEIA was observed at the same dilution ratio. MEIA had high negative rate in detection of specific IgG in the treated patients. Moreover, there was no cross reaction with the sera of paragonimiasis patients. These results suggested that MEIA based on rSj26GST is a simple, rapid, convenient assay for the diagnosis of schistosomiasis. PMID:22940100

  10. Detection and serogroup differentiation of Salmonella spp. in food within 30 hours by enrichment-immunoassay with a T6 monoclonal antibody capture enzyme-linked immunosorbent assay.

    PubMed Central

    Ng, S P; Tsui, C O; Roberts, D; Chau, P Y; Ng, M H

    1996-01-01

    We previously described an antigen capture enzyme-linked immunosorbent assay which makes use of monoclonal antibody T6, which recognizes an epitope on the outer core polysaccharide of Salmonella lipopolysaccharide molecules that is common to almost all Salmonella serovars. In this paper, we show that this assay can detect between 10(5) and 10(7) Salmonella cells per ml even in the presence of excess Escherichia coli. A total of 153 of 154 (99%) serogroup A to E strains and 51 of 78 (71%) serogroup F to 67 strains were reactive as determined by this assay. This corresponds to a detection rate of approximately 98% of all salmonellae known to affect humans. None of the 65 strains of non-Salmonella bacteria tested positive. Taking advantage of the O-factor polysaccharides also present on the antigen captured by the immobilized T6 antibody, we showed that 136 of 154 Salmonella serogroup A to E strains (88%) were correctly differentiated according to their serogroups by use of enzyme conjugates of a panel of O-factor-specific monoclonal antibodies. We evaluated this assay for the detection and serogroup differentiation of salmonellae directly from enrichment cultures of simulated food, eggs, pork, and infant formula milk. All 26 samples which had been contaminated with Salmonella spp. were detected by T6 (100% sensitivity), with only one false-positive result from 101 samples not contaminated by Salmonella spp. (99% specificity). The detection time was substantially reduced to between 17 and 29 h, depending on the enrichment methods used. Since there were no false-negative results, we concluded that this enrichment-immunoassay method can afford rapid screening for Salmonella spp. in food samples. PMID:8779567

  11. Performance of a Limiting-Antigen Avidity Enzyme Immunoassay for Cross-Sectional Estimation of HIV Incidence in the United States

    PubMed Central

    Konikoff, Jacob; Brookmeyer, Ron; Longosz, Andrew F.; Cousins, Matthew M.; Celum, Connie; Buchbinder, Susan P.; Seage, George R.; Kirk, Gregory D.; Moore, Richard D.; Mehta, Shruti H.; Margolick, Joseph B.; Brown, Joelle; Mayer, Kenneth H.; Koblin, Beryl A.; Justman, Jessica E.; Hodder, Sally L.; Quinn, Thomas C.; Eshleman, Susan H.; Laeyendecker, Oliver

    2013-01-01

    Background A limiting antigen avidity enzyme immunoassay (HIV-1 LAg-Avidity assay) was recently developed for cross-sectional HIV incidence estimation. We evaluated the performance of the LAg-Avidity assay alone and in multi-assay algorithms (MAAs) that included other biomarkers. Methods and Findings Performance of testing algorithms was evaluated using 2,282 samples from individuals in the United States collected 1 month to >8 years after HIV seroconversion. The capacity of selected testing algorithms to accurately estimate incidence was evaluated in three longitudinal cohorts. When used in a single-assay format, the LAg-Avidity assay classified some individuals infected >5 years as assay positive and failed to provide reliable incidence estimates in cohorts that included individuals with long-term infections. We evaluated >500,000 testing algorithms, that included the LAg-Avidity assay alone and MAAs with other biomarkers (BED capture immunoassay [BED-CEIA], BioRad-Avidity assay, HIV viral load, CD4 cell count), varying the assays and assay cutoffs. We identified an optimized 2-assay MAA that included the LAg-Avidity and BioRad-Avidity assays, and an optimized 4-assay MAA that included those assays, as well as HIV viral load and CD4 cell count. The two optimized MAAs classified all 845 samples from individuals infected >5 years as MAA negative and estimated incidence within a year of sample collection. These two MAAs produced incidence estimates that were consistent with those from longitudinal follow-up of cohorts. A comparison of the laboratory assay costs of the MAAs was also performed, and we found that the costs associated with the optimal two assay MAA were substantially less than with the four assay MAA. Conclusions The LAg-Avidity assay did not perform well in a single-assay format, regardless of the assay cutoff. MAAs that include the LAg-Avidity and BioRad-Avidity assays, with or without viral load and CD4 cell count, provide accurate incidence estimates. PMID:24386116

  12. Identification of specific antinuclear antibodies in dogs using a line immunoassay and enzyme-linked immunosorbent assay.

    PubMed

    Bremer, Hanna D; Lattwein, Erik; Renneker, Stefanie; Lilliehöök, Inger; Rönnelid, Johan; Hansson-Hamlin, Helene

    2015-12-15

    Circulating antinuclear antibodies (ANA) are commonly present in the systemic autoimmune disease Systemic Lupus Erythematosus (SLE) and in other systemic rheumatic diseases, in humans as well as in dogs. The indirect immunofluorescence (IIF)-ANA test is the standard method for detecting ANA. Further testing for specific ANA with immunoblot techniques or ELISAs is routinely performed in humans to aid in the diagnosis and monitoring of disease. Several specific ANA identified in humans have been identified also in suspected canine SLE but, in contrast to humans, investigation of autoantibodies in canine SLE is mainly restricted to the IIF-ANA test. Our aim was to identify both known and novel specific ANA in dogs and to investigate if different IIF-ANA patterns are associated with different specific ANA in dogs. Sera from 240 dogs with suspicion of autoimmune disease (210 IIF-ANA positive (ANA(pos)) and 30 IIF-ANA negative (ANA(neg))) as well as sera from 27 healthy controls were included. The samples were analysed with a line immunoassay, LIA (Euroline ANA Profile 5, Euroimmun, Lübeck, Germany) and four different ELISAs (Euroimmun). The ANA(pos) dogs were divided in two groups depending on the type of IIF-ANA pattern. Of the 210 ANA(pos) samples 68 were classified as ANA homogenous (ANA(H)) and 141 as ANA speckled (ANA(S)), one sample was not possible to classify. Dogs in the ANA(H) group had, compared to the other groups, most frequently high levels of anti-double stranded deoxyribonucleic acid (dsDNA) and anti-nucleosome ANA. Anti-dsDNA antibodies were confirmed in some dogs with the Crithidia luciliae indirect immunofluorescence test (CLIFT). The frequency of ANA(H) dogs with values above those observed in the healthy group was significantly higher compared to ANA(S) dogs for anti-dsDNA, anti-nucleosome, and anti-histone reactivity. Dogs in the ANA(S) group had, compared to the other groups, most frequently high levels of anti-ribonucleoproteins (RNP) and/or anti-Smith (Sm) antibodies. Reactivity against Sjögren's syndrome related antigens (SS)-A (including the Ro-60 and Ro-52 subcomponents), SS-B, histidyl tRNA synthetase (Jo-1), topoisomerase I antigen (Scl-70), polymyositis-scleroderma antigen (PM-Scl) and proliferating cell nuclear antigen (PCNA) was also noted in individual dogs. In conclusion, by using a commercial LIA and different ELISAs originally developed for detection of human ANA, we identified several specific ANA in serum samples from dogs sampled for IIF-ANA testing. Further, we found that the types of IIF-ANA pattern were associated with reactivity against some particular nuclear antigens. PMID:26547884

  13. Use of the INNO-LIA syphilis score assay in the resolution of discordant positive screening enzyme immunoassay results for the serological diagnosis of syphilis.

    PubMed

    Lam, T K; Lau, H Y; Lee, Y P; Fung, S M; Leung, W L; Kam, K M

    2014-01-01

    We studied the use of the INNO-LIA syphilis score assay in the resolution of discordant positive screening results of the Murex ICE Syphilis enzyme immunoassay (EIA) with the confirmatory results of both the Serodia Treponema pallidum particle agglutination (TPPA) and the fluorescent treponemal antibody-absorption (FTA-Abs) assays, for the serological diagnosis of syphilis. This was an observational study on the serum samples received by the Syphilis Laboratory, Hong Kong, during the period from January 2006 to December 2012. A total of 801 serum samples with discordant positive screening EIA results were used. Consensus results of such serum samples were derived from results of the EIA, TPPA and FTA-abs assays. The age range of the individuals was 14 to 104 years (median of 52). There were 369 males and 432 females. Of 378 serum samples, 139 showed agreement among positive results, 23 of 310 showed agreement among indeterminate results and 277 of 465 showed agreement among negative results. The proportions of agreement among positive, indeterminate and negative results were 0.37 (95% CI 0.32-0.42), 0.07 (95% CI 0.05-0.11) and 0.60 (95% CI 0.55-0.64), respectively; kappa 0.55 (95% CI 0.49-0.60). There were 60 serum samples with positive consensus results but negative INNO-LIA syphilis score results and 10 with negative consensus results but positive INNO-LIA syphilis score results. Although the INNO-LIA syphilis score assay can be considered a valid alternative confirmatory test for the serological diagnosis of syphilis, the present study showed that its use in the resolution of discordant positive screening EIA results was moderate. A more extensive characterization of serum samples with discordant reactive screening treponemal test results is necessary. PMID:23970631

  14. Assessment of the stress response in Columbian ground squirrels: laboratory and field validation of an enzyme immunoassay for fecal cortisol metabolites.

    PubMed

    Bosson, Curtis O; Palme, Rupert; Boonstra, Rudy

    2009-01-01

    Stress responses play a critical role in the ecology and demography of wild animals, and the analysis of fecal hormone metabolites is a powerful noninvasive method to assess the role of stress. We characterized the metabolites of injected radiolabeled cortisol in the urine and feces of Columbian ground squirrels and validated an enzyme immunoassay for measuring fecal cortisol metabolites (FCM) with a 5 alpha-3beta,11 beta-diol structure by stimulation and suppression of adrenocortical activity and by evaluation of the circadian pattern of FCM excretion. In addition, we also evaluated the impact of capture, handling, and acclimation to the laboratory on FCM. Cortisol is highly metabolized, with virtually none being excreted, and of the radiolabeled cortisol injected, 31% was recovered in urine and 6.5% in feces. The lag time between cortisol injection and its appearance in urine and feces was 4.5 +/- 0.82 (SE) h and 7.0 +/- 0.53 (SE) h, respectively. FCM levels varied over the day, reflecting circadian variation in endogenous cortisol. Dexamethasone decreased FCM levels by 33%, and ACTH increased them by 255%. Trapping and housing initially increased FCM levels and decreased body mass, but these reversed within 3-7 d, indicating acclimation. Finally, FCM levels were modestly repeatable over time (r=0.57) in wild, live trapped, nonbreeding animals, indicating that FCMs provide a measure of the squirrel's stress-axis state. This assay provides a robust noninvasive assessment of the stress response of the Columbian ground squirrel and will facilitate an integration of its life history and physiology. PMID:19335228

  15. Characterisation and validation of an enzyme-immunoassay for the non-invasive assessment of faecal glucocorticoid metabolites in cheetahs (Acinonyx jubatus).

    PubMed

    Ludwig, C; Wachter, B; Silinski-Mehr, S; Ganswindt, A; Bertschinger, H; Hofer, H; Dehnhard, M

    2013-01-01

    The non-invasive measurement of adrenocortical function in cheetahs is an important tool to assess stress in captive and free-ranging individuals, because stress has been suggested to be one of the causes of poor reproductive performance of captive cheetahs. We tested four enzyme immunoassays (EIA) in two captive cheetahs in Germany using adrenocorticotropic hormone (ACTH) challenges and identified the corticosterone-3-CMO EIA to be most sensitive to the increase in faecal glucocorticoid metabolite (fGCM) concentrations after the ACTH challenge. This EIA performed also well in five captive cheetahs in South Africa. The fGCM concentrations across all seven cheetahs increased within 24h by 681% compared to the baseline levels prior to ACTH. Storage of faecal samples at 0-4°C did not strongly affect fGCM concentrations within 24h, simplifying sample collection when immediate storage at -20°C is not feasible. The two cheetahs in Germany also received an injection of [(3)H]cortisol to characterise fGCMs in faecal extracts using high-performance liquid chromatography (HPLC) immunograms. HPLC fractions were measured for their radioactivity and immunoreactive fGCM concentrations with the corticosterone-3-CMO EIA, respectively. The results revealed a polar peak of radiolabelled cortisol metabolites co-eluting with the major peak of immunoreactive fGCMs. Thus, our EIA measured substantial amounts of fGCMs corresponding to the radioactive peaks. The peaks were of higher polarity than native cortisol and corticosterone, suggesting that the metabolites were conjugated, which was confirmed by solvolysis of the HPLC fractions. Our results show that the corticosterone-3-CMO EIA is a reliable tool to assess fGCMs in cheetahs. PMID:23108105

  16. Validation of an enzyme immunoassay for the measurement of faecal glucocorticoid metabolites in Eurasian (Lynx lynx) and Iberian lynx (Lynx pardinus).

    PubMed

    Pribbenow, Susanne; Jewgenow, Katarina; Vargas, Astrid; Serra, Rodrigo; Naidenko, Sergey; Dehnhard, Martin

    2014-09-15

    Stress hormone levels are important indicator of an animal's well-being, as stress has harmful effects on reproduction, growth and immune function. The development of enzyme immunoassays (EIA) to monitor faecal glucocorticoid metabolites (fGM) contributes a powerful tool to assess an animal's adrenal status non-invasively. We aimed to identify a suitable EIA for monitoring fGM by assessing the suitability of six different EIAs for detecting quantitative changes in fGM concentrations in response to an ACTH challenge test in Eurasian lynx. FGM were characterised in a male Eurasian lynx that received an injection of (3)H-cortisol. Using HPLC analyses radiolabeled metabolites were compared with immunoreactive metabolites. The second aim was to biologically validate the established EIA for monitoring adrenocortical activity of captive Iberian lynxes after a translocation to new enclosures in relation to behaviour. Additionally faecal samples of ten pregnant Iberian lynxes from the peripartal period were analysed. The ACTH challenge revealed an 11β-hydroxyetiocholanolone EIA as the most sensitive assay to reflect acute fGM elevations in the Eurasian lynx. HPLC immunograms demonstrated that the 11β-hydroxyetiocholanolone EIA measured significant amounts of immunoreactivities corresponding to radiolabeled metabolites with strong similarities across both lynx species. Additionally, HPLC and GC-MS analyses confirmed the presence of 11β-hydroxyetiocholanolone in faeces of both, the Eurasian and the Iberian lynx. Longitudinal fGM profiles of Iberian lynx revealed increases in concentrations associated with management events. During the peripartal period, however, fGM concentrations were not significantly elevated. Our results show that the 11β-hydroxyetiocholanolone EIA is a reliable tool to assess fGM in both lynx species. PMID:25066418

  17. Quantifying estrogen metabolism: an evaluation of the reproducibility and validity of enzyme immunoassays for 2-hydroxyestrone and 16alpha-hydroxyestrone in urine.

    PubMed Central

    Ziegler, R G; Rossi, S C; Fears, T R; Bradlow, H L; Adlercreutz, H; Sepkovic, D; Kiuru, P; Wahala, K; Vaught, J B; Donaldson, J L; Falk, R T; Fillmore, C M; Siiteri, P K; Hoover, R N; Gail, M H

    1997-01-01

    Rapid and simple enzyme immunoassays (EIAs) were recently developed to measure 2-hydroxyestrone and 16alpha-hydroxyestrone in unextracted urine. The balance between these competing estrogen metabolism pathways may serve as a biomarker of breast cancer risk. Before testing these assays in epidemiologic studies, we evaluated their reproducibility, and validity relative to gas chromatography-mass spectroscopy (GC-MS). Overnight 12-hr urine collections from five midfollicular premenopausal women, five midluteal premenopausal women, and five postmenopausal women were aliquoted and stored at -70 degrees C. Two aliquots from each woman were assayed with the EIAs in a random, blinded order, monthly over 4 months and 1 year later. Reproducibility over 4 months was good for both metabolites in premenopausal women (coefficient of variation = 8-14%) and satisfactory in postmenopausal women (approximately 19%). Reproducibility over 12 months remained good in premenopausal women, but was poor in postmenopausal women, with mean readings increasing 50 to 100%. Wide variation in estrogen metabolite levels enabled a single EIA measurement to characterize individual differences among premenopausal women in midfollicular (intraclass correlation coefficient = 98-99%) and midluteal phase (85-91%). A narrower range in metabolite levels among postmenopausal women reduced discrimination (78-82%). The correlation between EIA and GC-MS measurement was excellent for both metabolites (r>0.9), except for 2-hydroxyestrone in postmenopausal women (r=0.6). Analysis of absolute agreement suggested that both EIAs were less sensitive than GC-MS, and each detected nonspecific background. The low concentration of estrogen metabolites in urine from postmenopausal women may explain the problems with reproducibility and validity in this menstrual group. Accordingly, more sensitive EIAs have been developed and are now being evaluated. PMID:9168003

  18. Characterization and Comparison of Galactomannan Enzyme Immunoassay and Quantitative Real-Time PCR Assay for Detection of Aspergillus fumigatus in Bronchoalveolar Lavage Fluid from Experimental Invasive Pulmonary Aspergillosis

    PubMed Central

    Francesconi, Andrea; Kasai, Miki; Petraitiene, Ruta; Petraitis, Vidmantas; Kelaher, Amy M.; Schaufele, Robert; Hope, William W.; Shea, Yvonne R.; Bacher, John; Walsh, Thomas J.

    2006-01-01

    Bronchoalveolar lavage (BAL) is widely used for evaluation of patients with suspected invasive pulmonary aspergillosis (IPA). However, the diagnostic yield of BAL for detection of IPA by culture and direct examination is limited. Earlier diagnosis may be facilitated by assays that can detect Aspergillus galactomannan antigen or DNA in BAL fluid. We therefore characterized and compared the diagnostic yields of a galactomannan enzyme immunoassay (GM EIA), quantitative real-time PCR (qPCR), and quantitative cultures in experiments using BAL fluid from neutropenic rabbits with experimentally induced IPA defined as microbiologically and histologically evident invasion. The qPCR assay targeted the rRNA gene complex of Aspergillus fumigatus. The GM EIA and qPCR assay were characterized by receiver operator curve analysis. With an optimal cutoff of 0.75, the GM EIA had a sensitivity and specificity of 100% in untreated controls. A decline in sensitivity (92%) was observed when antifungal therapy (AFT) was administered. The optimal cutoff for qPCR was a crossover of 36 cycles, with sensitivity and specificity of 80% and 100%, respectively. The sensitivity of qPCR also decreased with AFT to 50%. Quantitative culture of BAL had a sensitivity of 46% and a specificity of 100%. The sensitivity of quantitative culture decreased with AFT to 16%. The GM EIA and qPCR assay had greater sensitivity than culture in detection of A. fumigatus in BAL fluid in experimentally induced IPA (P ± 0.04). Use of the GM EIA and qPCR assay in conjunction with culture-based diagnostic methods applied to BAL fluid could facilitate accurate diagnosis and more-timely initiation of specific therapy. PMID:16825367

  19. Validation of an enzyme immunoassay for assessing adrenocortical activity and evaluation of factors that affect levels of fecal glucocorticoid metabolites in two New World primates.

    PubMed

    Rimbach, Rebecca; Heymann, Eckhard W; Link, Andrés; Heistermann, Michael

    2013-09-15

    Non-invasive methods to assess stress hormone output via fecal glucocorticoid metabolites (FGCMs) have become a powerful tool in behavioral studies and conservation biology because they allow exploring the link between behavior, an animal's socio-ecological environment and its adrenocortical activity. However, FGCM levels are influenced by numerous other factors which often confound their interpretation. Thus, before applying these methods, knowledge on the impact of these factors is important. In this study we investigated the effect of (1) time of day, (2) age, (3) sex and (4) female reproductive state on FGCM levels in brown spider monkeys (Ateles hybridus) and red howler monkeys (Alouatta seniculus). Initially, we validated a 11β-hydroxyetiocholanolone enzyme immunoassay for monitoring the physiological stress response via fecal analysis in both species. We determined FGCM levels in fecal samples collected from two and six groups of wild spider monkeys (n=461 samples) and howler monkeys (n=166 samples), respectively. Our analyses revealed a strong effect of time of day on FGCM levels in spider monkeys, but no effect in howler monkeys. Adults of both species had significantly higher FGCM levels than subadults. In neither of the two species we found a sex-effect on FGCM output. Reproductive condition strongly affected FGCM levels in female spider monkeys which showed increasing concentrations with progressing gestation. This was not investigated in female howler monkeys due to an insufficient sample size. Our data indicate that the influence of the tested factors on fecal glucocorticoid metabolite output is species-specific, and that these variables need to be considered when interpreting FGCM levels in the species. PMID:23707497

  20. Development of highly sensitive chemiluminescence enzyme immunoassay based on the anti-recombinant H(C) subunit of botulinum neurotoxin type A monoclonal antibodies.

    PubMed

    Liu, Zhijia; Song, Chaojun; Li, Yongming; Liu, Fei; Zhang, Kui; Sun, Yuanjie; Li, Haitao; Wei, Yuying; Xu, Zhuwei; Zhang, Chunmei; Yang, Angang; Xu, Zhikai; Yang, Kun; Jin, Boquan

    2012-07-20

    Botulinum neurotoxins (BoNTs) are the most poisonous substances ever known. The early detection of these toxins could bear more time for appropriate medical intervention. The standard method for detecting BoNTs is the mouse bioassay, which is time consuming (up to 4 days) and requires a large number of laboratory animals. The immunologic detection methods could detect the toxins within a day, but most of these methods are less sensitive compared with the mouse bioassay due to the lack of high-affinity antibodies. Recently, the recombinant H(C) subunit of botulinum neurotoxin type A (rAH(C)) was expressed as an effective vaccine against botulism, indicating that the rAH(C) could be an effective immunogen that raises the monoclonal antibody (mAb) for detecting BoNT/A. After immunized BALB/c mice with rAH(C), 56 mAbs were generated. Two of these mAbs were selected to establish a highly sensitive sandwich chemiluminescence enzyme immunoassay (CLEIA), in which FMMU-BTA-49 and FMMU-BTA-22 were used as capture antibody and detection antibody, respectively. The calculated limit of detection (LOD) based on molecular weight of rAH(C) and BoNT/A reached 0.45 pg mL(-1). This CLEIA can be used in the detection of BoNT/A in matrices such as milk and beef extract. This method has 20-40 fold lower LOD than that of the mouse bioassay and takes only 3 h to complete the detection, indicating that it can be used as a valuable method to detect and quantify BoNT/A. PMID:22713913

  1. An Ultra-Sensitive Monoclonal Antibody-Based Competitive Enzyme Immunoassay for Sterigmatocystin in Cereal and Oil Products

    PubMed Central

    Li, Min; Li, Peiwu; Wu, Hui; Zhang, Qi; Ma, Fei; Zhang, Zhaowei; Ding, Xiaoxia; Wang, Hengling

    2014-01-01

    Sterigmatocystin (STG), a biosynthesis precursor of aflatoxin B1, is well known for its toxic and carcinogenic effects in humans and animals. STG derivatives and protein conjugates are needed for generation of monoclonal antibodies (mAbs). This work describes a reliable and fast synthesis of novel STG derivatives, based on which novel STG bovine serum albumin conjugates were prepared. With the novel STG bovine serum albumin conjugates, three sensitive and specific mAbs against STG, named VerA 3, VerA 4, and VerA 6, were prepared by semi-solid hypoxanthine/aminopterin/thymidine (HAT) medium using a modified two-step screening procedure. They exhibited high affinity for STG and no cross-reactivity (CR) with aflatoxins B1, B2, G1, G2, and M1. Based on the most sensitive antibody VerA 3, an ultra-sensitive competitive enzyme-linked immunosorbent assay (ELISA) was developed for STG in wheat, maize, and peanuts. Assays were performed in the STG-GA-BSA-coated (0.5 µg·mL−1) ELISA format, in which the antibody was diluted to 1∶80,000. Several physicochemical factors influencing assay performance, such as pH, ionic strength, blocking solution, and diluting solution, were optimized. The final results showed that the assays had the detection limits of 0.08 ng·g−1 for wheat, 0.06 ng·g−1 for maize, and 0.1 ng·g−1 for peanuts, inter-assay and intra-assay variations of less than 10%, and recoveries ranging from 83% to 110%. These recoveries were in good agreement with those obtained by using HPLC-MS/MS method (90–104%), indicating the importance of the mAb VerA 3 in the study of STG in crude agricultural products. PMID:25184275

  2. An ultra-sensitive monoclonal antibody-based competitive enzyme immunoassay for sterigmatocystin in cereal and oil products.

    PubMed

    Li, Min; Li, Peiwu; Wu, Hui; Zhang, Qi; Ma, Fei; Zhang, Zhaowei; Ding, Xiaoxia; Wang, Hengling

    2014-01-01

    Sterigmatocystin (STG), a biosynthesis precursor of aflatoxin B1, is well known for its toxic and carcinogenic effects in humans and animals. STG derivatives and protein conjugates are needed for generation of monoclonal antibodies (mAbs). This work describes a reliable and fast synthesis of novel STG derivatives, based on which novel STG bovine serum albumin conjugates were prepared. With the novel STG bovine serum albumin conjugates, three sensitive and specific mAbs against STG, named VerA 3, VerA 4, and VerA 6, were prepared by semi-solid hypoxanthine/aminopterin/thymidine (HAT) medium using a modified two-step screening procedure. They exhibited high affinity for STG and no cross-reactivity (CR) with aflatoxins B1, B2, G1, G2, and M1. Based on the most sensitive antibody VerA 3, an ultra-sensitive competitive enzyme-linked immunosorbent assay (ELISA) was developed for STG in wheat, maize, and peanuts. Assays were performed in the STG-GA-BSA-coated (0.5 µg · mL(-1)) ELISA format, in which the antibody was diluted to 1:80,000. Several physicochemical factors influencing assay performance, such as pH, ionic strength, blocking solution, and diluting solution, were optimized. The final results showed that the assays had the detection limits of 0.08 ng · g(-1) for wheat, 0.06 ng · g(-1) for maize, and 0.1 ng · g(-1) for peanuts, inter-assay and intra-assay variations of less than 10%, and recoveries ranging from 83% to 110%. These recoveries were in good agreement with those obtained by using HPLC-MS/MS method (90-104%), indicating the importance of the mAb VerA 3 in the study of STG in crude agricultural products. PMID:25184275

  3. Survey of immunoassay techniques for biological analysis

    SciTech Connect

    Burtis, C.A.

    1986-10-01

    Immunoassay is a very specific, sensitive, and widely applicable analytical technique. Recent advances in genetic engineering have led to the development of monoclonal antibodies which further improves the specificity of immunoassays. Originally, radioisotopes were used to label the antigens and antibodies used in immunoassays. However, in the last decade, numerous types of immunoassays have been developed which utilize enzymes and fluorescent dyes as labels. Given the technical, safety, health, and disposal problems associated with using radioisotopes, immunoassays that utilize the enzyme and fluorescent labels are rapidly replacing those using radioisotope labels. These newer techniques are as sensitive, are easily automated, have stable reagents, and do not have a disposal problem. 6 refs., 1 fig., 2 tabs.

  4. Measurement of Chlamydia pneumoniae-Specific Immunoglobulin A (IgA) Antibodies by the Microimmunofluorescence (MIF) Method: Comparison of Seven Fluorescein-Labeled Anti-Human IgA Conjugates in an In-House MIF Test Using One Commercial MIF and One Enzyme Immunoassay Kit

    PubMed Central

    Paldanius, Mika; Bloigu, Aini; Leinonen, Maija; Saikku, Pekka

    2003-01-01

    For the serological diagnosis of acute Chlamydia pneumoniae infection, the microimmunofluorescence (MIF) test is the most commonly used method and also the “gold standard” for the measurement of immunoglobulin G (IgG) and IgM antibodies. The role of IgA antibodies in diagnosis has not been established. Commercially available fluorescein-labeled anti-human IgA conjugates have not been systematically compared to each other, and this situation may cause considerable variations in IgA results. Therefore, we tested 261 serum samples from 122 patients with pneumonia for IgA antibodies by using six α-chain-specific anti-IgA conjugates in our in-house MIF test, one commercial MIF test, and one enzyme immunoassay (EIA). Interfering IgG antibodies were removed with Gullsorb reagent before the measurement of IgA antibodies. Altogether, 14 significant IgA antibody increases in serum samples between the acute phase and the convalescent phase were detected by at least one of the conjugates in the MIF test, while no increases were found in the IgA EIA. Only one patient showed a significant IgA antibody increase with all of the fluorescein-labeled conjugates. Five significant titer changes were detected by at least two conjugates, and in nine instances, the titer increase was detected by one conjugate only. The titer agreement indicated by kappa coefficients was very good or good for all of the fluorescein-labeled conjugates and the EIA with low antibody titers but decreased with increasing titers. PMID:12522032

  5. Establishment of an Algorithm Using prM/E- and NS1-Specific IgM Antibody-Capture Enzyme-Linked Immunosorbent Assays in Diagnosis of Japanese Encephalitis Virus and West Nile Virus Infections in Humans.

    PubMed

    Galula, Jedhan U; Chang, Gwong-Jen J; Chuang, Shih-Te; Chao, Day-Yu

    2016-02-01

    The front-line assay for the presumptive serodiagnosis of acute Japanese encephalitis virus (JEV) and West Nile virus (WNV) infections is the premembrane/envelope (prM/E)-specific IgM antibody-capture enzyme-linked immunosorbent assay (MAC-ELISA). Due to antibody cross-reactivity, MAC-ELISA-positive samples may be confirmed with a time-consuming plaque reduction neutralization test (PRNT). In the present study, we applied a previously developed anti-nonstructural protein 1 (NS1)-specific MAC-ELISA (NS1-MAC-ELISA) on archived acute-phase serum specimens from patients with confirmed JEV and WNV infections and compared the results with prM/E containing virus-like particle-specific MAC-ELISA (VLP-MAC-ELISA). Paired-receiver operating characteristic (ROC) curve analyses revealed no statistical differences in the overall assay performances of the VLP- and NS1-MAC-ELISAs. The two methods had high sensitivities of 100% but slightly lower specificities that ranged between 80% and 100%. When the NS1-MAC-ELISA was used to confirm positive results in the VLP-MAC-ELISA, the specificity of serodiagnosis, especially for JEV infection, was increased to 90% when applied in areas where JEV cocirculates with WNV, or to 100% when applied in areas that were endemic for JEV. The results also showed that using multiple antigens could resolve the cross-reactivity in the assays. Significantly higher positive-to-negative (P/N) values were consistently obtained with the homologous antigens than those with the heterologous antigens. JEV or WNV was reliably identified as the currently infecting flavivirus by a higher ratio of JEV-to-WNV P/N values or vice versa. In summary of the above-described results, the diagnostic algorithm combining the use of multiantigen VLP- and NS1-MAC-ELISAs was developed and can be practically applied to obtain a more specific and reliable result for the serodiagnosis of JEV and WNV infections without the need for PRNT. The developed algorithm should provide great utility in diagnostic and surveillance activities in which test accuracy is of utmost importance for effective disease intervention. PMID:26659204

  6. Application of a Newly Developed High-Sensitivity HBsAg Chemiluminescent Enzyme Immunoassay for Hepatitis B Patients with HBsAg Seroclearance

    PubMed Central

    Shinkai, Noboru; Matsuura, Kentaro; Sugauchi, Fuminaka; Watanabe, Tsunamasa; Murakami, Shuko; Iio, Etsuko; Ogawa, Shintaro; Nojiri, Shunsuke; Joh, Takashi

    2013-01-01

    We modified and automated a highly sensitive chemiluminescent enzyme immunoassay (CLEIA) for surface antigen (HBsAg) detection using a combination of monoclonal antibodies, each for a specific epitope of HBsAg, and by improving an earlier conjugation technique. Of 471 hepatitis B virus (HBV) carriers seen in our hospital between 2009 and 2012, 26 were HBsAg seronegative as determined by the Abbott Architect assay. The Lumipulse HBsAg-HQ assay was used to recheck those 26 patients who demonstrated seroclearance by the Abbott Architect assay. The performance of the Lumipulse HBsAg-HQ assay was compared with that of a quantitative HBsAg detection system (Abbott Architect) and the Roche Cobas TaqMan HBV DNA assay (CTM) (lower limit of detection, 2.1 log copies/ml) using blood serum samples from patients who were determined to be HBsAg seronegative by the Abbott Architect assay. Ten patients had spontaneous HBsAg loss. Of 8 patients treated with nucleotide analogues (NAs), two were HBsAg seronegative after stopping lamivudine therapy and 6 were HBsAg seronegative during entecavir therapy. Eight acute hepatitis B (AH) patients became HBsAg seronegative. Of the 26 patients, 16 were HBsAg positive by the Lumipulse HBsAg-HQ assay but negative by the Abbott Architect assay. The differences between the two assays in terms of detectable HBsAg persisted over the long term in the spontaneous loss group (median, 10 months), the NA-treated group (2.5 months), and the AH group (0.5 months). In 9 patients, the Lumipulse HBsAg-HQ assay detected HBsAg when HBV DNA was negative by the CTM assay. HBsAg was also detected by the Lumipulse HBsAg-HQ assay in 4 patients with an anti-HBs concentration of >10 mIU/ml, 3 of whom had no HBsAg escape mutations. The automatic, highly sensitive HBsAg CLEIA Lumipulse HBsAg-HQ is a convenient and precise assay for HBV monitoring. PMID:23946517

  7. Application of a newly developed high-sensitivity HBsAg chemiluminescent enzyme immunoassay for hepatitis B patients with HBsAg seroclearance.

    PubMed

    Shinkai, Noboru; Matsuura, Kentaro; Sugauchi, Fuminaka; Watanabe, Tsunamasa; Murakami, Shuko; Iio, Etsuko; Ogawa, Shintaro; Nojiri, Shunsuke; Joh, Takashi; Tanaka, Yasuhito

    2013-11-01

    We modified and automated a highly sensitive chemiluminescent enzyme immunoassay (CLEIA) for surface antigen (HBsAg) detection using a combination of monoclonal antibodies, each for a specific epitope of HBsAg, and by improving an earlier conjugation technique. Of 471 hepatitis B virus (HBV) carriers seen in our hospital between 2009 and 2012, 26 were HBsAg seronegative as determined by the Abbott Architect assay. The Lumipulse HBsAg-HQ assay was used to recheck those 26 patients who demonstrated seroclearance by the Abbott Architect assay. The performance of the Lumipulse HBsAg-HQ assay was compared with that of a quantitative HBsAg detection system (Abbott Architect) and the Roche Cobas TaqMan HBV DNA assay (CTM) (lower limit of detection, 2.1 log copies/ml) using blood serum samples from patients who were determined to be HBsAg seronegative by the Abbott Architect assay. Ten patients had spontaneous HBsAg loss. Of 8 patients treated with nucleotide analogues (NAs), two were HBsAg seronegative after stopping lamivudine therapy and 6 were HBsAg seronegative during entecavir therapy. Eight acute hepatitis B (AH) patients became HBsAg seronegative. Of the 26 patients, 16 were HBsAg positive by the Lumipulse HBsAg-HQ assay but negative by the Abbott Architect assay. The differences between the two assays in terms of detectable HBsAg persisted over the long term in the spontaneous loss group (median, 10 months), the NA-treated group (2.5 months), and the AH group (0.5 months). In 9 patients, the Lumipulse HBsAg-HQ assay detected HBsAg when HBV DNA was negative by the CTM assay. HBsAg was also detected by the Lumipulse HBsAg-HQ assay in 4 patients with an anti-HBs concentration of >10 mIU/ml, 3 of whom had no HBsAg escape mutations. The automatic, highly sensitive HBsAg CLEIA Lumipulse HBsAg-HQ is a convenient and precise assay for HBV monitoring. PMID:23946517

  8. Evaluation of Two Commercial Enzyme Immunoassays, Testing Immunoglobulin G (IgG) and IgA Responses, for Diagnosis of Helicobacter pylori Infection in Children

    PubMed Central

    Kindermann, Angelika; Konstantopoulos, Nikolaos; Lehn, Norbert; Demmelmair, Hans; Koletzko, Sibylle

    2001-01-01

    Serological testing to diagnose Helicobacter pylori infection in children is still controversial, although commonly used in clinical practice. We compared the immunoglobulin G (IgG) and IgA results of two commercially available enzyme immunoassays (EIAs) (Pyloriset IgG and IgA and Enzygnost II IgG and IgA) for 175 children with abdominal symptoms divided into three age groups (0 to ≤6 years, n = 47; >6 to ≤12 years, n = 77; >12 years, n = 51). A child was considered H. pylori infected if at least two of three tests (histology, rapid urease test, 13C-urea breath test) or culture were positive and noninfected if all results were concordantly negative. Of 175 children, 93 (53%) were H. pylori negative and 82 were H. pylori positive. With the recommended cutoff values, the overall specificity was excellent for all four EIAs (95.7 to 97.8%) regardless of age. Sensitivity varied markedly between tests and was 92.7, 70.7, 47.5, and 24.4% for Enzygnost II IgG, Pyloriset IgG, Enzygnost II IgA, and Pyloriset IgA, respectively. Sensitivity was low in the youngest age group (25 to 33.3%), except for Enzygnost II IgG (91.6%). Receiver-operating curve analyses revealed that lower cutoff values would improve the accuracy of all of the tests except Enzygnost II IgG. Measurement of specific IgA, in addition to IgG, antibodies hardly improved the sensitivity. The specificity of commercial serological tests is high in children when the cutoff values obtained from adults are used. In contrast, sensitivity is variable, with a strong age dependence in some, but not all, tests. We speculate that young children may have a different immune response to H. pylori, with preferable responses to certain antigens, as well as lower titers than adults. The Pyloriset test may fail to recognize these specific antibodies. PMID:11574578

  9. Target-induced nano-enzyme reactor mediated hole-trapping for high-throughput immunoassay based on a split-type photoelectrochemical detection strategy.

    PubMed

    Zhuang, Junyang; Tang, Dianyong; Lai, Wenqiang; Xu, Mingdi; Tang, Dianping

    2015-09-15

    Photoelectrochemical (PEC) detection is an emerging and promising analytical tool. However, its actual application still faces some challenges like potential damage of biomolecules (caused by itself system) and intrinsic low-throughput detection. To solve the problems, herein we design a novel split-type photoelectrochemical immunoassay (STPIA) for ultrasensitive detection of prostate specific antigen (PSA). Initially, the immunoreaction was performed on a microplate using a secondary antibody/primer-circular DNA-labeled gold nanoparticle as the detection tag. Then, numerously repeated oligonucleotide sequences with many biotin moieties were in situ synthesized on the nanogold tag via RCA reaction. The formed biotin concatamers acted as a powerful scaffold to bind with avidin-alkaline phosphatase (ALP) conjugates and construct a nanoenzyme reactor. By this means, enzymatic hydrolysate (ascorbic acid) was generated to capture the photogenerated holes in the CdS quantum dot-sensitized TiO2 nanotube arrays, resulting in amplification of the photocurrent signal. To elaborate, the microplate-based immunoassay and the high-throughput detection system, a semiautomatic detection cell (installed with a three-electrode system), was employed. Under optimal conditions, the photocurrent increased with the increasing PSA concentration in a dynamic working range from 0.001 to 3 ng mL(-1), with a low detection limit (LOD) of 0.32 pg mL(-1). Meanwhile, the developed split-type photoelectrochemical immunoassay exhibited high specificity and acceptable accuracy for analysis of human serum specimens in comparison with referenced electrochemiluminescence immunoassay method. Importantly, the system was not only suitable for the sandwich-type immunoassay mode, but also utilized for the detection of small molecules (e.g., aflatoxin B1) with a competitive-type assay format. PMID:26291091

  10. Immunoassays for pesticide monitoring

    NASA Astrophysics Data System (ADS)

    Wengatz, Ingrid; Szurdoki, Ferenc; Swamy, Anand R.; Evans, Lawrence, III; Patonay, Gabor; Stimmann, Eric; Delwiche, Michael; Stoutamire, Donald; Gee, Shirley J.; Hammock, Bruce D.

    1995-05-01

    This study compares two formats of rapid assays for the detection of pesticides (bromacil and pyrethroid based metabolites): enzyme linked immunosorbent assay (ELISA) and immunoassay with near-infrared (NIR) fluorescence detection. NIR dye immunoassay (NIRDIA) measurements were carried out by using two different instruments, both having a silicon photodiode as the detector and a laser diode for excitation. ELISA and NIRDIA were performed in a tracer format, where the specific antibody is bound to the surface of a microtiter plate well and the tracer with enzyme or fluorescent dye label competes with the analyte for the antibody binding site. It was demonstrated that the NIRDIA is at least as sensitive as the ELISA. Both assays detect pesticides in the (mu) g/L (ppb) range. Hapten- macromolecule-NIR dye-conjugates have been synthesized with various biopolymers (e.g., proteins) as carriers. The use of carrier macromolecules enables convenient purification of the cyanine dye derivatives. The mild conjugation method of the dye is based on isothiocyanate chemistry.

  11. A point-of-care test for measles diagnosis: detection of measles-specific IgM antibodies and viral nucleic acid

    PubMed Central

    Slibinskas, Rimantas; Chua, Kaw Bing; Nigatu, Wondatir; Brown, Kevin E; Sasnauskas, Kestutis; Samuel, Dhanraj; Brown, David

    2011-01-01

    Abstract Objective To evaluate the performance of a newly developed point-of-care test (POCT) for the detection of measles-specific IgM antibodies in serum and oral fluid specimens and to assess if measles virus nucleic acid could be recovered from used POCT strips. Methods The POCT was used to test 170 serum specimens collected through measles surveillance or vaccination programmes in Ethiopia, Malaysia and the Russian Federation: 69 were positive for measles immunoglobulin M (IgM) antibodies, 74 were positive for rubella IgM antibodies and 7 were positive for both. Also tested were 282 oral fluid specimens from the measles, mumps and rubella (MMR) surveillance programme of the United Kingdom of Great Britain and Northern Ireland. The Microimmune measles IgM capture enzyme immunoassay was the gold standard for comparison. A panel of 24 oral fluids was used to investigate if measles virus haemagglutinin (H) and nucleocapsid (N) genes could be amplified by polymerase chain reaction directly from used POCT strips. Findings With serum POCT showed a sensitivity and specificity of 90.8% (69/76) and 93.6% (88/94), respectively; with oral fluids, sensitivity and specificity were 90.0% (63/70) and 96.2% (200/208), respectively. Both H and N genes were reliably detected in POCT strips and the N genes could be sequenced for genotyping. Measles virus genes could be recovered from POCT strips after storage for 5 weeks at 20–25 °C. Conclusion The POCT has the sensitivity and specificity required of a field-based test for measles diagnosis. However, its role in global measles control programmes requires further evaluation. PMID:21897488

  12. Bioelectrochemical Immunoassay of Polychlorinated Biphenyl

    SciTech Connect

    Lin, Ying-Ying; Liu, Guodong; Wai, Chien M.; Lin, Yuehe

    2008-04-01

    A simple, rapid, and highly sensitive bioelectrochemical immunoassay method based on magnetic beads (MBs) and disposable screen-printed electrodes (SPE) has been developed to detect polychlorinated biphenyls (PCBs). The principle of this bioassay is based on a direct competitive enzyme-linked immunosorbent assay using PCB-antibody-coated MBs and horseradish peroxidase (HRP)-labeled PCB (HRP-PCB). A magnetic process platform was used to mix and shake the samples during the immunoreactions and to separate free and unbound reagents after the liquid-phase competitive immunoreactions among PCB-antibody-coated MBs, PCB analyte, and HRP-PCB. After a complete immunoassay, the HRP tracers attached to MBs were transferred to a substrate solution containing o-aminophenol and hydrogen peroxide for electrochemical detection. The different parameters, including the amount of HRP-PCB conjugates, immunoreaction time, and the concentration of substrate that governs the analytical performance of the immunoassay have been studied in detail and optimized. The detection limit of 5 pg mL-1 was obtained under optimum experimental conditions. The performance of this bioelectrochemical immunoassay was successfully evaluated with untreated river water spiked with PCBs, and the results were validated by commercial PCB enzyme-linked immunosorbent assay kit, indicating that this convenient and sensitive technique offers great promise for decentralized environmental application and trace PCBs monitoring.

  13. Evaluation of Enzyme Immunoassays and Real-Time PCR for Detecting Shiga Toxin-Producing Escherichia coli in Southern Alberta, Canada

    PubMed Central

    Patterson-Fortin, Laura; Kuo, Julie; Li, Vincent; Boras, Valerie

    2015-01-01

    Two immunoassays (Shiga Toxin Chek and Shiga Toxin Quik Chek) and real-time PCR were used to detect Shiga toxin-producing Escherichia coli. For enriched culture, the sensitivity and specificity of the three methods ranged from 80.0% to 98.2% and 98.0% to 100.0%, respectively. STEC isolates were identified in 2.6% of the 784 samples. PMID:25588656

  14. Enzyme

    MedlinePlus

    Enzymes are complex proteins that cause a specific chemical change in all parts of the body. For ... use them. Blood clotting is another example of enzymes at work. Enzymes are needed for all body ...

  15. Multiplexed single molecule immunoassays.

    PubMed

    Rissin, David M; Kan, Cheuk W; Song, Linan; Rivnak, Andrew J; Fishburn, Matthew W; Shao, Qichao; Piech, Tomasz; Ferrell, Evan P; Meyer, Raymond E; Campbell, Todd G; Fournier, David R; Duffy, David C

    2013-08-01

    We have developed a method that enables the multiplexed detection of proteins based on counting single molecules. Paramagnetic beads were labeled with fluorescent dyes to create optically distinct subpopulations of beads, and antibodies to specific proteins were then immobilized to individual subpopulations. Mixtures of subpopulations of beads were then incubated with a sample, and specific proteins were captured on their specific beads; these proteins were then labeled with enzymes via immunocomplex formation. The beads were suspended in enzyme substrate, loaded into arrays of femtoliter wells--or Single Molecule Arrays (Simoa)--that were integrated into a microfluidic device (the Simoa disc). The wells were then sealed with oil, and imaged fluorescently to determine: a) the location and subpopulation identity of individual beads in the femtoliter wells, and b) the presence or absence of a single enzyme associated with each bead. The images were analyzed to determine the average enzyme per bead (AEB) for each bead subpopulation that provide a quantitative parameter for determining the concentration of each protein. We used this approach to simultaneously detect TNF-α, IL-6, IL-1α, and IL-1β in human plasma with single molecule resolution at subfemtomolar concentrations, i.e., 200- to 1000-fold more sensitive than current multiplexed immunoassays. The simultaneous, specific, and sensitive measurement of several proteins using multiplexed digital ELISA could enable more reliable diagnoses of disease. PMID:23719780

  16. Triclosan and methyl-triclosan monitoring study in the northeast of Spain using a magnetic particle enzyme immunoassay and confirmatory analysis by gas chromatography mass spectrometry

    NASA Astrophysics Data System (ADS)

    Kantiani, Lina; Farré, Marinella; Asperger, Danijela; Rubio, Fernando; González, Susana; López de Alda, Maria J.; Petrović, Mira; Shelver, Weilin L.; Barceló, Damià

    2008-10-01

    SummaryFor the first time, the occurrence of triclosan and its metabolite methyl-triclosan was investigated in a typical Mediterranean area using a two-step methodology based on screening using a magnetic particle immunoassay (IA) and confirmatory analysis by solid phase extraction (SPE) followed by gas chromatography-mass spectrometry (GC-MS). In this study, 95 environmental samples were analyzed. A commercial immunoassay was assessed for use in the different types of water selected for this study. A large monitoring study was performed on the influent and the effluent of eight wastewater treatment plants (WWTPs), water samples from Ebro and Llobregat rivers, and drinking water. All wastewater samples tested in this study (influents and effluents) showed the presence of triclosan, with concentrations for raw influents being high (10 μg/L as average value). The percentages of triclosan removal for the WWTPs were evaluated (30-70%) along the different treatment processes showing that the best removal rates were obtained by the processes equipped with membrane bioreactors (MBRs). However, important concentrations of triclosan were detected even after treatment by MBRs. The presence of this biocide was confirmed in 50% of the river samples analyzed. Twenty two drinking water samples from the Barcelona city area were investigated, and in this case no triclosan was detected. Due to its properties and the widespread usage of triclosan, there is a need for monitoring and controlling the amounts present in wastewater effluents, river water, drinking water catchments areas, and drinking water. To this end, we present a feasible methodology using a magnetic particle-based immunoassay as a screening, followed by confirmatory analysis using solid phase extraction-gas chromatography-mass spectrometry (SPE-GC-MS).

  17. Recognition of Porphyromonas gingivalis Gingipain Epitopes by Natural IgM Binding to Malondialdehyde Modified Low-Density Lipoprotein

    PubMed Central

    Turunen, S. Pauliina; Kummu, Outi; Harila, Kirsi; Veneskoski, Marja; Soliymani, Rabah; Baumann, Marc; Pussinen, Pirkko J.; Hörkkö, Sohvi

    2012-01-01

    Objective Increased risk for atherosclerosis is associated with infectious diseases including periodontitis. Natural IgM antibodies recognize pathogen-associated molecular patterns on bacteria, and oxidized lipid and protein epitopes on low-density lipoprotein (LDL) and apoptotic cells. We aimed to identify epitopes on periodontal pathogen Porphyromonas gingivalis recognized by natural IgM binding to malondialdehyde (MDA) modified LDL. Methods and Results Mouse monoclonal IgM (MDmAb) specific for MDA-LDL recognized epitopes on P. gingivalis on flow cytometry and chemiluminescence immunoassays. Immunization of C57BL/6 mice with P. gingivalis induced IgM, but not IgG, immune response to MDA-LDL and apoptotic cells. Immunization of LDLR−/− mice with P. gingivalis induced IgM, but not IgG, immune response to MDA-LDL and diminished aortic lipid deposition. On Western blot MDmAb bound to P. gingivalis fragments identified as arginine-specific gingipain (Rgp) by mass spectrometry. Recombinant domains of Rgp produced in E. coli were devoid of phosphocholine epitopes but contained epitopes recognized by MDmAb and human serum IgM. Serum IgM levels to P. gingivalis were associated with anti-MDA-LDL levels in humans. Conclusion Gingipain of P. gingivalis is recognized by natural IgM and shares molecular identity with epitopes on MDA-LDL. These findings suggest a role for natural antibodies in the pathogenesis of two related inflammatory diseases, atherosclerosis and periodontitis. PMID:22496875

  18. A new dual immunoassay for tumor markers based on chemiluminescence signal amplification by magnetic mesoporous silica and enzyme modified gold nanoparticles.

    PubMed

    Lin, Jiehua; Chu, Pengfei; Wei, Zhijing

    2012-01-01

    A sensitive dual immunoassay was proposed for the determination of carcinoembryonic antigen (CEA) and α-fetoprotein (AFP) based on signal amplification. Monoclonal antibodies immobilized on magnetic mesoporous silica particles (Fe(3)O(4)/SiO(2)) were prepared as the primary probe. Horseradish peroxidase (HRP) labeled antibodies co-coated with HRP on gold nanoparticles (AuNPs) were used as the secondary probe to achieve signal amplification. HRP tags were retained in the flow cells after a sandwich immunoassay. By controlling two switches on the two channels, chemiluminescent substrates were injected orderly man way, and then signals for CEA and AFP were sequentially detected by HRP-luminol-H(2)O(2). Due to the increased amount of HRP on AuNPs and the increased amount of monoclonal antibodies on Fe(3)O(4)/SiO(2), the signals were largely amplified. Under the optimal conditions, CEA and AFP could be detected in the linear ranges of 1.0 - 80 and 1.0 - 75 ng mL(-1) with detection limits of 0.25 and 0.5 ng mL(-1), respectively. PMID:22232219

  19. A new bioluminescent assay for studies of protein G and protein A binding to IgG and IgM.

    PubMed

    Zatta, P F

    1996-04-01

    It has been reported that protein G (PrG) specifically binds to IgG, but not to IgM. In the present paper, I report that using a bioluminescent immunoassay, that utilizes the recombinant calcium-dependent protein, Aequorin, as a photoprobe, IgM as well as IgG binds to PrG. The method presented here may also be used in general to study the glycosylation of proteins. PMID:8773543

  20. IgM, IgG and IgA rheumatoid factors and circulating immune complexes in patients with AIDS and AIDS-related complex with serological abnormalities.

    PubMed Central

    Procaccia, S; Lazzarin, A; Colucci, A; Gasparini, A; Forcellini, P; Lanzanova, D; Foppa, C U; Novati, R; Zanussi, C

    1987-01-01

    To investigate some humoral aspects which may reflect the involvement of B lymphocytes in the acquired immunodeficiency syndrome (AIDS), we used an enzyme-linked immunoassay (ELISA) to determine the levels of IgM, IgG and IgA rheumatoid factors (RF) in 16 patients suffering from full-blown AIDS and 32 patients with AIDS-related complex (ARC), in the clinical form of lymphoadenopathy syndrome (LAS), compared with 40 healthy, young heterosexual subjects. Both AIDS and ARC patients showed a greater incidence of high IgM RF levels, with mean values significantly higher than controls, but with no differences between the two pathological groups. IgG RF behaviour was similar in the two patient populations and the healthy subjects. IgA RF were significantly raised in AIDS and ARC. Further information on RF was obtained by determination of the immunoglobulin levels of the respective isotypes in the same patients. Mean IgG levels were above normal in AIDS and ARC patients, but the latter group showed a higher incidence of increased values and higher mean levels. The IgA isotype was significantly increased mainly in AIDS patients. The behaviour of IgM was virtually the same in the three groups studied. A difference between AIDS and ARC patients was established by the detection of circulating immune-complexes (IC) by the C1q-binding and CIC-conglutinin assays. IC were significantly high, by both methods, only in the ARC group, but normal or very low in AIDS. These overall findings suggest once again the impairment of B cell function in AIDS, with prevalent hyperactivation in ARC and exhaustion in full-blown AIDS, and apparent preservation, in the latter group, of the antibody responses which are more closely related to the activity of subsets of T helper cells. PMID:3608224

  1. Use of biotinylated 17beta-estradiol in enzyme-immunoassay development: spacer length and chemical structure of the bridge are the main determinants in simultaneous streptavidin-antibody binding.

    PubMed

    Lacorn, Markus; Fleischer, Kerstin; Willig, Stephanie; Gremmel, Sabine; Steinhart, Hans; Claus, Rolf

    2005-02-01

    17beta-estradiol (E2) concentrations are in the low pg/ml range in plasma. To develop a sensitive enzyme immunoassay (EIA) for E2-determination a highly specific antibody raised against a 6-carboxymethyl (CMO)-E2-bovine serum albumine conjugate was used. Based on 6-CMO-E2 and 6-amino-E2, four biotinylated tracers with two different spacer lengths between E2 and biotin were synthesized using biotinylation reagents in one step reactions. All amino-based tracers were unsuitable for assay development because the antibody binding was too weak compared to the analyte E2. For 6-CMO-based tracers the simultaneous binding of the tracer to the antibody and streptavidin seems to be the determining step in the procedure depending on incubation temperature and spacer lengths. While a short spacer of 9 carbon atoms was susceptible to room temperature, a longer spacer of 16 carbon atoms showed nearly the same results for incubation at 4 degrees C or at room temperature. The absolute detection limit of this system was 0.63 pg/well. For sample clean-up, porcine plasma was solvent-extracted and depending on the initial plasma volume further purified by solvent partition. Determination of reproducibility resulted in intraassay coefficients of variation of 13% and 5.3% for samples with E2-levels of 15 pg/ml and 236 pg/ml, respectively. Measurement of E2-spiked blood plasma revealed recoveries of 83% up to 100% for E2 concentrations between 50 pg/ml and 1000 pg/ml. Only for the lowest concentration (20 pg/ml) a recovery of 58% was observed. Correlation of the EIA with an established radio immunoassay resulted in r=0.991 using the same antibody. PMID:15777945

  2. Association of mycobacterial-specific and Mycobacterium leprae specific antibody levels with clinical activity in tuberculoid leprosy: a comparative study of three serological enzyme-immunoassays.

    PubMed

    Chaturvedi, V; Sinha, S; Girdhar, B K; Katoch, K; Bhatia, A S; Sengupta, U

    1991-06-01

    The ELISAs for polyclonal antibodies against Mycobacterium leprae (ML-ELISA) and specific antibodies against epitopes on 35 kDa protein (SACT-ELISA) and phenolic glycolipid I (PG-ELISA) of M. leprae were evaluated comparatively in a group of 88 tuberculoid leprosy patients. The overall seropositivity rate with a battery of 3 tests (68%) was not significantly higher than that obtained with ML-ELISA alone (55%) for IgG class of antibodies. Seropositivities for SACT-ELISA and PG-ELISA were, respectively, 38% and 26%. ML-ELISA for IgM class of antibodies was least sensitive, showing only 8% positivity. A significant correlation was noted between individual values of the three assays, but the positive proportions overlapped maximally in the case of ML-ELISA (IgG) and SACT-ELISA. Further, positivity for the latter two assays, particularly SACT-ELISA, showed significant associations with the extent of 'active' (largely untreated) infection. Immunoblotting revealed that the main antibody response was directed towards M. leprae antigens in the molecular weight range of 20-40 kDa and the densitometry results of this zone correlated significantly with corresponding SACT-ELISA and ML-ELISA (IgG) values. PMID:1870374

  3. Determination of Glutamic Acid Decarboxylase (GAD65) in Pancreatic Islets and Its In Vitro and In Vivo Degradation Kinetics in Serum Using a Highly Sensitive Enzyme Immunoassay

    PubMed Central

    Schlosser, Michael; Walschus, Uwe; Klöting, Ingrid; Walther, Reinhard

    2008-01-01

    Glutamic acid decarboxylase GAD65 autoantibodies (GADA) are an established marker for autoimmune diabetes. Recently, the autoantigen GAD65 itself was proposed as biomarker of beta-cell loss for prediction of autoimmune diabetes and graft rejection after islet transplantation. Therefore, the GAD65 content in pancreatic islets of different species and its serum degradation kinetics were examined in this study using a sensitive immunoassay. GAD65 was found in quantities of 78 (human), 43.7 (LEW.1A rat) and 37.4 (BB/OK rat) ng per 1,000 islets, respectively, but not in mouse islets. The in vitro half-life of porcine GAD65 and human recombinant GAD65 ranged from 1.27 to 2.35 hours at 37°C in human serum, plasma and blood, and was unaffected by presence of GAD65 autoantibodies. After injecting 2,000 ng recombinant human GAD65 into LEW.1A rats, the in vivo half-life was 2.77 hours. GAD65 was undetectable after 24 hours in these animals, and for up to 48 hours following diabetes induction by streptozotocin in LEW.1A rats. Estimated from these data, at least 13 islets in rat and 1,875 in human must be simultaneously destroyed to detect GAD65 in circulation. These results should be taken into consideration in further studies aimed at examining the diagnostic relevance of GAD65. PMID:18334741

  4. Determination of glutamic acid decarboxylase (GAD65) in pancreatic islets and its in vitro and in vivo degradation kinetics in serum using a highly sensitive enzyme immunoassay.

    PubMed

    Schlosser, Michael; Walschus, Uwe; Klöting, Ingrid; Walther, Reinhard

    2008-01-01

    Glutamic acid decarboxylase GAD65 autoantibodies (GADA) are an established marker for autoimmune diabetes. Recently, the autoantigen GAD65 itself was proposed as biomarker of beta-cell loss for prediction of autoimmune diabetes and graft rejection after islet transplantation. Therefore, the GAD65 content in pancreatic islets of different species and its serum degradation kinetics were examined in this study using a sensitive immunoassay. GAD65 was found in quantities of 78 (human), 43.7 (LEW.1A rat) and 37.4 (BB/OK rat) ng per 1,000 islets, respectively, but not in mouse islets. The in vitro half-life of porcine GAD65 and human recombinant GAD65 ranged from 1.27 to 2.35 hours at 37 degrees C in human serum, plasma and blood, and was unaffected by presence of GAD65 autoantibodies. After injecting 2,000 ng recombinant human GAD65 into LEW.1A rats, the in vivo half-life was 2.77 hours. GAD65 was undetectable after 24 hours in these animals, and for up to 48 hours following diabetes induction by streptozotocin in LEW.1A rats. Estimated from these data, at least 13 islets in rat and 1,875 in human must be simultaneously destroyed to detect GAD65 in circulation. These results should be taken into consideration in further studies aimed at examining the diagnostic relevance of GAD65. PMID:18334741

  5. Kinase Activity Studied in Living Cells Using an Immunoassay

    ERIC Educational Resources Information Center

    Bavec, Aljos?a

    2014-01-01

    This laboratory exercise demonstrates the use of an immunoassay for studying kinase enzyme activity in living cells. The advantage over the classical method, in which students have to isolate the enzyme from cell material and measure its activity in vitro, is that enzyme activity is modulated and measured in living cells, providing a more

  6. Kinase Activity Studied in Living Cells Using an Immunoassay

    ERIC Educational Resources Information Center

    Bavec, Aljos?a

    2014-01-01

    This laboratory exercise demonstrates the use of an immunoassay for studying kinase enzyme activity in living cells. The advantage over the classical method, in which students have to isolate the enzyme from cell material and measure its activity in vitro, is that enzyme activity is modulated and measured in living cells, providing a more…

  7. Immunoassays in Biotechnology

    EPA Science Inventory

    Immunoassays have broad applications for a wide variety of important biological compounds and environmental contaminants. Immunoassays can detect the presence of an antigen in the human body, a pollutant in the environment, or a critical antibody in a patient’s serum to develop a...

  8. Immunoassays in Biotechnology

    EPA Science Inventory

    Immunoassays have broad applications for a wide variety of important biological compounds and environmental contaminants. Immunoassays can detect the presence of an antigen in the human body, a pollutant in the environment, or a critical antibody in a patients serum to develop a...

  9. Microfluidic paper-based analytical device for photoelectrochemical immunoassay with multiplex signal amplification using multibranched hybridization chain reaction and PdAu enzyme mimetics.

    PubMed

    Lan, Feifei; Sun, Guoqiang; Liang, Linlin; Ge, Shenguang; Yan, Mei; Yu, Jinghua

    2016-05-15

    Combining multibranched hybridization chain reaction (mHCR), the photoelectrochemical (PEC) immunosensor was fabricated with a microfluidic paper-based analytical devices using different sizes of CdTe quantum dots (QDs) sensitized flower-like 3D ZnO superstructures as photoactive materials. Firstly, 4-aminothiophenol (PATP) functioned ZnO was anchored on gold-paper working electrode. With the aid of PATP, large-sized CdTe-COOH QDs (QDs1) were conjugated onto the ZnO surface because of the formation of a strong bond (Zn-S) between the thiol of PATP molecule and the ZnO, and the remaining amino group formed an amide bond with carboxylic acid group capping CdTe. Then the small-sized CdTe-NH2 QDs (QDs2) were modified on the QDs1 by forming amide bond, which leaded to a very strong photocurrent response because of the formation of cosensitized structure. The designed mHCR produced long products with multiple branched arms, which could attached multiple PdAu nanoparticles and catalyze the oxidation of hydroquinone (HQ) using H2O2 as anoxidant. Double strands DNA with multiple branched arms (mdsDNA) was formed by mHCR. In the presence of carcinoembryonic antigen (CEA), PdAu-mdsDNA conjugates-labeled CEA antibody was captured. The concentrations of CEA were measured through the decrease in photocurrent intensity resulting from the increase in steric hindrance of the immunocomplex and the polymeric oxidation product of HQ. In addition, the oxidation product of HQ deposited on the as-obtained electrode, which could efficiently inhibit the photoinduced electron transfer. Under optimal conditions, the PEC immunosensor exhibited excellent analytical performance: the detection range of CEA was from 0.001 to 90ngmL(-1) with low detection limit of 0.33pgmL(-1). The as-obtained immunosensor exhibited excellent precision, prominent specificity, acceptable stability and reproducibility, and could be used for the detection of CEA in real samples. The proposed assay opens a promising platform of clinical immunoassay for other biomolecules. PMID:26735876

  10. Surface second-harmonic generation (SSHG): a new scheme for immunoassay

    NASA Astrophysics Data System (ADS)

    Yang, Liqun; McStay, Daniel; Quinn, Peter J.

    1996-04-01

    A new optical immunoassay scheme based on surface second harmonic generation (SHG) is proposed. The utility of the technique has been investigated by deposition of sample antigen and antibody (bovine serum albumin & mouse anti-bovine serum albumin IgG antibody) on glass surfaces. The laser-induced second harmonic signals generated from these sample surfaces were monitored using a photomultiplier tube (Thorn EMI, 9524A). The surface second harmonic generation characteristics of an enzyme-linked immunoassay process were investigated, which is parallely monitored by a conventional ELISA method. The SSHG immunoassay experiment was performed using a conventional sandwich immunoassay scheme on ELISA immunoassay plates. The laser-induced surface-second harmonic signals generated from sample wells of different populations of the antigen-antibody complex were subsequently measured using the photomultiplier tube. To verify the new SSHG immunoassay results, a conventional ELISA sandwich immunoassay was performed, and their results compared. The new SSHG immunoassay results showed a virtually identical conclusion as that of conventional ELISA sandwich immunoassay. The SSHG readings are promotional to that of the ELISA data. However, the readings from the new SSHG immunoassay were found much larger, which is in agreement with the theoretical expectation. In this paper, detailed experimental results of the new SSHG immunoassay are described, and subsequently compared with those of a conventional ELISA immunoassay. The feasibility and advantages of the SSHG as a new immunoassay technique also are discussed.

  11. Evaluation of third-generation RIDASCREEN enzyme immunoassay for the detection of norovirus antigens in stool samples of hospitalized children in Belém, Pará, Brazil.

    PubMed

    Siqueira, Jones Anderson Monteiro; Linhares, Alexandre da Costa; Oliveira, Darleise de Souza; Soares, Luana da Silva; Lucena, Maria Silvia Sousa; Wanzeller, Ana Lúcia Monteiro; Mascarenhas, Joana D'Arc Pereira; Gabbay, Yvone Benchimol

    2011-12-01

    Noroviruses (NoVs) are major agents of gastroenteritis outbreaks and hospitalization worldwide. This study evaluated the sensitivity and specificity of the commercially available third-generation RIDASCREEN® Norovirus Enzyme Immunoassay (EIA) kit in comparison to the reverse transcription-polymerase chain reaction (RT-PCR) to detect NoVs in hospitalized children with gastroenteritis. An agreement of 88% (81/92) was observed when comparing EIA with RT-PCR. A sensitivity of 92% and a specificity of 83.3% were demonstrated. Eleven samples were positive by 1 method only (4 RT-PCR/7 EIA). Fourteen samples were sequenced and all classified as NoV genogroup GII-4. The 7 positive only by EIA were also evaluated by electron microscopy, and in 3 (42.9%) samples viral particles with a suggestive morphology of NoVs were visualized. These same samples were tested by seminested-RT-PCR with a positivity of 85.7%. The results obtained in this study demonstrated a significant improvement in the sensitivity and specificity of this updated assay. PMID:22001621

  12. Utility of galactomannan enzyme immunoassay and (1,3) beta-D-glucan in diagnosis of invasive fungal infections: low sensitivity for Aspergillus fumigatus infection in hematologic malignancy patients.

    PubMed

    Hachem, R Y; Kontoyiannis, D P; Chemaly, R F; Jiang, Y; Reitzel, R; Raad, I

    2009-01-01

    Previous studies have reported that galactomannan (GM) enzyme immunoassay and 1,3 beta-glucan (BG) assay may be useful diagnostic tools, but their sensitivities are variable. We compared the performances of both tests. Between October 2002 and May 2005, 82 patients were prospectively monitored for 12 weeks. A total of 414 samples were tested by GM assay and 409 samples were tested by BG assay for the following four groups of patients: those with invasive aspergillosis (IA), those with other mold infections (Fusarium, scedosporium, zygomycosis, etc.), those with candidemia, and control patients. Blood samples were obtained twice on week 1 and once every other week for a total of 12 weeks. Patients in the invasive fungal infection groups had comparable risk factors. The sensitivity of the GM test was significantly higher for patients with IA due to non-fumigatus Aspergillus species than for patients with IA due to Aspergillus fumigatus (49% versus 13%; P < 0.0001) or with other mold infections (49% versus 6%; P < 0.0001). However, the sensitivity range (47% to 64%) and specificity (88%) of the BG assay were comparable among all patients tested, regardless of the infecting pathogen. The performance of GM-based diagnosis appears to be better for detecting non-fumigatus Aspergillus species. The diagnostic marker BG was shown to have a higher sensitivity than that of GM in detecting IA and other mold infections in hematologic malignancy patients. PMID:19005145

  13. Production of monoclonal antibodies and enzyme immunoassay to bovine retinol-binding protein and determination of retinol-binding protein serum levels and retinol concentrations in serum and liver in dairy cows before and after parturition.

    PubMed

    Lindberg, L A; Sinkkonen, H; Pösö, A R; Tesfa, A T; Schröder, J

    1999-06-01

    To study vitamin A transport in dairy cows and heifers around parturition, an enzyme immunoassay for bovine retinol binding protein (RBP) was developed and serum levels determined. Serum and liver concentrations of retinol were assayed by HPLC. Four weeks before expected calving the cows and heifers were divided into two groups each, and half of the animals received a protein supplementation during the dry period. The mean serum RBP concentration 4 weeks before calving was 42 mg l-1 for the cows and 44 mg l-1 for the heifers. The serum retinol concentrations were 0.53 mg l-1 for the cows and 0.42 mg l-1 for the heifers, and the liver retinol concentrations 0.30 mg l-1 and 0.13 mg g-1, respectively. In the groups without protein supplementation there was a significant decrease in serum RBP at sampling 1 week before parturition compared to initial values. The measurement of serum RBP may prove useful in assessment of amino acid availability in dairy cows. PMID:10333469

  14. Enzyme-linked Immunoassay Index for Anti-NC16a IgG and IgE Auto-antibodies Correlates with Severity and Activity of Bullous Pemphigoid.

    PubMed

    Kalowska, Monika; Ciepiela, Olga; Kowalewski, Cezary; Demkow, Urszula; Schwartz, Robert A; Wozniak, Katarzyna

    2016-03-01

    Bullous pemphigoid (BP) is characterized by IgG and IgE autoantibodies to the NC16a domain of BP180. This study evaluated the correlation between body surface area (BSA), total serum IgE and enzyme-linked immunoassay (ELISA) for IgG and IgE anti-NC16a in 77 patients with BP in the active stage, and the degree of conversion to negative of the studied parameters in clinical remission. Statistically positive correlations were observed between BSA and examined parameters (correlation index 0.2548, 0.2491, 0.311, respectively). Originally, patients with BP with positive ELISAs for both IgG and IgE autoantibodies presented twice as extensive skin lesions as those with positive IgG and negative IgE ELISAs. Conversion of ELISAs for IgG and IgE to negative in clinical remission occurred in 9.3% and 81% of patients with BP, respectively. This confirmed that ELISA for IgE anti-NC16a is a helpful parameter in the modification of current treatment and the assessment of risk of relapse in BP. PMID:25791894

  15. Clostridium difficile testing algorithms using glutamate dehydrogenase antigen and C. difficile toxin enzyme immunoassays with C. difficile nucleic acid amplification testing increase diagnostic yield in a tertiary pediatric population.

    PubMed

    Ota, Kaede V; McGowan, Karin L

    2012-04-01

    We evaluated the performance of the rapid C. diff Quik Chek Complete's glutamate dehydrogenase antigen (GDH) and toxin A/B (CDT) tests in two algorithmic approaches for a tertiary pediatric population: algorithm 1 entailed initial testing with GDH/CDT followed by loop-mediated isothermal amplification (LAMP), and algorithm 2 entailed GDH/CDT followed by cytotoxicity neutralization assay (CCNA) for adjudication of discrepant GDH-positive/CDT-negative results. A true positive (TP) was defined as positivity by CCNA or positivity by LAMP plus another test (GDH, CDT, or the Premier C. difficile toxin A and B enzyme immunoassay [P-EIA]). A total of 141 specimens from 141 patients yielded 27 TPs and 19% prevalence. Sensitivity, specificity, positive predictive value, and negative predictive value were 56%, 100%, 100%, and 90% for P-EIA and 81%, 100%, 100%, and 96% for both algorithm 1 and algorithm 2. In summary, GDH-based algorithms detected C. difficile infections with superior sensitivity compared to P-EIA. The algorithms allowed immediate reporting of half of all TPs, but LAMP or CCNA was required to confirm the presence or absence of toxigenic C. difficile in GDH-positive/CDT-negative specimens. PMID:22259201

  16. Enzyme immunoassay and proteomic characterization of troponin I as a marker of mammalian muscle compounds in raw meat and some meat products.

    PubMed

    Zvereva, Elena A; Kovalev, Leonid I; Ivanov, Alexei V; Kovaleva, Marina A; Zherdev, Anatoly V; Shishkin, Sergey S; Lisitsyn, Andrey B; Chernukha, Irina M; Dzantiev, Boris B

    2015-07-01

    The skeletal muscle protein troponin I (TnI) has been characterized as a potential thermally stable and species-specific biomarker of mammalian muscle tissues in raw meat and meat products. This study proposed a technique for the quantification of TnI comprising protein extraction and sandwich enzyme-linked immunosorbent assay (ELISA). The technique is characterized by a TnI detection limit of 4.8 ng/ml with quantifiable concentrations ranging from 8.7 to 52 ng/ml. The method was shown to be suitable for detection of TnI in mammalian (beef, pork, lamb, and horse) meat but not in poultry (chicken, turkey, and duck) meat. In particular, the TnI content in beef was 0.40 3 ± 0.058 mg/g of wet tissue. The TnI estimations obtained for the pork and beef samples using ELISA were comparable to the proteomic analysis results. Thus, the quantitative study of TnI can be a convenient way to assess the mammalian muscle tissue content of various meat products. PMID:25777979

  17. Novel enzyme immunoassay and optimized DNA extraction for the detection of polymerase-chain-reaction-amplified viral DNA from paraffin-embedded tissue.

    PubMed Central

    Merkelbach, S.; Gehlen, J.; Handt, S.; Füzesi, L.

    1997-01-01

    Four different DNA extraction methods were compared to determine their ability to provide DNA for amplification of viral sequences from paraffin-embedded human tissue samples by polymerase chain reaction (PCR). The suitability of extraction methods was assessed using parameters like DNA yield, length of recovered DNA fragments, and duration. Furthermore, the efficiency of amplifying a human single-copy gene, the beta-globin gene, from DNA samples was tested. The best preservation of DNA molecules could be achieved by binding the DNA onto a silica column before further purification. Viral DNA sequences could be amplified by PCR in DNA extracted from routinely processed paraffin blocks from cases with clinically or morphologically suspected cytomegalovirus or Epstein-Barr virus infections. The PCR products were specified by a novel liquid hybridization assay called PCR-enzyme-linked immunosorbent assay. Using this assay, the time-consuming Southern hybridization could be replaced and the time requirement for the detection of PCR products could be reduced from 1 day to 4 hours. The assay system described here represents a reliable, sensitive, and specific method for the detection of viral DNA from paraffin-embedded tissue samples. Images Figure 2 Figure 3 Figure 4 Figure 5 PMID:9137080

  18. Ultrasensitive enzyme-free electrochemical immunoassay for free thyroxine based on three dimensionally ordered macroporous chitosan-Au nanoparticles hybrid film.

    PubMed

    Zhang, Qi; Chen, Xiaojun; Tu, Fulai; Yao, Cheng

    2014-09-15

    The measurement of free thyroxine concentration in serum is considered to be an essential indicator of thyroid function. Here, a novel enzyme-free sandwich electrochemical immunosensor for the detection of FT4 antigen based on the immobilization of primary antibody (Ab1) on three dimensional ordered macroporous chitosan-Au nanoparticles hybrid (3DOM CS-AuNPs) film electrode, and magnetic multiwall carbon nanotubes (MMWCNTs) were used as label of secondary antibody (Ab2). The 3DOM CS-AuNPs film electrode was constructed by one-step electrodeposition of CS-AuNPs composite onto Au electrode with silica opal template. MMWCNTs were prepared by chemical co-precipitation of Fe(2+) and Fe(3+) salts on carboxylated MWCNTs. Ru(bpy)3(2+) labeled anti-FT4 (Ru(bpy)3(2+)-Ab2) was covalently attached to MMWCNTs through the formation of amide bond between the carboxylic groups of MWCNTs and the amine groups of antibody. Under the optimal conditions, FT4 was detected in a concentration range from 0.71 fg mL(-1) to 1.15 pg mL(-1) with a correlation coefficient of 0.998 and a detection limit of 0.20 fg mL(-1). Moreover, the immunosensor showed excellent selectivity, good stability, satisfactory reproducibility and regeneration. Importantly, the developed method was used to assay clinical serum specimens, achieving a good relation with those obtained from the commercialized electrochemiluminescent method. PMID:24752149

  19. Comparison of premier CAMPY enzyme immunoassay (EIA), ProSpecT Campylobacter EIA, and ImmunoCard STAT! CAMPY tests with culture for laboratory diagnosis of Campylobacter enteric infections.

    PubMed

    Granato, Paul A; Chen, Li; Holiday, Iris; Rawling, Russell A; Novak-Weekley, Susan M; Quinlan, Tammy; Musser, Kimberlee A

    2010-11-01

    Campylobacter enteritis is a food-borne or waterborne illness caused almost exclusively by Campylobacter jejuni and, to a lesser extent, by Campylobacter coli. These organisms produce indistinguishable clinical diseases and together represent the second most common cause of bacterial diarrhea in the United States and the leading cause of enteric infection throughout the world. The conventional approach to the laboratory diagnosis of Campylobacter enteritis is based on the recovery of the organism from a stool specimen, which requires the use of a specialized medium incubated at 42°C for several days in an artificially created microaerophilic environment. Recently, several commercially available enzyme immunoassays (EIAs) have been developed for the direct detection of C. jejuni and C. coli in stool specimens. This study compared conventional culture with three EIA methods, the Premier CAMPY EIA (Meridian Bioscience, Cincinnati, OH), the ProSpecT Campylobacter EIA (Remel, Lenexa, KS), and the ImmunoCard STAT! CAMPY test (Meridian Bioscience, Cincinnati, OH), for the detection of C. jejuni and C. coli in 485 patient stool samples. Discordant results were arbitrated by using an in-house, real-time PCR assay that was developed and validated by a public health reference laboratory. Following analyses of the discrepant specimens by PCR, the sensitivity and specificity of both the Premier CAMPY and ProSpecT Campylobacter EIAs were 99.3% and 98%, respectively, while the ImmunoCard STAT! CAMPY test had a sensitivity of 98.5% and a specificity of 98.2%. By use of the PCR test as the reference standard, culture detected 127 of 135 Campylobacter-positive stool specimens, yielding a sensitivity of 94.1%. These results showed that the three EIAs evaluated in this study provide a rapid and reliable alternative for the laboratory diagnosis of enteric infections with C. jejuni and C. coli and that conventional culture may no longer be recognized as the "gold standard" for diagnosis. PMID:20810765

  20. Comparison of the Syva MicroTrak enzyme immunoassay and Gen-Probe PACE 2 with cell culture for diagnosis of cervical Chlamydia trachomatis infection in a high-prevalence female population.

    PubMed Central

    Clarke, L M; Sierra, M F; Daidone, B J; Lopez, N; Covino, J M; McCormack, W M

    1993-01-01

    Culture is currently considered the "gold standard" for detecting Chlamydia trachomatis infections. We evaluated the Syva MicroTrak enzyme immunoassay (EIA) and Gen-Probe PACE 2 tests, which detect chlamydial antigens and rRNA, respectively. These assays were compared with each other and with culture for the detection of C. trachomatis in cervical specimens obtained from 217 women attending a clinic for sexually transmitted diseases. The prevalence of infection was 22.1% by culture. The sensitivity, specificity, and positive and negative predictive values were 79.2, 98.2, 92.6, and 94.3%, respectively, for EIA. For PACE 2, the respective values were 77.1, 97.6, 90.1, and 93.7%. After corrections for two false-negative cultures, the sensitivities and specificities were 80 and 99.4%, respectively, for the EIA and 78 and 98.8%, respectively, for the probe assay. Quantitative evaluation of the results showed that false-negative results with either assay were associated with cultures that had low inclusion counts or were negative without subpassage. Analysis of nonculture results revealed that 2.3% of the EIA results and 4.6% of the probe assay results were within +/- 30% of the respective assay cutoff values. These included four false-negative (one EIA and three probe) and two false-positive (one EIA and one probe) results. The Syva MicroTrak EIA and the Gen-Probe PACE 2 assay are comparable to but significantly less sensitive than culture. Use of a grey zone may help identify the need for repeat or confirmatory testing. PMID:7681852

  1. Detection of hepatitis C virus by PCR in second-generation enzyme immunoassay-seropositive blood donors by using matched pairs of fresh frozen plasma and pilot tube sera.

    PubMed Central

    Krajden, M; Zhao, J; Bourke, C; Scalia, V; Gill, P; Lau, W

    1996-01-01

    Between April 1993 and March 1995, 429 of 334,454 (0.13%) blood donations at the Toronto Centre of the Canadian Red Cross were reactive for hepatitis C virus (HCV) by second-generation enzyme immunoassay (EIA-2). Of the 429 EIA-2-positive donations, 189 (44%), 138 (32%), and 102 (24%) were positive, indeterminate, and negative by Second-Generation Recombinant Immunoblot Assay (RIBA-2). To assess HCV viremia and minimize the risk that specimen handling affected PCR-based detection, the qualitative AMPLICOR HCV test was performed on both pilot tube sera (PTS) and the corresponding fresh frozen plasma (FFP) from 294 EIA-2-reactive donations. AMPLICOR PCR results for PTS and FFP were 100% concordant and were confirmed by nested HCV PCR for 27 of 294 donations. The AMPLICOR HCV test was positive for 127 of 140 (91%) of RIBA-2-positive donations (81, 91, and 96% of donations with two, three, and four reactive bands, respectively), 5 of 88 (5.7%) indeterminate donations, and 0 of 66 (0%) RIBA-2-negative donations. The Third-Generation Recombinant Immunoblot Assay (RIBA-3) was performed on RIBA-2-negative, -indeterminate, and -positive, PCR-negative donations. RIBA-3 demonstrated enhanced specificity and resolved 18 of 88 (20%) of RIBA-2-indeterminate samples as HCV antibody positive. The study demonstrates that PTS are as suitable as FFP for PCR-based detection of HCV and can be used to determine if EIA-2-reactive blood donors are viremic at the time of donation. PMID:8862583

  2. Long-term follow-up of Hepatitis B Surface antibody levels in subjects receiving universal Hepatitis B vaccination in infancy in an area of hyperendemicity: correlation between radioimmunoassay and enzyme immunoassay.

    PubMed

    Wang, Ching-Wen; Wang, Li-Chieh; Chang, Mei-Hwei; Ni, Yen-Hsuan; Chen, Huey-Ling; Hsu, Hong-Yuan; Chen, Ding-Shin

    2005-12-01

    The aims of the present study were to determine (i) the long-term immunogenicity and the decay rate of hepatitis B virus (HBV) surface antibody (anti-HBs) from universal hepatitis B vaccination at infancy for a healthy population in an area of hyperendemicity and (ii) whether the anti-HBs levels measured by enzyme immunoassay (EIA) were closely correlated with those assayed by radioimmunoassay (RIA) methods during long-term monitoring. A total of 1,337 apparently healthy children (696 boys and 641 girls) who were vaccinated against HBV at infancy and monitored for anti-HBs annually from 7 to 16 years of age entered the study. Serum samples were analyzed for anti-HBs by RIA at 7 to 15 years of age and were also analyzed by EIA at 13 to 16 years of age. Antibody titers were quantified in mIU/ml by EIA as well as by the ratio of the count in the sample to the count for a negative control (S/N) by RIA. In non-boosted children, the average decay of anti-HBs from 7 to 16 years of ages indicated that approximately 20% of the geometric mean titer decays per year. There was a good correlation between serum anti-HBs levels measured by the RIA and the EIA methods (r=0.91; P<0.0001). An equation for RIA to EIA level conversion was established: log EIA titer=-0.12+ (1.31 . log RIA S/N). The anti-HBs titers measured by EIA correlate well with the S/N assayed by RIA. The annual decay rate of the log anti-HBs level may help in planning booster immunizations for hypo-responders or individuals at risk in adolescence. PMID:16339069

  3. Reliable confirmation of antibodies to human immunodeficiency virus type 1 (HIV-1) with an enzyme-linked immunoassay using recombinant antigens derived from the HIV-1 gag, pol, and env genes.

    PubMed Central

    Ng, V L; Chiang, C S; Debouck, C; McGrath, M S; Grove, T H; Mills, J

    1989-01-01

    An enzyme-linked immunoassay (ELISA) using six recombinant proteins corresponding to large segments of the human immunodeficiency virus type 1 (HIV-1) gag, pol, and env gene products (HIVAGEN; SmithKline Bio-Science Laboratories, Van Nuys, Calif.) was developed to confirm the presence of antibodies to HIV-1 in sera reactive in the whole-cell-derived virion screening ELISAs. Serum samples for testing were obtained from healthy seronegative blood donors and from the different categories of HIV-infected individuals (asymptomatic, acquired immunodeficiency syndrome [AIDS]-related complex, and AIDS). A positive reaction was defined as reactivity against an env and at least one other (either gag or pol) HIV-1 gene product; negative was defined as no reaction with any antigen; and indeterminate was defined as reactivity with gag or pol (or both) or with env alone. None of the 1,180 serum samples from healthy seronegative blood donors gave a positive result, and only 49 of these samples (4%) gave indeterminate results. The recombinant HIV-1 antigen ELISA panel identified seropositive individuals with a high degree of accuracy, as a positive reaction was seen with 99.3% of asymptomatic healthy seropositive individuals, 98.1% of patients with AIDS-related complex, and 90.4% of patients with AIDS. None of the 725 HIV-1-seropositive subjects had a negative test result. Reactivity with the Kp41 antigen, corresponding to an amino-terminal portion of the gp41 envelope glycoprotein, by itself demonstrated 100% sensitivity and specificity in distinguishing seronegative from seropositive sera. A subset of seronegative and seropositive samples were tested both with the recombinant HIV-1 antigen ELISA panel and by Western blot (Du Pont Co.). The recombinant HIV-1 antigen ELISA panel accurately identified more seropositive and seronegative samples and had fewer indeterminate results than did Western blot (interpreted by Du Pont criteria). PMID:2787334

  4. Validity and Reliability of Enzyme Immunoassays Using Leishmania major or L. infantum Antigens for the Diagnosis of Canine Visceral Leishmaniasis in Brazil

    PubMed Central

    de Arruda, Mauro Maciel; Figueiredo, Fabiano Borges; Cardoso, Fernanda Alvarenga; Hiamamoto, Roberto Mitsuyoshi; Brazuna, Júlia Cristina Macksoud; de Oliveira, Maria Regina Fernandes; Noronha, Elza Ferreira; Romero, Gustavo Adolfo Sierra

    2013-01-01

    Background American visceral leishmaniasis is caused by the protozoan Leishmania infantum. Dogs are the main reservoirs in the domestic transmission cycle. The limited accuracy of diagnostic tests for canine leishmaniasis may contribute to the lack of impact of control measures recommended by the Brazilian Ministry of Health. The objective of this study was to estimate the accuracy of two enzyme-linked immunosorbent assays employing L. major or L. infantum antigens and their reliability between three laboratories of different levels of complexity. Methods A validation study of ELISA techniques using L. major or L. infantum antigens was conducted. Direct visualization of the parasite in hematoxylin/eosin-stained histopathological sections, immunohistochemistry, and isolation of the parasite in culture.were used as gold standard. An animal that was positive in at least one of the tests was defined as infected with L. infantum. Serum samples collected from 1,425 dogs were analyzed. Samples were separated in three aliquots and tested in three different laboratories. Sensitivity, specificity and the area under de ROC curve were calculated and the reliability was evaluated between the participant laboratories. Results The sensitivity was 91.8% and 89.8% for the L. major and L. infantum assays, respectively. The specificity was 83.75% and 82.7% for the L. major and L. infantum assays, respectively. The area under de ROC curve was 0.920 and 0.898 for L. major and L. infantum, respectively. The mean intraclass correlation coefficients between laboratories ranged from 0.890 to 0.948 when L. major was used as antigen, and from 0.818 to 0.879 when L. infantum was used. Interpretation ELISA tests using L. major or L. infantum antigens have similar accuracy and reliability. Our results do not support the substitution of the L. major antigen of the ELISA test currently used for the diagnosis of canine visceral leishmaniasis in Brazil. PMID:23922884

  5. Detection of human rotavirus by using an alkaline phosphatase-conjugated synthetic DNA probe in comparison with enzyme-linked immunoassay and polyacrylamide gel analysis.

    PubMed Central

    Olive, D M; Sethi, S K

    1989-01-01

    An alkaline phosphatase-conjugated synthetic oligodeoxyribonucleotide probe was compared with polyacrylamide gel electrophoresis (PAGE) detection of rotavirus RNA as well as an enzyme-linked immunosorbent assay (ELISA) for the detection of rotavirus in stools from young children with gastroenteritis. The synthetic probe did not cross-react with bacterial causative agents of diarrheal disease. Extraction of viral RNA from stool samples with a phenol-chloroform mixture was suitable for most samples. In some cases fecal pigments interfered with the reaction of the probe with viral RNA. The use of ion-exchange chromatography to further purify viral RNA removed contaminating pigments and increased the sensitivity of the probe assay. Of 260 stool specimens, 77 (30%) were positive for rotavirus when tested by PAGE analysis of rotavirus RNA. The synthetic probe identified 71 rotavirus specimens when RNA obtained by phenol-chloroform extraction followed by chromatographic purification was used (sensitivity, 91.0%; specificity, 96.7%). The ELISA results also agreed well with the electrophoretic analysis (sensitivity, 98.7%; specificity 94%) and the probe assay (sensitivity, 90%; specificity, 100%). Discordant results between the ELISA and the probe assay were examined further by electron microscopy and PAGE analysis of viral RNA. The positive and negative predictive values of the probe assay in comparison with PAGE were 92.2 and 96.1%, respectively. Rotaviruses showing both long and short RNA electrophoretic patterns were detected by the probe. The probe assay coupled with chromatographic purification of rotavirus RNA is an effective method for detecting rotavirus and compares favorably with PAGE analysis and ELISA. Images PMID:2536392

  6. Hydrogel nanoparticle based immunoassay

    DOEpatents

    Liotta, Lance A; Luchini, Alessandra; Petricoin, Emanuel F; Espina, Virginia

    2015-04-21

    An immunoassay device incorporating porous polymeric capture nanoparticles within either the sample collection vessel or pre-impregnated into a porous substratum within fluid flow path of the analytical device is presented. This incorporation of capture particles within the immunoassay device improves sensitivity while removing the requirement for pre-processing of samples prior to loading the immunoassay device. A preferred embodiment is coreshell bait containing capture nanoparticles which perform three functions in one step, in solution: a) molecular size sieving, b) target analyte sequestration and concentration, and c) protection from degradation. The polymeric matrix of the capture particles may be made of co-polymeric materials having a structural monomer and an affinity monomer, the affinity monomer having properties that attract the analyte to the capture particle. This device is useful for point of care diagnostic assays for biomedical applications and as field deployable assays for environmental, pathogen and chemical or biological threat identification.

  7. Duplex Microsphere-Based Immunoassay for Detection of Anti-West Nile Virus and Anti-St. Louis Encephalitis Virus Immunoglobulin M Antibodies

    PubMed Central

    Johnson, Alison J.; Noga, Amanda J.; Kosoy, Olga; Lanciotti, Robert S.; Johnson, Alicia A.; Biggerstaff, Brad J.

    2005-01-01

    West Nile (WN) virus was introduced into the United States in 1999, when the first human cases of WN fever and encephalitis appeared in New York City. From there, the virus has spread throughout North America, in some areas cocirculating with the related flavivirus St. Louis encephalitis (SLE) virus. Public health laboratories currently use an immunoglobulin M (IgM) antibody capture enzyme-linked immunosorbent assay (MAC-ELISA) as a primary test for human serodiagnosis, followed by a confirmatory plaque-reduction neutralization test (PRNT). The MAC-ELISAs take 2 days to perform; therefore there is a need for a more rapid test. This report describes a duplex microsphere-based immunoassay (MIA) that shortens the test processing time to about 4.5 h. The assay employs two sets of microspheres coupled to a single flavivirus group-reactive antibody, which are used to capture the WN and SLE viral antigens independently. Immunoglobulin G-depleted serum is concurrently assayed for IgM antibodies to each of the viral antigens. The results are standardized and classified by using quadratic discriminant analysis so that a single result, anti-WN IgM-positive, anti-SLE IgM-positive, negative, or nonspecific, can be determined. The duplex MIA results compared favorably to those of the plaque-reduction neutralization test and MAC-ELISA. The assay proved to be reproducible, produced accurate classifications as to the infecting virus, and was specific. PMID:15879016

  8. IgG and IgM autoantibody differences in discoid and systemic lupus patients

    PubMed Central

    Chong, Benjamin F.; Tseng, Lin-chiang; Lee, Thomas; Vasquez, Rebecca; Li, Quan Z.; Zhang, Song; Karp, David R.; Olsen, Nancy J.; Mohan, Chandra

    2012-01-01

    Systemic lupus (SLE) patients with discoid lupus (DLE) were reported to have milder disease. To test this observation, we employed sandwich arrays containing 98 autoantigens to compare autoantibody profiles of SLE subjects without DLE (DLE−SLE+) (N=9), SLE subjects with DLE (DLE+SLE+) (N=10), DLE subjects without SLE (DLE+SLE−) (N=11), and healthy controls (N=11). We validated differentially expressed autoantibodies using immunoassays in DLE−SLE+ (N=18), DLE+SLE+ (N=17), DLE+SLE− (N=23), and healthy subjects (N=22). Arrays showed 15 IgG autoantibodies (ten against nuclear antigens) and four IgM autoantibodies that were differentially expressed (q-value<0.05). DLE−SLE+ subjects had higher IgG autoantibodies against dsDNA, ssDNA, dsRNA, histone H2A and H2B, and SS-A (52 kDa) than all other groups including DLE+SLE+ subjects (p<0.05). Immunoassays measuring anti-dsDNA, -ssDNA, and -SS-A (52 kDa) IgG autoantibodies showed similar trends (p<0.05). Healthy and DLE+SLE−subjects expressed higher IgM autoantibodies against alpha beta crystallin, lipopolysaccharide, heat shock cognate 70, and desmoglein-3 than DLE+SLE+ and DLE−SLE+ subjects. IgG:IgM ratios of autoantibodies against nuclear antigens progressively rose from healthy to DLE−SLE+ subjects. In conclusion, lower IgG autoantibodies against nuclear antigens in DLE+SLE+ versus DLE−SLE+ subjects suggest that DLE indicates lower disease severity. Higher IgM autoantibodies against selected antigens in healthy and DLE+SLE−subjects may be non-pathogenic. PMID:22763789

  9. Role of signal-to-cut-off ratios of anti-hepatitis C virus antibody by enzyme immunoassays along with ID-NAT for screening of whole blood donors in India

    PubMed Central

    Arora, Satyam; Doda, Veena

    2016-01-01

    Background: The use of elevated signal-to-cut off ratios (S/CO) as an alternate to further supplemental testing (i.e., RIBA) has been included in the guidelines provided by the Centres for Disease Control and Prevention for HCV diagnostic purposes since 2003. With availability of screening by NAT and non availability of RIBA, further confirmation of HCV infection has been possible at the molecular level (RNA). Aims: To study the role of S/CO ratios of anti hepatitis C virus antibody detection by enzyme immunoassays (EIA) along with ID-NAT for screening of whole blood donors. Methods: In this study we reviewed the donor screening status for anti HCV from January 2013 to May 2014. All the donations were screened for anti HCV with fourth generation ELISA (BioRad Monolisa Ag-Ab Ultra) as well as with ID NAT (Procleix Ultrio). The S/CO ratio of all the anti-HCV reactive samples were analysed for their presence of HCV RNA. Results: On screening 21,115 donors for HCV, 83 donors (0.39%) were found reactive on pilot tube and repeat plasma bag testing (S/Co ratio ≥1) by ELISA. 41 donors were HCV RNA reactive with ID-NAT. 4 samples out of 41 were NAT yields and 37 were concordant reactive with ELISA. The S/Co ratio of anti-HCV reactive samples ranged from 0.9-11.1 [mean = 5.1; SD ± 2.9] whereas S/Co ratio of anti HCV and NAT reactive samples (concordant positives) ranged from 4.1-11.1 [mean 7.3]. In our analysis we found that S/CO ratio of 4 showed positive predictive value (PPV) and sensitivity of 100%. Summary/Conclusions: Our study showed that S/CO of 4 for anti HCV on ELISA would have maximum positive predictive value of having donor with HCV RNA. S/CO ratio of 4 is very close to 3.8 which was the CDC guideline. The presence of anti-HCV does not distinguish between current or past infections but a confirmed anti-HCV-positive result indicates the need for counseling and medical evaluation for HCV infection. PMID:27011676

  10. Mass spectrometric immunoassay

    DOEpatents

    Nelson, Randall W.; Williams, Peter; Krone, Jennifer Reeve

    2005-12-13

    Rapid mass spectrometric immunoassay methods for detecting and/or quantifying antibody and antigen analytes utilizing affinity capture to isolate the analytes and internal reference species (for quantification) followed by mass spectrometric analysis of the isolated analyte/internal reference species. Quantification is obtained by normalizing and calibrating obtained mass spectrum against the mass spectrum obtained for an antibody/antigen of known concentration.

  11. Mass spectrometric immunoassay

    DOEpatents

    Nelson, Randall W; Williams, Peter; Krone, Jennifer Reeve

    2007-12-04

    Rapid mass spectrometric immunoassay methods for detecting and/or quantifying antibody and antigen analytes utilizing affinity capture to isolate the analytes and internal reference species (for quantification) followed by mass spectrometric analysis of the isolated analyte/internal reference species. Quantification is obtained by normalizing and calibrating obtained mass spectrum against the mass spectrum obtained for an antibody/antigen of known concentration.

  12. Mass spectrometric immunoassay

    DOEpatents

    Nelson, Randall W; Williams, Peter; Krone, Jennifer Reeve

    2013-07-16

    Rapid mass spectrometric immunoassay methods for detecting and/or quantifying antibody and antigen analytes utilizing affinity capture to isolate the analytes and internal reference species (for quantification) followed by mass spectrometric analysis of the isolated analyte/internal reference species. Quantification is obtained by normalizing and calibrating obtained mass spectrum against the mass spectrum obtained for an antibody/antigen of known concentration.

  13. Development of an ELISA to detect Sin Nombre virus-specific IgM from deer mice (Peromyscus maniculatus).

    PubMed

    Bego, Mariana G; Bawiec, Darcy; Dandge, Deepa; Martino, Benjamin; Dearing, Denise; Wilson, Eric; St Jeor, Stephen

    2008-08-01

    Peromyscus maniculatus (deer mouse) is the primary reservoir for Sin Nombre virus (SNV). Although the presence of IgG antibodies is often used as a marker of infection, it provides little information on active infections in a population but usually is an indicator of past infections. The presence of IgM antibodies is a much better marker for determining whether active infections are present in a population. A mu-capture SNV-specific IgM enzyme linked immunosorbent assay (ELISA) was developed. From live-trap and release studies a total of 68 rodent sera were studied for the presence of Sin Nombre virus-specific IgG and IgM antibodies. In these studies, IgM responses were detected in a number of animals. In some cases early SNV infection was determined through the presence of anti-SNV IgM before IgG antibodies could be detected. From the set of animals analyzed, it was concluded that the IgM response against SNV can persist anywhere from 1 to up to over 2 months, with a median of less than 1 month. Most importantly, it was demonstrated that anti-Sin Nombre virus IgM is an important tool for detection of early infections in rodents and should be considered as a key diagnostic tool. PMID:18586333

  14. Photon budget: constraints from the IGM

    NASA Astrophysics Data System (ADS)

    Kollmeier, Juna

    2015-08-01

    The intergalactic medium serves as an instantaneous calorimeter recording ionizing photon production over time. As such it is sensitive to the different types of photon sources, their amplitude and their evolution over time. In this talk, I will discuss what we are learning about different photon sources over cosmic time by studying the IGM. I will highlight new work at low and high redshift and, in particular, some puzzles and challenges that have come to the fore only recently.

  15. Sequential injection immunoassay for environmental measurements.

    PubMed

    Soh, Nobuaki; Tanaka, Mayumi; Hirakawa, Koji; Zhang, RuiQi; Nakajima, Hizuru; Nakano, Koji; Imato, Toshihiko

    2011-01-01

    Sequential injection immunoassay systems for environmental measurements based on the selective immunoreaction between antigen and antibody were described. A sequential injection analysis (SIA) technique is suitable to be applied for the procedure of enzyme-linked immunosorbent assay (ELISA), because the washing and the addition of reagent solutions can be automated by using a computer-controlled syringe pump and switching valve. We selected vitellogenin (Vg), which is a biomarker for evaluating environmental risk caused by endocrine-disrupting chemicals in the hydrosphere, and linear alkylbenzene sulfonates (LAS) and alkylphenol polyethoxylates (APEO), which are versatile surfactants, as target analytes in the flow immunoassay systems. For Vg monitoring, SIA systems based on spectrophotometric, chemiluminescence, and electrochemical determinations were constructed. On the other hand, chemiluminescence determination was applied to the detection of LAS and APEO. For APEO, an SIA system combined with surface plasmon resonance (SPR) sensor was also developed. These new sequential injection immunoassay systems are expected to be useful systems for environmental analysis. PMID:22076332

  16. Homogeneous Immunoassays: Historical Perspective and Future Promise

    NASA Astrophysics Data System (ADS)

    Ullman, Edwin F.

    1999-06-01

    The founding and growth of Syva Company is examined in the context of its leadership role in the development of homogeneous immunoassays. The simple mix and read protocols of these methods offer advantages in routine analytical and clinical applications. Early homogeneous methods were based on insensitive detection of immunoprecipitation during antigen/antibody binding. The advent of reporter groups in biology provided a means of quantitating immunochemical binding by labeling antibody or antigen and physically separating label incorporated into immune complexes from free label. Although high sensitivity was achieved, quantitative separations were experimentally demanding. Only when it became apparent that reporter groups could provide information, not only about the location of a molecule but also about its microscopic environment, was it possible to design practical non-separation methods. The evolution of early homogenous immunoassays was driven largely by the development of improved detection strategies. The first commercial spin immunoassays, developed by Syva for drug abuse testing during the Vietnam war, were followed by increasingly powerful methods such as immunochemical modulation of enzyme activity, fluorescence, and photo-induced chemiluminescence. Homogeneous methods that quantify analytes at femtomolar concentrations within a few minutes now offer important new opportunities in clinical diagnostics, nucleic acid detection and drug discovery.

  17. Multiplex Electrochemical Immunoassay Using Gold Nanoparticle Probes and Immunochromatographic Strips

    SciTech Connect

    Mao, Xun; Baloda, Meenu; Gurung, Anant; Lin, Yuehe; Liu, Guodong

    2008-10-20

    We describe a multiplex electrochemical immunoassay based on the use of gold nanoparticle (Au-NP) probes and immunochromatographic strips (ISs). The approach takes advantage of the speed and low cost of the conventional IS tests and the high sensitivities of the nanoparticle-based electrochemical immunoassays. Rabbit IgG(R-IgG) and human IgM (H-IgM) were used as model targets for the demonstration of the proof of concept. The Au-NPs based sandwich immunoreactions were performed on the IS, and the captured gold nanoparticle labels on the test zones were determined by highly-sensitive stripping voltammetric measurement of the dissolved gold ions (III) with a carbon paste electrode. The detection limits are 1.0 and 1.5 ng/mL with the linear ranges of 2.5-250 ng/mL for quantitative detection of R-IgG and H-IgM, respectively. The total assay time is around 25 minutes. Such multiplex electrochemical immunoassay could be readily highly multiplexed to allow simultaneous parallel detection of numerous proteins and is expected to open new opportunities for protein diagnostics and biosecurity.

  18. Enzyme-linked immunosorbent assays for the detection of antibody to Crimean-Congo haemorrhagic fever virus in the sera of livestock and wild vertebrates.

    PubMed Central

    Burt, F. J.; Swanepoel, R.; Braack, L. E.

    1993-01-01

    IgM antibody response to Crimean-Congo haemorrhagic fever (CCHF) virus was monitored in experimentally infected sheep and cattle by an IgM capture enzyme-linked immunoassay (ELISA). Specific binding of antigen was detected by a rabbit anti-CCHF horseradish peroxidase conjugate or a sandwich technique with hyperimmune mouse anti-CCHF ascitic fluid and commercially available anti-mouse immunoglobulin peroxidase conjugate. The persistence of IgM antibody activity was found to be of shorter duration than in humans, and this may be a function of the relative lack of susceptibility of these animals to infection with CCHF virus. IgG antibody responses in the sheep could be monitored by sandwich ELISA using commercially available anti-sheep immunoglobulin peroxidase conjugates. Total antibody activity in the sera of experimentally infected sheep, cattle and small mammals could be monitored in a competitive ELISA (CELISA) using rabbit anti-CCHF peroxidase conjugate. The CELISA was applied to the sera of 960 wild vertebrates from a nature reserve in South Africa, and the prevalence of antibody was found to be greatest in large mammals such as rhinoceros, giraffe and buffalo, which are known to be the preferred hosts of the adult tick (Hyalomma) vectors of the virus. PMID:8270014

  19. Protein microchips : use for immunoassay and enzymatic reactions.

    SciTech Connect

    Arenkov, P.; Kukhtin, A.; Gemmell, A.; Voloschuk, S.; Chupeeva, V.; Mirzabekov, A.; Biochip Technology Center; Russian Academy of Sciences

    2000-02-15

    Different proteins such as antibodies, antigens, and enzymes were immobilized within the 100 x 100 x 20-{mu}m gel pads of protein microchips. A modified polyacrylamide gel has been developed to accommodate proteins of a size up to 400,000 daltons. Electrophoresis in the microchip reaction chamber speeded up antigen-antibody interactions within the gel. Protein microchips were used in immunoassays for detection of antigens or antibodies, as well as to carry out enzymatic reactions and to measure their kinetics in the absence or presence of an inhibitor. A protein microchip can be used several times in different immunoassays and enzymatic kinetic measurements.

  20. A Wash-Free Homogeneous Colorimetric Immunoassay Method

    PubMed Central

    Liu, Huiqiao; Rong, Pengfei; Jia, Hongwei; Yang, Jie; Dong, Bo; Dong, Qiong; Yang, Cejun; Hu, Pengzhi; Wang, Wei; Liu, Haitao; Liu, Dingbin

    2016-01-01

    Rapid and convenient biosensing platforms could be beneficial to timely diagnosis and treatment of diseases in virtually any care settings. Sandwich immunoassays, the most commonly used methods for protein detection, often rely on expensive tags such as enzyme and tedious wash and incubation procedures operated by skilled labor. In this report, we revolutionized traditional sandwich immunoassays by providing a wash-free homogeneous colorimetric immunoassay method without requirement of any separation steps. The proposed strategy was realized by controlling the growth of gold nanoparticles (AuNPs) to mediate the interparticle spacing in the protein-AuNP oligomers. We have demonstrated the successful in vitro detection of cancer biomarker in serum samples from patients with high clinical sensitivity and specificity. PMID:26722373

  1. A Wash-Free Homogeneous Colorimetric Immunoassay Method.

    PubMed

    Liu, Huiqiao; Rong, Pengfei; Jia, Hongwei; Yang, Jie; Dong, Bo; Dong, Qiong; Yang, Cejun; Hu, Pengzhi; Wang, Wei; Liu, Haitao; Liu, Dingbin

    2016-01-01

    Rapid and convenient biosensing platforms could be beneficial to timely diagnosis and treatment of diseases in virtually any care settings. Sandwich immunoassays, the most commonly used methods for protein detection, often rely on expensive tags such as enzyme and tedious wash and incubation procedures operated by skilled labor. In this report, we revolutionized traditional sandwich immunoassays by providing a wash-free homogeneous colorimetric immunoassay method without requirement of any separation steps. The proposed strategy was realized by controlling the growth of gold nanoparticles (AuNPs) to mediate the interparticle spacing in the protein-AuNP oligomers. We have demonstrated the successful in vitro detection of cancer biomarker in serum samples from patients with high clinical sensitivity and specificity. PMID:26722373

  2. IgM in the kidney: a multiple personality disorder.

    PubMed

    Platt, Jeffrey L; Cascalho, Marilia

    2015-09-01

    IgM in the blood of normal individuals consists mainly of 'natural' polyreactive antibodies. Natural IgM is thought to provide an initial defense against infection and to promote the healing of wounded cells. Yet, as Panzer and colleagues show, these benefits can be eclipsed when the IgM binds to damaged cells of the glomerulus, activating complement. IgM in glomeruli thus signifies cellular damage and may warn that the pace of that damage exceeds the capacity for repair. PMID:26323070

  3. A rapid lateral flow immunoassay for the detection of fungal alpha-amylase at the workplace.

    PubMed

    Koets, Marjo; Sander, Ingrid; Bogdanovic, Jelena; Doekes, Gert; van Amerongen, Aart

    2006-09-01

    Fungal alpha-amylase is a flour supplement which is added to improve the quality of bakery products. Various studies have shown that exposure to this enzyme is an important risk factor for the development of bakers' allergy and this allergy is reported to be one of the most frequent causes of occupational asthma. A rapid assay was developed to monitor exposure to occupational allergens directly at the workplace. The sensitivity of the developed assay is 0.32 ng amylase mL(-1) in a buffer system with the commercially available alpha-amylase preparation Fungamyl 1600S as the standard. Initial validation tests (n = 33) were performed with airborne and settled dust from an industrial bakery. The new lateral flow immunoassay detected amylase in 22 of the 26 samples regarded as positive in an enzyme immunoassay, and was negative for all seven enzyme immunoassay-negative samples, while the four lateral flow immunoassay-negative/enzyme immunoassay-positive samples all had levels below 2 ng mL(-1). The sensitivity of 2 ng mL(-1) of the amylase lateral flow immunoassay is sufficient for first screening purposes and, therefore, this simple and rapid assay may allow direct on-site demonstration of work-related hazards of bio-allergen exposure. This would be particularly useful in occupational hygiene practice, especially in traditional or small-scale bakeries which lack the technological skills for testing the exposure to respiratory allergens. PMID:16951754

  4. Lateral Flow Immunoassay.

    PubMed

    Ching, Kathryn H

    2015-01-01

    Lateral flow immunoassays (LFIAs) are a staple in the field of rapid diagnostics. These small handheld devices require no specialized training or equipment to operate, and generate a result within minutes of sample application. They are an ideal format for many types of home test kits, for emergency responders and for food manufacturers and producers looking for a quick evaluation of a given sample. LFIAs rely on high quality monoclonal antibodies that recognize the analyte of interest. As monoclonal antibody technology becomes more accessible to smaller laboratories, there has been increased interest in developing LFIA prototypes for potential commercial manufacture. In this chapter, the basics of designing and building an LFIA prototype are described. PMID:26160571

  5. IgM in Microbial Infections: Taken for Granted?

    PubMed Central

    Racine, Rachael; Winslow, Gary M.

    2009-01-01

    Much has been learned about the structure, function, and production of IgM, since the antibody's initial characterization. It is widely accepted that IgM provides a first line of defense during microbial infections, prior to the generation of adaptive, high affinity IgG responses that are important for long-lived immunity and immunological memory. Although IgM responses are commonly used as a measure of exposure to infectious diseases, it is perhaps surprising that the role of and requirement for IgM in many microbial infections has not been well explored in vivo. This is in part due to the lack of capabilities, until relatively recently, to evaluate the requirement for IgM in the absence of coincident IgG responses. Such evaluations are now possible, using gene-targeted mouse strains that produce only IgM, or isotype-switched IgG. A number of studies have revealed that IgM, produced either innately, or in response to antigen challenge, plays an important and perhaps underappreciated role in many microbial infections. Moreover, the characterization of the roles of various B cell subsets, in the production of IgM, and in host defense, has revealed important and divergent roles for B-1a and B-1b cells. This review will highlight studies in which IgM, in its own right, has been found to play an important role, not only in early immunity, but also in long-term protection, against a variety of microbial pathogens. Observations that long-lived IgM responses can be generated in vivo suggest that it may be feasible to target IgM production as part of vaccination strategies. PMID:19539648

  6. Morphological resonances for multicomponent immunoassays

    NASA Astrophysics Data System (ADS)

    Whitten, W. B.; Shapiro, M. J.; Ramsey, J. M.; Bronk, B. V.

    1995-06-01

    An immunoassay technique capable of detecting and identifying a number of species of microorganisms in a single analysis is described. The method uses optical-resonance size discrimination of microspheres to identify antibodies to which stained microorganisms are bound.

  7. Rituximab impairs immunoglobulin (Ig)M and IgG (subclass) responses after influenza vaccination in rheumatoid arthritis patients

    PubMed Central

    Westra, J; van Assen, S; Wilting, K R; Land, J; Horst, G; de Haan, A; Bijl, M

    2014-01-01

    Rituximab (RTX) treatment in rheumatoid arthritis (RA) patients severely hampers humoral response after influenza vaccination as determined by haemagglutination inhibition assay (HI). It is not known whether HI reflects both immunoglobulin (Ig)M and IgG (subclass) influenza response, and whether IgM antibodies contribute to the low rate of influenza infection seen in RA patients. Twenty RA patients on methotrexate (MTX), 23 on RTX and 28 healthy controls (HC) received trivalent influenza subunit vaccination. Before and 28 days after vaccination, H1N1- and H3N2-specific antibodies were measured by HI and by IgM and IgG (subclass) enzyme-linked immunosorbent assay (ELISA). B cell activating factor (BAFF) levels were determined in serum samples before vaccination. Vaccination induced a significant increase of IgM and IgG (IgG1 and IgG3) antibodies against both strains in the HC and MTX groups (all P < 0·01), but not in the RTX group. HI correlated significantly in all cases with IgG (IgG1) but not with IgM. In RTX late patients (RTX treatment 6–10 months before vaccination), IgG (IgG1 and IgG3) response to vaccination was restored, but not IgM response. BAFF levels were significantly increased in RA-RTX patients and correlated with total IgG levels. Haemagglutination inhibition assay, used as gold standard, detects primarily IgG (IgG1) responses. IgM- and IgG influenza-specific antibodies increase after vaccination in HC and RA patients except in patients on RTX treatment. BAFF levels are increased in both early and late RTX-treated patients, but do not correlate with an influenza-specific antibody response. PMID:24889761

  8. IgG and IgM antibodies to viral glycoproteins in respiratory syncytial virus infections of graded severity.

    PubMed Central

    Toms, G L; Webb, M S; Milner, P D; Milner, A D; Routledge, E G; Scott, R; Stokes, G M; Swarbrick, A; Taylor, C E

    1989-01-01

    Serum antibodies to the fusion (F) and large glycoprotein (G) of respiratory syncytial virus in the serum of 57 infected infants were measured by enzyme linked immunosorbent assay (ELISA). Most serum samples taken at the time of admission to hospital contained antibodies to both glycoproteins, and overall there was no significant evidence of a selective deficiency of antibody to either viral antigen. Less than a quarter of the infants showed rising IgG antibody titres to either glycoprotein after infection, whereas over threequarters produced an IgM response. There was a significant correlation between IgG response to viral glycoproteins and the age of the infant. The correlation of age with the IgM response was less pronounced, and there was no correlation between serum IgG antibody derived transplancentally in the acute phase of infection and IgM response to either glycoprotein. Neither IgG or IgM responses correlated with a clinical assessment of the severity of infection in the infants. IgM responses, however, were weakly correlated with reduced secretion of infectious virus in the upper respiratory tract. PMID:2624472

  9. Development of an enzyme-immunoassay (EIA) for the measurement of follicle-stimulating-hormone (FSH) in Callitrichid primates using a monoclonal antibody against the human-FSH-beta-subunit.

    PubMed

    Rosenbusch, J; Dias, J A; Hodges, J K

    1997-01-01

    Despite the importance of Callitrichid primates in both biomedical and conservation research, practical and reliable immunoassays for the measurement of follicle-stimulating hormone (FSH) have not yet been described. A panel of monoclonal antibodies against specific peptide fragments within either the alpha or beta subunit of human FSH was evaluated for their ability to recognize FSH from Callitrichid and other New World primates. One of these, monoclonal antibody 46.3h6.b7 raised against human FSH, was selected due to its ability to recognize marmoset monkey FSH and its low crossreactivity with other gonadotrophins. The antibody formed the basis of an enzymeimmunoassay using a highly purified human urinary FSH (Metrodin, Serono) preparation coupled to biotin as label and unmodified as standard. After 24 h incubation, antibody bound label was visualized by addition of streptavidin-peroxidase followed by the appropriate substrate. Parallelism was obtained between the standard and dilutions of pituitary extracts, urine and plasma from the common marmoset (Callithrix jacchus) as well as from two tamarin species (Saguinus fuscicollis and S. oedipus) and one squirrel monkey (Saimiri sciureus). Profiles of plasma and urinary FSH during the follicular phase are shown for two individual marmosets. The ability to measure FSH in Callitrichidae provides new opportunities for studies of the reproductive biology of these New World primate species. PMID:9057964

  10. GRBs as Probes of the IGM

    NASA Astrophysics Data System (ADS)

    Cucchiara, Antonino; Totani, Tonomori; Tanvir, Nial

    2016-05-01

    Gamma-ray Bursts (GRBs) are the most powerful explosions known, capable of outshining the rest of gamma-ray sky during their short-lived prompt emission. Their cosmological nature makes them the best tool to explore the final stages in the lives of very massive stars up to the highest redshifts. Furthermore, studying the emission from their low-energy counterparts (optical and infrared) via rapid spectroscopy, we have been able to pin down the exact location of the most distant galaxies as well as placing stringent constraints on their host galaxies and intervening systems at low and high-redshift (e.g. metallicity and neutral hydrogen fraction). In fact, each GRB spectrum contains absorption features imprinted by metals in the host interstellar medium (ISM) as well as the intervening intergalactic medium (IGM) along the line of sight. In this chapter we summarize the progress made using a large dataset of GRB spectra in understanding the nature of both these absorbers and how GRBs can be used to study the early Universe, in particular to measure the neutral hydrogen fraction and the escape fraction of UV photons before and during the epoch of re-ionization.

  11. Variability in Telavancin Cross-Reactivity among Vancomycin Immunoassays

    PubMed Central

    McConeghy, Kevin W.; Liao, Siyun; Clark, Douglas; Worboys, Philip; Barriere, Steven L.

    2014-01-01

    Telavancin is a semisynthetic lipoglycopeptide with a dual mechanism of action against Gram-positive pathogens. Two brief reports have suggested potential cross-reactivity of telavancin with the vancomycin particle-enhanced turbidometric immunoassay (PETIA). The purpose of this study was to evaluate several commercially available vancomycin immunoassays (fluorescence polarization [FPIA], enzyme-multiplied immunoassays [EMIT], PETIA, and chemiluminescent immunoassay [CMIA]) for cross-reactivity with telavancin. Seven sites were selected to analyze serum samples for vancomycin. Each site received a set of samples (n = 18) which combined drug-free serum with telavancin, 7-OH telavancin metabolite, or vancomycin. Immunoassays demonstrating potential cross-reactivity were further evaluated by sending a duplicate sample set to multiple laboratories. Cross-reactivity was defined as the percent theoretical concentration (reported concentration/theoretical concentration × 100). No cross-reactivity was seen with FPIA or EMIT. Within the theoretical concentration range of 5 to 120 μg/ml of telavancin, the Synchron PETIA system reported vancomycin concentrations ranging from 4.7 to 54.2 μg/ml compared to vancomycin concentrations from 1.1 to 5.6 μg/ml for the Vista PETIA system. The Architect CMIA system reported vancomycin concentrations in the range of 0.27 to 0.97 μg/ml, whereas Advia Centaur XP CMIA reported vancomycin concentrations between 1.6 and 31.6 μg/ml. The Architect CMIA immunoassay had the lowest percent cross-reactivity (0.8 to 5.4%), while the Synchron PETIA immunoassay demonstrated the highest percent cross-reactivity (45.2 to 53.8%). Telavancin samples measured by liquid chromatography-mass spectroscopy were within 93.9 to 122% of theoretical concentrations. Vancomycin concentrations were not measured in any 7-OH telavancin-spiked sample. Vancomycin concentrations measured by liquid chromatography-mass spectroscopy were within 57.2 to 113% of theoretical concentrations. PETIA and CMIA measured vancomycin concentrations in telavancin-spiked samples. Significant variability in percent cross-reactivity was observed for each platform regardless of immunoassay method. PMID:25223996

  12. The importance of IgM positivity in laboratory diagnosis of gestational and congenital syphilis.

    PubMed

    Nemes-Nikodém, E; Vörös, E; Pónyai, K; Párducz, L; Kárpáti, S; Rozgonyi, F; Ostorházi, E

    2012-06-01

    From January 1, 2009 through December 31, 2011, from 33,753 blood samples for syphilis screening, Treponema pallidum infections were confirmed in 241 pregnant women at the Department of Dermatology, Venerology, and Dermatooncology of Semmelweis University Budapest. In this period, four children born to inadequately or untreated women were confirmed to have connatal syphilis. The height of rapid plasma reagin (RPR) titer was measured to determine the stage of the infection and to examine the success of the antilues therapy. The diagnosis of maternal syphilis infection was confirmed with enzyme linked immunosorbent assay (ELISA), T. pallidum particle agglutination (TPPA), and IgG and IgM immunoblots. Maternal IgM immunoblot results identify mothers at risk of delivering babies with connatal syphilis better than the height of maternal RPR titer. The standard serological tests are less useful in newborns because of IgG transfer across the placenta. IgM test which depends on the infant's response has more specificity in diagnosing connatal syphilis. PMID:24672684

  13. Purification of the Sm nuclear autoantigen. Detection and clinical significance of IgM antibody.

    PubMed Central

    Pollard, K M; Tan, E M

    1985-01-01

    Sm antigen from rabbit thymus acetone powder was purified using a combination of ammonium sulphate precipitation, DEAE-Sephacel and hydroxyapatite chromatography. This preparation was devoid of previously identified nuclear antigens including ribonucleoprotein (U1-RNP), proliferating cell nuclear antigen (PCNA), Sjgren's syndrome antigen A (SS-A/Ro), Sjgren's syndrome antigen B (SS-B/La), Sjgren's lupus antigen (SL), scleroderma antigen 70 (Scl-70), DNA and histones. The purified material was used in an enzyme linked immunosorbent assay (ELISA) to detect anti-Sm antibody. All sera with precipitating Sm antibody detected by immunodiffusion gave reactions in ELISA greater than 0.40 OD405 and contained predominantly IgG anti-Sm antibody. Of 112 sera which did not have anti-Sm by immunodiffusion there were five which gave reactions greater than 0.40 OD405. Four of these five sera contained only IgM antibody and the fifth contained both IgM and IgG. Of these five, one came from a 'normal' control who had a positive anti-nuclear antibody (ANA), facial rash and diabetes, two were from patients with systemic lupus erythematosus (SLE) and two were from patients with mixed connective tissue disease (MCTD). These findings demonstrate that there are patients whose anti-Sm response may be restricted to IgM and in some of these patients the clinical presentation may be different from that of classical SLE. PMID:4017287

  14. Fluorescence fluctuation immunoassay.

    PubMed

    Elings, V B; Nicoli, D F; Briggs, J

    1983-01-01

    The homogeneous fluorescent immunoassay described above allows one to measure the brightness of fluorescently tagged carrier particles that are suspended in a background of free, unbound fluorescent sources. We have demonstrated the feasibility of our technique using a gentamicin competitive assay as well as idealized model systems. We have seen that the fluctuation-correlation method is able to discriminate against free background sources because each fluorescing particle in solution contributes to the correlation peak [Eq. (4)] with a weighting equal to the square of its respective intensity. Hence, a few very bright sources contribute disproportionately to the "signal" relative to many weak ones. To take advantage of this property, one would therefore design an assay that uses relatively larger carrier particles, each of which is capable of binding on the order of 10(3) to 10(4) tagged antibodies or antigens. Unfortunately, the nonlinear dependence of the correlation peak on the brightness of the fluorescing species causes the technique to be perturbed by carrier particle aggregation; the apparent bound fluorescence intensity increases with the extent of aggregation. The latter may be an unavoidable consequence of performing assays using raw blood serum, for example. The ultimate usefulness of this method will depend on its sensitivity and speed when applied to "real" assays of clinical significance. These characteristics will be influenced by a number of technical details. Given our limited experience with the method thus far, it would appear that its principal drawback is its relatively slow speed. In order to decrease the time needed for a reliable measurement, one must average the random fluctuations in the fluorescent intensity to zero more quickly. In principle, this can be accomplished by decreasing the shot noise by collecting a larger fraction of the fluorescent light, and increasing the sampling rate. The method requires rather complicated instrumentation; it is by no means clear that this level of complexity is justified given the realistic level of sensitivity that will be obtained by this technique. PMID:6855625

  15. Kinetic analyses and performance of a colloidal magnetic nanoparticle based immunoassay dedicated to allergy diagnosis.

    PubMed

    Teste, Bruno; Kanoufi, Frédéric; Descroix, Stéphanie; Poncet, Pascal; Georgelin, Thomas; Siaugue, Jean-Michel; Petr, Jan; Varenne, Anne; Hennion, Marie-Claire

    2011-07-01

    In this paper, we demonstrate the possibility to use magnetic nanoparticles as immunosupports for allergy diagnosis. Most immunoassays used for immunosupports and clinical diagnosis are based on a heterogeneous solid-phase system and suffer from mass-transfer limitation. The nanoparticles' colloidal behavior and magnetic properties bring the advantages of homogeneous immunoassay, i.e., species diffusion, and of heterogeneous immunoassay, i.e., easy separation of the immunocomplex and free forms, as well as analyte preconcentration. We thus developed a colloidal, non-competitive, indirect immunoassay using magnetic core-shell nanoparticles (MCSNP) as immunosupports. The feasibility of such an immunoassay was first demonstrated with a model antibody and described by comparing the immunocapture kinetics using macro (standard microtiter plate), micro (microparticles) and nanosupports (MCSNP). The influence of the nanosupport properties (surface chemistry, antigen density) and of the medium (ionic strength, counter ion nature) on the immunocapture efficiency and specificity was then investigated. The performances of this original MCSNP-based immunoassay were compared with a gold standard enzyme-linked immunosorbent assay (ELISA) using a microtiter plate. The capture rate of target IgG was accelerated 200-fold and a tenfold lower limit of detection was achieved. Finally, the MCSNP-based immunoassay was successfully applied to the detection of specific IgE from milk-allergic patient's sera with a lower LOD and a good agreement (CV < 6%) with the microtiter plate, confirming the great potential of this analytical platform in the field of immunodiagnosis. PMID:21547429

  16. Nanobody medicated immunoassay for ultrasensitive detection of cancer biomarker alpha-fetoprotein.

    PubMed

    Chen, Jing; He, Qing-hua; Xu, Yang; Fu, Jin-heng; Li, Yan-ping; Tu, Zhui; Wang, Dan; Shu, Mei; Qiu, Yu-lou; Yang, Hong-wei; Liu, Yuan-yuan

    2016-01-15

    Immunoassay for cancer biomarkers plays an important role in cancer prevention and early diagnosis. To the development of immunoassay, the quality and stability of applied antibody is one of the key points to obtain reliability and high sensitivity for immunoassay. The main purpose of this study was to develop a novel immunoassay for ultrasensitive detection of cancer biomarker alpha-fetoprotein (AFP) based on nanobody against AFP. Two nanobodies which bind to AFP were selected from a phage display nanobody library by biopanning strategy. The prepared nanobodies are clonable, thermally stable and applied in both sandwich enzyme linked immunoassay (ELISA) and immuno-PCR assay for ultrasensitive detection of AFP. The limit detection of sandwich ELISA setup with optimized nanobodies was 0.48ng mL(-1), and the half of saturation concentration (SC50) value was 6.68±0.56ng mL(-1). These nanobodies were also used to develop an immuno-PCR assay for ultrasensitive detection of AFP, its limit detection values was 0.005ng mL(-1), and the linear range was 0.01-10,000ng mL(-1). These established immunoassays based on nanobodies were highly specific to AFP and with negligible cross reactivity with other tested caner biomarkers. Furthermore, this novel concept of nanobodies mediated immunoassay may provide potential applications in a general method for the ultrasensitive detection of various cancer biomarkers. PMID:26592642

  17. ELEGANT ENVIRONMENTAL IMMUNOASSAYS

    EPA Science Inventory

    Immunochemical methods are based on selective antibodies directed to a particular target analyte. The specific binding between antibody and analyte can be used for detection and quantitation. Methods such as the enzyme-linked immunosorbent assay (ELISA) can provide a sensitiv...

  18. Use of aptamers in immunoassays.

    PubMed

    Nezlin, Roald

    2016-02-01

    Aptamers, short single-chain DNA or RNA oligonucleotides, react specifically with small molecules, as well as with proteins. Unlike antibodies, they may be obtained relatively easily. Aptamers are now widely employed in immunological studies and could replace antibodies in immunoassays. In this short review, methods for immobilizing aptamers on various insoluble materials (so-called apta-sorbents) are described. Recent findings on their use in the detection and isolation of immunoglobulins and their application in various immunoassays are also discussed. PMID:26774749

  19. IRAK-4 and MyD88 deficiencies impair IgM responses against T-independent bacterial antigens

    PubMed Central

    Maglione, Paul J.; Simchoni, Noa; Black, Samuel; Radigan, Lin; Overbey, Jessica R.; Bagiella, Emilia; Bussel, James B.; Bossuyt, Xavier; Casanova, Jean-Laurent; Meyts, Isabelle; Cerutti, Andrea; Picard, Capucine

    2014-01-01

    IRAK-4 and MyD88 deficiencies impair interleukin 1 receptor and Toll-like receptor (TLR) signaling and lead to heightened susceptibility to invasive bacterial infections. Individuals with these primary immunodeficiencies have fewer immunoglobulin M (IgM)+IgD+CD27+ B cells, a population that resembles murine splenic marginal zone B cells that mount T-independent antibody responses against bacterial antigens. However, the significance of this B-cell subset in humans is poorly understood. Using both a 610 carbohydrate array and enzyme-linked immunosorbent assay, we found that patients with IRAK-4 and MyD88 deficiencies have reduced serum IgM, but not IgG antibody, recognizing T-independent bacterial antigens. Moreover, the quantity of specific IgM correlated with IgM+IgD+CD27+ B-cell frequencies. As with mouse marginal zone B cells, human IgM+CD27+ B cells activated by TLR7 or TLR9 agonists produced phosphorylcholine-specific IgM. Further linking splenic IgM+IgD+CD27+ B cells with production of T-independent IgM, serum from splenectomized subjects, who also have few IgM+IgD+CD27+ B cells, had reduced antibacterial IgM. IRAK-4 and MyD88 deficiencies impaired TLR-induced proliferation of this B-cell subset, suggesting a means by which loss of this activation pathway leads to reduced cell numbers. Thus, by bolstering the IgM+IgD+CD27+ B-cell subset, IRAK-4 and MyD88 promote optimal T-independent IgM antibody responses against bacteria in humans. PMID:25320238

  20. IgM, Fc mu Rs, and malarial immune evasion.

    PubMed

    Czajkowsky, Daniel M; Salanti, Ali; Ditlev, Sisse B; Shao, Zhifeng; Ghumra, Ashfaq; Rowe, J Alexandra; Pleass, Richard J

    2010-05-01

    IgM is an ancestral Ab class found in all jawed vertebrates, from sharks to mammals. This ancient ancestry is shared by malaria parasites (genus Plasmodium) that infect all classes of terrestrial vertebrates with whom they coevolved. IgM, the least studied and most enigmatic of the vertebrate Igs, was recently shown to form an intimate relationship with the malaria parasite Plasmodium falciparum. In this article, we discuss how this association might have come about, building on the recently determined structure of the human IgM pentamer, and how this interaction could affect parasite survival, particularly in light of the just-discovered Fc mu R localized to B and T cell surfaces. Because this parasite may exploit an interaction with IgM to limit immune detection, as well as to manipulate the immune response when detected, a better understanding of this association may prove critical for the development of improved vaccines or vaccination strategies. PMID:20410497

  1. Protein Adsorption in Microengraving Immunoassays

    PubMed Central

    Song, Qing

    2015-01-01

    Microengraving is a novel immunoassay forcharacterizing multiple protein secretions from single cells. During the immunoassay, characteristic diffusion and kinetic time scales τD and τK determine the time for molecular diffusion of proteins secreted from the activated single lymphocytes and subsequent binding onto the glass slide surface respectively. Our results demonstrate that molecular diffusion plays important roles in the early stage of protein adsorption dynamics which shifts to a kinetic controlled mechanism in the later stage. Similar dynamic pathways are observed for protein adsorption with significantly fast rates and rapid shifts in transport mechanisms when C0* is increased a hundred times from 0.313 to 31.3. Theoretical adsorption isotherms follow the trend of experimentally obtained data. Adsorption isotherms indicate that amount of proteins secreted from individual cells and subsequently captured on a clean glass slide surface increases monotonically with time. Our study directly validates that protein secretion rates can be quantified by the microengraving immunoassay. This will enable us to apply microengraving immunoassays to quantify secretion rates from 104–105 single cells in parallel, screen antigen-specific cells with the highest secretion rate for clonal expansion and quantitatively reveal cellular heterogeneity within a small cell sample. PMID:26501282

  2. IgM but not IgG monoclonal anti-Nocardia brasiliensis antibodies confer protection against experimental actinomycetoma in BALB/c mice.

    PubMed

    Gonzalez-Suarez, Maria L; Salinas-Carmona, Mario C; Pérez-Rivera, Isabel

    2009-10-01

    Nocardia brasiliensis is a facultative intracellular microorganism that produces a human chronic infection known as actinomycetoma. Human and mouse anti-N. brasiliensis antibody response identify P24, P26 and P61 immunodominant antigens. In this work, we generated immunoglobulin M (IgM) and IgG monoclonal antibodies (mAbs) specific to immunodominant P61 antigen. The monoclonal IgM (NbM1) and IgG2a (NbG1) antibodies were assessed for their in vitro bactericidal activity, in vivo protective effect and ability to block catalase activity. These mAbs specifically recognized P61, but they did not inhibit its enzyme activity. The in vitro bactericidal effect of NbG1 was higher than the killing ability of the IgM mAb. In vivo experiments with a murine model of experimental infection with N. brasiliensis injected into rear footpads was used to test the effect of NbM1 and NbG1. The negative untreated group developed a chronic actinomycetoma within 4 weeks. IgM mAbs conferred protection to BALB/c mice infected with N. brasiliensis. IgG mAb lacked this protective effect. IgM mAb showed a dose-response correlation between antibody concentration and lesion size. These results demonstrate that humoral immune response mediated by antigen-specific IgM antibody protects against an intracellular bacterial infection. PMID:19624737

  3. Determination of designer drug cross-reactivity on five commercial immunoassay screening kits.

    PubMed

    Regester, Laura E; Chmiel, Jeffrey D; Holler, Justin M; Vorce, Shawn P; Levine, Barry; Bosy, Thomas Z

    2015-03-01

    The detection of new designer drugs is often a difficult issue in forensic urine drug testing as immunoassays are the primary screening methodology for drugs of abuse in many of these laboratories. Cross-reactivity of compounds with immunoassay kits can either aid or complicate the detection of a variety of drug and drug metabolites. For instance, emerging designer drugs that share structural similarities to amphetamines and phencyclidine (PCP) have the potential to cross-react with assays designed to detect these compounds. This study evaluates the cross-reactivity of five commercially available immunoassay reagent kits for 94 designer drugs on a Roche/Hitachi Modular P automated screening instrument. The compounds used in this study are grouped by structural class as follows: 2,5-dimethoxyamphetamines, 2C (2,5-dimethoxyphenethylamines), β-keto amphetamines, substituted amphetamines, piperazines, α-pyrrolidinopropiophenones, tryptamines and PCP analogs. A drug concentration of 100 µg/mL was used to determine cross-reactivity for each assay and resulted in the following positive rates: Microgenics DRI(®) Ecstasy enzyme assay (19%), Microgenics DRI(®) Phencyclidine enzyme assay (20%), Lin-Zhi Methamphetamine enzyme immunoassay (39%), Siemens/Syva(®) EMIT(®)II Plus Amphetamines assay (43%) and CEDIA(®) DAU Amphetamine/Ecstasy assay (57%). Of the 94 designer drugs tested, 14% produced a negative response for all five kits. No designer drug used in this study generated a positive result for all five immunoassay kits. PMID:25492523

  4. Diagnostic value of detecting specific IgA and IgM with recombinant Trypanosoma cruzi antigens in congenital Chagas' disease.

    PubMed

    Lorca, M; Veloso, C; Munoz, P; Bahamonde, M I; Garcia, A

    1995-06-01

    The present study compares the early diagnosis of congenital Chagas' disease with a DOT assay using recombinant antigens with immunofluorescence antibody testing (IFAT) and an enzyme-linked immunosorbent assay (ELISA). The studies were performed using cord blood and sera of 12 infected newborns (group I) and 12 uninfected ones born to Trypanosoma cruzi-infected mothers (group II). Conventional IFAT and ELISA showed positive results for IgG at high titers, in infants and mothers of both groups; IgA antibodies were detected by ELISA in four of the infected infants and IgM was detected in two of them. All sera of the uninfected infants were negative for IgA and IgM in the ELISA. Application of a DOT assay using eight recombinant T. cruzi antigens allowed detection of specific IgA in the cord blood of six of the infected cases and IgM in eight of them. Repetition of these serologic tests in samples obtained during a monthly follow-up gave positive results for IgA in two of the initially negative infants of group I and for IgM in four of them. This means that diagnosis of congenital T. cruzi infection was confirmed, through demonstration of specific IgM, in all infected infants, and of IgA in eight of them. The importance of late detection of IgM in siblings born of infected mothers is discussed. The detection of IgM and IgA in sera obtained after birth is believed to be due to a congenital transmission of the parasite that occurred late in pregnancy. No IgA or IgM antibodies could be detected by the DOT assay in the sera of the negative controls.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:7611556

  5. IgM and IgG antibody response to Paracoccidioides brasiliensis in naturally infected wild armadillos (Dasypus novemcinctus).

    PubMed

    Fernandes, G F; Deps, P; Tomimori-Yamashita, J; Camargo, Z P

    2004-08-01

    We studied the extent to which wild nine-banded armadillos, Dasypus novemcinctus, produce immune humoral responses specifically directed against characteristic Paracoccidioides brasiliensis antigens. Such antibody production might reflect direct contact with the ecological microniche of P. brasiliensis, or might merely reflect inhalation of widely distributed airborne propagules. An enzyme-linked immunosorbent assay (ELISA) was designed containing purified glycoprotein gp43 and gp70 antigens from P. brasiliensis as well as cross-reactive antisera originally targeted against human IgM (mu chain) and armadillo anti-IgG (gamma-chain). It was used to detect and classify IgM and IgG antibodies to P. brasiliensis in the armadillo. In a serological survey of 47 wild armadillos, IgM antibodies to gp43 were detected in seven animals (14.8%), and IgG antibodies were detected in 20 (42.5%). IgM antibodies to gp70 were detected in 10 (21.3%) animals and IgG antibodies were detected in 18 (38.3%). These results, showing a pattern consistent with infection, suggest that P. brasiliensis is enzootic in armadillos. How the animals became exposed could not be determined. PMID:15473362

  6. Isolation and Characterization of IgM and IgY Antibodies from Plasma of Magellanic Penguins (Spheniscus magellanicus).

    PubMed

    Bizelli, Camila C; Silva, A Sandriana R; da Costa, Jessica D; Vanstreels, Ralph E T; Atzingen, Marina V; Santoro, Marcelo L; Fernandes, Irene; Catão-Dias, José L; Faquim-Mauro, Eliana L

    2015-03-01

    Infectious diseases such as aspergillosis, avian malaria, and viral infections are significant threats to the conservation of penguins, leading to morbidity and mortality of these birds both in captivity and in the wild. The immune response to such infectious diseases is dependent on different mechanisms mediated by cells and soluble components such as antibodies. Antibodies or immunoglobulins are glycoproteins that have many structural and functional features that mediate distinct effector immune functions. Three distinct classes of antibodies have been identified in birds: immunoglobulin A (IgA), immunoglobulin M (IgM), and immunoglobulin Y (IgY). In this study we aim to establish an efficient laboratory method to obtain IgM and IgY antibodies from plasma samples of healthy adult Magellanic penguins (Spheniscus magellanicus). The protocol was developed combining plasma delipidation, sequential precipitation with caprylic acid and ammonium sulfate, and size-exclusion chromatography. The efficiency of the protocol and the identity of the purified IgM and IgY antibodies were confirmed through enzyme-linked immunosorbent assay, Western blotting, one-dimensional and two-dimensional polyacrylamide gel electrophoresis, and lectin binding assay. Structural and physicochemical properties of IgM and IgY from Magellanic penguins were consistent with those of other avian species. This purification protocol will allow for more detailed studies on the humoral immunity of penguins and for the development of high specificity serologic assays to test Magellanic penguins for infectious pathogens. PMID:26292539

  7. Comparison of Three Automated Immunoassay Methods for the Determination of Epstein-Barr Virus-Specific Immunoglobulin M ▿

    PubMed Central

    Berth, Mario; Bosmans, Eugene

    2010-01-01

    In this study we compared the performances of three commercially available Epstein-Barr virus (EBV) immunoglobulin M (IgM) assays on highly automated immunoassay platforms: BioPlex 2200 (Bio-Rad Laboratories), Immulite 2000 (Siemens Healthcare Diagnostics), and Liaison (DiaSorin). As a confirmatory method, immunoblotting was performed. The specificity of the three EBV IgM assays was evaluated by testing 293 selected sera from patients with various infectious and noninfectious diseases. After the exclusion of 30 samples, the specificities were 96.2% for Liaison, 98.1% for Immulite, and 97.0% for BioPlex. For evaluation of the sensitivity, samples from 70 consecutive patients with a positive heterophile antibody test were examined, irrespective of clinical or biological findings. After the exclusion of six samples, the sensitivities were 89.1% for Liaison, 84.4% for Immulite, and 89.1% for BioPlex. Finally, in a prospective study performed with 500 samples obtained from consecutive patients and sent in by general practitioners, we also determined Epstein-Barr nuclear antigen IgG and viral capsid antigen IgG in a two-phase approach. Concordance of the EBV serologic status was 96.2% between Liaison and Immulite, 96.4% between Immulite and BioPlex, and 97.8% between BioPlex and Liaison. The three EBV IgM immunoassays that we evaluated have acceptable and comparable performances. PMID:20147496

  8. Quantitative immunoassay of human deoxycytidine kinase in malignant cells.

    PubMed

    Kawasaki, H; Carrera, C J; Carson, D A

    1992-11-15

    Deoxycytidine kinase (dCK) is necessary for the activity of several nucleosides used for the chemotherapy of cancer and AIDS. However, the measurement of dCK catalytic activity in crude cell extracts may be imprecise, due to the presence of phosphatases and nucleotidases that degrade the enzyme products. We describe a simple immunoassay for dCK that can measure accurately as little as 5 ng enzyme protein in crude tissue extracts. The assay enabled us to show (i) that mutant cells deficient in dCK activity lack immunoreactive dCK protein, (ii) that dCK catalytic activity and immunoreactivity correlate closely in human tumors, and (iii) that immunoreactive dCK is particularly high in lymphocytes and lymphoid malignancies, although certain solid tumors may also contain the enzyme. The immunoassay of dCK could prove useful in the selection and monitoring of patients who are being treated with nucleosides that are activated by this enzyme. PMID:1489095

  9. A highly efficient colorimetric immunoassay using a nanocomposite entrapping magnetic and platinum nanoparticles in ordered mesoporous carbon.

    PubMed

    Kim, Moon Il; Ye, Youngjin; Woo, Min-Ah; Lee, Jinwoo; Park, Hyun Gyu

    2014-01-01

    Nanocomposite to achieve ultrafast immunoassay: a new synergistically integrated nanocomposite consisting of magnetic and platinum nanoparticles, simultaneously entrapped in mesoporous carbon, is developed as a promising enzyme mimetic candidate to achieve ultrafast colorimetric immunoassays. Using new assay system, clinically important target molecules, such as human epidermal growth factor receptor 2 (HER2) and diarrhea-causing rotavirus, can be detected in only 3 min at room temperature with high specificity and sensitivity. PMID:23832855

  10. Intrathecal somatic hypermutation of IgM in multiple sclerosis and neuroinflammation.

    PubMed

    Beltrán, Eduardo; Obermeier, Birgit; Moser, Markus; Coret, Francisco; Simó-Castelló, María; Boscá, Isabel; Pérez-Miralles, Francisco; Villar, Luisa M; Senel, Makbule; Tumani, Hayrettin; Hohlfeld, Reinhard; Casanova, Bonaventura; Dornmair, Klaus

    2014-10-01

    Intrathecal oligoclonal bands of the cerebrospinal fluid are considered the most important immunological biomarkers of multiple sclerosis. They typically consist of clonally expanded IgG antibodies that underwent affinity maturation during sustained stimulation by largely unknown antigens. In addition, ∼40% of patients with multiple sclerosis have oligoclonal bands that consist of expanded IgM antibodies. We investigated the molecular composition of IgM- and IgG-chains from cerebrospinal fluid of 12 patients with multiple sclerosis, seven patients with other neurological diseases, and eight healthy control subjects by high-throughput deep-sequencing and single-cell PCR. Further, we studied the expression of activation-induced cytidine deaminase, the key enzyme for affinity maturation of antibodies, in cerebrospinal fluid samples of 16 patients. From the cerebrospinal fluid of two multiple sclerosis patients we isolated single B cells and investigated the co-expression of antibody chains with activation-induced cytidine deaminase. In striking contrast to IgM-chains from peripheral blood, IgM-chains from cerebrospinal fluid of patients with multiple sclerosis or neuroborreliosis showed a high degree of somatic hypermutation. We found a high content of mutations that caused amino acid exchanges as compared to silent mutations. In addition, more mutations were found in the complementarity determining regions of the IgM-chains, which interact with yet unknown antigens, as compared to framework regions. Both observations provide evidence for antigen-driven affinity maturation. Furthermore, single B cells from the cerebrospinal fluid of patients with multiple sclerosis co-expressed somatically hypermutated IgM-chains and activation-induced cytidine deaminase, an enzyme that is crucial for somatic hypermutation and class switch recombination of antibodies and is normally expressed during activation of B cells in germinal centres. Clonal tracking of particular IgM(+) B cells allowed us to relate unmutated ancestor clones in blood to hypermutated offspring clones in CSF. Unexpectedly, however, we found no evidence for intrathecal isotype switching from IgM to IgG. Our data suggest that the intrathecal milieu sustains a germinal centre-like reaction with clonal expansion and extensive accumulation of somatic hypermutation in IgM-producing B cells. PMID:25060097

  11. Regimes of transport (T-conduction) in IGM plasmas

    NASA Astrophysics Data System (ADS)

    Medvedev, Mikhail

    2015-11-01

    Galaxy clusters are the largest gravitationally bound systems of the universe. They contain large amounts (about 10 % by mass, whereas 90 % is dark matter) of thermal, T ~ few keV, plasmas. Thermal conduction in this intergalactic medium (IGM) is thought to play crucial role in plasma dynamics but its value is debated. Unlike collisional plasmas, energy transport in magnetized collisionless or weakly collisional plasmas of the IGM exhibit various regimes with vastly different values of the thermal conduction coefficient. Here we discuss these regimes and their implication for galaxy cluster physics. Supported by grant DOE grant DE-FG02-07ER54940 and NSF grant AST-1209665.

  12. [Advances in heavy metal ions immunoassay].

    PubMed

    Liu, Gong-Liang; Wang, Ju-Fang; Li, Zhi-Yong; Liang, Shi-Zhong

    2006-11-01

    Heavy metal leftover on farm and stock products has become a big threat to human. It is necessary to develop some fast and efficient detection methods. Heavy metal immunoassays are new methods for detection of heavy metal ions. Compared to the traditional chemical methods, immunoassays are not only fast, cheap, simple, but also reasonably portable, highly sensitive and selective. It can be used as preliminary screening for rapid determination of heavy metal ions. Except chemical chelators, phytochelatin and metallothionein can also be used for preparing immunogen, both of them can chelate heavy metal ions to carrier protein. There are two prototype assays: polyclonal antibody immunoassay and monoclonal antibody immunoassay. The former includes fluorescence polarization immunoassay; the latter includes indirectly competitive ELISA, one-step competitive immunoassay and KinExA immunoassay. Among these assays, indirectly competitive ELISA which was used for determining heavy metal ions in the early days was easy to be interfered and showed false positive. Fluorescence polarization immunoassay which used polyclonal antibody for determining heavy metal ions was simple and cheap. KinExA instrument could be functioned as an immunosensor for environmental samples. One-step immunoassay which avoided to the addition of second antibody and chromogenic substrate was simple and sensitive. Colloidal gold enhanced immunochromatography assay is a semi-quantitation for determining heavy metal ions. As an adjunctive way for chemical methods, it has the potential application in rapid determination of heavy metal ions. PMID:17168306

  13. IMMUNOASSAY METHODS FOR MEASURING ATRAZINE AND 3,5,6-TRICHLORO-2-PYRIDINOL IN FOODS

    EPA Science Inventory

    This chapter describes the use of enzyme-linked immunosorbent assay (ELISA) methods for the analysis of two potential environmental contaminants in food sample media, atrazine and 3,5,6-trichloro-2-pyridinol (3,5,6-TCP). Two different immunoassay formats are employed: a magnetic...

  14. Application and validation of polybrominated diphenyl ethers immunoassay for environmental and food matrices.

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A sensitive magnetic particle enzyme-linked immunoassay (ELISA) was developed to analyze polybrominated diphenyl ethers (PBDEs) in water, milk, fish, chicken and soil samples. The assay is rapid and can be used to analyze fifty samples in about one hour after sample cleanup. The assay has a limit ...

  15. Competitive chemiluminescent anzyme immunoassay for vitamin B12 analysis in human milk.

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Recent discoveries of matrix interferences by haptocorrin (HC) in human milk and serum show that past analyses of vitamin B12 in samples with high HC content might have been inaccurate (Lildballe et al., 2009; Carmel & Agrawal, 2012). We evaluated two competitive enzyme-binding immunoassays for seru...

  16. Serologic Cross-Reactivity of Human IgM and IgG Antibodies to Five Species of Ebola Virus

    PubMed Central

    MacNeil, Adam; Reed, Zachary; Rollin, Pierre E.

    2011-01-01

    Five species of Ebola virus (EBOV) have been identified, with nucleotide differences of 30–45% between species. Four of these species have been shown to cause Ebola hemorrhagic fever (EHF) in humans and a fifth species (Reston ebolavirus) is capable of causing a similar disease in non-human primates. While examining potential serologic cross-reactivity between EBOV species is important for diagnostic assays as well as putative vaccines, the nature of cross-reactive antibodies following EBOV infection has not been thoroughly characterized. In order to examine cross-reactivity of human serologic responses to EBOV, we developed antigen preparations for all five EBOV species, and compared serologic responses by IgM capture and IgG enzyme-linked immunosorbent assay (ELISA) in groups of convalescent diagnostic sera from outbreaks in Kikwit, Democratic Republic of Congo (n = 24), Gulu, Uganda (n = 20), Bundibugyo, Uganda (n = 33), and the Philippines (n = 18), which represent outbreaks due to four different EBOV species. For groups of samples from Kikwit, Gulu, and Bundibugyo, some limited IgM cross-reactivity was noted between heterologous sera-antigen pairs, however, IgM responses were largely stronger against autologous antigen. In some instances IgG responses were higher to autologous antigen than heterologous antigen, however, in contrast to IgM responses, we observed strong cross-reactive IgG antibody responses to heterologous antigens among all sets of samples. Finally, we examined autologous IgM and IgG antibody levels, relative to time following EHF onset, and observed early peaking and declining IgM antibody levels (by 80 days) and early development and persistence of IgG antibodies among all samples, implying a consistent pattern of antibody kinetics, regardless of EBOV species. Our findings demonstrate limited cross-reactivity of IgM antibodies to EBOV, however, the stronger tendency for cross-reactive IgG antibody responses can largely circumvent limitations in the utility of heterologous antigen for diagnostic assays and may assist in the development of antibody-mediated vaccines to EBOV. PMID:21666792

  17. Specific Noncompetitive Immunoassay for HT-2 Mycotoxin Detection.

    PubMed

    Arola, Henri O; Tullila, Antti; Kiljunen, Harri; Campbell, Katrina; Siitari, Harri; Nevanen, Tarja K

    2016-02-16

    Here we demonstrate a novel homogeneous one-step immunoassay, utilizing a pair of recombinant antibody antigen-binding fragments (Fab), that is specific for HT-2 toxin and has a positive readout. Advantages over the conventional competitive immunoassay formats such as enzyme-linked immunosorbent assay (ELISA) are the specificity, speed, and simplicity of the assay. Recombinant antibody HT2-10 Fab recognizing both HT-2 and T-2 toxins was developed from a phage display antibody library containing 6 × 10(7) different antibody clones. Specificity of the immunoassay was introduced by an anti-immune complex (IC) antibody binding the primary antibody-HT-2 toxin complex. When the noncompetitive immune complex assay was compared to the traditional competitive assay, an over 10-fold improvement in sensitivity was observed. Although the HT2-10 antibody has 100% cross-reactivity for HT-2 and T-2 toxins, the immune complex assay is highly specific for HT-2 alone. The assay performance with real samples was evaluated using naturally contaminated wheat reference material. The half-maximal effective concentration (EC50) value of the time-resolved fluorescence resonance energy transfer (TR-FRET) assay was 9.6 ng/mL, and the limit of detection (LOD) was 0.38 ng/mL (19 μg/kg). The labeled antibodies can be predried to the assay vials, e.g., microtiter plate wells, and readout is ready in 10 min after the sample application. PMID:26785138

  18. Development of immunoassays for detecting clothianidin residue in agricultural products.

    PubMed

    Li, Ming; Sheng, Enze; Cong, Lujing; Wang, Minghua

    2013-04-17

    Two enzyme-linked immunosorbent assays (ELISAs) based on polyclonal antibodies (PcAbs) for clothianidin are described: colorimetric detection format (ELISA) and pattern of chemiluminescent assay (CLEIA). Clothianidin hapten was synthesized and conjugated to bovine serum albumin (BSA) and ovalbumin (OVA) to produce immunogen and coating antigen. Anticlothianidin PcAbs were obtained from immunized New Zealand white rabbits. Under optimal conditions, the half-maximal inhibition concentration (IC₅₀) and the limit of detection (LOD, IC₂₀) of clothianidin were 0.046 and 0.0028 mg/L for the ELISA and 0.015 and 0.0014 mg/L for the CLEIA, respectively. There were no obvious cross-reactivities of the antibodies with its analogues except for dinotefuran. Recoveries of 76.4-116.4% for the immunoassays were achieved from spiked samples. The results of immunoassays for the spiked and authentic samples were largely consistent with gas chromatography. Therefore, the proposed immunoassays would be convenient and satisfactory analytical methods for the monitoring of clothianidin in agricultural products. PMID:23527939

  19. Enhancing immunoassay possibilities using magnetic carriers in biological fluids

    NASA Astrophysics Data System (ADS)

    Yu, Hao

    1997-05-01

    An antibody-based magnetic plate chemifluorescent immunoassay (MPFIA) for effective and rapid detection of bacteria and toxoid from biological fluids was developed. Streptavidin (SA)- magnetic particles and biotinylated antibody as a solid phase immunomagnetic carrier was used for antigen capture. An alkaline phosphatase-antibody conjugate as a secondary capture antibody to the antigen forms a sandwich with the primary antibody. The fluorgenic substrate, AttoPhos reacts with alkaline phosphatase that emits chemifluorescent intensities are proportional to captured antigens. Antigen separation and concentration from biological fluids using immunomagnetic carrier are the key step to reduce media interference for sensitive detection. Results of these efforts may actually enhance the immunoassay possibilities by concentration of specific antigen and reduction of background noise. Magnetic separation and chemifluorescent detection have been achieved by a multiple-well formatted magnetic plate separator and a fluorescent plate detector, respectively. Experiments were performed for virulent Escherichia coli cells, Staphylococcal enterotoxin B toxoid and Bacillus subtilus spore detection in biological fluids. In general, the fluorescent detection can be achieved at the same sensitivities as enzyme-linked immunoassay and the ECL detection is more sensitive than fluorescent assay. However, the unique features of MPFIA and MPECL are that the biological samples can be rapidly processed and detected on the same multiple sample formatted plate within one hour assay time.

  20. Validation of a Microsphere Immunoassay for Serological Leptospirosis Diagnosis in Human Serum by Comparison to the Current Gold Standard

    PubMed Central

    Wynwood, Sarah J.; Burns, Mary-Anne A.; Graham, Glenn C.; Weier, Steven L.; McKay, David B.; Craig, Scott B.

    2015-01-01

    A microsphere immunoassay (MIA) utilising Luminex xMap technology that is capable of determining leptospirosis IgG and IgM independently was developed. The MIA was validated using 200 human samples submitted for routine leptospirosis serology testing. The traditional microscopic agglutination (MAT) method (now 100 years old) suffers from a significant range of technical problems including a dependence on antisera which is difficult to source and produce, false positive reactions due to auto-agglutination and an inability to differentiate between IgG and IgM antibodies. A comparative validation method of the MIA against the MAT was performed and used to determine the ability of the MIA to detect leptospiral antibodies when compared with the MAT. The assay was able to determine samples in the reactive, equivocal and non-reactive ranges when compared to the MAT and was able to differentiate leptospiral IgG antibodies from leptospiral IgM antibodies. The MIA is more sensitive than the MAT and in true infections was able to detect low levels of antibody in the later stages of the acute phase as well as detect higher levels of IgM antibody earlier in the immune phase of the infection. The relatively low cost, high throughput platform and significantly reduced dependency on large volumes of rabbit antisera make this assay worthy of consideration for any microbiological assay that currently uses agglutination assays. PMID:25807009

  1. Pholcodine interference in the immunoassay for opiates in urine.

    PubMed

    Svenneby, G; Wedege, E; Karlsen, R L

    1983-01-01

    The excretion in urine after single oral therapeutic doses of morphine derivatives was analysed with radioimmunoassay (RIA) and homogeneous enzyme immunoassay (EMIT) for opiates. In contrast to the rapid excretion of ethylmorphine and codeine, pholcodine showed positive results for opiates 2-6 weeks after intake when the urines were analysed with the RIA-method. When analysed with the EMIT-method, positive results were obtained for pholcodine for approximately 10 days. As pholcodine is a common component in cough mixtures, its prolonged excretion could represent a hazard in interpreting the results from drug analyses of urines. PMID:6347841

  2. Multianalyte microspot immunoassay. The microanalytical 'compact disk' of the future.

    PubMed

    Ekins, R; Chu, F

    1992-01-01

    Throughout the 1970s, controversy centered both on immunoassay 'sensitivity' per se and on the relative sensitivities of labelled antibody and labelled analyte methods. Our own theoretical studies in this period revealed that radioimmunoassay (RIA) sensitivities could be surpassed only by the use of very high specific activity non-isotopic labels in 'non-competitive' designs, preferably based on the use of monoclonal antibodies. The time-resolved fluorescence methodology known as Delfia - developed in collaboration with the instrument manufacturer LKB/Wallac - represented the first commercial 'ultra-sensitive' non-isotopic technique based on these theoretical insights, the same concepts being subsequently adopted in comparable methodologies relying on the use of chemiluminescent and enzyme labels. However, a second advantage of high specific activity labels is that they permit the development of 'multi-analyte' immunoassay systems combining ultra-sensitivity with the simultaneous measurement of tens, hundreds or thousands of analytes in a small biological sample. This possibility relies on simple, albeit hitherto unexploited, physicochemical concepts. The first is that all immunoassays rely on measurement of Ab occupancy by analyte. The second is that, provided the Ab concentration used is 'vanishingly small', fractional Ab occupancy is independent of both Ab concentration and sample volume. This leads to the notion of 'ratiometric' immunoassay, involving measurement of the ratio of signals (eg fluorescent signals) emitted by two labelled Ab's, the first ('sensor' Ab) deposited as a microspot on a solid support, the second a 'developing' Ab directed against either occupied or unoccupied sensor Ab binding sites.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:1485691

  3. Multiple recurrent hordeola associated with selective IgM deficiency.

    PubMed

    Kiratli, H K; Akar, Y

    2001-02-01

    An external hordeolum is an acute, suppurative inflammation of the glands of Zeis and sweat glands or hair follicles most commonly caused by staphylococci, usually in the setting of a chronic blepharitis.(1) We report a case of a boy with unilateral multiple recurrent hordeola in association with selective IgM deficiency. PMID:11182678

  4. Mycobacterium tuberculosis Lipolytic Enzymes as Potential Biomarkers for the Diagnosis of Active Tuberculosis

    PubMed Central

    Brust, Belinda; Lecoufle, Mélanie; Tuaillon, Edouard; Dedieu, Luc; Canaan, Stéphane; Valverde, Viviane; Kremer, Laurent

    2011-01-01

    Background New diagnosis tests are urgently needed to address the global tuberculosis (TB) burden and to improve control programs especially in resource-limited settings. An effective in vitro diagnostic of TB based on serological methods would be regarded as an attractive progress because immunoassays are simple, rapid, inexpensive, and may offer the possibility to detect cases missed by standard sputum smear microscopy. However, currently available serology tests for TB are highly variable in sensitivity and specificity. Lipolytic enzymes have recently emerged as key factors in lipid metabolization during dormancy and/or exit of the non-replicating growth phase, a prerequisite step of TB reactivation. The focus of this study was to analyze and compare the potential of four Mycobacterium tuberculosis lipolytic enzymes (LipY, Rv0183, Rv1984c and Rv3452) as new markers in the serodiagnosis of active TB. Methods Recombinant proteins were produced and used in optimized ELISA aimed to detect IgG and IgM serum antibodies against the four lipolytic enzymes. The capacity of the assays to identify infection was evaluated in patients with either active TB or latent TB and compared with two distinct control groups consisting of BCG-vaccinated blood donors and hospitalized non-TB individuals. Results A robust humoral response was detected in patients with active TB whereas antibodies against lipolytic enzymes were infrequently detected in either uninfected groups or in subjects with latent infection. High specifity levels, ranging from 93.9% to 97.5%, were obtained for all four antigens with sensitivity values ranging from 73.4% to 90.5%, with Rv3452 displaying the highest performances. Patients with active TB usually exhibited strong IgG responses but poor IgM responses. Conclusion These results clearly indicate that the lipolytic enzymes tested are strongly immunogenic allowing to distinguish active from latent TB infections. They appear as potent biomarkers providing high sensitivity and specificity levels for the immunodiagnosis of active TB. PMID:21966416

  5. IMMUNOASSAYS FOR METAL IONS. (R824029)

    EPA Science Inventory

    Abstract

    Antibodies that recognize chelated forms of metal ions have been used to construct immunoassays for Cd(II), Hg(II), Pb(II), and Ni(II). In this paper, the format of these immunoassays is described and the binding properties of three monoclonal antibodies direc...

  6. Hydrophilic, bright CuInS2 quantum dots as Cd-free fluorescent labels in quantitative immunoassay.

    PubMed

    Speranskaya, Elena S; Beloglazova, Natalia V; Abé, Sofie; Aubert, Tangi; Smet, Philippe F; Poelman, Dirk; Goryacheva, Irina Y; De Saeger, Sarah; Hens, Zeger

    2014-07-01

    We report on the synthesis of core-shell CuInS2/ZnS quantum dots (QDs) in organic solution, their encapsulation with a PEG-containing amphiphilic polymer, and the application of the resulting water-soluble QDs as fluorescent label in quantitative immunoassay. By optimizing the methods for core synthesis and shell growth, CuInS2/ZnS QDs were obtained with a quantum yield of 50% on average after hydrophilization. After conjugation with an aflatoxin B1-protein derivative, the obtained QDs were used as fluorescent labels in microplate immunoassay for the quantitative determination of the mycotoxin aflatoxin B1. QDs-based immunoassay showed higher sensitivity compared to enzyme-based immunoassay. PMID:24892375

  7. Electrokinetic Microstrirring to Enhance Immunoassays

    NASA Astrophysics Data System (ADS)

    Feldman, Hope; Sigurdson, Marin; Meinhart, Carl

    2006-11-01

    Electrokinetic microstirring is used to improve the sensitivity of microfluidic heterogeneous immuno-sensors by enhancing the transport in diffusion-limited reactions. The AC electrokinetic force, Electrothermal Flow, is exploited to create a circular stirring fluid motion, thereby providing more binding opportunities between suspended and wall-immobilized molecules. This process can significantly reduce test times, important for both field-portable biosensors and for lab-based assays. A 2-D numerical simulation model is used to predict the effect of electrothermal flow on a heterogeneous immunoassay resulting from an AC potential applied to two parallel electrodes. The binding is increased by a factor of 7 for an applied voltage of 10 Vrms. The effect was investigated experimentally using a high affinity biotin-streptavidin reaction. Microstirred reaction rates were compared with passive reactions. The measurements show on average an order of magnitude increase in binding between immobilized biotin and fluorescently-labeled streptavidin after 5 minutes. Therefore, this technique shows significant promise for reducing incubation time and enhancing the sensitivity of immunoassays.

  8. A Reassessment of IgM Memory Subsets in Humans.

    PubMed

    Bagnara, Davide; Squillario, Margherita; Kipling, David; Mora, Thierry; Walczak, Aleksandra M; Da Silva, Lucie; Weller, Sandra; Dunn-Walters, Deborah K; Weill, Jean-Claude; Reynaud, Claude-Agnès

    2015-10-15

    From paired blood and spleen samples from three adult donors, we performed high-throughput VH sequencing of human B cell subsets defined by IgD and CD27 expression: IgD(+)CD27(+) ("marginal zone [MZ]"), IgD(-)CD27(+) ("memory," including IgM ["IgM-only"], IgG and IgA) and IgD(-)CD27(-) cells ("double-negative," including IgM, IgG, and IgA). A total of 91,294 unique sequences clustered in 42,670 clones, revealing major clonal expansions in each of these subsets. Among these clones, we further analyzed those shared sequences from different subsets or tissues for VH gene mutation, H-CDR3-length, and VH/JH usage, comparing these different characteristics with all sequences from their subset of origin for which these parameters constitute a distinct signature. The IgM-only repertoire profile differed notably from that of MZ B cells by a higher mutation frequency and lower VH4 and higher JH6 gene usage. Strikingly, IgM sequences from clones shared between the MZ and the memory IgG/IgA compartments showed a mutation and repertoire profile of IgM-only and not of MZ B cells. Similarly, all IgM clonal relationships (among MZ, IgM-only, and double-negative compartments) involved sequences with the characteristics of IgM-only B cells. Finally, clonal relationships between tissues suggested distinct recirculation characteristics between MZ and switched B cells. The "IgM-only" subset (including cells with its repertoire signature but higher IgD or lower CD27 expression levels) thus appear as the only subset showing precursor-product relationships with CD27(+) switched memory B cells, indicating that they represent germinal center-derived IgM memory B cells and that IgM memory and MZ B cells constitute two distinct entities. PMID:26355154

  9. The development of immunoassays for detection of chemical warfare agents

    SciTech Connect

    Lenz, D.E.

    1995-06-01

    With the advent of enzyme linked immunoabsorbant assays (ELISA) and monoclonal antibodies in the last two decades, there has been considerable effort devoted to the development of antibodies to detect and quantify low molecular weight toxic substances in environmental or biological fluids. Polyclonal antibodies against paraoxon (the toxic metabolite of parathion) were reported as capable of detecting paraoxon in body fluids at a level of 10{sup -9} M ({approximately}260 pg/mL) when used in a competitive inhibition enzyme immunoassay (CIEIA). Monoclonal antibodies developed against a structural analogue of the chemical warfare agent soman were capable of detecting soman in buffer solutions at a level of 10{sup -6} M ({approximately}180 ng/mL). In addition, these antibodies were highly specific for soman even in the presence of its major hydrolysis product. Subsequent studies with antisoman monoclonal antibodies reported an extension of the level of sensitivity to -80 ng/mL. Furthermore these antibodies did not cross react with other chemical warfare nerve agents such as sarin or tabun. In all cases, the time for a confirmatory test was two hours or less. Immunoassays for T-2 micotoxins have also been reported with a minimal detection range of 2 pg/assay to 50 ng/assay for the polyclonal and monoclonal T-2 antibodies respectively. These antibodies offer a sensitive, rapid and low cost approach to the diagnosis or detection of the presence of toxic chemical substances.

  10. Novel immunoassay formats for integrated microfluidic circuits: diffusion immunoassays (DIA)

    NASA Astrophysics Data System (ADS)

    Weigl, Bernhard H.; Hatch, Anson; Kamholz, Andrew E.; Yager, Paul

    2000-03-01

    Novel designs of integrated fluidic microchips allow separations, chemical reactions, and calibration-free analytical measurements to be performed directly in very small quantities of complex samples such as whole blood and contaminated environmental samples. This technology lends itself to applications such as clinical diagnostics, including tumor marker screening, and environmental sensing in remote locations. Lab-on-a-Chip based systems offer many *advantages over traditional analytical devices: They consume extremely low volumes of both samples and reagents. Each chip is inexpensive and small. The sampling-to-result time is extremely short. They perform all analytical functions, including sampling, sample pretreatment, separation, dilution, and mixing steps, chemical reactions, and detection in an integrated microfluidic circuit. Lab-on-a-Chip systems enable the design of small, portable, rugged, low-cost, easy to use, yet extremely versatile and capable diagnostic instruments. In addition, fluids flowing in microchannels exhibit unique characteristics ('microfluidics'), which allow the design of analytical devices and assay formats that would not function on a macroscale. Existing Lab-on-a-chip technologies work very well for highly predictable and homogeneous samples common in genetic testing and drug discovery processes. One of the biggest challenges for current Labs-on-a-chip, however, is to perform analysis in the presence of the complexity and heterogeneity of actual samples such as whole blood or contaminated environmental samples. Micronics has developed a variety of Lab-on-a-Chip assays that can overcome those shortcomings. We will now present various types of novel Lab- on-a-Chip-based immunoassays, including the so-called Diffusion Immunoassays (DIA) that are based on the competitive laminar diffusion of analyte molecules and tracer molecules into a region of the chip containing antibodies that target the analyte molecules. Advantages of this technique are a reduction in reagents, higher sensitivity, minimal preparation of complex samples such as blood, real-time calibration, and extremely rapid analysis.

  11. Oligopeptide-based enzyme immunoassay for ovine lentivirus antibody detection.

    PubMed Central

    Kwang, J; Torres, J V

    1994-01-01

    Ovine progressive pneumonia virus (OPPV) is a lentivirus which causes a progressive disease in sheep. Immunodominant epitopes have been identified in the envelope gp40 glycoprotein. Synthetic peptides representing these regions are able to detect the presence of OPPV antibodies in 96% of infected sheep. PMID:7929780

  12. Evaluation of Commercially Available Diagnostic Tests for the Detection of Dengue Virus NS1 Antigen and Anti-Dengue Virus IgM Antibody

    PubMed Central

    Hunsperger, Elizabeth A.; Yoksan, Sutee; Buchy, Philippe; Nguyen, Vinh Chau; Sekaran, Shamala Devi; Enria, Delia A.; Vazquez, Susana; Cartozian, Elizabeth; Pelegrino, Jose L.; Artsob, Harvey; Guzman, Maria G.; Olliaro, Piero; Zwang, Julien; Guillerm, Martine; Kliks, Susie; Halstead, Scott; Peeling, Rosanna W.; Margolis, Harold S.

    2014-01-01

    Commercially available diagnostic test kits for detection of dengue virus (DENV) non-structural protein 1 (NS1) and anti-DENV IgM were evaluated for their sensitivity and specificity and other performance characteristics by a diagnostic laboratory network developed by World Health Organization (WHO), the UNICEF/UNDP/World Bank/WHO Special Programme for Research and Training in Tropical Diseases (TDR) and the Pediatric Dengue Vaccine Initiative (PDVI). Each network laboratory contributed characterized serum specimens for the panels used in the evaluation. Microplate enzyme-linked immunosorbent assay (ELISA) and rapid diagnostic test (RDT formats) were represented by the kits. Each ELISA was evaluated by 2 laboratories and RDTs were evaluated by at least 3 laboratories. The reference tests for IgM anti-DENV were laboratory developed assays produced by the Armed Forces Research Institute for Medical Science (AFRIMS) and the Centers for Disease Control and Prevention (CDC), and the NS1 reference test was reverse transcriptase polymerase chain reaction (RT-PCR). Results were analyzed to determine sensitivity, specificity, inter-laboratory and inter-reader agreement, lot-to-lot variation and ease-of-use. NS1 ELISA sensitivity was 60–75% and specificity 71–80%; NS1 RDT sensitivity was 38–71% and specificity 76–80%; the IgM anti-DENV RDTs sensitivity was 30–96%, with a specificity of 86–92%, and IgM anti-DENV ELISA sensitivity was 96–98% and specificity 78–91%. NS1 tests were generally more sensitive in specimens from the acute phase of dengue and in primary DENV infection, whereas IgM anti-DENV tests were less sensitive in secondary DENV infections. The reproducibility of the NS1 RDTs ranged from 92-99% and the IgM anti-DENV RDTs from 88–94%. PMID:25330157

  13. IgM exacerbates glomerular disease progression in complement induced glomerulopathy

    PubMed Central

    Panzer, Sarah E.; Laskowski, Jennifer; Renner, Brandon; Kulik, Liudmila; Ljubanovic, Danica; Huber, Kendra M.; Zhong, Weixiong; Pickering, Matthew C.; Holers, V. Michael; Thurman, Joshua M.

    2015-01-01

    While glomerular IgM deposition occurs in a variety of glomerular diseases the mechanism of deposition and its clinical significance remain controversial. Some have theorized IgM becomes passively trapped in areas of glomerulosclerosis. However, recent studies found that IgM specifically binds damaged glomeruli. Therefore, we tested whether natural IgM binds to neo-epitopes exposed after insults to the glomerulus and exacerbate disease in mice deficient in the complement regulatory protein factor H; a model of non-sclerotic and nonimmune-complex glomerular disease. Immunofluorescence microscopy demonstrated mesangial and capillary loop deposition of IgM while ultrastructural analysis found IgM deposition on endothelial cells and subendothelial areas. Factor H deficient mice lacking B cells were protected from renal damage, as evidenced by milder histologic lesions on light and electron microscopy. IgM, but not IgG, from wild-type mice bound to cultured murine mesangial cells. Furthermore, injection of purified IgM into mice lacking B cells bound within the glomeruli and induced proteinuria. A monoclonal natural IgM recognizing phospholipids also bound to glomeruli in vivo and induced albuminuria. Thus, our results indicate specific IgM antibodies bind to glomerular epitopes and that IgM contributes to the progression of glomerular damage in this mouse model of non-sclerotic glomerular disease. PMID:25945405

  14. Human Merkel cell polyomavirus infection II. MCV is a common human infection that can be detected by conformational capsid epitope immunoassays.

    PubMed

    Tolstov, Yanis L; Pastrana, Diana V; Feng, Huichen; Becker, Jürgen C; Jenkins, Frank J; Moschos, Stergios; Chang, Yuan; Buck, Christopher B; Moore, Patrick S

    2009-09-15

    Merkel cell polyomavirus (MCV) is a newly-discovered human tumor virus found in approximately 80% of Merkel cell carcinoma (MCC). The rate of MCV infection among persons without MCC is unknown. We developed a MCV virus-like particle (VLP) enzyme-linked immunoassay (EIA) that does not cross-react with human BK or murine polyomaviruses. Peptide mapping of the MCV VP1 gene and immunoblotting with denatured MCV VLP are less sensitive than the MCV EIA in detecting MCV antibodies suggesting antibody reactivity in this assay primarily targets conformational but not linear epitopes. Among MCC patients, all 21 (100%) patients tested with MCV-positive tumors had high serum MCV IgG but not high MCV IgM levels. Only 3 of 6 (50%) MCC patients with MCV-negative tumors were positive for MCV antibodies. Sera from most adults, including 107 of 166 (64%) blood donors, 63 of 100 (63%) commercial donors and 37 of 50 (74%) systemic lupus erythematosus patients, show evidence for prior MCV exposure. Age-specific MCV prevalence was determined by examining a cross-sectional distribution of 150 Langerhans cell histiocytosis (an unrelated neoplasm) patient sera. MCV prevalence increases from 50% among children age 15 years or younger to 80% among persons older than 50 years. We did not find evidence for vertical transmission among infants. Although past exposure to MCV is common among all adult groups, MCC patients have a markedly elevated MCV IgG response compared with control patients. Our study demonstrates that MCV is a widespread but previously unrecognized human infection. PMID:19499548

  15. [FT4 immunoassay interference : A case report].

    PubMed

    Chaabouni, Khansa; Hargafi, Khaoula; Elleuch, Aida; Messedi, Mariem; Turki, Mouna; Lahyani, Amina; Ayedi, Fatma

    2015-04-01

    Measurement of thyrotropin and free thyroxin made using immunoassays are usually needed in clinical endocrinology. Here, we report a case of a patient with type 2 diabetes who presented a weight loss. To eliminate hyperthyroidism, thyroid function tests were performed. Free thyroxin (FT4) was decreased using two automated immunoassays TOSOH AIA 1800 and Roche ELECSYS 2010, with a normal thyrotropin value. Thyroid function tests repeated a month later were normal. The patient's history revealed contact with sheep, which may partly explain the interference. Investigations into the patient's serum were carried out using both the PEG test and dilution test. Interference factors were probably antibodies. Despite progress in immunoassays, we should be aware of interference occurrence since it can lead to false results, unnecessary investigations and incorrect treatment. Thus, simple tests must be carried out as if interference in immunoassays were suspected. Dilutions and PEG tests are generally performed as first line investigations. PMID:26375746

  16. Smart CuS Nanoparticles as Peroxidase Mimetics for the Design of Novel Label-Free Chemiluminescent Immunoassay.

    PubMed

    Yang, Zhanjun; Cao, Yue; Li, Juan; Lu, Mimi; Jiang, Zhikang; Hu, Xiaoya

    2016-05-18

    In the present work, a novel label-free chemiluminescent (CL) immunoassay method was designed by employing smart CuS nanoparticles (CuSNPs) as peroxidase mimetics. The CuSNPs were synthesized through a simple coprecipitation method, and showed high catalytic activity and stability. This efficient label-free CL immunoassay could be easily achieved through a simple strategy. First, CuSNPs dispersed in chitosan were modified on the epoxy-functionalized glass slide to form a solid CL signal interface. Streptavidin was then used to functionalize CuSNPs to capture the biotinylated antibody, further producing a sensing interface. After online incubation with antigen molecules, the formed antibody-antigen complex on the biosensing substrate could prevent the diffusion channel of CL substrate toward the signal interface, and restrained the mimic enzyme-catalyzed CL reaction, finally resulting in the decrease of CL signals of the assay system. Compared to the label-based CL immunoassay, the proposed label-free assay mode is more simple, cheap and fast. Using a model analyte alpha-fetoprotein, the label-free CL immunoassay method had a linear range of 0.1-60 ng/mL and a low detection limit of 0.07 ng/mL. Moreover, the peroxidase mimetic-based label-free CL immunoassay system showed good specificity, acceptable repeatability, and good accuracy. The study provided a promising strategy for the development of highly efficient label-free CL immunoassay system. PMID:27137349

  17. Fast and Sensitive Detection of Enteropathogenic Yersinia by Immunoassays

    PubMed Central

    Laporte, Jérôme; Savin, Cyril; Lamourette, Patricia; Devilliers, Karine; Volland, Hervé; Carniel, Elisabeth; Créminon, Christophe

    2014-01-01

    Yersinia enterocolitica and Yersinia pseudotuberculosis, the two Yersinia species that are enteropathogenic for humans, are distributed worldwide and frequently cause diarrhea in inhabitants of temperate and cold countries. Y. enterocolitica is a major cause of foodborne disease resulting from consumption of contaminated pork meat and is further associated with substantial economic cost. However, investigation of enteropathogenic Yersinia species is infrequently performed routinely in clinical laboratories because of their specific growth characteristics, which make difficult their isolation from stool samples. Moreover, current isolation procedures are time-consuming and expensive, thus leading to underestimates of the incidence of enteric yersiniosis, inappropriate prescriptions of antibiotic treatments, and unnecessary appendectomies. The main objective of the study was to develop fast, sensitive, specific, and easy-to-use immunoassays, useful for both human and veterinary diagnosis. Monoclonal antibodies (MAbs) directed against Y. enterocolitica bioserotypes 2/O:9 and 4/O:3 and Y. pseudotuberculosis serotypes I and III were produced. Pairs of MAbs were selected by testing their specificity and affinity for enteropathogenic Yersinia and other commonly found enterobacteria. Pairs of MAbs were selected to develop highly sensitive enzyme immunoassays (EIAs) and lateral flow immunoassays (LFIs or dipsticks) convenient for the purpose of rapid diagnosis. The limit of detection of the EIAs ranged from 3.2 × 103 CFU/ml to 8.8 × 104 CFU/ml for pathogenic serotypes I and III of Y. pseudotuberculosis and pathogenic bioserotypes 2/O:9 and 4/O:3 of Y. enterocolitica and for the LFIs ranged from 105 CFU/ml to 106 CFU/ml. A similar limit of detection was observed for artificially contaminated human feces. PMID:25355759

  18. A natural IgM antibody does inhibit polyclonal and antigen-specific IgM but not IgG B-cell responses.

    PubMed

    Kiss, K; Uher, F; Gergely, J

    1994-03-01

    Since a B-cell growth-inhibitory natural IgM antibody was identified in the culture supernatants of LPS-stimulated murine splenic B lymphocytes [11], attempts have been made to define other possible functional role(s) of this antibody. Here we show that this regulatory IgM is able to inhibit not only the proliferation of splenic B cells, but also their IgM secretion during LPS-induced polyclonal, as well as antigen (FITC-KLH)-specific antibody responses. In contrast, IgG1 production of hapten (FITC)-specific B cells neither during restimulation with LPS nor in the presence of carrier-specific T lymphocytes in vitro was affected by regulatory IgM. Therefore, whereas newly emerging naive B cells are highly susceptible, IgG-secreting B cells appear to be completely resistant to inactivation by the regulatory IgM autoantibody. PMID:7518418

  19. Diagnostic value of immunoassays for heparin-induced thrombocytopenia: a systematic review and meta-analysis.

    PubMed

    Nagler, Michael; Bachmann, Lucas M; Ten Cate, Hugo; Ten Cate-Hoek, Arina

    2016-02-01

    Immunoassays are essential in the workup of patients with suspected heparin-induced thrombocytopenia. However, the diagnostic accuracy is uncertain with regard to different classes of assays, antibody specificities, thresholds, test variations, and manufacturers. We aimed to assess diagnostic accuracy measures of available immunoassays and to explore sources of heterogeneity. We performed comprehensive literature searches and applied strict inclusion criteria. Finally, 49 publications comprising 128 test evaluations in 15?199 patients were included in the analysis. Methodological quality according to the revised tool for quality assessment of diagnostic accuracy studies was moderate. Diagnostic accuracy measures were calculated with the unified model (comprising a bivariate random-effects model and a hierarchical summary receiver operating characteristics model). Important differences were observed between classes of immunoassays, type of antibody specificity, thresholds, application of confirmation step, and manufacturers. Combination of high sensitivity (>95%) and high specificity (>90%) was found in 5 tests only: polyspecific enzyme-linked immunosorbent assay (ELISA) with intermediate threshold (Genetic Testing Institute, Asserachrom), particle gel immunoassay, lateral flow immunoassay, polyspecific chemiluminescent immunoassay (CLIA) with a high threshold, and immunoglobulin G (IgG)-specific CLIA with low threshold. Borderline results (sensitivity, 99.6%; specificity, 89.9%) were observed for IgG-specific Genetic Testing Institute-ELISA with low threshold. Diagnostic accuracy appears to be inadequate in tests with high thresholds (ELISA; IgG-specific CLIA), combination of IgG specificity and intermediate thresholds (ELISA, CLIA), high-dose heparin confirmation step (ELISA), and particle immunofiltration assay. When making treatment decisions, clinicians should be a aware of diagnostic characteristics of the tests used and it is recommended they estimate posttest probabilities according to likelihood ratios as well as pretest probabilities using clinical scoring tools. PMID:26518436

  20. IGM Overdensities towards Galaxies at High-Redshift

    NASA Astrophysics Data System (ADS)

    Frye, Brenda L.; Hurley, M.; Bowen, D.; Coe, D.; Fan, X.; Tripp, T.; Holden, B.; Egami, E.; Matsuda, Y.

    2011-01-01

    We present a high signal-to-noise spectrum of a bright galaxy at z = 4.9 in 14 h of integration on VLT FORS2. This galaxy is extremely bright, i_850 = 23.10 +/- 0.01, and is strongly-lensed by the foreground massive galaxy cluster Abell 1689 (z=0.18). Stellar continuum is seen longward of the Ly-alpha emission line at 7100 A, while intergalactic H I produces strong absorption shortward of Ly-alpha. Two transmission spikes at 6800 A and 7040 A are also visible, along with other structures at shorter wavelengths. Although fainter than a QSO, the absence of a strong central ultraviolet flux source in this star forming galaxy enables a measurement of the H I flux transmission in the intergalactic medium (IGM) in the vicinity of a high redshift object. We find that the effective H I optical depth of the IGM is remarkably high within a large 14 Mpc (physical) region surrounding the galaxy compared to that seen towards QSOs at similar redshifts. Evidently, this high-redshift galaxy is located in a region of space where the amount of H I is much larger than that seen at similar epochs in the diffuse IGM. We argue that observations of high-redshift galaxies like this one provide unique insights on the nascent stages of baryonic large-scale structures that evolve into the filamentary cosmic web of galaxies and clusters of galaxies observed in the present universe. Follow-up narrow-band imaging at Subaru Observatory has revealed an a coincident overdensity of Ly-alpha Emitters.

  1. A Rapid, Multiplexed, High-Throughput Flow-Through Membrane Immunoassay: A Convenient Alternative to ELISA

    PubMed Central

    Ramachandran, Sujatha; Singhal, Mitra; McKenzie, Katherine G.; Osborn, Jennifer L.; Arjyal, Amit; Dongol, Sabina; Baker, Stephen G.; Basnyat, Buddha; Farrar, Jeremy; Dolecek, Christiane; Domingo, Gonzalo J.; Yager, Paul; Lutz, Barry

    2013-01-01

    This paper describes a rapid, high-throughput flow-through membrane immunoassay (FMIA) platform. A nitrocellulose membrane was spotted in an array format with multiple capture and control reagents for each sample detection area, and assay steps were carried out by sequential aspiration of sample and reagents through each detection area using a 96-well vacuum manifold. The FMIA provides an alternate assay format with several advantages over ELISA. The high surface area of the membrane permits high label concentration using gold labels, and the small pores and vacuum control provide rapid diffusion to reduce total assay time to ~30 min. All reagents used in the FMIA are compatible with dry storage without refrigeration. The results appear as colored spots on the membrane that can be quantified using a flatbed scanner. We demonstrate the platform for detection of IgM specific to lipopolysaccharides (LPS) derived from Salmonella Typhi. The FMIA format provides analytical results comparable to ELISA in less time, provides integrated assay controls, and allows compensation for specimen-to-specimen variability in background, which is a particular challenge for IgM assays. PMID:26835678

  2. EVALUATION OF IMMUNOASSAY METHODS FOR DETERMINATION OF 3,5,6-TRICHLORO-2-PYRIDINOL IN MULTIPLE SAMPLE MEDIA

    EPA Science Inventory

    Two enzyme-linked immunosorbent assay (ELISA) methods were evaluated for the determination of 3,5,6-trichloro-2-pyridinol (3,5,6-TCP) in multiple sample media (dust, soil, food, and urine). The dust and soil samples were analyzed by the RaPID (TM) commercial immunoassay testing ...

  3. DEVELOPMENT OF A MAGNETIC PARTICLE IMMUNOASSAY FOR POLYBROMINATED DIPHENYL ETHERS AND APPLICATION TO ENVIRONMENTAL AND FOOD MATRICES.

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A sensitive magnetic particle enzyme-linked immunoassay (ELISA) was developed to analyze polybrominated diphenyl ethers (PBDEs) in water, milk, fish, and soil samples. The assay was rapid and can be used to analyze fifty samples in about one hour after sample cleanup. The assay has a limit of det...

  4. Seroprevalence of immunoglobulin M (IgM) and IgG antibodies to polysaccharides of Streptococcus pneumoniae in different age groups of Ecuadorian and German children.

    PubMed

    Brssow, H; Baensch, M; Sidoti, J

    1992-11-01

    The age-specific prevalence of serum immunoglobulin M (IgM) antibody to capsular polysaccharides of Streptococcus pneumoniae, as detected by enzyme-linked immunosorbent assay, was studied in 1,301 Ecuadorian children enrolled in a national nutrition and health survey. This prevalence was 6% in infants < 6 months old and increased to 28% in children 6 to 11 months old, 49% in those 12 to 17 months old, and 58% in those 18 to 23 months old. About 80% of the 5-year-old children had this antibody. When tested separately against six different capsular polysaccharides, serum IgM antibody reacted with decreasing frequency with serotype 3, 8, 19, 6, 23, and 1 capsular polysaccharides. We did not observe a broadening of the antibody response with increasing age in the sense that more and more serotypes were recognized. A similar age-related prevalence was found for IgM antibody to the species-specific C-polysaccharide of S. pneumoniae and for IgG antibody to capsular polysaccharides of S. pneumoniae. A smaller German serum collection showed a comparable age-related prevalence of pneumococcus-specific serum IgG and IgM antibodies. The highest incidence of respiratory diseases was observed in 1- and 2-year-old Ecuadorian children. It thus seems that acquisition of serum antibody to S. pneumoniae reflects more the developmental maturation of an immune response than an actual exposure to different pneumococcal serotypes. PMID:1452644

  5. Seroprevalence of immunoglobulin M (IgM) and IgG antibodies to polysaccharides of Streptococcus pneumoniae in different age groups of Ecuadorian and German children.

    PubMed Central

    Brssow, H; Baensch, M; Sidoti, J

    1992-01-01

    The age-specific prevalence of serum immunoglobulin M (IgM) antibody to capsular polysaccharides of Streptococcus pneumoniae, as detected by enzyme-linked immunosorbent assay, was studied in 1,301 Ecuadorian children enrolled in a national nutrition and health survey. This prevalence was 6% in infants < 6 months old and increased to 28% in children 6 to 11 months old, 49% in those 12 to 17 months old, and 58% in those 18 to 23 months old. About 80% of the 5-year-old children had this antibody. When tested separately against six different capsular polysaccharides, serum IgM antibody reacted with decreasing frequency with serotype 3, 8, 19, 6, 23, and 1 capsular polysaccharides. We did not observe a broadening of the antibody response with increasing age in the sense that more and more serotypes were recognized. A similar age-related prevalence was found for IgM antibody to the species-specific C-polysaccharide of S. pneumoniae and for IgG antibody to capsular polysaccharides of S. pneumoniae. A smaller German serum collection showed a comparable age-related prevalence of pneumococcus-specific serum IgG and IgM antibodies. The highest incidence of respiratory diseases was observed in 1- and 2-year-old Ecuadorian children. It thus seems that acquisition of serum antibody to S. pneumoniae reflects more the developmental maturation of an immune response than an actual exposure to different pneumococcal serotypes. PMID:1452644

  6. Detection of the Antimicrobial Triclosan in Environmental Samples by Immunoassay.

    PubMed

    Ahn, Ki Chang; Ranganathan, Anupama; Bever, Candace S; Hwang, Sung Hee; Holland, Erika B; Morisseau, Kevin; Pessah, Isaac N; Hammock, Bruce D; Gee, Shirley J

    2016-04-01

    A sensitive, competitive enzyme-linked immunosorbent assay (ELISA) for the detection of the antimicrobial triclosan (TCS; 2,4,4'-trichloro-2'-hydroxydiphenyl ether) was developed. Novel immunizing haptens were synthesized by derivatizing at the 4-Cl position of the TCS molecule. Compounds derived from substitutions at 4'-Cl and that replaced the 2'-OH with a Cl atom were designed as unique coating antigen haptens. Polyclonal rabbit antisera were screened against the coating antigen library to identify combinations of immunoreagents resulting in the most sensitive assays. The most sensitive assay identified was one utilizing antiserum no. 1155 and a heterologous competitive hapten, where the 2'-OH group was substituted with a Cl atom. An IC50 value and the detection range for TCS in assay buffer were 1.19 and 0.21-6.71 μg/L, respectively. The assay was selective for TCS, providing low cross-reactivity (<5%) to the major metabolites of TCS and to brominated diphenyl ether-47. A second assay utilizing a competitive hapten containing Br instead of Cl substitutions was broadly selective for both brominated and chlorinated diphenylethers. Using the most sensitive assay combination, we measured TCS concentrations in water samples following dilution. Biosolid samples were analyzed following the dilution of a simple solvent extract. The immunoassay results were similar to those determined by LC-MS/MS. This immunoassay can be used as a rapid and convenient tool to screen for human and environmental exposure. PMID:26937944

  7. Sensitive chemiluminescent immunoassay of triclopyr by digital image analysis.

    PubMed

    Díaz, Aurora N; Sánchez, Francisco G; Baro, Enrique N; Díaz, Ana F G; Aguilar, Alfonso; Algarra, Manuel

    2012-08-15

    An image based detection of chemiluminescence enzyme-linked immunosorbent assay (CL-ELISA) for the quantification of triclopyr has been developed. The immunoassay was an indirect competitive immunoassay with an anti-rabbit secondary antibody conjugated to horseradish peroxidase (HRP). Chemiluminescence was produced by the luminol/H(2)O(2)/HRP reaction, detected by a monochrome video CCD camera and digitized with an Imagraph IC-PCI frame grabber using a custom program developed in C(++) (Microsoft Visual C(++) 6.0). Two main improvements are reported in the data processing software: the implementation of a circular mesh covering the perimeter of each well, eliminating diffuse light from the neighboring wells, and the use of volume (the integration of light intensity of all pixels that define a well) as an analytical signal instead of CL intensity or area (as usual in commercial plate readers) to improve precision for normalization of the total light output. The standard curve was produced for 0.01-10 ng/L triclopyr. The limit of detection was 0.8 ng/L and the variation coefficient was 3.07% (n=10, P=0.05). PMID:22841045

  8. Multiplex Detection of Plant Pathogens Using a Microsphere Immunoassay Technology

    PubMed Central

    Charlermroj, Ratthaphol; Himananto, Orawan; Seepiban, Channarong; Kumpoosiri, Mallika; Warin, Nuchnard; Oplatowska, Michalina; Gajanandana, Oraprapai; Grant, Irene R.; Karoonuthaisiri, Nitsara; Elliott, Christopher T.

    2013-01-01

    Plant pathogens are a serious problem for seed export, plant disease control and plant quarantine. Rapid and accurate screening tests are urgently required to protect and prevent plant diseases spreading worldwide. A novel multiplex detection method was developed based on microsphere immunoassays to simultaneously detect four important plant pathogens: a fruit blotch bacterium Acidovorax avenae subsp. citrulli (Aac), chilli vein-banding mottle virus (CVbMV, potyvirus), watermelon silver mottle virus (WSMoV, tospovirus serogroup IV) and melon yellow spot virus (MYSV, tospovirus). An antibody for each plant pathogen was linked on a fluorescence-coded magnetic microsphere set which was used to capture corresponding pathogen. The presence of pathogens was detected by R-phycoerythrin (RPE)-labeled antibodies specific to the pathogens. The assay conditions were optimized by identifying appropriate antibody pairs, blocking buffer, concentration of RPE-labeled antibodies and assay time. Once conditions were optimized, the assay was able to detect all four plant pathogens precisely and accurately with substantially higher sensitivity than enzyme-linked immunosorbent assay (ELISA) when spiked in buffer and in healthy watermelon leaf extract. The assay time of the microsphere immunoassay (1 hour) was much shorter than that of ELISA (4 hours). This system was also shown to be capable of detecting the pathogens in naturally infected plant samples and is a major advancement in plant pathogen detection. PMID:23638044

  9. ABO (histo) blood group phenotype development and human reproduction as they relate to ancestral IgM formation: A hypothesis.

    PubMed

    Arend, Peter

    2016-01-01

    The formation of a histo (blood) group) ABO phenotype and the exclusion of an autoreactive IgM or isoagglutinin activity arise apparently in identical glycosylation of complementary domains on cell surfaces and plasma proteins. The fundamental O-glycan emptiness of the circulating IgM, which during the neonatal amino acid sequencing of the variable regions is exerting germline-specific O-GalNAc glycan-reactive serine/threonine residues that in the plasma of the adult human blood group O individuals apparently remain associated with the open glycosidic sites on the ABOH convertible red cell surface, must raise suggestions on a transient expression of developmental glycans, which have been "lost" over the course of maturation. In fact, while the mammalian non-somatic, embryogenic stem cell (ESC)- germ cell (GC) transformation is characterized by a transient and genetically as-yet-undefined trans-species-functional O-GalNAc glycan expression, in the C57BL/10 mouse such expression was potentially identified in growth-dependent, blood group A-like GalNAc glycan-bearing, ovarian glycolipids complementary with the syngeneic anti-A reactive IgM, which does not appear in early ovariectomized animals. This non-somatically encoded, polyreactive, ancestral IgM molecule has not undergone clonal selection and does primarily not differentiate between self and non-self and might, due to amino acid hydroxyl groups, highly suggest substrate competition with subsequent O-glycosylations in ongoing ESC-GC transformations and affecting GC maturation. However, the membrane-bound somatic N/O-glycotransferases, which initiate, after formation of the zygote, the complex construction of the human ABO phenotypes in the trans cisternae of the Golgi apparatus, are associated and/or completed with soluble enzyme versions exerting identical specificities in plasma and likely competing vice versa by glycosylation of neonatal IgM amino acids, where they suggest to accomplish the clearance of anti-A autoreactivity at germline serine and threonine residues. Sustaining the lineage-maintaining position of the classic A allele and the discovery of the OA hybrid alleles at the normal ABO locus and in heterozygous ESC lines have, together with clinical observations, raised discussions about a silent A-allelic support within blood group O reproduction. However, the question of whether a fictional "continued blood group O inbreeding" ultimately occurs without the A-allelic or somatic function remains unanswered because the genetic relationship between non-somatic O-GalNAc-glycosylations that operate before sperm-egg recognition and somatic O-GalNAc-glycosylations that arise after the formation of the zygote remains to be elucidated. PMID:26433867

  10. A novel immunosensor for detecting toxoplasma gondii-specific IgM based on goldmag nanoparticles and graphene sheets.

    PubMed

    Jiang, Shuting; Hua, Erhui; Liang, Mo; Liu, Bei; Xie, Guoming

    2013-01-01

    A novel electrochemical immunosensor for detecting toxoplasma gondii-specific IgM (Tg-IgM) was constructed based on goldmag (Au-Fe(3)O(4)) nanoparticles and graphene sheets (GS). Thionine (Thi), as a mediator, was first electropolymerized on a nafion-GS (Nf-GS) modified electrode. Subsequently, gold nanoparticles (AuNPs) were attached onto the poly-thionine film through π-stacking interactions, and then were used to immobilize toxoplasma gondii antigen (Tg-Ag) for immunosensor fabrication. A sandwich-type immunoassay for Tg-IgM was performed using Au-Fe(3)O(4) labeled anti-IgM-horseradish peroxidase (HRP) as trace label. Electrochemical detection was carried out in the presence of H(2)O(2) as HRP substrate. Using Au-Fe(3)O(4) provided a simple, non-chemical damaging method for regeneration, and enhanced the HRP reduction ability toward H(2)O(2). The AuNPs/Thi/Nf-GS nanocomposite also had good conductivity and biocompatibility, which effectively improved the immunosensor sensitivity. Under optimal conditions, the immunosensor can detect Tg-IgM in two linear ranges from 0.0375 to 1.2 AU mL(-1) and from 2.0 to 18 AU mL(-1) with a detection limit of 0.016 AU mL(-1) (S/N=3). The immunosensor exhibited good reproducibility, stability, and selectivity as well. PMID:23010058

  11. The hyper IgM syndrome--an evolving story.

    PubMed

    Etzioni, Amos; Ochs, Hans D

    2004-10-01

    The hyper IgM syndromes (HIGM) are a group of primary immune deficiency disorders characterized by defective CD40 signaling by B cells affecting class switch recombination and somatic hypermutation. As a consequence, patients with HIGM have decreased concentrations of serum IgG and IgA and normal or elevated IgM, leading to increased susceptibility to infections. The most common HIGM syndrome is X-linked and due to mutations of CD40 ligand (CD40L) expressed by activated CD4(+) T lymphocytes. Four other genes, expressed by B cells, have been associated with the HIGM phenotype. Mutations of CD40, the receptor for CD40L, cause a rare autosomal form of HIGM with a clinical phenotype similar to CD40L deficiency. Mutations of Activation-Induced Cytidine Deaminase (AICDA) and Uracil (DNA) Glycosylase (UNG), both expressed by follicular B lymphocytes, lead to defective class switch recombination and somatic hypermutation. Mutations of Nuclear Factor kappa B Essential Modulator (NEMO), an X-chromosome associated gene, result in hypohidrotic ectodermal dysplasia and immune deficiency. Thus, the molecular definition of these rare primary immune deficiency disorders has shed light on the complex events leading to the production of high-affinity, antigen-specific antibodies of different isotypes. PMID:15319456

  12. I. Studies on pyridine dinucleotide transhydrogenase in rat liver mitochondria. II. Identification of the tissue and cellular sites of catabolism of IgM in the rat

    SciTech Connect

    Weis, J.K.

    1988-01-01

    The orientation of the transmembrane enzyme, pyridine dinucleotide transhydrogenase, in the inner mitochondrial membrane of rat liver has been determined by evaluating effects of proteases on the integrity of the enzyme in mitoplasts and submitochondrial particles. Following treatment of these membranes with the non-specific protease, proteinase K, antigenic proteolytic products were detected by immunoblot analysis using polyclonal antibody prepared against purified bovine heart enzyme. Information from these proteolysis studies was used to construct a model of the orientation of transhydrogenase in the inner mitochondrial membrane. In this work, I have used the residualizing label, dilactitol-{sup 125}I-tyramine ({asterisk}I-DLT) to identify the tissue and cellular sites of catabolism of the immunoglobulin, IgM. Purified IgM was labeled conventionally with {sup 125}I or with the residualizing label, {asterisk}I-DLT. The circulating half-life of the protein, 2.7 {plus minus} 0.3 days, was the same when measured using either label, indicating that the residualizing label does not affect the kinetics of the protein's catabolism in vivo. At 2.4 or 5.1 days post injection, the liver contained the major fraction of catabolized protein compared to all the other organs in the body. Additionally, following collagenase digestion of the liver, the hepatocytes were shown to be 77% responsible for the catabolism of IgM by the liver. Autoradiography of the liver revealed that the remaining 23% of IgM catabolized by the liver was due to the Kupffer cells.

  13. Determination of dihydroergosine in sorghum ergot using an immunoassay.

    PubMed

    Molloy, John B; Moore, Chris J; Bruyeres, Anthea G; Murray, Sally-Ann; Blaney, Barry J

    2003-07-01

    Dihydroergosine (DHES) is the principal toxic alkaloid produced by sorghum ergot (Claviceps africana). It has recently been shown that DHES levels as low as 1 mg/kg in animal feed can cause significant production losses. Quantitative immunoassays for detecting the related rye ergot alkaloid, ergotamine, are described in the literature, but those assays are relatively insensitive for DHES. This paper describes competitive enzyme-linked immunosorbent assays (ELISA) for measuring the DHES concentration in grains and mixed animal feed. The assays were developed using a DHES specific mouse monoclonal antibody and rabbit polyclonal antibodies raised against DHES conjugated to bovine serum albumin. Recoveries of between 77 and 103% were obtained from spiked grain using a simple, one step extraction with 70% methanol. Both the monoclonal and the polyclonal assays are capable of detecting DHES concentrations above 0.01 mg/kg, but quantification is most reliable at concentrations of 0.1 mg/kg or higher. PMID:12822923

  14. Development of an Ultrasensitive Immunoassay for Detecting Tartrazine

    PubMed Central

    Li, Zhuokun; Song, Shanshan; Xu, Liguang; Kuang, Hua; Guo, Shidong; Xu, Chuanlai

    2013-01-01

    We have developed an ultrasensitive indirect competitive enzyme-linked immunosorbent assay for the determination of tartrazine. Two carboxylated analogues of tartrazine with different spacer lengths, and one derivative from commercial tartrazine after a little chemical modification, were synthesized as haptens in order to produce antibodies specific to tartrazine. The effect of sulfonic acid groups on the hapten structure of tartrazine was also studied carefully for the first time. A most specific monoclonal antibody against tartrazine was created and exhibited an IC50 value of 0.105 ng/mL and a limit of detection of 0.014 ng/mL, with no cross-reactivity to other structurally-related pigments. The established immunoassay was applied to the determination of tartrazine in fortified samples of orange juice and in real positive samples of carbonated beverages. PMID:23799494

  15. Nanoparticle-based immunosensors and immunoassays for aflatoxins.

    PubMed

    Wang, Xu; Niessner, Reinhard; Tang, Dianping; Knopp, Dietmar

    2016-03-17

    Aflatoxins are naturally existing mycotoxins produced mainly by Aspergillus flavus and Aspergillus parasiticus, present in a wide range of food and feed products. Because of their extremely high toxicity and carcinogenicity, strict control of maximum residue levels of aflatoxins in foodstuff is set by many countries. In daily routine, different chromatographic methods are used almost exclusively. As supplement, in several companies enzyme immunoassay-based sample testing as primary screening is performed. Recently, nanomaterials such as noble metal nanoparticles, magnetic particles, carbon nanomaterials, quantum dots, and silica nanomaterials are increasingly utilized for aflatoxin determination to improve the sensitivity and simplify the detection. They are employed either as supports for the immobilization of biomolecules or as electroactive or optical labels for signal transduction and amplification. Several nanoparticle-based electrochemical, piezoelectric, optical, and immunodipstick assays for aflatoxins have been developed. In this review, we summarize these recent advances and illustrate novel concepts and promising applications in the field of food safety. PMID:26920768

  16. Bicentric evaluation of Access Toxo immunoglobulin M (IgM) and IgG assays and IMx toxo IgM and IgG assays and comparison with Platelia Toxo IgM and IgG assays.

    PubMed Central

    Decoster, A; Lecolier, B

    1996-01-01

    The recent Access immunoanalysis system (Sanofi Diagnostics Pasteur) for the serological diagnosis of toxoplasmosis was compared with the Abbott Toxo IMx EIA system, taking the Platelia Toxo immunoglobulin G (IgG) and Platelia Toxo IgM systems as references and using as confirmation methods an indirect fluorescence assay or a dye test for IgG and an immunosorbent agglutination assay (ISAGA) for IgM. A total of 1,461 serum samples were studied, of which 128 were collected from 42 recently seroconverted patients. Sensitivity and specificity rates of the Access system were 97.7 and 99.5%, respectively, for IgM and 98.6 and 100%, respectively, for IgG. Sensitivity and specificity rates of the Abbott IMx EIA system were 91 and 100%, respectively, for IgM and 92.5 and 100%, respectively, for IgG. The Access Toxo IgG and IgM EIA systems were found to be more sensitive than the Abbott Toxo IgG and IgM IMx EIA systems. PMID:8784554

  17. Highly sensitive immunoassay based on immunogold-silver amplification and inductively coupled plasma mass spectrometric detection.

    PubMed

    Liu, Rui; Liu, Xing; Tang, Yurong; Wu, Li; Hou, Xiandeng; Lv, Yi

    2011-03-15

    In this work, we demonstrated a highly sensitive inductively coupled plasma mass spectrometric (ICPMS) method for the determination of human carcinoembryonic antigen (CEA), which combined the inherent high sensitivity of elemental mass spectrometric measurement with the signal amplification of catalytic silver deposition on immunogold tags. The silver amplification procedure was easy to handle and required cheap reagents, and the sensitivity was greatly enhanced to 60-fold after a 15 min silver amplification procedure. The experimental conditions, including detection of gold and silver by ICPMS, immunoassay parameters, silver amplification parameters, analytical performance, and clinical serum samples analysis, were investigated. The ICPMS Ag signal intensity depends linearly on the logarithm of the concentration of human CEA over the range of 0.07-1000 ng mL(-1) with a limit of detection (LOD, 3σ) of 0.03 ng mL(-1) (i.e., 0.15 pM). The LOD of the proposed method is around 2 orders of magnitude lower than that by the widely used enzyme-linked immunosorbent assay (ELISA) and 1 order of magnitude lower than that by clinical routine chemiluminescence immunoassay (CLIA) or time-resolved fluoroimmunoassay (TRFIA) and conventional ICPMS immunoassay. The present strategy was applied to the determination of human CEA in clinical human serum samples, and the results were in good agreement with those obtained by chemiluminescence immunoassay. PMID:21348438

  18. Silver deposition directed by self-assembled gold nanorods for amplified electrochemical immunoassay.

    PubMed

    Zhang, Hongfang; Ning, Danlei; Ma, Lina; Zheng, Jianbin

    2016-01-01

    A novel electrochemical immunoassay was developed based on the signal amplification strategy of silver deposition directed by gold nanorods (AuNRs), which was in-situ assembled on the sandwich immunocomplex. The superstructure formed by the self-assembly of AuNRs provided abundant active sites for the nucleation of silver nanoparticles. In this pathway, the stripping current of silver was greatly enhanced. Using human immunoglobulin G (HIgG) as a model analyte, the ultrasensitive immunoassay showed a wide linear range of six orders of magnitude from 0.1 fg mL(-1) to 100 pg mL(-1), with the low detection limit down to 0.08 fg mL(-1). The practicality of this electrochemical immunoassay for detection of HIgG in serum was validated with the average recovery of 93.9%. In addition, this enzyme-free immunoassay also has the advantages of acceptable reproducibility and specificity, and thus this immunosensing protocol can be extended to the detection of other low-abundant protein biomarkers. PMID:26703256

  19. The development of immunoassays for detection of chemical warfare agents

    SciTech Connect

    Lenz, D.E.; Brimfield, A.A.; Cook, L.

    1996-10-01

    With the advent of enzyme linked immunoabsorbent assays (ELISA) and monoclonal antibodies in the last two decades, there has been considerable effort devoted to the development of antibodies to detect and quantify low molecular weight toxic substances in environmental or biological fluids. Polyclonal antibodies against paraoxon (the toxic metabolite of parathion) were capable of detecting paraoxon in body fluids at a level of 10{sup -9} M ({approximately}260 pg/mL) when used in a competitive inhibition enzyme immunoassay (CIEIA). Monoclonal antibodies developed against a structural analogue of the chemical warfare agent soman were capable of detection soman in buffer solutions at a level of 10{sup -6} M ({approximately}180 ng/mL). In addition these antibodies were found to be highly specific for soman even in the presence of its major hydrolysis product. Subsequent studies with antisoman monoclonal antibodies extended the level of sensitivity to {approximately}80 ng/mL. Furthermore these antibodies did not cross react with other chemical warfare nerve agents such as sarin or tabun. In all cases, the time for a confirmatory test was two hours or less. Immunoassays for T-2 micotoxins have also been reported with a minimal detection range of 2 pg/assay to 50 ng/assay for the polyclonal and monoclonal T-2 antibodies respectively. These reagents offer a sensitive, rapid and low cost approach to the diagnosis or detection of the presence of toxic chemical substances. More recent efforts have focussed on developing antibodies specific for sulfur mustard a highly reactive vesicating agent.

  20. The contribution of naturally occurring IgM antibodies, IgM cross-reactivity and complement dependency in murine humoral responses to pneumococcal capsular polysaccharides.

    PubMed

    Jones, Hannah E; Taylor, Philip R; McGreal, Eamon; Zamze, Susanne; Wong, Simon Y C

    2009-09-25

    Immunogenicity of 12 capsular polysaccharides (CPS) from Streptococcus pneumoniae did not correlate with pre-existing levels of natural IgM anti-CPS antibodies in mice. Immunization of mice with individual CPS, with the exception of type 14 (the only neutral CPS tested), increased serum IgM that also bound other CPS serotypes independent of structural similarity or commonly known contaminants. Surprisingly only IgM response to type 4 (which has a small immunodominant epitope) was dependent on either complement C3 or complement receptors CD35/CD21. IgG anti-CPS responses were infrequently induced, but critically dependent on complement. Our results have clarified the role of complement in the induction of IgM and IgG anti-CPS antibody responses in mice and have implications for CPS vaccine development. PMID:19660585

  1. Investigation Of The Diffuse IGM By Cross-Correlation Studies

    SciTech Connect

    Farnsworth, Damon; Brown, Shea; Rudnick, Lawrence

    2009-12-18

    We present results from the first cross-correlation search for the synchrotron component of the diffuse intergalactic medium (IGM) in filamentary large scale structure (LSS). We used the low resolution (36') Bonn survey at 21cm, with the infrared 2MASS catalog as a tracer of the LSS. Synchrotron emission likely results from LSS formation shocks and feedback from AGN and galactic winds [2]. We determined 3{sigma} upper limits to the diffuse emission in units of flux per galaxy; these correspond to filament equipartition magnetic fields as low as 0.2 {mu}G. The detection threshold for the average (peak) filament brightness is 1 (7) mK for 0.03

  2. Low molecular weight IgM in selective IgA deficiency.

    PubMed Central

    Kwitko, A O; Roberts-Thomson, P J; Shearman, D J

    1982-01-01

    Thirty-nine persons with selective IgA deficiency were studied. These comprised 27 subjects found by population screening and 12 by other means. Low molecular weight (LMW) serum IgM was sought in 28 of the 39 persons. Nine of the 28 (32%) had LMW IgM detectable by a sensitive gel filtration technique. Of 17 patients discovered by screening, five (29%) had LMW IgM. In the nine positive persons, LMW IgM constituted up to 17% of the total serum IgM concentration. Eight of the nine IgA deficient persons with LMW IgM, had clinical disease while associated disease in the entire IgA deficient population was less frequent. Serum immune complexes were demonstrated in five of seven subjects with LMW IgM using a C1q-dependent radioimmunoassay; four of these had immune complex associated disorders, three with polyarthritis and one with glomerulonephritis. Because circulating immune complexes are frequently detected in IgA deficient persons without disease, it is proposed that the presence of LMW serum IgM in IgA deficiency may be associated with disease due to the formation of specific pathogenic immune complexes. PMID:7172505

  3. Epithelial cells are a source of natural IgM that contribute to innate immune responses.

    PubMed

    Shao, Wenwei; Hu, Fanlei; Ma, Junfan; Zhang, Chi; Liao, Qinyuan; Zhu, Zhu; Liu, Enyang; Qiu, Xiaoyan

    2016-04-01

    Currently, natural IgM antibodies are considered to be the constitutively secreted products of B-1 cells in mice and humans. In this study, we found that mouse epithelial cells, including liver epithelial cells and small intestinal epithelial cells (IECs), could express IgM that also showed natural antibody activity. Moreover, similar to the B-1 cell-derived natural IgM that can be upregulated by TLR9 agonists (mimicking bacterial infection), the expression of epithelial cell-derived natural IgM could also be significantly increased by TLR9 signaling. More importantly, the epithelial cell-derived IgM was polyreactive, and it could recognize single-stranded DNA (ssDNA), double-stranded DNA (dsDNA), lipopolysaccharide (LPS), and insulin with low affinity; additionally, TLR9 agonists could enhance it in a MyD88-dependent manner. Furthermore, epithelial cell-derived IgM could bind various bacteria; therefore, it could be involved in anti-infection responses. Together, these results highlight the fact that epithelial cells are an important source of natural IgM, in addition to that produced by B-1 cells, and IgM contributes to the innate immune responses in local tissues, further demonstrating that the epithelium is a first line of defense in the protection against invading microbes. PMID:26820901

  4. Hapten modification approach for switching immunoassay specificity from selective to generic.

    PubMed

    Burkin, Maksim A; Galvidis, Inna A

    2013-02-28

    The cross-reactivity profile of polyclonal antibodies against a low molecular weight analyte is strongly influenced by design of the coating or enzyme-linked hapten. The hapten modification effect on immunoassay specificity was studied. Heterology in hapten type and linking method were applied. The influence of these factors on analyses of two groups of antibiotics, 16-membered macrolides and glycopeptides was studied. This approach was used to convert the selective ELISAs to tylosin and eremomycin for group determination of tylosin\\tilmicosin, tylosin\\spiramycin and eremomycin\\vancomycin. It was shown that the analytical spectrum of the developed polyclonal antibody-based immunoassays could be expanded and depended mainly on the type of coating hapten but not on the linking method. Modification of the hapten type in coating conjugates applied in present study served as a mechanism for switching specificity of the ELISA between selective and group. PMID:23234756

  5. Anti-HDV IgM as a Marker of Disease Activity in Hepatitis Delta

    PubMed Central

    Wranke, Anika; Heidrich, Benjamin; Ernst, Stefanie; Calle Serrano, Beatriz; Caruntu, Florin Alexandru; Curescu, Manuela Gabriela; Yalcin, Kendal; Gürel, Selim; Zeuzem, Stefan; Erhardt, Andreas; Lüth, Stefan; Papatheodoridis, George V.; Bremer, Birgit; Stift, Judith; Grabowski, Jan; Kirschner, Janina; Port, Kerstin; Cornberg, Markus; Falk, Christine S.; Dienes, Hans-Peter; Hardtke, Svenja; Manns, Michael P.; Yurdaydin, Cihan; Wedemeyer, Heiner

    2014-01-01

    Background Hepatitis delta frequently leads to liver cirrhosis and hepatic decompensation. As treatment options are limited, there is a need for biomarkers to determine disease activity and to predict the risk of disease progression. We hypothesized that anti-HDV IgM could represent such a marker. Methods Samples of 120 HDV-infected patients recruited in an international multicenter treatment trial (HIDIT-2) were studied. Anti-HDV IgM testing was performed using ETI-DELTA-IGMK-2-assay (DiaSorin). In addition, fifty cytokines, chemokines and angiogenetic factors were measured using multiplex technology (Bio-Plex System). A second independent cohort of 78 patients was studied for the development of liver-related clinical endpoints (decompensation, HCC, liver transplantation or death; median follow up of 3.0 years, range 0.6–12). Results Anti-HDV IgM serum levels were negative in 18 (15%), low (OD<0.5) in 76 (63%), and high in 26 (22%) patients of the HIDIT-2 cohort. Anti-HDV IgM were significantly associated with histological inflammatory (p<0.01) and biochemical disease activity (ALT, AST p<0.01). HDV replication was independent from anti-HDV IgM, however, low HBV-DNA levels were observed in groups with higher anti-HDV IgM levels (p<0.01). While high IP-10 (CXCL10) levels were seen in greater groups of anti-HDV IgM levels, various other antiviral cytokines were negatively associated with anti-HDV IgM. Associations between anti-HDV IgM and ALT, AST, HBV-DNA were confirmed in the independent cohort. Clinical endpoints occurred in 26 anti-HDV IgM positive patients (39%) but in only one anti-HDV IgM negative individual (9%; p = 0.05). Conclusions Serum anti-HDV IgM is a robust, easy-to-apply and relatively cheap marker to determine disease activity in hepatitis delta which has prognostic implications. High anti-HDV IgM levels may indicate an activated interferon system but exhausted antiviral immunity. PMID:25072849

  6. Theoretical limitations of quantification for noncompetitive sandwich immunoassays.

    PubMed

    Woolley, Christine F; Hayes, Mark A; Mahanti, Prasun; Douglass Gilman, S; Taylor, Tom

    2015-11-01

    Immunoassays exploit the highly selective interaction between antibodies and antigens to provide a vital method for biomolecule detection at low concentrations. Developers and practitioners of immunoassays have long known that non-specific binding often restricts immunoassay limits of quantification (LOQs). Aside from non-specific binding, most efforts by analytical chemists to reduce the LOQ for these techniques have focused on improving the signal amplification methods and minimizing the limitations of the detection system. However, with detection technology now capable of sensing single-fluorescence molecules, this approach is unlikely to lead to dramatic improvements in the future. Here, fundamental interactions based on the law of mass action are analytically connected to signal generation, replacing the four- and five-parameter fittings commercially used to approximate sigmoidal immunoassay curves and allowing quantitative consideration of non-specific binding and statistical limitations in order to understand the ultimate detection capabilities of immunoassays. The restrictions imposed on limits of quantification by instrumental noise, non-specific binding, and counting statistics are discussed based on equilibrium relations for a sandwich immunoassay. Understanding the maximal capabilities of immunoassays for each of these regimes can greatly assist in the development and evaluation of immunoassay platforms. While many studies suggest that single molecule detection is possible through immunoassay techniques, here, it is demonstrated that the fundamental limit of quantification (precision of 10 % or better) for an immunoassay is approximately 131 molecules and this limit is based on fundamental and unavoidable statistical limitations. PMID:26342315

  7. Specific immunoassays for placental alkaline phosphatase as a tumor marker.

    PubMed

    Stinghen, Sérvio T; Moura, Juliana F; Zancanella, Patrícia; Rodrigues, Giovanna A; Pianovski, Mara A; Lalli, Enzo; Arnold, Dodie L; Minozzo, João C; Callefe, Luis G; Ribeiro, Raul C; Figueiredo, Bonald C

    2006-01-01

    Human placental (hPLAP) and germ cell (PLAP-like) alkaline phosphatases are polymorphic and heat-stable enzymes. This study was designed to develop specific immunoassays for quantifying hPLAP and PLAP-like enzyme activity (EA) in sera of cancer patients, pregnant women, or smokers. Polyclonal sheep anti-hPLAP antibodies were purified by affinity chromatography with whole hPLAP protein (ICA-PLAP assay) or a synthetic peptide (aa 57-71) of hPLAP (ICA-PEP assay); the working range was 0.1-11 U/L and cutoff value was 0.2 U/L EA for nonsmokers. The intra- and interassay coefficients of variation were 3.7%-6.5% (ICA-PLAP assay) and 9.0%-9.9% (ICA-PEP assay). An insignificant cross-reactivity was noted for high levels of unheated intestinal alkaline phosphatase in ICA-PEP assay. A positive correlation between the regression of tumor size and EA was noted in a child with embryonal carcinoma. It can be concluded that ICA-PEP assay is more specific than ICA-PLAP, which is still useful to detect other PLAP/PLAP-like phenotypes. PMID:17489017

  8. Age‐related aspects of human IgM+ B cell heterogeneity

    PubMed Central

    Martin, Victoria; Wu, Yu‐Chang; Kipling, David

    2015-01-01

    The CD27+IgD+ B cell population, known as IgM memory, reduces with age. It is thought that this population is responsible for pneumococcal polysaccharide T‐independent responses, and that the age‐related reduction might be partially responsible for the increased susceptibility of older people to bacterial pathogens. There are other IgM+ B cell populations that do not express IgD. We compared the different IgM populations using high‐throughput sequencing of the immunoglobulin (Ig) gene repertoire and multidimensional cell phenotyping and found that the different populations of IgM cells, defined by CD27 and IgD expression, have repertoire differences. Some of these differences are likely indicative of different selection pressures in an immune response, although the older individuals were found to have a changed repertoire in naive B cells, which may contribute to some of the changes seen in memory cells. In addition, even within the CD27+IgD+ IgM memory population there are multiple cell types. We show that the level of IgM expression varies substantially and hypothesize that this distinguishes between T‐dependent and T‐independent types of IgM memory cells. Significant age‐related changes in the relative proportions of these populations may exacerbate the reduction in T‐independent responders in old age. PMID:26152370

  9. The new ParaDIgm: IgM from bench to clinic

    PubMed Central

    Hanala, Sherif

    2012-01-01

    The inaugural IgM event entitled “The new ParaDIgm: IgM from bench to clinic” brought together the increasingly active and growing IgM antibody community to discuss recent advances and challenges facing the discovery and development of IgM antibody therapies and technologies. Researchers, clinicians and biomanufacturing experts delivered 21 talks on the basic science and isolation of IgM, upstream and downstream development, and formulation and clinical development of the molecules. Participants networked around topics aimed at exploring the full potential of IgM antibodies. The meeting was held at DECHEMA Gesellschaft für Chemische Technik und Biotechnologie e. V. (Society for Chemical Engineering and Biotechnology), a non-profit scientific and technical society based in Frankfurt am Main, Germany. The meeting was sponsored by Patrys, Laureate Biopharma, Bio-Rad Laboratories, BIA Separations, Percivia and the Bio Affinity Company (BAC). The second New ParaDIgm: IgM from bench to clinic meeting, will be held on April 23–24, 2013 in Frankfurt, Germany. PMID:22864407

  10. IgM nephropathy; can we still ignore it

    PubMed Central

    Vanikar, Aruna

    2013-01-01

    Context:IgM nephropathy (IgMN) is a relatively less recognized clinico-immunopathological entity in the domain of glomerulonephritis , often thought to be a bridge between minimal change disease and focal segmental glomerulosclerosis. Evidence Acquisitions: Directory of Open Access Journals (DOAJ), Google Scholar, Pubmed (NLM), LISTA (EBSCO) and Web of Science has been searched. Results: IgM nephropathy can present as nephritic syndrome or less commonly with subnephrotic proteinuria or rarely hematuria. About 30% patients respond to steroids whereas others are steroid dependent / resistant. They should be given a trial of Rituximab or stem cell therapy. Conclusions:IgM nephropathy (IgMN) is an important and rather neglected pathology responsible for renal morbidity in children and adults in developing countries as compared to developed nations with incidence of 2-18.5% of native biopsies. Abnormal T-cell function with hyperfunctioning suppressor T-cells are believed to be responsible for this disease entity. Approximately one third of the patients are steroid responders where as the remaining two thirds are steroid resistant or dependent. Therapeutic trials including cell therapies targeting suppressor T-cells are required. PMID:24475434

  11. Interaction between the IGM and a dwarf galaxy

    NASA Astrophysics Data System (ADS)

    Lora, V.; Raga, A. C.; Grebel, E. K.

    2015-04-01

    Dwarf Galaxies are the most common objects in the Universe and are believed to contain large amounts of dark matter. There are mainly three morphologic types of dwarf galaxies: dwarf ellipticals, dwarf spheroidals and dwarf irregulars. Dwarf irregular galaxies are particularly interesting in dwarf galaxy evolution, since dwarf spheroidal predecessors could have been very similar to them. Therefore, a mechanism linked to gas-loss in dwarf irregulars should be observed, i.e. ram pressure stripping. In this paper, we study the interaction between the ISM of a dwarf galaxy and a flowing IGM. We derive the weak-shock, plasmon solution corresponding to the balance between the post-bow shock pressure and the pressure of the stratified ISM (which we assume follows the fixed stratification of a gravitationally dominant dark matter halo). We compare our model with previously published numerical simulations and with the observed shape of the HI cloud around the Ho II and Pegasus dwarf irregular galaxies. We show that such a comparison provides a straightforward way for estimating the Mach number of the impinging flow.

  12. Transcriptional Heterogeneity of IgM+ Cells in Rainbow Trout (Oncorhynchus mykiss) Tissues

    PubMed Central

    Abós, Beatriz; Castro, Rosario; Pignatelli, Jaime; Luque, Alfonso; González, Lucia; Tafalla, Carolina

    2013-01-01

    Two major classes of B lymphocytes have been described to date in rainbow trout: IgM+ and IgT+ cells. IgM+ cells are mainly localized in the spleen, peripheral blood and kidney but are also found in other tissues. However, differences among IgM+ cell populations attending to its location are poorly defined in fish. Thus, the aim of this work was to characterize the expression of different immune molecules such as chemokine receptors, Toll-like receptors (TLRs) and transcription factors on sorted IgM+ lymphocytes from different rainbow trout tissues. IgM+ populations from blood, spleen, kidney, gills, intestine and liver were isolated by cell sorting and the constitutive levels of transcription of these genes evaluated by real-time PCR. To further characterize B cells, we identified an MS4A sequence. In humans, the MS4A family includes several genes with immune functions, such as the B cell marker CD20 or FcRβ. Subsequently, we have also evaluated the mRNA levels of this MS4A gene in the different IgM+ populations. The relevant differences in transcriptional patterns observed for each of these IgM+ populations analyzed, point to the presence of functionally different tissue-specific B cell populations in rainbow trout. The data shown provides a pattern of genes transcribed in IgM+ B cells not previously revealed in teleost fish. Furthermore, the constitutive expression of all the TLR genes analyzed in IgM+ cells suggests an important role for these cells in innate immunity. PMID:24324826

  13. Expression and glycoengineering of functionally active heteromultimeric IgM in plants

    PubMed Central

    Loos, Andreas; Gruber, Clemens; Altmann, Friedrich; Mehofer, Ulrich; Hensel, Frank; Grandits, Melanie; Oostenbrink, Chris; Stadlmayr, Gerhard; Furtmüller, Paul G.; Steinkellner, Herta

    2014-01-01

    IgM antibodies are an important player of the human’s innate defense mechanisms and increasingly have gained interest as therapeutics. Although the expression of IgM antibodies in mammalian cell culture is established, this approach remains costly and alternative methods have not been developed yet. Plants have a proven record for the production of therapeutically relevant recombinant proteins. However, whether they are able to express proteins like IgM antibodies, which range among the most complex human proteins, remains unknown so far. Here we report the in planta generation of the functionally active monoclonal antitumor IgM PAT-SM6 (SM6). SM6 efficiently accumulates in plant leaves and assembles correctly into heterooligomers (pentamers and hexamers). Detailed glycosylation analysis exhibited complex and oligomannosidic N-glycans in a site-specific manner on human-serum IgM and on plant- and human-cell-line–produced SM6. Moreover, extensive in planta glycoengineering allowed the generation of SM6 decorated with sialylated human-type oligosaccharides, comparable to plasma-derived IgM. A glycosylated model of pentameric IgM exhibits different accessibility of the glycosylation sites, explaining site-specific glycosylation. Biochemical and biophysical properties and importantly biological activities of plant-derived SM6 glycoforms are comparable to the human-cell–derived counterparts. The in planta generation of one of the most complex human proteins opens new pathways toward the production of difficult-to-express proteins for pharmaceutical applications. Moreover, the generation of IgMs with a controlled glycosylation pattern allows the study of the so far unknown contribution of sugar moieties to the function of IgMs. PMID:24706782

  14. Cytomegalovirus IgM Seroprevalence among Women of Reproductive Age in the United States.

    PubMed

    Wang, Chengbin; Dollard, Sheila C; Amin, Minal M; Bialek, Stephanie R

    2016-01-01

    Cytomegalovirus (CMV) IgM indicates recent active CMV infection. CMV IgM seroprevalence is a useful marker for prevalence of transmission. Using data from the National Health and Nutrition Examination Survey (NHANES) III 1988-1994, we present estimates of CMV IgM prevalence by race/ethnicity, provide a comparison of IgM seroprevalence among all women and among CMV IgG positive women, and explore factors possibly associated with IgM seroprevalence, including socioeconomic status and exposure to young children. There was no difference in IgM seroprevalence by race/ethnicity among all women (3.1%, 2.2%, and 1.6% for non-Hispanic white, non-Hispanic black and Mexican American, respectively; P = 0.11). CMV IgM seroprevalence decreased significantly with increasing age in non-Hispanic black women (P<0.001 for trend) and marginally among Mexican American women (P = 0.07), while no apparent trend with age was seen in non-Hispanic white women (P = 0.99). Among 4001 IgG+ women, 118 were IgM+, resulting in 4.9% IgM seroprevalence. In IgG+ women, IgM seroprevalence varied significantly by age (5.3%, 7.3%, and 3.7% for women of 12-19, 20-29, and 30-49 years; P = 0.04) and race/ethnicity (6.1%, 2.7%, and 2.0% for non-Hispanic white, non-Hispanic black, and Mexican American; P<0.001). The factors reported associated with IgG seroprevalence were not associated with IgM seroprevalence. The patterns of CMV IgM seroprevalence by age, race/ethnicity, and IgG serostatus may help understanding the epidemiology of congenital CMV infection as a consequence of vertical transmission and are useful for identifying target populations for intervention to reduce CMV transmission. PMID:26990759

  15. Cytomegalovirus IgM Seroprevalence among Women of Reproductive Age in the United States

    PubMed Central

    Wang, Chengbin; Dollard, Sheila C.; Amin, Minal M.; Bialek, Stephanie R.

    2016-01-01

    Cytomegalovirus (CMV) IgM indicates recent active CMV infection. CMV IgM seroprevalence is a useful marker for prevalence of transmission. Using data from the National Health and Nutrition Examination Survey (NHANES) III 1988–1994, we present estimates of CMV IgM prevalence by race/ethnicity, provide a comparison of IgM seroprevalence among all women and among CMV IgG positive women, and explore factors possibly associated with IgM seroprevalence, including socioeconomic status and exposure to young children. There was no difference in IgM seroprevalence by race/ethnicity among all women (3.1%, 2.2%, and 1.6% for non-Hispanic white, non-Hispanic black and Mexican American, respectively; P = 0.11). CMV IgM seroprevalence decreased significantly with increasing age in non-Hispanic black women (P<0.001 for trend) and marginally among Mexican American women (P = 0.07), while no apparent trend with age was seen in non-Hispanic white women (P = 0.99). Among 4001 IgG+ women, 118 were IgM+, resulting in 4.9% IgM seroprevalence. In IgG+ women, IgM seroprevalence varied significantly by age (5.3%, 7.3%, and 3.7% for women of 12–19, 20–29, and 30–49 years; P = 0.04) and race/ethnicity (6.1%, 2.7%, and 2.0% for non-Hispanic white, non-Hispanic black, and Mexican American; P<0.001). The factors reported associated with IgG seroprevalence were not associated with IgM seroprevalence. The patterns of CMV IgM seroprevalence by age, race/ethnicity, and IgG serostatus may help understanding the epidemiology of congenital CMV infection as a consequence of vertical transmission and are useful for identifying target populations for intervention to reduce CMV transmission. PMID:26990759

  16. Multiplexed Microsphere Suspension Array-Based Immunoassays.

    PubMed

    Lin, Andrew; Salvador, Alexandra; Carter, J Mark

    2015-01-01

    ELISA is an extremely powerful tool to detect analytes because of its sensitivity, selectivity, reproducibility and ease of use. Here we describe sandwich immunoassays performed in suspension on spectrally unique microspheres developed by Luminex. Luminex assays offer the benefit of multiplex analysis of large numbers of analytes in a single reaction. Because the microspheres are spectrally unique, many microspheres, each attached to various antibodies, can be added to a single sample. Luminex instruments can distinguish each microsphere and detect the intensity of a reporter signal for each microsphere. Results are reported in Median Fluorescent Intensities for each analyte. Luminex assays can be used to detect up to 500 analytes in a high-throughput format. Luminex refers to this technology as xMAP(®). Here we describe a routine protocol for a Luminex immunoassay. Other Luminex assays would have to be optimized for specific conditions according to their use. PMID:26160569

  17. Industrial fungal enzymes: an occupational allergen perspective.

    PubMed

    Green, Brett J; Beezhold, Donald H

    2011-01-01

    Occupational exposure to high-molecular-weight allergens is a risk factor for the development and pathogenesis of IgE-mediated respiratory disease. In some occupational environments, workers are at an increased risk of exposure to fungal enzymes used in industrial production. Fungal enzymes have been associated with adverse health effects in the work place, in particular in baking occupations. Exposure-response relationships have been demonstrated, and atopic workers directly handling fungal enzymes are at an increased risk for IgE-mediated disease and occupational asthma. The utilization of new and emerging fungal enzymes in industrial production will present new occupational exposures. The production of antibody-based immunoassays is necessary for the assessment of occupational exposure and the development of threshold limit values. Allergen avoidance strategies including personal protective equipment, engineering controls, protein encapsulation, and reduction of airborne enzyme concentrations are required to mitigate occupational exposure to fungal enzymes. PMID:21747869

  18. Industrial Fungal Enzymes: An Occupational Allergen Perspective

    PubMed Central

    Green, Brett J.; Beezhold, Donald H.

    2011-01-01

    Occupational exposure to high-molecular-weight allergens is a risk factor for the development and pathogenesis of IgE-mediated respiratory disease. In some occupational environments, workers are at an increased risk of exposure to fungal enzymes used in industrial production. Fungal enzymes have been associated with adverse health effects in the work place, in particular in baking occupations. Exposure-response relationships have been demonstrated, and atopic workers directly handling fungal enzymes are at an increased risk for IgE-mediated disease and occupational asthma. The utilization of new and emerging fungal enzymes in industrial production will present new occupational exposures. The production of antibody-based immunoassays is necessary for the assessment of occupational exposure and the development of threshold limit values. Allergen avoidance strategies including personal protective equipment, engineering controls, protein encapsulation, and reduction of airborne enzyme concentrations are required to mitigate occupational exposure to fungal enzymes. PMID:21747869

  19. Enzyme immunoassay by dynamic enhanced vibrational spectroscopy of the enzyme reaction product

    NASA Astrophysics Data System (ADS)

    Zhao, Haiying; Dou, Xiaoming

    2005-01-01

    This paper reports a kind of application of surface-enhanced Raman scattering (SERS) to immunology. In the proposed system, antibody immobilized on a solid substrate reacts with antigen, which binds with another antibody labeled with peroxidase. If this immunocomplex is subjected to reaction with o-phenylenediamine and hydrogenperoxide at 37°C, azoaniline is generated. This azo compound is adsorbed on a silver colloid and only the azo compound gives a strong surface-enhanced resonance Raman (SERRS) spectrum. A linear relationship was observed between the peak intensity of the N=N stretching band and the concentration of antigen, revealing that one can determine the concentration of antigen by the SERRS measurement of the reaction product.

  20. Suppressor T cells prevent in vitro expression of IgM rheumatoid factor in some healthy adults

    SciTech Connect

    Koopman, W.J.

    1981-12-01

    Peripheral blood mononuclear leukocytes (MNL) from 11 of 30 healthy adults elaborated detectable IgM RF when stimulated with pokeweed mitogen. The influence of T cells on IgM RF production by autologous B Cells prepared from donors whose unfractionated MNL synthesized IgM RF in response to PMW was investigated. Untreated T cells supported IgM RF production by autologous B cells with optimal synthesis observed at T:B cell ratios of 2:1 at higher T:B cell ratios a decline in IgM RF production occurred. In contrast, at higher T:B cell ratios irradiated T cells supported consistently higher levels of IgM RF production than untreated T cells suggesting the presence of radiosensitive suppressor T cells for IgM RF in these individuals. Irridiated T cells were compared to untreated T cells for capacity to support IgM RF production by autologous B cells from 12 randomly selected donors at T:B cell ratios of 3:1. Untreated T cells from 4 of 12 individuals were capable of cooperating in induction of IgM RF production by autologous B cells, whereas irradiated T cells supported IgM RF production in 6 of 12 individuals. Levels of IgM RF production in all 6 individuals were significantly higher with irradiated T cells than with untreated T cells; in 2 individuals IgM RF synthesis by autologous B cells was observed only in the presence of irradiated T cells. In 4 of 6 individuals increases in the ratio of IgM RF total IgM synthesis occured with irradiated T cells (when compared to untreated T cells), suggesting disproportionate suppression of RF production. These results indicate the presence of radiosensitive T cells capable of suppressing IgM RF production in a significant fraction of healthy adults and raise the possibility that these cells may regulate in vivo expression of RF.

  1. Flotation Immunoassay: Masking the Signal from Free Reporters in Sandwich Immunoassays

    PubMed Central

    Chen, Hui; Hagström, Anna E. V.; Kim, Jinsu; Garvey, Gavin; Paterson, Andrew; Ruiz-Ruiz, Federico; Raja, Balakrishnan; Strych, Ulrich; Rito-Palomares, Marco; Kourentzi, Katerina; Conrad, Jacinta C.; Atmar, Robert L.; Willson, Richard C.

    2016-01-01

    In this work, we demonstrate that signal-masking reagents together with appropriate capture antibody carriers can eliminate the washing steps in sandwich immunoassays. A flotation immunoassay (FI) platform was developed with horseradish peroxidase chemiluminescence as the reporter system, the dye Brilliant Blue FCF as the signal-masking reagent, and buoyant silica micro-bubbles as the capture antibody carriers. Only reporters captured on micro-bubbles float above the dye and become visible in an analyte-dependent manner. These FIs are capable of detecting proteins down to attomole levels and as few as 106 virus particles. This signal-masking strategy represents a novel approach to simple, sensitive and quantitative immunoassays in both laboratory and point-of-care settings. PMID:27075635

  2. Flotation Immunoassay: Masking the Signal from Free Reporters in Sandwich Immunoassays.

    PubMed

    Chen, Hui; Hagström, Anna E V; Kim, Jinsu; Garvey, Gavin; Paterson, Andrew; Ruiz-Ruiz, Federico; Raja, Balakrishnan; Strych, Ulrich; Rito-Palomares, Marco; Kourentzi, Katerina; Conrad, Jacinta C; Atmar, Robert L; Willson, Richard C

    2016-01-01

    In this work, we demonstrate that signal-masking reagents together with appropriate capture antibody carriers can eliminate the washing steps in sandwich immunoassays. A flotation immunoassay (FI) platform was developed with horseradish peroxidase chemiluminescence as the reporter system, the dye Brilliant Blue FCF as the signal-masking reagent, and buoyant silica micro-bubbles as the capture antibody carriers. Only reporters captured on micro-bubbles float above the dye and become visible in an analyte-dependent manner. These FIs are capable of detecting proteins down to attomole levels and as few as 10(6) virus particles. This signal-masking strategy represents a novel approach to simple, sensitive and quantitative immunoassays in both laboratory and point-of-care settings. PMID:27075635

  3. Age-Related Decline in Natural IgM Function: Diversification and Selection of the B-1a Cell Pool with Age.

    PubMed

    Holodick, Nichol E; Vizconde, Teresa; Hopkins, Thomas J; Rothstein, Thomas L

    2016-05-15

    Streptococcus pneumoniae is the most common cause of pneumonia, which claims the lives of people over the age of 65 y seven times more frequently than those aged 5-49 y. B-1a cells provide immediate and essential protection from S. pneumoniae through production of natural Ig, which has minimal insertion of N-region additions added by the enzyme TdT. In experiments with SCID mice infected with S. pneumoniae, we found passive transfer of IgG-depleted serum from aged (18-24 mo old) mice had no effect whereas IgG-depleted serum from young (3 mo old) mice was protective. This suggests protective natural IgM changes with age. Using single cell PCR we found N-region addition, which is initially low in fetal-derived B-1a cell IgM developing in the absence of TdT, increased in 7- to 24-mo-old mice as compared with 3-mo-old mice. To determine the mechanism responsible for the age related change in B-1a cell IgM, we established a mixed chimera system in which mice were reconstituted with allotype-marked mature peritoneal B-1a cells and adult bone marrow cells. We demonstrated even in the presence of mature peritoneal B-1a cells, adult bone marrow contributed to the mature B-1a cell pool. More importantly, using this system we found over a 10-mo-period peritoneal B-1a cell IgM changed, showing the number of cells lacking N-region additions at both junctions fell from 49 to 29% of sequences. These results strongly suggest selection-induced skewing alters B-1a cell-derived natural Ab, which may in turn be responsible for the loss of natural IgM-mediated protection against pneumococcal infection. PMID:27183643

  4. Comparative Accuracy of the InBios Scrub Typhus Detect IgM Rapid Test for the Detection of IgM Antibodies by Using Conventional Serology

    PubMed Central

    Kingston, Hugh W. F.; Blacksell, Stuart D.; Tanganuchitcharnchai, Ampai; Laongnualpanich, Achara; Basnyat, Buddha; Day, Nicholas P. J.

    2015-01-01

    This study investigated the comparative accuracy of a recombinant 56-kDa type-specific antigen-based rapid diagnostic test (RDT) for scrub typhus for the detection of IgM antibodies by using conventional serology in well-characterized serum samples from undifferentiated febrile illness patients. The RDT showed high specificity and promising comparative accuracy, with 82% sensitivity and 98% specificity for samples defined positive at an IgM indirect immunofluorescence assay positivity cutoff titer of ≥1:1,600 versus 92% and 95% at ≥1:6,400, respectively. PMID:26291089

  5. Comparative Accuracy of the InBios Scrub Typhus Detect IgM Rapid Test for the Detection of IgM Antibodies by Using Conventional Serology.

    PubMed

    Kingston, Hugh W F; Blacksell, Stuart D; Tanganuchitcharnchai, Ampai; Laongnualpanich, Achara; Basnyat, Buddha; Day, Nicholas P J; Paris, Daniel H

    2015-10-01

    This study investigated the comparative accuracy of a recombinant 56-kDa type-specific antigen-based rapid diagnostic test (RDT) for scrub typhus for the detection of IgM antibodies by using conventional serology in well-characterized serum samples from undifferentiated febrile illness patients. The RDT showed high specificity and promising comparative accuracy, with 82% sensitivity and 98% specificity for samples defined positive at an IgM indirect immunofluorescence assay positivity cutoff titer of ≥1:1,600 versus 92% and 95% at ≥1:6,400, respectively. PMID:26291089

  6. Vesiculovirus Neutralization by Natural IgM and Complement

    PubMed Central

    Tesfay, Mulu Z.; Ammayappan, Arun; Federspiel, Mark J.; Barber, Glen N.; Stojdl, David; Peng, Kah-Whye

    2014-01-01

    ABSTRACT Because of its very low human seroprevalence, vesicular stomatitis virus (VSV) has promise as a systemic oncolytic agent for human cancer therapy. However, as demonstrated in this report, the VSV infectious titer drops by 4 log units during the first hour of exposure to nonimmune human serum. This neutralization occurs relatively slowly and is mediated by the concerted actions of natural IgM and complement. Maraba virus, whose G protein is about 80% homologous to that of VSV, is relatively resistant to the neutralizing activity of nonimmune human serum. We therefore constructed and rescued a recombinant VSV whose G gene was replaced by the corresponding gene from Maraba virus. Comparison of the parental VSV and VSV with Maraba G substituted revealed nearly identical host range properties and replication kinetics on a panel of tumor cell lines. Moreover, in contrast to the parental VSV, the VSV with Maraba G substituted was resistant to nonimmune human serum. Overall, our data suggest that VSV with Maraba G substituted should be further investigated as a candidate for human systemic oncolytic virotherapy applications. IMPORTANCE Oncolytic virotherapy is a promising approach for the treatment of disseminated cancers, but antibody neutralization of circulating oncolytic virus particles remains a formidable barrier. In this work, we developed a pseudotyped vesicular stomatitis virus (VSV) with a glycoprotein of Maraba virus, a closely related but serologically distinct member of the family Rhabdoviridae, which demonstrated greatly diminished susceptibility to both nonimmune and VSV-immune serum neutralization. VSV with Maraba G substituted or lentiviral vectors should therefore be further investigated as candidates for human systemic oncolytic virotherapy and gene therapy applications. PMID:24648451

  7. COMPARISON OF IMMUNOASSAY AND GAS CHROMATOGRAPHY/MASS SPECTROMETRY METHODS FOR MEASURING 3,5,6-TRICHLORO-2PYRIDINOL IN MULTIPLE SAMPLE MEDIA

    EPA Science Inventory

    Two enzyme-linked immunosorbent assay (ELISA) methods were evaluated for the determination of 3,5,6-trichloro-2-pyridinol (3,5,6-TCP) in multiple sample media (dust, soil, food, and urine). The dust and soil samples were analyzed by a commercial RaPID immunoassay testing kit. ...

  8. Depletion of IgM xenoreactive natural antibodies by injection of anti-mu monoclonal antibodies.

    PubMed

    Latinne, D; Soares, M; Havaux, X; Cormont, F; Lesnikoski, B; Bach, F H; Bazin, H

    1994-10-01

    It is believed that IgM xenoreactive natural antibodies (XNA) and activation of complement are the two main effectors involved in the hyperacute rejection (HAR) of discordant xenografts, such as pig-to-primate kidney, liver or heart transplants. We have hypothesized that long-term depletion of circulating IgM XNA might be able to overcome HAR and induce the "accommodation" of pig-to-primate vascular discordant xenografts. Several techniques have been described to eliminate circulating XNA in primates but, up to now, none has been able to totally deplete these antibodies for a sufficiently long period of time in order to test the hypothesis of discordant xenograft "accommodation". Previous reports from our laboratory have shown that, in rodents, B-cell immunosuppression could be achieved by neonatal administration of anti-mu antibodies. Recently we have shown that administration of an anti-mu mAb, in adult rats, was able to totally deplete circulating IgM and IgM XNA, without immune complex disease. Furthermore, we have used different methods such as splenectomy, plasma exchange and an anti-B cell immunosuppressive agent mycophenylate mophetil (RS61443, Syntex, Palo Alto, USA) to pre-deplete circulating IgM before administration of anti-mu mAb (MARM-7) and showed that the effectiveness of anti-mu mAb to deplete circulating IgM was increased by 100-fold. Depletion of circulating IgM in adult rats by anti-mu mAb (MARM-7) was used as an experimental model to study the role of IgM XNA in the pathogenesis of HAR in guinea pig-to-rat cardiac xenografts. Our data show that IgM XNA play a major role in HAR, even if in this discordant combination direct activation of complement, probably through the alternative pathway, seems to be the main effector involved in HAR. We have analyzed the mechanisms of anti-mu depletion of circulating IgM in adult animals and shown that, besides anti-mu/IgM immune complex formation, depletion of circulating IgM results from the very significant inhibition of B-cell differentiation and secretion of IgM following in vivo crosslinking and internalization of surface IgM on B cells. As well, we provide evidence demonstrating that anti-mu mAb blocks B cells at an early stage of maturation, probably in the bone marrow. Furthermore, we have developed several rat anti-human and anti-baboon IgM mAb and tested their ability to deplete circulating IgM and IgM XNA in baboons, after splenectomy or splenectomy and plasma exchange.(ABSTRACT TRUNCATED AT 400 WORDS) PMID:7868159

  9. Circulating IgM Requires Plasma Membrane Disruption to Bind Apoptotic and Non-Apoptotic Nucleated Cells and Erythrocytes

    PubMed Central

    Hesketh, Emily E.; Dransfield, Ian; Kluth, David C.; Hughes, Jeremy

    2015-01-01

    Autoimmunity is associated with defective phagocytic clearance of apoptotic cells. IgM deficient mice exhibit an autoimmune phenotype consistent with a role for circulating IgM antibodies in apoptotic cell clearance. We have extensively characterised IgM binding to non-apoptotic and apoptotic mouse thymocytes and human Jurkat cells using flow cytometry, confocal imaging and electron microscopy. We demonstrate strong specific IgM binding to a subset of Annexin-V (AnnV)+PI (Propidium Iodide)+ apoptotic cells with disrupted cell membranes. Electron microscopy studies indicated that IgM+AnnV+PI+ apoptotic cells exhibited morphologically advanced apoptosis with marked plasma membrane disruption compared to IgM-AnnV+PI+ apoptotic cells, suggesting that access to intracellular epitopes is required for IgM to bind. Strong and comparable binding of IgM to permeabilised non-apoptotic and apoptotic cells suggests that IgM bound epitopes are 'apoptosis independent' such that IgM may bind any cell with profound disruption of cell plasma membrane integrity. In addition, permeabilised erythrocytes exhibited significant IgM binding thus supporting the importance of cell membrane epitopes. These data suggest that IgM may recognize and tag damaged nucleated cells or erythrocytes that exhibit significant cell membrane disruption. The role of IgM in vivo in conditions characterized by severe cell damage such as ischemic injury, sepsis and thrombotic microangiopathies merits further exploration. PMID:26121639

  10. Solid-phase competitive and sandwich-type erythro-immunoassays for human chorionic gonadotropin.

    PubMed

    Gupta, S K; Guesdon, J L; Avrameas, S; Talwar, G P

    1985-06-25

    A simple '1-step' competitive erythro-immunoassay for human chorionic gonadotropin (hCG) employing V-shaped well microtitration plates coated with monoclonal anti-beta-hCG antibody has been described. hCG of the test sample competes with the antigen-coupled sheep erythrocytes for binding to the antibody on the solid surface. The assay is able to detect up to 31.25 ng hCG/ml. A higher sensitivity enabling detection up to 0.25 ng hCG/ml is attained by the sandwich erythro-immunoassay using a chimera antibody prepared by coupling monoclonal anti-alpha-hCG antibody to an affinity-purified polyclonal antibody specific for sheep erythrocytes. This assay is amenable to the qualitative as well as quantitative use as described. The urinary components do not interfere in the assay. Results obtained by this assay on 47 human urine samples correlated well with the values obtained by '2-step' sandwich enzyme immunoassay and radioimmunoassay. PMID:2409175

  11. Comparison of nine commercial immunoassays for the detection of rotavirus in fecal specimens.

    PubMed Central

    Dennehy, P H; Gauntlett, D R; Tente, W E

    1988-01-01

    One hundred fecal specimens obtained from patients with acute gastroenteritis were tested for rotavirus with nine commercial immunoassays to evaluate the sensitivity, specificity, predictive value, and diagnostic accuracy of these assays. Kits evaluated included two monoclonal antibody-based enzyme immunoassays (EIAs) (Rotaclone and Pathfinder Rotavirus), three polyclonal antibody-based EIAs (Rotavirus Immunoassay, Rotazyme II, and Wellcozyme Rotavirus), and four latex agglutination assays (Rotastat, Virogen Rotatest, Meritec-Rotavirus, and The Wellcome Latex Test). Thirty-eight of the 100 specimens were found to contain rotavirus by a reference microplate EIA. The accuracy of the reference assay was determined by RNA electrophoresis and a blocking assay on discordant specimens. The two monoclonal antibody EIAs had superior sensitivities (100%) and identified two positive specimens which were negative by the reference method but positive by the blocking assay. Among the polyclonal EIAs, all had sensitivities of greater than 90%, but specificities were variable; Rotazyme II, with a specificity of 50%, showed considerable discrepancy from other polyclonal EIAs. The latex tests had sensitivities ranging from 70 to 90% and specificities of 80 to 100%. Latex agglutination tests were more rapid than EIAs and did not require expensive equipment. The final choice of assay system will depend on the cost, speed, and accuracy requirements of the clinical laboratory. PMID:2846645

  12. Physiological IgM Class Catalytic Antibodies Selective for Transthyretin Amyloid*

    PubMed Central

    Planque, Stephanie A.; Nishiyama, Yasuhiro; Hara, Mariko; Sonoda, Sari; Murphy, Sarah K.; Watanabe, Kenji; Mitsuda, Yukie; Brown, Eric L.; Massey, Richard J.; Primmer, Stanley R.; O'Nuallain, Brian; Paul, Sudhir

    2014-01-01

    Peptide bond-hydrolyzing catalytic antibodies (catabodies) could degrade toxic proteins, but acquired immunity principles have not provided evidence for beneficial catabodies. Transthyretin (TTR) forms misfolded β-sheet aggregates responsible for age-associated amyloidosis. We describe nucleophilic catabodies from healthy humans without amyloidosis that degraded misfolded TTR (misTTR) without reactivity to the physiological tetrameric TTR (phyTTR). IgM class B cell receptors specifically recognized the electrophilic analog of misTTR but not phyTTR. IgM but not IgG class antibodies hydrolyzed the particulate and soluble misTTR species. No misTTR-IgM binding was detected. The IgMs accounted for essentially all of the misTTR hydrolytic activity of unfractionated human serum. The IgMs did not degrade non-amyloidogenic, non-superantigenic proteins. Individual monoclonal IgMs (mIgMs) expressed variable misTTR hydrolytic rates and differing oligoreactivity directed to amyloid β peptide and microbial superantigen proteins. A subset of the mIgMs was monoreactive for misTTR. Excess misTTR was dissolved by a hydrolytic mIgM. The studies reveal a novel antibody property, the innate ability of IgMs to selectively degrade and dissolve toxic misTTR species as a first line immune function. PMID:24648510

  13. Emerging Functions of Natural IgM and Its Fc Receptor FCMR in Immune Homeostasis.

    PubMed

    Wang, Hongsheng; Coligan, John E; Morse, Herbert C

    2016-01-01

    Most natural IgM antibodies are encoded by germline Ig sequences and are produced in large quantities by both mice and humans in the absence of intentional immunization. Natural IgM are reactive with many conserved epitopes, including those shared by microorganisms and autoantigens. As a result, these antibodies play important roles in clearing intruding pathogens, as well as apoptotic/necrotic cells and otherwise damaged tissues. While natural IgM binds to target structures with low affinity due to a lack of significant selection by somatic hypermutation, its pentameric structure with 10 antigen-binding sites enables these antibodies to bind multivalent target antigens with high avidity. Opsonization of antigen complexed with IgM is mediated by cell surface Fc receptors. While the existence of Fc alpha/mu receptor has been known for some time, only recently has the Fc receptor specific for IgM (FCMR) been identified. In this review, we focus on our current understandings of how natural IgM and FCMR regulate the immune system and maintain homeostasis under physiological and pathological conditions. PMID:27014278

  14. Emerging Functions of Natural IgM and Its Fc Receptor FCMR in Immune Homeostasis

    PubMed Central

    Wang, Hongsheng; Coligan, John E.; Morse, Herbert C.

    2016-01-01

    Most natural IgM antibodies are encoded by germline Ig sequences and are produced in large quantities by both mice and humans in the absence of intentional immunization. Natural IgM are reactive with many conserved epitopes, including those shared by microorganisms and autoantigens. As a result, these antibodies play important roles in clearing intruding pathogens, as well as apoptotic/necrotic cells and otherwise damaged tissues. While natural IgM binds to target structures with low affinity due to a lack of significant selection by somatic hypermutation, its pentameric structure with 10 antigen-binding sites enables these antibodies to bind multivalent target antigens with high avidity. Opsonization of antigen complexed with IgM is mediated by cell surface Fc receptors. While the existence of Fc alpha/mu receptor has been known for some time, only recently has the Fc receptor specific for IgM (FCMR) been identified. In this review, we focus on our current understandings of how natural IgM and FCMR regulate the immune system and maintain homeostasis under physiological and pathological conditions. PMID:27014278

  15. Platelet antibodies of the IgM class in immune thrombocytopenic purpura

    SciTech Connect

    Cines, D.B.; Wilson, S.B.; Tomaski, A.; Schreiber, A.D.

    1985-04-01

    The clinical course and response to therapy of patients with immune thrombocytopenic purpura (ITP) are not completely determined by the level of IgG present on the platelet surface. It is possible that antibodies of other immunoglobulin classes also play a role in platelet destruction in some of these patients. Therefore, the authors studied 175 patients with ITP for the presence of IgM anti-platelet antibodies using radiolabeled polyclonal or monoclonal anti-IgM. They observed that 57% of patients with clinical ITP had increased levels of IgM on their platelets, compared with normal controls and patients with thrombocytopenia who did not have ITP. They obtained similar results using either radiolabeled polyclonal or monoclonal anti-IgM, reagents whose integrity was first characterized using erythrocytes coated with defined amounts of IgM antibody. Among patients with increased platelet-IgM there was a significant correlation both with the presence of increased platelet-C3 as well as the amount of platelet-C3. The authors demonstrated the presence of warm-reacting IgM anti-platelet antibodies in the plasma of two of these patients who were further studied. These studies demonstrate the presence of warm-reacting IgM anti-platelet antibodies in some patients with ITP. They suggest that the binding of complement to platelets by IgM antibodies may initiate platelet clearance as well as enhance the effect of IgG antibodies in ITP.

  16. Enzyme assays.

    PubMed

    Reymond, Jean-Louis; Fluxà, Viviana S; Maillard, Noélie

    2009-01-01

    Enzyme assays are analytical tools to visualize enzyme activities. In recent years a large variety of enzyme assays have been developed to assist the discovery and optimization of industrial enzymes, in particular for "white biotechnology" where selective enzymes are used with great success for economically viable, mild and environmentally benign production processes. The present article highlights the aspects of fluorogenic and chromogenic substrates, sensors, and enzyme fingerprinting, which are our particular areas of interest. PMID:19081993

  17. Multiplexed In-cell Immunoassay for Same-sample Protein Expression Profiling

    PubMed Central

    Shang, Jing; Zrazhevskiy, Pavel; Postupna, Nadia; Keene, C. Dirk; Montine, Thomas J.; Gao, Xiaohu

    2015-01-01

    In-cell immunoassays have become a valuable tool for protein expression analysis complementary to established assay formats. However, comprehensive molecular characterization of individual specimens has proven challenging and impractical due to, in part, a singleplex nature of reporter enzymes and technical complexity of alternative assay formats. Herein, we describe a simple and robust methodology for multiplexed protein expression profiling on the same intact specimen, employing a well-characterized enzyme alkaline phosphatase for accurate quantification of all targets of interest, while overcoming fundamental limitations of enzyme-based techniques by implementing the DNA-programmed release mechanism for segregation of sub-sets of target-bound reporters. In essence, this methodology converts same-sample multi-target labeling into a set of isolated singleplex measurements performed in a parallel self-consistent fashion. For a proof-of-principle, multiplexed detection of three model proteins was demonstrated on cultured HeLa cells, and two clinically-relevant markers of dementia, β-amyloid and PHF-tau, were profiled in formalin-fixed paraffin embedded brain tissue sections, uncovering correlated increase in abundance of both markers in the “Alzheimer’s disease” cohort. Featuring an analytically powerful yet technically simple and robust methodology, multiplexed in-cell immunoassay is expected to enable insightful same-sample protein profiling studies and become broadly adopted in biomedical research and clinical diagnostics. PMID:26328896

  18. Magnetic Electrochemical Immunoassays with Quantum Dot Labels for Detection of Phosphorylated Acetylcholinesterase in Plasma

    SciTech Connect

    Wang, Hua; Wang, Jun; Timchalk, Charles; Lin, Yuehe

    2008-11-01

    A new magnetic electrochemical immunoassay has been developed as a tool for biomonitoring exposures to organophosphate (OP) compounds, e.g., insecticides and chemical nerve agents, by directly detecting organophosphorylated acetylcholinesterase (OP-AChE). This immunoassay uniquely incorporates highly efficient magnetic separation with ultrasensitive square wave voltammetry (SWV) analysis with quantum dots (QDs) as labels. A pair of antibodies was used to achieve the specific recognition of OP-AChE that was prepared with paraoxon as an OP model agent. Antiphosphoserine polyclonal antibodies were anchored on amorphous magnetic particles preferably chosen to capture OP-AChE from the sample matrixes by binding their phosphoserine moieties that were exposed through unfolding the protein adducts. This was validated by electrochemical examinations and enzyme-linked immunosorbent assays. Furthermore, antihuman AChE monoclonal antibodies were labeled with cadmium-source QDs to selectively recognize the captured OP-AChE, as characterized by transmission electron microscopy. The subsequent electrochemical SWV analysis of the cadmium component released by acid from the coupled QDs was conducted on disposable screen-printed electrodes. Experimental results indicated that the SWV-based immunoassays could yield a linear response over a broad concentration range of 0.3-300 ng/mL OP-AChE in human plasma with a detection limit of 0.15 ng/mL. Such a novel electrochemical immunoassay holds great promise as a simple, selective, sensitive, and field-deployable tool for the effective biomonitoring and diagnosis of potential exposures to nerve agents and pesticides.

  19. Multiplexed electrochemical immunoassay of biomarkers using metal sulfide quantum dot nanolabels and trifunctionalized magnetic beads.

    PubMed

    Tang, Dianping; Hou, Li; Niessner, Reinhard; Xu, Mingdi; Gao, Zhuangqiang; Knopp, Dietmar

    2013-08-15

    A novel multiplexed stripping voltammetric immunoassay protocol was designed for the simultaneous detection of multiple biomarkers (CA 125, CA 15-3, and CA 19-9 used as models) using PAMAM dendrimer-metal sulfide quantum dot (QD) nanolabels as distinguishable signal tags and trifunctionalized magnetic beads as an immunosensing probe. The probe was prepared by means of co-immobilization of primary monoclonal anti-CA 125, anti-CA 15-3 and anti-CA 19-9 antibodies on a single magnetic bead. The PAMAM dendrimer-metal sulfide QD nanolabels containing CdS, ZnS and PbS were synthesized by using in situ synthesis method, which were utilized for the labeling of polyclonal rabbit anti-CA 125, anti-CA 15-3 and anti-CA 19-9 detection antibodies, respectively. A sandwich-type immunoassay format was adopted for the simultaneous determination of target biomarkers in a low-binding microtiter plate. The subsequent anodic stripping voltammetric analysis of cadmium, zinc, and lead components released by acid from the corresponding QD nanolabels was conducted at an in situ prepared mercury film electrode based on the difference of peak potentials. Experimental results indicated that the multiplexed immunoassay enabled the simultaneous detection of three cancer biomarkers in a single run with wide dynamic ranges of 0.01-50 U mL(-1) and detection limits (LODs) of 0.005 U mL(-1). Intra-assay and inter-assay coefficients of variation (CVs) were less than 7.2% and 10.4%, respectively. No significant differences at the 0.05 significance level were encountered in the analysis of 10 clinical serum specimens between the multiplexed immunoassay and a commercially available enzyme-linked immunosorbent assay (ELISA). PMID:23500474

  20. A nonenzymatic optical immunoassay strategy for detection of Salmonella infection based on blue silica nanoparticles.

    PubMed

    Sun, Qian; Zhao, Guangying; Dou, Wenchao

    2015-10-22

    A novel nonenzymatic optical immunoassay strategy was for the first time designed and utilized for sensitive detection of antibody to Salmonella pullorum and Salmonella gallinarum (S. pullorum and S. gallinarum) in serum. The optical immunoassay strategy was based on blue silica nanoparticles (Blue-SiNps) and magnetic beads (MB). To construct such an optical immunoassay system, the Blue-SiNPs were first synthesized by inverse microemulsion method, characterized by SEM, Zeta potential and FTIR. Two nanostructures including Blue-SiNPs and MB were both functionalized with antibody against S. pullorum and S. gallinarum (anti-PG) without using enzyme labeled antibody. Anti-PG functionalized blue silica nanoparticles (IgG-Blue-SiNps) were used as signal transduction labels, while anti-PG functionalized magnetic beads (IgG-MB) were selected to separate and enrich the final sandwich immune complexes. In the process of detecting negative serum, a sandwich immunocomplex is formed between the IgG-MB and IgG-Blue-SiNPs. With the separation of the immunocomplex using an external magnetic field, the final plaque displayed bright blue color. While in the detection of infected serum, IgG-MB and anti-PG formed sandwich immunocomplexes, IgG-Blue-SiNPs were unable to bind to the limited sites of the antigen, and a light brown plaque was displayed in the bottom of microplate well. Stable results were obtained with an incubation time of 60 min at room temperature, and different colors corresponding to different results can be directly detected with naked eye. The reaction of IgG-Blue-SiNPs with S. pullorum was inhibited by 1:100 dilution of positive chicken serum. Such a simple immunoassay holds great potential as sensitive, selective and point-of-care (POC) tool for diagnosis of other biological molecules. PMID:26526916

  1. Statistical approaches to developing a multiplex immunoassay for determining human exposure to environmental pathogens.

    PubMed

    Augustine, Swinburne A J; Simmons, Kaneatra J; Eason, Tarsha N; Griffin, Shannon M; Curioso, Clarissa L; Wymer, Larry J; Fout, G Shay; Grimm, Ann C; Oshima, Kevin H; Dufour, Al

    2015-10-01

    There are numerous pathogens that can be transmitted through water. Identifying and understanding the routes and magnitude of exposure or infection to these microbial contaminants are critical to assessing and mitigating risk. Conventional approaches of studying immunological responses to exposure or infection such as Enzyme-Linked Immunosorbent Assays (ELISAs) and other monoplex antibody-based immunoassays can be very costly, laborious, and consume large quantities of patient sample. A major limitation of these approaches is that they can only be used to measure one analyte at a time. Multiplex immunoassays provide the ability to study multiple pathogens simultaneously in microliter volumes of samples. However, there are several challenges that must be addressed when developing these multiplex immunoassays such as selection of specific antigens and antibodies, cross-reactivity, calibration, protein-reagent interferences, and the need for rigorous optimization of protein concentrations. In this study, a Design of Experiments (DOE) approach was used to optimize reagent concentrations for coupling selected antigens to Luminex™ xMAP microspheres for use in an indirect capture, multiplex immunoassay to detect human exposure or infection from pathogens that are potentially transmitted through water. Results from Helicobacter pylori, Campylobacter jejuni, Escherichia coli O157:H7, and Salmonella typhimurium singleplexes were used to determine the mean concentrations that would be applied to the multiplex assay. Cut-offs to differentiate between exposed and non-exposed individuals were determined using finite mixed modeling (FMM). The statistical approaches developed facilitated the detection of Immunoglobulin G (IgG) antibodies to H. pylori, C. jejuni, Toxoplasma gondii, hepatitis A virus, rotavirus and noroviruses (VA387 and Norwalk strains) in fifty-four diagnostically characterized plasma samples. Of the characterized samples, the detection rate was 87.5% for H. pylori, and 100% for T. gondii assays and 89% for HAV. Further, the optimized multiplex assay revealed exposure/infection to several other environmental pathogens previously uncharacterized in the samples. PMID:26070441

  2. Urchin-like (gold core)@(platinum shell) nanohybrids: A highly efficient peroxidase-mimetic system for in situ amplified colorimetric immunoassay.

    PubMed

    Gao, Zhuangqiang; Xu, Mingdi; Lu, Minghua; Chen, Guonan; Tang, Dianping

    2015-08-15

    The development of signal-amplified colorimetric immunoassay relies on the design of highly efficient signal-transduction tags. One promising route is to exploit a novel enzyme mimetic system as the signal label. Herein, we report that urchin-like (gold core)@(platinum shell) nanohybrids (Au@PtNHs) can be utilized as a highly efficient peroxidase mimetic system for in situ amplified colorimetric immunoassay of prostate-specific antigen (PSA, one kind of tumor marker). Initially, urchin-like Au@PtNHs were discovered to outperform horseradish peroxidase (HRP) by a vast margin in terms of the turnover number toward hydrogen peroxide (H2O2)-3,3',5,5'-tetramethylbenzidine (TMB) system and the stability against high temperatures and HRP inhibitors. Based on this discovery, the assay was simply carried out on a capture antibody-immobilized microplate by using the Au@PtNH-labeled detection antibody as a signal-transduction tag with a sandwich-type assay mode. The colorimetric signal stemmed from the labeled Au@PtNHs toward catalytic oxidation of TMB-H2O2 system. Experimental results indicated that the Au@PtNH-based colorimetric immunoassay could display a good colorimetric response toward PSA in the dynamic working range of 5-500 pg mL(-1) with a low detection limit of 2.9 pg mL(-1). Meanwhile, the developed immunoassay exhibited good precision and reproducibility, high specificity and acceptable accuracy for the detection of clinical serum samples. These results open up a new horizon for the development of highly sensitive, highly stable and inexpensive non-enzyme immunoassay platforms as an alternative to conventional enzyme-based immunoassay platforms. PMID:25814409

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

    PubMed Central

    Lindenschmidt, E G

    1986-01-01

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

  4. J chain synthesis and secretion of hexameric IgM is differentially regulated by lipopolysaccharide and interleukin 5.

    PubMed Central

    Randall, T D; Parkhouse, R M; Corley, R B

    1992-01-01

    Two functional polymeric forms of IgM can be produced by antibody-secreting B cells. Hexameric IgM lacks detectable J (joining) chain and activates complement 17-fold better than pentameric IgM, which usually contains one J chain per pentamer. Using the inducible B-cell lymphoma CH12, we determined if the synthesis of a particular polymeric form of IgM is a fixed property of B cells or can be altered. Lipopolysaccharide (LPS)-stimulated CH12 cells produced mixtures of IgM hexamers and pentamers, resulting in antibody with high complement-fixing activity. In contrast, interleukin-5-stimulated CH12 cells secreted predominantly pentameric IgM, with a correspondingly lower lytic activity. Differences in lytic activity were due only to the amount of hexameric IgM in the secreted antibody. Interleukin 5 stimulated higher production of J chain RNA and protein than LPS, while LPS induced the highest levels of the secretory form of mu protein. The amount of hexameric IgM secreted was therefore inversely proportional to the level of intracellular J chain protein in the responding B cells. We conclude that the biologic function of IgM produced by B cells differs depending on how they are stimulated and that this difference may be regulated by the relative availabilities of J chain and secretory mu proteins during IgM polymerization. Images PMID:1736312

  5. Nanomaterial Labels in Electrochemical Immunosensors and Immunoassays

    SciTech Connect

    Liu, Guodong; Lin, Yuehe

    2007-12-15

    This article reviews recent advances in nanomaterial labels in electrochemical immunosensors and immunoassays. Various nanomaterial labels are discussed, including colloidal gold/silver, semiconductor nanoparticles, and markers loaded nanocarriers (carbon nanotubes, apoferritin, silica nanoparticles, and liposome beads). The enormous signal enhancement associated with the use of nanomaterial labels and with the formation of nanomaterial–antibody-antigen assemblies provides the basis for ultrasensitive electrochemical detection of disease-related protein biomarkers, biothreat agents, or infectious agents. In general, all endeavors cited here are geared to achieve one or more of the following goals: signal amplification by several orders of magnitude, lower detection limits, and detecting multiple targets.

  6. Fluorescent immunoassay visualization of sorbed pollutants

    SciTech Connect

    Moore, W.K.; Mossman, D.J.; Schwab, A.P.; Feldbush, T.L.

    1994-12-31

    Current methods of detecting sorbed soil pollutants require that the contaminant be extracted from the soil. In an effort to make detection simpler and safer, standard fluorescent immunoassay techniques are being modified to allow fluorescent tags on the pollutant to be viewed and photographed with epifluorescent microscopy. Initial research focuses on detecting chlorinated benzenes on various soil types and developing a technique for tagging these pollutants with appropriate antibodies. This should lead to detection in actual soil cores and a better understanding of how contaminants progress through different soils.

  7. Gliadin Detection in Food by Immunoassay

    NASA Astrophysics Data System (ADS)

    Grant, Gordon; Sporns, Peter; Hsieh, Y.-H. Peggy

    Immunoassays are very sensitive and efficient tests that are commonly used to identify a specific protein. Examples of applications in the food industry include identification of proteins expressed in genetically modified foods, allergens, or proteins associated with a disease, including celiac disease. This genetic disease is associated with Europeans and affects about one in every 200 people in North America. These individuals react immunologically to wheat proteins, and consequently their own immune systems attack and damage their intestines. This disease can be managed if wheat proteins, specifically "gliadins," are avoided in foods.

  8. Nanomaterial Labels in Electrochemical Immunosensors and Immunoassays

    PubMed Central

    Liu, Guodong; Lin, Yuehe

    2009-01-01

    This article reviews recent advances in nanomaterial labels in electrochemical immunosensors and immunoassays. Various nanomaterial labels are discussed, including colloidal gold/silver, semiconductor nanoparticles, and markers loaded nanocarriers (carbon nanotubes, apoferritin, silica nanoparticles, and liposome beads). The enormous signal enhancement associated with the use of nanomaterial labels and with the formation of nanomaterialantibody-antigen assemblies provides the basis for ultrasensitive electrochemical detection of disease-related protein biomarkers, biothreat agents, or infectious agents. In general, all endeavors cited here are geared to achieve one or more of the following goals: signal amplification by several orders of magnitude, lower detection limits, and detecting multiple targets. PMID:18371644

  9. Sensitive, Fast, and Specific Immunoassays for Methyltestosterone Detection

    PubMed Central

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

    2015-01-01

    An indirect competitive enzyme-linked immunosorbent assay (icELISA) and an immunochromatographic strip assay using a highly specific monoclonal antibody, were developed to detect methyltestosterone (MT) residues in animal feed. The optimized icELISA had a half-inhibition concentration value of 0.26 ng/mL and a limit of detection value of 0.045 ng/mL. There was no cross-reactivity with eight analogues, revealing high specificity for MT. Based on icELISA results, the recovery rate of MT in animal feed was 82.4%–100.6%. The results were in accordance with those obtained by gas chromatography-mass spectrometry. The developed immunochromatographic strip assay, as the first report for MT detection, had a visual cut-off value of 1 ng/mL in PBS, 2.5 ng/g in fish feed, and 2.5 ng/g in pig feed. Therefore, these immunoassays are useful and fast tools for MT residue detection in animal feed. PMID:25938198

  10. Microfluidic immunoassays as rapid saliva-based clinical diagnostics

    PubMed Central

    Herr, Amy E.; Hatch, Anson V.; Throckmorton, Daniel J.; Tran, Huu M.; Brennan, James S.; Giannobile, William V.; Singh, Anup K.

    2007-01-01

    At present, point-of-care (POC) diagnostics typically provide a binary indication of health status (e.g., home pregnancy test strip). Before anticipatory use of diagnostics for assessment of complex diseases becomes widespread, development of sophisticated bioassays capable of quantitatively measuring disease biomarkers is necessary. Successful translation of new bioassays into clinical settings demands the ability to monitor both the onset and progression of disease. Here we report on a clinical POC diagnostic that enables rapid quantitation of an oral disease biomarker in human saliva by using a monolithic disposable cartridge designed to operate in a compact analytical instrument. Our microfluidic method facilitates hands-free saliva analysis by integrating sample pretreatment (filtering, enrichment, mixing) with electrophoretic immunoassays to quickly measure analyte concentrations in minimally pretreated saliva samples. Using 20 μl of saliva, we demonstrate rapid (<10 min) measurement of the collagen-cleaving enzyme matrix metalloproteinase-8 (MMP-8) in saliva from healthy and periodontally diseased subjects. In addition to physiologically measurable indicators of periodontal disease, conventional measurements of salivary MMP-8 were used to validate the microfluidic assays described in this proof-of-principle study. The microchip-based POC diagnostic demonstrated is applicable to rapid, reliable measurement of proteinaceous disease biomarkers in biological fluids. PMID:17374724

  11. Quantitative analysis of plasma interleiukin-6 by immunoassay on microchip

    NASA Astrophysics Data System (ADS)

    Abe, K.; Hashimoto, Y.; Yatsushiro, S.; Yamamura, S.; Tanaka, M.; Ooie, T.; Baba, Y.; Kataoka, M.

    2012-03-01

    Sandwich enzyme-linked immunoassay (ELISA) is one of the most frequently employed assays for clinical diagnosis, since this enables the investigator to identify specific protein biomarkers. However, the conventional assay using a 96-well microtitration plate is time- and sample-consuming, and therefore is not suitable for rapid diagnosis. To overcome these drawbacks, we performed a sandwich ELISA on a microchip. We employed the piezoelectric inkjet printing for deposition and fixation of 1st antibody on the microchannnel surface (300 μm width and 100 μm depth). Model analyte was interleukin-6 (IL-6) which was one of the inflammatory cytokine. After blocking the microchannel, antigen, biotin-labeled 2nd antibody, and avidin-labeled peroxidase were infused into the microchannel and incubated for 20 min, 10 min, and 5 min, respectively. This assay could detect 2 pg/ml and quantitatively measure the range of 0-32 pg/ml. Liner regression analysis of plasma IL-6 concentration obtained by microchip and conventional methods exhibited a significant relationship (R2 = 0.9964). This assay reduced the time for the antigen-antibody reaction to 1/6, and the consumption of samples and reagents to 1/50 compared with the conventional method. This assay enables us to determine plasma IL-6 with accuracy, high sensitivity, time saving ability, and low consumption of sample and reagents, and thus will be applicable to clinic diagnosis.

  12. Haptens and monoclonal antibodies for immunoassay of imidazolinone herbicides.

    PubMed

    Chin, Tina E; Wong, Rosie B; Pont, Joseph L; Karu, Alexander E

    2002-06-01

    A set of haptens structurally resembling the herbicide imazethapyr (PURSUIT) was synthesized and used to derive monoclonal antibodies (MAbs) and direct and indirect competition enzyme immunoassays (EIAs) which could detect imazethapyr, imazaquin (SCEPTER), imazapic (CADRE), and imazamox (RAPTOR) in the 3-30 ng/mL (parts per billion) range, and imazapyr (ARSENAL) and imazamethabenz-methyl (ASSERT) in the 300-500 ppb range. Two MAbs, 3A2 and 3A5, had affinities of 10-75 nM for imazethapyr. MAbs 1A5, 1D2, and 3A5 were specific for the S isomers of the herbicides. Some MAbs were stable in solutions containing up to 15% methanol and 5% acetonitrile in indirect EIAs. Plates coated with hapten conjugates for indirect EIA could be stored frozen. Selectivity for the imidazolinones by some MAbs varied with different coating conjugates. These MAbs and haptens should prove useful in immunochemical analysis and residue recovery methods for imazethapyr and other imidazolinone herbicides. PMID:12033799

  13. Serological diagnosis of Leptospirosis in bovine serum samples using a microsphere immunoassay.

    PubMed

    Wynwood, S J; Burns, M A; Graham, G C; Weier, S L; McKay, D B; Craig, S B

    2016-01-01

    Leptospirosis causes significant economic loss within the cattle industry worldwide. Current diagnostic methods are generally inadequate for dealing with large numbers of samples, are outdated, and provide little useful diagnostic and epidemiological information. This aim of this study was to apply a microsphere immunoassay (MIA), utilising Luminex xMap technology, to 200 bovine serum samples to determine this method's usefulness in leptospirosis diagnosis in comparison with the current gold standard, the microscopic agglutination test (MAT). Although MAT is the most widely used laboratory test for the diagnosis of leptospirosis, its reliance on live cultures, subjective interpretation of results and an inability to differentiate between antibody classes, suggest MAT is no longer the best method for the diagnosis of leptospirosis. The results presented in this paper show that MIA was able to determine reactive from non-reactive samples when compared with MAT, and was able to differentiate IgG and IgM classes of antibody. The results suggest increased sensitivity in MIA and the ability to multiplex up to 500 antigens at one time allows for significant improvements in cost-effectiveness as well as a reduced dependency on live cultures. The relatively low cost, high throughput platform and differentiation of antibody class, as shown in previous research, make this assay worthy of consideration for the diagnosis of leptospirosis in small-scale or large-scale bovine populations. PMID:26835139

  14. Serological diagnosis of Leptospirosis in bovine serum samples using a microsphere immunoassay

    PubMed Central

    Wynwood, S. J.; Burns, M. A.; Graham, G. C.; Weier, S. L.; McKay, D. B.; Craig, S. B.

    2016-01-01

    Leptospirosis causes significant economic loss within the cattle industry worldwide. Current diagnostic methods are generally inadequate for dealing with large numbers of samples, are outdated, and provide little useful diagnostic and epidemiological information. This aim of this study was to apply a microsphere immunoassay (MIA), utilising Luminex xMap technology, to 200 bovine serum samples to determine this method's usefulness in leptospirosis diagnosis in comparison with the current gold standard, the microscopic agglutination test (MAT). Although MAT is the most widely used laboratory test for the diagnosis of leptospirosis, its reliance on live cultures, subjective interpretation of results and an inability to differentiate between antibody classes, suggest MAT is no longer the best method for the diagnosis of leptospirosis. The results presented in this paper show that MIA was able to determine reactive from non-reactive samples when compared with MAT, and was able to differentiate IgG and IgM classes of antibody. The results suggest increased sensitivity in MIA and the ability to multiplex up to 500 antigens at one time allows for significant improvements in cost-effectiveness as well as a reduced dependency on live cultures. The relatively low cost, high throughput platform and differentiation of antibody class, as shown in previous research, make this assay worthy of consideration for the diagnosis of leptospirosis in small-scale or large-scale bovine populations. PMID:26835139

  15. Site-Specific N-Glycosylation of Recombinant Pentameric and Hexameric Human IgM

    NASA Astrophysics Data System (ADS)

    Moh, Edward S. X.; Lin, Chi-Hung; Thaysen-Andersen, Morten; Packer, Nicolle H.

    2016-04-01

    Glycosylation is known to play an important role in IgG antibody structure and function. Polymeric IgM, the largest known antibody in humans, displays five potential N-glycosylation sites on each heavy chain monomer. IgM can exist as a pentamer with a connecting singly N-glycosylated J-chain (with a total of 51 glycosylation sites) or as a hexamer (60 glycosylation sites). In this study, the N-glycosylation of recombinant pentameric and hexameric IgM produced by the same human cell type and culture conditions was site-specifically profiled by RP-LC-CID/ETD-MS/MS using HILIC-enriched tryptic and GluC glycopeptides. The occupancy of all putative N-glycosylation sites on the pentameric and hexameric IgM were able to be determined. Distinct glycosylation differences were observed between each of the five N-linked sites on the IgM heavy chains. While Asn171, Asn332, and Asn395 all had predominantly complex type glycans, differences in glycan branching and sialylation were observed between the sites. Asn563, a high mannose-rich glycosylation site that locates in the center of the IgM polymer, was only approximately 60% occupied in both the pentameric and hexameric IgM forms, with a difference in relative abundance of the glycan structures between the pentamer and hexamer. This study highlights the information obtained by characterization of the site-heterogeneity of a highly glycosylated protein of high molecular mass with quaternary structure, revealing differences that would not be seen by global glycan or deglycosylated peptide profiling.

  16. Fluorimetric Immunoassay for Multianalysis of Aflatoxins

    PubMed Central

    2013-01-01

    A sensitive fluorimetric ELISA was developed for the analysis of aflatoxins. The assay was performed in a 384 microwell plate, wherein high specificity monoclonal antibody against AFM1 (mAb-AFM1) was used as capture antibody and FITC conjugated secondary antibody was used for detection and quantification of the analyte. The linear range of the immunoassay was found to be 6.25–50 pg/mL. AFM1 as low as 1 pg/mL was detected by this method with assay volume 40 μL. The multi-analysis of different aflatoxins was also investigated in the microwell plate, based on the cross-reactivity (CR) approach. Real milk samples were tested along with certified reference material by standard addition method and recovery analysis was done. The mAb-AFM1 showed 23.2% CR with AFB1, 50% CR with respect to AFM2, and least CR towards AFG1 (<1%). Furthermore, mixture analysis of AFM2 and AFB1 was carried out at specific concentrations of AFM1. The advantages of this developed immunoassay are high sensitivity, high throughput, multianalyte detection, versatility, and ease of handling. PMID:24000318

  17. Comparative efficiency of commercial immunoassays for the diagnosis of rotavirus gastroenteritis during the course of infection.

    PubMed Central

    Miotti, P G; Eiden, J; Yolken, R H

    1985-01-01

    We evaluated the performance characteristics of three commercially available immunoassays for the detection of rotavirus antigens in stool samples obtained from infants during the course of rotavirus gastroenteritis. Two of the assays, Bio-EnzaBead (Litton Bionetics, Charleston, S.C.) and Rotazyme (Abbott Laboratories, North Chicago, Ill.), are enzyme immunoassays, while the third, Rotalex (Medical Technology Corporation, Somerset, N.J.), is a latex agglutination assay. We tested a total of 122 samples obtained from 26 children with gastroenteritis; 56 samples, obtained from 21 children, were found to contain rotavirus antigen by a reference microplate enzyme immunoassay. Rotavirus antigen was found by the Bio-EnzaBead, Rotazyme, and Rotalex assays in 53, 42, and 29 samples, respectively. The true positivity of samples which were positive by the reference microplate assay but negative by the other assays was confirmed by a specific neutralization assay or by the visualization of bands of double-stranded RNA by polyacrylamide gel electrophoresis or both. No false-positive assay results were noted with any of the commercial assays. The sensitivity of the assays was determined to a great extent by the time after the onset of illness at which the specimen was collected. Thus, the sensitivity of commercial assays with specimens collected early in the course of illness did not differ significantly from that of the microplate assay. However, significantly lower degrees of sensitivity were noted later in the course of illness. Results of our studies indicate that all three commercial assays can accurately detect rotavirus in stools from children with rotavirus gastroenteritis. However, the choice of assay systems for use in the clinical laboratory will depend on the conditions in which stool specimens are collected and tested in the laboratory. Images PMID:2997267

  18. Comparison between drug screening by immunoassay and ultra-high performance liquid chromatography/high-resolution time-of-flight mass spectrometry in post-mortem urine.

    PubMed

    Sundström, Mira; Pelander, Anna; Ojanperä, Ilkka

    2015-05-01

    Immunoassay is currently the most common approach for urine drug screening. However, the continuous emergence of new psychoactive substances (NPS) and their low urinary concentrations have challenged the scope and sensitivity of immunoassays. Consequently, specialized toxicology laboratories rely more and more on mass spectrometry (MS) based techniques. Ultra-high performance liquid chromatography/high-resolution time-of-flight mass spectrometry (UHPLC-HR-TOF-MS) is an especially attractive technique for comprehensive drug screening. The objective was to compare the performances of immunoassay and UHPLC-HR-TOF-MS in terms of scope, flexibility, sensitivity, and reliability of substance identification. A total of 279 post-mortem urine samples were analyzed using a method representative of each technique. The immunoassay method was an Emit II Plus enzyme immunoassay for the following drug groups: amphetamines, benzodiazepines, buprenorphine, cannabis, and opiates. The UHPLC-HR-TOF-MS method was a recently published method covering hundreds of drugs: conventional drugs of abuse, abused prescription drugs, and NPS of various classes. UHPLC-HR-TOF-MS produced a lower number of false positive (FP) results for the drug groups covered by immunoassay. Many of the false negative (FN, n = 40) and FP (n = 22) immunoassay results were obviously due to the higher cut-off concentrations and interfering matrix, respectively. Moreover, the wider scope of UHPLC-HR-TOF-MS allowed detection of NPS and prescription drugs. UHPLC-HR-TOF-MS gave FP results related to a few particular substances. The future option of adjusting all compound-specific reporting parameters individually would allow the method's sensitivity and specificity to be fully exploited. PMID:24953563

  19. Further insights into the anti-PF4/heparin IgM immune response.

    PubMed

    Krauel, Krystin; Schulze, Annika; Jouni, Rabie; Hackbarth, Christine; Hietkamp, Bernhard; Selleng, Sixten; Koster, Andreas; Jensch, Inga; van der Linde, Julia; Schwertz, Hansjörg; Bakchoul, Tamam; Hundt, Matthias; Greinacher, Andreas

    2016-04-01

    Anti-platelet factor 4 (PF4)/heparin antibodies are not only the cause of heparin-induced thrombocytopenia but might also play a role in the antibacterial host defence. Recently, marginal zone (MZ) B cells were identified to be crucial for anti-PF4/heparin IgG antibody production in mice. Combining human studies and a murine model of polymicrobial sepsis we further characterised the far less investigated anti-PF4/heparin IgM immune response. We detected anti-PF4/heparin IgM antibodies in the sera of paediatric patients < 6 months of age after cardiac surgery and in sera of splenectomised mice subjected to polymicrobial sepsis. In addition, PF4/heparin-specific IgM B cells were not only found in murine spleen, but also in peritoneum and bone marrow upon in vitro stimulation. Together, this indicates involvement of additional B cell populations, as MZ B cells are not fully developed in humans until the second year of life and are restricted to the spleen in mice. Moreover, PF4/heparin-specific B cells were detected in human cord blood upon in vitro stimulation and PF4-/- mice produced anti-PF4/heparin IgM antibodies after polymicrobial sepsis. In conclusion, the anti-PF4/heparin IgM response is a potential innate immune reaction driven by a B cell population distinct from MZ B cells. PMID:26467272

  20. Case Report: Acute Cerebellar Thrombosis in an Adult Patient with IgM Nephropathy

    PubMed Central

    Adike, Abimbola; Cherry, Mariyam; Awar, Melina

    2015-01-01

    IgM nephropathy is a relatively rare cause of idiopathic nephrotic syndrome.1 It was initially described by van de Putte,2 then by Cohen and Bhasin in 1978, as a distinctive feature of mesangial proliferative glomerulonephritis.2 It is typically characterized by diffuse IgM deposits on the glomeruli and diffuse mesangial hypercellularity. Little is known about the pathogenesis and treatment of this disease.1,3 We describe a patient who presented with nonspecific symptoms of epigastric pain, nausea, and early satiety. Abdominal imaging and endoscopies were unremarkable. She was found to have significant proteinuria (6.4 g/24 hours), hyperlipidemia, and edema consistent with a diagnosis of nephrotic syndrome. Kidney biopsy was performed and confirmed an IgM nephropathy. Less than 2 weeks after her diagnosis of IgM nephropathy, she presented with an acute cerebellar stroke. Thrombophilia is a well-known complication of nephrotic syndrome, but a review of the literature failed to show an association between IgM nephropathy and acute central nervous system thrombosis.

  1. The ontogeny of New Zealand groper (Polyprion oxygeneios) lymphoid organs and IgM.

    PubMed

    Parker, S; La Flamme, A; Salinas, I

    2012-10-01

    This study investigates the ontogeny of New Zealand groper (Polyprion oxygeneios) immune system, a new species for aquaculture in the Southern Pacific Ocean. In the eggs, both lysozyme and IgM were detected. Egg IgM was found at 1.07-1.56 μg/g wet weight and consisted of monomers compared to the polymerized IgM found in adult serum. In larvae, the head-kidney (HK) was first observed at 6 dph, followed by the spleen at 16 dph, and thymus at 20 dph, and within these organs IgM(+) cells were first detected in the HK (12 dph), then the spleen (32 dph) and finally in the thymus and the gastrointestinal tract (45 dph). Low levels of Igμ heavy chain transcripts were detected at 2 and 3 dph and they increased at 9 dph. Igμ expression further increased from day 45 onwards. In juveniles (115 dph), the HK and blood showed similar percentages of IgM(+) cells as the adult groper. These results highlight the important maturation steps that occur during the development of the immune system in the marine teleost P. oxygeneios. PMID:22766099

  2. West Nile virus IgM and IgG antibodies three years post- infection

    PubMed Central

    Papa, A; Anastasiadou, A; Delianidou, M

    2015-01-01

    Background: West Nile virus (WNV) causes to humans a variety of symptoms, from asymptomatic infection to severe neuroinvasive disease. In a previous study, it was shown that WNV IgM antibodies persisted in three of 26 (12%) patients, nine months after onset of the symptoms. The aim of the present study was to test 10 of these patients, three years post-infection for probable persistence of IgM antibodies and to investigate their IgG antibody patterns. Material and Methods: In summer 2013 serum samples were collected from 10 persons who were infected with WNV in 2010; 6 of them had a neuroinvasive disease. The three persons with detectable WNV IgM antibodies, nine months after onset of the symptoms, were included in the study. All samples were tested by ELISA in parallel with their stored paired samples taken in 2011. The positive results were confirmed by neutralization test. Results: WNV IgM antibodies were still detectable in the three persons, while high levels of WNV IgG and neutralizing antibodies were present in nine of the 10 persons, regardless the involvement of the nervous system. Conclusions: WNV IgM antibodies persist for more than three years in 12% of patients with WNV infection, while WNV IgG antibodies persist and even increase their levels, regardless the involvement of the nervous system, suggesting that the immune response in the symptomatic WNV infections is strong and long-lasting. Hippokratia 2015, 19 (1): 34-36. PMID:26435644

  3. Influenza Virus-Specific Neutralizing IgM Antibodies Persist for a Lifetime

    PubMed Central

    Skountzou, Ioanna; Satyabhama, Lakshmipriyadarshini; Stavropoulou, Anastasia; Ashraf, Zuhha; Esser, E. Stein; Vassilieva, Elena; Koutsonanos, Dimitrios; Compans, Richard

    2014-01-01

    Detection of immunoglobulin M (IgM) antibodies has long been used as an important diagnostic tool for identifying active viral infections, but their relevance in later stages has not been clearly defined in vivo. In this study, we followed the kinetics, longevity, and function of influenza virus-specific IgM antibodies for 2 years following sublethal infection of mice with live mouse-adapted A/PR/8/34 virus or immunization with formalin-inactivated virus. These groups mounted robust protective immune responses and survived lethal challenges with 50× 50% lethal dose (LD50) mouse-adapted A/PR/8/34 virus 600 days after the primary exposure. Surprisingly, the virus-specific IgM antibodies persisted along with IgG antibodies, and we found a significantly higher number of IgM-positive (IgM+) virus-specific plasma cells than IgG+ plasma cells that persisted for at least 9 months postexposure. The IgM antibodies were functional as they neutralized influenza virus in the presence of complement just as well as IgG antibodies did. PMID:25165027

  4. Roles of heavy and light chains in IgM polymerization.

    PubMed Central

    Bornemann, K D; Brewer, J W; Beck-Engeser, G B; Corley, R B; Haas, I G; Jäck, H M

    1995-01-01

    IgM antibodies are secreted as multisubunit polymers that consist of as many as three discrete polypeptides: mu heavy chains, light (L) chains, and joining (J) chains. We wished to determine whether L chains that are required to confer secretory competence on immunoglobulin molecules must be present for IgM to polymerize--that is, for intersubunit disulfide bonds to form between mu chains. Using a L-chain-loss variant of an IgM-secreting hybridoma, we demonstrated that mu chains were efficiently polymerized independent of L chains, in a manner similar to that observed for conventional microL complexes, and that the mu polymers incorporated J chain. These mu polymers were not secreted but remained associated with the endoplasmic reticulum-resident chaperone BiP (GRP78). This finding is consistent with the endoplasmic reticulum being the subcellular site of IgM polymerization. We conclude that mu chain alone has the potential to direct the polymerization of secreted IgM, a process necessary but not sufficient for IgM to attain secretory competence. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 PMID:7761423

  5. Identification of the minimal binding region of a Plasmodium falciparum IgM binding PfEMP1 domain

    PubMed Central

    Semblat, Jean-Philippe; Ghumra, Ashfaq; Czajkowsky, Daniel M.; Wallis, Russell; Mitchell, Daniel A.; Raza, Ahmed; Rowe, J.Alexandra

    2015-01-01

    Binding of host immunoglobulin is a common immune evasion mechanism demonstrated by microbial pathogens. Previous work showed that the malaria parasite Plasmodium falciparum binds the Fc-region of human IgM molecules, resulting in a coating of IgM on the surface of infected erythrocytes. IgM binding is a property of P. falciparum strains showing virulence-related phenotypes such as erythrocyte rosetting. The parasite ligands for IgM binding are members of the diverse P. falciparum Erythrocyte Membrane Protein One (PfEMP1) family. However, little is known about the amino acid sequence requirements for IgM binding. Here we studied an IgM binding domain from a rosette-mediating PfEMP1 variant, DBL4ζ of TM284var1, and found that the minimal IgM binding region mapped to the central region of the DBL domain, comprising all of subdomain 2 and adjoining parts of subdomains 1 and 3. Site-directed mutagenesis of charged amino acids within subdomain 2, predicted by molecular modelling to form the IgM binding site, showed no marked effect on IgM binding properties. Overall, this study identifies the minimal IgM binding region of a PfEMP1 domain, and indicates that the existing homology model of PfEMP1-IgM interaction is incorrect. Further work is needed to identify the specific interaction site for IgM within the minimal binding region of PfEMP1. PMID:26094597

  6. Differentiation of classical swine fever virus infection from CP7_E2alf marker vaccination by a multiplex microsphere immunoassay.

    PubMed

    Xia, Hongyan; Harimoorthy, Rajiv; Vijayaraghavan, Balaje; Blome, Sandra; Widén, Frederik; Beer, Martin; Belák, Sándor; Liu, Lihong

    2015-01-01

    Classical swine fever (CSF) is a highly contagious viral disease of pigs that has a tremendous socioeconomic impact. Vaccines are available for disease control. However, most industrialized countries are implementing stamping-out strategies to eliminate the disease and avoid trade restrictions. These restrictions can be avoided through the use of marker vaccines such as CP7_E2alf. Marker vaccines have to be accompanied by reliable and robust discriminatory assays. In this context, a multiplex microsphere immunoassay for serological differentiation of infected from vaccinated animals (DIVA) was developed to distinguish CSF virus (CSFV)-infected animals from CP7_E2alf-vaccinated animals. To this end, three viral proteins, namely, CSFV E2, CSFV E(rns), and bovine viral diarrhea virus (BVDV) E2, were produced in insect cells using a baculovirus expression system; they were used as antigens in a microsphere immunoassay, which was further evaluated by testing a large panel of pig sera and compared to a well-characterized commercial CSFV E2 antibody enzyme-linked immunosorbent assays (ELISAs) and a test version of an improved CSFV E(rns) antibody ELISA. Under a cutoff median fluorescence intensity value of 5,522, the multiplex microsphere immunoassay had a sensitivity of 98.5% and a specificity of 98.9% for the detection of antibodies against CSFV E2. The microsphere immunoassay and the CSFV E(rns) ELISA gave the same results for 155 out of 187 samples (82.8%) for the presence of CSFV E(rns) antibodies. This novel multiplex immunoassay is a valuable tool for measuring and differentiating immune responses to vaccination and/or infection in animals. PMID:25378351

  7. Differentiation of Classical Swine Fever Virus Infection from CP7_E2alf Marker Vaccination by a Multiplex Microsphere Immunoassay

    PubMed Central

    Xia, Hongyan; Harimoorthy, Rajiv; Vijayaraghavan, Balaje; Blome, Sandra; Widén, Frederik; Beer, Martin; Belák, Sándor

    2014-01-01

    Classical swine fever (CSF) is a highly contagious viral disease of pigs that has a tremendous socioeconomic impact. Vaccines are available for disease control. However, most industrialized countries are implementing stamping-out strategies to eliminate the disease and avoid trade restrictions. These restrictions can be avoided through the use of marker vaccines such as CP7_E2alf. Marker vaccines have to be accompanied by reliable and robust discriminatory assays. In this context, a multiplex microsphere immunoassay for serological differentiation of infected from vaccinated animals (DIVA) was developed to distinguish CSF virus (CSFV)-infected animals from CP7_E2alf-vaccinated animals. To this end, three viral proteins, namely, CSFV E2, CSFV Erns, and bovine viral diarrhea virus (BVDV) E2, were produced in insect cells using a baculovirus expression system; they were used as antigens in a microsphere immunoassay, which was further evaluated by testing a large panel of pig sera and compared to a well-characterized commercial CSFV E2 antibody enzyme-linked immunosorbent assays (ELISAs) and a test version of an improved CSFV Erns antibody ELISA. Under a cutoff median fluorescence intensity value of 5,522, the multiplex microsphere immunoassay had a sensitivity of 98.5% and a specificity of 98.9% for the detection of antibodies against CSFV E2. The microsphere immunoassay and the CSFV Erns ELISA gave the same results for 155 out of 187 samples (82.8%) for the presence of CSFV Erns antibodies. This novel multiplex immunoassay is a valuable tool for measuring and differentiating immune responses to vaccination and/or infection in animals. PMID:25378351

  8. Reversal of IgM deficiency following a gluten-free diet in seronegative celiac disease.

    PubMed

    Montenegro, Lucia; Piscitelli, Domenico; Giorgio, Floriana; Covelli, Claudia; Fiore, Maria Grazia; Losurdo, Giuseppe; Iannone, Andrea; Ierardi, Enzo; Di Leo, Alfredo; Principi, Mariabeatrice

    2014-12-14

    Selective IgM deficiency (sIGMD) is very rare; it may be associated with celiac disease (CD). We present the case of an 18-year-old man with sIGMD masking seronegative CD. Symptoms included abdominal pain, diarrhea and weight loss. Laboratory tests showed reduced IgM, DQ2-HLA and negative anti-transglutaminase. Villous atrophy and diffuse immature lymphocytes were observed at histology. Tissue transglutaminase mRNA mucosal levels showed a 6-fold increase. The patient was treated with a gluten-free diet (GFD) and six months later the symptoms had disappeared, the villous architecture was restored and mucosal tissue transglutaminase mRNA was comparable to that of healthy subjects. After 1 year of GFD, a complete restoration of normal IgM values was observed and duodenal biopsy showed a reduction of immature lymphocytes and normal appearance of mature immune cells. PMID:25516687

  9. Diagnostic and prognostic significance of the IgM antibody to the Hepatitis delta virus

    SciTech Connect

    Farci, P.; Gerin, J.L.; Aragona, M.; Lindsey, I.; Crivelli, O.; Balestrieri, A.; Smedile, A.; Thomas, H.C.; Rizzetto, M.

    1986-03-21

    The IgM class antibody to the hepatitis delta virus (HDV) was determined in different clinical categories of hepatitis B surface antigen carriers infected by the HDV (positive in the test for total antibody to HDV). The IgM antibody was found at high titers in each 70 patients with inflammatory liver disease and at a low titer in one six patients with inactive cirrhosis; it was not found in eight carriers with normal liver histology. Testing for Igm antibody to HDV distinguishes hepatitis B surface antigen carriers who have underlying inflammatory HDV liver disease from those with past HDV infection and provides prognostic information on the course of chronic HDV hepatitis.

  10. Antagonism of cannabinoid receptor 2 pathway suppresses IL-6-induced immunoglobulin IgM secretion

    PubMed Central

    2014-01-01

    Background Cannabinoid receptor 2 (CB2) is expressed predominantly in the immune system, particularly in plasma cells, raising the possibility that targeting the CB2 pathway could yield an immunomodulatory effect. Although the role of CB2 in mediating immunoglobulin class switching has been reported, the effects of targeting the CB2 pathway on immunoglobulin secretion per se remain unclear. Methods Human B cell line SKW 6.4, which is capable of differentiating into IgM-secreting cells once treated with human IL-6, was employed as the cell model. SKW 6.4 cells were incubated for 4 days with CB2 ligands plus IL-6 (100 U/ml). The amount of secreted IgM was determined by an ELISA. Cell proliferation was determined by the 3H-Thymidine incorporation assay. Signal molecules involved in the modulation of IgM secretion were examined by real-time RT-PCR and Western blot analyses or by using their specific inhibitors. Results We demonstrated that CB2 inverse agonists SR144528 and AM630, but not CB2 agonist HU308 or CB1 antagonist SR141716, effectively inhibited IL-6-induced secretion of soluble IgM without affecting cell proliferation as measured by thymidine uptake. SR144528 alone had no effects on the basal levels of IgM in the resting cells. These effects were receptor mediated, as pretreatment with CB2 agonist abrogated SR144528-mediated inhibition of IL-6 stimulated IgM secretion. Transcription factors relevant to B cell differentiation, Bcl-6 and PAX5, as well as the protein kinase STAT3 pathway were involved in the inhibition of IL-6-induced IgM by SR144528. Conclusions These results uncover a novel function of CB2 antagonists and suggest that CB2 ligands may be potential modulators of immunoglobulin secretion. PMID:24913620

  11. Environmental Immunoassays: Alternative Techniques for Soil and Water Analysis

    USGS Publications Warehouse

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

    1996-01-01

    Analysis of soil and water samples for environmental studies and compliance testing can be formidable, time consuming, and costly. As a consequence, immunochemical techniques have become popular for environmental analysis because they are reliable, rapid, and cost effective. During the past 5 years, the use of immunoassays for environmental monitoring has increased substantially, and their use as an integral analytical tool in many environmental laboratories is now commonplace. This chapter will present the basic concept of immunoassays, recent advances in the development of immunochemical methods, and examples of successful applications of immunoassays in environmental analysis.

  12. Detection by ELISA of IgA and IgM antibodies in secretion and IgM antibodies in serum in primary lower respiratory syncytial virus infection.

    PubMed

    Hornsleth, A; Friis, B; Grauballe, P C; Krasilnikof, P A

    1984-01-01

    Twenty-six infants and children with primary lower RS virus infection, diagnosed by the detection of RS virus in nasopharyngeal secretion (NPS) by use of immunofluorescent antibody (FA) technique, were studied with respect to the presence of IgA and IgM antibodies. Samples of NPS and serum obtained during the first 3-4 months following the beginning of illness, were investigated. Employing a reverse ELISA technique, we found IgM antibodies in the acute, but not during the convalescent, phase of illness in NPS from 20 of the patients and in serum from 21 of the patients. The majority of the IgM antibody conversions observed occurred in NPS as well as in serum on days 5-8 following the illness. RS virus IgA antibodies, also detected by a reverse ELISA technique, were demonstrated in NPS in 22 of the patients, with antibody conversions being found in 19 of the patients on days 5-8 following the beginning of the illness. Two patients still had IgA antibodies in NPS approximately 3 months FSOI. By comparison, RS virus was detected in acute-phase NPS by double-antibody sandwich ELISA in 25 of the 26 patients investigated. PMID:6363623

  13. Enzyme Kinetics.

    ERIC Educational Resources Information Center

    Moe, Owen; Cornelius, Richard

    1988-01-01

    Conveys an appreciation of enzyme kinetic analysis by using a practical and intuitive approach. Discusses enzyme assays, kinetic models and rate laws, the kinetic constants (V, velocity, and Km, Michaels constant), evaluation of V and Km from experimental data, and enzyme inhibition. (CW)

  14. Simultaneous detection of single molecules and singulated ensembles of molecules enables immunoassays with broad dynamic range.

    PubMed

    Rissin, David M; Fournier, David R; Piech, Tomasz; Kan, Cheuk W; Campbell, Todd G; Song, Linan; Chang, Lei; Rivnak, Andrew J; Patel, Purvish P; Provuncher, Gail K; Ferrell, Evan P; Howes, Stuart C; Pink, Brian A; Minnehan, Kaitlin A; Wilson, David H; Duffy, David C

    2011-03-15

    We report a method for combining the detection of single molecules (digital) and an ensemble of molecules (analog) that is capable of detecting enzyme label from 10(-19) M to 10(-13) M, for use in high sensitivity enzyme-linked immunosorbent assays (ELISA). The approach works by capturing proteins on microscopic beads, labeling the proteins with enzymes using a conventional multistep immunosandwich approach, isolating the beads in an array of 50-femtoliter wells (Single Molecule Array, SiMoA), and detecting bead-associated enzymatic activity using fluorescence imaging. At low concentrations of proteins, when the ratio of enzyme labels to beads is less than ∼1.2, beads carry either zero or low numbers of enzymes, and protein concentration is quantified by counting the presence of "on" or "off" beads (digital regime). (1) At higher protein concentrations, each bead typically carries multiple enzyme labels, and the average number of enzyme labels present on each bead is quantified from a measure of the average fluorescence intensity (analog regime). Both the digital and analog concentration ranges are quantified by a common unit, namely, average number of enzyme labels per bead (AEB). By combining digital and analog detection of singulated beads, a linear dynamic range of over 6 orders of magnitude to enzyme label was achieved. Using this approach, an immunoassay for prostate specific antigen (PSA) was developed. The combined digital and analog PSA assay provided linear response over approximately four logs of concentration ([PSA] from 8 fg/mL to 100 pg/mL or 250 aM to 3.3 pM). This approach extends the dynamic range of ELISA from picomolar levels down to subfemtomolar levels in a single measurement. PMID:21344864

  15. Nanoparticles for Enhanced Sensitivity in Electrochemical Immunoassays

    SciTech Connect

    Lin, Yuehe; Wang, Jun; Wang, Hua; Wu, Hong; Tang, Zhiwen

    2008-10-12

    In this manuscript, we report on electrochemical biosensors based on various nanoparticles (NPs) as labels for sensitive detection of protein biomarkers. We used silica nanoparticle as a carrier to loading a large amount of electroactive species such as poly(guanine) for sensitive immunoassay of tumor necrosis factor-alpha (TNF-a). We took the advantages of the unique hollow structure and reconstruction properties of apoferritin to prepare Cd3(PO4)2 nanoparticles as labels for sensitive assay of TNF-a. A novel immunochromatographic/electro-chemical biosensor based on quantum dots as labels has also been developed for rapid and sensitive detection of prostate-specific antigen (PSA) in human serum. These biosensors are quite sensitive with the detection limit at pM level and these approaches based on nanoparticle labels offer a new avenue for sensitive detection of protein biomarkers.

  16. Immunoassay procedures for fiber optic sensors

    NASA Astrophysics Data System (ADS)

    Ligler, Frances S.

    1988-04-01

    There is an increasing need for the development of an ultrasensitive immunoassay for use with fiber optic sensors. These detection systems can be used for such applications as disease diagnosis, detection of chemical and biological warfare agents or drugs of abuse, pollution control, therapeutic monitoring, and explosive detection. This specific program is designed to produce generic chemistries for use with existing fiber optic-based sensors to detect pathogens of particular threat to Army personnel as determined by USAMRIID. The detection system under development involves the attachment of antibodies to an optical fiber at high density. In addition, the immobilization must be achieved in a way which retains the antibody's ability to bind antigen. The functionality of the antibody will be tested through the binding of a labelled antigen. In the future, this assay could incorporate the antibodies developed by the Army for pathogens of particularly military concern.

  17. Flow immunoassay using solid-phase entrapment.

    PubMed

    Locascio-Brown, L; Martynova, L; Christensen, R G; Horvai, G

    1996-05-01

    A flow injection immunoassay was performed using a column packed with reversed-phase sorbents to effect separation of the immunoreacted species by entrapping free analyte and allowing antibody-conjugated analyte to pass unretained. Fluorescein-labeled analyte was measured in a competitive assay for the anticonvulsant drug phenytoin. The simplicity of the assay was the greatest advantage of the technique, which allowed for measurement of phenytoin in a 2-min assay time. The reliable detection limit for the assay was 5 nmol L(-)(1) of phenytoin in serum. The columns were regenerated with periodic injections of ethanol solutions to remove the entrapped analyte and prepare the column for subsequent analyses. PMID:21619134

  18. Detection of IgE, IgG, IgA and IgM antibodies against raw and processed food antigens

    PubMed Central

    Vojdani, Aristo

    2009-01-01

    Background Despite the first documented case of food allergy to cooked food in 1921 by Prausnitz and Kustner, all commercial food antigens are prepared from raw food. Furthermore, all IgE and IgG antibodies against dietary proteins offered by many clinical laboratories are measured against raw food antigens. Methods We developed an enzyme-linked immunosorbent assay for the measurement of IgE, IgG, IgA and IgM antibodies against raw and processed food antigens. Sera with low or high reactivity to modified food antigens were subjected to myelin basic protein, oxidized low density lipoprotein, and advanced glycation end products (AGE) such as AGE-human serum albumin and AGE-hemoglobin. Results Compared to raw food antigens, IgE antibodies showed a 3–8-fold increase against processed food antigens in 31% of the patients. Similarly, IgG, IgA and IgM antibodies against modified food antigens overall were found at much higher levels than antibody reactions against raw food antigens. Almost every tested serum with high levels of antibodies against modified food antigens showed very high levels of antibodies against myelin basic protein, oxidized low density lipoprotein, AGE-human serum albumin and AGE-hemoglobin. Conclusion We conclude that the determination of food allergy, intolerance and sensitivity would be improved by testing IgE, IgG, IgA and IgM antibodies against both raw and processed food antigens. Antibodies against modified food antigens, by reacting with AGEs and tissue proteins, may cause perturbation in degenerative and autoimmune diseases such as diabetes, atherosclerosis, inflammation, autoimmunity, neurodegeneration and neuroautoimmunity. PMID:19435515

  19. Comparison of immunoassay screening tests and LC-MS-MS for urine detection of benzodiazepines and their metabolites: results of a national proficiency test.

    PubMed

    Bertol, Elisabetta; Vaiano, Fabio; Borsotti, Maurizio; Quercioli, Massimo; Mari, Francesco

    2013-01-01

    For most diverse purposes, different immunoassay (IA) screening methods are usually used to detect benzodiazepines and their metabolites in urine. In this study, we compared the main IAs used in forensic toxicology (Cloned Enzyme Donor Immunoassay, CEDIA®; Enzyme-Multiplied Immunoassay Technique, EMIT®; Fluorescent Polarization ImmunoAssay, FPIA®; Kinetic Interaction of Microparticles in Solution, KIMS® and Immunochromatographic Techniques, IMC) with liquid chromatography-tandem mass spectrometry (LC-MS-MS). Twelve urine specimens were analyzed by 178 laboratories in Italy that participated in a National Proficiency Test, providing both qualitative and semi-quantitative results. Each IA was evaluated by the parameters: true positive, true negative, false positive (FP), false negative (FN), sensitivity (SENS), specificity (SPEC), positive predictive value, negative predictive value (NPV) and accuracy. SPEC was affected by a high FP rate for all IAs. The lowest SENS and NPV were provided by FPIA due to a high number of FN cases. Comparing IA semi-quantitative data with LC-MS-MS results, an overestimation of benzodiazepine amount is noted. This paper draws attention to the problem of the careless use of IA tests for forensic purposes as they may provide FP and/or FN results that can lead to errors of great severity. PMID:23943436

  20. Quantitative Immunoassay To Measure Plasma and Intracellular Atazanavir Levels: Analysis of Drug Accumulation in Cultured T Cells▿

    PubMed Central

    Roucairol, Camille; Azoulay, Stéphane; Nevers, Marie-Claire; Créminon, Christophe; Lavrut, Thibault; Garraffo, Rodolphe; Grassi, Jacques; Burger, Alain; Duval, Danièle

    2007-01-01

    We have developed an enzyme immunoassay to measure atazanavir (ATV) levels in plasma and cells. Anti-ATV polyclonal antibodies were raised in rabbits by using a synthetic ATV derivative coupled to bovine serum albumin as the immunogen, and the enzyme tracer was prepared by chemically coupling the ATV derivative with acetylcholinesterase. These reagents were used to develop a sensitive competitive enzyme immunoassay performed in microtitration plates, and the lowest limit of quantification was 150 pg/ml, which is about 10 times more sensitive than previously published techniques. The plasma assay was performed, after a simple methanol extraction, with a minimum of 30 μl of plasma. This assay showed good precision and efficiency, since the rates of recovery from human plasma and cell extracts spiked with ATV ranged form 93 to 113%, with coefficients of variation of less than 10%. ATV concentrations were measured in peripheral blood mononuclear cells incubated with various ATV concentrations and in CEM cells in the absence or presence of antiretroviral drugs and drug transporter inhibitors. The results indicated a dose-dependent uptake (intracellular concentration/extracellular concentration ratio range, 0.04 to 19). A significant increase in the accumulation of ATV was noticed in the presence of P-glycoprotein and MRP1 inhibitors (dipyridamole, inter alia). Interestingly, efavirenz significantly increased the baseline accumulation of ATV, whereas nevirapine induced a marked reduction. This new enzyme immunoassay for measuring plasma and intracellular ATV levels was fully validated and provides an inexpensive and useful tool for routine therapeutic drug monitoring. Moreover, in vitro results suggested the implication of drug transporters and interactions with other antiviral drugs that should be further explored in human immunodeficiency virus-infected patients. PMID:17116661

  1. Detection of narcotics with an immunoassay film badge

    SciTech Connect

    Lukens, H.R.

    1993-12-31

    Efficient personnel performance, a major requirement for a safe nuclear industry, is jeopardized where personnel use narcotics. However, detection of narcotics at nuclear plants is a challenge. The unique specificity and sensitivity of an immunoassay has been implemented in the form of a small, dry immunoassay film badge (IFB) for the detection of vapors emitted by narcotics. The device is suitable as an area monitor, and its characteristics are suitable for use as a breath monitor for the detection of drug use.

  2. Enzyme Informatics

    PubMed Central

    Alderson, Rosanna G.; Ferrari, Luna De; Mavridis, Lazaros; McDonagh, James L.; Mitchell, John B. O.; Nath, Neetika

    2012-01-01

    Over the last 50 years, sequencing, structural biology and bioinformatics have completely revolutionised biomolecular science, with millions of sequences and tens of thousands of three dimensional structures becoming available. The bioinformatics of enzymes is well served by, mostly free, online databases. BRENDA describes the chemistry, substrate specificity, kinetics, preparation and biological sources of enzymes, while KEGG is valuable for understanding enzymes and metabolic pathways. EzCatDB, SFLD and MACiE are key repositories for data on the chemical mechanisms by which enzymes operate. At the current rate of genome sequencing and manual annotation, human curation will never finish the functional annotation of the ever-expanding list of known enzymes. Hence there is an increasing need for automated annotation, though it is not yet widespread for enzyme data. In contrast, functional ontologies such as the Gene Ontology already profit from automation. Despite our growing understanding of enzyme structure and dynamics, we are only beginning to be able to design novel enzymes. One can now begin to trace the functional evolution of enzymes using phylogenetics. The ability of enzymes to perform secondary functions, albeit relatively inefficiently, gives clues as to how enzyme function evolves. Substrate promiscuity in enzymes is one example of imperfect specificity in protein-ligand interactions. Similarly, most drugs bind to more than one protein target. This may sometimes result in helpful polypharmacology as a drug modulates plural targets, but also often leads to adverse side-effects. Many cheminformatics approaches can be used to model the interactions between druglike molecules and proteins in silico. We can even use quantum chemical techniques like DFT and QM/MM to compute the structural and energetic course of enzyme catalysed chemical reaction mechanisms, including a full description of bond making and breaking. PMID:23116471

  3. Development of immunoassays for human urokinase

    NASA Technical Reports Server (NTRS)

    Atassi, M. Zouhair

    1988-01-01

    Radioimmune assays (RIA) and enzyme linked immune assays for measurement of pro-urokinase and the two active forms of the enzyme were developed. Polyclonal and monoclonal antibodies, with desired specificities against preselected synthetic regions of urokinase (UK), were obtained by immunization with the respective synthetic peptides and used to develop RIA for zymogen and the two activated forms of UK.

  4. Complete amino acid sequence of the Mu heavy chain of a human IgM immunoglobulin.

    PubMed

    Putnam, F W; Florent, G; Paul, C; Shinoda, T; Shimizu, A

    1973-10-19

    The amino acid sequence of the micro, chain of a human IgM immunoglobulin, including the location of all disulfide bridges and oligosaccharides, has been determined. The homology of the constant regions of immunoglobulin micro, gamma, alpha, and epsilon heavy chains reveals evolutionary relationships and suggests that two genes code for each heavy chain. PMID:4742735

  5. Spitzer Observations of Stephan's Quintet -- IGM Dust and Gas in a Multi-galaxy Collision

    NASA Astrophysics Data System (ADS)

    Xu, C. K.; Appleton, Ph.; Dopita, M.; Gao, Y.; Lu, N. Y.; Popescu, C.; Reach, W.; Tuffs, R.; Sulentic, J.; Yun, M.

    2005-12-01

    Stephan's Quintet (SQ) is the most famous and well studied compact group of galaxies. The rich literature archive of multi-wavelength data reveal one of the most fascinating pictures in the universe. We see a complex web of interactions between member galaxies and various constituents of the intragroup medium (IGM) which, in turn, have triggered spectacular activities such as a 40 kpc large scale shock and a strong IGM starburst. In this poster we will present our new Spitzer observations of SQ with unprecedented resolution and sensitivity. The observations impose new constraints on the physical conditions of the IGM gas and dust, the distribution and history of the star formation activity in member galaxies and in the IGM, as well as the effects of various interaction events that have occurred in the recent ( ˜ 1 Gyr) history of the group. Our results may have far-reaching inferences on studies of multi-nuclei ULIRGs and 'chain galaxies' found in high-z surveys. This work is based on observations made with the Spitzer Space Telescope, which is operated by the Jet Propulsion Laboratory, California Institute of Technology under a contract with NASA. Support for this work was provided by NASA through an award issued by JPL/Caltech

  6. Baryonic Content in the Warm-Hot IGM at Low Redshift

    NASA Technical Reports Server (NTRS)

    Sonneborn, George; Shull, M.; Danforth, C.; Moos, W.

    2007-01-01

    Baryons are 4.5% of the universe's mass/energy density; only 10% of these are in stars, galaxies, and clusters. At low-redshift 90% of baryons are in the IGM, 30% in Ly-alpha forest, but most are in hot gas (10(exp 5-7) K) produced by shocks during structure formation. O VI 1032-38 A are the best tracers of this gas. The distribution of O VI absorbers observed by FUSE rises as N(sup -2+/-0.2, down to 10(exp 13)/sq cm. Integrated to logN=13, 7% of baryons reside in the O VI-bearing IGM at 10% solar metallicity, T approx. 10(exp 5.5) K. At redshift z<0.1 metals have been transported less than 800/h kpc from L* galaxies and 200/h kpc from 0.1 L* galaxies. The steepness of dN/dz means that low-N absorbers contribute an equal mass of hot IGM as higher N gas. The total mass of O VI-bearing gas in the IGM depends on determining the turnover in dN/dz at low N(O VI). Future observations by FUSE are needed to reach lower N and to reduce the uncertainty in the dN/dz power law.

  7. Hyper IgM immunodeficiency. A primary dysfunction of B lymphocyte isotype switching.

    PubMed Central

    Levitt, D; Haber, P; Rich, K; Cooper, M D

    1983-01-01

    Immunological evaluations (lymphocyte markers, B cell differentiation, T cell function) were performed on peripheral blood mononuclear cells from four individuals with hyper IgM immunodeficiency. Number, proportion, and proliferation of T lymphocytes and T lymphocyte subpopulations were relatively normal in affected individuals. The percentage and number of B cells expressing surface IgM and IgD were either normal or elevated in both blood and lymph nodes. However, surface IgG- and IgA-bearing B lymphocytes were completely absent. In vitro stimulation of blood lymphocytes with both T cell-dependent and T-cell independent polyclonal B cell activators resulted in normal numbers of IgM plasma cells and IgM secretion in cultures, but failed to induce any IgG- or IgA-producing cells. This failure of isotype switching was intrinsic to the B cell population and did not involve aberrant T cell help or suppression. Therefore, individuals with this disorder possess an intrinsic B cell dysfunction that is not related to abnormal T cell regulation. PMID:6605368

  8. Clinical Applications of Capillary Electrophoresis-Based Immunoassays

    PubMed Central

    Moser, Annette C.; Willicott, Corey W.; Hage, David S.

    2014-01-01

    Immunoassays have long been an important set of tools in clinical laboratories for the detection, diagnosis and treatment of disease. Over the last two decades there has been growing interest in utilizing capillary electrophoresis (CE) as a means for conducting immunoassays with clinical samples. The resulting method is known as a CE immunoassay. This approach makes use of the selective and strong binding of antibodies for their targets, as is employed in a traditional immunoassay, and combines this with the speed, efficiency, and small sample requirements of CE. This review discusses the variety of ways in which CE immunoassays have been employed with clinical samples. An overview of the formats and detection modes that have been employed in these applications is first presented. A more detailed discussion is then given on the type of clinical targets and samples that have been measured or studied by using CE immunoassays. Particular attention is given to the use of this method in the fields of endocrinology, pharmaceutical measurements, protein and peptide analysis, immunology, infectious disease detection, and oncology. Representative applications in each of these areas are described, with these examples involving work with both traditional and microanalytical CE systems. PMID:24132682

  9. Magnetic bead-based reverse colorimetric immunoassay strategy for sensing biomolecules.

    PubMed

    Gao, Zhuangqiang; Xu, Mingdi; Hou, Li; Chen, Guonan; Tang, Dianping

    2013-07-16

    A novel reverse colorimetric immunoassay (RCIA) strategy was for the first time designed and utilized for sensitive detection of low-abundance protein (prostate-specific antigen, PSA, used in this case) in biological fluids by coupling highly catalytic efficient catalase with magnetic bead-based peroxidase mimics. To construct such a RCIA system, two nanostructures including magnetic beads and gold nanoparticles were first synthesized and functionalized with anti-PSA capture antibody and catalase/anti-PSA detection antibody, respectively. Thereafter, a specific sandwich-type immunoassay format was employed for determination of PSA by using functional gold nanoparticles as enzymatic bioreactors and anti-PSA-conjugated magnetic beads as a colorimetric developer. The carried catalase, followed by the sandwiched immunocomplex, partially consumed the added hydrogen peroxide in the detection solution, which slowed down the catalytic efficiency of magnetic bead-based peroxidase mimics toward TMB/H2O2, thereby weakening the visible color and decreasing the colorimetric density. Different from conventional colorimetric immunoassay, the RCIA method determined the residual hydrogen peroxide in the substrate after consumption. Under the optimal conditions, the developed RCIA exhibited a wide dynamic range of 0.05-20 ng mL(-1) toward PSA with a detection limit of 0.03 ng mL(-1) at the 3Sblank level. Intra- and interassay coefficients of variation were below 6.1% and 9.3%, respectively. Additionally, the methodology was further validated for the analysis of 12 PSA clinical serum specimens, giving results in good accordance with those obtained by the commercially available enzyme-linked immunosorbent assay (ELISA) method. PMID:23806145

  10. Hapten synthesis and antibody production for the development of a melamine immunoassay.

    PubMed

    Lei, Hongtao; Shen, Yudong; Song, Lijun; Yang, Jinyi; Chevallier, Olivier P; Haughey, Simon A; Wang, Hong; Sun, Yuanming; Elliott, Christopher T

    2010-04-14

    The incorporation of melamine into food products is banned but its misuse has been widely reported in both animal feeds and food. The development of a rapid screening immunoassay for monitoring of the substance is an urgent requirement. Two haptens of melamine were synthesized by introducing spacer arms of different lengths and structures on the triazine ring of the analyte molecular structure. 6-Aminocaproic acid and 3-mercaptopropionic acid were reacted with 2-chloro-4,6-diamino-1,3,5-triazine (CAAT) to produce hapten 1 [3-(4,6-diamino-1,6-dihydro-1,3,5-triazin-2-ylamino) hexanoic acid] and hapten 2 [3-(4,6-diamino-1,6-dihydro-1,3,5-triazin-2-ylthio) propanoic acid], respectively. The molecular structures of the two haptens were identified by (1)H nuclear magnetic resonance spectrometry, mass spectrometry and infrared spectrometry. An immunogen was prepared by coupling hapten 1 to bovine serum albumin (BSA). Two plate coating antigens were prepared by coupling both haptens to egg ovalbumin (OVA). A competitive indirect enzyme-linked immunosorbent assay (ciELISA) was developed to evaluate homogeneous and heterogeneous assay formats. The results showed that polyclonal antibodies with high titers were obtained, and the heterogeneous immunoassay format demonstrated a better performance with an IC(50) of 70.6 ng mL(-1), a LOD of 2.6 ng mL(-1) and a LOQ of 7.6 ng mL(-1). Except for cyromazine, no obvious cross-reactivity to common compounds was found. The data showed that the hapten synthesis was successful and the resultant antisera could be used in an immunoassay for the rapid and sensitive detection of this banned chemical. PMID:20381695

  11. Soil Enzymes

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The functionality and resilience of natural and managed ecosystems mainly rely on the metabolic abilities of microbial communities, the main source of enzymes in soils. Enzyme mediated reactions are critical in the decomposition of organic matter, cycling of nutrients, and in the breakdown of herbic...

  12. Enzymes, Industrial

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Enzymes serve key roles in numerous biotechnology processes and products that are commonly encountered in the forms of food and beverages, cleaning supplies, clothing, paper products, transportation fuels, pharmaceuticals, and monitoring devices. Enzymes can display regio- and stereo-specificity, p...

  13. Pre-cut Filter Paper for Detecting Anti-Japanese Encephalitis Virus IgM from Dried Cerebrospinal Fluid Spots

    PubMed Central

    Bharucha, Tehmina; Chanthongthip, Anisone; Phuangpanom, Soumphou; Phonemixay, Ooyanong; Sengvilaipaseuth, Onanong; Vongsouvath, Manivanh; Lee, Sue; Newton, Paul N.; Dubot-Pérès, Audrey

    2016-01-01

    Background The use of filter paper as a simple, inexpensive tool for storage and transportation of blood, ‘Dried Blood Spots’ or Guthrie cards, for diagnostic assays is well-established. In contrast, there are a paucity of diagnostic evaluations of dried cerebrospinal fluid (CSF) spots. These have potential applications in low-resource settings, such as Laos, where laboratory facilities for central nervous system (CNS) diagnostics are only available in Vientiane. In Laos, a major cause of CNS infection is Japanese encephalitis virus (JEV). We aimed to develop a dried CSF spot protocol and to evaluate its diagnostic performance using the World Health Organisation recommended anti-JEV IgM antibody capture enzyme-linked immunosorbent assay (JEV MAC-ELISA). Methodology and Principal Findings Sample volumes, spotting techniques and filter paper type were evaluated using a CSF-substitute of anti-JEV IgM positive serum diluted in Phosphate Buffer Solution (PBS) to end-limits of detection by JEV MAC-ELISA. A conventional protocol, involving eluting one paper punch in 200μl PBS, did not detect the end-dilution, nor did multiple punches utilising diverse spotting techniques. However, pre-cut filter paper enabled saturation with five times the volume of CSF-substitute, sufficiently improving sensitivity to detect the end-dilution. The diagnostic accuracy of this optimised protocol was compared with routine, neat CSF in a pilot, retrospective study of JEV MAC-ELISA on consecutive CSF samples, collected 2009–15, from three Lao hospitals. In comparison to neat CSF, 132 CSF samples stored as dried CSF spots for one month at 25–30°C showed 81.6% (65.7–92.3 95%CI) positive agreement, 96.8% (91.0–99.3 95%CI) negative agreement, with a kappa coefficient of 0.81 (0.70–0.92 95%CI). Conclusions/Significance The novel design of pre-cut filter paper saturated with CSF could provide a useful tool for JEV diagnostics in settings with limited laboratory access. It has the potential to improve national JEV surveillance and inform vaccination policies. The saturation of filter paper has potential use in the wider context of pathogen detection, including dried spots for detecting other analytes in CSF, and other body fluids. PMID:26986061

  14. Photoelectrochemical detection of enzymatically generated CdS nanoparticles: Application to development of immunoassay.

    PubMed

    Barroso, Javier; Saa, Laura; Grinyte, Ruta; Pavlov, Valeri

    2016-03-15

    We report an innovative photoelectrochemical process (PEC) based on graphite electrode modified with electroactive polyvinylpyridine bearing osmium complex (Os-PVP). The system relies on the in situ enzymatic generation of CdS quantum dots (QDs). Alkaline phosphatase (ALP) catalyzes the hydrolisis of sodium thiophosphate (TP) to hydrogen sulfide (H2S) which in the presence Cd(2+) ions yields CdS semiconductor nanoparticles (SNPs). Irradiation of SNPs with the standard laboratory UV-illuminator (wavelength of 365 nm) results in photooxidation of 1-thioglycerol (TG) mediated by Os-PVP complex on the surface of graphite electrode at applied potential of 0.31 V vs. Ag/AgCl. A novel immunoassay based on specific enzyme linked immunosorbent assay (ELISA) combined with the PEC methodology was developed. Having selected the affinity interaction between bovine serum albumine (BSA) with anti-BSA antibody (AB) as a model system, we built the PEC immunoassay for AB. The new assay displays a linear range up to 20 ngmL(-1) and a detection limit (DL) of 2 ngmL(-1) (S/N=3) which is lower 5 times that of the traditional chromogenic ELISA test employing p-nitro-phenyl phosphate (pNPP). PMID:26432195

  15. Protein immunoassay methods for detection of biotech crops: applications, limitations, and practical considerations.

    PubMed

    Stave, James W

    2002-01-01

    Immunoassay methods are available for detection and quantitation of proteins expressed by most biotechnology-derived crops in commercial production. The 2 most common test formats are enzyme-linked immunosorbent assay (ELISA) and immunochromatographic (lateral flow) strip tests. Two ELISA methods, one for Roundup Ready soybeans and one for MON810 CrylAb corn, were the subject of large international collaborative studies and were demonstrated to quantitatively determine the concentrations of biotech crops in samples of ground grain. Quantitative ELISA methods are also useful for analysis of processed fractions of agricultural commodities such as soybean toasted meal or corn flour. Both strip tests and ELISAs for biotech crops are currently being used on a large scale in the United States to manage the sale and distribution of grain. In these applications, tests are used to determine if the concentration of biotech grain is above or below specified threshold limits. Using existing U.S. Department of Agriculture sampling techniques, the reliability of the threshold determination is expressed in terms of statistical confidence rather than analytical precision. Combining the use of protein immunoassays with Identity Preservation systems provides an effective means of characterizing the raw and processed agricultural inputs to the food production system in a way that allows food producers to comply with labeling laws. PMID:12083275

  16. Clinical performance evaluation of four automated chemiluminescence immunoassays for hepatitis C virus antibody detection.

    PubMed

    Kim, Sinyoung; Kim, Jeong-Ho; Yoon, Seoyoung; Park, Youn-Hee; Kim, Hyon-Suk

    2008-12-01

    Various automated chemiluminescence immunoassay (CLIA) analyzers for the detection of antibodies to hepatitis C virus (HCV) are now commercially available in clinical laboratories and are replacing conventional enzyme immunoassays. We investigated the performance of four anti-HCV CLIAs (the Architect Anti-HCV assay on the Architect i2000 system, the Vitros Anti-HCV assay on the Vitros ECiQ Immunodiagnostic System, the Access HCV Ab PLUS assay on the UniCel DxI 800 analyzer, and the newly developed Elecsys Anti-HCV assay on the Cobas e 411 analyzer). The total percent coefficient of variation values of imprecision were 3.5 to 5.7% with positive control materials and 7.2 to 10.2% with negative control materials. The agreement between the results of the Elecsys, Architect, Vitros, and Access CLIAs ranged from 94.5 to 98.1%. The clinical sensitivity of all CLIAs was 100%. Each CLIA showed excellent reproducibility and clinical sensitivity. The Elecsys, Architect, Vitros, and Access CLIAs showed clinical specificities of 98.2, 98.8, 96.5, and 98.2%. PMID:18945839

  17. Ultrasensitive On-Chip Immunoassays with a Nanoparticle-Assembled Photonic Crystal

    PubMed Central

    Han, Jin-Hee; Sudheendra, L.; Kim, Hee-Joo; Gee, Shirley J.; Hammock, Bruce D.; Kennedy, Ian M.

    2012-01-01

    Electrophoretic particle entrapment system (EPES) is employed to generate 2D array of nanoparticles coated with biological molecules (i.e. antibodies). Phase matching of the excitation and the emission in the 2D arrays with particles produces a highly enhanced fluorescence signal that was shown to improve the limit of detection in immunoassays. The phase matching is achieved when the particle are in the sub-100 nm range. A comparison between different size particles shows that the sensitivity of an immunoassay is extended to a range that is difficult to achieve with standard technology (e.g. Enzyme-linked immunosorbent assay-ELISA). The effectiveness of this novel configuration of particle-in-a well was demonstrated with an assay for human epidermal growth factor receptor 2 (HER2; breast cancer biomarker), with a detection limit as low as 10 aM in less than 10 μl of serum-based sample. The limit of detection of HER2 indicated far superior assay performance compared to the corresponding standard 96-well plate-based ELISA. The particle-based photonic platform reduces the reagent volume and the time for performing an assay in comparison to competing methods. The simplicity of operation and the level of sensitivity demonstrated here can be used for rapid and early-stage detection of biomarkers. PMID:22957818

  18. Integrated optic immunoassay for virus detection

    NASA Astrophysics Data System (ADS)

    Boiarski, Anthony A.; Busch, James R.; Miller, Larry S.; Zulich, A. W.; Burans, James

    1995-05-01

    An integrated optic refractometer device was developed to perform a rapid one-step, label-free immunoassay. The device measures refractive index changes at the surface of a planar waveguide using interferometry. Antibodies were applied to the waveguide surface to provide a bioselective coating for detecting and quantifying a specific antigen of interest. The detection limit of this biosensor was determined for adenovirus as a model for other viral analytes of military, medical, and environmental interest. As binding of the antigen occurred on the sensor surface, a time-dependent phase shift of the helium-neon laser light beam was detected and was measured over a 10-minute time period. Adenovirus was detected at levels of 250 - 2500 viral particles/ml. This detection limit was obtained for a mono-layer of antibody attached to the sensor. Use of a high-density, multi-layer antibody coating approach resulted in improved detection limits for bacteria and protein analytes of general interest.

  19. NiCoBP-doped carbon nanotube hybrid: a novel oxidase mimetic system for highly efficient electrochemical immunoassay.

    PubMed

    Zhang, Bing; He, Yu; Liu, Bingqian; Tang, Dianping

    2014-12-01

    NiCoBP-doped multi-walled carbon nanotube (NiCoBP-MWCNT) was first synthesized by using induced electroless-plating method and functionalized with the biomolecules for highly efficient electrochemical immunoassay of prostate-specific antigen (PSA, used as a model analyte). We discovered that the as-synthesized NiCoBP-MWCNT had the ability to catalyze the glucose oxidization with a stable and well-defined redox peak. The catalytic current increased with the increment of the immobilized NiCoBP-MWCNT on the electrode. Transmission electron microscope (TEM) and energy dispersive X-ray spectrometry (EDX) were employed to characterize the as-prepared NiCoBP-MWCNT. Using the NiCoBP-MWCNT-conjugated anti-PSA antibody as the signal-transduction tag, a new enzyme-free electrochemical immunoassay protocol could be designed for the detection of target PSA on the capture antibody-functionalized immunosensing interface. Experimental results revealed that the designed immunoassay system could exhibit good electrochemical responses toward target PSA, and allowed the detection of PSA at a concentration as low as 0.035ngmL(-1). More importantly, the NiCoBP-MWCNT-based oxidase mimetic system could be further extended for the monitoring of other low-abundance proteins or disease-related biomarkers by tuning the target antibody. PMID:25440664

  20. AquaLite, a bioluminescent label for immunoassay and nucleic acid detection: quantitative analyses at the attomol level

    NASA Astrophysics Data System (ADS)

    Smith, David F.; Stults, Nancy L.

    1996-04-01

    AquaLiteR is a direct, bioluminescent label capable of detecting attomol levels of analyte in clinical immunoassays and assays for the quantitative measurement of nucleic acids. Bioluminescent immunoassays (BIAs) require no radioisotopes and avoid complex fluorescent measurements and many of the variables of indirect enzyme immunoassays (EIAs). AquaLite, a recombinant form of the photoprotein aequorin from a bioluminescent jellyfish, is coupled directly to antibodies to prepare bioluminescent conjugates for assay development. When the AquaLite-antibody complex is exposed to a solution containing calcium ions, a flash of blue light ((lambda) max equals 469 nm) is generated. The light signal is measured in commercially available luminometers that simultaneously inject a calcium solution and detect subattomol photoprotein levies in either test tubes or microtiter plates. Immunometric or 'sandwich' type assays are available for the quantitative measurement of human endocrine hormones and nucleic acids. The AquaLite TSH assay can detect 1 attomol of thyroid stimulating hormone (TSH) in 0.2 mL of human serum and is a useful clinical tool for diagnosing hyperthyroid patients. AquaLite-based nucleic acid detection permits quantifying attomol levels of specific nucleic acid markers and represents possible solution to the difficult problem of quantifying the targets of nucleic acid amplification methods.

  1. Detection of 3-phenoxybenzoic acid in river water with a colloidal gold-based lateral flow immunoassay.

    PubMed

    Liu, Yuan; Wu, Aihua; Hu, Jing; Lin, Manman; Wen, Mengtang; Zhang, Xiao; Xu, Chongxin; Hu, Xiaodan; Zhong, Jianfeng; Jiao, Lingxia; Xie, Yajing; Zhang, Cunzhen; Yu, Xiangyang; Liang, Ying; Liu, Xianjin

    2015-08-15

    3-Phenoxybenzoic acid (3-PBA) is a general metabolite of synthetic pyrethroids. It could be used as a generic biomarker for multiple pyrethroids exposure for human or pyrethroid residues in the environment. In this study, monoclonal antibodies (mAbs) against 3-PBA were developed by using PBA-bovine serum albumin (BSA) as an immunogen. In the competitive enzyme-linked immunosorbent assay (ELISA) format, the I50 and I10 values of purified mAbs were 0.63 and 0.13 ?g/ml, respectively, with a dynamic range between 0.19 and 2.04 ?g/ml. Then, the colloidal gold (CG)-based lateral flow immunoassay was established based on the mAbs. The working concentration of coating antigen and CG-labeled antibodies and the blocking effects were investigated to get optimal assay performance. The cutoff value for the assay was 1 ?g/ml 3-PBA, and the detection time was within 10 min. A total of 40 river water samples were spiked with 3-PBA at different levels and determined by the lateral flow immunoassay without any sample pretreatments. The negative false rate was 2.5%, and no positive false results were observed at these levels. This lateral flow immunoassay has the potential to be an on-site screening method for monitoring 3-PBA or pyrethroid residues in environmental samples. PMID:25957127

  2. Respiratory allergy to Aspergillus-derived enzymes in bakers' asthma.

    PubMed

    Quirce, S; Cuevas, M; Díez-Gómez, M; Fernández-Rivas, M; Hinojosa, M; González, R; Losada, E

    1992-12-01

    Baking and food industry workers are exposed to several powdered Aspergillus-derived enzymes with carbohydrate-cleaving activity that are commonly used to enhance baked products. We describe a retrospective study of sensitization to fungal alpha-amylase and cellulase on bakers. Five bakers in whom respiratory allergy symptoms developed when they were exposed to bread "improvers" that contained fungal alpha-amylase and cellulase were investigated by in vivo and in vitro tests. Type I hypersensitivity to these enzymes was demonstrated in the five patients by means of skin testing, histamine release test, positive reverse enzyme-immunoassay for specific IgE antibodies, and bronchial provocation test response to alpha-amylase or cellulase or both. Isolated immediate and dual responses to the bronchial challenge tests with these enzymes were observed. Immunoblot analysis with use of a pooled serum identified IgE-binding components in both enzymes. In the reverse-enzyme immunoassay-inhibition assays cross-reactivity between alpha-amylase and cellulase was not found, but some degree of cross-reactivity between alpha-amylase and A. oryzae, and between cellulase and A. niger was demonstrated. Four of the patients were also sensitized to cereal flour. Aspergillus-derived enzymes used as flour additives can elicit IgE-mediated respiratory allergy, and this fact has to be considered in the diagnosis and clinical management of bakers' asthma. PMID:1281180

  3. Concentration Gradient Immunoassay I. A Rapid Immunoassay Based on Interdiffusion and Surface Binding in a Microchannel

    PubMed Central

    Nelson, Kjell E.; Foley, Jennifer O.; Yager, Paul

    2008-01-01

    We describe a novel microfluidic immunoassay method based on the diffusion of a small molecule analyte into a parallel-flowing stream containing cognate antibody. This interdiffusion results in a steady-state gradient of antibody binding site occupancy transverse to convective flow. In contrast to the diffusion immunoassay (Hatch et al. Nature Biotechnology,19:461−465 (2001)), this antibody occupancy gradient is interrogated by a sensor surface coated with a functional analog of the analyte. Antibodies with at least one unoccupied binding site may specifically bind to this functionalized surface, leading to a quantifiable change in surface coverage by the antibody. SPR imaging is used to probe the spatial distribution of antibody binding to the surface and, therefore, the outcome of the assay. We show that the pattern of antibody binding to the SPR sensing surface correlates with the concentration of a model analyte (phenytoin) in the sample stream. Using an inexpensive disposable microfluidic device, we demonstrate assays for phenytoin ranging in concentration from 75 to 1000 nM in phosphate buffer. At a total volumetric flow rate of 90 nL/sec, the assays are complete within 10 minutes. Inclusion of an additional flow stream on the side of the antibody stream opposite to that of the sample enables simultaneous calibration of the assay. This assay method is suitable for rapid quantitative detection of low-molecular weight analytes for point-of-care diagnostic instrumentation. PMID:17437332

  4. Architecture of Human IgM in Complex with P. falciparum Erythrocyte Membrane Protein 1.

    PubMed

    Akhouri, Reetesh Raj; Goel, Suchi; Furusho, Hirotoshi; Skoglund, Ulf; Wahlgren, Mats

    2016-02-01

    Plasmodium falciparum virulence is associated with sequestration of infected erythrocytes. Microvascular binding mediated by PfEMP1 in complex with non-immune immunoglobulin M (IgM) is common among parasites that cause both severe childhood malaria and pregnancy-associated malaria. Here, we present cryo-molecular electron tomography structures of human IgM, PfEMP1 and their complex. Three-dimensional reconstructions of IgM reveal that it has a dome-like core, randomly oriented Fab2s units, and the overall shape of a turtle. PfEMP1 is a C- shaped molecule with a flexible N terminus followed by an arc-shaped backbone and a bulky C terminus that interacts with IgM. Our data demonstrate that the PfEMP1 binding pockets on IgM overlap with those of C1q, and the bulkiness of PfEMP1 limits the capacity of IgM to interact with PfEMP1. We suggest that P. falciparum exploits IgM to cluster PfEMP1 into an organized matrix to augment its affinity to host cell receptors. PMID:26776517

  5. Capillary Isoelectric Focusing Immunoassay for Fat Cell Differentiation Proteomics

    PubMed Central

    Johlfs, Mary G.; Gorjala, Priyatham; Urasaki, Yasuyo; Le, Thuc T.; Fiscus, Ronald R.

    2015-01-01

    Profiling cellular proteome is critical to understanding signal integration during cell fate determination. In this study, the capability of capillary isoelectric focusing (cIEF) immunoassays to detect post-translational modifications (PTM) of protein isoforms is demonstrated. cIEF immunoassays exhibit protein detection sensitivity at up to 5 orders of magnitude higher than traditional methods. This detection ultra-sensitivity permits proteomic profiling of several nanograms of tissue samples. cIEF immunoassays are employed to simultaneously profile three protein kinases during fat cell differentiation: cGMP-dependent protein kinase type I (PKG-I) of the nitric oxide (NO) signaling pathway, protein kinase B (Akt) of the insulin signaling pathway, and extracellular signal-regulated kinase (ERK) of the mitogen-activated protein kinase (MAPK) signaling pathway. Interestingly, a switch in the expression level of PKG- isoforms is observed during fat cell differentiation. While both PKG-Iα and PKG-Iβ isoforms are present in preadipocytes, only PKG-Iβ isoform is expressed in adipocytes. On the other hand, the phosphorylation level increases for Akt while decreases for ERK1 and ERK2 following the maturation of preadipocytes into adipocytes. Taken together, cIEF immunoassay provides a highly sensitive means to study fat cell differentiation proteomics. cIEF immunoassay should be a powerful proteomics tool to study complex protein signal integration in biological systems. PMID:26132171

  6. The Effects of Sample Matrices on Immunoassays to Detect Microcystin-LR in Water

    EPA Science Inventory

    Abstract: Immunoassays are widely used biochemical techniques to detect microcystins in environmental samples. The use of immunoassays for the detection of microcystins is vulnerable to matrix components and other interferents. This study is an evaluation of the effects of interf...

  7. PRNP variants in goats reduce sensitivity of detection of PrPSc by immunoassay

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Immunoassays are extensively utilized in disease diagnostics with monoclonal antibodies serving as critical tools within the assay. Detection of scrapie in sheep and goats relies heavily on immunoassays including immunohistochemistry, western blotting, and ELISA. In the United States, regulatory tes...

  8. Magnetic separation and immunoassay of multi-antigen based on surface enhanced Raman spectroscopy.

    PubMed

    Chen, Shuai; Yuan, Yaxian; Yao, Jianlin; Han, Sanyang; Gu, Renao

    2011-04-14

    A novel and highly sensitive immunoassay method based on surface enhanced Raman spectroscopy (SERS) and magnetic particles has been developed. This method exhibits great potential application in bio-separation and immunoassay. PMID:21359307

  9. SUPERFUND INNOVATIVE TECHNOLOGY EVALUATION (SITE) REPORT FOR THE WESTINGHOUSE BIOANALYTICAL SYSTEMS PENTACHLOROPHENOL (PCP) IMMUNOASSAYS

    EPA Science Inventory

    The results of the demonstration of two Westinghouse Bio-Analytic Systems (WBAS) immunoassay technologies are described in this report. The immunoassays measure parts per billion concentrations of pentachlorophenol in environmental water samples. The study was conducted under the...

  10. Toxicological Effects of Nickel Chloride on IgA+ B Cells and sIgA, IgA, IgG, IgM in the Intestinal Mucosal Immunity in Broilers

    PubMed Central

    Wu, Bangyuan; Cui, Hengmin; Peng, Xi; Fang, Jing; Zuo, Zhicai; Deng, Junliang; Huang, Jianying

    2014-01-01

    The objective of this study was to investigate the toxicological effects of dietary NiCl2 on IgA+ B cells and the immunoglobulins including sIgA, IgA, IgG and IgM in the small intestine and cecal tonsil of broilers by the methods of immunohistochemistry and enzyme-linked immunosorbent assay (ELISA). Two hundred and forty one-day-old avian broilers were randomly divided into four groups and fed on a control diet and three experimental diets supplemented with 300, 600, and 900 mg/kg NiCl2 for 42 days. Compared with the control group, the IgA+ B cell number and the sIgA, IgA, IgG, and IgM contents in the NiCl2-treated groups were significantly decreased (p < 0.05 or p < 0.01). It was concluded that dietary NiCl2 in the excess of 300 mg/kg had negative effects on the IgA+ B cell number and the abovementioned immunoglobulin contents in the small intestine and the cecal tonsil. NiCl2-reduced sIgA, IgA, IgG and IgM contents is due to decrease in the population and/or the activation of B cell. The results suggest that NiCl2 at high levels has intestinal mucosal humoral immunotoxicity in animals. PMID:25116637

  11. Inadequacy of IgM antibody tests for diagnosis of Rocky Mountain Spotted Fever.

    PubMed

    McQuiston, Jennifer H; Wiedeman, Caleb; Singleton, Joseph; Carpenter, L Rand; McElroy, Kristina; Mosites, Emily; Chung, Ida; Kato, Cecilia; Morris, Kevin; Moncayo, Abelardo C; Porter, Susan; Dunn, John

    2014-10-01

    Among 13 suspected Rocky Mountain spotted fever (RMSF) cases identified through an enhanced surveillance program in Tennessee, antibodies to Rickettsia rickettsii were detected in 10 (77%) patients using a standard indirect immunofluorescent antibody (IFA) assay. Immunoglobulin M (IgM) antibodies were observed for 6 of 13 patients (46%) without a corresponding development of IgG, and for 3 of 10 patients (30%) at least 1 year post-onset. However, recent infection with a spotted fever group rickettsiae could not be confirmed for any patient, based on a lack of rising antibody titers in properly timed acute and convalescent serologic specimens, and negative findings by polymerase chain reaction testing. Case definitions used in national surveillance programs lack specificity and may capture cases that do not represent current rickettsial infections. Use of IgM antibodies should be reconsidered as a basis for diagnosis and public health reporting of RMSF and other spotted fever group rickettsiae in the United States. PMID:25092818

  12. Immunochemical and clinical effects of immunosuppressive treatment in monoclonal IgM neuropathy.

    PubMed Central

    Ernerudh, J H; Vrethem, M; Andersen, O; Lindberg, C; Berlin, G

    1992-01-01

    A pathogenic role of the M protein in monoclonal IgM neuropathy has been suggested. This is based among other things on a close relation between immunosuppressive treatment, lowered concentration of M protein, and clinical effect. We studied five patients with monoclonal IgM and antibodies to peripheral nerve myelin. The immunosuppressive treatment was beneficial in three of the patients. In three patients there was a relationship between antibody concentration and clinical effect (in one there was no change in antibody concentrations and correspondingly no change in clinical status, and in two patients clinical improvement corresponded to decreased antibody concentrations). In two patients, however, there was no clear correlation, since one patient improved despite increasing antibody concentrations and one patient did not improve despite a lowered antibody concentration. It is therefore possible that other mechanisms may contribute to the effect of treatment. Images PMID:1279127

  13. Human cord blood contains an IGM antibody to the 41KD flagellar antigen of Borrelia burgdorferi.

    PubMed

    Cooke, W D; Orr, A S; Wiseman, B L; Rouse, S B; Murray, W C; Ranck, S G

    1993-10-01

    Natural antibodies are the IgM products of fetal and neonatal B cells. These are germline encoded low affinity antibodies with multiple specificities to self and exogenous antigens. Lyme borreliosis is the disease resulting from infection with the spirochete, Borrelia burgdorferi. The humoral response to this organism is brisk, directed at multiple proteins, and persistent. Antibody to the 41kd flagellar antigen is found early in disease, but may also be found in non-exposed individuals. These properties suggest that the anti-41kd antibody may be a natural antibody. We report here the finding of an IgM anti-41kd reactivity in 29% of cord blood samples from patients in an area non-endemic for Lyme disease. The results are consistent with the hypothesis that antibody to flagellin may be a germline encoded natural antibody, and could be important in the immunopathogenesis of Lyme arthritis and other arthritides. PMID:8211003

  14. Antibodies to steroids from a small human naive IgM library.

    PubMed

    Dörsam, H; Rohrbach, P; Kürschner, T; Kipriyanov, S; Renner, S; Braunagel, M; Welschof, M; Little, M

    1997-09-01

    Human antibodies specific for digoxigenin, estradiol, testosterone and progesterone have been isolated from a small combinatorial IgM repertoire (4 x 10(7)) of single chain antibodies (scFv). The affinities of both the anti-estradiol and antiprogesterone scFv were approximately 10(8) M(-1). Naive IgM genes appeared to be highly represented, since only the heavy chain variable domain of the anti estradiol antibody contained differences to corresponding germline sequences. The light chain variable domain of the progesterone receptor was also identical to a germline sequence, showing that it is possible for completely naive antibodies to bind steroids with affinities comparable to those obtained after a secondary immune response. PMID:9305722

  15. Pancreatic Enzymes

    MedlinePlus

    ... This fluid contains pancreatic enzymes to help with digestion and bicarbonate to neutralize stomach acid as it ... the formation of toxic substances due to incomplete digestion of proteins. Increased risk for intestinal infections. Amylase ...

  16. The Evolution of Multiple Isotypic IgM Heavy Chain Genes in the Shark1

    PubMed Central

    Lee, Victor; Huang, Jing Li; Lui, Ming Fai; Malecek, Karolina; Ohta, Yuko; Mooers, Arne; Hsu, Ellen

    2008-01-01

    The IgM H chain gene organization of cartilaginous fishes consists of 15–200 miniloci, each with a few gene segments (VH-D1-D2-JH) and one C gene. This is a gene arrangement ancestral to the complex IgH locus that exists in all other vertebrate classes. To understand the molecular evolution of this system, we studied the nurse shark, which has relatively fewer loci, and characterized the IgH isotypes for organization, functionality, and the somatic diversification mechanisms that act upon them. Gene numbers differ slightly between individuals (~15), but five active IgM subclasses are always present. Each gene undergoes rearrangement that is strictly confined within the minilocus; in B cells there is no interaction between adjacent loci located ≥120 kb apart. Without combinatorial events, the shark IgM H chain repertoire is based on junctional diversity and, subsequently, somatic hypermutation. We suggest that the significant contribution by junctional diversification reflects the selected novelty introduced by RAG in the early vertebrate ancestor, whereas combinatorial diversity coevolved with the complex translocon organization. Moreover, unlike other cartilaginous fishes, there are no germline-joined VDJ at any nurse shark μ locus, and we suggest that such genes, when functional, are species-specific and may have specialized roles. With an entire complement of IgM genes available for the first time, phylogenetic analyses were performed to examine how the multiple Ig loci evolved. We found that all domains changed at comparable rates, but VH appears to be under strong positive selection for increased amino acid sequence diversity, and surprisingly, so does Cμ2. PMID:18490746

  17. Normal human serum contains a natural IgM antibody cytotoxic for human neuroblastoma cells.

    PubMed

    Ollert, M W; David, K; Schmitt, C; Hauenschild, A; Bredehorst, R; Erttmann, R; Vogel, C W

    1996-04-30

    Neuroblastoma (NB) is characterized by the second highest spontaneous regression of any human malignant disorder, a phenomenon that remains to be elucidated. In this study, a survey of 94 normal human adult sera revealed a considerable natural humoral cytotoxicity against human NB cell lines in approximately one-third of the tested sera of both genders. Specific cell killing by these sera was in the range of 40% to 95%. Serum cytotoxicity was dependent on an intact classical pathway of complement. By several lines of evidence, IgM antibodies were identified as the cytotoxic factor in the sera. Further analyses revealed that a 260-kDa protein was recognized by natural IgM of cytotoxic sera in Western blots of NB cell extracts. The antigen was expressed on the surface of seven human NB cell lines but not on human melanoma or other control tumor cell lines derived from kidney, pancreas, colon, bone, skeletal muscle, lymphatic system, and bone marrow. Furthermore, no reactivity was observed with normal human fibroblasts, melanocytes, and epidermal keratinocytes. The antigen was expressed in vivo as detected by immunohistochemistry in both the tumor of a NB patient and NB tumors established in nude rats from human NB cell lines. Most interestingly, the IgM anti-NB antibody was absent from the sera of 11 human NB patients with active disease. The anti-NB IgM also could not be detected in tumor tissue obtained from a NB patient. Collectively, our data suggest the existence of a natural humoral immunological tumor defense mechanism, which could account for the in vivo phenomenon of spontaneous NB tumor regression. PMID:8633097

  18. The evolution of multiple isotypic IgM heavy chain genes in the shark.

    PubMed

    Lee, Victor; Huang, Jing Li; Lui, Ming Fai; Malecek, Karolina; Ohta, Yuko; Mooers, Arne; Hsu, Ellen

    2008-06-01

    The IgM H chain gene organization of cartilaginous fishes consists of 15-200 miniloci, each with a few gene segments (V(H)-D1-D2-J(H)) and one C gene. This is a gene arrangement ancestral to the complex IgH locus that exists in all other vertebrate classes. To understand the molecular evolution of this system, we studied the nurse shark, which has relatively fewer loci, and characterized the IgH isotypes for organization, functionality, and the somatic diversification mechanisms that act upon them. Gene numbers differ slightly between individuals ( approximately 15), but five active IgM subclasses are always present. Each gene undergoes rearrangement that is strictly confined within the minilocus; in B cells there is no interaction between adjacent loci located > or =120 kb apart. Without combinatorial events, the shark IgM H chain repertoire is based on junctional diversity and, subsequently, somatic hypermutation. We suggest that the significant contribution by junctional diversification reflects the selected novelty introduced by RAG in the early vertebrate ancestor, whereas combinatorial diversity coevolved with the complex translocon organization. Moreover, unlike other cartilaginous fishes, there are no germline-joined VDJ at any nurse shark mu locus, and we suggest that such genes, when functional, are species-specific and may have specialized roles. With an entire complement of IgM genes available for the first time, phylogenetic analyses were performed to examine how the multiple Ig loci evolved. We found that all domains changed at comparable rates, but V(H) appears to be under strong positive selection for increased amino acid sequence diversity, and surprisingly, so does Cmicro2. PMID:18490746

  19. Survivability of immunoassay reagents exposed to the space radiation environment on board the ESA BIOPAN-6 platform as a prelude to performing immunoassays on Mars.

    PubMed

    Derveni, Mariliza; Allen, Marjorie; Sawakuchi, Gabriel O; Yukihara, Eduardo G; Richter, Lutz; Sims, Mark R; Cullen, David C

    2013-01-01

    The Life Marker Chip (LMC) instrument is an immunoassay-based sensor that will attempt to detect signatures of life in the subsurface of Mars. The molecular reagents at the core of the LMC have no heritage of interplanetary mission use; therefore, the design of such an instrument must take into account a number of risk factors, including the radiation environment that will be encountered during a mission to Mars. To study the effects of space radiation on immunoassay reagents, primarily antibodies, a space study was performed on the European Space Agency's 2007 BIOPAN-6 low-Earth orbit (LEO) space exposure platform to complement a set of ground-based radiation studies. Two antibodies were used in the study, which were lyophilized and packaged in the intended LMC format and loaded into a custom-made sample holder unit that was mounted on the BIOPAN-6 platform. The BIOPAN mission went into LEO for 12 days, after which all samples were recovered and the antibody binding performance was measured via enzyme-linked immunosorbent assays (ELISA). The factors expected to affect antibody performance were the physical conditions of a space mission and the exposure to space conditions, primarily the radiation environment in LEO. Both antibodies survived inactivation by these factors, as concluded from the comparison between the flight samples and a number of shipping and storage controls. This work, in combination with the ground-based radiation tests on representative LMC antibodies, has helped to reduce the risk of using antibodies in a planetary exploration mission context. PMID:23286207

  20. Development of Indirect Competitive Immuno-Assay Method Using SPR Detection for Rapid and Highly Sensitive Measurement of Salivary Cortisol Levels

    PubMed Central

    Tahara, Yusuke; Huang, Zhe; Kiritoshi, Tetsuro; Onodera, Takeshi; Toko, Kiyoshi

    2014-01-01

    The monitoring of salivary cortisol as a key biomarker of an individuals stress response has been increasingly focused on. This paper describes the development of a novel cortisol immuno-assay method based on an indirect competitive method using a commercially available surface plasmon resonance instrument. The surface of an Au chip was modified with PEG6-COOH aromatic dialkanethiol self-assembled monolayers and hydrocortisone 3-(O-carboxymethyl) oxime (hydrocortisone 3-CMO) as a cortisol analog. A detection limit of 38?ppt range with a measurement range of 10?ppt100?ppb was accomplished without the incubation of a mixing solution consisting of standard cortisol and an anti-cortisol antibody, and the time for quantification of cortisol concentration was 8?min from the sample injection. We experimentally compared our immuno-assay with a commercialized salivary cortisol enzyme-linked immunosorbent assay (ELISA) kit using human saliva samples. It was found that the results obtained by the cortisol immuno-assay had a good correlation with those obtained by ELISA assay (R?=?0.96). Our findings indicate the potential utility of the cortisol immuno-assay for measurements of human salivary cortisol levels. PMID:25152888

  1. Development of a Bead Immunoassay To Measure Vi Polysaccharide-Specific Serum IgG after Vaccination with the Salmonella enterica Serovar Typhi Vi Polysaccharide ▿

    PubMed Central

    Staats, Herman F.; Kirwan, Shaun M.; Whisnant, Carol C.; Stephenson, James L.; Wagener, Diane K.; Majumder, Partha P.

    2010-01-01

    Vi polysaccharide from Salmonella enterica serotype Typhi is used as one of the available vaccines to prevent typhoid fever. Measurement of Vi-specific serum antibodies after vaccination with Vi polysaccharide by enzyme-linked immunosorbent assay (ELISA) may be complicated due to poor binding of the Vi polysaccharide to ELISA plates resulting in poor reproducibility of measured antibody responses. We chemically conjugated Vi polysaccharide to fluorescent beads and performed studies to determine if a bead-based immunoassay provided a reproducible method to measure vaccine-induced anti-Vi serum IgG antibodies. Compared to ELISA, the Vi bead immunoassay had a lower background and therefore a greater signal-to-noise ratio. The Vi bead immunoassay was used to evaluate serum anti-Vi IgG in 996 subjects from the city of Kolkata, India, before and after vaccination. Due to the location being one where Salmonella serotype Typhi is endemic, approximately 45% of the subjects had protective levels of anti-Vi serum IgG (i.e., 1 μg/ml anti-Vi IgG) before vaccination, and nearly 98% of the subjects had protective levels of anti-Vi serum IgG after vaccination. Our results demonstrate that a bead-based immunoassay provides an effective, reproducible method to measure serum anti-Vi IgG responses before and after vaccination with the Vi polysaccharide vaccine. PMID:20107010

  2. An electrochemical amplification immunoassay using bi-electrode signal transduction system.

    PubMed

    Chen, Zhao-Peng; Jiang, Jian-Hui; Zhang, Xiao-Bing; Shen, Guo-Li; Yu, Ru-Qin

    2007-03-30

    An electrochemical immunoassay technique has been developed based on the sensitive detection of the enzyme-generated product with a bi-electrode signal transduction system. The system uses two separate electrodes, an immunoelectrode and a detection electrode to form a galvanic cell to implement the redox reactions on two different electrodes, that is the enzyme-generated reductant in the anode region is electrochemically oxidized by an oxidant (silver ions) in the cathode apartment. Based on a sandwich procedure, after immunoelectrode with antibody immobilized on its surface bound with the corresponding antigen and alkaline phosphatase conjugated antibody successively, the immunoelectrode was placed in enzyme reaction solution and wired to the detection electrode which was immerged into a silver deposition solution. These two solutions are connected with a salt bridge. Thus a bi-electrode signal transduction system device is constructed in which the immunoelectrode acts as anode and the detection electrode serves as cathode. The enzyme bound on the anode surface initiates the hydrolysis of ascorbic acid 2-phosphate to produce ascorbic acid in the anode region. The ascorbic acid produced in the anodic apartment is electrochemically oxidized by silver ions coupled with the deposition of silver metal on the cathode. Via a period of 30min deposition, silver will deposited on the detection electrode in an amount corresponding to the quantity of ascorbic acid produced, leading to a great enhancement in the electrochemical stripping signal due to the accumulation of metallic silver by enzyme-generated product. Compared with the method using chemical deposition of silver, the electrochemical deposition of silver on a separate detection electrode apartment avoids the possible influence of silver deposition on the enzyme activity. PMID:19071559

  3. A patterned recombinant human IgM guides neurite outgrowth of CNS neurons

    NASA Astrophysics Data System (ADS)

    Xu, Xiaohua; Wittenberg, Nathan J.; Jordan, Luke R.; Kumar, Shailabh; Watzlawik, Jens O.; Warrington, Arthur E.; Oh, Sang-Hyun; Rodriguez, Moses

    2013-07-01

    Matrix molecules convey biochemical and physical guiding signals to neurons in the central nervous system (CNS) and shape the trajectory of neuronal fibers that constitute neural networks. We have developed recombinant human IgMs that bind to epitopes on neural cells, with the aim of treating neurological diseases. Here we test the hypothesis that recombinant human IgMs (rHIgM) can guide neurite outgrowth of CNS neurons. Microcontact printing was employed to pattern rHIgM12 and rHIgM22, antibodies that were bioengineered to have variable regions capable of binding to neurons or oligodendrocytes, respectively. rHIgM12 promoted neuronal attachment and guided outgrowth of neurites from hippocampal neurons. Processes from spinal neurons followed grid patterns of rHIgM12 and formed a physical network. Comparison between rHIgM12 and rHIgM22 suggested the biochemistry that facilitates anchoring the neuronal surfaces is a prerequisite for the function of IgM, and spatial properties cooperate in guiding the assembly of neuronal networks.

  4. Nomenclature of Toso, Fas apoptosis inhibitory molecule 3, and IgM FcR.

    PubMed

    Kubagawa, Hiromi; Carroll, Michael C; Jacob, Chaim O; Lang, Karl S; Lee, Kyeong-Hee; Mak, Tak; McAndrews, Monica; Morse, Herbert C; Nolan, Garry P; Ohno, Hiroshi; Richter, Günther H; Seal, Ruth; Wang, Ji-Yang; Wiestner, Adrian; Coligan, John E

    2015-05-01

    Hiromi Kubagawa and John E. Coligan coordinated an online meeting to define an appropriate nomenclature for the cell surface glycoprotein presently designated by different names: Toso, Fas apoptosis inhibitory molecule 3 (FAIM3), and IgM FcR (FcμR). FAIM3 and Faim3 are the currently approved symbols for the human and mouse genes, respectively, in the National Center for Biotechnology Information, Ensembl, and other databases. However, recent functional results reported by several groups of investigators strongly support a recommendation for renaming FAIM3/Faim3 as FCMR/Fcmr, a name better reflecting its physiological function as the FcR for IgM. Participants included 12 investigators involved in studying Toso/FAIM3(Faim3)/FμR, representatives from the Human Genome Nomenclature Committee (Ruth Seal) and the Mouse Genome Nomenclature Committee (Monica McAndrews), and an observer from the IgM research field (Michael Carroll). In this article, we provide a brief background of the key research on the Toso/FAIM3(Faim3)/FcμR proteins, focusing on the ligand specificity and functional activity, followed by a brief summary of discussion about adopting a single name for this molecule and its gene and a resulting recommendation for genome nomenclature committees. PMID:25888699

  5. Far-UV and UV Observations of the Low Redshift IGM

    SciTech Connect

    Savage, Blair D.

    2009-05-24

    Most of the normal baryonic matter of the Universe at high and at low redshift is found in the space between galaxies. The study of this matter at low redshift has required access to ultraviolet spectroscopic space observatories. HST and FUSE have revealed that at low redshift {approx}30% of the baryons reside in the cool (T{approx}2x10{sup 4} K) photoionized IGM traced by the Lyman {alpha} forest while another {approx}10-20% might exist in the warm (T{approx}1-9x10{sup 5} K) portion of the warm-hot IGM. Evidence for the warm gas is provided by some of the O VI absorbers, the Ne VIII absorbers and very broad Lyman alpha absorbers. Understanding the physical conditions in these absorbers is important for reliably determining their baryonic content. The low z IGM is likely to play a crucial role in the evolution of the {approx}8% of the baryons found in galaxies.

  6. Fenton reaction-based colorimetric immunoassay for sensitive detection of brevetoxin B.

    PubMed

    Lai, Wenqiang; Wei, Qiaohua; Zhuang, Junyang; Lu, Minghua; Tang, Dianping

    2016-06-15

    We designed a new colorimetric immunoassay for sensitive monitoring of brevetoxin B (BTB) using enzyme-controlled Fenton reaction with a high-resolution 3,3',5,5'-tetramethylbenzidine (TMB)-based visual colored system. Upon addition of hydrogen peroxide (H2O2), the equivalent iron(II) could be first converted into iron(III) and free hydroxyl radical (•OH) via the classical Fenton reaction. Then the as-produced iron(III) and •OH could cause a perceptible change from colorless to blue with the increasing H2O2 concentration in the presence of TMB. Based on Fenton reaction-triggered visual colored system, a novel competitive-type colorimetric enzyme immunoassay was developed for the quantitative screening of target BTB on the bovine serum albumin-BTB-modified magnetic bead using glucose oxidase/anti-BTB antibody-labeled gold nanoparticle as the signal-transduction tag. Upon target BTB introduction, the analyte competed with the conjugated BTB on the magnetic bead for anti-BTB antibody on gold nanoparticle. The carried glucose oxidase with the gold nanoparticle could implement the oxidation of glucose to produce H2O2, and the generated H2O2 promoted the above-mentioned Fenton reaction for color development. Under the optimal conditions, the absorbance decreased with the increasing target BTB in the range from 0.1 to 150ngkg(-1) with a low detection limit (LOD) of 0.076ngkg(-1). The LOD was 500-fold lower than that of commercialized Abraxis BTB ELISA kit. Non-specific adsorption was not observed. The precision, reproducibility and specificity were acceptable. Finally, the method accuracy was also validated for monitoring spiked seafood samples, giving results well matched with the referenced brevetoxin ELISA kit. PMID:26851583

  7. Immunoassay for mercury in seafood and animal tissues

    SciTech Connect

    Carlson, L.; Holmquist, B.; Ladd, R.; Riddell, M.

    1995-12-01

    Methylmercury accumulates to high levels in the tissues of fish and other animals through biomagnification. Since methylmercury is extremely toxic, it is important to identify fish or animal tissues with mercury levels too high for human consumption. Current methods for the analysis of mercury are expensive and time- consuming, and they must be performed in a laboratory setting. In this study, a rapid and inexpensive mercury-specific immunoassay developed by BioNebraska was used to measure total mercury in tissue following acid digestion and methylmercury decomposition. A good correlation was obtained between the immunoassay and cold vapor atomic absorption spectrophotometry (CVAAS). Use of the mercury immunoassay will facilitate the rapid screening of large numbers of tissue samples.

  8. Solid-phase enzyme immunoassay or radioimmunoassay for the detection of immune complexes based on their recognition by conglutinin: conglutinin-binding test. A comparative study with 125I-labelled C1q binding and Raji-cell RIA tests

    PubMed Central

    Casali, P.; Bossus, A.; Carpentier, Nicole A.; Lambert, P.-H.

    1977-01-01

    Bovine conglutinin was used in a solid-phase assay for the detection of immune complexes. In a first step, the tested serum sample is incubated in polypropylene tubes coated with conglutinin to allow C3-coated immune complexes to bind to solid-phase conglutinin. In a second step, the conglutinin-bound complexes are detected using an enzyme-conjugated or radiolabelled anti-immunoglobulin antibody. The conglutinin-binding (KgB) test does not suffer from the interference of DNA, heparin or endotoxins. Its limit of sensitivity for aggregated IgG is 3 μg/ml undiluted human serum. Immune complexes prepared in vitro using tetanus toxoid, or DNA, and corresponding antibodies in human sera could be detected at various antigen/antibody ratios and at antibody concentrations lower than 8 μg/ml. The KgB test allowed for the detection of immune complexes in sera from patients with systemic lupus erythematosus, rheumatoid arthritis, idiopathic vasculitis, leprosy and leukemia. These sera were also tested using the 125I-labelled Clq-binding activity (BA) test and the KgB test simultaneously, and a significant rank order correlation was observed. In patients with leukemia, a significant correlation was observed using three tests, KgB, 125I-labelled Clq BA and Raji-cell radioimmunoassay (RIA). Therefore, the KgB test appears as a simple and reproducible method, utilizing a very stable reagent, with a sensitivity and specificity comparable to the other tests studied and allowing for clinical application. PMID:332422

  9. Comparative biochemical characterization of a human IgM produced in both ascites and in vitro cell culture

    SciTech Connect

    Monica, T.J.; Goochee, C.F.; Maiorella, B.L. )

    1993-04-01

    Researchers have conducted a comparative analysis of a monoclonal human IgM obtained from cells cultured in nude-mouse ascites and from the same cells cultured in a bioreactor. We studied the glycosylation of the IgMs using lectin blotting and high-pH anion-exchange chromatography with pulsed amperometric detection (HPAE-PAD), and we also developed reverse phase liquid chromatography (RPLC) peptide maps of the IgM samples. The HPAE-PAD data indicate that the samples differ in both the type and distribution of oligosaccharides present on the IgMs. In addition, the proteins differ in their solubility behavior and in their RPLC peptide maps. We conclude that the method of cell culture of capable of significantly altering the characteristics of the glycoprotein product. 27 refs., 5 figs.

  10. Pathogenic Relevance of IgG and IgM Antibodies against Desmoglein 3 in Blister Formation in Pemphigus Vulgaris

    PubMed Central

    Tsunoda, Kazuyuki; Ota, Takayuki; Saito, Masataka; Hata, Tsuyoshi; Shimizu, Atsushi; Ishiko, Akira; Yamada, Taketo; Nakagawa, Taneaki; Kowalczyk, Andrew P.; Amagai, Masayuki

    2011-01-01

    Pemphigus vulgaris is an autoimmune disease caused by IgG antibodies against desmoglein 3 (Dsg3). Previously, we isolated a pathogenic mAb against Dsg3, AK23 IgG, which induces a pemphigus vulgaris-like phenotype characterized by blister formation. In the present study, we generated a transgenic mouse expressing AK23 IgM to examine B-cell tolerance and the pathogenic role of IgM. Autoreactive transgenic B cells were found in the spleen and lymph nodes, whereas anti-Dsg3 AK23 IgM was detected in the cardiovascular circulation. The transgenic mice did not develop an obvious pemphigus vulgaris phenotype, however, even though an excess of AK23 IgM was passively transferred to neonatal mice. Similarly, when hybridoma cells producing AK23 IgM were inoculated into adult mice, no blistering was observed. Immunoelectron microscopy revealed IgM binding at the edges of desmosomes or interdesmosomal cell membranes, but not in the desmosome core, where AK23 IgG binding has been frequently detected. Furthermore, in an in vitro dissociation assay using cultured keratinocytes, AK23 IgG and AK23 IgM F(ab?)2 fragments, but not AK23 IgM, induced fragmentation of epidermal sheets. Together, these observations indicate that antibodies must gain access to Dsg3 integrated within desmosomes to induce the loss of keratinocyte cell-cell adhesion. These findings provide an important framework for improved understanding of B-cell tolerance and the pathophysiology of blister formation in pemphigus. PMID:21718682

  11. An interference-free and rapid electrochemical lateral-flow immunoassay for one-step ultrasensitive detection with serum.

    PubMed

    Akanda, Md Rajibul; Joung, Hyou-Arm; Tamilavan, Vellaiappillai; Park, Seonhwa; Kim, Sinyoung; Hyun, Myung Ho; Kim, Min-Gon; Yang, Haesik

    2014-03-21

    Point-of-care testing (POCT) of biomarkers in clinical samples is of great importance for rapid and cost-effective diagnosis. However, it is extremely challenging to develop an electrochemical POCT technique retaining both ultrasensitivity and simplicity. We report an interference-free electrochemical lateral-flow immunoassay that enables one-step ultrasensitive detection with serum. The electrochemical-chemical-chemical (ECC) redox cycling combined with an enzymatic reaction of an enzyme label is used to obtain high signal amplification. The ECC redox cycling involving Ru(NH3)6(3+), enzyme product, and tris(3-carboxyethyl)phosphine (TCEP) depends on pH, because the formal potentials of an enzyme product and TCEP increase with decreasing pH although that of Ru(NH3)6(3+) is pH-independent. With consideration of the pH dependence of ECC redox cycling, a noble combination of enzyme label, substrate, and product [β-galactosidase, 4-amino-1-naphthyl β-D-galactopyranoside, and 4-amino-1-naphthol, respectively] is introduced to ensure fast and selective ECC redox cycling of the enzyme product along with a low background level. The selective ECC redox cycling at a low applied potential (0.05 V vs. Ag/AgCl) minimizes the interference effect of electroactive species (L-ascorbic acid, acetaminophen, and uric acid) in serum. A detection limit of 0.1 pg mL(-1) for troponin I is obtained only 11 min after serum dropping without the use of an additional solution. Moreover, the lateral-flow immunoassay is applicable to the analysis of real clinical samples. PMID:24482801

  12. Peroxidase-like activity of mesoporous silica encapsulated Pt nanoparticle and its application in colorimetric immunoassay.

    PubMed

    Wang, Zhifei; Yang, Xia; Yang, Jingjing; Jiang, Yanyun; He, Nongyue

    2015-03-01

    Nanomaterial-based artificial enzymes have received great attention in recent year due to their potential application in immunoassay techniques. However, such potential is usually limited by poor dispersion stability or low catalytic activity induced by the capping agent essentially required in the synthesis. In an attempt to address these challenges, here, we studied the novel Pt nanoparticles (NPs) based peroxidase-like mimic by encapsulating Pt NP in mesoporous silica (Pt@mSiO2 NPs). Compared with other nanomaterial-based artificial enzymes, the obtained Pt@mSiO2 NPs not only exhibit high peroxidase-like activity but also have good dispersion stability in buffer saline solution when grafted with spacer PEG. Results show that when the thickness of silica shell is about 9 nm the resulting Pt@mSiO2 NPs exhibit the catalytic activity similar to that of Pt NPs, which is approximately 26 times higher than that of Fe3O4 NPs (in terms of Kcat for H2O2). Due to the protection of silica shell, the subsequent surface modification with antibody has little effect on their catalytic activity. The analytical performance of this system in detecting hCG shows that after 5 min incubation the limit of detection can reach 10 ng mL(-1) and dynamic linear working range is 5-200 ng mL(-1). Our findings pave the way for design and development of novel artificial enzyme labeling. PMID:25682428

  13. Tyramine-based enzymatic conjugate repeats for ultrasensitive immunoassay accompanying tyramine signal amplification with enzymatic biocatalytic precipitation.

    PubMed

    Hou, Li; Tang, Yun; Xu, Mingdi; Gao, Zhuangqiang; Tang, Dianping

    2014-08-19

    A new impedimetric immunoassay protocol based on enzyme-triggered formation of tyramine-enzyme repeats on gold nanoparticle (AuNP) was designed for highly sensitive detection of carcinoembryonic antigen (CEA, as a model) by virtue of utilizing enzymatic biocatalytic precipitation toward 4-chloro-1-naphthol (4-CN) on anti-CEA antibody (Ab1)-modified immunosensor. Initially, AuNP was functionalized with horseradish peroxidase and detection antibody (HRP-AuNP-Ab2), and then HRP-tyramine conjugate was utilized for the formation of tyramine-HRP repeats through the triggering of the immobilized HRP on the AuNP with the aid of H2O2. In the presence of target CEA, the carried HRP-tyramine repeats accompanying the sandwiched immunocomplex catalyzed the 4-CN oxidation to produce an insoluble precipitation on the immunosensor, thus causing a local alteration of the conductivity. Three signal-transduction tags including HRP-Ab2, HRP-AuNP-Ab2, and HRP-AuNP-Ab2 with HRP-tyramine repeats were employed for target CEA evaluation, and improved analytical properties were achieved by HRP-AuNP-Ab2 with HRP-tyramine repeats. Using the unique signal-transduction tag, the analytical performance of the impedimetric immunoassay was studied in detail. Under the optimal conditions, the impedimetric immunosensor displayed a wide dynamic working range of between 0.5 pg mL(-1) and 40 ng mL(-1) with a detection limit (LOD) of 0.38 pg mL(-1) relative to target CEA. The coefficients of variation (CVs) were ≤9.3% and 13.3% for the intra-assay and interassay, respectively. The levels of CEA in eight clinical serum specimens were measured by using the developed impedimetric immunosensor. The obtained results correlated well with those from the electrochemiluminescent (ECL)-based immunoassay with a correlation coefficient of 0.998. PMID:25088522

  14. Development of an Enzyme-Linked Immunosorbent Assay for Immunoglobulin M Antibodies against Measles Virus

    PubMed Central

    Roodbari, F.; Roustai, M. H.; Mostafaie, A.; Soleimanjdahi, H.; Foroshani, R. Sarrami; Sabahi, F.

    2003-01-01

    Measles is a highly contagious respiratory virus infection, with typical clinical symptoms including maculopapular rash, fever, cough, coryza, and conjunctivitis. Despite implementation of widespread vaccination programs throughout the world, the rates of global morbidity and mortality are still considerable. This study was performed to design a reliable indirect enzyme-linked immunosorbent assay (ELISA) to measure measles-specific immunoglobulin M (IgM). First, human IgM was purified, and then an anti-IgM antibody was produced in rabbits and purified in a multistep process. The rabbit IgG against human IgM was conjugated with peroxidase. Measles virus-infected Vero cells produced viral antigen. One hundred serum samples from infants of 9 to 18 months of age, mostly vaccinated, were evaluated for determining the presence of specific IgM antibodies against measles virus. The samples were also evaluated for neutralizing antibodies against measles virus by a microneutralization test (MNT). By comparing the results of the ELISA with those of MNT, it was demonstrated that ELISA had a sensitivity and specificity of 100 and 92%, respectively. On the other hand, when the results obtained by our ELISA system were compared with those of an imported measles virus IgM ELISA kit (EIAgen; Adaltis Italia SPa, Bologna, Italy), a high level of agreement was shown (k = 0.926). PMID:12738645

  15. Zinc Enzymes.

    ERIC Educational Resources Information Center

    Bertini, I.; And Others

    1985-01-01

    Discusses the role of zinc in various enzymes concerned with hydration, hydrolysis, and redox reactions. The binding of zinc to protein residues, properties of noncatalytic zinc(II) and catalytic zinc, and the reactions catalyzed by zinc are among the topics considered. (JN)

  16. Food Enzymes

    ERIC Educational Resources Information Center

    McBroom, Rachel; Oliver-Hoyo, Maria T.

    2007-01-01

    Many students view biology and chemistry as two unrelated, separate sciences; how these courses are generally taught in high schools may do little to change that impression. The study of enzymes provide a great opportunity for both biology and chemistry teachers to share with students the interdisciplinary nature of science. This article describes

  17. Food Enzymes

    ERIC Educational Resources Information Center

    McBroom, Rachel; Oliver-Hoyo, Maria T.

    2007-01-01

    Many students view biology and chemistry as two unrelated, separate sciences; how these courses are generally taught in high schools may do little to change that impression. The study of enzymes provide a great opportunity for both biology and chemistry teachers to share with students the interdisciplinary nature of science. This article describes…

  18. CAPILLARY ELECTROPHORESIS IMMUNOASSAY FOR 2,4-DICHLOROPHENOXYACETIC ACID

    EPA Science Inventory

    A capillary electrophoresis (CE) immunoassay format for 2,4-dichlorophenoxyacetic acid (2,4-D) is demonstrated. A fluorescent labeled 2,4-D analog competes with the analyte of interest for a finite number of binding sites provided by anti-2,4-D monoclonal antibodies. CE then pr...

  19. Manipulation of Dispersed Magnetic Beads for On-Chip Immunoassay

    NASA Astrophysics Data System (ADS)

    Ishikawa, Tomohiro; Lee, Jaesung; Miyake, Ryo

    2012-04-01

    To provide a simple and low-cost mobile immunoassay platform, a test chip on which dispersed magnetic beads are manipulated was designed and fabricated by a 180 nm standard complementary metal-oxide-semiconductor (CMOS) process. In preliminary experiments, beads that have a diameter of 2.8 µm were successfully manipulated and their motion were captured and analyzed. Then, an immunoassay was conducted on the chip. First, the nonspecific binding of hydrophilic beads coated with an antibody was compared with that of hydrophobic beads that were used for the preliminary experiments. Next, comparison of an immunoassay of mouse IgG with a control assay and a test on the feasibility of the blocking process were conducted simultaneously. The beads coated with the antibody were successfully immobilized onto the chip surface in the presence of the target antigen, which was checked through bead manipulation. This indicates that an immunoassay on an inexpensive CMOS chip is feasible using an affordable amount of driving current.

  20. Effect of phenolic compounds on immunoassays of peanut allergens.

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Phenolic compounds (PCs) are antioxidants. Because of their health benefit, PCs may be added to some food products. Occasionally, these products may be subjected to screening for food allergens (i.e. peanuts). In this case, the screening (an immunoassay technique) may or may not be affected by the P...