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Sample records for assay elisa immunoassaying

  1. Comparing Assay Performance of ELISA and Chemiluminescence Immunoassay in Detecting Antibodies to Hepatitis B Surface Antigen

    PubMed Central

    Sagar, Siddharth; Vishwanath, Shashidhar; Banerjee, Barnini; Eshwara, Vandana Kalwaje; Chawla, Kiran

    2016-01-01

    Introduction Antibodies to Hepatitis B surface Antigen (Anti-HBs) levels are measured as markers for immune response to vaccination and in decision making for post-exposure prophylaxis against Hepatitis-B. Several immunoassay formats are used to measure Anti-HBs, thus carrying the possibility of variation in measured levels between different assays. This study compares the performance of Chemiluminescence Immunoassay (CLIA) against Enzyme-linked Immunosorbent Assay (ELISA) in measuring Anti-HBs titer by looking into concordance between the two test reports. Aim To compare the agreement between ELISA and CLIA in measurement of Anti–HBs antibody titers. Materials and Methods This prospective comparative study conducted at Kasturba Medical College, Manipal measured consecutive serum samples (69) sent for anti-HBs levels during May-June 2016 using both CLIA (Abbott Architect) and ELISA (Bio-Rad). Anti-HBs values of ≤10mIU/ml was considered as non-protective and >10mIU/ml as protective. The agreement between the tests in classifying the antibody titers as non-protective or protective was computed using Kappa coefficient, and the difference in individual titer values between the tests compared using Bland-Altman plot on SPSS (v.15). Results Out of the 69 samples analysed, 18 samples (26.1%) were of health-care personnel and remaining of patients. Agreement between ELISA and CLIA in identifying the antibody titers as protective and non-protective were 96.5% and 90.9% respectively, resulting in an agreement of 0.84. The coefficient-of-variation of ELISA and CLIA were 74.5% and 113.1%, respectively. Three value based discordant results were noted; two samples deemed protective by ELISA were reported as non-protective by CLIA. One non-protective titer by ELISA was reported as protective by CLIA. Conclusion Analytical agreement is good between the two immunoassays. However there are some discrepancies in quantitative measurement. This may have been due the variation in

  2. 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

  3. 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.

  4. 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.

  5. 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.

  6. Measurement of vasoactive intestinal peptide using a competitive fluorescent microsphere immunoassay or ELISA in human blood samples.

    PubMed

    Song, Eun Young; VanDunk, Cassandra; Kuddo, Thea; Nelson, Phillip G

    2005-05-01

    The concentration of Vasoactive Intestinal Peptide (VIP) as measured by recycling immunoaffinity chromatography (RIC) has been reported to be elevated in the blood of patients with autism as compared with normal subjects. In this study, we have developed a "Competitive Fluorescent Microsphere Immunoassay" (cFMI) in which VIP competes with biotinylated VIP in binding to polyclonal antibodies on microspheres. The results were obtained using the Luminex100 system. We measured VIP in serum, plasma, and material eluted from dried blood spots on filter paper with both the cFMI and an ELISA procedure. We found that a purification procedure was necessary for obtaining useful results from plasma and serum, however, a preincubation step was required with the blood eluates. This newly developed cFMI was more sensitive (2.5 vs. 20.0 pg/ml), and more reproducible than the ELISA. To get accurate measurements of VIP in eluted material high sensitivity is especially important. Thus, the cFMI using the Luminex system has definite advantages over a conventional ELISA including the possibility that samples can be assayed at higher dilutions. We have determined that the VIP concentrations of serum, plasma, and dried blood spot eluate specimens as measured with the cFMI assay system were similar to those measured with ELISA. Thus, the new cFMI using Luminex system may be useful for detection of VIP in human blood samples.

  7. AN ELISA ASSAY FOR HEME OXYGENASE (HO-1)

    EPA Science Inventory

    An ELISA assay for heme oxygenase (HO-l )

    Abstract

    A double antibody capture ELISA for the HO-l protein has been developed to separately quantitate HO-I protein. The use of 2.5% NP40 detergent greatly assists in freeing HO-l protein from membranes and/or other cel...

  8. Validation of an ELISA Synthetic Cannabinoids Urine Assay

    PubMed Central

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

    2015-01-01

    Background Synthetic cannabinoids are touted as legal alternatives to cannabis, at least when first released, and routine urine cannabinoid screening methods do not detect these novel psychoactive substances. Synthetic cannabinoids are widely available, are a major public health and safety problem, and a difficult challenge for drug testing laboratories. We evaluated performance of the NMS JWH-018 direct ELISA kit to sensitively, selectively, and rapidly screen urinary synthetic cannabinoids. Materials/ Methods The NMS ELISA kit targeting the JWH-018 N-(5-hydroxypentyl) metabolite was utilized to screen 2492 urine samples with 5 and 10µg/L cutoffs. A fully validated LC-MS/MS method for 29 synthetic cannabinoids markers confirmed all presumptive positive and negative results. Performance challenges at ±25 and ±50% of cutoffs determined intra- and inter-plate imprecision around proposed cutoffs. Result The immunoassay was linear from 1–500µg/L with intra- and inter-plate imprecision of ≤8.2% and <14.0%, respectively. No interferences were present from 93 common drugs of abuse, metabolites, co-administered drugs, over-the-counter medications or structurally similar compounds, and 19 of 73 individual, synthetic cannabinoids (26%) exhibited moderate to high cross-reactivity to JWH-018 N-(5-hydroxypentyl) metabolite. Sensitivity, specificity, and efficiency results were 83.7%, 99.4% and 97.6% and 71.6%, 99.7% and 96.4%, with the 5 and 10µg/L urine cutoffs, respectively. Conclusion This high throughput immunoassay exhibited good diagnostic efficiency and documented that the NMS JWH-018 direct ELISA is a viable method for screening synthetic cannabinoids in urine targeting the JWH-018 N-(5-hydroxypentyl) and related analytes. Optimal performance was achieved with a matrix-matched 5µg/L urine cutoff. PMID:25706046

  9. Comparison of electrochemiluminescence assay and ELISA for the detection of Clostridium botulinum type B neurotoxin.

    PubMed

    Guglielmo-Viret, V; Attrée, O; Blanco-Gros, V; Thullier, P

    2005-06-01

    We compared the ELISA and electrochemiluminescence (ECL) immunoassay technologies for the detection of botulinum type B neurotoxin (BotNT B), which requires highly sensitive techniques due to its potent biological activity. BotNT B complexes are the naturally secreted form of the toxin, approximately a third of which consists of the neurotoxin itself; they were aliquoted and frozen for this study. Results of both techniques were interpreted with the same standard statistical tests (ANOVA and Tukey). We first compared two commercial assays for BotNT B: the detection limit of the colorimetric ELISA was 1.56 ng/ml BotNT B complexes versus 0.39-0.78 ng/ml in the ECL test. We then used the same monoclonal antibody and the same polyclonal antibody, respectively purified by protein A and protein G chromatography, to optimize an in-house ELISA test and an in-house ECL test, making it possible to directly compare the two technologies without interference due to the properties of the antibodies used in the two tests. The colorimetric in-house ELISA had a detection threshold of 3.12 ng/ml versus the in-house ECL test whose detection threshold was 0.78-1.56 ng/ml. Thus, in both cases, the ECL assay was two to four times more sensitive than the colorimetric ELISA. The ECL assay was also more rapid (2.5 h for the in-house ECL versus 5 h for in-house ELISA with precoated wells). Overall, these elements can be used to compare the qualities of the two technologies, at least for the detection of protein antigens such as toxins.

  10. Cooperative Immunoassays: Ultrasensitive Assays with Mixed Monoclonal Antibodies

    NASA Astrophysics Data System (ADS)

    Ehrlich, Paul H.; Moyle, William R.

    1983-07-01

    Mixtures of certain monoclonal antibodies appear to bind human chorionic gonadotropin in a ``cooperative'' fashion because they form circular complexes with the hormone. Experiments illustrate how this property might be exploited to develop very sensitive immunoassays for human chorionic gonadotropin or any other antigen. Since the assays are not based on competitive inhibition between radiolabeled and unlabeled antigen, they are much more sensitive than a traditional radioimmunoassay in which either one of the same antibodies is used alone.

  11. Solution ELISA as a platform of choice for development of robust, drug tolerant immunogenicity assays in support of drug development.

    PubMed

    Mikulskis, Alvydas; Yeung, Dave; Subramanyam, Meena; Amaravadi, Lakshmi

    2011-02-28

    Humanized monoclonal antibody therapeutics are in many ways indistinguishable from the anti-therapeutic/anti-drug antibodies generated in humans. Therefore, immunogenicity assessments to such therapeutics pose unique challenges in clinical trials especially when significant drug interference is encountered. There are several technology platforms based on the bridging immunogenicity assay format, which have been successfully used for detection and quantification of anti-drug antibodies (ADA) in serum or plasma samples. Enzyme-Linked Immunosorbent Assay (ELISA) and Electrochemiluminescent (ECL) immunoassay formats are among the most popular technology platforms. Pretreatment of samples with acid can also be used to lower drug interference. While ECL technology platform offered many advantages over traditional solid-phase ELISA methods, reliance on a single (or limited) vendor source became a significant concern within the biopharmaceutical industry especially for immunogenicity assays that need to be implemented over a period of many years in support of a single drug development program. We describe herein a systematic evaluation of solid-phase ELISA, GYROS, AlphaLISA, ECL Immunoassay, and solution ELISA platforms for detection of anti-drug antibodies with the goal of selection and development of a robust technology platform that meets the desired performance characteristics for most immunogenicity assays and can be easily implemented in a typical immunoassay laboratory. As part of this effort the Design of Experiments (DOE) approach was utilized in optimization of sample acid treatment conditions in order to improve drug tolerance in the evaluated assay platforms. After the initial evaluation of various technology platforms, a solution ELISA format was chosen for further development to support clinical trials for a humanized therapeutic antibody. As part of the assay development, flexible use of digoxigenin and 6-(2,4-dinitrophenyl) aminohexanoic acid (DNP) for

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

    PubMed

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

    2013-06-07

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

  13. Comparison of LUCIO®-direct ELISA with CEDIA immunoassay for 'zero tolerance' drug screening in urine as required by the German re-licensing guidelines.

    PubMed

    Agius, Ronald; Nadulski, Thomas; Kahl, Hans-Gerhard; Dufaux, Bertin

    2013-06-01

    The performance of the previously validated LUCIO(®)-Direct-enzyme linked immunosorbent assay (direct ELISA) screening tests according to forensic guidelines is compared to that of cloned enzyme donor immunoassays (CEDIA) test for drugs of abuse in urine as defined in the new re-licensing German medical and psychological assessment (MPA) guidelines. The MPA screening cut-offs correspond to 10 ng/ml 11-nor-delta-9-tetrahydrocannabinol-9-carboxylic acid (THC-COOH), 50 ng/ml amphetamine and designer amphetamines, 25 ng/ml morphine, codeine and dihydrocodeine, 30 ng/ml benzoylecgonine, 50 ng/ml methadone metabolite, 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP) and metabolites of diazepam, oxazepam, bromazepam, alprazolam, flunitrazepam and lorazepam at 50 ng/ml. Average relative sensitivities and relative specificities were 99.7 % and 98.4 % for direct ELISA and 66 % and 91.4 % for CEDIA, respectively.

  14. Immunoassay screening of diphenhydramine (Benadryl®) in urine and blood using a newly developed assay.

    PubMed

    Rodrigues, Warren C; Castro, Catherine; Catbagan, Philip; Moore, Christine; Wang, Guohong

    2012-03-01

    Diphenhydramine (DPH) is a common over the counter antihistamine that produces drowsiness and has the potential to cause driving under the influence of drugs-related accidents. To date there are no commercially available immunoassay screening kits for its detection in biological fluids such as urine and/or blood. We describe a newly developed enzyme-linked immunosorbent assay (ELISA) screen and report on its utility in the analysis of authentic specimens taken from volunteers. The assay is specific for detection of DPH and does not detect closely related antihistamines like brompheniramine, chlorpheniramine, and doxylamine. There is a varying amount of cross-reactivity seen with certain tricyclic compounds, due to similarities in side chain structure with DPH. Intra- and interday precision of the assay were determined to be less than 10%. The assay is highly sensitive and has a working range from 1 to 500 ng/mL for urine and 1 to 250 ng/mL for blood. The assay was further validated with authentic urine and blood specimens obtained from volunteers and coroner's laboratories.

  15. Development of an ELISA assay for the quantification of soluble huntingtin in human blood cells

    PubMed Central

    2013-01-01

    Background Huntington’s disease (HD) is a monogenic disorder caused by an aberrant expansion of CAG repeats in the huntingtin gene (HTT). Pathogenesis is associated with expression of the mutant (mHTT) protein in the CNS, with its levels most likely related to disease progression and symptom severity. Since non-invasive methods to quantify HTT in the CNS do not exist, measuring amount of soluble HTT in peripheral cells represents an important step in development of disease-modifying interventions in HD. Results An ELISA assay using commercially available antibodies was developed to quantify HTT levels in complex matrices like mammalian cell cultures lysates and human samples. The immunoassay was optimized using a recombinant full-length HTT protein, and validated both on wild-type and mutant HTT species. The ability of the assay to detect significant variations of soluble HTT levels was evaluated using an HSP90 inhibitor that is known to enhance HTT degradation. Once optimized, the bioassay was applied to peripheral blood mononuclear cells (PBMCs) from HD patients, demonstrating good potential in tracking the disease course. Conclusions The method described here represents a validated, simple and rapid bio-molecular assay to evaluate soluble HTT levels in blood cells as useful tool in disease and pharmacodynamic marker identification for observational and clinical trials. PMID:24274906

  16. Development of an ELISA microarray assay for the sensitive and simultaneous detection of ten biodefense toxins.

    SciTech Connect

    Jenko, Kathryn; Zhang, Yanfeng; Kostenko, Yulia; Fan, Yongfeng; Garcia-Rodriguez, Consuelo; Lou, Jianlong; Marks, James D.; Varnum, Susan M.

    2014-10-21

    Plant and microbial toxins are considered bioterrorism threat agents because of their extreme toxicity and/or ease of availability. Additionally, some of these toxins are increasingly responsible for accidental food poisonings. The current study utilized an ELISA-based protein antibody microarray for the multiplexed detection of ten biothreat toxins, botulinum neurotoxins (BoNT) A, B, C, D, E, F, ricin, shiga toxins 1 and 2 (Stx), and staphylococcus enterotoxin B (SEB), in buffer and complex biological matrices. The multiplexed assay displayed a sensitivity of 1.3 pg/mL (BoNT/A, BoNT/B, SEB, Stx-1 and Stx-2), 3.3 pg/mL (BoNT/C, BoNT/E, BoNT/F) and 8.2 pg/mL (BoNT/D, ricin). All assays demonstrated high accuracy (75-120 percent recovery) and reproducibility (most coefficients of variation < 20%). Quantification curves for the ten toxins were also evaluated in clinical samples (serum, plasma, nasal fluid, saliva, stool, and urine) and environmental samples (apple juice, milk and baby food) with overall minimal matrix effects. The multiplex assays were highly specific, with little crossreactivity observed between the selected toxin antibodies. The results demonstrate a multiplex microarray that improves current immunoassay sensitivity for biological warfare agents in buffer, clinical, and environmental samples.

  17. 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.

  18. 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...

  19. Paraquat enzyme-immunoassays in biological samples: assessment of the effects of hapten-protein bridge structures on assay sensitivity.

    PubMed

    Abuknesha, Ramadan A; Luk, Connie

    2005-06-01

    Previously unreported paraquat derivatives were prepared and used to develop enzyme-immunoassay methods for paraquat in serum and urine matrices. The study involved comparison of the effects of novel paraquat derivatives made of methyl and ethyl-4,4'-bipyridinium and cyanuric chloride (heterologous bridges) or valeric acid (homologous bridges) on the ability of paraquat standards to inhibit binding of the antibody to adsorbed hapten-protein plate coating antigens prepared by coupling the derivatives to gelatine. The comparison showed striking differences in assay sensitivity due to the hapten bridge binding phenomenon where the heterologous bridge conjugates enabled achievement of sensitivity levels several orders of magnitude greater than the homologous structures. The constructed ELISA showed minimal detection limit in the range 4 pg mL(-1) in the buffer systems and less then 100 pg mL(-1) in charcoal-stripped human and horse sera and human urine. The study presents details of synthesis of novel paraquat derivatives and a highly sensitive ELISA. In addition the investigation demonstrates the critical importance of judicious selection of hapten-bridge structures to achieve improved levels of detection limits of paraquat immunoassays. The reported assay is suitable for use in monitoring of paraquat levels in exposed persons or animals and for emergency diagnostic tests.

  20. A double-sandwich ELISA for identification of monoclonal antibodies suitable for sandwich immunoassays

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The sandwich immunoassay (sIA) is an invaluable technique for concentrating, detecting, and quantifying target antigens. The two critical components required are a capture antibody and a detection antibody, each binding a different epitope on the target antigen. The specific antibodies incorporated...

  1. Yolk protein immunoassays (YP-ELISA) to assess diet and reproductive quality of mass-reared Orius insidiosus (Heteroptera: Anthocoridae).

    PubMed

    Shapiro, Jeffrey P; Ferkovich, Stephen M

    2002-10-01

    A yolk protein enzyme-linked immunosorbent assay (YP-ELISA) was developed for the predator Orius insidiosus (Say). The YP-ELISA is intended to assess reproductive response to dietary and other rearing conditions, and to assist in quality control and diet development for mass rearing. Hybridomas and monoclonal antibodies were produced against homogenates of eggs dissected from females. Hybridomas were selected for secretion of IgG that reacted with extracts of both females and their eggs, and that did not react with male extracts. Each cloned hybridoma produced a monoclonal antibody that specifically reacted on western blots against one of the two major yolk polypeptides, apoVn-I (180,000 molecular weight) or apoVn-II (40,000). Yolk protein ELISAs were developed with these antibodies to assess yolk protein content of female O. insidiosus as a measure of reproductive fitness and as a potential predictor of fecundity. Protocols for an indirect antigen ELISA and double antibody sandwich ELISA were developed to assess yolk protein contents of eggs and total contents in whole body homogenates. ELISA standards consisted of homogenates of eggs collected 0-24 h following oviposition. As determined with the sandwich ELISA, yolk protein contents of eggs declined with age before hatch, with a half-life of 32-34 h. Results were similar whether the detecting antibody-enzyme conjugate was anti-apoVn-I or anti-apoVn-II. Optimal conditions and sampling parameters were developed for the sandwich ELISA, which demonstrated minimal nonspecific interference in whole-insect extracts. In an initial application of the YP-ELISA, oviposition rates over a 10-d period were compared with yolk protein contents at the end of that period, dependent on diets of differing nutritional composition and quality. High and low yolk protein contents correlated with oviposition rates on respective diets, though oviposition showed more graded response to diets than did yolk protein. Improvements in sampling

  2. Sandwich ELISA Microarrays: Generating Reliable and Reproducible Assays for High-Throughput Screens

    SciTech Connect

    Gonzalez, Rachel M.; Varnum, Susan M.; Zangar, Richard C.

    2009-05-11

    The sandwich ELISA microarray is a powerful screening tool in biomarker discovery and validation due to its ability to simultaneously probe for multiple proteins in a miniaturized assay. The technical challenges of generating and processing the arrays are numerous. However, careful attention to possible pitfalls in the development of your antibody microarray assay can overcome these challenges. In this chapter, we describe in detail the steps that are involved in generating a reliable and reproducible sandwich ELISA microarray assay.

  3. 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...

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

    PubMed

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

    2006-06-30

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

  5. Comparison of conventional lateral-flow assays and a new fluorescent immunoassay to detect influenza viruses.

    PubMed

    Leonardi, Gary P; Wilson, Adele M; Zuretti, Alejandro R

    2013-05-01

    Sofia, a novel, fluorescent lateral-flow immunoassay was compared with two conventional colorimetric assays, Quickvue Influenza A+B and Directigen FLU A+B, to identify influenza viral antigen from patient nasopharyngeal specimens. A total of 118 frozen original influenza-positive specimens and 57 prospective specimens were examined. Using rt-PCR as a referee assay, sensitivity values (%) for influenza A/B of 80.0/74.8, 73.3/59.3 and 73.3/40.7 were obtained using the Sofia, Quickvue and Directigen assays, respectively. All assays demonstrated reduced sensitivity for influenza B as compared with influenza A virus. With respect to the Sofia assay, the sensitivity of influenza B for the Directigen assay was significantly diminished. False positive results were not observed in the Sofia and Directigen assays. The Quickvue assay produced 3 false-positive results (2 influenza A and 1 influenza B) resulting in a specificity (%) of 96 and 98 for influenza A and B, respectively. Cross-reactivity to other respiratory viruses was not observed among immunoassays. A sensitivity rank (highest to low) of rt-PCR>culture>Sofia>Quickvue>Directigen was established using dilutions of influenza A and B. Sofia provides enhanced sensitivity and objective result interpretation over conventional colorimetric immunoassays.

  6. Naked-eye sensitive ELISA-like assay based on gold-enhanced peroxidase-like immunogold activity.

    PubMed

    Wang, Shasha; Chen, Zhaopeng; Choo, Jaebum; Chen, Lingxin

    2016-02-01

    A naked-eye sensitive ELISA-like assay was developed based on gold-enhanced peroxidase-like activity of gold nanoparticles (AuNPs). Using human IgG (H-IgG) as an analytical model, goat anti-human IgG antibody (anti-IgG) adsorbed on microtiter plate and AuNPs-labeled anti-IgG acted as capture antibody and detection antibody, respectively. Because the surfaces of AuNPs were blocked by protein molecules, the peroxidase-like activity of AuNPs was almost inhibited, evaluated by the catalytic oxidation of peroxidase enzyme substrate 3,3',5,5'-tetramethylbenzidine (TMB), which could produce a bright blue color in the presence of H2O2. Fortunately, the catalytic ability of AuNPs was dramatically increased by the deposition of gold due to the formation of a new gold shell on immunogold. Under optimal reaction conditions, the colorimetric immunoassay presented a good linear relationship in the range of 0.7-100 ng/mL and the limit of detection (LOD) of 0.3 ng/mL calculated by 3σ/S for UV-vis detection, and obtained LOD of 5 ng/mL for naked-eye detection. The obtained results were competitive with conventional sandwich ELISA with the LOD of 1.6 ng/mL. Furthermore, this developed colorimetric immunoassay was successfully applied to diluted human serum and fetal bovine serum samples, and predicted a broad prospect for the use of peroxidase-like activity involving nanomaterials in bioassay and diagnostics.

  7. Performance evaluation of nine hormone assays on the Immulite 2000 immunoassay system.

    PubMed

    Tello, F L; Hernández, D M

    2000-10-01

    We evaluated the analytical performance of the Immulite 2000 immunoassay analyzer (Diagnostic Products Corporation, Los Angeles, USA) based on a new detection technology, electrochemical luminescence. The evaluated analytes were thyrotropin, triiodothyronine, free thyroxine, follitropin, lutropin, prolactin, cortisol, estradiol and progesterone. We tested the assay precision, linearity, recovery, and correlation with comparison methods for these analytes. For most assays, within-run and between-day imprecisions were less than 8% and 10%, respectively. The linearity and recovery were acceptable for all assays. The correlation between the Immulite 2000 assays and comparison methods showed satisfactory results.

  8. COMPARISONS OF ELISA AND WESTERN BLOT ASSAYS FOR DETECTION OF CRYPTOSPORIDIUM ANTIBODY

    EPA Science Inventory

    A seroprevalence survey was conducted using ELISA and Western blot (WB) assays for antibody to three Cryptosporidium antigens on 380 blood donors in Jackson County, Oregon. The purpose was to determine if either assay could detect serological evidence of an outbreak which occurre...

  9. Evaluation of Multiple Immunoassay Technology Platforms to Select the Anti-Drug Antibody Assay Exhibiting the Most Appropriate Drug and Target Tolerance

    PubMed Central

    Collet-Brose, Justine

    2016-01-01

    The aim of this study was, at the assay development stage and thus with an appropriate degree of rigor, to select the most appropriate technology platform and sample pretreatment procedure for a clinical ADA assay. Thus, ELISA, MSD, Gyrolab, and AlphaLISA immunoassay platforms were evaluated in association with target depletion and acid dissociation sample pretreatment steps. An acid dissociation step successfully improved the drug tolerance for all 4 technology platforms and the required drug tolerance was achieved with the Gyrolab and MSD platforms. The target tolerance was shown to be better for the ELISA format, where an acid dissociation treatment step alone was sufficient to achieve the desired target tolerance. However, inclusion of a target depletion step in conjunction with the acid treatment raised the target tolerance to the desired level for all of the technologies. A higher sensitivity was observed for the MSD and Gyrolab assays and the ELISA, MSD, and Gyrolab all displayed acceptable interdonor variability. This study highlights the usefulness of evaluating the performance of different assay platforms at an early stage in the assay development process to aid in the selection of the best fit-for-purpose technology platform and sample pretreatment steps. PMID:27243038

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

    PubMed

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

    2014-07-09

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

  11. ELISA assays and alcohol: increasing carbon chain length can interfere with detection of cytokines.

    PubMed

    von Maltzan, Kristine; Pruett, Stephen B

    2011-02-01

    Enzyme-linked immunosorbent assays (ELISAs) are frequently used in studies on cytokine production in response to treatment of cell cultures or laboratory animals. When an ELISA assay is performed on cell culture supernatants, samples often contain the treatment agents. The purpose of the present study was to determine if some of the agents evaluated might inhibit cytokine detection by interfering with the ELISA, leaving the question of whether cytokine production was inhibited unanswered. Mouse and human cytokine ELISA kits from BD Biosciences were used according to the manufacturer's instructions. Cytokine proteins were subjected to one to five carbon alcohols at 86.8mM (methanol, ethanol, 1-propanol, 2-propanol, n-butanol, and n-pentanol). After treating cell cultures with alcohols of different carbon chain lengths, we found that some of the alcohols interfered with measurement of some cytokines by ELISA, thus making their effects on cytokine production by cells in culture unclear. Increasing carbon chain length of straight chain alcohols positively correlated with their ability to inhibit detection of tumor necrosis factor alpha (TNF-α) and interleukin 10 (IL-10), but not with the detection of interleukin 6 (IL-6), interleukin 8, (IL-8), and interleukin 12 (IL-12). To avoid misinterpretation of treatment effects, ELISA assays should be tested with the reference protein and the treatment agent first, before testing biological samples. These results along with other recent results we obtained using circular dichroism indicate that alcohols with two or more carbons can directly alter protein conformation enough to disrupt binding in an ELISA (shown in the present study) or to inhibit ligand-induced conformational changes (results not shown). Such direct effects have not been given enough consideration as a mechanism of ethanol action in the immune system.

  12. Immunoassays for diagnosis of coagulation disorders.

    PubMed

    Kappel, A; Ehm, M

    2010-11-01

    Immunoassays play a pivotal role in the clinical laboratory. In the coagulation section of the laboratory, they are used as an aid for diagnosis of deep vein thrombosis or pulmonary embolism, thrombophilia screening, or detection of coagulation factor deficiencies, respectively. Enzyme-linked immunosorbent assay (ELISA) and latex agglutination immunoassay technologies are currently most widely used, while Luminescent Oxygen Channeling Immunoassay (LOCI®) and other chemiluminescence-based immunoassays are emerging technologies for the coagulation laboratory. However, not all immunoassay technologies employed are compatible with the workflow requirements of the coagulation laboratory, and, not all technologies are suitable for detection or quantification of every marker. This review focuses on technical and performance aspects of those immunoassay technologies that are most widely used in the coagulation laboratory, and provides a description of markers that are typically tested by immunoassays.

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

    ERIC Educational Resources Information Center

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

    2012-01-01

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

  14. A quantitative ELISA assay for the fragile x mental retardation 1 protein.

    PubMed

    Iwahashi, Christine; Tassone, Flora; Hagerman, Randi J; Yasui, Dag; Parrott, George; Nguyen, Danh; Mayeur, Greg; Hagerman, Paul J

    2009-07-01

    Non-coding (CGG-repeat) expansions in the fragile X mental retardation 1 (FMR1) gene result in a spectrum of disorders involving altered neurodevelopment (fragile X syndrome), neurodegeneration (late-onset fragile X-associated tremor/ataxia syndrome), or primary ovarian insufficiency. While reliable and quantitative assays for the number of CGG repeats and FMR1 mRNA levels are now available, there has been no scalable, quantitative assay for the FMR1 protein (FMRP) in non-transformed cells. Using a combination of avian and murine antibodies to FMRP, we developed a sensitive and highly specific sandwich enzyme-linked immunosorbent assay (ELISA) for FMRP in peripheral blood lymphocytes. This ELISA method is capable of quantifying FMRP levels throughout the biologically relevant range of protein concentrations and is specific for the intact FMRP protein. Moreover, the ELISA is well-suited for replicate protein determinations across serial dilutions in non-transformed cells and is readily scalable for large sample numbers. The FMRP ELISA is potentially a powerful tool in expanding our understanding of the relationship between FMRP levels and the various FMR1-associated clinical phenotypes.

  15. Comparison of GD2 Binding Capture ELISA Assays for Anti-GD2-Antibodies Using GD2-Coated Plates and a GD2-Expressing Cell-Based ELISA

    PubMed Central

    Soman, Gopalan; Yang, Xiaoyi; Jiang, Hengguang; Giardina, Steve; Mitra, Gautam

    2011-01-01

    Two assay methods for quantification of the disialoganglioside (GD2)-specific binding activities of anti-GD2 monoclonal antibodies and antibody immunofusion proteins, such as ch14.18 and hu14.18-IL2, were developed. The methods differed in the use of either microtiter plates coated with purified GD2 or plates seeded with GD2-expressing cell lines to bind the anti-GD2 molecules. The bound antibodies were subsequently detected using the reactivity of the antibodies to an HRP-labeled anti-IgG Fc or antibodies recognizing the conjugate IL-2 part of the Hu 14.18IL-2 fusion protein. The bound HRP was detected using reagents such as orthophenylene diamine, 2, 2’-azinobis [3-ethylbenzothiazoline-6-sulfonic acid] or tetramethylbenzidine. The capture ELISA using GD2-coated plates was developed earlier in assay development and used to demonstrate assay specificity and to compare lot-to-lot consistency and stability of ch14.18, and Hu14.18 IL-2 in clinical development. During this study, we found a number of issues related to plate-to-plate variability, GD2 lot variability, and variations due to GD2 storage stability, etc., that frequently lead to assay failure in plates coated with purified GD2. The cell-based ELISA (CbELISA) using the GD2 expressing melanoma cell line, M21/P6, was developed as an alternative to the GD2-coated plate ELISA. The results on the comparability of the capture ELISA on GD2-coated plates and the cell-based assay show that both assays give comparable results. However, the cell-based assay is more consistent and reproducible. Subsequently, the anti-GD2 capture ELISA using the GD2-coated plate was replaced with the CbELISA for product lot release testing and stability assessment. PMID:21893062

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

    PubMed

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

    2003-09-01

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

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

    PubMed

    Muller, R

    1997-10-01

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

  18. An ELISA DYRK1A non-radioactive assay suitable for the characterization of inhibitors

    PubMed Central

    Liu, Yong; Adayev, Tatyana; Hwang, Yu-Wen

    2017-01-01

    The DYRK1A (dual specificity tyrosine phosphorylation-regulated kinase 1A) gene encodes a proline-directed Ser/Thr kinase. Elevated expression and/or altered distribution of the kinase have been implicated in the neurological impairments associated with Down syndrome (DS) and Alzheimer’s disease (AD). Consequently, DYRK1A inhibition has been of significant interest as a potential strategy for therapeutic intervention of DS and AD. Many classes of novel inhibitors have been described in the past decade. Although non-radioactive methods for analyzing DYRK1A inhibition have been developed, methods employing radioactive tracers are still commonly used for quantitative characterization of DYRK1A inhibitors. Here, we present a non-radioactive ELISA assay based on the detection of DYRK1A-phosphorylated dynamin 1a fragment using a phosphorylation site-specific antibody. The assay was verified by the use of two well-characterized DYRK1A inhibitors, epigallocatechin gallate (EGCG) and harmine. The IC 50s for EGCG and harmine determined by the ELISA method were found to be comparable to those previously measured by radioactive tracing methods.  Furthermore, we determined the mode of inhibition for EGCG and harmine by a modification of the ELISA assay. This assay confirms the mode of inhibition of EGCG (non-ATP-competitive) and harmine (ATP-competitive), as previously determined. We conclude that the ELISA platform demonstrated here is a viable alternative to the traditional radioactive tracer assays for analyzing DYRK1A inhibitors. PMID:28163906

  19. Identifying the predator complex of Homalodisca vitripennis (Hemiptera: Cicadellidae): a comparative study of the efficacy of an ELISA and PCR gut content assay.

    PubMed

    Fournier, Valerie; Hagler, James; Daane, Kent; de León, Jesse; Groves, Russell

    2008-10-01

    A growing number of ecologists are using molecular gut content assays to qualitatively measure predation. The two most popular gut content assays are immunoassays employing pest-specific monoclonal antibodies (mAb) and polymerase chain reaction (PCR) assays employing pest-specific DNA. Here, we present results from the first study to simultaneously use both methods to identify predators of the glassy winged sharpshooter (GWSS), Homalodisca vitripennis (Germar) (Hemiptera: Cicadellidae). A total of 1,229 arthropod predators, representing 30 taxa, were collected from urban landscapes in central California and assayed first by means of enzyme-linked immunosorbent assay (ELISA) using a GWSS egg-specific mAb and then by PCR using a GWSS-specific DNA marker that amplifies a 197-base pair fragment of its cytochrome oxidase gene (subunit I). The gut content analyses revealed that GWSS remains were present in 15.5% of the predators examined, with 18% of the spiders and 11% of the insect predators testing positive. Common spider predators included members of the Salticidae, Clubionidae, Anyphaenidae, Miturgidae, and Corinnidae families. Common insect predators included lacewings (Neuroptera: Chrysopidae), praying mantis (Mantodea: Mantidae), ants (Hymenoptera: Formicidae), assassin bugs (Hemiptera: Reduviidae), and damsel bugs (Hemiptera: Nabidae). Comparison of the two assays indicated that they were not equally effective at detecting GWSS remains in predator guts. The advantages of combining the attributes of both types of assays to more precisely assess field predation and the pros and cons of each assay for mass-screening predators are discussed.

  20. An ELISA assay for cytochrome P4501A in fish liver cells

    SciTech Connect

    Brueschweiler, B.J.; Fent, K. |; Wuergler, F.E. |

    1996-04-01

    An enzyme-linked immunosorbent assay (ELISA) for measuring cytochrome P4501A (CYP1A) expression in vitro in fish hepatoma cells is described. Cells were cultured as monolayers in 96-microwell cell culture plates and exposed to polychlorinated biphenyl (PCB) congeners 77, 105, 153, and 169; 3-methylcholanthrene (3-MC), and {beta}-naphthoflavone (BNF) for 3 d. Relative CYP1A protein content, CYP1A enzymatic activity, and total protein content were determined directly within the wells. At low concentrations of PCB 77, PCB 169, and 3-MC, the ethoxyresorufin-O-deethylase (EROD) activity was induced, but it was inhibited at high concentrations of these compounds. However, CYP1A protein content measured in an ELISA performed with intact cells increased monotonically in response to the concentration. No CYP1A induction was observed for PCB 105 and PCB 153. Because comparison between EROD activity and CYP1A amount gives information about the catalytic efficiency of CYP1A in the cells, this noncompetitive, solid-phase ELISA is recommended as a complementary method to the EROD assay. This novel ELISA method may be an accurate in vitro technique for a rapid and sensitive screening of CYP1A-inducible compounds.

  1. Henipavirus microsphere immuno-assays for detection of antibodies against Hendra virus.

    PubMed

    McNabb, Leanne; Barr, J; Crameri, G; Juzva, S; Riddell, S; Colling, A; Boyd, V; Broder, C; Wang, L-F; Lunt, R

    2014-05-01

    Hendra and Nipah viruses (HeV and NiV) are closely related zoonotic pathogens of the Paramyxoviridae family. Both viruses belong to the Henipavirus genus and cause fatal disease in animals and humans, though only HeV is endemic in Australia. In general and due to the acute nature of the disease, agent detection by PCR and virus isolation are the primary tools for diagnostic investigations. Assays for the detection of antibodies against HeV are fit more readily for the purpose of surveillance testing in disease epidemiology and to meet certification requirements in the international movement of horses. The first generation indirect ELISA has been affected by non-specific reactions which must be resolved using virus neutralisation serology conducted at laboratory bio-safety level 4 containment (PC4). Recent developments have enabled improvements in the available serology assays. The production of an expressed recombinant truncated HeV G protein has been utilised in ELISA and in Luminex-based multiplexed microsphere assays. In the latter format, two Luminex assays have been developed for use in henipavirus serology: a binding assay (designed for antibody detection and differentiation) and a blocking assay (designed as a surrogate for virus neutralisation). Equine and canine field sera were used to evaluate the two Luminex assays relative to ELISA and virus neutralisation serology. Results showed that Luminex assays can be effective as rapid, sensitive and specific tests for the detection of HeV antibody in horse and dog sera. The tests do not require PC4 containment and are appropriate for high throughput applications as might be required for disease investigations and other epidemiological surveillance. Also, the results show that the Luminex assays detect effectively HeV vaccine-induced antibodies.

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

    SciTech Connect

    Greenberger, P.A.; Patterson, R.

    1982-02-01

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

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

    PubMed

    Greenberger, P A; Patterson, R

    1982-02-01

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

  4. Rapid determination of phenylethanolamine A in biological samples by enzyme-linked immunosorbent assay and lateral-flow immunoassay.

    PubMed

    Li, Xiangmei; Wang, Wenjun; Wang, Limiao; Wang, Qi; Pei, Xingyao; Jiang, Haiyang

    2015-10-01

    Phenylethanolamine A (PA) is a β-adrenergic agonist, which was first used in animal husbandry as a growth promoter in China in 2010. In this study, a monoclonal-antibody (mAb)-based indirect competitive enzyme-linked immunosorbent assay (icELISA) and lateral-flow immunoassay (LFA) for the detection of PA in swine urine and pork were developed. The immunogen was prepared by linking PA hapten with carrier protein via a diazotization method. The IC50 value of the optimized icELISA was 0.44 ng mL(-1). The limits of detection of the icELISA for PA in swine urine and pork were 0.13 ng mL(-1) and 0.39 ng g(-1), respectively. The recoveries of PA from spiked swine urine and pork were in the range 82.0-107.4 % and 81.8-113.3%, respectively, with the coefficients of variation in the range 4.1-16.2% and 1.2-6.3%, respectively. The mAbs had negligible cross reactivity with 10 other β-agonists. In contrast, the LFA had a cut-off level of 5 ng mL(-1) (g) in swine urine and pork, and the results could be achieved within 5 min. Ten blind samples of swine urine were analyzed simultaneously by icELISA, LFA, and ultra-high-performance liquid chromatography-tandem mass spectrometry, and the results of the three methods agreed well. Therefore, the combination of two immunoassays provides an effective and rapid screening method for detection of PA residues in biological samples.

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

    PubMed

    Gaskin, Ferdelie E; Taylor, Steve L

    2011-01-01

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

  6. A rapid method to improve protein detection by indirect ELISA

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The enzyme-linked immunosorbant assay (ELISA) is a rapid, high-throughput, quantitative immunoassay for the selective detection of target antigens. The general principle behind an ELISA is antibody mediated capture and detection of an antigen with a measureable substrate. Numerous incarnations of th...

  7. BrucELISA: an enzyme-antibody immunoassay for detection of Brucella abortus antibodies in milk: correlation with the Brucella ring test and with shedding of viable organisms.

    PubMed Central

    Boraker, D K; Stinebring, W R; Kunkel, J R

    1981-01-01

    An indirect enzyme-antibody immunosorbent assay (BrucELISA) is described for the detection of antibody to Brucella abortus in cow's milk. Three series of milk samples were obtained from an adult-vaccinated dairy herd infected with B. abortus. The BrucELISA system was used as a screening test for individual milks diluted 1:200 (BE 200 test), for undiluted bulk milks, and to determine antibody titer (BrucELISA titration assay). The BrucELISA results correlated highly with positive Brucella ring test reactions and culture positivity, eliminated false-positive Brucella ring test reactions, detected antibody in some samples which were Brucella ring test negative, and distinguished between vaccinated and infected animals. BrucELISA titration assay titers of greater than 1:800 were correlated with shedding, or were prognostic for animals which eventually became shedders. Binding of the enzyme-antibody conjugate to bovine immunoglobulin in the absence of rabbit anti-bovine immunoglobulin occurred with culture-positive or -negative milks showing titers of greater than 1:1,600 (the beta effect); the effect was also of predictive value in identifying eventual shedders. The BrucELISA system is a sensitive, specific, and inexpensive method for screening large numbers of individual or bulk milk samples for the presence of antibody to B. abortus. PMID:6793622

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

    PubMed Central

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

    1980-01-01

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

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

    PubMed Central

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

    2013-01-01

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

  10. Evaluation of the polyclonal ELISA HPV serology assay as a biomarker for HPV exposure

    PubMed Central

    Coseo, Sarah E.; Porras, Carolina; Dodd, Lori E.; Hildesheim, Allan; Rodriguez, Ana Cecilia; Schiffman, Mark; Herrero, Rolando; Wacholder, Sholom; Gonzalez, Paula; Sherman, Mark E.; Jimenez, Silvia; Solomon, Diane; Bougelet, Catherine; van Doorn, Leen-Jan; Quint, Wim; Safaeian, Mahboobeh

    2011-01-01

    Background Seropositivity to HPV16 and 18 antibodies is used as a measure of cumulative HPV exposure and as a stratifier of HPV exposure for vaccine efficacy analyses. Overall performance of these assays, as a measure of HPV exposure, has not been evaluated. Methods Using data from the enrollment phase of the HPV16/18 vaccine trial in Costa Rica, we evaluated the performance of the polyclonal ELISA HPV16 and 18 serological assays as a measure of HPV exposure. Biological (for eg. HPV infection at the cervix) and behavioral characteristics (for eg. lifetime number of sexual partners) with known associations with current and past HPV infection were used to define cases and controls (HPV exposed vs. not exposed). Pre-vaccination serum was measured for antibodies against HPV16 and HPV18 by ELISA; cervical samples were tested for HPV DNA using PCR SPF10/LiPA25. ELISA results were analyzed using receiver-operator-characteristic curves (ROC); performance was evaluated at the manufacturer set cutpoint (HPV16 =8, HPV18 =7) and at cutpoints chosen to optimize sensitivity and specificity (HPV16 =34, HPV18 =60). Results Defining cases as type-specific HPV DNA positive with high-grade abnormal cytolzogy (i.e. combined molecular and microscopic markers of infection), HPV16-ELISA gave sensitivity that was lower at the optimal cutpoint than the manufacturer cutpoint (62.2 compared with 75.7, respectively; p=0.44). However, specificity was higher (85.3 compared with 70.4, respectively; p<0.0001). Similarly, HPV18-ELISA gave sensitivity that was lower at the optimal cutpoint than the manufacturer cutpoint (34.5 compared with 51.7, respectively; p=0.40), with higher specificities (94.9 compared with 72.6, respectively; p<0.0001). Conclusions Modifying cutpoints did not improve the low sensitivity. The low sensitivity of this assay does not support its use for risk stratification or clinical settings. PMID:21934576

  11. Bisphenol A determination in baby bottles by chemiluminescence enzyme-linked immunosorbent assay, lateral flow immunoassay and liquid chromatography tandem mass spectrometry.

    PubMed

    Maiolini, Elisabetta; Ferri, Elida; Pitasi, Agata Laura; Montoya, Angel; Di Giovanni, Manuela; Errani, Ermanno; Girotti, Stefano

    2014-01-07

    Two immunoassays, a Lateral Flow ImmunoAssay (LFIA) based on colloidal gold nanoparticle labels and an indirect competitive chemiluminescence enzyme-linked immunosorbent assay (CL-ELISA), were developed and a high performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was optimized to assess the possible release of bisphenol A (BPA, 4,4'-isopropylidenediphenol) from different plastic baby bottles treated with simulating solutions. Coating conjugate concentration, anti-BPA antibody dilution, incubation time of the primary and secondary antibodies, and tolerance to different organic solvents were optimized to obtain the best performance of the ELISA with chemiluminescent end-point detection. The influence of different buffers on LFIA performance was also evaluated. Both methods showed good repeatability (mean CV value around 13%) and sensitivity. Reproducibility tests for CL-ELISA gave a mean CV value of about 25%. The IC50 and Limit of Detection (LOD) values of CL-ELISA were 0.2 and 0.02 ng mL(-1), respectively. The LOD of LFIA was 0.1 μg mL(-1). A LC-MS/MS method was also optimized. The separation was performed in a C18 column with a triple-quadrupole mass spectrometer with electrospray ionisation interface. The method showed a good linearity in the range 2 to 500 ng mL(-1), with a regression coefficient of 0.998. In the simulating solutions the detection and quantification limits, calculated by the signal to noise level of 3 (S/N = 3), were 5.8 ng mL(-1) and 17.4 ng mL(-1), respectively. This limit of quantification was about 3 and 35 times lower than the permitted limits set by the official method CEN/TS 13130-13 (0.05 μg mL(-1)) and by the Directive 2004/19/EC (0.6 μg mL(-1)), respectively. The methods were applied to determine BPA release from baby bottles, performing repeated procedures according to EU and national regulations. The results demonstrated that no BPA migration from the tested plastic materials occurred with only one

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

    PubMed

    Martin, Laurent; Chaabo, Ayman; Lasne, Françoise

    2015-06-01

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

  13. Predicting protein concentrations with ELISA microarray assays, monotonic splines and Monte Carlo simulation

    SciTech Connect

    Daly, Don S.; Anderson, Kevin K.; White, Amanda M.; Gonzalez, Rachel M.; Varnum, Susan M.; Zangar, Richard C.

    2008-07-14

    Background: A microarray of enzyme-linked immunosorbent assays, or ELISA microarray, predicts simultaneously the concentrations of numerous proteins in a small sample. These predictions, however, are uncertain due to processing error and biological variability. Making sound biological inferences as well as improving the ELISA microarray process require require both concentration predictions and creditable estimates of their errors. Methods: We present a statistical method based on monotonic spline statistical models, penalized constrained least squares fitting (PCLS) and Monte Carlo simulation (MC) to predict concentrations and estimate prediction errors in ELISA microarray. PCLS restrains the flexible spline to a fit of assay intensity that is a monotone function of protein concentration. With MC, both modeling and measurement errors are combined to estimate prediction error. The spline/PCLS/MC method is compared to a common method using simulated and real ELISA microarray data sets. Results: In contrast to the rigid logistic model, the flexible spline model gave credible fits in almost all test cases including troublesome cases with left and/or right censoring, or other asymmetries. For the real data sets, 61% of the spline predictions were more accurate than their comparable logistic predictions; especially the spline predictions at the extremes of the prediction curve. The relative errors of 50% of comparable spline and logistic predictions differed by less than 20%. Monte Carlo simulation rendered acceptable asymmetric prediction intervals for both spline and logistic models while propagation of error produced symmetric intervals that diverged unrealistically as the standard curves approached horizontal asymptotes. Conclusions: The spline/PCLS/MC method is a flexible, robust alternative to a logistic/NLS/propagation-of-error method to reliably predict protein concentrations and estimate their errors. The spline method simplifies model selection and fitting

  14. DETECTION OF HEAVY METALS BY IMMUNOASSAY: OPTIMIZATION AND VALIDATION OF A RAPID, PORTABLE ASSAY FOR IONIC CADMIUM (R824029)

    EPA Science Inventory

    An immunoassay is described that measured Cd(II) in aqueous samples at
    concentrations from approximately 7 to 500 ppb. The assay utilized a monoclonal
    antibody that bound tightly to a cadmium-ethylenediaminetetraacetic acid (EDTA)
    complex but not to metal-free EDTA...

  15. Investigation of Reagent Delivery Formats in a Multivalent Malaria Sandwich Immunoassay and Implications for Assay Performance.

    PubMed

    Liang, Tinny; Robinson, Robert; Houghtaling, Jared; Fridley, Gina; Ramsey, Stephen A; Fu, Elain

    2016-02-16

    Conventional lateral flow tests (LFTs), the current standard bioassay format used in low-resource point-of-care (POC) settings, have limitations that have held back their application in the testing of low concentration analytes requiring high sensitivity and low limits of detection. LFTs use a premix format for a rapid one-step delivery of premixed sample and labeled antibody to the detection region. We have compared the signal characteristics of two types of reagent delivery formats in a model system of a sandwich immunoassay for malarial protein detection. The premix format produced a uniform binding profile within the detection region. In contrast, decoupling the delivery of sample and labeled antibody to the detection region in a sequential format produced a nonuniform binding profile in which the majority of the signal was localized to the upstream edge of the detection region. The assay response was characterized in both the sequential and premix formats. The sequential format had a 4- to 10-fold lower limit of detection than the premix format, depending on assay conjugate concentration. A mathematical model of the assay quantitatively reproduced the experimental binding profiles for a set of rate constants that were consistent with surface plasmon resonance measurements and absorbance measurements of the experimental multivalent malaria system.

  16. Dextran, a hapten carrier in immunoassays for s-triazines. A comparison with ELISAs based on hapten-protein conjugates.

    PubMed

    Böcher, M; Giersch, T; Schmid, R D

    1992-07-06

    A conjugate of 2-aminohexylamino-4-ethylamino-6-isopropylamino-1,3,5-triazine (AHA), a derivative of the herbicide atrazine, with dextran as carrier has been synthesized and used as the coating antigen in ELISA procedures. The quantification of terbutryn, atrazine and prometryn in ELISA formats using monoclonal antibodies and the AHA-dextran conjugate was at least as sensitive as ELISAs using protein conjugates as immobilized antigens (sensitivity at 50% B/B0 was 0.4-0.6 micrograms/l for terbutryn). Formats with immobilized antibody and enzyme labelled AHA proved to be less sensitive (1.5 micrograms/l for terbutryn). The observed differences in sensitivity do not apparently result from structural effects of the carrier bound hapten since all conjugates were prepared with one form of the hapten, 2-aminohexylamino-atrazine, which was covalently linked via its amino function to the carriers or enzymes.

  17. Determination of protein levels in soy and peanut oils by colorimetric assay and ELISA.

    PubMed

    Jablonski, Joseph E; Fu, Tong-Jen; Jackson, Lauren S; Gendel, Steven M

    2010-01-01

    Analytical methods are needed for measuring the levels of protein from allergenic food transferred into cooking oil. A simple method for determination of total protein in cooking oils was developed. Oil was extracted with phosphate-buffered saline with 0.05% Tween (PBST) and the extracts were partitioned with hexane to remove residual oil. Total protein in the PBST extracts was assayed with bicinchoninic acid (BCA), micro-BCA, reducing-agent compatible BCA and CB-XT kits. These methods were used to measure recovery of protein from peanut butter spikes of soy and peanut oil in the range of 50-1000 ppm. Recoveries were generally above 70%. However, the BCA and micro-BCA assays were subject to interference and enhanced color formation which were probably due to co-extracted antioxidants present in oil. The reducing agent-compatible BCA and CB-X protein assays reduced interference and gave lower protein values in crude, cold-pressed, and refined peanut oils. Heating oil to 180 degrees C before extraction also reduced interference-induced color enhancement. A commercial ELISA test kit was also used to measure peanut protein in oil spiked with peanut butter. Recovery of peanut residues measured by ELISA was significantly decreased when the peanut butter-spiked oil was heated to 180 degrees C compared to unheated oil. Recovery of spiked peanut butter protein measured by the buffer extraction-colorimetric method was not decreased in heated oil. The method developed here could be used to determine protein levels in crude and refined oil, and to assess the potential for allergen cross-contact from reused cooking oil.

  18. Tuberculin purified protein derivative (PPD) immunoassay as an in vitro alternative assay for identity and confirmation of potency.

    PubMed

    Ho, Mei M; Kairo, Satnam K; Corbel, Michael J

    2006-01-01

    Tuberculin purified protein derivative (PPD) currently can only be standardised by delayed hypersensitivity skin reactions in sensitised guinea pigs. An in vitro dot blot immunoassay was developed for both identity and confirmation of potency estimation of PPD. Polyclonal antibodies (mainly IgG) were generated and immunoreacted with human, bovine and, to lesser extent, avian PPD preparations. Combining size exclusion chromatography (FPLC-SEC) and dot blot immunoassay, the results showed that PPD preparations were mixtures of very heterogeneous tuberculoproteins ranging in size from very large aggregates to very small degraded molecules. All individual fractions of PPD separated by size were immunoreactive, although those of the largest molecular sizes appeared the most immunoreactive in this in vitro dot blot immunoassay. This method is very sensitive and specific to tuberculoproteins and can be an in vitro alternative for the in vivo intradermal skin assay which uses guinea pigs for identity of PPD preparations. Although the capacity of PPD to elicit cell-mediated immune responses on intradermal testing has to be confirmed by in vivo assay, the dot blot immunoassay offers a rapid, sensitive and animal-free alternative to in vivo testing for confirming the identity of PPD preparations with appropriate potencies. This alternative assay would be particularly useful for national regulatory laboratories for confirming the data of manufacturers and thus reducing the use of animals.

  19. Identification of heme oxygenase-1 stimulators by a convenient ELISA-based bilirubin quantification assay.

    PubMed

    Rücker, Hannelore; Amslinger, Sabine

    2015-01-01

    The upregulation of heme oxygenase-1 (HO-1) has proven to be a useful tool for fighting inflammation. In order to identify new HO-1 inducers, an efficient screening method was developed which can provide new lead structures for drug research. We designed a simple ELISA-based HO-1 enzyme activity assay, which allows for the screening of 12 compounds in parallel in the setting of a 96-well plate. The well-established murine macrophage cell line RAW264.7 is used and only about 26µg of protein from whole cell lysates is needed for the analysis of HO-1 activity. The quantification of HO-1 activity is based on an indirect ELISA using the specific anti-bilirubin antibody 24G7 to quantify directly bilirubin in the whole cell lysate, applying a horseradish peroxidase-tagged antibody together with ortho-phenylenediamine and H2O2 for detection. The bilirubin is produced on the action of HO enzymes by converting their substrate heme to biliverdin and additional recombinant biliverdin reductase together with NADPH at pH 7.4 in buffer. This sensitive assay allows for the detection of 0.57-82pmol bilirubin per sample in whole cell lysates. Twenty-three small molecules, mainly natural products with an α,β-unsaturated carbonyl unit such as polyphenols, including flavonoids and chalcones, terpenes, an isothiocyanate, and the drug oltipraz were tested at typically 6 or 24h incubation with RAW264.7 cells. The activity of known HO-1 inducers was confirmed, while the chalcones cardamonin, flavokawain A, calythropsin, 2',3,4'-trihydroxy-4-methoxychalcone (THMC), and 2',4'-dihydroxy-3,4-dimethoxychalcone (DHDMC) were identified as new potent HO-1 inducers. The highest inductive power after 6h incubation was found at 10µM for DHDMC (6.1-fold), carnosol (3.9-fold), butein (3.1-fold), THMC (2.9-fold), and zerumbone (2.5-fold). Moreover, the time dependence of HO-1 protein production for DHDMC was compared to its enzyme activity, which was further evaluated in the presence of

  20. Detection of venom by enzyme linked immunosorbent assay (ELISA) in patients bitten by snakes in Thailand.

    PubMed Central

    Silamut, K; Ho, M; Looareesuwan, S; Viravan, C; Wuthiekanun, V; Warrell, D A

    1987-01-01

    The ability of an enzyme linked immunosorbent assay (ELISA) to detect venom was evaluated in 251 patients bitten by four of the commonest poisonous snakes in Thailand. Serum was tested only from patients who brought the snakes that had bitten them. About one third of all bitten patients had detectable venom antigenaemia, though a smaller proportion were symptomatic. Serum venom concentrations on admission correlated with the severity of clinical manifestations. The test was sensitive and specific even for specimens that had been collected and stored under suboptimal conditions. The technique is suitable for forensic use in cases of suspected snakebite. The combination of snake identification and venom antigen detection should be a more reliable means of studying the epidemiology of snakebite than the measurement of venom antibodies in a population. PMID:3101897

  1. General false positive ELISA reactions in visceral leishmaniasis. Implications for the use of enzyme immunoassay analyses in tropical Africa.

    PubMed

    Elshafie, Amir I; Mullazehi, Mohammed; Rönnelid, Johan

    2016-04-01

    Leishmaniasis is a neglected disease in tropical countries. Clinical and laboratory features may mimic autoimmune diseases and this can complicate the Leishmania diagnosis. Due to our previous investigation for false anti-CCP2 reactivity in Leishmania-infected subjects and our interest in immunity against the joint-specific collagen type II (CII) in rheumatoid arthritis (RA) we investigated the same cohort for anti-CII antibodies. We found elevated anti-CII reactivity in Leishmania-infected patients as compared to controls. When anti-CII OD values were compared with BSA-blocked control plates we found higher reactivity against BSA than in CII-coated plates in many Leishmania-infected patients. The percentage of such false positive anti-CII reactions increased with inflammatory activity, and was found in almost all Leishmania patients with highly active inflammatory disease, but was as low in Sudanese healthy controls as well as among Swedish RA patients. The correlation coefficients between false positive anti-CII and anti-CCP2 measured with a commercial ELISA were highest for patients with the most inflammatory disease but non-significant for Sudanese controls and Swedish RA patients, arguing that our findings may have general implications for ELISA measurements in leishmaniasis. ELISA investigations in areas endemic for leishmaniasis might benefit from individual-specific control wells for each serum sample. This approach might also be applicable to other geographical areas or patient groups with high incidence of inflammatory and infectious diseases.

  2. Comparative Analysis of Human Growth Hormone in Serum Using SPRi, Nano-SPRi and ELISA Assays

    PubMed Central

    Henrich, Vincent C.; Sandros, Marinella G.

    2016-01-01

    Sensitive and selective methods for the detection of human growth hormone (hGH) over a wide range of concentrations (high levels of 50-100 ng ml−1 and minimum levels of 0.03 ng ml−1) in circulating blood are essential as variable levels may indicate altered physiology. For example, growth disorders occurring in childhood can be diagnosed by measuring levels of hGH in blood. Also, the misuse of recombinant hGH in sports not only poses an ethical issue it also presents serious health threats to the abuser. One popular strategy for measuring hGH misuse, relies on the detection of the ratio of 22 kDa hGH to total hGH, as non-22 kDa endogenous levels drop after exogenous recombinant hGH (rhGH) administration.Surface plasmon resonance imaging (SPRi) is an analytical tool that allows direct (label-free) monitoring and visualization of biomolecular interactions by recording changes of the refractive index adjacent to the sensor surface in real time. In contrast, the most frequently used colorimetric method, enzyme-linked immunosorbent assay (ELISA) uses enzyme labeled detection antibodies to indirectly measure analyte concentration after the addition of a substrate that induces a color change. To increase detection sensitivity, amplified SPRi uses a sandwich assay format and near infrared quantum dots (QDs) to increase signal strength. After direct SPRi detection of recombinant rhGH in spiked human serum, the SPRi signal is amplified by the sequential injection of detection antibody coated with near-infrared QDs (Nano-SPRi). In this study, the diagnostic potential of direct and amplified SPRi was assessed for measuring rhGH spiked in human serum and compared directly with the capabilities of a commercially available ELISA kit. PMID:26780354

  3. Field Evaluation of an Enzyme-Linked Immunosorbent Assay (ELISA) for Plasmodium falciparum Sporozoite Detection in Anopheline Mosquitoes from Kenya

    DTIC Science & Technology

    1987-01-01

    Anopheles gambiae s.l. and An. funestus. The ELISA confirmed that 88% of 44 sporozoite-positive gland dissections were P. falciparuin. The ELISA...mosquitoes which did not contain salivary gland sporozoites. From a series of 379 Anopheles that were cut at the thorax. ELISA tests on "’head" and "body... Anopheles gainbiae s.l. mosquitoes in The Gambia and West Africa, "’ and recently, P. f/ciparum and P. vivax assays isseCtions were tested in Papua

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2016-07-21

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

  6. 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.

  7. Identifying the predator complex of Homalodisca vitripennis (Hemiptera: Cicadellidae): A comparative study of the efficacy of an ELISA and PCR gut content assay

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A growing number of ecologists are using molecular gut content assays to qualitatively measure predation. The two most popular gut content assays are immunoassays employing pest-specific monoclonal antibodies (MAb) and polymerase chain reaction (PCR) assays employing pest-specific DNA. Here we pre...

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

    ERIC Educational Resources Information Center

    Ott, Laura E.; Carson, Susan

    2014-01-01

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

  9. Enhanced immunoassay for porcine circovirus type 2 antibody using enzyme-loaded and quantum dots-embedded shell-core silica nanospheres based on enzyme-linked immunosorbent assay.

    PubMed

    Wu, Long; Li, Xuepu; Shao, Kang; Ye, Shiyi; Liu, Chen; Zhang, Chenjun; Han, Heyou

    2015-08-05

    Boosting the detection sensitivity of enzyme-linked immunosorbent assay (ELISA) is significant to the early clinical diagnosis of various diseases. Here, we developed a versatile immunosensor using silica nanospheres as carriers for sensitive detection of porcine circovirus type 2 (PCV2) antibody. With HRP enzyme covalently immobilized on the silica nanospheres and CdSe nanocrystals embedded inside, these signal probes were successfully utilized in the sensitive detection of PCV2 antibody by ELISA, fluorometry and square-wave voltammetry (SWV). To further demonstrate the performance of the immunosensor, Human IgG (HIgG) was used as a model analyte. Since more HRP and CdSe QDs were loaded, 5-, 200- and 400-fold enhancements in amplified ELISA, fluorometry and voltammetry responses for HIgG could be achieved compared to conventional ELISA. The respective detection limits of theses methods for HIgG were 3.9, 0.1 and 0.05 ng mL(-1) with a RSD below 5% for amplified ELISA, fluorescence and SWV measurements. Additionally, a 100-fold improvement was obtained in the detection sensitivity for PCV2 antibody immunoassay. The versatile immunosensor exhibits good sensitivity, stability and reproducibility, suggesting its potential applications in clinical diagnostics.

  10. Sporadic Creutzfeldt-Jakob disease diagnostic accuracy is improved by a new CSF ELISA 14-3-3γ assay.

    PubMed

    Leitão, M J; Baldeiras, I; Almeida, M R; Ribeiro, M H; Santos, A C; Ribeiro, M; Tomás, J; Rocha, S; Santana, I; Oliveira, C R

    2016-05-13

    Protein 14-3-3 is a reliable marker of rapid neuronal damage, specifically increased in cerebrospinal fluid (CSF) of sporadic Creutzfeldt-Jakob disease (sCJD) patients. Its detection is usually performed by Western Blot (WB), prone to methodological issues. Our aim was to evaluate the diagnostic performance of a recently developed quantitative enzyme-linked immunosorbent (ELISA) assay for 14-3-3γ, in comparison with WB and other neurodegeneration markers. CSF samples from 145 patients with suspicion of prion disease, later classified as definite sCJD (n=72) or Non-prion diseases (Non-CJD; n=73) comprised our population. 14-3-3 protein was determined by WB and ELISA. Total Tau (t-Tau) and phosphorylated Tau (p-Tau) were also evaluated. Apolipoprotein E gene (ApoE) and prionic protein gene (PRNP) genotyping was assessed. ELISA 14-3-3γ levels were significantly increased in sCJD compared to Non-CJD patients (p<0.001), showing very good accuracy (AUC=0.982; sensitivity=97%; specificity=94%), and matching WB results in 81% of all cases. It strongly correlated with t-Tau and p-Tau (p<0.0001), showing slightly higher specificity (14-3-3 WB - 63%; Tau - 90%; p-Tau/t-Tau ratio - 88%). From WB inconclusive results (n=44), ELISA 14-3-3γ correctly classified 41 patients. Additionally, logistic regression analysis selected ELISA 14-3-3γ as the best single predictive marker for sCJD (overall accuracy=93%). ApoE and PRNP genotypes did not influence ELISA 14-3-3γ levels. Despite specificity for 14-3-3γ isoform, ELISA results not only match WB evaluation but also help discrimination of inconclusive results. Our results therefore reinforce this assay as a single screening test, allowing higher sample throughput and unequivocal results.

  11. A rapid and simple assay for lamotrigine in serum/plasma by HPLC, and comparison with an immunoassay.

    PubMed

    Morgan, Phillip E; Fisher, Danielle S; Evers, Richard; Flanagan, Robert J

    2011-07-01

    Monitoring serum/plasma concentrations of lamotrigine may be useful under certain circumstances. An HPLC column packed with strong cation-exchange (SCX)-modified microparticulate silica together with a 100% methanol eluent containing an ionic modifier permits direct injection of sample extracts. An HPLC-UV method developed using this principle for the measurement of serum/plasma lamotrigine is simple, sensitive and selective. The analysis time is less than 5 min. Intra- and inter-assay precision and accuracy meet acceptance criteria, and sample stability, and potential interferences from other compounds have been evaluated. There was good agreement with consensus mean results from external quality assessment samples (n = 32). Analysis of patient samples (n = 115) using the HPLC method and the Seradyn QMS® Lamotrigine immunoassay showed that the immunoassay over-estimated lamotrigine by 21% on average.

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

    PubMed

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

    2010-08-01

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

  13. Blood Meal Identification in Field-Captured Sand flies: Comparison of PCR-RFLP and ELISA Assays

    PubMed Central

    Maleki-Ravasan, N; Oshaghi, MA; Javadian, E; Rassi, Y; Sadraei, J; Mohtarami, F

    2009-01-01

    Background We aimed to develop a PCR-RFLP assay based on available sequences of putative vertebrate hosts to identify blood meals ingested by field female sand fly in the northwest of Iran. In addition, the utility of PCR-RFLP was compared with ELISA as a standard method. Methods: This experimental study was performed in the Insect Molecular Biology Laboratory of School of Public Health, Tehran University of Medical Sciences, Iran in 2006–2007. For PCR-RFLP a set of conserved vertebrate primers were used to amplify a part of the host mitochondrial cytochrome b (cyt b) gene followed by digestion of the PCR products by Hae III enzyme. Results: The PCR-RFLP and ELISA assays revealed that 34% and 27% of field-collected sand flies had fed on humans, respectively. Additionally, PCR-RFLP assays could reveal specific host DNA as well as the components of mixed blood meals. Results of PCR-RFLP assay showed that the sand flies had fed on cow (54%), human (10%), dog (4%), human and cow (21%), dog and cow (14%), and human and dog (3%). Conclusion: The results can provide a novel method for rapid diagnosis of blood meal taken by sandflies. The advantages and limitations of PCR and ELISA assays are discussed. PMID:22808367

  14. Enhanced sensitive immunoassay: noncompetitive phage anti-immune complex assay for the determination of malachite green and leucomalachite green.

    PubMed

    Dong, Jie-Xian; Xu, Chao; Wang, Hong; Xiao, Zhi-Li; Gee, Shirley J; Li, Zhen-Feng; Wang, Feng; Wu, Wei-Jian; Shen, Yu-Dong; Yang, Jin-Yi; Sun, Yuan-Ming; Hammock, Bruce D

    2014-08-27

    To develop a more sensitive immunoassay for malachite green (MG) and leucomalachite green (LMG), we identified the immunocomplex binding phage-borne peptides for use in the noncompetitive phage anti-immunocomplex assay (PHAIA). An anti-LMG monoclonal antibody (mAb) was used to select immunocomplex binding peptides from a circular random eight-amino-acid phage-displayed library. After three rounds of panning-elution, five peptides that bound the LMG-mAb immunocomplex were obtained. One of the phage-borne peptide clones that resulted in an assay with the highest sensitivity was chosen for further research. The concentration of LMG producing 50% of the saturated signal and the limit of detection of the assay were 7.02 and 0.55 ng/mL, respectively, with a linear range of 1.35 to 21.56 ng/mL. The PHAIA based on the same antibody was 16 times more sensitive compared to the competitive immunoassay. PHAIA was used to analyze LMG, MG, and two mixtures of spiked fish samples, with validation by high-performance liquid chromatography (HPLC) with fluorescence detector. Results showed a good correlation (R(2)LMG = 0.9841; R(2)MG = 0.993; R(2)Mixture = 0.9903) between the data of PHAIA and HPLC, thus the assay was an efficient method for monitoring food safety.

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

    PubMed

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

    2008-07-28

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

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

    PubMed

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

    2003-01-01

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

  17. Direct replacement of antibodies with molecularly imprinted polymer nanoparticles in ELISA--development of a novel assay for vancomycin.

    PubMed

    Chianella, Iva; Guerreiro, Antonio; Moczko, Ewa; Caygill, J Sarah; Piletska, Elena V; De Vargas Sansalvador, Isabel M Perez; Whitcombe, Michael J; Piletsky, Sergey A

    2013-09-03

    A simple and straightforward technique for coating microplate wells with molecularly imprinted polymer nanoparticles (nanoMIPs) to develop assays similar to the enzyme-linked immunosorbent assay (ELISA) is presented here for the first time. NanoMIPs were synthesized by a solid-phase approach with an immobilized vancomycin (template) and characterized using Biacore 3000, dynamic light scattering, and electron microscopy. Immobilization, blocking, and washing conditions were optimized in microplate format. The detection of vancomycin was achieved in competitive binding experiments with a horseradish peroxidase-vancomycin conjugate. The assay was capable of measuring vancomycin in buffer and in blood plasma within the range of 0.001-70 nM with a detection limit of 0.0025 nM (2.5 pM). The sensitivity of the assay was 3 orders of magnitude better than a previously described ELISA based on antibodies. In these experiments, nanoMIPs have shown high affinity and minimal interference from blood plasma components. Immobilized nanoMIPs were stored for 1 month at room temperature without any detrimental effects to their binding properties. The high affinity of nanoMIPs and the lack of a requirement for cold chain logistics make them an attractive alternative to traditional antibodies used in ELISA.

  18. Superiority of radiobinding assay over ELISA for detection of IAAs in newly diagnosed type I diabetic children

    SciTech Connect

    Levy-Marchal, C.; Bridel, M.P.; Sodoyez-Goffaux, F.; Koch, M.; Tichet, J.; Czernichow, P.; Sodoyez, J.C. )

    1991-01-01

    Liquid- or solid-phase assays have been used for insulin autoantibody (IAA) determination, and the method of IAA measurement has not been standardized. IAAs were determined by radiobinding assay (RBA) and enzyme-linked immunosorbent assay (ELISA) in two large age-matched groups of nondiabetic and newly diagnosed insulin-dependent (type I) diabetic children. Positivity for IAA by RBA (greater than or equal to nondiabetic mean + 3SD) was 2 of 178 (1.1%) and 55 of 173 (32%) in nondiabetic and diabetic children, respectively. Prevalence of IAA by RBA was significantly higher in the youngest age-group (63% between 0-4 yr). Positivity for IAA by ELISA was 1 of 178 (0.6%) and 8 of 169 (4.7%) in nondiabetic and diabetic children, respectively. Concordance rates between both assays were 0 of 3 (0%) in control subjects and 5 of 58 (8.6%) in diabetic children. We conclude that RBA is more appropriate than ELISA for IAA detection at the onset of the disease. In addition, because available data suggest that IAAs detected by RBA only are high-affinity antibodies, it is tempting to speculate that IAAs reflect a mature immune reaction against endogenous insulin.

  19. Informative prior distributions for ELISA analyses.

    PubMed

    Klauenberg, Katy; Walzel, Monika; Ebert, Bernd; Elster, Clemens

    2015-07-01

    Immunoassays are capable of measuring very small concentrations of substances in solutions and have an immense range of application. Enzyme-linked immunosorbent assay (ELISA) tests in particular can detect the presence of an infection, of drugs, or hormones (as in the home pregnancy test). Inference of an unknown concentration via ELISA usually involves a non-linear heteroscedastic regression and subsequent prediction, which can be carried out in a Bayesian framework. For such a Bayesian inference, we are developing informative prior distributions based on extensive historical ELISA tests as well as theoretical considerations. One consideration regards the quality of the immunoassay leading to two practical requirements for the applicability of the priors. Simulations show that the additional prior information can lead to inferences which are robust to reasonable perturbations of the model and changes in the design of the data. On real data, the applicability is demonstrated across different laboratories, for different analytes and laboratory equipment as well as for previous and current ELISAs with sigmoid regression function. Consistency checks on real data (similar to cross-validation) underpin the adequacy of the suggested priors. Altogether, the new priors may improve concentration estimation for ELISAs that fulfill certain design conditions, by extending the range of the analyses, decreasing the uncertainty, or giving more robust estimates. Future use of these priors is straightforward because explicit, closed-form expressions are provided. This work encourages development and application of informative, yet general, prior distributions for other types of immunoassays.

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

    PubMed

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

    2016-07-01

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

  1. Validation and comparison of two commercial ELISA kits and three in-house developed real-time PCR assays for the detection of potentially allergenic mustard in food.

    PubMed

    Palle-Reisch, Monika; Hochegger, Rupert; Štumr, Stepan; Korycanova, Kveta; Cichna-Markl, Margit

    2015-05-01

    The study compares the applicability of two commercial mustard ELISA kits (Mustard ELISA Kit-specific and Mustard ELISA Kit-total) and three in-house developed real-time PCR assays (singleplex assay for white mustard, singleplex assay for black/brown mustard and duplex assay for the detection of white, black and brown mustard). Analyses of raw and brewed model sausages containing white and black/brown mustard in the range from 1 to 50 ppm indicate that both ELISAs and the three real-time PCR assays allow the detection of traces of mustard in raw and in brewed sausages. The ELISAs were found to be more sensitive than the real-time PCR assays. When the ELISAs and real-time PCR assays were applied to the analysis of 15 commercial foodstuffs differing in their labelling concerning mustard, in one sample mustard was detected with both ELISAs and the three real-time PCR assays although mustard was not indicated on the food ingredient list.

  2. Direct ELISA.

    PubMed

    Lin, Alice V

    2015-01-01

    First described by Engvall and Perlmann, the enzyme-linked immunosorbent assay (ELISA) is a rapid and sensitive method for detection and quantitation of an antigen using an enzyme-labeled antibody. Besides routine laboratory usage, ELISA has been utilized in medical field and food industry as diagnostic and quality control tools. Traditionally performed in 96-well or 384-well polystyrene plates, the technology has expanded to other platforms with increase in automation. Depending on the antigen epitope and availability of specific antibody, there are variations in ELISA setup. The four basic formats are direct, indirect, sandwich, and competitive ELISAs. Direct ELISA is the simplest format requiring an antigen and an enzyme-conjugated antibody specific to the antigen. This chapter describes the individual steps for detection of a plate-bound antigen using a horseradish peroxidase (HRP)-conjugated antibody and luminol-based enhanced chemiluminescence (ECL) substrate. The methodological approach to optimize the assay by chessboard titration is also provided.

  3. Colloidal nanomaterial-based immunoassay.

    PubMed

    Teste, Bruno; Descroix, Stephanie

    2012-06-01

    Nanomaterials have been widely developed for their use in nanomedicine, especially for immunoassay-based diagnosis. In this review we focus on the use of nanomaterials as a nanoplatform for colloidal immunoassays. While conventional heterogeneous immunoassays suffer from mass transfer limitations and consequently long assay time, colloidal immunosupports allow target capture in the entire volume, thus speeding up reaction kinetics and shortening assay time. Owing to their wide range of chemical and physical properties, nanomaterials are an interesting candidate for immunoassay development. The most popular colloidal nanomaterials for colloidal immunoassays will be discussed, as well as their influence on immune reactions. Recent advances in nanomaterial applications for different formats of immunoassays will be reported, such as nanomaterial-based indirect immunoassays, optical-based agglutination immunoassays, resonance energy transfer-based immunoassays and magnetic relaxation-based immunoassays. Finally, the future of using nanomaterials for homogeneous immunoassays dedicated to clinical diagnosis will be discussed.

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

    PubMed

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

    2013-01-01

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

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

    PubMed Central

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

    2013-01-01

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

  6. Design-of-experiment approach for HPLC analysis of 25-hydroxyvitamin D: a comparative assay with ELISA.

    PubMed

    Abu el Maaty, Mohamed Abdulla; Hanafi, Rasha Sayed; Aboul-Enein, Hassan Y; Gad, Mohamed Zakaria

    2015-01-01

    Although high-performance liquid chromatography-mass spectrometry (HPLC-MS) is adopted as the method of choice for the determination of vitamin D and its metabolites in plasma, yet the unavailability of this expensive detection technique in many clinical laboratories makes ultraviolet (UV) detection the alternative of choice in many places worldwide. In this regard, determination of parameters affecting HPLC separation of vitamins D2, D3 and their hydroxyl metabolites in plasma in a systematic way would put an end to irrelevant trials for more optimization. A new robust HPLC-UV was developed, optimized using DryLab(®)2000 and validated for the determination of vitamins D2 and D3 and their 25-hydroxyl metabolites in plasma to achieve best resolution and least runtime where the metabolites elute in <10 min, where vitamin D2 is considered a feasible internal standard. Chromatographic parameters affecting resolution of the four peaks were specifically defined by a two-dimensional resolution map. Forty-six plasma samples were analyzed by the optimized method as well as by an ELISA kit to compare results and to judge validity of ELISA as a technique of clinical importance. Statistical analyses proved that the investigated assays were incomparable. Variation among subjects was detected by HPLC but not ELISA, concluding that HPLC-UV is the better tool in determining vitamin D status than ELISA.

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

    PubMed

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

    2012-01-31

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

  8. Development of a high-throughput ELISA assay for platelet function testing using platelet-rich plasma or whole blood.

    PubMed

    Salles, Isabelle; Broos, Katleen; Fontayne, Alexandre; Szántó, Tímea; Ruan, Changgeng; Nurden, Alan T; Vanhoorelbeke, Karen; Deckmyn, Hans

    2010-08-01

    Platelets play an essential role in the development of cardiovascular diseases and are the target of several agents that can inhibit their function. Despite the existence of a wide array of techniques to study platelet function, an assay to evaluate several platelet signalling pathways in a high-throughput fashion, combined with minimal blood volume and handling is still needed. We have developed a sensitive assay in the form of a sandwich ELISA where monoclonal antibodies against P-selectin or alphaIIbbeta3 and GPIbalpha were used to capture and detect platelets, respectively, in the presence of five different agonists [ADP, TRAP (thrombin receptor agonist), U46619 (thromboxane A2 analogue), collagen-related-peptide, and arachidonic acid]. Binding of platelets to the antibodies increased dose-dependently with the concentration of either agonist, while binding of ADP-activated platelets was abrogated when inhibitors of platelet activation were concomitantly added. The test showed good sample reproducibility in 15 healthy donors with conserved platelet response to agonists throughout the assay. Healthy subjects could be identified as normal-, hypo- or hyper-responders for each agonist, which for most cases (73%) was confirmed upon retesting. Finally, we demonstrated that the platelet ELISA assay can not only be used in platelet-rich plasma but also in whole blood; it now awaits large scale studies to assess its full screening and diagnostic values.

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

    PubMed

    Xiao, Yuhong; Isaacs, Stuart N

    2012-10-31

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

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

    PubMed Central

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

    2017-01-01

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

  11. DEVELOPMENT AND EVALUATION OF AN ENZYME-LINKED IMMUNOASSAY (ELISA) METHOD FOR THE MEASUREMENT OF 2,4-DICHLOROPHENOXYACETIC ACID IN HUMAN URINE

    EPA Science Inventory

    This paper describes the development of a 96-microwell high sample capacity ELISA method for measuring 2,4-D in urine; the analysis of 2,4-D in real-world urine samples by both ELISA and GC/MS methods; and compares the ELISA and GC/MS results in several key areas: accuracy, preci...

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

    EPA Science Inventory

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

  13. A simple micro-ELISA method for the assay of antithyroglobulin autoantibodies in human serum

    PubMed Central

    Goodburn, Richard; Williams, David L; Marks, Vincent

    1981-01-01

    An indirect, enzyme-linked immunosorbent assay is described for the assay of thyroid autoantibodies, particularly those directed against thyroglobulin. The method is specific, sensitive and precise, and may be automated. The results are shown to correlate well with those obtained by the haemagglutination method. PMID:6974179

  14. Development of an enzyme-linked immunosorbent assay (ELISA) using highly-specific monoclonal antibodies against plumbagin.

    PubMed

    Sakamoto, Seiichi; Putalun, Waraporn; Tsuchihashi, Ryota; Morimoto, Satoshi; Kinjo, Junei; Tanaka, Hiroyuki

    2008-01-21

    Plumbagin (PL; 5-hydroxy-2-methyl-1,4-naphthoquinone) is a natural compound mainly isolated from Plumbago zeylanica. This plant is distributed in Southeast Asia, and well known as Ayurvedic medicine in India for its medicinal properties. PL has been shown to have various pharmacological activities. We have successfully prepared monoclonal antibodies against PL, and developed an enzyme-linked immunosorbent assay (ELISA) system for determination of PL. 3-(5-Hydroxy-2-methyl-1,4-naphthoquinone-3-yl) propanoic acid was synthesized and purified to prepare PL-bovine serum albumin conjugate (PL-BSA), which was used as an immunogen. PL-BSA conjugate was administered into BALB/c male mice for production of monoclonal antibodies against PL. The monoclonal antibody against PL which is secreted from established hybridoma cell line 3A3 (MAb 3A3) has been proven to have highly-specific to PL resulting from cross-reactivities test. The range for calibration of PL by ELISA was 0.2-25 microg mL(-1). Based on validation analysis, this analytical method by ELISA is a precise, accurate, and sensitive method for the determination of PL in plant.

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

    PubMed

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

    1996-01-01

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

  16. Development of hen antihepatitis B antigen IgY-based conjugate for ELISA assay

    PubMed Central

    Nafea, Najat Muayed; Sabbah, Majeed Arsheed; AL-Suhail, Raghad; Mahdavi, Amir Hossein; Asgary, Sedigheh

    2015-01-01

    Background: Chicken antibodies have many advantages to the mammalian antibodies and have several important differences against mammalian IgG with regard to their specificity and large-scale production. In this study, the production, purification, and HRP conjugation of polyclonal IgY against hepatitis virus surface antigen (HBsAg) were carried out. Materials and Methods: Single Comb White Leghorn hens were immunized intramuscularly with hepatitis B vaccine in combination with Freund's adjuvants. Blood and eggs were collected before and during ten weeks after the first immunization. Results: A highly purified of 180 KDa with specific activity of 200 mIU/ml was obtained by our purification protocol. One milligram of the purified IgY was labeled with horseradish peroxidase (HRP). Sandwich ELISA was used to determine the optimum titer of anti-HbsAg IgY-conjugate which was found to be 1:20. Conclusions: This study showed that laying hens can be used as an alternative source for production of polyclonal antibodies against HBsAg and anti-HBs IgY could be labeled with HRP enzyme and could subsequently be used successfully as secondary antibody in ELISA for detection of HBsAg in the patients sera. PMID:26015926

  17. Assessing Immunity to Rubella Virus: a Plea for Standardization of IgG (Immuno)assays

    PubMed Central

    Bouthry, Elise; Huzly, Daniela; Ogee-Nwankwo, Adaeze; Hao, LiJuan; Adebayo, Adebola; Icenogle, Joseph; Sarasini, Antonella; Revello, Maria Grazia; Grangeot-Keros, Liliane

    2016-01-01

    Immunity to rubella virus (RV) is commonly determined by measuring specific immunoglobulin G (RV IgG). However, RV IgG results and their interpretation may vary, depending on the immunoassay, even though most commercial immunoassays (CIAs) have been calibrated against an international standard and results are reported in international units per milliliter. A panel of 322 sera collected from pregnant women that tested negative or equivocal for RV IgG in a prior test (routine screening) was selected. This panel was tested with two reference tests, immunoblotting (IB) and neutralization (Nt), and with 8 CIAs widely used in Europe. IB and Nt gave concordant results on 267/322 (82.9%) sera. Of these, 85 (26.4%) sera were negative and 182 (56.5%) sera were positive for both tests. All 85 IB/Nt-negative samples were classified as negative with all CIAs. Of the 182 IB/Nt-positive samples, 25.3 to 61.5% were classified as equivocal and 6 to 64.8% were classified as positive with the CIAs. Wide variations in titers in international units per milliliter were observed. In our series, more than half of the women considered susceptible to RV based on CIA results tested positive for RV antibodies by IB/Nt. Our data suggest that (i) sensitivity of CIAs could be increased by considering equivocal results as positive and (ii) the definition of immunity to RV as the 10-IU/ml usual cutoff as well as the use of quantitative results for clinical decisions may warrant reconsideration. A better standardization of CIAs for RV IgG determination is needed. PMID:27147722

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

    PubMed Central

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

    2013-01-01

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

  19. Molecular mass dependence of hyaluronan detection by sandwich ELISA-like assay and membrane blotting using biotinylated hyaluronan binding protein

    PubMed Central

    Yuan, Han; Tank, Mihir; Alsofyani, Abeer; Shah, Naman; Talati, Nishant; LoBello, Jaclyn C; Kim, Jin Ryoun; Oonuki, Yoji; de la Motte, Carol A; Cowman, Mary K

    2013-01-01

    Hyaluronan (HA) is widely detected in biological samples and its concentration is most commonly determined by the use of a labeled specific HA binding protein (aggrecan G1-IGD-G2, HABP), employing membrane blotting and sandwich enzyme-linked immunosorbent assay (ELISA)-like methods. However, the detected signal intensity or the quantified value obtained by using these surface-based methods is related to the molecular mass (M) of HA, especially for HA in the low M range below ∼150 kDa. At the same mass or mass concentration, higher M HA gives a higher signal than lower M HA. We have experimentally determined the quantitative relationship between the M of HA (in the range 20–150 kDa) and the relative signal intensity in comparison with a standard HA, in a sandwich ELISA-like assay. An M-dependent signal correction factor (SCF) was calculated and used to correct the signal intensity, so that the corrected concentration value would more accurately reflect the true HA concentration in solution. The SCF for polydisperse low M HA was also calculated and compared with experimental results. When the molecular mass distribution of an HA sample is determined by a method such as gel electrophoresis, then its appropriately averaged SCF can be calculated and used to correct the signal in sandwich ELISA to obtain a more accurate concentration estimation. The correction method works for HA with M between ∼150 and 20 kDa, but lower M HA is too poorly detected for useful analysis. The physical basis of the M-dependent detection is proposed to be the increase in detector-accessible fraction of each surface-bound molecule as M increases. PMID:23964097

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

    PubMed Central

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

    2012-01-01

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

  1. SNAP Assay Technology.

    PubMed

    O'Connor, Thomas P

    2015-12-01

    The most widely used immunoassay configuration is the enzyme-linked immunosorbent assay (ELISA) because the procedure produces highly sensitive and specific results and generally is easy to use. By definition, ELISAs are immunoassays used to detect a substance (typically an antigen or antibody) in which an enzyme is attached (conjugated) to one of the reactants and an enzymatic reaction is used to amplify the signal if the substance is present. Optimized ELISAs include several steps that are performed in sequence using a defined protocol that typically includes application of sample and an enzyme-conjugated antibody or antigen to an immobilized reagent, followed by wash and enzyme reaction steps. The SNAP assay is an in-clinic device that performs each of the ELISA steps in a timed sequential fashion with little consumer interface. The components and mechanical mechanism of the assay device are described. Detailed descriptions of features of the assay, which minimize nonspecific binding and enhance the ability to read results from weak-positive samples, are given. Basic principles used in assays with fundamentally different reaction mechanisms, namely, antigen-detection, antibody-detection, and competitive assays are given. Applications of ELISA technology, which led to the development of several multianalyte SNAP tests capable of testing for up to 6 analytes using a single-sample and a single-SNAP device are described.

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

    PubMed

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

    2013-01-01

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

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

    USGS Publications Warehouse

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

    1997-01-01

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

  4. Determination of Cutoff of ELISA and Immunofluorescence Assay for Scrub Typhus

    PubMed Central

    Gupta, Nitin; Chaudhry, Rama; Thakur, Chandan Kumar

    2016-01-01

    Background: The most common method employed for diagnosis of scrub typhus is serology. It is widely known that demonstration of ≥4-fold rise in titers of antibody in paired sera is required for diagnosis. However, for guidance of initial treatment, there is a need for rapid diagnosis at the time of admission. Therefore, there is a need for standardized region specific cutoff titers at the time of admission. Materials and Methods: A total of 258 patients of all age groups with clinically suspected scrub typhus over a period of 24 months (October 2013-October 2015) were enrolled. Serum samples of these patients were subjected to immunofluorescent antibody (IFA) for immunoglobulin M (IgM) (Fuller Labs, USA) with dilutions of 1:64, 1:128, 1:256, and 1:512. Serum samples of all 258 patients were subjected to IgM ELISA (Inbios Inc., USA). Any patient with response to antibiotics within 48 h accompanied by either presence of an eschar or positivity by polymerase chain reaction was taken as positive. Receiver operating characteristic (ROC) curve was drawn to generate cutoff for these tests. Results: A total of 20 patients were diagnosed as cases of scrub typhus. The ROC curve analysis revealed a cutoff optical density value of 0.87 with sensitivity and specificity of 100% and 94.12%, respectively. ROC curve analysis of IFA revealed sensitivity and specificity of 100% and 93.5%, respectively at 1:64 dilution. Conclusion: Considering cost constraints, centers in and around New Delhi region can use the cutoffs we determined for the diagnosis of scrub typhus. PMID:27621559

  5. Comparative Multi-Donor Study of IFNγ Secretion and Expression by Human PBMCs Using ELISPOT Side-by-Side with ELISA and Flow Cytometry Assays.

    PubMed

    Hagen, Jodi; Zimmerman, Ryan; Goetz, Christine; Bonnevier, Jody; Houchins, Jeffrey P; Reagan, Kevin; Kalyuzhny, Alexander E

    2015-02-11

    ELISPOT, ELISA and flow cytometry techniques are often used to study the function of immune system cells. It is tempting to speculate that these assays can be used interchangeably, providing similar information about the cytokine secreting activity of cells: the higher the number of cytokine-positive cells measured by flow cytometry, the higher the number of cytokine-secreting cells expected to be detected by ELISPOT and the larger the amount of secreted cytokine expected to be measured by ELISA. We have analyzed the expression level and secretion capacity of IFNγ from peripheral blood mononuclear cells isolated from five healthy donors and stimulated by calcium ionomycin mixed with phorbol 12-myristate 13-acetate in a non-specific manner in side-by-side testing using ELISPOT, ELISA and flow cytometry assays. In our study, we observed a general correlation in donors' ranking between ELISPOT and flow cytometry; ELISA values did not correlate with either ELISPOT or flow cytometry. However, a detailed donor-to-donor comparison between ELISPOT and flow cytometry revealed significant discrepancies: donors who have similar numbers of IFNγ-positive cells measured by flow cytometry show 2-3-fold differences in the number of spot-forming cells (SFCs) measured by ELISPOT; and donors who have the same number of SFCs measured by ELISPOT show 30% differences in the number of IFNγ-positive cells measured by flow cytometry. Significant discrepancies between donors were also found when comparing ELISA and ELISPOT techniques: donors who secreted the same amount of IFNγ measured by ELISA show six-fold differences in the number of SFCs measured by ELISPOT; and donors who have 5-7-times less secreted IFNγ measured by ELISA show a two-fold increase in the number of SFCs measured by ELISPOT compared to donors who show a more profound secretion of IFNγ measured by ELISA. The results of our study suggest that there can be a lack of correlation between IFNγ values measured by

  6. 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

  7. 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...

  8. 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.

  9. A Colorimetric Enzyme-Linked Immunosorbent Assay (ELISA) Detection Platform for a Point-of-Care Dengue Detection System on a Lab-on-Compact-Disc.

    PubMed

    Thiha, Aung; Ibrahim, Fatimah

    2015-05-18

    The enzyme-linked Immunosorbent Assay (ELISA) is the gold standard clinical diagnostic tool for the detection and quantification of protein biomarkers. However, conventional ELISA tests have drawbacks in their requirement of time, expensive equipment and expertise for operation. Hence, for the purpose of rapid, high throughput screening and point-of-care diagnosis, researchers are miniaturizing sandwich ELISA procedures on Lab-on-a-Chip and Lab-on-Compact Disc (LOCD) platforms. This paper presents a novel integrated device to detect and interpret the ELISA test results on a LOCD platform. The system applies absorption spectrophotometry to measure the absorbance (optical density) of the sample using a monochromatic light source and optical sensor. The device performs automated analysis of the results and presents absorbance values and diagnostic test results via a graphical display or via Bluetooth to a smartphone platform which also acts as controller of the device. The efficacy of the device was evaluated by performing dengue antibody IgG ELISA on 64 hospitalized patients suspected of dengue. The results demonstrate high accuracy of the device, with 95% sensitivity and 100% specificity in detection when compared with gold standard commercial ELISA microplate readers. This sensor platform represents a significant step towards establishing ELISA as a rapid, inexpensive and automatic testing method for the purpose of point-of-care-testing (POCT) in resource-limited settings.

  10. Utility of Loop-mediated Isothermal Amplification Assay, Polymerase Chain Reaction, and ELISA for Diagnosis of Leptospirosis in South Indian Patients

    PubMed Central

    Sengupta, Mallika; Prabhakar, Abhilash Kundavaram Paul; Satyendra, Sowmya; Thambu, David; Abraham, Ooriapadickal Cherian; Balaji, Veeraraghavan; Chen, Hua-Wei; Chao, Chien-Chung; Ching, Wei-Mei; Prakash, John Antony Jude

    2017-01-01

    Background: Leptospirosis is a zoonotic disease which requires laboratory diagnosis for confirmation. Materials and Methods: In this study serum samples from adults with acute undifferentiated fever (duration ≤15 days) were tested for IgM antibodies to Leptospira by ELISA, PCR for rrs gene and loop-mediated isothermal amplification (LAMP) assay for LipL32 and LipL41. Results: Among the 150 sera tested, three were positive by PCR, LAMP and IgM ELISA/modified Faines’ criteria, two by only PCR; seven only by LAMP assay and forty fulfilled modified Faine's criteria (illness clinically compatible and IgM ELISA positive for leptospirosis). Clinical correlation revealed renal compromise, low platelet count and severe jaundice were significantly related to leptospirosis (P < 0.05). Conclusion: This study suggests that LAMP assay could be useful for diagnosis of leptospirosis during the 1st week of illness whereas IgM ELISA forms the mainstay of diagnosis from the 2nd week onward. Further studies especially community based, comparing ELISA, PCR, LAMP, culture and microscopic agglutination test are required to evaluate the veracity of these findings. PMID:28250618

  11. Free cortisol in serum assayed by temperature-controlled ultrafiltration before fluorescence polarization immunoassay.

    PubMed

    Lentjes, E G; Romijn, F; Maassen, R J; de Graaf, L; Gautier, P; Moolenaar, A J

    1993-12-01

    A method is described for a temperature-controlled ultrafiltration procedure to measure free cortisol in serum. A special thermometer with a sensor was developed, measuring the temperature directly in the ultrafiltration device. The sensor is screwed on the axis of the centrifuge rotor, and the centrifuge is placed in a temperature-controlled box so that the temperature of the sample is kept at 37 degrees C +/- 0.1 degrees C. The overall CV of the free cortisol assay ranges from 2.2% to 11.4%, of which the ultrafiltration contributes only 2.2-3.6%. Increasing amounts of cortisol-binding protein, as found in women using estrogen-containing oral contraceptives, have minor but significant effects on the free cortisol concentrations in serum. Serum free cortisol concentrations in a reference population (n = 114; central 95 percentiles) were 12-43 nmol/L (4-9.5% of total cortisol); in the group of the oral-contraceptive users (n = 27), the reference interval was 11-53 nmol/L (1.5-4.5%).

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

    PubMed

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

    2009-10-15

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

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

    PubMed

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

    2002-03-01

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

  14. Development of a cell culture/ELISA assay to detect anticoagulant rodenticides and its application to analysis of rodenticide treated grain.

    PubMed

    Lawley, Wendy J; Charlton, Andrew J A; Hughson, Elaine J; Grundy, Helen H; Brown, Peter M; Jones, Ainsley

    2006-03-08

    This study describes a generic biological screening assay designed to detect anticoagulant rodenticides based on their inhibitory action on the vitamin K epoxide reductase protein complex, resulting in an accumulation of under-carboxylated prothrombin or proteins induced by vitamin K antagonism (PIVKA-II). A combined cell culture/ELISA assay was optimized to measure PIVKA-II production by the human hepatoma HepG2 cell line cultured in the presence of anticoagulant rodenticides. The specificity and sensitivity of the assay was validated using 41 grain extracts containing representative concentrations of rodenticide or appropriate nonrodenticide control compounds. In all cases, PIVKA-II produced by HepG2 cells in response to grain extracts spiked with rodenticides was detected by ELISA, while PIVKA-II was not detected in supernatants collected from cells exposed to nonrodenticide controls. This represents a novel, class-specific biological assay for the detection of anticoagulant rodenticides present in contaminated grain.

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

    EPA Science Inventory

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

  16. Indirect enzyme-linked immunosorbent assay for immunoglobulin G and four immunoassays for immunoglobulin M to Toxoplasma gondii in a series of heart transplant recipients.

    PubMed Central

    Sluiters, J F; Balk, A H; Essed, C E; Mochtar, B; Weimar, W; Simoons, M L; Ijzerman, E P

    1989-01-01

    Toxoplasma gondii infections in heart transplant recipients were monitored by indirect enzyme-linked immunosorbent assay for immunoglobulin G (ELISA-IgG), indirect ELISA-IgM in serum IgM fractions, antibody capture ELISA-IgM, IgM-immunosorbent agglutination assay (ISAGA), and IgM immunoblotting. Basic immunosuppression consisted of cyclosporine and low-dose steroids. Before transplantation, 26 of 43 recipients showed serological evidence of infection. In serum samples from 15 (35%) recipients, specific antibodies were not detected. Approximately 50% of the heart donors, were toxoplasma seropositive. Eight of the fifteen seronegative recipients received hearts from toxoplasma-seropositive donors. In four of the eight recipients, seroconversion could be demonstrated with all tests used. In three of these four patients, clinical disease developed. One patient with strong serological evidence of toxoplasmosis died, but toxoplasma parasites and antigens were not detected at autopsy. In two patients, toxoplasma cysts were found in cardiac biopsies. Seroconversion was not prevented by the use of spiramycin prophylaxis in two recipients. Reactivations of latent infections or reinfections were detected by indirect ELISA in six (23%) seropositive recipients, but symptoms and signs of active T. gondii infection were not seen. Seroconversion and reactivation of infection were readily found by a combined use of indirect ELISA-IgG and ELISA-IgM and antibody capture ELISA-IgM. Discrepancies in results could be examined by immunoblotting. IgM-ISAGA retained stable positive values longer than IgM-ELISAs did. Cyclosporine treatment did not hamper detection of seroconversion but could cause antibody levels to remain relatively low in primary infections. Seronegative recipients should receive antitoxoplasma treatment on seroconversion. PMID:2654182

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

    PubMed Central

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

    1984-01-01

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

  18. Broad-specificity immunoassay for O,O-diethyl organophosphorus pesticides: Application of molecular modeling to improve assay sensitivity and study antibody recognition

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A monoclonal antibody (MAb) against 4-(diethoxyphosphorothioyloxy)benzoic acid (hapten 1) was raised and used to develop a broad-specificity competitive indirect enzyme-linked immunosorbent assay (ciELISA) for 14 O,O-diethyl organophosphorus pesticides (OPs). Computer-assisted molecular modeling was...

  19. Quantitation of IgE and carcinoembryonic antigen (CEA) by optical beam deflection (OBD) measurement of dot-immunobinding assay patterns visualized by an ELISA technique.

    PubMed

    Matsuzawa, S; Kimura, H; Tu, C Y; Kitamori, T; Sawada, T

    1993-05-05

    Dot-immunobinding assays of IgE and CEA were performed by a conventional dot-ELISA technique with diaminobenzidine staining, and the quantitative results were compared by densitometry and a new, spectroscopic, optical beam deflection (OBD) method using the same membrane. It was possible with the OBD method to detect quantities of these substances at least ten times smaller than with densitometry. Better intra-assay reproducibility for IgE and CEA measurements was obtained by the OBD method. The measurable ranges of the OBD method was broader than that of densitometry, because dark bands caused OBD in proportion to their color densities. When the dot-immunobinding assay with OBD measurement for CEA was also compared with a microtube ELISA using biotin-avidin conjugates, the sensitivities and reproducibilities of the two methods were found to be similar, with a correlation coefficient of 0.991.

  20. Utility of IgM ELISA, TaqMan real-time PCR, reverse transcription PCR, and RT-LAMP assay for the diagnosis of Chikungunya fever.

    PubMed

    Reddy, Vijayalakshmi; Ravi, Vasanthapuram; Desai, Anita; Parida, Manmohan; Powers, Ann M; Johnson, Barbara W

    2012-11-01

    Chikungunya fever a re-emerging infection with expanding geographical boundaries, can mimic symptoms of other infections like dengue, malaria which makes the definitive diagnosis of the infection important. The present study compares the utility of four laboratory diagnostic methods viz. IgM capture ELISA, an in house reverse transcription PCR for the diagnosis of Chikungunya fever, TaqMan real-time PCR, and a one step reverse transcription-loop mediated isothermal amplification assay (RT-LAMP). Out of the 70 serum samples tested, 29 (41%) were positive for Chikungunya IgM antibody by ELISA and 50 (71%) samples were positive by one of the three molecular assays. CHIKV specific nucleic acid was detected in 33/70 (47%) by reverse transcription PCR, 46/70 (66%) by TaqMan real-time PCR, and 43/70 (62%) by RT-LAMP assay. A majority of the samples (62/70; 89%) were positive by at least one of the four assays used in the study. The molecular assays were more sensitive for diagnosis in the early stages of illness (2-5 days post onset) when antibodies were not detectable. In the later stages of illness, the IgM ELISA is a more sensitive diagnostic test. In conclusion we recommend that the IgM ELISA be used as an initial screening test followed one of the molecular assays in samples that are collected in the early phase of illness and negative for CHIKV IgM antibodies. Such as approach would enable rapid confirmation of the diagnosis and implementation of public health measures especially during outbreaks.

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

    PubMed

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

    2003-01-01

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

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

    PubMed

    Obaldia, N

    1991-04-01

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

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

    PubMed

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

    2011-05-31

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

  4. 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 ...

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

    PubMed

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

    1981-09-01

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

  6. Development and Application of an ELISA Assay Using Excretion/Secretion Proteins from Epimastigote Forms of T. cruzi (ESEA Antigens) for the Diagnosis of Chagas Disease

    PubMed Central

    Berrizbeitia, Mariolga; Figueroa, Milagros; Ward, Brian J.; Rodríguez, Jessicca; Jorquera, Alicia; Figuera, Maria A.; Romero, Leomerys; Ndao, Momar

    2012-01-01

    An indirect enzyme-linked immunoabsorbent assay (ELISA) for Trypanosoma cruzi was developed using epimastigote secretion/excretion proteins (ESEA antigens) obtained from axenic culture supernatants. A panel of 120 serum samples from subjects with confirmed Chagas disease (n = 50), healthy controls (n = 50), and patients with other parasitic diseases (n = 20) was used to evaluate the new ESEA-based ELISA (ELISAESEA). This new test had excellent sensitivity (98%) and acceptable specificity (88%). Cross-reactivity was observed largely in sera from subjects with Leishmania and Ascaris infections. Using Western blotting and epimastigotes from two distinct T. cruzi isolates, several polypeptide bands with molecular masses ranging from 50 to 220 kDa were detected in pooled chagasic sera. However, the band pattern for each isolate was different. These data suggest that an inexpensive and technically simple ELISA based on ESEA antigens is a promising new tool for the diagnosis of Chagas disease. PMID:23049572

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

    PubMed

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

    2014-12-01

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

  8. Increased sensitivity of 3D-Well enzyme-linked immunosorbent assay (ELISA) for infectious disease detection using 3D-printing fabrication technology.

    PubMed

    Singh, Harpal; Shimojima, Masayuki; Fukushi, Shuetsu; Le Van, An; Sugamata, Masami; Yang, Ming

    2015-01-01

    Enzyme-linked Immunosorbent Assay or ELISA -based diagnostics are considered the gold standard in the demonstration of various immunological reaction including in the measurement of antibody response to infectious diseases and to support pathogen identification with application potential in infectious disease outbreaks and individual patients' treatment and clinical care. The rapid prototyping of ELISA-based diagnostics using available 3D printing technologies provides an opportunity for a further exploration of this platform into immunodetection systems. In this study, a '3D-Well' was designed and fabricated using available 3D printing platforms to have an increased surface area of more than 4 times for protein-surface adsorption compared to those of 96-well plates. The ease and rapidity in designing-product development-feedback cycle offered through 3D printing platforms provided an opportunity for its rapid assessment, in which a chemical etching process was used to make the surface hydrophilic followed by validation through the diagnostic performance of ELISA for infectious disease without modifying current laboratory practices for ELISA. The higher sensitivity of the 3D-Well (3-folds higher) compared to the 96-well ELISA provides a potential for the expansion of this technology towards miniaturization platforms to reduce time, volume of reagents and samples needed for laboratory or field diagnosis of infectious diseases including applications in other disciplines.

  9. Production of monoclonal antibodies for detection of Citrus leprosis virus C in enzyme-linked immuno-assays and immunocapture reverse transcription-polymerase chain reaction.

    PubMed

    Choudhary, Nandlal; Roy, Avijit; Govindarajulu, A; Nakhla, M K; Levy, L; Brlansky, R H

    2014-09-01

    Citrus leprosis virus C (CiLV-C) causes damage in citrus production in the South and Central America. Since closely related types of citrus viruses have recently been described monoclonal antibodies (MAbs) are needed for accurate and sensitive diagnosis of CiLV-C. In this study, MAbs to the expressed coat protein of CiLV-C were produced for serological detection of CiLV-C in crude extracts of infected tissues in double antibody sandwich enzyme-linked immunosorbent assays (DAS-ELISA), dot blot immunosorbent assays (DBIA) and immuonocapture-reverse transcription-polymerase chain reaction (IC-RT-PCR) procedures. Monoclonal antibodies were developed in mice to the purified expressed coat protein of CiLV-C. The published standard protocols of DAS-ELISA, DBIA and IC-RT-PCR were followed for the detection of coat protein p29 of CiLV-C in the crude extracts of CiLV-C infected tissues. Two monoclonal antibodies, designated G10 and C11, were identified from four potential candidates for the specific and sensitive detection of coat protein p29 of CiLV-C in the crude citrus extracts of CiLV-C infected tissues in DAS-ELISA, whereas G10 was also selected based on performance for use in the DBIA and IC-RT-PCR diagnostic assays. Sensitivity analysis comparing the three methods for detection of coat protein p29 of CiLV-C determined that IC-RT-PCR was more sensitive than DAS-ELISA and DBIA. The creation of MAbs to CiLV-C allows for the sensitive and accurate detection of the virus from CiLV-C infected citrus leaf tissues. Successful detection of the virus in three diagnostic assays formats provides flexibility to diagnosticians who can use either ELISA or DBIA for screening large numbers of samples, and IC-RT-PCR for rapid, sensitive confirmation testing.

  10. Development of a highly sensitive chemiluminescent assay for hydrogen peroxide under neutral conditions using acridinium ester and its application to an enzyme immunoassay.

    PubMed

    Arakawa, Hidetoshi; Tsuruoka, Keiko; Ohno, Ken-ichi; Tajima, Noriko; Nagano, Hiromi

    2014-06-01

    We developed a highly sensitive chemiluminescent (CL) assay for hydrogen peroxide using 10-methyl-9-(phenoxycarbonyl) acridinium fluorosulfonate (PMAC) that produced chemiluminescence under neutral conditions and applied it to an enzyme immunoassay (EIA). One picomole of hydrogen peroxide could be detected using the optimized PMAC-CL method and 6.2 × 10(-20) mol β-D-galactosidase (β-gal) could be detected by combining an indoxyl derivative substrate and the proposed PMAC-CL method. This highly sensitive CL β-gal assay was applied to an EIA for thyroid-stimulating hormone (TSH) using β-gal as a label enzyme; 0.02-100.0 μU/mL TSH in human serum could be assayed directly and with high reproducibility.

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

    PubMed

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

    2015-07-08

    Enzyme-linked Immunosorbent Assay (ELISA)-based diagnosis is the mainstay for measuring antibody response in infectious diseases and to support pathogen identification of potential use in infectious disease outbreaks and clinical care of individual patients. The development of laboratory diagnostics using readily available 3D printing technologies provides a timely opportunity for further expansion of this technology into immunodetection systems. Utilizing available 3D printing platforms, a '3D well' was designed and developed to have an increased surface area compared to those of 96-well plates. The ease and rapidity of the development of the 3D well prototype provided an opportunity for its rapid validation through the diagnostic performance of ELISA in infectious disease without modifying current laboratory practices for ELISA. The improved sensitivity of the 3D well of up to 2.25-fold higher compared to the 96-well ELISA provides a potential for the expansion of this technology towards miniaturization and Lab-On-a-Chip platforms to reduce time, volume of reagents and samples needed for such assays in the laboratory diagnosis of infectious and other diseases including applications in other disciplines.

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

    PubMed

    Pasquet, Camille; Vulliet, Emmanuelle

    2011-10-15

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

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  14. Development of an incurred cornbread model for gluten detection by immunoassays.

    PubMed

    Sharma, Girdhari M; Khuda, Sefat E; Pereira, Marion; Slate, Andrew; Jackson, Lauren S; Pardo, Christopher; Williams, Kristina M; Whitaker, Thomas B

    2013-12-11

    Gluten that is present in food as a result of cross-contact or misbranding can cause severe health concerns to wheat-allergic and celiac patients. Immunoassays, such as enzyme-linked immunosorbent assay (ELISA) and lateral flow device (LFD), are commonly used to detect gluten traces in foods. However, the performance of immunoassays can be affected by non-assay-related factors, such as food matrix and processing conditions. Gluten (0-500 ppm) and wheat flour (20-1000 ppm) incurred cornbread was prepared at different incurred levels and baking conditions (204.4 °C for 20, 27, and 34 min) to study the accuracy and precision of gluten measurement by seven immunoassay kits (three LFD and four ELISA kits). The stability and immunoreactivity of gluten proteins, as measured by western blot using three different antibodies, were not adversely affected by the baking conditions. However, the gluten recovery varied depending upon the ELISA kit and the gluten source used to make the incurred cornbread, affecting the accuracy of gluten quantification (BioKits, 9-77%; Morinaga, 91-137%; R-Biopharm, 61-108%; and Romer Labs, 113-190%). Gluten recovery was reduced with increased baking time for most ELISA kits analyzed. Both the sampling and analytical variance increased with an increase in the gluten incurred level. The predicted analytical coefficient of variation associated with all ELISA kits was below 12% for all incurred levels, indicative of good analytical precision.

  15. Immunodiagnosis of Fasciola gigantica Infection Using Monoclonal Antibody-Based Sandwich ELISA and Immunochromatographic Assay for Detection of Circulating Cathepsin L1 Protease

    PubMed Central

    Anuracpreeda, Panat; Chawengkirttikul, Runglawan; Sobhon, Prasert

    2016-01-01

    Background Tropical fasciolosis caused by Fasciola gigantica infection is one of the major diseases infecting ruminants in the tropical regions of Africa and Asia including Thailand. Parasitological diagnosis of fasciolosis is often unreliable and possesses low sensitivity. Therefore, the detection of circulating parasite antigens is thought to be a better alternative for diagnosis of fasciolosis, as it reflects the real parasite burden. Methods In this study, we have produced a monoclonal antibody (MoAb) against recombinant F. gigantica cathepsin L1 (rFgCatL1), and developed both sandwich enzyme-linked immunosorbent assay (sandwich ELISA) and immunochromatographic (IC) test for rapid detection of circulating cathepsin L1 protease (CatL1) in the sera from mice experimentally and cattle naturally infected with Fasciola gigantica. MoAb 4E3 and biotinylated rabbit anti-recombinant CatL1 antibody were selected due to their high reactivities and specificities. Results The lower detection limits of sandwich ELISA and IC test were 3 pg/ml and 0.256 ng/ml, respectively. Sandwich ELISA and IC test could detect F. gigantica infection from day 1 to 35 post infection. In experimental mice, the sensitivity, specificity and accuracy were 95%, 100% and 98.6% (for sandwich ELISA), and 93%, 100% and 98.2% (for IC test), while in natural cattle they were 98.3%, 100% and 99.5% (for sandwich ELISA), and 96.7%, 100% and 99.1% (for IC test). Conclusions These two assay methods showed high efficiencies and precisions for diagnosis of fasciolosis by F. gigantica. PMID:26731402

  16. Evaluation of a commercial rabies ELISA as a replacement for serum neutralization assays as part of the pet travel scheme and oral vaccination campaigns of foxes.

    PubMed

    Knoop, Eva V; Freuling, Conrad M; Kliemt, Jeannette; Selhorst, Thomas; Conraths, Franz J; Müller, Thomas

    2010-01-01

    EU Regulation 998/2003 requires the serological testing of rabies-vaccinated dogs and cats in approved laboratories using serum neutralization tests prior to movement of pet animals between certain EU member states and before pet animals are imported from unlisted third countries. Serum neutralisation tests are also used for measuring the efficacy of oral rabies vaccination programmes conducted in wild carnivore populations. In this study we evaluated an OIE-listed commercial ELISA as a potential replacement for serum neutralization assays under routine conditions as a diagnostic tool for both the serological testing of dog and cat sera as part of pet travel schemes and for follow-up investigations as part of oral vaccination campaigns. When dog and cat sera were analyzed by ELISA, a sensitivity compared to the standard serological test of 36.9-82.0% and 44.4-88.9%, respectively, was calculated depending on the method used. For fox field samples from oral vaccination areas the sensitivity compared to the Rapid Fluorescent Focus Inhibition Test (RFFIT) was 32.4% (95% CI 24.8-40.0%). In its present format, the ELISA cannot replace standard serological assays neither in the pet travel scheme nor in follow-up investigations of oral vaccination campaigns. The results obtained resemble those of other rabies ELISAs recently evaluated for the same purpose and may therefore exemplify a general misconception (binding versus neutralization) in rabies serology rather than a failure of this ELISA test per se. Also, problems with technical and legislative issues associated with the serological testing of dog and cat sera for non-commercial movement and related to the outcome of this study are addressed.

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

    PubMed

    Li, Ye; Cassone, Vincent M

    2015-09-01

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

  18. Development of a novel fluorescent microbead-based immunoassay and comparison with three enzyme-linked immunoassays for detection of anti-Erysipelothrix spp. IgG antibodies in pigs with known and unknown exposure.

    PubMed

    Giménez-Lirola, L G; Xiao, C T; Halbur, P G; Opriessnig, T

    2012-10-01

    A novel fluorescent microbead immunoassay (FMIA) using the recombinant polypeptide SpaA415 was developed for detection of anti-Erysipelothrix spp. IgG in pig sera. The diagnostic performance of the FMIA was evaluated on samples from pigs with known and unknown Erysipelothrix spp. exposure and compared to an in-house enzyme-linked immunosorbent assay (ELISA-1) based on the same capture antigen, and two commercially available ELISAs (ELISA-2 and ELISA-3). Sera from pigs experimentally infected with Erysipelothrix rhusiopathiae serotype 1a (n=60) or 19 (n=12), sera from pigs vaccinated with a commercial attenuated-live vaccine based on serotype 1a (n=12) or a commercial bacterin based on serotype 2 (n=12), and 90 field samples were utilized. The sensitivity on 22 true positive samples collected in the later stages of infection/post-vaccination was 100% for the FMIA and ELISA-1, 63.6% for ELISA-2 and 81.8% for ELISA-3. The earliest antibody response was detected 7days post inoculation with the FMIA (77.8%) and ELISA-1 (11.1%), and at 14days post-vaccination (dpv) with FMIA (50%) and ELISA-1 (50%). On field samples, a higher seroprevalence was found in pigs older than 21days with all four assays. Kappa analysis indicated that the FMIA and ELISA-1 had almost complete agreement whereas the agreement was slight with ELISA-2 and fair with ELISA-3. The sensitivity of both immunoassays based on the rSpaA415 antigen was higher compared to that of the two commercial ELISAs. The rSpaA415 FMIA has great potential as an inexpensive ELISA alternative for detection of antibodies against E. rhusiopathiae in the future.

  19. Fabricating a UV-Vis and Raman Spectroscopy Immunoassay Platform.

    PubMed

    Hanson, Cynthia; Israelsen, Nathan D; Sieverts, Michael; Vargis, Elizabeth

    2016-11-10

    Immunoassays are used to detect proteins based on the presence of associated antibodies. Because of their extensive use in research and clinical settings, a large infrastructure of immunoassay instruments and materials can be found. For example, 96- and 384-well polystyrene plates are available commercially and have a standard design to accommodate ultraviolet-visible (UV-Vis) spectroscopy machines from various manufacturers. In addition, a wide variety of immunoglobulins, detection tags, and blocking agents for customized immunoassay designs such as enzyme-linked immunosorbent assays (ELISA) are available. Despite the existing infrastructure, standard ELISA kits do not meet all research needs, requiring individualized immunoassay development, which can be expensive and time-consuming. For example, ELISA kits have low multiplexing (detection of more than one analyte at a time) capabilities as they usually depend on fluorescence or colorimetric methods for detection. Colorimetric and fluorescent-based analyses have limited multiplexing capabilities due to broad spectral peaks. In contrast, Raman spectroscopy-based methods have a much greater capability for multiplexing due to narrow emission peaks. Another advantage of Raman spectroscopy is that Raman reporters experience significantly less photobleaching than fluorescent tags(1). Despite the advantages that Raman reporters have over fluorescent and colorimetric tags, protocols to fabricate Raman-based immunoassays are limited. The purpose of this paper is to provide a protocol to prepare functionalized probes to use in conjunction with polystyrene plates for direct detection of analytes by UV-Vis analysis and Raman spectroscopy. This protocol will allow researchers to take a do-it-yourself approach for future multi-analyte detection while capitalizing on pre-established infrastructure.

  20. Immuno-PCR assay for sensitive detection of proteins in real time

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The immuno-PCR (IPCR) assay combines the versatility and robustness of immunoassays with the exponential signal amplification power of the polymerase chain reaction (PCR). Typically, IPCR allows a 10–1,000-fold increase in sensitivity over the analogous enzyme-linked immunosorbent assay (ELISA). Thi...

  1. Evaluation of fully automated assays for the detection of Rubella IgM and IgG antibodies by the Elecsys(®) immunoassay system.

    PubMed

    van Helden, Josef; Grangeot-Keros, Liliane; Vauloup-Fellous, Christelle; Vleminckx, Renaud; Masset, Frédéric; Revello, Maria-Grazia

    2014-04-01

    Screening for acute rubella infection in pregnancy is an important element of antenatal care. This study compared the sensitivity, specificity and reproducibility of two new, fully automated Elecsys(®) Rubella IgM and IgG immunoassays designed for the Elecsys 2010, Modular Analytics E170, COBAS e-411 and COBAS e-601 and e602 analytical platforms, with current assays using serum from patients with primary rubella infections, vaccinated patients, patients with potentially cross-reacting infections and on routine samples in clinical laboratories in France, Germany and Italy. Both assays showed good within-run and within-laboratory precision. A sensitivity of 79.8-96.0% was demonstrated for Elecsys IgM in primary, early acute infection, consistent with existing assays. In samples obtained from routine antenatal screening, the Elecsys Rubella IgM assay revealed high specificity (98.7-99.0%). A significantly (p<0.0001) lower reactivity was demonstrated in samples from previously infected patients where acute rubella infection was excluded, and the incidence of false positives in patients with potentially cross-reacting infections was lower with Elecsys Rubella IgM compared with other. The Elecsys Rubella IgG assay exhibited a relative sensitivity of 99.9-100.0% and specificity of 97.4-100.0% in samples from routine antenatal screening. The Elecsys Rubella IgM and IgG assays allow convenient, rapid and reliable determination of anti-rubella antibodies. Sensitivity, specificity and reproducibility were comparable with existing assay systems. Assay results were available in approximately half the time required for currently employed methods and the assays are compatible with widely used analytical platforms.

  2. Chagas disease-specific antigens: characterization of epitopes in CRA/FRA by synthetic peptide mapping and evaluation by ELISA-peptide assay

    PubMed Central

    2013-01-01

    Background The identification of epitopes in proteins recognized by medically relevant antibodies is useful for the development of peptide-based diagnostics and vaccines. In this study, epitopes in the cytoplasmic repetitive antigen (CRA) and flagellar repetitive antigen (FRA) proteins from Trypanosoma cruzi were identified using synthetic peptide techniques and pooled sera from Chagasic patients. The epitopes were further assayed with an ELISA assay based on synthetic peptides. Methods Twenty-two overlapping synthetic peptides representing the coding sequence of the T. cruzi CRA and FRA proteins were assessed by a Spot-synthesis array analysis using sera donated by patients with Chagas disease. Shorter peptides were selected that represented the determined epitopes and synthesized by solid phase synthesis to evaluate the patterns of cross-reactivities and discrimination through an ELISA-diagnostic assay. Results The peptide Spot-synthesis array successfully identified two IgG antigenic determinants in the CRA protein and four in FRA. Bioinformatics suggested that the CRA antigens were unique to T. cruzi while the FRA antigen showed similarity with sequences present within various proteins from Leishmania sp. Subsequently, shorter peptides representing the CRA-1, CRA-2 and FRA-1 epitopes were synthesized by solid phase synthesis and assayed by an ELISA-diagnostic assay. The CRA antigens gave a high discrimination between Chagasic, Leishmaniasis and T. cruzi-uninfected serum. A sensitivity and specificity of 100% was calculated for CRA. While the FRA antigen showed a slightly lower sensitivity (91.6%), its specificity was only 60%. Conclusions The epitopes recognized by human anti-T. cruzi antibodies have been precisely located in two biomarkers of T. cruzi, CRA and FRA. The results from screening a panel of patient sera through an ELISA assay based on peptides representing these epitopes strongly suggest that the sequences from CRA would be useful for the

  3. Updates in immunoassays: parasitology.

    PubMed

    Josko, Deborah

    2012-01-01

    Although most clinical laboratories use microscopy and routine O&P procedures when identifying parasitic infections, there are several parasites that are better detected through serological means. Toxoplasma, Giardia, and Cryptosporidium were discussed along with immunoassays used for their detection. Immunoassays provide quick results and are less labor intensive than specimen concentration and slide preparation for microscopic examination. These assays are easy to use and provide sensitive and specific results. Some clinical laboratories no longer perform O&Ps in house and refer specimens to reference laboratories for evaluation. By using immunoassays, some of the more common parasites can be identified in a timely manner reducing turn-around times. Some controversy exists over the use of IIF and EIA tests used for ANA testing along with measuring CRPs and PCT as predictors of bacterial sepsis and septic shock. Regardless of the methodology discussed in this series of articles, there are pros and cons to the various immunoassays available. Determining the most appropriate assay based on patient population and volume is governed by the institution and its patients' needs. In conclusion, immunoassays, whether manual or automated, are easy to use, cost effective and allow the medical laboratory professional to provide quick and accurate results to the clinician so the most appropriate treatment can be administered to the patient. The ultimate goal of healthcare professionals is to provide the highest quality of medical care in a timely manner. The use of immunoassays in the clinical laboratory allows the healthcare team to successfully achieve this goal.

  4. Development of an Assay for the Detection of PrPres in Blood and Urine Based on PMCA Assay and ELISA Methods

    DTIC Science & Technology

    2007-09-01

    diagnostic assay. The assay in the pilot form is developed with plasma from hamsters infected with the 263K strain of scrapie . The same assay can be...adapted to human PrP test. In this funding period we completed the optimization of the conditions for proteinase K (PK) digestion of PrPres in scrapie ...alone10. We have been developing a prototype assay for TSE infection using hamsters infected with the 263K stain of scrapie . In our current assay

  5. Evaluating concentration estimation errors in ELISA microarray experiments

    SciTech Connect

    Daly, Don S.; White, Amanda M.; Varnum, Susan M.; Anderson, Kevin K.; Zangar, Richard C.

    2005-01-26

    Enzyme-linked immunosorbent assay (ELISA) is a standard immunoassay to predict a protein concentration in a sample. Deploying ELISA in a microarray format permits simultaneous prediction of the concentrations of numerous proteins in a small sample. These predictions, however, are uncertain due to processing error and biological variability. Evaluating prediction error is critical to interpreting biological significance and improving the ELISA microarray process. Evaluating prediction error must be automated to realize a reliable high-throughput ELISA microarray system. Methods: In this paper, we present a statistical method based on propagation of error to evaluate prediction errors in the ELISA microarray process. Although propagation of error is central to this method, it is effective only when comparable data are available. Therefore, we briefly discuss the roles of experimental design, data screening, normalization and statistical diagnostics when evaluating ELISA microarray prediction errors. We use an ELISA microarray investigation of breast cancer biomarkers to illustrate the evaluation of prediction errors. The illustration begins with a description of the design and resulting data, followed by a brief discussion of data screening and normalization. In our illustration, we fit a standard curve to the screened and normalized data, review the modeling diagnostics, and apply propagation of error.

  6. Immunoassay as an analytical tool in agricultural biotechnology.

    PubMed

    Grothaus, G David; Bandla, Murali; Currier, Thomas; Giroux, Randal; Jenkins, G Ronald; Lipp, Markus; Shan, Guomin; Stave, James W; Pantella, Virginia

    2006-01-01

    Immunoassays for biotechnology engineered proteins are used by AgBiotech companies at numerous points in product development and by feed and food suppliers for compliance and contractual purposes. Although AgBiotech companies use the technology during product development and seed production, other stakeholders from the food and feed supply chains, such as commodity, food, and feed companies, as well as third-party diagnostic testing companies, also rely on immunoassays for a number of purposes. The primary use of immunoassays is to verify the presence or absence of genetically modified (GM) material in a product or to quantify the amount of GM material present in a product. This article describes the fundamental elements of GM analysis using immunoassays and especially its application to the testing of grains. The 2 most commonly used formats are lateral flow devices (LFD) and plate-based enzyme-linked immunosorbent assays (ELISA). The main applications of both formats are discussed in general, and the benefits and drawbacks are discussed in detail. The document highlights the many areas to which attention must be paid in order to produce reliable test results. These include sample preparation, method validation, choice of appropriate reference materials, and biological and instrumental sources of error. The article also discusses issues related to the analysis of different matrixes and the effects they may have on the accuracy of the immunoassays.

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

    PubMed

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

    2011-03-01

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

  8. Application of linear discriminant analysis in performance evaluation of extractable nuclear antigen immunoassay systems in the screening and diagnosis of systemic autoimmune rheumatic diseases.

    PubMed

    Pi, David; de Badyn, Monika Hudoba; Nimmo, Mike; White, Rick; Pal, Jason; Wong, Patrick; Phoon, Carmen; O'Connor, Deidre; Pi, Steven; Shojania, Kam

    2012-10-01

    This study applied a linear discriminant analysis model to evaluate the performance of 2 types of commercially available extractable nuclear antigen (ENA) immunoassays for the screening and diagnosis of systemic autoimmune rheumatic diseases (SARDs) in a large tertiary hospital reference laboratory: (1) an enzyme-linked immunosorbent assay (ELISA) and (2) a multiplex bead-based immunoassay (MPBI). The results of the study showed both ENA immunoassays had comparable sensitivity for the detection of SARDs compared with the antinuclear antigen immunofluorescence (ANA-IF) method (ANA-IF: 85.6%, ENA-ELISA: 91.5%, ENA-MPBI: 83.1%, pairwise comparisons with ANA-IF: P > .05). However, both ENA immunoassays offered improved specificity compared with the ANA-IF (ANA-IF: 24.2%; ENA-ELISA: 39.8%; ENA-MPBI: 53.1%; pairwise comparison with ANA-IF: P < .001). The use of a more specific screening immunoassay with comparable sensitivity to ANA-IF is important in a tertiary hospital with high prevalence of non-SARD immune diseases. Diagnostic performance of the ENA/dsDNA components by the MPBI and ELISA methods did not differ significantly (area under the curve [AUC], 81.0% vs 83.0%, respectively, P > .05), but the key ENA/dsDNA variables contributing to the discriminating power of the assays for the diagnosis of specific SARDs were reagent/method dependent.

  9. 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. ...

  10. Multiplex detection of plant pathogens using a microsphere immunoassay technology.

    PubMed

    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.

  11. Comparison of drugs of abuse detection in meconium by EMIT II and ELISA.

    PubMed

    Marin, Stephanie J; Keith, Lindsay; Merrell, Miles; McMillin, Gwendolyn A

    2009-04-01

    The results of meconium specimens and fortified samples screened for drugs of abuse by both enzyme multiplied immunoassay technique (EMIT((R) )II) and enzyme-linked immunosorbent assay (ELISA) methods were compared. The sample preparation for the ELISA screen was a simple buffer extraction versus a lengthy and more laborious sample preparation procedure for the EMIT II screen. The ELISA method was automated using a TECAN Genesis. The EMIT II analysis was automated with an Olympus AU400e. The opioid screen was calibrated with hydromorphone and the benzodiazepine screen was calibrated with clonazepam to maximize detection for these analytes. Previously validated gas chromatography-mass spectrometry (GC-MS), two-dimensional GC-MS, or liquid chromatography-tandem MS methods were used for confirmation. Results from the two techniques compared well. Agreement of the ELISA assay was greater than 90% when compared to EMIT II for all drug classes except barbiturates and benzodiazepines. ELISA appears to be more sensitive than EMIT II for the detection of amphetamines, methadone, propoxyphene, and cocaine. ELISA compared well to EMIT II for cannabinoids, opioids, and PCP. Specificity of the ELISA assay was slightly better for PCP and opioids. EMIT II appears to be more sensitive for the detection of barbiturates and benzodiazepines. The ELISA method reduced turnaround time by 50% compared to the EMIT II method.

  12. 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.

  13. The Dot Blot ELISA.

    ERIC Educational Resources Information Center

    Gerbig, Donald G., Jr.; Fenk, Christopher J.; Goodhart, Amy S.

    2000-01-01

    Uses two laboratory techniques, Enzyme Linked Immunosorbent Assay (ELISA) and Western Blot, to demonstrate antibody-antigen binding concepts. Includes a list of required materials and directions for the procedure, and makes suggestions for classroom applications. (Contains 13 references.) (YDS)

  14. High-throughput enzyme-linked immunoabsorbant assay (ELISA) electrochemiluminescent detection of botulinum toxins in foods for food safety and defence purposes.

    PubMed

    Phillips, R W; Abbott, D

    2008-09-01

    Clostridum species produce seven serotypes (A-G) of botulinum toxin, four of which (A, B, E, and F) are normally associated with human illness. To date, the most reliable test for botulinum toxin is the mouse bioassay. The authors' laboratory has been exploring the use of an antibody-based assay similar to an enzyme-linked immunoabsorbant assay (ELISA) but utilizing electrochemiluminescent technology (BioVerify assay) as an alternative to the mouse bioassay for testing food samples. The detection limit of this assay is as low as 10 ng g(-1) depending on the food matrix and the serotype detected. Detection of botulinum toxin between 10 and 200 ng g(-1) is a linear curve allowing for the possibility of performing quantitative as well as qualitative testing of samples. The ease of the assay, limited sample preparation, and low detection limit make the BioVerify assay and instrument an excellent, high-throughput option for detecting botulinum toxins in food matrices.

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

    PubMed Central

    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

  16. 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.

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

    PubMed

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

    1992-05-01

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

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

    PubMed Central

    Pschenitza, Michael; Hackenberg, Rudolf; Niessner, Reinhard; Knopp, Dietmar

    2014-01-01

    This paper describes the development of a molecularly imprinted polymer-based solid phase extraction (MISPE) method coupled with enzyme-linked immunosorbent assay (ELISA) for determination of the PAH benzo[a]pyrene (B[a]P) in vegetable oils. Different molecularly imprinted polymers (MIPs) were prepared using non-covalent 4-vinylpyridine/divinylbenzene co-polymerization at different ratios and dichloromethane as porogen. Imprinting was done with a template mixture of phenanthrene and pyrene yielding a broad-specific polymer for PAHs with a maximum binding capacity (Q) of ∼32 μg B[a]P per 50 mg of polymer. The vegetable oil/n-hexane mixture (1:1, (v/v)) was pre-extracted with acetonitrile, the solvent evaporated, the residue reconstituted in n-hexane and subjected to MISPE. The successive washing with n-hexane and isopropanol revealed most suitable to remove lipid matrix constituents. After elution of bound PAHs from MISPE column with dichloromethane, the solvent was evaporated, the residue reconstituted with dimethyl sulfoxide and diluted 100-fold with methanol/water (10:90, (v/v)) for analysis of B[a]P equivalents with an ELISA. The B[a]P recovery rates in spiked vegetable oil samples of different fatty acid composition were determined between 63% and 114%. The presence of multiple PAHs in the oil sample, because of MIP selectivity and cross-reactivity of the ELISA, could yield overestimated B[a]P values. PMID:24887045

  19. Development of a combined technique using a rapid one-step immunochromatographic assay and indirect competitive ELISA for the rapid detection of baicalin.

    PubMed

    Paudel, Madan Kumar; Putalun, Waraporn; Sritularak, Boonchoo; Morinaga, Osamu; Shoyama, Yukihiro; Tanaka, Hiroyuki; Morimoto, Satoshi

    2011-09-09

    A colloidal gold conjugated anti-baicalin monoclonal antibody (anti-BA MAb) was prepared and used in an immunochromatographic assay (ICA) for BA in Scutellariae Radix and Kampo medicines. This competitive ICA uses an anti-BA MAb which shows a high specificity for BA and baicalein. Its advantages include a short assay time (15 min), no dependence on any instrumental systems, and it can detect BA in plant materials and Kampo medicines. The limit of detection for the ICA was found to be around 0.6 μg mL(-1)of baicalin. Moreover, the usefulness of the combination of indirect competitive ELISA and the ICA using anti-BA MAb as a quality control method was confirmed for analysis of BA in Scutellariae Radix and Kampo medicines with a sufficient sensitivity (200 ng mL(-1) to 2 μg mL(-1)), obtainable in an easy and timely manner.

  20. How-to-do-it: Immunological Assays for the Classroom 1. Enzyme Linked Immunosorbent Assay (ELISA): A Laboratory Tool for Demonstration of Antibody-Antigen Interaction.

    ERIC Educational Resources Information Center

    Russo, A. J.; And Others

    1984-01-01

    Background information, list of required materials, and procedures are provided for an immunological assay which has been modified for use as a classroom/laboratory demonstration of antigen-antibody reaction. The assay is designed for a two and one-half hour laboratory period but may be modified for one hour laboratories. (JN)

  1. Evaluation of two immunoassay procedures for drug testing in hair samples.

    PubMed

    Musshoff, F; Kirschbaum, K M; Graumann, K; Herzfeld, C; Sachs, H; Madea, B

    2012-02-10

    A preliminary initial enzyme-linked immunosorbent assay (LUCIO-Direct ELISA kit) and a preliminary DRI enzyme immunoassay were evaluated for drug detection in head hair with respect to lowered cutoff values recommended in Germany for the control of abstinence in cases of re-granting of drivers' licences. Following drug classes were included: cannabinoids, opiates, cocaine like substances, amphetamine, methamphetamine (and methylenedioxyamphetamines), methadone, and benzodiazepines. 759 analyses were performed using LUCIO-Direct ELISA kits and 936 analyses using DRI enzyme immunoassay tests. Sample size for each drug group and immunoassay test reached from 74 to 178. The LUCIO-Direct ELISA kit revealed a sensitivity of 91% for amphetamine up to 98% for methadone (methamphetamine 92%, cocaine 94%, opiates 94%, benzodiazepines 96%) and values of specificity of 72% for methadone up to 89% for amphetamine and benzodiazepines. The test was not useful for a preliminary screening for tetrahydrocannabinol (sensitivity of 65%) in consideration of a suggested cutoff of 0.02 ng/mg. The DRI enzyme immunoassay test was only useful for morphine and cocaine testing at low recommended new cutoff values (0.1 ng/mg) revealing sensitivities of 94% and 99%, respectively.

  2. Interferences in Immunoassay

    PubMed Central

    Tate, Jill; Ward, Greg

    2004-01-01

    Substances that alter the measurable concentration of the analyte or alter antibody binding can potentially result in immunoassay interference. Interfering, endogenous substances that are natural, polyreactive antibodies or autoantibodies (heterophiles), or human anti-animal antibodies together with other unsuspected binding proteins that are unique to the individual, can interfere with the reaction between analyte and reagent antibodies in immunoassay. Lipaemia, cross-reactivity, and exogenous interferences due to pre-analytical variation, matrix and equipment reaction also affect immunoassay. Interfering substances may lead to falsely elevated or falsely low analyte concentration in one or more assay systems depending on the site of the interference in the reaction and possibly result in discordant results for other analytes. The prevalence of interference is generally low in assays containing blocking agents that neutralise or inhibit the interference but is often higher in new, untested immunoassays. A wide range of analytes measured by immunoassay including hormones, tumour markers, drugs, cardiac troponin and microbial serology may be affected. Interference in immunoassay may lead to the misinterpretation of a patient's results by the laboratory and the wrong course of treatment being given by the physician. Laboratories should put processes in place to detect, test and report suspected interferences. It is equally important that physicians communicate any clinical suspicion of discordance between the clinical and the laboratory data to the laboratory. The detection of interference may require the use of an alternate assay or additional measurements, before and after treatment with additional blocking reagent, or following dilution of the sample in non-immune serum. It is imperative that laboratories inform physicians of the follow-up procedure and report on the presence of any interference. The establishment of on-going laboratory-physician contact is

  3. Preprogrammed, parallel on-chip immunoassay using system-level capillarity control.

    PubMed

    Kim, Sung-Jin; Paczesny, Sophie; Takayama, Shuichi; Kurabayashi, Katsuo

    2013-07-16

    Fully manual use of conventional multiwell plates makes enzyme-linked immunosorbent assay (ELISA)-based immunoassays highly time-consuming and labor-intensive. Here, we present a capillarity-driven on-chip immunoassay that greatly saves time and labor with an inexpensive setup. Our immunoassay process starts with pipetting multiple solutions into multiwells constructed on a microfluidic device chip. Subsequently, capillarity spontaneously transports multiple sample solutions and common reagent solutions into assigned detection channels on the chip in a purely passive and preprogrammed manner. Our device implements capillarity-driven immunoassays involving four sample and six reagent solutions within 30 min by orchestrating the functions of on-chip passive components. Notably, our immunoassay technique reduces the total number of pipetting processes by ~5 times, as compared to assays on multiwell plates (48 vs 10). This assay technique allows us to quantify the concentrations of C-reactive protein and suppressor of tumorigenicity 2 with a detection limit of 8 and 90 pM, respectively. This device should be useful for sophisticated, parallel biochemical microfluidic processing in point-of-care settings under limited resources.

  4. Development of an Assay for the Detection of PrPres in Blood and Urine Based on PMCA Assay an ELISA Methods

    DTIC Science & Technology

    2005-09-01

    animal models: the hamster infected with the 263K strain of scrapie and the sheep either naturally or experimentally infected with scrapie . Using hamster...prototype assay can be developed using blood from animal models, in our case: hamsters infected with the 263K stain of scrapie and sheep naturally and...experimentally infected with scrapie . In addition to the usual assay diagnostic requirements of reproducibly, reliability and robustness, the TSE blood

  5. Development of an Assay for the Detection of PrPres in Blood and Urine Based on PMCA Assay and ELISA Methods

    DTIC Science & Technology

    2008-09-01

    based TSE diagnostic assay. This is the final report for this project. The assay was developed with plasma from hamsters infected with scrapie (263K...236K hamster strain11. The second report indicated that only mice with kidney infection and infected with scrapie excreted infectivity in urine. Mice...with scrapie alone did not have infectivity in their urine12. The third study was in deer infected with chronic wasting disease and showed no

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

    PubMed Central

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

    1996-01-01

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

  7. An immunoassay-based reverse-transcription loop-mediated isothermal amplification assay for the rapid detection of avian influenza H5N1 virus viremia.

    PubMed

    Tang, Yi; Yu, Xu; Chen, Hao; Diao, Youxiang

    2016-12-15

    Avian influenza virus (AIV) subtype H5N1 attracts particular consideration because it is a continuous threat to animals and public health systems. The viremia caused by AIV H5N1 infection may increase the risk of blood-borne transmission between humans. Therefore, there is a need to rapidly evaluate and implement screening measures for AIV H5N1 viremia that allows for rapid response to this potentially pandemic threat. The present report describes an immunoassay-based reverse-transcription loop-mediated isothermal amplification (immuno-RT-LAMP) assay for the rapid detection of AIV H5N1 in whole blood samples. Using PCR tubes coated with an H5 subtype monoclonal antibody, AIV H5N1 virions were specifically captured from blood samples. After a thermal lysis step, the released viral N1 gene was exponentially amplified using RT-LAMP on either a real-time PCR instrument for quantitative analysis, or in a water bath system for endpoint analysis. The detection limit of the newly developed immuno-RT-LAMP assay was as low as 1.62×10(1) 50% embryo infectious dose/mL of virus in both regular samples and simulated viremia samples. There were no cross-reactions with non-H5N1 influenza viruses or other avian viruses. The reproducibility of the assay was confirmed using intra- and inter-assay tests with variability ranging from 1.05% to 3.37%. Our results indicate that immuno-RT-LAMP is a novel, effective point-of-care virus identification solution for the rapid diagnosis and monitoring of AIV H5N1 in blood samples.

  8. Sensitivity of the Quidel Sofia Fluorescent Immunoassay Compared With 2 Nucleic Acid Assays and Viral Culture to Detect Pandemic Influenza A(H1N1)pdm09.

    PubMed

    Arbefeville, Sophie S; Fickle, Ann R; Ferrieri, Patricia

    2015-01-01

    To confirm a diagnosis of influenza at the point of care, healthcare professionals may rely on rapid influenza diagnostic tests (RIDTs). RIDTs have low to moderate sensitivity compared with viral culture or real-time reverse-transcription polymerase chain reaction (rRT-PCR). With the resurgence of the influenza A (Flu A; subtype H1N1) pandemic 2009 (pdm09) strain in the years 2013 and 2014, we evaluated the accuracy of the United State Food and Drug Administration (FDA)-approved Sofia Influenza A+B Fluorescent Immunoassay to detect epidemic Flu A(H1N1)pdm09 in specimens from the upper-respiratory tract. During a 3-month period, we collected 40 specimens that tested positive via PCR and/or culture for Flu A of the H1N1 pdm09 subtype. Of the 40 specimens, 27 tested positive (67.5%) via Sofia assay for Flu A. Of the 13 specimens with a negative result via Sofia testing, 4 had coinfection, as detected by the GenMark Diagnostics eSensor Respiratory Viral Panel. This sensitivity of the RIDT Sofia assay to detect Flu A(H1N1) pdm09 was comparable to previously reported sensitivities ranging from 10% to 75% for older RIDTs.

  9. Comparison of nested and ELISA based polymerase chain reaction assays for detecting Chlamydia trachomatis in pregnant women with preterm complications.

    PubMed

    Sulaiman, S; Chong, P P; Mokhtarudin, R; Lye, M S; Wan Hassan, W H

    2014-03-01

    Identification of pregnant women infected with Chlamydia trachomatis is essential to allow early antibiotic treatment in order to prevent adverse pregnancy outcomes. In this study, two nucleic acid amplification tests (NAAT) namely nested PCR (BioSewoom, Korea) and Amplicor CT/NG (Roche Diagnostic, USA) were evaluated in terms of sensitivity and specificity for the detection of C. trachomatis DNA in pregnant women with preterm complications. A cross-sectional study was carried out in two public hospitals in Southern Selangor, Malaysia. Endocervical swabs obtained were subjected to DNA amplification using nested PCR (BioSewoom, Korea) and Amplicor CT/NG (Roche Diagnostic, USA). A total of 83 endocervical swabs obtained from pregnant women of less than 37 weeks gestation and presented with preterm complications were subjected to chlamydial DNA detection using both assays. The study shows that Amplicor CT/NG assay is more effective in the detection of C. trachomatis DNA from endocervical swabs compared to Biosewoom nested PCR kit. Agreement between the two assays were poor (kappa=0.094) with nested PCR showing a low sensitivity of 10.81% and a 97.83% specificity when compared to Amplicor CT/NG. The results obtained indicated that BioSewoom nested PCR was less sensitive than Amplicor CT/ NG for detecting C. trachomatis in endocervical specimens and that another more reliable test is required for confirmatory result.

  10. Production of mono- and polyclonal antibodies to Citrus leprosis virus C2 and their application in triple antibody sandwich ELISA and immunocapture RT-PCR diagnostic assays.

    PubMed

    Choudhary, Nandlal; Roy, Avijit; Leon, M G; Wei, G; Nakhla, M K; Levy, L; Brlansky, R H

    2017-05-01

    The newly discovered Citrus leprosis virus cytoplasmic type 2 (CiLV-C2) is one of the causal virus of citrus leprosis disease complex; which leads to substantial loss of citrus production in the states of Meta and Casanare of Colombia. Specific and sensitive detection methods are needed to monitor the dissemination of CiLV-C2 in Colombia, and to prevent introduction of CiLV-C2 to other citrus growing countries. Toward this end, putative coat protein gene (CPG) of CiLV-C2 was amplified from CiLV-C2 infected citrus tissues. The CPG was cloned, expressed and purified a recombinant coat protein of ∼31kDa which used to generate monoclonal antibodies and polyclonal antisera. Four monoclonal antibodies and two polyclonal antisera were selected as being specific following Western blotting. The monoclonal antibody MAb E5 and polyclonal antiserum PAb UF715 were selected testing with an extract of CiLV-C2 infected leaves using triple antibody sandwich enzyme-linked immunosorbent assay (TAS-ELISA). In addition, an immunocapture RT-PCR was standardized using MAb E5 for specific and sensitive detection of CiLV-C2. The standardized TAS-ELISA and IC-RT-PCR were able to detect CiLV-C2 in the extracts of symptomatic citrus leprosis tissues up to the dilutions of 1:160 and 1:2580, respectively. Result demonstrated that CiLV-C2 is present in citrus orchards in Meta and Casanare citrus growing areas of Colombia. TAS-ELISA could be used for routine detection of CiLV-C2, epidemiological studies, and for border inspections for quarantine purposes. IC-RT-PCR could be valuable for CiLV-C2 validation and viral genome analysis.

  11. Undergraduate Laboratory Module for Implementing ELISA on the High Performance Microfluidic Platform.

    PubMed

    Giri, Basant; Peesara, Ravichander R; Yanagisawa, Naoki; Dutta, Debashis

    Implementing enzyme-linked immunosorbent assays (ELISA) in microchannels offers several advantages over its traditional microtiter plate-based format, including a reduced sample volume requirement, shorter incubation period, and greater sensitivity. Moreover, microfluidic ELISA platforms are inexpensive to fabricate and allow integration of analytical procedures, such as sample preconcentration, that further enhance the performance of the immunoassay. In view of the scientific potential of microfluidic ELISAs, inclusion of this technique into an undergraduate curriculum is valuable in preparing the next generation of scientists and engineers. Here, an experimental module is presented for this immunoassay method that can be completed in an undergraduate laboratory setting within two 3-h periods (including all incubation and data analyses procedures) using only a microliter of sample and reagents per assay. In addition to acquainting students with the microfluidic technology, the reported module provides training in quantitating ELISAs using the kinetic format of the assay. Furthermore, it offers a useful educational tool for introducing undergraduates to basic image analysis techniques, as well as signal-to-noise ratio and limit of detection calculations that are valuable in characterizing any analytical method.

  12. Undergraduate Laboratory Module for Implementing ELISA on the High Performance Microfluidic Platform

    PubMed Central

    Giri, Basant; Peesara, Ravichander R.; Yanagisawa, Naoki; Dutta, Debashis

    2015-01-01

    Implementing enzyme-linked immunosorbent assays (ELISA) in microchannels offers several advantages over its traditional microtiter plate-based format, including a reduced sample volume requirement, shorter incubation period, and greater sensitivity. Moreover, microfluidic ELISA platforms are inexpensive to fabricate and allow integration of analytical procedures, such as sample preconcentration, that further enhance the performance of the immunoassay. In view of the scientific potential of microfluidic ELISAs, inclusion of this technique into an undergraduate curriculum is valuable in preparing the next generation of scientists and engineers. Here, an experimental module is presented for this immunoassay method that can be completed in an undergraduate laboratory setting within two 3-h periods (including all incubation and data analyses procedures) using only a microliter of sample and reagents per assay. In addition to acquainting students with the microfluidic technology, the reported module provides training in quantitating ELISAs using the kinetic format of the assay. Furthermore, it offers a useful educational tool for introducing undergraduates to basic image analysis techniques, as well as signal-to-noise ratio and limit of detection calculations that are valuable in characterizing any analytical method. PMID:26052160

  13. Dual Electrophoresis Detection System for Rapid and Sensitive Immunoassays with Nanoparticle Signal Amplification

    NASA Astrophysics Data System (ADS)

    Zhang, Fangfang; Ma, Junjie; Watanabe, Junji; Tang, Jinlong; Liu, Huiyu; Shen, Heyun

    2017-02-01

    An electrophoretic technique was combined with an enzyme-linked immunosorbent assay (ELISA) system to achieve a rapid and sensitive immunoassay. A cellulose acetate filter modified with polyelectrolyte multilayer (PEM) was used as a solid substrate for three-dimensional antigen-antibody reactions. A dual electrophoresis process was used to induce directional migration and local condensation of antigens and antibodies at the solid substrate, avoiding the long diffusion times associated with antigen-antibody reactions in conventional ELISAs. The electrophoretic forces drove two steps in the ELISA process, namely the adsorption of antigen, and secondary antibody-labelled polystyrene nanoparticles (NP-Ab). The total time needed for dual electrophoresis-driven detection was just 4 min, nearly 2 h faster than a conventional ELISA system. Moreover, the rapid NP-Ab electrophoresis system simultaneously achieved amplification of the specific signal and a reduction in noise, leading to a more sensitive NP-Ab immunoassay with a limit of detection (LOD) of 130 fM, and wide range of detectable concentrations from 0.13 to 130 pM. These results suggest that the combination of dual electrophoresis detection and NP-Ab signal amplification has great potential for future immunoassay systems.

  14. Dual Electrophoresis Detection System for Rapid and Sensitive Immunoassays with Nanoparticle Signal Amplification.

    PubMed

    Zhang, Fangfang; Ma, Junjie; Watanabe, Junji; Tang, Jinlong; Liu, Huiyu; Shen, Heyun

    2017-02-15

    An electrophoretic technique was combined with an enzyme-linked immunosorbent assay (ELISA) system to achieve a rapid and sensitive immunoassay. A cellulose acetate filter modified with polyelectrolyte multilayer (PEM) was used as a solid substrate for three-dimensional antigen-antibody reactions. A dual electrophoresis process was used to induce directional migration and local condensation of antigens and antibodies at the solid substrate, avoiding the long diffusion times associated with antigen-antibody reactions in conventional ELISAs. The electrophoretic forces drove two steps in the ELISA process, namely the adsorption of antigen, and secondary antibody-labelled polystyrene nanoparticles (NP-Ab). The total time needed for dual electrophoresis-driven detection was just 4 min, nearly 2 h faster than a conventional ELISA system. Moreover, the rapid NP-Ab electrophoresis system simultaneously achieved amplification of the specific signal and a reduction in noise, leading to a more sensitive NP-Ab immunoassay with a limit of detection (LOD) of 130 fM, and wide range of detectable concentrations from 0.13 to 130 pM. These results suggest that the combination of dual electrophoresis detection and NP-Ab signal amplification has great potential for future immunoassay systems.

  15. Dual Electrophoresis Detection System for Rapid and Sensitive Immunoassays with Nanoparticle Signal Amplification

    PubMed Central

    Zhang, Fangfang; Ma, Junjie; Watanabe, Junji; Tang, Jinlong; Liu, Huiyu; Shen, Heyun

    2017-01-01

    An electrophoretic technique was combined with an enzyme-linked immunosorbent assay (ELISA) system to achieve a rapid and sensitive immunoassay. A cellulose acetate filter modified with polyelectrolyte multilayer (PEM) was used as a solid substrate for three-dimensional antigen-antibody reactions. A dual electrophoresis process was used to induce directional migration and local condensation of antigens and antibodies at the solid substrate, avoiding the long diffusion times associated with antigen-antibody reactions in conventional ELISAs. The electrophoretic forces drove two steps in the ELISA process, namely the adsorption of antigen, and secondary antibody-labelled polystyrene nanoparticles (NP-Ab). The total time needed for dual electrophoresis-driven detection was just 4 min, nearly 2 h faster than a conventional ELISA system. Moreover, the rapid NP-Ab electrophoresis system simultaneously achieved amplification of the specific signal and a reduction in noise, leading to a more sensitive NP-Ab immunoassay with a limit of detection (LOD) of 130 fM, and wide range of detectable concentrations from 0.13 to 130 pM. These results suggest that the combination of dual electrophoresis detection and NP-Ab signal amplification has great potential for future immunoassay systems. PMID:28198385

  16. Immunoassay based water quality analysis: A new tool for drinking water supply management

    SciTech Connect

    Kostyshyn, C.R.; Brown, W.; Hervey, E.; Hull, C.

    1996-11-01

    The recent availability of enzyme-linked immunosorbent assay (ELISA) tests for the analysis of organic environmental contaminants provides drinking water utility managers and operators with a new tool for managing treatment operations and monitoring source watersheds. Immunoassay technology permits rapid, inexpensive and accurate in-plant testing of many SDWA regulated organic contaminants at concentrations well below established MCL`s. Analytical testing which would not be practicable due to the high cost or long turnaround time limitations of conventional testing methods is now being performed using immunoassay based analysis. Water quality data generated using immunoassay based methods are being utilized by drinking water utilities as an integral part of source watershed management programs, process operations optimization efforts, pro-active raw and finished water testing programs, and flood and incident response management.

  17. Results of an International Interlaboratory Trial to Determine Twelve Allergens Using Real-time PCR- and ELISA-based Assays.

    PubMed

    Köppel, René; Rentsch, Jürg; Ruf, Jürg; Eugster, Albert; Graf, Christoph; Felderer, Nora; Pietsch, Klaus; Ilg, Evelyn

    2014-10-01

    To elucidate the capability of laboratories to determine allergen contents, an international interlaboratory trial was conducted using meat products spiked with 12 allergens. The measurement uncertainty was calculated independent of the applied method simulating realistic situations when comparing analysis certificates from different laboratories. The measurement uncertainty was revealed to be in the best cases +/-100%, in the worst cases quantification exhibited a measurement uncertainty of higher than 200% making quantitative analysis impossible. The measurement uncertainty seemed to depend on the analyte and assays used.

  18. Design, preparation and use of ligated phosphoproteins: a novel approach to study protein phosphatases by dot blot array, ELISA and Western blot assays.

    PubMed

    Sun, Luo; Ghosh, Inca; Barshevsky, Tanya; Kochinyan, Samvel; Xu, Ming-Qun

    2007-07-01

    The study of substrate specificity of protein phosphatases (PPs) is very challenging since it is difficult to prepare a suitable phosphorylated substrate. Phosphoproteins, phosphorylated by a protein kinase, or chemically synthesized phosphopeptides are commonly used substrates for PPs. Both types of these substrates have their advantages and limitations. Phosphoproteins mimic more closely the physiologically relevant PP substrates, but their preparation is technically demanding. Synthetic phosphopeptides present advantages over proteins because they can be easily produced in large quantity and their amino acid sequence can be designed to contain potential determinants of substrate specificity. However, short peptides are less optimal compared to in vivo PP substrates and often display poor and variable binding to different matrices, resulting in low sensitivity in analysis of PP activity on solid support. In this work we utilize the intein-mediated protein ligation (IPL) technique to generate substrates for PPs, combining the advantages of proteins and synthetic peptides in one molecule. The ligation of a synthetic phosphopeptide to an intein-generated carrier protein (CP) with a one-to-one stoichiometry results in the formation of a ligated phosphoprotein (LPP). Three widely used assays, dot blot array, Western blot and ELISA were employed to study the PP activity on LPP substrates. Dephosphorylation was measured by detection of the remaining phosphorylation, or lack of it, with a phospho-specific antibody. The data show the advantage of LPPs over free peptides in assays on solid supports. LPPs exhibited enhanced binding to the matrices used in the study, which significantly improved sensitivity and consistency of the assays. In addition, saturation of the signal was circumvented by serial dilution of the assay samples. This report describes detailed experimental procedures for preparation of LPP substrates and their use in PP assays based on immobilization on

  19. Feasibility of a simple microsieve-based immunoassay platform.

    PubMed

    Zweitzig, Daniel R; Tibbe, Arjan G; Nguyen, Ai T; van Rijn, Cees J M; Kopnitsky, Mark J; Cichonski, Kathleen; Terstappen, Leon W M M

    2016-10-01

    The intrinsic properties of silicon microsieves, such as an optically flat surface, high overall porosity, and low flow resistance have led to an increasing number of biotechnology applications. In this report, the feasibility of creating a microsieve-based immunoassay platform was explored. Microsieves containing 5μm pores were coupled with poly-acrylic acid chains, and then mounted into a plastic holder to enable rapid reagent exchanges via a wicking mechanism. The mounted microsieves were coated with infectious disease-related antigens at [2.5 and 25μg/mL], [20 and 50μg/mL], and [20 and 100μg/mL] to facilitate detection of serum-derived human antibodies against Rubella (3-day measles), B. burgdorferi (Lyme disease), or T. pallidum (syphilis), respectively. The prototype microsieve-based immunoassay platform was able to distinguish positive control sera containing antibodies against Rubella, T. pallidum, and B. burgdorferi from negative control sera with similar qualitative results as FDA-approved ELISA tests. Testing of a WHO IgG syphilitic standard at 0.3, 0.15, 0.075, 0.0375, and 0.01875IU/mL demonstrated that the T. pallidum microsieve assay is able to distinguish disease-specific IgG signal from background signal at similar, and possibly lower, levels than the corresponding ELISA. The T. pallidum microsieve assay prototype also differentiated positive clinical serum samples from negative donor samples, and the results were in good agreement with ELISA (R(2)=0.9046). These feasibility studies demonstrate the potential for utilizing microsieves, along with a reagent wicking device, as a simple diagnostic immunoassay platform.

  20. Biosensor, ELISA, and frog embryo teratogenesis assay: Xenopus (FETAX) analysis of water associated with frog malformations in Minnesota

    NASA Astrophysics Data System (ADS)

    Garber, Eric A. E.; Erb, Judith L.; Downward, James G.; Priuska, Eric M.; Wittliff, James L.; Feng, Wenke; Magner, Joseph; Larsen, Gerald L.

    2001-03-01

    Between 1995 and 1997 over 62% of the counties in Minnesota reported the presence of malformed frogs. While most sites have recently shown a decline in malformed frog populations, one site in northeastern Minnesota with no prior history of containing malformed frogs was recently discovered to contain > 67% malformed Rana pipiens (northern leopard frogs). As part of an effort to study the presence of hormonally active agents in fresh water sources, water samples were collected from lakes in Minnesota containing malformed frogs and analyzed for the presence of hormonally active compounds using a novel evanescent field fluorometric biosensor and the frog embryo teratogenesis assay: Xenopus (FETAX) bioassay. The waveguide based biosensor developed by ThreeFold Sensors (TFS biosensor, Ann Arbor, MI) detects the presence of estrogenic compounds capable of interacting with free human ER-a and by inhibiting binding to an immobilized estrogen. The FETAX bioassay is a developmental assay, which measures teratogenicity, mortality, and inhibition of growth during the first 96 hours of organogenesis and thereby provides a universal screen for endocrine disruptors. TFS biosensor and FETAX screening of the water samples suggest a relationship between estrogenic activity, mineral supplementation, and the occurrence of malformed frogs.

  1. Development of an Assay for the Detection of PrPres in Blood and Urine Based on PMCA Assay and ELISA Methods

    DTIC Science & Technology

    2006-09-01

    plasma from hamsters infected with the 263K strain of scrapie . The assay has shown high sensitivity and specificity and good reproducibility. In this...long term, “limiting dilution” titration of untreated, whole urine from scrapie infected hamsters, we have now conclusively shown that urine from...the 263K stain of scrapie . Great efforts have been directed towards the development of a pre- mortem preclinical diagnostic test using blood as the

  2. Novel signal-enhancing immunoassay for ultrasensitive biomarker detection based on laser-induced fluorescence.

    PubMed

    Zhang, Ji; Wang, Shuai; Liu, Kunping; Wei, Yin; Wang, Xu; Duan, Yixiang

    2015-03-03

    An innovative signal-enhancing immunoassay for ultrasensitive biomarker detection based on laser-induced fluorescence (LIF) has been developed. A novel LIF optical system with high collection efficiency was constructed by using a parabolic mirror. Carboxyl-functionalized magnetic beads were used to immobilize antibody for achieving a conventional sandwich assay. Fluorescence from Rhodamine 6G (R6G)-labeled antibody was collected by the newly designed optical system. To reduce photobleaching of R6G under laser irradiation, ethanol instead of commonly used aqueous solution was used as assay buffer in the last stage. The newly developed LIF immunoassay displayed excellent analytical performance for α-fetoprotein (AFP) detection in the concentration range from 0.005 to 1.0 ng/mL with a detection limit of 0.0016 ng/mL. The detection limit obtained in this work is about 3 orders of magnitude better than that of conventional enzyme-linked immunosorbent assay (ELISA). In addition, the proposed method exhibited excellent precision, acceptable stability, and good reproducibility. Furthermore, the proposed immunoassay was successfully applied to AFP determination in real serum specimens. Therefore, the present immunoassay was demonstrated to be a powerful tool for ultrasensitive biomarker detection.

  3. Clinical comparison of the Treponema pallidum CAPTIA syphilis-G enzyme immunoassay with the fluorescent treponemal antibody absorption immunoglobulin G assay for syphilis testing.

    PubMed

    Halling, V W; Jones, M F; Bestrom, J E; Wold, A D; Rosenblatt, J E; Smith, T F; Cockerill, F R

    1999-10-01

    Recently, a treponema-specific immunoglobulin G (IgG) enzyme immunoassay (EIA), the CAPTIA Syphilis-G (Trinity Biotech, Jamestown, N.Y.), has become available as a diagnostic test for syphilis. A total of 89 stored sera previously tested by the fluorescent treponemal antibody absorption (FTA-ABS) IgG assay were evaluated by the CAPTIA EIA. The FTA-ABS IgG procedure was performed by technologists unblinded to results of rapid plasmid reagin (RPR) testing of the same specimens. Borderline CAPTIA-positive samples (antibody indices of >/=0.650 and 0.900, the sample was considered positive. Thirteen of 89 (15%) samples had discrepant results. Compared to the FTA-ABS assay, the CAPTIA EIA had a sensitivity and specificity and positive and negative predictive values of 70.7, 97.9, 96.7, and 79.7%, respectively. In another analysis, discrepancies between results were resolved by repeated FTA-ABS testing (technologists were blinded to previous RPR results) and patient chart reviews. Seven CAPTIA-negative samples which were previously interpreted (unblinded) as minimally reactive by the FTA method were subsequently interpreted (blinded) as nonreactive. One other discrepant sample (CAPTIA negative and FTA-ABS positive [at an intensity of 3+], unblinded) was FTA negative with repeated testing (blinded). For the five remaining discrepant samples, chart reviews indicated that one patient (CAPTIA negative and FTA-ABS positive [minimally reactive], blinded) had possible syphilis. These five samples were also evaluated and found to be negative by another treponema-specific test, the Treponema pallidum microhemagglutination assay. Therefore, after repeated testing and chart reviews, 2 of the 89 (2%) samples had discrepant results; the adjusted sensitivity, specificity, and positive and negative predictive values were 96.7, 98.3, 96.7, and 98.3%, respectively. This study demonstrates that the CAPTIA IgG EIA is a

  4. Sensitive, fast, and specific immunoassays for methyltestosterone detection.

    PubMed

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

    2015-04-29

    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.

  5. 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

  6. Leishmania infantum mimotopes and a phage-ELISA assay as tools for a sensitive and specific serodiagnosis of human visceral leishmaniasis.

    PubMed

    Salles, Beatriz C S; Costa, Lourena E; Alves, Patrícia T; Dias, Ana C S; Vaz, Emília R; Menezes-Souza, Daniel; Ramos, Fernanda F; Duarte, Mariana C; Roatt, Bruno M; Chávez-Fumagalli, Miguel A; Tavares, Carlos A P; Gonçalves, Denise U; Rocha, Regina L; Goulart, Luiz R; Coelho, Eduardo A F

    2017-03-01

    Serological methods used to diagnose visceral leishmaniasis (VL) are considered minimally invasive, but they present problems related with their sensitivity and/or specificity. In this study, a subtractive selection using the phage display technology against antibodies from healthy subjects living in endemic and non-endemic areas of disease, as well as from Chagas disease patients and those developing active VL, was developed. The aim of this study was to select bacteriophage-fused epitopes to be used in the serodiagnosis of human VL. Eight phage clones were selected after the bio-panning rounds, and their reactivity was evaluated in a phage-ELISA assay against a human serological panel. A wild-type clone and the recombinant K39-based immunochromatographic test were used as controls. In the results, it was shown that all clones showed an excellent performance to serologically identify VL patients, demonstrating the feasibility of the isolated phages for developing a specific and sensitive serodiagnosis of human VL.

  7. 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.

  8. Evaluation of a new nanoparticle-based lateral-flow immunoassay for the exclusion of heparin-induced thrombocytopenia (HIT).

    PubMed

    Sachs, Ulrich J; von Hesberg, Jakob; Santoso, Sentot; Bein, Gregor; Bakchoul, Tamam

    2011-12-01

    Heparin-induced thrombocytopenia (HIT) is an adverse complication of heparin caused by HIT antibodies (abs) that recognise platelet factor 4-heparin (PF4/hep) complexes. Several laboratory tests are available for the confirmation and/or refutation of HIT. A reliable and rapid single-sample test is still pending. It was the objective of this study to evaluate a new lateral-flow immunoassay based on nanoparticle technology. A cohort of 452 surgical and medical patients suspected of having HIT was evaluated. All samples were tested in two IgG-specific ELISAs, in a particle gel immunoassay (PaGIA) and in a newly developed lateral-flow immunoassay (LFI-HIT) as well as in a functional test (HIPA). Clinical pre-test probability was determined using 4T's score. Platelet-activating antibodies were present in 34/452 patients, all of whom had intermediate to high clinical probability. PF4/hep abs were detected in 79, 87, 86, and 63 sera using the four different immunoassays. The negative predictive values (NPV) were 100% for both ELISA tests and LFI-HIT but only 99.2% for PaGIA. There were less false positives (n=29) in the LFI-HIT compared to any other test. Additionally, significantly less time was required to perform LFI-HIT than to perform the other immunoassays. In conclusion, a newly developed lateral-flow assay, LFI-HIT, was capable of identifying all HIT patients in a cohort in a short period of time. Beside an NPV of 100%, the rate of false-positive signals is significantly lower with LFI-HIT than with other immunoassay(s). These performance characteristics suggest a high potency in reducing the risk and costs in patients suspected of having HIT.

  9. From Interface to Solution: Integrating Immunoassay with Netlike Rolling Circle Amplification for Ultrasensitive Detection of Tumor Biomarker

    PubMed Central

    Feng, Chang; Bo, Bing; Mao, Xiaoxia; Shi, Hai; Zhu, Xiaoli; Li, Genxi

    2017-01-01

    Both the 3D solution and the 2D interface play important roles in bioanalysis. For the former, reactions can be carried out adequately; while for the latter, interfering substance can be eliminated simply through wash. It is a challenge to integrate the advantages of solution-based assays and the interface-based assays. Here, we report an immuno-NRCA (netlike rolling circle amplification) strategy, which integrates immunoassay with NRCA for the ultrasensitive detection of tumor biomarker, by taking the assay of a tumour marker as an example. In this strategy, immunoreactions occur on interface, while the target-induced signal amplification can be completed totally in solution. As a result, this system has the merits of both solution- and interface-based assays. The whole procedure of this novel strategy is similar to the conventional ELISA, inheriting the usability. But in comparison with ELISA, the performance is greatly improved. The detection limit can be lowered to 5.5 fg/L, making it possible to detect the target tumour marker in one drop of blood. Also, in comparison with established immuno-PCR method, which integrates immunoassay with the commonly used nucleic acid amplification approach, this system has no requirement for thermal cycler owing to the isothermal amplification, and it has the ability to retain the immunoreactivities. So, the new immunoassay method proposed in this study may have more feasible applications in the future. PMID:28042314

  10. Occurrence and Distribution of Pesticides in the St. Lucie River Watershed, South-Central Florida, 2000-01, Based on Enzyme-Linked Immunosorbent Assay (ELISA) Screening

    USGS Publications Warehouse

    Lietz, A.C.

    2003-01-01

    The St. Lucie River watershed is a valuable estuarine ecosystem and resource in south-central Florida. The watershed has undergone extensive changes over the last century because of anthropogenic activities. These activities have resulted in a complex urban and agricultural drainage network that facilitates the transport of contaminants, including pesticides, to the primary canals and then to the estuary. Historical data indicate that aquatic life criteria for selected pesticides have been exceeded. To address this concern, a reconnaissance was conducted to assess the occurrence and distribution of selected pesticides within the St. Lucie River watershed. Numerous water samples were collected from 37 sites among various land-use categories (urban/built-up, citrus, cropland/pastureland, and inte-grated). Samples were collected at inflow points to primary canals (C-23, C-24, and C-44) and at control structures along these canals from October 2000 to September 2001. Samples were screened for four pesticide classes (triazines, chloroacetanilides, chlorophenoxy compounds, and organophosphates) by using Enzyme-Linked Immunosorbent Assay (ELISA) screening. A temporal distribution of pesticides within the watershed was made based on samples collected at the integrated sites during different rainfall events between October 2000 and September 2001. Triazines were detected in 32 percent of the samples collected at the integrated sites. Chloroacetanilides were detected in 60 percent of the samples collected at the integrated sites, with most detections occurring at one site. Chlorophenoxy compounds were detected in 17 percent of the samples collected at the integrated sites. Organophosphates were detected in only one sample. A spatial distribution and range of concentration of pesticides at the 37 sampling sites in the watershed were determined among land-use categories. Triazine concentrations ranged from highest to lowest in the citrus, urban/built-up, and integrated areas

  11. 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...

  12. A Competitive Bio-Barcode Amplification Immunoassay for Small Molecules Based on Nanoparticles

    PubMed Central

    Du, Pengfei; Jin, Maojun; Chen, Ge; Zhang, Chan; Jiang, Zejun; Zhang, Yanxin; Zou, Pan; She, Yongxin; Jin, Fen; Shao, Hua; Wang, Shanshan; Zheng, Lufei; Wang, Jing

    2016-01-01

    A novel detection method of small molecules, competitive bio-barcode amplification immunoassay, was developed and described in this report. Through the gold nanoparticles (AuNPs) probe and magnetic nanoparticles (MNPs) probe we prepared, only one monoclonal antibody can be used to detect small molecules. The competitive bio-barcode amplification immunoassay overcomes the obstacle that the bio-barcode assay cannot be used in small molecular detection, as two antibodies are unable to combine to one small molecule due to its small molecular structure. The small molecular compounds, triazophos, were selected as targets for the competitive bio-barcode amplification immunoassay. The linear range of detection was from 0.04 ng mL−1 to 10 ng mL−1, and the limit of detection (LOD) was 0.02 ng mL−1, which was 10–20 folds lower than ELISA (Enzyme Linked Immunosorbent Assay). A practical application of the proposed immunoassay was evaluated by detecting triazophos in real samples. The recovery rate ranged from 72.5% to 110.5%, and the RSD was less than 20%. These results were validated by GC-MS, which indicated that this convenient and sensitive method has great potential for small molecular in real samples. PMID:27924952

  13. ELISA reader does not interfere by mobile phone radiofrequency radiation

    PubMed Central

    Mortazavi, Seyyed Mohammad Javad; Baradaran-Ghahfarokhi, Hamid Reza; Abdi, Mohammad Reza; Baradaran-Ghahfarokhi, Milad; Mostafavi, Nayyer Sadat; Mahmoudi, Golshan; Berenjkoub, Nafiseh; Akmali, Zahra; Hossein-Beigi, Fahimeh; Arsang, Vajiheh

    2016-01-01

    Background: The increasing number of mobile phones can physically cause electromagnetic interference (EMI) in medical environments; can also cause errors in immunoassays in laboratories. The ELISA readers are widely used as a useful diagnostic tool for Enzymun colorimetric assay in medicine. The aim of this study was to investigate whether the ELISA reader could be interfered by the exposure to the 900 MHz cell phones in the laboratory. Materials and Methods: Human serum samples were collected from 14 healthy donors (9 women and 5 men) and each sample was divided into four aliquots and was placed into four batches for the in-vitro quantitative determination of human chorionic gonadotropin (hCG). During colorimetric reading of the first, second, and third batches, the ELISA reader (Stat Fax 2100, Awareness Technology, Inc., USA) was exposed to 0.5, 1.0, and 2.0 W exposure of 900 MHz radiation, respectively. For the forth batch (control group), no radiation was applied. All experiments were performed comparing ELISA read out results of the I, II, and III batches with the control batch, using the Wilcoxon test with criterion level of P = 0.050. Results: The final scores in the exposed batches I, II, and III were not statistically significant relative to the control batch (P > 0.05). The results showed that 900 MHz radiation exposure did not alter the ELISA measured levels of hCG hormone in I (P = 0.219), II (P = 0.909), and III (P = 0.056) batches compared to the control batch. Conclusion: This study showed that ELISA reader does not interfere by mobile phone RF radiation at a closed contact (less than 5 cm distance). However, we recommend that medical institutions discuss these issues in the context of their specific use of technologies and frame a policy that is clear and straightforward to guide staff, patients, and visitors. PMID:27376040

  14. 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.

  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.

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

    PubMed

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

    2012-09-01

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

  17. Detection of the Antimicrobial Triclosan in Environmental Samples by Immunoassay

    PubMed Central

    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.

    2016-01-01

    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 were designed as unique coating antigen haptens. Polyclonal rabbit antisera were screened against the coating antigen library in order to identify combinations of immunoreagents resulting in the most sensitive assays. The most sensitive assay identified was one utilizing antiserum #1155 and a heterologous competitive hapten where the 2’–OH group was substituted with a Cl. 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, TCS concentrations were measured in water samples following dilution. Biosolid samples were analyzed following 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

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

    PubMed

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

    2013-10-15

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

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

    PubMed

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

    2015-01-01

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

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

    PubMed

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

    2017-01-01

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

  1. Polypropylene microtitre plates modified with [Cr(OH)6](3-) for enhanced ELISA sensitivity.

    PubMed

    Welch, Nicholas G; Lebot, Cedric J; Easton, Christopher D; Scoble, Judith A; Pigram, Paul J; Muir, Benjamin W

    2017-03-30

    Chromium solutions have been used as wet chemical modifiers for polymer microtitre plates used in improving immunoassay performance. However, polypropylene has been excluded from the list of potentially modifiable substrates (AnteoTechnologies, 2015). Here we show that untreated polypropylene microtitre plates can indeed be modified using a [Cr(OH)6](3-) complex. Compared to unmodified polypropylene, the chromium modified surfaces demonstrate an up to 4-fold improvement in both assay sensitivity and signal intensity in an antigen capture ELISA. Atomic force microscope (AFM) images indicate that the chromium complex facilitates dispersion of the antibody, reducing aggregation.

  2. Optimizing ELISAs for precision and robustness using laboratory automation and statistical design of experiments.

    PubMed

    Joelsson, Daniel; Moravec, Phil; Troutman, Matthew; Pigeon, Joseph; DePhillips, Pete

    2008-08-20

    Transferring manual ELISAs to automated platforms requires optimizing the assays for each particular robotic platform. These optimization experiments are often time consuming and difficult to perform using a traditional one-factor-at-a-time strategy. In this manuscript we describe the development of an automated process using statistical design of experiments (DOE) to quickly optimize immunoassays for precision and robustness on the Tecan EVO liquid handler. By using fractional factorials and a split-plot design, five incubation time variables and four reagent concentration variables can be optimized in a short period of time.

  3. Study on the use of an enzyme-linked immunosorbent assay in determining human antibodies to diphtheria toxin as compared with a reference toxin neutralization assay.

    PubMed

    Skoura, L; Efstratiou, A; Tsakris, A; Pournaras, S; George, R C; Douboyas, J

    1999-07-01

    Serum samples from 156 Greek persons were assessed by an IgG-specific enzyme-linked immunosorbent assay (ELISA) and a reference tissue culture toxin-neutralization (TN) assay for the quantitation of diphtheria toxin antibodies. By the reference method, 7.7% of the persons were susceptible to diphtheria (antitoxin < 0.01 IU/ml), 28.8% had basic protection (antitoxin 0.01-0.09 IU/ml) and 63.5% were fully protective (antitoxin > or = 0.1 IU/ ml), while the corresponding figures were 17.9, 36.5 and 45.5% when they were tested by the immunoassay. None of the samples been susceptible by the TN assay were found to have some protection when tested by ELISA. However, three (6.7%) of the 45 samples showing a basic protection with TN, were fully protective when titrated by the immunoassay. In addition, 31 (31.3%) of the 99 samples been fully protective by the bioassay, were found to be either basically protective or susceptible by means of the ELISA. Overall, validity features of the immunoassay were: sensitivity 68.7%, specificity 94.7%, positive predictive value 95.8% and negative predictive value 63.5%. The ELISA tested in our study could be used to determine diphtheria antitoxin in individuals needed a booster immunization (susceptible or basic protective samples), although it might falsely include in the above categories samples that are within the fully protective levels of antibodies.

  4. Development of a fluorescent microbead-based immunoassay for the detection of hepatitis E virus IgG antibodies in pigs and comparison to an enzyme-linked immunoassay.

    PubMed

    Owolodun, Olajide A; Giménez-Lirola, Luis G; Gerber, Priscilla F; Sanford, Brenton J; Feagins, Alicia R; Meng, Xiang-Jin; Halbur, Patrick G; Opriessnig, Tanja

    2013-11-01

    Swine hepatitis E virus (HEV) is a zoonotic virus and pigs are considered as an important reservoir. Swine HEV infection is widespread and most pig herds are infected. Humans can be infected with swine HEV via consumption of undercooked pork or through direct contact with infected pigs. To minimize the risk of zoonotic transmission, sensitive tools to assess the HEV infection status of pigs and pork products are needed. The objective of this study was to develop a fluorescent microbead-based immunoassay (FMIA) for the detection of IgG antibodies against swine HEV and compare it to an in-house enzyme-linked immunoassay (ELISA). Three sets of samples were utilized: (A) samples from pigs infected experimentally with different strains of HEV (positive controls, n=72), (B) samples from known HEV-negative pigs (negative controls, n=62) and (C) samples from pigs of unknown HEV infection status (n=182). All samples were tested by both ELISA and FMIA. The results on the experimental samples with known HEV exposure indicate that both assays have a specificity of 100% while the sensitivity ranges from 84.6% (ELISA) to 92.3% (FMIA). The overall prevalence of HEV IgG antibodies in field samples from pigs with unknown HEV exposure was 21.9% (40/182) for the ELISA and 21.4% (39/182) for the FMIA. The two assays had an almost perfect overall agreement (Kappa=0.92).

  5. Evaluation of Quantitative Anti-F1 IgG and Anti-V IgG ELISAs for use as an in Vitro-Based Potency Assay of Plague Vaccine in Mice

    DTIC Science & Technology

    2008-04-01

    protein, rF1-V, which is being developed as a plague vaccine. Several fundamental parameters of the ELISA were evaluated: specificity, precision, accuracy...assay1. Introduction Two major proteins of Yersinia pestis are associated with protection against plague . The first is Fraction 1 (F1) capsular protein...and translocation of Yersinia outer proteins (Yops) [11], inhibits chemotaxis [12], and modulates the cytokine response [13,14]. Two early plague

  6. 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.

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

    PubMed

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

    1997-01-01

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

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

    PubMed Central

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

    2015-01-01

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

  9. A comparison of a blocking ELISA and a haemagglutination inhibition assay for the detection of antibodies to Avibacterium (Haemophilus) paragallinarum in sera from artificially infected chickens.

    PubMed

    Sun, H; Miao, D; Zhang, P; Gong, Y; Blackall, P J

    2007-10-01

    The ability of blocking ELISAs and haemagglutination-inhibition (HI) tests to detect antibodies in sera from chickens challenged with either Avibacterium (Haemophilus) paragallinarum isolate Hp8 (serovar A) or H668 (serovar C) was compared. Serum samples were examined weekly over the 9 weeks following infection. The results showed that the positive rate of serovar A specific antibody in the B-ELISA remained at 100% from the second week to the ninth week. In chickens given the serovar C challenge, the highest positive rate of serovar C specific antibody in the B-ELISA appeared at the seventh week (60% positive) and was then followed by a rapid decrease. The B-ELISA gave significantly more positives at weeks 2, 3, 7, 8 and 9 post-infection for serovar A and at week 7 post-infection for serovar C. In qualitative terms, for both serovar A and serovar C infections, the HI tests gave a lower percentage of positive sera at all time points except at 9 weeks post-infection with serovar C. The highest positive rate for serovar A HI antibodies was 70% of sera at the fourth and fifth weeks post-infection. The highest rate of serovar C HI antibodies was 20% at the fifth and sixth weeks post-infection. The results have provided further evidence of the suitability of the serovar A and C B-ELISAs for the diagnosis of infectious coryza.

  10. Influence of affinity on antibody determination in microtiter ELISA systems

    SciTech Connect

    Peterman, J.H.; Voss, E.W. Jr.; Butler, J.E.

    1986-03-01

    Theoretically, all immunoassays are affinity (Ka) dependent when the product of the antibody (Ab) Ka and the free epitope concentration is less than 10. Thus, the degree of dependence on Ka depends on the concentration of available antigen in the system. The authors examined the binding of /sup 125/I-anti-fluorescein (a-FLU) monoclonal antibodies of different affinities to FLU-gelatin adsorbed on Immunlon 2 microtiter plates. Data obtained were in general agreement with our theoretical predictions; the percent of /sup 125/I-a-FLU which bound correlated with Ka, as did the shape of the titration curves. Measurement of 5 a-FLU monoclonals by the ELISA showed that the determination of Ab concentrations depends on the FLU-gelatin concentration, epitope density, and on the relationship between the Kas of test samples and the reference standard Ab preparation. Thus the ELISA is Ka dependent and should not be used routinely to estimate the absolute amount to Ab in unknown samples. However, the Ka dependency of the ELISA might provide a convenient assay for the estimation of the relative functional Ka (rfKa) of antibody preparations.

  11. Trace analysis of pollutants by use of honeybees, immunoassays, and chemiluminescence detection.

    PubMed

    Girotti, S; Ghini, S; Maiolini, E; Bolelli, L; Ferri, E N

    2013-01-01

    Specific and sensitive analysis to reveal and monitor the wide variety of chemical contaminants polluting all environment compartments, feed, and food is urgently required because of the increasing attention devoted to the environment and health protection. Our research group has been involved in monitoring the presence and distribution of agrochemicals by monitoring beehives distributed throughout the area studied. Honeybees have been used both as biosensors, because the pesticides affect their viability, and as "contaminant collectors" for all environmental pollutants. We focused our research on the development of analytical procedures able to reveal and quantify pesticides in different samples but with a special attention to the complex honeybee matrix. Specific extraction and purification procedures have been developed and some are still under optimization. The analytes of interest were determined by gas or liquid chromatographic methods and by compound-specific or group-specific immunoassays in the ELISA format, the analytical performance of which was improved by introducing luminescence detection. The range of chemiluminescent immunoassays developed was extended to include the determination of completely different pollutants, for example explosives, volatile organic compounds (including benzene, toluene, ethylbenzene, xylenes), and components of plastics, for example bisphenol A. An easier and portable format, a lateral flow immunoassay (LFIA) was added to the ELISA format to increase application flexibility in these assays. Aspects of the novelty, the specific characteristics, the analytical performance, and possible future development of the different chromatographic and immunological methods are described and discussed.

  12. Detection of alpha-fetoprotein in magnetic immunoassay of thin channels using biofunctional nanoparticles

    NASA Astrophysics Data System (ADS)

    Tsai, H. Y.; Gao, B. Z.; Yang, S. F.; Li, C. S.; Fuh, C. Bor

    2014-01-01

    This paper presents the use of fluorescent biofunctional nanoparticles (10-30 nm) to detect alpha-fetoprotein (AFP) in a thin-channel magnetic immunoassay. We used an AFP model biomarker and s-shaped deposition zones to test the proposed detection method. The results show that the detection using fluorescent biofunctional nanoparticle has a higher throughput than that of functional microparticle used in previous experiments on affinity reactions. The proposed method takes about 3 min (versus 150 min of previous method) to detect 100 samples. The proposed method is useful for screening biomarkers in clinical applications, and can reduce the run time for sandwich immunoassays to less than 20 min. The detection limits (0.06 pg/ml) and linear ranges (0.068 pg/ml-0.68 ng/ml) of AFP using fluorescent biofunctional nanoparticles are the same as those of using functional microparticles within experimental errors. This detection limit is substantially lower and the linear range is considerably wider than those of enzyme-linked immunosorbent assay (ELISA) and other methods in sandwich immunoassay methods. The differences between this method and an ELISA in AFP measurements of serum samples were less than 12 %. The proposed method provides simple, fast, and sensitive detection with a high throughput for biomarkers.

  13. Development of an Heterologous Immunoassay for Ciprofloxacin Residue in Milk

    NASA Astrophysics Data System (ADS)

    Jinqing, Jiang; Haitang, Zhang; Zhixing, An; Zhiyong, Xu; Xuefeng, Yang; Huaguo, Huang; Ziliang, Wang

    A heterologous immunoassay has been developed for the determination of Ciprofloxacin (CPFX) residues in milk. For this reason, carbodiimide active ester method was employed to synthesize the artificial antigen of CPFX-BSA, and mixed anhydride reaction was used to prepare the coating antigen of CPFX-OVA to pursue the heterologous sensitivity. Based on the square matrix titration, an icELISA method was developed for the quantitative detection of CPFX in cattle milk. The dynamic range was from 0.036 to 92.5 ng/mL, with LOD and IC50 value of 0.019 ng/mL and 1.8 ng/mL, respectively. Except for a high cross-reactivity (89.7%) to Enrofloxacin, negligible cross-reactivity to the other compounds was observed. After optimization, 0.03 mol/L of HCl, or 10% of methanol was used in the assay buffer. 20-fold dilution in cattle milk gave an inhibition curve almost the same as that in PBS buffer. The regression equation for this assay was y = 0.9036 x + 1.4574, with a correlation coefficient (R2) of 0.9844. The results suggest the veracity of the heterologous immunoassay for detecting CPFX residue in milk.

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

    PubMed

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

    2014-11-15

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

  15. Development of a Microsphere-based Immunoassay for Serological Detection of African Horse Sickness Virus and Comparison with Other Diagnostic Techniques.

    PubMed

    Sánchez-Matamoros, A; Beck, C; Kukielka, D; Lecollinet, S; Blaise-Boisseau, S; Garnier, A; Rueda, P; Zientara, S; Sánchez-Vizcaíno, J M

    2016-12-01

    African horse sickness (AHS) is a viral disease that causes high morbidity and mortality rates in susceptible Equidae and therefore significant economic losses. More rapid, sensitive and specific assays are required by diagnostic laboratories to support effective surveillance programmes. A novel microsphere-based immunoassay (Luminex assay) in which beads are coated with recombinant AHS virus (AHSV) structural protein 7 (VP7) has been developed for serological detection of antibodies against VP7 of any AHSV serotype. The performance of this assay was compared with that of a commercial enzyme-linked immunosorbent assay (ELISA) and commercial lateral flow assay (LFA) on a large panel of serum samples from uninfected horses (n = 92), from a reference library of all AHSV serotypes (n = 9), on samples from horses experimentally infected with AHSV (n = 114), and on samples from West African horses suspected of having AHS (n = 85). The Luminex assay gave the same negative results as ELISA when used to test the samples from uninfected horses. Both assays detected antibodies to all nine AHSV serotypes. In contrast, the Luminex assay detected a higher rate of anti-VP7 positivity in the West African field samples than did ELISA or LFA. The Luminex assay detected anti-VP7 positivity in experimentally infected horses at 7 days post-infection, compared to 13 days for ELISA. This novel immunoassay provides a platform for developing multiplex assays, in which the presence of antibodies against multiple ASHV antigens can be detected simultaneously. This would be useful for serotyping or for differentiating infected from vaccinated animals.

  16. Development of a plasma panel test for detection of human myocardial proteins by capillary immunoassay.

    PubMed

    Torabi, Fereidon; Mobini Far, Hamid Reza; Danielsson, Bengt; Khayyami, Masoud

    2007-02-15

    A chemiluminescence immunoassay for the detection of four heart marker proteins: myoglobin, creatine kinase mb [CKmb], troponin I [TnI], and fatty acid-binding protein [FABP], was designed. The immunoassay was based on enzyme-linked immunosorbent assay [ELISA] and antibodies immobilized in glass capillaries pre-treated with 3-aminopropyltriethoxysilane. The protein bound to the antibody was detected by using an anti-protein-horseradish peroxidase [HRP] conjugate. The reaction of the HRP with luminal and hydrogen peroxide-based substrate generated the chemiluminescence and a photodiode detector was used to measure the light intensity. The same assay protocol was used to detect all four proteins. Ultrasound waves were used to improve the silanization of glass and the antibody immobilization process. The optimization of the duration and intensity of the ultrasound was performed for the myoglobin assay. Ultrasound improved the silanization procedure and the capillaries gave an approximately 2.5 times greater ELISA response. Ultrasound also improved the sensitivity by approximately 100% when monoclonal antibody was immobilized on a glass capillary. Calibration curves corresponding to analyte concentrations ranging from 2.4 to 2400 ng/ml in plasma samples were recorded. The detection limits were in the region of 1.2 myoglobin, 0.6 CKmb, 5.6 TnI, and 4 ng/ml FABP in plasma with a coefficient of variation of 3-9.9%.

  17. Evaluation of a new automated chemiluminescence immunoassay for FGF23.

    PubMed

    Shimizu, Yuichiro; Fukumoto, Seiji; Fujita, Toshiro

    2012-03-01

    Fibroblast growth factor 23 (FGF23) is a hormone regulating phosphate and vitamin D metabolism. We have previously established a sandwich enzyme-linked immunosorbent assay (ELISA) for FGF23 and reported that FGF23 values are useful for the differential diagnosis of chronic hypophosphatemia. However, this ELISA has a rather narrow assay range of 3-800 pg/ml, and it was pointed out that the assay performance is not satisfactory when automatic washing is used. Here we evaluated a new automated chemiluminescence immunoassay for FGF23. This assay uses 10 μl sera or plasma samples and requires 20 min to obtain the first result. The assay was linear up to about 15,000 pg/ml and had a detection limit of 1 pg/ml. In addition, this assay showed coefficients of variation of less than 5% using samples with average FGF23 levels of 43.2-2,454.0 pg/ml. When FGF23 levels in 210 samples from chronic hypophosphatemic patients were evaluated by both the previous ELISA and this new assay, there was a good correlation of R (2) = 0.96. However, FGF23 levels by the new assay showed lower values, especially in samples with high FGF23 levels. Given that the lowest FGF23 level in patients with FGF23-related hypophosphatemia was 30.8 pg/ml and that the highest FGF23 levels in patients with non-FGF23-related hypophosphatemia was 20.8 pg/ml by this novel assay, the sensitivity and specificity were 100% when the cutoff was set between 20.8 and 30.8 pg/ml. From the aspect of convenience and the coefficients of variation of this assay, we propose that the cutoff be 25 pg/ml. There results indicate that this new assay is ideal for both clinical use and clinical studies, especially when measuring many samples with high FGF23 levels.

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

    PubMed Central

    Nekouei, Omid; Durocher, Jean; Keefe, Greg

    2016-01-01

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

  19. A new immunoassay to quantify fungal antigens from the indoor mould Aspergillus versicolor.

    PubMed

    Zahradnik, Eva; Kespohl, Sabine; Sander, Ingrid; Schies, Ursula; Khosravie-Hohn, Janett; Lorenz, Wolfgang; Engelhart, Steffen; Kolk, Annette; Schneider, Gerd; Brüning, Thomas; Raulf-Heimsoth, Monika

    2013-06-01

    Aspergillus versicolor is among the most commonly found moulds in moisture-damaged buildings and can be associated with adverse health effects in humans. This paper reports the development, validation and application of an enzyme immunoassay to quantify A. versicolor antigens. A sandwich ELISA was developed using polyclonal antibodies that recognize a broad range of A. versicolor proteins present in fungal spores and in mycelia fragments. To validate the new method, A. versicolor antigens were quantified in samples collected from homes with visible mould growth, including dust from vacuumed walls and bulk samples of building materials. Antigen concentrations were compared to the results of a commercial ELISA based on monoclonal antibodies (AveX ELISA, Indoor Biotechnologies, Charlottesville, USA) and correlated with colony forming units (CFU) of A. versicolor. The A. versicolor ELISA was very sensitive with a lower detection limit of 120 pg ml(-1). The assay also showed some reactivity to other moulds with strongest reactions with other Aspergillus species (1-3% reactivity). The new assay detected A. versicolor antigens in a much higher percentage of dust samples (88% vs. 27%) and bulk samples (89% vs. 24%) than the AveX assay. A significant correlation (r = 0.67, and p < 0.0001) was found between antigen concentrations and CFU of A. versicolor. Based on its low detection limit and good correlation with the culture-based method, this new immunoassay seems to be a useful tool for the measurement of A. versicolor exposure levels and a reliable complement to the traditional monitoring techniques, such as mould cultivation or microscopy.

  20. Nanoparticle-based sandwich electrochemical immunoassay for carbohydrate antigen 125 with signal enhancement using enzyme-coated nanometer-sized enzyme-doped silica beads.

    PubMed

    Tang, Dianping; Su, Biling; Tang, Juan; Ren, Jingjing; Chen, Guonan

    2010-02-15

    A novel nanoparticle-based electrochemical immunoassay of carbohydrate antigen 125 (CA125) as a model was designed to couple with a microfluidic strategy using anti-CA125-functionalized magnetic beads as immunosensing probes. To construct the immunoassay, thionine-horseradish peroxidase conjugation (TH-HRP) was initially doped into nanosilica particles using the reverse micelle method, and then HRP-labeled anti-CA125 antibodies (HRP-anti-CA125) were bound onto the surface of the synthesized nanoparticles, which were used as recognition elements. Different from conventional nanoparticle-based electrochemical immunoassays, the recognition elements of the immunoassay simultaneously contained electron mediator and enzyme labels and simplified the electrochemical measurement process. The sandwich-type immunoassay format was used for the online formation of the immunocomplex in an incubation cell and captured in the detection cell with an external magnet. The electrochemical signals derived from the carried HRP toward the reduction of H(2)O(2) using the doped thionine as electron mediator. Under optimal conditions, the electrochemical immunoassay exhibited a wide working range from 0.1 to 450 U/mL with a detection limit of 0.1 U/mL CA125. The precision, reproducibility, and stability of the immunoassay were acceptable. The assay was evaluated for clinical serum samples, receiving in excellent accordance with results obtained from the standard enzyme-linked immunosorbent assay (ELISA) method. Concluding, the nanoparticle-based assay format provides a promising approach in clinical application and thus represents a versatile detection method.

  1. Rapid and sensitive detection of Escherichia coli O157:H7 in milk and ground beef using magnetic bead-based immunoassay coupled with tyramide signal amplification.

    PubMed

    Aydin, Muhsin; Herzig, Gene P D; Jeong, Kwang Cheol; Dunigan, Samantha; Shah, Parth; Ahn, Soohyoun

    2014-01-01

    Escherichia coli O157:H7 is a major foodborne pathogen that has posed serious problems for food safety and public health. Recent outbreaks and recalls associated with various foods contaminated by E. coli O157:H7 clearly indicate its deleterious effect on food safety. A rapid and sensitive detection assay is needed for this harmful organism to prevent foodborne illnesses and control outbreaks in a timely manner. We developed a magnetic bead-based immunoassay for detection of E. coli O157:H7 (the most well-known Shiga toxigenic E. coli strain) with a 96-well microplate as an assay platform. Immunomagnetic separation (IMS) and tyramide signal amplification were coupled to the assay to increase its sensitivity and specificity. This immunoassay was able to detect E. coli O157:H7 in pure culture with a detection limit of 50 CFU/ml in less than 3 h without an enrichment step. The detection limit was decreased 10-fold to 5 CFU/ml with addition of a 3-h enrichment step. When this assay was tested with other nontarget foodborne pathogens and common enteric bacteria, no cross-reactivity was found. When tested with artificially contaminated ground beef and milk samples, the assay sensitivity decreased two- to fivefold, with detection limits of 250 and 100 CFU/ml, respectively, probably because of the food matrix effect. The assay results also were compared with those of a sandwich-type enzyme-linked immunosorbent assay (ELISA) and an ELISA coupled with IMS; the developed assay was 25 times and 4 times more sensitive than the standard ELISA and the IMS-ELISA, respectively. Tyramide signal amplification combined with IMS can improve sensitivity and specificity for detection of E. coli O157:H7. The developed assay could be easily adapted for other foodborne pathogens and will contribute to improved food safety and public health.

  2. Transferability of antibody pairs from ELISA to fiber optic surface plasmon resonance for infliximab detection

    NASA Astrophysics Data System (ADS)

    Van Stappen, Thomas; Lu, Jiadi; Bloemen, Maarten; Geukens, Nick; Spasic, Dragana; Delport, Filip; Verbiest, Thierry; Lammertyn, Jeroen; Gils, Ann

    2015-03-01

    Tumor necrosis factor (TNF)-alpha is a pleiotropic cytokine up-regulated in inflammatory bowel disease, rheumatoid arthritis and psoriasis. The introduction of anti-TNF drugs such as infliximab has revolutionized the treatment of these diseases. Recently, therapeutic drug monitoring (TDM) of infliximab has been introduced in clinical decision making to increase cost-efficiency. Nowadays, TDM is performed using radio-immunoassays, homogeneous mobility shift assays or ELISA. Unfortunately, these assays do not allow for in situ treatment optimization, because of the required sample transportation to centralized laboratories and the subsequent assay execution time. In this perspective, we evaluated the potential of fiber optic-surface plasmon resonance (FO-SPR). To achieve this goal, a panel of 55 monoclonal anti-infliximab antibodies (MA-IFX) was developed and characterized in-house, leading to the identification of nine different clusters. Based on this high diversity, 22 antibody pairs were selected and tested for their reactivity towards IFX, using one MA-IFX as capture and one MA-IFX for detection, in a sandwich type ELISA and FO-SPR. This study showed that the reactivity towards IFX of each antibody pair in ELISA is highly similar to its reactivity on FO-SPR, indicating that antibody pairs are easily transferable between both platforms. Given the fact that FO-SPR shows the potential for miniaturization and fast assay time, it can be considered a highly promising platform for on-site infliximab monitoring.

  3. 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

  4. 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.

  5. Rapid method for the detection of storage mites in cereals: feasibility of an ELISA based approach.

    PubMed

    Dunn, J A; Thind, B B; Danks, C; Chambers, J

    2008-04-01

    This paper describes the development of rapid immunodiagnostic tests for the detection of storage mite infestations in cereals and cereal products. The study's first phase (proof of concept) involved the production of a species-specific enzyme-linked immunoassay (ELISA) for the flour mite, Acarus siro (L.), a major pest of stored commodities. The specificity of this new assay was assessed against key stored product contaminants (13 species of mites of which three were predatory, five species of insects and five species of fungi) in the presence and absence of grain. The assay was species-specific (no cross-reactivity to other storage contaminants) and was unaffected by the presence of cereal antigens in the extract. In the study's second phase, species- and genera-specific ELISAs were developed for a range of key storage mite pests: the cosmopolitan food mite (Lepidoglyphus destructor), the grocers' itch mite (Glycyphagus domesticus), the grainstack mite (Tyrophagus longior), mites of the Tyrophagus and Glycyphagus generas, and all storage mites. All tests were demonstrably specific to target species or genera, with no cross-reactions observed to other storage pest contaminants or cereals. The final, validation phase, involved a comparative assessment of the species-specific A. siro and the genus-specific Tyrophagus ELISAs with the flotation technique using laboratory and field samples. Both ELISAs were quantitative (0-30 mites per 10 g wheat) and produced good comparative data with the flotation technique (A. siro r(2)=0.91, Tyrophagus spp. r(2)=0.99).

  6. Detection of c-reactive protein based on a magnetic immunoassay by using functional magnetic and fluorescent nanoparticles in microplates.

    PubMed

    Yang, S F; Gao, B Z; Tsai, H Y; Fuh, C Bor

    2014-11-07

    We report the preparation and application of biofunctional nanoparticles to detect C-reactive protein (CRP) in magnetic microplates. A CRP model biomarker was used to test the proposed detection method. Biofunctional magnetic nanoparticles, CRP, and biofunctional fluorescent nanoparticles were used in a sandwich nanoparticle immunoassay. The CRP concentrations in the samples were deduced from the reference plot, using the fluorescence intensity of the sandwich nanoparticle immunoassay. When biofunctional nanoparticles were used to detect CRP, the detection limit was 1.0 ng ml(-1) and the linear range was between 1.18 ng ml(-1) and 11.8 μg ml(-1). The results revealed that the method involving biofunctional nanoparticles exhibited a lower detection limit and a wider linear range than those of the enzyme-linked immunosorbent assay (ELISA) and most other methods. For CRP measurements of serum samples, the differences between this method and ELISA in CRP measurements of serum samples were less than 13%. The proposed method can reduce the analysis time to one-third that of ELISA. This method demonstrates the potential to replace ELISA for rapidly detecting biomarkers with a low detection limit and a wide dynamic range.

  7. Development of IgY based sandwich ELISA for the detection of staphylococcal enterotoxin G (SEG), an egc toxin.

    PubMed

    Nagaraj, Sowmya; Ramlal, Shylaja; Kingston, Joseph; Batra, Harsh Vardhan

    2016-11-21

    Staphylococcal food poisoning (SFP) is a major foodborne illness caused by staphylococcal enterotoxins (SEs). It is a well known fact that foodborne outbreak investigations are solely characterized by commercially available immunoassay kits. However, these assays encompass only few enterotoxins such as SEA-SEE which are renowned as "classical" enterotoxins and unable to detect any other novel enterotoxins even though their involvement is predicted. In this context, the present study involved development of a sandwich ELISA immunoassay for the specific detection of "non-classical" enterotoxin G (SEG). The toxin belongs to enterotoxin gene cluster (egc) which comprises a bunch of five toxin genes that are known to co-express. Thus, the developed assay might indirectly speculate the presence of other toxins in the cluster. The efficiency of ELISA was compared with PCR analysis where all strains possessing seg were found positive for toxin production. Additionally, analogous to other studies which reported the co-occurrence of seg and sei, the PCR analysis accomplished in the study evinced the same. The sandwich format allowed sensitive detection with a detection limit of 1ng/mL. High specificity was achieved in presence of non-target protein as well as bacteria. Likewise, staphylococcal protein A (SpA) interference that is inevitably associated with immunoassays was eliminated by implementation of anti-SEG IgY in our study. Consequently, chicken IgY were used to capture target antigen in developed sandwich ELISA. Further, spiking studies and analysis on natural samples emphasized the robustness as well as applicability of developed method. Altogether, the established assay could be a reliable detection tool for the routine investigation of SEG as well as to predict other egc toxins in samples from food and clinical sources.

  8. Development of rapid flow-through-based dot-immunoassay for serodiagnosis of leptospirosis in dogs.

    PubMed

    Subathra, M; Senthilkumar, T M A; Ramadass, P; Dhinakar Raj, G

    2011-01-01

    An IgG-ELISA used recombinant antigen and a rapid flow-through enzyme immunoassay were developed for rapid screening of leptospiral antibodies in dogs using recombinant LipL41, which is one of the conserved outer membrane proteins in pathogenic leptospires as the coating antigen. Results from this study were compared with the standard microscopic agglutination test and found that the sensitivity and specificity of the enzyme-linked immunosorbent assay were 75.46% and 93.29% and whereas that of flow-through-based dot-immunobinding assay were 87.73% and 89.63%, respectively. Relative merits of these tests were also assessed. The flow-through-based dot-immunobinding assay was thus proved to be a valid screening test for canine leptospirosis.

  9. Development and Validation of a Fluorescent Multiplexed Immunoassay for Measurement of Transgenic Proteins in Cotton (Gossypium hirsutum).

    PubMed

    Yeaman, Grant R; Paul, Sudakshina; Nahirna, Iryna; Wang, Yongcheng; Deffenbaugh, Andrew E; Liu, Zi Lucy; Glenn, Kevin C

    2016-06-22

    In order to provide farmers with better and more customized alternatives to improve yields, combining multiple genetically modified (GM) traits into a single product (called stacked trait crops) is becoming prevalent. Trait protein expression levels are used to characterize new GM products and establish exposure limits, two important components of safety assessment. Developing a multiplexed immunoassay capable of measuring all trait proteins in the same sample allows for higher sample throughput and savings in both time and expense. Fluorescent (bead-based) multiplexed immunoassays (FMI) have gained wide acceptance in mammalian research and in clinical applications. In order to facilitate the measurement of stacked GM traits, we have developed and validated an FMI assay that can measure five different proteins (β-glucuronidase, neomycin phosphotransferase II, Cry1Ac, Cry2Ab2, and CP4 5-enolpyruvyl-shikimate-3-phosphate synthase) present in cotton leaf from a stacked trait product. Expression levels of the five proteins determined by FMI in cotton leaf tissues have been evaluated relative to expression levels determined by enzyme-linked immunosorbent assays (ELISAs) of the individual proteins and shown to be comparable. The FMI met characterization requirements similar to those used for ELISA. Therefore, it is reasonable to conclude that FMI results are equivalent to those determined by conventional individual ELISAs to measure GM protein expression levels in stacked trait products but with significantly higher throughput, reduced time, and more efficient use of resources.

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

    PubMed Central

    2013-01-01

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

  11. 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).

  12. Development of an integrase-based ELISA for specific diagnosis of individuals infected with HIV.

    PubMed

    Rikhtegaran Tehrani, Zahra; Azadmanesh, Kayhan; Mostafavi, Ehsan; Soori, Shahrzad; Azizi, Mohammad; Khabiri, Alireza

    2015-04-01

    Currently, enzyme immunoassays (EIAs) are the most common immunological diagnostic methods that are used as the screening tool in HIV detection. Among all three major genes of HIV, the products of gag and env are usually used in EIAs (ELISAs and rapid tests). Hence, the presence of cross reacting antibodies against these antigens leads to the appearance of repetitive false positive results in screening tests. Re-testing the primary reactive samples with EIAs using other HIV antigens can considerably reduce the rate of false positive results. The products of pol gene may act as an appropriate candidate in this context. Integrase is a conserved and immunogenic product of HIV, encoded by the pol gene. The aim of this research was to determine the sensitivity and specificity of an ELISA detecting integrase antibodies. Recombinant integrase was produced in Escherichia coli to develop the integrase-based ELISA. Assay performance was evaluated by HIV positive and negative sera and an HIV panel of BBI (PRB-601). The sensitivity and specificity of assay was determined as 96.7 [95% confidence interval: 91.3-98.9%] and 100% [95% CI: 96.1-100%], respectively. High specificity of this assay may suggest its possible use in the detection of HIV.

  13. Development of a bead-based multiplex immunoassay for simultaneous quantitative detection of IgG serum antibodies against measles, mumps, rubella, and varicella-zoster virus.

    PubMed

    Smits, Gaby P; van Gageldonk, Pieter G; Schouls, Leo M; van der Klis, Fiona R M; Berbers, Guy A M

    2012-03-01

    Enzyme-linked immunosorbent assay (ELISA) is normally used to quantify the amount of serum IgG antibodies against measles, mumps, rubella, and varicella-zoster virus (MMRV). However, this method is time- and material-consuming. Therefore, a multiplex immunoassay for the simultaneous quantitative detection of antibodies against MMRV was developed. In-house as well as commercially available antigens can be used, making the assay available for all laboratories. The multiplex assay is much more sensitive than the separate ELISAs and has a high specificity, and only 5 μl of serum is needed. Heterologous inhibition did not exceed 11.5%, while homologous inhibition varied between 91.3 and 97.9%. Good correlations with the in-house ELISAs for measles (R(2) = 0.98), mumps (R(2) = 0.97), and rubella (R(2) = 0.97) virus as well as with the ELISA kit for varicella-zoster virus (R(2) = 0.95) were obtained. In conclusion, the MMRV multiplex assay is a good alternative to the conventional ELISAs and suitable for use in serosurveillance and vaccine studies.

  14. Determination of ANA specificity using multiplexed fluorescent microsphere immunoassay in patients with ANA positivity at high titres after infliximab treatment: preliminary results.

    PubMed

    Caramaschi, Paola; Ruzzenente, Orazio; Pieropan, Sara; Volpe, Alessandro; Carletto, Antonio; Bambara, Lisa Maria; Biasi, Domenico

    2007-05-01

    To evaluate ANA specificity using the fully automated multiplexed fluorescent microsphere immunoassay in patients affected either by rheumatoid arthritis or ankylosing spondylitis who developed strong positivity for ANA as assessed by indirect immunofluorescent method on HEp-2 cells during infliximab treatment. Three men affected by ankylosing spondylitis and 12 women affected by rheumatoid arthritis who developed ANA positivity at high titres during infliximab treatment underwent the identification of ANA specificity by multiplexed fluorescent microsphere immunoassay; moreover anti-DNA and anti-ENA antibodies were tested by indirect immunofluorescence and ELISA method, respectively. In 4 out of 15 cases, the determination of ANA reactivity by multiplexed fluorescent microsphere immunoassay was also performed on the serum collected before infliximab administration. One patient affected by rheumatoid arthritis showed multiple ANA reactivities against SS-A, SS-B, RNP, Sm, Jo-1 and histones; one patient affected by ankylosing spondylitis resulted positive for the same autoantibodies, except for anti-Sm antibody. Moreover, two patients, one with rheumatoid arthritis and one with ankylosing spondylitis, showed single antibody specificity to SS-B and RNP, respectively. The remaining 11 cases did not show any positivity. Instead, all the patients resulted negative for anti-ENA antibodies by the ELISA method. In the four cases tested for ANA specificity by multiplexed fluorescent microsphere immunoassay before and after infliximab administration no difference was found. The search for anti-DNA antibody always resulted negative by both the traditional immunofluorescent assay and the novel technique. The use of multiplexed fluorescent microsphere immunoassay in patients treated with infliximab with ANA positivity at high titres allowed to find some ANA specificities which were not revealed by ELISA method. Nevertheless, the majority of patients resulted negative in spite of

  15. CONTINUOUS MONITORING OF INFLAMMATION BIOMARKERS DURING SIMULATED CARDIOPULMONARY BYPASS USING A MICROFLUIDIC IMMUNOASSAY DEVICE – A PILOT STUDY

    PubMed Central

    Sasso, Lawrence A.; Aran, Kiana; Guan, Yulong; Ündar, Akif; Zahn, Jeffrey D.

    2012-01-01

    This work demonstrates the use of a continuous online monitoring system for tracking systemic inflammation biomarkers during cardiopulmonary bypass (CPB) procedures. The ability to monitor inflammation biomarkers during CPB will allow surgical teams to actively treat inflammation and reduce harmful effects on postoperative morbidity and mortality, enabling improved patient outcomes. A microfluidic device has been designed which allows automation of the individual processing steps of a microbead immunoassay to allow continuous tracking of antigen concentrations. Preliminary experiments have demonstrated that the results produced by the micro-immunoassay are comparable to results produced from a standard ELISA (r=0.98). Additionally, integration of the assay with a simulated CPB circuit has been demonstrated with temporal tracking of C3a concentrations within blood continuously sampled from the circuit. The presented work describes the motivation, design challenges, and preliminary experimental results of this project. PMID:23305589

  16. Novel potentiometry immunoassay with amplified sensitivity for diphtheria antigen based on Nafion, colloidal Ag and polyvinyl butyral as matrixes.

    PubMed

    Tang, Dianping; Yuan, Ruo; Chai, Yaqin; Zhang, Linyan; Zhong, Xia; Dai, Jianyuan; Liu, Yan

    2004-11-30

    A novel potentiometry immunoassay with amplified sensitivity has been developed for the detection of diphtheria antigen (Diph) via immobilizing diphtheria antibody (anti-Diph) on a platinum electrode based on Nafion, colloidal Ag (Ag), and polyvinyl butyral (PVB) as matrixes in this study. The modified procedure was further characterized by electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV). The influence and factors influencing the performance of resulting immunosensor were studied in detail. The resulting immunosensor exhibited sigmoid curve with log Diph concentrations, high sensitivity (51.4 mV/decade), wide linear range from 8 to 800 ng ml(-1) with a detection limit of 1.5 ng ml(-1), rapid potentiometric response (<3 min) and long-term stability (>6 months). Analytical results of clinical samples show that the developed immunoassay is comparable with the enzyme-linked immunosorbent assays (ELISAs) method, implying a promising alternative approach for detecting diphtheria antigen in the clinical diagnosis.

  17. 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.

  18. 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 μg/ml, respectively, with a dynamic range between 0.19 and 2.04 μg/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 μg/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.

  19. Monoclonal antibodies specific for immunorecessive epitopes of glucuronoxylomannan, the major capsular polysaccharide of Cryptococcus neoformans, reduce serotype bias in an immunoassay for cryptococcal antigen.

    PubMed

    Percival, Ann; Thorkildson, Peter; Kozel, Thomas R

    2011-08-01

    Immunoassay for detection of glucuronoxylomannan (GXM), the major capsular polysaccharide of Cryptococcus neoformans, is an important tool for diagnosis of cryptococcosis. However, immunoassays that are based solely or in part on detection with polyclonal antibodies may show serotype bias in detection of GXM, particularly limited sensitivity for serotype C. In this study, we describe detection of GXM in an antigen capture sandwich enzyme-linked immunosorbent assay (ELISA) that used a cocktail of two monoclonal antibodies (MAbs). MAb F12D2 was previously produced by immunization with GXM that had been treated to remove O-acetyl groups, a major source of serotype specificity. MAb F12D2 has a high degree of reactivity with GXM of serotypes A, B, C, and D, but the reactivity with serotype D was less than was found with other MAbs. MAb 339 is highly reactive with GXM of serotypes A and D. Use of a combination of the two MAbs produced an immunoassay that had the best properties of both MAbs, including good reactivity with serotype C, which is an emerging threat in sub-Saharan Africa. These results suggest that next-generation immunoassays for diagnosis of cryptococcosis may be formulated by (i) use of immunization and hybridoma screening strategies that are designed to prospectively meet the needs of immunoassay performance and (ii) careful selection of MAbs that span the expected polysaccharide serotypes in the subject patient population.

  20. Development of an Immunoassay for the Detection of the Phenylpyrazole Insecticide Fipronil

    PubMed Central

    Vasylieva, Natalia; Ahn, Ki Chang; Barnych, Bogdan; Gee, Shirley J.; Hammock, Bruce D.

    2015-01-01

    Phenylpyrazole insecticides such as fipronil have been used as replacements for organophosphates. The wide application of fipronil raises concern about environmental contamination and risk for fish, birds, other non-targeted beings and human health. A sensitive, competitive indirect heterologous enzyme-linked immunosorbent assay (ELISA) was developed. Antibodies with different specificities to fipronil and its metabolites were produced. Two ELISAs having IC50 values of 0.58 ± 0.06 and 2.6 ± 0.4 ng/mL were developed. Design of different haptens and coating antigens resulted in two assays with distinct cross-reactivity patterns for structurally related compounds: 96%, 38% and 101% vs 39%, 1.4% and 25% for fipronil-sulfide, fipronil-detrifluoromethylsulfonyl and fipronil-desulfinyl, respectively. Performance of the immunoassays was demonstrated by a recovery study from spiked water, human serum and urine matrices, giving recovery values in the range of 85–111% for different concentrations. The assays demonstrated good correlation in fipronil recovery with conventional LC-MS/MS analysis. The generic assay 2265 has the sensitivity to measure fipronil and its analogs in serum at levels relevant for exposure monitoring. The assays were used to analyze human urine samples obtained from exposure studies and serum samples from rats treated with fipronil-containing diet. PMID:26196357

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

    USGS Publications Warehouse

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

    1991-01-01

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

  2. Detection of antibodies against therapeutic proteins in the presence of residual therapeutic protein using a solid-phase extraction with acid dissociation (SPEAD) sample treatment prior to ELISA.

    PubMed

    Smith, Holly W; Butterfield, Anthony; Sun, Deqin

    2007-12-01

    The evaluation of the potential immunogenicity of therapeutic proteins in nonclinical safety studies has become complicated by the development of biopharmaceuticals that are dosed at high concentrations and/or have long half lives. These products remain in the circulation of the test system for extended periods of time, resulting in samples containing high concentrations of drug that interfere with standard immunogenicity assays. This protocol describes a novel solid-phase extraction with acid dissociation (SPEAD) sample treatment that removes the interfering therapeutic protein "drug" from the sample prior to performance of a direct immunoassay for detection of anti-drug antibodies (ADA). A biotin-avidin capture technique is used to physically separate ADA and ADA:Drug complexes from the drug and the sample matrix. The acid dissociation step removes the ADA from the biotin-avidin complex, and detection is performed by simple direct enzyme-linked immunoassay (ELISA). The SPEAD treatment allows for detection of ADA in an ELISA format and results in a >10-100-fold increase in residual drug tolerance as compared to an immunoassay without the sample treatment. The method can be used for serum samples from all species, but is presented here as an assay for detection of anti-drug antibodies in cynomolgus monkey serum.

  3. Evaluation of an inhouse rapid ELISA test for detection of giardia in domestic sheep (Ovis aries).

    PubMed

    Wilson, Jolaine M; Hankenson, F Claire

    2010-11-01

    Sheep (Ovis aries) are increasingly used at our institution as models of human disease. Within the research environment, routine husbandry and handling of sheep has potential for transmission of zoonotic agents, including Giardia. The prevalence of Giardia in sheep may approach 68%. Classic diagnostic testing involves microscopic examination for fecal cysts or trophozoites; however, limitations of microscopy include time, labor, and potential false-negative results due to intermittent shedding. We wished to determine whether a commercial rapid ELISA used for Giardia detection in dogs and cats could be used in sheep. Fecal samples collected from sheep (n = 93) were tested with a combination of 6 methods: reference laboratory fecal flotation, reference laboratory ELISA, inhouse fecal flotation, and commercially available tests (enzyme immunoassay, direct fluorescence antibody assay, and rapid ELISA). Prevalence of Giardia infection in facility sheep was 11.8% (11 of 93 animals). Of the 11 samples considered positive, 3 were confirmed by multiple testing methods, and 5 were positive by microscopy alone. Inhouse fecal flotation for 8 samples was positive on only 1 of 2 consecutive testing days. The rapid ELISA test exhibited 0% sensitivity for sheep giardiasis. Overall, the examined methods had low sensitivities and low positive predictive values. Despite limitations, microscopic analysis of repeat fecal samples remained the most accurate diagnostic method for ovine giardiasis among the methods tested.

  4. Miniaturized immunoassays: moving beyond the microplate.

    PubMed

    Verch, Thorsten; Bakhtiar, Ray

    2012-01-01

    After more than 40 years, immunoassays are still the backbone of protein biomarker analysis in clinical diagnostics and drug development. They have come a long way since their inception, incorporating technical developments including monoclonal antibodies, novel labels and lately microfluidics. A number of microfluidic platforms have been tested, such as centrifugational compact disc assays, lab-on-a-chip, arrays and digital electrochemical assays. This review focuses on commercial applications of microfluidic immunoassays with reference to some applied academic examples of interest. Advantages and disadvantages of the platform technologies are discussed in general.

  5. Rapid detection of autoantibodies to dsDNA with the particle gel immunoassay (ID-PaGIA)

    PubMed Central

    Seltsam, A; Schwind, P; Abraham, K; Hiepe, F; Cygan, S; Aebischer, I; Salama, A

    2002-01-01

    Patients and methods: Serum samples were collected from 40 unselected healthy blood donors and 200 patients with systemic lupus erythematosus (SLE) or a positive antinuclear antibody screen, or both. The samples were tested in the presence of red high density polystyrene particles coated with purified human dsDNA using the gel technique (Micro Typing System, ID-PaGIA, particle gel immunoassay). The results were compared with those obtained by the two standard anti-dsDNA antibody detection methods, Crithidia luciliae immunofluorescence test (CLIF) and enzyme linked immunosorbent assay (ELISA). Results: The three anti-dsDNA assays exhibited an overall agreement of 87% and significant correlation with each other (p<0.0001). In the SLE group (n=71), 45 patients (63%) were found to be positive by ID-PaGIA compared with only 11/129 (9%) patients in the non-SLE group. Thus the ID-PaGIA had a sensitivity of 63%, and a specificity of 92% for SLE. In comparison, the standard detection methods showed sensitivities of 62% (CLIF) and 70% (ELISA) and specificities of 99% (CLIF) and 84% (ELISA) for SLE. Anti-dsDNA reactivity in the agglutination assay correlated closely with the quantities of antibody obtained by CLIF (r=0.81, p<0.0001) and ELISA (r=0.73, p<0.0001). Conclusions: The new particle gel agglutination test is a sensitive and specific immunoassay. It is a simple test procedure that might be well suited as a rapid screening method. PMID:11874846

  6. Fully mechanized latex immunoassay for serum lipoprotein(a).

    PubMed

    Abe, A; Yoshimura, Y; Sekine, T; Maeda, S; Yamashita, S; Noma, A

    1994-03-01

    We have developed a fully automated system to quantify lipoprotein(a) (Lp(a)) in human serum, based on the latex-enhanced turbidimetric immunoassay by application of the Immuno Chemistry Analyzer 501X. This assay was carried out with undiluted serum and was able to detect at Lp(a) levels higher than 4.0 mg/l. When judged to be out of range of the calibration (> 600 mg/l), the sample was automatically re-tested after automatic 10-fold dilution. Within-run C.V.s ranged from 1.9 to 2.1% and between-run C.V.s from 2.7 to 3.9%. Results by the present method were in good agreement with those by the in-house ELISA (r = 0.978) and the commercial ELISA (r = 0.990). The distribution of Lp(a) levels in sera from 508 healthy donors was highly skewed; the mean and median were 158 mg/l and 105 mg/l, respectively.

  7. A Multiplex Microsphere Immunoassay for Zika Virus Diagnosis.

    PubMed

    Wong, Susan J; Furuya, Andrea; Zou, Jing; Xie, Xuping; Dupuis, Alan P; Kramer, Laura D; Shi, Pei-Yong

    2017-02-01

    Rapid and accurate diagnosis of infectious agents is essential for patient care, disease control, and countermeasure development. The present serologic diagnosis of Zika virus (ZIKV) infection relies mainly on IgM-capture ELISA which is confounded with the flaw of cross-reactivity among different flaviviruses. In this communication, we report a multiplex microsphere immunoassay (MIA) that captures the diagnostic power of viral envelope protein (that elicits robust, yet cross-reactive antibodies to other flaviviruses) and the differential power of viral nonstructural proteins NS1 and NS5 (that induce more virus-type specific antibodies). Using 153 patient specimens with known ZIKV and/or dengue virus (DENV; a closely related flavivirus) infections, we showed that (i) ZIKV envelope-based MIA is equivalent or more sensitive than IgM-capture ELISA in diagnosing ZIKV infection, (ii) antibody responses to NS1 and NS5 proteins are more ZIKV-specific than antibody response to envelope protein, (iii) inclusion of NS1 and NS5 in the MIA improves the diagnostic accuracy when compared with the MIA that uses envelope protein alone. The multiplex MIA achieves a rapid diagnosis (turnaround time<4h) and requires small specimen volume (10μl) in a single reaction. This serologic assay could be developed for use in clinical diagnosis of ZIKV infection and for monitoring immune responses in vaccine trials.

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

    PubMed Central

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

    1993-01-01

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

  9. A Simple ELISA Exercise for Undergraduate Biology.

    ERIC Educational Resources Information Center

    Baker, William P.; Moore, Cathy R.

    Understanding of immunological techniques such as the Enzyme Linked Immuno Sorbent Assay (ELISA) is an important part of instructional units in human health, developmental biology, microbiology, and biotechnology. This paper describes a simple ELISA exercise for undergraduate biology that effectively simulates the technique using a paper model.…

  10. Assessment of an ELISA Laboratory Exercise

    ERIC Educational Resources Information Center

    Robinson, David L.; Lau, Joann M.

    2012-01-01

    The enzyme-linked immunosorbent assay (ELISA) is a powerful immunological technique for quantifying small amounts of compounds and has been used in research and clinical settings for years. Although there are laboratory exercises developed to introduce the ELISA technique to students, their ability to promote student learning has not been…

  11. Modification of a deoxynivalenol-antigen-mimicking nanobody to improve immunoassay sensitivity by site-saturation mutagenesis.

    PubMed

    Qiu, Yu-Lou; He, Qing-Hua; Xu, Yang; Wang, Wei; Liu, Yuan-Yuan

    2016-01-01

    A nanobody (N-28) which can act as a deoxynivalenol (DON) antigen has been generated, and its residues Thr102-Ser106 were identified to bind with anti-DON monoclonal antibody by alanine-scanning mutagenesis. Site-saturation mutagenesis was used to analyze the plasticity of five residues and to improve the sensitivity of the N-28-based immunoassay. After mutagenesis, three mutants were selected by phage immunoassay and were sequenced. The half-maximal inhibitory concentrations of the immunoassay based on mutants N-28-T102Y, N-28-V103L, and N-28-Y105F were 24.49 ± 1.0, 51.83 ± 2.5, and 35.65 ± 1.6 ng/mL, respectively, showing the assay was, respectively, 3.2, 1.5, and 2.2 times more sensitive than the wild-type-based assay. The best mutant, N-28-T102Y, was used to develop a competitive phage ELISA to detect DON in cereals with high specificity and accuracy. In addition, the structural properties of N-28-T102Y and N-28 were investigated, revealing that the affinity of N-28-T102Y decreased because of increased steric hindrance with the large side chain. The lower-binding-affinity antigen mimetic may contribute to the improvement of the sensitivity of competitive immunoassays. These results demonstrate that nanobodies would be a favorable tool for engineering. Moreover, our results have laid a solid foundation for site-saturation mutagenesis of antigen-mimicking nanobodies to improve immunoassay sensitivity for small molecules.

  12. Development of ELISA for the detection of transgenic vegetative insecticidal protein in GM crops/produce.

    PubMed

    Kumar, R

    2012-01-11

    In the process of the development of insect-resistant genetically modified (GM) crops and also to evaluate the consistency in the expression of toxin under field conditions, immunological assays are commonly being used. An immunoassay was developed to support the labelling of vegetative insecticidal protein (Vip3A)-based GM produce. The developed ELISA for the measurement of Vip3A is a triple antibody sandwich procedure utilising a polyclonal capture antibody (mouse anti-Vip3A) and a polyclonal detection antibody (rabbit anti-Vip3A) followed by use of a third HRP-conjugated anti-species antibody (goat anti-rabbit IgG). The limit of detection limit of the ELISA assay was 16 ng ml(-1) with a linear quantification range from approximately 31 to 500 ng ml(-1) of Vip3A protein. Furthermore, the assay was in-house validated with GM brinjal samples. The assay was specific, sensitive and reproducible, which can be helpful to detect and track down the spread of unapproved and intentionally/unintentionally released GM produce harbouring Vip protein.

  13. Serodiagnosis of Zika virus (ZIKV) infections by a novel NS1-based ELISA devoid of cross-reactivity with dengue virus antibodies: a multicohort study of assay performance, 2015 to 2016

    PubMed Central

    Steinhagen, Katja; Probst, Christian; Radzimski, Christiane; Schmidt-Chanasit, Jonas; Emmerich, Petra; van Esbroeck, Marjan; Schinkel, Janke; Grobusch, Martin P; Goorhuis, Abraham; Warnecke, Jens M; Lattwein, Erik; Komorowski, Lars; Deerberg, Andrea; Saschenbrecker, Sandra; Stöcker, Winfried; Schlumberger, Wolfgang

    2016-01-01

    Serological diagnosis of Zika virus (ZIKV) infections is challenging due to high cross-reactivity between flaviviruses. We evaluated the diagnostic performance of a novel anti-ZIKV ELISA based on recombinant ZIKV non-structural protein 1 (NS1). Assay sensitivity was examined using sera from 27 patients with reverse transcription (RT)-PCR-confirmed and 85 with suspected ZIKV infection. Specificity was analysed using sera from 1,015 healthy individuals. Samples from 252 patients with dengue virus (n = 93), West Nile virus (n = 34), Japanese encephalitis virus (n = 25), chikungunya virus (n = 19) or Plasmodium spp. (n = 69) infections and from 12 yellow fever-vaccinated individuals were also examined. In confirmed ZIKV specimens collected ≥ 6 days after symptom onset, ELISA sensitivity was 58.8% (95% confidence interval (CI): 36.0–78.4) for IgM, 88.2% (95% CI: 64.4–98.0) for IgG, and 100% (95% CI: 78.4–100) for IgM/IgG, at 99.8% (95% CI: 99.2–100) specificity. Cross-reactivity with high-level dengue virus antibodies was not detected. Among patients with potentially cross-reactive antibodies anti-ZIKV positive rates were 0.8% (95% CI: 0–3.0) and 0.4% (95% CI: 0–2.4) for IgM and IgG, respectively. Providing high specificity and low cross-reactivity, the NS1-based ELISA has the potential to aid in counselling patients, pregnant women and travellers after returning from ZIKV-endemic areas. PMID:28006649

  14. A direct immunoassay for detecting diatoms in groundwater as an indicator of the direct influence of surface water

    USGS Publications Warehouse

    Walker, C.E.; Schrock, R.M.; Reilly, T.J.; Baehr, A.L.

    2005-01-01

    Groundwater under the direct influence of surface water (GWUDISW) is of concern in communities where growing public demand on groundwater resources has resulted in increased withdrawals and hydraulic stress near surface water bodies. Under these conditions, contaminants such as methyl-tert butyl ether (MTBE) and biological materials have been detected in domestic wells. Other contaminants and pathogens associated with surface water are not routinely tested for in groundwater-supplied systems. To address the need for methods to easily identify potentially vulnerable supplies, a direct immunoassay for the quantitative detection of diatoms in raw water samples was developed as a measure of surface water influence on groundwater. Cell wall preparations from Nitzschia palea Ku??tzing, a freshwater diatom found throughout North America, were used to produce a polyclonal antibody that was applied in a direct enzyme-linked immunosorbent assay (ELISA) developed to detect the presence of N. palea cell wall components. The direct immunoassay allows detection at 500 cells L-1, a level similar to diatom concentrations observed in samples of groundwater collected near the test site. This investigation was the first attempt to utilize an ELISA as an indicator of surface water influence on groundwater. Further research is needed to develop more specific diatom-based monoclonal antibodies, determine cross-reactivity, and optimize sample processing and ELISA procedures for development of a standardized method. ?? Springer 2005.

  15. A direct immunoassay for detecting diatoms in groundwater as an indicator of the direct influence of surface water

    USGS Publications Warehouse

    Walker, C.E.; Schrock, R.M.; Reilly, T.J.; Baehr, A.L.

    2005-01-01

    Groundwater under the direct influence of surface water (GWUDISW) is of concern in communities where growing public demand on groundwater resources has resulted in increased withdrawals and hydraulic stress near surface water bodies. Under these conditions, contaminants such as methyl-tert butyl ether (MTBE) and biological materials have been detected in domestic wells. Other contaminants and pathogens associated with surface water are not routinely tested for in groundwater-supplied systems. To address the need for methods to easily identify potentially vulnerable supplies, a direct immunoassay for the quantitative detection of diatoms in raw water samples was developed as a measure of surface water influence on groundwater. Cell wall preparations from Nitzschia palea Kützing, a freshwater diatom found throughout North America, were used to produce a polyclonal antibody that was applied in a direct enzyme-linked immunosorbent assay (ELISA) developed to detect the presence of N. palea cell wall components. The direct immunoassay allows detection at 500 cells L−1, a level similar to diatom concentrations observed in samples of groundwater collected near the test site. This investigation was the first attempt to utilize an ELISA as an indicator of surface water influence on groundwater. Further research is needed to develop more specific diatom-based monoclonal antibodies, determine cross-reactivity, and optimize sample processing and ELISA procedures for development of a standardized method.

  16. ELISA-BASE: An Integrated Bioinformatics Tool for Analyzing and Tracking ELISA Microarray Data

    SciTech Connect

    White, Amanda M.; Collett, James L.; Seurynck-Servoss, Shannon L.; Daly, Don S.; Zangar, Richard C.

    2009-06-15

    ELISA-BASE is an open-source database for capturing, organizing and analyzing protein enzyme-linked immunosorbent assay (ELISA) microarray data. ELISA-BASE is an extension of the BioArray Soft-ware Environment (BASE) database system, which was developed for DNA microarrays. In order to make BASE suitable for protein microarray experiments, we developed several plugins for importing and analyzing quantitative ELISA microarray data. Most notably, our Protein Microarray Analysis Tool (ProMAT) for processing quantita-tive ELISA data is now available as a plugin to the database.

  17. Novel ELISAs for screening of the biogenic amines GABA, glycine, beta-phenylethylamine, agmatine, and taurine using one derivatization procedure of whole urine samples.

    PubMed

    Huisman, Han; Wynveen, Paul; Nichkova, Mikaela; Kellermann, Gottfried

    2010-08-01

    The inhibitory neurotransmitters GABA, glycine and agmatine and neuromodulators beta-phenylethylamine (beta-PEA) and taurine are important biogenic amines of the sympathetic and parasympathetic nervous systems in the body. Abnormalities in the metabolism of these biomarkers have been implicated in a vast number of neurological diseases. Novel competitive immunoassays, using one unique whole urine derivatization procedure applicable for all five biomarkers, have been developed. The determination of these biomarkers was highly reproducible: the coefficient of variance of inter- and intra-assay variation is between 3.9% and 9.8% for all assays. The assays show a good linearity in urine samples within the range of 100-400 mg Cr/dL and specificity when urine samples are spiked with biogenic amines. The recoveries are between 76 and 154%. The correlation between HPLC and ELISA for glycine and taurine (n = 10) showed regression coefficients of 0.97 and 0.98, respectively. An in vivo study on the urinary clearance of beta-PEA, agmatine and taurine after oral intake by healthy individuals demonstrated the specificity and clinical significance of these new immunoassays. The immunoassays are useful for clinical and basic research where a fast and accurate assay for the screening of biogenic amines in urine is required, without preclearance of the sample.

  18. Direct construction of an open-sandwich enzyme immunoassay for one-step noncompetitive detection of thyroid hormone T4.

    PubMed

    Islam, Kamrun Nahar; Ihara, Masaki; Dong, Jinhua; Kasagi, Noriyuki; Mori, Toshihiro; Ueda, Hiroshi

    2011-02-01

    To establish a sensitive noncompetitive immunoassay for thyroxine (T4), we attempted to isolate anti-T4 antibodies from a phage display library based on a phagemid pDong1 ( Dong et al. Anal. Biochem.2009, 36, 386 ), which was designed to enable open-sandwich enzyme-linked immunosorbent assay (OS-ELISA) after selection on immobilized antigen. After the Fab-displaying phage library made from the splenocytes of T4-KLH immunized mice was subjected to biopanning on T4-BSA, two T4-specific clones were obtained. When they were assayed by indirect competitive ELISA, both clones showed low IC(50) (5-13 ng/mL), indicating their high affinity to T4. When they were used for OS-ELISA that detects antigen-dependency of the interaction between variable domains V(H) and V(L), a clone successfully detected 1 ng/mL of T4 with a working range superior to that of competitive IA. OS-ELISA was also performed with maltose binding protein (MBP)-fused V(H)/V(L) of this clone, which showed a detection limit less than 0.1 ng/mL T4. Moreover, the assay showed cross-reactivity with T3 similar to that of competitive ELISA, and also gave a reasonable total serum T4 concentration (90 ng/mL) from ethanol-extracted sample serum using the recombinant proteins. This is the first direct construction of an OS-ELISA system bypassing hybridoma, which will be applicable to the detection of many other small molecule antigens.

  19. A versatile SERS-based immunoassay for immunoglobulin detection using antigen-coated gold nanoparticles and malachite green-conjugated protein A/G.

    PubMed

    Neng, Jing; Harpster, Mark H; Zhang, Hao; Mecham, James O; Wilson, William C; Johnson, Patrick A

    2010-11-15

    A surface enhanced Raman scattering (SERS) immunoassay for antibody detection in serum is described in the present work. The developed assay is conducted in solution and utilizes Au nanoparticles coated with the envelope (E) protein of West Nile Virus (WNV) as the SERS-active substrate and malachite green (MG)-conjugated protein A/G (MG-pA/G) as a bi-functional Raman tag/antibody binding reporter. Upon incubation of these reagents with serum collected from rabbits inoculated with E antigen, laser interrogation of the sandwiched immunocomplex revealed a SERS signaling response diagnostic for MG. The intensification of signature spectral peaks is shown to be proportionate to the concentration of added serum and the limit of antibody detection is 2 ng/ml of serum. To assess assay performance relative to more a traditional immunoassay, indirect enzyme-linked immunosorbent assays conducted using the same concentrations of reagents were found to be >400-fold less sensitive. Quartz crystal microbalance with dissipation (QCM-D) monitoring of immunocomplex film deposition on solid Au surfaces also confirmed the formation of antigen-antibody-protein A/G trilayers and provided quantitative measurements of film thickness which likely position MG within the sensing distance of laser-elicited, enhanced electromagnetic fields. The sensitivity and inherent versatility of the assay, which is provided by the binding of pA/G to a broad spectrum of immunoglobulins in different mammalian species, suggest that it could be developed as an alternative immunoassay format to the ELISA.

  20. Ultrasensitive and accelerated detection of ciguatoxin by capillary electrophoresis via on-line sandwich immunoassay with rotating magnetic field and nanoparticles signal enhancement.

    PubMed

    Zhang, Zhaoxiang; Zhang, Chaoying; Luan, Wenxiu; Li, Xiufeng; Liu, Ying; Luo, Xiliang

    2015-08-12

    A sensitive and rapid on-line immunoassay for the determination of ciguatoxin CTX3C was developed based on a capillary mixing system, which was integrated with capillary electrophoresis (CE) separation and electrochemical (EC) detection. In the sandwich immunoassay system, anti-CTX3C-functionalized magnetic nanoparticles were used as immunosensing probes, and horseradish peroxidase (HRP) and anti-CTX3C antibody were bound onto the surface of gold nanoparticles (AuNPs) and used as recognition elements. Online formation of immunocomplex was realized in capillary inlet end with an external rotating magnetic field. Compared with classical HPLC-MS and ELISA, the assay adopting AuNPs as multienzyme carriers and online sandwich immunoassay format with rotating magnetic field exhibited higher sensitivity and shorter assay time. The linear range of the assay for CTX3C was from 0.6 to 150 ng/L with a correlation coefficient of 0.9948 (n = 2), and the detection limit (S/N = 3) was 0.09 ng/L. The developed assay showed satisfying reproducibility and stability, and it was successfully applied for the quantification of CTX3C in fish samples.

  1. Competitive Electrochemiluminescence Wash and No-Wash Immunoassays for Detection of Serum Antibodies to Smooth Brucella Strains▿

    PubMed Central

    Thompson, Iain; McGiven, John; Sawyer, Jason; Thirlwall, Rachel; Commander, Nicola; Stack, Judy

    2009-01-01

    Brucellosis is a bacterial zoonotic disease of major global importance. Natural hosts for Brucella species include animals of economic significance, such as cattle and small ruminants. Controlling brucellosis in natural hosts by high-throughput serological testing followed by the slaughter of seropositive animals helps to prevent disease transmission. This study aimed to convert an existing competitive enzyme-linked immunosorbent assay (cELISA), used for the serodiagnosis of brucellosis in ruminants, to two electrochemiluminescence (ECL) immunoassays on the Meso Scale Discovery (MSD) platform. The first assay employed a conventional plate washing step as part of the protocol. The second was a no-wash assay, made possible by the proximity-based nature of ECL signal generation by the MSD platform. Both ECL wash and no-wash assays closely matched the parent cELISA for diagnostic sensitivity and specificity. The results also demonstrated that both ECL assays met World Organization for Animal Health (OIE) standards, as defined by results for the OIE standard serum (OIEELISASPSS). This report is the first to describe an ECL assay incorporating lipopolysaccharide, an ECL assay for serodiagnosis of a bacterial infectious disease, a separation-free (no-wash) ECL assay for the detection of serum antibodies, and the use of the MSD platform for serodiagnosis. The simple conversion of the cELISA to the MSD platform suggests that many other serodiagnostic tests could readily be converted. Furthermore, the alignment of these results with the multiplex capability of the MSD platform offers the potential of no-wash multiplex assays to screen for several diseases. PMID:19261777

  2. Evaluation of Newcastle disease virus immunoassays for waterfowl using a monoclonal antibody specific for the duck immunoglobulin light chain.

    PubMed

    Kothlow, Sonja; Haüslaigner, Rafaela; Kaspers, Bernd; Grund, Christian

    2008-06-01

    In the present study a monoclonal antibody (mAb 14A3) was tested for its reactivity against serum immunoglobulin Y (IgY) of several waterfowl species, and subsequently for its applicability as anti-species antibody in common immunoassays. Western blot analyses demonstrated its broad cross-reactivity with the serum IgY light chain of different duck species: Muscovy duck (Cairina moschata), Mallard (Anas platyrhynchos), white-winged wood duck (Asarcornis scutulatus), common pintail (Dafila acuta). Reactivity was also evident with IgY of two swan species--mute swan (Cygnus olor) and black-necked swan (Sthenelides melanocoryphus)--and two goose species--domestic goose (Anser anser var. domestica) and red-breasted goose (Rufibrenta ruficollis). Applying the mAb for Newcastle disease virus (avian paramyxovirus serotype 1 [APMV-1]) test systems, its functionality within indirect immunoassays was evaluated. Using APMV-1-positive sera of domestic geese and Muscovy ducks, mAb 14A3 facilitated specific staining of APMV-1-infected cells in an immunofluorescence test. In addition, it proved to be functional in an indirect enzyme-linked immunosorbent assay (ELISA) and a western blot assay. Thus, the analysed mAb represents an attractive and versatile reagent that offers the opportunity to develop serological tests for waterfowl, allowing a high sample throughput using the ELISA technique or the fine analysis of humoral immune responses using the western blot.

  3. 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...

  4. Competitive Protein-binding assay-based Enzyme-immunoassay Method, Compared to High-pressure Liquid Chromatography, Has a Very Lower Diagnostic Value to Detect Vitamin D Deficiency in 9–12 Years Children

    PubMed Central

    Zahedi Rad, Maliheh; Neyestani, Tirang Reza; Nikooyeh, Bahareh; Shariatzadeh, Nastaran; Kalayi, Ali; Khalaji, Niloufar; Gharavi, Azam

    2015-01-01

    Background: The most reliable indicator of Vitamin D status is circulating concentration of 25-hydroxycalciferol (25(OH) D) routinely determined by enzyme-immunoassays (EIA) methods. This study was performed to compare commonly used competitive protein-binding assays (CPBA)-based EIA with the gold standard, high-pressure liquid chromatography (HPLC). Methods: Concentrations of 25(OH) D in sera from 257 randomly selected school children aged 9–11 years were determined by two methods of CPBA and HPLC. Results: Mean 25(OH) D concentration was 22 ± 18.8 and 21.9 ± 15.6 nmol/L by CPBA and HPLC, respectively. However, mean 25(OH) D concentrations of the two methods became different after excluding undetectable samples (25.1 ± 18.9 vs. 29 ± 14.5 nmol/L, respectively; P = 0.04). Based on predefined Vitamin D deficiency as 25(OH) D < 12.5 nmol/L, CPBA sensitivity and specificity were 44.2% and 60.6%, respectively, compared to HPLC. In receiver operating characteristic curve analysis, the best cut-offs for CPBA was 5.8 nmol/L, which gave 82% sensitivity, but specificity was 17%. Conclusions: Though CPBA may be used as a screening tool, more reliable methods are needed for diagnostic purposes. PMID:26330983

  5. A sensitive enzyme-linked immunosorbent assay amplified by biotin-streptavidin system for detecting non-steroidal anti-inflammatory drug ketoprofen.

    PubMed

    Bu, Dan; Zhuang, Hui S; Yang, Guang X

    2014-01-01

    A sensitive biotin-streptavidin-amplified enzyme-linked immunosorbent assay (BA-ELISA) method was developed for detecting non-steroidal anti-inflammatory drug ketoprofen. Compared with traditional ELISA method, the sensitivity of proposed immunoassay was enhanced by the biotin-streptavidin system. Under the optimal condition, the median inhibitory concentration (IC50) was 0.25 ng mL(-1), with minor cross-reactivity to a number of structural analogs. This developed assay was successfully applied to detect the ketoprofen residues in different fish samples, and good recoveries (72.6-105.5%) were obtained. The results indicated that this immunoassay method could specifically detect trace ketoprofen residues and could be widely used for routine monitoring of food samples.

  6. Clinical performance of a commercial real-time PCR assay for Aspergillus DNA detection in serum samples from high-risk patients: comparison with a galactomannan enzyme immunoassay.

    PubMed

    Pini, P; Bettua, C; Orsi, C F; Venturelli, C; Faglioni, L; Forghieri, F; Bigliardi, S; Luppi, F; Girardis, M; Blasi, E

    2015-01-01

    We investigated the clinical performance of a polymerase chain reaction (PCR)-based commercial platform, the Myconostica MycAssay™ Aspergillus (MAP), for fungal DNA detection in the serum of patients at risk of invasive aspergillosis (IA). Sixty-four hospitalized patients were prospectively enrolled and a total of 71 different episodes were investigated (30 episodes were clinically/microbiologically classified as IA and 41 as control episodes). When MAP was compared to the galactomannan (GM) assay, no significant differences were found in terms of sensitivity (46.7% vs. 50.0%), specificity (97.6% vs. 95.1%), positive predictive value (PPV) (93.3% vs. 88.2%), and negative predictive value (NPV) (71.4% vs. 72.2%). The corresponding areas under the curve (AUC) of the receiver operating characteristic (ROC) curves were also superimposable. Overall, because of the good agreement between the two assays and considering the high specificity and PPV of the MAP, we suggest the use of this PCR-based platform as a second-level examination for the evaluation of clinically undefined cases where culture or GM have provided positive results.

  7. Investigating the quantitative structure-activity relationships for antibody recognition of two immunoassays for polycyclic aromatic hydrocarbons by multiple regression methods.

    PubMed

    Zhang, Yan-Feng; Zhang, Li; Gao, Zhi-Xian; Dai, Shu-Gui

    2012-01-01

    Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous contaminants found in the environment. Immunoassays represent useful analytical methods to complement traditional analytical procedures for PAHs. Cross-reactivity (CR) is a very useful character to evaluate the extent of cross-reaction of a cross-reactant in immunoreactions and immunoassays. The quantitative relationships between the molecular properties and the CR of PAHs were established by stepwise multiple linear regression, principal component regression and partial least square regression, using the data of two commercial enzyme-linked immunosorbent assay (ELISA) kits. The objective is to find the most important molecular properties that affect the CR, and predict the CR by multiple regression methods. The results show that the physicochemical, electronic and topological properties of the PAH molecules have an integrated effect on the CR properties for the two ELISAs, among which molar solubility (S(m)) and valence molecular connectivity index ((3)χ(v)) are the most important factors. The obtained regression equations for Ris(C) kit are all statistically significant (p < 0.005) and show satisfactory ability for predicting CR values, while equations for RaPID kit are all not significant (p > 0.05) and not suitable for predicting. It is probably because that the Ris(C) immunoassay employs a monoclonal antibody, while the RaPID kit is based on polyclonal antibody. Considering the important effect of solubility on the CR values, cross-reaction potential (CRP) is calculated and used as a complement of CR for evaluation of cross-reactions in immunoassays. Only the compounds with both high CR and high CRP can cause intense cross-reactions in immunoassays.

  8. Immunoassays for the measurement of IGF-II, IGFBP-2 and -3, and ICTP as indirect biomarkers of recombinant human growth hormone misuse in sport. Values in selected population of athletes.

    PubMed

    Abellan, Rosario; Ventura, Rosa; Palmi, Ilaria; di Carlo, Simonetta; Bacosi, Antonella; Bellver, Montse; Olive, Ramon; Pascual, Jose Antonio; Pacifici, Roberta; Segura, Jordi; Zuccaro, Piergiorgio; Pichini, Simona

    2008-11-04

    Insulin-like growth factor-II (IGF-II), insulin-like growth factor binding proteins (IGFBPs) -2 and -3 and C-terminal telopeptide of type I collagen (ICTP) have been proposed, among others, as indirect biomarkers of the recombinant human growth hormone misuse in sport. An extended intra- and inter-laboratory validation of commercially available immunoassays for biomarkers detection was performed. ELISA assays for total IGF-II, IGFBP-2 and IGFBP-3 (IGF-II/ELISA1: DSLabs, IGFBP-2/ELISA2: Biosource, and IGFBP-3/ELISA3: BioSource) and an EIA assay for ICTP (ICTP/EIA: Orion Diagnostica) were evaluated. The inter- and intra-laboratory precision values were acceptable for all evaluated assays (maximum imprecision of 30% and 66% were found only for the lowest quality control samples of IGF-II and IGFBP-3). Correct accuracy was obtained for all inter-laboratory immunoassays and for IGFBP-2 intra-laboratory immunoassay. The range of concentrations found in serum samples under investigation was always covered by the calibration curves of the studied immunoassays. However, 11% and 15% of the samples felt below the estimated LOQ for IGF-II and ICTP, respectively, in the zone where lower precision was obtained. Although the majority of evaluated assays showed an overall reliability not always suitable for antidoping control analysis, relatively high concordances between laboratory results were obtained for all assays. Evaluated immunoassays were used to measure serum concentrations of IGF-II, IGFBP-2 and -3 and ICTP in elite athletes of various sport disciplines at different moments of the training season; in recreational athletes at baseline conditions and finally in sedentary individuals. Serum IGF-II was statistically higher both in recreational and elite athletes compared to sedentary individuals. Elite athletes showed lower IGFBP-2 and higher IGFBP-3 concentration with respect to recreational athletes and sedentary people. Among elite athletes, serum IGFBP-3 (synchronized

  9. [Comparison of antibody responses to hepatitis B surface antigen among four recipient groups of hepatitis B vaccines that have been approved in Japan: evaluation using passive hemagglutination assay and chemiluminescent immunoassay].

    PubMed

    Ogata, Norio

    2009-10-01

    In hepatitis B virus (HBV) infection-preventing programs, serum or plasma levels of antibody to hepatitis B surface antigen (anti-HBs) are important to determine whether individuals are protective or not. We compared anti-HBs responses using passive hemagglutination assay (Mycell) and chemiluminescent immunoassay (Architect) among four recipient groups of HB vaccines, Meinyu, HBY, Bimmugen and Heptavax II, that have been approved in Japan. Overall, in a total of 1875 vaccinees Mycell results showed recipient groups of Meinyu and HBY acquired higher anti-HBs levels than those of Bimmugen and Heptavax II. Comparison of anti-HBs responses by both Mycell and Architect in recipient groups of Meinyu (n=150), HBY (n=218), Bimmugen (n=260), and Heptavax II (n=47) demonstrated the order of vaccinees' responses, such as geometric mean titers, ratios of acquiring high antibody levels (Mycell titers over 1024, Architect measurements over 1000 mIU/mL), and ratios of having unsuccessful antibody responses (Mycell titers under 8, Architect measurements under 10 mIU/mL), were somewhat different between the two assays. Comparison of Architect measurements at given Mycell titers revealed Bimmugen-recipients showed significantly lower values than HBY- or Heptavax II-recipients. Around critical protective levels, 5 of 22 Bimmugen-recipients with Mycell titers 16 or 32 showed Architect measurements under 10 mIU/mL, while 8 of 11 Heptavax II-recipients with Mycell titers below 8 demonstrated Architect measurements over 10 mIU/mL. Thus, discrepancies in anti-HBs evaluation between Mycell and Architect seemed to partly depend on administered vaccines. These results indicate anti-HBs concentration should be evaluated carefully so that we could completely prevent HBV infection.

  10. Using an electro-microchip, a nanogold probe, and silver enhancement in an immunoassay.

    PubMed

    Yeh, Chia-Hsien; Huang, Hao-Hsuan; Chang, Tsung-Chain; Lin, Hong-Ping; Lin, Yu-Cheng

    2009-02-15

    This paper presents a novel immunoassay that uses an electro-microchip to detect the immuno-reaction signal, gold nanoparticles (ANPs) as a label of antigen or antibody and as a catalyst for silver precipitation, and the silver enhancement reaction to magnify the detection signal. This study is based on the direct immunoassay (two-layer format) and the sandwich immunoassay (three-layer format). The ANPs were introduced into the electro-microchip by the specific binding of the antibodies-ANPs conjugates and then were coupled with silver enhancement to produce black spots of silver metal. The silver precipitation constructs a "bridge" between two electrodes of the electro-microchip allowing electrons to pass. The variation of impedance can be easily measured with a commercial LCR meter. Various gap sizes (20, 50, 100, and 200 microm) of the electrodes of electro-microchips were designed for the sensitivity study. The experimental data show that a chip with a 20microm gap has the highest sensitivity. There was a significant difference in impedance between the experiment sample and the negative control after 10 min of reaction time. The proposed method requires less time and fewer steps than the conventional enzyme-linked immunosorbent assay (ELISA). In addition, it shows a high detection sensitivity (10 microg/mL of 1st antibody (IgG) immobilized on slides and 1 ng/mL of antigen (protein A)). There is a clear distinction between the signal intensity and the logarithm of the sample concentration. The proposed new immunoassay method has potential applications in proteomics research and clinical diagnosis.

  11. Development of a Nanobody-Based Lateral Flow Immunoassay for Detection of Human Norovirus

    PubMed Central

    Doerflinger, Sylvie Y.; Tabatabai, Julia; Schnitzler, Paul; Farah, Carlo; Rameil, Steffen; Sander, Peter; Koromyslova, Anna

    2016-01-01

    ABSTRACT Human noroviruses are the dominant cause of outbreaks of acute gastroenteritis. These viruses are usually detected by molecular methods, including reverse transcriptase PCR (RT-PCR) and enzyme-linked immunosorbent assay (ELISA). Human noroviruses are genetically and antigenically diverse, with two main genogroups that are further subdivided into over 40 different genotypes. During the past decade, genogroup 2 genotype 4 (GII.4) has dominated in most countries, but recently, viruses belonging to GII.17 have increased in prevalence in a number of countries. A number of commercially available ELISAs and lateral flow immunoassays were found to have lower sensitivities to the GII.17 viruses, indicating that the antibodies used in these methods may not have a high level of cross-reactivity. In this study, we developed a rapid Nanobody-based lateral flow immunoassay (Nano-immunochromatography [Nano-IC]) for the detection of human norovirus in clinical specimens. The Nano-IC assay detected virions from two GII.4 norovirus clusters, which included the current dominant strain and a novel variant strain. The Nano-IC method had a sensitivity of 80% and specificity of 86% for outbreak specimens. Norovirus virus-like particles (VLPs) representing four genotypes (GII.4, GII.10, GII.12, and GII.17) could be detected by this method, demonstrating the potential in clinical screening. However, further modifications to the Nano-IC method are needed in order to improve this sensitivity, which may be achieved by the addition of other broadly reactive Nanobodies to the system. IMPORTANCE We previously identified a Nanobody (termed Nano-85) that bound to a highly conserved region on the norovirus capsid. In this study, the Nanobody was biotinylated and gold conjugated for a lateral flow immunoassay (termed Nano-IC). We showed that the Nano-IC assay was capable of detecting at least four antigenically distinct GII genotypes, including the newly emerging GII.17. In the clinical

  12. Development of a Nanobody-Based Lateral Flow Immunoassay for Detection of Human Norovirus.

    PubMed

    Doerflinger, Sylvie Y; Tabatabai, Julia; Schnitzler, Paul; Farah, Carlo; Rameil, Steffen; Sander, Peter; Koromyslova, Anna; Hansman, Grant S

    2016-01-01

    Human noroviruses are the dominant cause of outbreaks of acute gastroenteritis. These viruses are usually detected by molecular methods, including reverse transcriptase PCR (RT-PCR) and enzyme-linked immunosorbent assay (ELISA). Human noroviruses are genetically and antigenically diverse, with two main genogroups that are further subdivided into over 40 different genotypes. During the past decade, genogroup 2 genotype 4 (GII.4) has dominated in most countries, but recently, viruses belonging to GII.17 have increased in prevalence in a number of countries. A number of commercially available ELISAs and lateral flow immunoassays were found to have lower sensitivities to the GII.17 viruses, indicating that the antibodies used in these methods may not have a high level of cross-reactivity. In this study, we developed a rapid Nanobody-based lateral flow immunoassay (Nano-immunochromatography [Nano-IC]) for the detection of human norovirus in clinical specimens. The Nano-IC assay detected virions from two GII.4 norovirus clusters, which included the current dominant strain and a novel variant strain. The Nano-IC method had a sensitivity of 80% and specificity of 86% for outbreak specimens. Norovirus virus-like particles (VLPs) representing four genotypes (GII.4, GII.10, GII.12, and GII.17) could be detected by this method, demonstrating the potential in clinical screening. However, further modifications to the Nano-IC method are needed in order to improve this sensitivity, which may be achieved by the addition of other broadly reactive Nanobodies to the system. IMPORTANCE We previously identified a Nanobody (termed Nano-85) that bound to a highly conserved region on the norovirus capsid. In this study, the Nanobody was biotinylated and gold conjugated for a lateral flow immunoassay (termed Nano-IC). We showed that the Nano-IC assay was capable of detecting at least four antigenically distinct GII genotypes, including the newly emerging GII.17. In the clinical setting, the

  13. One-step kinetics-based immunoassay for the highly sensitive detection of C-reactive protein in less than 30 min.

    PubMed

    Vashist, Sandeep Kumar; Czilwik, Gregor; van Oordt, Thomas; von Stetten, Felix; Zengerle, Roland; Marion Schneider, E; Luong, John H T

    2014-07-01

    This article reveals a rapid sandwich enzyme-linked immunosorbent assay (ELISA) for the highly sensitive detection of human C-reactive protein (CRP) in less than 30 min. It employs a one-step kinetics-based highly simplified and cost-effective sandwich ELISA procedure with minimal process steps. The procedure involves the formation of a sandwich immune complex on capture anti-human CRP antibody-bound Dynabeads in 15 min, followed by two magnet-assisted washings and one enzymatic reaction. The developed sandwich ELISA detects CRP in the dynamic range of 0.3 to 81 ng ml(-1) with a limit of detection of 0.4 ng ml(-1) and an analytical sensitivity of 0.7 ng ml(-1). It detects CRP spiked in diluted human whole blood and serum with high analytical precision, as confirmed by conventional sandwich ELISA. Moreover, the results of the developed ELISA for the determination of CRP in the ethylenediaminetetraacetic acid plasma samples of patients are in good agreement with those obtained by the conventional ELISA. The developed immunoassay has immense potential for the development of rapid and cost-effective in vitro diagnostic kits.

  14. Evaluation of qualitative and quantitative immunoassays to detect barley contamination in gluten-free beer with confirmation using LC/MS/MS.

    PubMed

    Allred, Laura K; Sealey Voyksner, Jennifer A; Voyksner, Robert D

    2014-01-01

    To meet the need for the detection and quantitation of barley gluten in beer, qualitative screening and quantitative immunoassays based on the monoclonal antigluten antibody 401/21 (Skerritt) were validated in a single laboratory. Sample replicates were tested at each stage of beer production using multiple yeast strains and methods of end-stage protein removal. Quantitation was performed using barley-specific standards based on barley flour extracts. Immunoassay results were confirmed using LC/MS/MS for barley-specific peptides. The LOD for the qualitative screening test was 5 mg/L barley gluten. Recovery for the barley-spiked worts ranged from 81 to 128% in the quantitative ELISA assay; the LOD was <1 mg/L, and the LOQ was 5 mg/L. Both screening and confirmation methods were found to be fit for the purposes of detection of low levels of barley gluten in beer.

  15. Comparison of Focus HerpesSelect® and Kalon™ HSV-2 gG2 ELISA serological assays to detect herpes simplex virus type 2 (HSV-2) antibodies in a South African population

    PubMed Central

    Delany, Sinéad; Jentsch, Ute; Weiss, Helen; Moyes, Jocelyn; Ashley-Morrow, Rhoda; Stevens, Wendy; Mayaud, Philippe

    2010-01-01

    Introduction Sero-epidemiological studies of herpes simplex virus type-2 (HSV-2) infection in Africa remain difficult to interpret owing to the high rate of false-positive results observed when using the new recombinant gG2 HSV-2 ELISA tests. We compared the performance of two widely used gG2 ELISAs to derive an appropriate testing algorithm for use in South Africa. Methods Sera from 210 women attending family planning clinics in Johannesburg were tested using HerpeSelect® and Kalon™ HSV-2 gG2 assays. Sera from 20 discordant pairs, 44 concordant positive and 33 concordant negative samples were further tested by HSV Western Blot (WB). Sensitivity and specificity of each test and of combination algorithms compared to WB were calculated. Results HerpeSelect® had a sensitivity of 98% (95% confidence interval [CI]: 95–100) and specificity of 61% (95%CI: 48–74). Kalon™ was less sensitive (89%, 95%CI: 83–94) but more specific (85%, 95%CI: 61–100). Seroprevalence may have been overestimated by as much as 14% by HerpeSelect®. Specificity was improved by raising the cut-off index for determination of a positive result for HerpeSelect® (to ≥3.5), but not for Kalon™. HIV-1 infection reduced the specificity of HerpeSelect® to 30%. Improved sensitivity and specificity were obtained by a two-test algorithm using HerpeSelect® (≥3.5) as the first test and Kalon™ to resolve equivocal results (sensitivity 92%, 95%CI: 82–98; specificity 91%, 95%CI: 79–98). Conclusion Newer HSV-2 serological tests have low specificity in this South African population with high HIV-1 prevalence. Two-step testing strategies could provide rational testing alternatives to WB. PMID:19837726

  16. An Immunoassay to Evaluate Human/Environmental Exposure to the Antimicrobial Triclocarban

    PubMed Central

    Ahn, Ki Chang; Kasagami, Takeo; Tsai, Hsing-Ju; Schebb, Nils Helge; Ogunyoku, Temitope; Gee, Shirley J.; Young, Thomas M.; Hammock, Bruce D.

    2011-01-01

    A sensitive, competitive indirect enzyme linked immunosorbent assay (ELISA) for the detection of the antimicrobial triclocarban (TCC) was developed. The haptens were synthesized by derivatizing the para position of a phenyl moiety of TCC. The rabbit antisera were screened and the combination of antiserum #1648 and a heterologous competitive hapten containing a piperidine was further characterized. The IC50 and the detection range for TCC in buffer were 0.70 and 0.13–3.60 ng/mL, respectively. The assay was selective for TCC, providing only low cross-reactivity to TCC-related compounds and its major metabolites except for the closely related antimicrobial 3-trifluoromethyl-4,4′-dichlorocarbanilide. A liquid-liquid extraction for sample preparation of human body fluids resulted in an assay that measured low part per billion levels of TCC in small volumes of the samples. The limits of quantification of TCC were 5 ng/mL in blood/serum, and 10 ng/mL in urine, respectively. TCC in human urine was largely the N- or N′-glucuronide. TCC concentrations of biosolids measured by the ELISA were similar to those determined by LC-MS/MS. This immunoassay can be used as a rapid, inexpensive and convenient tool to aid researchers monitoring human/environmental exposure to TCC to better understand the health effects. PMID:22077920

  17. Potentiometric enzyme immunoassay using miniaturized anion-selective electrodes for detection

    PubMed Central

    Szűcs, Júlia; Pretsch, Ernö; Gyurcsányi, Róbert E.

    2010-01-01

    An enzyme-linked immunosorbent assay (ELISA) for prostate specific antigen (PSA) detection in human serum was developed based on the potentiometric detection of 6,8-difluoro-4-methylumbelliferone (DiFMU). The assays were carried out in anti-human PSA capture-antibody modified microtiter plates (150 µl volume). After incubation in the PSA containing serum samples, β-galactosidase-labeled PSA tracer antibody was added. The β-galactosidase label catalyzed the hydrolysis of 6,8-difluoro-4-methylumbelliferyl-β-D-galactopyranoside (DiFMUG) and the resulting DiFMU− anion was detected by potentiometric microelectrodes with anion-exchanger membrane. The selectivity of the anion-exchanger electrode is governed by the lipophilicity of the anions in the sample. Since DiFMU− is much more lipophilic (log P = 1.83) than any of the inorganic anions normally present in the working buffers and occurs in its anionic form at the physiological pH (pKa = 4.19), it was chosen as the species to be detected. The potentiometric ELISA-based method detects PSA in serum with a linear concentration range of 0.1–50 ng/mL. These results confirm the applicability of potentiometric detection in diagnostic PSA assays. Owing to simple methodology and low cost, potentiometric immunoassays seem to offer a feasible alternative to the development of in vitro diagnostic platforms. PMID:20448926

  18. Comparison of a new serum topiramate immunoassay to fluorescence polarization immunoassay.

    PubMed

    Snozek, Christine L H; Rollins, Lisa A; Peterson, Paul W; Langman, Loralie J

    2010-02-01

    Topiramate is a newer anticonvulsant used to treat epilepsy, migraines, bipolar disorder, posttraumatic stress, and other conditions. Serum topiramate concentrations are measured to determine optimal levels, address therapeutic failure or drug-drug interactions, and assess compliance. Two high-throughput assays for serum topiramate measurement were compared: the Seradyn fluorescence polarization immunoassay (FPIA) on an Abbott TDx/FLx instrument and a new immunoassay from ARK Diagnostics performed on an Olympus AU680 automated analyzer. Precision, linearity, limit of quantitation, carryover, spike recovery, and endogenous interferences were found to be acceptable for the ARK assay. These studies were complemented by comparison of 120 patient samples analyzed using both methods. The ARK immunoassay performed comparably to FPIA with minimal difference in serum topiramate concentrations within the therapeutic range (2.0-20 microg/mL). A slight systematic discordance was observed at higher concentrations (greater than 30 microg/mL) with ARK immunoassay results being on average 6% higher than FPIA. Thus, the ARK immunoassay appears to provide acceptable analytical performance and comparability to FPIA; furthermore, the assay is compatible with high-throughput autoanalyzers.

  19. Evaluation of the One-Step ELISA kit for the detection of buprenorphine in urine, blood, and hair specimens.

    PubMed

    Cirimele, V; Etienne, S; Villain, M; Ludes, B; Kintz, P

    2004-07-16

    A solid-phase enzyme immunoassay involving microtiter plates was recently proposed by International Diagnostic Systems corporation (IDS) to screen for buprenorphine in human serum. The performance of the kit led us to investigate its applicability in other biological matrices such as urine or blood, and also hair specimens. Low concentrations of buprenorphine were detected with the ELISA test and confirmed by HPLC/MS (buprenorphine concentrations measured by HPLC/MS: 0.3 ng/mL in urine, 0.2 ng/mL in blood, and 40 pg/mg in hair). The intra-assay precision values were 8.7% at 1 ng/mL of urine (n = 8), 11.5% at 2 ng/mL in serum (n = 8), and 11.5% at 250 pg/mg of hair (n = 8), respectively. The immunoassay had no cross-reactivity with dihydrocodeine, ethylmorphine, 6-monoacetylmorphine, pholcodine, propoxyphene, dextromoramide, dextrometorphan at 1 and 10 mg/L, or codeine, morphine, methadone, and its metabolite EDDP. A 1% cross-reactivity was measured for a norbuprenorphine concentration of 50 ng/mL. Finally, the immunoassay was validated by comparing authentic specimens results with those of a validated HPLC/MS method. From the 136 urine samples tested, 93 were positive (68.4%) after the ELISA screening test (cutoff: 0.5 ng/mL) and confirmed by HPLC/MS (buprenorphine concentrations: 0.3-2036 ng/mL). From the 108 blood or serum samples screened, 27 were positive (25%) after the ELISA test with a cutoff value of 0.5 ng/mL (buprenorphine concentrations: 0.2-13.3 ng/mL). Eighteen hair specimens were positive (72%) after the screening (cutoff: 10 pg/mg) and confirmed by LC/MS (buprenorphine concentrations: 40-360 pg/mg). The ELISA method produced false positive results in less than 21% of the cases, but no false negative results were observed with the immunological test. Four potential adulterants (hypochloride 50 mL/L, sodium nitrite 50 g/L, liquid soap 50 mL/L, and sodium chloride 50 g/L) that were added to 10 positive urine specimens (buprenorphine concentrations in

  20. Defining the smallest analyte concentration an immunoassay can measure.

    PubMed

    Brown, E N; McDermott, T J; Bloch, K J; McCollom, A D

    1996-06-01

    An immunoassay's minimal detectable concentration (MDC), the smallest analyte concentration the assay can reliably measure, is one of its most important properties. Bayes' theorem is used to unify the five current mathematical MDC definitions. The unified definition has significant implications for defining positive results for screening and diagnostic tests, setting criteria for immunoassay quality control and optimal design, reliably measuring biological substances at low concentrations, and, in general, measuring small analyte concentrations with calibrated analytic methods. As an illustration, we apply the unified definition to the microparticle capture enzyme immunoassay for prostate-specific antigen (PSA) developed for the Abbott IMx automated immunoassay system. The MDC of this assay as estimated by our unifying approach is shown to be 4.1-7.1 times greater than currently reported. As a consequence, the ability of the assay to measure reliably small concentrations of PSA to detect early recurrences of prostate cancer is probably overstated.

  1. Photonic crystal enhanced cytokine immunoassay.

    PubMed

    Mathias, Patrick C; Ganesh, Nikhil; Cunningham, Brian T

    2009-01-01

    Photonic crystal surfaces are demonstrated as a means for enhancing the detection sensitivity and resolution for assays that use a fluorescent tag to quantify the concentration of an analyte protein molecule in a liquid test sample. Computer modeling of the spatial distribution of resonantly coupled electromagnetic fields on the photonic crystal surface are used to estimate the magnitude of enhancement factor compared to performing the same fluorescent assay on a plain glass surface, and the photonic crystal structure is fabricated and tested to experimentally verify the performance using a sandwich immunoassay for the protein Tumor Necrosis Factor-alpha (TNF-alpha). The demonstrated photonic crystal fabrication method utilizes a nanoreplica molding technique that allows for large-area inexpensive fabrication of the structure in a format that is compatible with confocal microarray laser scanners. The signal-to-noise ratio for fluorescent spots on the photonic crystal is increased by at least five-fold relative to the glass slide, allowing a TNF-alpha concentration of 1.6 pg/ml to be distinguished from noise on a photonic crystal surface. In addition, the minimum quantitative limit of detection on the photonic crystal surface is one-third the limit on the glass slide - a decrease from 18 pg/ml to 6 pg/ml. The increased performance of the immunoassay allows for more accurate quantitation of physiologically relevant concentrations of TNF-alpha in a protein microarray format that can be expanded to multiple cytokines.

  2. Development of a flow cytometric bead immunoassay and its assessment as a possible aid to potency evaluation of enterotoxaemia vaccines.

    PubMed

    Buys, Angela; Macdonald, Raynard; Crafford, Jannie; Theron, Jacques

    2014-03-10

    Enterotoxaemia, an economically important disease of sheep, goats and calves, is caused by systemic effects of the epsilon toxin produced by the anaerobic bacterium Clostridium perfringens type D. The only practical means of controlling the occurrence of enterotoxaemia is to immunise animals by vaccination. The vaccine is prepared by deriving a toxoid from the bacterial culture filtrate and the potency of the vaccine is tested with the in vivo mouse neutralisation test (MNT). Due to ethical, economic and technical reasons, alternative in vitro assays are needed. In this study an indirect cytometric bead immunoassay (I-CBA) was developed for use in vaccine potency testing and the results were compared with those obtained using an indirect enzyme-linked immunosorbent assay (I-ELISA) and the MNT. Sera were collected from guinea pigs immunised with three different production batches of enterotoxaemia vaccine and the levels of anti-epsilon toxin antibodies were determined. Although the intra- and inter-assay variability was satisfactory, epsilon antitoxin levels determined by both the I-ELISA and indirect cytometric bead immunoassay (I-CBA) tests were higher than those of the MNT assay. In contrast to the MNT, all of the serum samples were identified as having antitoxin levels above the required minimum (not less than 5 U/mL). These results indicate that the respective in vitro tests in their current formats are not yet suitable alternatives to the in vivo MNT. The growing demand for a more humane, cost-effective and efficient method for testing the potency of enterotoxaemia vaccines, however, provides a strong impetus for further optimisation and standardisation of the I-CBA assay but further analytical research is required.

  3. 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.

  4. Validity and reliability of a novel salivary immunoassay for individual profiling in applied sports science.

    PubMed

    Coad, Sam; Mclellan, Christopher; Whitehouse, Timothy; Gray, Bon

    2015-01-01

    The aim of this study was to examine the validity and reliability of a novel immunoassay, developed to assess salivary Immunoglobulin A (s-IgA). Validity and reliability of the Individual Profiling Lateral Flow Device (IPRO LFD) for s-IgA concentrations ([s-IgA]) was assessed in males (n = 12) and females (n =13) who were involved in recreational activities. Reliability of the IPRO LFD method was assessed by comparing [s-IgA] of two saliva samples collected concurrently, while validity was assessed by comparing with an enzyme-linked immunosorbent assay (ELISA) method. The IPRO LFD had a strong positive correlation (r = 0.93, p < 0.001), with no difference in [s-IgA] compared with the ELISA. The IPRO LFD was considered reliable (ICC r = 0.89, p < 0.001 and CV = 9.40 %) for measures of [s-IgA]. We concluded that the IPRO LFD method may be a substitute to the ELISA method for measurements of [s-IgA].

  5. Production of monoclonal antibodies for sandwich immunoassay detection of Pacific ciguatoxins.

    PubMed

    Tsumuraya, Takeshi; Fujii, Ikuo; Hirama, Masahiro

    2010-10-01

    Ciguatoxins are the major causative toxins of ciguatera seafood poisoning. Limited availability of ciguatoxins has hampered the development of a reliable and specific immunoassay for detecting these toxins in contaminated fish. Monoclonal antibodies (mAbs) specific against both ends of Pacific ciguatoxins CTX3C and 51-hydroxyCTX3C were prepared by immunization of mice with the protein conjugates of rationally designed synthetic haptens in place of the natural toxin. Haptenic groups that possess a surface area larger than 400 A(2) were required to produce mAbs that can bind strongly to CTX3C or 51-hydroxyCTX3C. A direct sandwich enzyme-linked immunosorbent assay (ELISA) using these mAbs was established to detect CTX3C and 51-hydroxyCTX3C at the ppb level with no cross-reactivity against the other marine toxins, including brevetoxin A, brevetoxin B, okadaic acid, or maitotoxin.

  6. Field detection capability of immunochemical assays during criminal investigations involving the use of TNT.

    PubMed

    Romolo, Francesco Saverio; Ferri, Elida; Mirasoli, Mara; D'Elia, Marcello; Ripani, Luigi; Peluso, Giuseppe; Risoluti, Roberta; Maiolini, Elisabetta; Girotti, Stefano

    2015-01-01

    The capability to collect timely information about the substances employed on-site at a crime scene is of fundamental importance during scientific investigations in crimes involving the use of explosives. TNT (2,4,6-trinitrotoluene) is one of the most employed explosives in the 20th century. Despite the growing use of improvised explosives, criminal use and access to TNT is not expected to decrease. Immunoassays are simple and selective analytical tests able to detect molecules and their immunoreactions can occur in portable formats for use on-site. This work demonstrates the application of three immunochemical assays capable of detecting TNT to typical forensic samples from experimental tests: an indirect competitive ELISA with chemiluminescent detection (CL-ELISA), a colorimetric lateral flow immunoassay (LFIA) based on colloidal gold nanoparticles label, and a chemiluminescent-LFIA (CL-LFIA). Under optimised working conditions, the LOD of the colorimetric LFIA and CL-LFIA were 1 μg mL(-1) and 0.05 μg mL(-1), respectively. The total analysis time for LFIAs was 15 min. ELISA proved to be a very effective laboratory approach, showing very good sensitivity (LOD of 0.4 ng mL(-1)) and good reproducibility (CV value about 7%). Samples tested included various materials involved in controlled explosions of improvised explosive devices (IEDs), as well as hand swabs collected after TNT handling tests. In the first group of tests, targets covered with six different materials (metal, plastic, cardboard, carpet fabric, wood and adhesive tape) were fixed on top of wooden poles (180 cm high). Samples of soil from the explosion area and different materials covering the targets were collected after each explosion and analysed. In the second group of tests, hand swabs were collected with and without hand washing after volunteers simulated the manipulation of small charges of TNT. The small amount of solution required for each assay allows for several analyses. Results of

  7. 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

  8. 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.

  9. Immobilized carboxymethylated dextran coatings for enhanced ELISA.

    PubMed

    Liberelle, Benoît; Merzouki, Abderrazzak; De Crescenzo, Gregory

    2013-03-29

    We here report the development of a new generation of enzyme-linked immunosorbent assay (ELISA) that takes advantage of a low-fouling carboxymethylated dextran (CMD) layer chemically grafted on ELISA wells. In our approach, the overnight capture antibody adsorption step found in classical ELISA was replaced by a covalent attachment step to the CMD layer completed in 15 min. As a model, the potential of our approach was highlighted using commercially available anti-human epidermal growth factor (EGF) antibodies to quantify EGF present in various samples. Of interest, the grafted CMD layer was found to be as efficient as the commonly used bovine serum albumine (BSA) to reduce non-specific adsorption, thus eliminating the need of a time-consuming BSA blocking step normally required in classical ELISA. Our results demonstrated similar specificity, affinity, and intra- and inter-assay variations regardless of the diluent used in the assay (BSA-based diluent or protein-free buffer solution) when compared to standard ELISA. Finally, accuracy and precision of the CMD-based ELISA were verified by a spike and recovery test. Dilutions of recombinant human EGF in serum from healthy human volunteers showed almost-perfect linearity and mean recovery rates ranging between 90 and 110%.

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

    PubMed

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

    2016-09-01

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

  11. Rapid, automated, parallel quantitative immunoassays using highly integrated microfluidics and AlphaLISA.

    PubMed

    Yu, Zeta Tak For; Guan, Huijiao; Cheung, Mei Ki; McHugh, Walker M; Cornell, Timothy T; Shanley, Thomas P; Kurabayashi, Katsuo; Fu, Jianping

    2015-06-15

    Immunoassays represent one of the most popular analytical methods for detection and quantification of biomolecules. However, conventional immunoassays such as ELISA and flow cytometry, even though providing high sensitivity and specificity and multiplexing capability, can be labor-intensive and prone to human error, making them unsuitable for standardized clinical diagnoses. Using a commercialized no-wash, homogeneous immunoassay technology ('AlphaLISA') in conjunction with integrated microfluidics, herein we developed a microfluidic immunoassay chip capable of rapid, automated, parallel immunoassays of microliter quantities of samples. Operation of the microfluidic immunoassay chip entailed rapid mixing and conjugation of AlphaLISA components with target analytes before quantitative imaging for analyte detections in up to eight samples simultaneously. Aspects such as fluid handling and operation, surface passivation, imaging uniformity, and detection sensitivity of the microfluidic immunoassay chip using AlphaLISA were investigated. The microfluidic immunoassay chip could detect one target analyte simultaneously for up to eight samples in 45 min with a limit of detection down to 10 pg mL(-1). The microfluidic immunoassay chip was further utilized for functional immunophenotyping to examine cytokine secretion from human immune cells stimulated ex vivo. Together, the microfluidic immunoassay chip provides a promising high-throughput, high-content platform for rapid, automated, parallel quantitative immunosensing applications.

  12. Rapid, automated, parallel quantitative immunoassays using highly integrated microfluidics and AlphaLISA

    NASA Astrophysics Data System (ADS)

    TakYu, Zeta; Guan, Huijiao; Ki Cheung, Mei; McHugh, Walker M.; Cornell, Timothy T.; Shanley, Thomas P.; Kurabayashi, Katsuo; Fu, Jianping

    2015-06-01

    Immunoassays represent one of the most popular analytical methods for detection and quantification of biomolecules. However, conventional immunoassays such as ELISA and flow cytometry, even though providing high sensitivity and specificity and multiplexing capability, can be labor-intensive and prone to human error, making them unsuitable for standardized clinical diagnoses. Using a commercialized no-wash, homogeneous immunoassay technology (‘AlphaLISA’) in conjunction with integrated microfluidics, herein we developed a microfluidic immunoassay chip capable of rapid, automated, parallel immunoassays of microliter quantities of samples. Operation of the microfluidic immunoassay chip entailed rapid mixing and conjugation of AlphaLISA components with target analytes before quantitative imaging for analyte detections in up to eight samples simultaneously. Aspects such as fluid handling and operation, surface passivation, imaging uniformity, and detection sensitivity of the microfluidic immunoassay chip using AlphaLISA were investigated. The microfluidic immunoassay chip could detect one target analyte simultaneously for up to eight samples in 45 min with a limit of detection down to 10 pg mL-1. The microfluidic immunoassay chip was further utilized for functional immunophenotyping to examine cytokine secretion from human immune cells stimulated ex vivo. Together, the microfluidic immunoassay chip provides a promising high-throughput, high-content platform for rapid, automated, parallel quantitative immunosensing applications.

  13. Rapid, automated, parallel quantitative immunoassays using highly integrated microfluidics and AlphaLISA

    PubMed Central

    Tak For Yu, Zeta; Guan, Huijiao; Ki Cheung, Mei; McHugh, Walker M.; Cornell, Timothy T.; Shanley, Thomas P.; Kurabayashi, Katsuo; Fu, Jianping

    2015-01-01

    Immunoassays represent one of the most popular analytical methods for detection and quantification of biomolecules. However, conventional immunoassays such as ELISA and flow cytometry, even though providing high sensitivity and specificity and multiplexing capability, can be labor-intensive and prone to human error, making them unsuitable for standardized clinical diagnoses. Using a commercialized no-wash, homogeneous immunoassay technology (‘AlphaLISA’) in conjunction with integrated microfluidics, herein we developed a microfluidic immunoassay chip capable of rapid, automated, parallel immunoassays of microliter quantities of samples. Operation of the microfluidic immunoassay chip entailed rapid mixing and conjugation of AlphaLISA components with target analytes before quantitative imaging for analyte detections in up to eight samples simultaneously. Aspects such as fluid handling and operation, surface passivation, imaging uniformity, and detection sensitivity of the microfluidic immunoassay chip using AlphaLISA were investigated. The microfluidic immunoassay chip could detect one target analyte simultaneously for up to eight samples in 45 min with a limit of detection down to 10 pg mL−1. The microfluidic immunoassay chip was further utilized for functional immunophenotyping to examine cytokine secretion from human immune cells stimulated ex vivo. Together, the microfluidic immunoassay chip provides a promising high-throughput, high-content platform for rapid, automated, parallel quantitative immunosensing applications. PMID:26074253

  14. A Rapid Lateral Flow Immunoassay for the Detection of Tyrosine Phosphatase-Like Protein IA-2 Autoantibodies in Human Serum

    PubMed Central

    Kikkas, Ingrid; Mallone, Roberto; Larger, Etienne; Volland, Hervé; Morel, Nathalie

    2014-01-01

    Type 1 diabetes (T1D) results from the destruction of pancreatic insulin-producing beta cells and is strongly associated with the presence of islet autoantibodies. Autoantibodies to tyrosine phosphatase-like protein IA-2 (IA-2As) are considered to be highly predictive markers of T1D. We developed a novel lateral flow immunoassay (LFIA) based on a bridging format for the rapid detection of IA-2As in human serum samples. In this assay, one site of the IA-2As is bound to HA-tagged-IA-2, which is subsequently captured on the anti-HA-Tag antibody-coated test line on the strip. The other site of the IA-2As is bound to biotinylated IA-2, allowing the complex to be visualized using colloidal gold nanoparticle-conjugated streptavidin. For this study, 35 serum samples from T1D patients and 44 control sera from non-diabetic individuals were analyzed with our novel assay and the results were correlated with two IA-2A ELISAs. Among the 35 serum samples from T1D patients, the IA-2A LFIA, the in-house IA-2A ELISA and the commercial IA-2A ELISA identified as positive 21, 29 and 30 IA-2A-positive sera, respectively. The major advantages of the IA-2A LFIA are its rapidity and simplicity. PMID:25047039

  15. A rapid lateral flow immunoassay for the detection of tyrosine phosphatase-like protein IA-2 autoantibodies in human serum.

    PubMed

    Kikkas, Ingrid; Mallone, Roberto; Larger, Etienne; Volland, Hervé; Morel, Nathalie

    2014-01-01

    Type 1 diabetes (T1D) results from the destruction of pancreatic insulin-producing beta cells and is strongly associated with the presence of islet autoantibodies. Autoantibodies to tyrosine phosphatase-like protein IA-2 (IA-2As) are considered to be highly predictive markers of T1D. We developed a novel lateral flow immunoassay (LFIA) based on a bridging format for the rapid detection of IA-2As in human serum samples. In this assay, one site of the IA-2As is bound to HA-tagged-IA-2, which is subsequently captured on the anti-HA-Tag antibody-coated test line on the strip. The other site of the IA-2As is bound to biotinylated IA-2, allowing the complex to be visualized using colloidal gold nanoparticle-conjugated streptavidin. For this study, 35 serum samples from T1D patients and 44 control sera from non-diabetic individuals were analyzed with our novel assay and the results were correlated with two IA-2A ELISAs. Among the 35 serum samples from T1D patients, the IA-2A LFIA, the in-house IA-2A ELISA and the commercial IA-2A ELISA identified as positive 21, 29 and 30 IA-2A-positive sera, respectively. The major advantages of the IA-2A LFIA are its rapidity and simplicity.

  16. 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.

  17. Comparative evaluation of enzyme-linked immunoassay and reference methods for the detection of shellfish hydrophilic toxins in several presentations of seafood.

    PubMed

    Garet, Elina; González-Fernández, Africa; Lago, Jorge; Vieites, Juan M; Cabado, Ana G

    2010-02-10

    A comparative study was conducted to determine the feasibility of enzyme-linked immunosorbent assays (ELISAs) for the detection of amnesic shellfish poisoning (ASP) and paralytic shellfish poisoning (PSP) toxins in nine naturally contaminated species in fresh, frozen, boiled and canned fish and shellfish. PSP and ASP were analyzed in 138 shellfish samples (mussels, clams, barnacles, razor shells, scallops and cockles) and anchovies by mouse bioassay (MBA) and high performance liquid chromatography with ultraviolet detection (HPLC-UV), respectively. Results were compared with toxin concentrations obtained using two commercial competitive ELISAs, saxitoxin and ASP kits. Immunoassays were able to quantify toxins in different matrices showing excellent Pearson's correlation coefficients (r = 0.974 for saxitoxin ELISA and r = 0.973 for ASP ELISA) and to detect PSP and ASP with a lower limit of detection (LOD), namely, 50 microg saxitoxin equivalent/kg shellfish meat for PSP and 60 microg/kg domoic acid in shellfish flesh for ASP, than the reference methods (350 microg saxitoxin equivalent/kg shellfish meat and 1.6 mg/kg domoic acid in shellfish flesh, respectively). These results suggest that the ELISA method could be used as screening systems in a variety of species without matrix interference.

  18. 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.

  19. Comparison of Antibody Responses to Human Papillomavirus Vaccination as Measured by Three Assays

    PubMed Central

    Robbins, Hilary A.; Kemp, Troy J.; Porras, Carolina; Rodriguez, Ana Cecilia; Schiffman, Mark; Wacholder, Sholom; Gonzalez, Paula; Schiller, John; Lowy, Douglas; Poncelet, Sylviane; Esser, Mark; Matys, Katie; Hildesheim, Allan; Pinto, Ligia A.; Herrero, Rolando; Safaeian, Mahboobeh

    2014-01-01

    Background: Different assays, including the competitive Luminex immunoassay (cLIA), secreted alkaline phosphatase neutralization assay (SEAP-NA), and virus-like particle-based ELISA, are commonly used to measure antibody responses after human papillomavirus (HPV) vaccination. Direct assay comparisons aid interpretation of immunogenicity data evaluated by different assays. Methods: We compared cLIA to SEAP-NA and ELISA among 51 HPV16/18-vaccinated women enrolled in the Costa Rica Vaccine Trial. We tested replicate serum samples collected at months 0, 1, and 12 by HPV16/18 cLIA, SEAP-NA, and ELISA. For a subset (N = 10), we further tested month 6, 24 and 36 samples. We calculated seroprevalence estimates and Spearman rank correlation coefficients comparing cLIA to SEAP-NA and ELISA. Results: After one vaccine dose, seroprevalence by SEAP-NA and ELISA was 100% (both HPV16 and HPV18), and by cLIA was 96% (95% CI 87–100%) for HPV16 and 71% (95% CI 56–83%) for HPV18. Seroprevalence was 100% by all assays after three doses. Correlation between assays was high after one vaccine dose [cLIA/SEAP-NA ρ = 0.91 (HPV16) and ρ = 0.86 (HPV18); cLIA/ELISA ρ = 0.84 (HPV16) and ρ = 0.74 (HPV18); all p < 0.001] and remained high through month 36. Ratios of mean antibody levels to seropositivity cutoffs at month 36 were lower for cLIA than for SEAP-NA or ELISA, particularly for HPV18 (HPV18 ratio for cLIA 1.9, SEAP-NA 3.5, ELISA 3.4). Conclusion: Though correlation between cLIA and SEAP-NA/ELISA is high and stable after vaccination, the assays differ in scale and sensitivity, with notable differences after one vaccine dose and for HPV18. Our results demonstrate that comparisons of antibody responses to HPV vaccination measured by different assays are approximate, and must consider biological and technical differences between assays. PMID:24455487

  20. 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

  1. Fifth-Generation Digital Immunoassay for Prostate Specific Antigen Using Single Molecule Arrays

    PubMed Central

    Wilson, D.H.; Hanlon, D.W.; Provuncher, G.K.; Chang, L.; Song, L.; Patel, P.P.; Ferrell, E.P.; Lepor, H; Partin, A.W.; Chan, D.W.; Sokoll, L.J.; Cheli, C.D.; Thiel, R.P.; Fournier, D.R.; Duffy, D.C.

    2012-01-01

    BACKGROUND Measurement of prostate specific antigen (PSA) in prostate cancer patients following radical prostatectomy (RP) has been limited by the sensitivity of available assays. Because radical prostatectomy removes the tissue responsible for PSA production, post-surgical PSA is typically undetectable with current assay methods. However, evidence suggests that more sensitive determination of PSA status following RP could improve assessment of patient prognosis, response to treatment, and better target secondary therapy to those who may benefit most. We report the development and validation of an investigational digital immunoassay with two logs greater sensitivity than today’s ultrasensitive third-generation PSA assays. METHODS Reagents were developed for a paramagnetic bead-based ELISA for use with high-density arrays of femtoliter-volume wells. Anti-PSA capture-beads with immunocomplexes and associated enzyme labels were singulated within the wells of the arrays and interrogated for the presence of enzymatic product. Analytical performance of the assay was characterized, its accuracy compared with a commercially available test, and longitudinal serum samples from a pilot study of 33 RP patients were analyzed. RESULTS The assay exhibited a functional sensitivity (20% inter-assay CV) of less than 0.00005 ng/mL (0.05 pg/mL), total imprecision of less than 10% from 1 to 50 pg/mL, and excellent agreement with the comparator method. All RP samples were well within the assay measurement capability. PSA values following surgery were examined in the context of five-year biochemical cancer recurrence. CONCLUSION The assay demonstrated a robust two-log advance in measurement sensitivity relative to current ultrasensitive assays, and the analytical performance for accurate assessment of PSA status after RP. PMID:21998342

  2. Development of Prototype Filovirus Recombinant Antigen Immunoassays

    PubMed Central

    Boisen, Matt L.; Oottamasathien, Darin; Jones, Abigail B.; Millett, Molly M.; Nelson, Diana S.; Bornholdt, Zachary A.; Fusco, Marnie L.; Abelson, Dafna M.; Oda, Shun-ichiro; Hartnett, Jessica N.; Rowland, Megan M.; Heinrich, Megan L.; Akdag, Marjan; Goba, Augustine; Momoh, Mambu; Fullah, Mohammed; Baimba, Francis; Gbakie, Michael; Safa, Sadiki; Fonnie, Richard; Kanneh, Lansana; Cross, Robert W.; Geisbert, Joan B.; Geisbert, Thomas W.; Kulakosky, Peter C.; Grant, Donald S.; Shaffer, Jeffery G.; Schieffelin, John S.; Wilson, Russell B.; Saphire, Erica Ollmann; Branco, Luis M.; Garry, Robert F.; Khan, S. Humarr; Pitts, Kelly R.

    2015-01-01

    Background. Throughout the 2014–2015 Ebola outbreak in West Africa, major gaps were exposed in the availability of validated rapid diagnostic platforms, protective vaccines, and effective therapeutic agents. These gaps potentiated the development of prototype rapid lateral flow immunodiagnostic (LFI) assays that are true point-of-contact platforms, for the detection of active Ebola infections in small blood samples. Methods. Recombinant Ebola and Marburg virus matrix VP40 and glycoprotein (GP) antigens were used to derive a panel of monoclonal and polyclonal antibodies. Antibodies were tested using a multivariate approach to identify antibody-antigen combinations suitable for enzyme-linked immunosorbent assay (ELISA) and LFI assay development. Results. Polyclonal antibodies generated in goats were superior reagents for capture and detection of recombinant VP40 in test sample matrices. These antibodies were optimized for use in antigen-capture ELISA and LFI assay platforms. Prototype immunoglobulin M (IgM)/immunoglobulin G (IgG) ELISAs were similarly developed that specifically detect Ebola virus–specific antibodies in the serum of experimentally infected nonhuman primates and in blood samples obtained from patients with Ebola from Sierra Leone. Conclusions. The prototype recombinant Ebola LFI assays developed in these studies have sensitivities that are useful for clinical diagnosis of acute ebolavirus infections. The antigen-capture and IgM/IgG ELISAs provide additional confirmatory assay platforms for detecting VP40 and other ebolavirus-specific immunoglobulins. PMID:26232440

  3. Development and validation of a multiplex immunoassay for the simultaneous determination of serum antibodies to Bordetella pertussis, diphtheria and tetanus.

    PubMed

    van Gageldonk, Pieter G M; van Schaijk, Frank G; van der Klis, Fiona R; Berbers, Guy A M

    2008-06-01

    To increase testing of vaccine induced humoral immunity in immune surveillance studies and vaccine trials, a rapid and simple microsphere-based multiplex assay (pentaplex) was developed for the quantitation of IgG serum antibodies directed against the Bordetella pertussis antigens: Pertussis Toxin (Ptx), Filamentous hemagglutinin (FHA), Pertactin (Prn) and to Diphtheria toxin and Tetanus toxin. All individual antigens were covalently linked to carboxylated microspheres. The method was validated with different serum panels (n=60-78 samples). With the Multiplex Immunoassay (MIA) no evidence for bead interference between monoplex and pentaplex was found. The specificity of the method was shown by a heterologous inhibition of <16% and homologous inhibition of >92%. The pentaplex MIA appeared sensitive with lower limits of quantitation (LLOQ) well below those for ELISA (enzyme-linked immuno-sorbant assay). Assay reproducibility was high with intra-assay variability less than 10% and inter-assay variability below 14%. The reproducibility of the bead conjugation was good and beads could be stored up to at least 6 months without quality reduction. Importantly, the correlation of the pentaplex MIA with the individual ELISAs was excellent, R>0.98 for the Pertussis antigens and R=0.95 for Diphtheria and R=0.98 for Tetanus. Serum IgG antibodies to B. pertussis, Diphtheria and Tetanus can be measured easily, specific and reproducible using the pentaplex MIA. The pentaplex MIA shares features of the ELISA with the additional advantages of high sample throughput and small sample volumes and antigen required.

  4. A new approach to ELISA-based anti-glycolipid antibody evaluation of highly adhesive serum samples

    PubMed Central

    Usuki, Seigo; O’Brien, Dawn; Rivner, Michael H.; Yu, Robert K.

    2014-01-01

    The enzyme-linked immunosorbent assay (ELISA) is a standard immunoassay used in measuring antibody reactivity (expressed as titers) for glycosphingolipids (GSLs) such as gangliosides and sulfoglycolipids in the sera of patients with Guillain-Barré syndrome (GBS), variants of GBS, and chronic inflammatory demyelinating polyneuropathy (CIDP). In the present study, anti-GSL antibodies were evaluated using a new formula of affinity parametric complex (APC), calculated from limiting-dilution serum assay data, followed by affinity parametric complex criterion (APCC). Using assay results based on APCC, we analyzed serum samples categorized into acute inflammatory demyelinating polyneuropathy (AIDP), acute motor-sensory axonal neuropathy (AMSAN), CIDP, CIDP with Myasthenia Gravis (MG), and Amyotrophic Lateral Sclerosis (ALS). We were able to determine the affinity strength of antibodies otherwise hidden in the non-specific background activity in highly adhesive serum samples. The thin-layer chromatography (TLC)-immuno-overlay method assured us that this new method is an accurate and reliable way for evaluating anti-GSL antibodies using ELISA serum sample data. PMID:24861939

  5. HPV16 Seropositivity and Subsequent HPV16 Infection Risk in a Naturally Infected Population: Comparison of Serological Assays

    PubMed Central

    Lin, Shih-Wen; Ghosh, Arpita; Porras, Carolina; Markt, Sarah C.; Rodriguez, Ana Cecilia; Schiffman, Mark; Wacholder, Sholom; Kemp, Troy J.; Pinto, Ligia A.; Gonzalez, Paula; Wentzensen, Nicolas; Esser, Mark T.; Matys, Katie; Meuree, Ariane; Quint, Wim; van Doorn, Leen-Jan; Herrero, Rolando; Hildesheim, Allan; Safaeian, Mahboobeh

    2013-01-01

    Background Several serological assays have been developed to detect antibodies elicited against infections with oncogenic human papillomavirus (HPV) type 16. The association between antibody levels measured by various assays and subsequent HPV infection risk may differ. We compared HPV16-specific antibody levels previously measured by a virus-like particle (VLP)-based direct enzyme-linked immunoassay (ELISA) with levels measured by additional assays and evaluated the protection against HPV16 infection conferred at different levels of the assays. Methodology/Principal Findings Replicate enrollment serum aliquots from 388 unvaccinated women in the control arm of the Costa Rica HPV vaccine trial were measured for HPV16 seropositivity using three serological assays: a VLP-based direct ELISA; a VLP-based competitive Luminex immunoassay (cLIA); and a secreted alkaline phosphatase protein neutralization assay (SEAP-NA). We assessed the association of assay seropositivity and risk of subsequent HPV16 infection over four years of follow-up by calculating sampling-adjusted odds ratios (OR) and HPV16 seropositivity based on standard cutoff from the cLIA was significantly associated with protection from subsequent HPV16 infection (OR = 0.48, CI = 0.27–0.86, compared with seronegatives). Compared with seronegatives, the highest seropositive tertile antibody levels from the direct ELISA (OR = 0.53, CI = 0.28–0.90) as well as the SEAP-NA (OR = 0.20, CI = 0.06, 0.64) were also significantly associated with protection from HPV16 infection. Conclusions/Significance Enrollment HPV16 seropositivity by any of the three serological assays evaluated was associated with protection from subsequent infection, although cutoffs for immune protection were different. We defined the assays and seropositivity levels after natural infection that better measure and translate to protective immunity. PMID:23301022

  6. Variants of ELISA in plant virus diagnosis.

    PubMed

    Koenig, R; Paul, H L

    1982-10-01

    Variations of enzyme-linked immunosorbent assay (ELISA) were compared with respect to their ability to detect and to differentiate serologically related plant viruses. The broadest range of serologically related viruses was detected by an indirect ELISA on unprecoated plates. Coating the plates with F(ab')2 fragments led to narrowing of the specificity in heterologous reactions of tymo-, tombus- and tobamoviruses in indirect ELISA. With Andean potato latent virus (APLV) heterologous reactions were weaker on plates precoated with F(ab')2 fragments than on those precoated with intact antibodies. Even on plates precoated with F(ab')2 fragments the indirect ELISA detected a broader range of serologically related viruses than the direct double antibody sandwich method. Heterologous reactions in indirect ELISA procedures on plates precoated with either intact antibodies or F(ab')2 fragments were always weaker than homologous reactions independent of the concentration of coating reactants and detecting antibodies. Attempts to differentiate closely related strains of APLV or radish mosaic virus by direct ELISA using F(ab')2 fragments either for coating the plates or after labelling with alkaline phosphatase for detecting the trapped antigens failed. Under suitable conditions, the additional working step usually necessary for indirect ELISA could be avoided by using a short procedure which at low concentrations of detecting antibodies was more sensitive than the conventional procedure.

  7. Quantitation of estrogen receptor in seventy-five specimens of breast cancer: comparison between an immunoassay (Abbott ER-EIA monoclonal) and a (3H)estradiol binding assay based on isoelectric focusing in polyacrylamide gel

    SciTech Connect

    Pousette, A.; Gustafsson, S.A.; Thoernblad, A.M.N.; Nordgren, A.; Saellstroem, J.Li.; Lindgren, A.; Sundelin, P.; Gustafsson, J.A.

    1986-08-01

    Quantitation of estrogen receptor has been performed in cytosol prepared from 75 specimens of breast cancer tissue from patients who had not received hormonal therapy. The study was performed in order to compare an immunoassay (Abbott Laboratories, North Chicago, IL) with our currently used method for estrogen receptor analysis based on isoelectric focusing of (/sup 3/H)estradiol-receptor complex in polyacrylamide gels. Using linear regression analysis, a regression coefficient (slope) of 1.30 and a correlation coefficient of 0.75 were calculated. The differences in results between the two methods are probably partly explained by the fact that the ligand-based method only measures unoccupied receptor, whereas the immunoassay detects the total amount of receptor, resulting in generally slightly higher concentrations with the latter method. However, in five of 75 specimens the ligand-based method gave a considerably higher concentration of estrogen receptor. This was most probably explained by partial proteolysis resulting in the formation of receptor fragment(s), which was undetectable with the immunoassay but detectable with the ligand-based method. These observations underline the importance of careful handling of specimens during the whole immunoassay procedure.

  8. 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.

  9. 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.

  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. Antibodies for immunoassays.

    PubMed

    Newman, D J

    2000-01-01

    What is an immunoassay without an antibody? Clearly the name provides the answer to this question; without antibodies there would be no immunoassays. An immunoassay is an analytical technique, quantitative or qualitative, that relies absolutely on the specificity and affinity of the interaction between epitope and paratope for generation of a detectable response. The actual detection of this binding interaction can be via one of literally hundreds of different signal transduction mechanisms, e.g., fluorimetry, chemiluminescence, agglutination (turbidimetry or nephelometry) enzyme reactions, and so forth (1 -4), but these are simply transducing systems for the primary binding interaction. Antibodies thus provide us with an exquisitely sensitive and specific analytical technology for detecting and quantifying epitopic structures. These structures include amino-acid derivatives, e.g., thyroid hormones, peptides, e.g., vasopressin, proteins, e.g., cytokines, as well as carbohydrate structures, e.g., CA-125. Immunoassay technology has developed to such an extent that it is probably the most versatile analytical tool available able to identify and quantify epitopic structures across the milli- to zeptomolar concentration ranges (2).

  12. One-sample measurement in laser nephelometric immunoassay using magnetic nanoparticles

    NASA Astrophysics Data System (ADS)

    Yang, S. Y.; Wu, R. M.; Jian, Z. F.; Horng, H. E.; Hong, Chin-Yih; Yang, H. C.

    2006-12-01

    In contrast to the type of two-sample measurement used in conventional nephelometric immunoassay, a methodology to achieve a one-sample nephelometric immunoassay is developed in this work. Magnetic nanoparticles instead of latex particles are used as scattering centers. The experimental results show that the sensitivity of assaying avidin or c-reactive protein increased about three times under the action of the magnetic field in nephelometric immunoassay using magnetic nanoparticles.

  13. 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.

  14. Surface modification by electron irradiation for improved immunoassay

    NASA Astrophysics Data System (ADS)

    Safrany, Agnes; Deelder, André

    1999-08-01

    Polystyrene microtitration (ELISA) plates modified by electron beam irradiation were used for a monoclonal antibody based sandwich immunoassay for quantitation of circulating anodic antigen levels in Schistosoma-infected individuals. The plates irradiated with 15 kGy showed 2-4-fold lower detection level compared to untreated plates, and a 10-fold lower antibody coating concentration than usually used was still detectable. These results were reproducible and the modified surfaces were stable even after 2 years when kept at room temperature.

  15. Assays of Serum Testosterone.

    PubMed

    Herati, Amin S; Cengiz, Cenk; Lamb, Dolores J

    2016-05-01

    The diagnosis of male hypogonadism depends on an assessment of the clinical signs and symptoms of hypogonadism and serum testosterone level. Current clinical laboratory testosterone assay platforms include immunoassays and mass spectrometry. Despite significant advances to improve the accuracy and precision of the currently available assays, limited comparability exists between assays at the lower and upper extremes of the testosterone range. Because of this lack of comparability, there is no current gold standard assay for the assessment of total testosterone levels.

  16. Development of an innovative immunoassay for CP4EPSPS and Cry1AB genetically modified protein detection and quantification.

    PubMed

    Ermolli, M; Prospero, A; Balla, B; Querci, M; Mazzeo, A; Van Den Eede, G

    2006-09-01

    An innovative immunoassay, called enzyme-linked immunoabsorbant assay (ELISA) Reverse, based on a new conformation of the solid phase, was developed. The solid support was expressly designed to be immersed directly in liquid samples to detect the presence of protein targets. Its application is proposed in those cases where a large number of samples have to be screened simultaneously or when the simultaneous detection of different proteins is required. As a first application, a quantitative immunoassay for Cry1AB protein in genetically modified maize was optimized. The method was tested using genetically modified organism concentrations from 0.1 to 2.0%. The limit of detection and limit of quantitation of the method were determined as 0.0056 and 0.0168 (expressed as the percentage of genetically modified organisms content), respectively. A qualitative multiplex assay to assess the presence of two genetically modified proteins simultaneously was also established for the case of the Cry1AB and the CP4EPSPS (5-enolpyruvylshikimate-3-phosphate synthase) present in genetically modified maize and soy, respectively.

  17. Description of a Nanobody-based Competitive Immunoassay to Detect Tsetse Fly Exposure

    PubMed Central

    Caljon, Guy; Hussain, Shahid; Vermeiren, Lieve; Van Den Abbeele, Jan

    2015-01-01

    Background Tsetse flies are the main vectors of human and animal African trypanosomes. The Tsal proteins in tsetse fly saliva were previously identified as suitable biomarkers of bite exposure. A new competitive assay was conceived based on nanobody (Nb) technology to ameliorate the detection of anti-Tsal antibodies in mammalian hosts. Methodology/Principal Findings A camelid-derived Nb library was generated against the Glossina morsitans morsitans sialome and exploited to select Tsal specific Nbs. One of the three identified Nb families (family III, TsalNb-05 and TsalNb-11) was found suitable for anti-Tsal antibody detection in a competitive ELISA format. The competitive ELISA was able to detect exposure to a broad range of tsetse species (G. morsitans morsitans, G. pallidipes, G. palpalis gambiensis and G. fuscipes) and did not cross-react with the other hematophagous insects (Stomoxys calcitrans and Tabanus yao). Using a collection of plasmas from tsetse-exposed pigs, the new test characteristics were compared with those of the previously described G. m. moristans and rTsal1 indirect ELISAs, revealing equally good specificities (> 95%) and positive predictive values (> 98%) but higher negative predictive values and hence increased sensitivity (> 95%) and accuracy (> 95%). Conclusion/Significance We have developed a highly accurate Nb-based competitive immunoassay to detect specific anti-Tsal antibodies induced by various tsetse fly species in a range of hosts. We propose that this competitive assay provides a simple serological indicator of tsetse fly presence without the requirement of test adaptation to the vertebrate host species. In addition, the use of monoclonal Nbs for antibody detection is innovative and could be applied to other tsetse fly salivary biomarkers in order to achieve a multi-target immunoprofiling of hosts. In addition, this approach could be broadened to other pathogenic organisms for which accurate serological diagnosis remains a

  18. Using the MMPB technique to confirm microcystin concentrations in water measured by ELISA and HPLC (UV, MS, MS/MS).

    PubMed

    Foss, Amanda J; Aubel, Mark T

    2015-09-15

    Microcystins have been detected in raw and finished drinking water using a variety of techniques, including assays (immunoassay, phosphatase inhibition) and HPLC (UV, MS/(MS)). The principal challenge to microcystin analysis is accounting for the over 150 variants that have been described. A confirmatory individual variant HPLC analysis is prone to under-reporting total microcystins due to method specificity. One method that allows for total microcystin quantitation is the MMPB technique. In this study, water samples with native microcystins were oxidized to cleave the Adda moiety, common to all microcystin variants. LC-MS/MS analysis was conducted on the subsequent MMPB (3-methoxy-2-methyl-4-phenylbutyric acid) molecule and calibrated using a certified reference standard (microcystin-LR) and 4-phenylbutyric acid. Total microcystin concentrations from MMPB were compared to Adda ELISA and individual variant analyses (LC-UV, LC-MS/(MS)). Variants of microcystin, including [DAsp(3)]MC-RR, [Dha(7)]MC-RR, MC-RR, MC-YR, MC-LR, [DAsp(3)]MC-LR, [Dha(7)]MC-LR, MC-WR, MC-LA, and MC-LY were detected and quantified in samples. The individual variant analyses did not account for total microcystins present in samples, as indicated by ELISA and MMPB data. Results demonstrated the MMPB technique is a simple and valuable approach to confirm ELISA data when analyzing microcystins, with method detection limits of 0.05 μg L(-1) for total microcystins.

  19. Rapid sandwich ELISA-based in vitro diagnostic procedure for the highly-sensitive detection of human fetuin A.

    PubMed

    Vashist, Sandeep Kumar; Schneider, E Marion; Luong, John H T

    2015-05-15

    A rapid sandwich enzyme-linked immunosorbent assay (ELISA)-based in vitro diagnostic (IVD) procedure has been developed for human fetuin A (HFA), an important disease biomarker for inflammatory diseases as well as malignancies. In this simplified and cost-effective procedure, the EDC-activated anti-HFA antibody (Ab) was admixed with 1% (v/v) 3-aminopropyltriethoxysilane (APTES) in 1:1 (v/v) and dispensed in a KOH-pretreated microtiter plate (MTP). APTES formed a stable complex with the capture antibody that was in turn covalently bonded on the KOH-treated surface in 30 min. The resulting immunoassay (IA) format detects HFA with a dynamic range of 0.1-243 ng mL(-1), and a limit of detection (LOD) and analytical sensitivity of 0.3 ng mL(-1) and 1.0 ng mL(-1), respectively. For the determination of HFA spiked in diluted human whole blood and serum, and HFA in ethylenediaminetetraacetic acid (EDTA)-plasma of patients, the obtained analytical precision is similar to that of the conventional sandwich ELISA. The anti-HFA Ab-bound MTPs, stored at 4 °C in 0.1M PBS, pH 7.4, retained its biological activity for 8 weeks, thereby demonstrating excellent storage stability. This generic sandwich ELISA procedure can be extended for rapid, simplified and cost-effective detection of other disease biomarkers.

  20. Development of a barcode-style lateral flow immunoassay for the rapid semi-quantification of gliadin in foods.

    PubMed

    Yin, Hsin-Yi; Chu, Pei-Tzu; Tsai, Wen-Che; Wen, Hsiao-Wei

    2016-02-01

    In this work, a barcode-style lateral flow immunoassay is developed using two cut-off values (10 and 50 mg kg(-1) gliadin) to provide a semi-quantification for identifying "gluten-free" and "very low gluten" foods, based on the international Codex Alimentarius Standard. This developed assay exhibits favorable specificity in differentiating wheat from seven commonly used grains, with only a slight cross-reaction with barely. The intra-assay and inter-assay CV values of this assay were 1.5-1.7% and 2.5-4.5%, respectively, revealing high reproducibility. In the analysis of 48 food samples, the results of this assay closely agreed with those obtained using AOAC-approved ELISA or strip kits, as the Cohen's kappa coefficients for both comparisons exceeded 0.8. Thus, this developed assay can be used to quickly estimate the gliadin content in foods in order to protect people with wheat allergy or celiac disease from the accidental ingestion of gliadin.

  1. Indirect competitive assays on DVD for direct multiplex detection of drugs of abuse in oral fluids.

    PubMed

    Zhang, Lingling; Li, Xiaochun; Li, Yunchao; Shi, Xiaoli; Yu, Hua-Zhong

    2015-02-03

    On-site oral fluid testing for drugs of abuse has become prominent in order to take immediate administrative action in an enforcement process. Herein, we report a DVD technology-based indirect competitive immunoassay platform for the quantitative detection of drugs of abuse. A microfluidic approach was adapted to prepare multiplex immunoassays on a standard DVD-R, an unmodified multimode DVD/Blu-Ray drive to read signal, and a free disc-quality analysis software program to process the data. The DVD assay platform was successfully demonstrated for the simultaneous, quantitative detection of drug candidates (morphine and cocaine) in oral fluids with high selectivity. The detection limit achieved was as low as 1.0 ppb for morphine and 5.0 ppb for cocaine, comparable with that of standard mass spectrometry and ELISA methods.

  2. Quality assurance in immunoassay performance--comparison of different enzyme immunoassays for the determination of caffeine in consumer products.

    PubMed

    Grandke, Julia; Oberleitner, Lidia; Resch-Genger, Ute; Garbe, Leif-Alexander; Schneider, Rudolf J

    2013-02-01

    Enzyme immunoassays with optical detection are amongst the most widely used bioanalytical tools. We defined seven parameters for the quality assessment of immunoassays that were addressed in a systematic study of direct and indirect immunoassays, using the enzymes horseradish peroxidase (HRP) and alkaline phosphatase (AP), the chromogenic substrates 3,3',5,5'-tetramethylbenzidine (TMB) and para-nitrophenyl phosphate, and the fluorescent substrates 3-(4-hydroxyphenyl)propionic acid and 4-methylumbelliferyl phosphate. The same monoclonal antibody against caffeine was used throughout the study. The four quality parameters regarding the standard curve were the test midpoint (sensitivity), the measurement range, the relative dynamic range of the signal, and the goodness of fit of the adjusted four-parameter logistic function. All HRP immunoassays showed a higher sensitivity compared to the AP assays. On the basis of all four criteria, it was established that the direct assay format is superior to the indirect format, the immunoassay using HRP TMB fulfilling all requirements best. In a second step, caffeine concentrations in 24 beverage and cosmetics samples were determined and three more quality parameters were assessed with this application. The direct HRP TMB assay showed one of the best intra- and inter-plate precisions and the best accuracy, defined by the correlation of results with those from the chosen reference method liquid chromatography tandem mass spectrometry (LC-MS/MS). Considering all criteria, HRP TMB seems to be the enzyme substrate system of choice preferably used in the direct assay format.

  3. Diagnosing Human Anisakiasis: Recombinant Ani s 1 and Ani s 7 Allergens versus the UniCAP 100 Fluorescence Enzyme Immunoassay

    PubMed Central

    Anadón, A. M.; Rodríguez, E.; Gárate, M. T.; Cuéllar, C.; Romarís, F.; Chivato, T.; Rodero, M.; González-Díaz, H.; Ubeira, F. M.

    2010-01-01

    Commercially available serological methods for serodiagnosis of human anisakiasis either are poorly specific or do not include some of the most relevant Anisakis allergens. The use of selected recombinant allergens may improve serodiagnosis. To compare the diagnostic and clinical values of enzyme-linked immunosorbent assay (ELISA) methods based on Ani s 1 and Ani s 7 recombinant allergens and of the UniCAP 100 fluorescence enzyme immunoassay (CAP FEIA) system, we tested sera from 495 allergic and 25 non-food-related allergic patients. The decay in specific IgE antibodies in serum was also investigated in 15 positive patients over a period of 6 to 38 months. Considering sera that tested positive by either Ani s 1 or Ani s 7 ELISA, the CAP FEIA classified 25% of sera as falsely positive, mainly in the group of patients with the lowest levels of anti-Anisakis IgE antibodies, and 1.28% of positive sera as falsely negative. Considering allergens individually, the overall sensitivities of Ani s 7 ELISA and Ani s 1 ELISA were 94% and 61%, respectively. The results also showed that anti-Anisakis IgE antibodies can be detected in serum for longer with Ani s 1 ELISA than with Ani s 7 ELISA and CAP FEIA (P < 0.01). Our findings suggest that ELISA methods with Ani s 7 and Ani s 1 allergens as targets of IgE antibodies are currently the best option for serodiagnosis of human anisakiasis, combining specificity and sensitivity. The different persistence of anti-Ani s 1 and anti-Ani s 7 antibodies in serum may help clinicians to distinguish between recent and old Anisakis infections. PMID:20107002

  4. Research highlights: digital assays on chip.

    PubMed

    Kim, Donghyuk; Wei, Qingshan; Kong, Janay Elise; Ozcan, Aydogan; Di Carlo, Dino

    2015-01-07

    The ability to break up a volume of fluid into smaller pieces that are confined or separated to prevent molecular communication/transport is a key capability intrinsic to microfluidic systems. This capability has been used to develop or implement digital versions of traditional molecular analysis assays, including digital PCR and digital immunoassays/ELISA. In these digital versions, the concentration of the target analyte is in a range such that, when sampled into smaller fluid volumes, either a single molecule or no molecule may be present. Subsequent amplification is sensitive enough to obtain a digital readout of the presence of these target molecules. Advantages of such approaches that are claimed include quantification without calibration and robustness to variations in reaction conditions or times because the digital readout is less sensitive to absolute signal intensity levels. Weaknesses of digital approaches include a lower dynamic range of concentrations over which the assay is sensitive, which depends on the total volume that can be analyzed. We highlight recent efforts to expand the dynamic range of digital assays based on exploiting reaction/diffusion phenomena. A side-by-side study that evaluates the strengths of digital assays reveals that the majority of these claims are supported, with specific caveats. Finally, we highlight approaches to apply digital assays to analyze new types of reactions, including the active transport of protons across membranes by ATPases at the single protein level - perhaps opening up new biophysical understanding and screening opportunities, similar to widely deployed single-molecule ion channel analysis.

  5. Development and validation of a monoclonal based immunoassay for the measurement of fungal alpha-amylase: focus on peak exposures.

    PubMed

    Elms, J; Denniss, S; Smith, M; Evans, P G; Wiley, K; Griffin, P; Curran, A D

    2001-03-01

    The inhalation of flour dust has been implicated in the induction of sensitisation and elicitation of respiratory symptoms, such as asthma in bakers. In addition to the cereal allergens present in wheat flour, enzymes in flour improvers, in particular fungal alpha-amylase, are now known to be a significant cause of respiratory allergy in the baking industry.A monoclonal antibody based enzyme-linked immunoassay (ELISA) was developed using two monoclonal antibodies that recognised two distinct epitopes of the fungal alpha-amylase enzyme. The ELISA had an inter-assay variation of 12.0% at 1360 pg/ml and 12.8% at 564 pg/ml and intra-assay variation of 4.9% at 1340 pg/ml and 6.1% at 504 pg/ml. The assay had a sensitivity of 200 pg/ml. Competitive inhibition assays confirmed that the monoclonal antibodies had no cross reactivity with other enzymes used in the baking industry and could distinguish added fungal alpha-amylase from cereal amylase. We assessed the levels of exposure to dust, total protein and fungal alpha-amylase in four UK bakeries ranging in size and technical capabilities. Within the bakeries we surveyed, workers were exposed to variable levels of inhalable dust (0.8-39.8 mg/m3), total protein (0-5.7 mg/m3) and fungal alpha-amylase (0-29.8 ng/m3). Consecutive 15 min personal samples taken over a 1 h period demonstrated that the ELISA could measure fungal alpha-amylase exposure in such a 15 min period. Short term peak exposures to fungal alpha-amylase could be identified which may contribute to the sensitisation in individuals who appear to have low exposure levels if measured over a full shift period.

  6. NGF and proNGF Reciprocal Interference in Immunoassays: Open Questions, Criticalities, and Ways Forward

    PubMed Central

    Malerba, Francesca; Paoletti, Francesca; Cattaneo, Antonino

    2016-01-01

    The homeostasis between mature neurotrophin NGF and its precursor proNGF is thought to be crucial in physiology and in pathological states. Therefore, the measurement of the relative amounts of NGF and proNGF could serve as a footprint for the identification of disease states, for diagnostic purposes. Since NGF is part of proNGF, their selective identification with anti-NGF antibodies is not straightforward. Currently, many immunoassays for NGF measurement are available, while the proNGF assays are few and not validated by published information. The question arises, as to whether the commercially available assays are able to distinguish between the two forms. Also, since in biological samples the two forms coexist, are the measurements of one species affected by the presence of the other? We describe experiments addressing these questions. For the first time, NGF and proNGF were measured together and tested in different immunoassays. Unexpectedly, NGF and proNGF were found to reciprocally interfere with the experimental outcome. The interference also calls into question the widely used NGF ELISA methods, applied to biological samples where NGF and proNGF coexist. Therefore, an immunoassay, able to distinguish between the two forms is needed. We propose possible ways forward, toward the development of a selective assay. In particular, the use of the well validated anti-NGF αD11 antibody in an alphaLISA assay with optimized incubation times would be a solution to avoid the interference in the measurement of a mixed sample containing NGF and proNGF. Furthermore, we explored the possibility of measuring proNGF in a biological sample. But the available commercial kit for the detection of proNGF does not allow the measurement of proNGF in mouse brain tissues. Therefore, we validated an SPR approach for the measurement of proNGF in a biological sample. Our experiments help in understanding the technical limits in the measurement of the NGF/proNGF ratio in biological

  7. A rapid sandwich immunoassay for human fetuin A using agarose-3-aminopropyltriethoxysilane modified microtiter plate.

    PubMed

    Vashist, Sandeep Kumar; Schneider, E Marion; Luong, John H T

    2015-07-09

    A rapid sandwich immunoassay (IA) with enhanced signal response for human fetuin A (HFA) was developed by modifying the surface of a KOH-treated polystyrene microtiter plate (MTP) with agarose and 3-aminopropyltriethoxysilane (APTES). The agarose-APTES complex binds covalently to the hydroxyl moiety of the MTP plate to serve as a binding platform for bioconjugation of EDC-activated anti-HFA antibody (Ab) via carbodiimide coupling. The one-step kinetics-based sandwich enzyme-linked immunosorbent assay (ELISA) enabled the detection of HFA in 30 min with a limit of detection (LOD) and a linear range of 0.02 ng mL(-1) and 1-243 ng mL(-1), respectively. It detected HFA spiked in diluted human whole blood and serum, and HFA in ethylenediaminetetraacetic acid (EDTA)-plasma of patients with high precision similar to that of conventional ELISA. The anti-HFA Ab-bound agarose-functionalized MTPs retained their functional activity after 6 weeks of storage in 0.1 M PBS, pH 7.4 at 4 °C.

  8. European sero-epidemiology network: standardisation of the results of diphtheria antitoxin assays.

    PubMed

    von Hunolstein, C; Aggerbeck, H; Andrews, N; Berbers, G; Fievet-Groyne, F; Maple, P A; Olander, R M; Raux, M; Tischer, A

    2000-08-01

    A European Sero-Epidemiological Network (ESEN) was established with the aim to co-ordinate and harmonise serological surveillance of immunity to communicable diseases in Europe. In this study the inter-laboratory standardisation of diphtheria toxin antibody measurements is reported. A standard panel of 162 sera was tested by the participating laboratories using an in vitro assay of their choice: VERO cell toxin neutralisation assay (NT), double-antigen delayed time-resolved fluorescence immuno-assay (DA-DELFIA), double-antigen enzyme-linked immunosorbent assay (DAE), toxin binding inhibition test (ToBI) and an indirect enzyme-linked immunosorbent assay (ELISA). The results were standardised using regression against the NT. The variations due to inter-laboratory and inter-assay variation, which would otherwise make it difficult directly to compare the main serum bank results by the different laboratories and the various assays were successfully minimised by the standardisation. The regression equations obtained will be used to transform the respective local results of testing the main serum bank into the reference test unitages. This study also gave the opportunity to compare the various assays within and between laboratories. This demonstrated a very high correlation between DA-DELFIA, DAE, ToBI and the NT. The ELISA showed a good correlation, too, however sera below some 0.1 IU/ml were overestimated.

  9. Peptide-Recombinant VP6 Protein Based Enzyme Immunoassay for the Detection of Group A Rotaviruses in Multiple Host Species

    PubMed Central

    Sircar, Subhankar; Saurabh, Sharad; Gulati, Baldev R.; Singh, Neeraj; Singh, Arvind Kumar; Joshi, Vinay G.; Banyai, Krisztian; Dhama, Kuldeep

    2016-01-01

    We developed a novel enzyme immunoassay for the detection of group A rotavirus (RVA) antigen in fecal samples of multiple host species. The assay is based on the detection of conserved VP6 protein using anti-recombinant VP6 antibodies as capture antibodies and anti-multiple antigenic peptide (identified and constructed from highly immunodominant epitopes within VP6 protein) antibodies as detector antibodies. The clinical utility of the assay was evaluated using a panel of 914 diarrhoeic fecal samples from four different host species (bovine, porcine, poultry and human) collected from diverse geographical locations in India. Using VP6- based reverse transcription-polymerase chain reaction (RT-PCR) as the gold standard, we found that the diagnostic sensitivity (DSn) and specificity (DSp) of the new assay was high [bovine (DSn = 94.2% & DSp = 100%); porcine (DSn = 94.6% & DSp = 93.3%); poultry (DSn = 74.2% & DSp = 97.7%) and human (DSn = 82.1% & DSp = 98.7%)]. The concordance with RT-PCR was also high [weighted kappa (k) = 0.831–0.956 at 95% CI = 0.711–1.0] as compared to RNA-polyacrylamide gel electrophoresis (RNA-PAGE). The performance characteristics of the new immunoassay were comparable to those of the two commercially available ELISA kits. Our results suggest that this peptide-recombinant protein based assay may serve as a preliminary assay for epidemiological surveillance of RVA antigen and for evaluation of vaccine effectiveness especially in low and middle income settings. PMID:27391106

  10. Optimization of a lateral flow immunoassay for the ultrasensitive detection of aflatoxin M1 in milk.

    PubMed

    Anfossi, Laura; Baggiani, Claudio; Giovannoli, Cristina; Biagioli, Flavia; D'Arco, Gilda; Giraudi, Gianfranco

    2013-04-15

    A high sensitive immunoassay-based lateral flow device for semi-quantitatively determine aflatoxin M1 (AFM1) in milk was developed. Investigation and optimization of the competitor design and of the gold-labelling strategy allowed the attainment of the ultra-sensitive assessment of AFM1 contamination at nanograms per litre level (LOD 20 ng L(-1), IC50 99 ng L(-1)), as requested by European regulations. A one order of magnitude detectability enhancement in comparison to previously reported gold colloid immunochromatographic assays for this toxin was obtained. Direct detection of the target toxin in milk could be obtained by acquiring images of the strips and correlating intensities of the coloured lines with analyte concentrations. The one-step assay can be completed in 17 min, including a very simple and rapid sample preparation, which allowed the application of the assay to milk samples which differ in fat and protein contents. Although imprecise (mean RSD about 30%), the method proved to be accurate and sensitive enough to allow the correct attribution of sample as compliant or non-compliant according to EU legislation in force. Agreeing results to those of a reference ELISA were obtained on 40 milk samples by matrix-matched calibration in pasteurized milk.

  11. Unconventional application of conventional enzymatic substrate: first fluorogenic immunoassay based on enzymatic formation of quantum dots.

    PubMed

    Malashikhina, Natalia; Garai-Ibabe, Gaizka; Pavlov, Valeri

    2013-07-16

    In this study, a simple fluorogenic immunoassay based on in situ formation of semiconductor quantum dots (QDs) is described. We discovered that alkaline phosphatase (ALP), the enzyme broadly used in enzyme-linked immuno-sorbent assay (ELISA), is able to trigger formation of fluorescent CdS QDs. ALP-catalyzed hydrolysis of p-nitrophenyl phosphate (pNPP) leads to the formation of p-nitrophenol and inorganic phosphate. The latter stabilizes CdS QDs produced in situ through interaction of Cd(2+) with S(2-) ions. So, the specific interaction of analyte (antibody) with ALP-labeled antibody can be detected through formation of CdS QDs, monitored by recording emission spectra at λex = 290 nm. The fluorescence intensity showed to be dependent on the concentration of target antibody. This method allowed us to detect as low as 0.4 ng mL(-1) of analyte antibody with a linear range up to 10 ng mL(-1). The sensitivity of this novel assay showed to be 1 order of magnitude better than that of the standard method based on colorimetric p-nitrophenyl phosphate assay.

  12. ELISA in the multiplex era: potentials and pitfalls.

    PubMed

    Tighe, Patrick J; Ryder, Richard R; Todd, Ian; Fairclough, Lucy C

    2015-04-01

    Multiplex immunoassays confer several advantages over widely adopted singleplex immunoassays including increased efficiency at a reduced expense, greater output per sample volume ratios and higher throughput predicating more resolute, detailed diagnostics and facilitating personalised medicine. Nonetheless, to date, relatively few protein multiplex immunoassays have been validated for in vitro diagnostics in clinical/point-of-care settings. This review article will outline the challenges, which must be ameliorated prior to the widespread integration of multiplex immunoassays in clinical settings: (i) biomarker validation; (ii) standardisation of immunoassay design and quality control (calibration and quantification); (iii) availability, stability, specificity and cross-reactivity of reagents; (iv) assay automation and the use of validated algorithms for transformation of raw data into diagnostic results. A compendium of multiplex immunoassays applicable to in vitro diagnostics and a summary of the diagnostic products currently available commercially are included, along with an analysis of the relative states of development for each format (namely planar slide based, suspension and planar/microtitre plate based) with respect to the aforementioned issues.

  13. A fast and sensitive immunoassay of avian influenza virus based on label-free quantum dot probe and lateral flow test strip.

    PubMed

    Li, Xuepu; Lu, Donglian; Sheng, Zonghai; Chen, Kun; Guo, Xuebo; Jin, Meilin; Han, Heyou

    2012-10-15

    A novel fluorescence immunoassay method for fast and ultrasensitive detection of avian influenza virus (AIV) was developed. The immunoassay method which integrated lateral flow test strip technique with fluorescence immunoassay used the label-free and high luminescent quantum dots (QDs) as signal output. By the sandwich immunoreaction performed on lateral flow test strip, the gold nanoparticle (NP) labels were captured in the test zone and further dissolved to release a large number of gold ions as a signal transduction bridge that was detected by the QDs-based fluorescence quenching method. Under the optimal conditions, the relative fluorescence intensity of QDs was linear over the range of 0.27-12 ng mL(-1) AIV, and the limit of detection was estimated to be 0.09 ng mL(-1) which was 100-fold greater than enzyme-linked immunosorbent assay (ELISA). The sensitive and specific response was also coupled with high reproducibility in the proposed method. A series of six parallel measurements produced reproducible fluorescent signals with a relative standard deviation of 4.7%. The proposed method can be used to directly detect clinical sample without any pretreatment, and showed high efficiency (90.0%), sensitivity (100.0%) and specificity (88.2%) compared with virus isolation (gold method). The new method shows great promise for rapid, sensitive, and quantitative detection of AIV in-field or point-of-care diagnosis.

  14. Updates in immunoassays: virology.

    PubMed

    Josko, Deborah

    2012-01-01

    Virus identification is a challenge to the clinical microbiologist since growing viruses in traditional cell culture is labor intensive, time consuming, and subject to contamination. The advent of rapid and automated immunoassays has eliminated this problem by generating positive results in minutes to hours. For example, testing for infectious mononucleosis can yield a positive result in 3-8 minutes as seen with the Beckman Coulter, Inc. ICON Mono test or in 5-15 minutes with the MONO Mononucleosis Rapid Test Device marketed by ACON Laboratories, Inc. Fully automated immunoassay analyzers provide fast, accurate, sensitive results that aid in a prompt and accurate diagnosis for the patient. Turnaround times are shortened, allowing for timely medical intervention and treatment. The priority in any hospital or medical facility is to treat the patient as quickly and appropriately as possible. By using immunoassays, clinical laboratory professionals are able to report out correct results in a timely manner, ensuring overall positive patient outcomes and improved quality of healthcare.

  15. Multi-Laboratory Validation of Estrone (E1) ELISA Methods

    EPA Science Inventory

    This project is a round-robin evaluation of commercially available Enzyme-Linked Immunosorbent Assay (ELISA) technology to quantitatively or qualitatively measure the hormone estrone (E1) in combined animal feeding operation (CAFO) receiving streams. ELISA is meant to be a simpl...

  16. Implementation of microfluidic sandwich ELISA for superior detection of plant pathogens.

    PubMed

    Thaitrong, Numrin; Charlermroj, Ratthaphol; Himananto, Orawan; Seepiban, Channarong; Karoonuthaisiri, Nitsara

    2013-01-01

    Rapid and economical screening of plant pathogens is a high-priority need in the seed industry. Crop quality control and disease surveillance demand early and accurate detection in addition to robustness, scalability, and cost efficiency typically required for selective breeding and certification programs. Compared to conventional bench-top detection techniques routinely employed, a microfluidic-based approach offers unique benefits to address these needs simultaneously. To our knowledge, this work reports the first attempt to perform microfluidic sandwich ELISA for Acidovorax citrulli (Ac), watermelon silver mottle virus (WSMoV), and melon yellow spot virus (MYSV) screening. The immunoassay occurs on the surface of a reaction chamber represented by a microfluidic channel. The capillary force within the microchannel draws a reagent into the reaction chamber as well as facilitates assay incubation. Because the underlying pad automatically absorbs excess fluid, the only operation required is sequential loading of buffers/reagents. Buffer selection, antibody concentrations, and sample loading scheme were optimized for each pathogen. Assay optimization reveals that the 20-folds lower sample volume demanded by the microchannel structure outweighs the 2- to 4-folds higher antibody concentrations required, resulting in overall 5-10 folds of reagent savings. In addition to cutting the assay time by more than 50%, the new platform offers 65% cost savings from less reagent consumption and labor cost. Our study also shows 12.5-, 2-, and 4-fold improvement in assay sensitivity for Ac, WSMoV, and MYSV, respectively. Practical feasibility is demonstrated using 19 real plant samples. Given a standard 96-well plate format, the developed assay is compatible with commercial fluorescent plate readers and readily amendable to robotic liquid handling systems for completely hand-free assay automation.

  17. Fluidic Force Discrimination Assays: A New Technology for Tetrodotoxin Detection

    PubMed Central

    Yakes, Betsy Jean; Etheridge, Stacey M.; Mulvaney, Shawn P.; Tamanaha, Cy R.

    2010-01-01

    Tetrodotoxin (TTX) is a low molecular weight (~319 Da) neurotoxin found in a number of animal species, including pufferfish. Protection from toxin tainted food stuffs requires rapid, sensitive, and specific diagnostic tests. An emerging technique for the detection of both proteins and nucleic acids is Fluidic Force Discrimination (FFD) assays. This simple and rapid method typically uses a sandwich immunoassay format labeled with micrometer-diameter beads and has the novel capability of removing nonspecifically attached beads under controlled, fluidic conditions. This technique allows for near real-time, multiplexed analysis at levels of detection that exceed many of the conventional transduction methods (e.g., ELISAs). In addition, the large linear dynamic range afforded by FFD should decrease the need to perform multiple sample dilutions, a common challenge for food testing. By applying FFD assays to an inhibition immunoassay platform specific for TTX and transduction via low magnification microscopy, levels of detection of ~15 ng/mL and linear dynamic ranges of 4 to 5 orders of magnitude were achieved. The results from these studies on the first small molecule FFD assay, along with the impact to detection of seafood toxins, will be discussed in this manuscript. PMID:20411115

  18. A novel enzyme immunoassay based on potentiometric measurement of molecular adsorption events by an extended-gate field-effect transistor sensor.

    PubMed

    Kamahori, Masao; Ishige, Yu; Shimoda, Maki

    2007-06-15

    We developed a novel enzyme immunoassay based on a potentiometric measurement of molecular adsorption events by using an extended-gate field-effect transistor (FET) sensor. The adsorbing rate of a thiol compound on a gold surface was found to depend on the concentration of the compound. To construct an electrochemical enzyme immunoassay system by using the sensor, the enzyme chemistry of acetylcholinesterase (AChE) to generate a thiol compound was used and combined with the enzyme-linked immunosorbent assays (ELISA). After the AChE-catalyzed reaction, the amount of the antigen was obtained by detecting the adsorbing rate of the generated thiol compound on the gold electrode using the FET sensor. The measurement stability was also found to improve when a high frequency voltage of 10 kHz or more was superimposed to the reference electrode. The signal corresponding to a range between 1 and 250 pg/mL of Interleukin 1 beta was obtained by the FET sensor when a voltage of 1 MHz was superimposed onto the reference electrode. The FET sensor based ELISA used in this measurement technique can successfully detect Interleukin 1 beta at concentrations as low as 1 pg/mL.

  19. Rapid Wuchereria bancrofti-specific antigen Wb123-based IgG4 immunoassays as tools for surveillance following mass drug administration programs on lymphatic filariasis.

    PubMed

    Steel, Cathy; Golden, Allison; Kubofcik, Joseph; LaRue, Nicole; de Los Santos, Tala; Domingo, Gonzalo J; Nutman, Thomas B

    2013-08-01

    The Global Programme to Eliminate Lymphatic Filariasis has an urgent need for rapid assays to detect ongoing transmission of lymphatic filariasis (LF) following multiple rounds of mass drug administration (MDA). Current WHO guidelines support using the antigen card immunochromatographic test (ICT), which detects active filarial infection but does not detect early exposure to LF. Recent studies found that antibody-based assays better serve this function. In the present study, two tests, a rapid IgG4 enzyme-linked immunosorbent assay (ELISA) and a lateral-flow strip immunoassay, were developed based on the highly sensitive and specific Wuchereria bancrofti antigen Wb123. A comparison of W. bancrofti-infected and -uninfected patients (with or without other helminth infections) demonstrated that both tests had high sensitivities and specificities (93 and 97% [ELISA] and 92 and 96% [strips], respectively). When the W. bancrofti-uninfected group was separated into those with other filarial/helminth infections (i.e., onchocerciasis, loiasis, and strongyloidiasis) and those who were parasite uninfected, the specificities of the assays varied between 91 and 100%. In addition, the geometric mean response by ELISA of W. bancrofti-infected patients was significantly higher than the response of those without W. bancrofti infection (P < 0.0001). Furthermore, the Wb123 ELISA and the lateral-flow strips had high positive and negative predictive values, giving valuable information on the size of survey population needed to be reasonably certain whether or not transmission is ongoing. These highly sensitive and specific IgG4 tests to the W. bancrofti Wb123 protein give every indication that they will serve as useful tools for post-MDA monitoring.

  20. Analysis of High-Throughput ELISA Microarray Data

    SciTech Connect

    White, Amanda M.; Daly, Don S.; Zangar, Richard C.

    2011-02-23

    Our research group develops analytical methods and software for the high-throughput analysis of quantitative enzyme-linked immunosorbent assay (ELISA) microarrays. ELISA microarrays differ from DNA microarrays in several fundamental aspects and most algorithms for analysis of DNA microarray data are not applicable to ELISA microarrays. In this review, we provide an overview of the steps involved in ELISA microarray data analysis and how the statistically sound algorithms we have developed provide an integrated software suite to address the needs of each data-processing step. The algorithms discussed are available in a set of open-source software tools (http://www.pnl.gov/statistics/ProMAT).

  1. Microspot-based ELISA in microfluidics: chemiluminescence and colorimetry detection using integrated thin-film hydrogenated amorphous silicon photodiodes.

    PubMed

    Novo, Pedro; Prazeres, Duarte Miguel França; Chu, Virginia; Conde, João Pedro

    2011-12-07

    Microfluidic technology has the potential to decrease the time of analysis and the quantity of sample and reactants required in immunoassays, together with the potential of achieving high sensitivity, multiplexing, and portability. A lab-on-a-chip system was developed and optimized using optical and fluorescence microscopy. Primary antibodies are adsorbed onto the walls of a PDMS-based microchannel via microspotting. This probe antibody is then recognised using secondary FITC or HRP labelled antibodies responsible for providing fluorescence or chemiluminescent and colorimetric signals, respectively. The system incorporated a micron-sized thin-film hydrogenated amorphous silicon photodiode microfabricated on a glass substrate. The primary antibody spots in the PDMS-based microfluidic were precisely aligned with the photodiodes for the direct detection of the antibody-antigen molecular recognition reactions using chemiluminescence and colorimetry. The immunoassay takes ~30 min from assay to the integrated detection. The conditions for probe antibody microspotting and for the flow-through ELISA analysis in the microfluidic format with integrated detection were defined using antibody solutions with concentrations in the nM-μM range. Sequential colorimetric or chemiluminescence detection of specific antibody-antigen molecular recognition was quantitatively detected using the photodiode. Primary antibody surface densities down to 0.182 pmol cm(-2) were detected. Multiplex detection using different microspotted primary antibodies was demonstrated.

  2. 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.

  3. Anti-HCV immunoassays based on a multiepitope antigen and fluorescent lanthanide chelate reporters.

    PubMed

    Salminen, Teppo; Juntunen, Etvi; Khanna, Navin; Pettersson, Kim; Talha, Sheikh M

    2016-02-01

    There is a need for simple to produce immunoassays for hepatitis C virus (HCV) antibody capable of detecting all genotypes worldwide. Current commonly used third generation immunoassays use three to six separate recombinant proteins or synthetic peptides. We have developed and expressed in Escherichia coli a single recombinant antigen incorporating epitopes from different HCV proteins. This multiepitope protein (MEP) was used to develop two types of HCV antibody immunoassays: a traditional antibody immunoassay using a labeled secondary antibody (indirect assay) and a double-antigen assay with the same MEP used as capture binder and labeled binder. The secondary antibody assay was evaluated with 171 serum/plasma samples and double-antigen assay with 148 samples. These samples included an in-house patient sample panel, two panels of samples with different HCV genotypes and a seroconversion panel. The secondary antibody immunoassay showed 95.6% sensitivity and 100% specificity while the double-antigen assay showed 91.4% sensitivity and 100% specificity. Both assays detected samples from all six HCV genotypes. The results showed that combining a low-cost recombinant MEP binder antigen with a high sensitivity fluorescent lanthanide reporter can provide a sensitive and specific immunoassay for HCV serology. The results also showed that the sensitivity of HCV double-antigen assays may suffer from the low avidity immune response of acute infections.

  4. 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.

  5. Cobalt-Porphyrin-Platinum-Functionalized Reduced Graphene Oxide Hybrid Nanostructures: A Novel Peroxidase Mimetic System For Improved Electrochemical Immunoassay

    PubMed Central

    Shu, Jian; Qiu, Zhenli; Wei, Qiaohua; Zhuang, Junyang; Tang, Dianping

    2015-01-01

    5,10,15,20-Tetraphenyl-21H,23H-porphine cobalt flat stacking on the reduced graphene oxide with platinum nanoparticles (PtNPs/CoTPP/rGO) were first synthesized and functionalized with monoclonal rabbit anti-aflatoxin B1 antibody (anti-AFB1) for highly efficient electrochemical immunoassay of aflatoxin B1 (AFB1) in this work. Transmission electron microscopy (TEM), atomic force microscope (AFM) and spectral techniques were employed to characterize the PtNPs/CoTPP/rGO hybrids. Using anti-AFB1-conjugated PtNPs/CoTPP/rGO as the signal-transduction tag, a novel non-enzymatic electrochemical immunosensing system was designed for detection of target AFB1 on the AFB1-bovine serum albumin-functionalized sensing interface. Experimental results revealed that the designed immunoassay could exhibit good electrochemical responses for target analyte and allowed the detection of AFB1 at a concentration as low as 5.0 pg mL−1 (5.0 ppt). Intra- and inter-assay coefficients of variation were below 10%. Importantly, the methodology was further validated for analyzing naturally contaminated or spiked blank peanut samples with consistent results obtained by AFB1 ELISA kit, thus providing a promising approach for quantitative monitoring of organic pollutants. PMID:26462136

  6. Cobalt-Porphyrin-Platinum-Functionalized Reduced Graphene Oxide Hybrid Nanostructures: A Novel Peroxidase Mimetic System For Improved Electrochemical Immunoassay

    NASA Astrophysics Data System (ADS)

    Shu, Jian; Qiu, Zhenli; Wei, Qiaohua; Zhuang, Junyang; Tang, Dianping

    2015-10-01

    5,10,15,20-Tetraphenyl-21H,23H-porphine cobalt flat stacking on the reduced graphene oxide with platinum nanoparticles (PtNPs/CoTPP/rGO) were first synthesized and functionalized with monoclonal rabbit anti-aflatoxin B1 antibody (anti-AFB1) for highly efficient electrochemical immunoassay of aflatoxin B1 (AFB1) in this work. Transmission electron microscopy (TEM), atomic force microscope (AFM) and spectral techniques were employed to characterize the PtNPs/CoTPP/rGO hybrids. Using anti-AFB1-conjugated PtNPs/CoTPP/rGO as the signal-transduction tag, a novel non-enzymatic electrochemical immunosensing system was designed for detection of target AFB1 on the AFB1-bovine serum albumin-functionalized sensing interface. Experimental results revealed that the designed immunoassay could exhibit good electrochemical responses for target analyte and allowed the detection of AFB1 at a concentration as low as 5.0 pg mL-1 (5.0 ppt). Intra- and inter-assay coefficients of variation were below 10%. Importantly, the methodology was further validated for analyzing naturally contaminated or spiked blank peanut samples with consistent results obtained by AFB1 ELISA kit, thus providing a promising approach for quantitative monitoring of organic pollutants.

  7. One-step synthesis of redox-active polymer/AU nanocomposites for electrochemical immunoassay of multiplexed tumor markers.

    PubMed

    Liu, Zhimin; Rong, Qinfeng; Ma, Zhanfang; Han, Hongliang

    2015-03-15

    In this work, a simple and sensitive multiplexed immunoassay protocol for simultaneous electrochemical determination of alpha-fetoprotein (AFP) and carcinoembryonic antigen (CEA) was designed using redox-active nanocomposites. As the redox-active species, the poly(o-phenylenediamine) (POPD)/Au nanocomposite and poly(vinyl ferrocene-2-aminothiophenol) (poly(VFc-ATP))/Au nanocomposite were obtained by one-step method which HAuCl4 was used as the oxidant. With Au nanoparticles (AuNPs), the nanocomposites were successful to immobilize labeled anti-CEA and anti-AFP as the immunosensing probes. The proposed electrochemical immunoassay enabled the simultaneous monitoring of AFP and CEA in a wide range of 0.01-100ngmL(-1). The detection limits was 0.006ngmL(-1) for CEA and 0.003ngmL(-1) for AFP (S/N=3). The assay results of serum samples with the proposed method were well consistent with the reference values from standard ELISA method. And the negligible cross-reactivity between the two analytes makes it possesses potential promise in clinical diagnosis.

  8. Microarray-integrated optoelectrofluidic immunoassay system.

    PubMed

    Han, Dongsik; Park, Je-Kyun

    2016-05-01

    A microarray-based analytical platform has been utilized as a powerful tool in biological assay fields. However, an analyte depletion problem due to the slow mass transport based on molecular diffusion causes low reaction efficiency, resulting in a limitation for practical applications. This paper presents a novel method to improve the efficiency of microarray-based immunoassay via an optically induced electrokinetic phenomenon by integrating an optoelectrofluidic device with a conventional glass slide-based microarray format. A sample droplet was loaded between the microarray slide and the optoelectrofluidic device on which a photoconductive layer was deposited. Under the application of an AC voltage, optically induced AC electroosmotic flows caused by a microarray-patterned light actively enhanced the mass transport of target molecules at the multiple assay spots of the microarray simultaneously, which reduced tedious reaction time from more than 30 min to 10 min. Based on this enhancing effect, a heterogeneous immunoassay with a tiny volume of sample (5 μl) was successfully performed in the microarray-integrated optoelectrofluidic system using immunoglobulin G (IgG) and anti-IgG, resulting in improved efficiency compared to the static environment. Furthermore, the application of multiplex assays was also demonstrated by multiple protein detection.

  9. Reducing heterophilic antibody interference in immunoassays using single chain antibodies

    SciTech Connect

    Baird, Cheryl L.; Tan, Ruimin; Fischer, Christopher J.; Victry, Kristin D.; Zangar, Richard C.; Rodland, Karin D.

    2011-12-15

    Sandwich ELISA microarrays have the potential to simultaneously quantify the levels of multiple diagnostic targets in a biological sample. However, as seen with traditional ELISA diagnostics, heterophilic antibodies (HA) in patient sera have the potential to cause interference in these assays. We demonstrate here that reducing the diagnostic capture antibody to its minimal functional unit, the variable heavy and light domains artificially connected with a short polypeptide linker (scFv), is an effective strategy for reducing the HA assay interference.

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

    USGS Publications Warehouse

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

    2000-01-01

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

  11. Gluten fragment detection with a competitive ELISA.

    PubMed

    Haas-Lauterbach, Sigrid; Immer, Ulrike; Richter, Mareike; Koehler, Peter

    2012-01-01

    The second generation of a competitive ELISA for prolamin quantification based on the R5 antibody was studied for method performance and suitability to detect partially hydrolyzed prolamins in food. To be able to convert signal intensities to gluten concentrations, as required by the Codex Alimentarius Standard, a new calibrator consisting of a peptic-tryptic digest of wheat, rye, and barley prolamins was used for the first time. LOD and LOQ of the assay were 1.36 and 5.0 mg prolamin/kg food, respectively. Analysis of beer samples and a hydrolyzed wheat product showed that the assay provided significantly higher prolamin concentrations, compared to the sandwich ELISA based on the same antibody, which is only suitable for the detection of intact prolamins. Spiking experiments with defined concentrations of partially hydrolyzed prolamins gave recoveries ranging from 92 to 136%.

  12. 3-(4-Hydroxyphenyl)propionic acid: the forgotten detection substrate for ligand-binding assay-based bioanalysis.

    PubMed

    Jordan, Gregor; Stubenrauch, Kay-Gunnar; Heinrich, Julia; Staack, Roland F

    2017-02-01

    Ligand-binding assays are ideal for routine bioanalysis, but we reason that the straightforward replacement of the conventional chromogenic horseradish peroxidase substrate, 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid, of a routinely used preclinical immunoassay to detect hIgG, with the fluorogenic 3-(4-hydroxyphenyl)propionic acid would broaden the narrow dynamic range. The replacement leads to a sensitivity of 0.47 (minimum required dilution [MRD] 10) and 1.02 (MRD 50) ng/ml, and dynamic ranges of 3.3 (MRD 10) and 3.6 (MRD 50) orders of magnitude, and thereby had improved sensitivity and dynamic range compared with other conventional colorimetric ELISAs, other ligand-binding assay technologies or LC-MS assays. Improvements in sensitivity and dynamic range were achieved for the sera of horse, mice and monkeys without assay optimization.

  13. Data mining strategies to improve multiplex microbead immunoassay tolerance in a mouse model of infectious diseases.

    PubMed

    Mani, Akshay; Ravindran, Resmi; Mannepalli, Soujanya; Vang, Daniel; Luciw, Paul A; Hogarth, Michael; Khan, Imran H; Krishnan, Viswanathan V

    2015-01-01

    Multiplex methodologies, especially those with high-throughput capabilities generate large volumes of data. Accumulation of such data (e.g., genomics, proteomics, metabolomics etc.) is fast becoming more common and thus requires the development and implementation of effective data mining strategies designed for biological and clinical applications. Multiplex microbead immunoassay (MMIA), on xMAP or MagPix platform (Luminex), which is amenable to automation, offers a major advantage over conventional methods such as Western blot or ELISA, for increasing the efficiencies in serodiagnosis of infectious diseases. MMIA allows detection of antibodies and/or antigens efficiently for a wide range of infectious agents simultaneously in host blood samples, in one reaction vessel. In the process, MMIA generates large volumes of data. In this report we demonstrate the application of data mining tools on how the inherent large volume data can improve the assay tolerance (measured in terms of sensitivity and specificity) by analysis of experimental data accumulated over a span of two years. The combination of prior knowledge with machine learning tools provides an efficient approach to improve the diagnostic power of the assay in a continuous basis. Furthermore, this study provides an in-depth knowledge base to study pathological trends of infectious agents in mouse colonies on a multivariate scale. Data mining techniques using serodetection of infections in mice, developed in this study, can be used as a general model for more complex applications in epidemiology and clinical translational research.

  14. Pretreatment-free lateral flow enzyme immunoassay for progesterone detection in whole cows' milk.

    PubMed

    Samsonova, J V; Safronova, V A; Osipov, A P

    2015-01-01

    New rapid method of lateral flow enzyme immunoassay (LFEIA) for progesterone detection in whole cows' milk was developed. The test system utilized horseradish peroxidase as a label along with the substrate solution containing 3,3',5,5'-tetramethylbenzidine and dextran sulfate to obtain an insoluble blue colored product of the enzyme reaction on a surface of analytical membrane (test and control lines). Several aspects of LFEIA were optimized: time of the signal detection, membrane materials and assay conditions. Resulting competitive LFEIA can be performed within 15 minutes with the limit of progesterone detection of 0.8 ng/ml. Progesterone concentration in whole milk samples was determined by LFEIA and enzyme-linked immunosorbent assay (ELISA). The results obtained were in good correlation (R=0.97, n=46). Thus new sensitive LFEIA can be successfully used for on-site monitoring of oestrus status of cows' reproductive system and for early none-pregnancy detection. The method is fast, easy to perform and needs no preliminary sample preparation.

  15. Ultrasensitive fluorescence immunoassay for detection of ochratoxin A using catalase-mediated fluorescence quenching of CdTe QDs

    NASA Astrophysics Data System (ADS)

    Huang, Xiaolin; Zhan, Shengnan; Xu, Hengyi; Meng, Xianwei; Xiong, Yonghua; Chen, Xiaoyuan

    2016-04-01

    Herein, for the first time we report an improved competitive fluorescent enzyme linked immunosorbent assay (ELISA) for the ultrasensitive detection of ochratoxin A (OTA) by using hydrogen peroxide (H2O2)-induced fluorescence quenching of mercaptopropionic acid-modified CdTe quantum dots (QDs). In this immunoassay, catalase (CAT) was labeled with OTA as a competitive antigen to connect the fluorescence signals of the QDs with the concentration of the target. Through the combinatorial use of H2O2-induced fluorescence quenching of CdTe QDs as a fluorescence signal output and the ultrahigh catalytic activity of CAT to H2O2, our proposed method could be used to perform a dynamic linear detection of OTA ranging from 0.05 pg mL-1 to 10 pg mL-1. The half maximal inhibitory concentration was 0.53 pg mL-1 and the limit of detection was 0.05 pg mL-1. These values were approximately 283- and 300-folds lower than those of horseradish peroxidase (HRP)-based conventional ELISA, respectively. The reported method is accurate, highly reproducible, and specific against other mycotoxins in agricultural products as well. In summary, the developed fluorescence immunoassay based on H2O2-induced fluorescence quenching of CdTe QDs can be used for the rapid and highly sensitive detection of mycotoxins or haptens in food safety monitoring.Herein, for the first time we report an improved competitive fluorescent enzyme linked immunosorbent assay (ELISA) for the ultrasensitive detection of ochratoxin A (OTA) by using hydrogen peroxide (H2O2)-induced fluorescence quenching of mercaptopropionic acid-modified CdTe quantum dots (QDs). In this immunoassay, catalase (CAT) was labeled with OTA as a competitive antigen to connect the fluorescence signals of the QDs with the concentration of the target. Through the combinatorial use of H2O2-induced fluorescence quenching of CdTe QDs as a fluorescence signal output and the ultrahigh catalytic activity of CAT to H2O2, our proposed method could be used to

  16. Robust detection of peak signals for lateral flow immunoassays

    NASA Astrophysics Data System (ADS)

    Kim, Jongwon; Kim, Jong Dae; Nahm, Kie Bong; Choi, Eui Yul; Lee, Geumyoung

    2011-02-01

    Template matching method is presented to identify the peaks from the scanned signals of lateral flow immunoassay strips. The template is composed of two pulses separated by the distance of the control and the target ligand line in the assay, and is convolved with the scanned signal to deliver the maximum at the center of the two peaks. The peak regions were identified with the predefined distances from the center. Glycosylated haemoglobin immunoassay strips and fluorescent strip readers from Boditechmed Inc. were tested to estimate the lot and reader variations of the concentration measurands. The results showed the robustness of the propose method.

  17. Monitoring of individual human exposure to aflatoxins (AF) and N-nitrosamines (NNO) by immunoassays

    SciTech Connect

    Wild, C.P.; Umbenhauer, D.; Chapot, B.; Montesano, R.

    1986-01-01

    Highly sensitive immunoassays have been used to quantitate aflatoxins (AF) and N-nitrosamines (NNO) in human body fluids and tissues, respectively. This approach was taken in order to quantitate environmental exposure to these agents at an individual level to facilitate the investigation of their role in the etiology of human cancer. In order to analyse AF in human urine, an immunopurification step has been developed by using AF-specific antibody bound to AH-Sepharose 4B gel in a small (4-ml gel volume) affinity column prior to enzyme-linked immunosorbent assay (ELISA). The ELISA can be used to quantitate aflatoxin B1 (AFB1) over the range 0.01 ng/ml to 10 ng/ml and the assay system has been validated by using human urine samples spiked with AFB1 over this concentration range. In addition, 29 urine samples from the Philippines have been analyzed and found to contain a range of levels from zero to 4.25 ng/ml AFB1 equivalent with a mean of 0.875 ng/ml. This compared with a mean of 0.066 ng/ml AFB1 equivalent in samples from France. Radioimmunoassay of O6-methyldeoxyguanosine (O6-medG) has been performed on human esophageal and cardiac stomach mucosal DNA from tissue samples obtained during surgery in Linxian County, People's Republic of China, an area of high risk for both esophageal and stomach cancer. Using the methodology described and having 1 mg of hydrolyzed DNA allows the detection of approximately 25 fmol O6medG per mg DNA.

  18. Application of real-time PCR and ELISA assays for equine influenza virus to determine the duration of viral RNA shedding and onset of antibody response in naturally infected horses.

    PubMed

    Read, A J; Finlaison, D S; Gu, X; Davis, R J; Arzey, K E; Kirkland, P D

    2011-07-01

    During the equine influenza (EI) outbreak, two assays were used in parallel to diagnose the disease, to demonstrate freedom from infection in disease control zones and ultimately to demonstrate that EI virus had been eliminated from the Australian horse population. A longitudinal study of a population of naturally infected horses was established to determine the performance characteristics of these assays.

  19. Quantum dots as FRET acceptors for highly sensitive multiplexing immunoassays

    NASA Astrophysics Data System (ADS)

    Geissler, Daniel; Hildebrandt, Niko; Charbonnière, Loïc J.; Ziessel, Raymond F.; Löhmannsröben, Hans-Gerd

    2009-02-01

    Homogeneous immunoassays have the benefit that they do not require any time-consuming separation steps. FRET is one of the most sensitive homogeneous methods used for immunoassays. Due to their extremely strong absorption over a broad wavelength range the use of quantum dots as FRET acceptors allows for large Foerster radii, an important advantage for assays in the 5 to 10 nm distance range. Moreover, because of their size-tunable emission, quantum dots of different sizes can be used with a single donor for the detection of different analytes (multiplexing). As the use of organic dyes with short fluorescence decay times as donors is known to be inefficient with quantum dot acceptors, lanthanide complexes with long luminescence decays are very efficient alternatives. In this contribution we present the application of commercially available biocompatible CdSe/ZnS core/shell quantum dots as multiplexing FRET acceptors together with a single terbium complex as donor in a homogeneous immunoassay system. Foerster radii of 10 nm and FRET efficiencies of 75 % are demonstrated. The high sensitivity of the terbium-toquantum dot FRET assay is shown by sub-100-femtomolar detection limits for two different quantum dots (emitting at 605 and 655 nm) within the same biotin-streptavidin assay. Direct comparison to the FRET immunoassay "gold standard" (FRET from Eu-TBP to APC) yields a three orders of magnitude sensitivity improvement, demonstrating the big advantages of quantum dots not only for multiplexing but also for highly sensitive nanoscale analysis.

  20. Mass spectrometric immunoassay

    SciTech Connect

    Nelson, R.W.; Krone, J.R.; Bieber, A.L.; Williams, P.

    1995-04-01

    A new, general method of immunoassay is demonstrated. The approach is based on the microscale immunoaffinity capture of target antigens followed by mass-specific identification and quantitation using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Immunoaffinity capture of antigens effectively overcomes signal suppression effects typically encountered during traditional matrix-assisted laser desorption/ionization analysis of complex biological mixtures while simultaneously concentrating the analyte into a small volume. Sample incubation and processing methods were such that a typical analysis could be performed in less than 1 h while subnanomolar sensitivities were maintained. The technique has been used for the rapid, selective, and quantitative screening of human blood for the presence of myotoxin a, and Mojave toxin from the venoms of the prairie rattlesnake, Crotalus virdis virdis, and the Mojave rattlesnake, Crotalus scutulatus scutulatus. 18 refs., 8 figs.

  1. Development of sandwich ELISA for testing bovine β-lactoglobulin allergenic residues by specific polyclonal antibody against human IgE binding epitopes.

    PubMed

    He, Shengfa; Li, Xin; Gao, Jinyan; Tong, Ping; Chen, Hongbing

    2017-07-15

    Bovine β-lactoglobulin (BLG) is the main allergen in cows' milk, and the most commonly used method for detecting BLG is enzyme-linked immunosorbent assay (ELISA). However, antibodies used in commercial ELISA kits do not recognize specifically BLG IgE epitopes. Here, an antibody specific to IgE linear epitopes for BLG was used to develop a sandwich ELISA using a rabbit anti-BLG polyclonal antibody. The linear range for BLG detection was 31.25-8000ng/mL and limit of detection was 1.96ng/mL. BLG content in dairy samples was determined, and there was a good agreement between this immunoassay and reversed-phase high-performance liquid chromatography with high recovery. Additionally, BLG content in food samples had an average recovery of 104.25%. Allergenic residues were also detected in hydrolyzed infant formulas. The method developed could be a practical approach to determine BLG and its allergenic residues in food with a high degree of sensitivity, reliability and recovery.

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

    PubMed Central

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

    1997-01-01

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

  3. 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.

  4. Preparation of antibodies and development of a sensitive immunoassay with fluorescence detection for triazine herbicides.

    PubMed

    Herranz, Sonia; Ramón-Azcón, Javier; Benito-Peña, Elena; Marazuela, María Dolores; Marco, María Pilar; Moreno-Bondi, María Cruz

    2008-07-01

    Specific polyclonal antibodies against s-triazine herbicides were obtained by preparing immunogens coupling home-synthesized haptens derivatives of simazine (6-chloro-N-ethyl-N'-ethyl-1,3,5-triazine-2,4-diamine) to lysine groups of hemocyanin from keyhole limpets and bovine serum albumin carrier proteins. Three highly sensitive rabbit antisera were obtained and evaluated with a battery of six enzyme tracers derived from triazine structures in an optimized ELISA format. The antiserum As8 and the HRP-2f tracer, which yield the best assay sensitivity for simazine (detection limit 0.11 +/- 0.02 microg L(-1), IC(50) 0.88 +/- 0.04 microg L(-1)), were applied to the development of a sensitive flow-through immunoassay for the analysis of this herbicide. The automated assay was based on a direct competitive immunosorbent assay and fluorescence detection. The optimized method presents an IC(50) value of 0.35 +/- 0.04 microg L(-1) with a detection limit of 1.3 +/- 0.9 ng L(-1) and a dynamic range from 0.010 to 7.5 microg L(-1) simazine. The generic nature of the antiserum was shown by good relative cross-reactivities with other triazines such as atrazine (420%) or propazine (130%) and a lower response to terbutylazine (6.4%) and desethyl-atrazine (2.2%). No cross-reactivity was obtained for nonrelated pesticides such as 2,4-dichlorophenoxyacetic acid or linuron and the assay could be applied as a screening method for triazine herbicides. The total analysis time was 30 min per determination and the immunosensor could be reused for more than 150 cycles without significant loss of activity. The immunosensor has been successfully applied to the direct analysis of simazine in surface water samples at the nanogram per liter level. The results obtained by comparative analysis of the immunosensor with a chromatographic procedure for triazines showed a close correspondence.

  5. Detection of pregnancy and fertility status in big cats using an enzyme immunoassay based on 5α-pregnan-3α-ol-20-one.

    PubMed

    Umapathy, Govindhaswamy; Kumar, Vinod; Wasimuddin; Kabra, Meha; Shivaji, S

    2013-01-01

    Development of non-invasive steroid hormone assays using fecal samples is crucial for detection of pregnancy and monitoring of fertility status in big cats and thus facilitates conservation and management of wild animals. Due to changes in metabolism and excretory pattern, animals excrete different steroid metabolites in feces and urine. The present study is an attempt to develop a common enzyme immunoassay for 5α-pregnan-3α-ol-20-one one of the predominant progestogen metabolites in the feces samples of big cats. The developed ELISA showed a high sensitivity and low cross reactivity to other hormones compared to commercially available RIA kits based on progesterone antibody. It could be used in a wide range of animals for monitoring fertility status and pregnancy detection by measuring fecal steroid metabolites.

  6. Color encoded microbeads-based flow cytometric immunoassay for polycyclic aromatic hydrocarbons in food.

    PubMed

    Meimaridou, Anastasia; Haasnoot, Willem; Noteboom, Linda; Mintzas, Dimitrios; Pulkrabova, Jana; Hajslová, Jana; Nielen, Michel W F

    2010-07-05

    Food contamination caused by chemical hazards such as persistent organic pollutants (POPs) is a worldwide public health concern and requires continuous monitoring. The chromatography-based analysis methods for POPs are accurate and quite sensitive but they are time-consuming, laborious and expensive. Thus, there is a need for validated simplified screening tools, which are inexpensive, rapid, have automation potential and can detect multiple POPs simultaneously. In this study we developed a flow cytometry-based immunoassay (FCIA) using a color-encoded microbeads technology to detect benzo[a]pyrene (BaP) and other polycyclic aromatic hydrocarbons (PAHs) in buffer and food extracts as a starting point for the future development of rapid multiplex assays including other POPs in food, such as polychlorinated biphenyls (PCBs) and polybrominated diphenyl ethers (PBDEs). A highly sensitive assay for BaP was obtained with an IC(50) of 0.3 microg L(-1) using a monoclonal antibody (Mab22F12) against BaP, similar to the IC(50) of a previously described enzyme-linked immunosorbent assay (ELISA) using the same Mab. Moreover, the FCIA was 8 times more sensitive for BaP compared to a surface plasmon resonance (SPR)-based biosensor immunoassay (BIA) using the same reagents. The selectivity of the FCIAs was tested, with two Mabs against BaP for 25 other PAHs, including two hydroxyl PAH metabolites. Apart from BaP, the FCIAs can detect PAHs such as indenol[1,2,3-cd]pyrene (IP), benz[a]anthracene (BaA), and chrysene (CHR) which are also appointed by the European Food Safety Authority (EFSA) as suitable indicators of PAH contamination in food. The FCIAs results were in agreement with those obtained with gas chromatography-mass spectrometry (GC-MS) for the detection of PAHs in real food samples of smoked carp and wheat flour and has great potential for the future routine application of this assay in a simplex or multiplex format in combination with simplified extraction procedure which

  7. A novel double antibody sandwich-lateral flow immunoassay for the rapid and simple detection of hepatitis C virus.

    PubMed

    Xiang, Tingxiu; Jiang, Zheng; Zheng, Jian; Lo, Chaoyu; Tsou, Harry; Ren, Guosheng; Zhang, Jun; Huang, Ailong; Lai, Guoqi

    2012-11-01

    The objective of this study was to screen for antigens of the hepatitis C virus (HCV) to establish a new double antibody sandwich-lateral flow immunoassay (DAS-LFIA) method for testing the presence of anti-HCV antibodies in human serum or plasma. A series of different recombinant HCV proteins in Escherichia coli cells were constructed, expressed, purified and the new DAS-LFIA strip was developed. The sensitivity and specificity of new the DAS-LFIA strip were evaluated by detecting 23 HCV-positive sera, a set of quality control references for anti-HCV detection that contain known amounts of anti-HCV antibodies, and 8 HCV-negative sera. A total of 300 clinical serum samples was examined by both the new DAS-LFIA strip and enzyme-linked immunosorbent assay (ELISA). Data were analyzed using SPSS 11.5 software. The sensitivity and specificity of the new DAS-LFIA strip were 100%. The lowest test line of the HCV DAS-LFIA strips was 2 NCU/ml. Additionally, the concordance between the new DAS-LFIA strip and ELISA methods was 94.33%. In conclusion, our new testing method is rapid, simple, sensitive and specifically detects the presence of anti-HCV antibodies in human serum or plasma. Therefore, it may be used for monitoring HCV.

  8. A sensitive three monoclonal antibodies based automatic latex particle-enhanced turbidimetric immunoassay for Golgi protein 73 detection

    PubMed Central

    Xia, Yanyan; Shen, Han; Zhu, Yefei; Xu, Hongpan; Li, Zhiyang; Si, Jin

    2017-01-01

    Golgi protein 73 (GP73) is a novel and potential marker for diagnosing hepatocellular carcinoma (HCC) that has been found to be abnormally elevated in liver disease. A latex particle-enhanced turbidimetric immunoassay (LTIA) was recently introduced and licensed for application in a variety of automated clinical chemistry analyzers. However, no studies have reported sufficient data on analytical performance of this method when using 3 monoclonal antibodies for GP73 measurement. The experimental conditions were firstly optimized and range of linearity, diagnostic potential, clinical relevance were compared with the LTIA based on polyclonal antibodies and ELISA. Dilution tests for the LTIA using 3 monoclonal antibodies produced a calibration curve from 10 to 350 ng/mL while the polyclonal antibodies produced the curve from 20 to 320 ng/mL. The detection limit was achieved at 1.82 ng/mL concentration. Within-run CV was obtained in the range of 1.5–2.9% and ROC curves indicated sensitivity and specificity of the LTIA based on 3 monoclonal antibodies were 96.7% and 93.3%, respectively, higher than for the polyclonal antibodies (94.6% and 72.4%) and ELISA (70.0% and 83.3%). Therefore, the LTIA assay based on 3 monoclonal antibodies is thus applicable in quantification of GP73 concentration in automated biochemistry analyzers. PMID:28054632

  9. Use of a whole blood competitive immunoassay for the assessment of worker exposures to propylene oxide at three manufacturing facilities.

    PubMed

    Jones, Alan L; Van der Woord, Miriam; Bourrillon, François

    2005-04-01

    The level of N-(2-hydroxypropyl)valine adducts in haemoglobin has been shown to correlate well with workplace exposure to propylene oxide (PO). However, the analytical method, using the modified Edman degradation procedure, is prohibitively time-consuming and expensive for use as a routine workplace exposure measurement tool. As an alternative, AB Biomonitoring Ltd of Cardiff, Wales, developed a competitive immunoassay for the determination of N-(2-hydroxypropyl)valine adducts in human haemoglobin. Studies showed that whole blood samples analysed using an enzyme linked immunosorbent assay (ELISA) and the modified Edman degradation procedure over the concentration range 3.7-992 nmol N-(2-hydroxypropyl)valine g(-1) haemoglobin are in good agreement (correlation coefficient 0.998, n = 10). The intervariance and intravariance data indicate the repeatability of the ELISA method over the assay conditions employed and show that it is robust over its working range [2-200 pmol N-(2-hydroxypropyl)valine g(-1) haemoglobin]. The assay employs a whole blood matrix and has a working range of 2-6000 pmol g(-1) Hb (equivalent to up to 5 ppm PO exposure, 8 h per day, 5 days per week, over 4 months). The practicality of the assay was tested by assessing exposures to PO at three world-scale manufacturing sites in France and The Netherlands. Over 800 samples were taken over a 2 year period from operators, maintenance fitters and office staff. The data, typically <50 pmol g(-1) globin, indicate that exposures were significantly <0.1 ppm at all times (The Dutch occupational exposure limit is 2.5 ppm over 8 h). Samples were taken after a major turnaround and also before and after the start-up of a newly commissioned plant. All data indicated that high levels of control were effective in minimizing exposure. This study has shown that the immunoassay is a powerful tool for the exposure component of future epidemiology studies, as well as a definitive demonstration of the effectiveness of

  10. Evaluation of two novel chemiluminescence immunoassays for the detection of Clostridium difficile glutamate dehydrogenase and toxin A&B.

    PubMed

    Blaich, Annette; Frei, Reno; Castellano, Carine; Kiessling, Christine; Geschke, Angelika; Rentsch, Katharina M; Egli, Adrian

    2017-04-01

    A novel immunoassay for Clostridium difficile glutamate dehydrogenase (GDH) and toxin A&B (LIAISON, DiaSorin) was compared to another GDH assay (Alere), PCR and toxigenic culture. The GDH-DiaSorin is slightly more sensitive than the GDH-Alere. Sensitivity of the Toxin-Diasorin test is in accordance to the sensitivity of other immunoassays in literature.

  11. Fasciola hepatica saposin-like-2 protein based ELISA for the serodiagnosis of chronic human fascioliasis

    PubMed Central

    Figueroa-Santiago, Olgary; Delgado, Bonnibel; Espino, Ana M.

    2011-01-01

    An indirect enzyme-linked immunosorbent assay (ELISA) was developed and evaluated for its diagnostic ability to detect human IgG antibodies against Fasciola hepatica saposin-like protein-2. The assay was compared with an indirect ELISA with excretory-secretory products (FhES) from adult F. hepatica. In an analysis of the sera of 37 patients infected with F. hepatica, 40 patients with other parasitic infections, and 50 healthy controls, the sensitivity of both ELISA assays was 100%. However, the FhSAP2-based ELISA was more specific (95.6%) than the FhES-ELISA (91.9%). These results demonstrated that FhSAP2 can be used in the serodiagnosis of chronic human fascioliasis with additional advantage that is relative cheap and easy to produce. Studies are in progress to evaluate this FhSAP2-ELISA assay in a large-scale prevalence surveys in endemic areas. PMID:21683266

  12. Rapid dioxin screening by enzyme immunoassay

    SciTech Connect

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

    1995-12-31

    A system has been developed for rapid screening of 2,3,7,8-Tetra-ChloroDibenzo-p-Dioxin (TCDD). The system uses a competitive inhibition Enzyme ImmunoAssay (EIA) based on a mouse monoclonal antibody which is specific for TCDD and related congeners. Sample preparation can be performed with a programmable automated extraction and cleanup system which uses disposable Teflon clad columns. The extraction and cleanup system has been extensively validated by GC-MS for a variety of sample types. The sample preparation system allows immunoassay analysis of soil, serum, water, and other matrices by taking each sample type to the same sample preparation endpoint. Concentration factors and endpoint conditions are completely flexible and programmable. Immunoassay analysis is performed by the addition of a prepared sample extract in organic solvent to an antibody coated microwell containing an aqueous sample diluent. This is mixed and incubated for 30 minutes to allow the immobilized antibody to capture analyte from the sample. The liquid is then removed and the well is washed to remove unbound materials. The well is then incubated with a competitor-HRP conjugate capable of binding specifically to the antibody sites not occupied by TCDD. After 30 minutes, the unbound conjugate is washed away and enzyme substrate is added for color development. The color generated is directly related to the amount of competitor-HRP bound in the second step, which is inversely related to the amount of analyte bound in the first step. After 30 minutes, a stop solution is added and the developed color is read on a microplate reader. The total time required for the EIA analysis of a prepared extract is less than 2 hours.

  13. Comparison of electron microscopy, ELISA, real time RT-PCR and insulated isothermal RT-PCR for the detection of Rotavirus group A (RVA) in feces of different animal species.

    PubMed

    Soltan, Mohamed A; Tsai, Yun-Long; Lee, Pei-Yu A; Tsai, Chuan-Fu; Chang, Hsiao-Fen G; Wang, Hwa-Tang T; Wilkes, Rebecca P

    2016-09-01

    There is no gold standard for detection of Rotavirus Group A (RVA), one of the main causes of diarrhea in neonatal animals. Sensitive and specific real-time RT-PCR (rtRT-PCR) assays are available for RVA but require submission of the clinical samples to diagnostic laboratories. Patient-side immunoassays for RVA protein detection have shown variable results, particularly with samples from unintended species. A sensitive and specific test for detection of RVA on the farm would facilitate rapid management decisions. The insulated isothermal RT-PCR (RT-iiPCR) assay works in a portable machine to allow sensitive and specific on-site testing. The aim of this investigation was to evaluate a commercially available RT-iiPCR assay for RVA detection in feces from different animal species. This assay was compared to an in-house rtRT-PCR assay and a commercially available rtRT-PCR kit, as well as an ELISA and EM for RVA detection. All three PCR assays targeted the well-conserved NSP5 gene. Clinical fecal samples from 108 diarrheic animals (mainly cattle and horses) were tested. The percentage of positive samples by ELISA, EM, in-house rtRT-PCR, commercial rtRT-PCR, and RT-iiPCR was 29.4%, 31%, 36.7%, 51.4%, 56.9%, respectively. The agreement between different assays was high (81.3-100%) in samples containing high viral loads. The sensitivity of the RT-iiPCR assay appeared to be higher than the commercially available rtRT-PCR assay, with a limit of detection (95% confidence index) of 3-4 copies of in vitro transcribed dsRNA. In conclusion, the user-friendly, field-deployable RT-iiPCR system holds substantial promise for on-site detection of RVA.

  14. Detection of toxigenic strains of Bacillus cereus and other Bacillus spp. with an improved cytotoxicity assay.

    PubMed

    Beattie, S H; Williams, A G

    1999-03-01

    An improved qualitative cell cytotoxicity assay for the detection of Bacillus cereus emetic and enterotoxin is described. The presence of toxin in culture supernatant fluids was detected by measurement with the tetrazolium salt MTT, as it adversely affects the metabolic status of cultured CHO cells. Psychrotrophic B. cereus isolates (65) were assessed for toxin production using the cytotoxicity assay, and 91% of culture supernatant fluids were cytotoxic. Toxin assessment using BCET-RPLA and ELISA immunoassays indicated that 51% and 85% of the cultures, respectively, were toxigenic. There were pronounced strain differences in the amount of toxin produced by the B. cereus isolates. Some isolates of B. circulans, B. laterosporus/cereus, B. lentus, B. licheniformis, B. mycoides, B. subtilis and B. thuringiensis were also toxigenic.

  15. Demonstration of four immunoassay formats using the array biosensor

    NASA Technical Reports Server (NTRS)

    Sapsford, Kim E.; Charles, Paul T.; Patterson, Charles H Jr; Ligler, Frances S.

    2002-01-01

    The ability of a fluorescence-based array biosensor to measure and quantify the binding of an antigen to an immobilized antibody has been demonstrated using the four different immunoassay formats: direct, competitive, displacement, and sandwich. A patterned array of antibodies specific for 2,4,6-trinitrotoluene (TNT) immobilized onto the surface of a planar waveguide and used to measure signals from different antigen concentrations simultaneously. For direct, competitive, and displacement assays, which are one-step assays, measurements were obtained in real time. Dose-response curves were calculated for all four assay formats, demonstrating the array biosensor's ability to quantify the amount of antigen present in solution.

  16. 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.

  17. An inexpensive, fast and sensitive quantitative lateral flow magneto-immunoassay for total prostate specific antigen.

    PubMed

    Barnett, Jacqueline M; Wraith, Patrick; Kiely, Janice; Persad, Raj; Hurley, Katrina; Hawkins, Peter; Luxton, Richard

    2014-09-01

    We describe the detection characteristics of a device the Resonant Coil Magnetometer (RCM) to quantify paramagnetic particles (PMPs) in immunochromatographic (lateral flow) assays. Lateral flow assays were developed using PMPs for the measurement of total prostate specific antigen (PSA) in serum samples. A detection limit of 0.8 ng/mL was achieved for total PSA using the RCM and is at clinically significant concentrations. Comparison of data obtained in a pilot study from the analysis of serum samples with commercially available immunoassays shows good agreement. The development of a quantitative magneto-immunoassay in lateral flow format for total PSA suggests the potential of the RCM to operate with many immunoassay formats. The RCM has the potential to be modified to quantify multiple analytes in this format. This research shows promise for the development of an inexpensive device capable of quantifying multiple analytes at the point-of-care using a magneto-immunoassay in lateral flow format.

  18. An Inexpensive, Fast and Sensitive Quantitative Lateral Flow Magneto-Immunoassay for Total Prostate Specific Antigen

    PubMed Central

    Barnett, Jacqueline M.; Wraith, Patrick; Kiely, Janice; Persad, Raj; Hurley, Katrina; Hawkins, Peter; Luxton, Richard

    2014-01-01

    We describe the detection characteristics of a device the Resonant Coil Magnetometer (RCM) to quantify paramagnetic particles (PMPs) in immunochromatographic (lateral flow) assays. Lateral flow assays were developed using PMPs for the measurement of total prostate specific antigen (PSA) in serum samples. A detection limit of 0.8 ng/mL was achieved for total PSA using the RCM and is at clinically significant concentrations. Comparison of data obtained in a pilot study from the analysis of serum samples with commercially available immunoassays shows good agreement. The development of a quantitative magneto-immunoassay in lateral flow format for total PSA suggests the potential of the RCM to operate with many immunoassay formats. The RCM has the potential to be modified to quantify multiple analytes in this format. This research shows promise for the development of an inexpensive device capable of quantifying multiple analytes at the point-of-care using a magneto-immunoassay in lateral flow format. PMID:25587419

  19. Evaluation of the performance of 5 commercialized enzyme immunoassays for the detection of Taenia solium antibodies and for the diagnosis of neurocysticercosis.

    PubMed

    Carod, Jean-François; Randrianarison, Mickaël; Razafimahefa, Julien; Ramahefarisoa, Rondro Mamitiana; Rakotondrazaka, Mahenintsoa; Debruyne, Monique; Dautigny, Mélanie; Cazal, Pierrette; Andriantseheno, Marcellin Lalaoarisoa; Charles, Emile Ramarokoto

    2012-01-01

    This study aimed to evaluate 5 enzyme immunoassays for detecting human antibodies against Taenia solium in human serum and for the diagnosis of neurocysticercosis (NCC): DRG™, RIDASCREEN™, NOVATECH™, CYPRESS™, and IVD™. A collection of 114 reference serum samples were used. All sera were tested both by ELISA and by an immunoblot method (enzyme-linked immunoelectrotransfer blot [EITB]). When compared with EITB, the Ridascreen™ test had the best positive concordance rate (85.1-91.2%) and the NovaLisa test™ showed the optimal negative concordance rate (93.7-95.6%). All tests had a sensitivity under 72% and a specificity above 60%. The best sensitivity was obtained using Ridascreen™ test (71.4%). An optimal specificity was achieved by the NovaLisa test™. T. solium-positive sera all cross-reacted with E. granulosus positive samples. In the commercial assays evaluated here, the most appropriate ELISA test for screening may be the Ridascreen™ assay. Antibody detection seems to be not appropriate for NCC diagnosis because of its overall lack of sensitivity.

  20. Effects of particle characteristics on magnetic immunoassay in a thin channel.

    PubMed

    Tsai, H Y; Hsieh, Y C; Su, Y M; Chan, J R; Chang, Y C; Fuh, C Bor

    2011-10-15

    The effects of size and porosity of particles on magnetic immunoassay in a thin channel were studied. Experimental parameters were investigated and compared using a model immunoassay complex of carcinoembryonic antigen (CEA)/anti-CEA. The rate constant for the affinity reaction between functional particles increased as the size of magnetic nanoparticles (800-80 nm) decreased. The affinity reaction between functional particles had no significant effect on the sizes of microparticles (1.0-4.4 μm) at commonly used thin channel flow-rates of 0.001-0.025 ml/min. Competitive and sandwich reactions of CEA/anti-CEA were studied for CEA detection. Microparticles of different porosities produced similar linear ranges of detection and limits of detection. The limits of detection for CEA were 0.29 pg/ml and 0.21 pg/ml for competitive and sandwich reactions, respectively. The linear ranges of detection were from 0.49 pg/ml to 4.9 ng/ml for both competitive and sandwich reactions. The detection limits were lower, and the linear ranges were wider than those of literature. There was a 9% difference in CEA detection measurements between competitive and sandwich magnetic immunoassay. The measurements of two magnetic immunoassays differed by less than 13% from the ELISA reference measurements. The running time was less than 30 min. Magnetic immunoassay in a thin channel has great potential for biochemical analysis and immunoassay-related applications.

  1. An Immunoassay for Dibutyl Phthalate Based on Direct Hapten Linkage to the Polystyrene Surface of Microtiter Plates

    PubMed Central

    Wei, Chenxi; Ding, Shumao; You, Huihui; Zhang, Yaran; Wang, Yao; Yang, Xu; Yuan, Junlin

    2011-01-01

    Background Dibutyl phthalate (DBP) is predominantly used as a plasticizer inplastics to make them flexible. Extensive use of phthalates in both industrial processes and other consumer products has resulted in the ubiquitous presence of phthalates in the environment. In order to better determine the level of pollution in the environment and evaluate the potential adverse effects of exposure to DBP, immunoassay for DBP was developed. Methodology/Principal Findings A monoclonal antibody specific to DBP was produced from a stable hybridoma cell line generated by lymphocyte hybridoma technique. An indirect competitive enzyme-linked immunosorbent assay (icELISA) employing direct coating of hapten on polystyrene microtiter plates was established for the detection of DBP. Polystyrene surface was first oxidized by permanganate in dilute sulfuric acid to generate carboxyl groups. Then dibutyl 4-aminophthalate, which is an analogue of DBP, was covalently linked to the carboxyl groups of polystyrene surface with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC). Compared with conjugate coated format (IC50 = 106 ng/mL), the direct hapten coated format (IC50 = 14.6 ng/mL) improved assay sensitivity after careful optimization of assay conditions. The average recovery of DBP from spiked water sample was 104.4% and the average coefficient of variation was 9.95%. Good agreement of the results obtained by the hapten coated icELISA and gas chromatography-mass spectrometry further confirmed the reliability and accuracy of the icELISA for the detection of DBP in certain plastic and cosmetic samples. Conclusions/Significance The stable and efficient hybridoma cell line obtained is an unlimited source of sensitive and specific antibody to DBP. The hapten coated format is proposed as generally applicable because the carboxyl groups on modified microtiter plate surface enables stable immobilization of aminated or hydroxylated hapten with EDC. The developed hapten

  2. Direct competitive chemiluminescence immunoassays based on gold-coated magnetic particles for detection of chloramphenicol.

    PubMed

    Liang, Xiaohui; Fang, Xiangyi; Yao, Manwen; Yang, Yucong; Li, Junfeng; Liu, Hongjun; Wang, Linyu

    2016-02-01

    Direct competitive chemiluminescence immunoassays (CLIA) based on gold-coated magnetic nanospheres (Au-MNPs) were developed for rapid analysis of chloramphenicol (CAP). The Au-MNPs were modified with carboxyl groups and amino groups by 11-mercaptoundecanoic acid (MUA) and cysteamine respectively, and then were respectively conjugated with CAP base and CAP succinate via an activating reaction using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS). NSP-DMAE-NHS, a new and effective luminescence reagent, was employed to label anti-CAP antibody (mAb) as a tracer in direct CLIA for CAP detection using a 'homemade' luminescent measurement system that was set up with a photomultiplier tube (PMT) and a photon counting unit linked to a computer. The sensitivities and limits of detection (LODs) of the two methods were obtained and compared according to the inhibition curves. The 50% inhibition concentration (IC50 ) values of the two methods were about 0.044 ng/mL and 0.072 ng/mL respectively and LODs were approximately 0.001 ng/mL and 0.006 ng/mL respectively. To our knowledge, they were much more sensitive than any traditional enzyme-linked immunosorbent assay (ELISA) ever reported. Moreover, the new luminescence reagent NSP-DMAE-NHS is much more sensitive and stable than luminol and its derivatives, contributing to the sensitivity enhancement.

  3. 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.

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

    DTIC Science & Technology

    2013-07-12

    parasitaemia detection for both methods was determined for reference clones and IEV isolates. Since human white blood cell (WBC) DNA in clinical samples is...that may reduce sensitivity of parasite detection [8]. Since 2004, Armed Forces Research Institute of Medical Sciences (AFRIMS) routinely applies the HRP...susceptibilities of P. falciparum la- boratory reference strains showed that the HRP-2 assay provides a similar limit of detection in either whole blood

  5. A new ELISA for determination of potency in snake antivenoms.

    PubMed

    Rial, A; Morais, V; Rossi, S; Massaldi, H

    2006-09-15

    A competitive ELISA for potency determination of bothropic equine antivenom was developed and compared to the conventional in vivo ED(50) assay, with the aim of partially substituting the in vivo assay in the monitoring of antivenom immunoglobulin levels. On this purpose, blood samples were taken at different times during and after the immunization protocol of the lot of horses used for production of snake antivenom at the Instituto de Higiene, Uruguay. Both the competitive ELISA and the ED(50) assay were performed on those samples. In addition, a group of five commercial pepsin-digested antivenoms were tested by both methods. A significant (P<0.001) correlation (Pearson's r=0.957) was found between the ELISA titres and the corresponding ED(50) values, indicating that the in vitro test can estimate the neutralizing antibody capacity of the sera as well as the in vivo assay. By means of this new ELISA, it was found that the immunized animals maintained good venom antibody titres, in the order of 20-50% of the maximum achieved, even 10 month after the end of the immunization schedule. The main advantage of our ELISA design is its ability to correctly estimate the neutralization capacity of crude hyperimmune plasma and antivenom sera independently of their antibody composition in terms of whole IgG or F(ab')(2) fragment.

  6. High sensitive immunoassay for multiplex mycotoxin detection with photonic crystal microsphere suspension array.

    PubMed

    Deng, Guozhe; Xu, Kun; Sun, Yue; Chen, Yu; Zheng, Tiesong; Li, Jianlin

    2013-03-05

    A novel, sensitive, and high throughput competitive immunoassay for multiplex mycotoxins was established by immobilizing the artificial antigens (Ags) of mycotoxins on the surfaces of three kinds of silica photonic crystal microsphere (SPCM) suspension arrays. The SPCMs were encoded by their reflectance peak positions. Aflatoxin B1 (AFB1), fumonisin B1 (FB1), and citrinin (CIT) spiked in the cereals were extracted, and the fluorescein isothiocyanate (FITC) labeled antibodies (Abs) of these mycotoxins were added into the centrifuge tube which contained the SPCMs of the modified artificial antigens (Ags). The fluorescence signal was collected by an array fluorescent scanner. The limit of detection (LOD) was as low as 0.5, 1, and 0.8 pg/mL for AFB1, FB1, and CIT, respectively. The new method provided a wide linear detection range from 0.001 to 10, 0.001 to 10, and 0.001 to 1 ng/mL for AFB1, FB1, and CIT, respectively. The mean recovery rates are in range of 74.7 ± 4.0% to 127.9 ± 4.4% for the three mycotoxins in corn, peanuts, and wheat. The developed method for mycotoxins was used to assay the AFB1, FB1, and CIT level in 10 naturally contaminated cereal samples, and the results of detection were in agreement with that of a classic enzyme-linked immunosorbent assay (ELISA) method. This method saves a large amount of reagents (10 μL volume) and detection time (<3 h) for multiplex mycotoxin assay.

  7. Particle counting immunoassay for urinary cotinine. Comparison with chromatography, enzyme-linked immunoassay and fluorescence polarization immunoassay.

    PubMed

    Galanti, L M; Dell'Omo, J; Vanbeckbergen, D; Dubois, P; Masson, P L; Cambiaso, C L

    1999-07-01

    Urinary cotinine was measured according to its inhibitory activity on the agglutination of cotinine-coated latex particles by anti-cotinine antibodies, the agglutination being measured by optical counting of the remaining non-agglutinated particles (particle counting, PaC). The detection limit was 0.03 microgram/ml and the practical range extended from 0.03 to 3.9 micrograms/ml. The correlation results of 320 urine samples with those of high pressure liquid chromatography, enzyme-linked (Coti-Tracq EIA, Serex Inc., Maywood, NJ, USA), and fluorescence polarization immunoassay (TDX instrument, Abbott, Abbott Park, IL, USA) were r = 0.90, r = 0.69, r = 0.87, respectively, whereas the correlation coefficients between the assays other than particle counting ranged from 0.62 to 0.88. PaC does not require any separation step and can thus be easily automated.

  8. Molecular Form Differences Between Prostate-Specific Antigen (PSA) Standards Create Quantitative Discordances in PSA ELISA Measurements

    NASA Astrophysics Data System (ADS)

    McJimpsey, Erica L.

    2016-02-01

    The prostate-specific antigen (PSA) assays currently employed for the detection of prostate cancer (PCa) lack the specificity needed to differentiate PCa from benign prostatic hyperplasia and have high false positive rates. The PSA calibrants used to create calibration curves in these assays are typically purified from seminal plasma and contain many molecular forms (intact PSA and cleaved subforms). The purpose of this study was to determine if the composition of the PSA molecular forms found in these PSA standards contribute to the lack of PSA test reliability. To this end, seminal plasma purified PSA standards from different commercial sources were investigated by western blot (WB) and in multiple research grade PSA ELISAs. The WB results revealed that all of the PSA standards contained different mass concentrations of intact and cleaved molecular forms. Increased mass concentrations of intact PSA yielded higher immunoassay absorbance values, even between lots from the same manufacturer. Standardization of seminal plasma derived PSA calibrant molecular form mass concentrations and purification methods will assist in closing the gaps in PCa testing measurements that require the use of PSA values, such as the % free PSA and Prostate Health Index by increasing the accuracy of the calibration curves.

  9. Parts per trillion detection of 7-aminonitrazepam by nano-enhanced ELISA.

    PubMed

    Peng, Chifang; Duan, Xiaohui; Song, Shanshan; Xue, Feng

    2013-09-25

    It is challenging to detect 7-aminonitrazepam (7-ANZP) residue in animal tissues simply and sensitively by the enzyme-linked sorbent immunoassay (ELISA) method. This paper demonstrates that utilizing a bioconjugate of gold nanoparticles and enzyme-labeled antibody as a signal probe increases the sensitivity of a traditional ELISA for 7-ANZP by nearly 20 times. The sensitivity of this ELISA for 7-ANZP was 5.6 pg/mL in buffer, and the limit of detection (LOD) of 0.18 µg/kg for 7-ANZP in urine could be achieved after the urine samples were simply hydrolyzed and diluted by buffer. This simple and sensitive method has potential application for improving the sensitivity of ELISA methods against various small molecules.

  10. 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.

  11. Europium nanoparticle-based high performing immunoassay for the screening of treponemal antibodies.

    PubMed

    Talha, Sheikh M; Hytönen, Jukka; Westhorpe, Adam; Kumar, Sushil; Khanna, Navin; Pettersson, Kim

    2013-01-01

    Treponema pallidum subspecies pallidum (Tp) is the causative agent of syphilis which mainly spreads through sexual contact, blood transfusion and perinatal route. In order to curtail the spread of the infection and to clinically manage the disease, timely, accurate and reliable diagnosis is very important. We have developed an immunoassay for the detection of treponemal antibodies in human serum or plasma samples. In vivo biotinylated and non-biotinylated versions of the recombinant antigen were designed by the fusion of three Tp-specific antigens namely Tp15, Tp17 and Tp47. These fusion antigens were expressed in E. coli and purified using single-step metal affinity chromatography. Biotinylated fusion antigen immobilized on streptavidin coated plate was used to capture the treponemal antibodies and the non-biotinylated antigen coated on europium nanoparticles was used as tracer. Assays with two different incubation times of 10 min and 1 h were developed, and following the incubation the europium fluorescence was measured using time-resolved fluorometry. The developed time-resolved fluorometric (TRF) immunoassays were evaluated with in-house and commercial serum/plasma sample panels. For well-established treponemal antibodies positive or negative samples, the sensitivity of TRF immunoassay with 10 min incubation time was 97.4%, and of TRF immunoassay with 1 h incubation time was 98.7%, and the specificities of both the TRF immunoassays were 99.2%. For the samples with discordant results with the reference assays, both the TRF immunoassays showed better specificity than the Enzygnost syphilis enzyme immunoassay as a screening test. The two different incubation times did not have any significant effect on the signal to cutoff (S/Co) ratios obtained with the two immunoassays (p=0.06). Our results indicate that the developed immunoassay with a short incubation time of 10 min has the potential to be used in clinical laboratories and in blood-bank settings as a screening

  12. Europium Nanoparticle-Based High Performing Immunoassay for the Screening of Treponemal Antibodies

    PubMed Central

    Talha, Sheikh M.; Hytönen, Jukka; Westhorpe, Adam; Kumar, Sushil; Khanna, Navin; Pettersson, Kim

    2013-01-01

    Treponema pallidum subspecies pallidum (Tp) is the causative agent of syphilis which mainly spreads through sexual contact, blood transfusion and perinatal route. In order to curtail the spread of the infection and to clinically manage the disease, timely, accurate and reliable diagnosis is very important. We have developed an immunoassay for the detection of treponemal antibodies in human serum or plasma samples. In vivo biotinylated and non-biotinylated versions of the recombinant antigen were designed by the fusion of three Tp-specific antigens namely Tp15, Tp17 and Tp47. These fusion antigens were expressed in E. coli and purified using single-step metal affinity chromatography. Biotinylated fusion antigen immobilized on streptavidin coated plate was used to capture the treponemal antibodies and the non-biotinylated antigen coated on europium nanoparticles was used as tracer. Assays with two different incubation times of 10 min and 1 h were developed, and following the incubation the europium fluorescence was measured using time-resolved fluorometry. The developed time-resolved fluorometric (TRF) immunoassays were evaluated with in-house and commercial serum/plasma sample panels. For well-established treponemal antibodies positive or negative samples, the sensitivity of TRF immunoassay with 10 min incubation time was 97.4%, and of TRF immunoassay with 1 h incubation time was 98.7%, and the specificities of both the TRF immunoassays were 99.2%. For the samples with discordant results with the reference assays, both the TRF immunoassays showed better specificity than the Enzygnost syphilis enzyme immunoassay as a screening test. The two different incubation times did not have any significant effect on the signal to cutoff (S/Co) ratios obtained with the two immunoassays (p = 0.06). Our results indicate that the developed immunoassay with a short incubation time of 10 min has the potential to be used in clinical laboratories and in blood-bank settings as a

  13. A commercial rapid optical immunoassay detects Streptococcus agalactiae from aquatic cultures and clinical specimens

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The BioStar STREPT B Optical ImmunoAssay (OIA) (BioStar® OIA® Strep B Assay Kit; Biostar Incorporation; Louisville, CO, USA) was used to identify 32 known group B streptococcus (GBS) isolates of fish, dolphin, bovine, and human origin. Thirteen non-GBS isolates from fish and other animals were test...

  14. 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.

  15. Development of a highly sensitive monoclonal antibody based ELISA for detection of benzo[a]pyrene in potable water.

    PubMed

    Matschulat, Diana; Deng, Anping; Niessner, Reinhard; Knopp, Dietmar

    2005-07-01

    In Europe, a limit value of 10 ng L(-1) was set by the European Commission for benzo[a]pyrene (B[a]P) in water intended for human consumption (Council Directive 98/83/EC) and, therefore, sensitive and reliable methods are needed to evaluate its presence. We report here on the development of a highly sensitive indirect competitive ELISA for the detection of B[a]P in potable water. Fourteen monoclonal antibodies were generated in mice using novel B[a]P derivatives. The immunoassay with the least interference and the best sensitivity was optimized and characterized. As co-solvent, ten percent methanol (v/v) was determined as the optimum concentration for B[a]P solubilization for use with the developed ELISA. With the purified antibody (clone 22F12) the average IC50 for B[a]P and corresponding detection limit at a signal:noise (S/N) ratio of 3 was 65 ng L(-1) and 24 ng L(-1), respectively. From the 16 EPA-designated PAHs, only chrysene, indeno[1,2,3-cd]pyrene, and benzo[b]fluoranthene showed a cross-reactivity (CR) higher than 20%. No CR was observed for two- and three-ringed aromatics as well as dibenz[ah]anthracene and benzo[ghi]perylene. The effect of pH value (range 6.5-9.5), ionic strength (specific electric conductivity 1 microS cm(-1)-2.5 mS cm(-1)), and inorganic ions (sodium, copper, iron, aluminium, manganese, chloride, sulfate, nitrate, and nitrite at maximum permissible levels according to the Council Directive) on both signal and sensitivity of the ELISA was studied. No significant influence of these parameters on the ELISA competition curve was found. We suggest that the optimized ELISA can be used to monitor potable water samples without previous extraction from the samples. The assay should facilitate the cleanup of B[a]P contaminated sites where B[a]P levels fall close to the limit value of the new drinking water directive.

  16. Quantification of xylitol in foods by an indirect competitive immunoassay.

    PubMed

    Sreenath, Kundimi; Venkatesh, Yeldur P

    2010-01-27

    Sugar alcohols are widely used as food additives and drug excipients. d-Xylitol (INS 967), an important five-carbon sugar alcohol, is a natural constituent of many fruits and vegetables. The critical reagent for an immunoassay of haptens is the requirement of hapten-specific antibodies. Here, affinity-purified xylitol-specific antibodies generated earlier [Sreenath, K.; Venkatesh, Y. P. Reductively aminated D-xylose-albumin conjugate as the immunogen for generation of IgG and IgE antibodies specific to D-xylitol, a haptenic allergen. Bioconjugate Chem. 2007, 18, 1995-2003] have been utilized for developing an indirect competitive ELISA for xylitol. With xylitol-BSA conjugate as the coating antigen, a working range of 5-400 ng of xylitol could be determined in the immunoassay; the limit of detection was 1 ng of xylitol. Onion (Allium cepa) and strawberry (Fragaria nilgerrensis) were selected as the food sources containing D-xylitol. The amount of D-xylitol was found to be 12.6 and 44 mg/100 g fresh weight of onion and strawberry, respectively, and the results are in good agreement with the reported values by HPLC and GC. The recovery analyses showed that added amounts of D-xylitol were recovered fairly accurately with recoveries in the range of 89.2 to 94.9% in the case of onion, and 88.4 to 95.9% in the case of strawberry. The indirect competitive ELISA for xylitol quantification is a simple method using a 3 kDa ultrafiltrate of whole food extract, and does not require extensive sample preparation and derivatization as in the case of GC and HPLC analyses. This is the first immunoassay developed for the sugar alcohol, xylitol.

  17. Development and validation of an ELISA method for the quantification of nivolumab in plasma from non-small-cell lung cancer patients.

    PubMed

    Puszkiel, Alicja; Noé, Gaëlle; Boudou-Rouquette, Pascaline; Cossec, Chloé Le-; Arrondeau, Jennifer; Giraud, Jean-Stephane; Thomas-Schoemann, Audrey; Alexandre, Jérôme; Vidal, Michel; Goldwasser, François; Blanchet, Benoit

    2017-02-24

    Nivolumab, an anti PD-1 monoclonal antibody, has been approved for the treatment of previously treated advanced or metastatic non-small-cell lung cancer (NSCLC). The aim of this study was to develop and validate an ELISA method for the quantification of nivolumab in plasma from patients with NSCLC in order to perform future pharmacokinetic/pharmacodynamic (PK/PD) studies. A home-made ELISA was developed and validated according to the general recommendations for the immunoassays. Then, the ELISA method was applied to quantify plasma trough levels (Cmin) of nivolumab (3mg/kg every two weeks) in 27 NSCLC patients at days 14, 28 and 42 after start of treatment. Blood samples were collected just before the infusion on days 0 (baseline), 14, 28 and 42 after start of treatment. The dynamic calibration range for nivolumab assay was 5-100μg/mL. Within- and between-day imprecision for quality controls (5, 20 and 75μg/mL) were less than 5 and 12%, respectively. The mean (±standard deviation) nivolumab Cmin was 17.3±4.8μg/mL (coefficient of variation, CV=27.8%), 25.0±9.7μg/mL (CV=38.8%) and 33.0±12.9μg/mL (CV=39.1%) on days 14, 28 and 42, respectively. IgG (p=0.002) and ALT (p=0.041) were independently associated with plasma nivolumab Cmin at day 42. The present ELISA method for quantification of nivolumab in plasma from NSCLC patients is sensitive and accurate enough to be used for further PK/PD investigations.

  18. 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

  19. Lateral flow immunoassay with upconverting nanoparticle-based detection for indirect measurement of interferon response by the level of MxA.

    PubMed

    Juntunen, Etvi; Salminen, Teppo; Talha, Sheikh M; Martiskainen, Iida; Soukka, Tero; Pettersson, Kim; Waris, Matti

    2017-04-01

    Myxovirus resistance protein A (MxA) is a biomarker of interferon-induced gene expression state involved in many viral infections and some autoimmune disorders. It has a variety of potential utilities in clinical diagnostics, including distinguishing between bacterial and viral infections. Currently, MxA-assays are used for monitoring of IFN-β therapy in multiple sclerosis (MS) patients. As a proof-of-concept for rapid quantitative measurement of interferon response, a lateral flow immunoassay (LFIA) with upconverting nanoparticle (UCNP) reporters was developed and evaluated with clinical whole blood samples to assess the potential for a rapid and user-friendly quantitative assay for MxA, since the currently available rapid test for MxA (FebriDX) produces only qualitative result. The high detection sensitivity enabled by the UCNP reporter technology allowed the sample pre-treatment with dilution of whole blood into lysis buffer at a detectable analyte concentration. The assay can be done within 2 hr and the results correlate with the reference MxA-ELISA, which requires an overnight incubation. With 36 samples, R(2) for linear regression was 0.86. The assay detected 96% of the IFN-β responders with 89% specificity using a cut-off level of 100 μg/L for an elevated MxA-concentration. J. Med. Virol. 89:598-605, 2017. © 2016 Wiley Periodicals, Inc.

  20. Highly sensitive homogenous chemiluminescence immunoassay using gold nanoparticles as label

    NASA Astrophysics Data System (ADS)

    Luo, Jing; Cui, Xiang; Liu, Wei; Li, Baoxin

    2014-10-01

    Homogeneous immunoassay is becoming more and more attractive for modern medical diagnosis because it is superior to heterogeneous immunoassay in sample and reagent consumption, analysis time, portability and disposability. Herein, a universal platform for homogeneous immunoassay, using human immunoglobulin G (IgG) as a model analyte, has been developed. This assay relies upon the catalytic activity of gold nanoparticles (AuNPs) on luminol-AgNO3 chemiluminescence (CL) reaction. The immunoreaction of antigen and antibody can induce the aggregation of antibody-functionalized AuNPs, and after aggregation the catalytic activity of AuNPs on luminol-AgNO3 CL reaction is greatly enhanced. Without any separation steps, a CL signal is generated upon addition of a trigger solution, and the CL intensity is directly correlated to the quantity of IgG. The detection limit of IgG was estimated to be as low as 3 pg/mL, and the sensitivity was better than that of the reported AuNPs-based CL immunoassay for IgG.

  1. Lateral flow colloidal gold-based immunoassay for pesticide.

    PubMed

    Wang, Shuo; Zhang, Can; Zhang, Yan

    2009-01-01

    In recent years, immunochromatographic lateral flow test strips are used as a popular diagnostic tool. There are two formats (noncompetitive and competitive) in gold-based immunoassay. Noncompetitive gold-based immunoassay also called sandwich assay is applied for the detection of large molecular mass. For small molecular mass such as pesticide, competitive format of lateral flow colloidal gold-based immunoassay is described in this chapter. The preparation of gold colloidal and the conjugation between antibody and gold colloidal are described. Hi-flow plus nitrocellulose membranes are separately coated with goat anti-rabbit IgG (control line) and hapten-OVA conjugate (test line). Thus, the degree of intensity of color of the test line is the reverse of the concentration of pesticide in the sample and the visual result is immediately observable. Colloidal gold-based immunoassay can also be applied for multianalysis in one test strip if the detected targets show different physico-chemical properties and their haptens show great differences in chemical structure.

  2. Magnetic Beads-based Bioelectrochemical Immunoassay of Polycyclic Aromatic Hydrocarbons

    SciTech Connect

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

    2007-07-01

    A simple, rapid, and sensitive bioelectrochemical immunoassay method based on magnetic beads (MBs) has been developed to detect polycyclic aromatic hydrocarbons (PAHs). The principle of this bioassay is based on a direct competitive enzyme-linked immunosorbent assay using PAH-antibody-coated MBs and horseradish peroxidase (HRP)-labeled PAH (HRP-PAH). 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 immunoreaction among PAH-antibody-coated MBs, PAH analyte, and HRP-PAH. After a complete immunoassay, the HRP tracers attached to MBs were transferred to a substrate solution containing 3, 3´, 5, 5´- tetramethylbenzidine (TMB) and hydrogen peroxide (H2O2) for electrochemical detection. The voltammetric characteristics of the substrate were investigated, and the reduction peak current of TMB was used to quantify the concentration of PAH. The different parameters, including the amount of HRP-PAH conjugates, the enzyme catalytic reaction time, and the pH of the supporting electrolyte that governs the analytical performance of the immunoassay have been studied in detail and optimized. The detection limit of 50 pg mL-1 was obtained under optimum experimental conditions. The performance of this bioelectrochemical magnetic immunoassay was successfully evaluated with tap water spiked with PAHs, indicating that this convenient and sensitive technique offers great promise for decentralized environmental applications.

  3. 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.

  4. Detection of African Swine Fever Virus Antibodies in Serum and Oral Fluid Specimens Using a Recombinant Protein 30 (p30) Dual Matrix Indirect ELISA.

    PubMed

    Giménez-Lirola, Luis G; Mur, Lina; Rivera, Belen; Mogler, Mark; Sun, Yaxuan; Lizano, Sergio; Goodell, Christa; Harris, D L Hank; Rowland, Raymond R R; Gallardo, Carmina; Sánchez-Vizcaíno, José Manuel; Zimmerman, Jeff

    2016-01-01

    In the absence of effective vaccine(s), control of African swine fever caused by African swine fever virus (ASFV) must be based on early, efficient, cost-effective detection and strict control and elimination strategies. For this purpose, we developed an indirect ELISA capable of detecting ASFV antibodies in either serum or oral fluid specimens. The recombinant protein used in the ELISA was selected by comparing the early serum antibody response of ASFV-infected pigs (NHV-p68 isolate) to three major recombinant polypeptides (p30, p54, p72) using a multiplex fluorescent microbead-based immunoassay (FMIA). Non-hazardous (non-infectious) antibody-positive serum for use as plate positive controls and for the calculation of sample-to-positive (S:P) ratios was produced by inoculating pigs with a replicon particle (RP) vaccine expressing the ASFV p30 gene. The optimized ELISA detected anti-p30 antibodies in serum and/or oral fluid samples from pigs inoculated with ASFV under experimental conditions beginning 8 to 12 days post inoculation. Tests on serum (n = 200) and oral fluid (n = 200) field samples from an ASFV-free population demonstrated that the assay was highly diagnostically specific. The convenience and diagnostic utility of oral fluid sampling combined with the flexibility to test either serum or oral fluid on the same platform suggests that this assay will be highly useful under the conditions for which OIE recommends ASFV antibody surveillance, i.e., in ASFV-endemic areas and for the detection of infections with ASFV isolates of low virulence.

  5. Detection of African Swine Fever Virus Antibodies in Serum and Oral Fluid Specimens Using a Recombinant Protein 30 (p30) Dual Matrix Indirect ELISA

    PubMed Central

    Giménez-Lirola, Luis G.; Mur, Lina; Rivera, Belen; Mogler, Mark; Sun, Yaxuan; Lizano, Sergio; Goodell, Christa; Harris, D. L. Hank; Rowland, Raymond R. R.; Gallardo, Carmina; Sánchez-Vizcaíno, José Manuel; Zimmerman, Jeff

    2016-01-01

    In the absence of effective vaccine(s), control of African swine fever caused by African swine fever virus (ASFV) must be based on early, efficient, cost-effective detection and strict control and elimination strategies. For this purpose, we developed an indirect ELISA capable of detecting ASFV antibodies in either serum or oral fluid specimens. The recombinant protein used in the ELISA was selected by comparing the early serum antibody response of ASFV-infected pigs (NHV-p68 isolate) to three major recombinant polypeptides (p30, p54, p72) using a multiplex fluorescent microbead-based immunoassay (FMIA). Non-hazardous (non-infectious) antibody-positive serum for use as plate positive controls and for the calculation of sample-to-positive (S:P) ratios was produced by inoculating pigs with a replicon particle (RP) vaccine expressing the ASFV p30 gene. The optimized ELISA detected anti-p30 antibodies in serum and/or oral fluid samples from pigs inoculated with ASFV under experimental conditions beginning 8 to 12 days post inoculation. Tests on serum (n = 200) and oral fluid (n = 200) field samples from an ASFV-free population demonstrated that the assay was highly diagnostically specific. The convenience and diagnostic utility of oral fluid sampling combined with the flexibility to test either serum or oral fluid on the same platform suggests that this assay will be highly useful under the conditions for which OIE recommends ASFV antibody surveillance, i.e., in ASFV-endemic areas and for the detection of infections with ASFV isolates of low virulence. PMID:27611939

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

    SciTech Connect

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

    1990-09-01

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

  7. Integration of an optical CMOS sensor with a microfluidic channel allows a sensitive readout for biological assays in point-of-care tests.

    PubMed

    Van Dorst, Bieke; Brivio, Monica; Van Der Sar, Elfried; Blom, Marko; Reuvekamp, Simon; Tanzi, Simone; Groenhuis, Roelf; Adojutelegan, Adewole; Lous, Erik-Jan; Frederix, Filip; Stuyver, Lieven J

    2016-04-15

    In this manuscript, a microfluidic detection module, which allows a sensitive readout of biological assays in point-of-care (POC) tests, is presented. The proposed detection module consists of a microfluidic flow cell with an integrated Complementary Metal-Oxide-Semiconductor (CMOS)-based single photon counting optical sensor. Due to the integrated sensor-based readout, the detection module could be implemented as the core technology in stand-alone POC tests, for use in mobile or rural settings. The performance of the detection module was demonstrated in three assays: a peptide, a protein and an antibody detection assay. The antibody detection assay with readout in the detection module proved to be 7-fold more sensitive that the traditional colorimetric plate-based ELISA. The protein and peptide assay showed a lower limit of detection (LLOD) of 200 fM and 460 fM respectively. Results demonstrate that the sensitivity of the immunoassays is comparable with lab-based immunoassays and at least equal or better than current mainstream POC devices. This sensitive readout holds the potential to develop POC tests, which are able to detect low concentrations of biomarkers. This will broaden the diagnostic capabilities at the clinician's office and at patient's home, where currently only the less sensitive lateral flow and dipstick POC tests are implemented.

  8. Visible paper chip immunoassay for rapid determination of bacteria in water distribution system.

    PubMed

    Ma, Sai; Tang, Yanyan; Liu, Jingqing; Wu, Jianmin

    2014-03-01

    Paper chips for immunoassay were patterned by screen printing of polydimethylsiloxane (PDMS) or wax pencil drawing. The methods for paper chip patterning are cheap, convenient, rapid and suitable for most laboratories. The whole time for patterning a paper chip is no more than 10 min. Visible immunoassay for the detection of bacteria (Escherichia coli ) has been realized using the paper chip, on which the antibody for capturing E. Coli was immobilized on the detection zones of the paper chip, while the detection antibody was labeled with gold nanoparticles (AuNPs) as a signal reporter. After an immunological reaction, the AuNPs bound on the paper chip can effectively catalyse the reduction of silver ions during the silver enhancing step, generating a visible result that can be read by naked eyes. The quantitative results can be acquired by scanning the silver stained paper chip with a commercial scanner/or digital camera. The density of E. coli in water samples can be measured after calibrating the gray value of silver stained spots with the logarithmic number of bacteria. The time and reagents consumed on the paper chip immunoassay is much smaller than those of conventional ELISA, while the sensitivity of the paper chip immunoassay is comparable to conventional ELISA. The technology proposed in this work displays a great potential in the in-situ analysis when daily monitoring of water quality are required.

  9. Cross-reactivity of steroid hormone immunoassays: clinical significance and two-dimensional molecular similarity prediction

    PubMed Central

    2014-01-01

    Background Immunoassays are widely used in clinical laboratories for measurement of plasma/serum concentrations of steroid hormones such as cortisol and testosterone. Immunoassays can be performed on a variety of standard clinical chemistry analyzers, thus allowing even small clinical laboratories to do analysis on-site. One limitation of steroid hormone immunoassays is interference caused by compounds with structural similarity to the target steroid of the assay. Interfering molecules include structurally related endogenous compounds and their metabolites as well as drugs such as anabolic steroids and synthetic glucocorticoids. Methods Cross-reactivity of a structurally diverse set of compounds were determined for the Roche Diagnostics Elecsys assays for cortisol, dehydroepiandrosterone (DHEA) sulfate, estradiol, progesterone, and testosterone. These data were compared and contrasted to package insert data and published cross-reactivity studies for other marketed steroid hormone immunoassays. Cross-reactivity was computationally predicted using the technique of two-dimensional molecular similarity. Results The Roche Elecsys Cortisol and Testosterone II assays showed a wider range of cross-reactivity than the DHEA sulfate, Estradiol II, and Progesterone II assays. 6-Methylprednisolone and prednisolone showed high cross-reactivity for the cortisol assay, with high likelihood of clinically significant effect for patients administered these drugs. In addition, 21-deoxycortisol likely produces clinically relevant cross-reactivity for cortisol in patients with 21-hydroxylase deficiency, while 11-deoxycortisol may produce clinically relevant cross-reactivity in 11β-hydroxylase deficiency or following metyrapone challenge. Several anabolic steroids may produce clinically significant false positives on the testosterone assay, although interpretation is limited by sparse pharmacokinetic data for some of these drugs. Norethindrone therapy may impact immunoassay measurement

  10. Evaluation of lateral flow assay as a field test for investigation of brucellosis outbreak in an organized buffalo farm: A pilot study

    PubMed Central

    Shome, R.; Filia, G.; Padmashree, B. S.; Krithiga, N.; Sahay, Swati; Triveni, K.; Shome, B. R.; Mahajan, V.; Singh, Amarjit; Rahman, H.

    2015-01-01

    Aim: The aim was to evaluate lateral flow assay (LFA) as a field test for investigation of brucellosis outbreak in organized buffalo farm. Materials and Methods: A total of 153 serum samples were tested to detect the presence of brucella antibodies by LFA and three other serological tests i.e. rose bengal plate test (RBPT), protein G based indirect enzyme-linked immunoassay (iELISA), and competitive ELISA (cELISA). The performances of LFA and other serological tests were evaluated using OIE complaint cELISA as the gold standard. Results: Serological tests revealed 50% of the animals were seropositive for Brucella antibodies and correlated with clinical history of abortions, infertility, and productive failures. The newly developed assay showed 87.1% and 92.6% sensitivity and specificity, which was even higher than the specificity of RBPT. Conclusions: The investigation proved the potential usefulness of LFA for field diagnosis of brucellosis in the regions where laboratory facilities are limited. PMID:27047121

  11. Bupropion interference with immunoassays for amphetamines and LSD.

    PubMed

    Vidal, Christian; Skripuletz, Thomas

    2007-06-01

    A 50-year-old male patient suddenly had lost consciousness, although he had previously been healthy. On arrival at hospital seizures arose. The authors investigated a urine sample of the patient, and performed toxicological drug screening with immunochemical Cloned Enzyme Donor Immunoassay (CEDIA) assays. Positive findings for amphetamines and LSD could not be confirmed. Using gas chromatography/mass spectrometry (GC/MS), and liquid chromatography/mass spectrometry (LC/MS), the authors identified bupropion, a drug used to aid in smoking cessation, as the interfering compound, which may cause false-positive results for amphetamines and LSD using the CEDIA assays.

  12. Evaluation of the usefulness of an oxycodone immunoassay in combination with a traditional opiate immunoassay for the screening of opiates in urine.

    PubMed

    Gingras, Marie; Laberge, Marie-Hélène; Lefebvre, Michel

    2010-03-01

    Oxycodone is a semisynthetic opioid analgesic largely prescribed for post-operative and chronic pain management. The introduction of a slow release formulation of oxycodone has led to its frequent abuse and to an increase in emergency cases related to oxycodone overdose. Until recently, oxycodone testing has been confined to gas chromatography-mass spectrometry (GC-MS) analysis because the widely used automated opiate immunoassays poorly react to this compound. We investigated the utility of a new oxycodone immunoassay as a screening procedure to eliminate inappropriate GC-MS testing of negative urine specimens. We analyzed 96 urine specimens using GC-MS and two immunoassays, CEDIA((R)) opiates and DRI((R)) oxycodone assays from Microgenics, on a Hitachi 917 analyzer. The GC-MS allowed us to detect codeine, hydrocodone, hydromorphone, morphine, oxycodone, and oxymorphone following enzymatic hydrolysis and derivation by acetylation. The combination of the two immunoassays gave the best performance (98% sensitivity and specificity) when considering a positive result from GC-MS for any of the opiates. Considering positive GC-MS results for oxycodone or oxymorphone only, the oxycodone immunoassay resulted in two false-positives and one false-negative (50 ng/mL cutoff). Using these immunoassays for screening before GC-MS analysis provides a reduced opiate GC-MS workload without compromising quality.

  13. Fast, antigen-saving multiplex immunoassay to determine levels and avidity of mouse serum antibodies to pertussis, diphtheria, and tetanus antigens.

    PubMed

    Stenger, Rachel M; Smits, Mieke; Kuipers, Betsy; Kessen, Sabine F M; Boog, Claire J P; van Els, Cécile A C M

    2011-04-01

    To enhance preclinical evaluation of serological immune responses to the individual diphtheria, tetanus, and pertussis (DTP) components of DTP combination vaccines, a fast hexavalent bead-based method was developed. This multiplex immunoassay (MIA) can simultaneously determine levels of specific mouse serum IgG antibodies to P antigens P.69 pertactin (P.69 Prn), filamentous hemagglutinin (FHA), pertussis toxin (Ptx), and combined fimbria type 2 and 3 antigens (Fim2/3) and to diphtheria toxin (Dtx) and tetanus toxin (TT) in a single well. The mouse DTP MIA was shown to be specific and sensitive and to correlate with the six single in-house enzyme-linked immunosorbent assays (ELISAs) for all antigens. Moreover, the MIA was expanded to include avidity measurements of DTP antigens in a multivalent manner. The sensitivities of the mouse DTP avidity MIA per antigen were comparable to those of the six individual in-house avidity ELISAs, and good correlations between IgG concentrations obtained by both methods for all antigens tested were shown. The regular and avidity mouse DTP MIAs were reproducible, with good intra- and interassay coefficients of variability (CV) for all antigens. Finally, the usefulness of the assay was demonstrated in a longitudinal study of the development and avidity maturation of specific IgG antibodies in mice having received different DTP vaccines. We conclude that the hexaplex mouse DTP MIA is a specific, sensitive, and high-throughput alternative for ELISA to investigate the quantity and quality of serological responses to DTP antigens in preclinical vaccine studies.

  14. Development of a Specimen-Sparing Multichannel Bead Assay to Detect Antiparasite IgG4 for the Diagnosis of Schistosoma and Wuchereria Infections on the Coast of Kenya

    PubMed Central

    DuVall, Adam S.; Fairley, Jessica K.; Sutherland, Laura; Bustinduy, Amaya L.; Mungai, Peter L.; Muchiri, Eric M.; Malhotra, Indu; Kitron, Uriel; King, Charles H.

    2014-01-01

    To better delineate the impact of parasitic coinfection in coastal Kenya, we developed a novel specimen-sparing bead assay using multiplex flow immunoassay (MFI) technology to simultaneously measure serum or plasma immunoglobulin G4 (IgG4) against Brugia malayi antigen (BMA) and Schistosoma haematobium soluble worm antigen (SWAP). Properties of the bead assay were estimated by latent class analysis using data from S. haematobium egg counts/filarial rapid diagnostic cards (RDTs), parasite-specific enzyme-linked immunosorbent assays (ELISAs), and the multichannel IgG4 assay. For schistosomiasis, the bead assay had an estimated sensitivity of 81% and a specificity of 45%, and it was more sensitive than ELISA or urine egg counts for diagnosing infection. For filariasis, it had a sensitivity of 86% and a specificity of 39%, and it was more sensitive than ELISA or RDT. Measuring antibody by MFI is feasible and may provide more accurate epidemiological information than current parasitological tests, especially in the setting of low-intensity infections. PMID:24515945

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

    PubMed

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

    2014-02-06

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

  16. Development of an immunoassay to detect benzene adducts in hemoglobin

    SciTech Connect

    Grassman, J.A.

    1993-01-01

    The purpose of this project was to develop an immunoassay to detect the adducts formed in hemoglobin after exposure to benzene, which is known to cause bone marrow degeneration and acute myelogenous leukemia. The use of benzene-adduct detection as a biological monitoring method would permit measurement of low exposures and exposures sustained weeks earlier. The reactivity of hydroquinone, an important benzene metabolite, with blood proteins and amino acids was investigated in order to decide which antigens and analytes were likely to be suitable for immunoassay development. The second section determined the combination of benzene-metabolite and antigen need to produce an immunoassay with the requisite low detection limit and specificity. The immunoassays with the best performance were tested on hemoglobin from benzene-exposed mice. In vitro studies showed that hydroquinone efficiently formed adducts with erythrocyte membranes and hemoglobin but not with albumin. Adduction efficiency was greater in incubations using purified hemoglobin than whole blood. Cysteine accounted for 15 to 27% of the adducts formed by hydroquinone. The site of the other adducts were not identified although there was evidence that the hemoglobin heme was adducted. Adducts were found on only 1 of the 2 globin chains. Tryptic digestion of the globin failed to associate the adducts with a specific peptide. Antigens made from hydroquinone-adducted hemoglobin but not hydroquinone-adducted cysteines coupled to carrier proteins effectively elicited adduct-specific antibodies. Interference due to reactivity to hemoglobin was controlled by using uniform quantities of hemoglobin in all wells. The mid-range of the best assays were approximately 12 pmoles HQ per well. Antibodies directed toward hemoglobin adducted with the benzene metabolites phenol, catechol and 1,2,4-trihydroxybenzene were also made. The performance of the anti-1,2,4-trihydroxybenzene were suitable for quantitative immunoassays.

  17. Immunoassay for Detection of Infliximab in Whole Blood Using a Fiber-Optic Surface Plasmon Resonance Biosensor.

    PubMed

    Lu, Jiadi; Spasic, Dragana; Delport, Filip; Van Stappen, Thomas; Detrez, Iris; Daems, Devin; Vermeire, Séverine; Gils, Ann; Lammertyn, Jeroen

    2017-03-21

    Monitoring the concentration of a therapeutic drug antibody, infliximab (IFX), is recommended for enhancing its efficacy in patients with inflammatory bowel disease (IBD). However, IFX concentrations are currently determined in patients' serum/plasma, which requires sample preparation from blood, hence hampering the turnaround time. In this paper, we present a short immunoassay (10 min) using a fiber-optic surface plasmon resonance (FO-SPR) biosensor for detection of IFX spiked in 100-fold diluted serum, plasma, and whole blood. The calculated limits of detection (LOD) based on calibration curves were 1.42, 1.00, and 1.34 ng/mL, respectively, which coincides with expected IFX concentrations in diluted samples from IBD patients. A linear correlation was established among different matrixes, indicating that the matrix effect was insignificant. The established point-of-care (POC) FO-SPR bioassay was also used to measure IFX in 100-fold diluted extracts of dried blood spots (DBS), and LOD achieved was below 2 ng/mL. Although DBS might be ideal for POC, this is the first report of using an SPR biosensor for measuring DBS samples. Finally, the POC FO-SPR immunoassay was validated by using matching serum and plasma samples from five IBD patients. A Pearson correlation of 0.968 was obtained between serum and plasma samples. IFX concentrations determined with FO-SPR were compared to a clinically validated enzyme-linked immunosorbent assay (ELISA), resulting in excellent Pearson correlation and intraclass correlation coefficient, both being 0.99 for serum and plasma samples. In conclusion, this paper demonstrates that our FO-SPR biosensor can be used as a true POC diagnostic tool for determining IFX concentrations in a variety of matrixes.

  18. 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.

  19. Five-Antigen Fluorescent Bead-Based Assay for Diagnosis of Lyme Disease

    PubMed Central

    Hasenkampf, Nicole R.; Barnes, Mary B.; Didier, Elizabeth S.; Philipp, Mario T.; Tardo, Amanda C.

    2016-01-01

    The systematically difficult task of diagnosing Lyme disease can be simplified by sensitive and specific laboratory tests. The currently recommended two-tier test for serology is highly specific but falls short in sensitivity, especially in the early acute phase. We previously examined serially collected serum samples from Borrelia burgdorferi-infected rhesus macaques and defined a combination of antigens that could be utilized for detection of infection at all phases of disease in humans. The five B. burgdorferi antigens, consisting of OspC, OspA, DbpA, OppA2, and the C6 peptide, were combined into a fluorescent cytometric bead-based assay for the detection of B. burgdorferi antigen-specific IgG antibodies. Samples from Lyme disease patients and controls were used to determine the diagnostic value of this assay. Using this sample set, we found that our five-antigen multiplex IgG assay exhibited higher sensitivity (79.5%) than the enzyme immunoassay (EIA) (76.1%), the two-tier test (61.4%), and the C6 peptide enzyme-linked immunosorbent assay (ELISA) (77.2%) while maintaining specificity over 90%. When detection of IgM was added to the bead-based assay, the sensitivity improved to 91%, but at a cost of reduced specificity (78%). These results indicate that the rational combination of antigens in our multiplex assay may offer an improved serodiagnostic test for Lyme disease. PMID:26843487

  20. Production of monoclonal antibodies against hop-derived (Humulus lupulus L.) prenylflavonoids and the development of immunoassays.

    PubMed

    Wyns, Ciska; Derycke, Lara; Soenen, Bram; Bolca, Selin; Deforce, Dieter; Bracke, Marc; Heyerick, Arne

    2011-07-15

    Monoclonal antibodies against the hop-derived prenylated chalcone xanthohumol (X) and the prenylated flavonoids isoxanthohumol (IX) and 8-prenylnaringenin (8-PN) were developed. Carboxylic acid haptens of X, IX and 8-PN were synthesized by linking a spacer to their C4'-OH group followed by subsequent coupling to bovine serum albumin (BSA) to form conjugates that were employed as immunogens in BALB/c mice to raise antibodies. The monoclonal antibodies that were secreted from the established hybridoma cell lines proved, in cross-reactivity studies, to possess highly specific binding capacities in an optimized competitive indirect ELISA. The immunoassays make use of immunogen-coated microtiterplates and a peroxidase-labeled anti-mouse IgG(1) secondary antibody with ABTS as a chromogenic substrate. For X the IC(50) value derived from the standard curve was 62.91 ng mL(-1), and for both IX and 8-PN 37.15 ng mL(-1). The assay was validated for the quantitative analysis of X, IX and 8-PN in urine and serum. A simple sample pretreatment procedure using a diethyl ether extraction was optimized and the recoveries and matrix effects were assessed. The validity of the established assay was tested and mean inter- and intra-assay variations in urine were 2.32% and 1.91%, respectively for X, 6.24% and 2.39%, respectively for IX and 7.18% and 0.74%, respectively for 8-PN. In serum, the mean inter- and intra-assay variations were 8.90% and 1.37%, respectively for X, 6.13% and 1.57%, respectively for IX and 6.13% and 2.43%, respectively for 8-PN. Furthermore, the method demonstrated excellent accuracy and significant correlation with measurements by an established and validated HPLC-MS method.

  1. [Sensitivity and specificity of the ELISA Kit for the detection of antidobies to Junin virus].

    PubMed

    Pirozhkov, A P; Timofeev, M A; Borisevich, I V; Syromiatnikova, S I; Shatokhina, I V; Pantyukhov, V B; Kovalchuk, A V; Borisevich, S V

    2015-01-01

    The goal of this work was to describe methodological approaches to determination of sensitivity and specificity of the enzyme-linked immunosorbent assay kit (ELISA Kit) for detection of the specific anti-Junin virus (JV) antibody. Comparison of ELISA to plaque reduction neutralization test (PRNT) showed direct relationship between antibody titers in the samples of serum of immunized animals, determined by either PRNT or ELISA methods. The obtained results provided an opportunity to form the panels of positive and negative serum samples to determine the sensitivity and specificity of the ELISA Kit. Sensitivity of the ELISA Kit was at least 98% when studying the samples of serum of immunized guinea pigs and rabbits (determined as positive in PRNT). The sensitivity of the ELISA Kit was at least 68% when studying the samples determined by PNRT as uncertain positive. The specificity was 98%. The specificity of the ELISA Kit was 98%.

  2. A multiplexed immunoassay system based upon reciprocating centrifugal microfluidics

    NASA Astrophysics Data System (ADS)

    Noroozi, Zahra; Kido, Horacio; Peytavi, Régis; Nakajima-Sasaki, Rie; Jasinskas, Algimantas; Micic, Miodrag; Felgner, Philip L.; Madou, Marc J.

    2011-06-01

    A novel, centrifugal disk-based micro-total analysis system (μTAS) for low cost and high throughput semi-automated immunoassay processing was developed. A key innovation in the disposable immunoassay disk design is in a fluidic structure that enables very efficient micro-mixing based on a reciprocating mechanism in which centrifugal acceleration acting upon a liquid element first generates and stores pneumatic energy that is then released by a reduction of the centrifugal acceleration, resulting in a reversal of direction of flow of the liquid. Through an alternating sequence of high and low centrifugal acceleration, the system reciprocates the flow of liquid within the disk to maximize incubation/hybridization efficiency between antibodies and antigen macromolecules during the incubation/hybridization stage of the assay. The described reciprocating mechanism results in a reduction in processing time and reagent consumption by one order of magnitude.

  3. Enzyme immunoassays and related procedures in diagnostic medical virology

    PubMed Central

    Kurstak, Edouard; Tijssen, Peter; Kurstak, Christine; Morisset, Richard

    1986-01-01

    This review article describes several applications of the widely used enzyme immunoassay (EIA) procedure. EIA methods have been adapted to solve problems in diagnostic virology where sensitivity, specificity, or practicability is required. Concurrent developments in hybridoma and conjugation methods have increased significantly the use of these assays. A general overview of EIA methods is given together with typical examples of their use in diagnostic medical virology; attention is drawn to possible pitfalls. Recent advances in recombinant DNA technology have made it possible to produce highly specific nucleic acid probes that have a sensitivity approximately 100 times greater than that of EIA. Some applications of these probes are described. Although the non-labelled nucleic acid probes for use in the field are not as refined as non-labelled immunoassays, their range of applications is expected to expand rapidly in the near future. ImagesFig. 4 PMID:3533302

  4. Fluorescence polarization immunoassays for the quantification of caffeine in beverages.

    PubMed

    Oberleitner, Lidia; Grandke, Julia; Mallwitz, Frank; Resch-Genger, Ute; Garbe, Leif-Alexander; Schneider, Rudolf J

    2014-03-19

    Homogeneous fluorescence polarization immunoassays (FPIAs) were developed and compared for the determination of caffeine in beverages and cosmetics. FPIAs were performed in cuvettes in a spectrometer for kinetic FP measurements as well as in microtiter plates (MTPs) on a multimode reader. Both FPIAs showed measurement ranges in the μg/L range and were performed within 2 and 20 min, respectively. For the application on real samples, high coefficients of variations (CVs) were observed for the performance in MTPs; the CVs for FPIAs in cuvettes were below 4%. The correlations between this method and reference methods were satisfying. The sensitivity was sufficient for all tested samples including decaffeinated coffee without preconcentration steps. The FPIA in cuvettes allows a fast, precise, and automated quantitative analysis of caffeine in consumer products, whereas FPIAs in MTPs are suitable for semiquantitative high-throughput screenings. Moreover, specific quality criteria for heterogeneous assays were applied to homogeneous immunoassays.

  5. A multiplexed immunoassay system based upon reciprocating centrifugal microfluidics

    PubMed Central

    Noroozi, Zahra; Kido, Horacio; Peytavi, Régis; Nakajima-Sasaki, Rie; Jasinskas, Algimantas; Micic, Miodrag; Felgner, Philip L.; Madou, Marc J.

    2011-01-01

    A novel, centrifugal disk-based micro-total analysis system (μTAS) for low cost and high throughput semi-automated immunoassay processing was developed. A key innovation in the disposable immunoassay disk design is in a fluidic structure that enables very efficient micro-mixing based on a reciprocating mechanism in which centrifugal acceleration acting upon a liquid element first generates and stores pneumatic energy that is then released by a reduction of the centrifugal acceleration, resulting in a reversal of direction of flow of the liquid. Through an alternating sequence of high and low centrifugal acceleration, the system reciprocates the flow of liquid within the disk to maximize incubation/hybridization efficiency between antibodies and antigen macromolecules during the incubation/hybridization stage of the assay. The described reciprocating mechanism results in a reduction in processing time and reagent consumption by one order of magnitude. PMID:21721711

  6. A Practical Guide to Immunoassay Method Validation.

    PubMed

    Andreasson, Ulf; Perret-Liaudet, Armand; van Waalwijk van Doorn, Linda J C; Blennow, Kaj; Chiasserini, Davide; Engelborghs, Sebastiaan; Fladby, Tormod; Genc, Sermin; Kruse, Niels; Kuiperij, H Bea; Kulic, Luka; Lewczuk, Piotr; Mollenhauer, Brit; Mroczko, Barbara; Parnetti, Lucilla; Vanmechelen, Eugeen; Verbeek, Marcel M; Winblad, Bengt; Zetterberg, Henrik; Koel-Simmelink, Marleen; Teunissen, Charlotte E

    2015-01-01

    Biochemical markers have a central position in the diagnosis and management of patients in clinical medicine, and also in clinical research and drug development, also for brain disorders, such as Alzheimer's disease. The enzyme-linked immunosorbent assay (ELISA) is frequently used for measurement of low-abundance biomarkers. However, the quality of ELISA methods varies, which may introduce both systematic and random errors. This urges the need for more rigorous control of assay performance, regardless of its use in a research setting, in clinical routine, or drug development. The aim of a method validation is to present objective evidence that a method fulfills the requirements for its intended use. Although much has been published on which parameters to investigate in a method validation, less is available on a detailed level on how to perform the corresponding experiments. To remedy this, standard operating procedures (SOPs) with step-by-step instructions for a number of different validation parameters is included in the present work together with a validation report template, which allow for a well-ordered presentation of the results. Even though the SOPs were developed with the intended use for immunochemical methods and to be used for multicenter evaluations, most of them are generic and can be used for other technologies as well.

  7. Comparison of monolisa HCV Ag/Ab ULTRA with two anti-HCV assays for the detection of HCV infection in hospital setting.

    PubMed

    Yagci, Server; Padalko, Elizaveta

    2012-02-01

    In this study, we compared the performance of three serological assays (Monolisa HCV Ag/Ab ULTRA, Innotest HCV Ab IV enzyme immunoassay--EIA, and Ortho HCV 3.0 enzyme-linked immunosorbent assay--ELISA) for the detection of HCV infection. Ninety plasma samples were collected, representing 63 samples from groups at risk for acquiring HCV infection and 27 HCV RNA-positive samples. The results of Ortho HCV 3.0 ELISA, Innotest HCV Ab IV, and Monolisa HCV Ag/Ab ULTRA were fully concordant for 27 HCV RNA-positive samples. Ortho HCV 3.0 ELISA test and Innotest HCV Ab IV also gave the same results for risk groups, while three samples were found to be reactive by Monolisa HCV Ag/Ab ULTRA and were consequently found negative for HCV RNA. As two of the solely Monolisa HCV Ag/Ab ULTRA-positive samples were also hepatitis B s antigen (HBsAg)-positive, neutralization of HBsAg was performed but no arguments for the HBsAg interference were observed. In conclusion, the non-specific reactive signal was observed, in three samples using Monolisa HCV Ag/Ab ULTRA, to be negative by other serological assays, and observed to be negative in an HCV RNA assessment, a result that could not be attributed to the interference with HBsAg. In the context of diagnostic testing, no test for various HCV genotypes was observed to be superior to any other.

  8. New Commercially Available IgG Kits and Time-Resolved Fluorometric IgE Assay for Diagnosis of Allergic Bronchopulmonary Aspergillosis in Patients with Cystic Fibrosis

    PubMed Central

    Barrera, Coralie; Richaud-Thiriez, Bénédicte; Rocchi, Steffi; Rognon, Bénédicte; Roussel, Sandrine; Grenouillet, Frédéric; Laboissière, Audrey; Dalphin, Jean-Charles; Reboux, Gabriel

    2015-01-01

    Allergic bronchopulmonary aspergillosis (ABPA) is difficult to diagnose; diagnosis relies on clinical, radiological, pathological, and serological criteria. Our aim was to assess the performance of two new commercially available kits and a new in-house assay: an Aspergillus fumigatus enzyme-linked immunosorbent assay (ELISA) IgG kit (Bordier Affinity Products), an Aspergillus Western blotting IgG kit (LDBio Diagnostics), and a new in-house time-resolved fluorometric IgE assay (dissociation-enhanced lanthanide fluorescent immunoassay, or DELFIA) using recombinant proteins from an Aspergillus sp. recently developed by our laboratory for ABPA diagnosis in a retrospective study that included 26 cystic fibrosis patients. Aspergillus fumigatus-specific IgG levels measured by a commercial ELISA kit were in accordance with the level of precipitins currently used in our lab. The ELISA kit could accelerate and help standardize ABPA diagnosis. Aspergillus fumigatus-specific IgE levels measured by ImmunoCAP (Phadia) with A. fumigatus M3 antigen and by DELFIA with a purified protein extract of A. fumigatus were significantly correlated (P < 10−6). The results with recombinant antigens glucose-6-phosphate isomerase and mannitol-1-phosphate dehydrogenase were encouraging but must be confirmed with sera from more patients. The DELFIA is an effective tool that can detect specific IgE against more fungal allergens than can be detected with other commercially available tests. PMID:26698651

  9. A cell-based time-resolved fluorescence assay for selection of antibody reagents for G protein-coupled receptor immunohistochemistry.

    PubMed

    Su, Jui-Lan; Fornwald, Jim; Rivers, Philip; Goldsworthy, Susan; Looney, Noeleen A; Hanvey, Jeff; Plumpton, Chris; Parham, Janet; Romanos, Michael; Kost, Thomas A; Kull, Frederick C

    2004-08-01

    A cell-based time-resolved fluorescence (celTRF) immunoassay is described for pre-screening antibodies to G protein-coupled receptor (GPCR) peptides that predicts suitability for immunohistochemistry (IHC). Rat GPCRs were expressed in Saos-2 human osteosarcoma cells via recombinant baculoviruses designed for mammalian cell expression, i.e., the transduced cells were used as a "screening lawn". The lawn was fixed and permeabilized similarly to IHC tissue. The celTRF, a dissociation-enhanced lanthanide fluorescence immunoassay (DELFIA), employed Eu-labelled goat anti-rabbit IgG. It exhibited a broad dynamic range upon which enzyme-linked immunosorbant assay (ELISA)-positive affinity-purified anti-peptide antibody reagents were examined for specificity and potency. Over 150 anti-peptide reagents to 27 GPCRs were characterized. All celTRF-positive antibodies were found to be suitable for IHC, whereas ELISA alone did not predict IHC utility. Examples are illustrated with five rabbit anti-neuropeptide FF receptor 1 (NPFF1) antibodies, where a strong correlation between celTRF potency and IHC utility was observed in both applications. In contrast, two high anti-peptide ELISA titer but celTRF-negative antibodies failed to recognize the NPFF1 receptor in IHC. The celTRF assay was performed manually and in an automated fashion, in our case, using a Biomek FX station and Sami scheduling software. The celTRF is the first in vitro automated assay that offers confident pre-selection of antibodies for IHC and the versatility to accommodate the rapid screening of large numbers of GPCRs. The celTRF is readily applicable to other protein target classes.

  10. Measurement of urinary N-telopeptides and serum C-telopeptides from type I collagen using a lateral flow-based immunoassay.

    PubMed

    Lee, Kyoung Min; Lee, Min Ho; Chung, Chin Youb; Seong, Woo Kyeong; Lee, Sang Dae; Park, Moon Seok

    2012-12-24

    Measuring bone turnover markers could detect early stages of osteoporosis and early responses to anti-osteoporotic treatments. Currently, commonly used bone turnover markers, N-telopeptides (NTx) and C-telopeptides (CTx), are measured using ELISA tests, which demands time and increases cost. Bone turnover markers need to be measured more easily for general use. Lateral flow-based immunoassay would be an appropriate method for this context. This study was performed to investigate the precision of a newly developed lateral flow-based immunoassay for measuring the urinary NTx and serum CTx, and their correlations with ELISA measurements. Urine NTx and serum CTx concentrations were determined by photoscan of newly developed strips, using a lateral flow-based immunoassay for 36 subjects (mean age 66.2 years, SD 7.5 years; four males and 32 females). Repeated measurement of urinary NTx and serum CTx were performed three times, using this technology for a precision test. The correlation of the lateral flow-based immunoassay with the ELISA measurements was analyzed. Precision of the newly developed lateral flow based immunoassay was 0.974 (ICC, 95% confidence interval, 0.955 to 0.986) and 0.995 (ICC, 95% confidence interval, 0.991 to 0.997) for urinary NTx and serum CTx, respectively. The correlation of lateral flow based immunoassay with ELISA was 0.913 for urinary NTx and 0.872 for serum CTx. These results suggest that measuring the urinary NTx and serum CTx, using a lateral flow-based immunoassay, is a relevant method for point-of-care testing and screening of bone resorption markers.

  11. Undergraduate Laboratory Module for Implementing ELISA on the High Performance Microfluidic Platform

    ERIC Educational Resources Information Center

    Giri, Basant; Peesara, Ravichander R.; Yanagisawa, Naoki; Dutta, Debashis

    2015-01-01

    Implementing enzyme-linked immunosorbent assays (ELISA) in microchannels offers several advantages over its traditional microtiter plate-based format, including a reduced sample volume requirement, shorter incubation period, and greater sensitivity. Moreover, microfluidic ELISA platforms are inexpensive to fabricate and allow integration of…

  12. A Sensitive Amphotericin B Immunoassay for Pharmacokinetic and Distribution Studies

    PubMed Central

    Machard, Sophie; Theodoro, Frederic; Benech, Henri; Grognet, Jean-Marc; Ezan, Eric

    2000-01-01

    Since currently used assays of amphotericin B (AMB) lack sensitivity or are not easily adaptable in all laboratories, we have developed an enzyme immunoassay for AMB in biological fluids and tissues. Antibodies to AMB were raised in rabbits after administration of an AMB-bovine serum albumin conjugate. The enzymatic tracer was obtained by coupling AMB via its amino group to acetylcholinesterase (EC 3.1.1.7). These reagents were used for the development of a competitive immunoassay performed on microtitration plates. The limit of quantification was 100 pg/ml in plasma and 1 ng/g in tissues. The plasma assay was performed directly without extraction on a minimal volume of 0.1 ml. The intra- and interassay coefficients of variation were in the range of 5 to 17%, and the recoveries were 92 to 111% for AMB added to human plasma. The assay was applied to a pharmacokinetic study with mice given AMB intraperitoneally at the dose of 1 mg/kg. The drug distribution in selected compartments (plasma, liver, spleen, lung, and brain) was monitored until 72 h after administration. In conclusion, our assay is at least 100-fold more sensitive than previously described bioassays or chromatographic determinations of AMB and may be useful in studying the tissue pharmacokinetics of new AMB formulations and in drug monitoring in clinical situations. PMID:10681316

  13. Preparation of protein-like silver-cysteine hybrid nanowires and application in ultrasensitive immunoassay of cancer biomarker.

    PubMed

    Chen, Wenjuan; Zheng, Liyan; Wang, Meilan; Chi, Yuwu; Chen, Guonan

    2013-10-15

    Novel protein-like silver-cysteine hybrid nanowires (p-SCNWs) have been synthesized by a green, simple, nontemplate, seedless, and one-step aqueous-phase approach. AgNO3 and l-cysteine were dissolved in distilled water, forming Ag-cysteine precipitates and HNO3. Under vigorous stirring, the pH of the solution was rapidly adjusted to 9.0 by addition of concentrated sodium hydroxide solution, leading to quick dissolution of the Ag-cysteine precipitates and sudden appearance of white precipitates of p-SCNWs. The p-SCNWs are monodispersed nanowires with diameter of 100 nm and length of tens of micrometers, and have abundant carboxyl (-COOH) and amine (-NH2) groups at their surfaces, large amounts of peptide-linkages and S-bonding silver ions (Ag(+)) inside, making them look and act like Ag-hybrid protein nanostructures. The abundant -COOH and -NH2 groups at the surfaces of p-SCNWs have been found to facilitate the reactions between the p-SCNWs and proteins including antibodies. Furthermore, the fact that the p-SCNWs contain large amounts of silver ions enables biofunctionalized p-SCNWs to be excellent signal amplifying chemiluminescence labels for ultrasensitive and highly selective detection of important antigens, such as cancer biomarkers. In this work, the immunoassay of carcinoembryonic antigen (CEA) in human serum was taken as an example to demonstrate the immunoassay applications of antibody-functionalized p-SCNWs. By the novel p-SCNW labels, CEA can be detected in the linear range from 5 to 400 fg/mL with a limit of detection (LOD) of 2.2 fg/mL (at signal-to-noise ratio of 3), which is much lower than that obtained by commercially available enzyme-linked immunosorbent assay (ELISA). Therefore, the synthesized p-SCNWs are envisioned to be an excellent carrier for proteins and related immunoassay strategy would have promising applications in ultrasensitive clinical screening of cancer biomarkers for early diagnostics of cancers.

  14. Analysis of urinary drugs of abuse by a multianalyte capillary electrophoretic immunoassay.

    PubMed

    Caslavska, J; Allemann, D; Thormann, W

    1999-04-09

    This paper characterizes a novel multianalyte competitive binding, electrokinetic capillary-based immunoassay for urinary methadone, opiates, benzoylecgonine (cocaine metabolite) and amphetamines. After incubation of 25 microliters urine with the reactants for several minutes in the presence of an internal standard, a small aliquot of the mixture is applied onto a fused-silica capillary and the unbound fluorescein labelled drug tracers are monitored by capillary electrophoresis with on-column laser induced fluorescence detection. The multianalyte assay is shown to be rapid, simple, quantitative, capable of recognizing urinary drug concentrations > or = 30 ng/ml and suitable for screening of patient urines. Data are demonstrated to compare well with those obtained by routine screening methods based on enzyme multiplied immunoassay techniques and fluorescence polarization immunoassays. The electrokinetic capillary assay has been validated via analysis of external quality control urines and confirmation analysis of patient urines using GC-MS.

  15. Development and validation of a sensitive enzyme immunoassay for surveillance of Cry1Ab toxin in bovine blood plasma of cows fed Bt-maize (MON810).

    PubMed

    Paul, Vijay; Steinke, Kerstin; Meyer, Heinrich H D

    2008-01-21

    The increasing global adoption of genetically modified (GM) plant derivatives in animal feed has provoked a strong demand for an appropriate detection method to evaluate the existence of transgenic protein in animal tissues and animal by-products derived from GM plant fed animals. A highly specific and sensitive sandwich enzyme immunoassay for the surveillance of transgenic Cry1Ab protein from Bt-maize in the blood plasma of cows fed on Bt-maize was developed and validated according to the criteria of EU-Decision 2002/657/EC. The sandwich assay is based on immuno-affinity purified polyclonal antibody raised against Cry1Ab protein in rabbits. Native and biotinylated forms of this antibody served as capture antibody and detection antibody for the ELISA, respectively. Streptavidin-horseradish peroxidase conjugate and TMB substrate provided the means for enzymatic colour development. The immunoassay allowed Cry1Ab protein determination in bovine blood plasma in an analytical range of 0.4-100 ng mL(-1) with a decision limit (CCalpha) of 1.5 ng mL(-1) and detection capability (CCbeta) of 2.3 ng mL(-1). Recoveries ranged from 89 to 106% (mean value of 98%) in spiked plasma. In total, 20 plasma samples from cows (n=7) fed non-transgenic maize and 24 samples from cows (n=8) fed transgenic maize (collected before and, after 1 and 2 months of feeding) were investigated for the presence of the Cry1Ab protein. There was no difference amongst both groups (all the samples were below 1.5 ng mL(-1); CCalpha). No plasma sample was positive for the presence of the Cry1Ab protein at CCalpha and CCbeta of the assay.

  16. Immunoassay of haemoglobin-acrylonitrile adduct in rat as a biomarker of exposure.

    PubMed

    L Wong Yu Ting Zheng Junyu Li Carlo H Tamburro Frederick W Benz, J

    1998-01-01

    Acrylonitrile (AN) is a rat carcinogen. Human exposure may come from chemical industries and smoking. A haemoglobin adduct of acrylonitrile (Hb-AN) has been used as a biomarker of exposure by means of gas chromatography-mass spectrometry (GC-MS) analysis. We have developed specific monoclonal antibodies (Mab) to human Hb-AN and wish to report evaluation of an immunoassay in rats using an Mab that cross-reacts with rat Hb-AN. A dose response study of LD 0, 10, 50, and 90 in Sprague-Dawley rats was undertaken, with each rat receiving \\[2,3-14C]AN at 50 Ci kg-1 sc, and Hb from an aliquot of blood was taken for covalent binding analysis by liquid scintillation spectrometry and fluorescence ELISA. The dose responses of rats at 0.25, 0.5, 1.0, and 2.0 h after AN doses of 20, 50, 80, 115 mg kg-1 were compared by both methods with Hb and globin samples. Regression analysis showed a linear relationship between immunoassay and 14C-AN binding. This indicates that an antigenic form of Hb-AN may be used as a surrogate of Hb-AN adduct. The sensitivity of ELISA was tested in rats exposed for 1 h to sub-toxic doses of AN (10-1.1 mg kg-1). Quantification of Hb-AN by immunoassay was achieved by calibration with a synthetic adduct HbAN4h, a reference adduct prepared by treating rat Hb with excess AN for 4 h. ELISA and GC-MS analysis of N-terminal valine-AN in the Hb-AN adduct were compared and similar detection levels were found. This rat study appears to have validated the new immunoassay method for biomonitoring of AN exposure.

  17. Detection of Shiga toxin-producing Escherichia coli by sandwich enzyme-linked immunosorbent assay using chicken egg yolk IgY antibodies

    PubMed Central

    Parma, Y. R.; Chacana, P. A.; Lucchesi, P. M. A.; Rogé, A.; Granobles Velandia, C. V.; Krüger, A.; Parma, A. E.; Fernández-Miyakawa, M. E.

    2012-01-01

    Enterohemorrhagic Escherichia coli (EHEC), a subset of Shiga toxin producing E. coli (STEC) is associated with a spectrum of diseases that includes diarrhea, hemorrhagic colitis and a life-threatening hemolytic-uremic syndrome (HUS). Regardless of serotype, Shiga toxins (Stx1 and/or Stx2) are uniformly expressed by all EHEC, and so exploitable targets for laboratory diagnosis of these pathogens. In this study, a sandwich ELISA for determination of Shiga toxin (Stx) was developed using anti-Stx2B subunit antibodies and its performance was compared with that of the Vero cell assay and a commercial immunoassay kit. Chicken IgY was used as capture antibody and a HRP-conjugated rabbit IgG as the detection antibody. The anti-Stx2B IgY was harvested from eggs laid by hens immunized with a recombinant protein fragment. Several parameters were tested in order to optimize the sandwich ELISA assay, including concentration of antibodies, type and concentration of blocking agent, and incubation temperatures. Supernatants from 42 STEC strains of different serotypes and stx variants, including stx2EDL933, stx2vha, stx2vhb, stx2g, stx1EDL933, and stx1d were tested. All Stx variants were detected by the sandwich ELISA, with a detection limit of 115 ng/ml Stx2. Twenty three strains negative for stx genes, including different bacteria species, showed no activity in Vero cell assay and produced negative results in ELISA, except for two strains. Our results show that anti-Stx2B IgY sandwich ELISA could be used in routine diagnosis as a rapid, specific and economic method for detection of Shiga toxin-producing E. coli. PMID:22919675

  18. Method evaluation study of a new generation of vitamin D assays

    PubMed Central

    Kriegshäuser, Gernot; Stolba, Robert; Worf, Elfriede; Halwachs-Baumann, Gabriele

    2015-01-01

    Introduction Recently several diagnostic manufacturers have launched new 25-hydroxy-vitamin D (25[OH]D) assays, which are aligned to the National Institute of Standards and Technology (NIST) Standard Reference Materials (SRM) (NIST, Gaithersburg, Maryland). The aim of this study was to compare the performance of one liquid chromatography-tandem mass spectrometry (LC-MS/MS) method, one enzyme linked immunosorbent assay (ELISA), and one recalibrated and previous version of a chemiluminescence immunoassay (CLIA). Material and methods Serum-aliquots of 198 patient samples from routine 25(OH)D analysis were measured by the ClinMass® LC-MS/MS Complete Kit (RECIPE Chemicals + Instruments GmbH, Munich, Germany), the ORGENTEC 25(OH)D3/D2 ELISA (ORGENTEC Diagnostika GmbH, Mainz, Germany), the recalibrated Immunodiagnostic Systems (IDS)-iSYS 25(OH)DS and the previous used IDS-iSYS 25(OH)D CLIA (Immunodiagnostic Systems Ltd, Boldon, United Kingdom). Bland-Altman and Deming regression analyses were calculated for methods comparison of all tested 25(OH)D assays. The LC-MS/MS method was defined as the reference method. Within-run and between-run precision measurements were performed for all methods with three different concentration levels. Results Compared to the LC-MS/MS method, the new IDS-iSYS 25(OH)DS and ORGENTEC 25(OH)D3/D2 assay demonstrated mean relative biases of 16.3% and 17.8%. The IDS-iSYS 25(OH)D assay showed the lowest mean bias of 1.5%. Deming regression analyses of the recalibrated IDS-iSYS 25(OH)DS and the ORGENTEC 25(OH)D3/D2 assay showed proportional differences, when compared to the reference method. All assays showed a within-run and between-run imprecision of ≤ 20% at each of the evaluated concentration levels. Conclusions The evaluated standardized immunoassays and LC-MS/MS are useful methods for measuring 25(OH)D serum-levels in clinical laboratories. PMID:26110032

  19. Immunoassays as high-throughput tools: monitoring spatial and temporal variations of carbamazepine, caffeine and cetirizine in surface and wastewaters.

    PubMed

    Bahlmann, Arnold; Carvalho, José João; Weller, Michael G; Panne, Ulrich; Schneider, Rudolf J

    2012-11-01

    Carbamazepine (CBZ), caffeine and cetirizine were monitored by enzyme-linked immunosorbent assays (ELISAs) in surface and wastewaters from Berlin, Germany. This fast and cost-efficient method enabled to assess the spatial and temporal variation of these anthropogenic markers in a high-throughput screening. CBZ and cetirizine were detected by the same antibody, which selectively discriminates between both compounds depending on the pH value used in the incubation step. To our best knowledge, this is the first dual-analyte immunoassay working with a single antibody. The frequent sampling with 487 samples being processed allowed for the repeated detection of unusually high concentrations of CBZ and caffeine. ELISA results correlate well with the ones obtained by liquid chromatography tandem mass spectrometry (LC-MS/MS). Caffeine concentrations found in surface waters were elevated by combined sewer overflows after stormwater events. During the hay fever season, the concentrations of the antihistamine drug cetirizine increased in both surface and wastewaters. Caffeine was almost completely removed during wastewater treatment, while CBZ and cetirizine were found to be more persistent. The maximum concentrations of caffeine, CBZ and cetirizine found in influent wastewater by LC-MS/MS were 470, 5.0 and 0.49 μg L(-1), while in effluent wastewater the concentrations were 0.22, 4.5 and 0.51 μg L(-1), respectively. For surface waters, concentrations up to 3.3, 4.5 and 0.72 μg L(-1) were found, respectively.

  20. A Protein Microarray ELISA for the Detection of Botulinum neurotoxin A

    SciTech Connect

    Varnum, Susan M.

    2007-06-01

    An enzyme-linked immunosorbent assay (ELISA) microarray was developed for the specific and sensitive detection of botulinum neurotoxin A (BoNT/A), using high-affinity recombinant monoclonal antibodies against the receptor binding domain of the heavy chain of BoNT/A. The ELISA microarray assay, because of its sensitivity, offers a screening test with detection limits comparable to the mouse bioassay, with results available in hours instead of days.

  1. Lateral flow assays

    PubMed Central

    Koczula, Katarzyna M.

    2016-01-01

    Lateral flow assays (LFAs) are the technology behind low-cost, simple, rapid and portable detection devices popular in biomedicine, agriculture, food and environmental sciences. This review presents an overview of the principle of the method and the critical components of the assay, focusing on lateral flow immunoassays. This type of assay has recently attracted considerable interest because of its potential to provide instantaneous diagnosis directly to patients. The range and interpretation of results and parameters used for evaluation of the assay will also be discussed. The main advantages and disadvantages of LFAs will be summarized and relevant future improvements to testing devices and strategies will be proposed. Finally, the major recent advances and future diagnostic applications in the LFA field will be explored. PMID:27365041

  2. Heterogeneous immunoassays using magnetic beads on a digital microfluidic platform.

    PubMed

    Sista, Ramakrishna S; Eckhardt, Allen E; Srinivasan, Vijay; Pollack, Michael G; Palanki, Srinivas; Pamula, Vamsee K

    2008-12-01

    A digital microfluidic platform for performing heterogeneous sandwich immunoassays based on efficient handling of magnetic beads is presented in this paper. This approach is based on manipulation of discrete droplets of samples and reagents using electrowetting without the need for channels where the droplets are free to move laterally. Droplet-based manipulation of magnetic beads therefore does not suffer from clogging of channels. Immunoassays on a digital microfluidic platform require the following basic operations: bead attraction, bead washing, bead retention, and bead resuspension. Several parameters such as magnetic field strength, pull force, position, and buffer composition were studied for effective bead operations. Dilution-based washing of magnetic beads was demonstrated by immobilizing the magnetic beads using a permanent magnet and splitting the excess supernatant using electrowetting. Almost 100% bead retention was achieved after 7776-fold dilution-based washing of the supernatant. Efficient resuspension of magnetic beads was achieved by transporting a droplet with magnetic beads across five electrodes on the platform and exploiting the flow patterns within the droplet to resuspend the beads. All the magnetic-bead droplet operations were integrated together to generate standard curves for sandwich heterogeneous immunoassays on human insulin and interleukin-6 (IL-6) with a total time to result of 7 min for each assay.

  3. Validation of two new immunoassays for sensative detection of a broad range of shiga toxins

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The objective of this study was to evaluate two newly developed commercial assays, Abraxis Stx1 and Stx2 enzyme-linked immunosorbent assays (ELISAs), for their ability to detect Shiga toxin (Stx) produced by Escherichia coli (E. coli). The performance of these two assays were compared to a widely us...

  4. Development and evaluation of a gamma-interferon assay for tuberculosis in badgers (Meles meles).

    PubMed

    Dalley, Deanna; Davé, Dipesh; Lesellier, Sandrine; Palmer, Simonette; Crawshaw, Timothy; Hewinson, R Glyn; Chambers, Mark

    2008-05-01

    In this paper we report the development of a sensitive and specific assay for the detection of tuberculosis (TB) in European badgers (Meles meles), based on the stimulation of lymphocytes in whole-blood culture and the subsequent detection of gamma-interferon (IFNgamma) by sandwich ELISA. The comparative levels of IFNgamma produced to bovine and avian tuberculin (B-A) was used as the basis of determining the TB status of badgers, resulting in a more sensitive test than that based on the defined Mycobacterium bovis antigens ESAT6 and CFP10. The assay was evaluated using 235 badgers. The IFNgamma EIA (enzyme immunoassay) based on a monoclonal pair (mEIA) was more sensitive than one using a rabbit polyclonal antiserum (pEIA). At a specificity of 93.6%, the mEIA was 80.9% sensitive, compared to a sensitivity of 74.5% for the pEIA. At the same specificity as the EIA, the current serological ELISA test for TB in badgers (Brock test) had a sensitivity of 48.9%. Only one of the culture positive badgers missed by the mEIA was correctly diagnosed by the Brock test, suggesting that the combination of both a T-cell and serological test has little diagnostic advantage.

  5. CCQM-P58.1: Immunoassay Quantitation of Human Cardiac Troponin I.

    NASA Astrophysics Data System (ADS)

    Bunk, David; Noble, James; Knight, Alex E.; Wang, Lili; Klauenberg, Katy; Walzel, Monika; Elster, Clemens

    2015-01-01

    The CCQM study P58.1 assessed the equivalence of immunoassay measurements between participating NMIs. The aim of P58.1 was to demonstrate the equivalence of immunoassay measurements to determine the mass concentration of the clinically-relevant protein human cardiac troponin I (cTnI) present at low concentration relative to the protein concentration of the sample matrix. The measurement equivalence was assessed using traceability to a common certified reference material. To quantify cTnI, participants used a homogeneous sandwich-based immunoassay with an enzymatic amplification step. The antibody format consisted of a single capture and single detection antibody (referred to as 1 + 1), both were supplied to study participants. In the previous P58 study, ELISA measurement results were compared between laboratories which all used common ELISA reagents (including 96-well plates), samples, a standard for the production of calibrants, and a detailed ELISA protocol, which were supplied by a single laboratory. The P58.1 study only utilized common samples, a standard of the production of calibrants, and a set of monoclonal antibodies (mAbs). Because much of the experimental procedure for the P58 study was essentially standardized across participating labs, the study primarily highlighted between-laboratory differences in plate sampling designs and in plate reader response. As the participants in the P58.1 study had to produce most of their own analytical reagents and develop their own measurement procedure, the study provides a better evaluation of the equivalence of ELISA measurements between the participating laboratories. Main text. To reach the main text of this paper, click on Final Report The final report has been peer-reviewed and approved for publication by CCQM.

  6. An evaluation of two commercially available ELISAs and one in-house reference laboratory ELISA for the determination of human anti-rabies virus antibodies.

    PubMed

    Welch, Ryan J; Anderson, Brian L; Litwin, Christine M

    2009-06-01

    The envelope glycoprotein G of rabies virus in vaccines induces the production of neutralizing antibodies important in the protection against the disease. The measurement of anti-envelope glycoprotein antibodies is a good predictor of the degree of humoral immunity in people during anti-rabies treatment or after vaccination. Several assays exist for the serological determination of antibody protection against rabies virus infection. Antibody neutralization by the rapid fluorescent focus inhibition test (RFFIT) or the fluorescent antibody virus neutralization (FAVN) test is currently the gold standard. Performance of the highly complex RFFIT and FAVN tests, however, requires specialized reference laboratories with expertise with this assay. Although not widely used, ELISA test kits are available and may be an additional option for testing that is more accessible. The aim of the present study was to evaluate available ELISA assays for the determination of anti-rabies antibodies. We compared the Bio-Rad Platelia Rabies II ELISA, DRG Rabies Virus IgG Ab ELISA and Focus Diagnostics Rabies Antibody Detection by ELISA to RFFIT. Bland-Altman plots comparing the Bio-Rad Platelia assay and the Focus Diagnostics assay to RFFIT showed a low degree of variability between the ELISA assays and RFFIT results except in samples with high RFFIT values. The agreement, sensitivity and specificity of Bio-Rad Platelia Rabies II ELISA when compared to RFFIT were 95.1 %, 94.1 % and 95.8 %, respectively. The DRG Rabies assay compared to RFFIT had an agreement of 77.7 %, a sensitivity of 86.7 % and a specificity of 69.4 %. The agreement, sensitivity and specificity of Focus Diagnostics Rabies Detection by ELISA when compared to RFFIT were 82.2 %, 91.7 % and 73.0 %, respectively. Overall, the Bio-Rad Platelia assay showed higher accuracy and specificity than either the DRG or Focus assays. All of these ELISAs, however, measure all antibody types and do not discriminate the neutralizing

  7. Production of anti-idiotype antibodies for deoxynivalenol and their evaluation with three immunoassay platforms

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Immunoassays for deoxynivalenol (DON) that involve the competition for binding to DON-specific antibodies have been widely developed. In such assays, the responses of samples are generally compared to calibration curves generated by using DON in competition with labeled reagents such as enzymatic or...

  8. Fluorescence polarization immunoassays for rapid, accurate and sensitive determination of mycotoxins

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Fluorescence polarization immunoassay (FPIA) is a type of homogeneous assay. For low molecular weight antigens, such as mycotoxins, it is based on the competition between an unlabeled antigen and its fluorescent-labeled derivative (tracer) for an antigen-specific antibody. The antigen content is det...

  9. Clinical evaluation of a chemiluminescence immunoassay for determination of immunoglobulin g avidity to human cytomegalovirus.

    PubMed

    Revello, Maria Grazia; Gorini, Giovanna; Gerna, Giuseppe

    2004-07-01

    Clinical evaluation of a novel fully automated chemiluminescence immunoassay for determination of immunoglobulin G avidity to human cytomegalovirus (HCMV) showed 92.8% sensitivity and 84.7% specificity in detecting a recent (< or =90 days) primary HCMV infection. The assay appears useful for accurately diagnosing recent primary HCMV infections.

  10. 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...

  11. Determination of alachlor and its sulfonic acid metabolite in water by solid-phase extraction and enzyme-linked immunosorbent assay

    USGS Publications Warehouse

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

    1994-01-01

    Solid-phase extraction (SPE) and enzyme-linked immunosorbent assay (ELISA) were combined for the trace analysis of the herbicide alachlor and its major soil metabolite, ethanesulfonic acid (ESA). The anti-alachlor antibody cross-reacted with ESA, which produced false-positive detections of alachlor in water samples by immunoassay screens. Alachlor and ESA were isolated from water by SPE on a C18 resin and eluted sequentially with ethyl acetate and methanol. Alachlor is soluble in ethyl acetate while the anionic ESA is not. Thus ESA remained adsorbed on the C18 resin and was eluted later with methanol. The combination of SPE with ELISA effectivety separated and quantified both alachlor and ESA using the same antibody for two ELISA methods. The general method may have applicability for the separation of other herbicides and their ionic metabolites. The SPE-ELISA method has a, detection limit of 0.01 ??g/L for alachlor and 0.05 ??g/L for ESA, with a precision of ?? 10%. Analyses of surface and ground water samples were confirmed by gas chromatography/mass spectrometry and high-performance liquid chromatography with photodiode-array detection. Results showed widespread occurrence of ESA in surface and ground water of the midwestern United States, with concentrations ranging from 10 ??g/L.

  12. Study of two different enzyme immunoassays for the detection of Mayaro virus antibodies.

    PubMed

    Figueiredo, L T; Nogueira, R M; Cavalcanti, S M; Schatzmayr, H; da Rosa, A T

    1989-01-01

    This paper presents the evaluation of an enzyme immunoassay in which Mayaro virus-infected cultured cells are used as antigen (EIA-ICC) and an IgM antibody capture ELISA (MAC-ELISA) for Mayaro serologic diagnosis using 114 human sera obtained during a Mayaro outbreak occurred in Bolivia, in 1987. Results were compared with those obtained by haemagglutination-inhibition test (HAI). MAC-ELISA was the most sensitive technique for anti-Mayaro IgM detection. MAC-ELISA was twice as sensitive as IgM EIA-ICC. The data shows that MAC-ELISA is a practical and valid technique for diagnosis of recent Mayaro infection. IgG EIA-ICC showed high sensitivity and high specificity compared to HAI. The combination of anti-Mayaro IgG and IgM EIA-ICC results presented the highest sensitivity of the study. Anti-Mayaro IgG and IgM simultaneous detection by EIA-ICC can be used for recent infection diagnosis (in spite of a less sensitive IgM detection than by MAC-ELISA), for surveillance and sero-epidemiologic studies, and for studies of IgG and IgM responses to Mayaro infection.

  13. A Novel Signal-Amplified Immunoassay for the Detection of C-Reactive Protein Using HRP-Doped Magnetic Nanoparticles as Labels with the Electrochemical Quartz Crystal Microbalance as a Detector.

    PubMed

    Gan, Ning; Xiong, Ping; Wang, Ji; Li, Tianhua; Hu, Futao; Cao, Yuting; Zheng, Lei

    2013-01-01

    A novel horseradish peroxidase- (HPR-) doped magnetic core-shell Fe3O4@SiO2@Au nanocomposites (Fe-Au MNPs) were employed on immunoassay for the determination of C-reactive protein (CRP) based on a electrochemical quartz crystal microbalance detector (EQCM). Firstly, the secondary CRP antibody and HRP were both immobilized on the Fe-Au MNPs (Fe-Au MNPs-anti-CRP2/HRP) as a signal tag. Secondly, the above tag and the primary antibody (anti-CRP1) in the bottom of 96-well microtiter plate were employed to conjugate with a serial of CRP concentrations to produce a sandwich immunocomplex. Thirdly, the immunocomplex solution was subsequently exposed to 3, 3'-diaminobenzidine (DAB) in the presence of H2O2, resulting in an insoluble product. When the precipitation solution was dripped on EQCM, it can achieve a decrease of frequency of crystal (Δf). The amount of Δf was proportional to (CRP) from 0.003 to 200 ng mL(-1) with a low detection limit of 1 pg mL(-1). Compared with the enzyme-linked immunosorbent assay (ELISA), the immunoassay shows greatly improved sensitivity due to the significant amount of HRP labeled on signal tag. It also has good specificity and low sample consumption, which is expected to be a benefit for the CRP screening in early diagnosis of cardiovascular disease.

  14. Stool diagnosis of giardiasis using a commercially available enzyme immunoassay to detect Giardia-specific antigen 65 (GSA 65).

    PubMed

    Rosoff, J D; Sanders, C A; Sonnad, S S; De Lay, P R; Hadley, W K; Vincenzi, F F; Yajko, D M; O'Hanley, P D

    1989-09-01

    A commercially available enzyme immunoassay for the diagnosis of giardiasis was evaluated in a clinical trial. The ProSpecT/Giardia diagnostic test (Alexon, Inc., Mountain View, Calif.) was compared with the standard ova and parasite (O&P) microscopic examination. Additionally, several widely used stool fixatives and a commonly used transport medium were assessed for compatibility with the immunoassay. A total of 325 stool specimens were collected and used to evaluate assay performance. Of those, 93 specimens were collected from symptomatic Giardia O&P-positive patients and 232 specimens were randomly collected from patients as part of a routine health screening procedure. All 93 Giardia O&P-positive stool specimens were strongly positive by visual and spectrophotometric examination using the immunoassay. Of the 232 randomly collected specimens, 16 were positive by O&P examination and immunoassay, 6 were negative by O&P examination but positive by immunoassay, and 1 was positive by O&P examination and negative by immunoassay. There was substantial supportive evidence that indicated that the six immunoassay-positive, O&P-negative specimens were true-positives. When these six specimens were accepted as true-positives, the immunoassay detected almost 30% more cases of Giardia infection than did O&P examination. Its sensitivity and specificity were 96 and 100%, respectively, while the sensitivity and specificity of O&P examination were 74 and 100%, respectively. The immunoassay also performed well on specimens treated with 10% neutral Formalin, sodium acetate-Formalin fixative, and Cary-Blair transport medium. However, the test was not compatible with polyvinyl alcohol-treated specimens. Overall, the ProSpecT/Giardia test was a sensitive, specific immunoassay which was easy to run and interpret. It offers a simple solution to traditional difficulties encountered in diagnosing Giardia infection.

  15. Application Of Laser Fluorimetry To Enzyme-Linked Immunoassay

    NASA Astrophysics Data System (ADS)

    Hinsberg, William D.; Milby, Kristin H.; Lidofsky, Steven D.; Zare, Richard N.

    1981-09-01

    An enzyme-linked sandwich immunoassay for insulin is described. Horseradish peroxidase is employed as an enzyme label for antibody, and enzyme activity is measured via the fluorogenic substrate, p-hydroxyphenylacetic acid. The product is detected by excitation of fluorescence with the 325 nm line of a cw helium-cadmium ion laser on-line with reverse phase high performance liquid chromatography. The method requires a total incubation time of 45 minutes, and the limit of insulin detection is 1.1 μU/ml (6.6 pM). This assay is applicable to the analysis of human serum samples.

  16. Measuring Hsp72 (HSPA1A) by indirect sandwich ELISA.

    PubMed

    Ireland, H Elyse; Williams, John H H

    2011-01-01

    The enzyme-linked immunosorbent assay (ELISA) is an immunological technique which is used to determine the presence or quantity of an antigen within a sample. ELISAs rely on the use of at least one antibody (Ab) specific for the antigen being measured. This antibody is covalently linked to an enzyme which is detected through the use of an enzymatic substrate, which can be colorimetric, fluorogenic, or chemiluminescent. The ELISA for Hsp72 described here is a typical indirect sandwich ELISA, which can be used for measuring Hsp72 from cellular/tissue extracts, tissue culture supernatant, and serum. Typically, a 96-well ELISA plate is coated with a specific antibody which captures Hsp72 from the sample, and another antibody specific for a different Hsp72 epitope is used to detect Hsp72. An enzyme-labelled species-specific antibody conjugate is then applied which is consequently detected using a colorimetric enzyme substrate. The quantity of Hsp72 present in the samples is interpolated using a standard curve of known amounts of pure Hsp72.

  17. Development of a highly sensitive noncompetitive electrochemical immunosensor for the detection of atrazine by phage anti-immunocomplex assay.

    PubMed

    González-Techera, Andrés; Zon, María Alicia; Molina, Patricia Gabriela; Fernández, Héctor; González-Sapienza, Gualberto; Arévalo, Fernando Javier

    2015-02-15

    The development of immunosensors for the detection of small molecules is of great interest because of their simplicity, high sensitivity and extended analytical range. Due to their size, small compounds cannot be simultaneously recognized by two antibodies impeding their detection by noncompetitive two-site immunoassays, which are superior to competitive ones in terms of sensitivity, kinetics, and working range. In this work, we combine the advantages of magneto-electrochemical immunosensors with the improved sensitivity and direct proportional signal of noncompetitive immunoassays to develop a new Phage Anti-Immunocomplex Electrochemical Immunosensor (PhAIEI) for the detection of the herbicide atrazine. The noncompetitive assay is based on the use of recombinant M13 phage particles bearing a peptide that specifically recognizes the immunocomplex of atrazine with an anti-atrazine monoclonal antibody. The PhAIEI performed with a limit of detection (LOD) of 0.2 pg mL(-1), which is 200-fold better than the LOD obtained using the same antibody in an optimized conventional competitive ELISA, with a large increase in working range. The developed PhAIEI was successfully used to assay undiluted river water samples with no pretreatment and excellent recoveries. Apart from the first demonstration of the benefits of integrating phage anti-immunocomplex particles into electrochemical immunosensors, the extremely low and environmentally relevant detection limits of atrazine attained with the PhAIEIS may have direct applicability to fast and sensitive detection of this herbicide in the environment.

  18. Developing a Salivary Antibody Multiplex Immunoassay to ...

    EPA Pesticide Factsheets

    The etiology and impacts of human exposure to environmental pathogens are of major concern worldwide and, thus, the ability to assess exposure and infections using cost effective, high-throughput approaches would be indispensable. The principal objective of this work is to develop an immunoassay capable of measuring the presence of antibodies in human saliva to multiple pathogens simultaneously. Saliva is particularly attractive in this application because it is noninvasive, cheaper and easier to collect than serum. Antigens from environmental pathogens were coupled to carboxylated microspheres (beads) and used to measure antibodies in very small volumes of human saliva samples using the Luminex xMAP solution-phase assay. Beads were coupled to antigens from Campylobacter jejuni, Helicobacter pylori, Toxoplasma gondii, noroviruses (G I.1 and G II.4) and hepatitis A virus. To ensure that the antigens were sufficiently coupled to the beads, coupling was confirmed using species-specific, animal-derived primary detection antibodies, followed by incubation with biotinylated anti-species secondary detection antibodies and streptavidin-R-phycoerythrin reporter (SAPE). As a control to measure non-specific binding, one bead set was treated identically to the others except it was not coupled to any antigen. The antigen coupled and control beads were then incubated with prospectively-collected human saliva samples, analyzed on a Luminex 100 platform, and the presence

  19. Buflomedil interference with the monoclonal EMIT d.a.u. amphetamine/methamphetamine immunoassay.

    PubMed

    Papa, P; Rocchi, L; Mainardi, C; Donzelli, G

    1997-05-01

    The interference of buflomedil with the monoclonal and polyclonal EMIT d.a.u. amphetamine immunoassays was investigated. Urine samples collected from 20 patients taking 600 mg of buflomedil daily gave false positive results with the monoclonal EMIT d.a.u. assay, as did urine specimens collected 2 hours after the first oral dose of buflomedil. Conversely, no false positive results occurred with the polyclonal EMIT d.a.u. amphetamine assay. Urine samples with buflomedil added at concentrations greater than 100 mg/l gave false positive results with the monoclonal immunoassay. Buflomedil concentrations found in the patient urines (56-400 mg/l) failed to correlate to EMIT assay responses: this result suggests that one or more buflomedil metabolites, besides the unchanged drug, probably interfere in the monoclonal EMIT d.a.u. assay.

  20. A new sandwich immunoassay for detection of the α-secretase cleaved, soluble amyloid-β protein precursor in cerebrospinal fluid and serum.

    PubMed

    Taverna, Mara; Straub, Tobias; Hampel, Harald; Rujescu, Dan; Lichtenthaler, Stefan F

    2013-01-01

    Alzheimer's disease (AD) is the most common neurodegenerative disorder. Frequently used diagnostic biomarkers are amyloid-β42 (Aβ42), tau, and phospho-tau, which are measured in cerebrospinal fluid (CSF), and allow a reasonable, but not full, separation of AD patients and controls. Besides Aβ42, additional proteolytic cleavage products of the amyloid-β protein precursor (AβPP) have been investigated as potential biomarkers. This includes the α-secretase cleaved soluble AβPP ectodomain (sAβPPα). However, some studies found a reduction of sAβPPα, whereas other studies reported an increase of sAβPPα in the CSF of AD patients. The divergent findings may result from the detection of sAβPPα with antibodies, such as 6E10, which do not exclusively detect sAβPPα, but also the alternative β-secretase cleavage product sAβPPβ'. Here, we used the sAβPPα-specific antibody 14D6 and developed an ELISA-like sandwich immunoassay. The assay specifically detected sAβPPα in cell culture supernatants, in human CSF and even in serum, which is more readily accessible than CSF. The assay was used to analyze sA