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…

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...

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

USDA-ARS?s Scientific Manuscript database

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

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

PubMed

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

2015-05-01

For detection of infectious laryngotracheitis virus (ILTV) antibody, glycoprotein B-, C-, and D-based enzyme-linked immunosorbent assays (B-, C-, and D-ELISAs, respectively) were developed. The B- and D-ELISAs showed enhanced detection of anti-ILTV antibodies in infected chickens compared to that of the commercial ELISA. Furthermore, the D-ELISA was efficient in detecting seroconversion with vectored vaccine, using recombinant Newcastle disease virus (rNDV) expressing glycoprotein D (gD) as the vaccine vector. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

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

PubMed Central

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

2005-01-01

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

Comparison of 2 Luminex-based Multiplexed Protein Assays for Quantifying Microglia Activation and Inflammatory Proteins

DTIC Science & Technology

2016-04-01

streptavidin-phycoerythrin (PE) similar to sandwich enzyme-linked immunosorbent assays ( ELISAs ). The 3 fluorescent markers (2 beads plus PE) allow for...within the kit, this worked out to a set of expensive, problematic, and subjective ELISA . The space on the black-96 well plate was split between cell...ARL US Army Research Laboratory BBB blood–brain barrier CSF cerebral spinal fluid DOD US Department of Defense ELISA enzyme-linked immunosorbent

Immunoglobulin M (IgM)-Glycoinositolphospholipid Enzyme-Linked Immunosorbent Assay: an Immunoenzymatic Assay for Discrimination between Patients with Acute Toxoplasmosis and Those with Persistent Parasite-Specific IgM Antibodies

PubMed Central

Giraldo, Mónica; Portela, Ricardo W. D.; Snege, Mirian; Leser, Paulo G.; Camargo, Mário E.; Mineo, José Roberto; Gazzinelli, Ricardo T.

2002-01-01

In the present study we developed an enzyme-linked immunosorbent assay (ELISA) to measure immunoglobulin M (IgM) specific for glycoinositolphospholipids (GIPL) derived from tachyzoite membrane (IgM-GIPL ELISA). The sensitivity and specificity of the assay were compared with those of commercially available Toxoplasma-specific IgM serological tests, namely, immunofluorescence assay (IFA) with fixed tachyzoites and capture ELISA employing tachyzoite extracts. Our results show that all patients with acute toxoplasmosis, as determined by clinical data and conventional serological tests, were also positive by the IgM-GIPL ELISA. Interestingly, many patients that were classified as indeterminate, who had IgG with high avidity but positive results in the IgM-specific IFA and capture ELISA, were negative by the IgM-GIPL ELISA. Finally, we tested the sera from patients with rheumatoid arthritis and various parasitic infections and found no evidence of false positives in the IgM-GIPL ELISA. PMID:11923364

Monoclonal antibodies against human angiotensinogen, their characterization and use in an angiotensinogen enzyme linked immunosorbent assay.

PubMed

Rubin, I; Lykkegaard, S; Olsen, A A; Selmer, J; Ballegaard, M

1988-01-01

Monoclonal antibodies were produced against human angiotensinogen. An enzyme linked immunosorbent assay (ELISA) was developed using a high affinity monoclonal antibody as catching antibody and a polyclonal rabbit anti human angiotensinogen antibody as detecting antibody in a "sandwich" ELISA. Linear range of the ELISA was 15-450 pmol/l of human angiotensinogen. Intra- and inter- assay variation coefficients were in the range of 2% to 8%. A correlation coefficient, r = 0.97, (n = 20), with values obtained by radioimmunoassay. This correlation coefficient, obtained by using both normal and pregnant sera, confirmed that the ELISA fulfill the requirements for clinical useful assay. Characterization of the antibodies were performed with respect to affinity constant and epitopes.

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.

Comparison of commercial enzyme-linked immunosorbent assay kits with agar gel precipitation and hemagglutination-inhibition tests for detecting antibodies to avian influenza viruses.

PubMed

Shiraishi, Rikiya; Nishiguchi, Akiko; Tsukamoto, Kenji; Muramatsu, Masatake

2012-09-01

We evaluated the utility of 5 commercial enzyme-linked immunosorbent assay (ELISA) kits for detecting antibodies to avian influenza viruses. The sensitivities and specificities of the ELISA kits were compared with those of the agar gel precipitation (AGP) and hemagglutination-inhibition (HI) tests. The results suggest that some ELISA kits might not be suitable for monitoring during the early stages of avian influenza virus infections. Therefore, ELISA kits should only be used in conjunction with a profound knowledge about monitoring of avian influenza.

Evaluation of a caprine arthritis-encephalitis virus/maedi-visna virus indirect enzyme-linked immunosorbent assay in the serological diagnosis of ovine progressive pneumonia virus in U.S. sheep

USDA-ARS?s Scientific Manuscript database

Serological diagnostic testing of sheep and goats using enzyme immunosorbent assays (ELISAs) is the most common method of determining small ruminant lentivirus (SRLV) infection. A caprine arthritis-encephalitis virus (CAEV)/maedi-visna virus (MVV) indirect (i) ELISA, which utilizes MVV EV1 capsid a...

Detection of Antibodies to the Biofilm Exopolysaccharide of Histophilus somni following Infection in Cattle by Enzyme-Linked Immunosorbent Assay

PubMed Central

Pan, Yu; Fisher, Taylor; Olk, Christina

2014-01-01

An enzyme-linked immunosorbent assay (ELISA) was developed to detect bovine antibodies to Histophilus somni exopolysaccharide (EPS), which is created during biofilm formation. When an index value of 0.268 was used, the sensitivity of the assay for infected calves was 90.5% at 3 weeks postinfection, but the number of positive animals increased by week 4. The specificity of the assay for healthy calves was 92.5%. The EPS ELISA may aid in identifying calves with H. somni diseases. PMID:25143338

Using an enzyme linked immunosorbent assay (ELISA) and a protein phosphatase inhibition assay (PPIA) for the detection of microcystins and nodularins.

PubMed

Carmichael, W W; An, J

1999-01-01

Cyanotoxins produced by cyanobacteria (blue-green algae) include potent neurotoxins and hepatotoxins. The hepatotoxins include cyclic peptide microcystins and nodularins plus the alkaloid cylindrospermopsins. Among the cyanotoxins the microcystins have proven to be the most widespread, and are most often implicated in animal and human poisonings. This paper presents a practical guide to two widely used methods for detecting and quantifying microcystins and nodularins in environmental samples-the enzyme linked immunosorbant assay (ELISA) and the protein phosphatase inhibition assay (PPIA).

Effects of Early Altitude Exposure Following Traumatic Injury and Hemorrhagic Shock

DTIC Science & Technology

2017-06-27

chemokines by multiplex enzyme-linked immunosorbent assay ( ELISA ) (Quansys, Logan, UT), including the following: interleukin 1 alpha and beta (IL...Tissue Cytokine Profiles Fourteen cytokines and chemokines were analyzed from serum and intestinal tissues via multiplex ELISA . There were no...2017-3567, 25 Jul 2017. LIST OF ABBREVIATIONS AND ACRONYMS AE aeromedical evacuation BCA bicinchoninic acid ELISA enzyme-linked immunosorbent

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

PubMed

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

2016-02-01

For detection of Aleutian mink disease virus (AMDV) antibodies, an enzyme-linked immunosorbent assay (ELISA) was developed using the recombinant VP2332-452 protein as an antigen. Counterimmunoelectrophoresis (CIEP) was used as a reference test to compare the results of the ELISA and Western blotting (WB); the specificity and sensitivity of the VP2332-452 ELISA were 97.9% and 97.3%, respectively, which were higher than those of WB. Therefore, this VP2332-452 ELISA may be a preferable method for detecting antibodies against AMDV. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Enzyme-linked immunosorbent assay compared with neutralization tests for evaluation of live mumps vaccines.

PubMed Central

Sakata, H; Hishiyama, M; Sugiura, A

1984-01-01

Mumps-specific antibody levels before and after vaccination with live mumps vaccines were determined by enzyme-linked immunosorbent assay (ELISA) and neutralization tests. A correlation was found between neutralization titers and optical density in ELISA. However, postvaccination sera from some vaccinees who failed to seroconvert by neutralization contained significant levels of mumps-specific antibody detectable by ELISA. In some of these serum specimens, the antibody directed to the F polypeptide of mumps virus was predominant. Most sera positive in ELISA neutralized mumps virus upon the addition of fresh guinea pig serum to the virus-serum mixture. Images PMID:6361060

Quantum dot-linked immunosorbent assay (QLISA) using orientation-directed antibodies.

PubMed

Suzuki, Miho; Udaka, Hikari; Fukuda, Takeshi

2017-09-05

An approach similar to the enzyme-linked immunosorbent assay (ELISA), with the advantage of saving time and effort but exhibiting high performance, was developed using orientation-directed half-part antibodies immobilized on CdSe/ZnS quantum dots. ELISA is a widely accepted assay used to detect the presence of a target substance. However, it takes time to quantify the target with specificity and sensitivity owing to signal amplification. In this study, CdSe/ZnS quantum dots are introduced as bright and photobleaching-tolerant fluorescent materials. Since hydrophilic surface coating of quantum dots rendered biocompatibility and functional groups for chemical reactions, the quantum dots were modified with half-sized antibodies after partial reduction. The half-sized antibody could be bound to a quantum dot through a unique thiol site to properly display the recognition domain for the core process of ELISA, which is an antigen-antibody interaction. The reducing conditions were investigated to generate efficient conjugates of quantum dots and half-sized antibodies. This was applied to IL-6 detection, as the quantification of IL-6 is significant owing to its close relationships with various biomedical phenomena that cause different diseases. An ELISA-like assay with CdSe/ZnS quantum dot institution (QLISA; Quantum dot-linked immunosorbent assay) was developed to detect 0.05ng/mL IL-6, which makes it sufficiently sensitive as an immunosorbent assay. Copyright © 2017 Elsevier B.V. All rights reserved.

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

USDA-ARS?s Scientific Manuscript database

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

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

USGS Publications Warehouse

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

2014-01-01

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

Design of a compact disk-like microfluidic platform for enzyme-linked immunosorbent assay.

PubMed

Lai, Siyi; Wang, Shengnian; Luo, Jun; Lee, L James; Yang, Shang-Tian; Madou, Marc J

2004-04-01

This paper presents an integrated microfluidic device on a compact disk (CD) that performs an enzyme-linked immunosorbent assay (ELISA) for rat IgG from a hybridoma cell culture. Centrifugal and capillary forces were used to control the flow sequence of different solutions involved in the ELISA process. The microfluidic device was fabricated on a plastic CD. Each step of the ELISA process was carried out automatically by controlling the rotation speed of the CD. The work on analysis of rat IgG from hybridoma culture showed that the microchip-based ELISA has the same detection range as the conventional method on the 96-well microtiter plate but has advantages such as less reagent consumption and shorter assay time over the conventional method.

9 CFR 146.13 - Testing.

Code of Federal Regulations, 2012 CFR

2012-01-01

...) Enzyme-linked immunosorbent assay (ELISA). ELISA must be conducted using test kits approved by the... conducted on all ELISA-positive samples. (B) The AGID test must be conducted using reagents approved by the...

9 CFR 146.13 - Testing.

Code of Federal Regulations, 2013 CFR

2013-01-01

...) Enzyme-linked immunosorbent assay (ELISA). ELISA must be conducted using test kits approved by the... conducted on all ELISA-positive samples. (B) The AGID test must be conducted using reagents approved by the...

9 CFR 146.13 - Testing.

Code of Federal Regulations, 2010 CFR

2010-01-01

...) Enzyme-linked immunosorbent assay (ELISA). ELISA must be conducted using test kits approved by the... conducted on all ELISA-positive samples. (B) The AGID test must be conducted using reagents approved by the...

9 CFR 146.13 - Testing.

Code of Federal Regulations, 2014 CFR

2014-01-01

...) Enzyme-linked immunosorbent assay (ELISA). ELISA must be conducted using test kits approved by the... conducted on all ELISA-positive samples. (B) The AGID test must be conducted using reagents approved by the...

9 CFR 146.13 - Testing.

Code of Federal Regulations, 2011 CFR

2011-01-01

...) Enzyme-linked immunosorbent assay (ELISA). ELISA must be conducted using test kits approved by the... conducted on all ELISA-positive samples. (B) The AGID test must be conducted using reagents approved by the...

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

USGS Publications Warehouse

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

2003-01-01

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

Absorption of p,p'-dichlorodiphenyldichloroethylene and dieldrin in largemouth bass from a 60-D slow-release pellet and detection using a novel enzyme-linked immunosorbent assay method for blood plasma

USGS Publications Warehouse

Muller, Jennifer K.; Sepulveda, Maria S.; Borgert, Christopher J.; Gross, Timothy S.

2005-01-01

This work describes the uptake of two organochlorine pesticides from slow-release pellets by largemouth bass and the utility of a blood plasma enzyme-linked immunosorbent assay (ELISA) method for exposure verification. We measured blood and tissue levels by gas chromatography/mass spectrometry and by a novel ELISA method, and present a critical comparison of the results.

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

PubMed Central

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

2014-01-01

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

Enzyme-linked immunosorbent assay for detection of antibodies to Epstein-Barr virus antigens.

PubMed

Voevodin, A F; Pácsa, A S

1983-01-01

Enzyme-Linked Immunosorbent Assay (ELISA) was standardized for measurement of antibody activity of reference human and baboon (Papio hamadryas) sera to soluble Epstein-Barr virus (EBV) antigens. A comparison with the immunofluorescent (IF) method showed that ELISA detects antibody specifically and sensitivity. In ELISA, Herpesvirus Papio (HVP) nuclear antigen (HUPNA) positive baboon serum reacted with EBV nuclear antigen (EBNA), as a further indication of the antigenic similarity between HVP and EBV. Forty-two baboon sera were tested with EBV antigens in both ELISA and IF test. The results showed an agreement between the two methods and also that by the use of EBV antigens, ELISA measures anti-HVP activity of baboon sera. ELISA did not reveal significant difference in antibody activity of 23 baboons with lymphoma and that of 24 healthy baboons. Results provide further data that ELISA can be used effectively in the field of EBV serology.

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

PubMed

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

2014-09-01

We validated an antigen capture enzyme-linked immunosorbent assay (ELISA) in Colombian persons with AIDS and proven histoplasmosis and evaluated the correlation between antigenuria and clinical improvement during follow-up. The sensitivity of the Histoplasma capsulatum ELISA was 86%, and the overall specificity was 94%. The antigen test successfully monitored the response to therapy. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Enzyme-linked immunosorbent assay for total sennosides using anti-sennside A and anti-sennoside B monoclonal antibodies.

PubMed

Morinaga, Osamu; Uto, Takuhiro; Sakamoto, Seiichi; Tanaka, Hiroyuki; Shoyama, Yukihiro

2009-01-01

Total sennosides concentration is a very important factor when rhubarb and senna will be used as crude drugs. However, one-step analytical technique for total sennosides has not been reported except HPLC. An enzyme-linked immunosorbent assay (ELISA) for total sennosides concentration by using the combination of anti-sennoside A (SA) and anti-sennoside B (SB) monoclonal antibodies (MAbs) in a single assay has been investigated. Total sennosides concentration in rhubarb and senna samples determined by newly developed assay system showed good agreement with those analyzed by ELISA using anti-SA MAb and anti-SB MAb, respectively.

Thermometric enzyme linked immunosorbent assay: TELISA.

PubMed

Mattiasson, B; Borrebaeck, C; Sanfridson, B; Mosbach, K

1977-08-11

A new method, thermometric enzyme linked immunosorbent assay (TELISA), for the assay of endogenous and exogenous compounds in biological fluids is described. It is based on the previously described enzyme linked immunosorbent assay technique, ELISA, but utilizes enzymic heat formation which is measured in an enzyme thermistor unit. In the model system studied determination of human serum albumin down to a concentration of 10(-10) M (5 ng/ml) was achieved, with both normal and catalase labelled human serum albumin competing for the binding sites on the immunosorbent, which was rabbit antihuman serum albumin immobilized onto Sepharose CL-4B.

Enzyme-linked immunosorbent assay for Escherichia coli heat-stable enterotoxin type II.

PubMed Central

Handl, C; Rönnberg, B; Nilsson, B; Olsson, E; Jonsson, H; Flock, J I

1988-01-01

The gene for Escherichia coli heat-stable enterotoxin type II (STII) was fused to the genes for protein A from Staphylococcus aureus and beta-galactosidase in two different expression systems. Antibodies raised in rabbits against the protein A-STII fusion protein recognized the beta-galactosidase-STII fusion protein. The latter fusion protein was used as the immobilized antigen in an enzyme-linked immunosorbent assay (ELISA) for detection of STII. The correlation between the results of the ELISA and the intestinal loop test in piglets was 95%, suggesting that the ELISA can be used to reliably detect STII. Images PMID:3049659

Enzyme-linked immunosorbent assays for determination of plasma aldosterone using highly specific polyclonal antibodies.

PubMed

Schwartz, F; Hadas, E; Harnik, M; Solomon, B

1990-01-01

Two enzyme-linked immunosorbent assays were established and compared for the estimation of plasma aldosterone. In the first method immobilized aldosterone-protein complexes on the ELISA plates compete with aldosterone to be determined for the binding of certain amount of anti-aldosterone antibodies. The sensitivity of this method depends on the protein carrier used to conjugate with aldosterone. In the second method, anti-aldosterone antibodies adsorbed on ELISA plates compete for binding of known amount of the enzyme-labeled aldosterone and aldosterone to be determined. The highly specific rabbit anti-aldosterone antibodies were obtained by injection of aldosterone-oxime thyroglobulin. The detection limit of aldosterone in both methods ranged between 2-20 pg. The proposed assays are suitable for the determination of aldosterone in biological fluids compared with other reported ELISA assays, as well as with RIA.

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

PubMed Central

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

2013-01-01

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

9 CFR 52.1 - Definitions.

Code of Federal Regulations, 2011 CFR

2011-01-01

... Test; Fluorescent Antibody Tissue Section Test; Enzyme-Linked Immunosorbent Assay (ELISA) Test, except for approved differential pseudorabies tests other than the glycoprotein I (gpI) ELISA test; Latex...

9 CFR 52.1 - Definitions.

Code of Federal Regulations, 2014 CFR

2014-01-01

... Test; Fluorescent Antibody Tissue Section Test; Enzyme-Linked Immunosorbent Assay (ELISA) Test, except for approved differential pseudorabies tests other than the glycoprotein I (gpI) ELISA test; Latex...

9 CFR 52.1 - Definitions.

Code of Federal Regulations, 2013 CFR

2013-01-01

... Test; Fluorescent Antibody Tissue Section Test; Enzyme-Linked Immunosorbent Assay (ELISA) Test, except for approved differential pseudorabies tests other than the glycoprotein I (gpI) ELISA test; Latex...

9 CFR 52.1 - Definitions.

Code of Federal Regulations, 2010 CFR

2010-01-01

... Test; Fluorescent Antibody Tissue Section Test; Enzyme-Linked Immunosorbent Assay (ELISA) Test, except for approved differential pseudorabies tests other than the glycoprotein I (gpI) ELISA test; Latex...

9 CFR 52.1 - Definitions.

Code of Federal Regulations, 2012 CFR

2012-01-01

... Test; Fluorescent Antibody Tissue Section Test; Enzyme-Linked Immunosorbent Assay (ELISA) Test, except for approved differential pseudorabies tests other than the glycoprotein I (gpI) ELISA test; Latex...

Evaluation of ethanol vortex ELISA for detection of bovine tuberculosis in cattle and deer

USDA-ARS?s Scientific Manuscript database

Background The use of serological assays for diagnosis of bovine tuberculosis (TB) has been intensively studied and use of specific antigens have aided in improving the diagnostic accuracy of the assays. In the present study, we report an in-house enzyme linked immunosorbent assay (ELISA), developed...

9 CFR 85.1 - Definitions.

Code of Federal Regulations, 2013 CFR

2013-01-01

... Immunosorbent Assay (ELISA) Test, except for approved differential pseudorabies tests other than the glycoprotein I (gpI) ELISA test; 5. Latex Agglutination Test (LAT); and 6. Particle Concentration Fluorescence...

9 CFR 85.1 - Definitions.

Code of Federal Regulations, 2012 CFR

2012-01-01

... Immunosorbent Assay (ELISA) Test, except for approved differential pseudorabies tests other than the glycoprotein I (gpI) ELISA test; 5. Latex Agglutination Test (LAT); and 6. Particle Concentration Fluorescence...

9 CFR 85.1 - Definitions.

Code of Federal Regulations, 2014 CFR

2014-01-01

... Immunosorbent Assay (ELISA) Test, except for approved differential pseudorabies tests other than the glycoprotein I (gpI) ELISA test; 5. Latex Agglutination Test (LAT); and 6. Particle Concentration Fluorescence...

9 CFR 85.1 - Definitions.

Code of Federal Regulations, 2011 CFR

2011-01-01

... Immunosorbent Assay (ELISA) Test, except for approved differential pseudorabies tests other than the glycoprotein I (gpI) ELISA test; 5. Latex Agglutination Test (LAT); and 6. Particle Concentration Fluorescence...

9 CFR 85.1 - Definitions.

Code of Federal Regulations, 2010 CFR

2010-01-01

... Immunosorbent Assay (ELISA) Test, except for approved differential pseudorabies tests other than the glycoprotein I (gpI) ELISA test; 5. Latex Agglutination Test (LAT); and 6. Particle Concentration Fluorescence...

Evaluation of Chicken IgY Generated Against Canine Parvovirus Viral-Like Particles and Development of Enzyme-Linked Immunosorbent Assay and Immunochromatographic Assay for Canine Parvovirus Detection.

PubMed

He, Jinxin; Wang, Yuan; Sun, Shiqi; Zhang, Xiaoying

2015-11-01

Immunoglobulin Y (IgY) antibodies were generated against canine parvovirus virus-like particles (CPV-VLPs) antigen using chickens. Anti-CPV-VLPs-IgY was extracted from hen egg yolk and used for developing enzyme-linked immunosorbent assay (ELISA) and immunochromatographic assay (ICA) for the detection of CPV in dog feces. The cutoff negative values for anti-CPV-VLPs-IgY were determined using negative fecal samples (already confirmed by polymerase chain reaction [PCR]). In both ELISA and ICA, there was no cross-reaction with other diarrheal pathogens. Thirty-four fecal samples were collected from dogs with diarrhea, of which 26.47% were confirmed as CPV-positive samples by PCR, while 29.41% and 32.35% of the samples were found to be positive by ELISA and ICA, respectively. The developed ELISA and ICA exhibited 97.06% and 94.12% conformity with PCR. Higher sensitivity and specificity were observed for IgY-based ELISA and ICA. Thus, they could be suitable for routine use in the diagnosis of CPV in dogs.

Enzyme-Linked Immunosorbent Assay (ELISA).

PubMed

Konstantinou, George N

2017-01-01

Food allergy is a public health concern especially after recognizing its constantly increased prevalence and severity. Despite careful reading of food ingredient statements, food allergic individuals may experience reactions caused by "hidden", "masked", or "contaminated" proteins that are known major allergens. Many techniques have been developed to detect even small traces of food allergens, for clinical or laboratory purposes. Enzyme-linked immunosorbent assay (ELISA) is one of the best validated and most routinely used immunoassay in allergy research, in allergy diagnosis in allergy-related quality control in various industries. Although as a technique it has been implemented for the last 45 years, the evolution in biochemistry allowed the development of ultrasensitive ELISA variations that are capable of measuring quantities in the scale of picograms, rendering ELISA attractive, robust, and very famous.

Evaluation of a sensitive reverse transcription PCR-enzymelinked immunosorbent assay for detection of hepatitis A virus in oysters (Saccostrea glomerata) on the east coast of the Gulf of Thailand.

PubMed

Intamaso, Uraiwan; Ketkhunthod, Sitthisak

2014-05-01

Hepatitis A virus (HAV) contamination in food can lead to major health problems. We developed a combination reverse transcription (RT) PCR method plus enzyme-linked immunosorbent assay (ELISA) to detect HAV in fresh oysters harvested along the east coast of the Gulf of Thailand. Viral nucleic acid was extracted via the glycine-arginine-polyethylene glycol method followed by RT-PCR amplification with specifically designed primers against HAV and an ELISA to detect the digoxigenin-labeled RT-PCR products. The ELISA in concert with the RT-PCR protocol further increased the detection sensitivity by 100-fold for the HAV genome and 10-fold in artificially contaminated oysters. The overall sensitivity of the RT-PCR in combination with the ELISA was 31.88 pg and 16 PFU/g, respectively. The ELISA increases the specificity of the RT-PCR assay for detecting naturally occurring HAV in oysters. This combined RT-PCR-ELISA approach is a practical and sensitive method for HAV detection and can be utilized in routine screening for HAV in shellfish.

Development and evaluation of ELISA and qRT-PCR for identification of Squash vein yellowing virus in cucurbits

USDA-ARS?s Scientific Manuscript database

Enzyme linked-immunosorbent assay (ELISA) and quantitative reverse transcription-PCR (qRT-PCR) assays were developed for identification of Squash vein yellowing virus (SqVYV), the cause of viral watermelon vine decline. Both assays were capable of detecting SqVYV in a wide range of cucurbit hosts. ...

[Comparison between anti-ouabain egg yolk(IgY) and rabbit antibody(IgG) in enzyme-linked immunosorbent assay].

PubMed

Zhang, Ming-juan; Yang, Jun; Ge, Heng; Qiang, Lei; Duan, Zong-ming; Wang, Cong-xia; Wang, Rong; Lu, Zhuo-rern

2007-11-01

To improve specificity and accuracy of endogenous ouabain measurement assay. Anti-ouabain polyclonal antibody egg yolk (IgY) and anti-ouabain rabbit antibody (IgG) were prepared respectively. In the presence of two kinds of antibody, then the specificity and accuracy of enzyme-linked immunosorbent assay (ELISA) were compared. The ELISA, in the presence of IgY, provided a sensitivity of the average intraassay coefficient of variation(CV) was 2.03%, and the inter-assay CV was 2.34% respectively. In contrast, IgG were 2.83% and 3.29%. No significant interferences were observed with hydrocortisone and dexamethasone. There was 3.45% vs. 5.95%, 3.20% vs. 5.20% of crossreaction with cedilanid and digoxin. The specificity and accuracy of ELISA, in which IgY was used, were more better than IgG.

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

PubMed

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

1990-01-01

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

Comparability of the effect of storage time and temperature on serum anti-Müllerian hormone measurement between original and modified enzyme-linked immunosorbent assay.

PubMed

Yue, Chao-Yan; Ying, Chun-Mei

2017-01-01

To explore the effect of modified enzyme-linked immunosorbent assay on the AMH results is increased or decreased, and to investigate the effect of storage time and temperature on AMH measurements with and without sample premixing assay buffer using the Kangrun ELISA method. Serum AMH concentration were measured by ELISA, consistency between two kits, and comparability between original and the modified assay under different stored conditions were analyzed by Passing-Bablok regression analysis and Bland-Altman bias evaluation. There was a strong consistency between AMH concentrations measured in Kangrun ELISA and Ansh Labs ultra-sensitive AMH ELISA. Pre-mixing serum specimens with assay buffer gave consistent results compared with original assay. Modified protocol can reduce the amplitude of increase affected by sample aged and give the most consistent results regardless of storage conditions. Pre-mixing protocol did not influence the results of fresh serum or frozen serum incubation <3days at 4°C and -80°C, but when specimens detected after collection and stored in other storage conditions, should be pre-mixed with assay buffer to insure its accuracy. Copyright © 2016 Elsevier B.V. All rights reserved.

An enzyme-linked immunosorbent assay for detection of botulinum toxin-antibodies.

PubMed

Dressler, Dirk; Gessler, Frank; Tacik, Pawel; Bigalke, Hans

2014-09-01

Antibodies against botulinum neurotoxin (BNT-AB) can be detected by the mouse protection assay (MPA), the hemidiaphragm assay (HDA), and by enzyme-linked immunosorbent assays (ELISA). Both MPA and HDA require sacrifice of experimental animals, and they are technically delicate and labor intensive. We introduce a specially developed ELISA for detection of BNT-A-AB and evaluate it against the HDA. Thirty serum samples were tested by HDA and by the new ELISA. Results were compared, and receiver operating characteristic analyses were used to optimize ELISA parameter constellation to obtain either maximal overall accuracy, maximal test sensitivity, or maximal test specificity. When the ELISA is optimized for sensitivity, a sensitivity of 100% and a specificity of 55% can be reached. When it is optimized for specificity, a specificity of 100% and a sensitivity of 90% can be obtained. We present an ELISA for BNT-AB detection that can be-for the first time-customized for special purposes. Adjusted for optimal sensitivity, it reaches the best sensitivity of all BNT-AB tests available. Using the new ELISA together with the HDA as a confirmation test allows testing for BNT-AB in large numbers of patients receiving BT drugs in an economical, fast, and more animal-friendly way. © 2014 International Parkinson and Movement Disorder Society.

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

EPA Science Inventory

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

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

EPA Science Inventory

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

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

PubMed Central

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

2013-01-01

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

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...

Development of an Antigen Capture Enzyme-Linked Immunosorbent Assay for Virus Detection Based on Porcine Epidemic Diarrhea Virus Monoclonal Antibodies

PubMed Central

Wang, Zanyu; Jiyuan, Yin; Su, Chen; Xinyuan, Qiao

2015-01-01

Abstract Porcine epidemic diarrhea virus (PEDV), a coronavirus, can cause acute diarrhea and dehydration in pigs. In the current study, two positive monoclonal cell lines (5D7 and 3H4) specific for PEDV were established, and the immunoreactivity of the monoclonal antibodies was confirmed by immunofluorescence and dot-immunobinding assays. A method, termed antigen capture enzyme-linked immunosorbent assay (AC-ELISA), which used the monoclonal antibody 5D7 as the detecting antibody and rabbit antiserum of PEDV protein S as the capture antibody, was developed. Compared with the reverse transcription polymerase chain reaction method of detecting PEDV in fecal samples, AC-ELISA showed similar sensitivity and specificity. These results suggested that AC-ELISA would be useful for the diagnosis and epidemiological studies of PEDV. PMID:25658793

Development of a sensitive enzyme-linked immunosorbent assay for the measurement of biologically active etanercept in patients with ankylosing spondylitis.

PubMed

Wang, Lei; Wang, Xiaoxia; Li, Ying; Cheng, Zeneng

2016-01-01

Etanercept is the first tumor necrosis factor inhibitor to be approved for rheumatic disease treatment. Its in vivo concentration is usually detected with commercial enzyme-linked immunosorbent assay (ELISA) kits; specifically, previous researchers have mostly used double-antibody sandwich ELISA technology. Double-antibody sandwich ELISA is employed to detect the total etanercept rather than biologically active etanercept, which is more relevant in terms of therapeutic drug monitoring. In this work, a sensitive ELISA that employed its antigen TNF-α to capture biologically active etanercept for concentration detection was established and validated for etanercept pharmacokinetic (PK) study in patients with ankylosing spondylitis (AS). The proposed assay was demonstrated to be precise and accurate over the linear range of 12.5-400pg/mL. The intra- and inter-assay relative standard deviation ranged from 3.9 to 12.2% and 6.2 to 11.1%, respectively, and recovery varied between 90.1 and 99.7%, confirming the assay's reliability. The effectiveness and accuracy of the assay was also validated according to quality samples containing etanercept with different TNF-α concentrations, and with plasma samples from patients with AS. To complete the study, both the proposed assay and double-antibody sandwich ELISA were applied to the PK study of etanercept in patients and compared. The multiple-dose results of both analytical methods were consistent, while the drug exposure of the first dose as-detected by the proposed assay was lower than that detected by double-antibody sandwich ELISA. In conclusion, the proposed ELISA was shown to provide more accurate concentration data for therapeutic drug monitoring in comparison to commercial ELISA kits. Copyright © 2015 Elsevier B.V. All rights reserved.

Improvement of an enzyme immunosorbent assay for detecting antibodies against Dioctophyma renale.

PubMed

Pedrassani, Daniela; do Nascimento, Adjair Antonio; André, Marcos Rogério; Machado, Rosangela Zacarias

2015-09-15

An available enzyme-linked immunosorbent assay (ELISA) was studied for the detection of anti-Dioctophyma renale antibodies in the sera of dogs using, detection of parasite eggs in urine sediment as a reference test. ELISA uses a soluble antigenic preparation of esophagus of D. renale and the optimal dilutions of the antigen, serum and conjugate were determined by means of checker board titration, using positive (n=13) and negative (n=27) reference serum. The specificity and sensitivity of the ELISA were 93.8% and 92.3% respectively and the kappa index was good (0.76). These results suggest that ELISA described may prove to be an effective serological test for detecting dogs infected and exposed to this parasite mainly dogs that are not eliminating parasite eggs through their urine. Copyright © 2015 Elsevier B.V. All rights reserved.

Improvement of an enzyme-linked immunosorbent assay for equine herpesvirus type 4 by using a synthetic-peptide 24-mer repeat sequence of glycoprotein G as an antigen

PubMed Central

BANNAI, Hiroshi; NEMOTO, Manabu; TSUJIMURA, Koji; YAMANAKA, Takashi; MAEDA, Ken; KONDO, Takashi

2015-01-01

To increase the sensitivity of an enzyme-linked immunosorbent assay (ELISA) for equine herpesvirus type 4 (EHV-4) that uses a 12-mer peptide of glycoprotein G (gG4-12-mer: MKNNPIYSEGSL) [4], we used a longer peptide consisting of a 24-mer repeat sequence (gG4-24-mer: MKNNPIYSEGSLMLNVQHDDSIHT) as an antigen. Sera of horses experimentally infected with EHV-4 reacted much more strongly to the gG4-24-mer peptide than to the gG4-12-mer peptide. We used peptide ELISAs to test paired sera from horses naturally infected with EHV-4 (n=40). gG4-24-mer ELISA detected 37 positive samples (92.5%), whereas gG4-12-mer ELISA detected only 28 (70.0%). gG4-24-mer ELISA was much more sensitive than gG4-12-mer ELISA. PMID:26424485

Improvement of an enzyme-linked immunosorbent assay for equine herpesvirus type 4 by using a synthetic-peptide 24-mer repeat sequence of glycoprotein G as an antigen.

PubMed

Bannai, Hiroshi; Nemoto, Manabu; Tsujimura, Koji; Yamanaka, Takashi; Maeda, Ken; Kondo, Takashi

2016-02-01

To increase the sensitivity of an enzyme-linked immunosorbent assay (ELISA) for equine herpesvirus type 4 (EHV-4) that uses a 12-mer peptide of glycoprotein G (gG4-12-mer: MKNNPIYSEGSL) [4], we used a longer peptide consisting of a 24-mer repeat sequence (gG4-24-mer: MKNNPIYSEGSLMLNVQHDDSIHT) as an antigen. Sera of horses experimentally infected with EHV-4 reacted much more strongly to the gG4-24-mer peptide than to the gG4-12-mer peptide. We used peptide ELISAs to test paired sera from horses naturally infected with EHV-4 (n=40). gG4-24-mer ELISA detected 37 positive samples (92.5%), whereas gG4-12-mer ELISA detected only 28 (70.0%). gG4-24-mer ELISA was much more sensitive than gG4-12-mer ELISA.

Enzyme-linked immunosorbent assay using a recombinant baculovirus-expressed Bacillus anthracis protective antigen (PA): measurement of human anti-PA antibodies.

PubMed Central

Iacono-Connors, L C; Novak, J; Rossi, C; Mangiafico, J; Ksiazek, T

1994-01-01

We developed an antigen capture enzyme-linked immunosorbent assay (ELISA) which does not require purified protective antigen (PA) for detection of human antibodies to Bacillus anthracis PA. Lysates of Spodoptera frugiperda (Sf-9) cells infected with recombinant baculovirus containing the PA gene were used as the source of PA to develop the ELISA. Recombinant PA from crude Sf-9 cell lysates or PA purified from B. anthracis Sterne strain was captured by an anti-PA monoclonal antibody coated onto microtiter plates. We demonstrated that human serum antibody titers to PA were identical in the ELISA whether we used crude Sf-9 cell lysates containing recombinant baculovirus-expressed PA or purified Sterne PA. Finally, false-positive results observed in a direct ELISA were eliminated with this antigen capture ELISA. Thus, the antigen capture ELISA with crude preparations of baculovirus-expressed PA is reliable, safe, and inexpensive for determining anti-PA antibody levels in human sera. PMID:7496927

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

PubMed

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

2016-12-01

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

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. © 2016 Institute of Food Technologists®

Sandwich enzyme-linked immunosorbent assay (ELISA) for measuring the concentration of, and detection of antibodies to, Aujeszky's disease virus.

PubMed

Kardi, V; Szegletes, E; Perényi, T; Pergel, I; Smal, Z

1990-01-01

A double antibody sandwich enzyme-linked immunosorbent assay (ELISA) was developed for measuring Aujeszky's disease virus (ADV) antigen concentration and an inhibition technique based on the former was developed for detection of antibodies to ADV. The results were checked by determining the cytopathic and serum neutralization titres. The correlation was satisfactory in both cases, with correlation coefficients above 0.8. When measuring ADV antigen concentration, the lower limit of detection was 10(3) TCID 50/0.2 ml. The sensitivity of ELISA in detecting antibodies to ADV was found to be superior to that of the serum neutralization test and, thus, enabled the testing of rabbit and guinea-pig sera.

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

PubMed Central

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

1997-01-01

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

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

USDA-ARS?s Scientific Manuscript database

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

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

EPA Science Inventory

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

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

USDA-ARS?s Scientific Manuscript database

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

9 CFR 147.7 - Standard test procedures for mycoplasma. 5

Code of Federal Regulations, 2011 CFR

2011-01-01

... plate agglutination test, the tube agglutination test, and the enzyme-linked immunosorbent assay (ELISA... accurate however, and are useful in evaluating serum samples that react with the ELISA, plate, and/or tube...

9 CFR 147.7 - Standard test procedures for mycoplasma. 5

Code of Federal Regulations, 2012 CFR

2012-01-01

... plate agglutination test, the tube agglutination test, and the enzyme-linked immunosorbent assay (ELISA... accurate however, and are useful in evaluating serum samples that react with the ELISA, plate, and/or tube...

9 CFR 147.7 - Standard test procedures for mycoplasma. 5

Code of Federal Regulations, 2010 CFR

2010-01-01

... plate agglutination test, the tube agglutination test, and the enzyme-linked immunosorbent assay (ELISA... accurate however, and are useful in evaluating serum samples that react with the ELISA, plate, and/or tube...

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)

9 CFR 147.6 - Procedure for determining the status of flocks reacting to tests for Mycoplasma gallisepticum...

Code of Federal Regulations, 2014 CFR

2014-01-01

... agglutination test, enzyme-labeled immunosorbent assay (ELISA), official molecular examination procedure, or... the tube agglutination, ELISA, or serum plate test is positive, the hemaglutination inhibition (HI...

9 CFR 147.6 - Procedure for determining the status of flocks reacting to tests for Mycoplasma gallisepticum...

Code of Federal Regulations, 2012 CFR

2012-01-01

... agglutination test, enzyme-labeled immunosorbent assay (ELISA), official molecular examination procedure, or... the tube agglutination, ELISA, or serum plate test is positive, the hemaglutination inhibition (HI...

9 CFR 147.7 - Standard test procedures for mycoplasma. 5

Code of Federal Regulations, 2014 CFR

2014-01-01

... plate agglutination test, the tube agglutination test, and the enzyme-linked immunosorbent assay (ELISA... accurate however, and are useful in evaluating serum samples that react with the ELISA, plate, and/or tube...

9 CFR 147.6 - Procedure for determining the status of flocks reacting to tests for Mycoplasma gallisepticum...

Code of Federal Regulations, 2013 CFR

2013-01-01

... agglutination test, enzyme-labeled immunosorbent assay (ELISA), official molecular examination procedure, or... the tube agglutination, ELISA, or serum plate test is positive, the hemaglutination inhibition (HI...

9 CFR 147.7 - Standard test procedures for mycoplasma. 5

Code of Federal Regulations, 2013 CFR

2013-01-01

... plate agglutination test, the tube agglutination test, and the enzyme-linked immunosorbent assay (ELISA... accurate however, and are useful in evaluating serum samples that react with the ELISA, plate, and/or tube...

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…

Paeonol attenuates lipopolysaccharide-induced depressive-like behavior in mice.

PubMed

Tao, Weiwei; Wang, Hanqing; Su, Qiang; Chen, Yanyan; Xue, Wenda; Xia, Baomei; Duan, Jinao; Chen, Gang

2016-04-30

The present study was designed to detect the anti-depressant effects of paeonol and the possible mechanisms in the lipopolysaccharide-induced depressive-like behavior. Open-field test(OFT), tail suspension test(TST) and forced swimming test(FST) were used to evaluate the behavioral activity. The contents of 5-hydroxytryptamine (5-HT) and norepinephrine (NE) in mice hippocampus were determined by HPLC-ECD. Serum interleukin (IL)-1β, IL-6 and tumor necrosis factor (TNF)-α levels were evaluated by enzyme-linked immunosorbent assay (ELISA). Our results showed that LPS significantly decreased the levels of 5-HT and NE in the hippocampus. LPS also reduced open-field activity, as well as increased immobility duration in FST and TST. Paeonol administration could effectively reverse the alterations in the concentrations of 5-HT, NE and reduce the IL-6 and TNF-α levels. Moreover, paeonol effectively downregulated brain-derived neurotrophic factor (BDNF), tropomyosin-related kinase B (TrkB) and Nuclear factor-κB (NF-κB) in hippocampal. In conclusion, paeonol administration exhibited significant antidepressant-like effects in mice with LPS-induced depression. Copyright © 2016. Published by Elsevier Ireland Ltd.

Development of a Quantitative Sandwich Enzyme-Linked Immunosorbent Assay for Detecting the MPT64 Antigen of Mycobacterium tuberculosis

PubMed Central

Ji, Mijung; Cho, Byungki; Cho, Young Shik; Park, Song-Yong; Cho, Sang-Nae

2014-01-01

Purpose Tuberculosis (TB) is a major infectious disease and is responsible for two million deaths annually. For the identification and quantitation of Mycobacterium tuberculosis (M. tuberculosis), a causative agent of TB, a sandwich enzyme-linked immunosorbent assay (ELISA) against the MPT64 protein of M. tuberculosis, an antigen marker of the M. tuberculosis complex, was developed. Materials and Methods The MPT64 protein was expressed, and anti-MPT64 monoclonal antibodies were prepared. A sandwich ELISA was established using recombinant MPT64 protein and anti-MPT64 monoclonal antibodies. The sandwich MPT64 ELISA was evaluated using reference and clinical mycobacterial strains. Results The sandwich MPT64 ELISA detected MPT64 protein from 2.1 ng/mL to 250 ng/mL (equivalent to 1.7×104 CFU/mL and 2.0×106 CFU/mL). All 389 clinical M. tuberculosis isolates tested positive in the sandwich MPT64 ELISA (sensitivity, 100%), and the assay showed no cross reactivity to any tested nontuberculous mycobacterial strain (specificity, 100%). Conclusion The sandwich MPT64 ELISA is a highly sensitive and quantitative test for MPT64 protein, which can identify M. tuberculosis. PMID:24719143

Development of a quantitative sandwich enzyme-linked immunosorbent assay for detecting the MPT64 antigen of Mycobacterium tuberculosis.

PubMed

Ji, Mijung; Cho, Byungki; Cho, Young Shik; Park, Song-Yong; Cho, Sang-Nae; Jeon, Bo-Young; Yoon, Byoung-Su

2014-05-01

Tuberculosis (TB) is a major infectious disease and is responsible for two million deaths annually. For the identification and quantitation of Mycobacterium tuberculosis (M. tuberculosis), a causative agent of TB, a sandwich enzyme-linked immunosorbent assay (ELISA) against the MPT64 protein of M. tuberculosis, an antigen marker of the M. tuberculosis complex, was developed. The MPT64 protein was expressed, and anti-MPT64 monoclonal antibodies were prepared. A sandwich ELISA was established using recombinant MPT64 protein and anti-MPT64 monoclonal antibodies. The sandwich MPT64 ELISA was evaluated using reference and clinical mycobacterial strains. The sandwich MPT64 ELISA detected MPT64 protein from 2.1 ng/mL to 250 ng/mL (equivalent to 1.7×10⁴ CFU/mL and 2.0×10⁶ CFU/mL). All 389 clinical M. tuberculosis isolates tested positive in the sandwich MPT64 ELISA (sensitivity, 100%), and the assay showed no cross reactivity to any tested nontuberculous mycobacterial strain (specificity, 100%). The sandwich MPT64 ELISA is a highly sensitive and quantitative test for MPT64 protein, which can identify M. tuberculosis.

Correlation Between Iron and alpha and pi Glutathione-S-Transferase Levels in Humans

DTIC Science & Technology

2012-09-01

assays were performed as described in the Biotrin High Sensitivity Alpha GST EIA kit protocol. First, serum samples were diluted 1:10 with wash solution...immunosorbent assays were performed as described in the Biotrin Pi GST EIA kit protocol. First, plasma samples were diluted 1:5 with sample diluent...immunosorbent assays were performed as described in the AssayMax Human Transferrin ELISA kit protocol. First, serum samples were diluted 1:2000 with MIX

Enhanced Enzyme-Linked Immunosorbent Assay for Detection of Antibodies to Virus-Like Particles of Human Papillomavirus

PubMed Central

Studentsov, Yevgeniy Y.; Schiffman, Mark; Strickler, Howard D.; Ho, Gloria Y. F.; Pang, Yuk-Ying Susana; Schiller, John; Herrero, Rolando; Burk, Robert D.

2002-01-01

Measurement of antibodies to human papillomavirus (HPV) is complicated by many factors. Although enzyme-linked immunosorbent assays (ELISAs) that use virus-like particles (VLPs) have proved useful, the assays have, in general, had moderate sensitivities and low signal-to-noise ratios. To enhance the performance of the assay, a systematic investigation was undertaken to examine key variables used in ELISAs for the detection of antibodies to VLPs of HPV. Incorporation of two vinyl polymers, polyvinyl alcohol (molecular weight, 50,000) (PVA-50) and polyvinylpyrrolidone (molecular weight, 360,000) (PVP-360), was found to increase the sensitivity as well as the specificity of the assay for the detection of antibodies to VLPs of HPV. In particular, the addition of PVA-50 to the blocking solution reduced the amount of nonspecific binding of antibodies to VLPs and the microplate surface, whereas the addition of PVP-360 increased the sensitivity of antibody detection. The new ELISA demonstrated increased sensitivity and specificity for the detection of cervical HPV type 16 infection compared to those of a prototype assay with coded clinical serum samples from women with known cervicovaginal HPV infection status. It is anticipated that the enhanced ELISA conditions will have wide application to a large number of clinical diagnostic assays. PMID:11980956

Reliability and clinical utility of Enzyme-linked immunosorbent assay for detection of anti-aminoacyl-tRNA synthetase antibody.

PubMed

Abe, Takeo; Tsunoda, Shinichiro; Nishioka, Aki; Azuma, Kouta; Tsuboi, Kazuyuki; Ogita, Chie; Yokoyama, Yuichi; Furukawa, Tetsuya; Maruoka, Momo; Tamura, Masao; Yoshikawa, Takahiro; Saito, Atsushi; Sekiguchi, Masahiro; Azuma, Naoto; Kitano, Masayasu; Matsui, Kiyoshi; Hosono, Yuji; Nakashima, Ran; Ohmura, Koichiro; Mimori, Tsuneyo; Sano, Hajime

2016-01-01

Anti-aminoacyl-tRNA synthetase (ARS) antibody is one of the myositis-specific autoantibodies to make a diagnosis of polymyositis (PM) and dermatomyositis (DM). Recently a new enzyme-linked immunosorbent assay (ELISA) kit of concurrently detected anti-ARS antibodies (anti-Jo-1, anti-PL-7, anti-PL-12, anti-EJ and anti-KS) have become to measure in the clinical setting. To evaluate the reliability of this ELISA kit, we measured anti-ARS antibodies in 75 PM and DM patients using by this ELISA assay and compared them with the results by RNA immunoprecipitation assay. Between the measurements of anti-PL-7, anti-PL-12, anti-EJ and anti-KS autoantibodies by ELISA assay and RNA-IP assay, the concordance rate of reproducibility is 95.1% and the positive agreement rate is 90.9% and negative agreement rate is 96.0% and kappa statistic is 0.841. Between the measurements of existing anti-Jo-1 antibody ELISA kit and anti-ARS antibody ELISA kit, the concordance rate of reproducibility is 96.9%, the positive agreement rate is 100%, negative agreement rate is 96.1% and kappa statistic is 0.909. The lung involvement in patients with PM and DM patients are positive of anti-ARS antibodies and anti-melanoma differentiation associated gene5 (MDA5) antibody at a rate around 70%. Then most life-threatening ILD with anti-MDA5 positive clinically amyopathic dermatomyositis patients could be highly guessed when anti-ARS antibodies are negative.

A New Highly Selective and Specific Anti-puerarin polyclonal Antibody for Determination of Puerarin Using a Mannich Reaction Hapten Conjugate

PubMed Central

Udomsin, Orapin; Krittanai, Supaluk; Kitisripanya, Tharita; Tanaka, Hiroyuki; Putalun, Waraporn

2017-01-01

Background: Puerarin (PUE) is a phytoestrogen found in Pueraria candollei and Pueraria lobata. These plants are substantial for traditional medicine in various Asian countries. PUE is a key marker that can be found only in the Pueraria species. Objective: To establish the method for determination of PUE content which is required for quality control of pharmaceutical products. Materials and Methods: PUE-cationized bovine serum albumin conjugate was created via Mannich reaction. After the rabbit immunization, the obtain anti-PUE polyclonal antibody (PAb) was used to develop an enzyme-linked immunosorbent assay (ELISA). Results: An anti-PUE PAb possess a great sensitivity and specificity. The cross-reactivity analysis shows no cross-reaction of an established antibody against other substances. In addition, we successfully developed an indirect competitive ELISA (icELISA) for the quantitative analysis of PUE. The result of method validation conforms to acceptance criteria and correlates with high-performance liquid chromatography, the reference method. The icELISA was applied to determine PUE content in Pueraria spp. plant samples and its derived pharmaceutical products. Conclusion: This highly specific immunogen was created from the Mannich reaction. An icELISA can also be applied to other research propose in the further studies. SUMMARY The new immunogen conjugated (puerarin-cBSA) via Mannich reaction was successfully in rising of antibody against puerarin (PUE)The obtained anti-PUE polyclonal antibody (PAb) was high sensitivity and specificity to PUEAn indirect competitive enzyme-linked immunosorbent assay (icELISA) was developed and validated using anti-PUE PAbThe established icELISA was applied to determine PUE content in various tuberous root of Pueraria sppMoreover, icELISA method can be applicable in Pueraria spp. derived products. Abbreviations used: PUE: Puerarin; PAb: Polyclonal antibody; ELISA: Enzyme-linked immunosorbent assay; icELISA: Indirect competitive ELISA; cBSA: Cationized bovine serum albumin. PMID:29491643

DARPA Antibody Technology Program. Standardized Test Bed for Antibody Characterization: Characterization of an MS2 ScFv Antibody Produced by Illumina

DTIC Science & Technology

2016-08-01

platforms. 15. SUBJECT TERMS Antibody Antibody Technology Program (ATP) Quality Enzyme-linked immunosorbent assay ( ELISA ) Biosurveillance Single-chain...2.6 Thermal Stress Test............................................................................................4 2.7 ELISA ...3.5 ELISA Results .................................................................................................11 3.6 SPR Results

42 CFR 71.53 - Requirements for importers of nonhuman primates.

Code of Federal Regulations, 2014 CFR

2014-10-01

... Trade in Endangered Species. ELISA means enzyme-linked immunosorbent assay, a type of laboratory test... serum for immunoglobulin G (IgG) antibodies to filovirus by using an ELISA methodology, or other method... for filovirus antigen by using the antigen-capture ELISA method must be submitted to a qualified...

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…

42 CFR 71.53 - Requirements for importers of nonhuman primates.

Code of Federal Regulations, 2013 CFR

2013-10-01

... Trade in Endangered Species. ELISA means enzyme-linked immunosorbent assay, a type of laboratory test... serum for immunoglobulin G (IgG) antibodies to filovirus by using an ELISA methodology, or other method... for filovirus antigen by using the antigen-capture ELISA method must be submitted to a qualified...

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

EPA Science Inventory

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

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

PubMed

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

2017-11-01

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

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

USDA-ARS?s Scientific Manuscript database

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

Evaluation of a Commercial Sandwich Enzyme-Linked Immunosorbent Assay for the Quantification of Beta-Casomorphin 7 in Yogurt Using Solid-Phase Extraction Coupled to Liquid Chromatography-Tandem Mass Spectrometry as the "Gold Standard" Method.

PubMed

Nguyen, Duc Doan; Busetti, Francesco; Johnson, Stuart Keith; Solah, Vicky Ann

2018-03-01

This study investigated beta-casomorphin 7 (BCM7) in yogurt by means of LC-tandem MS (MS/MS) and enzyme-linked immunosorbent assay (ELISA) and use LC-MS/MS as the "gold standard" method to evaluate the applicability of a commercial ELISA. The level of BCM7 in milk obtained from ELISA analysis was much lower than that obtained by LC-MS/MS analysis and trended to increase during fermentation and storage of yogurt. Meanwhile, the results obtained from LC-MS/MS showed that BCM7 degraded during stages of yogurt processing, and its degradation may have been caused by X-prolyl dipeptidyl aminopeptidase activity. As a result, the commercial sandwich ELISA kit was not suitable for the quantification of BCM7 in fermented dairy milk.

A Protein Microarray ELISA for the Detection of Botulinum neurotoxin A

DOE Office of Scientific and Technical Information (OSTI.GOV)

Varnum, Susan M.

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.

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

USGS Publications Warehouse

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

1996-01-01

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

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

PubMed

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

2001-12-01

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

Review on enzyme-linked immunosorbent assays for sulfonamide residues in edible animal products.

PubMed

Zhang, Hongyan; Wang, Shuo

2009-10-31

The current status of enzyme-linked immunosorbent assays (ELISAs) for sulfonamides in edible animal products is reviewed. The attention was focused on the design and synthesis of haptens, conjugation to carrier protein, production of antibody, application of homologous and heterologous systems, as well as the molecular modeling of the haptens and sulfonamides. Researches have shown that sulfonamides seem to be particularly resistant to attempts to produce broad specificity antibodies. By summarizing the available research on sulfonamide ELISAs, it is hoped that it can be considered as a basis for further investigation aimed at developing the most efficient approaches for detection.

Determination of antibody response to influenza virus surface glycoproteins by kinetic enzyme-linked immunosorbent assay.

PubMed Central

Snyder, M H; Banks, S; Murphy, B R

1988-01-01

We modified an existing enzyme-linked immunosorbent assay (ELISA) to be able to use new spectrophotometers which can measure the rate of color development in microtiter wells. This new kinetic-based ELISA (KELISA) required only a single dilution of specimen rather than the multiple dilutions required with endpoint ELISA. In addition, 10- to 100-fold-less specimen was required to perform the KELISA than the ELISA. The level of serum or nasal wash antibody against surface glycoproteins of influenza A or influenza B viruses determined by KELISA was reproducible and correlated highly with the results of endpoint ELISA or hemagglutination inhibition tests. The difference between the KELISA rates, which indicated than an antibody response to infection had occurred, was defined and was analogous to a 2.2-fold rise in titer for serum and a 3.4-fold rise in titer for nasal wash determined by endpoint ELISA. The KELISA was similar to endpoint ELISAs in its ability to detect rises in antibody level in paired serum or nasal wash specimens obtained from volunteers who received live attenuated influenza A reassortant virus vaccines. By eliminating the need for multiple dilutions, the use of KELISA offers the advantage of increasing the number of assays that can be performed by the same personnel compared with endpoint ELISA, while it maintains sensitivity and specificity. PMID:3182992

Development and Application of an Indirect Enzyme-Linked Immunosorbent Assay Using Recombinant Mag1 for Serodiagnosis of Toxoplasma gondii In Dogs.

PubMed

Zhuo, Xunhui; Sun, Hongchao; Zhang, Zhi; Luo, Jiaqing; Shan, Ying; Du, Aifang

2017-06-01

Serologic tests are widely accepted and applied as means to detect anti- Toxoplasma gondii immunoglobulin G antibodies. In this study, recombinant matrix antigen (rMAG1) was induced by isopropyl-β-d-thiogalactoside and purified by nickel-nitrilotriacetic acid purification system. We then developed and optimized an indirect enzyme-linked immunosorbent assay (ELISA) through checkerboard assays using serial dilutions of antigens and sera to assess the potential use of rMAG1 in serologic detection of T. gondii infection in dogs. Serum samples from 93 domestic dogs were analyzed by western blot and rMAG1-ELISA. The results were compared with those obtained from an ELISA with the soluble Toxoplasma lysate antigens (TLA). We found that although yielding an excellent agreement (96.7%) with western blot data (κ = 0.9659), rMAG1-ELISA produced higher sensitivity (93.9% vs. 87.8%) and specificity (98.3% vs. 96.7%) than TLA-ELISA. In addition, receiver operating characteristic analysis also revealed that rMAG1-ELISA is in more agreement with western blot (area under the curve [AUC] = 0.985) relative to TLA-ELISA (AUC = 0.955). These results indicated that the rMAG1-ELISA established in this study provides a promising and reliable tool for serologic detection of T. gondii infection in dogs.

Development and evaluation of an avian influenza (AI) neuraminidase subtype 1 (N1) based serological ELISA for poultry using the differentiation of infected and vaccinated animals (DIVA)approach

USDA-ARS?s Scientific Manuscript database

An indirect enzyme-linked immunosorbent assay (ELISA) was developed using baculovirus expressed N1 protein from the A/CK/Indonesia/PA/2003 (H5N1) virus. The specificity of the assay was tested with a panel of chicken anti-sera raised against N1 to N9 virus subtypes. The N1-ELISA was specific for t...

Amplification of the enzyme-linked immunosorbent assay (ELISA) in the detection of class-specific antibodies.

PubMed

Butler, J E; McGivern, P L; Swanson, P

1978-01-01

A modification of the standard enzyme-linked immunosorbent assay (ELISA) is described which circumvents the requirement for specifically purified antibodies from which antibody-enzyme complexes are made. The assay utilizes the principle of a soluble anti-alkaline phosphatase immune complex (AP-A-AP) and has been called the amplified ELISA. Methods for preparing and evidence for the specificity of rabbit anti-rat gamma-FC, IgM (mu) and IgA (alpha) are presented. These reagents are used to measure anti-DNP antibodies belonging to classes IgG, IgM and IgA in rat serum. Using antiglobulin and anti-enzyme reagents prepared in guinea pigs, anti-ovalbumin antibodies are measured in rabbit serum. Titration curves are similar when the amplified ELISA is compared to the standard ELISA. A change in slope suggesting an effect of saturation of antigen sites, occurs at the same input antibody concentration for both assays. Determination of the anti-DNP concentration of unknown sera by extrapopulation from titration graphs of a known serum suggests that the value is overestimated, i.e., amplified when the amplified ELISA is used. In addition, the amplified ELISA has an improved ability to detect low levels of antibody. Evidence is presented which illustrates how the use of optimally conjugated DNP-proteins, age of conjugates, and optimal dilutions of secondary antiglobulins and the AP-A-AP reduce non-specific binding in the amplified ELISA. The amplified ELISA is capable of detecting 2.4 ng of antibody to ovalbumin in a one: one million dilution of rabbit serum with high reproducibility and low background.

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

USDA-ARS?s Scientific Manuscript database

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

DARPA Antibody Technology Program Standardized Test Bed for Antibody Characterization: Characterization of an MS2 Human IgG Antibody Produced by AnaptysBio, Inc.

DTIC Science & Technology

2016-02-01

Enzyme-linked immunosorbent assay ( ELISA ) Quality Testing MS2 coat protein (MS2CP) 16. SECURITY CLASSIFICATION OF: 17. LIMITATION OF... ELISA ................................................................................................................4 2.8 SPR Method...3.5 ELISA Results .................................................................................................11 3.6 SPR Results

Desomorphine Screening Using Commercial Enzyme-Linked Immunosorbent Assays.

PubMed

Winborn, Jessica; Kerrigan, Sarah

2017-06-01

Desomorphine ("Krokodil") is a semi-synthetic opioid that has drawn attention as a recreational drug, particularly in Russia, neighboring former Soviet Republics, Eastern and Central Europe. It has no accepted medicinal uses and is currently a schedule I drug in the United States. In clandestine environments, desomorphine is synthesized from codeine using red phosphorous, hydroiodic acid and gasoline. Residual starting materials in illicit preparations have been associated with severe dermatological effects and extensive tissue necrosis. Desomorphine is not well studied, and there are limited reports concerning its pharmacology or detection in biological matrices. Immunoassays are widely relied upon for both antemortem and postmortem toxicology screening. Although desomorphine is an opioid of the phenanthrene-type, its ability to bind to conventional opioid antibodies has not been described. In this report we describe the cross-reactivity of desomorphine using six commercially available enzyme-linked immunosorbent assays (Immunalysis Opiates Direct ELISA, Immunalysis Oxycodone/Oxymorphone Direct ELISA, Randox Opiate ELISA, OraSure Technologies OTI Opiate Micro-plate EIA, Neogen Opiate Group ELISA and Neogen Oxycodone/Oxymorphone ELISA). Cross-reactivites were highly variable between assays, ranging from 77 to <2.5%. In general, assays directed towards morphine produced greater cross-reactivity with desomorphine than those directed towards oxycodone. The Immunalysis Opiates Direct ELISA produced the greatest cross-reactivity, although several of the assays evaluated produced cross-reactivity of a sufficient magnitude to be effective for desomorphine screening. © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

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

PubMed

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

2014-09-01

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

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.

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

PubMed Central

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

2015-01-01

Enzyme-linked Immunosorbent Assay (ELISA)-based diagnosis is the mainstay for measuring antibody response in infectious diseases and to support pathogen identification of potential use in infectious disease outbreaks and clinical care of individual patients. The development of laboratory diagnostics using readily available 3D printing technologies provides a timely opportunity for further expansion of this technology into immunodetection systems. Utilizing available 3D printing platforms, a ‘3D well’ was designed and developed to have an increased surface area compared to those of 96-well plates. The ease and rapidity of the development of the 3D well prototype provided an opportunity for its rapid validation through the diagnostic performance of ELISA in infectious disease without modifying current laboratory practices for ELISA. The improved sensitivity of the 3D well of up to 2.25-fold higher compared to the 96-well ELISA provides a potential for the expansion of this technology towards miniaturization and Lab-On-a-Chip platforms to reduce time, volume of reagents and samples needed for such assays in the laboratory diagnosis of infectious and other diseases including applications in other disciplines. PMID:26184194

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.

Influence of the incubation temperature and the batch components on the sensitivity of an enzyme-linked immunosorbent assay to detect Aujeszky's disease virus glycoprotein E (gE).

PubMed

Cay, A B; Van der Stede, Y

2010-12-01

Although licensed batches of an enzyme-linked immunosorbent assay (ELISA) for Aujeszky's disease virus (ADV) were used, and the assays were performed within an ISO/IEC 17025 accredited quality control system, certain routine runs of the ADV ELISA were not validated using the quality system criteria, even when all technical parameters were controlled. Incubation at different temperatures and batch composition were identified as parameters that could result in non-validated assays/runs. Therefore, the effect of incubation temperature and batch composition on the analytical sensitivity of the ELISA was investigated. The World Organisation for Animal Health (OIE) standard reference serum ADV1 was diluted 1:8 and tested in 94 different glycoprotein E ELISA runs performed with different batches and different incubation temperatures. The incubation temperature and batch components had a significant influence on the qualitative result for the OIE standard reference serum. An incubation temperature of at least 22 degrees C was recommended, based on the results of this analysis. Which of the batch components caused these differences in sensitivity was not investigated further.

An Evaluation Study of Enzyme-Linked Immunosorbent Assay (ELISA) Using Recombinant Protein Pap31 for Detection of Antibody against Bartonella bacilliformis Infection among the Peruvian Population

PubMed Central

Angkasekwinai, Nasikarn; Atkins, Erin H.; Romero, Sofia; Grieco, John; Chao, Chien Chung; Ching, Wei Mei

2014-01-01

Reliable laboratory testing is of great importance to detect Bartonella bacilliformis infection. We evaluated the sensitivity and specificity of the enzyme-linked immunosorbent assay (ELISA) using recombinant protein Pap31 (rPap31) for the detection of antibodies against B. bacilliformis as compared with immunofluorescent assay (IFA). Of the 302 sera collected between 1997 and 2000 among an at-risk Peruvian population, 103 and 34 samples tested positive for IFA-immunoglobulin G (IgG) and IFA-IgM, respectively. By using Youden's index, the cutoff values of ELISA-IgG at 0.915 gave a sensitivity of 84.5% and specificity of 94%. The cutoff values of ELISA-IgM at 0.634 gave a sensitivity of 88.2% and specificity of 85.1%. Using latent class analysis, estimates of sensitivity and specificity of almost all the assays were slightly higher than those of a conventional method of calculation. The test is proved beneficial for discriminating between infected and non-infected individuals with the advantage of low-cost and high-throughput capability. PMID:24515944

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

PubMed

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

2014-11-01

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

77 FR 6471 - Bacillus thuringiensis Cry2Ae Protein in Cotton; Exemption from the Requirement of a Tolerance

Federal Register 2010, 2011, 2012, 2013, 2014

2012-02-08

... analytical method using enzyme- linked immunosorbent assay (ELISA) analyses for the qualitative detection of... study conducted by an independent third party laboratory to evaluate the ELISA test kit's performance as...

Smartphone instrument for portable enzyme-linked immunosorbent assays

PubMed Central

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

2014-01-01

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

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

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

PubMed

Johnson, J C; Van Emon, J M

1996-01-01

An enzyme-linked immunosorbent assay (ELISA) for the quantitative determination of Aroclors 1242, 1248, 1254, and 1260 in soil and sediments was developed and its performance compared with that of gas chromatography (GC). The detection limits for Aroclors 1242 and 1248 in soil are 10.5 and 9 ng/g, respectively. The assay linear dynamic range is 50-1333 ng/g. Cross-reactivity of the assay with 37 structurally related potential cocontaminants in environmental soil samples was examined; none of the chlorinated anisoles, benzenes, or phenols exhibited >3% cross-reactivity, with <0.1% cross-reactivity being the norm. Soil spike recoveries of 107% and 104% were obtained for Aroclors 1242 and 1248, respectively, for a spike level of 5 mg/kg, with corresponding relative standard deviations of 14% and 17%. One hundred forty-eight environmental soil, sediment, and paper pulp samples, obtained from two EPA listed Superfund sites, were analyzed by ELISA and standard GC methods. Samples were extracted for ELISA analysis by shaking with methanol. Additional extractions of the same samples were performed either with supercritical carbon dioxide or by Soxhlet extraction with methanol. ELISA results for both the supercritical fluid and the Soxhlet extracts were in close agreement with the GC results, while the ELISA results for the methanol shake extracts were not. The data for the environmental samples demonstrated the capability of the ELISA to provide accurate results and reinforced the dependence of any detection method, including ELISA, on appropriate extraction procedures.

Comparison of Laboratory-Developed and Commercial Monoclonal Antibody-Based Sandwich Enzyme-Linked Immunosorbent Assays for Almond (Prunus dulcis) Detection and Quantification.

PubMed

Liu, Changqi; Chhabra, Guneet S; Zhao, Jing; Zaffran, Valerie D; Gupta, Sahil; Roux, Kenneth H; Gradziel, Thomas M; Sathe, Shridhar K

2017-10-01

A commercially available monoclonal antibody (mAb)-based direct sandwich enzyme-linked immunosorbent assay (ELISA) kit (BioFront Technologies, Tallahassee, Fla., U.S.A.) was compared with an in-house developed mAb 4C10-based ELISA for almond detection. The assays were comparable in sensitivity (limit of detection < 1 ppm full fat almond, limit of quantification < 5 ppm full fat almond), specificity (no cross-reactivity with 156 tested foods at a concentration of 100000 ppm whole sample), and reproducibility (intra- and interassay variability < 15% CV). The target antigens were stable and detectable in whole almond seeds subjected to autoclaving, blanching, frying, microwaving, and dry roasting. The almond recovery ranges for spiked food matrices were 84.3% to 124.6% for 4C10 ELISA and 81.2% to 127.4% for MonoTrace ELISA. The almond recovery ranges for commercial and laboratory prepared foods with declared/known almond amount were 30.9% to 161.2% for 4C10 ELISA and 38.1% to 207.6% for MonoTrace ELISA. Neither assay registered any false-positive or negative results among the tested commercial and laboratory prepared samples. Ability to detect and quantify trace amounts of almonds is important for improving safety of almond sensitive consumers. Two monoclonal antibody-based ELISAs were compared for almond detection. The information is useful to food industry, regulatory agencies, scientific community, and almond consumers. © 2017 Institute of Food Technologists®.

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

PubMed

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

2014-08-01

The enzyme-linked immunosorbent assay (ELISA) is the diagnostic test most commonly used in efforts to control paratuberculosis in domestic ruminants. However, commercial ELISAs have not been validated for detecting antibodies against Mycobacterium avium subsp. paratuberculosis in wild animals. In this study, we compared the sensitivities and specificities of five ELISAs using individual serum samples collected from 41 fallow deer with or without histopathological lesions consistent with paratuberculosis. Two target antigenic preparations were selected, an ethanol-treated protoplasmic preparation obtained from a fallow deer M. avium subsp. paratuberculosis isolate (ELISAs A and B) and a paratuberculosis protoplasmic antigen (PPA3) (ELISAs C and D). Fallow deer antibodies bound to the immobilized antigens were detected by using a horseradish peroxidase (HRP)-conjugated anti-fallow deer IgG antibody (ELISAs A and C) or HRP-conjugated protein G (ELISAs B and D). A commercially available assay, ELISA-E, which was designed to detect M. avium subsp. paratuberculosis antibodies in cattle, sheep, and goats, was also tested. Although ELISAs A, C, and E had the same sensitivity (72%), ELISAs A and C were more specific (100%) for detecting fallow deer with lesions consistent with paratuberculosis at necropsy than was the ELISA-E (87.5%). In addition, the ELISA-A was particularly sensitive for detecting fallow deer in the latent stages of infection (62.5%). The antibody responses detected with the ELISA-A correlated with both the severity of enteric lesions and the presence of acid-fast bacteria in gut tissue samples. In summary, our study shows that the ELISA-A can be a cost-effective diagnostic tool for preventing the spread of paratuberculosis among fallow deer populations. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

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

PubMed Central

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

2014-01-01

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

Enzyme-Linked Immunosorbent Assay and Serologic Responses to Pneumocystis jiroveci

PubMed Central

Koch, Judy; Levin, Linda; Walzer, Peter D.

2004-01-01

Seroepidemiologic studies of Pneumocystis pneumonia (PCP) in humans have been limited by inadequate reagents. We have developed an enzyme-linked immunosorbent assay (ELISA) using three overlapping recombinant fragments of the human Pneumocystis major surface glycoprotein (MsgA, MsgB, and MsgC) for analysis of antibody responses in HIV-positive patients and healthy blood donors. HIV-positive patients had significantly higher antibody levels to all Msg fragments. Furthermore, HIV-positive patients who experienced a previous episode of PCP (PCP-positive) had higher level of antibodies to MsgC than patients who never had PCP. A significant association was found between ELISA antibody level and reactivity by Western blot in HIV-positive patients, especially those who were PCP-positive. Thus, this ELISA will be useful in studying serum antibody responses to Pneumocystis in different human populations. PMID:15200818

Definition of purified enzyme-linked immunosorbent assay antigens from the culture filtrate protein of Mycobacterium bovis by proteomic analysis.

PubMed

Cho, Yun Sang; Lee, Sang-Eun; Ko, Young Joon; Cho, Donghee; Lee, Hyang Shim; Hwang, Inyeong; Nam, Hyangmi; Heo, Eunjung; Kim, Jong Man; Jung, Sukchan

2009-01-01

Enzyme-linked immunosorbent assay (ELISA) has been developed as the ancillary diagnosis of bovine tuberculosis at ante-mortem to overcome the disadvantages of intradermal skin test. In this study, the antigenic proteins were purified, applied to bTB ELISA, and identified through proteomic analysis. Culture filtrate protein of Mycobacterium bovis was fractionated by MonoQ column chromatography, and examined the antigenicity by immunoblotting. The antigenic 20 kDa protein was in-gel digested and identified the antigenome by LTQ mass spectrometer and peptide match fingerprinting, which were MPB64, MPB70, MPB83, Fas, Smc, Nrp, RpoC, Transposase, LeuA, and MtbE. The 20 kDa protein exhibited the highest antigenicity to bTB positive cattle in ELISA and would be useful for bTB serological diagnosis.

Celiac Disease Testing (for Health Care Professionals)

MedlinePlus

... diet. tTG The tTG-IgA test is an enzyme-linked immunosorbent assay (ELISA) test. The tTG-IgA test is the preferred ... a more expensive and time-consuming process than ELISA testing. In addition, the EMA test is qualitative, ...

Evaluation of a recombinant LigB protein of Leptospira interrogans serovar Canicola in an enzyme-linked immunosorbent assay for the serodiagnosis of bovine leptospirosis.

PubMed

Sankar, Surya; Harshan, Hiron M; Somarajan, S R; Srivastava, S K

2010-06-01

A recombinant leptospiral lipoprotein, LigB, was evaluated for use in the diagnosis of bovine leptospirosis by enzyme-linked immunosorbent assay (rLigB IgG ELISA). The standard reference test (Microscopic agglutination test, MAT) of 200 serum samples from cattle suspected of leptospirosis showed that 95 (47.5%) samples had positive agglutination titres, which ranged from 100 to 1600. In rLigB IgG ELISA, 49% of the samples were positive. Sensitivity of IgG ELISA for 95 bovine sera, which had MAT titres of greater than or equal to 100, were 100%. ELISA showed a specificity of 97.1% with 105 bovine sera, which were negative at a 1:50 dilution in MAT for Leptospira interrogans serovars. The results of ELISA and MAT correspond very good. When analytical specificity of IgG ELISA was evaluated using bovine serum samples from animals showing the serum antibodies to other pathogens, no cross-reaction was observed. Thus the recombinant LigB IgG ELISA can be used instead of the MAT as an aid to the diagnosis of bovine leptospirosis. Copyright 2009. Published by Elsevier India Pvt Ltd.

Development of an enzyme-linked immunosorbent assay (ELISA) for residue analysis of the fungicide azoxystrobin in agricultural products.

PubMed

Kondo, Mika; Tsuzuki, Kazuyuki; Hamada, Hiroshi; Yamaguchi Murakami, Yukie; Uchigashima, Mikiko; Saka, Machiko; Watanabe, Eiki; Iwasa, Seiji; Narita, Hiroshi; Miyake, Shiro

2012-02-01

A direct competitive enzyme-linked immunosorbent assay (dc-ELISA) was developed for residue analysis of azoxystrobin in garden crops, for which the maximum residue limits (MRLs) are 0.5-50 mg/kg in Japan. For hapten synthesis, an ethyl carboxyl group was introduced to the 4-position of the 2-cyanophenoxy group in azoxystrobin, and its cyano group was changed to a methyl group. An anti-azoxystrobin monoclonal antibody was prepared from mice immunized with hapten-keyhole limpet hemocyanin conjugate. The dc-ELISA using prepared antibody showed 50-250-fold higher sensitivity compared to the MRLs. The working range of the dc-ELISA was 10-200 ng/mL. The dc-ELISA showed high specificity to azoxystrobin. When methanol extracts from nine kinds of garden crops spiked with azoxystrobin ranging near the MRLs were analyzed, the determined results by the dc-ELISA agreed well with the results of their controls. In addition, azoxystrobin spiked in garden crops homogenates was satisfactorily extracted by methanol solution and easily analyzed. The recovery rate of dc-ELISA was 96-109% and correlated well with the results obtained by HPLC analysis.

Development of 316v antibody enzyme-linked immunosorbent assay for detection of paratuberculosis in sheep.

PubMed

Gurung, R B; Begg, D J; Purdie, A C; Eamens, G J; Whittington, R J

2015-12-01

An enzyme-linked immunosorbent assay (ELISA) was developed and optimised using a Mycobacterium avium subspecies paratuberculosis (MAP) antigen prepared from a C strain (316v) passed through a French press. The optimised assay was evaluated with a panel of sera from MAP infected (n = 66) and uninfected (n = 1,092) sheep. Animals in the MAP infected category were positive on either tissue culture or histopathology but were of unknown serum antibody status. The diagnostic performance and cost of the assay were compared with those of a commercial ELISA (IDEXX). At 99.8% diagnostic specificity the assay showed a diagnostic sensitivity of 23% (95% CI: 15.1-35.8) compared with 36.4% (95% CI: 25.8-48.4) for the commercial ELISA (McNemar's test: chi-square 5.82, p < 0.05). The sensitivities were 5.9% (95% CI: 1-26.9), 27.9% (95% CI: 14.7-45.7) and 35% (95% CI: 18.1-56.7), for low grade, paucibacillary and multibacillary lesion grades, respectively. The cost of the commercial assay kit was 2.7 to 5.2 times greater than that of the 316v ELISA for an equivalent number of tests, the multiple depending on the number of plates processed per run. For flock-level surveillance, to account for the lower sensitivity of the 316v ELISA compared with the commercial ELISA, sample sizes would be increased but the test cost would still be lower. The 316v assay will be useful for diagnosis of Johne's disease in sheep flocks, particularly in developing countries where labour costs are low relative to the cost of consumables.

Analysis of pirlimycin residues in beef muscle, milk and honey by a biotin-streptavidin-amplified enzyme-linked immunosorbent assay

USDA-ARS?s Scientific Manuscript database

Food contamination caused by veterinary drug residues is a world-wide public health concern and requires continuous monitoring. In this paper, we describe a biotin–streptavidin-amplified ELISA (BA-ELISA) for detecting pirlimycin residues in beef, milk, and honey. The IC50 value of the BA-ELISA was...

DARPA Antibody Technology Program Standardized Test Bed for Antibody Characterization: Characterization of an MS2 sdAb Produced by U.S. Naval Research Laboratory

DTIC Science & Technology

2016-10-01

Program (ATP) Quality MS2 coat protein (MS2CP) Enzyme-linked immunosorbent assay ( ELISA ) 16. SECURITY CLASSIFICATION OF: 17. LIMITATION OF...3 2.6 Thermal Stress Test............................................................................................4 2.7 ELISA ...7 3.4 DSC Results .....................................................................................................10 3.5 ELISA

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

PubMed

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

2010-02-01

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

Enzyme-linked immunosorbent assay for the quantitative/qualitative analysis of plant secondary metabolites.

PubMed

Sakamoto, Seiichi; Putalun, Waraporn; Vimolmangkang, Sornkanok; Phoolcharoen, Waranyoo; Shoyama, Yukihiro; Tanaka, Hiroyuki; Morimoto, Satoshi

2018-01-01

Immunoassays are antibody-based analytical methods for quantitative/qualitative analysis. Since the principle of immunoassays is based on specific antigen-antibody reaction, the assays have been utilized worldwide for diagnosis, pharmacokinetic studies by drug monitoring, and the quality control of commercially available products. Berson and Yalow were the first to develop an immunoassay, known as radioimmunoassay (RIA), for detecting endogenous plasma insulin [1], a development for which Yalow was awarded the Nobel Prize in Physiology or Medicine in 1977. Even today, after half a century, immunoassays are widely utilized with some modifications from the originally proposed system, e.g., radioisotopes have been replaced with enzymes because of safety concerns regarding the use of radioactivity, which is referred to as enzyme immunoassay/enzyme-linked immunosorbent assay (ELISA). In addition, progress has been made in ELISA with the recent advances in recombinant DNA technology, leading to increase in the range of antibodies, probes, and even systems. This review article describes ELISA and its applications for the detection of plant secondary metabolites.

Aggregated silver nanoparticles based surface-enhanced Raman scattering enzyme-linked immunosorbent assay for ultrasensitive detection of protein biomarkers and small molecules.

PubMed

Liang, Jiajie; Liu, Hongwu; Huang, Caihong; Yao, Cuize; Fu, Qiangqiang; Li, Xiuqing; Cao, Donglin; Luo, Zhi; Tang, Yong

2015-06-02

Lowering the detection limit is critical to the design of bioassays required for medical diagnostics, environmental monitoring, and food safety regulations. The current sensitivity of standard color-based analyte detection limits the further use of enzyme-linked immunosorbent assays (ELISAs) in research and clinical diagnoses. Here, we demonstrate a novel method that uses the Raman signal as the signal-generating system of an ELISA and combines surface-enhanced Raman scattering (SERS) with silver nanoparticles aggregation for ultrasensitive analyte detection. The enzyme label of the ELISA controls the dissolution of Raman reporter-labeled silver nanoparticles through hydrogen peroxide and generates a strong Raman signal when the analyte is present. Using this assay, prostate-specific antigen (PSA) and the adrenal stimulant ractopamine (Rac) were detected in whole serum and urine at the ultralow concentrations of 10(-9) and 10(-6) ng/mL, respectively. The methodology proposed here could potentially be applied to other molecules detection as well as PSA and Rac.

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

PubMed

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

2014-03-01

Tetrabromobisphenol A (TBBPA) is a widely used brominated flame retardant. A sensitive and selective indirect competitive biotin-streptavidin-amplified enzyme-linked immunosorbent assay (BA-ELISA) was developed for detecting TBBPA. The optimal hapten of TBBPA was 2-(2,6-dibromo-4-(2-(3,5-dibromo-4-hydroxyphenly)propan-2-yl)) acetic acid. Several physiochemical factors that influence assay performance, such as optimal coupling concentration of immunogen and antibody, organic solvent, ionic strength, and pH, were studied and optimized. The limit of detection (IC10) was 0.027 ng/mL and the median inhibitory concentration (IC50) was 0.58 ng/mL. The BA-ELISA was highly selective, with low cross-reactivity with TBBPA analogs. Finally, the assay was used to detect TBBPA in electronic waste samples. The results are consistent with those using liquid chromatography, which proves that the proposed immunoassay is accurate and receptive. This BA-ELISA method is suitable for the rapid and sensitive screening of TBBPA in environmental monitoring. © 2013 Published by Elsevier B.V.

Microbubble Enzyme-Linked Immunosorbent Assay for the Detection of Targeted Microbubbles in in Vitro Static Binding Assays.

PubMed

Wischhusen, Jennifer; Padilla, Frederic

2017-07-01

Targeted microbubbles (MBs) are ultrasound contrast agents that are functionalized with a ligand for ultrasound molecular imaging of endothelial markers. Novel targeted MBs are characterized in vitro by incubation in protein-coated wells, followed by binding quantification by microscopy or ultrasound imaging. Both methods provide operator-dependent results: Between 3 and 20 fields of view from a heterogeneous sample are typically selected for analysis by microscopy, and in ultrasound imaging, different acoustic settings affect signal intensities. This study proposes a new method to reproducibly quantify MB binding based on enzyme-linked immunosorbent assay (ELISA), in which bound MBs are revealed with an enzyme-linked antibody. MB-ELISA was adapted to in vitro static binding assays, incubating the MBs in inverted position or by agitation, and compared with microscopy. The specificity and sensitivity of MB-ELISA enable the reliable quantification of MB binding in a rapid, high-throughput and whole-well analysis, facilitating the characterization of new targeted contrast agents. Copyright © 2017 World Federation for Ultrasound in Medicine & Biology. Published by Elsevier Inc. All rights reserved.

COMPARISON OF ELISAS FOR DETECTING VITELLOGENIN IN THE FATHEAD MINNOW (PIMEPHALES PROMELAS)

EPA Science Inventory

Measurement of vitellogenin (VTG) concentrations in the fathead minnow is currently being evaluated and considered for screening of endocrine active substances. One of the proposed methods, an enzyme-linked immunosorbent assay (ELISA) based on VTG from carp, was recently evaluate...

Validation of 2 commercial Neospora caninum antibody enzyme linked immunosorbent assays

PubMed Central

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

2002-01-01

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

Comparison of Three Enzyme-Linked Immunosorbent Assays for Detection of Immunoglobulin G Antibodies to Tetanus Toxoid with Reference Standards and the Impact on Clinical Practice▿

PubMed Central

van Hoeven, Karen H.; Dale, Connie; Foster, Phil; Body, Barbara

2008-01-01

Accurate determination of the concentrations of immunoglobulin G (IgG) antibody to tetanus toxoid is important in order to evaluate the immunogenicity of tetanus toxoid vaccines, determine immune competence in individual patients, and measure the prevalence of immunity in populations. The performance of three commercially available enzyme-linked immunosorbent assays (ELISAs) for IgG antibodies to tetanus toxoid were evaluated. Serially diluted NIBSC 76/589 and TE-3 human tetanus IgG immunoglobulin international reference standards were analyzed in quadruplicate using ELISAs manufactured by The Binding Site, Inc. (VaccZyme); Scimedx; and Euroimmun. In addition, IgG antibodies to tetanus toxoid were measured in 83 deidentified serum specimens using each manufacturer's ELISA. Each ELISA provided linear results when evaluated with the reference preparations. The Binding Site ELISA provided results that closely corresponded to the reference preparations (y = 1.09x − 0.08), whereas the Scimedx ELISA gave results that were consistently lower (y = 0.21x − 0.07) and the Euroimmun ELISA gave results that were consistently higher (y = 1.5x + 0.30) than the reference preparation concentrations. Using the recommended cutoff for each ELISA (<0.10 IU/ml), the overall agreement of all of the ELISA methods was 78%. Three of eighty-three (3.6%) human serum samples demonstrated inadequate immunity with all three assays. The Binding Site ELISA yielded nonprotective antibody concentrations in only these 3 samples, whereas 19 samples (22.9%) according to the Scimedx ELISA and 6 samples (7.2%) according to the Euroimmun ELISA demonstrated nonprotective concentrations. The performance characteristics of ELISAs for tetanus immunoglobulin titers were manufacturer dependent, and the differences translated into important disparities in reported results. PMID:18845832

The Effect of Different Methods of Fermentation on the Detection of Milk Protein Residues in Retail Cheese by Enzyme-Linked Immunosorbent Assay (ELISA).

PubMed

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

2017-11-01

Milk and milk products are among the most important allergenic food ingredients, both in the United States and throughout the world; cheeses are among the most important of these milk products. Milk contains several major antigenic proteins, each with differing susceptibilities to proteolytic enzymes. The extent of proteolysis in cheese varies as a result of conditions during manufacture and ripening. Proteolysis has the potential to degrade antigenic and allergenic epitopes that are important for residue detection and elicitation of allergic reactions. Commercial enzyme-linked immunosorbent assays (ELISAs) are not currently validated for use in detecting residues in hydrolyzed or fermented food products. Eighteen retail cheeses produced using 5 different styles of fermentation were investigated for detectable milk protein residues with 4 commercial ELISA kits. Mozzarella, Swiss, Blue, Limburger, and Brie cheeses were assessed. The Neogen Veratox® Casein and Neogen Veratox® Total Milk kits were capable of detecting milk residues in most cheeses evaluated, including blue-veined cheeses that exhibit extensive proteolysis. The other 2 ELISA kits evaluated, r-Biopharm® Fast Casein and ELISA Systems™ Casein, can detect milk residues in cheeses other than blue-veined varieties. ELISA results cannot be quantitatively compared among kits. The quantitative reliability of ELISA results in detection of cheese residues is questionable, but some methods are sufficiently robust to use as a semi-quantitative indication of proper allergen control for the validation of cleaning programs in industry settings. Many commercially available enzyme-linked immunosorbent assays (ELISAs) are not validated for detection of allergenic residues in fermented or hydrolyzed products. This research seeks to determine if commercial milk ELISAs can detect milk residues in varieties of cheese that have undergone different styles of fermentation and different degrees of proteolysis. Only certain milk ELISA kits are capable of detecting residues in all varieties of cheese. However, commercial milk ELISA kits are capable of semiquantitative detection of cheese residues in foods, or in industry settings for the validation of allergen cleaning programs. © 2017 Institute of Food Technologists®.

Immunosorbent analysis of ricin contamination in milk using colorimetric, chemiluminescence, and electrochemiluminescence detection

USDA-ARS?s Scientific Manuscript database

Analytical methodology to detect ricin in food matrices is important because of the potential use of foodborne ricin as a terrorist weapon. Monoclonal antibodies (mAbs) that bind ricin were used for both capture and detection in sandwich enzyme-linked immunosorbent assay (ELISA) and electrochemilumi...

An Application of Outer Membrane Protein P6-Specific Enzyme-Linked Immunosorbent Assay for Detection of Haemophilus influenzae in Middle Ear Fluids and Nasopharyngeal Secretions

PubMed Central

Hotomi, Muneki; Togawa, Akihisa; Kono, Masamitsu; Sugita, Gen; Sugita, Rinya; Fujimaki, Yutaka; Kamide, Yosuke; Uchizono, Akihiro; Kanesada, Keiko; Sawada, Shoichi; Okitsu, Naohiro; Masuda, Hisayo; Tanaka, Hideaki; Tanaka, Yumi; Yamanaka, Noboru

2013-01-01

An enzyme-linked immunosorbent assay specific to outer membrane protein P6 (P6-ELISA) was applied for detecting Haemophilus influenzae in middle ear fluids (MEFs) from acute otitis media (AOM) patients and in nasopharyngeal secretions (NPSs) from acute rhinosinusitis patients. P6-ELISA had a sensitivity of 83.3% for MEFs and 71.5% for NPSs and a specificity of 85.6% for MEFs and 92.5% for NPSs, respectively. Real-time PCR exhibited significant differences in the number of ompP1 gene copies among samples determined by P6-ELISA to be positive and negative for H. influenzae. However, because the P6-ELISA test has the reactivity in Haemophilus species include two commensals H. haemolyticus and H. parainfluenzae, it is thus a weak method in order to detect only NTHi correctly. Consequently, diagnosis using the P6-ELISA should be based on an overall evaluation, including the results of other related examinations and clinical symptoms to prevent misleading conclusions in clinical setting. PMID:24015192

An application of outer membrane protein p6-specific enzyme-linked immunosorbent assay for detection of haemophilus influenzae in middle ear fluids and nasopharyngeal secretions.

PubMed

Hotomi, Muneki; Togawa, Akihisa; Kono, Masamitsu; Sugita, Gen; Sugita, Rinya; Fujimaki, Yutaka; Kamide, Yosuke; Uchizono, Akihiro; Kanesada, Keiko; Sawada, Shoichi; Okitsu, Naohiro; Masuda, Hisayo; Tanaka, Hideaki; Tanaka, Yumi; Yamanaka, Noboru

2013-01-01

An enzyme-linked immunosorbent assay specific to outer membrane protein P6 (P6-ELISA) was applied for detecting Haemophilus influenzae in middle ear fluids (MEFs) from acute otitis media (AOM) patients and in nasopharyngeal secretions (NPSs) from acute rhinosinusitis patients. P6-ELISA had a sensitivity of 83.3% for MEFs and 71.5% for NPSs and a specificity of 85.6% for MEFs and 92.5% for NPSs, respectively. Real-time PCR exhibited significant differences in the number of ompP1 gene copies among samples determined by P6-ELISA to be positive and negative for H. influenzae. However, because the P6-ELISA test has the reactivity in Haemophilus species include two commensals H. haemolyticus and H. parainfluenzae, it is thus a weak method in order to detect only NTHi correctly. Consequently, diagnosis using the P6-ELISA should be based on an overall evaluation, including the results of other related examinations and clinical symptoms to prevent misleading conclusions in clinical setting.

Antibody screening by enzyme-linked immunosorbent assay using pooled soluble HLA in renal transplant candidates.

PubMed

Zaer, F; Metz, S; Scornik, J C

1997-01-15

The enzyme-linked immunosorbent assay (ELISA) using HLA class I molecules purified from pooled platelets has the potential to detect HLA antibodies with increased efficiency without sacrificing sensitivity or specificity. This test, which was originally developed in our institution, has been independently validated by recent studies and is now commercially available. We now present evidence of its usefulness as a routine HLA antibody screening test for renal transplant patients. A total of 515 patients were tested monthly by ELISA (13.9 tests/patient) and by antiglobulin-enhanced panel reactivity (6.3 tests/patient). In patients found to be unsensitized, the incidence of false-positive results was less for ELISA than for the panel studies. In patients who were highly sensitized, both tests performed equally well, whereas discordant results were registered mainly in cases of mild sensitization. Because 66% of our patients were not sensitized, the ELISA was effective in reducing the number of more involved tests aimed at characterizing the antibodies. These results provide a foundation to use the pooled platelet HLA ELISA on a routine basis for HLA antibody screening.

Antigen-capture blocking enzyme-linked immunosorbent assay based on a baculovirus recombinant antigen to differentiate Transmissible gastroenteritis virus from Porcine respiratory coronavirus antibodies.

PubMed

López, Lissett; Venteo, Angel; García, Marga; Camuñas, Ana; Ranz, Ana; García, Julia; Sarraseca, Javier; Anaya, Carmen; Rueda, Paloma

2009-09-01

A new commercially available antigen-capture, blocking enzyme-linked immunosorbent assay (antigen-capture b-ELISA), based on baculovirus truncated-S recombinant protein of Transmissible gastroenteritis virus (TGEV) and 3 specific monoclonal antibodies, was developed and evaluated by examining a panel of 453 positive Porcine respiratory coronavirus (PRCoV), 31 positive TGEV, and 126 negative field sera by using another commercially available differential coronavirus b-ELISA as the reference technique to differentiate TGEV- from PRCoV-induced antibodies. The recombinant S protein-based ELISA appeared to be 100% sensitive for TGEV and PRCoV detection and highly specific for TGEV and PRCoV detection (100% and 92.06%, respectively), when qualitative results (positive or negative) were compared with those of the reference technique. In variability experiments, the ELISA gave consistent results when the same serum was evaluated on different wells and different plates. These results indicated that truncated recombinant S protein is a suitable alternative to the complete virus as antigen in ELISA assays. The use of recombinant S protein as antigen offers great advantages because it is an easy-to-produce, easy-to-standardize, noninfectious antigen that does not require further purification or concentration. Those advantages represent an important improvement for antigen preparation, in comparison with other assays in which an inactivated virus from mammalian cell cultures is used.

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

PubMed Central

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

2011-01-01

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

Sensitivity and specificity enhanced enzyme-linked immunosorbent assay by rational hapten modification and heterogeneous antibody/coating antigen combinations for the detection of melamine in milk, milk powder and feed samples.

PubMed

Cao, Biyun; Yang, Hong; Song, Juan; Chang, Huafang; Li, Shuqun; Deng, Anping

2013-11-15

The adulteration of food products with melamine has led to an urgent requirement for sensitive, specific, rapid and reliable quantitative/screening methods. To enhance the sensitivity and specificity of the enzyme-linked immunosorbent assay (ELISA) for the detection of melamine in milk, milk powder and feed samples, rational hapten modification and heterogeneous antibody/coating antigen combinations were adopted. Three melamine derivatives with different length of carboxylic spacer at the end were synthesized and linked to carrier proteins for the production of immunogens and coating antigens. Monoclonal antibody against melamine was produced by hybridoma technology. Under optimal experimental conditions, the standard curves of the ELISAs for melamine were constructed in range of 0.1-100 ng mL(-1). The sensitivity was 10-300 times enhanced compared to those in the published literatures. The cross-reactivity values of the ELISAs also demonstrated the assays exhibited high specificity. Five samples were spiked with melamine at different concentrations and detected by the ELISA. The recovery rates of 72.8-123.0% and intra-assay coefficients of variation of 0.8-18.9% (n=3) were obtained. The ELISA for milk sample was confirmed by high-performance liquid chromatography with a high correlation coefficient of 0.9902 (n=6). The proposed ELISA was proven to be a feasible quantitative/screening method for melamine analysis. Copyright © 2013 Elsevier B.V. All rights reserved.

Synergistic Use of Gold Nanoparticles (AuNPs) and “Capillary Enzyme-Linked Immunosorbent Assay (ELISA)” for High Sensitivity and Fast Assays

PubMed Central

Kim, Wan-Joong; Cho, Hyo Young; Jeong, Bongjin; Byun, Sangwon; Huh, JaeDoo; Kim, Young Jun

2017-01-01

Using gold nanoparticles (AuNPs) on “capillary enzyme-linked immunosorbent assay (ELISA)”, we produced highly sensitive and rapid assays, which are the major attributes for point-of-care applications. First, in order to understand the size effect of AuNPs, AuNPs of varying diameters (5 nm, 10 nm, 15 nm, 20 nm, 30 nm, and 50 nm) conjugated with Horseradish Peroxidase (HRP)-labeled anti-C reactive protein (antiCRP) (AuNP•antiCRP-HRP) were used for well-plate ELISA. AuNP of 10 nm produced the largest optical density, enabling detection of 0.1 ng/mL of CRP with only 30 s of incubation, in contrast to 10 ng/mL for the ELISA run in the absence of AuNP. Then, AuNP of 10 nm conjugated with antiCRP-HRP (AuNP•antiCRP-HRP) was used for “capillary ELISA” to detect as low as 0.1 ng/mL of CRP. Also, kinetic study on both 96-well plates and in a capillary tube using antiCRP-HRP or AuNP•antiCRP-HRP showed a synergistic effect between AuNP and the capillary system, in which the fastest assay was observed from the “AuNP capillary ELISA”, with its maximum absorbance reaching 2.5 min, while the slowest was the typical well-plate ELISA with its maximum absorbance reaching in 13.5 min. PMID:29278402

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

USGS Publications Warehouse

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

1993-01-01

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

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

PubMed

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

2013-01-01

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

Determination of the folate content in cladodes of nopal (Opuntia ficus indica) by microbiological assay utilizing Lactobacillus casei (ATCC 7469) and enzyme-linked immunosorbent assay.

PubMed

Ortiz-Escobar, Tania Breshkovskaya; Valverde-González, Maria Elena; Paredes-López, Octavio

2010-05-26

Prickly pear cactus has been an important food source in Mexico since ancient times due to its economical and ecological benefits and potential nutraceutical value. Nevertheless, studies on the nutritional aspects and health benefits have been scarce. The purpose of this study was to assess, apparently for the first time, the folate contents of cladodes of nopal by a microbiological assay, using Lactobacillus casei (ATCC 7469) in extracts that were enzymatically treated to release the bound vitamin, employing single, dual, and trienzymatic procedures, and using the enzyme-linked immunosorbent assay (ELISA). We used Opuntia cladodes of different length sizes. The microbiological assay showed some differences among enzyme treatments and sizes of nopal; the trienzyme treatment (alpha-amylase-protease-conjugase) was more efficient in determining the folate content in nopal, giving 5.0 ng/g in the small size cladodes at 54 h of testing time, while ELISA showed no significant differences in the folate content among sizes of cladodes (5.5-5.62 ng/g at 0 min testing time). Both techniques may be used for the assessment of folate content in cladodes, but ELISA is more rapid and reliable.

Simple Identification of Human Taenia Species by Multiplex Loop-Mediated Isothermal Amplification in Combination with Dot Enzyme-Linked Immunosorbent Assay

PubMed Central

Nkouawa, Agathe; Sako, Yasuhito; Okamoto, Munehiro; Ito, Akira

2016-01-01

For differential detection of Taenia solium, Taenia saginata, and Taenia asiatica, loop-mediated isothermal amplification (LAMP) assay targeting the cytochrome c oxidase subunit 1 gene has been recently developed and shown to be sensitive, specific, and effective. However, to achieve differential identification, one specimen requires three reaction mixtures containing a primer set of each Taenia species separately, which is complex and time consuming and increases the risk of cross-contamination. In this study, we developed a simple differential identification of human Taenia species using multiplex LAMP (mLAMP) in combination with dot enzyme-linked immunosorbent assay (dot-ELISA). Forward inner primers of T. solium, T. saginata, and T. asiatica labeled with fluorescein isothiocyanate (FITC), digoxigenin (DIG), and tetramethylrhodamine (TAMRA), respectively, and biotin-labeled backward inner primers were used in mLAMP. The mLAMP assay succeeded in specific amplification of each respective target gene in a single tube. Furthermore, the mLAMP product from each species was easily distinguished by dot-ELISA with an antibody specific for FITC, DIG, or TAMRA. The mLAMP assay in combination with dot-ELISA will make identification of human Taenia species simpler, easier, and more practical. PMID:27044566

An integrated target sequence and signal amplification assay, reverse transcriptase-PCR-enzyme-linked immunosorbent assay, to detect and characterize flaviviruses.

PubMed Central

Chang, G J; Trent, D W; Vorndam, A V; Vergne, E; Kinney, R M; Mitchell, C J

1994-01-01

We previously described a reverse transcriptase-PCR using flavivirus genus-conserved and virus species-specific amplimers (D. W. Trent and G. J. Chang, p. 355-371, in Y. Becker and C. Darai; ed., Frontiers of Virology, vol. 1, 1992). Target amplification was improved by redesigning the amplimers, and a sensitive enzyme-linked immunosorbent assay (ELISA) technique has been developed to detect amplified digoxigenin (DIG)-modified DNA. A single biotin motif and multiple DIG motifs were incorporated into each amplicon, which permitted amplicon capture by a biotin-streptavidin interaction and detection with DIG-specific antiserum in a colorimetric ELISA. We evaluated the utility of this assay for detecting St. Louis encephalitis (SLE) viral RNA in infected mosquitoes and dengue viral RNA in human serum specimens. The reverse transcriptase-PCR-ELISA was as sensitive as isolation of SLE virus by cell culture in detecting SLE viral RNA in infected mosquitoes. The test was 89% specific and 95 to 100% sensitive for identification of dengue viral RNA in serum specimens compared with isolation of virus by Aedes albopictus C6/36 cell culture and identification by the indirect immunofluorescence assay. PMID:7512096

An integrated target sequence and signal amplification assay, reverse transcriptase-PCR-enzyme-linked immunosorbent assay, to detect and characterize flaviviruses.

PubMed

Chang, G J; Trent, D W; Vorndam, A V; Vergne, E; Kinney, R M; Mitchell, C J

1994-02-01

We previously described a reverse transcriptase-PCR using flavivirus genus-conserved and virus species-specific amplimers (D. W. Trent and G. J. Chang, p. 355-371, in Y. Becker and C. Darai; ed., Frontiers of Virology, vol. 1, 1992). Target amplification was improved by redesigning the amplimers, and a sensitive enzyme-linked immunosorbent assay (ELISA) technique has been developed to detect amplified digoxigenin (DIG)-modified DNA. A single biotin motif and multiple DIG motifs were incorporated into each amplicon, which permitted amplicon capture by a biotin-streptavidin interaction and detection with DIG-specific antiserum in a colorimetric ELISA. We evaluated the utility of this assay for detecting St. Louis encephalitis (SLE) viral RNA in infected mosquitoes and dengue viral RNA in human serum specimens. The reverse transcriptase-PCR-ELISA was as sensitive as isolation of SLE virus by cell culture in detecting SLE viral RNA in infected mosquitoes. The test was 89% specific and 95 to 100% sensitive for identification of dengue viral RNA in serum specimens compared with isolation of virus by Aedes albopictus C6/36 cell culture and identification by the indirect immunofluorescence assay.

HAPTEN DESIGN FOR COMPOUND-SELECTIVE ANTIBODIES: ELISAS FOR ENVIRONMENTALLY DELETERIOUS SMALL MOLECULES. (R825433)

EPA Science Inventory

We have developed an enzyme-linked immunosorbent assay (ELISA) for the herbicide simazine with virtually no recognition of propazine and very low (8%) recognition of atrazine. In this research we have developed a generalized "size-exclusion" concept for designing immun...

DETERMINATION OF 3,5,6-TRICHLORO-2-PYRIDINOL (TCP) BY ELISA

EPA Science Inventory

A sensitive, competitive enzyme-linked immunosorbent assay (ELISA) for 3,5,6-trichloro-2pyridinol (TCP) has been developed to quantitate parts per billion (ppb) amounts of the analyte in urine. TCP is a major metabolite and environmental degradation product of the insecticide c...

Dot enzyme-linked immunosorbent assay (ELISA) for the detection of Toxocara infection using a rat model.

PubMed

Paller, Vachel Gay V; Besana, Cyrelle M; Valdez, Isabel Kristine M

2017-12-01

Toxocariasis is a zoonotic disease usually caused by dog and cat roundworms, Toxocara canis and T. cati. Detection and diagnosis is difficult in paratenic and accidental hosts, including humans, as they cannot be detected through conventional methods such as fecal examination. Diagnosis therefore relies on immunological methods and molecular methods such as enzyme-linked immunosorbent assay (ELISA) and Western Blot, which are both time-consuming and requires sophisticated equipment. In the Philippines, only a few studies are available on Toxocara seroprevalence. Therefore, there is a need to adapt methods for serodiagnosis of Toxocara infection in humans for the Philippine setting. A dot enzyme linked immunosorbent assay (dot-ELISA) was standardized using T. canis excretory-secretory antigens. Test sera were collected from laboratory rats (Sprague-Dawley strain) experimentally infected with embryonated eggs of T. canis and Ascaris suum as well as rice field rats naturally infected with Taenia taeniaeformis and Nippostrongylus sp. Optimum conditions used were 20 µg/ml antigen concentration and 1:10 serum dilution. The sensitivity, specificity, positive, and negative predictive values were 90% (95% CI 55.5-99.7%), 100% (95% CI 69.2-100.0%), 100% (95% CI 66.4-100%), and 90.9% (95% CI 58.7-99.8%), respectively. Dot-ELISA has the potential to be developed as a cheaper, simpler, and more practical method for detection of anti- Toxocara antibodies on accidental hosts. This is a preliminary study conducted on experimental animals before optimization and standardization for human serum samples.

Validation of 2 commercially available enzyme-linked immunosorbent assays for adiponectin determination in canine serum samples.

PubMed

Tvarijonaviciute, Asta; Martínez-Subiela, Silvia; Ceron, José J

2010-10-01

The aim of this study was to validate 2 commercially available enzyme-linked immunosorbent assays (ELISAs) for adiponectin in dogs, 1 canine-specific and 1 originally designed for measurements in humans. Intra-assay and interassay precision was evaluated by multiple measurements in canine serum samples, and assay accuracy was indirectly determined by linearity under dilution. Interference caused by hemolysis and lipemia was also studied. Both assays were subsequently used for measuring adiponectin concentrations in clinically healthy dogs and those with different grades of obesity. The intra-assay and inter-assay precision was less than 7.5% and 13.5% in serum samples with low and high adiponectin concentrations, respectively. Lipemia and hemolysis did not affect the results of any of the assays. Both assays were able to differentiate lean dogs from those that were overweight or obese on the basis of the measured adiponectin concentrations. From these results it can be concluded that canine adiponectin concentrations can be measured reliably by means of the 2 ELISAs evaluated in this study.

Validation of 2 commercially available enzyme-linked immunosorbent assays for adiponectin determination in canine serum samples

PubMed Central

Tvarijonaviciute, Asta; Martínez-Subiela, Silvia; Ceron, José J.

2010-01-01

The aim of this study was to validate 2 commercially available enzyme-linked immunosorbent assays (ELISAs) for adiponectin in dogs, 1 canine-specific and 1 originally designed for measurements in humans. Intra-assay and interassay precision was evaluated by multiple measurements in canine serum samples, and assay accuracy was indirectly determined by linearity under dilution. Interference caused by hemolysis and lipemia was also studied. Both assays were subsequently used for measuring adiponectin concentrations in clinically healthy dogs and those with different grades of obesity. The intra-assay and inter-assay precision was less than 7.5% and 13.5% in serum samples with low and high adiponectin concentrations, respectively. Lipemia and hemolysis did not affect the results of any of the assays. Both assays were able to differentiate lean dogs from those that were overweight or obese on the basis of the measured adiponectin concentrations. From these results it can be concluded that canine adiponectin concentrations can be measured reliably by means of the 2 ELISAs evaluated in this study. PMID:21197228

Citrullinated Chemokines in Rheumatoid Arthritis

DTIC Science & Technology

2016-12-01

immunosorbent assay ( ELISA ) in RA and normal (NL) sera and in RA, osteoarthritis (OA), and other inflammatory rheumatic disease (OD) synovial fluids (SFs... ELISA ) Epithelial Neutrophil Chemoattractant Peptide-78 (ENA-78/CXCL5) Monocyte Chemoattractant Protein-1 (MCP-1/CCL2) Macrophage Inflammatory...Citrullinated chemokines are highly expressed in RA sera and SFs. Citrullinated chemokines were measured using an ELISA in which chemokines were captured on

9 CFR 85.6 - Interstate movement of pseudorabies vaccinate swine, except swine from qualified negative gene...

Code of Federal Regulations, 2010 CFR

2010-01-01

... enzyme-linked immunosorbent assay (ELISA) or a gpI Particle Concentration Fluorescence Immunoassay (PCFIA... negative; (iv) The date of the gpI ELISA or the gpI PCFIA approved differential pseudorabies test; and (v) The name of the laboratory that conducted the gpI ELISA or the gpI PCFIA approved differential...

9 CFR 85.6 - Interstate movement of pseudorabies vaccinate swine, except swine from qualified negative gene...

Code of Federal Regulations, 2011 CFR

2011-01-01

... enzyme-linked immunosorbent assay (ELISA) or a gpI Particle Concentration Fluorescence Immunoassay (PCFIA... negative; (iv) The date of the gpI ELISA or the gpI PCFIA approved differential pseudorabies test; and (v) The name of the laboratory that conducted the gpI ELISA or the gpI PCFIA approved differential...

9 CFR 85.6 - Interstate movement of pseudorabies vaccinate swine, except swine from qualified negative gene...

Code of Federal Regulations, 2012 CFR

2012-01-01

... enzyme-linked immunosorbent assay (ELISA) or a gpI Particle Concentration Fluorescence Immunoassay (PCFIA... negative; (iv) The date of the gpI ELISA or the gpI PCFIA approved differential pseudorabies test; and (v) The name of the laboratory that conducted the gpI ELISA or the gpI PCFIA approved differential...

9 CFR 85.6 - Interstate movement of pseudorabies vaccinate swine, except swine from qualified negative gene...

Code of Federal Regulations, 2014 CFR

2014-01-01

... enzyme-linked immunosorbent assay (ELISA) or a gpI Particle Concentration Fluorescence Immunoassay (PCFIA... negative; (iv) The date of the gpI ELISA or the gpI PCFIA approved differential pseudorabies test; and (v) The name of the laboratory that conducted the gpI ELISA or the gpI PCFIA approved differential...

9 CFR 85.6 - Interstate movement of pseudorabies vaccinate swine, except swine from qualified negative gene...

Code of Federal Regulations, 2013 CFR

2013-01-01

... enzyme-linked immunosorbent assay (ELISA) or a gpI Particle Concentration Fluorescence Immunoassay (PCFIA... negative; (iv) The date of the gpI ELISA or the gpI PCFIA approved differential pseudorabies test; and (v) The name of the laboratory that conducted the gpI ELISA or the gpI PCFIA approved differential...

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

PubMed

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

2009-01-28

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

[Trypanosoma cruzi in triatomines from Nuevo Leon, Mexico].

PubMed

Molina-Garza, Zinnia Judith; Rosales-Encina, José Luis; Galaviz-Silva, Lucio; Molina-Garza, Daniel

2007-01-01

To determine the prevalence of Trypanosoma cruzi in triatomines from Nuevo León using the standardization of an improved enzyme-linked immunosorbent assay test. From July to September 2005, 52 triatomines were captured in General Terán, a municipality located in Nuevo León. They were analyzed using optical microscopy (OM) and a polymerase chain reaction (PCR), as standards of reference, to develop a technique for detecting the parasite using enzyme-linked immunosorbent assay (ELISA). Using OM and PCR, 31 triatomines were found to be positive and 21 negative. Using ELISA, 27 samples were identified as positive and 25 negative (specificity 100%, sensitivity 87%, negative predictive value 84%, and positive predictive value 100%). The prevalence of infected triatomines was 59.61% with OM and PCR, and 51.92% with ELISA. Our data confirm that the ELISA assay in triatomines is a fast, reliable and useful tool. Since it was possible to simultaneously analyze a large number of samples with high sensibility and specificity values, the ELISA test proves to be useful for new epidemiologic studies having a high number of vectors. It is also less expensive than PCR. It is therefore recommended for epidemiological and preventive surveillance programs as a first screening test before conducting a confirmatory test using PCR.

Detection of Plasmodium Aldolase Using a Smartphone and Microfluidic Enzyme Linked Immunosorbent Assay

PubMed Central

2017-01-01

Background Malaria control efforts are limited in rural areas. A low-cost system to monitor response without the use of electricity is needed. Plasmodium aldolase is a malaria biomarker measured using enzyme linked immunosorbent assay (ELISA) techniques. A three-part system using ELISA was developed consisting of a microfluidic chip, hand crank centrifuge, and a smartphone. Methods A circular microfluidic chip was fabricated using clear acrylic and a CO2 laser. A series of passive valves released reagents at precise times based upon centrifugal force. Color change was measured via smartphone camera using an application programmed in Java. The microchip was compared to a standard 96-well sandwich ELISA. Results Results from standard ELISA were compared to microchip at varying concentrations (1–10 ng/mL). Over 15 different microfluidic patterns were tested, and a final prototype of the chip was created. The prototype microchip was compared to standard sandwich ELISA (n = 20) using samples of recombinant aldolase. Color readings of standard ELISA and microfluidic microchip showed similar results. Conclusion A low-cost microfluidic system could detect and follow therapeutic outcomes in rural areas and identify resistant strains. PMID:29057138

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

USGS Publications Warehouse

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

1998-01-01

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

Analysis of castor by ELISAs that distinguish Ricin and Ricinus communis agglutinin (RCA)

USDA-ARS?s Scientific Manuscript database

To facilitate the analysis of castor (Ricinus communis L.) seed fractions and germplasm for ricin content, we investigated the use of enzyme-linked immunosorbent assay (ELISA) methods to differentiate between ricin toxin and the related Ricinus communis agglutinin (RCA). Both proteins are based on ...

Depletion of Urinary Zilpaterol Residues in Horses as Measured by ELISA and UPLC-MS/MS

USDA-ARS?s Scientific Manuscript database

Three horses were dosed with dietary zilpaterol and the urine concentration measured from withdrawal day 0 to withdrawal day 21. The analyses were carried out using both enzyme linked immunosorbent assay (ELISA) and an ultra-performance liquid chromatography with triple-quadrupole-tandem mass spect...

QUANTITATIVE ELISA OF POLYCHLORINATED BIPHENYLS IN AN OILY SOIL MATRIX USING SUPERCRITICAL FLUID EXTRACTION

EPA Science Inventory

Soil samples from the GenCorp Lawrence Brownfields site were analyzed with a commercial semi-quantitative enzyme-linked immunosorbent assay (ELISA) using a methanol shake extraction. Many of the soil samples were extremely oily, with total petroleum hydrocarbon levels up to 240...

ELISA MEASUREMENT OF STACHYLYSIN (TM) IN SERUM TO QUANTIFY HUMAN EXPOSURES TO THE INDOOR MOLD STACHYBOTRYS CHARTARUM

EPA Science Inventory

Antibodies were produced against the hemolytic agent stachylysin obtained from the mold Stachybotryis chartarum. These antibodies were used to develop two enzyme-linked immunosorbent assay (ELISA) methods for the analysis of stachylysin in human and rat sera and environmental sa...

Transmission of a Viral Disease (AIDS) Detected by a Modified ELISA Reaction: A Laboratory Simulation.

ERIC Educational Resources Information Center

Grimes, William J.; Chambers, Linda; Kubo, Kenneth M.; Narro, Martha L.

1998-01-01

Describes a laboratory exercise that simulates the spread of an infectious agent among students in a classroom. Uses a modified Enzyme Linked ImmunoSorbent Assay (ELISA) to provide students with experience using an authentic diagnostic tool for detecting human infections. (DDR)

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

White, Amanda M.; Collett, James L.; Seurynck-Servoss, Shannon L.

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.

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

PubMed

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

2005-09-01

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

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

PubMed Central

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

2005-01-01

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

Direct Competitive Enzyme-Linked Immunosorbent Assay (ELISA).

PubMed

Kohl, Thomas O; Ascoli, Carl A

2017-07-05

The competitive enzyme-linked immunosorbent assay (ELISA) (cELISA; also called an inhibition ELISA) is designed so that purified antigen competes with antigen in the test sample for binding to an antibody that has been immobilized in microtiter plate wells. The same concept works if the immobilized molecule is antigen and the competing molecules are purified labeled antibody versus antibody in a test sample. Direct cELISAs incorporate labeled antigen or antibody, whereas indirect assay configurations use reporter-labeled secondary antibodies. The cELISA is very useful for determining the concentration of small-molecule antigens in complex sample mixtures. In the direct cELISA, antigen-specific capture antibody is adsorbed onto the microtiter plate before incubation with either known standards or unknown test samples. Enzyme-linked antigen (i.e., labeled antigen) is also added, which can bind to the capture antibody only when the antibody's binding site is not occupied by either the antigen standard or antigen in the test samples. Unbound labeled and unlabeled antigens are washed away and substrate is added. The amount of antigen in the standard or the test sample determines the amount of reporter-labeled antigen bound to antibody, yielding a signal that is inversely proportional to antigen concentration within the sample. Thus, the higher the antigen concentration in the test sample, the less labeled antigen is bound to the capture antibody, and hence the weaker is the resultant signal. © 2017 Cold Spring Harbor Laboratory Press.

Development of a biomimetic enzyme-linked immunosorbent assay based on molecularly imprinted polymers on paper for the detection of carbaryl.

PubMed

Zhang, Can; Cui, Hanyu; Han, Yufeng; Yu, Fangfang; Shi, Xiaoman

2018-02-01

A biomimetic enzyme-linked immunosorbent assay (BELISA) which was based on molecularly imprinted polymers on paper (MIPs-paper) with specific recognition was developed. As a detector, the surface of paper was modified with γ-MAPS by hydrolytic action and anchored the MIP layer on γ-MAPS modified-paper by copolymerization to construct the artificial antibody Through a series of experimentation and verification, we successful got the MIPs-paper and established BELISA for the detection of carbaryl. The development of MIPs-paper based on BELISA was applied to detect carbaryl in real samples and validated by an enzyme-linked immunosorbent assay (ELISA) based on anti-carbaryl biological antibody. The results of these two methods (BELISA and ELISA) were well correlated (R 2 =0.944). The established method of MIPs-paper BELISA exhibits the advantages of low cost, higher stability and being re-generable, which can be applied as a convenient tool for the fast and efficient detection of carbaryl. Copyright © 2017. Published by Elsevier Ltd.

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

USDA-ARS?s Scientific Manuscript database

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...

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

PubMed

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

2013-11-01

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

Detection of liver kidney microsomal type 1 antibody using molecularly based immunoassays

PubMed Central

Kerkar, N; Ma, Y; Davies, E T; Cheeseman, P; Mieli-Vergani, G; Vergani, D

2002-01-01

Aims: To assess the diagnostic value of two commercial molecularly based immunoassays detecting liver kidney microsomal type 1 antibody (LKM1). Methods: The performance of Varelisa and LKM1 enzyme linked immunosorbent assay (ELISA) was compared with immunofluorescence, and two validated research techniques—an in house ELISA and a radioligand assay measuring antibodies to P4502D6. Thirty serum samples from three patients with autoimmune hepatitis type 2 covering immunofluorescence titres of 1/10 to 1/10 240 and 55 LKM1 negative controls were tested. Results: All 30 sera that were LKM1 positive by immunofluorescence were positive by the in house ELISA, the radioligand assay, and LKM1-ELISA, and 29 were also positive by Varelisa. None of the 55 sera negative for LKM1 by immunofluorescence was positive by the in house ELISA and radioligand assay, but one was positive by Varelisa and 14 were positive using the LKM1-ELISA. Agreement between immunofluorescence, the in house ELISA, the radioligand assay, and Varelisa was high (κ > 0.8), and agreement between immunofluorescence and LKM1-ELISA was moderate (κ = 0.63). Conclusion: The assay kit marketed as Varelisa allows accurate detection of LKM1. PMID:12461054

Detection of liver kidney microsomal type 1 antibody using molecularly based immunoassays.

PubMed

Kerkar, N; Ma, Y; Davies, E T; Cheeseman, P; Mieli-Vergani, G; Vergani, D

2002-12-01

To assess the diagnostic value of two commercial molecularly based immunoassays detecting liver kidney microsomal type 1 antibody (LKM1). The performance of Varelisa and LKM1 enzyme linked immunosorbent assay (ELISA) was compared with immunofluorescence, and two validated research techniques-an in house ELISA and a radioligand assay measuring antibodies to P4502D6. Thirty serum samples from three patients with autoimmune hepatitis type 2 covering immunofluorescence titres of 1/10 to 1/10 240 and 55 LKM1 negative controls were tested. All 30 sera that were LKM1 positive by immunofluorescence were positive by the in house ELISA, the radioligand assay, and LKM1-ELISA, and 29 were also positive by Varelisa. None of the 55 sera negative for LKM1 by immunofluorescence was positive by the in house ELISA and radioligand assay, but one was positive by Varelisa and 14 were positive using the LKM1-ELISA. Agreement between immunofluorescence, the in house ELISA, the radioligand assay, and Varelisa was high (kappa > 0.8), and agreement between immunofluorescence and LKM1-ELISA was moderate (kappa = 0.63). The assay kit marketed as Varelisa allows accurate detection of LKM1.

Moderate reagent mixing on an orbital shaker reduces the incubation time of enzyme-linked immunosorbent assay.

PubMed

Kumar, Saroj; Ahirwar, Rajesh; Rehman, Ishita; Nahar, Pradip

2017-07-01

Rapid diagnostic tests can be developed using ELISA for detection of diseases in emergency conditions. Conventional ELISA takes 1-2 days, making it unsuitable for rapid diagnostics. Here, we report the effect of reagents mixing via shaking or vortexing on the assay timing of ELISA. A 48-min protocol of ELISA involving 12-min incubations with reagent mixing at 750 rpm for every step was optimized. Contrary to this, time-optimized control ELISA performed without mixing produced similar results in 8 h, leaving a time gain of 7 h using the developed protocol. Collectively, the findings suggest the development of ELISA-based rapid diagnostics. Copyright © 2017 Elsevier Inc. All rights reserved.

Indirect immunoglobulin G (IgG) and IgM enzyme-linked immunosorbent assays (ELISAs) and IgM capture ELISA for detection of antibodies to lipopolysaccharide in adult typhoid fever patients in Pakistan.

PubMed

Sippel, J; Bukhtiari, N; Awan, M B; Krieg, R; Duncan, J F; Karamat, K A; Malik, I A; Igbal, L M; Legters, L

1989-06-01

Sera from 339 adult febrile patients in Pakistan were tested for antibodies to Salmonella typhi lipopolysaccharide by indirect immunoglobulin G (IgG) and IgM enzyme-linked immunosorbent assay (ELISA) and IgM capture ELISA. A total of 55 patients had S. typhi cultured from their blood, 20 had S. typhi cultured from their stool, 24 were blood or stool culture positive for S. paratyphi A, 41 were culture negative but clinically diagnosed as having enteric fever, 41 had gastrointestinal or urinary tract infections, 41 were clinically diagnosed as having malaria, 20 were smear-positive patients with malaria, 58 had respiratory infections, and the remaining 39 individuals were placed in a miscellaneous group who did not have Salmonella infection. The sensitivities of the indirect IgG ELISA, indirect IgM ELISA, and IgM capture ELISA determined with specimens obtained from the blood culture-positive patients with typhoid fever (positive controls) were 80, 64, and 62%, respectively. The specificities of the assays determined with sera from the patients with respiratory infections (negative controls) were 95, 95, and 97%, respectively. The percentage of smear-positive patients with malaria who were positive by these assays was lower than that in the negative control group. The percentages of individuals in the other patient categories who were positive by these tests were between those obtained with the positive and negative controls. Of the positive controls, 26 were positive by both IgM assays, 9 were IgM positive only by indirect ELISA, and 8 were IgM positive only by IgM capture ELISA. A total of 70% of the positive control patients who were tested for O agglutinins by the Widal tube agglutination assay were positive; however, 29% of the negative control patients were also positive. The indirect IgG ELISA was the single most effective test for the serodiagnosis of typhoid fever in this population.

Indirect immunoglobulin G (IgG) and IgM enzyme-linked immunosorbent assays (ELISAs) and IgM capture ELISA for detection of antibodies to lipopolysaccharide in adult typhoid fever patients in Pakistan.

PubMed Central

Sippel, J; Bukhtiari, N; Awan, M B; Krieg, R; Duncan, J F; Karamat, K A; Malik, I A; Igbal, L M; Legters, L

1989-01-01

Sera from 339 adult febrile patients in Pakistan were tested for antibodies to Salmonella typhi lipopolysaccharide by indirect immunoglobulin G (IgG) and IgM enzyme-linked immunosorbent assay (ELISA) and IgM capture ELISA. A total of 55 patients had S. typhi cultured from their blood, 20 had S. typhi cultured from their stool, 24 were blood or stool culture positive for S. paratyphi A, 41 were culture negative but clinically diagnosed as having enteric fever, 41 had gastrointestinal or urinary tract infections, 41 were clinically diagnosed as having malaria, 20 were smear-positive patients with malaria, 58 had respiratory infections, and the remaining 39 individuals were placed in a miscellaneous group who did not have Salmonella infection. The sensitivities of the indirect IgG ELISA, indirect IgM ELISA, and IgM capture ELISA determined with specimens obtained from the blood culture-positive patients with typhoid fever (positive controls) were 80, 64, and 62%, respectively. The specificities of the assays determined with sera from the patients with respiratory infections (negative controls) were 95, 95, and 97%, respectively. The percentage of smear-positive patients with malaria who were positive by these assays was lower than that in the negative control group. The percentages of individuals in the other patient categories who were positive by these tests were between those obtained with the positive and negative controls. Of the positive controls, 26 were positive by both IgM assays, 9 were IgM positive only by indirect ELISA, and 8 were IgM positive only by IgM capture ELISA. A total of 70% of the positive control patients who were tested for O agglutinins by the Widal tube agglutination assay were positive; however, 29% of the negative control patients were also positive. The indirect IgG ELISA was the single most effective test for the serodiagnosis of typhoid fever in this population. PMID:2754002

Usefulness of Enzyme-linked Immunosorbent Assay Using Recombinant BP180 and BP230 for Serodiagnosis and Monitoring Disease Activity of Bullous Pemphigoid

PubMed Central

Lee, Eui Hyung; Kim, Yeon Hee; Kim, Sinyoung; Kim, Song-ee

2012-01-01

Background Bullous pemphigoid (BP) is an autoimmune subepidermal bullous disease associated with autoantibodies against BP180 and BP230. Enzyme-linked immunosorbent assay (ELISA) is a sensitive tool for the detection of immunoglobulin G (IgG) anti-BP180 and anti-BP230 autoantibodies. Objective The aim of this study was to evaluate the usefulness of ELISA for diagnosing and monitoring the disease activity of BP. Methods We evaluated serum IgG levels of anti-BP180 and anti-BP230 autoantibodies in 47 BP patients, 16 epidermolysis bullosa aquisita patients, and 15 healthy volunteers using ELISA. Through retrospective review of the medical records, the clinical characteristics of BP including disease activity, duration, pruritus severity and peripheral blood eosinophil counts were assessed. Results The sensitivity of BP180 ELISA was 97.9%, BP230 ELISA 72.3%, and a combination of the two was 100%. The specificity of BP180 ELISA was 90.3%, BP230 ELISA 100%, and a combination of the two was 90.3%. BP180 ELISA scores showed strong associations with disease activity, pruritus severity, peripheral blood eosinophil counts, and disease duration, whereas BP230 ELISA scores did not. Conclusion BP180 and BP230 ELISAs are highly sensitive methods for the diagnosis of BP, and BP180 ELISA, in particular, is a sensitive tool for monitoring the disease activity of BP. PMID:22363155

Accurate Determination of Soluble Axl by Enzyme-Linked Immunosorbent Assay.

PubMed

Dengler, Mirko; Huber, Heidemarie; Müller, Christian J; Zellmer, Angela; Rauch, Peter; Mikulits, Wolfgang

2016-11-01

Levels of soluble Axl (sAxl) are routinely assessed in human sera by sandwich enzyme-linked immunosorbent assay (ELISA). Although sAxl values are suggested to diagnose different types of disorders, no uniform ELISA method is available, allowing the reliable interassay comparison between results. Furthermore, little is known about the stability of sAxl under storage conditions, which is a relevant parameter for biomedical trials. The evaluation of sAxl stability under various stress conditions and the determination of proper conditions to use the sAxl ELISA for routine clinical applications are of great interest. In this study, serum samples were subjected to freeze-thaw cycles and incubation at different temperatures to analyze the stability of sAxl by ELISA. Dilution and spike-in experiments were carried out to examine the impact of serum and diluent components on the ELISA performance. Various diluents and media were employed to resolve masking effects of the serum. The assay components were further optimized for long-term usability by treatment with stabilizers and validation under temperature stress. Indeed, sAxl showed long-term stability in serum during freeze-thaw cycles and incubation under temperature stress conditions. The dilution experiments revealed that unknown components in the serum caused masking effects that can be reduced by proper dilutions. The assay performance was further increased by using a standardized buffer system to dilute serum samples. Stabilization of coated plates and of streptavidin-horseradish peroxidase allowed long-term storage for up to 6 months. In sum, our data demonstrate proper ELISA conditions, allowing the accurate analysis of sAxl levels in human serum.

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

PubMed

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

2014-12-01

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

Design and characterization of a direct ELISA for the detection and quantification of leucomalachite green

PubMed Central

Singh, Gurmit; Koerner, Terence; Gelinas, Jean-Marc; Abbott, Michael; Brady, Beth; Huet, Anne-Catherine; Charlier, Caroline; Delahaut, Philippe; Godefroy, Samuel Benrejeb

2011-01-01

Malachite green (MG), a member of the N-methylated triphenylmethane class of dyes, has long been used to control fungal and protozoan infections in fish. MG is easily absorbed by fish during waterborne exposure and is rapidly metabolized into leucomalachite green (LMG), which is known for its long residence time in edible fish tissue. This paper describes the development of an enzyme-linked immunosorbent assay (ELISA) for the detection and quantification of LMG in fish tissue. This development includes a simple and versatile method for the conversion of LMG to monodesmethyl-LMG, which is then conjugated to bovine serum albumin (BSA) to produce an immunogenic material. Rabbit polyclonal antibodies are generated against this immunogen, purified and used to develop a direct competitive enzyme-linked immunosorbent assay (ELISA) for the screening and quantification of LMG in fish tissue. The assay performed well, with a limit of detection (LOD) and limit of quantification (LOQ) of 0.1 and 0.3 ng g−1 of fish tissue, respectively. The average extraction efficiency from a matrix of tilapia fillets was approximately 73% and the day-to-day reproducibility for these extractions in the assay was between 5 and 10%. PMID:21623496

Assessing protein oxidation by inorganic nanoparticles with enzyme-linked immunosorbent assay (ELISA).

PubMed

Sun, Wenjie; Luna-Velasco, Antonia; Sierra-Alvarez, Reyes; Field, Jim A

2013-03-01

Growth in the nanotechnology industry is leading to increased production of engineered nanoparticles (NPs). This has given rise to concerns about the potential adverse and toxic effects to biological system and the environment. An important mechanism of NP toxicity is oxidative stress caused by the formation of reactive oxygen species (ROS) or via direct oxidation of biomolecules. In this study, a protein oxidation assay was developed as an indicator of biomolecule oxidation by NPs. The oxidation of the protein, bovine serum albumin (BSA) was evaluated with an enzyme-linked immunosorbent assay (ELISA) to measure the protein carbonyl derivatives formed from protein oxidation. The results showed that some NPs such as Cu(0), CuO, Mn(2)O(3), and Fe(0) caused oxidation of BSA; whereas, many of the other NPs tested were not reactive or very slowly reactive with BSA. The mechanisms involved in the oxidation of BSA protein by the reactive NPs could be attributed to the combined effects of ROS-dependent and direct protein oxidation mechanisms. The ELISA assay is a promising method for the assessment of protein oxidation by NPs, which can provide insights on NP toxicity mechanisms. Copyright © 2012 Wiley Periodicals, Inc.

Development of a direct competitive enzyme-linked immunosorbent assay for parathion residue in food samples.

PubMed

Gui, Wen-Jun; Liu, Yi-Hua; Wang, Chun-Mei; Liang, Xiao; Zhu, Guo-Nian

2009-10-01

A heterologous direct competitive enzyme-linked immunosorbent assay (ELISA) for parathion residue determination is described based on a monoclonal antibody and a new competitor. The effects of several physicochemical factors, such as methanol concentration, ionic strength, pH value, and sample matrix, on the performance of the ELISA were optimized for the sake of obtaining a satisfactory assay sensitivity. Results showed that when the assay medium was in the optimized condition (phosphate buffer solution [PBS] containing 10% [v/v] methanol and 0.2 mol/L NaCl at a pH value of 5.0), the sensitivity (estimated as the IC(50) value) and the limit of detection (LOD, estimated as the IC(10) value) were 1.19 and 0.08 ng/ml, respectively. The precision investigation indicated that the intraassay precision values all were below 10% and that the interassay precision values ranged from 4.89 to 19.12%. In addition, the developed ELISA showed a good linear correlation (r(2)=0.9962) to gas chromatography within the analyte's concentration range of 0.1 to 16 ng/ml. When applied to the fortified samples (parathion adding level: 5-15 microg/kg), the developed ELISA presented mean recoveries of 127.46, 122.52, 91.92, 124.01, 129.72, 99.37, and 87.17% for tomato, cucumber, banana, apple, orange, pear, and sugarcane, respectively. Results indicated that the established ELISA is a potential tool for parathion residue determination.

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

PubMed

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

2013-11-13

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

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

PubMed Central

Blacksell, Stuart D.; Tanganuchitcharnchai, Ampai; Jintaworn, Suthatip; Kantipong, Pacharee; Richards, Allen L.; Day, Nicholas P. J.

2016-01-01

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

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

PubMed Central

Stiffler-Rosenberg, G; Fey, H

1978-01-01

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

Improved quantification of a commercial enzyme-linked immunosorbent assay kit for measuring anti-MDA5 antibody.

PubMed

Gono, Takahisa; Okazaki, Yuka; Murakami, Akihiro; Kuwana, Masataka

2018-04-09

To compare the quantitative performance for measuring anti-MDA5 antibody titer of two enzyme-linked immunosorbent assay (ELISA) systems: an in-house ELISA and the commercial MESACUP TM anti-MDA5 test. Anti-MDA5 antibody titer was measured in sera from 70 patients with dermatomyositis using an in-house ELISA and the MESACUP TM anti-MDA5 test side-by-side. For the commercial ELISA kit, serum samples diluted 1:101 were used according to the manufacturer's protocol, but serial dilutions of sera were also examined to identify the optimal serum dilution for quantification. The anti-MDA5 antibody titers measured by the in-house and commercial ELISAs were positively correlated with each other (r = 0.53, p = .0001), but the antibody titer measured by the commercial ELISA was less sensitive to change after medical treatment, and 37 (80%) of 46 anti-MDA5-positive sera had antibody titer exceeding the quantification range specified by the manufacturer (≥150 index). Experiments using diluted serum samples revealed that diluting the sera 1:5050 improved the quantitative performance of the MESACUP TM anti-MDA5 test, including a better correlation with the in-house ELISA results and an increased sensitivity to change. We improved the ability of the commercial ELISA kit to quantify anti-MDA5 antibody titer by altering its protocol.

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.

Rapid determination of ractopamine residues in edible animal products by enzyme-linked immunosorbent assay: development and investigation of matrix effects.

PubMed

Zhang, Yan; Wang, Fengxia; Fang, Li; Wang, Shuo; Fang, Guozhen

2009-01-01

To determine ractopamine residues in animal food products (chicken muscle, pettitoes, pig muscle, and pig liver), we established a rapid direct competitive enzyme-linked immunosorbent assay (ELISA) using a polyclonal antibody generated from ractopamine-linker-BSA. The antibody showed high sensitivity and specificity in phosphate buffer, with an IC(50) of 0.6 ng/mL, and the limit of detection was 0.04 ng/mL. The matrix effect of the samples was easily eliminated by one-step extraction with PBS, without any organic solution or clean-up procedure such as SPE or liquid-liquid extraction, making it a much more simple and rapid method than previously reported ones. The detection limit in blank samples was 0.2 mug/kg. To validate this new RAC (ractopamine hydrochloride) ELISA, a RAC-free pig liver sample spiked at three different concentrations was prepared and analyzed by HPLC and ELISA. The results showed a good correlation between the data of ELISA and HPLC (R(2) > 0.95), which proves that the established ELISA is accurate enough to quantify the residue of RAC in the animal derived foods.

Development of an indirect enzyme-linked immunosorbent assay (ELISA) to differentiate antibodies against wild-type porcine reproductive and respiratory syndrome from the vaccine strain TJM-F92 based on a recombinant Nsp2 protein.

PubMed

Wang, X X; Wang, F X; Li, Z G; Wen, Y J; Wang, X; Song, N; Wu, H

2018-01-01

An accurate ELISA method to differentiate pigs infected with wild-type porcine reproductive and respiratory syndrome (PRRSV) strains from vaccinated ones would help to monitor PRRSV vaccination compliance. The recombinant protein GST-d120aa derived from the continuous deletion of 120 amino acids in the non-structural protein 2 region of the modified-live vaccine strain TJM-F92 was used to develop an indirect enzyme-linked immunosorbent assay (d120-ELISA) for differentiating serum antibodies against TJM-F92 from other PRRSV strains. At the optimized cut-off value which was calculated at an S/P of 0.25, it yielded a sensitivity of 90.7% and a specificity of 95.1%. Cross-reactivity tests suggested that the d120-ELISA was PRRSV-specific. Coefficient of variations of the repeatability tests ranged between 1.41-17.02%. The results suggest that the d120-ELISA is suitable for differentiating animals infected with wild-type strains from those immunized with MLV TJM-F92. Copyright © 2017. Published by Elsevier B.V.

Validation of a Competitive Enzyme-Linked Immunosorbent Assay for Detection of Babesia bigemina Antibodies in Cattle

USDA-ARS?s Scientific Manuscript database

A competitive ELISA (cELISA) based on a broadly conserved, species-specific, B-cell epitope within the C-terminus of Babesia bigemina rhoptry-associated protein-1a was validated for international use. Receiver Operating Characteristic (ROC) analysis revealed 16% inhibition as the threshold for a neg...

Evaluation of ELISA tests specific for Shiga toxin 1 and 2 in food and water samples

USDA-ARS?s Scientific Manuscript database

Two enzyme-linked immunosorbent assay (ELISA) kits were evaluated for their effectiveness in detecting and differentiating between Shiga toxin 1 and 2 (Stx1 and Stx2) produced by Shiga toxin-producing E. coli (STEC) inoculated into food and water samples. Each kit incorporated monoclonal antibodies ...

Detecting quinoxaline-2-carboxylic acid in animal tissues by using sensitive rapid enzyme-linked immunosorbent assay and time-resolved fluoroimmunoassay.

PubMed

Le, Tao; Yu, Huan; Niu, Xiaodong

2015-05-15

An indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) and time-resolved fluoroimmunoassay (TR-FIA) based on an anti-N-butylquinoxaline-2-carboxamide (BQCA) monoclonal antibody were standardized and validated for quinoxaline-2-carboxylic acid (QCA) screening in animal tissues and its performance were compared to HPLC. The sensitivities obtained for edible tissue extracts were 1.62 and 1.12 ng ml(-1) for ic-ELISA and TR-FIA detection, respectively. Two samples were spiked with QCA and analyzed by both methods. The recovery values ranged from 92.6% to 112.2% and the coefficients of variation were less than 15% for QCA spiking into swine tissue samples at concentrations of 2.5-50.0 μg kg(-1). Excellent correlations (r(2)=0.987-0.996) of the ic-ELISA/HPLC and TR-FIA/HPLC data were observed for processed samples. The results demonstrated that the ic-ELISA and TR-FIA methods were rapid and accurate for the residue detection of QCA in animal tissues. Copyright © 2014 Elsevier Ltd. All rights reserved.

Assessment of maternal antibody decay and response to canine parvovirus vaccination using a clinic-based enzyme-linked immunosorbent assay.

PubMed

Waner, T; Naveh, A; Wudovsky, I; Carmichael, L E

1996-10-01

Interference caused by maternal antibodies is considered a major cause of canine parvovirus (CPV) vaccination failure. In this study, an immunoblot clinic-based enzyme-linked immunosorbent assay (ELISA) method was used to detect CPV antibodies in sera of pregnant bitches and their offspring to study the response of pups to vaccination. With a easily accessible procedure for CPV antibody determination, the veterinarian should be able to gauge the response of pups after vaccination. The validity of the technique was tested in parallel against the standard hemagglutination inhibition (HI) test. Results of the ELISA were correlated with those of the standard HI method for quantification of CPV antibodies. With the ELISA, successfully immunized pups were identified, allowing for a more reliable and cost-effective program of vaccination. This simple clinic-based test could be used for the assessment of vaccination status of pups during the critical phase of 6 to about 16 weeks of age. This study is the first in which vaccination response to CPV in pups was followed, using a clinic-based ELISA for CPV antibody monitoring.

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. Copyright © 2014 John Wiley & Sons, Ltd.

A novel double recognition enzyme-linked immunosorbent assay based on the nucleocapsid protein for early detection of European porcine reproductive and respiratory syndrome virus infection.

PubMed

Venteo, A; Rebollo, B; Sarraseca, J; Rodriguez, M J; Sanz, A

2012-04-01

Precise and rapid detection of porcine reproductive respiratory syndrome virus (PRRSV) infection in swine farms is critical. Improvement of control procedures, such as testing incoming gilt and surveillance of seronegative herds requires more rapid and sensitive methods. However, standard serological techniques detect mainly IgG antibodies. A double recognition enzyme-linked immunosorbent assay (DR-ELISA) was developed for detection of antibodies specific to European PRRSV. This new assay can recognize both IgM and IgG antibodies to PRSSV which might be useful for detecting in routine surveillance assays pigs that are in the very early stages of infection and missed by conventional assays detecting only IgG antibodies. DR-ELISA is based on the double recognition of antigen by antibody. In this study, the recombinant nucleocapsid protein (N) of PRRSV was used both as the coating and the enzyme-conjugated antigen. To evaluate the sensitivity of the assay at early stages of the infection, sera from 69 pigs infected with PRRSV were collected during successive days post infection (pi) and tested. While standard methods showed low sensitivity rates before day 14 pi, DR-ELISA detected 88.4% seropositive samples at day 7 showing greater sensitivity at early stages of the infection. Further studies were carried out to assess the efficiency of the new assay, and the results showed DR-ELISA to be a sensitive and accurate method for early diagnosis of EU-PRRSV infection. Copyright © 2012 Elsevier B.V. All rights reserved.

Simple Identification of Human Taenia Species by Multiplex Loop-Mediated Isothermal Amplification in Combination with Dot Enzyme-Linked Immunosorbent Assay.

PubMed

Nkouawa, Agathe; Sako, Yasuhito; Okamoto, Munehiro; Ito, Akira

2016-06-01

For differential detection of Taenia solium, Taenia saginata, and Taenia asiatica, loop-mediated isothermal amplification (LAMP) assay targeting the cytochrome c oxidase subunit 1 gene has been recently developed and shown to be sensitive, specific, and effective. However, to achieve differential identification, one specimen requires three reaction mixtures containing a primer set of each Taenia species separately, which is complex and time consuming and increases the risk of cross-contamination. In this study, we developed a simple differential identification of human Taenia species using multiplex LAMP (mLAMP) in combination with dot enzyme-linked immunosorbent assay (dot-ELISA). Forward inner primers of T. solium, T. saginata, and T. asiatica labeled with fluorescein isothiocyanate (FITC), digoxigenin (DIG), and tetramethylrhodamine (TAMRA), respectively, and biotin-labeled backward inner primers were used in mLAMP. The mLAMP assay succeeded in specific amplification of each respective target gene in a single tube. Furthermore, the mLAMP product from each species was easily distinguished by dot-ELISA with an antibody specific for FITC, DIG, or TAMRA. The mLAMP assay in combination with dot-ELISA will make identification of human Taenia species simpler, easier, and more practical. © The American Society of Tropical Medicine and Hygiene.

Competitive Enzyme-Linked Immunosorbent Assay Based on a Rhoptry-Associated Protein 1 Epitope Specifically Identifies Babesia bovis-Infected Cattle

PubMed Central

Goff, Will L.; McElwain, Terry F.; Suarez, Carlos E.; Johnson, Wendell C.; Brown, Wendy C.; Norimine, Junzo; Knowles, Donald P.

2003-01-01

The competitive enzyme-linked immunosorbent assay (cELISA) format has proven to be an accurate, reliable, easily standardized, and high-throughput method for detecting hemoparasite infections. In the present study, a species-specific, broadly conserved, and tandemly repeated B-cell epitope within the C terminus of the rhoptry-associated protein 1 of the hemoparasite Babesia bovis was cloned and expressed as a histidine-tagged thioredoxin fusion peptide and used as antigen in a cELISA. The assay was optimized with defined negative and positive bovine sera, where positive sera inhibited the binding of the epitope-specific monoclonal antibody BABB75A4. The cELISA accurately differentiated animals with B. bovis-specific antibodies from uninfected animals and from animals with antibodies against other tick-borne hemoparasites (98.7% specificity). In addition, B. bovis-specific sera from Australia, Argentina, Bolivia, Puerto Rico, and Morocco inhibited the binding of BABB75A4, confirming conservation of the epitope. The assay first detected experimentally infected animals between 13 and 17 days postinfection, and with sera from naturally infected carrier cattle, was comparable to indirect immunofluorescence (98.3% concordance). The assay appears to have the characteristics necessary for an epidemiologic and disease surveillance tool. PMID:12522037

Development and evaluation of an avian influenza, neuraminidase subtype 1, indirect enzyme-linked immunosorbent assay for poultry using the differentiation of infected from vaccinated animals control strategy

USDA-ARS?s Scientific Manuscript database

An indirect ELISA was developed using baculovirus expressed N1 protein from the A/chicken/Indonesia/7/2003 (H5N1) virus. The specificity of the assay was tested with a panel of chicken antisera raised against N1 to N9 virus subtypes. The N1-ELISA was specific for the detection of N1 antibodies in ...

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

PubMed Central

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

2014-01-01

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

A novel enzyme-linked immuno-sorbent assay (ELISA) for the quantification of total and free polysaccharide in Haemophilus influenzae b-Tetanus toxoid conjugate vaccines in monovalent and combined vaccine formulations.

PubMed

Saydam, Manolya; Rigsby, Peter; Mawas, Fatme

2014-01-01

Current Haemophilus influenzae b conjugate vaccines (Hib), which are made of purified capsular polysaccharide (poly-ribosyl-ribitol-phosphate; PRP) conjugated to a carrier protein, are almost completely evaluated by physico-chemical methods to ensure the integrity and stability of the vaccine and consistency of manufacture of batches. The absence of a potency assay makes the quantification of total PRP content (in SI units) and of % free polysaccharide in final fills or bulk components of Hib vaccines critical release tests for both manufacturers and national control authorities. Here we describe a simple and sensitive Enzyme-Linked Immuno-sorbent Assay (ELISA) which has been developed to quantify total and free PRP content in Hib-TT vaccine alone or when in combination with other vaccines. The assay is robust, specific and highly sensitive. Copyright © 2013 The International Alliance for Biological Standardization. Published by Elsevier Ltd. All rights reserved.

Antibody-capture enzyme-linked immunosorbent assays that use enzyme-labelled antigen for detection of virus-specific immunoglobulin M, A and G in patients with varicella or herpes zoster.

PubMed Central

van Loon, A. M.; van der Logt, J. T.; Heessen, F. W.; Heeren, M. C.; Zoll, J.

1992-01-01

Antibody-capture enzyme-linked immunosorbent assays (AC-ELISA) which use enzyme-labelled antigen were developed for detection of varicella-zoster virus-(VZV) specific IgM, IgA and IgG antibody in patients with varicella or herpes zoster and in sera from healthy individuals. All 18 patients with varicella developed a VZV-IgM and a VZV-IgG response, 17 also a VZV-IgA response. In contrast, all 19 patients with herpes zoster were shown to be positive for VZV-IgA whereas only 13 of these reacted positively for VZV-IgM. A VZV-IgM response was detected in only two sera from 100 healthy individuals and an IgA response in only one. The presence of virus-specific IgA and IgG in the cerebrospinal fluid as determined by AC-ELISA was a useful indicator of VZV infection of the central nervous system. By AC-ELISA, VZV-IgG was detected predominantly in sera from patients with acute or recent VZV infection. Only 14 sera from 100 healthy individuals were positive for VZV-IgG by AC-ELISA, whereas all were positive by an indirect ELISA. These results indicate that AC-ELISA's may be useful assays for determination for acute or recurrent VZV infection, but are not suitable for determination of past infection with this virus. PMID:1312479

[Establishment of enzyme-linked immunosorbent assay (ELISA) for measuring human urinary uromodulin and application of the method in patients with IgA nephropathy].

PubMed

Liu, Ying; Chen, Yu-qing; Zhou, Jing-jing; Han, Jia; Liang, Yu; Li, Xue-ying; Zhang, Hong

2012-04-18

To establish a method of enzyme-linked immunosorbent assay (ELISA) to measure urinary uromodulin and explore the urinary uromudulin level in IgA nephropathy. The rabbit anti-human uromodulin polyclonal antibodies were coated on plates to capture uromodulin and the mouse anti-human uromodulin monoclonal antibody was used as detecting antibody to set up ELISA procedure. The precision and repeatability of this ELISA method were evaluated, and then this method was compared with the commercialized Tamm-Horsfall Glycoprotein ELISA Kit by examining urinary uromodulin levels in 55 individuals. Finally, the urinary uromodulin level in 166 IgA nephropathy patients were detected as well as 48 normal controls with this established method. The detecting range of uromodulin was 0.78-12.5 μg/L by this method. The coefficient of variation within-run was 7.5%, and between-run of coefficient of variation was 7.9%. Correlation of this method and comercialized kit was good (r=0.615, P<0.001). The urinary uromodulin/urinary creatinine ratio in IgA nephropathy was significantly lower than that in normal controls. The established ELISA method is sensitive and repeatable, and can be used in further studies.

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

PubMed

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

2005-08-10

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

Development of an enzyme-linked immunosorbent assay and a beta-1 adrenergic receptor-based assay for monitoring the drug atenolol.

PubMed

Sapir, A; Shalev, A Hariton; Skalka, N; Bronshtein, A; Altstein, M

2013-03-01

Two approaches for monitoring atenolol (ATL) were applied: an immunochemical assay and a competitive-binding assay, based on the interaction between ATL and its target receptor, β1 adrenergic receptor (β1AR). Polyclonal antibodies (Abs) for ATL were generated, and a highly specific microplate immunochemical assay, that is, an enzyme-linked immunosorbent assay (ELISA), for its detection was developed. The ATL ELISA exhibited I50 and limit of detection (I20) values of 0.15 ± 0.048 and 0.032 ± 0.016 ng/ml, respectively, and the Abs did not cross-react with any of the tested beta-blocker drugs. Furthermore, a human β1AR (h-β1AR) was stably expressed in Spodoptera frugiperda cells (Sf9). The receptor was employed to develop a competitive-binding assay that monitored binding of ATL in the presence of isoproteranol by quantification of secondary messenger, cyclic adenosine monophosphate (cAMP), levels in the transfected cells. The assay showed that the recombinant h-β1AR was functional, could bind the agonistic ligand isoproterenol as well as the antagonist ATL, as indicated by a dose-dependent elevation of cAMP in the presence of isoproteranol, and decrease after ATL addition. The highly efficient and sensitive ELISA and the receptor assay represent two methods suitable for efficient and cost-effective large-scale, high-throughput monitoring of ATL in environmental, agricultural, and biological samples. Copyright © 2012 SETAC.

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

PubMed

Sudarshana, M R; Reddy, D V

1989-10-01

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

Preparation of antibodies and development of an enzyme-linked immunosorbent assay (ELISA) for the determination of doxycycline antibiotic in milk samples.

PubMed

Adrian, Javier; Fernández, Fátima; Sánchez-Baeza, Francisco; Marco, M-Pilar

2012-04-18

This paper reports the development of an immunoassay for the specific analysis of doxycycline (DC), a congener of the tetracycline antibiotic family (TCs), in milk samples. This is the first time that DC antibody production is reported, based on a rationally designed and well-characterized immunizing hapten. The chemical structure of the immunizing hapten (13-[(2-carboxyethyl)thiol]-5-hydroxy-6-α-deoxytetracycline, TC1) was designed to maximize recognition of the tetracycline characteristic moiety defined as lower periphery of the TCs plus the region of the upper periphery composed by the hydroxyl group at position C(5) (B ring) and the dimethylamino group in ring A. Polyclonal antibodies raised against TC1 coupled to horseshoe crab hemocianyn (HCH) were used to develop a homologous indirect competitive enzyme-linked immunosorbent assay (ELISA). The microplate ELISA can detect DC in buffer down to 0.1 μg L(-1). The ELISA has been proven to tolerate a wide range of ionic strengths and pH values. The assay is very selective for DC with a minor recognition of methacycline (32% of cross-reactivity). Experiments performed with whole milk samples demonstrate that samples can be directly analyzed after a simple treatment method, reaching detectability values below 5 μg L(-1).

Determination of trace amount of cyanobacterial toxin in water by microchip based enzyme-linked immunosorbent assay.

PubMed

Pyo, Dongjin; Hahn, Jong Hoon

2009-01-01

Routine monitoring of microcystin in natural waters is difficult because the concentration of the toxin is usually lower than the detection limits. As a more sensitive detection method for microcystin, we developed a microchip based enzyme-linked immunosorbent assay (ELISA) based on monoclonal antibodies. New monoclonal antibodies against the microcystin leucine-arginine variant (MCLR), a cyclic peptide toxin of the freshwater cyanobacterium Microcystis aeruginosa, were prepared from cloned hybridoma cell lines. We used keyhole limpet hemocyanin(KLH)-conjugated MCLR as an immunogen for the production of mouse monoclonal antibody. The immunization, cell fusion, and screening of hybridoma cells producing anti-MCLR antibody were conducted. Since the ELISA test was highly sensitive, the newly developed microchip based ELISA can be suitable for the trace analysis of cyanobacterial hepatotoxins, microcystins in water. The linear responses of monoclonal antibodies with different concentrations of microcystin LR were established between 0.025 and 0.3 ng/mL.

Discrimination between active and inactive neurocysticercosis by metacestode excretory/secretory antigens of Taenia solium in an enzyme-linked immunosorbent assay.

PubMed

Molinari, José L; García-Mendoza, Esperanza; de la Garza, Yazmin; Ramírez, José A; Sotelo, Julio; Tato, Patricia

2002-06-01

To detect IgG antibodies to Taenia solium, a controlled double-blind study was conducted using 91 coded cerebrospinal fluid samples from patients with neurocysticercosis (NCC) and other neurologic disorders. Samples were tested in an enzyme-linked immunosorbent assay (ELISA) using metacestode excretion/secretion antigens. The results were correlated with data from medical records on the diagnosis of NCC (based on computed tomography and magnetic resonance imaging criteria) and other neurologic disorders. The ELISA results were positive in 22 of the 24 cases with active NCC. In contrast, six cases with calcified cysts (inactive NCC), as well as one case in a transitional stage, were negative. One case with a calcified granuloma and another with a granuloma plus calcifications (classified as inactive NCC) had positive results. The remaining negative results corresponded to other neurologic disorders (58 cases). The results of the ELISA showed a significant difference between active and inactive NCC (P = 0.0034).

Comparison of Enzyme-Linked Immunosorbent Assay, Surface Plasmon Resonance and Biolayer Interferometry for Screening of Deoxynivalenol in Wheat and Wheat Dust.

PubMed

Sanders, Melanie; McPartlin, Daniel; Moran, Kara; Guo, Yirong; Eeckhout, Mia; O'Kennedy, Richard; De Saeger, Sarah; Maragos, Chris

2016-04-11

A sample preparation method was developed for the screening of deoxynivalenol (DON) in wheat and wheat dust. Extraction was carried out with water and was successful due to the polar character of DON. For detection, an enzyme-linked immunosorbent assay (ELISA) was compared to the sensor-based techniques of surface plasmon resonance (SPR) and biolayer interferometry (BLI) in terms of sensitivity, affinity and matrix effect. The matrix effects from wheat and wheat dust using SPR were too high to further use this screenings method. The preferred ELISA and BLI methods were validated according to the criteria established in Commission Regulation 519/2014/EC and Commission Decision 2002/657/EC. A small survey was executed on 16 wheat lots and their corresponding dust samples using the validated ELISA method. A linear correlation (r = 0.889) was found for the DON concentration in dust versus the DON concentration in wheat (LOD wheat: 233 μg/kg, LOD wheat dust: 458 μg/kg).

Sensitive detection of respiratory syncytial virus based on a dual signal amplified plasmonic enzyme-linked immunosorbent assay.

PubMed

Zhan, Lei; Wu, Wen Bi; Yang, Lin; Huang, Cheng Zhi

2017-04-15

The timely detection of infectious pathogen is critical in clinical early diagnosis and treatment of infectious diseases. Plasmonic enzyme-linked immunosorbent assay (ELISA), by means of enzyme-mediated growth or aggregation of AuNPs, has received considerable attention because it allows a naked-eye detection of target in very low numbers. In this work, a dual-signal amplified plasmonic ELISA combined the high loading capacity of magnetic beads with the establishing stimulation effect of zinc ion has been developed to detect RSV as a model pathogen based on alkaline phosphatase-triggered dispersion of aggregated AuNPs. In ideal conditions, the proposed immunoassay can conveniently distinguish the concentration of RSV in a range of 0.1-30 pg/mL. In addition, the limit of detection of RSV of this immunoassay exceeds that of conventional ELISA by about 50 times. The high sensitivity makes this approach a good alternative to existing colorimetric immunoassays for pathogen detection. Copyright © 2017 Elsevier B.V. All rights reserved.

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

PubMed Central

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

2013-01-01

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

EVALUATION OF A COMMERCIAL COMPETITIVE ENZYME-LINKED IMMUNOSORBENT ASSAY FOR DETECTION OF AVIAN INFLUENZA VIRUS SUBTYPE H5 ANTIBODIES IN ZOO BIRDS.

PubMed

Jensen, Trine Hammer; Andersen, Jannie Holmegaard; Hjulsager, Charlotte Kristiane; Chriél, Mariann; Bertelsen, Mads Frost

2017-09-01

The hemagglutination inhibition (HI) test is the current gold standard for detecting antibodies to avian influenza virus (AIV). Enzyme-linked immunosorbent assays (ELISAs) have been explored for use in poultry and certain wild bird species because of high efficiency and lower cost. This study compared a commercial ELISA for detection of AIV subtype H5 antibodies with HI test of 572 serum samples from zoo birds. There was no significant difference between the results of the two tests when statistically compared by a McNemar χ 2 test (P = 0.86) and assessment of κ (κ = 0.87). With a specificity of 94.2% (95% confidence interval [CI], 0.92-0.97), a sensitivity of 93.9% (95% CI, 0.91-0.97), and an excellent correlation between the two tests, this ELISA can be recommended as an alternative to the HI test for preliminary screening of zoo bird sera for antibodies to AIV subtype H5.

Parameters for measurement of oxidative stress in diabetes mellitus: applicability of enzyme-linked immunosorbent assay for clinical evaluation.

PubMed

Noiri, Eisei; Tsukahara, Hirokazu

2005-05-01

Investigations of the mechanisms involved in the onset and progression of diabetes have recently confronted the role of reactive oxygen species (ROS) and oxidative stress. Prolonged exposure to hyperglycemic conditions induces nonenzymatic glycation of protein via the so-called Maillard reaction, resulting in Schiff-base products and Amadori products that engender ROS production. These processes initiate and exacerbate micro- and macrovascular complications in diabetes. Increased oxidative stress is induced by excessive ROS production and inadequate antioxidant defenses. Recently, oxidative stress status markers have been associated directly with the severity and prognosis of diabetes. To examine oxidative stress, reliable and high-throughput methods are needed to examine large numbers of clinical samples. The emerging availability of enzyme-linked immunosorbent assay (ELISA) for oxidative stress status markers allows its application to assessment of various pathophysiologic conditions, including diabetes. This review outlines the recent achievements of ELISA application for clinical studies elucidating oxidative stress. It introduces the potential applicability of ELISA for investigating oxidative stress in diabetes.

Validation of an enzyme-linked immunosorbent assay developed for measuring cortisol concentration in human saliva and serum for its applicability to analyze cortisol in pig saliva.

PubMed

Thomsson, Ola; Ström-Holst, Bodil; Sjunnesson, Ylva; Bergqvist, Ann-Sofi

2014-09-06

The purpose of this study was to validate a commercially available enzyme-linked immunosorbent assay (ELISA) developed for measuring free cortisol in human saliva and total cortisol concentration in diluted human serum, for its applicability in measuring cortisol concentration in pig saliva. Collection of saliva is less stressful than e.g. blood sampling, and is a non-invasive method. Saliva was collected by allowing sows to chew on cotton swabs held by forceps. Thereafter, the swabs were centrifuged to retrieve the saliva. The ELISA was performed according to instructions provided by the manufacturer. To validate the ELISA, determination of the intra-assay coefficient of variation (CV), inter-assay CV, recovery, linearity and parallelism was performed. The intra-assay CV was below 10% and inter-assay CV below 15% for samples of high, medium and low cortisol concentrations. The mean recovery was 117% and the linearity and parallelism showed an r2-value of 0.994 and 0.993, respectively. For biological assessment of induced social stress, two saliva samples were collected in the morning from 6 primiparous and 21 multiparous sows. One sample was collected when the sows were individually housed in a farrowing pen and a second sample was collected when the sows were group housed. The primiparous sows had a significant higher cortisol concentration compared to the multiparous sows when group housed. The results obtained in this validation study indicate that the ELISA is suitable for measuring cortisol concentration in porcine saliva.

Optimized enzyme-linked immunosorbent assay for detecting cytomegalovirus infections during clinical trials of recombinant vaccines.

PubMed

Pagnon, Anke; Piras, Fabienne; Gimenez-Fourage, Sophie; Dubayle, Joseline; Arnaud-Barbe, Nadège; Hessler, Catherine; Caillet, Catherine

2017-11-01

In clinical trials of cytomegalovirus (CMV) glycoprotein B (gB) vaccines, CMV infection is detected by first depleting serum of anti-gB antibodies and then measuring anti-CMV antibodies with a commercially available enzyme-linked immunosorbent assay (ELISA) kit, with confirmation of positive findings by immunoblot. Identification of CMV immunoantigens for the development of an ELISA that detects specifically CMV infection in clinical samples from individuals immunized with gB vaccines. Sensitivity and specificity of ELISAs using antigenic regions of CMV proteins UL83/pp65, UL99/pp28, UL44/pp52, UL80a/pp38, UL57, and UL32/pp150 were measured. An IgG ELISA using a UL32/pp150 [862-1048] capture peptide was the most specific (93.7%) and sensitive (96.4%) for detecting CMV-specific antibodies in sera. The ELISA successfully detected CMV-specific antibodies in 22 of 22 sera of subjects who had been vaccinated with a gB vaccine but who had later been infected with CMV. The ELISA was linear over a wide range of CMV concentrations (57-16,814 ELISA units/mL) and was reproducible as indicated by a 5% intra-day and 7% inter-day coefficients of variation. The signal was specifically competed by UL32/pp150 [862-1048] peptide but not by CMV-gB or herpes simplex virus 2 glycoprotein D. Lipid and hemoglobin matrix did not interfere with the assay. The UL32/pp150 [862-1048] IgG ELISA can be used for the sensitive and specific detection of CMV infection in gB-vaccinated individuals. Copyright © 2017 Elsevier B.V. All rights reserved.

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

PubMed

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

2015-06-01

Nucleocapsid (N) protein gene of turkey coronavirus (TCoV) was expressed in a prokaryotic system and used to develop an enzyme-linked immunosorbent assay (ELISA) for detection of antibody to TCoV. Anti-TCoV hyperimmune turkey serum and normal turkey serum were used as positive or negative controls for optimization of the ELISA. Goat anti-turkey IgG (H+L) conjugated with horseradish peroxidase was used as detector antibody. Three hundred and twenty two turkey sera from the field were used to evaluate the performance of ELISA and determine the cut-off point of ELISA. The established ELISA was also examined with serum samples obtained from turkeys experimentally infected with TCoV. Those serum samples were collected at various time intervals from 1 to 63 days post-infection. The optimum conditions for differentiation between anti-TCoV hyperimmune serum and normal turkey serum were recombinant TCoV N protein concentration at 20 μg/ml, serum dilution at 1:800, and conjugate dilution at 1:10,000. Of the 322 sera from the field, 101 were positive for TCoV by immunofluorescent antibody assay (IFA). The sensitivity and specificity of the ELISA relative to IFA test were 86.0% and 96.8%, respectively, using the optimum cut-off point of 0.2 as determined by logistic regression method. Reactivity of anti-rotavirus, anti-reovirus, anti-adenovirus, or anti-enterovirus antibodies with the recombinant N protein coated on the ELISA plates was not detected. These results indicated that the established antibody-capture ELISA in conjunction with recombinant TCoV N protein as the coating protein can be utilized for detection of antibodies to TCoV in turkey flocks. Copyright © 2015 Elsevier B.V. All rights reserved.

Antibody binding in altered gravity: implications for immunosorbent assay during space flight

NASA Technical Reports Server (NTRS)

Maule, Jake; Fogel, Marilyn; Steele, Andrew; Wainwright, Norman; Pierson, Duane L.; McKay, David S.

2003-01-01

A single antibody-incubation step of an indirect, enzyme-linked immunosorbent assay (ELISA) was performed during microgravity, Martian gravity (0.38 G) and hypergravity (1.8 G) phases of parabolic flight, onboard the NASA KC-135 aircraft. Antibody-antigen binding occurred within 15 seconds; the level of binding did not differ between microgravity, Martian gravity and 1 G (Earth's gravity) conditions. During hypergravity and 1 G, antibody binding was directly proportional to the fluid volume (per microtiter well) used for incubation; this pattern was not observed during microgravity. These effects in microgravity may be due to "fluid spread" within the chamber (observed during microgravity with digital photography), leading to greater fluid-surface contact and subsequently antibody-antigen contact. In summary, these results demonstrate that: i) ELISA antibody-incubation and washing steps can be successfully performed by human operators during microgravity, Martian gravity and hypergravity; ii) there is no significant difference in antibody binding between microgravity, Martian gravity and 1 G conditions; and iii) a smaller fluid volume/well (and therefore less antibody) was required for a given level of binding during microgravity. These conclusions indicate that reduced gravity would not present a barrier to successful operation of immunosorbent assays during spaceflight.

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)

Validation of two new immunoassays for sensative detection of a broad range of shiga toxins

USDA-ARS?s Scientific Manuscript database

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...

Monoclonal antibody-based serological assays and immunocapture-RT-PCR for detecting Rice dwarf virus in field rice plants and leafhopper vectors.

PubMed

Wu, Jianxiang; Ni, Yuequn; Liu, Huan; Ding, Ming; Zhou, Xueping

2014-01-01

Rice dwarf virus (RDV) causes Rice dwarf disease, which leads to considerable losses in rice production in Asia. Purified RDV virions were used as the immunogen to prepare monoclonal antibodies (mAbs). Three murine mAbs against RDV were prepared. Plate-trapped antigen enzyme-linked immunosorbent assay (PTA-ELISA), dot enzyme-linked immunosorbent assay (dot-ELISA) and immunocapture-RT-PCR (IC-RT-PCR) were then developed for sensitive, specific, and rapid detection of RDV in rice and leafhopper samples obtained in the field using the mAbs. The PTA-ELISA, dot-ELISA and IC-RT-PCR detected the virus in infected tissue crude extracts diluted at 1:81,920, 1:10,240 and 1:655,360 (w/v, g mL(-1)), in individual viruliferous rice green leafhopper crude extracts diluted at 1:25,600, 1:6400 and 1:3,276,800 (individual leafhopper/μL), respectively. 878 rice field samples and 531 leafhopper field samples from ten provinces of China were screened for the presence of RDV using the two serological assays and the IC-RT-PCR and the results indicated that 37 of the 878 rice samples and 22 of the 531 leafhopper samples were infected by RDV. All positive samples were from Yunnan Province, indicating that RDV is prevalent in this province, but not in the other nine provinces. The dot-ELISA is suitable for routine detection of large-scale rice and leafhopper samples in field surveys. Copyright © 2013 Elsevier B.V. All rights reserved.

Development of a highly sensitive and specific monoclonal antibody based enzyme-linked immunosorbent assay for the detection of a new β-agonist, phenylethanolamine A, in food samples.

PubMed

Jiang, Danni; Cao, Biyun; Wang, Meiyu; Yang, Hong; Zhao, Kang; Li, Jianguo; Li, Mingxin; Sun, Lulu; Deng, Anping

2017-02-01

All β-agonists are banned as feed additives for growth promotion in animals due to toxic effects on humans after consuming the β-agonist contaminated meats. Phenylethanolamine A (PA) is a newly emerged β-agonist. Thus there is a need to develop highly sensitive and specific analytical methods for the detection of PA in food samples. In this study, the monoclonal antibody (mAb) against PA was produced by hybridoma technology and used for the development of enzyme-linked immunosorbent assay (ELISA). The IC 50 values and limits of detection (LODs) of the ELISA using homogeneous combination of coating antigen/antibody for PA were 0.16 ng mL -1 and 0.011 ng mL -1 , respectively. The cross-reactive (CR) values of the assay with 14 structurally related β-agonists were lower than 0.59%. Swine liver and meat samples were spiked with PA at different content and analysed by ELISA. Acceptable recovery rates of 91.40-105.51% and intra-assay coefficients of variation of 1.56-9.92% (n = 3) were obtained. The ELISA for seven spiked samples was confirmed by LC-MS/MS with a high correlation coefficient of 0.9881. The proposed mAb-based ELISA was highly sensitive and specific for PA and could be used as a quantitative/screening method for PA analysis in food samples. © 2016 Society of Chemical Industry. © 2016 Society of Chemical Industry.

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

PubMed Central

Khatami, Alireza; Seyedin, Zahra; Daneshpazhooh, Maryam

2015-01-01

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

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

PubMed Central

Thiha, Aung; Ibrahim, Fatimah

2015-01-01

The enzyme-linked Immunosorbent Assay (ELISA) is the gold standard clinical diagnostic tool for the detection and quantification of protein biomarkers. However, conventional ELISA tests have drawbacks in their requirement of time, expensive equipment and expertise for operation. Hence, for the purpose of rapid, high throughput screening and point-of-care diagnosis, researchers are miniaturizing sandwich ELISA procedures on Lab-on-a-Chip and Lab-on-Compact Disc (LOCD) platforms. This paper presents a novel integrated device to detect and interpret the ELISA test results on a LOCD platform. The system applies absorption spectrophotometry to measure the absorbance (optical density) of the sample using a monochromatic light source and optical sensor. The device performs automated analysis of the results and presents absorbance values and diagnostic test results via a graphical display or via Bluetooth to a smartphone platform which also acts as controller of the device. The efficacy of the device was evaluated by performing dengue antibody IgG ELISA on 64 hospitalized patients suspected of dengue. The results demonstrate high accuracy of the device, with 95% sensitivity and 100% specificity in detection when compared with gold standard commercial ELISA microplate readers. This sensor platform represents a significant step towards establishing ELISA as a rapid, inexpensive and automatic testing method for the purpose of point-of-care-testing (POCT) in resource-limited settings. PMID:25993517

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

PubMed

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

2004-01-01

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

Development of an enzyme-linked immunosorbent assay for the detection of dicamba.

PubMed

Clegg, B S; Stephenson, G R; Hall, J C

2001-05-01

A competitive indirect enzyme-linked immunosorbent assay (CI-ELISA) was developed to quantitate the herbicide dicamba (3,6-dichloro-2-methoxybenzoic acid) in water. The CI-ELISA has a detection limit of 2.3 microg L(-1) and a linear working range of 10--10000 microg L(-1) with an IC(50) value of 195 microg L(-1). The dicamba polyclonal antisera did not cross-react with a number of other herbicides tested but did cross-react with a dicamba metabolite, 5-hydroxydicamba, and structurally related chlorobenzoic acids. The assay was used to estimate quantitatively dicamba concentrations in water samples. Water samples were analyzed directly, and no sample preparation was required. To improve detection limits, a C(18) (reversed phase) column concentration step was devised prior to analysis, and the detection limits were increased by at least by 10-fold. After the sample preconcentration, the detection limit, IC(50), and linear working range were 0.23, 19.5, and 5-200 microg L(-1), respectively. The CI-ELISA estimations in water correlated well with those from gas chromatography-mass spectrometry (GC-MS) analysis (r(2) = 0.9991). This assay contributes to reducing laboratory costs associated with the conventional GC-MS residue analysis techniques for the quantitation of dicamba in water.

Issues encountered in development of enzyme-linked immunosorbent assay for use in detecting Influenza A virus subtype H5N1 exposure in swine.

PubMed

Buehler, Jason; Lager, Kelly; Vincent, Amy; Miller, Cathy; Thacker, Eileen; Janke, Bruce

2014-03-01

A potential mechanism by which highly pathogenic avian Influenza A virus subtype H5N1 could more readily infect human beings is through the infection of and adaptation in pigs. To detect the occurrence of such infection, monitoring of pig populations through serological screening would be highly desirable. In the current study, hemagglutination inhibition assays were able to detect antibodies against H5N1 developed in pigs, but because of antigenic variation between clades, the use of multiple virus strains were required. Whole recombinant virus and recombinant hemagglutinin antigen enzyme-linked immunosorbent assays (ELISAs) were generated that could detect antibody against multiple H5N1 strains, but which also detected antibody against endemic swine influenza viruses. A recombinant hemagglutinin antigen-based ELISA was as effective as the whole virus antigen ELISAs in detecting antibody against the H5N1 virus strains used and eliminated nearly all of the cross-reactivity with non-H5N1 virus antibody. The current study also highlighted the difficulty in establishing a decision (cutoff) value that would effectively counterbalance nonspecific reactivity against sensitivity. The results provide important information and considerations for the development of serological screening assays for highly pathogenic avian H5N1 viruses.

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

PubMed

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

2016-01-01

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

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

PubMed Central

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

2016-01-01

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

Development of cathepsin-L cysteine proteinase based Dot-enzyme-linked immunosorbent assay for the diagnosis of Fasciola gigantica infection in buffaloes.

PubMed

Varghese, Anju; Raina, O K; Nagar, Gaurav; Garg, Rajat; Banerjee, P S; Maharana, B R; Kollannur, Justin D

2012-02-10

Native cathepsin-L cysteine proteinase (28 kDa) was purified from the excretory secretory products of Fasciola gigantica and was used for sero-diagnosis of F. gigantica infection in buffaloes by Dot-enzyme-linked immunosorbent assay (Dot-ELISA). The test detected F. gigantica field infection in these animals with a sensitivity of ∼ 90%. No specific IgG antibody binding was displayed by sera obtained from 76 buffaloes considered to be Fasciola and other parasite-free by microscopic examination of faeces and necropsy examination of liver, rumen and intestine. Additionally, sera from 156 Fasciola-free buffaloes, yet infected with Gigantocotyle explanatum, Paramphistomum epiclitum, Gastrothylax spp., Strongyloides papillosus and hydatid cyst were all negative, indicating that F. gigantica cathepsin-L cysteine proteinase does not cross-react with these helminth parasites in natural infection of the host. The data indicated that cathepsin-L cysteine proteinase based Dot-ELISA reached ∼ 90% sensitivity and 100% specificity with relation to above parasites in the detection of bubaline fasciolosis. The present Dot-ELISA diagnostic assay is relevant to the field diagnosis of F. gigantica infection in buffaloes. Copyright © 2011 Elsevier B.V. All rights reserved.

Competitive Enzyme-Linked Immunosorbent Assay for Detection of Leptospira interrogans Serovar pomona Antibodies in Bovine Sera

PubMed Central

Surujballi, Om; Mallory, Maria

2001-01-01

A competitive enzyme-linked immunosorbent assay (ELISA) using a specific monoclonal antibody (M898) was developed for detection of bovine antibodies to Leptospira interrogans serovar pomona. This assay was evaluated using field sera (n = 190) with serovar pomona microscopic agglutination test (MAT) titers of ≥100 as the positive population (group A); field sera (n = 1,445) which were negative in the MAT (1:100 dilution) for serovar pomona (group B); and sera (from a specific-pathogen-free cattle herd [n = 210]) which were negative in the MAT (1:100 dilution) for serovars canicola, copenhageni, grippotyphosa, hardjo, pomona, and sejroe (group C). At the cutoff point recommended by receiver operating characteristic (ROC) curve analysis of the combined ELISA results of serum groups A, B, and C, the sensitivity and specificity values were 93.7 and 96.3%, respectively. The value for the area under this ROC curve was 0.977, indicating a high level of accuracy for the ELISA. Similar results were obtained from the analysis of the combined results of serum groups A and B and from the analysis of the combined results of serum groups A and C. PMID:11139193

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)

Removal of Lipid from Serum Increases Coherence between Brucellosis Rapid Agglutination Test and Enzyme-linked Immunosorbent Assay in Bears in Alaska, USA.

PubMed

Godfroid, Jacques; Beckmen, Kimberlee; Helena Nymo, Ingebjørg

2016-10-01

In cases of chronic Brucella spp. infection, results of the rose bengal plate test (RBPT) and indirect enzyme-linked immunosorbent assay (ELISA) should be coherent, as reported in controlled conditions in the literature. We compared RBPT and ELISA results in 58 Alaska grizzly bears ( Ursus arctos horribilis), eight Kodiak brown bears ( Ursus arctos middendorffi), and six Alaska Peninsula brown bears ( Ursus arctos gyas). Of the 72 bears tested, 42 (58%) were ELISA positive and 53 (73%) were RBPT positive. However, the coherence between the tests was only fair (K=0.37, SE=0.11), suggesting that either the serologic results were not compatible with Brucella spp. infection or that there was a technical problem with the tests. To address a potential technical problem, we performed a 30-min chloroform/centrifugation cleanup. Following cleanup, the ELISA identified 43 positives (59%) and the RBPT identified 47 (65%), and the coherence between the tests was much improved (K=0.80, SE=0.07). We recommend cleaning wildlife sera with a high lipid content before performing RBPT and performing RBPT and ELISA in parallel to assess coherence. Our results suggest that Alaskan brown bears have been exposed to Brucella spp.

Cross-reactivity of antibodies with phenolic compounds in pistachios during quantification of ochratoxin A by commercial enzyme-linked immunosorbent assay kits.

PubMed

Lee, Hyun Jung; Meldrum, Alexander D; Rivera, Nicholas; Ryu, Dojin

2014-10-01

Ochratoxin A (OTA), a nephrotoxic mycotoxin, naturally occurs in wide range of agricultural commodities. Typical screening of OTA involves various enzyme-linked immunosorbent assay (ELISA) methods. Pistachio (Pistacia vera L.) is a rich source of phenolic compounds that may result in a false positive due to structural similarities to OTA. The present study investigated the cross-reactivity profiles of phenolic compounds using two commercial ELISA test kits. High-performance liquid chromatography was used to confirm the concentration of OTA in the pistachio samples and compared with the results obtained from ELISA. When the degree of interaction and 50 % inhibitory concentration of phenolic compounds were determined, the cross-reactivity showed a pattern similar to that observed with the commercial ELSIA kits, although quantitatively different. In addition, the degree of interaction increased with the increasing concentration of phenolic compounds. The ELISA value had stronger correlations with the content of total phenolic compound, gallic acid, and catechin (R(2) = 0.757, 0.732, and 0.729, respectively) compared with epicatechin (R(2) = 0.590). These results suggest that phenolic compounds in pistachio skins may cross-react with the OTA antibody and lead to a false positive or to an overestimation of OTA concentration in ELISA-based tests.

High sensitivity, high surface area Enzyme-linked Immunosorbent Assay (ELISA).

PubMed

Singh, Harpal; Morita, Takahiro; Suzuki, Yuma; Shimojima, Masayuki; Le Van, An; Sugamata, Masami; Yang, Ming

2015-01-01

Enzyme-linked immunosorbent assays (ELISA) are considered the gold standard in the demonstration of various immunological reactions with an application in the detection of infectious diseases such as during outbreaks or in patient care. This study aimed to produce an ELISA-based diagnostic with an increased sensitivity of detection compared to the standard 96-well method in the immunologic diagnosis of infectious diseases. A '3DStack' was developed using readily available, low cost fabrication technologies namely nanoimprinting and press stamping with an increased surface area of 4 to 6 times more compared to 96-well plates. This was achieved by stacking multiple nanoimprinted polymer sheets. The flow of analytes between the sheets was enhanced by rotating the 3DStack and confirmed by Finite-Element (FE) simulation. An Immunoglobulin G (IgG) ELISA for the detection of antibodies in human serum raised against Rubella virus was performed for validation. An improved sensitivity of up to 1.9 folds higher was observed using the 3DStack compared to the standard method. The increased surface area of the 3DStack developed using nanoimprinting and press stamping technologies, and the flow pattern between sheets generated by rotating the 3DStack were potential contributors to a more sensitive ELISA-based diagnostic device.

Development of a nanogold slot blot inhibition assay for the detection of antibodies against bovine herpesvirus type 1.

PubMed

Japolla, Greice; Cunha-Junior, Jair Pereira; Pajuaba, Ana Claudia Arantes Marquez; Taketomi, Ernesto Akio; Bührer-Sékula, Samira; Bataus, Luiz Artur Mendes; de Souza, Guilherme Rocha Lino

2018-06-01

Bovine herpesvirus type 1 (BoHV-1) is recognized as an important pathogen causing respiratory, reproductive, and neurological disorders in cattle and is associated with economic losses to animal industry. Accurate diagnostic methods are needed for prevention of disease transmission. While the virus neutralization test is considered the gold standard method, it requires maintenance of the virus and cell cultures, which is time consuming and expensive. Serological techniques such as enzyme-linked immunosorbent assay (ELISA) are widely applied, as these are easy to perform and provide quick results. In the present study, a nanogold slot blot inhibition assay was developed for the serological diagnosis of BoHV-1 and compared with standard ELISA and horseradish peroxidase (HRP) slot blot assays. Of 42 serum samples tested by ELISA, 32 (76.2%) were positive and 10 (23.8%), were negative. The sensitivity and specificity of the nanogold slot blot inhibition assay was similar to that observed for ELISA and HRP slot blot assays, and a strong correlation was observed between the tests. Thus, the nanogold slot blot inhibition assay may serve as an efficient and rapid alternative to ELISA in settings, where plate-reading equipment is lacking.

Production of monoclonal antibody for okadaic acid and its utilization in an ultrasensitive enzyme-linked immunosorbent assay and one-step immunochromatographic strip.

PubMed

Liu, Biing-Hui; Hung, Chun-Tse; Lu, Chuan-Chen; Chou, Hong-Non; Yu, Feng-Yih

2014-02-12

Okadaic acid (OA) is a common marine biotoxin that accumulates in bivalves and causes diarrhetic shellfish poisoning (DSP). This study generated a monoclonal antibody (mAb) specific to OA from a hybridoma cell line, 6B1A3, which was obtained by fusion of myeloma cells (P3/NS1/1-AG4-1) with spleen cells isolated from a BALB/c mouse immunized with OA-γ-globulin. The 6B1A3 mAb belongs to the immunoglobulin G1 (κ chain) isotype. Both competitive direct and indirect enzyme-linked immunosorbent assays (ELISAs) were established for characterization of the antibody. The concentrations causing 50% inhibition of binding of OA-horseradish peroxidase to the antibody by OA were calculated to be 0.077 ng/mL in the cdELISA. A rapid and sensitive mAb-based gold nanoparticle immunochromatographic strip was also established. This proposed strip has a detection limit of 5 ng/mL for OA and can be finished in 10 min. Extensive analyses of 20 seafood samples with ELISA revealed that 10 were slightly contaminated with OA, with a mean concentration of 0.892 ng/g. Analysis of OA in shellfish samples showed that data acquired by the immunochromatographic strip agreed well with those acquired by the ELISA. The mAb-based ELISA and immunochromatographic strip assay developed in this study have adequate sensitivity and accuracy for rapid screening of OA in shellfish samples.

Iodoacetyl-functionalized pullulan: A supplemental enhancer for single-domain antibody-polyclonal antibody sandwich enzyme-linked immunosorbent assay for detection of survivin.

PubMed

Matsushita, Takahiko; Arai, Hidenao; Koyama, Tetsuo; Hatano, Ken; Nemoto, Naoto; Matsuoka, Koji

2017-11-01

Survivin, an inhibitor of the apoptosis protein family, is a potent tumor marker for diagnosis and prognosis. The enzyme-linked immunosorbent assay (ELISA) is one of the methods that has been used for detection of survivin. However, ELISA has several disadvantages caused by the use of conventional antibodies, and we have therefore been trying to develop a novel ELISA system using camelid single-domain antibodies (VHHs) as advantageous replacements. Here we report a supplemental approach to improve the VHH-polyclonal antibody sandwich ELISA for survivin detection. Iodoacetyl-functionalized pullulan was synthesized, and its thiol reactivity was characterized by a model reaction with l-cysteine. The thiophilic pullulan was applied to an immunoassay asan additive upon coating of standard assay plates with an anti-survivin VHH fusion protein with C-terminal cysteine. The results showed that the mole ratio of the additive to VHH had a significant effect on the consequent response. Mole ratios of 0.07, 0.7, and 7 led to 90% lower, 15% higher, and 69% lower responses, respectively, than the response of a positive control in which no additive was used. The background levels observed in any additive conditions were as low as that of a negative control lacking both VHH and the additive. These results indicate the applicability of the thiol-reactive pullulan as a response enhancer to VHH-based ELISA. Copyright © 2017 Elsevier Ltd. All rights reserved.

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.

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

PubMed

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

2014-07-01

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

Validation and use of an indirect enzyme-linked immunosorbent assay for detection of antibodies to West Nile virus in American Alligators (Alligator mississippiensis) in Florida.

PubMed

Jacobson, Elliott R; Johnson, April J; Hernandez, Jorge A; Tucker, Sylvia J; Dupuis, Alan P; Stevens, Robert; Carbonneau, Dwayne; Stark, Lillian

2005-01-01

In October 2002, West Nile virus (WNV) was identified in farmed American alligators (Alligator mississippiensis) in Florida showing clinical signs and having microscopic lesions indicative of central nervous system disease. To perform seroepidemiologic studies, an indirect enzyme-linked immunosorbent assay (ELISA) was developed to determine exposure of captive and wild alligators to WNV. To validate the test, a group of WNV-seropositive and -seronegative alligators were identified at the affected farm using hemagglutination inhibition (HAI) and the plaque reduction neutralization test (PRNT). The indirect ELISA utilized a rabbit anti-alligator immunoglobulins polyclonal antibody as the secondary antibody, and inactivated WNV-infected Vero cells were used as the coating antigen. For all samples (n=58), the results of the ELISA were consistent with the HAI and PRNT findings. Plasma was collected from 669 free-ranging alligators from 21 sites across Florida in April and October 2003. Four samples collected in April and six in October were positive for WNV antibodies using HAI, PRNT, and the indirect ELISA. This indicated that wild alligators in Florida have been exposed to WNV. These findings can be used as a baseline for future surveys.

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. Copyright © 2013 Elsevier B.V. All rights reserved.

Diagnostic accuracy of enzyme-linked immunosorbent assay (ELISA) and immunoblot (IB) for the detection of antibodies against Neospora caninum in milk from dairy cows.

PubMed

Chatziprodromidou, I P; Apostolou, T

2018-04-01

The aim of the study was to estimate the sensitivity and specificity of enzyme-linked immunosorbent assay (ELISA) and immunoblot (IB) for detecting antibodies of Neospora caninum in dairy cows, in the absence of a gold standard. The study complies with STRADAS-paratuberculosis guidelines for reporting the accuracy of the test. We tried to apply Bayesian models that do not require conditional independence of the tests under evaluation, but as convergence problems appeared, we used Bayesian methodology, that does not assume conditional dependence of the tests. Informative prior probability distributions were constructed, based on scientific inputs regarding sensitivity and specificity of the IB test and the prevalence of disease in the studied populations. IB sensitivity and specificity were estimated to be 98.8% and 91.3%, respectively, while the respective estimates for ELISA were 60% and 96.7%. A sensitivity analysis, where modified prior probability distributions concerning IB diagnostic accuracy applied, showed a limited effect in posterior assessments. We concluded that ELISA can be used to screen the bulk milk and secondly, IB can be used whenever needed.

Development and Validation of Sandwich ELISA Microarrays with Minimal Assay Interference

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gonzalez, Rachel M.; Servoss, Shannon; Crowley, Sheila A.

Sandwich enzyme-linked immunosorbent assay (ELISA) microarrays are emerging as a strong candidate platform for multiplex biomarker analysis because of the ELISA’s ability to quantitatively measure rare proteins in complex biological fluids. Advantages of this platform are high-throughput potential, assay sensitivity and stringency, and the similarity to the standard ELISA test, which facilitates assay transfer from a research setting to a clinical laboratory. However, a major concern with the multiplexing of ELISAs is maintaining high assay specificity. In this study, we systematically determine the amount of assay interference and noise contributed by individual components of the multiplexed 24-assay system. We findmore » that non-specific reagent cross-reactivity problems are relatively rare. We did identify the presence of contaminant antigens in a “purified antigen”. We tested the validated ELISA microarray chip using paired serum samples that had been collected from four women at a 6-month interval. This analysis demonstrated that protein levels typically vary much more between individuals then within an individual over time, a result which suggests that longitudinal studies may be useful in controlling for biomarker variability across a population. Overall, this research demonstrates the importance of a stringent screening protocol and the value of optimizing the antibody and antigen concentrations when designing chips for ELISA microarrays.« less

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

PubMed Central

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

2010-01-01

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

Echinococcus multilocularis coproantigen detection by enzyme-linked immunosorbent assay in fox, dog, and cat populations.

PubMed

Deplazes, P; Alther, P; Tanner, I; Thompson, R C; Eckert, J

1999-02-01

A sandwich enzyme-linked immunosorbent assay (ELISA) for the detection of Echinococcus multilocularis coproantigens (EM-ELISA) was developed with polyclonal rabbit (solid phase) and chicken egg (catching) antibodies that were directed against E. multilocularis coproantigens and somatic worm antigens, respectively. In experimentally infected dogs and cats, coproantigens were first detectable 6-17 days postinfection (PI) in samples of 8 dogs (worm burdens at necropsy: 6,330-43,200) and from 11 days PI onward in samples of 5 cats infected with 20-6,833 worms. After anthelmintic treatment of 4 dogs and 5 cats at day 20 PI, coproantigen excretion disappeared within 3-5 days. The sensitivity of the ELISA was 83.6% in 55 foxes infected with 4-60,000 E. multilocularis, but reached 93.3% in the 45 foxes harboring more than 20 worms. The EM-ELISA was used in surveys of "normal" dog and cat populations in Switzerland. Among 660 dogs and 263 cats, 5 dogs and 2 cats exhibited a positive reaction. In 2 of these dogs (0.30%) and 1 cat (0.38%), intestinal E. multilocularis infections were confirmed by necropsy, polymerase chain reaction PCR, or both. The specificites of the ELISA in these groups were found to be 99.5% and 99.6%, respectively, if positive ELISA results that could not be confirmed by other methods were classified as "false positive" reactions.

Plasmonic Enzyme-Linked Immunosorbent Assay Using Nanospherical Brushes as a Catalase Container for Colorimetric Detection of Ultralow Concentrations of Listeria monocytogenes.

PubMed

Chen, Rui; Huang, Xiaolin; Xu, Hengyi; Xiong, Yonghua; Li, Yanbin

2015-12-30

Plasmonic enzyme-linked immunosorbent assay (pELISA) based on catalase (CAT)-mediated gold nanoparticle growth exhibits ultrahigh sensitivity for detecting disease-related biomarkers using sandwich formats. However, the limit of detection (LOD) of this strategy for Listeria monocytogenes is only around 10(3) CFU/mL, which considerably exceeds the amount of L. monocytogenes commonly present in food products (<100 CFU/g). Herein, we report an improved pELISA method for detection of L. monocytogenes at ultralow concentrations with the sandwich formats using silica nanoparticles carrying poly(acrylic acid) brushes as a "CAT container" to increase enzyme loading for enhancing the detection signal. Under optimal conditions, the proposed pELISA exhibits good specificity and excellent sensitivity for L. monocytogenes with a LOD of 8 × 10(1) CFU/mL in 0.01 M phosphate-buffered saline, via a reaction that can be discriminated by the naked eye. The LOD obtained by this method was 2 and 5 orders of magnitude lower than that of conventional CAT-based pELISA and horseradish peroxidase (HRP)-based conventional ELISA, respectively. Coupled with large-volume immunomagnetic separation, the LOD for L. monocytogenes-spiked lettuce samples reached 8 × 10(1) CFU/g. The improved pELISA also exhibited a great potential in detecting a single cell of L. monocytogenes in 100 μL of solution.

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.

Mobile phone based ELISA (MELISA).

PubMed

Zhdanov, Arsenii; Keefe, Jordan; Franco-Waite, Luis; Konnaiyan, Karthik Raj; Pyayt, Anna

2018-04-30

Enzyme-linked immunosorbent assay (ELISA) is one of the most important technologies for biochemical analysis critical for diagnosis and monitoring of many diseases. Traditional systems for ELISA incubation and reading are expensive and bulky, thus cannot be used at point-of-care or in the field. Here, we propose and demonstrate a new miniature mobile phone based system for ELISA (MELISA). This system can be used to complete all steps of the assay, including incubation and reading. It weighs just 1 pound, can be fabricated at low cost, portable, and can transfer test results via mobile phone. We successfully demonstrated how MELISA can be calibrated for accurate measurements of progesterone and demonstrated successful measurements with the calibrated system. Copyright © 2017 Elsevier B.V. All rights reserved.

Development of 2 types of competitive enzyme-linked immunosorbent assay for detecting antibodies to the rinderpest virus using a monoclonal antibody for a specific region of the hemagglutinin protein.

PubMed

Khamehchian, S; Madani, R; Rasaee, M J; Golchinfar, F; Kargar, R

2007-06-01

A competitive enzyme-linked immunosorbent assay (C-ELISA) has been developed and standardized for the detection of antibodies to the rinderpest virus (RPV) in sera from cattle, sheep, and goats. The test is specific for rinderpest because it does not detect antibodies to peste-des-petits-ruminants virus (PPRV). The test depends on the ability of the monoclonal antibody (MAb) directed against the hemagglutinin (H) protein of RPV to compete with the binding of RPV antibodies in the positive serum to the H protein of this virus. This MAb recognized a region from amino acids 575 to 583 on the H protein of RPV that is unique to the RPV H protein and is not present on the hemagglutinin-neuraminidase protein of PPRV. Another C-ELISA (peptide C-ELISA) was set up using this specific region as an antigen. A threshold value of 64.4% inhibition was established for the RPV C-ELISA, with 90 known RPV-negative and 30 RPV-positive serum samples. Using common serum samples, a cutoff value of 43.0% inhibition for the peptide C-ELISA was established. Based on statistical analysis, the overall sensitivity and specificity of the RPV C-ELISA, relative to those of a commercial kit, were found to be 90.00% and 103.33%, respectively. However, the sensitivity and specificity of the peptide C-ELISA were found to be 180.00% and 73.33%, respectively. Although a common MAb in 2 new C-ELISA systems was used, variation in their percent inhibition, due to the use of different antigens, was observed. Taking into consideration the difference in percent inhibition of the 2 described assays and the commercial kit (50%), it was found that the RPV C-ELISA and the peptide C-ELISA are more specific and sensitive tools than the commercial kit for assessing herd immune status and for epidemiologic surveillance.

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

PubMed Central

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

2016-01-01

Objective Autoantibodies to melanoma differentiation-associated gene 5 (MDA5) are specifically expressed in patients with dermatomyositis (DM) and are associated with a subset of DM patients with rapidly progressive interstitial lung disease (RP-ILD). Here, we examined the clinical utility of a newly developed enzyme-linked immunosorbent assay (ELISA) system for detecting these antibodies. Methods Here we developed an improved ELISA for detecting anti-MDA5 antibodies. We then performed a multicenter clinical study involving 8 medical centers and enrolled 242 adult patients with polymyositis (PM)/DM, 190 with non-PM/DM connective tissue disease (CTD), 154 with idiopathic interstitial pneumonia (IIP), and 123 healthy controls. Anti-MDA5 antibodies in the patients’ serum samples were quantified using our newly developed ELISA, and the results were compared to those obtained using the gold-standard immunoprecipitation (IP) assay. In addition, correlations between the ELISA-quantified anti-MDA5 antibodies and clinical characteristics were evaluated. Results In patients with PM/DM, the anti-MDA5 antibody measurements obtained from the ELISA and IP assay were highly concordant; the ELISA exhibited an analytical sensitivity of 98.2%, specificity of 100%, positive predictive value of 100%, and negative predictive value of 99.5% (compared to the IP assay). Anti-MDA5 antibodies were detected in 22.7% of the DM patients, but not in any of the patients with PM, non-PM/DM CTD, or IIP. Clinically amyopathic DM, RP-ILD, arthritis, and fever were more prevalent in DM patients who were anti-MDA5 antibody-positive than in those who were antibody-negative (P ≤ 0.0002 for all comparisons). In addition, anti-MDA5 antibody-positive patients with RP-ILD exhibited higher antibody levels than those without RP-ILD (P = 0.006). Conclusion Our newly developed ELISA can detect anti-MDA5 antibodies as efficiently as the gold standard IP assay and has the potential to facilitate the routine clinical measurement of anti-MDA5 antibodies in patients who suspected to have DM. PMID:27115353

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

PubMed

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

2016-01-01

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

EVOLUTION OF ENVIRONMENTAL IMMUNOCHEMISTRY

EPA Science Inventory

Enzyme-linked immunosorbent assays (ELISAs), initially developed for clinical applications, have made a tremendous impact as clinical diagnostic indicators. Pesticide chemists became attracted to the potential of these sensitive and selective methods in the 1970s. Thus, beg...

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...

Enzyme-linked immunosorbent assay for Potomac horse fever disease.

PubMed

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

1987-01-01

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

Nanoswitch-linked immunosorbent assay (NLISA) for fast, sensitive, and specific protein detection.

PubMed

Hansen, Clinton H; Yang, Darren; Koussa, Mounir A; Wong, Wesley P

2017-09-26

Protein detection and quantification play critical roles in both basic research and clinical practice. Current detection platforms range from the widely used ELISA to more sophisticated, and more expensive, approaches such as digital ELISA. Despite advances, there remains a need for a method that combines the simplicity and cost-effectiveness of ELISA with the sensitivity and speed of modern approaches in a format suitable for both laboratory and rapid, point-of-care applications. Building on recent developments in DNA structural nanotechnology, we introduce the nanoswitch-linked immunosorbent assay (NLISA), a detection platform based on easily constructed DNA nanodevices that change conformation upon binding to a target protein with the results read out by gel electrophoresis. NLISA is surface-free and includes a kinetic-proofreading step for purification, enabling both enhanced sensitivity and reduced cross-reactivity. We demonstrate femtomolar-level detection of prostate-specific antigen in biological fluids, as well as reduced cross-reactivity between different serotypes of dengue and also between a single-mutation and wild-type protein. NLISA is less expensive, uses less sample volume, is more rapid, and, with no washes, includes fewer hands-on steps than ELISA, while also achieving superior sensitivity. Our approach also has the potential to enable rapid point-of-care assays, as we demonstrate by performing NLISA with an iPad/iPhone camera for imaging.

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

USDA-ARS?s Scientific Manuscript database

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...

Monoclonal antibodies against colonization factor antigen I pili from enterotoxigenic Escherichia coli.

PubMed

Worobec, E A; Shastry, P; Smart, W; Bradley, R; Singh, B; Paranchych, W

1983-09-01

Hybridomas secreting monoclonal antibodies directed against intact colonization factor antigen I pili have been produced by the fusion of spleen cells from immunized BALB/c mice with NS1/SP2 myeloma cells. The four monoclones with the highest antibody titer, as detected by enzyme-linked immunosorbant assay (ELISA), were chosen for antibody amplification by production of mouse ascitic fluid. These four were examined for antibody specificity by ELISA and immunoblot assays, using six different pilus types. Three of the four monoclonal isolates were specific for only colonization factor antigen I pili in both assays, whereas the remaining isolate showed a distinct cross-reactivity with K99 pili in the ELISA assay but not in immunoblot analysis. These results indicate that this monoclone may be recognizing a common structural element between the two adhesive pilus types.

Monoclonal antibodies against colonization factor antigen I pili from enterotoxigenic Escherichia coli.

PubMed Central

Worobec, E A; Shastry, P; Smart, W; Bradley, R; Singh, B; Paranchych, W

1983-01-01

Hybridomas secreting monoclonal antibodies directed against intact colonization factor antigen I pili have been produced by the fusion of spleen cells from immunized BALB/c mice with NS1/SP2 myeloma cells. The four monoclones with the highest antibody titer, as detected by enzyme-linked immunosorbant assay (ELISA), were chosen for antibody amplification by production of mouse ascitic fluid. These four were examined for antibody specificity by ELISA and immunoblot assays, using six different pilus types. Three of the four monoclonal isolates were specific for only colonization factor antigen I pili in both assays, whereas the remaining isolate showed a distinct cross-reactivity with K99 pili in the ELISA assay but not in immunoblot analysis. These results indicate that this monoclone may be recognizing a common structural element between the two adhesive pilus types. Images PMID:6136463

On optimizing the blocking step of indirect enzyme-linked immunosorbent assay for Epstein-Barr virus serology.

PubMed

Lim, Chun Shen; Krishnan, Gopala; Sam, Choon Kook; Ng, Ching Ching

2013-01-16

Because blocking agent occupies most binding surface of a solid phase, its ability to prevent nonspecific binding determines the signal-to-noise ratio (SNR) and reliability of an enzyme-linked immunosorbent assay (ELISA). We demonstrate a stepwise approach to seek a compatible blocking buffer for indirect ELISA, via a case-control study (n=176) of Epstein-Barr virus (EBV)-associated nasopharyngeal carcinoma (NPC). Regardless of case-control status, we found that synthetic polymer blocking agents, mainly Ficoll and poly(vinyl alcohol) (PVA) were able to provide homogeneous backgrounds among samples, as opposed to commonly used blocking agents, notably nonfat dry milk (NFDM). The SNRs for NPC samples that correspond to blocking using PVA were approximately 3-fold, on average, higher than those blocking using NFDM. Both intra- and inter-assay precisions of PVA-based assays were <14%. A blocking agent of choice should have tolerable sample backgrounds from both cases and controls to ensure the reliability of an immunoassay. Copyright © 2012 Elsevier B.V. All rights reserved.

Role of specific immunoglobulin E in diagnosis of acute toxoplasma infection and toxoplasmosis.

PubMed

Wong, S Y; Hajdu, M P; Ramirez, R; Thulliez, P; McLeod, R; Remington, J S

1993-11-01

Toxoplasma immunoglobulin E (IgE) antibodies were evaluated in an immunosorbent agglutination assay (ISAGA) and an enzyme-linked immunosorbent assay (ELISA) to determine their usefulness in the diagnosis of acute infection with Toxoplasma gondii. IgE antibodies were not detected in serum specimens from otherwise seronegative individuals, individuals with chronic toxoplasma infection, or infants without congenital toxoplasmosis. In contrast, they were detected in pregnant women who seroconverted during gestation (100% by ELISA, 63% by ISAGA), patients with toxoplasmic lymphadenopathy (96% by ELISA, 88% by ISAGA), infants with signs of congenital toxoplasmosis which prompted serologic testing in the postnatal period (92% by ELISA, 67% by ISAGA), children and adults with toxoplasmic chorioretinitis (36% by ELISA, 18% by ISAGA), and adult patients with AIDS and toxoplasmic encephalitis (33% by ELISA, 25% by ISAGA). In many of the serum specimens, the titer of IgE antibodies detected by the ISAGA were close to or at the positive cutoff value. The duration of detectable IgE antibodies in patients with acute infections varied considerably among individuals but showed a trend toward a briefer duration by the ISAGA than by the ELISA. These results reveal that recrudescence of IgE antibodies in patients with reactivated chronic infection (toxoplasmic chorioretinitis and toxoplasmic encephalitis) may be useful diagnostically and that demonstration of toxoplasma IgE antibodies is a useful adjunct to currently available serologic tests for the diagnosis of acute toxoplasma infection and toxoplasmosis.

Role of specific immunoglobulin E in diagnosis of acute toxoplasma infection and toxoplasmosis.

PubMed Central

Wong, S Y; Hajdu, M P; Ramirez, R; Thulliez, P; McLeod, R; Remington, J S

1993-01-01

Toxoplasma immunoglobulin E (IgE) antibodies were evaluated in an immunosorbent agglutination assay (ISAGA) and an enzyme-linked immunosorbent assay (ELISA) to determine their usefulness in the diagnosis of acute infection with Toxoplasma gondii. IgE antibodies were not detected in serum specimens from otherwise seronegative individuals, individuals with chronic toxoplasma infection, or infants without congenital toxoplasmosis. In contrast, they were detected in pregnant women who seroconverted during gestation (100% by ELISA, 63% by ISAGA), patients with toxoplasmic lymphadenopathy (96% by ELISA, 88% by ISAGA), infants with signs of congenital toxoplasmosis which prompted serologic testing in the postnatal period (92% by ELISA, 67% by ISAGA), children and adults with toxoplasmic chorioretinitis (36% by ELISA, 18% by ISAGA), and adult patients with AIDS and toxoplasmic encephalitis (33% by ELISA, 25% by ISAGA). In many of the serum specimens, the titer of IgE antibodies detected by the ISAGA were close to or at the positive cutoff value. The duration of detectable IgE antibodies in patients with acute infections varied considerably among individuals but showed a trend toward a briefer duration by the ISAGA than by the ELISA. These results reveal that recrudescence of IgE antibodies in patients with reactivated chronic infection (toxoplasmic chorioretinitis and toxoplasmic encephalitis) may be useful diagnostically and that demonstration of toxoplasma IgE antibodies is a useful adjunct to currently available serologic tests for the diagnosis of acute toxoplasma infection and toxoplasmosis. PMID:8263181

Visual Detection of Canine Parvovirus Based on Loop-Mediated Isothermal Amplification Combined with Enzyme-Linked Immunosorbent Assay and with Lateral Flow Dipstick

PubMed Central

SUN, Yu-Ling; YEN, Chon-Ho; TU, Ching-Fu

2013-01-01

ABSTRACT Loop-mediated isothermal amplification (LAMP) combined with enzyme-linked immunosorbent assay (LAMP–ELISA) and with lateral flow dipstick (LAMP–LFD) are rapid, sensitive and specific methods for the visual detection of clinical pathogens. In this study, LAMP–ELISA and LAMP–LFD were developed for the visual detection of canine parvovirus (CPV). For LAMP, a set of four primers (biotin-labeled forward inner primers) was designed to specifically amplify a region of the VP2 gene of CPV. The optimum time and temperature for LAMP were 60 min and 65°C, respectively. The specific capture oligonucleotide probes, biotin-labeled CPV probe for LAMP–ELISA and fluorescein isothiocyanate-labeled CPV probe for LAMP–LFD were also designed for hybridization with LAMP amplicons on streptavidin-coated wells and LFD strips, respectively. For the comparison of detection sensitivity, conventional PCR and LAMP for CPV detection were also performed. The CPV detection limits by PCR, PCR–ELISA, LAMP, LAMP–ELISA and LAMP–LFD were 102, 102, 10−1, 10−1 and 10−1 TCID50/ml, respectively. In tests using artificially contaminated dog fecal samples, the samples with CPV inoculation levels of ≥1 TCID50/ml gave positive results by both LAMP–ELISA and LAMP–LFD. Our data indicated that both LAMP–ELISA and LAMP–LFD are promising as rapid, sensitive and specific methods for an efficient diagnosis of CPV infection. PMID:24334855

Development and Evaluation of Recombinant Nucleocapsid Protein Based Diagnostic ELISA for Detection of Nipah Virus Infection in Pigs.

PubMed

Kulkarni, Diwakar D; Venkatesh, Govindarajalu; Tosh, Chakradhar; Patel, Priyanka; Mashoria, Anita; Gupta, Vandana; Gupta, Sourabh; D, Senthilkumar

2016-01-01

The recombinant viral protein-based indirect enzyme-linked immunosorbent assay (ELISA) is a cost-effective, safe, specific, and rapid tool to diagnose the viral infection. Nipah virus nucleocapsid (NiV-N) protein was expressed in Escherichia coli and purified by histidine tag-based affinity chromatography. The N protein was selected based on its immuno dominance and conservation among different NiV strains. An indirect immunoglobulin G (IgG) enzyme-linked immunosorbent assay (ELISA) for swine sera was optimized using the recombinant NiV-N protein as an antigen along with negative and positive controls. The background reading was blocked using skim milk powder and chicken serum. A total number of 1709 swine serum samples from various states of India were tested with indirect ELISA and Western blot. The test was considered positive only when its total reactivity reading was higher than 0.2 cut-off value and the ratio of the total reactivity to the background reading was more than 2.0. Since specificity is high for Western blotting it was used as standard test for comparison of results of indirect ELISA. Sensitivity and specificity of indirect ELISA was 100% and 98.7%, respectively, in comparison with Western blotting. Recombinant N protein-based ELISA can be used in screening large number of serum samples for epidemiological investigations in developing countries where high containment laboratories are not available to handle this zoonotic virus.

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. Copyright © 2015 Elsevier B.V. All rights reserved.

Development of an enzyme-linked immunosorbent assay for detection of chicken osteocalcin and its use in evaluation of perch effects on bone remodeling in caged White Leghorns

USDA-ARS?s Scientific Manuscript database

Osteocalcin (OC) is a sensitive biochemical marker for evaluating bone turnover in mammals. The role of avian OC is less clear because of a need for a chicken assay. Our objectives were to develop an assay using indirect competitive ELISA for detecting chicken serum OC and use the assay to examine t...

Evaluation of diagnostic assays for the serological detection of Actinobacillus pleuropneumoniae on samples of known or unknown exposure.

PubMed

Opriessnig, Tanja; Hemann, Michelle; Johnson, John K; Heinen, Sheila; Giménez-Lirola, Luis G; O'Neill, Kevin C; Hoang, Hai; Yoon, Kyoung-Jin; Gottschalk, Marcelo; Halbur, Patrick G

2013-01-01

Accurate diagnosis of exposure to Actinobacillus pleuropneumoniae is important for maintaining negative farms. In the present study, the ability of a dual-plate complement fixation (CF) assay and 3 commercially available enzyme-linked immunosorbent assays (ELISAs; quad-plate ELISA-1, single-plate ELISA-2, and single-plate ELISA-3) in detecting serological evidence of A. pleuropneumoniae exposure was compared using serum samples of experimentally infected or vaccinated pigs, or field samples from the United States. Forty-two pigs were divided into groups of 2 pigs and were inoculated with 1 of 15 A. pleuropneumoniae strains representing all known serovars of A. pleuropneumoniae, or with Actinobacillus suis, or were vaccinated with a bacterin containing A. pleuropneumoniae serovar 1, 3, 5, or 7. Serum samples collected at the day of inoculation or vaccination and 7, 14, 21, and 28 days later were used to compare the assays. On samples from experimentally infected pigs, the dual-plate CF assay, quad-plate ELISA-1, single-plate ELISA-2, and single-plate ELISA-3 had sensitivities of 0.46, 0.74, 0.13, and 0.13 and specificities of 0.90, 1.0, 1.0, and 1.0, respectively. Vaccinated pigs were identified only by the dual-plate CF assay and the quad-plate ELISA-1. In addition, 90 serum samples with unknown A. pleuropneumoniae exposure collected under field conditions were tested with all assays. The agreement of the 4 assays on field samples was slight to fair. While several assays are available for demonstration of A. pleuropneumoniae exposure, differences in assay targets complicate test choices. Decisions on which assay or combination of assays to use depend on the specific reasons for running the assays.

Optimization and Validation of ELISA for Pre-Clinical Trials of Influenza Vaccine.

PubMed

Mitic, K; Muhandes, L; Minic, R; Petrusic, V; Zivkovic, I

2016-01-01

Testing of every new vaccine involves investigation of its immunogenicity, which is based on monitoring its ability to induce specific antibodies in animals. The fastest and most sensitive method used for this purpose is enzyme-linked immunosorbent assay (ELISA). However, commercial ELISA kits with whole influenza virus antigens are not available on the market, and it is therefore essential to establish an adequate assay for testing influenza virusspecific antibodies. We developed ELISA with whole influenza virus strains for the season 2011/2012 as antigens and validated it by checking its specificity, accuracy, linearity, range, precision, and sensitivity. The results show that we developed high-quality ELISA that can be used to test immunogenicity of newly produced seasonal or pandemic vaccines in mice. The pre-existence of validated ELISA enables shortening the time from the process of vaccine production to its use in patients, which is particularly important in the case of a pandemic.

Comparison of the performance of IFA, CFA, and ELISA assays for the serodiagnosis of acute Q fever by quality assessment.

PubMed

Herremans, Tineke; Hogema, Boris M; Nabuurs, Marrigje; Peeters, Marcel; Wegdam-Blans, Marjolijn; Schneeberger, Peter; Nijhuis, Carla; Notermans, Daan W; Galama, Joep; Horrevorts, Anton; van Loo, Inge H M; Vlaminckx, Bart; Zaaijer, Hans L; Koopmans, Marion P; Berkhout, Hanneke; Socolovschi, Cristina; Raoult, Didier; Stenos, John; Nicholson, William; Bijlmer, Henk

2013-01-01

The indirect immunofluorescence assay (IFA) is considered the reference method for diagnosing Q fever, but serology is also performed by complement fixation assay (CFA) or enzyme-linked immunosorbent assay (ELISA). However, comparability between these assays is not clear, and therefore a quality assessment was performed. A total of 25 serum samples from negative controls, Q fever patients, and a serial diluted high-positive sample were analyzed in 10 Dutch laboratories. Six laboratories performed CFA, 5 performed IFA, and 5 performed ELISAs. Three international reference laboratories from Australia, France, and the USA also participated in this study. Qualitative values between laboratories using the same methods were within close range, and all 3 methods correctly identified acute Q fever patients. The IFA, ELISA, and CFA are all suitable serodiagnostic assays to diagnose acute Q fever, but the IFA remains an important tool in the follow-up of patients and in identifying patients at risk for developing chronic Q fever. Copyright © 2013 Elsevier Inc. All rights reserved.

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

PubMed Central

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

2014-01-01

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

Development of two antibody detection enzyme-linked immunosorbent assays for serodiagnosis of human chronic fascioliasis.

PubMed

Cabán-Hernández, Kimberly; Gaudier, José F; Ruiz-Jiménez, Caleb; Espino, Ana M

2014-03-01

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

Detection of anti-Yta antibodies using a sensitive and specific enzyme-linked immunosorbent assay.

PubMed

Geen, J; Hullin, D A; Hogg, S I

1999-01-01

A specific, sensitive and semi-quantitative enzyme-linked immunosorbent assay (ELISA) is described to detect anti-Yta antibodies in human serum. Recombinant acetylcholinesterase (AChE E.C.3.1.1.7) was employed as the coating antigen in the microtitre plate and horseradish peroxidase (HRP)-conjugated specific antibody (IgG) was used as the secondary antibody. The method developed showed excellent sensitivity, detecting a titre > 1 in 600,000 (3.5 ng/mL mouse IgG protein) for mouse monoclonal (mMAb) anti-AChE antibody. No cross-reaction was seen with other common blood group antibodies, confirming the specificity of the method. The recombinant antigen's AChE phenotype was confirmed as Yta, as no reaction was detected with anti-Ytb-positive sera. The ELISA method correlated closely with the established serological grading system used routinely in blood transfusion laboratories.

Detection of Candida albicans Sap2 in cancer patient serum samples by an indirect competitive enzyme-linked immunosorbent assay for the diagnosis of candidiasis.

PubMed

Wang, Yicun; Gao, Xiang; Zhi Gang, J U; Liu, Jingyuan; Dong, Shuai; Wang, Li

2013-01-01

The secreted aspartyl proteinases 2 (Sap2) of Candida albicans (C. albicans) is a potential marker of candididasis. It is a virulence factor associated with adherence and tissue invasion. In order to detect Sap2 in clinical sera, we developed an indirect competitive enzyme-linked immunosorbent assay (ELISA). Polyclonal antibodies were produced for Sap2 by injecting Sap2 into a New Zealand White inbred rabbit. They could be used at a dilution exceeding 1:1200 in an indirect ELISA, and detected Sap2 concentration up to 1 ng/mL. Of the 286 cancer serum samples tested, 16.8% were found as candidiasis. The test was simple and economical to perform and had a level of sensitivity for detection of low-titer positive sera; thus, it may be proven to be of value in epidemiological studies on candidiasis.

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

NASA Astrophysics Data System (ADS)

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

2016-03-01

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

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

PubMed

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

2016-07-01

ICOSL (B7-H2, CD275), a co-stimulatory molecule of the B7 superfamily, functions as a positive signal in immune response. To investigate whether ICOSL could be released into sera and the possible biological function of soluble ICOS (sICOSL), we generated and characterized a functional anti-human ICOSL monoclonal antibody (mAb), 20B10, and developed a novel enzyme-linked immunosorbent assay (ELISA) based on two anti-human ICOSL antibodies with different epitope specificities. Using the ELISA system, we found that sICOSL in the serum of healthy donors increases in an age-dependent manner and that the matrix metalloproteinase inhibitor (MMPI) could suppress sICOSL production. Together, these data demonstrate that the existence of circulating sICOSL in human serum might play an important role in immunoregulation. Copyright © 2016. Published by Elsevier B.V.

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

NASA Astrophysics Data System (ADS)

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

2014-01-01

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

Practical colorimeter for direct measurement of microplates in enzyme immunoassay systems.

PubMed

Clem, T R; Yolken, R H

1978-01-01

A colorimeter capable of measuring results of enzyme-linked immunosorbent assay (ELISA) reactions directly in the wells of a microtiter plate is described. This colorimeter proved to be as accurate as a conventional spectrophotometer in assessing ELISA reactions, but had the advantage of not requiring transfer of the specimen to a separate chamber. With this colorimeter, 96 specimens can be read in approximately 5 min. A practical colorimeter such as this can make the use of ELISA tests more feasible for many laboratories.

Monoclonal antibody passive hemagglutination and capture enzyme-linked immunosorbent assays for direct detection and quantitation of F41 and K99 fimbrial antigens in enterotoxigenic Escherichia coli.

PubMed Central

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

1987-01-01

Production of diarrhea in neonatal calves by enterotoxigenic Escherichia coli depends on its ability to attach to the epithelial cells of the intestine via surface adhesins called pili or fimbriae and to secrete enterotoxins. The most important of these fimbriae are designated K99 and F41. We produced and characterized a murine monoclonal antibody specific to F41. This monoclonal antibody and a K99-specific monoclonal antibody were used to develop sensitive and specific passive hemagglutination and capture enzyme-linked immunosorbent assays (ELISAs) for detection and quantitation of F41 and K99 antigens in E. coli cultures and culture supernatants. The capture ELISA systems exhibited excellent sensitivity and specificity, whereas the passive hemagglutination systems appeared to be oversensitive. The ability of the capture ELISAs to detect K99 and F41 fimbrial antigens in fecal specimens from calves was evaluated. Fimbrial antigens were detected in six of six specimens from scouring calves but not in four of four specimens from nonscouring calves. PMID:2880866

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

PubMed

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

2016-07-01

Equine coronavirus (EqCoV) infection has been documented in most reports through quantitative qPCR analysis of feces and viral genome sequencing. Although qPCR is used to detect antigen during the acute disease phase, there is no equine-specific antibody test available to study EqCoV seroprevalence in various horse populations. We developed an enzyme-linked immunosorbent assay (ELISA) targeting antibodies to the spike (S) protein of EqCoV and validated its use, using acute and convalescent sera from 83 adult horses involved in 6 outbreaks. The EqCoV S protein-based ELISA was able to reliably detect antibodies to EqCoV in naturally infected horses. The greatest seroconversion rate was observed in horses with clinical signs compatible with EqCoV infection and EqCoV qPCR detection in feces. The EqCoV S protein-based ELISA could be used effectively for seroepidemiologic studies in order to better characterize the overall infection rate of EqCoV in various horse populations. © 2016 The Author(s).

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

PubMed

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

2014-07-01

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

Development of a sensitive and specific indirect enzyme-linked immunosorbent assay based on a baculovirus recombinant antigen for detection of specific antibodies against Ehrlichia canis.

PubMed

López, Lissett; Venteo, Angel; Aguirre, Enara; García, Marga; Rodríguez, Majosé; Amusátegui, Inmaculada; Tesouro, Miguel A; Vela, Carmen; Sainz, Angel; Rueda, Paloma

2007-11-01

An indirect enzyme-linked immunosorbent assay (ELISA) based on baculovirus recombinant P30 protein of Ehrlichia canis and the 1BH4 anticanine IgG monoclonal antibody was developed and evaluated by examining a panel of 98 positive and 157 negative sera using the indirect fluorescent antibody (IFA) test as the reference technique. The P30-based ELISA appeared to be sensitive and specific (77.55% and 95.54%, respectively) when qualitative results (positive/negative) were compared with those of the IFA test; the coefficient of correlation (R) between the 2 tests was 0.833. Furthermore, it was possible to establish a mathematical formula for use in comparing the results of both techniques. These results indicate that recombinant P30 antigen-based ELISA is a suitable alternative of the IFA test for simple, consistent, and rapid serodiagnosis of canine ehrlichiosis. Moreover, the use of this recombinant protein as antigen offers a great advantage for antigen preparation in comparison with other techniques in which the whole E. canis organism is used as antigen.

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

NASA Technical Reports Server (NTRS)

Hejtmancik, Kelly E.

1987-01-01

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

Comparison of Enzyme-Linked Immunosorbent Assay, Surface Plasmon Resonance and Biolayer Interferometry for Screening of Deoxynivalenol in Wheat and Wheat Dust

PubMed Central

Sanders, Melanie; McPartlin, Daniel; Moran, Kara; Guo, Yirong; Eeckhout, Mia; O’Kennedy, Richard; De Saeger, Sarah; Maragos, Chris

2016-01-01

A sample preparation method was developed for the screening of deoxynivalenol (DON) in wheat and wheat dust. Extraction was carried out with water and was successful due to the polar character of DON. For detection, an enzyme-linked immunosorbent assay (ELISA) was compared to the sensor-based techniques of surface plasmon resonance (SPR) and biolayer interferometry (BLI) in terms of sensitivity, affinity and matrix effect. The matrix effects from wheat and wheat dust using SPR were too high to further use this screenings method. The preferred ELISA and BLI methods were validated according to the criteria established in Commission Regulation 519/2014/EC and Commission Decision 2002/657/EC. A small survey was executed on 16 wheat lots and their corresponding dust samples using the validated ELISA method. A linear correlation (r = 0.889) was found for the DON concentration in dust versus the DON concentration in wheat (LOD wheat: 233 μg/kg, LOD wheat dust: 458 μg/kg). PMID:27077883

Evaluation of enzyme-linked immunosorbent assays performed on milk and serum samples for detection of neosporosis and leukosis in lactating dairy cows

PubMed Central

Walsh, Robert B.; Kelton, David F.; Hietala, Sharon K.; Duffield, Todd F.

2013-01-01

Serum and milk samples from 1229 cows on 22 Ontario dairy farms were individually tested for antibodies specific for bovine leukosis virus (BLV) and Neospora caninum by enzyme-linked immunosorbent assay (ELISA). Antibodies against BLV were present in 361 serum samples (29.4%) and 369 milk samples (30.0%). Comparing the 2 tests, agreement was almost perfect (k = 0.86; 95% CI = 0.83 to 0.90) and the proportions of samples positive were not significantly different (P = 0.56). Both tests identified the same 3 herds free of bovine leukosis virus. Antibodies against N. caninum were detected in 138 serum samples (11.2%), and 111 milk samples (9.0%). Agreement between the 2 tests was moderate (k = 0.52; 95% CI = 0.43 to 0.59). Four herds were free of neosporosis by the serum test, while 10 herds were negative by the milk test. The ELISA on milk samples facilitates sample collection to classify herds free of BLV; the milk N. caninum ELISA was less reliable in predicting herd-level infection. PMID:24082160

Pertussis serology: assessment of IgG anti-PT ELISA for replacement of the CHO cell assay*

PubMed Central

DALBY, TINE; SØRENSEN, CHARLOTTE; PETERSEN, JESPER WESTPHAL; KROGFELT, KAREN ANGELIKI

2010-01-01

Dalby T, Sørensen C, Petersen JW, Krogfelt KA. Pertussis serology: assessment of IgG anti-PT ELISA for replacement of the CHO cell assay. APMIS 2010; 118: 968–72. Two types of serological assays are commonly used for the assessment of pertussis vaccine-induced antibodies; the Chinese hamster ovary cell (CHO cell) assay and the immunoglobulin G (IgG) anti pertussis toxin (PT) enzyme-linked immunosorbent assay (IgG anti-PT ELISA). Recently, both the techniques have been modified to improve performance with sera with interfering activity (CHO cell assay) or with heat-treated sera (IgG anti-PT ELISA). These two improved techniques were compared by the analysis of 100 individual serum samples from a previous clinical trial and 213 sera from a longitudinal serum collection from 20 Danish adults recently vaccinated with the Danish acellular pertussis vaccine. The comparison showed a significant linear correlation between the results of the two assays with a p-value of <0.0001 for the 100 individual samples. We, therefore, conclude that the improved IgG anti-PT ELISA can be used as a replacement for the often troublesome and time-consuming CHO cell assay for the measurement of vaccine-induced human antibodies to PT. PMID:21091778

Comparison of ELISA and LC-MS/MS for the measurement of flunixin plasma concentrations in beef cattle after intravenous and subcutaneous administration

USDA-ARS?s Scientific Manuscript database

The feasibility of using an enzyme linked immunosorbant assay (ELISA) with only simple preparation steps to determine flunixin plasma concentrations in healthy beef cattle was explored. Eight cattle (288 ± 22 kg) were treated with 2.2 mg/kg bw flunixin free acid in a cross-over design by subcutaneo...

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

PubMed

Nekouei, Omid; Durocher, Jean; Keefe, Greg

2016-07-01

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.

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

An enzyme-linked immunosorbent assay for the determination of dioxins in contaminated sediment and soil samples

PubMed Central

Van Emon, Jeanette M.; Chuang, Jane C.; Lordo, Robert A.; Schrock, Mary E.; Nichkova, Mikaela; Gee, Shirley J.; Hammock, Bruce D.

2010-01-01

A 96-microwell enzyme-linked immunosorbent assay (ELISA) method was evaluated to determine PCDDs/PCDFs in sediment and soil samples from an EPA Superfund site. Samples were prepared and analyzed by both the ELISA and a gas chromatography/high resolution mass spectrometry (GC/HRMS) method. Comparable method precision, accuracy, and detection level (8 ng kg−1) were achieved by the ELISA method with respect to GC/HRMS. However, the extraction and cleanup method developed for the ELISA requires refinement for the soil type that yielded a waxy residue after sample processing. Four types of statistical analyses (Pearson correlation coefficient, paired t-test, nonparametric tests, and McNemar’s test of association) were performed to determine whether the two methods produced statistically different results. The log-transformed ELISA-derived 2,3,7,8-tetrachlorodibenzo-p-dioxin values and logtransformed GC/HRMS-derived TEQ values were significantly correlated (r = 0.79) at the 0.05 level. The median difference in values between ELISA and GC/HRMS was not significant at the 0.05 level. Low false negative and false positive rates (<10%) were observed for the ELISA when compared to the GC/HRMS at 1000 ng TEQ kg−1. The findings suggest that immunochemical technology could be a complementary monitoring tool for determining concentrations at the 1000 ng TEQ kg−1 action level for contaminated sediment and soil. The ELISA could also be used in an analytical triage approach to screen and rank samples prior to instrumental analysis. PMID:18313102

Comparison of enzyme-linked immunosorbent assay and gas chromatography procedures for the detection of cyanazine and metolachlor in surface water samples

USGS Publications Warehouse

Schraer, S.M.; Shaw, D.R.; Boyette, M.; Coupe, R.H.; Thurman, E.M.

2000-01-01

Enzyme-linked immunosorbent assay (ELISA) data from surface water reconnaissance were compared to data from samples analyzed by gas chromatography for the pesticide residues cyanazine (2-[[4-chloro-6-(ethylamino)-l,3,5-triazin-2-yl]amino]-2-methylpropanenitrile ) and metolachlor (2-chloro-N-(2-ethyl-6-methylphenyl)-N-(2-methoxy-1-methylethyl)acetamide). When ELISA analyses were duplicated, cyanazine and metolachlor detection was found to have highly reproducible results; adjusted R2s were 0.97 and 0.94, respectively. When ELISA results for cyanazine were regressed against gas chromatography results, the models effectively predicted cyanazine concentrations from ELISA analyses (adjusted R2s ranging from 0.76 to 0.81). The intercepts and slopes for these models were not different from 0 and 1, respectively. This indicates that cyanazine analysis by ELISA is expected to give the same results as analysis by gas chromatography. However, regressing ELISA analyses for metolachlor against gas chromatography data provided more variable results (adjusted R2s ranged from 0.67 to 0.94). Regression models for metolachlor analyses had two of three intercepts that were not different from 0. Slopes for all metolachlor regression models were significantly different from 1. This indicates that as metolachlor concentrations increase, ELISA will over- or under-estimate metolachlor concentration, depending on the method of comparison. ELISA can be effectively used to detect cyanazine and metolachlor in surface water samples. However, when detections of metolachlor have significant consequences or implications it may be necessary to use other analytical methods.

Diagnosis of murine mycoplasmal infections by enzyme-linked immunosorbent assay (ELISA).

PubMed

Davis, J; Cassell, G H; Gambill, G; Cox, N; Watson, H; Davidson, M

1987-06-01

ELISA is the currently accepted method for screening rodent colonies for Mycoplasma pulmonis infection. While this assay has greatly improved mycoplasmal detection, it suffers from major defects. Cross-reactions with M. arthritidis are the major technical problem, and prevent definitive diagnosis. Current methods for obtaining a definitive diagnosis are accurate in about 80% of cases, and include ELISA testing for both organisms, immunoblot analysis, and blocking of the murine reaction with heterologous serum. Another technical difficulty is the inherent variability in the assay, which can be overcome by rigid quality control measures and careful attention to detail. The difficulties that arise from the natural history of mycoplasmal infection in barrier-maintained colonies, i.e., low incidence of infected animals and delayed antibody response in animals infected with low numbers of organisms, seriously limit the usefulness of the ELISA. While the assay can be extremely useful in screening breeding colonies and in eliminating mycoplasmas from such colonies, it cannot easily be used to screen potential sources of weanling animals for experimental use.

Development of a Sensitive Luciferase-Based Sandwich ELISA System for the Detection of Human Extracellular Matrix 1 Protein.

PubMed

Li, Ya; Li, Yanqing; Zhao, Junli; Zheng, Xiaojing; Mao, Qinwen; Xia, Haibin

2016-12-01

Enzyme-linked immunosorbent assay (ELISA) has been one of the main methods for detecting an antigen in an aqueous sample for more than four decades. Nowadays, one of the biggest concerns for ELISA is still how to improve the sensitivity of the assay, and the luciferase-luciferin reaction system has been noticed as a new detection method with high sensitivity. In this study, a luciferin-luciferase reaction system was used as the detection method for a sandwich ELISA system. It was shown that this new system led to an increase in the detection sensitivity of at least two times when compared with the traditional horseradish peroxidase (HRP) detection method. Lastly, the serum levels of the human extracellular matrix 1 protein of breast cancer patients were determined by the new system, which were overall similar to the HRP chemiluminescent system. Furthermore, this new luciferase reporter can be implemented into other ELISA systems for the purpose of increasing the assay sensitivity.

Combining two serological assays optimises sensitivity and specificity for the identification of Streptococcus equi subsp. equi exposure.

PubMed

Robinson, Carl; Steward, Karen F; Potts, Nicola; Barker, Colin; Hammond, Toni-ann; Pierce, Karen; Gunnarsson, Eggert; Svansson, Vilhjálmur; Slater, Josh; Newton, J Richard; Waller, Andrew S

2013-08-01

The detection of anti-Streptococcus equi antibodies in the blood serum of horses can assist with the identification of apparently healthy persistently infected carriers and the prevention of strangles outbreaks. The aim of the current study was to use genome sequencing data to develop an indirect enzyme linked immunosorbent assay (iELISA) that targets two S. equi-specific protein fragments. The sensitivity and specificity of the antigen A and antigen C iELISAs were compared to an SeM-based iELISA marketed by IDvet - diagnostic Vétérinaire (IDvet). Individually, each assay compromised specificity in order to achieve sufficient sensitivity (SeM iELISA had a sensitivity of 89.9%, but a specificity of only 77.0%) or sensitivity to achieve high specificity. However, combining the results of the antigen A and antigen C iELISAs permitted optimisation of both sensitivity (93.3%) and specificity (99.3%), providing a robust assay for the identification of horses exposed to S. equi. Copyright © 2013 Elsevier Ltd. All rights reserved.

Development and application of an indirect enzyme-linked immunosorbent assay using recombinant truncated Cap protein for the diagnosis of porcine circovirus-like virus P1.

PubMed

Wen, Li-bin; Wen, Shi-fu; He, Kong-wang

2016-01-19

Porcine circovirus-like virus P1 is a newly discovered virus. To date, there has been no specific serological assay for use in the diagnosis of P1 infection. Because P1 has high homology to porcine circovirus type 2 (PCV2) at the nucleotide level, the C-terminal portion of the capsid protein (amino acids 73-114), a discriminative antigen, was expressed in a prokaryotic expression system. The recombinant product (rctCap), composed of three identical repeated domains, was shown to be strongly immunoreactive to P1-specific serum. This assay was validated by comparison with an indirect immunofluorescence assay (IFA). The diagnostic sensitivity and specificity of the rctCap enzyme-linked immunosorbent assay (ELISA) developed in this study are 93.6% and 98.3%, respectively, compared with the results from IFAs on 450 sera samples from pigs. The indirect ELISA that we developed with rctCap, the recombinant capsid fragment containing the 217-342 nt repeat domain, was sensitive, specific, and suitable for the large-scale detection of P1 infections in swine.

Computer-assisted enzyme immunoassays and simplified immunofluorescence assays: applications for the diagnostic laboratory and the veterinarian's office.

PubMed

Jacobson, R H; Downing, D R; Lynch, T J

1982-11-15

A computer-assisted enzyme-linked immunosorbent assay (ELISA) system, based on kinetics of the reaction between substrate and enzyme molecules, was developed for testing large numbers of sera in laboratory applications. Systematic and random errors associated with conventional ELISA technique were identified leading to results formulated on a statistically validated, objective, and standardized basis. In a parallel development, an inexpensive system for field and veterinary office applications contained many of the qualities of the computer-assisted ELISA. This system uses a fluorogenic indicator (rather than the enzyme-substrate interaction) in a rapid test (15 to 20 minutes' duration) which promises broad application in serodiagnosis.

An Enzyme-linked Immunosorbent Assay for Genistein 7-O-[α-rhamnopyranosyl-(1→6)]-β-glucopyranoside Determination in Derris scandens using a Polyclonal Antibody.

PubMed

Jutathis, Kamonthip; Kitisripanya, Tharita; Udomsin, Orain; Inyai, Chadathorn; Sritularak, Boonchoo; Tanaka, Hiroyuki; Putalun, Waraporn

2016-11-01

Genistein 7-O-[α-rhamnopyranosyl-(1→6)]-β-glucopyranoside (GTG) is a major bioactive compound in Derris scandens. It is responsible for anti-inflammatory activity by inhibition of cyclooxygenase and lipoxygenase. There are many commercial products of D. scandens available in Thailand. To develop an enzyme-linked immunosorbent assay (ELISA) for the quantitative analysis of GTG in plant material and derived products using a polyclonal antibody. An immunogen was synthesised by conjugating GTG with a carrier protein. The polyclonal antibody against GTG (GTG-PAb) was produced in New Zealand white rabbits. The ELISA method was validated for specificity, sensitivity, accuracy, precision and correlation with HPLC. The polyclonal antibody was specific to GTG and genistin within the range of compounds tested. The GTG ELISA was applied in the range 0.04-10.00 μg/mL with a limit of detection of 0.03 μg/mL. The recovery of GTG in spiked Derris scandens extracts ranged from 100.7 to 107.0%, with a coefficient of variation less than 7.0%. The intra- and inter-assay variations were less than 5.0%. The ELISA showed a good correlation with HPLC-UV analysis for GTG determination in samples, with a coefficient of determination (r 2 ) of 0.9880. An ELISA was established for GTG determination in Derris scandens. The GTG-PAb can react with GTG and genistin, but genistin has not been found in the plant. Therefore, the ELISA can be used for high throughput quality control of GTG content in D. scandens and its products. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

Comparison of the presence of Shiga toxin 1 in food matrices as determined by an enzyme-linked immunosorbent assay and a biological activity assay.

PubMed

Lumor, Stephen E; Fredrickson, Neal R; Ronningen, Ian; Deen, Bronwyn D; Smith, Kenneth; Diez-Gonzalez, Francisco; Labuza, Theodore P

2012-06-01

This study was conducted to compare the identification of Shiga toxin 1 (Stx1) based on its specific biological activity and based on results of a commercial enzyme-linked immunosorbent assay (ELISA) kit. Stx1 was thermally treated for various periods in phosphate-buffered saline, milk, and orange juice. The residual Stx1 concentration was determined with the commercial ELISA kit, and its residual enzymatic activity (amount of adenine released from a 2,551-bp DNA substrate) was determined with a biological activity assay (BAA). Regression analysis indicated that the inactivation of Stx1 as a function of time followed first-order kinetics. The half-lives determined at 60, 65, 70, 75, 80, and 85°C were 9.96, 3.19, 2.67, 0.72, 0.47, and 0.29 min, respectively, using the BAA. The half-lives determined by the ELISA with thermal treatments at 70, 75, 80, and 85°C were 40.47, 11.03, 3.64, and 1.40 min, respectively. The Z, Q(10), and Arrhenius activation energy values derived by both assays were dissimilar, indicating that the rate of inactivation of the active site of Stx1 was less sensitive to temperature change than was denaturation of the epitope(s) used in the ELISA. These values were 10.28°C and 9.40 and 54.70 kcal/mol, respectively, with the ELISA and 16°C and 4.11 and 34 kcal/mol, respectively, with the BAA. Orange juice enhanced Stx1 inactivation as a function of increasing temperature, whereas inactivation in 2% milk was not very much different from that in phosphate-buffered saline. Our investigation indicates that the ELISA would be a reliable method for detecting the residual toxicity of heat-treated Stx1 because the half-lives determined with the ELISA were greater than those determined with the BAA (faster degradation) at all temperatures and were highly correlated (R(2) = 0.994) with those determined with the BAA.

Test/QA Plan for Verification of Microcystin Test Kits

EPA Science Inventory

Microcystin test kits are used to quantitatively measure total microcystin in recreational waters. These test kits are based on enzyme-linked immunosorbent assays (ELISA) with antibodies that bind specifically to microcystins or phosphate activity inhibition where the phosphatas...

Novel use of a N2-specific enzyme-linked immunosorbent assay for differentiation of infected from vaccinated animals (DIVA)-based identification of avian influenza.

PubMed

Kwon, Ji-Sun; Kim, Min-Chul; Jeong, Ok-Mi; Kang, Hyun-Mi; Song, Chang-Seon; Kwon, Jun-Hun; Lee, Youn-Jeong

2009-05-21

Proper vaccination with validated companion differentiation of infected from vaccinated animals (DIVA) tests using a vaccine containing a heterologous neuraminidase to the field virus can be effective to control avian influenza (AI). However, indirect immunofluorescent assay, the only field validated DIVA test, has limitations to be set up as high throughput screening test and the assay requires subjective interpretation of the results. To apply the DIVA strategy to the Korean H9N2 low pathogenic AI (LPAI) vaccine program and overcome these limitations, we generated a reassortant H9N8 virus (rgH9N8) vaccine using plasmid-based reverse genetics and developed a novel N2-specific enzyme-linked immunosorbent assay (N2-ELISA). The rgH9N8 vaccine showed adequate immunogenicity and protection, and the optimized N2-ELISA showed that the sensitivity was 97.0% and specificity was 96.4% compared with a hemagglutination inhibition test. In vaccination-challenge experiments in specific pathogen-free chickens, the sera of chickens vaccinated with rgH9N8 vaccine and uninfected were negative by the N2-ELISA (S/P< or =0.4), whereas infected sera with H9N2 were positive (S/P>0.4). These results suggest that the rgH9N8 vaccine and the companion DIVA test, N2-ELISA, allow the utilization of the DIVA strategy for the control of H9N2 LPAI infections in Korea.

Generation of E. coli-derived virus-like particles of porcine circovirus type 2 and their use in an indirect IgG enzyme-linked immunosorbent assay.

PubMed

Zhang, Yan; Wang, Zhanfeng; Zhan, Yang; Gong, Qian; Yu, Wanting; Deng, Zhibang; Wang, Aibing; Yang, Yi; Wang, Naidong

2016-06-01

Porcine circovirus type 2 (PCV2) causes increased mortality and poor growth or weight loss in apparently healthy swine. Therefore, methods to detect PCV2-specific antibodies in swine serum are important for prevention, diagnosis, and control of PCV2-associated diseases (PCVAD). In this study, PCV2 virus-like particles (VLPs) were used to develop a rapid, simple and economical indirect enzyme-linked immunosorbent assay to detect (with high sensitivity) PCV2-specific antibodies in swine serum. The PCV2 capsid protein (Cap) was overexpressed in E. coli after optimizing the cap gene. Subsequently, the soluble Cap was rapidly purified in one step by automated fast protein liquid chromatography (FPLC). The purified PCV2 Cap was shown by transmission electron microscopy and gel filtration chromatography to be capable of self-assembling into VLPs in vitro. Using the purified VLPs as antigens, optimal operating conditions for the VLP ELISA were determined. The concentration of PCV2 VLPs was 1 µg/ml per well, and the dilution factors for swine serum and horseradish peroxidase (HRP)-labeled goat anti-pig antibody were 1:150 and 1:4000, respectively. Out of 241 serum samples tested with this assay, 83.4 % were found to be positive. Importantly, the VLP ELISA had a total coincidence rate of 97.4 % (74/76) compared to an Ingezim PCV2 ELISA IgG assay. In summary, this rapid, inexpensive VLP ELISA has the potential to greatly facilitate large-scale investigations of PCV2-associated serotypes.

Detection of serum antibodies against Ehrlichia risticii in Potomac horse fever by enzyme-linked immunosorbent assay.

PubMed

Dutta, S K; Rice, R M; Hughes, T D; Savage, P K; Myrup, A C

1987-01-01

An indirect enzyme-linked immunosorbent assay (ELISA) was developed which was specific and sensitive in detecting antibodies to Ehrlichia risticii in Potomac horse fever (PHF). The ELISA antibody titers were correlated with the indirect fluorescent antibody (IFA) titers. E. risticii propagated in human histiocyte culture was purified on renografin gradient and the band of the organisms at a density of 1.182 g/ml was used as antigen. ELISA antibody titers were determined through computer assisted analysis, the observed antibody titers were derived by serial serum dilutions and using a resultant standard curve the predicted antibody titers were obtained from a single serum dilution. The standard curve had a correlation coefficient of 0.8975. The observed and predicted antibody titers were in good agreement, as the respective titers fell within a two-fold range. There was a good correlation between ELISA and IFA test results, but the ELISA titers were several times higher. In experimental infections of horses produced with the infected equine whole blood and the Ehrlichia infected macrophage culture, the antibodies were first detected in two weeks and one week postinoculation (PI), respectively. In both cases the titers reached a peak in about 4 weeks PI with a mean titer of 1:16558 and 1:4030, respectively. The antibody titers of the convalescent sera of field cases of PHF were comparatively lower than the experimentally infected horses.

Comparison of Immunofluorescence and Desmoglein Enzyme-linked Immunosorbent Assay in the Diagnosis of Pemphigus: A Prospective, Cross-sectional Study in a Tertiary Care Hospital

PubMed Central

Ravi, Deepthi; Prabhu, S Smitha; Rao, Raghavendra; Balachandran, C; Bairy, Indira

2017-01-01

Background: Pemphigus is an acquired immunobullous disorder in which antibodies are directed against epidermal cadherins. Despite the commercial availability and less cost of enzyme-linked immunosorbent assays (ELISAs) to detect antidesmoglein 1 (Dsg1) and anti-Dsg3, immunofluorescence is still widely used for confirmation of diagnosis. Aims: (1) To compare the usefulness of indirect immunofluorescence (IIF) and ELISA tests in the diagnosis of pemphigus. (2) To find the clinical correlation between the tests and severity of the disease. Materials and Methods: Sixty-one patients (27 women and 34 men, age distribution from 20 to 75) were clinically diagnosed as pemphigus (pemphigus foliaceus - 11, pemphigus vulgaris - 50) and were recruited for the study. IIF and Dsg ELISA were performed and the findings were compared with each other and with the pemphigus area activity score. Data were entered in SPSS and were analyzed using Kruskal–Wallis test. Results: There was a moderate positive correlation between the cutaneous score and Dsg1 titer, and mucosal score and Dsg3 titer. The titer of IIF showed statistically significant positive correlation with the cutaneous score but not the mucosal score. Dsg ELISA showed higher sensitivity (90.2%) than IIF (75.4%) in the diagnosis of pemphigus. Conclusions: Dsg ELISA is a more sensitive method than IIF and shows more correlation with the disease severity. PMID:28400637

Enzyme linked immunosorbent assay for detecting antibody to Trichomonas vaginalis: use of whole cells and aqueous extract as antigen.

PubMed

Alderete, J F

1984-06-01

An enzyme linked immunosorbent assay (ELISA) for detecting antibody to antigenic Trichomonas vaginalis macromolecules has been identified using whole cells or an aqueous protein extract as antigen. The test was developed under optimum conditions using serum samples from experimental animals. The sensitivity of the ELISA was equal to or greater than that obtained by radioimmunoprecipitation and electrophoresis-fluorography techniques. The ELISA was capable of assessing antibody responses during the development of lesions in animals inoculated subcutaneously and it reproducibly measured the individual classes immunoglobulins directed at T vaginalis. The colorimetric assay was also suitable for showing cross reactivity between trichomonal species as well as between different strains of T vaginalis. Conditions established for monitoring antibody to trichomanads in immunised rabbits or infected mice were equally effective for human materials, such as serum or vaginal washes. Serum from experimental animals or infected people showed high concentrations of IgG, IgA, and IgM antibody to trichomonads. Only antibodies of the IgG and IgA class were detected in vaginal washes from women with acute trichomoniasis. No IgE antibody to trichomonads was found under a variety of conditions in serum samples from patients or experimental animals.

Analysis of High-Throughput ELISA Microarray Data

DOE Office of Scientific and Technical Information (OSTI.GOV)

White, Amanda M.; Daly, Don S.; Zangar, Richard C.

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).

Reverse enzyme-linked immunosorbent assay using monoclonal antibodies against SAG1-related sequence, SAG2A, and p97 antigens from Toxoplasma gondii to detect specific immunoglobulin G (IgG), IgM, and IgA antibodies in human sera.

PubMed

Carvalho, Fernando R; Silva, Deise A O; Cunha-Júnior, Jair P; Souza, Maria A; Oliveira, Taísa C; Béla, Samantha R; Faria, Gabriele G; Lopes, Carolina S; Mineo, José R

2008-08-01

The present study aimed to evaluate the performance of three monoclonal antibodies (MAbs) in reverse enzyme-linked immunosorbent assays (ELISAs) for detecting immunoglobulin G (IgG), IgM, and IgA antibodies against Toxoplasma gondii in 175 serum samples from patients at different stages of T. gondii infection, as defined by both serological and clinical criteria, as follows: recent (n = 45), transient (n = 40), and chronic (n = 55) infection as well as seronegative subjects (n = 35). The results were compared with those obtained by indirect ELISA using soluble Toxoplasma total antigen (STAg). Our data demonstrated that MAb A3A4 recognizes a conformational epitope in SAG1-related-sequence (SRS) antigens, while A4D12 and 1B8 recognize linear epitopes defined as SAG2A surface antigen and p97 cytoplasmatic antigen, respectively. Reverse ELISA for IgG with A3A4 or A4D12 MAbs was highly correlated with indirect ELISA for anti-STAg IgG, whereas only A4D12 reverse ELISA showed high correlation with indirect ELISA for IgM and IgA isotypes. To our knowledge, this is the first report analyzing the performance of a reverse ELISA for simultaneous detection of IgG, IgM, and IgA isotypes active toward native SAG2A, SRS, and p97 molecules from STAg, using a panel of human sera from patients with recent and chronic toxoplasmosis. Thus, reverse ELISA based on the capture of native SAG2A and SRS antigens of STAg by MAbs could be an additional approach for strengthening the helpfulness of serological tests assessing the stage of infection, particularly in combination with highly sensitive and specific assays that are frequently used nowadays for diagnosis of toxoplasmosis during pregnancy or congenital infection in newborns.

Diagnostic accuracy of the anti-glutamic acid decarboxylase antibody in type 1 diabetes mellitus: Comparison between radioimmunoassay and enzyme-linked immunosorbent assay.

PubMed

Murata, Takashi; Tsuzaki, Kokoro; Nirengi, Shinsuke; Watanabe, Tomokazu; Mizutani, Yukako; Okada, Hayami; Tsukamoto, Masami; Odori, Shinji; Nakagawachi, Reiko; Kawaguchi, Yaeko; Yoshioka, Fumi; Yamada, Kazunori; Shimatsu, Akira; Kotani, Kazuhiko; Satoh-Asahara, Noriko; Sakane, Naoki

2017-07-01

The distributer of the anti-glutamic acid decarboxylase antibody assay kit using radioimmunoassay (RIA) recently announced its discontinuation, and proposed an alternative kit using enzyme-linked immunosorbent assay (ELISA). The aim of the present study was to investigate the diagnostic values of the anti-glutamic acid decarboxylase antibody by RIA and ELISA among type 1 diabetes mellitus patients and control participants. A total of 79 type 1 diabetes mellitus patients and 79 age-matched controls were enrolled and assessed using RIA and ELISA. Sensitivity, specificity, positive predictive values and negative predictive values were calculated for cut-off values (RIA = 1.5 U/mL and ELISA = 5.0 U/mL, respectively). Kappa coefficients were used to test for agreements between the RIA and ELISA methods regarding the diagnosis of type 1 diabetes mellitus. The sensitivity, specificity, positive predictive values, and negative predictive values for diagnosing type 1 diabetes mellitus were 57.0, 97.5, 95.7, and 69.4% by RIA, and 60.8, 100.0, 100.0 and 71.8% by ELISA, respectively. The diagnosis of type 1 diabetes mellitus using the RIA and ELISA methods showed substantial agreement with the kappa values of 0.74 for all participants, and of 0.64 for the acute type; however, there was moderate agreement with the kappa value of 0.56 for the slowly progressive type. The present study suggests that both anti-glutamic acid decarboxylase antibody by RIA and ELISA was useful for diagnosing type 1 diabetes mellitus. However, in the slowly progressive type, the degree of agreement of these two kits was poorer compared with those in all participants or in the acute type. © 2016 The Authors. Journal of Diabetes Investigation published by Asian Association for the Study of Diabetes (AASD) and John Wiley & Sons Australia, Ltd.

Commercial enzyme-linked immunosorbent assay versuspolymerase chain reaction for the diagnosis of chronic Chagas disease: a systematic review and meta-analysis.

PubMed

Brasil, Pedro Emmanuel Alvarenga Americano do; Castro, Rodolfo; Castro, Liane de

2016-01-01

Chronic Chagas disease diagnosis relies on laboratory tests due to its clinical characteristics. The aim of this research was to review commercial enzyme-linked immunosorbent assay (ELISA) and polymerase chain reaction (PCR) diagnostic test performance. Performance of commercial ELISA or PCR for the diagnosis of chronic Chagas disease were systematically searched in PubMed, Scopus, Embase, ISI Web, and LILACS through the bibliography from 1980-2014 and by contact with the manufacturers. The risk of bias was assessed with QUADAS-2. Heterogeneity was estimated with the I2 statistic. Accuracies provided by the manufacturers usually overestimate the accuracy provided by academia. The risk of bias is high in most tests and in most QUADAS dimensions. Heterogeneity is high in either sensitivity, specificity, or both. The evidence regarding commercial ELISA and ELISA-rec sensitivity and specificity indicates that there is overestimation. The current recommendation to use two simultaneous serological tests can be supported by the risk of bias analysis and the amount of heterogeneity but not by the observed accuracies. The usefulness of PCR tests are debatable and health care providers should not order them on a routine basis. PCR may be used in selected cases due to its potential to detect seronegative subjects.

Commercial enzyme-linked immunosorbent assay versus polymerase chain reaction for the diagnosis of chronic Chagas disease: a systematic review and meta-analysis

PubMed Central

do Brasil, Pedro Emmanuel Alvarenga Americano; Castro, Rodolfo; de Castro, Liane

2016-01-01

Chronic Chagas disease diagnosis relies on laboratory tests due to its clinical characteristics. The aim of this research was to review commercial enzyme-linked immunosorbent assay (ELISA) and polymerase chain reaction (PCR) diagnostic test performance. Performance of commercial ELISA or PCR for the diagnosis of chronic Chagas disease were systematically searched in PubMed, Scopus, Embase, ISI Web, and LILACS through the bibliography from 1980-2014 and by contact with the manufacturers. The risk of bias was assessed with QUADAS-2. Heterogeneity was estimated with the I2 statistic. Accuracies provided by the manufacturers usually overestimate the accuracy provided by academia. The risk of bias is high in most tests and in most QUADAS dimensions. Heterogeneity is high in either sensitivity, specificity, or both. The evidence regarding commercial ELISA and ELISA-rec sensitivity and specificity indicates that there is overestimation. The current recommendation to use two simultaneous serological tests can be supported by the risk of bias analysis and the amount of heterogeneity but not by the observed accuracies. The usefulness of PCR tests are debatable and health care providers should not order them on a routine basis. PCR may be used in selected cases due to its potential to detect seronegative subjects. PMID:26814640

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.

Comparison of a neutralization enzyme immunoassay and an enzyme-linked immunosorbent assay for evaluation of immune status of children vaccinated for mumps.

PubMed Central

Harmsen, T; Jongerius, M C; van der Zwan, C W; Plantinga, A D; Kraaijeveld, C A; Berbers, G A

1992-01-01

A 50% neutralization enzyme immunoassay (N50-EIA) was compared with an indirect enzyme-linked immunosorbent assay (ELISA) for determining mumps virus antibodies in three consecutive serum samples from 138 children vaccinated with a live mumps vaccine at the age (in years) of 1.5. By the N50-EIA, most (132 of 138) preserum samples did not show neutralizing activity. Eight to 12 weeks after vaccination, 17 of the children were still negative, but only 7 remained so after 2.5 years, resulting in a seroconversion rate of 125 of 132 (95%). Over the same period, the neutralization geometric mean titer rose from 3.6 to 9.9. By an indirect ELISA, 128 of 138 preserum samples were found negative. The early and late postvaccination sera of 8 children were ELISA negative, resulting in a seroconversion rate of 120 of 128 (94%). Only two children remained seronegative by both methods. Seven of the late postvaccination serum samples yielded noncorresponding results, reflecting 95% correlation between both methods. Due to cross-reactivity with parainfluenza viruses, the ELISA proved to be less specific, but on the other hand, it showed a greater sensitivity than the N50-EIA. PMID:1500523

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

PubMed

Ott, Laura E; Carson, Susan

2014-01-01

Flow cytometry and enzyme-linked immunosorbent assay (ELISA) are commonly used techniques associated with clinical and research applications within the immunology and medical fields. The use of these techniques is becoming increasingly valuable in many life science and engineering disciplines as well. Herein, we report the development and evaluation of a novel half-semester course that focused on introducing undergraduate and graduate students to advance conceptual and technical skills associated with flow cytometry and ELISA, with emphasis on applications, experimental design, and data analysis. This course was offered in the North Carolina State University Biotechnology Program over three semesters and consisted of weekly lectures and laboratories. Students performed and/or analyzed flow cytometry and ELISA in three separate laboratory exercises: (1) identification of transgenic zebrafish hematopoietic cells, (2) analysis of transfection efficiency, and (3) analysis of cytokine production upon lipopolysaccharide stimulation. Student learning outcomes were achieved as demonstrated by multiple means of assessment, including three laboratory reports, a data analysis laboratory practicum, and a cumulative final exam. Further, anonymous student self-assessment revealed increased student confidence in the knowledge and skill sets defined in the learning outcomes. Copyright © 2014 The International Union of Biochemistry and Molecular Biology.

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. © 2013 Elsevier B.V. All rights reserved.

Comparison of nonstructural protein-1 antigen detection by rapid and enzyme-linked immunosorbent assay test and its correlation with polymerase chain reaction for early diagnosis of dengue

PubMed Central

Gaikwad, Seema; Sawant, Sandhya S.; Shastri, Jayanthi S.

2017-01-01

INTRODUCTION: Early diagnosis of dengue is important for appropriate clinical management and vector control. Different serological tests based on the principle of immunochromatography and enzyme-linked immunosorbent assay (ELISA) are commonly used for detection of antigen and antibodies of dengue virus. The performance of these tests depends on the sensitivity and specificity. Hence, the study was undertaken to compare nonstructural protein-1 (NS1) antigen detection by rapid and ELISA with real-time polymerase chain reaction (RT-PCR) for diagnosis of dengue. MATERIALS AND METHODS: Prospective laboratory study was carried out on sera samples (n = 200) from clinically suspected cases of dengue. The sera samples were subjected for NS1 antigen detection test by rapid test, NS1 ELISA, and RT-PCR. The results of rapid and ELISA tests were compared with real Time PCR. RESULTS: The sensitivity, specificity, positive, and negative predictive value of rapid dengue NS1 antigen test were 81.5%, 66.7%, 78.2%, and 71.1%, respectively whereas that of NS1 ELISA were 89.9%, 100%, 100%, and 94%, respectively. Concordance of Rapid NS1 and NS1 ELISA with PCR was 75.5% and 94%. DISCUSSION AND CONCLUSION: NS1 antigen ELISA can be implemented in diagnostic laboratories for diagnosis of dengue in the acute phase of illness. The test also has great potential value for use in epidemic situations, as it could facilitate the early screening of patients and limit disease expansion. PMID:28706387

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...

Expression and Refolding of Truncated Recombinant Major Outer Membrane Protein Antigen (r56) of Orientia tsutsugamushi and Its Use in Enzyme-Linked Immunosorbent Assays

PubMed Central

Ching, W.-M.; Wang, H.; Eamsila, C.; Kelly, D. J.; Dasch, G. A.

1998-01-01

The variable 56-kDa major outer membrane protein of Orientia tsutsugamushi is the immunodominant antigen in human scrub typhus infections. The gene encoding this protein from Karp strain was cloned into the expression vector pET11a. The recombinant protein (r56) was expressed as a truncated nonfusion protein (amino acids 80 to 456 of the open reading frame) which formed an inclusion body when expressed in Escherichia coli BL21. Refolded r56 was purified and compared to purified whole-cell lysate of the Karp strain of O. tsutsugamushi by immunoglobulin G (IgG) enzyme-linked immunosorbent assay (ELISA) for reactivity with rabbit sera prepared against eight antigenic prototypes of O. tsutsugamushi as well as several other species of Rickettsiales and nonrickettsial antigens. Refolded r56 exhibited broad reactivity with the rabbit antisera against the Orientia prototypes, and the ELISA reactions with the r56 and Karp whole-cell lysate antigens correlated well (r = 0.81, n = 22, sensitivity compared to that of standard ELISA of 91%). Refolded r56 did not react with most antisera against other rickettsial species or control antigens (specificity = 92%, n = 13) using a positive cutoff value determined with eight uninfected rabbit sera. Refolded r56 was evaluated further by ELISA, using 128 sera obtained from patients with suspected scrub typhus from Korat, Thailand, and 74 serum specimens from healthy Thai soldiers. By using the indirect immunoperoxidase assay as the reference assay, the recombinant antigen exhibited a sensitivity and specificity of 93% or greater for detection of both IgG and IgM in the ELISA at 1:400 serum dilution. These results strongly suggest that purified r56 is a suitable candidate for replacing the density gradient-purified, rickettsia-derived, whole-cell antigen currently used in the commercial dipstick assay available in the United States. PMID:9665960

Expression and refolding of truncated recombinant major outer membrane protein antigen (r56) of Orientia tsutsugamushi and its use in enzyme-linked immunosorbent assays.

PubMed

Ching, W M; Wang, H; Eamsila, C; Kelly, D J; Dasch, G A

1998-07-01

The variable 56-kDa major outer membrane protein of Orientia tsutsugamushi is the immunodominant antigen in human scrub typhus infections. The gene encoding this protein from Karp strain was cloned into the expression vector pET11a. The recombinant protein (r56) was expressed as a truncated nonfusion protein (amino acids 80 to 456 of the open reading frame) which formed an inclusion body when expressed in Escherichia coli BL21. Refolded r56 was purified and compared to purified whole-cell lysate of the Karp strain of O. tsutsugamushi by immunoglobulin G (IgG) enzyme-linked immunosorbent assay (ELISA) for reactivity with rabbit sera prepared against eight antigenic prototypes of O. tsutsugamushi as well as several other species of Rickettsiales and nonrickettsial antigens. Refolded r56 exhibited broad reactivity with the rabbit antisera against the Orientia prototypes, and the ELISA reactions with the r56 and Karp whole-cell lysate antigens correlated well (r = 0.81, n = 22, sensitivity compared to that of standard ELISA of 91%). Refolded r56 did not react with most antisera against other rickettsial species or control antigens (specificity = 92%, n = 13) using a positive cutoff value determined with eight uninfected rabbit sera. Refolded r56 was evaluated further by ELISA, using 128 sera obtained from patients with suspected scrub typhus from Korat, Thailand, and 74 serum specimens from healthy Thai soldiers. By using the indirect immunoperoxidase assay as the reference assay, the recombinant antigen exhibited a sensitivity and specificity of 93% or greater for detection of both IgG and IgM in the ELISA at 1:400 serum dilution. These results strongly suggest that purified r56 is a suitable candidate for replacing the density gradient-purified, rickettsia-derived, whole-cell antigen currently used in the commercial dipstick assay available in the United States.

Development of an Enzyme-Linked Immunosorbent Assay Using a Recombinant LigA Fragment Comprising Repeat Domains 4 to 7.5 as an Antigen for Diagnosis of Equine Leptospirosis

PubMed Central

Yan, Weiwei; Saleem, Muhammad Hassan; McDonough, Patrick; McDonough, Sean P.; Divers, Thomas J.

2013-01-01

Leptospira immunoglobulin (Ig)-like (Lig) proteins are a novel family of surface-associated proteins in which the N-terminal 630 amino acids are conserved. In this study, we truncated the LigA conserved region into 7 fragments comprising the 1st to 3rd (LigACon1-3), 4th to 7.5th (LigACon4-7.5), 4th (LigACon4), 4.5th to 5.5th (LigACon4.5–5.5), 5.5th to 6.5th (LigACon5.5–6.5), 4th to 5th (LigACon4-5), and 6th to 7.5th (LigACon6-7.5) repeat domains. All 7 recombinant Lig proteins were screened using a slot-shaped dot blot assay for the diagnosis of equine leptospirosis. Our results showed that LigACon4-7.5 is the best candidate diagnostic antigen in a slot-shaped dot blot assay. LigACon4-7.5 was further evaluated as an indirect enzyme-linked immunosorbent assay (ELISA) antigen for the detection of Leptospira antibodies in equine sera. This assay was evaluated with equine sera (n = 60) that were microscopic agglutination test (MAT) negative and sera (n = 220) that were MAT positive to the 5 serovars that most commonly cause equine leptospirosis. The indirect ELISA results showed that at a single serum dilution of 1:250, the sensitivity and specificity of ELISA were 80.0% and 87.2%, respectively, compared to those of MAT. In conclusion, an indirect ELISA was developed utilizing a recombinant LigA fragment comprising the 4th to 7.5th repeat domain (LigACon4-7.5) as a diagnostic antigen for equine leptospirosis. This ELISA was found to be sensitive and specific, and it yielded results that concurred with those of the standard MAT. PMID:23720368

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.

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

Evaluation of a Rapid Fecal PCR Test for Detection of Mycobacterium avium subsp. paratuberculosis in Dairy Cattle▿

PubMed Central

Wells, Scott J.; Collins, Michael T.; Faaberg, Kay S.; Wees, Carrie; Tavornpanich, Saraya; Petrini, Kristine R.; Collins, James E.; Cernicchiaro, Natalia; Whitlock, Robert H.

2006-01-01

A high-throughput TaqMan PCR assay for detection of bovine paratuberculosis was evaluated by using fecal samples from 1,808 dairy cattle in seven naturally infected herds and 347 dairy cattle in seven herds considered free of paratuberculosis. Fecal, blood, and milk samples were submitted to laboratories where the PCR-based assay, three different fecal culture procedures for Mycobacterium avium subsp. paratuberculosis (centrifugation, sedimentation, and the BACTEC filter concentration method), two serologic enzyme-linked immunosorbent assays (ELISAs), and one milk ELISA were performed. Results from testing of dairy cattle in herds free of M. avium subsp. paratuberculosis showed that the PCR assay's specificity was 99.7%. Twenty-three percent of the dairy cows that were fecal culture positive by at least one of the three methods were positive by the PCR assay. By Bayesian non-“gold standard” analysis methods, the TaqMan PCR assay had a higher specificity than the serum ELISAs (99.3%; 95% confidence interval [CI] = 98.6 to 99.7%) and a test sensitivity similar to that of the serum ELISAs (29%; 95% CI = 24 to 35%). By classical methods, the estimated relative sensitivity of the fecal PCR assay was 4% for light and moderate fecal shedders (compared to 12 to 13% for the ELISAs) and 76% for heavy fecal shedders (compared to 67% for the milk ELISA). The PCR assay has higher sensitivity for detection of heavy fecal shedders than the evaluated milk ELISA but lower sensitivity than a serum or milk ELISA for detection of light and moderate fecal shedders. This assay can be used as a quick test for detection of cattle with heavy fecal shedding, those cattle with the highest risk of transmitting infection to susceptible cattle. PMID:16928884

Validation of an enzyme-linked immunosorbent assay for the quantification of human IgG directed against the repeat region of the circumsporozoite protein of the parasite Plasmodium falciparum

PubMed Central

2012-01-01

Background Several pre-erythrocytic malaria vaccines based on the circumsporozoite protein (CSP) antigen of Plasmodium falciparum are in clinical development. Vaccine immunogenicity is commonly evaluated by the determination of anti-CSP antibody levels using IgG-based assays, but no standard assay is available to allow comparison of the different vaccines. Methods The validation of an anti-CSP repeat region enzyme-linked immunosorbent assay (ELISA) is described. This assay is based on the binding of serum antibodies to R32LR, a recombinant protein composed of the repeat region of P. falciparum CSP. In addition to the original recombinant R32LR, an easy to purify recombinant His-tagged R32LR protein has been constructed to be used as solid phase antigen in the assay. Also, hybridoma cell lines have been generated producing human anti-R32LR monoclonal antibodies to be used as a potential inexhaustible source of anti-CSP repeats standard, instead of a reference serum. Results The anti-CSP repeats ELISA was shown to be robust, specific and linear within the analytical range, and adequately fulfilled all validation criteria as defined in the ICH guidelines. Furthermore, the coefficient of variation for repeatability and intermediate precision did not exceed 23%. Non-interference was demonstrated for R32LR-binding sera, and the assay was shown to be stable over time. Conclusions This ELISA, specific for antibodies directed against the CSP repeat region, can be used as a standard assay for the determination of humoral immunogenicity in the development of any CSP-based P. falciparum malaria vaccine. PMID:23173602

Novel enzyme-linked immunosorbent assay for determination of fluvastatin in plasma at picogram level.

PubMed

Darwish, Ibrahim A; Al-Obaid, Abdul-Rahman M; Al-Malaq, Hamoud A

2009-11-15

For the first time, an enzyme-linked immunosorbent assay (ELISA) has been developed and validated for the determination of fluvastatin (FLV) in plasma samples at picogram level. The assay employed a polyclonal antibody that specifically recognizes FLV with high affinity, and FLV conjugate of bovine serum albumin (FLV-BSA) immobilized onto microplate wells as a solid-phase. The assay involved a competitive binding reaction between FLV, in plasma sample, and the immobilized FLV-BSA for the binding sites on a limited amount of the anti-FLV antibody. The bound anti-FLV antibody was quantified with horseradish peroxidase-labeled second anti-rabbit IgG antibody (HRP-IgG) and 3,3',5,5'-tetramethylbenzidine (TMB) as a substrate for the peroxidase enzyme. The concentration of FLV in the sample was quantified by its ability to inhibit the binding of the anti-FLV antibody to the immobilized FLV-BSA and subsequently the color intensity in the assay wells. The conditions for the proposed ELISA were investigated and the optimum conditions were employed in the determination of FLV in plasma samples. The assay limit of detection was 10 pg mL(-1) and the effective working range at relative standard deviations (RSD) of

Introducing MINA--The Molecularly Imprinted Nanoparticle Assay.

PubMed

Shutov, Roman V; Guerreiro, Antonio; Moczko, Ewa; de Vargas-Sansalvador, Isabel Perez; Chianella, Iva; Whitcombe, Michael J; Piletsky, Sergey A

2014-03-26

A new ELISA- (enzyme-linked immunosorbent assay)-like assay is demonstrated in which no elements of biological origin are used for molecular recognition or signaling. Composite imprinted nanoparticles that contain a catalytic core and which are synthesized by using a solid-phase approach can simultaneously act as recognition/signaling elements, and be used with minimal modifications to standard assay protocols. This assay provides a new route towards replacement of unstable biomolecules in immunoassays. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Adaptive Focused Acoustics (AFA) Improves the Performance of Microtiter Plate ELISAs.

PubMed

Green, David J; Rudd, Edwin A; Laugharn, James A

2014-08-01

We investigated the use of Adaptive Focused Acoustics (AFA) technology to improve the performance of microtiter plate enzyme-linked immunosorbent assays (ELISAs). Experiments were performed with commercially available AFA instrumentation and off-the-shelf 96-well microtiter plate sandwich ELISAs. AFA was applied over a range of acoustic energies, temperatures, and durations to the antigen/antibody binding step of an ELISA for measuring HIV-1 p24 in tissue culture samples. AFA-mediated antigen/antibody binding was enhanced up to 2-fold over passive binding at comparable temperatures and was superior or comparable at low temperature (8-10 °C) to passive binding at 37 °C. Lower nonspecific binding (NSB), lower inter- and intra-assay coefficients of variation (CVs), higher Z' factors, and lower limits of detection (LODs) were measured in AFA-mediated assays compared with conventional passive binding. In a more limited study, AFA enhancement of antigen/antibody binding and lower NSB was measured in an ELISA for measuring IGFBP-3 in human plasma. We conclude from this study that application of AFA to antigen/antibody binding steps in microtiter plate ELISAs can enhance key assay performance parameters, particularly Z' factors and LODs. These features render AFA-mediated binding assays potentially more useful in applications such as high-throughput screening and in vitro diagnostics than assays processed with conventional passive antigen/antibody binding steps. © 2014 Society for Laboratory Automation and Screening.

An rK28-Based Immunoenzymatic Assay for the Diagnosis of Canine Visceral Leishmaniasis in Latin America

PubMed Central

Lauricella, Marta Alicia; Maidana, Cristina Graciela; Frias, Victoria Fragueiro; Romagosa, Carlo M.; Negri, Vanesa; Benedetti, Ruben; Sinagra, Angel J.; Luna, Concepcion; Tartaglino, Lilian; Laucella, Susana; Reed, Steven G.; Riarte, Adelina R.

2016-01-01

Direct observation of Leishmania parasites in tissue aspirates has shown low sensitivity for the detection of canine visceral leishmaniasis (VL). Therefore in the last quarter century immunoenzymatic tests have been developed to improve diagnosis. The purpose of this study was to develop a fast recombinant K28 antigen, naked-eye qualitative enzyme-linked immunosorbent assay (VL Ql-ELISA) and a quantitative version (VL Qt-ELISA), and to display it in a kit format, whose cutoff value (0.156) was selected as the most adequate one to differentiate reactive from nonreactive samples. Considering 167 cases and 300 controls, sensitivity was 91% for both assays and specificity was 100% and 98.7% in Ql-ELISA and Qt-ELISA, respectively. Positive predictive values were 100% and 97.4% for Ql-ELISA and Qt-ELISA, respectively, and negative predictive values were 95.2% for both ELISAs. Reagent stability, reliability studies, including periodic repetitions and retest of samples, cutoff selection, and comparison of rK28 ELISAs with rK39 immunochromatographic test, were the international criteria that supported the quality in both kits. The performance of both ELISA kits in this work confirmed their validity and emphasized their usefulness for low-to-medium complexity laboratories. PMID:27162270

Practical colorimeter for direct measurement of microplates in enzyme immunoassay systems.

PubMed Central

Clem, T R; Yolken, R H

1978-01-01

A colorimeter capable of measuring results of enzyme-linked immunosorbent assay (ELISA) reactions directly in the wells of a microtiter plate is described. This colorimeter proved to be as accurate as a conventional spectrophotometer in assessing ELISA reactions, but had the advantage of not requiring transfer of the specimen to a separate chamber. With this colorimeter, 96 specimens can be read in approximately 5 min. A practical colorimeter such as this can make the use of ELISA tests more feasible for many laboratories. Images PMID:342540

Comparison of PanBio Dengue Duo Enzyme-Linked Immunosorbent Assay (ELISA) and MRL Dengue Fever Virus Immunoglobulin M Capture ELISA for Diagnosis of Dengue Virus Infections in Southeast Asia

PubMed Central

Cuzzubbo, Andrea J.; Vaughn, David W.; Nisalak, Ananda; Solomon, Tom; Kalayanarooj, Siripen; Aaskov, John; Dung, Nguyen Minh; Devine, Peter L.

1999-01-01

The performances of the MRL dengue fever virus immunoglobulin M (IgM) capture enzyme-linked immunosorbent assay (ELISA) and the PanBio Dengue Duo IgM capture and IgG capture ELISA were compared. Eighty sera from patients with dengue virus infections, 24 sera from patients with Japanese encephalitis (JE), and 78 sera from patients with nonflavivirus infections, such as malaria, typhoid, leptospirosis, and scrub typhus, were used. The MRL test showed superior sensitivity for dengue virus infections (94 versus 89%), while the PanBio test showed superior specificity for JE (79 versus 25%) and other infections (100 versus 91%). The PanBio ELISA showed better overall performance, as assessed by the sum of sensitivity and specificity (F value). When dengue virus and nonflavivirus infections were compared, F values of 189 and 185 were obtained for the PanBio and MRL tests, respectively, while when dengue virus infections and JE were compared, F values of 168 and 119 were obtained. The results obtained with individual sera in the PanBio and MRL IgM ELISAs showed good correlation, but this analysis revealed that the cutoff value of the MRL test was set well below that of the PanBio test. Comparing the sensitivity and specificity of the tests at different cutoff values (receiver-operator analysis) revealed that the MRL and PanBio IgM ELISAs performed similarly in distinguishing dengue virus from nonflavivirus infections, although the PanBio IgM ELISA showed significantly better distinction between dengue virus infections and JE. The implications of these findings for the laboratory diagnosis of dengue are discussed. PMID:10473522

Establishment of a sandwich enzyme-linked immunosorbent assay for specific detection of Bacillus thuringiensis (Bt) Cry1Ab toxin utilizing a monoclonal antibody produced with a novel hapten designed with molecular model.

PubMed

Dong, Sa; Zhang, Xiao; Liu, Yuan; Zhang, Cunzheng; Xie, Yajing; Zhong, Jianfeng; Xu, Chongxin; Liu, Xianjin

2017-03-01

Cry1Ab toxin is commonly expressed in genetically modified crops in order to control chewing pests. At present, the detection method with enzyme-linked immunosorbent assay (ELISA) based on monoclonal antibody cannot specifically detect Cry1Ab toxin for Cry1Ab's amino acid sequence and spatial structure are highly similar to Cry1Ac toxin. In this study, based on molecular design, a novel hapten polypeptide was synthesized and conjugated to keyhole limpet hemocyanin (KLH). Then, through animal immunization with this antigen, a monoclonal antibody named 2C12, showing high affinity to Cry1Ab and having no cross reaction with Cry1Ac, was produced. The equilibrium dissociation constant (K D ) value of Cry1Ab toxin with MAb 2C12 was 1.947 × 10 -8  M. Based on this specific monoclonal antibody, a sandwich enzyme-linked immunosorbent assay (DAS-ELISA) was developed for the specific determination of Cry1Ab toxin and the LOD and LOQ values were determined as 0.47 ± 0.11 and 2.43 ± 0.19 ng mL -1 , respectively. The average recoveries of Cry1Ab from spiked rice leaf and rice flour samples ranged from 75 to 115%, with coefficient of variation (CV) less than 8.6% within the quantitation range (2.5-100 ng mL -1 ), showing good accuracy for the quantitative detection of Cry1Ab toxin in agricultural samples. In conclusion, this study provides a new approach for the production of high specific antibody and the newly developed DAS-ELISA is a useful method for Cry1Ab monitoring in agriculture products. Graphical Abstract Establishment of a DAS-ELISA for the specific detecting of Bacillus thuringiensis (Bt) Cry1Ab toxin.

LDRD final report on microencapsulated immunoreagents for development of one-step ELISA

DOE Office of Scientific and Technical Information (OSTI.GOV)

Henderson, C.C.; Singh, A.K.

1997-08-01

Microencapsulation of biological macromolecules was investigated as a method for incorporating the necessary immunoreagents into an improved enzyme-linked immunosorbant assay (ELISA) package that would self-develop. This self-contained ELISA package would eliminate the need for a trained technician to perform multiple additions of immunoreagent to the assay. Microencapsulation by insolution drying was selected from the many available microencapsulation methods, and two satisfactory procedures for microencapsulation of proteins were established. The stability and potential for rapid release of protein from these microencapsulates was then evaluated. The results suggest that the chosen method for protein entrapment produces microcapsules with a considerable amount ofmore » protein in the walls making these particular microcapsules unsuitable for their intended use.« less

Evaluation of a broad-ranging and convenient enzyme-linked immunosorbent assay using the lysate of infected cells with five serotypes of Orientia tsutsugamushi, a causative agent of scrub typhus.

PubMed

Ogawa, Motohiko; Satoh, Masaaki; Saijo, Masayuki; Ando, Shuji

2017-01-05

Scrub typhus is a mite-borne rickettsiosis caused by infection of Orientia tsutsugamushi, which is endemic to several Asia-Pacific Rim countries, including Japan. Although micro-indirect immunofluorescent assay (micro-IFA) is the standard method for the serological diagnosis of scrub typhus, enzyme-linked immunosorbent assay (ELISA) is considered to be more objective, by providing digitized results as opposed to being subject to the judgment of the evaluator as in micro-IFA. Therefore, the aim of this study was to develop a broad-ranging ELISA using the five major prevalent serotypes of O. tsutsugamushi in Japan as the antigens. Furthermore, in contrast to previous studies that used purified microorganisms via ultracentrifugation, we directly used the infected cells, and evaluated the diagnostic accuracy of this simplified method to that of micro-IFA. Evaluation of paired patient sera against the five serotypes showed that the accuracy of ELISA relative to micro-IFA was 87.4 and 79.5% for immunoglobulin (Ig)M and IgG assays, respectively, at the optimized cut-off value. Further evaluation of patient sera against the expected serotype of the infecting strain showed that the accuracy of ELISA compared to micro-IFA increased to 100 and 97.4% in the IgM and IgG assays, respectively. This suggests that use of the five prevalent serotypes contributed to the increase of the accuracy of ELISA. When applying the criteria of serological diagnosis for paired sera samples to ELISA, all 19 patients were diagnosed as positive; a ≥4-fold elevation of the antibody titer was observed in 15 of 19 patients that were positive, and very high antibody titers were observed in both paired sera samples of the remaining four patients. In addition, all samples of healthy subjects and patients with other types of rickettsiosis were diagnosed as negative using these criteria. Our results suggest the excellent performance of the new broad-ranging and convenient ELISA, which appears to be applicable for the diagnosis of scrub typhus patients infected with the wide variety of prevalent strains in Japan. Furthermore, the ELISA is more objective than the micro-IFA, and can therefore provide more accurate diagnoses in Japan.

Development and evaluation of monoclonal antibodies for paxilline

USDA-ARS?s Scientific Manuscript database

Paxilline (PAX) is a tremorgenic mycotoxin that has been found in perennial ryegrass infected with Acremonium lolii. To facilitate screening for this toxin, four murine monoclonal antibodies (mAbs) were developed. In competitive indirect enzyme-linked immunosorbent assays (CI-ELISAs) the concentrati...

Development and analytical validation of an enzyme linked immunosorbent assay for the measurement of canine gastric lipase immunoreactivity in serum

PubMed Central

2004-01-01

Abstract The objective of this study was to develop and analytically validate an enzyme linked immunosorbent assay (ELISA) for measurement of canine gastric lipase immunoreactivity (cGLI). A sandwich ELISA was developed using canine gastric lipase (cGL) purified from canine stomachs and polyclonal antibodies directed against cGL, raised in rabbits and purified by affinity chromatography. The assay was validated by determination of sensitivity, working range, linearity, accuracy, precision, reproducibility, and the upper limit of the control range by determining the 97.5th percentile of serum cGLI concentration in 74 healthy canines. Sensitivity and working range in serum were 200 ng/L and 200 to 39 160 ng/L, respectively. Observed to expected ratios for dilutional parallelism for 3 serum samples and 3 dilutions ranged from 86.1% to 244.2% (mean ± standard deviation [s]; 125.4% ± 48.2%). Observed to expected ratios for spiking recoveries for 3 serum samples and 6 spiking concentrations ranged from 66.4% to 152.5% (mean ± s; 104.5% ± 22.9%). Intra-assay and interassay variabilities for 3 different serum samples were 25.5%, 9.4%, and 13.4% and 26.0%, 17.2%, and 14.4%, respectively. The upper limit of the control range for serum cGLI was 662 ng/L. We concluded that the ELISA for cGLI described here is highly sensitive and shows a wide working range. However, the validation characteristics for this assay are suboptimal and below values of approximately 2.000 ng/L the assay is more semiquantitative in nature. Despite its limitations, whether this assay is useful for the diagnosis of canine gastric disorders remains to be determined. PMID:15352539

Serologic Diagnosis of Lyme Borreliosis by Using Enzyme-Linked Immunosorbent Assays with Recombinant Antigens

PubMed Central

Magnarelli, Louis A.; Ijdo, Jacob W.; Padula, Steven J.; Flavell, Richard A.; Fikrig, Erol

2000-01-01

Class-specific enzyme-linked immunosorbent assays (ELISAs) with purified recombinant antigens of Borrelia burgdorferi sensu stricto and Western blot analyses with whole cells of this spirochete were used to test human sera to determine which antigens were diagnostically important. In analyses for immunoglobulin M (IgM) antibodies, 14 (82%) of 17 serum samples from persons who had erythema migrans reacted positively by an ELISA with one or more recombinant antigens. There was frequent antibody reactivity to protein 41-G (p41-G), outer surface protein C (OspC), and OspF antigens. In an ELISA for IgG antibodies, 13 (87%) of 15 serum samples had antibodies to recombinant antigens; reactivity to p22, p39, p41-G, OspC, and OspF antigens was frequent. By both ELISAs, serum specimens positive for OspB, OspE, and p37 were uncommon. Analyses of sera obtained from persons who were suspected of having human granulocytic ehrlichiosis (HGE) but who lacked antibodies to ehrlichiae revealed IgM antibodies to all recombinant antigens of B. burgdorferi except OspB and IgG antibodies to all antigens except OspE. Immunoblotting of sera from the study group of individuals suspected of having HGE reaffirmed antibody reactivity to multiple antigens of B. burgdorferi. There was minor cross-reactivity when sera from healthy subjects or persons who had syphilis, oral infections, or rheumatoid arthritis were tested by ELISAs with p37, p41-G, OspB, OspC, OspE, and OspF antigens. Although the results of class-specific ELISAs with recombinant antigens were comparable to those recorded for assays with whole-cell antigen and for individuals with confirmed clinical diagnoses of Lyme borreliosis, immunoblotting is still advised as an adjunct procedure, particularly when there are low antibody titers by an ELISA. PMID:10790090

Comparison of an IgG-Specific Enzyme-Linked Immunosorbent Assay Cutoff of 0.4 Versus 0.8 and 1.0 Optical Density Units for Heparin-Induced Thrombocytopenia.

PubMed

Ritchie, Brianne M; Connors, Jean M; Sylvester, Katelyn W

2017-04-01

Previous studies have demonstrated optimized diagnostic accuracy in utilizing higher antiheparin-platelet factor 4 (PF4) enzyme-linked immunosorbent assay (ELISA) optical density (OD) thresholds for diagnosing heparin-induced thrombocytopenia (HIT). We describe the incidence of positive serotonin release assay (SRA) results, as well as performance characteristics, for antiheparin-PF4 ELISA thresholds ≥0.4, ≥0.8, and ≥1.0 OD units in the diagnosis of HIT at our institution. Following institutional review board approval, we conducted a single-center retrospective chart review on adult inpatients with a differential diagnosis of HIT evaluated by both antiheparin-PF4 ELISA and SRA from 2012 to 2014. The major endpoints were to assess incidence of positive SRA results, sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and accuracy at antiheparin-PF4 ELISA values ≥0.4 OD units when compared to values ≥0.8 and ≥1.0 OD units. Clinical characteristics, including demographics, laboratory values, clinical and safety outcomes, length of stay, and mortality, were collected. A total of 140 patients with 140 antiheparin-PF4 ELISA and SRA values were evaluated, of which 23 patients were SRA positive (16.4%) and 117 patients were SRA negative (83.6%). We identified a sensitivity of 91.3% versus 82.6% and 73.9%, specificity of 61.5% versus 87.2% and 91.5%, PPV of 31.8% versus 55.9% and 63.0%, NPV of 97.3% versus 96.2% and 94.7%, and accuracy of 66.4% versus 86.4% and 88.6% at antiheparin-PF4 ELISA thresholds ≥0.4, ≥0.8, and ≥1.0 OD units, respectively. Our study suggests an increased antiheparin-PF4 ELISA threshold of 0.8 or 1.0 OD units enhances specificity, PPV, and accuracy while maintaining NPV with decreased sensitivity.

Protein microarray with horseradish peroxidase chemiluminescence for quantification of serum α-fetoprotein.

PubMed

Zhao, Yuanshun; Zhang, Yonghong; Lin, Dongdong; Li, Kang; Yin, Chengzeng; Liu, Xiuhong; Jin, Boxun; Sun, Libo; Liu, Jinhua; Zhang, Aiying; Li, Ning

2015-10-01

To develop and evaluate a protein microarray assay with horseradish peroxidase (HRP) chemiluminescence for quantification of α-fetoprotein (AFP) in serum from patients with hepatocellular carcinoma (HCC). A protein microarray assay for AFP was developed. Serum was collected from patients with HCC and healthy control subjects. AFP was quantified using protein microarray and enzyme-linked immunosorbent assay (ELISA). Serum AFP concentrations determined via protein microarray were positively correlated (r = 0.973) with those determined via ELISA in patients with HCC (n = 60) and healthy control subjects (n = 30). Protein microarray showed 80% sensitivity and 100% specificity for HCC diagnosis. ELISA had 83.3% sensitivity and 100% specificity. Protein microarray effectively distinguished between patients with HCC and healthy control subjects (area under ROC curve 0.974; 95% CI 0.000, 1.000). Protein microarray is a rapid, simple and low-cost alternative to ELISA for detecting AFP in human serum. © The Author(s) 2015.

Identification of Proteinaceous Binders in Ancient Tripitaka by the Use of an Enzyme-linked Immunosorbent Assay.

PubMed

Liu, Yi; Li, Yi; Chang, Runxing; Zheng, Hailing; Li, Menglu; Hu, Zhiwen; Zhou, Yang; Wang, Bing

2016-01-01

Proteinaceous materials, such as ovabumin and collagen, were commonly used as binding media, and as adhesives and protective coatings. However, the identification of ancient proteinaceous binders is a great challenge for archaeologists, due to their limited sample size, complex combinations of various ingredients and reduced availability of the binder during the process of protein degradation. In this paper, an enzyme-linked immunosorbent assay (ELISA) provides to be a particularly promising method for the detection of proteinaceous binding materials in ancient relics. The present work focused on the specific identification of proteins in archaeological binders, which was brushed on the Tripitaka. Two samples, the adhesion area (S1) and the ink area (S2), were tested by ELISA. The results showed that both S1 and S2 reacted positively when treated with an anti-collagen-I antibody. It proved the existence of proteinaceous binders in Ancient Tripitaka, and the percentage of collagen in S1 and S2 was 61.44 and 15.4%, respectively. Compared with other conventional techniques, ELISA has advantages of high specificity, sensitivity, rapidity and low cost, making it especially suitable for the protein detection in the archaeological field.

Development and validation of a sensitive monoclonal antibody-based indirect competitive enzyme-linked immunosorbent assay for the determination of the aflatoxin M1 levels in milk.

PubMed

Peng, Dapeng; Yang, Bijia; Pan, Yuanhu; Wang, Yulian; Chen, Dongmei; Liu, Zhenli; Yang, Wenxiang; Tao, Yanfei; Yuan, Zonghui

2016-04-01

A sensitive monoclonal antibody (mAb) against aflatoxin M1 (AFM1) was generated to quickly monitor the AFM1 residues in milk. Then, a mAb-based indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) was established that utilizes simple sample preparation and clean-up methods. The obtained 3D8 mAb, which is an IgG1 isotype mAb, displayed an IC50 value of 64.75 ng L(-1) for AFM1 and did not exhibit measurable cross-reactivity with other aflatoxins and antibiotics. The decision limit (CCα, α = 1%), detection capability (CCβ, β = 5%), and LOQ value for the AFM1 matrix calibration method were 24 ng L(-1), 27.5 ng L(-1), and 35 ng L(-1) in the milk matrices, respectively. The AFM1 recovery ranged from 85.3% to 107.6%. The CVs were less than 13.8%. A positive correlation (r > 0.99) was observed between the ic-ELISA and HPLC-MS/MS results. This ic-ELISA would be a useful tool for screening the AFM1 residues in milk. Copyright © 2016 Elsevier Ltd. All rights reserved.

Preparation of a monoclonal antibody against amantadine and rimantadine and development of an indirect competitive enzyme-linked immunosorbent assay for detecting the same in chicken muscle and liver.

PubMed

Peng, Dapeng; Wei, Wei; Pan, Yuanhu; Wang, Yulian; Chen, Dongmei; Liu, Zhenli; Wang, Xu; Dai, Menghong; Yuan, Zonghui

2017-01-30

A monoclonal antibody (mAb) was produced in order to monitor the illegal use of amantadine and rimantadine in animals. The produced mAb 2G3 exhibited an IC 50 value of 15.8μgL -1 for amantadine and exhibited cross-reactivity to both amantadine (100%) and rimantadine (70.6%). Standard curves ranged from 5 to 80μgL -1 for 2G3. The limits of detection of the developed indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) ranged from 5.0μgkg -1 to 5.4μgkg -1 in chicken muscle and liver. The recoveries were 81.3% to 98.1% with a coefficient of variation less than 15.7%. Good correlations were observed between the results of the ic-ELISA and liquid chromatography-mass spectrometry in the incurred tissues. These results suggest that ic-ELISA is a sensitive, accurate, and low-cost method that could be a useful tool for screening the residues of amantadine and rimantadine in chicken muscle and liver. Copyright © 2016 Elsevier B.V. All rights reserved.

Enzyme-linked immunosorbent assay with monoclonal and single-chain variable fragment antibodies selective to coplanar polychlorinated biphenyls.

PubMed

Inui, Hideyuki; Takeuchi, Tetsuya; Uesugi, Akari; Doi, Fumito; Takai, Mikio; Nishi, Kosuke; Miyake, Shiro; Ohkawa, Hideo

2012-02-22

Coplanar polychlorinated biphenyls (Co-PCBs) consisting of non-ortho and mono-ortho-chlorinated PCBs are dioxin-like compounds and cause wide contamination in the environment. To monitor Co-PCB residues, it was attempted to establish an enzyme-linked immunosorbent assay (ELISA) with monoclonal and recombinant antibodies selective to Co-PCBs. When 3,3',5,5'-tetrachlorobiphenoxybutyric acid (PCBH)-keyhole limpet hemocyanin conjugate was immunized into mice, two monoclonal antibodies, Mab-0217 and Mab-4444, were obtained. 3,3',5,5'-Tetrachlorobiphenyl (PCB80) was determined with an IC(50) value of 2.6 and 0.46 ng mL(-1) in ELISA based on Mab-0217 and Mab-4444, respectively. Mab-4444 cross-reacted with Co-PCB congeners, except for PCB77 and PCB81. Mab-0217 reacted with PCB80 and cross-reacted with PCB111. A single-chain variable fragment (scFv) antibody derived from Mab-4444 was produced in recombinant Escherichia coli cells. The scFv antibody showed nearly the same sensitivity toward PCBH as the parent monoclonal antibody in ELISA. These results clearly suggested that Mab-4444 and its scFv antibodies were suitable for monitoring the representative congeners of Co-PCBs.

Molecular detection of genotype II grass carp reovirus based on nucleic acid sequence-based amplification combined with enzyme-linked immunosorbent assay (NASBA-ELISA).

PubMed

Zeng, Weiwei; Yao, Wei; Wang, Yingying; Li, Yingying; Bermann, Sven M; Ren, Yan; Shi, Cunbin; Song, Xinjian; Huang, Qiwen; Zheng, Shuchen; Wang, Qing

2017-05-01

Grass carp reovirus (GCRV) is the causative agent of the grass carp hemorrhagic disease that has resulted in severe economic losses in the grass carp (Ctenopharyngodon idella) farming industry in China. Early diagnosis and vaccine administration are important priorities for GCRV control. In this study, a nucleic acid sequence-based amplification with enzyme-linked immunosorbent assay (NASBA-ELISA) was developed for to detect genotype II GCRV (GCRV- II). Primers specifically targeting viral RNA genome segment 6 were utilized for amplification in an isothermal digoxigenin-labeling NASBA process, resulting in DIG-labeled RNA amplicons. The amplicons were hybridized to specific biotinylated DNA probes and the products were detected colorimetrically using horseradish peroxidase and a microplate reader. The new method is able to detect GCRV at 14 copies/μL within 5h and had a diagnostic sensitivity and a specificity of 100% when GCRV-II and non-target virus were tested. This NASBA-ELISA was evaluated using a panel of clinical samples (n=103) to demonstrate that it is a rapid, effective and sensitive method for GCRV detection in grass carp aquaculture. Copyright © 2017 Elsevier B.V. All rights reserved.

A digital enzyme-linked immunosorbent assay for ultrasensitive measurement of amyloid-β 1-42 peptide in human plasma with utility for studies of Alzheimer's disease therapeutics.

PubMed

Song, Linan; Lachno, D Richard; Hanlon, David; Shepro, Adam; Jeromin, Andreas; Gemani, Dipika; Talbot, Jayne A; Racke, Margaret M; Dage, Jeffrey L; Dean, Robert A

2016-12-15

Amyloid-β 1-42 peptide (Aβ 1-42 ) is associated with plaque formation in the brain of patients with Alzheimer's disease (AD). Pharmacodynamic studies of AD therapeutics that lower the concentrations of Aβ 1-42 in peripheral blood require highly sensitive assays for its measurement. A digital enzyme-linked immunosorbent assay (ELISA) using single molecule array (Simoa) technology has been developed that provides improved sensitivity compared with conventional ELISA methods using the same antibody reagents. A sensitive digital ELISA for measurement of Aβ 1-42 using antibodies 3D6 and 21F12 was developed. Assay performance was evaluated by repeated testing of pooled human plasma and buffer diluent quality control samples to determine relative accuracy, intra- and inter-assay precision, limit of detection (LOD), lower limit of quantification (LLOQ), dilutional linearity, and spike recovery. The optimized assay was used to quantify Aβ 1-42 in clinical samples from patients treated with the β-site amyloid precursor protein cleaving enzyme 1 inhibitor LY2886721. The prototype assay measured Aβ 1-42 with an LOD of 0.3 pg/ml and an LLOQ of 2.8 pg/ml in plasma, calibrated using an Aβ 1-42 peptide standard from Fujirebio. Assay precision was acceptable with intra- and inter-assay coefficients of variation both being ≤10%. Dilutional linearity was demonstrated in sample diluent and immunodepleted human plasma. Analyte spike recovery ranged from 51% to 93% with a mean of 80%. This assay was able to quantify Aβ 1-42 in all of the 84 clinical samples tested. A rapid reduction in levels of Aβ 1-42 was detected within 1 h after drug treatment, and a dose-dependent decrease of Aβ 1-42 levels was also observed over the time course of sample collection. This digital ELISA has potential utility in clinical applications for quantification of Aβ 1-42 in plasma where high sensitivity and precision are required.

Comparison of six commercial tick-borne encephalitis IgM and IgG ELISA kits and the molecular characterization of their antigenic design.

PubMed

Velay, Aurélie; Solis, Morgane; Barth, Heidi; Sohn, Véronique; Moncollin, Anne; Neeb, Amandine; Wendling, Marie-Josée; Fafi-Kremer, Samira

2018-04-01

Tick-borne encephalitis virus (TBEV) diagnosis is mainly based on the detection of viral-specific antibodies in serum. Several commercial assays are available, but published data on their performance remain unclear. We assessed six IgM and six IgG commercial enzyme-linked immunosorbent assay (ELISA) kits (ELISA-1 through ELISA-6) using 94 samples, including precharacterized TBEV-positive samples (n=50) and -negative samples (n=44). The six manufacturers showed satisfactory sensitivity and specificity and high overall agreement for both IgM and IgG. Three manufacturers showed better reproducibility and were the most sensitive (100%) and specific (95.5-98.1%) for both IgM and IgG. Two of them were also in agreement with the clinical interpretation in more than 90% of the cases. All the assays use inactivated virus as antigen, with strains showing approximately 94% homology at the amino acid level. The antigenic format of the assays was discussed to further improve this TBEV diagnostic tool. Copyright © 2017 Elsevier Inc. All rights reserved.

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

PubMed

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

1997-01-01

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

Pistachio (Pistacia vera L.) Detection and Quantification Using a Murine Monoclonal Antibody-Based Direct Sandwich Enzyme-Linked Immunosorbent Assay.

PubMed

Liu, Changqi; Chhabra, Guneet S; Sathe, Shridhar K

2015-10-21

A commercially available direct sandwich enzyme-linked immunosorbent assay (ELISA) (BioFront Technologies, Tallahassee, FL, USA) using murine anti-pistachio monoclonal antibodies (mAbs) as capture and detection antibodies was evaluated. The assay was sensitive (limit of detection = 0.09 ± 0.02 ppm full fat pistachio, linear detection range = 0.5-36 ppm, 50% maximum signal concentration = 7.9 ± 0.7 ppm), reproducible (intra- and inter-assay variability < 24% CV), and rapid (post-extraction testing time ∼ 1.5 h). The target antigen was stable and detectable in whole pistachio seeds subjected to autoclaving (121 °C, 15 psi, 15, 30 min), blanching (100 °C, 5, 10 min), frying (191 °C, 1 min), microwaving (500, 1000 W, 3 min), and dry roasting (140 °C, 30 min; 168 °C, 12 min). No cross-reactivity was observed in 156 food matrices, each tested at 100,000 ppm, suggesting the ELISA to be pistachio specific. The pistachio recovery ranges for spiked (10 ppm) and incurred (10-50000 ppm) food matrices were 93.1-125.6% and 35.7-112.2%, respectively. The assay did not register any false-positive or -negative results among the tested commercial and laboratory prepared samples.

Development of an ELISA to detect circulating anti-asparaginase antibodies in dogs with lymphoid neoplasia treated with Escherichia coli l-asparaginase.

PubMed

Kidd, J A; Ross, P; Buntzman, A S; Hess, P R

2015-06-01

Resistance to Escherichia coli l-asparaginase in canine lymphoma occurs frequently with repeated administration, a phenomenon often attributed, without substantiation, to the induction of neutralizing antibodies. To test the hypothesis that treated dogs develop antibodies against the drug, we created an enzyme-linked immunosorbent assay (ELISA) to measure plasma anti-asparaginase immunoglobulin G responses. Using samples from dogs that had received multiple doses, specific reactivity against l-asparaginase was demonstrated, while naïve patients' samples were negative. The optimized ELISA appeared sensitive, with endpoint titers >1 600 000 in positive control dogs. Intra- and inter-assay coefficients of variation were 3.6 and 14.5%. The assay was supported by the observation that ELISA-positive plasma could immunoprecipitate asparaginase activity. When clinical patients were evaluated, 3/10 dogs developed titers after a single injection; with repeated administration, 4/7 dogs were positive. l-asparaginase antibodies showed reduced binding to the PEGylated drug formulation. The ELISA should prove useful in investigating the potential correlation of antibody responses with resistance. © 2012 Blackwell Publishing Ltd.

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. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

Evaluation of Two Enzyme-Linked Immunosorbent Assay Kits for Chikungunya Virus IgM Using Samples from Deceased Organ and Tissue Donors

PubMed Central

Altrich, Michelle L.; Nowicki, Marek J.

2016-01-01

The identification of nearly 3,500 cases of chikungunya virus (CHIKV) infection in U.S. residents returning in 2014 and 2015 from areas in which it is endemic has raised concerns within the transplant community that, should recently infected individuals become organ and/or tissue donors, CHIKV would be transmitted to transplant recipients. Thus, tests designed to detect recent CHIKV infection among U.S. organ and tissue donors may become necessary in the future. Accordingly, we evaluated 2 enzyme-linked immunosorbent assays (ELISAs) for CHIKV IgM readily available in the United States using 1,000 deidentified serum or plasma specimens collected from donors between November 2014 and March 2015. The Euroimmun indirect ELISA identified 38 reactive specimens; however, all 38 were negative for CHIKV IgG and IgM in immunofluorescence assays (IFAs) conducted at a reference laboratory and, thus, were falsely reactive in the Euroimmun CHIKV IgM assay. The InBios IgM-capture ELISA identified 26 reactive samples, and one was still reactive (index ≥ 1.00) when retested using the InBios kit with a background subtraction modification to identify false reactivity. This reactive specimen was CHIKV IgM negative but IgG positive by IFAs at two reference laboratories; plaque reduction neutralization testing (PRNT) demonstrated CHIKV-specific reactivity. The IgG and PRNT findings strongly suggest that the InBios CHIKV IgM-reactive result represents true reactivity, even though the IgM IFA result was negative. If testing organ/tissue donors for CHIKV IgM becomes necessary, the limitations of the currently available CHIKV IgM ELISAs and options for their optimization must be understood to avoid organ/tissue wastage due to falsely reactive results. PMID:27535838

An enzyme-linked immunosorbant assay using monoclonal antibody against bacoside A₃ for determination of jujubogenin glycosides in Bacopa monnieri (L.) Wettst.

PubMed

Tothiam, Charinrat; Phrompittayarat, Watoo; Putalun, Waraporn; Tanaka, Hiroyuki; Sakamoto, Seiichi; Khan, Ikhlas A; Ingkaninan, Kornkanok

2011-01-01

In Ayurvedic medicines, Bacopa monnieri (L.) Wettst. (brahmi) is known as a medicinal plant used for memory enhancement. Its active compounds are classified as pseudojujubogenin and jujubogenin glycosides. Owing to the lack of chromophore in the saponin glycoside structures, HPLC-UV-vis gives low sensitivity for determination of such compounds. In the case of the detection of small amounts of saponin glycosides, immunological assay could be a suitable method. To develop and validate a sensitive enzyme-linked immunosorbant assay (ELISA) using monoclonal antibody (MAb) against bacoside A₃, the major jujubogenin glycoside found in brahmi. An immunogen was prepared by conjugating bacoside A₃ with a bovine serum albumin (BSA). To determine its immunogenicity, the ratio of hapten in bacoside A₃-BSA conjugate was determined by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF-MS). After immunisation in mice, hybridomas secreting MAbs against bacoside A₃ were produced by fusing the immunised splenocytes with SP2/0- Ag14 myeloma cells. The antibody was raised specifically against jujubogenin glycosides. The ELISA using anti-bacoside A₃ MAb was developed. Bacoside A₃ in the range of 3.05-97.70 ng mL⁻¹ could be detected by ELISA using anti-bacoside A₃ MAb. The assay showed a detection limit of 0.48 ng mL⁻¹ (0.517 nm). The validation study showed that the method was precise, accurate and sensitive. Interestingly, the MAb showed cross-reactivity with the other jujubogenin glycosides, bacopaside X and IV. However, it did not show cross-reactivity with any of pseudojujubogenin glycosides. The study demonstrated that ELISA using anti-bacoside A₃ MAb can be used for determination of total jujubogenin glycosides in brahmi. Copyright © 2011 John Wiley & Sons, Ltd.

Serodiagnosis of infectious mononucleosis by using recombinant Epstein-Barr virus antigens and enzyme-linked immunosorbent assay technology.

PubMed Central

Gorgievski-Hrisoho, M; Hinderer, W; Nebel-Schickel, H; Horn, J; Vornhagen, R; Sonneborn, H H; Wolf, H; Siegl, G

1990-01-01

Four recombinant, diagnostically useful Epstein-Barr virus (EBV) proteins representative of the viral capsid antigen (p150), diffuse early antigen (p54), the major DNA-binding protein (p138), and the EBV nuclear antigen (p72) (W. Hinderer, H. Nebel-Schickel, H.H. Sonneborn, M. Motz, R. Kühbeck, and H. Wolf, J. Exp. Clin. Cancer Res. 7[Suppl.]:132, 1988) were used to set up individual enzyme-linked immunosorbent assays (ELISAs) for the qualitative and quantitative detection of immunoglobulin M (IgM) and IgG antibodies. In direct comparison with results obtained by standard immunofluorescence or immunoperoxidase assays, it was then shown that the recombinant EBV ELISAs provide the means for specific and sensitive serodiagnosis of infectious mononucleosis (IM) caused by EBV. The most useful markers in sera from such patients proved to be IgM antibodies against p54, p138, and p150. Additional positive markers for recent or ongoing IM apparently were IgG antibodies against p54 and p138. In contrast, anti-p72 IgG had a high preference for sera from healthy blood donors and, therefore, can be considered indicative of past exposure to the virus. Altogether, the individual ELISAs proved to be as specific and at least as sensitive for the diagnosis of IM as the currently available standard techniques are. Moreover, our findings suggest that, by combining individual test antigens, a workable ELISA system consisting of three assays (IgM against p54, p138, and p150; IgG against p54 and p138; and IgG against p72) can be established for the standardized rapid diagnosis of acute EBV infections. PMID:2172287

Enhancement in the sensitivity of microfluidic enzyme-linked immunosorbent assays through analyte preconcentration.

PubMed

Yanagisawa, Naoki; Dutta, Debashis

2012-08-21

In this Article, we describe a microfluidic enzyme-linked immunosorbent assay (ELISA) method whose sensitivity can be substantially enhanced through preconcentration of the target analyte around a semipermeable membrane. The reported preconcentration has been accomplished in our current work via electrokinetic means allowing a significant increase in the amount of captured analyte relative to nonspecific binding in the trapping/detection zone. Upon introduction of an enzyme substrate into this region, the rate of generation of the ELISA reaction product (resorufin) was observed to increase by over a factor of 200 for the sample and 2 for the corresponding blank compared to similar assays without analyte trapping. Interestingly, in spite of nonuniformities in the amount of captured analyte along the surface of our analysis channel, the measured fluorescence signal in the preconcentration zone increased linearly with time over an enzyme reaction period of 30 min and at a rate that was proportional to the analyte concentration in the bulk sample. In our current study, the reported technique has been shown to reduce the smallest detectable concentration of the tumor marker CA 19-9 and Blue Tongue Viral antibody by over 2 orders of magnitude compared to immunoassays without analyte preconcentration. When compared to microwell based ELISAs, the reported microfluidic approach not only yielded a similar improvement in the smallest detectable analyte concentration but also reduced the sample consumption in the assay by a factor of 20 (5 μL versus 100 μL).

Human Exhaled Breath Condensate (EBC) Media: Implementation of Automated Quanterix SIMOA Immunochemistry Instrumentation

EPA Science Inventory

Immunochemistry is an important clinical tool for observing biological pathways leading to disease. Standard enzyme-linked immunosorbent assays (ELISA) for cytokines are typically labor intensive and lack sensitivity at sub pg/ml concentrations. Here we report on emerging tec...

Bayesian Validation of the Indirect Immunofluorescence Assay and Its Superiority to the Enzyme-Linked Immunosorbent Assay and the Complement Fixation Test for Detecting Antibodies against Coxiella burnetii in Goat Serum.

PubMed

Muleme, Michael; Stenos, John; Vincent, Gemma; Campbell, Angus; Graves, Stephen; Warner, Simone; Devlin, Joanne M; Nguyen, Chelsea; Stevenson, Mark A; Wilks, Colin R; Firestone, Simon M

2016-06-01

Although many studies have reported the indirect immunofluorescence assay (IFA) to be more sensitive in detection of antibodies to Coxiella burnetii than the complement fixation test (CFT), the diagnostic sensitivity (DSe) and diagnostic specificity (DSp) of the assay have not been previously established for use in ruminants. This study aimed to validate the IFA by describing the optimization, selection of cutoff titers, repeatability, and reliability as well as the DSe and DSp of the assay. Bayesian latent class analysis was used to estimate diagnostic specifications in comparison with the CFT and the enzyme-linked immunosorbent assay (ELISA). The optimal cutoff dilution for screening for IgG and IgM antibodies in goat serum using the IFA was estimated to be 1:160. The IFA had good repeatability (>96.9% for IgG, >78.0% for IgM), and there was almost perfect agreement (Cohen's kappa > 0.80 for IgG) between the readings reported by two technicians for samples tested for IgG antibodies. The IFA had a higher DSe (94.8%; 95% confidence interval [CI], 80.3, 99.6) for the detection of IgG antibodies against C. burnetii than the ELISA (70.1%; 95% CI, 52.7, 91.0) and the CFT (29.8%; 95% CI, 17.0, 44.8). All three tests were highly specific for goat IgG antibodies. The IFA also had a higher DSe (88.8%; 95% CI, 58.2, 99.5) for detection of IgM antibodies than the ELISA (71.7%; 95% CI, 46.3, 92.8). These results underscore the better suitability of the IFA than of the CFT and ELISA for detection of IgG and IgM antibodies in goat serum and possibly in serum from other ruminants. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Bayesian Validation of the Indirect Immunofluorescence Assay and Its Superiority to the Enzyme-Linked Immunosorbent Assay and the Complement Fixation Test for Detecting Antibodies against Coxiella burnetii in Goat Serum

PubMed Central

Stenos, John; Vincent, Gemma; Campbell, Angus; Graves, Stephen; Warner, Simone; Devlin, Joanne M.; Nguyen, Chelsea; Stevenson, Mark A.; Wilks, Colin R.; Firestone, Simon M.

2016-01-01

Although many studies have reported the indirect immunofluorescence assay (IFA) to be more sensitive in detection of antibodies to Coxiella burnetii than the complement fixation test (CFT), the diagnostic sensitivity (DSe) and diagnostic specificity (DSp) of the assay have not been previously established for use in ruminants. This study aimed to validate the IFA by describing the optimization, selection of cutoff titers, repeatability, and reliability as well as the DSe and DSp of the assay. Bayesian latent class analysis was used to estimate diagnostic specifications in comparison with the CFT and the enzyme-linked immunosorbent assay (ELISA). The optimal cutoff dilution for screening for IgG and IgM antibodies in goat serum using the IFA was estimated to be 1:160. The IFA had good repeatability (>96.9% for IgG, >78.0% for IgM), and there was almost perfect agreement (Cohen's kappa > 0.80 for IgG) between the readings reported by two technicians for samples tested for IgG antibodies. The IFA had a higher DSe (94.8%; 95% confidence interval [CI], 80.3, 99.6) for the detection of IgG antibodies against C. burnetii than the ELISA (70.1%; 95% CI, 52.7, 91.0) and the CFT (29.8%; 95% CI, 17.0, 44.8). All three tests were highly specific for goat IgG antibodies. The IFA also had a higher DSe (88.8%; 95% CI, 58.2, 99.5) for detection of IgM antibodies than the ELISA (71.7%; 95% CI, 46.3, 92.8). These results underscore the better suitability of the IFA than of the CFT and ELISA for detection of IgG and IgM antibodies in goat serum and possibly in serum from other ruminants. PMID:27122484

Seroprevalence study of Equine rhinitis B virus (ERBV) in Australian weanling horses using serotype-specific ERBV enzyme-linked immunosorbent assays.

PubMed

Horsington, Jacquelyn; Hartley, Carol A; Gilkerson, James R

2013-09-01

Respiratory infections are a major burden in the performance horse industry. Equine rhinitis B virus (ERBV) has been isolated from horses displaying clinical respiratory disease, and ERBV-neutralizing antibodies have been detected in 50-80% of horses in reported surveys. Current ERBV isolation and detection methods may underestimate the number of ERBV-positive animals and do not identify multiple serotype infections. The aim of the current study was to develop a serotyping ERBV antibody-detection enzyme-linked immunosorbent assay (ELISA) and examine the seroprevalence of ERBV in a group of Australian weanling horses. ELISAs with high sensitivity and specificity were developed. The seroprevalence of ERBV in the weanling horses was high (74-86%); ERBV-3 antibodies were most prevalent (58-62%) and ERBV-2 antibodies were least prevalent (10-16%). Many horses were seropositive to 2 or more serotypes. All 3 serotypes of ERBV were detected, and concurrent positivity to multiple serotypes was common.

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

PubMed

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

2015-08-01

Basement membrane collagens or derived fragments are measured in biological fluids such as blood and urine of patients and appear to be useful for diagnosis, prognostication, or treatment monitoring as proposed for endostatin, a fragment of collagen XVIII, or tumstatin, a fragment of collagen IV. Tetrastatin, the NC1 alpha 4 collagen IV domain, was previously reported to inhibit tumor growth and angiogenesis. The aim of this study was to develop and validate a method to measure tetrastatin concentrations in human fluids. We developed a competitive enzyme-linked immunosorbent assay (ELISA). It allowed measuring tetrastatin levels in human serum, bronchial aspiration and bronchoalveolar lavage fluids, and lung tissue extracts. The tetrastatin level was significantly higher in tumor tissues than in healthy lung tissues. Tetrastatin competitive ELISA could be useful to quantify tetrastatin in tissues and biological fluids for the diagnosis or prognostication of diseases in which basement membrane metabolism may be altered, especially tumor progression. Copyright © 2015 Elsevier Inc. All rights reserved.

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

PubMed

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

2013-08-01

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

Evaluation of a newly designed sandwich enzyme linked immunosorbent assay for the detection of hydatid antigen in serum, urine and cyst fluid for diagnosis of cystic echinococcosis.

PubMed

Chaya, Dr; Parija, Subhash Chandra

2013-07-01

Cystic echinococcosis (CE) is a zoonotic disease of humans with variable clinical manifestations. Imaging and immunological methods are currently the mainstay of diagnosis of this disease. Although the immunological tests for detection of anti-echinococcal antibodies have several disadvantages, they are widely being used. Antigen is far more superior than antibody detection test as they can provide a specific parasitic diagnosis. A sandwich enzyme linked immunosorbent assay (ELISA) was designed using antibodies to 24 kDa urinary hydatid antigen for the detection of hydatid antigens in urine, serum and cyst fluid specimens. The performance of this novel test was compared with that of other hydatid antibody detection ELISA and enzyme immune transfer blot (EITB) using radiological and surgical confirmation as the gold standard. The antigen detection ELISA showed 100% sensitivity and specificity when tested with cyst fluid. On testing urine and serum, the antigen detection ELISA was found to be more specific than antibody detection ELISA. EITB was found to be the most sensitive and specific test. ELISA using polyclonal antibodies against 24 kDa urinary hydatid protein was moderately sensitive to detect hydatid antigen in serum and urine. Hence polyclonal antibodies to 24 kDa urinary hydatid antigen can be used as an alternative source of antibody to detect hydatid antigen in serum, urine and cyst fluid. In the present study, EITB was found to be highly specific test for detection of hydatid antibodiesin serum. 24 kDa protein was found to be specific and of diagnostic value in CE.

Recombinant Nucleoprotein-Based Enzyme-Linked Immunosorbent Assay for Detection of Immunoglobulin G Antibodies to Crimean-Congo Hemorrhagic Fever Virus

PubMed Central

Saijo, Masayuki; Qing, Tang; Niikura, Masahiro; Maeda, Akihiko; Ikegami, Tetsuro; Prehaud, Christophe; Kurane, Ichiro; Morikawa, Shigeru

2002-01-01

The full-length nucleoprotein of Crimean-Congo hemorrhagic fever virus (CCHFV; 482 amino acid residues) was expressed as a His-tagged recombinant protein (His-CCHFV rNP) in the baculovirus system. The His-CCHFV rNP was efficiently expressed in insect cells and purified by Ni2+ column chromatography. Using this substrate, an immunoglobulin G (IgG) enzyme-linked immunosorbent assay (ELISA) was developed. We evaluated the sensitivity and specificity of the IgG ELISA, using serum samples previously determined to be antibody positive or negative by immunofluorescence tests on CCHFV-infected Vero E6 cells. We found very good correlation between the two tests: 87% for the positive sera (13 of 15) and 99% for the negative sera (107 of 108). These results indicate that the new IgG ELISA using His-CCHFV rNP has high sensitivity and specificity for detecting CCHFV antibodies. The CCHF patients' sera with high titers reacted only with the NP fragment containing amino acid residues between 201 and 306 in Western blotting. It is known that amino acid homologies are high in this region among various isolates. Thus, it is expected that this ELISA can detect antibodies not only for Chinese strains of CCHFV but also for other strains circulating in the world. These results suggest that the IgG ELISA system developed with the recombinant CCHFV NP is a valuable tool for diagnosis and epidemiological investigations of CCHFV infections. PMID:11980926

Infectious hematopoietic necrosis (IHN) and viral hemorrhagic septicemia (VHS): Detection of the trout antibodies to the causative viruses by means of plaque neutralization, immunofluorescence, and enzyme-linked immunosorbent assay

USGS Publications Warehouse

Vestergard Jorgensen, P. E.; Olesen, N.J.; Lorenzen, N.; Winton, J.R.; Ristow, S.S.

1991-01-01

Sera collected from cultured rainbow trout Oncorhynchus mykiss surviving outbreaks of infectious hematopoietic necrosis (IHN) or viral hemorrhagic septicemia (VHS) were examined for the presence of antibodies to both of the causative viruses, infectious hematopoietic necrosis virus (IHNV) and Egtved virus (viral hemorrhagic septicemia virus: VHSV). Sera were screened with three serological tests: 50% plaque neutralization test (PNT), immunofluorescence (IF), and enzyme-linked immunosorbent assay (ELISA). In sera from 20 rainbow trout surviving IHN, antibodies to IHNV were detected in 9 fish by PNT, in 12 fish by IF, and in 9 fish by ELISA. In these sera, antibodies cross-reacting with VHSV were rare (detected in 0 fish by PNT, in 1 by IF, and in 1 by ELISA). In sera from 20 rainbow trout surviving VHS, antibodies to VHSV were detected in 9 fish by PNT, in 16 fish by IF, and in 18 fish by ELISA. A considerable percentage of the VHS-survivor sera contained antibodies that cross-reacted with IHNV, as detected by ELISA (16 fish) and 1F (7 fish) but not by PNT (0 fish). The three serological tests appear to be useful tools for IHNV and VHSV epidemiology; however, the presence of cross-reacting antibodies in some sera suggests caution when farms require specific pathogen-free certification for one of the viruses in the presence of the other.

Validation of the World Health Organization Enzyme-Linked Immunosorbent Assay for the Quantitation of Immunoglobulin G Serotype-Specific Anti-Pneumococcal Antibodies in Human Serum

PubMed Central

2017-01-01

The World Health Organization (WHO) enzyme-linked immunosorbent assay (ELISA) guideline is currently accepted as the gold standard for the evaluation of immunoglobulin G (IgG) antibodies specific to pneumococcal capsular polysaccharide. We conducted validation of the WHO ELISA for 7 pneumococcal serotypes (4, 6B, 9V, 14, 18C, 19F, and 23F) by evaluating its specificity, precision (reproducibility and intermediate precision), accuracy, spiking recovery test, lower limit of quantification (LLOQ), and stability at the Ewha Center for Vaccine Evaluation and Study, Seoul, Korea. We found that the specificity, reproducibility, and intermediate precision were within acceptance ranges (reproducibility, coefficient of variability [CV] ≤ 15%; intermediate precision, CV ≤ 20%) for all serotypes. Comparisons between the provisional assignments of calibration sera and the results from this laboratory showed a high correlation > 94% for all 7 serotypes, supporting the accuracy of the ELISA. The spiking recovery test also fell within an acceptable range. The quantification limit, calculated using the LLOQ, for each of the serotypes was 0.05–0.093 μg/mL. The freeze-thaw stability and the short-term temperature stability were also within an acceptable range. In conclusion, we showed good performance using the standardized WHO ELISA for the evaluation of serotype-specific anti-pneumococcal IgG antibodies; the WHO ELISA can evaluate the immune response against pneumococcal vaccines with consistency and accuracy. PMID:28875600

Validation of the World Health Organization Enzyme-Linked Immunosorbent Assay for the Quantitation of Immunoglobulin G Serotype-Specific Anti-Pneumococcal Antibodies in Human Serum.

PubMed

Lee, Hyunju; Lim, Soo Young; Kim, Kyung Hyo

2017-10-01

The World Health Organization (WHO) enzyme-linked immunosorbent assay (ELISA) guideline is currently accepted as the gold standard for the evaluation of immunoglobulin G (IgG) antibodies specific to pneumococcal capsular polysaccharide. We conducted validation of the WHO ELISA for 7 pneumococcal serotypes (4, 6B, 9V, 14, 18C, 19F, and 23F) by evaluating its specificity, precision (reproducibility and intermediate precision), accuracy, spiking recovery test, lower limit of quantification (LLOQ), and stability at the Ewha Center for Vaccine Evaluation and Study, Seoul, Korea. We found that the specificity, reproducibility, and intermediate precision were within acceptance ranges (reproducibility, coefficient of variability [CV] ≤ 15%; intermediate precision, CV ≤ 20%) for all serotypes. Comparisons between the provisional assignments of calibration sera and the results from this laboratory showed a high correlation > 94% for all 7 serotypes, supporting the accuracy of the ELISA. The spiking recovery test also fell within an acceptable range. The quantification limit, calculated using the LLOQ, for each of the serotypes was 0.05-0.093 μg/mL. The freeze-thaw stability and the short-term temperature stability were also within an acceptable range. In conclusion, we showed good performance using the standardized WHO ELISA for the evaluation of serotype-specific anti-pneumococcal IgG antibodies; the WHO ELISA can evaluate the immune response against pneumococcal vaccines with consistency and accuracy. © 2017 The Korean Academy of Medical Sciences.

Detection of Human Epididymis Protein 4 (HE4) in Human Serum Samples Using a Specific Monoclonal Antibody-Based Sandwich Enzyme-Linked Immunosorbent Assay (ELISA).

PubMed

Zhou, Lijun; Lv, Zhiqiang; Shao, Jing; Xu, Ying; Luo, Xiaohong; Zhang, Yuming; Hu, Yang; Zhang, Wenji; Luo, Shuhong; Fang, Jianmin; Wang, Ying; Duan, Chaohui; Huang, Ruopan

2016-09-01

The human epididymis protein 4 (HE4) may have high specificity in the detection of malignant diseases, making the development of an immunoassay for HE4 essential. In our study, a fusion gene was constructed encoded with the HE4 protein. This protein was then produced in the bacterial cells (Escherichia coli) and used to immunize mice in order to eventually generate hybridomas specific to HE4. The hybridoma supernatants were then screened, and four positive anti-HE4 cell lines were selected. These cell lines produce monoclonal antibodies against HE4 epitopes, as demonstrated in the Western blot as well as by direct enzyme-linked immunosorbent assay (ELISA). Using the developed antibodies, we successfully identified several good antibody pairs from the hybridomas, which allowed for the development of a sandwich ELISA to measure HE4 levels. By using the HE4 ELISA, we measured HE4 levels of 60 clinical human serum samples. Compared with the Food and Drug Administration (FDA) approved kit (Roche), our results showed a strong positive correlation to those of the FDA-approved kit. In summary, highly sensitive antibody pairs were screened against HE4, and a sandwich ELISA was developed as an accurate analytical tool for the detection of HE4 in human serum, which could be especially valuable for diagnosing ovarian carcinomas. © 2015 Wiley Periodicals, Inc.

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.

Multi-analyte validation in heterogeneous solution by ELISA.

PubMed

Lakshmipriya, Thangavel; Gopinath, Subash C B; Hashim, Uda; Murugaiyah, Vikneswaran

2017-12-01

Enzyme Linked Immunosorbent Assay (ELISA) is a standard assay that has been used widely to validate the presence of analyte in the solution. With the advancement of ELISA, different strategies have shown and became a suitable immunoassay for a wide range of analytes. Herein, we attempted to provide additional evidence with ELISA, to show its suitability for multi-analyte detection. To demonstrate, three clinically relevant targets have been chosen, which include 16kDa protein from Mycobacterium tuberculosis, human blood clotting Factor IXa and a tumour marker Squamous Cell Carcinoma antigen. Indeed, we adapted the routine steps from the conventional ELISA to validate the occurrence of analytes both in homogeneous and heterogeneous solutions. With the homogeneous and heterogeneous solutions, we could attain the sensitivity of 2, 8 and 1nM for the targets 16kDa protein, FIXa and SSC antigen, respectively. Further, the specific multi-analyte validations were evidenced with the similar sensitivities in the presence of human serum. ELISA assay in this study has proven its applicability for the genuine multiple target validation in the heterogeneous solution, can be followed for other target validations. Copyright © 2017 Elsevier B.V. All rights reserved.

Development of an indirect enzyme-linked immunosorbent assay test for detecting antibodies to chicken astrovirus in chicken sera.

PubMed

Skibinska, A; Lee, A; Wylie, M; Smyth, V J; Welsh, M D; Todd, D

2015-01-01

The development of an indirect enzyme-linked immunosorbent assay (ELISA) for the serological diagnosis of Group B chicken astrovirus (CAstV) infections is described. The test was based on the use of an affinity-purified capsid antigen, specific to CAstV isolate 11672, produced as a glutathione-S-transferase N-terminal fusion protein by a recombinant baculovirus. Strongly positive ELISA signals were elicited against experimentally produced antisera raised to CAstVs from Group B (subgroups i and ii) but were negative for antisera raised to a Group A CAstV. Using a panel of 240 selected serum samples, 99% agreement was observed when the results obtained by ELISA were compared to those from an indirect immunofluorescence test for CAstV 11672. The ELISA test was applied to 68 serum sets comprising 1864 samples, which were obtained from parent and grandparent flocks originating mainly in the UK. Of the 52 sets containing ELISA-positive samples, 24 sets had >75% samples positive and nine sets had <25% samples positive and were regarded as having high and low seropositivities, respectively. Of the 1864 serum samples tested 1090 (58.5%) were ELISA positive and of these, 234 sera (21.5%) produced strongly positive signals, whereas moderately positive and weakly positive signals were produced by 562 (51.5%) and 294 (27%) sera. When used for flock screening purposes, this ELISA test can be used to (i) investigate the occurrence of first-time CAstV infections of parent flocks during lay and the possible adverse effects caused by vertically transmitted CAstV infections on broiler hatchability and performance and (ii) diagnose Group B CAstV infections within specific pathogen free flocks.

Evaluation of inapparent nosocomial severe acute respiratory syndrome coronavirus infection in Vietnam by use of highly specific recombinant truncated nucleocapsid protein-based enzyme-linked immunosorbent assay.

PubMed

Yu, Fuxun; Le, Mai Quynh; Inoue, Shingo; Thai, Hong Thi Cam; Hasebe, Futoshi; Del Carmen Parquet, Maria; Morita, Kouichi

2005-07-01

Severe acute respiratory syndrome (SARS) is a recently emerged human disease associated with pneumonia. Inapparent infection with SARS coronavirus (CoV) is not well characterized. To develop a safe, simple, and reliable screening method for SARS diagnosis and epidemiological study, two recombinant SARS-CoV nucleocapsid proteins (N' protein and (N)Delta(121) protein) were expressed in Escherichia coli, purified by affinity chromatography, and used as antigens for indirect, immunoglobulin G enzyme-linked immunosorbent assays (ELISA). Serum samples collected from healthy volunteers and SARS patients in Vietnam were used to evaluate the newly developed methods. The N' protein-based ELISA showed a highly nonspecific reaction. The (N)Delta(121) protein-based ELISA, with a nonspecific reaction drastically reduced compared to that of the nearly-whole-length N' protein-based ELISA, resulted in higher rates of positive reactions, higher titers, and earlier detection than the SARS-CoV-infected cell lysate-based ELISA. These results indicate that our newly developed SARS-CoV (N)Delta(121) protein-based ELISA is not only safe but also a more specific and more sensitive method to diagnose SARS-CoV infection and hence a useful tool for large-scale epidemiological studies. To identify inapparent SARS-CoV infections, serum samples collected from health care workers (HCWs) in Vietnam were screened by the (N)Delta(121) protein-based ELISA, and positive samples were confirmed by a virus neutralization test. Four out of 149 HCWs were identified to have inapparent SARS-CoV infection in Vietnam, indicating that subclinical SARS-CoV infection in Vietnam is rare but does exist.

Evaluation of Inapparent Nosocomial Severe Acute Respiratory Syndrome Coronavirus Infection in Vietnam by Use of Highly Specific Recombinant Truncated Nucleocapsid Protein-Based Enzyme-Linked Immunosorbent Assay

PubMed Central

Yu, Fuxun; Le, Mai Quynh; Inoue, Shingo; Thai, Hong Thi Cam; Hasebe, Futoshi; del Carmen Parquet, Maria; Morita, Kouichi

2005-01-01

Severe acute respiratory syndrome (SARS) is a recently emerged human disease associated with pneumonia. Inapparent infection with SARS coronavirus (CoV) is not well characterized. To develop a safe, simple, and reliable screening method for SARS diagnosis and epidemiological study, two recombinant SARS-CoV nucleocapsid proteins (N′ protein and NΔ121 protein) were expressed in Escherichia coli, purified by affinity chromatography, and used as antigens for indirect, immunoglobulin G enzyme-linked immunosorbent assays (ELISA). Serum samples collected from healthy volunteers and SARS patients in Vietnam were used to evaluate the newly developed methods. The N′ protein-based ELISA showed a highly nonspecific reaction. The NΔ121 protein-based ELISA, with a nonspecific reaction drastically reduced compared to that of the nearly-whole-length N′ protein-based ELISA, resulted in higher rates of positive reactions, higher titers, and earlier detection than the SARS-CoV-infected cell lysate-based ELISA. These results indicate that our newly developed SARS-CoV NΔ121 protein-based ELISA is not only safe but also a more specific and more sensitive method to diagnose SARS-CoV infection and hence a useful tool for large-scale epidemiological studies. To identify inapparent SARS-CoV infections, serum samples collected from health care workers (HCWs) in Vietnam were screened by the NΔ121 protein-based ELISA, and positive samples were confirmed by a virus neutralization test. Four out of 149 HCWs were identified to have inapparent SARS-CoV infection in Vietnam, indicating that subclinical SARS-CoV infection in Vietnam is rare but does exist. PMID:16002634

Serological assessment of neutrophil elastase activity on elastin during lung ECM remodeling.

PubMed

Kristensen, Jacob H; Karsdal, Morten A; Sand, Jannie Mb; Willumsen, Nicholas; Diefenbach, Claudia; Svensson, Birte; Hägglund, Per; Oersnes-Leeming, Diana J

2015-05-03

During the pathological destruction of lung tissue, neutrophil elastase (NE) degrades elastin, one of the major constituents of lung parenchyma. However there are no non-invasive methods to quantify NE degradation of elastin. We selected specific elastin fragments generated by NE for antibody generation and developed an ELISA assay (EL-NE) for the quantification of NE-degraded elastin. Monoclonal antibodies were developed against 10 NE-specific cleavage sites on elastin. One EL-NE assay was tested for analyte stability, linearity and intra- and inter-assay variation. The NE specificity was demonstrated using elastin cleaved in vitro with matrix metalloproteinases (MMPs), cathepsin G (CatG), NE and intact elastin. Clinical relevance was assessed by measuring levels of NE-generated elastin fragments in serum of patients diagnosed with idiopathic pulmonary fibrosis (IPF, n = 10) or lung cancer (n = 40). Analyte recovery of EL-NE for human serum was between 85% and 104%, the analyte was stable for four freeze/thaw cycles and after 24 h storage at 4°C. EL-NE was specific for NE-degraded elastin. Levels of NE-generated elastin fragments for elastin incubated in the presence of NE were 900% to 4700% higher than those seen with CatG or MMP incubation or in intact elastin. Serum levels of NE-generated elastin fragments were significantly increased in patients with IPF (137%, p = 0.002) and in patients with lung cancer (510%, p < 0.001) compared with age- and sex-matched controls. The EL-NE assay was specific for NE-degraded elastin. The EL-NE assay was able to specifically quantify NE-degraded elastin in serum. Serum levels of NE-degraded elastin might be used to detect excessive lung tissue degradation in lung cancer and IPF.

Development of an enzyme-linked immunosorbent assay for detection of CDCP1 shed from the cell surface and present in colorectal cancer serum specimens.

PubMed

Chen, Yang; Harrington, Brittney S; Lau, Kevin C N; Burke, Lez J; He, Yaowu; Iconomou, Mary; Palmer, James S; Meade, Brian; Lumley, John W; Hooper, John D

2017-05-30

CUB domain containing protein 1 (CDCP1) is a transmembrane protein involved in progression of several cancers. When located on the plasma membrane, full-length 135kDa CDCP1 can undergo proteolysis mediated by serine proteases that cleave after two adjacent amino acids (arginine 368 and lysine 369). This releases from the cell surface two 65kDa fragments, collectively termed ShE-CDCP1, that differ by one carboxyl terminal residue. To evaluate the function of CDCP1 and its potential utility as a cancer biomarker, in this study we developed an enzyme-linked immunosorbent assay (ELISA) to reliably and easily measure the concentration of ShE-CDCP1 in biological samples. Using a reference standard we demonstrate that the developed ELISA has a working range of 0.68-26.5ng/ml, and the limit of detection is 0.25ng/ml. It displays high intra-assay (repeatability) and high inter-assay (reproducibility) precision with all coefficients of variation ≤7%. The ELISA also displays high accuracy detecting ShE-CDCP1 levels at ≥94.8% of actual concentration using quality control samples. We employed the ELISA to measure the concentration of ShE-CDCP1 in human serum samples with our results suggesting that levels are significantly higher in serum of colorectal cancer patients compared with serum from individuals with benign conditions (p<0.05). Our data also suggest that colorectal cancer patients with stage II-IV disease have at least 50% higher serum levels of ShE-CDCP1 compared with stage I cases (p<0.05). We conclude that the developed ELISA is a suitable method to quantify ShE-CDCP1 concentration in human serum. Copyright © 2017 Elsevier B.V. All rights reserved.

Comparative analysis of toxin detection in biological and enviromental samples

NASA Astrophysics Data System (ADS)

Ogert, Robert A.; Burans, James; O'Brien, Tom; Ligler, Frances S.

1994-03-01

The basic recognition schemes underlying the principles of standard enzyme-linked immunosorbent assay (ELISA) and radioimmunoassay (RIA) protocols are increasingly being adapted for use with new detection devices. A direct comparison was made using a fiber optic biosensor that employs evanescent wave detection and an ELISA using avidin-biotin. The assays were developed for the detection of Ricinus communis agglutinin II, also known as ricin or RCA60. Detection limits between the two methods were comparable for ricin in phosphate buffered saline (PBS), however results in complex samples differed slightly. In PBS, sensitivity for ricin was 1 ng/ml using the fiber optic device and 500 pg/ml using the ELISA. The fiber optic sensor could not detect ricin directly in urine or serum spiked with 5 ng/ml ricin, however, the ELISA showed detection but at reduced levels to the PBS control.

Impacts of Extraction Methods in the Rapid Determination of Atrazine Residues in Foods using Supercritical Fluid Chromatography and Enzyme-Linked Immunosorbent Assay: Microwave Solvent vs. Supercritical Fluid Extractions

PubMed Central

El-Saeid, Mohamed H.; Kanu, Ijeoma; Anyanwu, Ebere C.; Saleh, Mahmoud A.

2005-01-01

It is an accepted fact that many food products that we eat today have the possibility of being contaminated by various chemicals used from planting to processing. These chemicals have been shown to cause illnesses for which some concerned government agencies have instituted regulatory mechanisms to minimize the risks and the effects on humans. It is for these concerns that reliable and accurate rapid determination techniques are needed to effect proper regulatory standards for the protection of people's nutritional health. This paper, therefore, reports the comparative evaluation of the extraction methods in the determination of atrazine (commonly used in agricultural as a herbicide) residues in foods using supercritical fluid chromatography (SFC) and enzyme-linked immunosorbent assay (ELISA) techniques. Supercritical fluid extraction (SFE) and microwave solvent extraction (MSE) methods were used to test samples of frozen vegetables, fruit juice, and jam from local food markets in Houston. Results showed a high recovery percentage of atrazine residues using supercritical fluid coupled with ELISA and SFC than with MSE. Comparatively, however, atrazine was detected 90.9 and 54.5% using SFC and ELISA techniques, respectively. ELISA technique was, however, less time consuming, lower in cost, and more sensitive with low detection limit of atrazine residues than SFC technique. PMID:15674445

A heterogeneous biotin-streptavidin-amplified enzyme-linked immunosorbent assay for detecting tris(2,3-dibromopropyl) isocyanurate in natural samples.

PubMed

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

2014-10-01

Tris(2,3-dibromopropyl) isocyanurate (TBC) is a novel brominated flame retardant (BFR) that is widely used to substitute the prohibited BFRs throughout the world. With the development of research, the potential environmental and ecological harms of TBC have been revealed. For sensitive and selective detecting TBC, an indirect competitive biotin-streptavidin-amplified enzyme-linked immunosorbent assay (BA-ELISA) has been established in this study. The small molecular TBC-hapten was synthesized first; it mimicked the chemical structure of TBC and possessed a secondary amine group. The as-obtained hapten was then conjugated with carrier proteins to prepare artificial antigen. After immunization, the anti-TBC polyclonal antibody was obtained from separating rabbit serum. The procedures of this BA-ELISA were optimized. Under the optimal conditions, the limit of detection (IC10) was 0.0067 ng/ml and the median inhibitory concentration (IC50) was 0.66 ng/ml. Cross-reactivity values of the BA-ELISA with the tested TBC analogues were ⩽5%. This immunoassay was successfully applied to determine the TBC residue in river water samples that were collected near a BFR manufacturing plant. Satisfactory recoveries (92.1-109.2%) were obtained. The results indicated that this proposed BA-ELISA is suitable for the rapid and sensitive determining of TBC in environmental monitoring. Copyright © 2014 Elsevier Inc. All rights reserved.

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

PubMed Central

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

2015-01-01

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

Evaluation of Enzyme-Linked Immunosorbent Assay for Diagnosis of Post-Kala-Azar Dermal Leishmaniasis with Crude or Recombinant k39 Antigen

PubMed Central

Salotra, P.; Sreenivas, G.; Nasim, A. A.; Subba Raju, B. V.; Ramesh, V.

2002-01-01

The diagnosis of post-kala-azar dermal leishmaniasis (PKDL), a dermatosis that provides the only known reservoir for the parasite Leishmania donovani in India, remains a problem. Timely recognition and treatment of PKDL would contribute significantly to the control of kala-azar. We evaluated here the potential of the enzyme-linked immunosorbent assay (ELISA) as a diagnostic tool for PKDL. Antigen prepared from promastigotes and axenic amastigotes with parasite isolates that were derived from skin lesions of a PKDL patient gave sensitivities of 86.36 and 92%, respectively, in the 88 PKDL cases examined. The specificity of the ELISA test was examined by testing groups of patients with other skin disorders (leprosy and vitiligo) or coendemic infections (malaria and tuberculosis), as well as healthy controls from areas where this disease is endemic or is not endemic. A false-positive reaction was obtained in 14 of 144 (9.8%) of the controls with the promastigote antigen and in 14 of 145 (9.7%) of the controls with the amastigote antigen. Evaluation of the serodiagnostic potential of recombinant k39 by ELISA revealed a higher sensitivity (94.5%) and specificity (93.7%) compared to the other two antigens used. The data demonstrate that ELISA with crude or recombinant antigen k39 provides a relatively simple and less-invasive test for the reliable diagnosis of PKDL. PMID:11874880

Development of an indirect ELISA with epitope on nonstructural protein of Muscovy duck parvovirus for differentiating between infected and vaccinated Muscovy ducks.

PubMed

Yan, B; Ma, J-Z; Yu, T-F; Shao, S-L; Li, M; Fan, X-D

2014-12-01

The aim of this study was to develop an indirect enzyme-linked immunosorbent assay (i-ELISA) based on epitope AA503-509 (RANEPKE), which is on nonstructural protein of Muscovy duck parvovirus (MDPV). Sera (100) from negative and vaccinated Muscovy ducks were compared with infected sera (240) to establish the cut-off value of this i-ELISA. There was a significant difference between the positive and negative populations (P < 0·05). The adoption of this positive-negative threshold value for this i-ELISA assay resulted in specificity of 98·0%. This i-ELISA could be used as a diagnostic tool for differentiating infected Muscovy ducks from Muscovy ducks vaccinated with inactivated virus. In this study, we developed an i-ELISA based on epitope AA503-509 (RANEPKE), which is on nonstructural protein of MDPV. This i-ELISA could be used as a diagnostic tool for differentiating infected Muscovy ducks from Muscovy ducks vaccinated with inactivated virus. © 2014 The Society for Applied Microbiology.

Immunochemistry for high-throughput screening of human exhaled breath condensate (EBC) media: implementation of automated quanterix SIMOA instrumentation

EPA Science Inventory

Immunochemistry is an important clinical tool for indicating biological pathways leading towards disease. Standard enzyme-linked immunosorbent assays (ELISA) are labor intensive and lack sensitivity at low-level concentrations. Here we report on emerging technology implementing f...

COMPARISON OF IMMUNOASSAY AND GAS CHROMATOGRAPHY/MASS SPECTROMETRY FOR MEASUREMENT OF POLYCYCLIC AROMATIC HYDROCARBONS IN CONTAMINATED SOIL

EPA Science Inventory

Polycyclic aromatic hydrocarbons (PAHs) are frequently encountered in the environment and may pose health concerns due to their carcinogenicity. A commercial enzyme-linked immunosorbent assay (ELISA), was evaluated as a screening method for monitoring PAHs at contaminated site...

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

PubMed

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

2015-01-01

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

Enzyme immunoassay of Chlamydia in birds.

PubMed

Ruppanner, R; Behymer, D E; DeLong, W J; Franti, C E; Schulz, T

1984-01-01

Serum samples from 192 free-living birds (27 species) were tested for antibodies against Chlamydia using the enzyme-linked immunosorbent assay (ELISA); 97 (51%) were seropositive. The highest antibody prevalence was among pheasants (96%), ducks (88%), and blackbirds (86%). None of 41 starlings tested were seropositive. The serotesting of 42 confined pheasants indicated 100% exposure to the organism. The ELISA is a sensitive, rapid serologic method that can be of epidemiologic and diagnostic value for detecting exposure to Chlamydia. The ELISA could also be used for mass-screening of pet birds where chlamydiosis may be considered a potential public health hazard.

Replacement of Antibodies in Pseudo-ELISAs: Molecularly Imprinted Nanoparticles for Vancomycin Detection.

PubMed

Canfarotta, Francesco; Smolinska-Kempisty, Katarzyna; Piletsky, Sergey

2017-01-01

The enzyme-linked immunosorbent assay (ELISA) is a widely employed analytical test used to quantify a given molecule. It relies on the use of specific antibodies, linked to an enzyme, to target the desired molecule. The reaction between the enzyme and its substrate gives rise to the analytical signal that can be quantified. Thanks to their robustness and low cost, molecularly imprinted polymer nanoparticles (nanoMIPs) are a viable alternative to antibodies. Herein, we describe the synthesis of nanoMIPs imprinted for vancomycin and their subsequent application in an ELISA-like format for direct replacement of antibodies.

Optimization and validation of indirect ELISA using truncated TssB protein for the serodiagnosis of glanders amongst equines.

PubMed

Singha, Harisankar; Malik, Praveen; Goyal, Sachin K; Khurana, Sandip K; Mukhopadhyay, Chiranjay; Eshwara, Vandana K; Singh, Raj K

2014-01-01

To express truncated TssB protein of Burkholderia mallei and to evaluate its diagnostic efficacy for serological detection of glanders among equines. In an attempt to develop recombinant protein based enzyme-linked immunosorbent assay (ELISA), N-terminal 200 amino acid sequences of B. mallei TssB protein-a type 6 secretory effector protein--were expressed in prokaryotic expression system. Diagnostic potential of recombinant TssB protein was evaluated in indirect ELISA using a panel of glanders positive (n = 49), negative (n = 30), and field serum samples (n = 1811). Cross-reactivity of the assay was assessed with equine disease control serum and human melioidosis positive serum. In comparison to CFT, diagnostic sensitivity and specificity of ELISA were 99.7% and 100%, respectively. The indirect ELISA method using the truncated TssB offered safer and more rapid and efficient means of serodiagnosis of glanders in equines. These data highlight the use of TssB as potential diagnostic antigen for serological diagnosis of glanders.

Comparison and Correlation of Neisseria meningitidis Serogroup B Immunologic Assay Results and Human Antibody Responses following Three Doses of the Norwegian Meningococcal Outer Membrane Vesicle Vaccine MenBvac

PubMed Central

Findlow, Jamie; Taylor, Stephen; Aase, Audun; Horton, Rachel; Heyderman, Robert; Southern, Jo; Andrews, Nick; Barchha, Rita; Harrison, Ewan; Lowe, Ann; Boxer, Emma; Heaton, Charlotte; Balmer, Paul; Kaczmarski, Ed; Oster, Philipp; Gorringe, Andrew; Borrow, Ray; Miller, Elizabeth

2006-01-01

The prediction of efficacy of Neisseria meningitidis serogroup B (MenB) vaccines is currently hindered due to the lack of an appropriate correlate of protection. For outer membrane vesicle (OMV) vaccines, immunogenicity has primarily been determined by the serum bactericidal antibody (SBA) assay and OMV enzyme-linked immunosorbent assay (ELISA). However, the opsonophagocytic assay (OPA), surface labeling assay, whole blood assay (WBA), and salivary antibody ELISA have been developed although correlation with protection is presently undetermined. Therefore, the aim of the study was to investigate further the usefulness of, and relationships between, MenB immunologic assays. A phase II trial of the OMV vaccine, MenBvac, with proven efficacy was initiated to compare immunologic assays incorporating the vaccine and six heterologous strains. Correlations were achieved between the SBA assay, OMV ELISA, and OPA using human polymorphonuclear leukocytes and human complement but not between an OPA using HL60 phagocytic cells and baby rabbit complement. Correlations between the surface labeling assay, the SBA assay, and the OMV ELISA were promising, although target strain dependent. Correlations between the salivary antibody ELISA and other assays were poor. Correlations to the WBA were prevented since many samples had results greater than the range of the assay. The study confirmed the immunogenicity and benefit of a third dose of MenBvac against the homologous vaccine strain using a variety of immunologic assays. These results emphasize the need for standardized methodologies that would allow a more robust comparison of assays between laboratories and promote their further evaluation as correlates of protection against MenB disease. PMID:16861642

Cross-validation of commercial enzyme-linked immunosorbent assay and radioimmunoassay for porcine C-peptide concentration measurements in non-human primate serum.

PubMed

Gresch, Sarah C; Mutch, Lucas A; Janecek, Jody L; Hegstad-Davies, Rebecca L; Graham, Melanie L

2017-09-01

C-peptide concentration is widely used as a marker of insulin secretion and is especially relevant in evaluating islet graft function following transplantation, because its measurement is not confounded by the presence of exogenous insulin. To address the shortage of human islet donors, the use of porcine islets has been proposed as a possible solution and the stringent pig-to-non-human primate (NHP) model is often the most relevant for pre-clinical evaluation of the potential for diabetes reversal resulting from an islet xenograft. The Millipore radioimmunoassay (RIA) was exclusively used to measure porcine C-peptide (PCP) until 2013 when the assay was discontinued and subsequently a commercially available enzyme-linked immunosorbent assay (ELISA) from Mercodia has been widely adopted. Both assays have been used in pre-clinical trials evaluating the therapeutic potential of xenograft products in reversing diabetes in the pig-to-NHP model, to interpret data in a comparable way it may be useful to perform a harmonization of C-peptide measurements. We performed a method comparison by determining the PCP concentration in 620 serum samples collected from 20 diabetic cynomolgus macaques transplanted with adult porcine islets. All analyses were performed according to manufacturer instructions. With both assays, we demonstrated an acceptable detection limit, precision, and recovery. Linearity of the ELISA met acceptance criteria at all concentrations tested while linearity of the RIA only met acceptance criteria at five of the eight concentrations tested. The RIA had a detection limit of 0.16 ng/mL, and recovery ranged from 82% to 96% and met linearity acceptance criteria at 0.35 ng/mL and from 0.78 to 2.33 ng/mL. The ELISA had a detection limit of 0.03 ng/mL, and recovery ranged from 81% to 115% and met linearity acceptance criteria from 0.08 to 0.85 ng/mL. Both assays had intra-assay precision <11% and inter-assay precision <14%. PCP concentration measured by ELISA demonstrated a significant correlation with RIA (R 2 =.9721, P<.0001). This strong correlation supports use of the regression equation y=2.029x+0.0897 to transform ELISA data to RIA or inversely y=0.4930x-0.0456 to convert RIA data to ELISA for direct comparison between assays in the concentration range of 0-3.0 ng/mL. Measured C-peptide concentration was lower with the ELISA than with the RIA; individual measurements plotted against the averages of the pair demonstrated that the variability from the mean strongly depended on increasing concentration. Porcine C-peptide can be reliably measured in NHP serum using the Mercodia ELISA, making this assay interchangeable with the Millipore RIA. Inherent differences in antibody affinity and calibration factors may explain the lower ELISA values as compared to the RIA; however without access to a traceable reference standard, it is not possible to determine which assay is most accurate. Regression modeling resulted in a correction factor appropriate for conversion of ELISA data to RIA-equivalent data facilitating comparison of assay results longitudinally and between groups. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Development and evaluation of an enzyme-linked immunosorbent assay (ELISA) method for the measurement of 2,4-dichlorophenoxyacetic acid in human urine.

PubMed

Chuang, Jane C; Emon, Jeanette M Van; Durnford, Joyce; Thomas, Kent

2005-09-15

An enzyme-linked immunosorbent assay (ELISA) method was developed to quantitatively measure 2,4-dichlorophenoxyacetic acid (2,4-D) in human urine. Samples were diluted (1:5) with phosphate-buffered saline containing 0.05% Tween and 0.02% sodium azide, with analysis by a 96-microwell plate immunoassay format. No clean up was required as dilution step minimized sample interferences. Fifty urine samples were received without identifiers from a subset of pesticide applicators and their spouses in an EPA pesticide exposure study (PES) and analyzed by the ELISA method and a conventional gas chromatography/mass spectrometry (GC/MS) procedure. For the GC/MS analysis, urine samples were extracted with acidic dichloromethane (DCM); methylated by diazomethane and fractionated by a Florisil solid phase extraction (SPE) column prior to GC/MS detection. The percent relative standard deviation (%R.S.D.) of the 96-microwell plate triplicate assays ranged from 1.2 to 22% for the urine samples. Day-to-day variation of the assay results was within +/-20%. Quantitative recoveries (>70%) of 2,4-D were obtained for the spiked urine samples by the ELISA method. Quantitative recoveries (>80%) of 2,4-D were also obtained for these samples by the GC/MS procedure. The overall method precision of these samples was within +/-20% for both the ELISA and GC/MS methods. The estimated quantification limit for 2,4-D in urine was 30ng/mL by ELISA and 0.2ng/mL by GC/MS. A higher quantification limit for the ELISA method is partly due to the requirement of a 1:5 dilution to remove the urine sample matrix effect. The GC/MS method can accommodate a 10:1 concentration factor (10mL of urine converted into 1mL organic solvent for analysis) but requires extraction, methylation and clean up on a solid phase column. The immunoassay and GC/MS data were highly correlated, with a correlation coefficient of 0.94 and a slope of 1.00. Favorable results between the two methods were achieved despite the vast differences in sample preparation. Results indicated that the ELISA method could be used as a high throughput, quantitative monitoring tool for human urine samples to identify individuals with exposure to 2,4-D above the typical background levels.

Aspergillus antigen testing in bone marrow transplant recipients

PubMed Central

Williamson, E; Oliver, D; Johnson, E; Foot, A; Marks, D; Warnock, D

2000-01-01

Aims—To assess the clinical usefulness of a commercial aspergillus antigen enzyme linked immunosorbent assay (ELISA) in the diagnosis of invasive aspergillosis (IA) in bone marrow transplant recipients, and to compare it with a commercial latex agglutination (LA) test. Methods—In total, 2026 serum samples from 104 bone marrow transplant recipients were tested. These comprised 67 sera from seven patients who had died with confirmed IA, 268 sera from nine patients who had died with suspected IA, and 1691 sera from 88 patients with no clinical, radiological, or microbiological signs of IA. Results—The ELISA was more sensitive than the LA test. All patients who were ELISA positive were also LA positive, and a positive LA result never preceded a positive ELISA. Twelve of 16 patients with confirmed or suspected IA were ELISA positive on two or more occasions, compared with 10 of 15 who were LA positive. ELISA was positive before LA in five patients (range, 2–14 days), and became positive on the same day in the remainder. Aspergillus antigen was detected by ELISA a median of 15 days before death (range, 4–233). Clinical and/or radiological evidence of IA was noted in all patients, and a positive ELISA was never the sole criterion for introduction of antifungal treatment. Two samples (one from each of two patients without IA) gave false positive results. Conclusions—The aspergillus ELISA is a specific indicator of invasive aspergillosis if the criterion of two positive samples is required to confirm the diagnosis. However, the test is insufficiently sensitive to diagnose aspergillosis before other symptoms or signs are apparent, and hence is unlikely to lead to earlier initiation of antifungal treatment. It is therefore unsuitable for screening of asymptomatic patients at risk of invasive aspergillosis, but does have a useful role in confirming the diagnosis in symptomatic patients. Key Words: invasive aspergillosis • aspergillus antigen • Platelia enzyme linked immunosorbent assay PMID:10889818

DEVELOPMENT OF AN ENZYME-LINKED IMMUNOSORBENT ASSAY (ELISA) FOR VITELLOGENIN OF THE MOSQUITOFISH (GAMBUSIA AFFINIS). (R826130)

EPA Science Inventory

The perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Concl...

AN ENZYME-LINKED IMMUNOSORBENT ASSAY (ELISA) FOR DETERMINING DIOXINS IN SEDIMENT AND SOIL SAMPLES

EPA Science Inventory

The dioxins comprise a family of compounds chemically referred to as polychlorinated dibenzo-p-dioxins (PCDDs) and polychlorinated dibenzofurans (PCDFs). The most toxic of these compounds is 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), a known human carcinogen. Dioxins are formed ...

Environmental Technology Verification Report for Abraxis Ecologenia® Ethynylestradiol (EE2) Microplate Enzyme-Linked Immunosorbent Assay (ELISA) Test Kits

EPA Science Inventory

The EPA's National Risk Management Research Laboratory (NRMRL) and its verification organization partner, Battelle, operate the Advanced Monitoring Systems (AMS) Center under ETV. The AMS Center recently evaluated the performance of the Abraxis Ecologenia Ethynylestradiol (EE2) ...

Method 546: Determination of Total Microcystins and Nodularins in Drinking Water and Ambient Water by Adda Enzyme-Linked Immunosorbent Assay

EPA Pesticide Factsheets

This method is listed as Tier I for presumptive analysis of microcystins in water samples and Tier II all other environmental sample types. The method determines total microcystins and nodularins using an indirect competitive immunoassay (ELISA).

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...

Prevalence of antibodies to Sarcocystis neurona and Neospora hughesi in horses from Mexico

USDA-ARS?s Scientific Manuscript database

The risk of equine protozoal myeloencephalitis (EPM) to horses in Mexico has not been established. Serum samples from 495 horses in Durango State, Mexico were examined for the presence of antibodies to Sarcocystis neurona and Neospora hughesi using enzyme-linked immunosorbent assays (ELISAs) based o...

Purification and Characterization of Recombinant Vaccinia L1R Protein from Escherichia coli

DTIC Science & Technology

2016-08-01

Solubilization .................................................2  2.4  Denaturing Chromatography (Purification Step 1...Concentration Determination ................................................................4  2.10  Enzyme -Linked Immunosorbent Assay (ELISA...the preparation of the recombinant VACV L1R protein fragment by denaturing , refolding, and purifying material expressed into inclusion bodies in

Development of a monoclonal antibody-based, congener-specific and solvent-tolerable direct enzyme-linked immunosorbgent assay for the detection of 2,2',4,4'-tetrabromodiphenyl ether in environmental samples

USDA-ARS?s Scientific Manuscript database

A sensitive direct enzyme-linked immunosorbent assay (ELISA) for the detection of 2,2',4,4'-tetrabromodiphenyl ether (BDE-47) in environmental samples was developed. A hapten mimicking the whole structure of BDE-47 was synthesized by introducing a butyric acid spacer to 5-hydroxy-BDE-47 and coupled ...

Specific and sensitive enzyme-linked immunosorbent assays for analysis of residual allergenic food proteins in commercial bottled wine fined with egg white, milk, and nongrape-derived tannins.

PubMed

Rolland, Jennifer M; Apostolou, Effie; de Leon, Maria P; Stockley, Creina S; O'Hehir, Robyn E

2008-01-23

Regulations introduced by the Food Standards Australia New Zealand in December 2002 require all wine and wine product labels in Australia to identify the presence of a processing aid, additive or other ingredient, which is known to be a potential allergen. The objective of this study was to establish sensitive assays to detect and measure allergenic proteins from commonly used processing aids in final bottled wine. Sensitive and specific enzyme-linked immunosorbent assays (ELISA) were developed and established for the proteins casein, ovalbumin, and peanut. Lower limits of detection of these proteins were 8, 1, and 8 ng/mL, respectively. A panel of 153 commercially available bottled Australian wines were tested by these ELISA, and except for two red wines known to contain added whole eggs, residuals of these food allergens were not detected in any wine. These findings are consistent with a lack of residual potentially allergenic egg-, milk-, or nut-derived processing aids in final bottled wine produced in Australia according to good manufacturing practice at a concentration that could cause an adverse reaction in egg, milk, or peanut/tree-nut allergic adult consumers.

Use of a commercial enzyme-linked immunosorbent assay for rapid detection of Giardia duodenalis in dog stools in the environment: a Bayesian evaluation.

PubMed

Papini, Roberto; Carreras, Giulia; Marangi, Marianna; Mancianti, Francesca; Giangaspero, Annunziata

2013-05-01

Giardia duodenalis is considered a potentially zoonotic protozoan. Some animal species, including infected dogs, may play an important role in the spread of Giardia cysts through environmental contamination with their feces. In the present study, a commercial enzyme-linked immunosorbent assay (ELISA) was used to examine 143 samples of dog feces collected in urban areas as an indicator of the risk of field contamination. Using a Bayesian statistical approach, the ELISA showed a sensitivity of 88.9% and a specificity of 95.8% with positive and negative predictive values of 89.6% and 95.4%, respectively. The test affords the advantage of rapid processing of fecal samples without a complex technical structure and extensive costly labor. Moreover, the present results show that the assay provides public health veterinarians with a practical tool that can be used in screening programs, as a valid alternative or as an adjunct to other tests, in order to assess the biological risk of exposure to G. duodenalis cysts from dogs in human settlements. However, the test may not be completely accurate for human health risk evaluation, as it does not discriminate between zoonotic and non-zoonotic isolates.

Prevalence of bovine brucellosis in organized dairy farms, using milk ELISA, in quetta city, balochistan, pakistan.

PubMed

Shafee, Muhammad; Rabbani, Masood; Sheikh, Ali Ahmad; Ahmad, Mansoor Din; Razzaq, Abdul

2011-01-24

A total of 200 milk samples from cattle (n = 86) and buffalo (n = 114) were evaluated using milk ring test (MRT) and indirect enzyme-linked immunosorbent assay (i-ELISA). The overall prevalence was found to be 3% and 8.5% in cattle and buffaloes using MRT and i-ELISA, respectively. The prevalence was 4.6% and 1.7% in cattle and buffalo using MRT, respectively, while i-ELISA exhibited 20% and 0% in cattle and buffalo, respectively. The prevalence was higher in government dairy farm, compared to privately owned dairy farm. This paper points out an alarming situation in the target area with respect to the public health significance.

Prevalence of Bovine Brucellosis in Organized Dairy Farms, Using Milk ELISA, in Quetta City, Balochistan, Pakistan

PubMed Central

Shafee, Muhammad; Rabbani, Masood; Sheikh, Ali Ahmad; Ahmad, Mansoor din; Razzaq, Abdul

2011-01-01

A total of 200 milk samples from cattle (n = 86) and buffalo (n = 114) were evaluated using milk ring test (MRT) and indirect enzyme-linked immunosorbent assay (i-ELISA). The overall prevalence was found to be 3% and 8.5% in cattle and buffaloes using MRT and i-ELISA, respectively. The prevalence was 4.6% and 1.7% in cattle and buffalo using MRT, respectively, while i-ELISA exhibited 20% and 0% in cattle and buffalo, respectively. The prevalence was higher in government dairy farm, compared to privately owned dairy farm. This paper points out an alarming situation in the target area with respect to the public health significance. PMID:21331157

Homologous ELISA for detection of oligomeric human TNF: properties of the assay.

PubMed

Petyovka, N; Lyach, L; Voitenok, N N

1995-10-26

In order to quantify oligomeric human tumor necrosis factor-alpha (TNF), we have developed a sensitive homologous enzyme-linked immunosorbent assay (Hm-ELISA) using the same monoclonal antibody (MoAb) for both solid and liquid phase. Different anti-TNF MoAb have been compared in terms of their efficacy in the Hm-ELISA, affinity, neutralization capacity and epitope specificity. The data suggest, that effectiveness in the Hm-ELISA may represent a novel characteristic of MoAb. Of the MoAbs tested, 5 N was capable of recognizing oligomeric TNF in the Hm-ELISA with a detection limit of 15 pg/ml. Furthermore, using Hm-ELISA against human TNF, interleukin-8 (IL-8) and lymphotoxin, we have demonstrated that these cytokines are oligomeric in physiological solutions, but are converted into monomeric forms in the presence of the non-ionic detergent Tween 20. High salt buffer was employed to abrogate a nonspecific false positive reaction in the Hm-ELISA found in nearly half of the plasma samples obtained from healthy subjects. Finally, a good correlation between the Hm-ELISA and the L929 bioassay was observed for natural and recombinant TNF measured in human plasma.

Nonstructural protein 1 antibody-based epitope-blocking enzyme-linked immunosorbent assay to differentiate Japanese encephalitis virus from dengue virus infections in humans.

PubMed

Konishi, Eiji; Konishi, Mayu

2011-01-01

Japanese encephalitis virus (JEV) and the four dengue viruses (DENV1-4) are co-distributed in Southeast and South Asia. Since JEV is antigenically cross-reactive with DENV1-4, the differentiation between these viruses using antibody assays may be difficult. Herein, we describe the development of an epitope-blocking enzyme-linked immunosorbent assay (ELISA) using a monoclonal antibody specific for the nonstructural protein 1 (NS1) of JEV (JEV-NS1) to differentiate antibodies against JEV from those against DENV1-4. Hyperimmune mouse sera against JEV-NS1 showed >60% inhibition, whereas those against NS1 of DENV1-4 showed <30% inhibition. The present assay could therefore detect antibodies specific for JEV. For testing of human sera, a temporary cutoff value (30.8%) was calculated the average and standard deviation obtained for sera of control humans negative for JEV antibodies. Human sera positive for antibodies to any of DENV1-4 NS1 but negative for antibodies to JEV-NS1 showed a lower percentage inhibition than the cutoff value. On the other hand, sera with JEV-NS1 antibody levels of ≥0.400, as determined by the conventional ELISA (medially/strongly positive for JEV-NS1 antibodies), showed percentage inhibition greater than the cutoff. Although this blocking ELISA afforded false-negative results for most sera that were weakly positive for JEV-NS1 antibodies, it may be useful for investigating the seroepidemiology of JEV antibodies in dengue-endemic areas.

Development of an enzyme-linked immunosorbent assay for seven sulfonamide residues and investigation of matrix effects from different food samples.

PubMed

Zhang, Hongyan; Wang, Lei; Zhang, Yan; Fang, Guozhen; Zheng, Wenjie; Wang, Shuo

2007-03-21

Direct competitive enzyme-linked immunosorbent assays (ELISA) were developed to detect a broad range of sulfonamides in various matrices. Screening for this class of antibiotics in pig muscle, chicken muscle, fish, and egg extracts was accomplished by simple, rapid extraction methods carried out with only phosphate-buffered saline (PBS) buffer. Twenty milliliters of extract solution was added to 4 g of sample to extract the sulfonamide residues, and sample extracts diluted with assay buffer were directly analyzed by ELISA; matrix effects could be avoided with 1:5 dilution of pig muscle, chicken muscle, and egg extracts with PBS and 1:5 dilution of fish extract with 1% bovine serum albumin (BSA)-PBS. For liver sample, the extraction method was a little more complicated; 2 g of sample was added to 20 mL of ethanol, mixed, and then centrifuged. The solvent of 10 mL of the upper liquid was removed, and the residues were dissolved in 10 mL of PBS and then filtered; the filtrate was diluted two-fold with 0.5% BSA-PBS for ELISA. These common methods were able to detect seven sulfonamide residues such as sulfisozole, sulfathiazole, sufameter, sulfamethoxypyridazine, sulfapyridine, sulfamethizole, and sulfachlorpyridazine in pig muscle, liver, chicken muscle, egg, and fish. The assay's detection limits for these compounds were less than 100 microg kg-1. Various extraction methods were tested, and the average recovery (n=3) of 100 microg kg-1 for the matrices was found to range from 77.3 to 123.7%.

A comparison of the sensitivity, specificity, and molecular weight accuracy of three different commercially available Hyaluronan ELISA-like assays.

PubMed

Haserodt, Sarah; Aytekin, Metin; Dweik, Raed A

2011-02-01

Hyaluronan (HA) is a glycosaminoglycan found in the extracellular matrix and ranges from several thousand to millions of daltons in size. HA has importance in various pathological conditions and is known to be elevated in several diseases. Three commonly used, commercially available HA enzyme-linked immunosorbent assay (ELISA)-like assays (from Corgenix, Echelon and R&D) were compared on the basis of accuracy, sample variability and ability to measure a range of HA sizes. The Corgenix HA ELISA-like assay displayed the lowest intra-assay variability [coefficient of variation (CV) = 11.7 ± 3.6%], followed by R&D (CV = 12.3 ± 4.6%) and Echelon (CV = 18.9 ± 9.2%). Interassay variability was also lowest for the Corgenix assay (CV = 6.0%), intermediate for the Echelon assay (9.5%) and highest for the R&D assay (CV = 34.1%). The high interassay variability seen for the R&D assay may have been due to the effect of dilution, since the dilution-independent interassay variability was 15.5%. The concentration of the standard HA was overestimated by the Echelon assay by 85% and underestimated by the R&D and Corgenix assays by 34 and 32%, respectively. The Echelon HA ELISA-like assay was the most effective at measuring all sizes of HA tested (2 MDa and 132, 66 and 6.4 kDa), whereas the Corgenix and R&D assays were unable to detect 6.4 kDa HA. These findings suggest that the Echelon HA ELISA-like assay is better suited for size-sensitive HA measurements but has a relatively high variability. The Corgenix and R&D HA ELISA-like assays have low variability and high accuracy but are not suitable for detecting low-molecular-weight HA.

Enzyme-linked immunosorbent assay by enhanced chemiluminescence detection for the standardization of estrogenic miroestrol in Pueraria candollei Graham ex Benth.

PubMed

Yusakul, Gorawit; Udomsin, Orapin; Tanaka, Hiroyuki; Morimoto, Satoshi; Juengwatanatrakul, Thaweesak; Putalun, Waraporn

2015-08-01

Miroestrol (ME) is a potent phytoestrogen from the P. candollei tuberous root. It has been approved for use in clinical trials due to its beneficial effect on disorders associated with estrogen deficiency. To ensure medical efficacy and safety, high performance analytical methods for ME analysis are required to standardize products from the P. candollei root. An enhanced chemiluminescence enzyme-linked immunosorbent assay (ECL-ELISA) was developed and validated using a polyclonal antibody against ME and a chemiluminescent system of luminol-H2 O2 -horseradish peroxidase-4-(1-imidazolyl) phenol. The ECL-ELISA system exhibited linearity over a concentration range of 0.31-10.00 ng mL(-1) , for which the relative standard variation (%RSD) was less than 10% for both intra- and interplate determinations. The ECL-ELISA is reliable for the determination of ME as reflected by the high recovery percentage (101.22-103.06%). As a comparative analysis, the ME content in each sample determined by ECL-ELISA was correlated with high coefficients of determination with colorimetric ELISA (R(2)  = 0.998) and high performance liquid chromatography (HPLC) (R(2)  = 0.998) methods. The ECL-ELISA method could be applied to all of the commercial products containing P. candollei root, when the products contain between 0.706 ± 0.046 and 13.123 ± 0.794 µg g(-1) dry wt. of ME. This method is useful as a high performance analytical method for the quantity control of ME in raw materials and end products at both the research and industrial levels. Copyright © 2014 John Wiley & Sons, Ltd.

Performance of Commercial Enzyme-Linked Immunosorbent Assays for Detection of Antibodies to Bordetella pertussis▿

PubMed Central

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

2010-01-01

Measuring antibodies to Bordetella pertussis antigens is mostly done by enzyme-linked immunosorbent assays (ELISAs). We compared the performance of ELISA kits that were commercially available in Germany. Eleven measured IgG antibodies, and nine measured IgA antibodies. An in-house ELISA with purified antigens served as a reference method. Samples included two WHO reference preparations, the former Food and Drug Administration (FDA)/Center for Biologics Evaluation and Research (CBER) reference preparations, serum samples from patients with clinically suspected pertussis, and serum samples from patients having received a combined tetanus, diphtheria, and pertussis (Tdap) vaccination. Kits using pertussis toxin (PT) as an antigen showed linearity compared to the WHO Reference preparation (r2 between 0.82 and 0.99), and these kits could quantify antibodies according to the reference preparation. ELISA kits using mixed antigens showed no linear correlation to the reference preparations. Patient results were compared to results of in-house ELISAs using a dual cutoff of either ≥100 IU/ml anti-PT IgG or ≥40 IU/ml anti-PT IgG together with ≥12 IU/ml anti-PT IgA. The sensitivities of kits measuring IgG antibodies ranged between 0.84 and 1.00. The specificities of kits using PT as an antigen were between 0.81 and 0.93. The specificities of kits using mixed antigens were between 0.51 and 0.59 and were thus not acceptable. The sensitivities of kits measuring IgA antibodies ranged between 0.53 and 0.73, and the specificities were between 0.67 and 0.94, indicating that IgA antibodies may be of limited diagnostic value. Our data suggest that ELISAs should use purified PT as an antigen and be standardized to the 1st International Reference preparation. PMID:20943873

Application of IgY to sandwich enzyme-linked immunosorbent assays, lateral flow devices, and immunopillar chips for detecting staphylococcal enterotoxins in milk and dairy products.

PubMed

Jin, Wanchun; Yamada, Keiko; Ikami, Mai; Kaji, Noritada; Tokeshi, Manabu; Atsumi, Yusuke; Mizutani, Makoto; Murai, Atsushi; Okamoto, Akira; Namikawa, Takao; Baba, Yoshinobu; Ohta, Michio

2013-03-01

Staphylococcal enterotoxins (SEs), produced by Staphylococcus aureus, are a major cause of staphylococcal food poisoning. Traditionally, sandwich enzyme-linked immunosorbent assay (ELISA) and reverse passive latex agglutination with rabbit antibody IgG have been used to detect SEs. However, most of these kits require a long processing time and there is a risk of false-positive results since IgG reacts nonspecifically with protein A produced by S. aureus. In this study, we prepared antienterotoxin chicken IgY antibodies specific for each SE (SEA to SEE) without reaction to protein A, which enabled a drastic reduction in nonspecific reactions. ELISAs, lateral flow device (LFDs), and IgY-based immunopillar chips were developed for SE detection. All the ELISAs developed were as sensitive as commercially available kits. The SEs in milk were successfully detected by the ELISAs, LFDs, and immunopillar chips without any sample pretreatment. The LFD could detect SEA even at the low concentration of 0.2 ng/ml within 15 min in milk. The detection limit of the immunopillar chips for the SEs ranged from 0.01 to 0.1 ng/ml in milk; the SEs were detected within 12 min and specialized skills were not required. The ELISA and LFD detected SEA in dairy products artificially contaminated with S. aureus, including ice cream, yogurt, and café au lait, in a dose-dependent manner. In conclusion, IgY allows highly specific detection of SEs, and ELISAs, LFDs, and immunopillar chips should be useful tools for screening SEs in milk and dairy products. Copyright © 2013 Elsevier B.V. All rights reserved.

Evaluation of a newly designed sandwich enzyme linked immunosorbent assay for the detection of hydatid antigen in serum, urine and cyst fluid for diagnosis of cystic echinococcosis

PubMed Central

Chaya, DR; Parija, Subhash Chandra

2013-01-01

Introduction: Cystic echinococcosis (CE) is a zoonotic disease of humans with variable clinical manifestations. Imaging and immunological methods are currently the mainstay of diagnosis of this disease. Although the immunological tests for detection of anti-echinococcal antibodies have several disadvantages, they are widely being used. Antigen is far more superior than antibody detection test as they can provide a specific parasitic diagnosis. Materials and Methods: A sandwich enzyme linked immunosorbent assay (ELISA) was designed using antibodies to 24 kDa urinary hydatid antigen for the detection of hydatid antigens in urine, serum and cyst fluid specimens. The performance of this novel test was compared with that of other hydatid antibody detection ELISA and enzyme immune transfer blot (EITB) using radiological and surgical confirmation as the gold standard. Results: The antigen detection ELISA showed 100% sensitivity and specificity when tested with cyst fluid. On testing urine and serum, the antigen detection ELISA was found to be more specific than antibody detection ELISA. EITB was found to be the most sensitive and specific test. Conclusions: ELISA using polyclonal antibodies against 24 kDa urinary hydatid protein was moderately sensitive to detect hydatid antigen in serum and urine. Hence polyclonal antibodies to 24 kDa urinary hydatid antigen can be used as an alternative source of antibody to detect hydatid antigen in serum, urine and cyst fluid. In the present study, EITB was found to be highly specific test for detection of hydatid antibodiesin serum. 24 kDa protein was found to be specific and of diagnostic value in CE. PMID:24470996

Development of a Competitive Enzyme-Linked Immunosorbent Assay for Detection of Antibodies against the 3B Protein of Foot-and-Mouth Disease Virus

PubMed Central

Yang, Ming; Parida, Satya; Salo, Tim; Hole, Kate; Velazquez-Salinas, Lauro

2015-01-01

Foot-and-mouth disease (FMD) is one of the most highly contagious and economically devastating diseases, and it severely constrains the international trade of animals. Vaccination against FMD is a key element in the control of FMD. However, vaccination of susceptible animals raises critical issues, such as the differentiation of infected animals from vaccinated animals. The current study developed a reliable and rapid test to detect antibodies against the conserved, nonstructural proteins (NSPs) of the FMD virus (FMDV) to distinguish infected animals from vaccinated animals. A monoclonal antibody (MAb) against the FMDV NSP 3B was produced. A competitive enzyme-linked immunosorbent assay (cELISA) for FMDV/NSP antibody detection was developed using a recombinant 3ABC protein as the antigen and the 3B-specific MAb. Sera collected from naive, FMDV experimentally infected, vaccinated carrier, and noncarrier animals were tested using the 3B cELISA. The diagnostic specificity was 99.4% for naive animals (cattle, pigs, and sheep) and 99.7% for vaccinated noncarrier animals. The diagnostic sensitivity was 100% for experimentally inoculated animals and 64% for vaccinated carrier animals. The performance of this 3B cELISA was compared to that of four commercial ELISA kits using a panel of serum samples established by the World Reference Laboratory for FMD at The Pirbright Institute, Pirbright, United Kingdom. The diagnostic sensitivity of the 3B cELISA for the panel of FMDV/NSP-positive bovine serum samples was 94%, which was comparable to or better than that of the commercially available NSP antibody detection kits. This 3B cELISA is a simple, reliable test to detect antibodies against FMDV nonstructural proteins. PMID:25651918

A novel sandwich enzyme-linked immunosorbent assay with covalently bound monoclonal antibody and gold probe for sensitive and rapid detection of bovine β-lactoglobulin.

PubMed

He, Shengfa; Li, Xin; Wu, Yong; Wu, Shandong; Wu, Zhihua; Yang, Anshu; Tong, Ping; Yuan, Juanli; Gao, Jinyan; Chen, Hongbing

2018-06-01

Bovine milk is a recognized allergenic food source with β-lactoglobulin (BLG) as its major allergen. Reliable detection of BLG epitopes can, therefore, be a useful marker for the presence of milk in processed food products, and for potential allergenicity. At the present, enzyme-linked immunosorbent assays (ELISA) for the detection of BLG are time-consuming and generally not specific to BLG IgE epitopes. In this study, the 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide-activated anti-BLG IgE epitope monoclonal antibody (mAb 1G9) was covalently bound onto the KOH-treated microtiter plate surface. Using this mAb-bound plate in sandwich combination with biotinylated anti-BLG polyclonal antibody-labeled gold nanoparticles, a linear dynamic range between 31.25 and 64 × 10 3  ng mL -1 with a limit of detection for BLG of 0.49 ng mL -1 was obtained, which is 32 times wider and 16 times more sensitive than conventional sandwich ELISA (sELISA). Total recovery of BLG in spiked food samples was found, without matrix effects. Also in partially hydrolyzed infant formulas, the allergenic BLG residues were detected quantitatively. Compared with conventional and commercial BLG detection sELISAs, our sELISA is reliable, highly BLG epitope-specific, user-friendly, and time-saving and allows accurate detection of potentially allergenic residues in different types of processed foods. This improved sELISA protocol can be easily extended to detect other well-identified and characterized food allergens. Graphical abstract IgE epitope mAb-bound plate in sandwich combination with gold probe for sensitive and rapid detection of bovine β-lactoglobulin and its potentially allergenic residues.

Seroprevalence of Fasciola gigantica infection in bovines using cysteine proteinase dot enzyme-linked immunosorbent assay

PubMed Central

Kumar, Niranjan; Varghese, Anju; Solanki, J. B.

2017-01-01

Aim: The objective of the present study was to know the seroprevalence status of Fasciola gigantica infection in cattle and buffaloes using cysteine proteinase (CP) antigen in dot enzyme-linked immunosorbent assay (ELISA) format under field conditions. Materials and Methods: As per the standard protocol, the sera were collected from the blood of 112 cattle and 38 buffaloes of coastal areas of Navsari district, South Gujarat, India. The indirect ELISA was performed on the strip of nitrocellulose paper blotted with 1 µl of CP antigen, to detect F. gigantica seropositive animals. Results: The native CP of F. gigantica revealed a single visible band on 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. There was no any noted cross-reaction between the selected antigen and sera of Gastrothylax crumenifer-infected animals in ELISA. Out of 150 screened bovines, the sera of 47 (31.33%) were found to be reactive in dot-ELISA, with a prevalence rate of 31.25% and 31.58% in cattle and buffaloes, respectively. The seropositive bovines with heavy, moderate, and light level of infection were 44.68%, 34.04%, and 21.28%, respectively (p<0.05 between heavy and light; p>0.05 between moderate and heavy or light). The share of F. gigantica seropositive and negative animals was 31% and 69%, respectively. The optical density at 450 nm of pooled sera of seropositive bovines with heavy, moderate, and light reactivity in plate-ELISA was significantly higher with field or reference ­negative sera. Conclusion: The CP-based dot-ELISA can be useful for field veterinarians for quick and timely isolation of the animals requiring urgent flukicide therapy. PMID:29184364

Diagnostic specificity of the African swine fever virus antibody detection enzyme-linked immunosorbent assay in feral and domestic pigs in the United States.

PubMed

Bergeron, H C; Glas, P S; Schumann, K R

2017-12-01

African swine fever (ASF) is a highly contagious haemorrhagic disease of pigs that has the potential to cause mortality nearing 100% in naïve animals. While an outbreak of ASF in the United States' pig population (domestic and feral) has never been reported, an introduction of the disease has the potential to cause devastation to the pork industry and food security. During the recovery phase of an outbreak, an antibody detection diagnostic assay would be required to prove freedom of disease within the previously infected zone and eventually nationwide. Animals surviving an ASF infection would be considered carriers and could be identified through the persistence of ASF viral antibodies. These antibodies would demonstrate exposure to the disease and not vaccination, as there is no ASF vaccine available. A well-established commercial enzyme-linked immunosorbent assay (ELISA) detects antibodies against ASF virus (ASFV), but the diagnostic specificity of the assay had not been determined using serum samples from the pig population of the United States. This study describes an evaluation of the World Organization for Animal Health (OIE)-recommended Ingezim PPA COMPAC ELISA using a comprehensive cohort (n = 1791) of samples collected in the United States. The diagnostic specificity of the assay was determined to be 99.4% (95% confidence interval (CI): [98.9, 99.7]). The result of this study fills a gap in understanding the performance of the Ingezim PPA COMPAC ELISA in the ASF naïve pig population of the United States. Published 2017. This article is a U.S. Government work and is in the public domain in the USA.

Preparation of anti-Sudan red monoclonal antibody and development of an indirect competitive enzyme-linked immunosorbent assay for detection of Sudan red in chilli jam and chilli oil.

PubMed

Xu, Jing; Zhang, Yuanyang; Yi, Jian; Meng, Meng; Wan, Yuping; Feng, Caiwei; Wang, Shanliang; Lu, Xiao; Xi, Rimo

2010-10-01

Sudan dyes are banned to be used in food additives because of the carcinogenicity of their metabolites. A rapid and sensitive indirect competitive enzyme-linked immunosorbent assay (ELISA) was developed to detect the residues of Sudan dyes. Novel immunogen and coating antigen were synthesized via glutaraldehyde linking. The hapten-bovine serum albumin (BSA) was applied as immunogen and the hapten-ovalbumin (OVA) was served as coating antigen. The monoclonal antibody obtained showed high sensitivity to Sudan I with an IC(50) value of 1.7 μg L(-1) in buffer and was suitable to detect the residues of Sudan red in food products. The specificity of the assay was studied by measuring cross-reactivity of the antibody with the structurally related compounds of Sudan II (<1%), Sudan IV (<1%) and para red (120%). Chilli jam and chilli oil samples spiked with Sudan dyes were analyzed by the method. The detection limit (LOD) of the ELISA method applied in chilli jam and chilli oil was 9.0 μg L(-1) and 19.6 μg L(-1), respectively. The recovery rates of Sudan-I in chilli oil and chilli jam were in the range of 80%-110% with coefficients of variation <25%. The intra-assay variation and inter-assay variation in buffer were both <9%.

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

PubMed Central

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

2009-01-01

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

Influenza A virus H5-specific antibodies in mute swans (Cygnus olor) in the USA.

PubMed

Kistler, Whitney M; Stallknecht, David E; Lebarbenchon, Camille; Pedersen, Kerri; Marks, David R; Mickley, Randy; DeLiberto, Thomas J; Yabsley, Michael J

2015-04-01

The use of serologic assays for influenza A virus (IAV) surveillance in wild birds has increased because of the availability of commercial enzyme-linked immunosorbent assays (ELISAs). Recently, an H5-specific blocking ELISA (bELISA) was shown to reliably detect H5-specific antibodies to low- and high-pathogenic H5 viruses in experimentally infected waterfowl. Mute Swans (Cygnus olor) were frequently associated with highly pathogenic H5N1 outbreaks in Europe and may have a similar role if highly pathogenic H5N1 is introduced into North America. We measured the prevalence of antibodies to the nucleoprotein and H5 protein in Mute Swans using three serologic assays. We collected 340 serum samples from Mute Swans in Michigan, New Jersey, New York, and Rhode Island, US. We detected antibodies to the IAV nucleoprotein in 66.2% (225/340) of the samples. We detected H5-specific antibodies in 62.9% (214/340) and 18.8% (64/340) using a modified H5 bELISA protocol and hemagglutination inhibition (HI) assay, respectively. The modified H5 bELISA protocol detected significantly more positive samples than did the manufacturer's protocol. We also tested 46 samples using virus neutralization. Neutralization results had high agreement with the modified H5 bELISA protocol and detected a higher prevalence than did the HI assay. These results indicate that North American Mute Swans have high nucleoprotein and H5 antibody prevalences.

Sandwich enzyme-linked immunosorbent assay for naringin.

PubMed

Qu, Huihua; Wang, Xueqian; Qu, Baoping; Kong, Hui; Zhang, Yue; Shan, Wenchao; Cheng, Jinjun; Wang, Qingguo; Zhao, Yan

2016-01-15

Among the currently used immunoassay techniques, sandwich ELISA exhibits higher specificity, lower cross-reactivity, and a wider working range compared to the corresponding competitive assays. However, it is difficult to obtain a pair of antibodies that can simultaneously bind to two epitopes of a molecule with a molecular weight of less than 1000 Da. Naringin (Nar) is a flavonoid with a molecular mass of 580 Da. The main aim of this study was to develop a sandwich ELISA for detecting Nar. Two hybridomas secreting anti-Nar monoclonal antibodies (mAbs) were produced by fusing splenocytes from a mouse immunised against Nar-bovine serum albumin (BSA) conjugated with a hypoxanthine-aminopterin-thymidine (HAT)-sensitive mouse myeloma cell line; a sandwich ELISA for detecting Nar was developed using these two well-characterised anti-Nar mAbs. The performance of the sandwich assay was further evaluated by limit of detection (LOD), limit of quantification (LOQ), recovery, and interference analyses. A dose-response curve to Nar was obtained with an LOD of 6.78 ng mL(-1) and an LOQ of 13.47 ng mL(-1). The inter-assay and intra-assay coefficients of variation were 4.32% and 7.48%, respectively. The recovery rate of Nar from concentrated Fructus aurantii granules was 83.63%. A high correlation was obtained between HPLC and sandwich ELISA. These results demonstrate that the sandwich ELISA method has higher specificity for Nar than indirect competitive ELISA. Copyright © 2015 Elsevier B.V. All rights reserved.

Plasma osteopontin levels in patients with head and neck cancer and cervix cancer are critically dependent on the choice of ELISA system.

PubMed

Vordermark, Dirk; Said, Harun M; Katzer, Astrid; Kuhnt, Thomas; Hänsgen, Gabriele; Dunst, Jürgen; Flentje, Michael; Bache, Matthias

2006-08-15

The tumor-associated glycoprotein osteopontin (OPN) is discussed as a plasma surrogate marker of tumor hypoxia and as an indicator of the presence of pleural mesothelioma in asbestos-exposed individuals. The clinical introduction of plasma OPN measurements requires the availability of a reliable enzyme-linked immunosorbence assay (ELISA). We compared previously described and currently available ELISA systems on 88 archival plasma samples obtained from patients with head and neck or cervix cancer between 20 days before and 171 after the start of radiotherapy. Median (range) plasma OPN levels were 667 (148.8-2095) ng/ml and 9.8 (3.5-189.5) ng/ml for a previously described and a newly marketed assay, respectively. Although results for different assays were significantly correlated (r = 0.38, p < 0.05, Spearman rank test), between-assay factors ranged from 2.0 to 217.9 (median 74.6) in individual patients. OPN levels in cervix cancer patients were comparable to those of head and neck cancer patients. Commercially available OPN ELISA systems produce different absolute plasma OPN levels, compromising a comparison of individual patient data with published results. However, different assays appear to have a similar capacity to rank patients according to plasma OPN level. A review of literature data suggests that plasma OPN levels measured even with identical ELISA systems can only be compared with caution.

Enzyme-Linked Immunosorbent Assay Testing of Shoots Grown In Vitro and the Use of Immunocapture-Reverse Transcription-Polymerase Chain Reaction Improve the Detection of Prunus necrotic ringspot virus in Rose.

PubMed

Moury, B; Cardin, L; Onesto, J P; Candresse, T; Poupet, A

2000-05-01

We developed and evaluated two different methods to improve the detection of the most prevalent virus of rose in Europe, Prunus necrotic ring-spot virus (PNRSV). Immunocapture-reverse transcription-polymerase chain reaction was estimated to be about 100 times more sensitive than double-antibody sandwich-enzyme-linked immunosorbent assay (DAS-ELISA) and showed an equivalent specificity. Based on the observation that PNRSV multiplies actively in young growing tissues (axillary shoots and cuttings), an in vitro culture method allowing rapid (about 15 days) and homogeneous development of dormant axillary buds with high virus titers was standardized. ELISA tests of these young shoots showed, in some cases, a 10(4) to 10(5) increase in sensitivity in comparison to adjacent leaf tissues from the rose mother plants. Between 21 and 98% (depending on the season) more samples were identified as positive by using ELISA on samples from shoot tips grown in vitro rather than on leaves collected directly from the PNRSV-infected mother plants. This simple method of growing shoot tips in vitro improved the confidence in the detection of PNRSV and eliminated problems in sampling appropriate tissues.

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

PubMed

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

2016-05-01

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

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

PubMed

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

2009-08-01

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

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wang, Liming; Du, Dan; Lu, Donglai

2011-05-05

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

Evaluation of a new serological technique for detecting rabies virus antibodies following vaccination.

PubMed

Ma, Xiaoyue; Niezgoda, Michael; Blanton, Jesse D; Recuenco, Sergio; Rupprecht, Charles E

2012-08-03

Two major techniques are currently used to estimate rabies virus antibody values: neutralization assays, such as the rapid fluorescent focus inhibition test (RFFIT), and enzyme-linked immunosorbent assays (ELISAs). The RFFIT is considered the gold standard assay and has been used to assess the titer of rabies virus neutralizing antibodies for more than three decades. In the late 1970s, ELISA began to be used to estimate the level of rabies virus antibody and has recently been used by some laboratories as an alternate screening test for animal sera. Although the ELISA appears simpler, safer and more efficient, the assay is less sensitive in detecting low values of rabies virus neutralizing antibodies than neutralization tests. This study was designed to evaluate a new ELISA-based method for detecting rabies virus binding antibody. This new technique uses electro-chemi-luminescence labels and carbon electrode plates to detect binding events. In this comparative study, the RFFIT and the new ELISA-based technique were used to evaluate the level of rabies virus antibodies in human and animal serum samples. By using a conservative approximation of 0.15 IU/ml as a cutoff point, the new ELISA-based technique demonstrated a sensitivity of 100% and a specificity of 95% for human samples and for experimental animal samples. The sensitivity and specificity for field animal samples was 96% and 95%, respectively. The preliminary results from this study appear promising and demonstrate a higher sensitivity than traditional ELISA methods. Published by Elsevier Ltd.

Identification and strain differentiation of Vibrio cholerae by using polyclonal antibodies against outer membrane proteins.

PubMed

Martínez-Govea, A; Ambrosio, J; Gutiérrez-Cogco, L; Flisser, A

2001-07-01

Cholera is caused only by O1 and O139 Vibrio cholerae strains. For diagnosis, 3 working days are needed for bacterial isolation from human feces and for biochemical characterization. Here we describe the purification of bacterial outer membrane proteins (OMP) from V. cholerae O1 Ogawa, O1 Inaba, and O139 strains, as well as the production of specific antisera and their use for fecal Vibrio antigen detection. Anti-OMP antisera showed very high reactivity and specificity by enzyme-linked immunosorbent assay (ELISA) and dot-ELISA. An inmunodiagnostic assay for V. cholerae detection was developed; this assay avoids preenrichment and costly equipment and can be used for epidemiological surveillance and clinical diagnosis of cases, considering that prompt and specific identification of bacteria is mandatory in cholera.

Recombinant truncated nucleocapsid protein as antigen in a novel immunoglobulin M capture enzyme-linked immunosorbent assay for diagnosis of severe acute respiratory syndrome coronavirus infection.

PubMed

Yu, Fuxun; Le, Mai Quynh; Inoue, Shingo; Hasebe, Futoshi; Parquet, Maria del Carmen; Morikawa, Shigeru; Morita, Kouichi

2007-02-01

We report the development of an immunoglobulin M (IgM) antibody capture enzyme-linked immunosorbent assay (MAC-ELISA) for severe acute respiratory syndrome coronavirus (SARS-CoV) by using recombinant truncated SARS-CoV nucleocapsid protein as the antigen. The newly developed MAC-ELISA had a specificity and sensitivity of 100% as evaluated by using sera from healthy volunteers and patients with laboratory-confirmed SARS. Using serial serum samples collected from SARS patients, the times to seroconversion were determined by IgM antibody detection after SARS-CoV infection. The median time to seroconversion detection was 8 days (range, 5 to 17 days) after disease onset, and the seroconversion rates after the onset of illness were 33% by the first week, 97% by the second week, and 100% by the third week. Compared with the results of our previous report on the detection of IgG, the median seroconversion time by IgM detection was 3 days earlier and the seroconversion rate by the second week after the illness for IgM was significantly higher than by IgG assay. Our results indicating that the IgM response appears earlier than IgG after SARS-CoV infection in consistent with those for other pathogens. Our newly developed MAC-ELISA system offers a new alternative for the confirmation of SARS-CoV infection.

Recombinant Truncated Nucleocapsid Protein as Antigen in a Novel Immunoglobulin M Capture Enzyme-Linked Immunosorbent Assay for Diagnosis of Severe Acute Respiratory Syndrome Coronavirus Infection▿

PubMed Central

Yu, Fuxun; Le, Mai Quynh; Inoue, Shingo; Hasebe, Futoshi; Parquet, Maria del Carmen; Morikawa, Shigeru; Morita, Kouichi

2007-01-01

We report the development of an immunoglobulin M (IgM) antibody capture enzyme-linked immunosorbent assay (MAC-ELISA) for severe acute respiratory syndrome coronavirus (SARS-CoV) by using recombinant truncated SARS-CoV nucleocapsid protein as the antigen. The newly developed MAC-ELISA had a specificity and sensitivity of 100% as evaluated by using sera from healthy volunteers and patients with laboratory-confirmed SARS. Using serial serum samples collected from SARS patients, the times to seroconversion were determined by IgM antibody detection after SARS-CoV infection. The median time to seroconversion detection was 8 days (range, 5 to 17 days) after disease onset, and the seroconversion rates after the onset of illness were 33% by the first week, 97% by the second week, and 100% by the third week. Compared with the results of our previous report on the detection of IgG, the median seroconversion time by IgM detection was 3 days earlier and the seroconversion rate by the second week after the illness for IgM was significantly higher than by IgG assay. Our results indicating that the IgM response appears earlier than IgG after SARS-CoV infection in consistent with those for other pathogens. Our newly developed MAC-ELISA system offers a new alternative for the confirmation of SARS-CoV infection. PMID:17202310

Management of bacterial kidney disease in Chinook Salmon hatcheries based on broodstock testing by enzyme-linked immunosorbent assay: A multiyear study

USGS Publications Warehouse

Munson, A. Douglas; Elliott, Diane G.; Johnson, Keith

2010-01-01

From the mid-1980s through the early 1990s, outbreaks of bacterial kidney disease (BKD) caused by Renibacterium salmoninarum continued in Chinook salmon Oncorhynchus tshawytscha in Idaho Department of Fish and Game (IDFG) hatcheries despite the use of three control methods: (1) injection of returning adult fish with erythromycin to reduce prespawning BKD mortality and limit vertical transmission of R. salmoninarum, (2) topical disinfection of green eggs with iodophor, and (3) prophylactic treatments of juvenile fish with erythromycin-medicated feed. In addition, programs to manage BKD through measurement of R. salmoninarum antigen levels in kidney tissues from spawning female Chinook salmon by an enzyme-linked immunosorbent assay (ELISA) were tested over 13–15 brood years at three IDFG hatcheries. The ELISA results were used for either (1) segregated rearing of progeny from females with high ELISA optical density (OD) values (usually ≥0.25), which are indicative of high R. salmoninarum antigen levels, or (2) culling of eggs from females with high ELISA OD values. The ELISA-based culling program had the most profound positive effects on the study populations. Mortality of juvenile fish during rearing was significantly lower at each hatchery for brood years derived from culling compared with brood years for which culling was not practiced. The prevalence of R. salmoninarum in juvenile fish, as evidenced by detection of the bacterium in kidney smears by the direct fluorescent antibody test, also decreased significantly at each hatchery. In addition, the proportions of returning adult females with kidney ELISA OD values of 0.25 or more decreased 56–85% for fish reared in brood years during which culling was practiced, whereas the proportions of ELISA-negative adults increased 55–58%. This management strategy may allow IDFG Chinook salmon hatcheries to reduce or eliminate prophylactic erythromycin-medicated feed treatments. We recommend using ELISA-based management of BKD in Chinook salmon hatcheries where it is a concern.

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

PubMed Central

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

2017-01-01

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

Development and Characterization of Monoclonal Antibodies to Yellow Fever Virus and Application in Antigen Detection and IgM Capture Enzyme-Linked Immunosorbent Assay

PubMed Central

Adungo, Ferdinard; Kamau, David; Inoue, Shingo; Hayasaka, Daisuke; Posadas-Herrera, Guillermo; Sang, Rosemary; Mwau, Matilu

2016-01-01

Yellow fever (YF) is an acute hemorrhagic viral infection transmitted by mosquitoes in Africa and South America. The major challenge in YF disease detection and confirmation of outbreaks in Africa is the limited availability of reference laboratories and the persistent lack of access to diagnostic tests. We used wild-type YF virus sequences to generate recombinant envelope protein in an Escherichia coli expression system. Both the recombinant protein and sucrose gradient-purified YF vaccine virus 17D (YF-17D) were used to immunize BALB/c mice to generate monoclonal antibodies (MAbs). Eight MAbs were established and systematically characterized by indirect enzyme-linked immunosorbent assay (ELISA), Western blot analysis, and immunofluorescence assay (IFA). The established MAbs showed strong reactivity with wild-type YF virus and recombinant protein with no detectable cross-reactivity to dengue virus or Japanese encephalitis virus. Epitope mapping showed strong binding of three MAbs to amino acid positions 1 to 51, while two MAbs mapped to amino acid positions 52 to 135 of the envelope protein. The remaining three MAbs did not show reactivity to envelope fragments. The established MAbs exert no neutralization against wild-type YF and 17D viruses (titer of <10 for both strains). The applicability of MAbs 8H3 and 3F4 was further evaluated using IgM capture ELISA. A total of 49 serum samples were analyzed, among which 12 positive patient and vaccinee samples were correctly identified. Using serum samples that were 2-fold serially diluted, the IgM capture ELISA was able to detect all YF-positive samples. Furthermore, MAb-based antigen detection ELISA enabled the detection of virus in culture supernatants containing titers of about 1,000 focus-forming units. PMID:27307452

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.

Medical Surveillance Monthly Report (MSMR). Volume 21, Number 11, November 2014

DTIC Science & Technology

2014-11-01

enzyme -linked immu- nosorbent assay (ELISA) utilizing an outer membrane protein antigen from C. jejuni Penner serotypes 1, 2, and 3.20 Acute and...Human serum antibody response to Campylobacter jejuni infection as measured in an enzyme -linked immunosorbent assay. Infect Immun. 1984;44: 292–298...Georgia: USD, Inc., 1990. 23. Coates D, Hutchinson DN, Bolton FJ. Survival of thermophilic Campylobacters on fi ngertips and their elimination by

Dual-mode lensless imaging device for digital enzyme linked immunosorbent assay

NASA Astrophysics Data System (ADS)

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

2014-03-01

Digital enzyme linked immunosorbent assay (ELISA) is an ultra-sensitive technology for detecting biomarkers and viruses etc. As a conventional ELISA technique, a target molecule is bonded to an antibody with an enzyme by antigen-antibody reaction. In this technology, a femto-liter droplet chamber array is used as reaction chambers. Due to its small volume, the concentration of fluorescent product by single enzyme can be sufficient for detection by a fluorescent microscopy. In this work, we demonstrate a miniaturized lensless imaging device for digital ELISA by using a custom image sensor. The pixel array of the sensor is coated with a 20 μm-thick yellow filter to eliminate excitation light at 470 nm and covered by a fiber optic plate (FOP) to protect the sensor without resolution degradation. The droplet chamber array formed on a 50μm-thick glass plate is directly placed on the FOP. In the digital ELISA, microbeads coated with antibody are loaded into the droplet chamber array, and the ratio of the fluorescent to the non-fluorescent chambers with the microbeads are observed. In the fluorescence imaging, the spatial resolution is degraded by the spreading through the glass plate because the fluorescence is irradiated omnidirectionally. This degradation is compensated by image processing and the resolution of ~35 μm was achieved. In the bright field imaging, the projected images of the beads with collimated illumination are observed. By varying the incident angle and image composition, microbeads were successfully imaged.

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

Evaluation of immunity to varicella zoster virus with a novel double antigen sandwich enzyme-linked immunosorbent assay.

PubMed

Liu, Jian; Chen, Chunye; Zhu, Rui; Ye, Xiangzhong; Jia, Jizong; Yang, Lianwei; Wang, Yongmei; Wang, Wei; Ye, Jianghui; Li, Yimin; Zhu, Hua; Zhao, Qinjian; Zhang, Jun; Cheng, Tong; Xia, Ningshao

2016-11-01

Varicella is a highly contagious disease caused by primary infection of Varicella zoster virus (VZV). Varicella can be severe or even lethal in susceptible adults, immunocompromised patients and neonates. Determination of the status of immunity to VZV is recommended for these high-risk populations. Furthermore, measurement of population immunity to VZV can help in developing proper varicella vaccination programmes. VZV glycoprotein E (gE) is an antigen that has been demonstrated to be a highly accurate indicator of VZV-specific immunity. In this study, recombinant gE (rgE) was used to establish a double antigen sandwich enzyme-linked immunosorbent assay (ELISA). The established sandwich ELISA showed high specificity and sensitivity in the detection of human sera, and it could detect VZV-specific antibodies at a concentration of 11.25 m IU/mL with a detection linearity interval of 11.25 to 360 m IU/mL (R 2  = 0.9985). The double gE antigen sandwich ELISA showed a sensitivity of 95.08 % and specificity of 100 % compared to the fluorescent-antibody-to-membrane-antigen (FAMA) test, and it showed a sensitivity of 100 % and a specificity of 94.74 % compared to a commercial neutralizing antibody detection kit. Thus, the established double antigen sandwich ELISA can be used as a sensitive and specific quantitative method to evaluate immunity to VZV.

Development of duplex RT-PCR-ELISA for the simultaneous detection of hepatitis A virus and hepatitis E virus.

PubMed

Tahk, Hongmin; Lee, Min Hwa; Lee, Kang Bum; Cheon, Doo-Sung; Choi, Changsun

2011-07-01

This study aimed to develop a specific and sensitive duplex reverse transcription polymerase chain reaction enzyme-linked immunosorbent assay (duplex RT-PCR-ELISA) for hepatitis A virus (HAV) and hepatitis E virus (HEV). Duplex RT-PCR-ELISA could detect and differentiate HAV and HEV with specific probes. When ELISA technique was used to detect probe-bound RT-PCR products, duplex RT-PCR-ELISA could detect as little as 0.1 ng/μL HAV and HEV from clinical samples. Human norovirus, enterovirus, poliovirus, murine norovirus and feline calicivirus were used for the specificity test; all were negative. Therefore duplex RT-PCR-ELISA can be used for the simultaneous detection of HAV and HEV in contaminated fecal samples. Copyright © 2011 Elsevier B.V. All rights reserved.

A highly sensitive and selective immunoassay for the detection of tetrabromobisphenol A in soil and sediment

USDA-ARS?s Scientific Manuscript database

Tetrabromobisphenol A (TBBPA) is the most widely used brominated flame retardant. A sensitive and selective enzyme-linked immunosorbent assay (ELISA) for the detection of TBBPA was developed. Six haptens (T1-T6) mimicking different structural elements of TBBPA were synthesized and coupled to keyhole...

Environmental Technology Verification Report for Abraxis 17β-Estradiol (E2) Magnetic Particle Enzyme-Linked Immunosorbent Assay (ELISA) Test Kits

EPA Science Inventory

The EPA's National Risk Management Research Laboratory (NRMRL) and its verification organization partner, Battelle, operate the Advanced Monitoring Systems (AMS) Center under ETV. The AMS Center recently evaluated the performance of the Abraxis 17(beta)-estradiol (E2) magnetic p...

Enzyme immunoassays for detection of gypsy moth nuclear polyhedrosis virus

Treesearch

Michael Ma

1985-01-01

Enzyme-linked immunosorbent assays (ELISA) were developed for detecting gypsy moth (Lymantria dispar L.) nuclear polyhedrosis virus (NPV). They were used to detect the presence of NPV in hemoplymph samples collected from infected larvae. The incorporation of hybridoma antibodies with these procedures would make them even more specific for gypsy moth...

Spatial and temporal distribution of imidacloprid in eastern hemlock

Treesearch

Richard M. Turcotte

2007-01-01

Enzyme-linked immunosorbent assay (ELISA) and gas chromatography/mass spectrometry (GC/MS) techniques were used to measure imidacloprid and metabolite concentrations in xylem fluid extracted from eastern hemlock (Tsuga canadensis) trees treated in the spring or fall with soil or trunk applications of the systemic insecticides Merit® 2F and...

COMPARATIVE ESTROGENICITY OF ESTRADIOL, ETHYNYL ESTRADIOL AND DIETHYLSTILBESTROL IN AN IN VIVO, MALE SHEEPSHEAD MINNOW (CYPRINODON VARIEGATUS), VITELLOGENIN BIOASSAY

EPA Science Inventory

An in vivo bioassay for vitellogenin (VTG) synthesis was developed to screen individual chemicals or mixtures of chemicals for potentially estrogenic effects in a marine teleost model. An enzyme-linked immunosorbent assay (ELISA) was used to quantitate VTG synthesis in male sheep...

A serologic survey of Mycoplasma spp. in farmed bison (Bison bison) herds in western Canada

USDA-ARS?s Scientific Manuscript database

Mycoplasma bovis is emerging as an important pathogen of farmed bison in North America, associated with high morbidity and mortality. An in-house enzyme-linked immunosorbent assay (ELISA) was developed to detect antibodies against Mycoplasma sp. in bison sera. The aims of the study were to estimate ...

Discerning Silk Produced by Bombyx mori from Those Produced by Wild Species Using an Enzyme-Linked Immunosorbent Assay Combined with Conventional Methods.

PubMed

You, Qiushi; Li, Qingqing; Zheng, Hailing; Hu, Zhiwen; Zhou, Yang; Wang, Bing

2017-09-06

Recently, much interest has been paid to the separation of silk produced by Bombyx mori from silk produced by other species and tracing the beginnings of silk cultivation from wild silk exploitation. In this paper, significant differences between silks from Bombyx mori and other species were found by microscopy and spectroscopy, such as morphology, secondary structure, and amino acid composition. For further accurate identification, a diagnostic antibody was designed by comparing the peptide sequences of silks produced by Bombyx mori and other species. The results of the noncompetitive indirect enzyme-linked immunosorbent assay (ELISA) indicated that the antibody that showed good sensitivity and high specificity can definitely discern silk produced by Bombyx mori from silk produced by wild species. Thus, the antibody-based immunoassay has the potential to be a powerful tool for tracing the beginnings of silk cultivation. In addition, combining the sensitive, specific, and convenient ELISA technology with other conventional methods can provide more in-depth and accurate information for species identification.

Use of enzyme-linked immunosorbent assay to screen for aflatoxins, ochratoxin A, and deoxynivalenol in dry pet foods.

PubMed

Okuma, Tara A; Huynh, Thu P; Hellberg, Rosalee S

2018-03-01

The objective of this study was to perform a market survey on dry pet foods using enzyme-linked immunosorbent assay (ELISA) to detect total aflatoxins (AFs), ochratoxin A (OTA), and deoxynivalenol (DON). Pet food products (n = 58) marketed for dogs, cats, birds, and rabbits were tested in duplicate with ELISA, and results above the limit of quantitation were confirmed using liquid chromatography tandem mass spectrometry (LC-MS/MS). OTA was detected in one product (rabbit food) and AFs were detected in two products (one dog treat and one bird treat). In contrast, DON was detected in the majority (74%) of products tested. Bird and rabbit products were the most affected by DON, with levels above 0.5 μg/g in 50 and 80% of samples, respectively. One rabbit sample tested positive for both OTA and DON. Overall, the findings of this study revealed a low incidence of AFs and OTA in commercial pet food. Although DON was detected in numerous products, the levels were well below those associated with acute toxic effects.

Development of a monoclonal antibody-based sandwich-type enzyme-linked immunosorbent assay (ELISA) for detection of abrin in food samples.

PubMed

Zhou, Yu; Tian, Xiang-Li; Li, Yan-Song; Pan, Feng-Guang; Zhang, Yuan-Yuan; Zhang, Jun-Hui; Wang, Xin-Rui; Ren, Hong-Lin; Lu, Shi-Ying; Li, Zhao-Hui; Liu, Zeng-Shan; Chen, Qi-Jun; Liu, Jing-Qiu

2012-12-15

Abrin is a plant toxin, which can be easily isolated from the seeds of Abrus precatorius. It may be used as a biological warfare agent. In order to detect abrin in food samples, a two-layer sandwich format enzyme-linked immunosorbent assay based on the monoclonal antibody (mAb) (as capture antibody) and rabbit polyclonal serum (as detecting antibody) was developed and applied for the determination of abrin in some food matrices. The linear range of the mAb was 1-100 μg L(-1) with a detection limit of 0.5 μg L(-1) for abrin in phosphate buffered saline (PBS). The recoveries of abrin from sausage, beer and milk samples ranged 97.5-98.6%, 95.8-98.4% and 94.8-9.6%, respectively, with a coefficient of variation (CV) of 3.7% or less. The newly developed sandwich ELISA using the mAb appears to be a reliable and useful method for detection of abrin in sausage, beer and milk. Copyright © 2012 Elsevier Ltd. All rights reserved.

Production of a monoclonal antibody against serum immunoglobulin M of South American camelids and assessment of its suitability in two immunoassays.

PubMed

Friedrich, Adrián; Ledesma, Martín; Landone, Ignacio; Ferrari, Alejandro; Leoni, Juliana

2014-09-01

A monoclonal antibody (mAb) was produced against immunoglobulin M (IgM) of South American camelids. A single radial immunodiffusion (SRID) assay and a competitive enzyme-linked immunosorbent assay (ELISA) were developed to measure IgM in serum samples. Isotype and specificity of the mAb were assessed. The performance of the SRID assay was preliminarily evaluated in terms of working range, plate stability over a 4-week period, and initial intra- and interassay variation. The concentration of IgM was determined in 55 samples by SRID assay and ELISA, and results were not significantly different by t-test (0.64 ± 0.19 mg/ml for the SRID assay, and 0.58 ± 0.24 mg/ml for ELISA; P = 0.1489). The mAb was shown to be stable over the 4-week evaluation period, and the SRID assay was reproducible when tested in triplicate for intra-assay variability and in quadruplicate for interassay variability, with a percentage coefficient of variation of less than or equal to 5%. Also, the SRID assay proved to be sensitive enough to measure IgM levels in undiluted serum samples, and had a good correlation with ELISA. The current study is intended to submit a preliminary report of a mAb against IgM of South American camelids, and suggest the future potential of the mAb developed for diagnostic application, including use in the SRID assay. © 2014 The Author(s).

Immunoassays for pesticide monitoring

NASA Astrophysics Data System (ADS)

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

1995-05-01

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

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

Development of an indirect enzyme linked immunoassay for abscisic acid. [Pisum sativum

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ross, G.S.; Elder, P.A.; McWha, J.A.

1987-09-01

AN INDIRECT METHOD OF ENZYME-LINKED-IMMUNOSORBENT-ASSAY (ELISA) IS REPORTED FOR ABSCISIC ACID (ABA), UTILIZING A THYROGLOBULIN-ABA CONJUGATE FOR COATING WELLS. THE ASSAY CAN USE COMMERCIALLY AVAILABLE MONOCLONAL ANTIBODIES, IS SENSITIVE TO AS LITTLE AS 20 PICOGRAMS ABA PER WELL, AND IS MUCH MORE CONSERVATIVE OF ANTIBODY THAN DIRECT METHODS. THE MOST DILUTE ABA STANDARDS DID NOT RETAIN THEIR ANTIGENICITY DURING STORAGE, SO ABA STANDARD SETS WERE DILUTED IMMEDIATELY PRIOR TO USE. THE INDIRECT ELISA WAS USED SUCCESSFULLY TO ESTIMATE ABA CONCENTRATIONS IN DEVELOPING COTYLEDONS OF PISUM SATIVUM L., AFTER ONLY LITTLE PRELIMINARY PURIFICATION. IT WAS VALIDATED FOR THIS TISSUE THROUGH THEmore » USE OF GAS CHROMATOGRAPHY-ELECTRON CAPTURE DETECTION (GC-EC), AND CAPILLARY GC-SELECTED ION MONITORING (GC-MS-SIM) USING LABELLED ABA AS AN INTERNAL STANDARD. FULL SPECTRUM GC-MASS SPECTROMETRY WAS ALSO USED TO VERIFY THAT ABA WAS PRESENT IN A SAMPLE ASSAYED QUANTITATIVELY BY BOTH ELISA AND GC-MS-SIM.« less

Validation of high throughput screening of human sera for detection of anti-PA IgG by Enzyme-Linked Immunosorbent Assay (ELISA) as an emergency response to an anthrax incident

PubMed Central

Semenova, Vera A.; Steward-Clark, Evelene; Maniatis, Panagiotis; Epperson, Monica; Sabnis, Amit; Schiffer, Jarad

2017-01-01

To improve surge testing capability for a response to a release of Bacillus anthracis, the CDC anti-Protective Antigen (PA) IgG Enzyme-Linked Immunosorbent Assay (ELISA) was re-designed into a high throughput screening format. The following assay performance parameters were evaluated: goodness of fit (measured as the mean reference standard r2), accuracy (measured as percent error), precision (measured as coefficient of variance (CV)), lower limit of detection (LLOD), lower limit of quantification (LLOQ), dilutional linearity, diagnostic sensitivity (DSN) and diagnostic specificity (DSP). The paired sets of data for each sample were evaluated by Concordance Correlation Coefficient (CCC) analysis. The goodness of fit was 0.999; percent error between the expected and observed concentration for each sample ranged from −4.6% to 14.4%. The coefficient of variance ranged from 9.0% to 21.2%. The assay LLOQ was 2.6 μg/mL. The regression analysis results for dilutional linearity data were r2 = 0.952, slope = 1.02 and intercept = −0.03. CCC between assays was 0.974 for the median concentration of serum samples. The accuracy and precision components of CCC were 0.997 and 0.977, respectively. This high throughput screening assay is precise, accurate, sensitive and specific. Anti-PA IgG concentrations determined using two different assays proved high levels of agreement. The method will improve surge testing capability 18-fold from 4 to 72 sera per assay plate. PMID:27814939

Validation of high throughput screening of human sera for detection of anti-PA IgG by Enzyme-Linked Immunosorbent Assay (ELISA) as an emergency response to an anthrax incident.

PubMed

Semenova, Vera A; Steward-Clark, Evelene; Maniatis, Panagiotis; Epperson, Monica; Sabnis, Amit; Schiffer, Jarad

2017-01-01

To improve surge testing capability for a response to a release of Bacillus anthracis, the CDC anti-Protective Antigen (PA) IgG Enzyme-Linked Immunosorbent Assay (ELISA) was re-designed into a high throughput screening format. The following assay performance parameters were evaluated: goodness of fit (measured as the mean reference standard r 2 ), accuracy (measured as percent error), precision (measured as coefficient of variance (CV)), lower limit of detection (LLOD), lower limit of quantification (LLOQ), dilutional linearity, diagnostic sensitivity (DSN) and diagnostic specificity (DSP). The paired sets of data for each sample were evaluated by Concordance Correlation Coefficient (CCC) analysis. The goodness of fit was 0.999; percent error between the expected and observed concentration for each sample ranged from -4.6% to 14.4%. The coefficient of variance ranged from 9.0% to 21.2%. The assay LLOQ was 2.6 μg/mL. The regression analysis results for dilutional linearity data were r 2  = 0.952, slope = 1.02 and intercept = -0.03. CCC between assays was 0.974 for the median concentration of serum samples. The accuracy and precision components of CCC were 0.997 and 0.977, respectively. This high throughput screening assay is precise, accurate, sensitive and specific. Anti-PA IgG concentrations determined using two different assays proved high levels of agreement. The method will improve surge testing capability 18-fold from 4 to 72 sera per assay plate. Published by Elsevier Ltd.

The plasma interleukin-6 response to acute psychosocial stress in humans is detected by a magnetic multiplex assay: comparison to high-sensitivity ELISA.

PubMed

Quinn, Andrea M; Williams, Allison R; Sivilli, Teresa I; Raison, Charles L; Pace, Thaddeus W W

2018-03-13

Circulating concentrations of interleukin (IL)-6, an inflammatory biomarker widely assessed in humans to study the inflammatory response to acute psychological stress, have for decades been quantified using enzyme-linked immunosorbent assay (ELISA). However, biobehavioral researchers are increasingly using cytokine multiplex assays instead of ELISA to measure IL-6 and other cytokines. Despite this trend, multiplex assays have not been directly compared to ELISA for their ability to detect subtle stress-induced changes of IL-6. Here, we tested the prediction that a high-sensitivity multiplex assay (human Magnetic Luminex Performance Assay, R&D Systems, Minneapolis, MN) would detect changes in IL-6 as a result of acute stress challenge in a manner comparable to high-sensitivity ELISA. Blood was collected from 12 healthy adults immediately before and then 90 and 210 min after the start of the Trier Social Stress Test (TSST), an acute laboratory psychosocial stress challenge. In addition to quantifying IL-6 concentrations in plasma with both multiplex and ELISA, we also assessed concentrations of tumor necrosis factor-alpha, IL-8, IL-10, IL-5, and IL-2 with multiplex. The multiplex detected IL-6 in all samples. Concentrations strongly correlated with values determined by ELISA across all samples (r = 0.941, p < .001) as well as among samples collected at individual TSST time points. IL-6 responses to the TSST (i.e. area under the curve) captured by multiplex and ELISA were also strongly correlated (r s   = 0.937, p < .001). While other cytokines were detected by multiplex, none changed as a result of TSST challenge at time points examined. These results suggest high-sensitivity magnetic multiplex assay is able to detect changes in plasma concentrations of IL-6 as a result of acute stress in humans.

Evaluation of an Erns-based enzyme-linked immunosorbent assay to distinguish Classical swine fever virus-infected pigs from pigs vaccinated with CP7_E2alf.

PubMed

Pannhorst, Katrin; Fröhlich, Andreas; Staubach, Christoph; Meyer, Denise; Blome, Sandra; Becher, Paul

2015-07-01

Infections with Classical swine fever virus (CSFV) are a major economic threat to pig production. To combat CSF outbreaks and to maintain trade, new marker vaccines were developed that allow differentiation of infected from vaccinated animals (DIVA principle). The chimeric pestivirus CP7_E2alf was shown to be safe and efficacious. Its DIVA strategy is based on the detection of CSFV E(rns)-specific antibodies that are only developed on infection. However, for the new marker vaccine to be considered a valuable control tool, a validated discriminatory assay is needed. One promising candidate is the already commercially available enzyme-linked immunosorbent assay, PrioCHECK CSFV E(rns) ELISA (Prionics BV, Lelystad, The Netherlands). Four laboratories of different European Union member states tested 530 serum samples and country-specific field sera from domestic pigs and wild boar. The ELISA displayed a good robustness. However, based on its reproducibility and repeatability, ranges rather than single values for diagnostic sensitivity and specificity were defined. The ELISA displayed a sensitivity of 90-98% with sera from CSFV-infected domestic pigs. A specificity of 89-96% was calculated with sera from domestic pigs vaccinated once with CP7_E2alf. The ELISA detected CSFV infections in vaccinated domestic pigs with a sensitivity of 82-94%. The sensitivity was lower with sera taken ≤21 days post-challenge indicating that the stage of CSFV infection had a considerable influence on testing. Taken together, the PrioCHECK CSFV E(rns) ELISA can be used for detection of CSFV infections in CP7_E2alf-vaccinated and nonvaccinated domestic pig populations, but should only be applied on a herd basis by testing a defined number of animals. © 2015 The Author(s).

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

PubMed

Ciaurriz, Paula; Fernández, Fátima; Tellechea, Edurne; Moran, Jose F; Asensio, Aaron C

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.

Effects of grain species and cultivar, thermal processing, and enzymatic hydrolysis on gluten quantitation.

PubMed

Pahlavan, Autusa; Sharma, Girdhari M; Pereira, Marion; Williams, Kristina M

2016-10-01

Gluten from wheat, rye, and barley can trigger IgE-mediated allergy or Celiac disease in sensitive individuals. Gluten-free labeled foods are available as a safe alternative. Immunoassays such as the enzyme-linked immunosorbent assay (ELISA) are commonly used to quantify gluten in foods. However, various non-assay related factors can affect gluten quantitation. The effect of gluten-containing grain cultivars, thermal processing, and enzymatic hydrolysis on gluten quantitation by various ELISA kits was evaluated. The ELISA kits exhibited variations in gluten quantitation depending on the gluten-containing grain and their cultivars. Acceptable gluten recoveries were obtained in 200mg/kg wheat, rye, and barley-spiked corn flour thermally processed at various conditions. However, depending on the enzyme, gluten grain source, and ELISA kit used, measured gluten content was significantly reduced in corn flour spiked with 200mg/kg hydrolyzed wheat, rye, and barley flour. Thus, the gluten grain source and processing conditions should be considered for accurate gluten analysis. Published by Elsevier Ltd.

Evaluation of Leishmania species reactivity in human serologic diagnosis of leishmaniasis.

PubMed

Silvestre, Ricardo; Santarém, Nuno; Teixeira, Lúcia; Cunha, Joana; Schallig, Henk; Cordeiro-da-Silva, Anabela

2009-08-01

The sensitivities and specificities of IgG-ELISA and IgG flow cytometry based techniques using different Leishmania species were determined using sera collected from 40 cutaneous or visceral leishmaniasis patients. The flow cytometry technique, using promastigote parasite forms, performed better than total soluble extract IgG-ELISA. At the species level, the use of Leishmania amazonensis and Leishmania major as antigens in enzyme linked immunosorbent assay (ELISA) decreased the overall sensitivity. To assess the specificity of these tests, sera from malaria, toxoplasmosis, amoebiasis, schistosomiasis, and leprosy patients were used. We also included sera from Leishmania non-infected endemic individuals. The cutaneous species displayed a decreased specificity in both assays. Although more sensitive, flow cytometry using promastigote parasite forms generally presented lower levels of specificity when compared with total extract of IgG-ELISA. Overall, the results of the study show the potential of IgG flow cytometry for the diagnosis of leishmaniasis. Although highly sensitive, a refinement of the flow cytometry method should be performed to improve the overall specificity.

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

Evaluation of Two Enzyme-Linked Immunosorbent Assay Kits for Chikungunya Virus IgM Using Samples from Deceased Organ and Tissue Donors.

PubMed

Prince, Harry E; Altrich, Michelle L; Nowicki, Marek J

2016-10-01

The identification of nearly 3,500 cases of chikungunya virus (CHIKV) infection in U.S. residents returning in 2014 and 2015 from areas in which it is endemic has raised concerns within the transplant community that, should recently infected individuals become organ and/or tissue donors, CHIKV would be transmitted to transplant recipients. Thus, tests designed to detect recent CHIKV infection among U.S. organ and tissue donors may become necessary in the future. Accordingly, we evaluated 2 enzyme-linked immunosorbent assays (ELISAs) for CHIKV IgM readily available in the United States using 1,000 deidentified serum or plasma specimens collected from donors between November 2014 and March 2015. The Euroimmun indirect ELISA identified 38 reactive specimens; however, all 38 were negative for CHIKV IgG and IgM in immunofluorescence assays (IFAs) conducted at a reference laboratory and, thus, were falsely reactive in the Euroimmun CHIKV IgM assay. The InBios IgM-capture ELISA identified 26 reactive samples, and one was still reactive (index ≥ 1.00) when retested using the InBios kit with a background subtraction modification to identify false reactivity. This reactive specimen was CHIKV IgM negative but IgG positive by IFAs at two reference laboratories; plaque reduction neutralization testing (PRNT) demonstrated CHIKV-specific reactivity. The IgG and PRNT findings strongly suggest that the InBios CHIKV IgM-reactive result represents true reactivity, even though the IgM IFA result was negative. If testing organ/tissue donors for CHIKV IgM becomes necessary, the limitations of the currently available CHIKV IgM ELISAs and options for their optimization must be understood to avoid organ/tissue wastage due to falsely reactive results. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

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

PubMed

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

2016-11-01

Bile acids are promising chemical markers to assess the pollution of water samples with fecal material. This study describes the optimization and validation of a direct competitive enzyme-linked immunosorbent assay for the bile acid isolithocholic acid (ILA). The quantification range of the optimized assay was between 0.09 and 15 μg/L. The assay was applied to environmental water samples. Most studies until now were focused on bile acid fractions in the particulate phase of water samples. In order to avoid tedious sample preparation, we undertook to evaluate the dynamics and significance of ILA levels in the aqueous phase. Very low concentrations in tap and surface water samples made a pre-concentration step necessary for this matrix as well as for wastewater treatment plant (WWTP) effluent. Mean recoveries for spiked water samples were between 97% and 109% for tap water and WWTP influent samples and between 102% and 136% for WWTP effluent samples. 90th percentiles of intra-plate and inter-plate coefficients of variation were below 10% for influents and below 20% for effluents and surface water. ILA concentrations were quantified in the range of 33-72 μg/L in influent, 21-49 ng/L in effluent and 18-48 ng/L in surface water samples. During wastewater treatment the ILA levels were reduced by more than 99%. ILA concentrations of influents determined by ELISA and LC-MS/MS were in good agreement. However, findings in LC-ELISA experiments suggest that the true ILA levels in concentrated samples are lower due to interfering effects of matrix compounds and/or cross-reactants. Yet, the ELISA will be a valuable tool for the performance check and comparison of WWTPs and the localization of fecal matter input into surface waters. Copyright © 2016 Elsevier Ltd. All rights reserved.

Comparison of desmoglein ELISA and indirect immunofluorescence using two substrates (monkey oesophagus and normal human skin) in the diagnosis of pemphigus.

PubMed

Ng, Patricia P L; Thng, Steven T G; Mohamed, Khatija; Tan, Suat Hoon

2005-11-01

A prospective study was performed to assess the usefulness of desmoglein enzyme-linked immunosorbent assay testing compared with indirect immunofluorescence in the diagnosis of new cases of pemphigus, as well as to compare the relative sensitivities of monkey oesophagus and normal human skin as substrates for indirect immunofluorescence. These tests were performed on the sera of 29 consecutive new cases of pemphigus diagnosed over a 2-year period based on clinical, histological and direct immunofluorescence findings. Desmoglein enzyme-linked immunosorbent assay was positive in all patients whereas indirect immunofluorescence was positive in only 25 of 29 patients. All four patients with negative indirect immunofluorescence had positive antinuclear antibodies or cytoplasmic fluorescence that could have masked the anti-intercellular antibodies. Desmoglein enzyme-linked immunosorbent assay appeared to reflect the disease activity better than indirect immunofluorescence in a few patients who had active disease of recent onset. Monkey oesophagus was found to be superior or equal to human skin as a substrate for indirect immunofluorescence in both pemphigus vulgaris and foliaceus.

Effective surveillance for early classical swine fever virus detection will utilize both virus and antibody detection capabilities.

PubMed

Panyasing, Yaowalak; Kedkovid, Roongtham; Thanawongnuwech, Roongroje; Kittawornrat, Apisit; Ji, Ju; Giménez-Lirola, Luis; Zimmerman, Jeffrey

2018-03-01

Early recognition and rapid elimination of infected animals is key to controlling incursions of classical swine fever virus (CSFV). In this study, the diagnostic characteristics of 10 CSFV assays were evaluated using individual serum (n = 601) and/or oral fluid (n = 1417) samples collected from -14 to 28 days post inoculation (DPI). Serum samples were assayed by virus isolation (VI), 2 commercial antigen-capture enzyme-linked immunosorbent assays (ELISA), virus neutralization (VN), and 3 antibody ELISAs. Both serum and oral fluid samples were tested with 3 commercial real-time reverse transcription-polymerase chain reaction (rRT-PCR) assays. One or more serum samples was positive by VI from DPIs 3 to 21 and by antigen-capture ELISAs from DPIs 6 to 17. VN-positive serum samples were observed at DPIs ≥ 7 and by antibody ELISAs at DPIs ≥ 10. CSFV RNA was detected in serum samples from DPIs 2 to 28 and in oral fluid samples from DPIs 4 to 28. Significant differences in assay performance were detected, but most importantly, no single combination of sample and assay was able to dependably identify CSFV-inoculated pigs throughout the 4-week course of the study. The results show that effective surveillance for CSFV, especially low virulence strains, will require the use of PCR-based assays for the detection of early infections (<14 days) and antibody-based assays, thereafter. Copyright © 2018 Elsevier B.V. All rights reserved.

Evaluation of the Double Agar Gel Immunodiffusion Test and of the Enzyme-Linked Immunosorbent Assay in the Diagnosis and Follow-Up of Patients with Chronic Pulmonary Aspergillosis

PubMed Central

de Azevedo, Priscila Zacarias; Sylvestre, Tatiane Fernanda; Cavalcante, Ricardo de Souza; de Carvalho, Lídia Raquel; Moris, Daniela Vanessa; de Oliveira, Maria Luiza Cotrim Sartor; Mendes, Rinaldo Poncio

2015-01-01

The diagnosis of chronic pulmonary aspergillosis (CPA) depends on the radiologic image and the identification of specific antibodies. The present study aimed to evaluate accuracy parameters of enzyme-linked immunosorbent assay (ELISA) and of the determination of serum galactomannan level in the diagnosis of patients with CPA, comparing these results with the double agar gel immunodiffusion (DID) test. In addition, the prevalence of cross-reactivity and the serological progression after treatment were evaluated by comparing DID and ELISA. Six study groups were formed: G1: 22 patients with CPA, 17 of whom had Aspergillus fungus ball, one chronic cavitary pulmonary aspergillosis (CCPA) and four chronic fibrosing pulmonary aspergillosis (CFPA); G2: 28 patients with pulmonary tuberculosis (TB); G3: 23 patients with histoplasmosis (HST); G4: 50 patients with paracoccidioidomycosis (PCM); G5: 20 patients with cryptococcosis (CRC); and G6: 200 healthy controls. Serum antibodies were measured by DID and ELISA, with two antigen preparations—Aspergillus fumigatus (DID1, ELISA1) and a pool of A. fumigatus, A. flavus and A. niger antigens (DID2, ELISA2). The Platélia Aspergillus Enzyme Immunoassay (EIA) kit was used to measure galactomannan. The cut-off points of ELISA were determined for each antigen preparation and for the 95% and 99% confidence intervals. Despite the low sensitivity, DID was the technique of choice due to its specificity, positive and negative predictive values and positive likelihood ratio–especially with the antigen pool and due to the low frequency of cross-reactivity. ELISA1 and a 0.090 cut-off showed high sensitivity, specificity and negative predictive value, but a high frequency of cross-reactivity with CRC. The best degree of agreement was observed between ELISA1 and ELISA2. The detection of serum galactomannan showed high sensitivity, comparable to ELISA2. The immunodiffusion test showed an excellent relationship with the progression after treatment, which made it the reaction of choice for patient follow-up. PMID:26271000

Blood collected on filter paper for wildlife serology: detecting antibodies to Neospora caninum, West Nile virus, and five bovine viruses in reindeer.

PubMed

Curry, Patricia S; Ribble, Carl; Sears, William C; Hutchins, Wendy; Orsel, Karin; Godson, Dale; Lindsay, Robbin; Dibernardo, Antonia; Kutz, Susan J

2014-04-01

We compared Nobuto filter paper (FP) whole-blood samples to serum for detecting antibodies to seven pathogens in reindeer (Rangifer tarandus tarandus). Serum and FP samples were collected from captive reindeer in 2008-2009. Sample pairs (serum and FP eluates) were assayed in duplicate at diagnostic laboratories with the use of competitive enzyme-linked immunosorbent assays (cELISAs) for Neospora caninum and West Nile virus (WNV); indirect ELISA (iELISAs) for bovine herpesvirus type 1 (BHV-1), parainfluenza virus type 3 (PI-3), and bovine respiratory syncytial virus (BRSV); and virus neutralization (VN) for bovine viral diarrhea virus (BVDV) types I and II. Assay thresholds were evidence-based values employed by each laboratory. Comparable performance to serum was defined as FP sensitivity and specificity ≥ 80%. Filter-paper specificity estimates ranged from 92% in the cELISAs for N. caninum and WNV to 98% in the iELISAs for PI-3 and BRSV. Sensitivity was >85% for five tests (most ≥ 95%) but was insufficient (71-82%) for the PI-3 and BRSV iELISAs. Lowering the threshold for FP samples in these two ELISAs raised sensitivity to ≥ 87% and reduced specificity slightly (≥ 90% in three of the four test runs). Sample size limited the precision of some performance estimates. Based on the criteria of sensitivity and specificity ≥ 80%, and using adjusted FP thresholds for PI-3 and BRSV, FP sensitivity and specificity were comparable to serum in all seven assays. A potential limitation of FP is reduced sensitivity in tests that require undiluted serum (i.e., N. caninum cELISA and BVDV VNs). Possible toxicity to the assay cell layer in VN requires investigation. Results suggested that cELISA is superior to iELISA for detecting antibodies in FP samples from reindeer and other Rangifer tarandus subspecies. Our findings expand the potential utility of FP sampling from wildlife.

Chicken IgY-Fc antibody elicited in a rabbit as first coat on frosted glass beads in immunoassays.

PubMed

Polson, A

1993-07-01

The observation of Esteves and Binaghi that the Fc moieties of mammalian IgG are antigenically similar but antigenically unrelated to the Fc fraction of chicken IgG (IgY) was usefully applied in enzyme linked immuno-sorbent assay (ELISA). In this communication its use in ELISA of two plant viruses viz. tobacco mosaic virus (common strain) and grape vine A virus is illustrated. Frosted glass beads were used as reagent carriers and the reactions were conducted in the wells of multi-titre plates.

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. © 2011 Institute of Food Technologists®

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

PubMed Central

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

2012-01-01

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

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

PubMed Central

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

2007-01-01

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

A Functional Assay for GPR55: Envision Protocol.

PubMed

Anavi-Goffer, Sharon; Ross, Ruth A

2016-01-01

AlphaScreen(®) SureFire(®) assay is a novel technology that combines luminescent oxygen channeling technology, nano-beads, and monocloncal antibodies to detect the level of a selected protein in a volume lower than 5 μl. This method is more sensitive compared with the traditional enzyme-linked immunosorbent assays (ELISA), and can detect an increasing number of new targets. Here, we described a method for AlphaScreen(®) SureFire(®) assay that targets ERK1/2 phosphorylation, a primary downstream signaling pathway that conveys activation of GPR55 by L-α-lysophosphatidylinositol (LPI) and certain cannabinoids.

An enzyme-linked immunosorbent assay-based system for determining the physiological level of poly(ADP-ribose) in cultured cells.

PubMed

Ida, Chieri; Yamashita, Sachiko; Tsukada, Masaki; Sato, Teruaki; Eguchi, Takayuki; Tanaka, Masakazu; Ogata, Shin; Fujii, Takahiro; Nishi, Yoshisuke; Ikegami, Susumu; Moss, Joel; Miwa, Masanao

2016-02-01

PolyADP-ribosylation is mediated by poly(ADP-ribose) (PAR) polymerases (PARPs) and may be involved in various cellular events, including chromosomal stability, DNA repair, transcription, cell death, and differentiation. The physiological level of PAR is difficult to determine in intact cells because of the rapid synthesis of PAR by PARPs and the breakdown of PAR by PAR-degrading enzymes, including poly(ADP-ribose) glycohydrolase (PARG) and ADP-ribosylhydrolase 3. Artifactual synthesis and/or degradation of PAR likely occurs during lysis of cells in culture. We developed a sensitive enzyme-linked immunosorbent assay (ELISA) to measure the physiological levels of PAR in cultured cells. We immediately inactivated enzymes that catalyze the synthesis and degradation of PAR. We validated that trichloroacetic acid is suitable for inactivating PARPs, PARG, and other enzymes involved in metabolizing PAR in cultured cells during cell lysis. The PAR level in cells harvested with the standard radioimmunoprecipitation assay buffer was increased by 450-fold compared with trichloroacetic acid for lysis, presumably because of activation of PARPs by DNA damage that occurred during cell lysis. This ELISA can be used to analyze the biological functions of polyADP-ribosylation under various physiological conditions in cultured cells. Copyright © 2015 Elsevier Inc. All rights reserved.

Development and evaluation of flow through assay for detection of antibodies against porcine cysticercosis.

PubMed

Sreedevi, C; Hafeez, Md; Subramanyam, K V; Anand Kumar, P; Chengalva Rayulu, V

2011-04-01

A flow through assay (FTA) was developed on cellulose acetate membrane for the serodiagnosis of porcine cysticercosis using cyst fluid (CFA) and whole cyst antigens (WCA) of Taenia solium metacestode. The assay consisted of antigen of T. solium metacestode coated onto membrane, mounted on a flow-through test device to provide assay capture matrix. The optimum concentration of coating antigen was 250 ng. The protein A colloidal gold conjugate served as antigen-antibody detecting reagent. A total of 225 serum samples were tested using two antigens. Results were better with CFA (96.0% sensitivity; 96.0% specificity) compared to WCA (92.0% sensitivity; 96.0% specificity). The test was also compared with enzyme-linked immunosorbent assay. The ELISA showed 96 per cent sensitivity with both the antigens whereas; the specificity was 96 and 92 per cent with CFA and WCA respectively. The sensitivity and specificity of flow through assay agrees closely with those of the ELISA. The cross-reaction was observed in one out of eight hydatidosis positive pigs (12.5%) with CFA by both the assays. The highest diagnostic accuracy (96%) was obtained with CFA-FTA and CFA-ELISA. For its high sensitivity and sporadic cross-reactions, CFA-FTA appears to be suitable for practical use at field level without instrumentation.

Implementation of an Enzyme Linked Immunosorbent Assay for the Quantification of Allergenic Egg Residues in Red Wines Using Commercially Available Antibodies

PubMed Central

Koestel, Carole; Simonin, Céline; Belcher, Sandrine

2016-01-01

Abstract Since the early 2000s, labeling of potentially allergenic food components to protect people who suffer from food allergies is compulsory in numerous industrialized countries. In Europe, milk and egg components used during the winemaking process must be indicated on the label since July 1, 2012. Several ELISA procedures have been developed to detect allergenic residues in wines. However, the complexity of the wine matrix can inhibit the immunoenzymatic reaction. The aim of this study was to implement an ELISA assay for the detection of ovalbumin in red wines using commercially available antibodies. The specificity of the acquired antibodies and the absence of cross reactivity were assessed by immunoblotting and ELISA. An ELISA assay with a LOD of 14.2 μg/L and a LOQ of 56.4 μg/L of ovalbumin in aqueous solution was obtained. Differences in ELISA signals were observed when analyzing various fining agents, although reproducible conformation of the antigen could be reached for the comparison of ovalbumin and Ovicolle. The differences between samples in terms of pH could be leveled but the inhibition of the ELISA signal, positively correlated to the tannin content of the wines, could not be suppressed. Thus, standard curves of ovalbumin in several wines were obtained by relative quantification. The control steps and the difficulties encountered presented in this study should be considered by anybody working toward the development of ELISA assays for the detection of allergenic residues in complex food matrices. PMID:27356183

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 ...

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

PubMed

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

2011-09-01

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

Matrix effects in applying mono- and polyclonal ELISA systems to the analysis of weathered oils in contaminated soil.

PubMed

Pollard, S J T; Farmer, J G; Knight, D M; Young, P J

2002-01-01

Commercial mono- and polyclonal enzyme-linked immunosorbent assay (ELISA) systems were applied to the on-site analysis of weathered hydrocarbon-contaminated soils at a former integrated steelworks. Comparisons were made between concentrations of solvent extractable matter (SEM) determined gravimetrically by Soxhlet (dichloromethane) extraction and those estimated immunologically by ELISA determination over a concentration range of 2000-330,000 mg SEM/kg soil dry weight. Both ELISA systems tinder-reported for the more weathered soil samples. Results suggest this is due to matrix effects in the sample rather than any inherent bias in the ELISA systems and it is concluded that, for weathered hydrocarbons typical of steelworks and coke production sites, the use of ELISA requires careful consideration as a field technique. Consideration of the target analyte relative to the composition of the hydrocarbon waste encountered appears critical.

An enzyme-linked immunosorbent assay for monoester-type aconitic alkaloids and its application in the pharmacokinetic study of benzoylhypaconine in rats.

PubMed

Liu, Can-Can; Xu, Yun-Hui; Yuan, Shuai; Xu, Yu; Hua, Mo-Li

2018-04-01

A new enzyme-linked immunosorbent assay (ELISA) method for quantitative determination of monoester-type aconitic alkaloids was developed. The antibodies derived from the immunogen of benzoylmesaconine (BM) could be electively affined to benzoylaconitine-type alkaloids with an ester bond (14-benzoyl-), especially to benzoylhypaconine (BH, 140.02% of cross-reactivity). The effective working range of BH was 1 ng/ml to 5 μg/ml; the lower limit of detection and the quantification were 0.35 and 0.97 ng/ml, respectively. The values of CV for intra-day and inter-day assays and recovery ratios were in acceptable ranges. The results of stability experiments were also satisfactory. This validated method was employed for pharmacokinetic study of BH in rats and the bioavailability orally administered was estimated to be 16.3%.

Rapid screening of toxigenic vibrio cholerae O1 strains from south Iran by PCR-ELISA.

PubMed

Mousavi, Seyed Latif; Nazarian, Shahram; Amani, Jafar; Rahgerdi, Ahmad Karimi

2008-01-01

The ability to sensitively detect Vibrio cholera with PCR-ELISA method represents a considerable advancement over alternative more time-consuming methods for detection of this pathogen. The aim of this research is to evaluate the suitability of a PCR-enzyme-linked immunosorbent assay for sensitive and rapid detection of V. cholera O1. The 398-bp sequence of a gene that codes for the cholera toxin B subunit was amplified by PCR. The digoxigenin-labeled amplified products were coated on microplates and detected by ELISA. The PCR product was also hybridized with biotin labelled probe and detected by ELISA using streptavidin. The specificity of the PCR was determined using 10 bacterial strains and 50 samples from south Iran. The detection limit was 0.5 pg of the genomic DNA and five bacterial cells. Adaptation of PCR into PCR-ELISA assay format facilitates specific and sensitive detection and diagnosis of human cholera disease. We conclude that this PCR-ELISA is a diagnostic method that specifically detects toxin genes in V. cholera O1 strains. It is more rapid and less cumbersome than other diagnostic methods for detection of toxicity in these strains.

Preparation and Characteristic of Dextran-BSA Antibody and Establishment of its ELISA Immunoassay.

PubMed

Xie, Zhen-ming; Yu, Lin; Fang, Li-sha

2015-01-01

The enzyme-linked immunosorbent assay (ELISA) is a potential tool for the determination of dextran. In this study, dextran neoglycoprotein antigens were prepared by Reductive Amination method, and were confirmed by SDS-PAGE and free amino detection. The impact factors such as different oxidation degree of dextran, the conjugate reaction time to BSA were investigated. The best preparation conditions were obtained (n(dextran)/n(oxidant) of NaIO4 = 1/120, the reaction time of 24 h), and the antigen with best combination with standard was obtained. The antigens interacted with standard antibody and were evaluated through ELISA. The immunogen was immunized with white rabbits to obtained antibody, respectively. A general and broad class-specific ELISA immunoassay was developed for dextran detection according to ELISA theory. The optimized conditions of assay used coating antigen at 10 μg/mL, reaction time of antibody and rabbit-anti-bovine IgG in 45 min, blocking reagents with 5% calf serum. The developed ELISA detection method with good linear and accuracy was put to use for quantitative analysis of dextran T40 in commercial sugarpractical for detection of dextran.

Development of an epitope-blocking-enzyme-linked immunosorbent assay to differentiate between animals infected with and vaccinated against foot-and-mouth disease virus.

PubMed

Oem, Jae Ku; Chang, Byung Sik; Joo, Hoo Don; Yang, Mi Young; Kim, Gwang Jae; Park, Jee Yong; Ko, Young Joon; Kim, Yong Ju; Park, Jong Hyeon; Joo, Yi Seok

2007-06-01

An epitope-blocking ELISA (EB-ELISA) was developed to distinguish animals infected with foot-and-mouth-disease (FMDV) from those immunized with commercial vaccines. The assay used monoclonal antibodies to target the 3B core repeat motif (QKPLK) and purified recombinant 3AB proteins from the major B cell line epitopes of FMDV. Sera from uninfected and regularly vaccinated cattle, pigs, goats, and sheep (raised in FMDV free areas) were screened to evaluate the specificity of the EB-ELISA. The specificity scores of the assays were 99.8-100% and 100%, respectively. Reference sera from cattle, pigs, goats, and sheep experimentally infected with FMDV tested positive, with only a single exception. Antibodies formed in response to FMDV 3B appeared 1 week after infection and persisted at high levels for more than 8 weeks within the sera collected from serial bleeding of animals infected with FMDV O/SKR/2000. The EB-ELISA was used to differentiate between farms vaccinated against and those infected with FMDV (FMDV Asia serotype) during the 2005 epidemic in Mongolia by detecting antibodies against the FMDV Asia serotype in outbreak farms. This EB-ELISA method shows promise as an effective tool for FMDV control and eradication.