scholarly journals Generation and characterization of monoclonal antibodies against mature hepcidin and its application to neutralization and quantitative alteration assay

2021 ◽  
Vol 85 (2) ◽  
pp. 340-350
Author(s):  
Shinji Sakamoto ◽  
Mika Kirinashizawa ◽  
Yumi Mohara ◽  
Yoshihiro Watanabe
Keyword(s):  
Monoclonal Antibodies ◽  
Sandwich Elisa ◽  
High Sensitivity ◽  
Cellular Membrane ◽  
Average Concentration ◽  
Cellular Iron ◽  
Capture Antibody ◽  
Quantitative Alteration ◽  
Detection Reagent

ABSTRACT Hepcidin regulates the quantity of ferroportin (FPN) on cellular membrane. In our cell assay expressing ferroportin labeled with green fluorescence, FPN was internalized and degraded only after treatment with hepcidin-25, not hepcidin-22 or hepcidin-20, leading to accumulation of cellular iron. Thus we generated murine monoclonal antibodies (mAbs) against hepcidin-25, and then characterized and validated their functions. Among them, several mAbs showed a neutralizing activity that may prevent ferroportin internalization induced by hepcidin-25. To measure hepcidin level in various fluids, mAbs specific for human and rat hepcidin-25 were selected. As for rat, a sandwich ELISA developed using clone rHN1 as capture antibody and biotinylated clone mHW1 as a detection reagent had high sensitivity, allowing for the detection of 1-100 ng/mL of hepcidin-25. Rat hepcidin-25 level in plasma was measured at an average concentration of 63.0 ng/mL in healthy condition, and at 218.2 ng/mL after stimulation of lipopolysaccharide.

scholarly journals Production and Characterization of Two Monoclonal Antibodies Specific for Plasmopara halstedii

2000 ◽  
Vol 66 (8) ◽  
pp. 3277-3282 ◽  
Author(s):  
S. Bouterige ◽  
R. Robert ◽  
J. P. Bouchara ◽  
A. Marot-Leblond ◽  
V. Molinero ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Detection System ◽  
Sandwich Elisa ◽  
Plasmopara Viticola ◽  
Enzyme Linked Immunosorbent Assay ◽  
Plasmopara Halstedii ◽  
Race 1 ◽  
Molecular Masses ◽  
Fungal Antigens

ABSTRACT Sunflower downy mildew, caused by the fungus Plasmopara halstedii, is a potentially devastating disease. We produced two monoclonal antibodies (MAbs) (12C9 and 18E2) by immunizing mice with a partially purified extract of P. halstedii race 1. Both MAbs detected in enzyme-linked immunosorbent assay (ELISA) all races ofP. halstedii present in France. No cross-reactions were observed with Plasmopara viticola or with other fungi commonly associated with sunflowers. Both MAbs recognized the same three fungal antigens with molecular masses of 68, 140, and 192 kDa. However, the epitopes on the fungal antigens were distinct and repetitive. Seed homogenates from infected plants were incubated in wells coated with MAb 18E2. This resulted in the trapping of P. halstedii antigens that were identified with biotinylated MAb 12C9. No reactions were seen with seed homogenates from healthy plants. Thus, our results suggest that these MAbs might be used to develop a sandwich ELISA detection system for P. halstedii in infected seeds.

Rapid, sensitive two-site ELISA for detection of cows' milk in goats' or ewes' milk using monoclonal antibodies

1994 ◽  
Vol 61 (1) ◽  
pp. 91-99 ◽  
Author(s):  
Didier Levieux ◽  
Annie Venien
Keyword(s):  
Monoclonal Antibodies ◽  
Sandwich Elisa ◽  
High Specificity ◽  
Enzyme Linked Immunosorbent Assay ◽  
Factors Affecting ◽  
Assay Performance ◽  
Capture Antibody ◽  
Constant Quality ◽  
Species Specific ◽  
Β Lactoglobulin

SummaryA sandwich ELISA (enzyme-linked immunosorbent assay) of the two-site type has been successfully developed for the detection of cows' milk in goats' or ewes' milk. The assay uses two monoclonal antibodies (MAb) raised in mice against cows' β-lactoglobulin (β-lg). These MAb recognize different epitopes of the β-lg, which are sufficiently distinct to allow simultaneous binding of the corresponding antibodies. One of the MAb recognizes a species-specific epitope of the bovine β-lg and was adsorbed to a plastic microtitration plate (capture antibody). The second MAb was labelled with peroxidase and used to detect the captured cows' β-lg. Factors affecting assay performance were investigated. The optimized assay is highly specific, reproducible (intra- and inter-assay CV were 8 and 13% respectively) and sensitive: as little as 5 ng β-lg/ml or 1 part cows' milk per 100000 parts goats' or ewes' milk can be detected. The technique is robust, cheap, rapid, reliable and suitable for high sample throughput, semi-automation and screening surveys. The MAb used guarantee the high specificity of the assay and indefinite reagent supply of constant quality once approved by collaborative national or international trials.

Characterization of rotavirus subgroup-specific monoclonal antibodies and use in single-sandwich ELISA systems for rapid subgrouping of human strains

1989 ◽  
Vol 107 (3-4) ◽  
pp. 315-322 ◽  
Author(s):  
G. Gerna ◽  
Antonella Sarasini ◽  
Maria Torsellini ◽  
Angela di Matteo ◽  
F. Baldanti ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Sandwich Elisa

scholarly journals 645. Singulex Clarity Norovirus Assay (In Development) Provides Ultrasensitive Detection of Norovirus Genogroups I and II

2019 ◽  
Vol 6 (Supplement_2) ◽  
pp. S297-S298
Author(s):  
Gipshu Dave ◽  
Phoebe Katzenbach ◽  
Johanna Sandlund ◽  
Joel Estis ◽  
Ali Mukherjee ◽  
...  
Keyword(s):  
Single Molecule ◽  
Operating Characteristic ◽  
High Sensitivity ◽  
Excellent Performance ◽  
C Statistic ◽  
Stool Samples ◽  
Capture Antibody ◽  
Automated Platform ◽  
Paramagnetic Microparticles ◽  
Detection Reagent

Abstract Background Commercially available enzyme immunoassays (EIAs) for detection of norovirus antigen have poor sensitivity and are limited to use in investigations of a gastroenteritis outbreak. Hence, there remains a need for a standalone high-sensitivity assay that enables rapid and accurate detection of norovirus antigen. Methods The Singulex Clarity norovirus assay is currently in development for use on the Singulex Clarity® system (Singulex Inc., Alameda, CA, USA), a fully-automated platform powered by Single Molecule Counting technology (registered with the FDA and CE marked). The assay uses paramagnetic microparticles bound to capture antibody and a fluorescently labeled reporter antibody to detect virion capsid protein of norovirus genogroups I (GI) and II (GII) in the stool. For the development of Clarity Norovirus assay, diagnostic performance of 4 antibody pairs (as Capture and Detection reagent) were evaluated by testing 137 stool samples from patients with suspected norovirus infection. Samples were sourced from three providers: (1) 90 genotyped samples of which 75 were positive (19 different genotypes) and 15 were negative by the CDC assay, (2) 3 samples positive and 5 samples negative by the BioFire® FilmArray® Gastrointestinal Panel, and (3) 39 samples negative by a lab-developed test using Cepheid reagents (SmartCycler®). Results From all the antibody pairs tested, one of the pairs had best performance with the area under the receiver operating characteristic (AuROC) curve demonstrating a C-Statistic of 0.959 (95% CI 0.921–0.997), compared with AuROC C-statistic of 0.943 (95% CI 0.896–0.990), 0.871 (95% CI 0.807–0.936), and 0.914 (95% CI 0.863–0.964) for the three other pairs. The Clarity assay detected all 19 different genotypes tested (figures). Conclusion The ultrasensitive and rapid Clarity norovirus assay (in development) for detection of GI and GII demonstrated excellent performance with one of the antibody pairs tested and detected all 19 tested genotypes. The Clarity assay may offer a standalone solution for norovirus diagnostics. Disclosures All authors: No reported disclosures.

New separation method for monoclonal immunoradiometric assays and its application to assays for thyrotropin and human choriogonadotropin.

1984 ◽  
Vol 30 (9) ◽  
pp. 1457-1461 ◽  
Author(s):  
S J Rattle ◽  
D R Purnell ◽  
P I Williams ◽  
K Siddle ◽  
G C Forrest
Keyword(s):  
Monoclonal Antibodies ◽  
Magnetic Particles ◽  
Solid Phase ◽  
Reaction Times ◽  
Fluorescein Isothiocyanate ◽  
High Sensitivity ◽  
Separation Method ◽  
Separation Procedure ◽  
Human Choriogonadotropin ◽  
Capture Antibody

Abstract We describe a novel separation procedure for immunoradiometric assays involving monoclonal antibodies in which both radiolabeled and capture antibodies are used in solution, the capture antibody being labeled with fluorescein isothiocyanate (FITC). Separation is achieved by incubation with anti-FITC antibodies on magnetic particles. This technique enhances reaction kinetics relative to those of assays in which a solid-phase capture antibody is used, thus allowing faster reaction times and more economic use of the monoclonal antibodies. The use of anti-FITC magnetic solid phase produces an assay having a highly specific separation method, minimal nonspecific binding, and high sensitivity. The method is illustrated by application to assays for thyrotropin and human choriogonadotropin.

Development and Characterization of Six Monoclonal Antibodies to Hemagglutinin-70 ofClostridium botulinumand Their Application in a Sandwich ELISA

2013 ◽  
Vol 32 (1) ◽  
pp. 6-15 ◽  
Author(s):  
Miles C. Scotcher ◽  
Luisa W. Cheng ◽  
Kathryn Ching ◽  
Jeffery McGarvey ◽  
Robert Hnasko ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Sandwich Elisa

Use of chicken antibodies in enzyme immunoassays to avoid interference by rheumatoid factors

1991 ◽  
Vol 37 (3) ◽  
pp. 411-414 ◽  
Author(s):  
Anders Larsson ◽  
Alex Karlsson-Parra ◽  
J Sjöquist
Keyword(s):  
Monoclonal Antibodies ◽  
False Positive ◽  
Mammalian Species ◽  
Sandwich Elisa ◽  
Rheumatoid Factors ◽  
Igg Antibodies ◽  
False Positive Reaction ◽  
Avian Origin ◽  
Capture Antibody ◽  
Positive Results

Abstract Rheumatoid factor (RF) is a major source of interference in many immunoassays. Most immunoassays use mammalian polyclonal or monoclonal antibodies, and RF can react with IgG from mammalian species, thus causing false-positive results. In this work we have studied RF interference in a sandwich ELISA, where RF in the sample may react with both the capture antibody and the detection antibody to give a false-positive reaction. We show that rheumatoid factors do not react with chicken IgY; if the capture antibody or detection antibody (or both) is of avian origin, the interference of RF or other anti-IgG antibodies in sandwich ELISA can be avoided.

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