Peptide Microarrays for Determination of Cross-Reactivity

Author(s):  
Alexandra Thiele
1979 ◽  
Author(s):  
Daniel Walz ◽  
Thomas Brown

Human prothrombin activation is unique in that, in addition to the release of fragment 1.2 (FI.2) from the NH-terminus of prothrombin by factor Xa during the generation of thrombin, an additional 13 residue polypeptide, fragment 3 (F3), is autocatalytically removed from the amino-terminus of the thrombin A chain. We have developed a rapid radioimmunoassay for human F3 which incorporates short incubation times and the use of a preprecipitated second antibody; the assay can be performed in three hours. Specificity studies in buffer systems show prothrombin and prethrombin 1 cross-reacting at a level of 0.001; purified thrombin does not cross-react. In the presence of 5% BSA, prothrombin displays considerably less cross-reactivity. No immunoreactive material to F3 antibodies could be detected in 400 μL of plasma. Serum, obtained from whole blood clotting, contained measurable quantities of F3 (40-100 ng/mL). This amount in serum represents only 5-10% of the theoretical amount available should all of the fragment be hydrolytically cleaved during the conversion of prothrombin to thrombin. This assay procedure is currently being utilized to monitor the activation of purified human prothrombin in the absence and presence of selected plasma inhibitors. (Supported in part by NIH 05384-17 and the Michigan Heart Association).


2020 ◽  
Vol 58 (6) ◽  
pp. 1002-1009 ◽  
Author(s):  
Caroline Le Goff ◽  
Jordi Farre-Segura ◽  
Violeta Stojkovic ◽  
Patrice Dufour ◽  
Stéphanie Peeters ◽  
...  

AbstractHistorically, the determination of low concentration analytes was initially made possible by the development of rapid and easy-to-perform immunoassays (IAs). Unfortunately, typical problems inherent to IA technologies rapidly appeared (e.g. elevated cost, cross-reactivity, lot-to-lot variability, etc.). In turn, liquid chromatography tandem mass spectrometry (LC-MS/MS) methods are sensitive and specific enough for such analyses. Therefore, they would seem to be the most promising candidates to replace IAs. There are two main choices when implementing a new LC-MS/MS method in a clinical laboratory: (1) Developing an in-house method or (2) purchasing ready-to-use kits. In this paper, we discuss some of the respective advantages, disadvantages and mandatory requirements of each choice. Additionally, we also share our experiences when developing an in-house method for cortisol determination and the implementation of an “ready-to-use” (RTU) kit for steroids analysis.


1979 ◽  
Vol 91 (1) ◽  
pp. 158-166 ◽  
Author(s):  
Günter Emons ◽  
Peter Ball ◽  
Gertrud v. Postel ◽  
Rudolf Knuppen

ABSTRACT A bovine serum albumin conjugate of 2-methoxyoestrone was used for the preparation of highly specific antibodies in rabbits. Cross-reactivity for catecholoestrogens and monophenolic steroids was below 0.3 %. Only 2-methoxyoestradiol cross-reacted with 44 %. An assay procedure for the determination of unconjugated and conjugated 2-methoxyoestrone in human plasma is described. The following mean plasma concentrations (pg/ml) were found (unconjugated/conjugated): children 61/1130, young men 74/1320, elderly men 109/1260, cycling women 131/1040, post-menopausal women 102/1420, and pregnant women 3980/5850.


2019 ◽  
Vol 2019 ◽  
pp. 1-11 ◽  
Author(s):  
Jie Cao ◽  
Xiao-Ying Chen ◽  
Wu-Rong Zhao

A competitive fluorescence immunoassay for the identification and quantification of morphine has been developed on the basis of hapten-coated plate format. Hapten was prepared through covalent conjugating a morphine derivative with albumin bovine. In the immunoassay, the hapten was inoculated on a 96-well plate and then bound with monoclonal antibodies labeled with a signal indicating dye, fluorescein isothiocyanate (FITC). Unbound FITC-antibodies were rinsed off from the plate. The fluorescein intensity decreases in the presence of morphine molecules due to the competitively binding to antibodies against hapten. The intensity is inversely correlated with the concentration of morphine. In quantitative analysis for urine samples, we obtained a linearity range of 0.2 μg/mL∼2.5 μg/mL, along with a detection limit of c.a. 1 ng/mL. The fluorescence immunoassay shows low cross-reactivity (below 10%) to 6-acetylmorphine, 3-acetylmorphine, and heroine. The developed method produced comparable results to the standard GC-MS/MS method. In conclusion, a rapid and efficient screening tool for morphine in clinical human urine has been established.


2000 ◽  
Vol 83 (5) ◽  
pp. 1141-1148 ◽  
Author(s):  
Harvey E Indyk ◽  
Eileen A Evans ◽  
Malin C Bostrom Caselunghe ◽  
Bjorn S Persson ◽  
Paul M Finglas ◽  
...  

Abstract Biomolecular interaction analysis was evaluated for the automated analysis of biotin- and folate-supplemented infant formulas and milk powders. The technique was configured as a biosensor-based, nonlabeled inhibition immunoassay using monoclonal antibodies raised against analyte-conjugate. Sample extraction conditions were optimized and antibodies were evaluated for cross-reactivity. Performance parameters included a quantitation range of 2–70 ng/mL, recoveries of 86–102%, agreement against assigned reference values for National Institute of Standards and Technology Standard Reference Material 1846, between-laboratory reproducibility relative standard deviation of 9.1% for biotin and 8.1% for folate, respectively, and equivalence against reference microbiological assay methods for both analytes.


1987 ◽  
Vol 33 (12) ◽  
pp. 2225-2229 ◽  
Author(s):  
H F Schran ◽  
T G Rosano ◽  
A E Hassell ◽  
M A Pell

Abstract We measured cyclosporine in whole blood from normal volunteers administered single oral doses of the drug and from two renal-transplant patients on immunosuppressive maintenance therapy, by liquid chromatography (I) and by radioimmunoassay with use of nonspecific polyclonal (II), specific monoclonal (III), and nonspecific monoclonal (IV) antibodies. Concentrations determined by III were equivalent to I, irrespective of cyclosporine dose, concentration, time after dose, or time after transplant. Concentrations determined by II and IV were consistently higher than those by I, owing to cross reactivity with metabolites. Ratios of values by II and IV to those by I increased from less than 1.5 to about 3-4 between 0.5 and 12 h after a single cyclosporine dose, owing to differences in rates of appearance and disappearance of cyclosporine and cross-reacting metabolites, though for the constant 12-h dose intervals in the two renal-transplant patients at steady state these ratios (most within the range 3-4) were relatively stable. Ratios of concentrations measured by IV to those by II (mean of 1.2 for single-dose data, most within the range of 1.2 to 1.5 at steady state) were unaffected by time after dose or time after transplant, suggesting that, despite certain cross-reactivity differences between the two nonspecific antibodies, results are proportional throughout therapy. We therefore propose that III and IV offer alternatives, respectively, to the currently used I and II for cyclosporine monitoring.


2004 ◽  
Vol 2004 (3) ◽  
pp. 143-149 ◽  
Author(s):  
Juliana F. Moura ◽  
Luiz DeLacerda ◽  
Romolo Sandrini ◽  
Fernanda M. Borba ◽  
Denise N. Castelo ◽  
...  

Human growth hormone (hGH) signal transduction initiates with a receptor dimerization in which one molecule binds to the receptor through sites 1 and 2. A sandwich enzyme-linked immunosorbent assay was developed for quantifying hGH molecules that present helix 4 from binding site 1. For this, horse anti-rhGH antibodies were eluted by an immunoaffinity column constituted by sepharose-rhGH. These antibodies were purified through a second column with synthetic peptide correspondent tohGH helix 4, immobilized to sepharose, and used as capture antibodies. Those that did not recognize synthetic peptide were used as a marker antibody. The working range was of 1.95 to 31.25 ng/mL of hGH. The intra-assay coefficient of variation (CV) was between 4.53% and 6.33%, while the interassay CV was between 6.00% and 8.27%. The recovery range was between 96.0% to 103.8%. There was no cross-reactivity with human prolactin. These features show that our assay is an efficient method for the determination of hGH.


2003 ◽  
Vol 13 (18) ◽  
pp. 2997-3000 ◽  
Author(s):  
Mahesh Uttamchandani ◽  
Elaine W.S. Chan ◽  
Grace Y.J. Chen ◽  
Shao Q. Yao

2012 ◽  
Vol 461 ◽  
pp. 67-70 ◽  
Author(s):  
Chao Ying Li ◽  
Jin Qing Jiang

This paper reports an indirect competitive enzyme-linked immunosorbent assay (icELISA) using polyclonal antibody (pAb) for estradiol (E2) residues. After derivation, E2 haptens were conjugated to bovine serum albumin (BSA) and ovalbumin (OVA) through 1-Ethyl-3-(3-dimethylaminopropy) carbodiimide (EDC) method, and New Zealand white rabbits were immunized to produce anti-E2 pAb. The conjugation ratio of E2-BSA was proved to be 18.6:1 by an UV absorbance method. Based on the square matrix titration, an icELISA standard curve was developed. The dynamic range was from 0.16 to 128 ng/mL, with LOD and IC50 value of 0.08 ng/mL and 3.76 ng/mL, respectively. Except for a little cross-reactivity (16.2%) to estrone, this assay showed negligible cross-reactivity to other analogues tested. The results suggest that the produced anti-E2 pAb could be used to develop an icELISA method for the determination of E2 residues in animal-originally products.


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