Two different approaches for developing immunometric assays of haptens

1996 ◽  
Vol 42 (9) ◽  
pp. 1532-1536 ◽  
Author(s):  
J Grassi ◽  
C Créminon ◽  
Y Frobert ◽  
E Etienne ◽  
E Ezan ◽  
...  
Keyword(s):  
Amino Acids ◽  
Monoclonal Antibody ◽  
Solid Phase ◽  
Leukotriene C4 ◽  
Amino Groups ◽  
Small Peptides ◽  
Assay Sensitivity ◽  
Primary Amino ◽  
C And N

Abstract To improve immunoassays of small haptens, we developed two different approaches for their measurement in a non-competitive format. We first devised two-site immunometric assays for small peptides (8-11 amino acids) by selecting two sets of antibodies specifically directed against C- and N-terminal moieties of the peptides. In each case, assay sensitivity improved substantially over that of the corresponding competitive assays. More interestingly, all of these new immunometric assays were much more specific than the competitive assays. In a second approach, we developed a new procedure, solid-phase-immobilized epitope immunoassay (SPIE-IA), in which a single monoclonal antibody uses the same epitope for capture and tracer binding and the hapten is covalently cross-linked to solid-phase proteins. To date, SPIE-IA have been successfully applied to the determination of haptens bearing primary amino groups, including substance P, thyroxine, leukotriene C4, endothelin, and angiotensin II. In each case, assay sensitivity was significantly improved.

Measurement of proteolysis in cheese: relationship between phosphotungstic acid-soluble N fraction by Kjeldahl and 2,4,6- trinitrobenzenesulphonic acid-reactive groups in water-soluble N

1994 ◽  
Vol 61 (3) ◽  
pp. 437-440 ◽  
Author(s):  
Yvette Bouton ◽  
Remy Grappin
Keyword(s):  
Amino Acids ◽  
Free Amino Acids ◽  
Free Amino ◽  
Phosphotungstic Acid ◽  
Water Soluble ◽  
Hydroxyl Groups ◽  
Amino Groups ◽  
Reactive Groups ◽  
Primary Amino

Free amino groups produced during cheese ripening are used to indicate the extent of cheese proteolysis. Several studies have shown a high correlation between the level of free amino acids and the flavour of Gouda (Aston et al. 1983) or Comté (Grappin & Berdagué, 1989). Measurement of the level of free amino acids seems useful for the investigation of flavour chemistry in cheese (Lemieux et al. 1990). The determination of N fractions is often used to estimate the degree of proteolysis in cheese, but since this procedure is laborious and time consuming several attempts have been made to replace it by more rapid methods (Ardö & Meisel, 1991). Since its introduction by Satake et al. (1960), the 2,4,6-trinitrobenzenesulphonic acid (TNBS) method has been widely used for the determination of free amino groups. Because TNBS does not react with the imino groups of histidine and proline or the hydroxyl groups of tyrosine, serine or threonine, it has been accepted as a selective reagent for primary amino groups (Burger, 1974). Measurement of N by Kjeldahl in the phosphotungstic acid (PTA)–sulphuric acid extract (Gripon et al. 1975) estimates the N of free amino acids and low molecular mass peptides. The purpose of this study was to compare the TNBS and PTA-soluble N methods in order to find out whether the TNBS procedure can replace that of PTA-soluble N in the determination of a cheese proteolysis index.

A spectrophotometric assay for solid phase primary amino groups

1992 ◽  
Vol 36 (2) ◽  
pp. 81-85 ◽  
Author(s):  
Violeta G. Janolino ◽  
Harold E. Swaisgood
Keyword(s):  
Solid Phase ◽  
Spectrophotometric Assay ◽  
Amino Groups ◽  
Primary Amino

A solid-phase enzymeimmunoassay for the determination of progesterone in bovine, porcine and ovine plasma

1995 ◽  
Vol 75 (4) ◽  
pp. 525-529 ◽  
Author(s):  
R. P. Del Vecchio ◽  
W. D. Sutherland ◽  
M. L. Connor
Keyword(s):  
Bovine Serum Albumin ◽  
Serum Albumin ◽  
Petroleum Ether ◽  
Bovine Serum ◽  
Solid Phase ◽  
Plasma Samples ◽  
Assay Sensitivity ◽  
Coefficients Of Variation ◽  
Rabbit Igg

The purpose of this project was to develop a valid quantitative enzymeimmunoassay (EIA) for progesterone in blood plasma of cattle, pigs and sheep. Rabbit anti-progesterone, mouse monoclonal anti-rabbit IgG, authentic progesterone, and acetylcholine esterase bound covalently to progesterone were the principal reagents used to develop the EIA. Ninety-six well microliter plates were coated with mouse monoclonal anti-rabbit IgG and saturated with bovine serum albumin before use. Rabbit anti-progesterone was diluted to a working dilution of 1:2.0 × 106. Standard curves were linear and ranged from 1.56 to 400 pg of progesterone per well which allowed for the measurement of 0.03125 to 8.0 ng mL−1. Assay sensitivity averaged 1.56 pg well−1. Progesterone was extracted from plasma samples with petroleum ether. Plasma samples (n = 3 or 4 from each species) with unknown amounts of progesterone that were extracted and serially diluted with EIA buffer did not deviate from parallelism with progesterone standard curves in buffer. The correlation between EIA and radioimmunoassay (RIA) measurements of progesterone in the same plasma samples was high (P < 0.0001) for all three species (r = 0.96 for bovine; r = 0.96 for porcine; r = 0.94 for ovine). The regression of EIA data on RIA data produced the following equations:[Formula: see text]The intra- and inter-assay coefficients of variation were 5.4 and 10.6% for bovine, 5.8 and 11.0% for porcine and, 6.1 and 12.3% for ovine, respectively. These data show that this EIA is a valid and reliable memod for quantitating progesterone in extracts of bovine, porcine and ovine plasma. Key words: Enzymeimmunoassay, progesterone, plasma, bovine, porcine, ovine

Automated Determination of Free Amino Groups in Serum and Plasma Using 2,4,6-Trinitrobenzene Sulfonate

1969 ◽  
Vol 15 (9) ◽  
pp. 891-901 ◽  
Author(s):  
D W Palmer ◽  
T Peters
Keyword(s):  
Amino Acids ◽  
Free Amino Acids ◽  
Free Amino ◽  
Clinical Applications ◽  
Amino Groups ◽  
Automated Method ◽  
Total Free Amino Acids ◽  
Colored Complexes ◽  
Automated Determination

Abstract A simple automated method is described for determining the level of total free amino acids in the blood. The method utilizes the AutoAnalyzer, and is based on the formation of colored complexes by uniting free amino groups with 2,4,6-trinitrobenzene sulfonate (TNBS). Proteins do not interfere because the free amino acids are first separated by dialysis. Characteristics of the reaction and potential clinical applications of the procedure are discussed.

scholarly journals Quantitative determination of N-terminal amino acids of peptides and proteins with cobalt(III) chelates

1976 ◽  
Vol 153 (1) ◽  
pp. 137-138 ◽  
Author(s):  
K W Bentley
Keyword(s):  
Amino Acids ◽  
Quantitative Determination ◽  
Peptide Bond ◽  
Small Peptides ◽  
Acidic Hydrolysis ◽  
Terminal Amino ◽  
A Chain ◽  
Glucagon And Insulin

Quantitative N-terminal peptide-bond hydrolysis with the cis-beta-hydroxyaquo(triethylenetetramine) cobal (III) ion, i.e. β-[Co(trien)(OH)(OH2)]2+, is reported. The method has been demonstrated with 20 small peptides, a hexapeptide, bradykinin, insulin A chain (oxidized), glucagon and insulin. The procedure involves no acidic hydrolysis step and thus no destruction of labile amino acids.

scholarly journals Direct Assay for Cobalamin Bound to Transcobalamin (Holo-Transcobalamin) in Serum

2002 ◽  
Vol 48 (3) ◽  
pp. 526-532 ◽  
Author(s):  
Marius Ulleland ◽  
Ingar Eilertsen ◽  
Edward V Quadros ◽  
Sheldon P Rothenberg ◽  
Sergey N Fedosov ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Detection Limit ◽  
Quantitative Determination ◽  
Reliable Method ◽  
Solid Phase ◽  
Reference Interval ◽  
Magnetic Microspheres ◽  
Affinity Constant ◽  
Capture Assay

Abstract Background: Only cobalamin carried by transcobalamin (holo-transcobalamin) is available for cellular uptake and hence is physiologically relevant. However, no reliable or accurate methods for quantifying holo-transcobalamin are available. We report a novel holo-transcobalamin assay based on solid-phase capture of transcobalamin. Methods: A monoclonal antibody specific for human transcobalamin with an affinity constant &gt;1010 L/mol was immobilized on magnetic microspheres to capture and concentrate transcobalamin. The cobalamin bound to transcobalamin was then released and assayed by a competitive binding radioassay. The quantification of holo-transcobalamin was accomplished using calibrators composed of recombinant, human holo-transcobalamin. Results: The assay was specific for holo-transcobalamin and had a detection limit of 5 pmol/L. Within-run and total imprecision (CV) was 5% and 8–9%, respectively. The working range (CV &lt;20%) was 5–370 pmol/L. Dilutions of serum were linear in the assay range. The recovery of recombinant, human holo-transcobalamin added to serum was 93–108%. A 95% reference interval of 24–157 pmol/L was established for holo-transcobalamin in 105 healthy volunteers 20–80 years of age. For 72 of these sera, holo-haptocorrin and total cobalamin were also determined. Whereas holo-haptocorrin correlated well (r2 = 0.87) with total cobalamin, holo-transcobalamin correlated poorly (r2 = 0.23) with total cobalamin or holo-haptocorrin. Conclusions: The solid-phase capture assay provides a simple, reliable method for quantitative determination of holo-transcobalamin in serum.

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