Elimination of cross-reactivity by addition of an excess of cross-reactant for radioimmunoassay of 17α-hydroxypregnenolone

被引:24
作者
Hill, M
Hampl, R
Lukác, D
Lapcík, O
Pouzar, V
Sulcová, J
机构
[1] Res Inst Endocrinol, CR-11694 Prague 1, Czech Republic
[2] Acad Sci Czech Republ, Inst Organ Chem & Biochem, Prague, Czech Republic
关键词
RIA; polyclonal antibody; specificity; 17; alpha-hydroxypregnenolone; steroids;
D O I
10.1016/S0039-128X(99)00017-3
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
Polyclonal antiserum against 3 beta,17 alpha-dihydroxypregn-5-en-20-one-19-O-(carboxymethyl)-oxime bovine serum albumin (17 alpha-hydroxypregnenolone-19-CMO:BSA), was raised in rabbits. Its main structural determinants were the substituents on D-ring as demonstrated by its 107% cross-reaction with 17 alpha-hydroxyprogesterone. This unspecificity was almost completely eliminated by addition of the excess of the cross-reactant directly to the analytical system. The contribution of the cross-reactant from the sample in such a system became negligible due to saturation of the populations of polyclonal antibodies recognizing the analyte as well as the cross-reactant. The possible interference of 17 alpha-hydroxypregnenolone-3-sulfate was avoided by inserting ether extraction. The analytical system appeared to be stable to differences in cross-reactant concentrations even in samples from patients with pathologically elevated serum levels of 17 alpha-hydroxyprogesterone. The radioimmunoassay was compared with the system using the unspecific antiserum alone but after separation of the cross-reactants by HPLC. As demonstrated by parallel measurement of 125 samples of human plasma from both sexes and various ages either before and/or after adrenocorticotropin stimulation and 17 samples with elevated basal of human plasma 17 alpha-hydroxyprogesterone levels, an excellent correlation was achieved between both methods. The method, based on a simple addition of the cross-reactant, avoids the time-consuming chromatographic separation and, in comparison with the other approaches for improving the specificity of polyclonal antisera, is efficient and rapid. Mathematical analysis of the relations in equilibrium demonstrates that such a simple approach is an efficient way for improvement of immunoassay specificity using some polyclonal antisera. (C) 1999 Elsevier Science Inc. All rights reserved.
引用
收藏
页码:341 / 355
页数:15
相关论文
共 24 条
[1]   USE OF HOMOLOGOUS AND HETEROLOGOUS RADIOLIGANDS-I-125 IN RADIOIMMUNOASSAY OF PROGESTERONE [J].
ALLEN, RM ;
REDSHAW, MR .
STEROIDS, 1978, 32 (04) :467-486
[2]   ACTIVE IMMUNIZATION OF RABBITS AGAINST PROGESTERONE - INCREASE IN HORMONE LEVELS, AND CHANGES IN METABOLIC-CLEARANCE RATES AND IN GENITAL-TRACT TISSUES [J].
BOCHSKANL, R ;
THIE, M ;
KIRCHNER, C .
JOURNAL OF STEROID BIOCHEMISTRY AND MOLECULAR BIOLOGY, 1989, 33 (03) :349-355
[3]  
Cook B, 1987, STEROID HORMONES PRA, P1
[4]   MEASUREMENT OF AMINO GROUPS IN PROTEINS AND PEPTIDES [J].
FIELDS, R .
BIOCHEMICAL JOURNAL, 1971, 124 (03) :581-&
[5]   SPECIFICITY OF THE IMMUNE-RESPONSE TO THE ESTRONE-AZO-HAPTEN STRUCTURE [J].
FRANEK, M ;
HRUSKA, K .
JOURNAL OF STEROID BIOCHEMISTRY AND MOLECULAR BIOLOGY, 1985, 22 (03) :341-347
[6]  
Ghosh S K, 1988, Steroids, V52, P1, DOI 10.1016/0039-128X(88)90213-9
[7]   ISOLATION OF ANTIBODIES OF IMPROVED SPECIFICITY AFTER FRACTIONATION ON ANTIGEN-SEPHAROSE AFFINITY COLUMNS OF ANTISERA TO BOVINE SERUM-ALBUMIN CONJUGATES OF C-3-LINKED AND C-7-LINKED (O-CARBOXYMETHYL)OXIMOSUCCINAMIDO AND HEMISUCCINAMIDO DERIVATIVES OF 5-ALPHA-DIHYDROTESTOSTERONE [J].
GRENOT, C ;
CUILLERON, CY .
STEROIDS, 1979, 34 (01) :15-34
[8]   PREPARATION OF IODINE-131 LABELLED HUMAN GROWTH HORMONE OF HIGH SPECIFIC ACTIVITY [J].
HUNTER, WM ;
GREENWOOD, FC .
NATURE, 1962, 194 (4827) :495-&
[9]   Radioimmunological and chromatographic properties of tyrosine methyl ester conjugates with stereoisomeric steroid carboxy derivatives [J].
Lapcik, O ;
Hampl, R ;
Hill, M ;
Starka, L ;
Kasal, A ;
Pouzar, V ;
Putz, Z .
COLLECTION OF CZECHOSLOVAK CHEMICAL COMMUNICATIONS, 1996, 61 (05) :799-807
[10]   AFFINITY-CHROMATOGRAPHY USING COMPETITIVE ELUTION SEPARATES POLYCLONAL GLUCOCORTICOID ANTISERA INTO FRACTIONS OF VARYING CROSS-REACTIVITY [J].
LEWIS, JG ;
ELDER, PA .
JOURNAL OF STEROID BIOCHEMISTRY AND MOLECULAR BIOLOGY, 1988, 29 (02) :191-195