Application of avidin-biotin technology for the characterization of a model hapten-protein conjugate

被引:3
作者
Dotsikas, Y [1 ]
Loukas, YL [1 ]
机构
[1] Univ Athens, Sch Pharm, Dept Pharmaceut Chem, GR-15771 Athens, Greece
关键词
hapten-BSA conjugate; electrospray mass spectrometry; HABA; biotin;
D O I
10.1080/15321810500220845
中图分类号
Q5 [生物化学];
学科分类号
071010 ; 081704 ;
摘要
A simple method was developed for the rapid characterization of the covalent binding of haptens to proteins such as enzymes, bovine serum albumin (BSA), and other carrier-proteins and antibodies. In the present study, a commercially available fentanyl-BSA conjugate was characterized by a 4'-hydroxyazobenzene-2-carboxylic acid (HABA) dye assay that followed a biotinylation reaction. This protocol allowed the indirect observation of the average hapten number per BSA molecule. Such measurement is useful for optimizing reaction conditions to yield a more precisely defined product for immunological applications. The obtained result was within the limits suggested by the manufacturer of the conjugate.
引用
收藏
页码:285 / 293
页数:9
相关论文
共 25 条
[1]  
BARBARAKIS MS, 1991, CLIN CHEM, V37, P87
[2]  
COLE RB, 1997, ELECTROSPRAY IONIZAT, V55
[3]   Employment of 4-(1-imidazolyl)phenol as a luminol signal enhancer in a competitive-type chemiluminescence immunoassay and its comparison with the conventional antigen-horseradish peroxidase conjugate-based assay [J].
Dotsikas, Y ;
Loukas, YL .
ANALYTICA CHIMICA ACTA, 2004, 509 (01) :103-109
[4]   Kinetic degradation study of insulin complexed with methyl-beta cyclodextrin.: Confirmation of complexation with electrospray mass spectrometry and 1H NMR [J].
Dotsikas, Y ;
Loukas, YL .
JOURNAL OF PHARMACEUTICAL AND BIOMEDICAL ANALYSIS, 2002, 29 (03) :487-494
[5]   Determination of umbilical cord and maternal plasma concentrations of fentanyl by using novel spectrophotometric and chemiluminescence enzyme immunoassays [J].
Dotsikas, Y ;
Loukas, YL ;
Siafaka, I .
ANALYTICA CHIMICA ACTA, 2002, 459 (02) :177-185
[6]   A GENERAL-METHOD FOR DEVELOPING IMMUNOASSAYS TO CHLOROACETANILIDE HERBICIDES [J].
FENG, PCC ;
HORTON, SR ;
SHARP, CR .
JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY, 1992, 40 (02) :211-214
[7]   STUDIES ON THE BIOTIN-BINDING SITE OF AVIDIN - LYSINE RESIDUES INVOLVED IN THE ACTIVE-SITE [J].
GITLIN, G ;
BAYER, EA ;
WILCHEK, M .
BIOCHEMICAL JOURNAL, 1987, 242 (03) :923-926
[8]  
GOSSLING JP, 2000, IMMUNOASSAYS PRACTIC, V172
[9]   DETERMINATION OF FREE AMINO GROUPS IN PROTEINS BY TRINITROBENZENESULFONIC ACID [J].
HABEEB, AFS .
ANALYTICAL BIOCHEMISTRY, 1966, 14 (03) :328-&
[10]  
HENDERSON GL, 1975, J PHARMACOL EXP THER, V192, P489