Open AccessImprovement of an eia system for basic fibroblast growth factor by use of biotinylated antibody prepared with nhs‐lc‐biotin
Author(s) -
Takei Yoshifumi,
Higashira Hanae,
Hayashi Kyozo
Publication year - 1995
Publication title -
journal of clinical laboratory analysis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.536
H-Index - 50
eISSN - 1098-2825
pISSN - 0887-8013
DOI - 10.1002/jcla.1860090204
Subject(s) - biotinylation , basic fibroblast growth factor , chromatofocusing , biotin , chromatography , chemistry , immunoassay , recombinant dna , isoelectric focusing , antibody , isoelectric point , detection limit , reproducibility , microbiology and biotechnology , biochemistry , growth factor , enzyme , biology , immunology , receptor , gene
We improved our previously devised enzyme immunoassay (FIA) system for basic fibroblast growth factor (bFGF) using biotinylated antibody prepared with sulfosuccinimidyl‐6‐(biotinamido)hexanoate (NHS‐LC‐Biotin, Pierce), a water‐soluble biotin analogue, with an extended spacer arm. The discriminatory detection limit of the improved EIA was found to be 5 pg/ml (0.5 pg/assay tube), sixfold more sensitive than that of the previous system. The reproducibility of within‐ and between‐assay series was 5.10–8.61% and 5.99–8.69%, respectively; and recovery of exogenous bFGF from serum was approximately 102%. Employing the improved EIA system, we investigated by chromatofocusing chromatography the isoelectric points of two immunoreactive bFGFs (high‐molecular‐weight bFGF‐like immunoreactive substance, designated as HMW‐bFGF‐LI and 16‐kd bFGF‐LI, having the same molecular weight as recombinant bFGF) detected in serum from a breast cancer patient. As a result, the pl value of HMW‐bFGF‐LI was estimated to be 7.13, and that of 16 kd bFGF‐LI, 9.58.©1995 wiley‐Liss, inc.