Improvement of an EIA system for basic fibroblast growth factor by use of biotinylated antibody prepared with NHS-LC-biotin.
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 pI value of HMW-bFGF-LI was estimated to be 7.13, and that of 16 kd bFGF-LI, 9.58.
Takei
Y
Y
Department of Molecular Biology, Gifu Pharmaceutical University, Japan.
Higashira
H
H
Hayashi
K
K
D000328:Adult; D000906:Antibodies / Q000378:metabolism*; D001710:Biotin / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism*; D016222:Fibroblast Growth Factor 2 / Q000097:blood* / Q000276:immunology; D006801:Humans; D007124:Immunoenzyme Techniques; D007525:Isoelectric Focusing; D008297:Male; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013388:Succinimides / Q000737:chemistry*
D000906:Antibodies; D013388:Succinimides; D016222:Fibroblast Growth Factor 2; C061443:sulfosuccinimidyl 6-(biotinamido)hexanoate; D001710:Biotin