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.