Quantitation of basic fibroblast growth factor by immunoassay using BIAcore 2000.

A sensitive, accurate, and efficient immunoassay using a BIAcore 2000 biosensor instrument for the quantitation of basic fibroblast growth factor (bFGF) in HEPES-buffered saline containing 100 microg/ml heparin (HHBS) has been developed and validated. In this method, anti-bFGF monoclonal antibody 48.1 (MAb 48.1) was selected as a binding ligand and immobilized to the matrix surface of Sensor Chip CM5 by amine coupling. A high immobilization level of MAb 48.1 (12643+/-816 RU, mean +/- S.D., n = 5) was achieved with high reproducibility (i.e. coefficient of variation (CV) was 6.5%). This immobilized MAb 48.1 sensor surface was used to detect and quantity bFGF. This assay has a range of reliable BIAcore response from 5.65 to 1440 ng/ml bFGF in HHBS. which was well fitted with a sigmoidal model. The immobilized MAb 48.1 was found to be stable for at least 150 regeneration cycles and for at least 9 days at room temperature. Intra- and interassay CVs ranged from 0.9 to 5.9%, and from 2.7 to 8.5%, respectively. Matrices such as serum, bovine serum albumin (BSA), and two pharmaceutical excipients (Pluronic F127 surfactant and sodium carboxymethylcellulose) did not interfere with bFGF analysis over the sensor surface. Therefore, this validated assay has good precision, accuracy and specificity, and has been found useful in quantifying bFGF in several research and development studies.