Peroxidase-linked anti-basic fibroblast growth factor monoclonal antibody Fab' conjugates: application for two-site enzyme immunoassay and immunohistochemical detection.

Abstract

After conjugating thiol groups in the hinge region of monoclonal antibody (mAb) Fab' fragments specific for basic fibroblast growth factor (bFGF) with maleimido-horseradish peroxidase HRP) complexes synthesized by incubation of HRP with the heterobifunctional reagent N-succinimidyl-4-(maleimidomethyl)cyclohexane-1-carboxylate, we developed a fluorometric enzyme immunoassay method based on the sandwiching of the factor between anti-bFGF IgG-coated polystyrene beads and the conjugates, and also an immunohistochemical method for detection of the location of the factor. The discriminatory detection limit by the developed enzyme immunoassay (EIA) was as low as 30 pg/mL. The reproducibility of within- and between-assay series was 6.07-9.18% and 6.28-6.82%, respectively, and the recovery of exogenous bFGF from serum was approximately 98%. The curves generated by the concentrated fraction that eluted at the same position as standard bFGF by size-exclusion chromatography on a TSK 2000SW column were parallel to the curve for standard bFGF. From these results, we consider the developed EIA method to be acceptable in regard to sensitivity, precision, and specificity. Also, without the introduction of any additional signal amplification system, positive immunohistochemical reactions were successfully detected by the HRP-linked anti-bFGF mAb Fab' in fibroblastic and endothelial cells, which have already been shown to synthesize and secrete bFGF, indicating that these conjugates provide a useful means for direct immunohistochemical detection of the factor.