Characterization of polyclonal antibodies that distinguish acidic and basic fibroblast growth factors by using western immunoblotting and enzyme-linked immunosorbent assays

Abstract

Rabbit polyclonal antibodies were raised against ovalbumin conjugates of purified bovine brain acidic fibroblast growth factor (aFGF) and a synthetic peptide containing the N^α‐terminal 1–24 amino acid sequence of bovine basic fibroblast growth factor (bFGF). Theses antibodies were used to specifically detect 1‐ng quantities of aFGF and bFGF by using enzyme‐linked immunosorbent assay(ELISA)and Western immunoblot procedures. Antibodies raised against aFGF recognized bovine brain aFGF and bovine recombinant aFGF by very poorly recognized recombinant bFGF or purified porcine or bovine pituitary bFGF with ELISA and Western immunoblot procedures. Antibodies raised against bFGF (1–24) recognized purified bovine, procine, and recombinant human bFGF but only very poorly reconized aFGF with ELISA and Western immunoblot procedures. In vitro addition of anti‐bFGF antibodies was able to partially neutralize bFGF‐stimulated ³H‐thymidine incorporation by COMMA‐D mouse mammary epithelial cells while having no effect on aFGF or epidermal growth factor (EGF) stimulation. In vitro additionof anti‐aFGF antibodies had no effect on bFGF‐or EGF‐stimulated ³H‐thymidine incorporation, but surprisingly, had a potentiating effect on aFGF stimulation. Antibodies against aFGF immobilized on protein A‐Sepharose were able to specifically and completely remove mitogenic activity from solutions containing aFGF but had no effect on removal of mitogenic activity from control solutions containing bFGF or EGF. similarly, immobilized anti‐bFGF antibodies completely removed mitogenic activity from solutions of bFGF, but notaFGF or EGF controls. These antibodies have been useful for the identification and characterization of growth factors from tissue and recombinant sources.