The development of a quantitative RIA for basic fibroblast growth factor using polyclonal antibodies against the 157 amino acid form of human bFGF. The identification of bFGF in adherent elicited murine peritoneal macrophages.
Polyclonal antibodies which have the capacity to neutralize the biological activity of basic fibriblast growth factor (bFGF) in vitro, have been raised in rabbits against the 157 amino acid form of bFGF purified from human placenta. In a dot blot assay the anti-bFGF antibodies do not recognize the acidic form of FGF (aFGF) with which the basic form shares significant amino acid sequence homology. As determined by immunoblotting, bFGF antibodies recognized only bFGF in a mixture of placentally derived heparin-binding proteins, demonstrating the specificity of these antibodies. Using the anti-human bFGF antibodies, we have developed a solid-phase competitive radioimmunoassay sensitive to 7.8 ng/ml (0.4 pmol/ml) for bFGF. aFGF does not compete with bFGF for binding to the antibodies in the radioimmunoassay even at 2.04 micrograms/ml. The specificity of the assay was further demonstrated by a lack of competition of cytochrome C, myoglobin, epidermal growth factor or bovine serum albumin with bFGF for binding to the antibodies. We have identified bFGF in extracts of adherent thioglycollate-stimulated mouse peritoneal macrophages by immunological criteria including the ability of the extract to compete with 125I-bFGF for binding to affinity-purified anti-human bFGF antibodies in the RIA and the ability of these antibodies in inhibit the bFGF-like biological activity of the macrophage extract.
Joseph-Silverstein
J
J
Department of Cell Biology, New York University Medical Center 10016.
Moscatelli
D
D
Rifkin
D B
DB
D000596:Amino Acids; D000818:Animals; D000906:Antibodies / Q000032:analysis; D000918:Antibody Specificity; D001666:Binding Sites, Antibody; D001667:Binding, Competitive; D002448:Cell Adhesion; D005260:Female; D005346:Fibroblast Growth Factors / Q000032:analysis* / Q000276:immunology; D006801:Humans; D008262:Macrophage Activation; D008264:Macrophages / Q000032:analysis*; D051379:Mice; D008807:Mice, Inbred BALB C; D010529:Peritoneal Cavity / Q000166:cytology; D011817:Rabbits; D011863:Radioimmunoassay / Q000379:methods*
D000596:Amino Acids; D000906:Antibodies; D005346:Fibroblast Growth Factors