Detection of elevated basic fibroblast growth factor during early hours of in vitro angiogenesis using a fast ELISA immunoassay.
Basic FGF (bFGF) is a growth factor that is thought to play an important role in angiogenesis. Available assays that are used to detect bFGF are long and cumbersome. Here, we present a fast, easy and sensitive sandwich-type enzyme immunoassay for bFGF detection. Our method is a modification of the method described by Watanabe et al (Biochem. Biophys. Res. Commun. 1991; 175, 229). Two monoclonal antibodies for antigen capture and one noncongugated polyclonal antibody for antigen detection are used instead of using three monoclonal antibodies with the congugation of one of them for detection. There is no change in the sensitivity of the assay with average detection limit of 1 pg/well. Acidic fibroblast growth factor does not interfere with the assay. Using this method, samples from conditioned media of capillary endothelial cell culture before and after angiogenesis were measured. Associated with detection of start of tube formation, basic FGF was elevated at 8 hours from angiogenic stimulation and peaked at 48 hour (4 times control), showing for the first time in an in vitro system that there is a transient increase in endogenous bFGF accompanying early steps of angiogenesis which in turn may be the trigger for new capillary formation.
Gabra
N
N
Department of Medicine, Brown University School of Medicine, Providence, RI.
Khayat
A
A
Calabresi
P
P
Khiat A [corrected to Khayat
A ]
A]
D000311:Adrenal Glands / Q000098:blood supply; D000818:Animals; D000911:Antibodies, Monoclonal; D002196:Capillaries / Q000473:pathology / Q000502:physiology*; D002417:Cattle; D002478:Cells, Cultured; D003429:Cross Reactions; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D016222:Fibroblast Growth Factor 2 / Q000032:analysis* / Q000378:metabolism*; D006801:Humans; D009389:Neovascularization, Pathologic; D011994:Recombinant Proteins / Q000032:analysis; D012680:Sensitivity and Specificity
D000911:Antibodies, Monoclonal; D011994:Recombinant Proteins; D016222:Fibroblast Growth Factor 2