In situ assay of basic fibroblast growth factor in cultured cells with a specific monoclonal antibody.
The localization of basic fibroblast growth factor (bFGF) in various cultured bFGF-producing cells, including bovine capillary endothelial (BCE) cells and human cholangiocellular carcinoma (HuCC-T1) cells, was determined by measuring binding of 125I-labeled specific monoclonal antibody against bFGF. bFGF on the cell surface and within the cells was determined by counting the radioactivity of the labeled monoclonal antibody bound to the cells before and after increasing their permeability by treatment with alcohol or Triton X-100. The radioactivity bound to these cells decreased with increase in the concentration of unlabeled monoclonal antibody. Scatchard analyses of these data suggested the quantitative localization of bFGF and Kd values showing the specific binding. This method was designated as in situ assay with radio-labeled antibody (ISARA). bFGF detected on the cell surface and within the cells could be partially removed by treatment with heparin or heparinase and with heparin or DNase, respectively. Exogenous bFGF bound to cells not producing bFGF was also quantified by ISARA. Measurements of bFGF in extracts of cells producing bFGF suggested that 8.3-13.3% of the bFGF associated with the cells was detected by ISARA. This method should be useful for quantitative confirmation of immunohistochemical results on bFGF.
Minemura
M
M
Department of Biochemistry, Kanazawa Medical University, Ishikawa, Japan.
Yoshitake
Y
Y
Matsuzaki
K
K
Watanabe
A
A
Nishikawa
K
K
D000818:Animals; D000911:Antibodies, Monoclonal; D000918:Antibody Specificity; D016222:Fibroblast Growth Factor 2 / Q000032:analysis*; D006801:Humans; D007118:Immunoassay / Q000379:methods*; D007150:Immunohistochemistry; D007457:Iodine Radioisotopes; D011863:Radioimmunoassay; D051381:Rats; D013194:Staining and Labeling; D014407:Tumor Cells, Cultured / Q000737:chemistry
D000911:Antibodies, Monoclonal; D007457:Iodine Radioisotopes; D016222:Fibroblast Growth Factor 2