INSULIN BINDING TO HUMAN CULTURED LYMPHOCYTES MEASURED BY FLOW-CYTOMETRY USING 3 LIGANDS

The binding of insulin to cultured IM-9 human lymphocytes was studied by flow cytometry using FITC-insulin and biotinylated insulins coupled to streptavidin-phycoerythrin (N-alpha B1-biotinylinsulin (B-insulin) and N-alpha B1-(biotinyl-epsilon-aminocaproyl)insulin (NBC-insulin)). The reference methods were I-125-insulin binding and the insulin-anti insulin antibody complexes for flow cytometry. There was a close corre lation between I-125-insulin binding and increase in fluorescence for B-insuhn, NBC-insulin, and insulin-antiinsulin antibody complexes, but not for FITC-insulin. NBC-insulin gave the largest increase in fluore scence (79 +/- 9 channels) and the the insulin-antiinsulin antibody co mplexes the smallest (34 +/- 2 channels) (P < 0.05). FITC-insulin and B-insulin gave similar results: 47 +/- 6 and 59 +/- 6 channels. The co ncentration reducing I-125-insulin binding by 50% was 1.1 x 10(-9) M f or native insulin, 2.7 x 10(-9) M for B-insulin, 3.3 x 10(-9) M for NB C-insulin, and 6.6 x 10(-9) M for FITC-insulin (P < 0.05). Nonspecific binding was low for B-insulin and NBC-insulin but reached 75% for 10( -6) M FITC-insulin. These results suggest that B-insulin and NBC-insul in are suitable ligands for insulin binding studies using flow cytomet ry. This two-step procedure is easier than the insulin-antiinsulin ant ibody complex technique. Its poor affinity, specificity, and sensitivi ty make FITC-insulin less suitable. (C) 1994 Wiley-Liss, Inc.