Insulin binding to human cultured lymphocytes measured by flow cytometry using three ligands

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Abstract
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 (NαB1‐biotinylinsulin (B‐insulin) and NαB1‐(biotinyl‐ϵ‐aminocaproyl)insulin (NBC‐insulin)). The reference methods were 125I‐insulin binding and the insulin‐antiinsulin antibody complexes for flow cytometry. There was a close correlation between 125I‐insulin binding and increase in fluorescence for B‐insulin, NBC‐insulin, and insulin‐antiinsulin antibody complexes, but not for FITC‐insulin. NBC‐insulin gave the largest increase in fluorescence (79 ± 9 channels) and the the insulin‐antiinsulin antibody complexes the smallest (34 ± 2 channels) (P < 0.05). FITC‐insulin and B‐insulin gave similar results: 47 ± 6 and 59 ± 6 channels. The concentration reducing 125I‐insulin binding by 50% was 1.1 × 10−9 M for native insulin, 2.7 × 10−9 M for B‐insulin, 3.3 × 10−9 M for NBC‐insulin, and 6.6 × 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‐insulin are suitable ligands for insulin binding studies using flow cytometry. This two‐step procedure is easier than the insulin‐antiinsulin antibody complex technique. Its poor affinity, specificity, and sensitivity make FITC‐insulin less suitable.