Ziegler O, Cantin C, Germain L, Dupuis M, Sekaly R P, Drouin P, Chiasson J L
Institut de Recherches Cliniques de Montréal, Québec, Canada.
Cytometry. 1994 Aug 1;16(4):339-45. doi: 10.1002/cyto.990160408.
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 beta 1-biotinylinsulin (B-insulin) and N alpha B1-(biotinyl-epsilon-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-anti-insulin 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 x 10(-9) M for native insulin, 2.7 x 10(-9) M for B-insulin, 3.3 x 10(-9) M for NBC-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-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.
利用异硫氰酸荧光素标记的胰岛素（FITC -胰岛素）以及与链霉亲和素 - 藻红蛋白偶联的生物素化胰岛素（Nαβ1 - 生物素化胰岛素（B - 胰岛素）和NαB1 - （生物素 - ε - 氨基己酰）胰岛素（NBC - 胰岛素）），通过流式细胞术研究了胰岛素与培养的IM - 9人淋巴细胞的结合。参考方法是125I - 胰岛素结合以及用于流式细胞术的胰岛素 - 抗胰岛素抗体复合物。B - 胰岛素、NBC - 胰岛素和胰岛素 - 抗胰岛素抗体复合物的125I - 胰岛素结合与荧光增加之间存在密切相关性，但FITC - 胰岛素不存在这种相关性。NBC - 胰岛素的荧光增加最大（79±9个通道），胰岛素 - 抗胰岛素抗体复合物最小（34±2个通道）（P < 0.05）。FITC - 胰岛素和B - 胰岛素给出了相似的结果：47±6和59±6个通道。使天然胰岛素的125I - 胰岛素结合减少50%的浓度，对于天然胰岛素为1.1×10⁻⁹ M，对于B - 胰岛素为2.7×10⁻⁹ M，对于NBC - 胰岛素为3.3×10⁻⁹ M，对于FITC - 胰岛素为6.6×10⁻⁹ M（P < 0.05）。B - 胰岛素和NBC - 胰岛素的非特异性结合较低，但对于10⁻⁶ M的FITC - 胰岛素，非特异性结合达到75%。这些结果表明，B - 胰岛素和NBC - 胰岛素是使用流式细胞术进行胰岛素结合研究的合适配体。这种两步法比胰岛素 - 抗胰岛素抗体复合物技术更简便。FITC - 胰岛素的亲和力、特异性和敏感性较差，不太适合。
