Insulin binding to human cultured lymphocytes measured by flow cytometry using three 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 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.
Ziegler
O
O
Institut de Recherches Cliniques de Montréal, Québec, Canada.
Cantin
C
C
Germain
L
L
Dupuis
M
M
Sekaly
R P
RP
Drouin
P
P
Chiasson
J L
JL
D000818:Animals; D001710:Biotin / Q000031:analogs & derivatives* / Q000378:metabolism; D002417:Cattle; D005434:Flow Cytometry; D016650:Fluorescein-5-isothiocyanate / Q000031:analogs & derivatives; D006801:Humans; D007328:Insulin / Q000031:analogs & derivatives* / Q000378:metabolism*; D008024:Ligands; D008214:Lymphocytes / Q000378:metabolism*; D009101:Multiple Myeloma / Q000473:pathology; D011485:Protein Binding; D011972:Receptor, Insulin / Q000378:metabolism*; D013552:Swine; D014407:Tumor Cells, Cultured
C090793:B-insulin; C079861:B29-biotin insulin; D007328:Insulin; D008024:Ligands; C090794:NBC-insulin; C031573:insulin, fluorescein-isothiocyanated-; D001710:Biotin; D011972:Receptor, Insulin; D016650:Fluorescein-5-isothiocyanate