Validation of a competitive enzyme-linked immunosorbent assay for measuring the insulin-regulatable glucose transporter.
A novel competitive enzyme-linked immunosorbent assay (ELISA) for measuring the insulin-regulatable glucose transporter (GLUT4) has been developed in our laboratory. The purpose of the present study was to verify the competitive ELISA assay. Towards this goal, a time-course study was conducted in control Wistar rat hearts to determine the points at which insulin stimulated an initial and a maximal GLUT4 translocation. Plasma and intracellular membrane fractions were purified from heart ventricles isolated from rats either in the basal state or injected with insulin. GLUT4 content in the membrane fractions was quantified with both the competitive ELISA method and also with enhanced chemiluminescence (ECL) Western blot. It was shown that after insulin injection plasma membrane GLUT4 level increased about 60% at 15 min and intracellular GLUT4 decreased about 40-50% at 5 min and remained at this level throughout the remaining 25 min by both methods. In conclusion, the data from this study demonstrate that the ELISA assay is reliable as verified by the Western blot method.
Li
S H
SH
Division of Pharmacology and Toxicology, Faculty of Pharmaceutical Sciences, The University of British Columbia, 2146 East Mall, Vancouver, British Columbia, Canada V6T 1Z3.
McNeill
J H
JH
D000818:Animals; D001786:Blood Glucose / Q000378:metabolism; D015153:Blotting, Western; D002417:Cattle; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods* / Q000592:standards; D051275:Glucose Transporter Type 4; D006352:Heart Ventricles / Q000187:drug effects / Q000378:metabolism; D007328:Insulin / Q000097:blood / Q000493:pharmacokinetics / Q000494:pharmacology; D007425:Intracellular Membranes / Q000187:drug effects / Q000378:metabolism; D008163:Luminescent Measurements; D009004:Monosaccharide Transport Proteins / Q000378:metabolism*; D009124:Muscle Proteins; D009206:Myocardium / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D015203:Reproducibility of Results; D013347:Subcellular Fractions / Q000378:metabolism; D013552:Swine
D001786:Blood Glucose; D051275:Glucose Transporter Type 4; D007328:Insulin; D009004:Monosaccharide Transport Proteins; D009124:Muscle Proteins; C495694:Slc2a4 protein, rat