A rapid, sensitive, and easy-to-perform solid phase insulin radioimmunoassay.
Accurate measurement of basal insulin release in perifusion, perfusion and low-density beta-cell preparations has been difficult with present assays. A simple, competitive, equilibrium, 15-hour insulin assay using 125I-insulin with microtiter plate immobilized antibody, has been developed. This method, a Solid-phase-RadioImmunoAssay (SPRIA), is very sensitive and has a broad useful range (1 - 64 microU/ml). For a test series of 4 standard curves, interassay variation between controls of 1, 4, 16, and 64 microU/ml was +/- 5.2% (SEM) and intra-assay variation over the range of standards between 0.5 to 64 microU/ml was 5.1% (SEM). Nonspecific binding was not significantly different from empty borosilicate culture tubes; 4.0 +/- 0.4 and 3.5 +/- 0.5 counts/minute (mean +/- SEM; n = 54), respectively. This SPRIA can be used with existing gamma-counters, while reducing the radioactive and glass waste presently produced by RIA (test-tubes can be reused). The radioactivity of unused test-tubes was compared against test-tubes used for greater than 10 assays, values were 3.5 +/- 0.5 and 4.4 +/- 0.6 counts/minute mean +/- SEM; n = 54), respectively. Results of an oral glucose tolerance test (oGTT) performed on four male Wistar Furth rats showed a close correlation between SPRIA and RIA insulin values (linear regression, r2 = 0.990). This SPRIA measured plasma insulin levels from a human oGTT with a variation of less than or equal to 3.7% (SEM) between sample triplicates. Standard curves from three commonly measured insulin isoforms (human, rat and porcine) showed a high correlation (multiple linear regression, r2 = 0.998, n = 5 standard curves). In order to determine SPRIA's ability to measure acid extracts, insulin recovery from 2N acetic acid was compared against insulin recovery from Dulbecco's Modified Eagles medium (DME). The insulin recovery from 2N acetic acid was greater than 90% of that achieved with DME. In conclusion, an easy-to-perform assay which is ideal for the rapid quantification of insulin from isolated islets of Langerhans, isolated beta-cells, acetic acid extracts or plasma with greater sensitivity, and less waste than the conventional RIA has been developed.
Väänänen
J E
JE
Medical Research Council Regulatory Peptide Group, University of British Columbia, Faculty of Medicine, Vancouver, Canada.
Buchan
A M
AM
Pederson
R A
RA
D000328:Adult; D000818:Animals; D000918:Antibody Specificity; D005951:Glucose Tolerance Test; D006168:Guinea Pigs; D006801:Humans; D007328:Insulin / Q000032:analysis*; D008297:Male; D011863:Radioimmunoassay / Q000379:methods*; D051381:Rats; D011919:Rats, Inbred Strains; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
D007328:Insulin