A rapid, sensitive, and easy-to-perform solid phase insulin radioimmunoassay

Accurate measurement of basal insulin release in perifusion, perfusion and low-density β-cell preparations has been difficult with present assays. A simple competitive, equilibrium, 15-hour insulin assay using 125I-insulin with microtiter immobilized antubody, has been developed. This method, a Solid-phase-RadioImmunoAssay (SPRIA), is very sensitive and has a broad useful range (1 - 64 μU/ml). For a test series of 4 standard curves, interassay variation between controls of 1, 4, 16, and 64 μU/ml was ±5.2% (SEM) and intra-assay variation over the range of standards between 0.5 to 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 γ-counters, while reducing the radioactive and glass waste presently produced by RIA (test-tube can be reused). The radioactive of unused test-tubes was compared againts 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 Fursth rats showed a close correlation between SPRIA and RIA insulin values (linear regression, r 2 = 0.990). This SPRIA measured plasma insulin levels from a human oGTT with a variation of ≤3.7% (SSEM0 between sample triplicates. Standard curves from the three commonly measured insulin isoforms (human, rat and porcine) showed a high correlation (mulitiple linear regression, r 2 = 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 Dullbecco'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 deal for the rapid quantification of insulin from isolated islets of Langerhans, isolated β-cells, acetic acid extracts or plasma with greater sensitivity, and less waste than the conventional RIA has been developed.