A Rapid and Sensitive Radioimmunoassay for the Measurement of Proinsulin in Human Serum

Abstract

RIA methodology is used widely to measure proinsulin in human serum. However, some RIAs lack the sensitivity necessary to quantify proinsulin in unextracted serum and require long incubation periods. We developed an RIA with a sensitivity of 3.5 pM that permits the routine measurement of proinsulin in less than 48 h. This was accomplished by using a nonequilibrium binding reaction at room temperature and PEG-assisted second antibody precipitation as the method for separating bound and free proinsulin. We obtained a specific antiproinsulin antibody by absorbing the initial goat antiserum with human C-peptide-agarose. Proinsulin produced 50% displacement of tracer at 25.6 pM, whereas both human insulin and C-peptide failed to displace tracer at concentrations as high as 1 microM. We evaluated several cleaved derivatives of proinsulin for cross-reactivity with the antibody. B-chain-C-peptide cleaved derivatives (less than or equal to 50% cross-reactivity) were more potent than A-chain-C-peptide cleaved derivatives (less than 5% cross-reactivity). However, all derivatives cleaved in the region from 56-60 failed to cross-react with the antiserum. These data indicate that a major antigenic determinant is present on the C-peptide region of proinsulin adjacent to the A-chain-C-peptide junction. After administration of an oral glycemic challenge, the mean fasting serum concentration of proinsulin in normal adults rose from 4.1 +/- 0.28 to 23.6 +/- 3.8 pM. We found a significant difference in the proinsulin concentrations in 6 adults before and after a glycemic challenge when two different antibodies were used in the RIA.(ABSTRACT TRUNCATED AT 250 WORDS)

Total immunoreactive proinsulin, immunoreactive insulin and specific insulin in relation to conversion to NIDDM: the Mexico City Diabetes Study

“…Cross-reactivity with intact and des 31, 32 proinsulin (the major circulating form of split proinsulin) has been determined to be 0.2 % and less than 0.2 %, respectively [37]. The cross-reactivity of the insulin assay with des 64, 65 proinsulin is much higher ( ∼ 76 %), but des 64, 65 proinsulin comprises less than 5 % of total circulating insulin precursors [38]. Within-and between-assay coefficients of variation of the specific insulin assay ranged from 3 to 7 %.…”

“…The lower limit of detection of the assay was 14.4 pmol/l. Insulin precursors were measured by a non-equilibrium RIA method [38]. This method was modified slightly to improve the sensitivity at low concentrations of proinsulin.…”

Subclinical Coronary Atherosclerosis in Asymptomatic Filipino and White Women

“…Proinsulin and C-peptide were measured in the research laboratory of S. Edwin Fineberg (Indiana University). Proinsulin was measured by radioimmunoassay based on a method by Bowsher et al 18 C-peptide was measured by radioimmunoassay (Linco Research, Inc). Fasting plasma lipids and lipoproteins, total cholesterol, HDL cholesterol, and triglyceride levels were measured by enzymatic methods.…”

Hyperproinsulinemia is not a characteristic feature in the offspring of patients with different phenotypes of type II diabetes

“…Within-and between-assay coef®cients of variation of the speci®c insulin assay ranged from 3 to 7%. Proinsulin concentrations were measured by a non-equilibrium RIA method (human proinsulin RIA kit; Linco, St Louis, MO, USA) (32). The polyclonal antibody used in this assay recognizes a proinsulin speci®c epitope formed by the intact A-chain±C-peptide junction.…”