Structural Studies of Insulin and Insulin Derivatives Using Various Immunologic Indicators and Antibody Populations

Abstract

These studies suggest that the immunologic indicator in the radioimmune assay, 125I-iodoinsulin, selects antibody populations from within the antiserum that interact with determinants distant from the solvent surface on the insulin molecule to which iodine is substituted. Evidence is presented that the connecting peptide of proinsulin is in close proximity to regions on the solvent surface of the A-chain of insulin that include the tyrosyl residues at A-14 and A-19. A marked immunologic cross-reaction between derivatives of insulin with perturbations in the regions of tyrosyl A-14 and A-19 was noted in the radioimmune assay employing desalanine-(B-30)-desasparagine-(A-21)-insulin antiserum. This observation is consistent with the presence of a restricted population of antibodies in such antisera that is directed toward immunologic determinants in or near the insulin dimer site. The apparent immunologic activity of insulin derivatives depends on which antibody populations from the antiserum pool can react with the immunologic indicatory employed on the one hand and on the composition of antibodies in that antiserum on the other. These studies indicate that the specificity of antibody populations in a given antiserum can be identified and their levels quantitated with several assay systems, each employing one of a variety of indicators.

Effects of zinc ion on the conformation of antigenic determinants on insulin

“…Immune haemolysis-inhibition experiments These were basically the same as the immune haemolysis experiments, except that only Zninsulin-erythrocyte conjugates were used as the immunological indicator. Various amounts of inhibitor (antigen) were added to the antiserum immediately before addition of the Zn-insulinerythrocyte conjugates (Arquilla et al, 1976).…”

“…In this first group of experiments antiserum A was used at a dilution sufficient to lyse 80% of the 8.7 x 106 Zn-insulin-erythrocyte conjugates in the presence of an excess of complement (Brugman & Arquilla, 1973;Arquilla et al, 1976).…”

“…Experiments were therefore designed to study the effects of Zn2+ on the immunological activity of insulin at concentrations where it is thought to exist predominantly as a monomer. Two immunological systems, the immune haemolysis-inhibition assay and radioimmune assay, were used to measure the ability of antibodies to bind to different regions on the insulin molecule (Arquilla et al, 1976).…”

Scintigraphic distribution of complexes of antiinsulin antibodies and 123I-insulin. In vivo studies in rats.

“…The guinea pig IgGl and IgG2 fractions contain very similar levels of insulin antibodies as determined by superimposable radioimmune titration and inhibition curves generated by a previously described method (13)(14)(15). In the absence of inhibitor, 0.1 ml of each fraction diluted 1:2,300 bound 7.0 X 103 cpm 125I-ins.…”

“…1). The AlIgG2 fraction induced passive immune hemolysis of insulin-coated sheep red blood cells (13) As flow rate was 4 ml/h, 10 h were required for elution of IC. As discussed later, to minimize their dissociation during the process ofchromatography, we also determined IC size by a faster technique, using Superose 6 and a fast protein liquid chromatography (FPLC) system (Pharmacia, Uppsala, Sweden) connected with an ultraviolet (UV) detector.…”