F. Sodoyez-Goffaux

Advantages and pitfalls of radioimmune and enzyme linked immunosorbent assays of insulin antibodies

SummaryHuman sera were tested for insulin antibodies by fluid and solid phase assays. Radioimmune titres determined with 125-I Tyr A14 insulin were not correlated with those obtained using insulin coated microplates and enzyme linked immunodetection (n=60). Several reasons for this lack of correlation were found. Iodine substitution on the A14 residue of insulin may significantly alter the avidity of some insulin antibodies for their ligand; hence, disclosing a heretofore unsuspected pitfall for antibody determination by radio-immunoassay. Specificity for bovine insulin was easily demonstrable in fluid phase by comparing the binding of monoiodinated bovine, porcine and human insulin. By contrast, in solid phase assay, titres obtained with microplates coated with bovine or human insulin were almost equal, regardless of the serum specificity for bovine insulin. This lack of specificity of the solid phase assay is not due to denaturation or unavailability of the bovine specific epitope because: bovine specificity could be demonstrated by competitive assay, after preincubation of the serum with insulin of the different species; and, coating with crosslinked insulin dimers or oligomers instead of monomers did not unmask bovine specificity. It is concluded that radioimmune methods are best suited to study specificity but may be biased by the presence of the radioiodine label whereas solid phase assay detects low avidity antibodies with great efficiency but is less appropriate to study specificity.

Evidence for an Insulin-Induced Inhibition of Insulin Release by Isolated Islets of Langerhans∗

Summary Isolated islets of Langerhans were incubated in a medium containing different concentrations of glucose with or without different concentrations of insulin. The results suggest that insulin inhibits further insulin release and that the insulin level at which this feedback mechanism operates is determined by the intensity of the insulinogenic stimulus.

Effects of Gestational Age, Birth and Feeding on the Insulinogenic Response to Glucose and Tolbutamide by Fetal and Newborn Rat Pancreas

Perinatal maturation of the pancreatic beta cells was studied in the rat. Insulin synthesis proceeded rapidly be tween the twentieth gestational day and the third postnatal day. The insulin concentration in the pancreas and the amount of insulin available per gram of body weight reached a maximum sixty hours after birth and then decreased slowly toward the values observed in the adult rat. The pancreatic beta cells became responsive to glucose and to tolbutamide during the twenty-third day after fertilization, This phenomenon seemed to be related to the gestational age and not to act of birth or to feeding, although feeding appeared to increase the insulinogenic response to glucose.

Superiority of Radiobinding Assay Over ELISA for Detection of IAAs in Newly Diagnosed Type I Diabetic Children

Objective Liquid- or solid-phase assays have been used for insulin autoantibody (IAA) determination, and the method of IAA measurement has not been standardized. Research Design and Methods IAAs were determined by radiobinding assay (RBA) and enzyme-linked immunosorbent assay (ELISA) in two large age-matched groups of nondiabetic and newly diagnosed insulin-dependent (type I) diabetic children. Results Positivity for IAA by RBA (≥ to nondiabetic mean + 3SD) was 2 of 178 (1.1%) and 55 of 173 (32%) in nondiabetic and diabetic children, respectively. Prevalence of IAA by RBA was significantly higher in the youngest age-group (63% between 0-4 yr). Positivity for IAA by ELISA was 1 of 178 (0.6%) and 8 of 169 (4.7%) in nondiabetic and diabetic children, respectively. Concordance rates between both assays were 0 of 3 (0%) in control subjects and 5 of 58 (8.6%) in diabetic children. Conclusions We conclude that RBA is more appropriate than ELISA for IAA detection at the onset of the disease. In addition, because available data suggest that IAAs detected by RBA only are high-affinity antibodies, it is tempting to speculate that IAAs reflect a mature immune reaction against endogenous insulin.

Insulin Secretion and Metabolism during the Perinatal Period in the Rat: EVIDENCE FOR A PLACENTAL ROLE IN FETAL HYPERINSULINEMIA

To better understand why plasma immunoreactive insulin (IRI) concentration is high in the rat fetus during the last 3 d of gestation and why fetal hyperinsulinemia abruptly subsides after birth, insulin secretion and metabolic clearance rates were estimated in fetuses and nursed pups. Intravenously injected [(125)I]monoiodoinsulin was cleared from the plasma of prematurely delivered pups at least as rapidly as from that of 7- to 10-d-old pups, suggesting that fetal hyperinsulinemia is not a result of slow clearance of the hormone. The fetal liver bound 35% of the injected label within 3 min, and binding was saturable. The uptake of radioactivity by the fetal kidney was nonsaturable and much lower than that of adult rat kidney. Isolated fetal islets were already reactive to glucose on the 19th d of gestation. Pancreatic insulin secretory capacity was estimated by measuring (a) the insulin release of isolated islets incubated in the presence of 2.8 mM glucose, (b) the insulin content of the same islets, and (c) the total insulin extracted from the pancreas, using the formula (a x c)/b. 2 d before birth, the pancreatic insulin secretory capacity was high, accounting for fetal hyperinsulinemia. It was even higher after birth, not accounting for the postnatal decrease in plasma IRI concentration. Pups delivered by caesarian section 1 d before term exhibited a brisk decrease in plasma IRI concentration when the cord was cut. By contrast, if the feto-placental unit was removed from the dam, maintaining fetal blood circulation through the placenta, fetal plasma IRI concentration remained as high as in utero. These experiments suggest that a placental factor stimulates fetal insulin secretion. After birth, when the cord is cut, insulin secretion is rapidly turned off, and the pups switch from a state of unlimited fuel supply by the mother to a state of fuel saving.

Influence of affinity of antibodies upon their detection by liquid phase radiobinding assay and solid phase enzyme linked immunosorbent assay. Demonstration using monoclonal antibodies raised against rDNA human proinsulin

SummaryHybridomas producing proinsulin antibodies were cloned by limiting dilution of cell cultures obtained by fusion of splenocytes of immunized mice with immortal myeloma cells. Some proinsulin monoclonal antibodies crossreacted with labelled insulin but none did with labelled C-peptide indicating that the involved epitopes were at one of the insulin/C-peptide junctions or included in the insulin moiety. Hybridoma supernatants were assayed for IgG concentration by a solid phase assay and for ligand binding by a radiobinding assay and an enzyme linked immunosorbent assay. The half-life of immune complexes formed with radioligand was measured and, as expected, correlated with affinity as measured by the method of Scatchard. Antibody titres determined by enzyme linked immunosorbent assay did not correlate to those measured by radiobinding assay. IgG concentration correlated to enzyme linked immunosorbent assay titres but not to radiobinding assay titres. Finally, a significant correlation was found between radiobinding assay titre and the product of enzyme linked immunosorbent assay titre by the period of immune complexes. It is concluded that, except for very low affinity antibodies, enzyme linked immunosorbent assay is a capacity assay whereas radiobinding assay is influenced by both antibody concentration and affinity. The former assay is thus best suited to detecting low affinity antibodies whereas the latter is more efficient in the presence of low levels of high affinity antibodies.

Reduction in the Activity of the Pancreatic Islets Induced in Normal Rodents by Prolonged Treatment with Derivatives of Sulfonylurea

Normal Syrian hamsters were given daily intraperitoneal injections of tolbutamide sodium or 0.9 per cent NaCl solution. Normal albino rats were fed a control or a glyburide (HB-419) containing diet. After seven weeks of treatment, the animals were killed, the pancreatic islets were isolated by collagenase digestion and their diameter and secretory activity in vitro were measured. In other animals, the total pancreatic acid-alcohol extractable insulin was measured. In control animals, there was a linear relationship · between body weight on the one hand and pancreatic insulin content on the other. In animals previously treated with tolbutamide, at the dose of 63 mg./kg., the relationships between these two variables were altered: a slight increase in body weight was accompanied by a decrease in pancreatic insulin content. The islets of animals treated with either drug released less insulin in vitro than those of control animals; the degree of B cell depression was related to the dose of tolbutamide which the animals had received. All drug-induced abnormalities disappeared approximately one week after the cessation of treatment. The possible significance of these observations to human therapy is discussed.

Maturation of Liver Handling of Insulin in the Rat Fetus

Purified carrier-free 125I-Tyr insulin (125I-Tyr Ins) was injected into the vitelline vein of rat fetuses in utero after 17, 19, or 21 days of gestation. Three minutes later, a radioactivity concentration index (RCI) was calculated by dividing the specific activity of each organ by that of the feto-placental unit. The highest RCI were found in the liver (4.09, 5.97, and 6.48, respectively after 17, 19, and 21 days of gestation). Binding of 125I-Tyr Ins to the liver was inhibited by coinjection of excess unlabeled insulin. Sequential injections of 125I-Tyr Ins followed by excess unlabeled insulin demonstrated that 125I-Tyr Ins binding to the liver was but partly reversible and allowed us to calculate that the half-life of the tracer in the whole body receptor compartment was 6 min in 21-day postcoitum fetuses. After extraction and chromatographic analysis, liver radioactivity appeared composed of undamaged 125I-Tyr Ins and low mol. wt. degradation products (125I- and 125I-tyrosine). Liver maturation was characterized by an enhanced capacity to degrade 125I-Tyr Ins and to expel low mol. wt. radioactive products out of the cells. Autoradiographic studies demonstrated that 125I-Tyr Ins was bound in a saturable manner to the hepatocytes and, to a lesser extent, to the liver hematopoietic cells. Three minutes after the tracer alone, silver grains were predominantly associated with the hepatocytes′ plasma membranes. Later, the percentage of internalized grains increased. Because the percentage of liver radioactivity identified as low mol. wt. radioactive products was always smaller than that of internalized silver grains, true 125I-Tyr Ins was actually internalized in the hepatocytes. The rate of 125I-Tyr Ins translocation from the membrane into the cytoplasm increased with the degree of liver maturity. It is concluded that during the last 5 days of gestation, liver maturation concerns insulin internalization and degradation, i.e., postreceptor steps rather than receptor ontogenesis.

Function of the pancreatic B-cells in hamsters bearing a transplantable islet cell tumor

Abstract Hyperinsulinism produced in the Syrian hamster by a transplantable islet cell tumor caused a decrease in the amount of insulin extractable from the pancreas and in the amount of insulin released by the B-cell in vivo as well as in vitro. A comparable pancreatic suppression was obtained by daily injections of Lente insulin ( 1.5 U 100 Gm. ), but not by daily injections of phlorizin ( 20 mg. 100 Gm. ). These results suggest that insulin itself plays a mamor role in the islet cells inhibition of tumor-bearing animals.

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

Clearance of immune complexes made of antiinsulin antibodies and 123I-insulin was studied with scintillation scanning in anesthetized rats. Complexes made with purified guinea pig antiinsulin IgG2 (cytophilic isotype) were rapidly cleared by the liver whereas those made with IgG1 remained in the plasma, as did 123I-labeled IgG1 or IgG2 of control animals. Hepatic clearance of insulin-antiinsulin IgG complexes was not inhibited by either an excess of insulin or decomplementation, thereby ruling out interaction with insulin and C3b receptors. Insulin and guinea pig antiinsulin serum or its purified IgG isotypes formed large aggregates exceeding 5 IgG. Antiinsulin antibodies of diabetics, mostly IgG1 and IgG3 (cytophilic isotypes), formed complexes that either remained in plasma (small aggregates) or were cleared by the liver (large aggregates). In conclusion, clearance of insulin-antiinsulin IgG complexes is probably mediated by Fc gamma receptors on macrophages and requires cytophilic subclass composition and formation of large IgG aggregates.