First direct assay for intact human proinsulin.

We describe a sensitive two-site sandwich enzyme-linked immunosorbent assay for the measurement of intact human proinsulin in 100 microL of serum or plasma. The assay is based on the use of two monoclonal antibodies specific for epitopes at the C-peptide/insulin A chain junction and at the insulin B chain/C-peptide junction, respectively. Cross-reactivities with insulin, C-peptide, and the four proinsulin conversion intermediates were negligible.

Highly specific radioimmunoassay for human insulin based on immune exclusion of all insulin precursors.

We describe a rapid and simple insulin RIA in which proinsulin and conversion intermediates do not interfere. Three monoclonal antibodies (S1, S2, and S53) were selected for their specificity (directed, respectively, against the B10 region, the junction between A chain and C-peptide, and the junction between B chain and C-peptide), their affinity constant (approximately 10(10) L/mol), and their interactive properties in mixture. S2 and S53 were able to bind simultaneously to the same proinsulin molecule, whereas neither could bind simultaneously with S1.

Imaging of the buffering effect of insulin antibodies in the autoimmune hypoglycemic syndrome.

Insulin autoimmune hypoglycemia is characterized by recurrent hypoglycemia and high levels of immunoreactive insulin in the presence of insulin autoantibodies. The mechanisms inducing hypoglycemia are largely unknown. An [123I]insulin scintigraphic scanning was performed to directly demonstrate the effect of antibodies on insulin biodistribution in one patient with this syndrome both before and after treatment.

Proinsulin and its conversion intermediates in human pancreas and isolated islet tissue: kinetics and steady-state analysis.

In non-insulin-dependent diabetes, circulating insulin-related immunoreactivity (IRI) is often composed of a higher fraction of the incompletely converted forms proinsulin and des-31,32 proinsulin. The present study describes an immunoadsorption method for measuring the proportions of proinsulin, its two split products, and insulin in human pancreatic tissue and for determining their rates of formation in human isolated islets. The method uses two junction-specific monoclonal proinsulin antibodies in a protein G fractionation; it is validated by > or = 90% specificity and recovery.

Abnormal circulating pancreatic enzyme activities in more than twenty-five percent of recent-onset insulin-dependent diabetic patients: association of hyperlipasemia with high-titer islet cell antibodies. Belgian Diabetes Registry.

Pancreatic amylase and lipase activities were measured in sera of 307 Caucasian insulin-dependent diabetes mellitus patients (IDDM) at clinical onset, 303 nondiabetic siblings of registered patients, and 207 control subjects under age 40 years. In all subject groups lipasemia and pancreatic (but not salivary) amylasemia increased with age and were significantly correlated. Using age-dependent reference ranges, reduced pancreatic enzyme levels were measured in 18% of patients, 6% of siblings, and only 2% of control subjects (p < 0.

Immunogenicity of long-term intraperitoneal insulin administration with implantable programmable pumps. Metabolic consequences.

Objective: To assess immunogenicity of intraperitoneal insulin infusion via implanted pumps by two methods and to evaluate the possible influence of an increased antibody level on metabolic and clinical parameters.

Research Design And Methods: We studied insulin antibody levels in 17 type I diabetic patients before and until 24 months after implantation of a programmable pump delivering insulin intraperitoneally. Antibody levels were determined by radioimmunoassay (RIA) and enzyme-linked immunosorbent assay (ELISA).

Heterogeneous IgG subclass distribution of islet cell antibodies.

Islet cell antibodies (ICA)-IgG are the serological marker of type 1 diabetes, an organ-specific autoimmune disease. A proportion of patients also have thyro-gastric autoimmunity, implying a broader humoral autoreactivity in these cases. In order to determine whether this is also reflected within islet cell antibodies (ICA) we examined the ICA-IgG subclass distribution in type 1 diabetic patients with or without associated thyro-gastric autoantibodies.

Insulin autoantibodies and immune response to human insulin therapy in 24 type 1 (insulin-dependent) diabetic children: superiority of radio binding assay over solid phase assay.

To evaluate the immunization pattern against human insulin, 24 newly diagnosed diabetic children (12 females, 12 males; mean age: 7 +/- 4 years) were treated from diagnosis onwards with semisynthetic human insulin (NOVO). Informed consent was obtained from all parents. Blood samples were taken before, 1, 2, 3, 4, 6 and 8 weeks after the start of therapy and, thereafter, at monthly intervals for 2 years.

Evidence that insulin-like growth factor 2 (IGF2) is the dominant thymic peptide of the insulin superfamily.

The central T-cell tolerance of neuroendocrine functions has been proposed to be primarily induced by the thymic repertoire of neuroendocrine self antigens. The present study aimed at characterizing the human thymic insulin-related autoantigen able to represent the pancreatic B-cell function in face of the developing T-cells. Immunofluorescence studies were performed on human and rat thymic sections, as well as on the rat IT-45R1 thymic epithelial cell line using several antibodies to epitopes of the insulin peptide superfamily.

Detection of autoantibodies against islet amyloid polypeptide in human serum. Lack of association with type 1 (insulin-dependent) diabetes mellitus, or with conditions favouring amyloid deposition in islets. The Belgian Diabetes Registry.

A radiobinding assay for the detection of autoantibodies against islet amyloid polypeptide was developed, analytically validated, and--in parallel with a similar assay for the detection of autoantibodies against insulin--applied to sera from recent-onset Type 1 (insulin-dependent) diabetic patients and from age- and sex-matched control subjects. There was no difference in islet amyloid polypeptide autoantibody titres between patient groups and matched control subjects, nor within subject groups according to age. At onset of Type 1 diabetes, elevated islet amyloid polypeptide-autoantibody levels (> 97th percentile of control subjects) were only detected in 1 of 30 patients aged 0-19 years and in 2 of 35 patients aged 20-39 years.

[Effects of chronic administration of benfluorex on the pharmacokinetics of iodine 123 labelled insulin in Zucker obese rats (fa/fa)].

Zucker obese rats (fa/fa), aged 8 to 10 weeks, were treated orally by benfluorex (2 x 25 mg.kg-1, day-1) for 2 weeks and were compared with a control group of the same age treated with placebo. Benfluorex induced a break in the weight curve, a significant fall in serum triglycerides, blood glucose and plasma insulin and in the insulin content of the pancreas.

Polymorphism of insulin antibodies in six patients with insulin-immune hypoglycaemic syndrome.

Insulin antibodies in six patients with immune hypoglycaemic syndrome were studied. The antibodies displayed a higher affinity for bovine insulin in two patients, were specific for human insulin in one patient and non-species specific in the other three patients. The predominant IgG subclass of the insulin antibodies was IgG4 in two patients, IgG3 in two and IgG1 in two.

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.

Hybridomas 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.

[Insulin antibodies: methodology and clinical implications].

Anti-insulin antibodies detection is based on the demonstration of a specific and saturable binding to insulin either radiolabelled with 125 I (Radiobinding assay or RBA) or coated on a solid phase (Enzyme linked immunosorbent assay or EIA). The 2 assays are remarkably different by their sensitivity to the affinity of the antigen antibody reaction. In addition, RBA may be biased by the presence of the iodine atom on the radioiodinated insulin whereas, at least on theoretical grounds.

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 (greater than or equal to nondiabetic mean + 3SD) was 2 of 178 (1.

Clonally restricted insulin autoantibodies in a cohort of 2200 healthy blood donors.

Serum samples of 2200 blood donors were screened for anti-insulin IgG by enzyme-linked immunosorbent assay. Specificity of detected antibodies was verified by competition with an excess of insulin and observation that saturated anti-insulin IgG were no longer measurable. The upper limit of measured signal in the population was defined as the mean plus three SD.

Is quantitative assessment of insulin-antibodies and autoantibodies feasible?

Nine selected sera were studied using radioimmunoassay and enzyme linked immunosorbent assay; eight contained insulin antibodies and were from Type 1 (insulin-dependent) diabetic patients, one of whom had antibody-mediated insulin resistance, and one contained insulin-autoantibodies and was from an asymptomatic blood donor. Sera were assayed in serial dilution to assess their suitability for use as reference standards. Dilution curves were non-parallel in radioimmunoassay but were parallel in immunosorbent assay.

Scintigraphic distribution of 123 I labelled proinsulin, split conversion intermediates and insulin in rats.

Insulin, biosynthetic human proinsulin and 2 human proinsulin conversion intermediates, des (64, 65) human proinsulin and des (31, 32) human proinsulin, were labelled with 123 I and the derivatives monosubstituted on Tyr A14 were purified by reverse phase high performance liquid chromatography. The four tracers were injected into anaesthetized rats via a jugular or a portal vein and time activity curves were generated for the liver and kidneys using a gamma camera and an online computer. Liver extraction coefficients varied in the order insulin (38%), des (64, 65) human proinsulin (11.

Progesterone and synthetic steroids produce insulin resistance at the post-receptor level in adipocytes of female rats.

The effects of progesterone, its agonists (progestin RU-5020, glucocorticoid RU-26988) and antagonist (antiprogesterone, anti-glucocorticoid RU-486) were tested on isolated fat cells in vitro. When added to the incubation medium, all four steroids decreased basal glucose oxidation. The inhibitory effect of the steroids appeared early (20 min incubation) and was sustained during a 2-h incubation.

The diabetic intrauterine milieu has a long-lasting effect on insulin secretion by B cells and on insulin uptake by target tissues.

From our previous work, it appears that fetal development in the abnormal intrauterine milieu of a mother with diabetes results in impaired glucose tolerance in adult life. In adult Wistar rats that were the offspring of mildly or severely diabetic mothers, in vitro islet stimulation and in vivo insulin uptake studies were undertaken to distinguish between alterations in glucose sensitivity and insulin secretion at the B cell level and alterations in insulin sensitivity and uptake at the level of the peripheral tissues. Insulin output after glucose stimulation by isolated islets was lower than normal in the rats of mothers with mild diabetes and higher than normal in the animals of severely diabetic mothers, confirming the results of previous in vivo studies.

Glycerol-insulin raises circulating insulin antibodies in type I diabetic patients treated with permanently implanted devices.

The instability of insulin in the reservoirs of implantable insulin delivery devices has been a major obstacle in implementing this form of therapy. To overcome the problem of precipitation, a glycerol-insulin preparation has been used in large-scale long-term clinical trials. The aim of this study was to evaluate the stability of the glycerol-insulin solution and its effects on circulating insulin antibodies in eight type I diabetic patients who were implanted with an Infusaid pump (Infusaid Corporation, Norwood, MA) and followed for 1 year or more.

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

Human 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.

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.

Visualization of viral clearance in the living animal.

The early events in viral dissemination via the bloodstream were identified by monitoring the fate of 123I-radiolabeled reovirus after it was injected intravenously in rats. Continuous scintillation camera imaging showed that reovirus serotypes 1 and 3 were cleared from the circulation in less than 10 minutes by specific and distinct target organs. Reovirus serotype 1 accumulated predominantly in the lungs and the liver, whereas serotype 3 accumulated in the liver and the spleen with very little virus uptake by the lungs.