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  • 1
    ISSN: 1432-0428
    Keywords: Standardization ; insulin-antibodies ; insulin-auto-antibodies ; radioimmunoassay ; enzyme linked immunosorbent assay
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Summary Nine selected sera were studied using radioim-munoassay 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-auto-antibodies 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. In all sera, insulin antibodies were readily detected in both assays whereas the low avidity insulin autoantibodies were only detected by immunosorbent assay and not at all by radioimmunoassay, suggesting that the assays respond differently to antibodies of different avidity. Avidity was estimated in liquid phase from the dissociation rate of preformed complexes of antibody and 125-iodinated insulin. When high avidity antibodies are used as a reference in radioimmunoassay, lower avidity antibodies are underestimated and vice versa. In contrast, in immunosorbent assay, any serum could be used as a reference regardless of avidity; furthermore competition experiments comparing the highest avidity insulin antibodies, from the insulin-resistant patient, with the insulin autoantibodies from the asymptomatic blood donor yielded nearsuperimposable curves. We conclude that radioimmunoassay is selective for high avidity antibodies whereas enzyme linked immunosorbent assay is not; computer modelling of the two assays supports this conclusion. In practice immunosorbent assay can be standardized using a reference serum, whereas experimental findings and mathematical considerations preclude the use of a standard serum in radioimmunoassay.
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  • 2
    ISSN: 1432-0428
    Keywords: Insulin autoantibodies ; IgG isotype ; light chain ; species specificity ; blood donors ; affinity
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Summary 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. In the direct assay, 32 sera were positive. Among these, 22 (1%) contained saturable insulin antibodies (true positive) and 10 were non-saturable and considered as non-insulin-specific. The positive blood donors were requested to answer a questionnaire and according to their answers, none had ever received insulin, was a first degree relative of a Type 1 (insulin-dependent) diabetic patient or had experienced a hypoglycaemic episode. None of the 22 true positive sera detected by enzyme-immunosorbent assay bound 125-I-insulin to any significant extent. The nine sera yielding the highest signal were further characterized with regard to heavy and light chains as well as species specificity of ligand. Anti-insulin IgG from healthy blood donors contained only one heavy (γ1 2/9; γ3 7/9) and one light (κ 8/9; λ 1/9) chain. Three sera were human insulin specific; two were non-species-specific; the other four bound insulin in the order human = porcine 〉 bovine. These results indicate that low affinity clonally restricted antibodies against insulin are present in unselected blood donors with a prevalence of 1%.
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  • 3
    ISSN: 1432-0428
    Keywords: Proinsulin monoclonal antibodies ; affinity ; solid-liquid phase assay ; radiobinding assay
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Summary 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. 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.
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  • 4
    ISSN: 1432-0428
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
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  • 5
    ISSN: 1432-0428
    Keywords: Pregnancy ; rats ; insulin treatment ; hypoglycaemia ; neonatal rats ; pancreas ; insulin release ; B-cell activity ; glucoreceptors
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Summary Intermittent hypoglycaemia was induced by insulin injections into pregnant rats. At birth, the pups body weight and pancreatic insulin content were slightly but significantly decreased. In contrast, the B-cells of these newborn rats responded more markedly to a glucose challenge than the controls. The pancreases of the insulin injected rats were grearly depleted of insulin, possibly because of feedback inhibition of insulin synthesis by exogenous insulin. This inhibitory effect was restricted to the mother since the injected insulin did not cross the placental barrier. It is proposed that the nutrient supply to the fetus influenced body growth and pancreatic insulin accumulation whereas blood glucose variations, even intermittent hypoglycaemia, promoted precocious maturation of the B-cell glucoreceptor system.
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  • 6
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    Electronic Resource
    Springer
    Diabetologia 20 (1981), S. 563-567 
    ISSN: 1432-0428
    Keywords: Rat fetus ; feto-placental units ; placental extract ; glucose ; amino acids ; non esterified fatty acids ; insulin ; glucagon ; corticosterone ; isolated islets ; placento-insular axis
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Summary Rat pups delivered by caesarian section one day before term, either remained attached to (experimental group) or were separated from their placenta (control group). Both groups were transferred into an incubator and their metabolic parameters studied. In the control group, plasma amino acid concentration fell from 11.2 to 6.9 mmol/l, plasma insulin fell from 151 to 45 μU/ml and blood glucose fell from 3.5 to 2.6 mmol/l during the first hour of extrauterine life. In the breathing feto-placental units, plasma amino acid concentration remained at its birth level, plasma insulin remained high (109 μU/ ml) causing rapid hypoglycaemia (1.63 mmol/l). An immediately postnatal glucagon and corticosterone surge was visible only in the experimental group. A partially purified placental extract (molecular weight 6000 to 30000) stimulated insulin secretion in vivo (fasted adult rats) and in vitro (isolated neonatal islets). It is concluded that the placenta maintains fetal hyperinsulinaemia by creating fetal hyperaminoacidaemia and, possibly, by secreting a beta cytotropic factor.
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  • 7
    ISSN: 1432-0428
    Keywords: Fetal gastrointestinal epithelium ; insulin receptors ; rat fetus ; quantitative autoradiograph
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Summary Purified carrier-free 125I-insulin was injected into the vitelline vein of rat fetuses in utero after 17, 19 or 21 days of a 22-day gestation. Three minutes later, the weight and radioactivity of various organs and the remaining carcass were measured. A radioactivity concentration index was calculated by dividing the specific activity of each organ by that of the whole feto-placental unit. In each of the three age groups studied, the gastrointestinal tract radioactivity concentration indices were 1.7, 2 and 1.9 respectively, indicating that the gastrointestinal tract concentrated the labelled hormone. Three, 9 and 15 min after 125I-insulin injection, the gastrointestinal tract was removed, homogenized and chromatographed on a G-50 fine Sephadex column. At 3 min, 91.4% of gastrointestinal tract radioactivity co-eluted with a standard of 125I-insulin. At the later time intervals studied, the percentage of 125I-insulin decreased while that of low molecular weight degradation products increased. Quantitative autoradiographic study of the fetal gastrointestinal tract indicated that epithelial cells bound 125I-insulin and that this binding was inhibited by co-injection of large amounts of unlabelled insulin. 125I-insulin binding was highest in the proximal small bowel and lowest in the colon. Insulin binding did not appear to depend upon degree of cell maturation or cell type. These results indicate that the epithelium of the gastrointestinal tract is characterized by the presence of numerous insulin receptors and is a potentially important insulin target.
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  • 8
    ISSN: 1432-0428
    Keywords: 123I-Insulin ; Zucker rats ; receptors ; scintillation scanning ; computer analysis
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Summary Imaging and quantitative analysis of insulin-receptor interaction was studied in vivo in lean and obese Zucker rats, using a recently developed technique in which purified Tyr A14 123I-monoiodoinsulin is intravenously injected and the tracer followed by scintillation scanning. The obese rats were 72% overweight, had near normal blood glucose concentrations and an 11-fold increase in plasma insulin concentration. In both groups of rats, the tracer was rapidly taken up by the liver (by a receptor mediated mechanism) and the kidneys (by a non-receptor mediated process). Past this maximum, radioactivity decreased in both organs as 123I-insulin was degraded and free 123I-iodide was released into the plasma compartment. Heart radioactivity (i.e. blood pool) mirrored that of the liver and kidneys. The rapid initial decrease of blood radioactivity was concomitant with liver and kidney uptake of 123I-insulin. Release of free iodide from these organs induced a slow secondary rise of blood radioactivity followed by a final decline corresponding to clearance of plasma iodide, mainly by urinary excretion. Liver radioactivity profiles of lean and obese rats were parallel. When expressed per g weight, liver radioactivity was significantly decreased in obese rats. However, due to hepatomegaly in obese rats, total liver radioactivity was significantly higher in homozygous fa/fa rats than in lean littermates. Furthermore, if the marked hyperinsulinaemia of the obese rats is taken into account, total bound insulin was enhanced in the liver of fa/fa rats whatever reference is used, either g weight or total liver. The kidney profile of radioactivity of both rats was not significantly different. In conclusion: (1) obese rats are insulin resistant as near normal glycaemia is achieved at the price of a marked hyperinsulinaemia; (2) liver uptake of insulin is enhanced in obese rats, and (3) the insulin resistance syndrome of fa/fa rats is not due to a decrease in liver insulin receptor number and/or affinity but rather to as yet unknown event(s) subsequent to receptor binding.
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  • 9
    ISSN: 1432-0428
    Keywords: 123I-insulin ; anti-insulin antibodies ; insulin-dependent diabetes
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Summary Bovine insulin was labelled with carrier-free Na 123I and the species laibelled on the A14 tyrosyl residue was purified by reverse phase high pressure liquid chromatography. After sterilization by filtration through a 0.22 urn millipore filter, the labelled hormone (123I-insulin) was injected intravenously into normal volunteers and into two insulin-immunized insulin-dependent diabetic patients. Patient 1 was treated with 74 U insulin/day, whereas, despite daily insulin doses 〉 100 U, patient 2 remained hyperglycaemic and continued to lose weight. Plasma insulin binding capacity was 10 U/l in patient 1 and 〉 20 U/l in patient 2. In both diabetic patients, heart activity (i.e. blood pool) decreased more slowly than in control patients, an observation consistent with the reduction of plasma insulin clearance rate in immunized patients. Kidney activity was decreased in patient 1 and undetectable in patient 2, suggesting that most of the circulating 123I-insulin was bound to antibodies and not filtrable. Finally, in both patients, the profile of liver radioactivity was markedly altered. Maximum liver activity was delayed and the liver image persisted for 〉 45 min. According to analysis of the time activity profile in various organs, we consider that specific antibodies act predominantly either as ‘carrier-proteins’ retarding the action of soluble insulin as in patient 1 or as insulin ‘scavengers’, the immune complexes being cleared by the reticulo-endothelial system as in patient 2.
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  • 10
    ISSN: 1432-0428
    Keywords: Biosynthetic human proinsulin ; conversion intermediates ; 123-I-labelling ; scintillation scanning
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Summary 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 (11.7%), des (31, 32) human proinsulin (3.2%), human proinsulin (1.6%); whereas half-life of hepatic activity varied in the reverse order, from 6 min for insulin, to 45 min for human proinsulin. As expected for a non-receptor mediated process, kidney extraction varied conversely to liver extraction, being highest for human proinsulin and lowest for insulin. It is concluded that the kinetics of human proinsulin conversion intermediates depends upon the site of cleavage and deletion and is intermediate between those of insulin and intact human proinsulin.
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