Detection of autoantibodies to the 65-kD isoform of glutamate decarboxylase by radioimmunoassay.

Autoantibodies (AAb) to glutamate decarboxylase (GAD) occur with a high prevalence in sera of newly diagnosed type I (insulin-dependent) diabetic patients. The aim of this study was to establish a GAD-AAb radioimmunoassay using 125I-labelled GAD65 and to evaluate this assay in a cross-sectional study with newly diagnosed type I diabetic patients (diabetes duration < 6 weeks). Furthermore, subjects at high risk of developing type I diabetes and individuals suffering from other autoimmune diseases were examined in this assay. For GAD-AAb detection, 125I-labelled GAD65 was incubated with 10 microliters of human serum overnight on ice. Thirty of 51 (59%) type I diabetic patients but none of the 54 healthy blood donors tested were found to be positive. A displacement step using 100,000 g supernatant from rat brain containing or not containing GAD showed the specificity of the binding of 125I-GAD65. Concerning the individuals at high risk of developing diabetes. 9/12 (75%) islet cell antibody (ICA)-positive non-diabetic and 4/34 (12%) ICA-negative subjects with metabolic abnormalities were GAD-AAb positive. These results show the association between type I (insulin-dependent) diabetes mellitus and the occurrence of GAD65-AAb, which possibly predicts a risk of developing the disease.

Detection of antibodies against both isoforms of glutamate decarboxylase in BB/OK rats by western blotting and immuno trapping enzyme activity assay.

The GABA-producing enzyme glutamate decarboxylase (GAD) is a prominent autoantigen in insulin-dependent diabetes mellitus (IDDM). Autoantibodies against GAD were found with a high prevalence in IDDM patients and in animal models for IDDM. The aim of this study was to detect autoantibodies against both isoforms of GAD in diabetic and non-diabetic but diabetes-prone BB/OK rats by Western blotting and to test their specificity to GAD by an immuno-trapping enzyme activity assay. Eighteen diabetic and 18 non-diabetic BB/OK rats (age 121 +/- 20 days) were investigated. In 10/18 (56%) of the diabetic and 13/18 (72%) of the non-diabetic BB/OK rats autoantibodies against at least one GAD-isoform were detected by Western blotting. In the immunotrapping enzyme activity assay, the mean value of the diabetic (1151 +/- 552 cpm, n = 11) and nondiabetic BB/OK rats (1978 +/- 1213 cpm, n = 10) was significantly (p < 0.01) increased compared to the LEW. 1A control rats (581 +/- 274 cpm, n = 12). 7/10 (70%) individual sera of the non-diabetic and 5/11 (45%) of the diabetic BB/OK rats were positive in this test. In conclusion, the prevalence of GAD autoantibodies in BB/OK rat is connected with the genetic susceptibility to IDDM but is not a predictor for the onset of the disease in BB/OK rats.

Characterization of two porcine proinsulin reactive monoclonal antibodies by immunostaining of beta-cells in pancreatic sections of different species.

Murine monoclonal antibodies against porcine proinsulin were generated by somatic cell hybridization. As detected by radioimmunoassay, 2 monoclonal antibodies KSPI14D4 and KSPI13G10 showed a strong binding to 125I-labelled porcine proinsulin but not to insulin. The species specificity of these 2 monoclonals was found to be different as shown by indirect immunofluorescence using sections of Bouin-fixed pancreata of different species. The KSPI14D4 recognized the proinsulin of pig, mouse, man, cattle, rat, dog, and cat but not that of guinea pig, whereas the KSPI13G10 bound to porcine proinsulin only. From these results it is concluded that KSPI14D4 effectively recognizes a wide-spread epitope located in one of the insulin-C-peptide junctions of the proinsulin molecule, whereas KSPI13G10 is directed to a species-specific epitope of the porcine connecting peptide.

Mathematical modelling of competitive labelled-ligand assay systems. Theoretical re-evaluation of optimum assay conditions and precision data for some experimentally established radioimmunoassay systems.

A mathematical theory of competitive labelled-ligand assays was developed with the intention of theoretically re-evaluating the optimal assay conditions and precision data of assay systems established by experiment. Our theory is based upon the assumptions of a simple bimolecular reaction mechanism, homogeneous reactants, as well as kinetically indistinguishable labelled and non-labelled ligands. The general case of two-step (non-equilibrium) assay was considered including the one-step (equilibrium) assay as a special case. The solution of the system of corresponding kinetic differential equations was used to mathematically construct standard curves. Furthermore, intraassay precision profiles and indices as well as detection limits were calculated considering solely the pipetting error, epsilon, as a source of experimental error. A procedure was outlined to mathematically determine the optimal incubation conditions for any assay system targeted to a given analyte concentration, P, at which the standard deviation of assay results is to be minimized. Estimates of both the content of binding sites and the equilibrium constant, K, of the specific binding agent are necessary, and these can be derived from Scatchard plots. For six RIA systems, of which three were one-step and three were two-step assays, experimental assay conditions and precision data were compared with theoretical predictions. Experimentally determined antibody binding site concentrations agreed fairly well with those independently evaluated by mathematical optimization. Mean precision indices, defined as constituting an average over the complete precision profile, were found to be within the theoretically predicted range, i.e. two- to threefold the pipetting error.(ABSTRACT TRUNCATED AT 250 WORDS)

Autoantibodies against insulin (IAA), C-peptide (CAA), and glucagon (GAA) in new-onset type 1 diabetic patients.

Autoantibodies against insulin, C-peptide, and glucagon were determined by radio-binding assay in 63 new-onset Type 1 (insulin-dependent) diabetic patients as well as in 70 controls. Plasma peptide binding was determined by means of 125I-labeled peptides and charcoal-dextran separation technique. Binding values exceeding the mean plus three standard deviations of the controls were considered as antibody-positive. Sixteen patients (25%) were positive for IAA, as 6 (10%) were positive for CAA and 2 (3%) for GAA. Of all control subjects, none were positive for either IAA or CAA, whereas 2 (2%) had GAA. The mean 125I-glucagon binding in the patients' group was, however, slightly enhanced and could be suppressed to normal values by excess unlabeled glucagon. The presence of IAA and/or CAA was significantly associated with more severe symptoms at diabetes manifestation. These results indicate that in new-onset Type 1 diabetics autoimmunity arises against all the insular peptides tested but is predominantly directed against those antigens secreted from the beta cells. Nevertheless, extremely low-binding GAA seem to be common in these patients. The determination of IAA/CAA might be useful in detecting a possible heterogeneity of Type 1 diabetes with regard to its clinical mode of manifestation.

Radioiodination of peptide hormones and immunoglobulin preparations: comparison of the chloramine T and iodogen method.

Following optimization of the reaction conditions, e.g. concentration of oxidizing agents, reaction time, volume of reaction mixture, and pH, chloramine T and the new iodination reagent, Iodogen, were compared for their effectiveness in radioiodination of insulin, glucagon, human growth hormone (hGH), and rabbit anti-mouse IgG. The radioactive peptide hormones prepared were analyzed for the presence of aggregate and breakdown products by polyacrylamide gel electrophoresis (PAGE) at pH 8.9, the rabbit anti-mouse IgG was tested for the presence of low molecular weight damage products by gel filtration on Sephadex G-50. The results demonstrate that with respect to iodine incorporation, specific activity, and immunological reactivity either method can be used to prepare under carefully controlled conditions a wide range of tracers with high specific activity at minimal oxidation damage. These tracers are shown to be highly suitable in radioimmunoassays after previous purification by PAGE and gel filtration, respectively.

[Heterologous and homologous radioimmunoassays for human prolactin. 1. Development and critical evaluation of the method]. / Heterologe und homologe Radioimmunoassays für humanes Prolaktin. 1. Mitteilung: Aufbau und Methodenkritik.

Based on the production of a high specific antibody against ovine prolactin a heterologous prolactin RIA (self proposed method) is introduced. From this work both a homologous and a heterologous system have been developed and used for the PRL-RIA kit production. Substances necessary for setting up the kits, their investigations and applications are described. Statistical considerations about precision, sensitivity, specificity and accuracy show the validity of these heterologous PRL-systems.

Screening monoclonal islet cell surface antibodies (ICSA) by radioimmunoassay--detection of crossreactivity with ICSA from insulin-dependent (type 1) diabetic patients.

A radioimmunoassay for the detection of monoclonal islet cell antibodies was developed using rat insulinoma cells as antigen carriers and 125I-labeled affinity-chromatographically purified anti-mouse Ig antibodies for detecting cell-bound mouse Ig. Prior to the assay cells had been attached to glass tubes by poly-dimethyl-diallyl ammonium chloride thus allowing to perform the assay as easy as a solid-phase immunoassay. Incubation protocol and cell number were chosen to ensure a high sensitivity of the assay. Results compared well with immunofluorescence findings. Of seven monoclonal islet cell antibodies tested for crossreactivity only one was displaceable by islet cell surface antibodies from diabetic sera. This antibody was induced by immunization with human islets whereas all others were from mice which had been autoimmunized with streptozotocin and complete Freund's adjuvant.

Measurement of insulin in human sera using a new RIA kit. 1. Insulin determination in the absence of insulin antibodies--conventional assay and micro modification.

A sensitive and versatile radioimmunoassay (RIA) for insulin was established using human insulin standard, a specific guinea pig anti-insulin antiserum and rabbit anti-guinea pig serum. Radioiodination was performed according to a modified chloramine T method. Tracer preparations were used for as long as 6 weeks after iodination. The standard curve ranges from 0.044 to 1.2 nmol/l. The intra-assay coefficient of variation (CV) was 3-5% and the inter-assay CV was 6-9% in the optimal range between 0.4 and 0.9 nmol/l. The average recovery of human insulin added to plasma or serum samples was 100.2 +/- 2.0% (n = 38) and 100.1 +/- 1.9% (n = 42), respectively. In addition to human insulin, porcine, canine, rabbit and bovine insulin can also be determined but not rat or mouse insulin. The cross-reactivity of the antiserum with porcine proinsulin was found to be 40% on the molar basis. The range of mean fasting plasma insulin concentrations in healthy subjects and under various pathological conditions were estimated.

Measurement of insulin in human sera using a new RIA kit. 2. Determination of free and total insulin--correlations to insulin antibody levels.

Using the micro-scale modification of a newly developed RIA kit for insulin, we established methods for the determination of free and total insulin in serum of insulin-treated diabetics. Precipitation with polyethylene glycol 6000 or acid alcohol extraction of sera was carried out to remove or to dissociate antibody-bound insulin. Both assays permit precise and accurate measurement of either serum insulin fraction. In 50 diabetic sera with tracer insulin binding of 0-97%, free (after equilibration of the sera at 37 degrees C) and total insulin levels as well as insulin antibody binding parameters were determined. There was a good correlation of free to total insulin levels with maximally 10-fold higher values of total insulin. Both free and total insulin were found to be correlated with the ability of the serum to bind insulin. In detail, binding affinities (i.e. the reciprocal of equilibrium dissociation constants) and binding site concentrations were evaluated which were shown to be positively correlated with free and total insulin levels as well. From these data we conclude that insulin antibodies in the serum may accumulate therapeutic insulin and function as a depot for delivering insulin in insulinopenic episodes (Keilacker et al., 1982 and 1986).

The role of insulin antibodies in insulin treatment of type I diabetes.

We investigated equilibrium plasma binding patterns of insulin in 45 juvenile diabetics treated with conventional insulin preparations. Insulin binding parameters were evaluated by Scatchard analysis of the binding data. Stable diabetics had significantly lower equilibrium dissociation constants than labile, thus suggesting an enhanced insulin depot effect due to stronger insulin binding. Correlation of insulin binding data with a glycemic control index yielded a positive relationship between insulin antibody binding and the degree of glycemic control. Insulin neutralization as detected by a relationship between maximum binding capacity of high affinity antibodies and insulin requirement could only be found if patients with poor diabetes control were excluded. Similarly, the well-known promoting influence of residual beta-cell functional capacity (assessed by C-peptide levels) on diabetic stability was observed only after exclusion of patients with higher insulin antibody binding. These data suggest that insulin antibodies are influencing insulin treatment of diabetics in a dual way. They may neutralize therapeutic insulin but at the same time they exert an insulin-sparing action by improvement of diabetes control. Occasionally the latter effect may abolish the correlation between diabetes control and beta-cell functional capacity.

Kinetic properties of monoclonal insulin antibodies.

We investigated four insulin-specific hybridoma antibodies with respect to their kinetic properties as well as the binding behaviour of some combinations of them. From equilibrium binding data all but one antibodies were shown to bear homogeneous binding sites. They revealed homogeneity of binding sites also by kinetic experiments, thus with high probability being monoclonal. At 0 degree C, two of them showed discrepancies between kinetic and steady state binding data in as much as, at steady state, the measured bound-to-free ratio of tracer insulin was 3-4 times lower than calculated from kinetic data. Thus a simple bimolecular reaction mechanism could possibly not be applicable. Mixing two monoclonal insulin antibodies, neither cooperative nor additive binding to the insulin molecule could be observed but only competitive effects. Especially, no positive cooperativity between two or more antibodies could be detected, which would be able to account for the higher affinity usually observed for polyclonal vs. monoclonal antibodies.

Polyacrylamide gel electrophoretic preparation of labeled and non-labeled peptides for radioimmunoassay.

Radioiodinated polypeptide hormones, such as insulin, glucagon, human growth hormone, and human C-peptide are employed for radioimmunoassays for investigations of hormonal alterations in states of disturbed carbohydrate metabolism. Iodination was performed using chloramine T. Iodination products of these polypeptide hormones and, for preparation of standard material, native human C-peptide from cadaver pancreases were fractionated by polyacrylamide gel electrophoresis at pH 8.9. Disc electrophoresis in 24 cm-long gel rods resulted in stable tracers with high specific activity as well as homogeneous standard material being highly suitable for radioimmunoassays.

Measurement of plasma canine C-peptide by radioimmunoassay.

A sensitive radioimmunoassay (RIA) for canine C-peptide (CCP) was established using synthetic CCP, a specific antiserum, and rabbit anti-guinea pig serum. Radioiodination was performed according to a modified chloramine-T method. Tracer preparations were used for long as 6 weeks after iodination. The standard curve ranges from 0.028 to 3.0 nmol/l. The intra-assay coefficient of variation (CV) was 3-5% and the inter-assay CV was 6-9% in the optimal range between 0.3 and 0.8 nmol/l. The average recovery of CCP added to plasma samples was 100.6% (n = 9). Canine insulin, porcine proinsulin, bovine proinsulin, and human C-peptide exhibited no cross-reactivity. The mean fasting plasma CCP concentration was 0.089 +/- 0.021 nmol/l in normal dogs and -0.005 +/- 0.007 nmol/l (mean +/- SEM) in diabetic dogs, respectively.

Differences in molecular heterogeneity of glucagon-like immunoreactivity in plasma and liver metastases of a patient with alpha-cell tumor.

In the present study we characterized and compared the different molecular forms of glucagon-like immunoreactivity in extracts of peripheral plasma and hepatic metastases of a patient with pancreatic alpha-cell tumor. Plasma and tissue extracts were chromatographed on Sephadex G-50 columns. Immunoreactivity in the eluting fractions was assayed with an anti-glucagon antiserum that specifically recognizes the C-terminal region of the pancreas glucagon molecule. Total plasma glucagon-like immunoreactivity prior to surgery was 26.64 nmol/l and consisted of four peaks of immunoreactivity of apparent 9,000 mol wt, 5,800-5,400 mol wt, and 4,000 mol wt. Total glucagon-like immunoreactivity extracted from the hepatic metastasis was 47.41 nmol/g wet weight and eluted as two major peaks of immunoreactivity as follows: peak I, mol wt 3,800, corresponding to "true" 3,500 mol wt glucagon; peak II, mol wt 1,400, probably consisted of glucagon degradation products. The results clearly demonstrated that both plasma and glucagon-like immunoreactivity extracted from hepatic metastases were heterogeneous and comprised species corresponding not only to "true" glucagon but also to higher mol wt forms. The lack of higher mol wt forms of immunoreactivity in the hepatic metastases of the alpha-cell tumor suggests that this metastatic tumor tissue may contain an enzyme capable of converting the higher mol wt forms to smaller glucagon-like components whereas this degradative system seems to be defective in the primary tumor.

[Effect of mestranol and chlormadinone acetate on HGH secretion in Turner's syndrome]. / Einfluss von Mestranol und Chlormadinonazetat auf die HGH-Sekretion beim Turner-Syndrom.

In 19 patients aged from 12 to 24 years (average age 17.0 years) with Turner's syndrome the influence of the hormonal substitution with mestranol and chlormadinone acetate on both basal and arginine stimulated HGH secretion was investigated. This test was performed before, during and after treatment. HGH serum levels were determined by RIA. Both the basal values and the increase of HGH secretion after arginine stimulation were used for evaluation of the test results. Under estrogen-gestagen treatment in 15 of the 19 patients hypersomatotropic basal levels were determined. The basal HGH levels before and during as well as during and after treatment are significant different (p less than 0,01). The responsibility of the somatotrophs to the arginine stimulation was individually different and did not show any uniform trend.

[Effect of ethinyl estradiol sulfonate/norethisterone acetate on HGH secretion in pubertal girls]. / Einfluss von Athinylöstradiolsulfonat/Noräthisteronazetat auf die HGH-Sekretion bei puberalen Mädchen.

In 13 healthy tall girls the influence of the hormonal treatment with depotestrogen ethinylestradiolsulfonate and norethisterone acetate was investigated on basal and arginine stimulated HGH secretion. The investigations were performed before, during and after finishing the therapy. HGH serum levels were determined by RIA. During the hormonal therapy the mean basal HGH levels were significant higher (p less than 0.05) than before or after therapy. Independent from hormonal therapy there was no stimulation of the HGH secretion by arginine in about 2/3 of the girls.