Antibody response to islet antigens in anti-CD4/prednisolone immune intervention of type 1 diabetes.

Cytoplasmic islet cell antibodies, glutamic acid decarboxylase autoantibodies, spontaneous insulin autoantibodies, and insulin-induced antibodies were analyzed in a 1-year follow-up study of 12 newly diagnosed patients with insulin-dependent diabetes mellitus aged 14 +/- 2 years (range 7-20 years) who had been initially treated with either multiple injections of insulin alone (control group) or, in addition, anti-CD4 monoclonal antibody/prednisolone (treatment group). Despite individual variations in islet cell antibody titers, there were no significant differences in the prevalence or changes in the mean titers between the two groups. Glutamic acid decarboxylase autoantibodies remained almost unchanged, but correlated with levels of islet cell antibodies.

Autoantibodies to insulin and to proinsulin in type 1 diabetic patients and in at-risk probands differentiate only little between both antigens.

To answer the question whether insulin or proinsulin would be the true antigen for both insulin and proinsulin autoantibodies, displacement experiments of 125I-insulin and -proinsulin binding with both unlabeled antigens were performed in sera of four groups of antibody-positive probands: first-degree relatives of Type 1 diabetic patients, pre-Type 1 diabetic persons, recent-onset Type 1 diabetic patients, insulin-treated Type 1 diabetic patients. In subjects who were primarily screened to constitute these groups, prevalences of insulin and proinsulin autoantibodies were nearly identical. In antibody-positive sera, 125I-insulin and -proinsulin binding values in general were closely correlated to each other with regression coefficients near 1.

The detection of autoantibodies to pancreatic islet cells by immunoenzyme histochemistry.

Using horseradish peroxidase- or alkaline phosphatase-conjugated secondary antibodies, an immunohistochemical assay was established for the detection of islet cell cytoplasmic antibodies (ICA). Determination of end-point titers showed a significant correlation between the conventional immunofluorescence and either immunocytochemistry assay. The assays with the enzyme-conjugated antibodies were more sensitive than the indirect immunofluorescence assay.

The frequency of diabetes in children of type 1 diabetic parents.

There is little information in the literature about the actual diabetes risk of children of Type 1 diabetic parents. In a follow-up study of 48 children born from 35 Type 1 diabetic parents between 1955 and 1989, we have recorded all data relating to the incidence of Type 1 diabetes and of appearance of signs of subclinical diabetes by June, 1992. Information about the state of health was obtained during repeated stays of the patients in our hospital or by interviewing them by letter.

Proinsulin autoantibodies are more closely associated with type 1 (insulin-dependent) diabetes mellitus than insulin autoantibodies.

The disease association of autoantibodies to proinsulin and insulin was compared in patients with Type 1 (insulin-dependent) diabetes mellitus and first-degree relatives. Following the recommendation of the Fourth International Workshop on the Standardization of insulin autoantibodies, autoantibodies were determined by fluid-phase radioimmunoassay using equimolar concentrations of mono-125I-A14-insulin or -proinsulin to detect insulin or proinsulin autoantibodies, respectively. A higher prevalence of proinsulin autoantibodies vs insulin autoantibodies was found in 97 patients with Type 1 diabetes prior to insulin treatment (34.

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.

Glucose tolerance behaviour before the onset of type I (insulin-dependent) diabetes in young people as a predictor of the further course of the disease: a retrospective analysis of 33 cases.

A study was made of glucose tolerance and insulin secretion in 33 persons who later developed insulin-dependent diabetes (aged 4-24 years) and observation continued further in the first years after manifestation. Patients who developed the typical labile type of diabetes were of normal weight and had either normal glucose tolerance tests before diagnosis or had impaired glucose tolerance (IGT) for a short interval of 2-16 months. Subjects with IGT over a significantly (p less than 0.

[Enzyme immunoassay for the determination of albumin in urine].

Two modifications of a double antibody enzyme immunoassay for the determination of urinary albumin content are described. The method is simple, rapid and precise and can be carried out in test tubes and on microtiter plates as well. In 1:10 diluted urine samples albumin concentrations of 1.

A sensitive sandwich enzyme immunoassay for measurement of insulin on microtiter plates.

A sensitive enzyme immunoassay for the measurement of insulin in human sera on microtiter plates was established. The assay is based on the sandwich technique with guinea pig anti-insulin IgG adsorbed at microtiter plate wells, human insulin as standard and the same anti-insulin IgG labeled with horseradish peroxidase. Standards used cover a range from 0 to 1200 pmol/l with a detection limit of 10 pmol/l.

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.

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.

Influence of polyethylene glycol (PEG) extraction on the C-peptide determination in sera from insulin-dependent diabetic patients with circulating insulin antibodies.

To investigate whether the unexpectedly high C-peptide levels in some insulin-dependent diabetic (IDDM) patients are due to co-determination of proinsulin bound to circulating insulin antibodies, 36 randomly selected sera from IDDM patients were assayed for C-peptide immunoreactivity (CPR) after polyethylene glycol (PEG) extraction, preceding incubation with proinsulin binding antibodies (LAB + PEG) or without pretreatment of the sera. Recovery of proinsulin was checked by addition of 1 nmol/l proinsulin to all sera. Recovery was found to be 101.

Purification of mouse monoclonal antibodies by chromatography on sepharose 6 B.

By using Sepharose 6 B, a simple procedure for purification of mouse monoclonal antibodies (mcAbs) of the IgM and IgG class from ascites has been developed. The procedure which was applied to purify mcAbs against insulin and pancreatic islet cells permits either direct chromatographic separation from ascites protein components or after precipitating the immunoglobulins with ammonium sulphate. Recovery of the immunoglobulins was found to be approximately 80%, and the immunological reactivity, as tested by indirect immunofluorescence and ligand binding assay, was almost completely retained.

Diabetogenic effects of N-nitrosomethylurea in rats.

The influence of a single i.v. injection of 75 or 175 mg N-nitrosomethylurea (NMU)/kg into Wistar rats on plasma glucose, i.

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.

[Production and use of monoclonal glucagon and insulin antibodies--reduction of pancreatic insulin in rats by treatment with complete Freund's adjuvant].

Murine monoclonal antibodies against glucagon and insulin were generated by somatic cell hybridization and partially characterized. The monoclonal glucagon antibody K79bB10 exhibited no cross-reaction with gut glucagon. This antibody and the insulin antibody K36aC10 were found of very high concentration in ascites.

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.

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 effect of prehepatic insulin administration on alanine flux rates in diabetic dogs.

The in vivo flux rates of glucose (6-3H-glucose) and of alanine (U-14C-alanine) were measured in insulin-dependent chronically diabetic dogs which were infused with insulin employing a bedside-type artificial B cell and either the peripheral or the portal venous route. In comparison with non-diabetic control animals the diabetic dogs had near-normal patterns of glucose metabolism and pancreatic glucagon regardless of the route of insulin administration. They also showed reduced basal portal but moderately elevated peripheral insulin levels on peripheral and near-normal peripheral values on portal insulin infusion.

5-year follow-up study of C-peptide secretion in newly diagnosed type I diabetics: relations to HLA-phenotype, insulin requirement and metabolic control.

50 HLA-typed insulin-dependent diabetics were studied at the time of diabetes onset and after 1, 2, 3 and 5 years with regard to C-peptide secretion after combined stimulation with glucose and glucagon, insulin requirement and glycaemic control index. The mean decrease of the residual B-cell reserve was observed within two years. C-peptide secretion was correlated with better metabolic control and lower insulin requirement after more than one year of diabetes duration, but had no influence on this at the time of diabetes onset.

Measurement of insulin during isolation and purification from animal pancreas. A comparison of radioimmunoassay, radioreceptor assay and in-vivo bioassay.

For the purpose of monitoring the yield of the insulin extraction procedure from animal pancreas three methods of insulin determination were compared, i.e. the mouse convulsion test, a radioreceptor assay (RRA) on rat fat cells and a radioimmunoassay (RIA) which was especially laid out for high insulin concentrations.

Differences in the content of glucagon-like immunoreactivity in rat submaxillary glands.

Two different antisera which specifically reacted with either the C- or N-terminal region of pancreas glucagon were employed to determine the concentration of glucagon and glucagon-like immunoreactivity in extracts from submaxillary glands of rats. The content of glucagon and glucagon-like immunoreactivity increased with age and was found to be higher in adult males than in females. Pancreas glucagon immunoreactivity constituted only a small proportion of the total glucagon-like immunoreactivity.

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.