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)

Characterization of the F(ab')2 and Fab' fragments prepared from a mouse monoclonal insulin antibody.

In this report, optimum conditions were determined for the production of immunoreactive fragments from a mouse monoclonal insulin antibody, and their immunochemical characterization is described. Stable fragments can be obtained in good yield from the purified IgG 1, first by cleavage with pepsin and then by reducing the disulfide bonds with cysteine and subsequent alkylation with iodoacetamide. F(ab')2 and Fab' fragments having molecular weights (Mr) of about 108,000 (108 K) and 55 K, respectively, were produced. Ligand binding assay as well as indirect immunofluorescence on mouse pancreas section demonstrated their reactivity with free and tissue-bound insulin antigen. These results provide methods for the production and characterization of defined fragments of insulin antibody useful in experiments where non-specific interactions mediated by the Fc portion of the whole immunoglobulin may occur.

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. For purification of IgG from ascites, precipitation with ammonium sulphate is recommended prior to chromatography on Sepharose 6 B, whereas IgM can directly be subjected without any pretreatment.

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).

[Suitability of specific staining methods for the estimation of beta cell volume in rat pancreas with normal and reduced insulin content]. / Eignung spezifischer Färbemethoden für die Bestimmung des beta-Zellvolumens im Rattenpankreas mit normalem und reduziertem Insulingehalt.

Investigations were carried out on streptozotocin (SZ)-treated female Wistar rats (single i.v. injection of 30 mg/kg body weight) and control animals with normal pancreatic insulin content. After staining of the pancreatic slices with aldehyde fuchsin (AF), Victoria Blue (Ivic 1959), and FITC-labelled antiserum (indirect), the beta-cell volume was determined with a point sampling method. For control animals, all 3 selected beta-cell specific staining methods are equivalent. After reduction of the pancreatic and the beta-cell insulin content by SZ-treatment the sensitivity of the common histochemical methods is diminished in comparison to the immunofluorescence method. In normoglycemia SZ-treated animals (pancreatic insulin content about 30% of control values) 0.22% AF-stained beta-cells are demonstrable, after IVIC-staining 0.14% beta-cell volume is visible. In hyperglycemic SZ-treated rats (about 6% of pancreatic insulin content of controls) with AF-staining only 0.08% and with IVIC-staining 0.03% beta-cell volume could be measured whereas with fluorescence technique in both SZ treated groups 0.34% beta-cell volume is measurable. The reduction of pancreatic insulin content after the injection of 30 mg/kg SZ is caused by a reduction of the beta-cell volume and a diminution of the insulin content in the remaining beta-cells.