A prozone phenomenon interferes in islet cell antibody detection: direct comparison of two methods in subjects at risk of diabetes and in insulin dependent diabetics at onset.

A recent international workshop documented marked interlaboratory variation in end point titers of standard islet cell antibody (ICA) positive sera. End titers were lower using a modified assay which utilizes fluorescein labeled protein A (ICA-pA) rather than fluoresceinated anti-IgG (ICA-IgG) to detect antibody binding to islets. In this study we sought to compare directly two ICA assays with respect to future development of IDDM. Sera were obtained from 26 prospectively evaluated high risk subjects identified by family screening or history of transient hyperglycemia and 12 normal controls. As expected, end point titers for ICA positive sera were 10 times greater using the ICA-IgG assay than with the ICA-pA assay. However, despite higher end point titers, the ICA-IgG assay failed to detect more 'prediabetics' and showed a prozone effect. Fourteen subjects were positive at a 1:2 dilution using the ICA-pA assay. Only 10 of these 14 were positive at a 1:2 dilution using the ICA-IgG assay but all became positive at greater sera dilutions. No normal controls were positive using either assay. A similar prozone was observed with anti-islet monoclonal antibodies A2B5 and 4F2. Sera from 14 long-standing IDDM patients (where titers of ICA may have decreased relative to time of onset of diabetes) which were negative using ICA-pA were also assayed using ICA-IgG. Five sera positive for ICA-IgG but negative for ICA-pA were identified. In addition two sera in which a prozone effect was seen with ICA-pA were identified.(ABSTRACT TRUNCATED AT 250 WORDS)

Expression of cytoplasmic islet cell antigens by rat pancreas.

A major problem in standardization of the islet cell cytoplasmic antibody (ICA) assay is variation in sensitivity of the different human pancreas substrates used in individual laboratories. To circumvent this problem, we have developed an assay that utilizes Wistar-Furth rat pancreas as substrate, an anti-islet monoclonal antibody (A2B5) to identify islets and fluorescein-conjugated protein A to identify patient autoantibodies. Sera from 85 control subjects, 27 type I diabetics, and 17 subjects at high risk for developing type I diabetes were assayed in parallel with our standard ICA assay on human pancreas substrate and with Wistar-Furth rat pancreas as substrate. Two sera from control subjects (2 of 85) were ICA positive with rat pancreas compared to 1 of 85 with human pancreas substrate. Sera from 11 of 27 type I diabetics and 15 of 17 sera from high-risk subjects were ICA positive with either rat or human pancreas substrate. A correlation between the specific islet fluorescence readings on human and rat pancreas sections was found with sera from high-risk and control subjects. Furthermore, end-point titers of an ICA-positive serum were identical with both assays. Finally, incubation of an ICA-positive serum with glycolipids, extracted from either human or Wistar-Furth rat pancreas, blocked subsequent ICA binding. These findings suggest that Wistar-Furth rat pancreas expresses an identical or similar autoantigen to human pancreas.

First-degree relatives of patients with type I diabetes mellitus. Islet-cell antibodies and abnormal insulin secretion.

In a prospective study to evaluate the prevalence and predictive potential of circulating islet-cell antibodies, we have screened 1723 "normal" first-degree relatives (parents, siblings, and offspring) of patients with insulin-dependent diabetes mellitus. The prevalence of islet-cell antibodies on initial screening was 0.9 per cent (16 of 1723). Over a maximal follow-up period of two years, insulin-dependent diabetes mellitus developed in 2 of 16 relatives with islet-cell antibodies and in 1 of 1707 without antibodies. In addition, 6 of 12 nondiabetic relatives with islet-cell antibodies had abnormally low insulin responses--below the third percentile in 6 and below the first percentile in 4--on their initial intravenous glucose challenge. Thus, prospective islet-cell antibody screening of high-risk first-degree relatives, in combination with intravenous glucose-tolerance testing, is capable of identifying immunologically abnormal persons with profoundly diminished beta-cell function, who are presumably at increased risk of insulin-dependent diabetes mellitus.

"Cytoplasmic" islet cell antibodies. Evidence that the target antigen is a sialoglycoconjugate.

We have biochemically treated (periodate, borohydride, neuraminidase, organic solvents) frozen sections of human pancreas and studied the reactivity of islet-cell-antibody-positive human sera and monoclonal antibodies. The autoantigen of pancreatic sections has the properties of sialic acid containing glycolipid.

Assay for islet cell antibodies. Protein A--monoclonal antibody method.

Assays for islet cell antibodies (ICA) are finding increasing application in clinical diabetology. We have developed a new islet cell antibody assay system (ICA-pA), whose salient features include: (1) utilization of fluorescein-conjugated staphylococcal protein A as a standard second-step reagent, the advantages of this approach being improved "signal" (islet)/"noise" (acini) ratio due to reduction of interfering background acinar pancreatic staining, and facilitation of assay standardization provided by the use of a chemically pure conjugated protein A reagent; (2) monoclonal antibody counterstaining with rhodamine-conjugated BISL-32 for the rapid identification of islets in pancreatic sections; and (3) quantitation of circulating serum ICA by microimmunofluorometric techniques.

Characterization of monoclonal antibody 3G5 and utilization of this antibody to immobilize pancreatic islet cell gangliosides in a solid phase radioassay.

Monoclonal antibody 3G5, which was initially produced by immunization of mice with fetal rat brain, reacts specifically by indirect immunofluorescence with all cells of the pancreatic islets of human, rat, mouse, and bovine pancreas. This antibody reacts with the cell surface of isolated islet cells as well as the rat (RIN5F) insulinoma cell line. Antibody 3G5 reacts with islets, thyroid follicular cells, pituitary, and the adrenal medulla of a pattern similar to but distinct from those of antibody A2B5 and tetanus toxin, both of which react with complex gangliosides (sialic acid-containing glycosphingolipids). The antigen with which antibody 3G5 reacts also has the properties of ganglioside (neuraminidase sensitive, extracted into chloroform-methanol, partitioned into a methanol-water phase, soluble in water, and nondialyzable). Antibody 3G5, adsorbed to polyvinyl plates, can immobilize islet ganglioside micelles to which 125I-labeled 3G5, A2B5, and tetanus toxin all bind. The ability to immobilize micelles containing several complex gangliosides has led to a solid phase radioassay to detect antiganglioside antibodies. Monoclonal antibody 3G5 joins antibody A2B5 and tetanus toxin as markers for distinct complex gangliosides found on pancreatic islets and neurons.