Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
1997-09-25
2001-11-13
Wortman, Donna (Department: 1648)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C435S007400, C436S506000, C436S518000
Reexamination Certificate
active
06316209
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates generally to methods for identifying patients who are at risk of developing insulin-dependent diabetes mellitus (IDDM). More particularly, the present invention relates to the detection of an approximately 38 kD autoantigen associated with diabetes in serum of patients prior to clinical onset of the disease.
Insulin-dependent diabetes mellitus (IDDM) primarily afflicts young people. Although insulin is available for treatment, the several fold increased morbidity and mortality associated with this disease urge the development of early diagnostic and preventive methods. The destruction of pancreatic &bgr;-cells, which precedes the clinical onset of IDDM, is mediated by autoimmune mechanisms. Among the most thoroughly studied autoimmune abnormalities associated with the disease is the high incidence of circulating &bgr;-cell specific autoantibodies at the time of diagnosis. Family studies have shown that the autoantibodies to certain &bgr;-cell autoantigens appear prior to overt IDDM by a number of years, suggesting a long prodromal period of humoral autoimmunity before clinical symptoms emerge. The family studies have also documented a slow, progressive loss of insulin response to intravenous glucose in the years preceding diagnosis. The presence of &bgr;-cell specific autoantibodies in the prediabetic period is likely to reflect the ongoing autoimmune process, one that eventually leads to critical &bgr;-cell depletion and insulin dependency. It has been estimated that only 10% of the total &bgr;-cell mass remains at the time of clinical onset.
Thus, methods for early and accurate identification of susceptible individuals are needed. Assays that can detect autoantibodies associated with early humoral autoimmunity accompanying &bgr;-cell destruction are particularly desirable. The classical method for detecting islet cell autoantibodies (ICA) is by immunohistology using frozen pancreatic sections. Family studies, however, have shown that the &bgr;-cell cytoplasmic antibodies measured by this method are of insufficient specificity to serve as a single marker of susceptibility. Moreover, ICA are very difficult to standardize, and interpretation of the stained section is subject to observer bias. Thus, there has been no way to define what is a “positive” specimen. More accurate assays may be achieved by employing more specific markers, either alone or in combination with ICCA. Alternative markers include autoantibodies to an approximately 64 kD autoantigen, insulin autoantibodies, and MHC class II DR/DQ&bgr; haplotype.
The 64 kD autoantigen holds particular promise as a diagnostic marker for IDDM. Autoantibodies to the 64 kD autoantigen have an incidence of from about 70% to 80%. both at the time of clinical onset of the disease and the prediabetic period. The presence of the autoantibodies has been shown to precede overt IDDM by several years and familial studies. Recently, inventors herein together with another research group have discovered that the 64 kD autoantigen of pancreatic &bgr;-cells associated with IDDM is glutamic acid decarboxylase (GAD) (E.C. 4.1.1.15) which is an abundant protein of GABA-secreting neurons in the central nervous system (CNS). Based on this discovery, numerous conventional assay formats have become available for detecting autoantibodies to the 64 kD autoantigen (GAD) to permit patient screening.
Identification of autoantibodies to the 64 kD autoantigen, however, is insufficient by itself as a screening test to identify patients at risk for developing IDDM. As stated above, autoantibodies to the 64 kD autoantigen are present in only 70% to 80% of patients who later develop IDDM, thus failing to identify a significant number of susceptible individuals. It would therefore be desirable to provide additional and alternative markers which are able to predict IDDM susceptibility in at least a portion of those patients who do not develop antibody to the 64 kD autoantigen. Such markers should be present at an early stage of &bgr;-cell destruction prior to the clinical onset of IDDM, and should remain detectable from that early stage through the time of clinical onset. This marker should be identifiable in patient serum, thus facilitating screening, and should preferably be compatible with detection of autoantibodies to the 64 kD autoantigen.
2. Description of the Background Art
Antibodies to a 38 kD antigen present in human islets have been detected by immunoprecipitation with sera from patients suffering from insulin dependent diabetes mellitus. Baekkeskov et al. (1982)
Nature
298:167-169 and Aanstoot et al. (1991) Abstract 898
, Diabetes
40 Supp. 1, page 225A. A 38 kD islet cell protein has been reported in the BB-rat, an animal model of insulin dependent diabetes mellitus. Ko et al. (1991) Diabetologia 34:548-554. T-cell reactivity to an insulinoma protein of approximately 38 kD has been detected in newly diagnosed diabetic patients. Roep et al. (1990) Nature 345:632-634 and Lancet (1991) 337:1439-1441.
Antibodies to the 65 kD isoform of the enzyme glutamic acid decarboxylase (GAD
65
) have been identified in 70% to 80% of individuals experiencing &bgr;-cell destruction and development of insulin dependent diabetes mellitus. Baekkeskov et al. (1990)
Nature
347:151-156 and WO 92/04632. Some patients having islet cell antibodies (ICA) identified by immunofluorescence of frozen pancreas sections do not have antibodies to GAD
65
. Seissler et al. (1991)
Diabetologia
34:548-554.
SUMMARY OF THE INVENTION
The present invention comprises methods for assessing the risk of developing insulin-dependent diabetes mellitus (IDDM) in asymptomatic human patients. The methods rely on detecting the presence of autoantibodies to a 38 kD amphiphilic membrane-bound islet cell protein having a pI in the range from 5.6 to 6.1. It has been found that autoantibodies to the 38 kD autoantigen appear in a significant subgroup of patients who later develop IDDM, with the appearance occurring well before clinical onset of the disease, usually more than one year prior to clinical onset, and often two years or more prior to clinical onset. It has been also found that autoantibodies to the 38 kD autoantigen are present in a significant number of patients who do not display autoantibodies to the 64 kD autoantigen (GAD
65
). Thus, assays according to the present invention will preferably rely on detection of antibodies to both the 38 kD autoantigen and the 64 kD autoantigen (GAD
65
), with the presence of either autoantibody being predictive of IDDM.
A further understanding of the nature and advantages of the invention will become apparent by reference to the remaining portions of the specification and drawings.
REFERENCES:
patent: 5674692 (1997-10-01), Baekkeskov et al.
patent: WO 92 04632 (1992-03-01), None
DeAizpurua et al., “A 64 kDa Antigen/Glutamic Acid Decarboxylase (GAD) in Fetal PIG Pro-Islets: Co-Precipitation with a 38 kDa Protein and Recognition by T Cells in Human at Risk for Insulin-Dependent Diabetes” Journal of Autoimmunity 5:759-770 (1992).
Ko et al., “Studies on Autoimmunity for Initiation of Beta-Call Destruction VIII. Pancreatic Beta-Cell Dependent Autoantibody to a 38 Kilodalton Protein Precedes the Clinical Onset of Diabetes in BB Rats” Diabetologia 34:548-554 (1991).
Aanstoot Henk-Jan
Baekkeskov Steinunn
The Regents of the University of California
Townsend and Townsend / and Crew LLP
Wortman Donna
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