Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
1999-05-12
2002-03-05
Navarro, Mark (Department: 1645)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C435S007920, C435S007930, C435S007950, C435S007930, C514S866000, C424S078170, C424S078180
Reexamination Certificate
active
06352831
ABSTRACT:
TECHNICAL FIELD
The present invention relates to novel forms of glycolipids and novel methods for assaying anti-glycolipid antibodies. The preferred glycolipid is sulphatide. The novel assaying methods are particularly adapted for the diagnosis of any disease for which elevated levels of an anti-glycolipid autoantibody is a marker. The disease may be, for instance, neuropathy and insulin-dependent diabetes (IDDM, Type 1 diabetes) including diagnoses excluding Type 2 diabetes, late complications associated therewith and monitoring the treatment of IDDM. The novel forms of sulphatide may potentially find use as a medicament in the treatment of IDDM.
BACKGROUND OF THE INVENTION
Sulphatide, the most important antigen of the present invention is a glycosphingolipid having the structure:
(=galactosylceramide-3-sulphate).
The development of manifested IDDM involves an autoimmune process where the insulin producing cells in the islet of Langerhan in the pancreas gradually are knocked out. At the time of diagnoses 70-80% of the cells have been destroyed and the remaining cells will generally disappear within a 5 year period. Prediabetic individuals are those which have an ongoing destruction of insulin-producing cells but still without clinical symptoms. A particular type of prediabetic individuals are non-IDDM patients whose disease will switch to IDDM.
In the context of the invention the term IDDM include manifested IDDM as well as preforms thereof, if not otherwise specified.
The main marker autoantibodies found in IDDM are islet cell antibodies (ICA), anti-glutamic acid decarboxylase antibodies (anti-GAD), anti-insulin antibodies (IAA), antibodies against 37 kD protein (likely the same as tyrosine phosphatase) and anti-sulphatide antibodies. The main disadvantages of these markers are:
ICAs are determined by conventional immunohistochemistry which make them unsuitable for large screenings. They are normally considered good predictors of IDDM but their assay suffers from relatively high intra- and interassay variations.
Anti-GAD antibodies are easy to determine also in large scale screenings, but they may not be of a strong predictive value.
IAAs are fairly easy to determine but are not present in more than 40-50% of newly diagnosed type 1 diabetic children and is less common in adult patients.
Anti-37 kD protein antibodies have a significant lower frequency (about 50%) than ICA and anti-GAD antibodies.
Both ICAs and anti-GAD antibodies have been used for the diagnoses of prediabetics.
Anti-sulphatide antibodies have been recognized as a good marker for manifested IDDM (Buschard et al., Lancet 342 (1993) page 840-; and Buschard et al., APMIS 101 (1993) 963-970). Results supporting a diagnostic link between this type of antibodies and prediabetic forms of IDDM have not hitherto been published. Increased titers of anti-sulphatide antibody have been found in neuropathy patients and could be used for identification of individuals that might develop neuropathy. Part of diabetes type 2 patients also develop neuropathy. In a preliminary study one of the present inventors has shown that diabetes type 2 patients without neuropathy did not have anti-sulphatide antibody reactivity and there is thus a possibility that those with neuropathy development will be positive and thus anti-sulphatide antibody might be useful as a prognostic tool. See Fredman et al., J. Neurolog. 238 (1991) 75-79.
Serum levels of antibodies towards glycosphingolipids have often been assayed by a combination of thin layer chromatography (TLC) of the antigens and ELISA methodology (enzyme-linked immuno sorbent assay) employing the chromatographed material as antigen for assaying occurrence of serum antibodies (TLC-ELISA) (Fredman et al., J. Neurol. 238 (1991) 75-79; and Fredman et al., J. Neurol. 240 (1993) 381-387).
The assaying methods of anti-glycolipid antibodies have been problematic, in particular the methods for anti-sulphatide antibodies.
Sulphated glycolipids, in particular sulphatide, lactosylceramide-3-sulphate and seminolipid and corresponding antibodies have been suggested as diagnostic markers and therapeutic agents in the context of diabetes (Buschard K, WO-A-9219633).
We have tried to vary the attachment of sulphatide to plastic wells, and have clear evidences that direct adsorption results in a high coefficient of variation and often gives unspecific reactions without any correlation to earlier TLC-ELISA findings. The criticality of exposing correctly both the lipid moiety and the carbohydrate moiety of short carbohydrate chain glycolipids has also been illustrated with monoclonal anti-sulphatide antibodies. Thus, the monoclonal anti-sulphatide antibody described by Fredman et al (Biochem. J. 251 (1988) 17-22) is sensitive to changes in the lipid part. Another aspect is that galactose substituted with sulphate is a common epitope on several glycoproteins and glycolipids, and there are no results so far supporting that a general reactivity towards this epitope is relevant for the development of IDDM and its late complications. Accordingly, the main problem the invention sets out to solve relates to the presentation of glycolipid epitopes.
Another problem the invention sets out to solve is connected to the diagnoses of prediabetic forms of IDDM.
SUMMARY OF THE INVENTION
A first objective of the invention is to improve the presentation of glycolipid antigens/haptens in immunoassays and potentially also in therapy. The antigens/haptens primarily concerned exhibit short carbohydrate chains.
A second objective is to provide improved immunoassays for measuring anti-glycolipid antibodies or glycolipid antigens/haptens, in particular autoantibodies against glycosphingolipid antigens exhibiting mono- and/or disaccharide units that may be sulphated.
A third objective is to provide improved diagnostic methods utilizing as markers auto-antibodies binding to one or more of the previously mentioned glycolipid antigens/haptens for diagnosing IDDM including preforms thereof.
A fourth objective is to provide diagnostic methods for determining preforms of IDDM.
These objectives may be complied with by complexing the glycolipid antigens/haptens as defined above to a polymeric hydrophilic carrier exhibiting hydrophobic regions. Examples of suitable carrier molecules are delipidized forms of hydrophilic proteins, such as albumin, that are capable of associating to lipid compounds, such as fatty acids and derivatives thereof.
REFERENCES:
patent: 62-008055 (1987-01-01), None
patent: WO 9219633 (1992-11-01), None
patent: WO 9413311 (1994-06-01), None
Monti et al,Biochimica et Biophysica Acta, 1124 (1992):80-87.
Buschard et al,APMIS, 101 (1993):963-970.
Fredman et al,Biochem. J., 251 (1998):17-22.
Fredman et al,J. Neurol., 235 (1991):75-79.
Buschard et al,Lancet, 342 (1993):840.
Fredman et al,J. Neurol., 240 (1993):381-387.
Viani al,Biochimica et Biophysica Acta, 1002 (1989):20-27.
Buschard Karsten
Fredman Pam
Månsson Jan-Eric
A+ Science Invest AB
Baskar Padma
Dinsmore & Shohl LLP
Navarro Mark
LandOfFree
Glycolipid complexes and their uses does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Glycolipid complexes and their uses, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Glycolipid complexes and their uses will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-2850591