Reagents and methods for the diagnosis and treatment of diabetes and stiff man syndrome
AssigneeThe Regents of the University of California
InventorsSteinunn Baekkeskov, Wiltrud Richter, Yuguang Shi, Mark Namchuk, John Kim
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Abstract
The invention provides fragments of GAD.sub.65 that are specifically reactive with at least one class of GAD.sub.65 autoantibody. Most fragments are substantially free of N-terminal amino acids that would otherwise limit solubility. Different fragment contain epitopes for different classes of GAD.sub.65 autoantibodies. The fragments are used in methods of diagnosing and treating insulin dependent diabetes mellitus and stiff man syndrome.
Claims29
A method for detecting GAD.sub.65 autoantibodies, the method comprising:
- exposing a serum sample to a fragment of GAD.sub.65 that is specifically reactive with a GAD.sub.65 autoantibody and has a solubility of at least 50 .mu.g/ml in an aqueous solvent;
- and
- detecting a specific interaction between the GAD.sub.65 fragment and the autoantibody, wherein the soluble fragment is produced by expression of a truncated GAD.sub.65 DNA sequence encoding the fragment or by solid phase peptide synthesis and further wherein the soluble fragment is substantially free of N-terminal region amino acids that would otherwise limit solubility to less than 50 .mu.g/ml in an aqueous solvent.
A method for detecting GAD.sub.65 autoantibodies in sera, the method comprising:
- providing a fusion polypeptide comprising a GAD.sub.65 protein or fragment thereof, specifically reactive with a GAD.sub.65 autoantibody, and an extension polypeptide fused to the N-terminal of the protein or fragment;
- immobilizing the fusion polypeptide to a solid support via the extension polypeptide;
- exposing a serum sample to the fusion polypeptide;
- and
- detecting a specific interaction between the GAD.sub.65 protein or fragment and the autoantibody.
A method for detecting GAD.sub.65 autoantibodies in sera, the method comprising:
- providing a first antibody specifically reactive with an epitope occurring within amino acids 1 to 244 of a GAD.sub.65 polypeptide;
- immobilizing the first antibody to a solid support;
- exposing the GAD.sub.65 polypeptide to the first antibody to immobilize the protein to said support via the first antibody, wherein the GAD.sub.65 polypeptide comprises an epitope reactive with a GAD.sub.65 autoantibody;
- exposing a serum sample to the GAD.sub.65 fragment;
- and
- detecting specific interaction between the GAD.sub.65 fragment and the autoantibody.
A method for detecting GAD.sub.65 autoantibodies in sera, said method comprising:
- denaturing a soluble fragment of GAD.sub.65 produced by expression of a truncated GAD65 nucleic acid sequence encoding the fragment or by solid-phase peptide synthesis containing a nonconformational epitope, and free of N-terminal region amino acids that would otherwise limit solubility to less than 50 .mu.g/ml in an aqueous solvent;
- contacting the denature a soluble fragment with a serum sample;
- and
- detecting binding between said denatured GAD.sub.65 polypeptide and a GAD.sub.65 autoantibody.
A method for diagnosing or monitoring insulin-dependent diabetes mellitus in a patient, the method comprising:
- exposing a serum sample from the patient to a soluble GAD.sub.65 fragment produced by expression of a truncated GAD65 nucleic acid sequence encoding the fragment or by solid-phase peptide synthesis, wherein the soluble GAD.sub.65 fragment has an epitope specifically reactive with a GAD.sub.65 autoantibody diagnostic of insulin-dependent diabetes mellitus;
- and
- detecting interaction between the fragment and the autoantibody diagnostic of insulin-dependent diabetes mellitus.
A method for diagnosing or monitoring stiff man syndrome in a patient, the method comprising:
- exposing the serum sample to a GAD.sub.65 fragment having an epitope specifically reactive with a GAD.sub.65 autoantibody diagnostic of stiff man syndrome and free of amino acids forming segments reactive with GAD.sub.65 autoantibodies diagnostic of insulin dependent diabetes mellitus;
- and
- detecting interaction between the fragment and the autoantibody diagnostic of stiff man syndrome.
A method of monitoring the progression of insulin-dependent diabetes mellitus (IDDM) in a patient, the method comprising the steps of:
- exposing a serum sample from the patient to a first GAD.sub.65 polypeptide comprising a first epitope specifically reactive with a first GAD.sub.65 autoantibody;
- detecting specific interaction between the first polypeptide and the first autoantibody;
- exposing the serum sample to a second GAD.sub.65 polypeptide comprising a second epitope specifically reactive with a second GAD.sub.65 autoantibody;
- and
- detecting specific interaction between the second polypeptide and the second autoantibody
- wherein the detection of specific interaction in both detecting steps indicates greater progression toward IDDM than detection in only one of the detecting steps.
Dependencies are inferred from the claim text.