Detection of autoantibodies reactive with pancreatic islet cell antigenic molecules and/or insulin

1. A method of screening an undiluted sample of serum or plasma obtained from an animal subject for analyte autoantibodies reactive with one or more antigenic molecules selected from glutamic acid decarboxylase 65 (GAD65) or glutamic acid decarboxylase 67 (GAD67), said method comprising:

(a) immobilizing one or more first sources of antigenic molecules to a solid support in an amount so as to form a monovalent intermediate complex comprising [antigenic molecule of said first source]-[analyte autoantibody] capable of forming a divalent complex with antigenic molecules of one or more second sources, wherein said one of more first sources of antigenic molecules is capable of interacting with analyte autoantibodies in a sample of serum or plasma from said subject, or a calibrator and is selected from the group consisting of GAD65, GAD67, and fusion molecules comprising two or more directly or indirectly fused antigenic molecules selected from GAD65 or GAD67;

(b) contacting said immobilized one or more first sources of antigenic molecules with:

(1) an undiluted sample of serum or plasma from said subject, or said calibrator; and

(2) one or more second sources of antigenic molecules comprising a direct or indirect label, wherein said one of more second sources of antigenic molecules is capable of interacting with analyte autoantibodies in a sample of serum or plasma from said subject, or said calibrator and is selected from the group consisting of GAD65, GAD67, and fusion molecules comprising two or more directly or indirectly fused antigenic molecules selected from GAD65 or GAD67,

whereby analyte autoantibodies when present in said sample of serum or plasma interact with said first and second sources of antigenic molecules to form one or more divalent complexes comprising [antigenic molecule of said first source]-[analyte autoantibody]-[antigenic molecule of said second source]; and

(c) detecting the presence of complexes formed in (b) to provide an indication of analyte autoantibodies present in said sample.

2. The method according to claim 1 wherein the calibrator is diluted 16-fold or less.

3. The method according to claim 1 wherein the calibrator is diluted 8-fold or less.

4. The method according to claim 1 , which comprises providing a first source of antigenic molecules consisting essentially of GAD65 or GAD67.

5. The method according to claim 1 , wherein the antigenic molecules of the first and second sources when present in the one or more complexes comprise GAD65.

6. The method according to claim 1 , wherein the antigenic molecules of said first and second sources when present in the one or more complexes comprise GAD67.

7. The method according to claim 1 , wherein the antigenic molecules of the first source comprises GAD65 or the antigenic molecules of the second source comprises GAD67.

8. The method according to claim 1 , wherein the antigenic molecules of the first source comprise GAD67 or the antigenic molecules of the second source comprises GAD65.

9. The method according to claim 1 , wherein said immobilised antigenic molecules of said one or more the first sources is contacted with the sample of body fluid being screened so as to form the monovalent intermediate complex comprising [antigenic molecule of first source]-[analyte autoantibody] and the formed immobilised monovalent intermediate complex is subsequently contacted with said antigenic molecules of said one or more second sources, present in solution phase, so as to form said divalent complexes immobilised to the solid support via the antigenic molecule of the first source.

10. The method according to claim 1 , wherein the said sample of serum or plasma is obtained from an animal subject suspected of suffering from, susceptible to or having one or more of the following disease states-type 1 diabetes mellitus and/or stiff man syndrome, type 2 diabetes mellitus, one or more autoimmune thyroid diseases, celiac disease, one or more connective tissue diseases, adrenal autoimmunity, or a combination of two or more different autoimmune diseases.

11. The method according to claim 1 wherein the calibrator is diluted 32-fold or less.