Methods for detecting insulin autoantibody
View Patent ↗The present invention provides methods for detecting insulin autoantibody. Such methods can be used, for example, to predict susceptibility of and/or diagnose the presence of Type 1 diabetes in a subject. Some aspects of the invention also provide kits adapted for use in such methods. In particular, some aspects of the invention use proinsulin to detect the presence of insulin autoantibody.
1. A method of detecting whether an insulin autoantibody is present in a fluid sample of a subject, wherein the fluid sample comprises blood, serum, or plasma, the method comprising:
(i) contacting the fluid sample of the subject with a first proinsulin and a second proinsulin under conditions, whereby a first proinsulin-insulin autoantibody-second proinsulin complex is formed when the insulin autoantibody is present in the fluid sample, thereby forming a first system,
wherein the second proinsulin comprises proinsulin labeled with an electrochemical luminescence (ECL) labeling molecule, wherein the ECL labeling molecule comprises a ruthenium (II) tris-bipyridine-(4-methylsulfonate) group, and
wherein the first proinsulin comprises proinsulin labeled with a biotin tagging molecule;
(ii) contacting the first system formed in step (i) with a solid substrate comprising a surface-bound capture molecule which is bound to the surface of the solid substrate, wherein the surface-bound capture molecule comprises streptavidin,
whereby, if the first proinsulin-insulin autoantibody-second proinsulin complex is present in the first system, the biotin tagging molecule binds to the streptavidin on the surface of the solid substrate, thereby attaching the first proinsulin-insulin autoantibody-second proinsulin complex to the solid substrate; and
(iii) determining whether a first proinsulin-insulin autoantibody-second proinsulin complex is attached to the solid substrate, wherein the presence of the first proinsulin-insulin autoantibody-second proinsulin complex attached to the solid substrate indicates that insulin autoantibody is present in the fluid sample of the subject.
2. The method of claim 1 , further comprising removing any unbound first proinsulin from the surface of the solid substrate prior to step (iii).