Comparison of three assays for the detection of GAD65Ab-specific anti-idiotypic antibodies

Abstract

Anti-idiotypic antibodies (anti-Id) to autoantibodies are present in several autoimmune diseases and are hypothesized to have regulatory function. Recently we reported the presence of anti-Id to a major type 1 diabetes-associated autoantibody (GAD65Ab) in sera of healthy individuals. Our current assay for the detection of GAD65Ab-specific anti-Id requires the initial removal of anti-Id from the sera using immobilized monoclonal GAD65Ab, followed by detection of the now exposed GAD65Ab. However, anti-Id in samples that are GAD65Ab-negative cannot be detected in this assay. Furthermore, we cannot distinguish between serum GAD65Ab and the monoclonal GAD65Ab used in the absorption of anti-Id. In this study we evaluated two novel detection assays for GAD65Ab-specific anti-Id. The biotin/streptavidin based absorption assay utilizes the strong interaction of biotin and streptavidin to prevent possible leakage of the immobilized antibody. Moreover, this assay format allows to identify the origin of the detected GAD65Ab. The ECL-based assay allows the direct detection of anti-Id independent of the presence of GAD65Ab. We analyzed new-onset type 1 diabetes patients (n=133) and matched healthy controls (n=178) for the presence of GAD65Ab-specific anti-Id using both new detection assays and the original absorption assay. We found that all three assays can distinguish between the type 1 diabetes cohort and the healthy control samples. The biotin/streptavidin assay allowed us to positively exclude the monoclonal GAD65Ab as the source of the detected GAD65Ab. While the original absorption assay showed the highest sensitivity and specificity, the ECL format showed the highest peak signal-to-noise ratio and excellent linear correlation, making this assay our first choice for quantification of anti-Id.

Decline in Titers of Anti-Idiotypic Antibodies Specific to Autoantibodies to GAD65 (GAD65Ab) Precedes Development of GAD65Ab and Type 1 Diabetes

“…Interassay variation of the WHO standard was ∼10%. An anti-Id index was calculated as the observed increase in GAD65Ab levels after absorption of the anti-Id compared to the GAD65Ab level before absorption of the anti-Id as previously reported [22]. Antibody levels were expressed as a relative index to correct for interassay variation.…”

The preventive role of type 2 NKT cells in the development of type 1 diabetes

“…Oak et al. highlight the impact of anti‐idiotypic antibodies against GAD65‐specific antibodies on the pathogenesis of T1D. Comparably, autoantibodies against sulfatide and the corresponding anti‐idiotypic antibodies can be of importance.…”

“…Anti-idiotypic antibodies -Current discussions on the pathogenesis of T1D and other autoimmune diseases consider the presence of antibodies against autoantigens and the opportunity to induce anti-idiotypic antibodies against these (98). Oak et al (99,100) highlight the impact of anti-idiotypic antibodies against GAD65-specific antibodies on the pathogenesis of T1D. Comparably, autoantibodies against sulfatide and the corresponding anti-idiotypic antibodies can be of importance.…”

Changes in GAD65Ab-Specific Antiidiotypic Antibody Levels Correlate with Changes in C-Peptide Levels and Progression to Islet Cell Autoimmunity

“…Interassay variation on this sample was approximately 10%. Anti-Id were calculated as the observed increase in GAD65Ab levels after absorption compared with the GAD65Ab level before absorption (index after absorption Ϫ index before absorption) as previously reported (18). Antibody levels were expressed as a relative index to correct for interassay variation using an in-house serum sample obtained from a healthy donor.…”

“…Previously, we demonstrated the presence of GAD65Ab-specific antiidiotypic antibodies (anti-Id) in healthy individuals and their relative absence in T1D patients at clinical onset (17,18). These anti-Id bind the antigen-binding site of the GAD65Ab, preventing thereby the latter's binding to GAD65.…”