Genetic origin of human anti-insulin antibodies

Numerous studies have characterized the specificity of human anti-insulin antibodies; however, little is known about their genetic origin. To initiate molecular studies, B cells that bind human insulin were selected from the peripheral blood of diabetic donors and transformed with Epstein-Barr virus. The resulting anti-insulin B-cell lines were cultured at limiting dilution and examined for immunoglobulin heavy-chain gene rearrangements on Southern gels. These studies demonstrated the clonality of the B-cell lines and showed that multiple immunoglobulin heavy chain rearrangements are present. When the heavy-chain variable region (VH) gene from one of these Epstein-Barr virus cell lines was cloned, it was found to belong to the recently identified human VHV gene family that represents less than 1% of known human VH genes. Using the polymerase chain reaction, the germline VHV gene of the donor was amplified and sequenced. The sequences showed a high level of homology (98%) between the expressed and germline VHV gene of the donor. While antibodies reactive with autologous insulin (like other autoantibodies) are not extensively mutated from their germline configuration, two replacement substitutions are present in this IgM antibody.