V-H AND V-L GENE USAGE BY ANTI-BETA-AMYLOID AUTOANTIBODIES IN ALZHEIMERS-DISEASE - DETECTION OF HIGHLY MUTATED V-REGIONS IN BOTH HEAVY AND LIGHT-CHAINS

In a previous study, four human IgM(K) monoclonal antibodies (mAbs) secreted by Epstein-Barr-virus-transformed B cell lines independently derived from the peripheral blood of a patient with Alzheimer's disease (AD) were found to react with a conformational epitope of beta-amyloid protein and to stain amyloid plaques in AD brain. Three of these mAbs were studied further. They did not react with an additional panel of antigens and autoantigens. By a competitive inhibition ELISA, the K-d was determined to be 5.7 x 10(-8) M. The V-H and V-K of these mAbs were sequenced at the cDNA level and found to be identical. The corresponding V-H and V-K germline counterpart genes hsigghvm148 and hsiggkvm148 were identified to be analogous to the previous reported V(H)3 germline gene humighvf1/dp53 and the VKIV germline gene hsigkl8. DA1/4 and J(H)4b were used by the heavy chain and J(K)4 was used by the light chain. Multiple nucleotide substitutions were seen in both V-H and V-K when their sequences were compared to the germline sequences. High replacement/silent ratios in the CDR regions of both V-H and V-K indicate positive-selective pressure. Of a total of 22 amino acid replacements in V-H and V-K, 12 were nonconservative replacements. Furthermore, 7 of these 12 resulted in charge changes. The monoreactivity, the moderately high affinity constant, the clonal expansion evident by identical V-H and V-L used by these B cells secreting antibodies of interest, and the excessive replacement mutations in both V-H and V-L segments lead to the conclusion that these antibodies have been generated due to an antigen-driven process. (C) 1995 Academic Press, Inc.