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Specific binding to methylated polynucleotides in monoclonal anti-poly(dT) antibodies from autoimmune mice
1Department of Internal Medicine, Faculty of Medicine, Kyoto University, Japan.
Six monoclonal antibodies (mAbs) reactive to synthetic polynucleotide, poly(dT), were established from spontaneous autoimmune MRL/MpJ-lpr/lpr mice and male BXSB mice by spleen cell hybridization method, and were analyzed for cross-reactivity with polydeoxy-5-methylcytidylic acid [poly(dmC)] in comparison with its unmethylated counterpart poly(dC). By direct binding tests, these mAbs, all of which had preponderant binding activity to poly(dT) relative to poly(dU), were divided into two groups: (i) four mAbs showing reactivity to poly(dmC) as well as to natural DNA preparations and (ii) two mAbs with limited reactivity to poly(dT) but no binding to poly(dmC) or natural DNAs. Inhibition binding tests with these synthetic polynucleotides demonstrated that one mAb (TP-A9) in the first group reacted specifically to poly(dmC), as well as to poly(dT). In the second group, one mAb (TP-B5) showed highly specific reactivity to poly(dT) that could not be inhibited by poly(dU), poly(dC), or poly(dmC). However, another mAb (TP-C8) in the second group showed reactivity to poly(dT) that could be inhibited by poly(dU) as well as by poly(dT). Thus, these findings indicate that monoclonal anti-poly(dT) antibodies can cross-react with poly(dmC) with different specificities and suggest that the methylated base may be one of the major antigenic sites of DNA molecules recognized by anti-DNA antibodies spontaneously produced in autoimmune mice.
Six monoclonal antibodies (mAbs) reactive to synthetic polynucleotide, poly(dT), were established from spontaneous autoimmune MRL/MpJ-lpr/lpr mice and male BXSB mice by spleen cell hybridization method, and were analyzed for cross-reactivity with polydeoxy-5-methylcytidylic acid [poly(dmC)] in comparison with its unmethylated counterpart poly(dC). By direct binding tests, these mAbs, all of which had preponderant binding activity to poly(dT) relative to poly(dU), were divided into two groups: (i) four mAbs showing reactivity to poly(dmC) as well as to natural DNA preparations and (ii) two mAbs with limited reactivity to poly(dT) but no binding to poly(dmC) or natural DNAs. Inhibition binding tests with these synthetic polynucleotides demonstrated that one mAb (TP-A9) in the first group reacted specifically to poly(dmC), as well as to poly(dT). In the second group, one mAb (TP-B5) showed highly specific reactivity to poly(dT) that could not be inhibited by poly(dU), poly(dC), or poly(dmC). However, another mAb (TP-C8) in the second group showed reactivity to poly(dT) that could be inhibited by poly(dU) as well as by poly(dT). Thus, these findings indicate that monoclonal anti-poly(dT) antibodies can cross-react with poly(dmC) with different specificities and suggest that the methylated base may be one of the major antigenic sites of DNA molecules recognized by anti-DNA antibodies spontaneously produced in autoimmune mice.