| Budget Amount *help |
¥3,100,000 (Direct Cost: ¥3,100,000)
Fiscal Year 1999: ¥800,000 (Direct Cost: ¥800,000)
Fiscal Year 1998: ¥600,000 (Direct Cost: ¥600,000)
Fiscal Year 1997: ¥1,700,000 (Direct Cost: ¥1,700,000)
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| Research Abstract |
In 1997 and 1998, we tried to establish human-mouse heterohybridomas producing antibodies to the serotype-b specific antigen from Actinobacillus actinomycetemcomitans in order to analyze idiotype of the antibodies. However, none of the twenty established hybridomas showed positive reactions with the serotype-b specific antigen. In 1999, we tried idiotypic analysis of the mouse monoclonal antibody to the serotype-b specific antigen by preparing single-chain F-. Variable (ScFv) recombinant antigen-binding molecules. mRNA was isolated from two hybridomas (S5; specific for the serotype-b specific antigen, L2; specific for LPS from A. actinomycetemcomitans), and cDNAs of variable regions of H- and L-chains were amplified by RT-PCR. cDNA could not be amplified as for the L-chain of Mab S5, although we confirmed that the L-chain is kappa chain. Amplified cDNAs of H- and L-chains of Mab L2 were joined into a single chain (ScFv) with linker DNA. The ScFv was ligated to pGEM T-easy vector, and transformed to JM 109 competent cell. Two clones selected by insert check were analyzed for DNA sequence. In conclusion, we could not have the expected results in this study, however, the DNA sequence obtained in this study would be an important tool to proceed idiotypic analysis of serum anti-A. actinomycetemcomitans antibody.
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