Idiotypic analysis of serum anti-A. actinomycetemcomitans antibody from penodontitis patients

1997年和1998年，我们试图建立人-小鼠杂交瘤，产生对放线菌的血清型特异性抗原的抗体，以分析抗体的独特型。然而，20例已建立的杂交瘤均未显示血清型b特异性抗原阳性反应。1999年，我们尝试用制备单链F-对b血清型特异性抗原的小鼠单克隆抗体进行独特型分析。可变（ScFv）重组抗原结合分子。从两个杂交瘤（S5，对b血清型特异性抗原L2特异性，对A.放线菌temcomitans的LPS特异性）中分离mRNA，并通过RT-PCR扩增H链和l链可变区域的cdna。单抗S5的l链cDNA无法扩增，但我们确认该l链为kappa链。Mab L2的H链和l链扩增的cdna与连接DNA连接成单链（ScFv）。将ScFv连接到pGEM T-easy载体上，转化到jm109感受态细胞。对插入检查选择的两个克隆进行DNA序列分析。综上所述，本研究无法获得预期的结果，但本研究获得的DNA序列将是进行血清抗a独特型分析的重要工具。actinomycetemcomitans抗体。

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.