细菌脂多糖单克隆抗体Clone 20的多反应性及其抗独特型抗体的制备与特性

Preparation and Characterization of Antiidiotype Antibodies Specific for Clone 20,a Polyreactive Monoclonal Antibody against Bacterial Lipopolysaccharids

Clone20系由S．minnesotaR595死菌免疫Balb／c小鼠制备的IgM类单克隆抗体，曾报告它对脂多糖（LPS）中的末端αKDO（2-酮基-3-脱氧辛酸）特异。本文报告采用ELISA显示，Clone20尚能和LPS的毒性成分类脂A结合。用Clone20免疫的小鼠淋巴细胞制备杂交瘤并筛选出4个分泌抗独特型抗体的细胞株．编码为Clone47－50。ELISA抑制试验显示，单克隆抗体Clone49和Clone50与固相Clone20的结合能为E．coliJ5（Re-型）LPS、S．minnesotaR595（Re-型）LPS、类脂A及αKDO所抑制；Clone47和Clone48则否。另一方面，4个克隆均能抑制Clone20结合到团相Re－LPS及类脂A。在被动溶血试验中，Clone20与固相Re－LPS的结合，也为4个克隆所抑制。对于Clone20与αKDO的结合，仅Clone49与50显示抑制作用。这些结果提示，Clone20分子上与Clones47－50的结合部位均不同程度地接近乃至分享其与类脂A结合的独特型决定簇，Clones49与50则尚可结合Clone20上和。KDO结合的独特型决定簇或其邻近结构。此外，在Clone50的腹水中检出高滴度的抗Re－LPS和抗类脂A抗体。综合以上关于Clone50的免疫学特性，推断它至少是部分模拟了LPS的抗原结构。初步的动物实验显示，提前3hr给小鼠注射Clone50，对随后的致死性内毒素休克有明显对抗作用。

Clone 20 is a murlne IgM monoclonal antibody raised against Salmonella minnesota R595 and was reported specific for terminal α-3-deoxy-D-manno-octulosonic acid(αKDO) of the inner core of lipopolysacchride.We show in this report that Clone 20 is also able to bind with lipid A,the toxic part of LPS.Anti-Clone 20 hybriaomas were prepared and four antiidiotye antibody (AID)-secreting clones,Clones 47-50,were selected for further characterization.In ELISA inhibition studies,binding of Clones 49 and 50 to solid phase wbound Clone 20 were inhibited by E.coli J5 (Rc-) and S.minnesota R595 (Re-) LPS,lipid A,and αKDO,whereas Clones 47 and 48 were not inhibited.On the other hand,all of the four clones were able to inhibit the binding of Clone 20 to solid phase-bound lipid A (in ELISA) and Re -LPS(in ELISA and passive hemolysis assay).For binding of Clone 20 to αKDO,Clones 49 and 50 were inhibitory.These results indicate that Clones 47- 50 may bind to,or bind close to,the lipid A-binding site of Clone 20,and that Clones49 and 50 may also bind to .or bind close to,its KDO-binding site.High levels of anti -LPS and anti-lipid A antibodies were detected in Clone 50 ascites.Clone 50 was thus expected as an AID which at least partially mimics .the antigenic structure of Re -LPS.Preliminary animal study showed that intravenous injection of Clone 50 into the mice 3hr prior to K.pneumoniae challenge in mucin -hemoglobin compromised model protected the animal from lethal endotoximia.The protective outcomes raise the interest of development of anti -Clone 20 AIDs as LPS receptor antagonists and/or surrogates of LPS im-munogens in anti-endotoxin immune prophylaxis and therapy.