抗人μ链单克隆抗体的制备、鉴定及初步应用

目的:制备高效价的鼠源性抗人μ链单克隆抗体(mAb),并建立可用于感染性疾病早期血清学诊断的ELISA捕捉法。方法:以人IgM全分子免疫BALB/c小鼠,按常规方法进行细胞融合,用间接ELISA法筛选及克隆化,建立可稳定分泌抗μ链mAb的杂交瘤细胞株。mAb的特性(效价、Ig亚类、特异性及相对亲和力)采用ELISA及Westernblot法鉴定。以纯化的mAb包被建立ELISA捕捉法,并用于可疑乙脑患者标本中特异性IgM抗体的检测。结果:筛选到1株可稳定分泌抗人μ链mAb的细胞株2E5。mAb腹水的ELISA效价为1×10-6,Ig亚类(型)为IgG1(κ),相对亲和力为1×10-5。Westernblot结果显示mAb2E5仅与IgM的μ链结合,Mr为75000。以辛酸硫酸铵法纯化的mAb2E5包被,建立了ELISA捕捉法,用于30份乙脑患者血清IgM的检测,敏感性及特异性良好。结论:成功地制备1株抗人μ链mAb2E5,建立了可用于感染性疾病早期血清学诊断的ELISA捕捉法。

Preparation, characterization and preliminary application of monoclonal antibody against human μ chain

AIM: To prepare monoclonal antibody (mAb) against human mu chain with high titer and establish a capture ELISA for early serological diagnosis of infectious diseases. METHODS: BALB/c mice were immunized with human IgM. Hybridoma cell line which could stably secret the mAb to human IgM was established by routine cell fusion technique. mAb's characteristics (titer, Ig subclass, specificity and relative affinity) were identified by indirect ELISA and Western blot, respectively. A capture ELISA was established by using purified mAb to capture specific IgM for early diagnosis of Japanese encephalitis. RESULTS: One hybridoma cell line 2E5 stably secreting mAb against human IgMmu chain was obtained. The titer of ascites of the mAb was 1 x 10(-6) and the Ig subclass was IgG1(kappa). Relative affinity of 2E5 was 1 x 10 (-5). Western blot analysis showed that mAb 2E5 reacted specifically to mu chain. Both sensitivity and specificity of the capture ELISA in detecting specific IgM in Japanese encephalitis patients sera were high. CONCLUSION: mAb 2E5 against human mu chain was prepared successfully, and a capture ELISA for early serological diagnosis of Japanese encephalitis was set up.