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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。
OBJECTIVE: To prepare high titer murine anti-human chain monoclonal antibody (mAb) and establish an ELISA method for the early serological diagnosis of infectious diseases. Methods: BALB / c mice were immunized with all human IgM molecules. The cells were fused by routine methods and cloned by indirect ELISA. A hybridoma cell line stably secreting mAb against μ chain was established. The characteristics of mAb (titer, Ig subclass, specificity and relative affinity) were identified by ELISA and Western blotting. The ELISA capture method was established with purified mAb and used for the detection of specific IgM antibodies in specimens from patients with suspected Japanese encephalitis. Results: A strain of 2E5 that can stably secrete anti-human μ chain mAb was screened. The mAb ascites ELISA titer was 1 × 10-6, the Ig subclass (type) was IgG1 (κ), and the relative affinity was 1 × 10-5. Western blot results showed that mAb2E5 binds only to the μ chain of IgM, Mr 75000. The mAb2E5 coated with ammonium octanoate sulfate was coated by ELISA, and the ELISA method was established for the detection of serum IgM in 30 patients with JE. The sensitivity and specificity were good. CONCLUSION: An anti-human μ chain mAb2E5 was successfully prepared and an ELISA capture method was established for the early serological diagnosis of infectious diseases.