抗猪囊虫单克隆抗体的制备及应用

Preparation of monoclonal antibody against cysticerus cellulosae antigen and its application

目的利用杂交瘤技术制备抗猪囊虫单克隆抗体,建立抑制性酶联免疫吸附法I-ELISA并应用于临床。方法运用自提的猪囊虫囊液与福氏不完全佐剂加干扰素混合免疫Balb/c小鼠,取小鼠脾淋巴细胞与SP2/0骨髓瘤细胞融合,反复筛选及克隆化;用低温无水乙醇沉淀法分离纯化单抗IgG(腹水抗体效价1:3200);用改良的过碘酸钠法制备辣根过氧化物酶标单抗;用I-ELISA检测患者脑脊液和血清中囊虫特异性抗体。结果获得了3株稳定分泌抗猪囊虫单克隆抗体的杂交瘤细胞株,单抗的免疫球蛋白亚类鉴定为2株IgG3,1株IgM。标记的HRP-IgG酶标单抗,经临床病例标本验证,确诊的脑囊虫病患者脑脊液63例,抗体阳性61例,阳性率为96.83％;血清标本60例,抗体阳性53例,阳性率为88.33％;其他颅内感染性疾病患者脑脊液标本240例、血清标本240例,肝肺包虫病患者血清10例,正常脑脊液25例(阑尾炎手术腰麻患者),健康志愿者血清32例,检测结果均为阴性。结论本研究制备的HRP-IgG酶标单克隆抗体经临床应用,证实对脑囊虫病有良好诊断价值。

Objective To prepare monoclonal antibody (McAb) against cysticorus cellulosae antigen by hybridoma technique, and to establish I-ELISA method for clinical application. Methods Spleen cells of Balb/c mouse immunized subcutaneously with cysticercus fluid (mixture of Freund's incomplete adjuvant and interferon) were fused with myeloma cells SP2/0, and the resulting hybridoma cells were selected and cloned. The purification of McAbs IgG (The ascites antibody titre was 1:3200) was made by anhydrous ethanol sedimentation method at low temperature. Horseradish peroxidase (HRP) conjugated antibody was produced by treatment of sodium iodate; special antibody against cysticorus cellulosae antigen in cerebrospinal fluid and serum of patients were assayed by indirect enzyme-linked immunosorbent assay (I-ELISA). Results Three hybridoma cell lines that secreting specific McAbs against cysticercus fluid antigen were attained. Two McAbs belonged to IgG3, and another one belonged to IgM. HRP conjugated antibody was assayed. A total of 63 cases were diagnosed as cerebrocystise-rocosis, and positive antibody was found in 61 cases with a rate of 96.83%. A total of 53 cases of positive antibody were found in 60 serum samples with a rate of 88.33% . Negative outcomes were found in other samples. Conclusion HRP conjugated McAbs against cysticorus cellulosae antigen generated by our laboratory have clinical value in diagnosis of cerebrocystiserocosis.