抗大片吸虫分泌排泄抗原单克隆抗体制备

【目的】制备大片吸虫分泌排泄抗原单克隆抗体，为大片吸虫病的免疫诊断、防治等研究奠定基础。【方法】利用杂交瘤技术，以大片吸虫分泌排泄抗原为免疫原制备单克隆抗体，并用ELISA、硫氰酸盐洗脱法等方法鉴定其生物学特性。【结果】通过间接ELISA筛选及3次亚克隆后，共获得5株大片吸虫分泌排泄抗原单克隆抗体杂交瘤细胞株，分别命名为6D3、6B4、7D2、7D1和7D4。经鉴定，发现5株杂交瘤细胞染色体数为90～110条，均大于亲本细胞，其亚类鉴定分别属于IgG2b、IgM、IgM、IgG1和IgM，其轻链均为κ型；5株单克隆抗体（6D3、6B4、7D2、7D1、7D4）的上清效价分别为1∶400、1∶3200、1∶25600、1∶6400和1∶6400，腹水效价分别为1∶104、1∶104、1∶105、1∶107和1∶106；而表位测定结果表明，5株单克隆抗体中，6D3与7D1、7D2以及7D4与7D1、7D2作用于不同表位，6D3与6B4 可能识别同一表位或重叠表位，或同一表位有空间位阻，7D4与6D3以及6B4与7D4、7D1、7D2可能具有空间位阻；5株单克隆抗体的相对亲和力为：6B4＞7D4＞7D1＞6D3＞7D2；5株杂交瘤细胞株均能稳定地分泌单克隆抗体。【结论】制备获得的分泌排泄抗原单克隆抗体可用于大片吸虫病的诊断和免疫机理等方面的进一步研究。

Preparation of monoclonal antibodies against excretory-secretory antigen of Fasciola gigantica and identification of its biological characteristics

Objective]Fasciola gigantica excretory and secretory antigen monoclonal antibodies were prepared for further study on diagnosis and control of Fasciola gigantica diseases.Method]The monoclonal antibodies of Fasciola gigantica excretory and secretory antigen were made by hybridoma technology and its biological characteristics were identified by ELISA,thiocyanate elution.Result]After indirect ELISA selection and three times of subcloning,five hybridoma cell lines(6D3,6B4,7D2,7D1,and 7D4) of monoclonal antibodies against excretory-secretory antigen of Fasciola gigantic were obtained.The numbers of chromosome of hybridoma cell lines,ranging from 90 to 110,were larger than that of the parent cells.The prepared five McAbs were found to be the isotopes of Ig G2b,Ig M,Ig M,Ig G1 and Ig M,respectively,with κ-light chain.The supernate antibody titers of 6D3,6B4,7D2,7D1,and 7D4 were 1:400,1:3200,1:25600,1:6400,and 1:6400,respectively;the ascites titers of five hybridoma cell lines were 1:104,1:104,1:105,1:107,and 1:106,respectively.Epitope mapping indicated that 6D3 and 7D1,6D3 and 7D2,7D1 and 7D2,7D4 and 7D1,and 7D4 and 7D2 acted on different epitopes;6D3 and 6B4 probably acted on the same epitopes,or had steric hindrance on the same epitope;7D4 and 6D3,6B4 and 7D4,6B4 and 7D1,and 6B4 and 7D2 might have steric hindrance.Relative affinity sequence of the five lines was 6B4>7D4>7D1>6D3>7D2.All of the five cell lines could excrete monoclonal antibodies steadily.Conclusion]It was concluded that the produced monoclonal antibodies against excretory-secretory antigen could be applied in the follow-up research of Fasciola gigantica diagnosis and immune mechanism.