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目的:制备并鉴定抗T-2毒素单克隆抗体杂交瘤细胞株,以期为T-2毒素的快速检测提供有力的抗体工具。方法:采用琥珀酸酐法将T-2毒素活化为T-2HS,T-2HS在DCC与NHS作用下与牛血清白蛋白(BSA)或卵清白蛋白(OVA)偶联合成完全抗原。以T-2HS-BSA为抗原,免疫BALB/c小鼠,用ELISA筛选、建立分泌抗T-2毒素抗体的杂交瘤细胞株。测定单抗免疫球蛋白亚类及单抗效价,用间接竞争法检测单克隆抗体细胞株的特异性。结果:建立了1株稳定分泌抗T-2毒素抗体的杂交瘤细胞株T-1B3,该细胞株所分泌的单隆抗体Ig亚类为IgG1,ELISA检测结果表明抗T-2毒素抗体可以与T-2毒素发生特异性反应,工作浓度为1:6.5×104,IC50为3.6 ng/ml。该抗体与HT-2毒素的交叉反应为65.9%,与黄曲霉毒素无交叉。结论:制备出能分泌具有较高特异性和亲和力的抗T-2毒素单克隆抗体的杂交瘤细胞株。
OBJECTIVE: To prepare and identify monoclonal antibody against T-2 toxin hybridoma cell line, in order to provide a powerful antibody tool for the rapid detection of T-2 toxin. Methods: The T-2 toxin was activated to T-2HS by succinic anhydride method. T-2HS was conjugated with bovine serum albumin (BSA) or ovalbumin (OVA) under the action of DCC and NHS to synthesize complete antigen. BALB / c mice were immunized with T-2HS-BSA as an antigen and screened by ELISA to establish a hybridoma cell line secreting anti-T-2 toxin antibody. The monoclonal antibody immunoglobulin subclasses and monoclonal antibody titer were determined, and the specificity of monoclonal antibody cell lines was tested by indirect competition assay. Results: A hybridoma cell line T-1B3 secreting anti-T-2 toxin antibody was successfully established. The Ig subtype of monoclonal antibody secreted by this cell line was IgG1. The results of ELISA showed that anti-T-2 toxin antibody could compete with T-2 toxin specific reaction, the working concentration of 1: 6.5 × 104, IC50 of 3.6 ng / ml. The antibody cross-reacts with HT-2 toxin at 65.9% with no cross-reaction to aflatoxin. Conclusion: A hybridoma cell line secreting monoclonal antibody against T-2 toxin with high specificity and affinity was prepared.