T2毒素人工抗原的合成及鼠源多克隆抗血清的制备

Preparation of T-2 Toxin Artificial Antigen and Mouse Polyclonal Antiserum

合成并鉴定T 2毒素人工抗原,通过动物免疫制备敏感性高、特异性好的鼠源T 2毒素多克隆抗血清。采用琥珀酸酐法将T 2毒素活化为T 2HS,T 2HS在碳二亚胺(EDC)作用下与牛血清白蛋白(BSA)或卵清白蛋白(OVA)偶联,合成免疫抗原T 2 BSA和检测抗原T 2OVA,经鉴定后,分别按30、50μg/只的剂量免疫BALB/c小鼠,共免疫4次,每次间隔4周,最后1次免疫10d后,断尾采血,制备多抗血清。利用间接ELISA方法测定抗血清效价,间接竞争ELISA测定其敏感性和特异性。结果表明,免疫的6只小鼠效价均达到了1∶104以上,2号小鼠多抗血清的敏感性最好,其半数抑制质量浓度(IC50)为324ng/mL,且具有良好的特异性。成功合成了T 2毒素人工抗原,制备了多克隆抗体血清,为T 2毒素单克隆抗体的制备及其免疫学快速检测方法的建立奠定了基础。

To prepare high sensitive and specific polyclonal antiserum against T-2 toxin,T-2 toxin artificial antigens were synthesized and identified by animal immunization in this study.T-2 toxin was converted to T-2-hemisuccinate（T-2-HS） and coupled with carrier pro tein BSA and OVAusing EDC method,and then the immune antigen T-2-BSA and detection antigen T-2-OVA were obtained,respectively.To prepare the poly-antiserum against T-2,BALB/c mouse was immunized with T-2-BSA at the doses of 30 μg and 50 μg for four times at the intervals of four weeks.Ten days after the last immunization,the titer of antiserum was determined by indirect ELISA.The results showed that antiserum of high titers above 1∶104 were produced in six mice.The antiserum of mouse #2 had the best sensibility.Its IC50 was 324 ng/mL and it had good specificity.In this study,T-2 toxin artificial antigens were successful synthesized and the poly-antiserum with high sensibility and specificity was obtained,providing a foundation for the further study.