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6-苄基腺嘌呤人工抗原的合成及鼠源多克隆抗体的制备 被引量:1

Synthesis of 6-Benzylaminopurine Artificial Antigen and Preparation of Murine Polyclonal Antibodies
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摘要 为制备6-苄基腺嘌呤(6-benzylaminopurine,6-BA)的抗体,利用高碘酸盐法将6-BA的代谢产物6-苄基腺苷(6-benzylaminopurine riboside,6-BAR)分别与牛血清蛋白(BSA)和卵清蛋白(OVA)偶联,制备出免疫抗原6-BAR-BSA和包被抗原6-BAR-OVA。采用紫外扫描(UV)和凝胶电泳(SDS-PAGE)对人工抗原进行初步鉴定。用6-BAR-BSA以背部皮下多点注射的方式免疫4只BALB/c小鼠(编号1—4),免疫剂量50μg/只,间隔21 d,4免后10 d断尾采血制备鼠源多克隆抗体血清。间接ELISA测定血清效价,间接竞争ELISA测定血清敏感性及特异性。结果显示,6-BA人工抗原制备成功,免疫小鼠血清效价均达到1∶51200以上,2号小鼠血清效价最高为1∶204800,且其对6-BA的半数抑制浓度(IC50)为13.48μg/L,线性范围为0.26~701.45μg/L,与激动素、赤霉素、4-氯苯氧乙酸钠、腐霉利、呋喃苯烯酸钠、BSA、OVA交叉反应率均小于0.01%。综上,成功制备了6-BA人工抗原,并通过动物免疫得到高敏感性、高特异性鼠源多克隆抗体。 For developing antibodies against 6-benzylaminopurine(6-BA),6-benzylaminopurine riboside(6-BAR),the metabolite of 6-BA,was coupled with bovine serum albumin(BSA)and ovalbumin(OVA)separately by the periodate method to prepare the immune antigen 6-BAR-BSA and the envelope antigen 6-BAR-OVA.UV scanning and SDS-PAGE were used to identify the artificial antigens.Four BALB/c mice(No.1—4)were immunized with 6-BAR-BSA by subcutaneous multipoint injection on the back at a dose of 50μg each at an interval of 21 d,and tail-cut blood samples were collected 10 days after the fourth immunization to prepare rat polyclonal antibodies.Serum titer was determined by indirect ELISA,and serum sensitivity and specificity were determined by indirect competitive ELISA.The results showed that the artificial antigen of 6-BA was prepared successfully.The serum titers of immunized mice were all more than 1∶51200,among which the No.2 mouse was the highest with 1∶204800.Furthermore,the serum half inhibitory concentration(IC50)of No.2 mouse against 6-BA was 13.48μg/L,the liner range was 0.26—701.45μg/L,and the cross reaction rate with kinetin,gibberellin,sodium 4-chlorophenoxyacetate,procymidone,sodium furbenzenate,BSA,OVA was less than 0.01%.In conclusion,6-BA artificial antigen was successfully prepared,and mouse polyclonal antibodies with high sensitivity and specificity were obtained by animal immunization.
作者 孟继秋 胡骁飞 王耀 邢云瑞 曹金博 陈琳琳 孙亚宁 张改平 MENG Jiqiu;HU Xiaofei;WANG Yao;XING Yunrui;CAO Jinbo;CHEN Linlin;SUN Yaning;ZHANG Gaiping(Key Laboratory of Animal Immunology,Henan Academy of Agricultural Sciences,Zhengzhou 450002,China;Food and Bioengineering College,Henan University of Science and Technology,Luoyang 471003,China;Henan Agricultural University,Zhengzhou 450002,China)
出处 《河南农业科学》 北大核心 2021年第5期157-164,共8页 Journal of Henan Agricultural Sciences
基金 “十三五”国家重点研发计划项目(2018YFC1602903) 国家科技支撑计划项目(2014BAD13B05) 河南省农业科学院科技创新创意项目(2020CX20)。
关键词 6-苄基腺嘌呤 6-苄基腺苷 人工抗原 ELISA 多克隆抗体 6-benzylaminopurine 6-benzylaminopurine riboside Artificial antigen ELISA Polyclonal antibodies
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