Synthesis of Ethyl Vanillin Artificial Antigen and Preparation of Murine Polyclonal Antibodies

doi:10.15933/j.cnki.1004-3268.2024.06.019

Abstract

Abstract: In order to study the immunological methods for detecting ethyl vanillin(EV),ethyl vanillic acid(EVA)was coupled with bovine serum albumin(BSA)by the active ester(AE)and mixed anhydride(MA)methods to prepare the immune antigens BSA‑EV(AE)and BSA‑EV(MA).UV scanning and SDS‑PAGE were used to identify the artificial antigens.BALB/c mice were immunized with BSA‑EV(AE)or BSA‑EV(MA),tail‑cut blood samples were collected and serum were separated at 10 days after the third 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 EV was prepared successfully,and the coupling ratios of BSA‑EV(AE)and BSA‑EV(MA)were 1∶2.92 and 1∶37.95.The serum titer of the No.3 mouse of BSA‑EV(AE)immunization group was the highest with 1∶51 200,the serum half inhibitory concentration(IC₅₀)against EV was 173.78 μg/L,the liner range was 14.79—2 041.74 μg/L.The serum titer of the No.1 mouse of BSA‑EV(MA)immunization group was the highest with 1∶ 102 400,the serum half inhibitory concentration(IC₅₀)against EV was 617.00 μg/L,the liner range was 42.66—8 709.64 μg/L.The cross reaction rate of serum antibodies in the two immunization groups with vanillin,methyl vanillin,maltol,ethyl maltol,BSA,ovalbumin(OVA)was less than 1%.In conclusion,the artificial antigens of EV were successfully synthesized,and mouse polyclonal antibodies against EV with high sensitivity and specificity were prepared by animal immunization.

Key words: Ethyl vanillin, Artificial antigen, Active ester, Mixed anhydride, Polyclonal antibodies, Indirect competitive ELISA

摘要： 为研究检测乙基香兰素（EV）的免疫学方法，采用活性酯（AE）和混合酸酐（MA）2种方法，将乙基香草酸（EVA）与牛血清白蛋白（BSA）偶联，制备乙基香兰素人工免疫抗原BSA-EV（AE）和BSA-EV（MA），并采用紫外扫描（UV）和凝胶电泳（SDS-PAGE）对人工抗原进行鉴定。将BSA-EV（AE）和BSAEV（MA）分别免疫BALB/c 小鼠，3 免后10 d 断尾采血并分离血清制备鼠源多克隆抗体。采用间接ELISA法测定血清抗体效价，间接竞争ELISA法测定血清抗体敏感性和特异性。结果显示，乙基香兰素人工抗原制备成功，BSA-EV（AE）和BSA-EV（MA）的偶联比分别为1∶2.92和1∶37.95。BSA-EV（AE）免疫组3 号血清效价最高为1∶51 200，对EV 的半数抑制浓度（IC₅₀）为173.78 μg/L，线性范围为14.79~2 041.74 μg/L。BSA-EV（MA）免疫组1 号血清效价最高为1∶102 400，对EV的IC₅₀ 为617.00 μg/L，线性范围为42.66~8 709.64 μg/L。2个免疫组的血清抗体与香兰素、甲基香兰素、麦芽酚、乙基麦芽酚、BSA、鸡卵清蛋白（OVA）的交叉反应率均小于1%。综上，人工抗原BSA-EV 合成成功，并成功制备了对EV灵敏特异的鼠源多克隆抗体。

关键词: 乙基香兰素, 人工抗原, 活性酯法, 混合酸酐法, 多克隆抗体, 间接竞争ELISA

CLC Number:

S852.5
TS202

ZHAO Yinli, CHEN Panpan, WANG Jinrong, DUAN Erzhen, GAN Liping. Synthesis of Ethyl Vanillin Artificial Antigen and Preparation of Murine Polyclonal Antibodies[J]. Journal of Henan Agricultural Sciences, 2024, 53(6): 173-180.

赵银丽, 陈盼盼, 王金荣, 段二珍, 甘丽平. 乙基香兰素人工抗原的合成及鼠源多克隆抗体的制备[J]. 河南农业科学, 2024, 53(6): 173-180.

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