黄曲霉毒素B_1单克隆抗体的制备及基于该抗体的黄曲霉毒素B_1免疫学检测方法的建立

Establishment of Immunologic Detection Method for Aflatoxin B_1 Based on Preparation of Aflatoxin B_1 Monoclonal Antibody

为了快速准确检测粮食与饲料中黄曲霉毒素B_1(AFB_1)含量,本试验制备了具有高灵敏度的AFB_1单克隆抗体,并将其应用于AFB_1间接竞争酶联免疫吸附测定(ELISA)方法的建立。采用碳二亚胺(EDC)法制备AFB_1完全抗原[AFB_1-牛血清白蛋白(BSA)和AFB_1-鸡卵白蛋白(OVA)],并免疫B_ALB/c小鼠。经细胞融合,筛选出能够分泌高灵敏度AFB_1单克隆抗体的杂交瘤细胞株。采用体内诱生腹水法,大量制备AFB_1单克隆抗体,并对其免疫学特性进行鉴定。基于制备的AFB_1单克隆抗体,建立AFB_1间接竞争ELISA检测方法。结果显示:通过筛选,获得稳定产生抗体的杂交瘤细胞株9H1F5,经间接ELISA方法测定,获得的AFB_1单克隆抗体的效价高达5.12×105,亲和力常数(Ka)=6.72×107L/mol,抗体亚型为免疫球蛋白G_2(IgG_2)。采用间接竞争ELISA方法测得AFB_1的半数抑制浓度(IC_(50))为0. 232 ng/mL,检测范围为0.014～1.920 ng/mL,最低检测限为0.014 ng/mL。同时,该AFB_1单克隆抗体与黄曲霉毒素B_2(AFB_2)、黄曲霉毒素G_1(AFG_1)、黄曲霉毒素G_2(AFG_2)、黄曲霉毒素M1(AFM1)的交叉反应率分别为40.21%、33.19%、31.96%、4.40%,与其他霉菌毒素无交叉反应。由上述结果可知,本试验成功制备了灵敏度高、特异性强的AFB_1单克隆抗体,并基于该抗体建立了检测AFB_1的间接竞争ELISA方法,为粮食与饲料中AFB_1快速免疫学检测奠定了基础。

In order to detect aflatoxin B1 (AFB1) content in cereals and feeds rapidly and correctly,a highly sensitive AFB1 monoclonal antibody was prepared and an indirect competitive enzyme-linked immunosorbent assay (ELISA) for AFB1 was established. AFB1 complete antigens [AFB1-bovine serum albumin (BSA) and AFB1-ovalbumin (OVA) ]were prepared via carbodiimide (EDC) method and used to immunize the BALB/c mice. After the cell fusion,hybridoma cell line which could secret highly sensitive AFB1 monoclonal antibody was selected. Then a lot of AFB1 monoclonal antibodies were prepared using inducing ascites in vivo,and the AFB1 monoclonal antibody properties were identified. Finally,an indirect competitive ELISA method for detecting AFB1 based on AFB1 monoclonal antibody was established. A hybridoma cell line 9 H1 F5 was selected,and the titer of AFB1 monoclonal antibody was up to 5.12×105,the affinity constant (Ka) = 6.72×107 L/mol,and the subtype was immunoglobulin G2 (IgG2) measured by indirect ELISA method. According to the indirect competitive ELISA,the median inhibitory concentration (IC50) of AFB1 was 0.232 ng/mL,the detection range was 0.014 to 1. 920 ng/mL,and the limit of detection (LOD) of AFB1 was 0. 014 ng/mL. Besides,cross reaction rates of AFB1 monoclonal antibody with aflatoxin B2 (AFB2),aflatoxin G1 (AFG1),aflatoxin G2 (AFG2) and aflatoxin M1 (AFM1) were 40.21%,33.19%,31.96% and 4.40%,respectively,but there were no cross reactions with other mycotoxins. According to the above results,the highly sensitive and specific AFB1 monoclonal antibody is successfully prepared,and the indirect competitive ELISA method for AFB1 detection is established on the base of AFB1 monoclonal antibody,which can provide the foundation for AFB1 rapid immunological detection in cereals and feeds.