摘要
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 B_1 (AFB_1) content in cereals and feeds rapidly and correctly,a highly sensitive AFB_1 monoclonal antibody was prepared and an indirect competitive enzyme-linked immunosorbent assay (ELISA) for AFB_1 was established. AFB_1 complete antigens [AFB_1-bovine serum albumin (BSA) and AFB_1-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 AFB_1 monoclonal antibody was selected. Then a lot of AFB_1 monoclonal antibodies were prepared using inducing ascites in vivo,and the AFB_1 monoclonal antibody properties were identified. Finally,an indirect competitive ELISA method for detecting AFB_1 based on AFB_1 monoclonal antibody was established. A hybridoma cell line 9 H1 F5 was selected,and the titer of AFB_1 monoclonal antibody was up to 5.12×105,the affinity constant (Ka) = 6.72×107 L/mol,and the subtype was immunoglobulin G_2 (IgG_2) measured by indirect ELISA method. According to the indirect competitive ELISA,the median inhibitory concentration (IC_(50)) of AFB_1 was 0.232 ng/mL,the detection range was 0.014 to 1. 920 ng/mL,and the limit of detection (LOD) of AFB_1 was 0. 014 ng/mL. Besides,cross reaction rates of AFB_1 monoclonal antibody with aflatoxin B_2 (AFB_2),aflatoxin G_1 (AFG_1),aflatoxin G_2 (AFG_2) 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 AFB_1 monoclonal antibody is successfully prepared,and the indirect competitive ELISA method for AFB_1 detection is established on the base of AFB_1 monoclonal antibody,which can provide the foundation for AFB_1 rapid immunological detection in cereals and feeds.
引文
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