黄曲霉毒素B1单克隆抗体的制备及间接竞争ELISA检测技术研究

Preparation of anti-aflatoxin B1 monoclonal antibodies and its use in an indirect competitive ELISA for aflatoxin B1

摘要:

【目的】制备黄曲霉毒素B1单克隆(AFB1)抗体，建立间接竞争ELISA检测方法用于污染样品中AFB1的检测。【方法】用碳二亚胺法制备黄曲霉毒素B1的完全抗原AFB1-BSA后免疫Balb/c小鼠，经过细胞融合和克隆化筛选获得抗AFB1单克隆抗体的杂交瘤细胞株。采用体内诱生腹水法制备抗体，通过间接ELISA方法分别测定抗体亚类和效价。经优化实验条件，建立稳定的间接竞争ELISA检测方法，并用于检测饲料样品中的黄曲霉毒素B1。【结果】获得4株稳定分泌抗AFB1单克隆抗体的杂交瘤细胞株，选择3B9细胞株制备抗体，测定抗体亚类为IgG1，效价为1:204 800，与黄曲霉毒素B2、G1、G2和M1的交叉反应率分别为2.2%、33.9%、1.8%和4.1%，与赭曲霉毒素A、伏马毒素和玉米赤霉烯酮几乎不存在交叉反应。以此单抗构建了AFB1间接竞争ELISA检测方法，在AFB1浓度为1.04?25.00 μg/L范围内呈线性(R2=0.993 1)，检测限为1.04 μg/L，半数抑制率(IC50)为6.03 μg/L，平均加标回收率在线性范围内可达85%?120%，变异系数均小于10%。【结论】通过饲料样品检测证实，该方法与进口ELISA试剂盒检测一致性良好，可用于实际样品中黄曲霉毒素B1的快速筛检。

Abstract:

[Objective] Prepare anti-aflatoxin B1 monoclonal antibodies to develop an indirect competitive ELISA (ic-ELISA) applied to detect AFB1 in samples. [Methods] The aflatoxin B1 (AFB1) was conjugated with bovine serum albumin by the carbodiimide method and used to immunize Balb/c mice. Hybridoma cell strains could be obtained by clonal screening and monoclonal antibodies (mAb) could be obtained by inducing ascites in vivo. The subclass and titer of mAb were identificatied by indirect ELISA. After optimizing experimental conditions, a stable ic-ELISA would be developed and applied to detect AFB1 in feed samples. [Results] Four hybridoma cell strains stably secreting specific antibodies to AFB1 were obtained. One of the cell strains 3B9 was chosen to prepare mAb, The mAb belongs to IgG1 with a titer of 1: 204 800. Its cross-reactivity with aflatoxins B2, G1, G2 and M1 was 2.2%, 33.9%, 1.8% and 4.1% respectively. It did not react with ochratoxin A, fumonisin and zearalenone. An indirect-competitive ELISA (ic-ELISA) coated with the mAb had a detection range of 1.04?25.00 μg/L with the detection limit at 1.04 μg/L and the 50% inhibitory concentration (IC50) at 6.03 μg/L. The average recovery rate ranged from 85% to 120% with the variation coefficient less than 10%. [Conclusion] The ic-ELISA has good consistency compared with a commercial kit. We suppose that the ic-ELISA could be used to detect AFB1 from food or feed samples.