基于双特异性单克隆抗体的免疫亲和柱的制备用于黄曲霉毒素B和赭曲霉毒素A的检测并结合免疫竞争酶联免疫吸附测定法
Preparation of an Immunoaffinity Column Based on Bispecific Monoclonal Antibody for Aflatoxin B and Ochratoxin A Detection Combined with ic-ELISA.
作者信息
Lu Disha, Wang Xu, Su Ruijue, Cheng Yongjian, Wang Hong, Luo Lin, Xiao Zhili
机构信息
Guangdong Provincial Key Laboratory of Food Quality and Safety, College of Food Science, South China Agricultural University, Guangzhou 510642, China.
出版信息
Foods. 2022 Jan 25;11(3):335. doi: 10.3390/foods11030335.
A novel and efficient immunoaffinity column (IAC) based on bispecific monoclonal antibody (BsMAb) recognizing aflatoxin B (AFB) and ochratoxin A (OTA) was prepared and applied in simultaneous extraction of AFB and OTA from food samples and detection of AFB/OTA combined with ic-ELISA (indirect competitive ELISA). Two deficient cell lines, hypoxanthine guanine phosphoribosyl-transferase (HGPRT) deficient anti-AFB hybridoma cell line and thymidine kinase (TK) deficient anti-OTA hybridoma cell line, were fused to generate a hybrid-hybridoma producing BsMAb against AFB and OTA. The subtype of the BsMAb was IgG via mouse antibody isotyping kit test. The purity and molecular weight of BsMAb were confirmed by SDS-PAGE method. The cross-reaction rate with AFB was 37%, with AFG 15%, with AFM 48%, with AFM 10%, and with OTB 36%. Negligible cross-reaction was observed with other tested compounds. The affinity constant (Ka) was determined by ELISA. The Ka (AFB) and Ka (OTA) was 2.43 × 10 L/mol and 1.57 × 10 L/mol, respectively. Then the anti-AFB/OTA BsMAb was coupled with CNBr-Sepharose, and an AFB/OTA IAC was prepared. The coupling time and elution conditions of IAC were optimized. The coupling time was 1 h with 90% coupling rate, the eluent was methanol-water (60:40, :, pH 2.3) containing 1 mol/L NaCl, and the eluent volume was 4 mL. The column capacities of AFB and OTA were 165.0 ng and 171.3 ng, respectively. After seven times of repeated use, the preservation rates of column capacity for AFB and OTA were 69.3% and 68.0%, respectively. The ic-ELISA for AFB and OTA were applied combined with IAC. The IC (50% inhibiting concentration) of AFB was 0.027 ng/mL, the limit of detection (LOD) was 0.004 ng/mL (0.032 µg/kg), and the linear range was 0.006 ng/mL0.119 ng/mL. The IC of OTA was 0.878 ng/mL, the LOD was 0.126 ng/mL (1.008 µg/kg), and the linear range was 0.259 ng/mL6.178 ng/mL. Under optimum conditions, corn and wheat samples were pretreated with AFB-OTA IAC. The recovery rates of AFB and OTA were 95.4%~105.0% with ic-ELISA, and the correlations between the detection results and LC-MS were above 0.9. The developed IAC combined with ic-ELISA is reliable and could be applied to the detection of AFB and OTA in grains.
制备了一种基于双特异性单克隆抗体（BsMAb）识别黄曲霉毒素B（AFB）和赭曲霉毒素A（OTA）的新型高效免疫亲和柱（IAC），并将其应用于从食品样品中同时提取AFB和OTA以及结合间接竞争酶联免疫吸附测定（ic-ELISA）检测AFB/OTA。将两种缺陷细胞系，次黄嘌呤鸟嘌呤磷酸核糖转移酶（HGPRT）缺陷的抗AFB杂交瘤细胞系和胸苷激酶（TK）缺陷的抗OTA杂交瘤细胞系进行融合，以产生针对AFB和OTA的产生BsMAb的杂交杂交瘤。通过小鼠抗体分型试剂盒测试，BsMAb的亚型为IgG。通过SDS-PAGE方法确认了BsMAb的纯度和分子量。与AFB的交叉反应率为37%，与AFG为15%，与AFM为48%，与AFM为10%，与OTB为36%。与其他测试化合物的交叉反应可忽略不计。通过ELISA测定亲和常数（Ka）。Ka（AFB）和Ka（OTA）分别为2.43×10 L/mol和1.57×10 L/mol。然后将抗AFB/OTA BsMAb与CNBr-琼脂糖偶联，制备了AFB/OTA IAC。对IAC的偶联时间和洗脱条件进行了优化。偶联时间为1小时，偶联率为90%，洗脱液为含1 mol/L NaCl的甲醇-水（60:40，:，pH 2.3），洗脱液体积为4 mL。AFB和OTA的柱容量分别为165.0 ng和171.3 ng。重复使用七次后，AFB和OTA的柱容量保留率分别为69.3%和68.0%。将AFB和OTA的ic-ELISA与IAC联合应用。AFB的IC（50%抑制浓度）为0.027 ng/mL，检测限（LOD）为0.004 ng/mL（0.032 µg/kg），线性范围为0.006 ng/mL0.119 ng/mL。OTA的IC为0.878 ng/mL，LOD为0.126 ng/mL（1.008 µg/kg），线性范围为0.259 ng/mL6.178 ng/mL。在最佳条件下，用AFB-OTA IAC对玉米和小麦样品进行预处理。ic-ELISA法测定AFB和OTA的回收率为95.4%~105.0%，检测结果与液相色谱-质谱法的相关性高于0.9。所开发的IAC与ic-ELISA相结合是可靠的，可应用于谷物中AFB和OTA的检测。
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