氯霉素单克隆抗体的制备及ELISA检测方法的建立

doi:10.16431/j.cnki.1671-7236.2018.09.029

摘要/Abstract

摘要：

本研究旨在建立检测动物源性食品中氯霉素（CAP）残留的间接竞争ELISA（ci-ELISA）方法。在CAP羟基（-OH）位点上引入活性基团羧基（-COOH）得到氯霉素半琥珀酸酯（CAP-HS）；采用混合酸酐法将CAP-HS分别与BSA和OVA偶联合成人工免疫原CAP-HS-BSA和包被抗原CAP-HS-OVA，用CAP-HS-BSA免疫BALB/c小鼠，间接ELISA和ci-ELISA筛选细胞融合备用鼠；应用杂交瘤技术制备抗CAP单克隆抗体；以CAP单克隆抗体为基础、CAP-HS-OVA为检测原建立ci-ELISA方法。结果显示，试验成功筛选获得一株稳定分泌抗CAP抗体的杂交瘤细胞株（2C4），抗体效价为4.8×10^-5，利用2C4腹水优化得到ci-ELISA最佳试验条件：0.4 μg/mL CAP-HS-OVA 37℃包被2 h；5%猪血清37℃封闭1 h；1：6.4×10⁴ CAP单克隆抗体37℃孵育15 min；1：1 000羊抗鼠酶标二抗（GaMIgG-HRP）37℃孵育30 min；室温显色9 min。绘制的CAP残留ci-ELISA标准曲线为典型的S型，与4参数logit拟合曲线相吻合，半数抑制浓度（IC₅₀）为0.53 ng/mL。添加回收试验结果显示，阴性鱼肉、牛奶的回收率分别为93.3%~96.6%和93.7%~96.8%，批内变异系数分别为2.3%~5.0%和2.2%~4.6%，批间变异系数分别为2.7%~4.1%和2.3%~3.6%。HPLC对比试验结果显示，ci-ELISA与HPLC的测定结果无显著差异。本试验成功建立了CAP的ELISA残留检测方法，该方法具有较高的灵敏度、准确度和精密度，可满足动物源性食品中CAP残留检测要求。

关键词: 氯霉素; 残留检测; 单克隆抗体; ci-ELISA

Abstract:

This study was aimed to establish an indirect competition ELISA (ci-ELISA) method for determination of chloramphenicol (CAP) residue in animal-derived foods.An active carboxyl group (-COOH) was introduced to the hydroxy (-OH) locus of phenyl of CAP to form CAP-HS.Via the method of mixed anhydride,CAP-HS was conjugated with BSA and OVA to obtain artificial immunogen (CAP-HS-BSA) and the coating antigen (CAP-HS-OVA) was obtained in the same way.CAP-HS-BSA was used to immunize BALB/c mice.Cell fusion spare mice were screened by indirect ELISA and ci-ELISA.CAP mAb were established using hybridoma technology.ci-ELISA based on CAP mAb and CAP-HS-OVA for CAP was developed.The result showed that one hybridoma cell line (2C4) was isolated,which produced mAb that could bind CAP,the titer of mAb was 4.8×10^-5.The optimal conditions for ci-ELISA based on 2C4 for CAP were as follows:0.4 μg/mL CAP-HS-OVA coating at 37℃ for 2 h;5% pig serum blocking at 37℃ for 1 h; 1:6.4×10⁴ CAP mAb incubating at 37℃ for 15 min;1:1 000 goat anti-mouse enzyme-conjugated secondary antibody (GaMIgG-HRP) incubating at 37℃ for 30 min;TMB was used for color development at room temperature for 9 min.The calibration curve of ci-ELISA CAP showed typical sigmoid and fitted to the four parameters logistic equation.The IC₅₀ in ci-ELISA was 0.53 ng/mL.The recoveries of CAP in negative carp meat and pork were 93.3% to 96.6% and 93.7% to 96.8%,respectively;The coefficient variation of the variability of intra-assay were 2.3% to 5.0% and 2.2% to 4.6%,respectively;The coefficient variation of the variability of inter-assay were 2.7% to 4.1% and 2.3% to 3.6%, respectively.HPLC comparative test results showed no significant difference between ci-ELISA and HPLC.The ELISA method for residue detection of CAP was successfully established in this experiment.This method had high sensitivity,accuracy and precision,and could meet the requirements for CAP residue detection in animal-derived foods.

Key words: chloramphenicol; residue detection; monoclonal antibody (mAb); ci-ELISA

中图分类号:

S859.84

范素菊, 杨兴东, 高润. 氯霉素单克隆抗体的制备及ELISA检测方法的建立[J]. 《中国畜牧兽医》, 2018, 45(9): 2566-2574.

FAN Suju, YANG Xingdong, GAO Run. Preparation of Chloramphenicol Monoclonal Antibody and Establishment of ELISA Detection Method[J]. , 2018, 45(9): 2566-2574.

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