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Generic Hapten Synthesis, Broad-Specificity Monoclonal Antibodies Preparation, and Ultrasensitive ELISA for Five Antibacterial Synergists in Chicken and Milk
Journal of Agricultural and Food Chemistry ( IF 5.7 ) Pub Date : 2018-09-25 00:00:00 , DOI: 10.1021/acs.jafc.8b03834 Hongfang Li 1 , Shaoqin Ma 1 , Xiya Zhang 2 , Chenglong Li 1 , Baolei Dong 1 , Mari Ghulam Mujtaba 1 , Yujie Wei 1 , Xiao Liang 3 , Xuezhi Yu 1 , Kai Wen 1 , Wenbo Yu 1 , Jianzhong Shen 1 , Zhanhui Wang 1
Journal of Agricultural and Food Chemistry ( IF 5.7 ) Pub Date : 2018-09-25 00:00:00 , DOI: 10.1021/acs.jafc.8b03834 Hongfang Li 1 , Shaoqin Ma 1 , Xiya Zhang 2 , Chenglong Li 1 , Baolei Dong 1 , Mari Ghulam Mujtaba 1 , Yujie Wei 1 , Xiao Liang 3 , Xuezhi Yu 1 , Kai Wen 1 , Wenbo Yu 1 , Jianzhong Shen 1 , Zhanhui Wang 1
Affiliation
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An antibody with broad specificity and principally depending on hapten structure and size is a key reagent for developing a class-selective immunoassay. In the present study, three new generic haptens of antibacterial synergists (ASGs) were proposed using trimethoprim as the starting molecule. These haptens contained carboxyl groups on the meta position of trimethoxybenzene for conjugating to protein, while, the common moiety of ASGs, i.e., diaminopyrimidine, was intentionally and maximally exposed to the immune system in animals in order to induce antibodies with broad specificity against ASGs. Five monoclonal antibodies (mAbs) were finally obtained, and 5C4 from the hapten with a short spacer arm, named Hapten A, showed not only uniform broad specificity but also high affinity to all five ASGs. We further determined the possible recognition mechanism of mAbs in terms of conformational and electronic aspects. An indirect competitive ELISA (icELISA)-based 5C4 was established and exhibited IC50 values of 0.067–0.139 μg L–1 with cross-reactivity of 48.2%–418.7% for the five ASGs in buffer under optimal conditions. The calculated limits of detection of the icELISA for chicken and milk were 0.06–0.8 μg kg–1 and 0.05–0.6 μg L–1, respectively. The recoveries in spiked chicken and milk samples were 75.2%–101.4% with a coefficient of variation less than 14.3%. In summary, we have developed, for the first time, a rapid and reliable icELISA for ASGs with significantly improved sensitivity and class selectivity.
中文翻译：
鸡肉和牛奶中五种抗菌增效剂的通用半抗原合成，广谱单克隆抗体制备和超灵敏ELISA
具有广泛特异性并且主要取决于半抗原结构和大小的抗体是用于开发类选择性免疫测定法的关键试剂。在本研究中，使用甲氧苄啶为起始分子，提出了三种新的抗菌增效剂（ASG）通用半抗原。这些半抗原在间位上含有羧基在三甲氧基苯的位置结合蛋白质，而有意地和最大程度地将ASG的共同部分，即二氨基嘧啶暴露于动物的免疫系统中，以诱导具有针对ASG的广泛特异性的抗体。最终获得了五种单克隆抗体（mAb），带有短间隔臂的半抗原5C4命名为Hapten A，不仅显示出均一的广泛特异性，而且对所有五个ASG均显示出高亲和力。我们从构象和电子方面进一步确定了单克隆抗体的可能识别机制。建立了基于间接竞争ELISA（icELISA）的5C4，其IC 50值为0.067–0.139μgL –1在最佳条件下，缓冲液中的五个ASG的交叉反应率为48.2％–418.7％。鸡肉和牛奶的icELISA检测极限分别为0.06–0.8μgkg –1和0.05–0.6μgL –1。加标的鸡肉和牛奶样品的回收率为75.2％–101.4％，变异系数小于14.3％。总而言之，我们首次开发了一种快速，可靠的用于ASG的icELISA，其灵敏度和类别选择性大大提高。
更新日期：2018-09-25
中文翻译：
鸡肉和牛奶中五种抗菌增效剂的通用半抗原合成，广谱单克隆抗体制备和超灵敏ELISA
具有广泛特异性并且主要取决于半抗原结构和大小的抗体是用于开发类选择性免疫测定法的关键试剂。在本研究中，使用甲氧苄啶为起始分子，提出了三种新的抗菌增效剂（ASG）通用半抗原。这些半抗原在间位上含有羧基在三甲氧基苯的位置结合蛋白质，而有意地和最大程度地将ASG的共同部分，即二氨基嘧啶暴露于动物的免疫系统中，以诱导具有针对ASG的广泛特异性的抗体。最终获得了五种单克隆抗体（mAb），带有短间隔臂的半抗原5C4命名为Hapten A，不仅显示出均一的广泛特异性，而且对所有五个ASG均显示出高亲和力。我们从构象和电子方面进一步确定了单克隆抗体的可能识别机制。建立了基于间接竞争ELISA（icELISA）的5C4，其IC 50值为0.067–0.139μgL –1在最佳条件下，缓冲液中的五个ASG的交叉反应率为48.2％–418.7％。鸡肉和牛奶的icELISA检测极限分别为0.06–0.8μgkg –1和0.05–0.6μgL –1。加标的鸡肉和牛奶样品的回收率为75.2％–101.4％，变异系数小于14.3％。总而言之，我们首次开发了一种快速，可靠的用于ASG的icELISA，其灵敏度和类别选择性大大提高。
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