School of Agricultural Engineering and Food Science, Shandong University of Technology, Zibo 255049, China.
Shandong Lvdu Biotechnology Co., Ltd., Binzhou 256600, China.
Biosensors (Basel). 2022 Jul 24;12(8):560. doi: 10.3390/bios12080560.
To produce a sensitive monoclonal antibody (mAb) for the simultaneous detection of carbofuran, benfuracarb, carbosulfan and 3-hydroxy-carbofuran, 2,3-dihydro-2,2-dimethyl-7-benzofuranmethanamine (DDB) was conjugated to bovine serum albumin (BSA) to prepare the immunogen DDB-BSA and mice were immunized. Coating antigens were prepared by conjugating DDB and 5-methoxy-2,3-dihydrobenzofuran-3-acetic acid (MDA) to BSA and ovalbumin (OVA), respectively. Furthermore, the effect of different antibody-antigen pairs on the sensitivity of ELISA and LFIA methods for the detection of carbofuran was investigated. After the immunization, a high-affinity mAb 13C8 was obtained. The ability of the coating antigen to compete with carbofuran for binding antibodies was found to be significantly different between ELISA and LFIA methods. With the antibody-antigen pair 13C8-MDA-OVA, the IC values of the ELISA and QD-LFIA methods for carbofuran were 0.18 ng/mL and 0.67 ng/mL, respectively. The cross-reactivity (CR) values of the two methods for benfuracarb, carbosulfan and 3-hydroxy-carbofuran ranged from 72.0% to 83.7%, while, for other carbamate pesticides, the CR values were less than 1%. The spiked recoveries of carbofuran in vegetables by the QD-LFIA method were 83-111%, with a coefficient of variation below 10%, and the test results of the actual samples were consistent with the HPLC-MS method. Overall, this study provides key materials for the development of immunoassays for carbofuran and its analogues, and the antibody-antigen pair selection strategy established in this study provides useful insights for the development of sensitive immunoassays for other compounds.
为了制备能够同时检测克百威、涕灭威、丙硫克百威和 3-羟基克百威的高灵敏度单克隆抗体（mAb），将 2,3-二氢-2,2-二甲基-7-苯并呋喃甲胺（DDB）与牛血清白蛋白（BSA）偶联以制备免疫原 DDB-BSA，并对小鼠进行免疫。分别将 DDB 和 5-甲氧基-2,3-二氢苯并呋喃-3-乙酸（MDA）与 BSA 和卵清蛋白（OVA）偶联以制备包被抗原。此外，还研究了不同抗体-抗原对用于检测克百威的 ELISA 和 LFIA 方法的灵敏度的影响。免疫后，获得了高亲和力的 mAb 13C8。发现 ELISA 和 LFIA 方法中，包被抗原与克百威竞争结合抗体的能力有显著差异。使用抗体-抗原对 13C8-MDA-OVA，ELISA 和 QD-LFIA 方法对克百威的 IC 值分别为 0.18 ng/mL 和 0.67 ng/mL。两种方法对涕灭威、丙硫克百威和 3-羟基克百威的交叉反应（CR）值在 72.0%至 83.7%之间，而对于其他氨基甲酸酯农药，CR 值小于 1%。通过 QD-LFIA 方法在蔬菜中添加克百威的回收率为 83-111%，变异系数低于 10%，实际样品的测试结果与 HPLC-MS 方法一致。总的来说，本研究为克百威及其类似物的免疫分析的发展提供了关键材料，并且本研究中建立的抗体-抗原对选择策略为其他化合物的敏感免疫分析的发展提供了有用的见解。
Zotero 插件
