Monoclonal antibody-based fluorescence polarization immunoassay for sulfamethoxypyridazine and sulfachloropyridazine.
Monoclonal antibody-based fluorescence polarization immunoassay for sulfamethoxypyridazine and sulfachloropyridazine.
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DOI：
10.1021/jf070948d
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发表时间：
2007-07
影响因子：
6.1
通讯作者：
Zhanhui Wang;Suxia Zhang;I. S. Nesterenko;S. Eremin;Jianzhong Shen
中科院分区：
文献类型：
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作者：
Zhanhui Wang;Suxia Zhang;I. S. Nesterenko;S. Eremin;Jianzhong Shen
本文制备了一种新的磺胺甲氧哒嗪（SMP）单克隆抗体（Mab），并以此为基础建立了荧光偏振免疫分析法（FPIA）。Mab由用SMP免疫的小鼠产生，SMP通过碳二亚胺活化的酯形成与牛血清白蛋白（BSA）连接，在SMP和BSA之间使用琥珀酸酐间隔分子。合成了黄绿素标记的磺胺氯哒嗪（SCP）和SMP（示踪剂），并通过薄层色谱（TLC）进行了纯化。所建立的荧光偏振免疫分析方法可耐受高达20%的甲醇，在pH 4 ~ 8和较低的盐浓度下均可获得满意的检测灵敏度。抗SMP单克隆抗体与SCP具有较高的交叉反应性。研究了示踪剂结构对测定分析特性和抗原抗体结合常数的影响。缓冲液中SMP和SCP的检测限（LOD）分别为0.7 ng/mL和0.25 ng/mL，而相关磺胺类药物的交叉反应性可忽略不计。通过FPIA分析SMP和SCP强化的牛奶样品显示平均回收率为60%至145%。
In this paper, a new monoclonal antibody (Mab) against sulfamethoxypyridazine (SMP) was produced, and a fluorescence polarization immunoassay (FPIA) based on the produced Mab was developed and optimized for the qualitative screening analysis of SMP. The Mab was raised from mice immunized with SMP linked to bovine serum albumin (BSA) by carbodiimide activated ester formation, using a succinic anhydride spacer molecule between SMP and BSA. Fluorescein labeled sulfachloropyridazine (SCP) and SMP (tracer) were synthesized and purified by thin layer chromatography (TLC). The developed screening FPIA method can tolerate up to 20% methanol, and satisfactory assay sensitivity can be obtained between pH 4 and pH 8 and at lower salt concentration. The anti-SMP Mab exhibited a high cross-reactivity with SCP. The effect of the tracer structure on the analytical characteristic of the determination and on antigen-antibody binding constants was studied. The limits of detection (LOD) were 0.7 ng/mL for SMP and 0.25 ng/mL for SCP in buffer, respectively, whereas negligible cross-reactivities were exhibited by related sulfonamides. Analysis of SMP and SCP-fortified milk samples by the FPIA showed average recoveries from 60 to 145%.