Enzyme-linked immunosorbent assays based on rabbit polyclonal and rat monoclonal antibodies against isoproturon
Enzyme-linked immunosorbent assays based on rabbit polyclonal and rat monoclonal antibodies against isoproturon
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DOI：
10.1021/jf035498d
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发表时间：
2004-05-05
影响因子：
6.1
通讯作者：
Kremmer, E
中科院分区：
文献类型：
--
作者：
Krämer, PM;Goodrow, MH;Kremmer, E
本文描述了兔抗异proturon多克隆血清（pAb）和大鼠抗异proturon单克隆抗体（mAb）的制备和鉴定。开发了包被抗原和酶示踪剂格式。异proturon的标准曲线在40 mM磷酸盐缓冲盐水（PBS）或milliq水中进行。优化后的酶示踪剂（酶示踪剂格式）的检测中点为1.06 +/- 0.34马克杯/L （n = 19，以milliq水为标准），检出限约为0.1马克杯/L。与IOC 7E1单抗相比较的ELISA检测中点分别为0.07 +/- 0.04马克杯/升（n = 7，标准液为milliq水）和0.11 +/- 0.08马克杯/升（n = 33，标准液为40 mM IPBS）。在ml - q水和PBS中的检出限分别为0.003和0.01马克/升。与主要代谢物4-异丙基苯胺、4-异丙基苯基脲和1-（4-异丙基苯基）-3-甲基脲有明显的交叉反应。使用pAb 352时，这些CRs分别为5%、7%和31%；使用mAb IOC 7E1时，这些CRs分别为3%、5%和约19%。所有芳脲类除草剂的CR值都很小，从无CR值（如氯磺隆）到最高3.3%（氯脲隆）不等。评估了有机溶剂（甲醇、乙醇、乙腈和丙酮）的影响。基于pAb和mab的免疫分析显示对甲醇的耐受性最高，可达5%。乙醇和乙腈不能超过2%而不影响测定。丙酮也是如此，尽管仅在单克隆抗体为基础的试验中进行了测试。用这些单抗和单抗elisa对不同来源和基质的水样进行加标和分析。结果表明，这些免疫测定是有用的筛选工具。
This work describes the production and characterization of rabbit polyclonal antisera (pAb) and rat monoclonal antibodies (mAb) against isoproturon. Coating antigen and enzyme-tracer formats were developed. Standard curves for isoproturon were conducted either in 40 mM phosphate buffered saline (PBS) or in Milli-Q water. PAb 352 together with the best enzyme tracer revealed in the optimized ELISA (enzyme tracer format) a test midpoint of 1.06 +/- 0.34 mug/L (n = 19, standard set up in Milli-Q water) with a detection limit of about 0.1 mug/L. The comparable ELISA with mAb IOC 7E1 had test midpoints of 0.07 +/- 0.04 mug/L (n = 7, standards in Milli-Q water) and 0.11 +/- 0.08 mug/L (n = 33; standards in 40 mM IPBS). The limits of detection were about 0.003 and 0.01 mug/L in Milli-Q water and PBS, respectively. Noticeable cross reactivities (CRs) were seen with the major metabolites, namely 4-isopropylaniline, 4-isopropylphenylurea, and 1-(4-isopropylphenyl)-3-methylurea. With pAb 352, these CRs were 5%, 7%, and 31%, respectively, and with mAb IOC 7E1, they were 3%, 5%, and ca. 19%, respectively. All arylurea herbicides had only minor CRs, which ranged from no CR (e.g., chlorosulfuron) to a maximum of 3.3% (chlortoluron). Influences of organic solvents (methanol, ethanol, acetonitrile, and acetone) were evaluated. Both pAb- and mAb-based immunoassays showed the highest tolerance for methanol, up to 5%. Ethanol and acetonitrile could not be used above 2% without an influence on the assays. The same was true for acetone, although tested only in the mAb-based assay. Water samples of different origins and matrices were spiked and analyzed with these pAb and mAb ELISAs. The results demonstrated that these immunoassays are useful screening tools.