Production of monoclonal antibodies with broad specificity and development of an immunoassay for microcystins and nodularin in water
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- 作者：Huijuan Yang ; Rui Dai ; Huiyan Zhang ; Chenglong Li…
- 刊名：Analytical and Bioanalytical Chemistry
- 出版年：2016
- 出版时间：September 2016
- 年：2016
- 卷：408
- 期：22
- 页码：6037-6044
- 全文大小：1,180 KB
- 刊物类别：Chemistry and Materials Science
- 刊物主题：Chemistry
Analytical Chemistry
Food Science
Inorganic Chemistry
Physical Chemistry
Monitoring, Environmental Analysis and Environmental Ecotoxicology - 出版者：Springer Berlin / Heidelberg
- ISSN：1618-2650
- 卷排序：408
文摘
Microcystins (MCs) and nodularin (NOD) are cyanobacterial hepatotoxins that can greatly harm human health. Multi-analyte immunoassays provide efficient and cheap methods of screening these toxins. To develop a multi-analyte immunoassay, an antibody with both broad specificity and high affinity for structurally similar algal toxins is urgently needed. In this study, microcystin–leucine–arginine (MC-LR) and NOD were conjugated to carrier proteins using a one-step active ester (AE) method and multistep thiol-ene click chemistry and glutaraldehyde method, respectively. The immunogens obtained from these two conjugation methods were evaluated for their effectiveness in producing antibodies. The results demonstrated that the antisera derived from AE immunogens showed better performance in terms of affinity and titer. Using this simple AE method, we prepared a new immunogen for NOD and successfully produced a monoclonal antibody (mAb), 2G5, which could recognize not only NOD but also all eight of the tested MCs (MC-LR, MC-RR, MC-YR, MC-WR, MC-LA, MC-LF, MC-LY, and MC-LW) with high sensitivity and improved uniform affinities (0.23 ≤ IC50 ≤ 0.68 ng mL−1) compared with previously described mAbs. Under optimal conditions, one indirect competitive enzyme-linked immunosorbent assay was developed based on mAb2G5 for the detection of MC-LR and NOD, with limits of detection of 0.16 and 0.10 μg L−1, respectively, and a recovery of 62–86 % with a coefficient of variation below 12.6 % in water samples.KeywordsMicrocystinsNodularinMonoclonal antibodyBroad specificityConjugation methodsIcELISA