Production of monoclonal antibodies with broad specificity and development of an immunoassay for microcystins and nodularin in water.
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.
Beijing Advanced Innovation Center for Food Nutrition and Human Health, College of Veterinary Medicine, China Agricultural University, No. 2 Yuanmingyuan West Road, Beijing, 100193, China.
Huijuan
H
Yang
Beijing Laboratory for Food Quality and Safety, No. 2 Yuanmingyuan West Road, Beijing, 100193, China.
Rui
R
Dai
Beijing Laboratory for Food Quality and Safety, No. 2 Yuanmingyuan West Road, Beijing, 100193, China.
Huiyan
H
Zhang
Beijing Key Laboratory of Detection Technology for Animal-Derived Food Safety, No. 2 Yuanmingyuan West Road, Beijing, 100193, China.
Chenglong
C
Li
Beijing Key Laboratory of Detection Technology for Animal-Derived Food Safety, No. 2 Yuanmingyuan West Road, Beijing, 100193, China.
Xiya
X
Zhang
Beijing Advanced Innovation Center for Food Nutrition and Human Health, College of Veterinary Medicine, China Agricultural University, No. 2 Yuanmingyuan West Road, Beijing, 100193, China.
Jianzhong
J
Shen
Beijing Key Laboratory of Detection Technology for Animal-Derived Food Safety, No. 2 Yuanmingyuan West Road, Beijing, 100193, China.
Kai
K
Wen
Beijing Advanced Innovation Center for Food Nutrition and Human Health, College of Veterinary Medicine, China Agricultural University, No. 2 Yuanmingyuan West Road, Beijing, 100193, China. wangzhanhui@cau.edu.cn.
Zhanhui
Z
Wang
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D000917:Antibody Formation; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D005260:Female; D018796:Immunoconjugates / Q000737:chemistry / Q000276:immunology; D057230:Limit of Detection; D008807:Mice, Inbred BALB C; D052998:Microcystins / Q000032:analysis* / Q000276:immunology; D010456:Peptides, Cyclic / Q000032:analysis* / Q000276:immunology; D014867:Water / Q000032:analysis*; D014874:Water Pollutants, Chemical / Q000032:analysis* / Q000276:immunology
D000911:Antibodies, Monoclonal; D018796:Immunoconjugates; D052998:Microcystins; D010456:Peptides, Cyclic; D014874:Water Pollutants, Chemical; D014867:Water; C063998:nodularin; C078588:microcystin
Broad specificity; Conjugation methods; IcELISA; Microcystins; Monoclonal antibody; Nodularin