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Construction of an immunized rabbit phage display antibody library for screening microcystin-LR high sensitive single-chain antibody

Author:
Xu, Chongxin, Miao, Wenjie, He, Yan, Zu, Yao, Liu, Xiaoqin, Li, Jianhong, Liu, Xianjin
Source:
International journal of biological macromolecules 2019 v.123 pp. 369-378
ISSN:
0141-8130
Subject:
DNA libraries, Escherichia coli, agricultural products, bacteriophages, cross reaction, enzyme-linked immunosorbent assay, microcystin-LR, monitoring, polyclonal antibodies, rabbits, screening
Abstract:
Microcystin-LR (MC-LR) is one of the most common biotoxin that pollutes water and agricultural products. The study aims to obtain the high sensitive anti-MC-LR single-chain antibody (scFv) for detecting MC-LR. Here, a MC-LR-immunized rabbit phage display scFv library with its capacity of 3.26×109CFU/mL was constructed and used for anti-MC-LR phage scFv particles screening. After four rounds of biopanning, 18 positives were isolated and identified successfully. The most positive scFv (RscFv3) was expressed in Escherichia coli HB2151 and purified with a concentration of 796.7μg/mL, and the purified anti-MC-LR polyclonal antibodies (PAbs) were 3.6mg/mL. The PAbs and scFv based indirect competitive enzyme linked immunosorbent assay (IC-ELISAs) were established for MC-LR and its analogues, and the results showed they all had high sensitive for MC-LR with detection limits of 0.03 and 0.05μg/L, and had strong cross-reactivity for MC-RR, MC-WR and MC-YR, respectively. The average recovery rate was 91.9% with coefficient of variation <6.8% for scFv-based IC-ELISA to detect MC-LR spiked in water samples, which met the requirement of indoor testing. The present results indicate that we have obtained a high sensitive anti-MC-LR scFv by the MC-LR-immunized phage library construction and screening, and the scFv-based IC-ELISA can be used for monitoring MC-LR in water samples.
Agid:
6226849