High sensitive single chain variable fragment screening from a microcystin-LR immunized mouse phage antibody library and its application in immunoassay

Chongxin Xu; Xiaoqin Liu; Yuan Liu; Xiao Zhang; Cunzheng Zhang; Jianhong Li; Xianjin Liu

doi:10.1016/j.talanta.2019.01.064

High sensitive single chain variable fragment screening from a microcystin-LR immunized mouse phage antibody library and its application in immunoassay

Talanta. 2019 May 15:197:397-405. doi: 10.1016/j.talanta.2019.01.064. Epub 2019 Jan 17.

Authors

Chongxin Xu¹, Xiaoqin Liu², Yuan Liu³, Xiao Zhang³, Cunzheng Zhang³, Jianhong Li⁴, Xianjin Liu⁵

Affiliations

¹ Laboratory for Food Quality and Safety-State Key Laboratory Cultivation Base of Ministry of Science and Technology, Institute of Food Safety and Nutrition, Jiangsu Academy of Agricultural Sciences, Nanjing 210014, China; College of Life Sciences, Nanjing Normal University, Nanjing 210023, China.
² Huaihua Vocational and Technical College, Huaihua 418007, China.
³ Laboratory for Food Quality and Safety-State Key Laboratory Cultivation Base of Ministry of Science and Technology, Institute of Food Safety and Nutrition, Jiangsu Academy of Agricultural Sciences, Nanjing 210014, China.
⁴ College of Life Sciences, Nanjing Normal University, Nanjing 210023, China. Electronic address: lijianhong@njnu.edu.cn.
⁵ Laboratory for Food Quality and Safety-State Key Laboratory Cultivation Base of Ministry of Science and Technology, Institute of Food Safety and Nutrition, Jiangsu Academy of Agricultural Sciences, Nanjing 210014, China. Electronic address: jaasliu@jaas.ac.cn.

PMID: 30771953
DOI: 10.1016/j.talanta.2019.01.064

Abstract

Microcystin-LR (MC-LR) is one of common high-toxic biotoxins produced by cyanobacteria in waterbody. A high sensitive and convenient detection method is necessary for monitoring for MC-LR. To establish a high sensitive indirect competitive enzyme-linked immunosorbent assay (IC-ELISA) based on single chain variable fragment (scFv) for detecting MC-LR, 16 positive anti-MC-LR phage scFv particles were screened out from a MC-LR-immunized mouse phage scFv library, which was successfully constructed with the capacity of 8.67 × 10⁷ CFU/mL. The most positive anti-MC-LR phage scFv (MscFv7) was successfully expressed in Escherichia coli (E.coli) HB2151. The molecular weight (M.W.) of expressed protein was about 30 kDa, and the concentration of purified protein was 512.6 μg/mL analyzed by SDS-PAGE and protein quantitative respectively. The IC-ELISA based on MscFv7-scFv for MC-LR shows a half-maximum inhibition (IC₅₀) of 0.471 μg/L and a limit of detection (LOD) of 0.044 μg/L, which is below the maximum residue limit standard (MRLs) of 1.0 μg/L in drinking water. The MscFv7-scFv has a strong cross-recognition for MC-RR and MC-YR with cross-reactivity (CRs) of 93.1% and 85.9%, respectively, but weak for MC-LW with that of 9.7%, even non-recognition for MC-WR, MC-LF and MC-LY. The recovery rates of IC-ELISA to detect MC-LR spiked in different cleanliness of water samples were 81.2-106.3% with CVs of 2.62-10.22% at intra-assay and inter-assay. The results showed that we obtained a high sensitive anti-MC-LR scFv, and the established IC-ELISA based on MscFv7-scFv should be promising for ultrasensitive monitoring MC-LR, MC-RR and MC-YR in water samples.

Keywords: Immunoassay; Microcystin-LR; Phage antibody library; Single chain variable fragment.

MeSH terms

Animals
Antibodies, Monoclonal / chemistry*
Antibodies, Monoclonal / immunology
Bacteriophages / chemistry*
Enzyme-Linked Immunosorbent Assay*
Marine Toxins
Mice
Mice, Inbred BALB C
Microcystins / analysis*
Microcystins / immunology
Peptide Library

Substances

Antibodies, Monoclonal
Marine Toxins
Microcystins
Peptide Library
cyanoginosin LR