Improved sensitivity of the anti-microcystin-LR ELISA using phage-displayed alpha-type anti-idiotypic nanobody

Shu Liu; Manman Lin; Xiaodan Hu; Cheng Shen; Xiao Zhang; Chongxin Xu; Qin Zhu; Yajing Xie; Haiyan Lu; Yun Wang; Peng Lü; Ofentse Jacob Pooe; Yuan Liu; Aidong Sun; Xianjin Liu

doi:10.1016/j.ab.2022.115030

Improved sensitivity of the anti-microcystin-LR ELISA using phage-displayed alpha-type anti-idiotypic nanobody

Anal Biochem. 2023 Mar 1:664:115030. doi: 10.1016/j.ab.2022.115030. Epub 2022 Dec 23.

Authors

Shu Liu¹, Manman Lin², Xiaodan Hu², Cheng Shen², Xiao Zhang², Chongxin Xu², Qin Zhu², Yajing Xie², Haiyan Lu², Yun Wang³, Peng Lü⁴, Ofentse Jacob Pooe⁵, Yuan Liu⁶, Aidong Sun⁷, Xianjin Liu⁸

Affiliations

¹ Key Laboratory of Food Quality and Safety of Jiangsu Province, Nanjing, 210014, China; School of Food and Biological Engineering, Jiangsu University, Zhenjiang, 212013, China.
² Key Laboratory of Food Quality and Safety of Jiangsu Province, Nanjing, 210014, China.
³ School of Food and Biological Engineering, Jiangsu University, Zhenjiang, 212013, China.
⁴ School of Life Sciences, Jiangsu University, Zhenjiang, 212013, China.
⁵ School of Life Sciences, University of KwaZulu-Natal, Durban, 4000, South Africa.
⁶ Key Laboratory of Food Quality and Safety of Jiangsu Province, Nanjing, 210014, China; School of Food and Biological Engineering, Jiangsu University, Zhenjiang, 212013, China. Electronic address: liuyuan@jaas.ac.cn.
⁷ Key Laboratory of Food Quality and Safety of Jiangsu Province, Nanjing, 210014, China. Electronic address: idong.sun@qq.com.
⁸ Key Laboratory of Food Quality and Safety of Jiangsu Province, Nanjing, 210014, China; School of Food and Biological Engineering, Jiangsu University, Zhenjiang, 212013, China. Electronic address: jaasliu@jaas.ac.cn.

PMID: 36572217
DOI: 10.1016/j.ab.2022.115030

Abstract

Anti-idiotypic antibodies (Ab2) are valuable tools that can be used for a better understanding of molecular mimicry and the immunological network. In this work, we showed a new application of a phage-displayed alpha-type Ab2 (Ab2α) to improve the sensitivity of an enzyme-linked immunosorbent assay (ELISA) detecting cyanobacterial toxin microcystin-LR (MC-LR). A monoclonal antibody (mAb) against MC-LR was used as an antigen to isolate binders in a camelid nanobody library. After three rounds of panning, three unique clones with strong binding against anti-MC-LR mAbs were isolated. These clones could specifically bind to anti-MC-LR mAbs without influencing mAbs binding with MC-LR, meaning these clones were Ab2αs. Based on the signal amplification effect of phage coat proteins and the non-competitive nature of Ab2α, a novel competitive ELISA method for MC-LR was established with a phage-displayed Ab2α. It showed that the phage-displayed Ab2α greatly enhanced the ELISA signal and sensitivity of the method was improved 3.5-fold to the conventional one. Combining with the optimization of pre-incubation time, the optimized ELISA decreased its limit of detection (LOD) from 4.5 ng/mL to 0.8 ng/mL (5.6-fold improvement). This new application of Ab2α may potentially be employed to improve the sensitivity of immunoassays for other environmental pollutants.

Keywords: Anti-idiotypic antibody; Enzyme-linked immunosorbent assay; Microcystin-LR; Nanobody; Sensitivity.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Antibodies, Monoclonal
Bacteriophages*
Enzyme-Linked Immunosorbent Assay / methods
Immunoassay
Peptide Library*

Substances

Peptide Library
Antibodies, Monoclonal