Production and application of polyclonal and monoclonal antibodies against Spiroplasma eriocheiris.

Ying Zhang,Haixun Bao,Wen Wang,Wei Gu,Yaqin Peng,Qingguo Meng,Fengqin Miao,Yuqing Shen,Jianqiong Zhang

doi:10.1038/srep17871

Ying Zhang, Haixun Bao + Show 7 more

Open Access

https://doi.org/10.1038/srep17871

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Abstract

A new species of spiroplasma, Spiroplasma eriocheiris (S. eriocheiris), was identified as a lethal pathogen of tremor disease (TD) in Chinese mitten crab recently. In order to acquire appropriate biological and diagnostic tools for characterizing this newly discovered pathogen, 5 monoclonal antibodies (mAbs) and a polyclonal antibody (pAb) against S. eriocheiris were produced. Among the mAbs, 6F5, 7C8 and 12H5 lead to the deformation of S. eriocheiris. A peptide sequence, YMRDMQSGLPRY was identified as a mimic motif of MreB that is the cell shape determining protein of S. eriocheiris interacting with 3 mAbs. Furthermore, a double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) for detection of S. eriocheiris was established using the mAb and pAb we prepared. It detected as low as 0.1 μg/mL of S. eriocheiris. No cross-reaction was observed with three other common bacteria (Pseudomonas aeruginosa, Escherichia coli, and Bacillus subtilis) and the hemolymph samples of healthy Eriocheir sinensis. Collectively, our results indicated that the mAbs and pAb we prepared could be used in the analysis of S. eriocheiris membrane proteins mimotope and development of a diagnostic kit for S. eriocheiris infections.

Highlights

ELISA using pAb prepared for the rapid detection of S. eriocheiris was developed, but it is time-consuming and the sensitivity and specificity needs to be improved[8]
The cells that have strong ELISA reactivities with S. eriocheiris were subsequently subjected to cloning procedures
A polyclonal antibody against S. eriocheiris was developed soon after the spiroplasma was identified. It was used in the indirect ELISA to detect S. eriocheiris in crab hemolymph[8]

Summary

Introduction

ELISA using pAb prepared for the rapid detection of S. eriocheiris was developed, but it is time-consuming and the sensitivity and specificity needs to be improved[8]. The main objective of our study was to generate and characterize more mAbs and pAb against S. eriocheiris. These antibodies were used to analyse mimotopes of its membrane proteins and to develop DAS-ELISA, enhancing the ability to detect and confirm suspected cases of S. eriocheiris infection. This in turn may reduce TD mortality and direct strategies for controlling infection

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