Dataset Information

Novel sandwich immunoassay detects a shrimp AHPND-causing binary PirAB^Vp toxin produced by Vibrio parahaemolyticus.

ABSTRACT:

Introduction

The binary PirA/PirB toxin expressed by Vibrio parahaemolyticus (PirAB^Vp) is a virulent complex that causes acute hepatopancreatic necrosis disease (AHPND) in shrimps, affecting the global shrimp farming industry. AHPND is currently diagnosed by detecting pirA and pirB genes by PCR; however, several V. parahaemolyticus strains do not produce the two toxins as proteins. Thus, an immunoassay using antibodies may be the most effective tool for detecting toxin molecules. In this study, we report a sandwich ELISA-based immunoassay for the detection of PirAB^Vp.

Methods

We utilized a single-chain variable fragment (scFv) antibody library to select scFvs against the PirA or PirB subunits. Phage display panning rounds were conducted to screen and identify scFv antibodies directed against each recombinant toxin subunit. Selected scFvs were converted into IgGs to develop a sandwich immunoassay to detect recombinant and bacterial PirAB^Vp.

Results

Antibodies produced as IgG forms showed sub-nanomolar to nanomolar affinities (K_D), and a pair of anti-PirA antibody as a capture and anti-PirB antibody as a detector showed a limit of detection of 201.7 ng/mL for recombinant PirAB^Vp. The developed immunoassay detected PirAB^Vp in the protein lysates of AHPND-causing V. parahaemolyticus (Vp^AHPND) and showed a significant detectability in moribund or dead shrimp infected with a Vp^AHPND virulent strain compared to that in non-infected shrimp.

Discussion

These results indicate that the developed immunoassay is a reliable method for diagnosing AHPND by detecting PirAB^Vp at the protein level and could be further utilized to accurately determine the virulence of extant or newly identified Vp^AHPND in the global shrimp culture industry.

SUBMITTER: Jeon MY

PROVIDER: S-EPMC10711049 | biostudies-literature | 2023

REPOSITORIES: biostudies-literature

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Novel sandwich immunoassay detects a shrimp AHPND-causing binary PirABVp toxin produced by Vibrio parahaemolyticus.

Jeon Min-Young MY Han Jee Eun JE Lee Dong Gwang DG Cho Young-Lai YL Jang Ju-Hong JH Lee Jangwook J Park Jong-Gil JG Kwon Do Hyung DH Park Seon Young SY Kim Wantae W Lee Kyunglee K Kim Ji Hyung JH Lee Nam-Kyung NK

Frontiers in cellular and infection microbiology 20231127

<h4>Introduction</h4>The binary PirA/PirB toxin expressed by Vibrio parahaemolyticus (PirABVp) is a virulent complex that causes acute hepatopancreatic necrosis disease (AHPND) in shrimps, affecting the global shrimp farming industry. AHPND is currently diagnosed by detecting pirA and pirB genes by PCR; however, several V. parahaemolyticus strains do not produce the two toxins as proteins. Thus, an immunoassay using antibodies may be the most effective tool ...[more]

PMID: 38089817

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