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

Front Cell Infect Microbiol. 2023 Nov 27:13:1294801. doi: 10.3389/fcimb.2023.1294801. eCollection 2023.

Abstract

Introduction: 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 for detecting toxin molecules. In this study, we report a sandwich ELISA-based immunoassay for the detection of PirABVp.

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 PirABVp.

Results: Antibodies produced as IgG forms showed sub-nanomolar to nanomolar affinities (KD), 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 PirABVp. The developed immunoassay detected PirABVp in the protein lysates of AHPND-causing V. parahaemolyticus (VpAHPND) and showed a significant detectability in moribund or dead shrimp infected with a VpAHPND 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 PirABVp at the protein level and could be further utilized to accurately determine the virulence of extant or newly identified VpAHPND in the global shrimp culture industry.

Keywords: PirABVp toxin; acute hepato-pancreatic necrosis disease; sandwich immunoassay; shrimp; vibrio parahaemolyticus.

Publication types

  • Research Support, U.S. Gov't, Non-P.H.S.
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Acute Disease
  • Animals
  • Enzyme-Linked Immunosorbent Assay
  • Necrosis
  • Penaeidae* / microbiology
  • Toxins, Biological*
  • Vibrio parahaemolyticus* / genetics

Substances

  • Toxins, Biological

Grants and funding

The author(s) declare financial support was received for the research, authorship, and/or publication of this article. This research was supported by the Korea Research Institute of Bioscience and Biotechnology (KRIBB) Research Initiative Program (KGM5272323), by the National Research Foundation of Korea (NRF-2020R1I1A2068827) funded by the Ministry of Education, and by the Development of Technology for the Biomaterialization of Marine Fishery Byproducts of the Korea Institute of Marine Science and Technology Promotion (KIMST) funded by the Ministry of Oceans and Fisheries (KIMST-20220128). This research was also supported by the National Institute of Fisheries Science (R2023004) of the Ministry of Oceans and Fisheries.