Development of a monoclonal antibody for specific detection of Vibrio parahaemolyticus and analysis of its antigen

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Highlights

  • Generated mAbs detected all serogroups of V. parahaemolyticus with no cross-reaction.

  • The antigen of our mAbs was OM lipoprotein includes the species-specific sequence.

  • We developed an IC strip for rapid and specific detection of V. parahaemolyticus.

  • OM lipoprotein (lpp) may be a good target for detection of V. parahaemolyticus.

Abstract

Vibrio parahaemolyticus is a major foodborne pathogen worldwide. Contamination of V. parahaemolyticus in foods must be detected as quickly as possible because raw seafood, a major source of V. parahaemolyticus infection, is shipped immediately after production due to its short expiration date. In this study, we generated monoclonal antibodies (mAbs) against V. parahaemolyticus to develop a rapid and specific detection assay. Obtained mAbs were categorized into four groups according to their specificity. Of the groups, Group 1 (mAb VP7, VP11, and VP24) reacted to O1–O12 of V. parahaemolyticus without cross-reaction with human pathogenic Vibrio spp. (V. alginolyticus, V. cholerae, V. fluvialis, V. furnissii, V. mimicus, and V. vulnificus). We developed an immunochromatographic (IC) strip for the rapid detection of V. parahaemolyticus in the field using VP7 as a membrane-immobilized antibody and VP24 as a colloidal gold-conjugated antibody. The IC strip detected any and all serogroups (O1 to O12) or isolates (clinical, food, and environmental strains) of V. parahaemolyticus, regardless of the presence of virulence factors thermostable direct hemolysin (TDH) or TDH-related hemolysin (TRH). It did not cross-react with any other non-V. parahaemolyticus strains tested. To elucidate the target of the IC strip, we analyzed the antigen recognized by these mAbs. Group 1 mAbs showed two specific bands at molecular masses of approximately 11 and 16 kDa by western blotting analysis. Nano liquid chromatography mass spectrometry (LC-MS)/MS analysis revealed that the candidate antigen recognized by these mAbs was outer membrane (OM) lipoprotein Q87G48. We verified that mAb VP7 detected His-tagged OM lipoprotein synthesized by reconstituted cell-free protein synthesis reagent. Reactivity to an N-terminus deletion form and protease digestion form of the OM lipoprotein showed that the extent of epitope recognized by VP mAbs was 22nd–41st amino acids (AAs) from N-terminus of the OM lipoprotein, with the sequence “22SDDAATANAAKLDEL36.” This region was also confirmed to be a V. parahaemolyticus-specific sequence by comparing putative orthologs of OM lipoprotein among Vibrio spp. The C-terminus deletion form (1st–39th AAs) including the sequence primarily recognized by VP mAbs (22nd–36th AAs) showed poor reactivity, indicating that the sequence after 40 residues of OM lipoprotein is also important for recognition by VP mAbs and VP mAbs recognize a conformational epitope. Bioinformatics research demonstrated that the OM lipoprotein is an ortholog of the lpp protein conserved throughout many bacteria. Lpp is an abundant and constitutively expressed protein and exists on the bacterial surface, suggesting it may be a good target for detection of V. parahaemolyticus.

Introduction

Vibrio parahaemolyticus is a major foodborne pathogen that causes gastroenteritis from consumption of raw or inadequately cooked seafood. V. parahaemolyticus has been the cause of outbreaks and has been globally isolated clinically and environmentally, such as from nations like the United States (Newton et al., 2012), China (Li et al., 2016), and Japan (Hara-Kudo et al., 2012). Major pathogenic factors of V. parahaemolyticus are thermostable direct hemolysin (TDH) and TDH-related hemolysin (TRH) (Shinoda, 2011), but it is difficult to ensure food safety by simply testing for TDH and TRH in food samples. First, most food isolates or environmental strains of V. parahaemolyticus do not possess tdh or trh; 84% of clinical and 1.57% of seafood isolates of V. parahaemolyticus were tdh-positive, and 12% of clinical and 3.66% of seafood isolates were positive for trh gene, indicating that majority of clinical isolates harbor tdh or trh genes (Vongxay et al., 2008). Second, it is difficult to avoid misdetection of samples contaminated with toxin-producing strains because detection of toxic protein requires high sensitivity so that the amounts of toxins produced by a strain depend on its ability and culture conditions. Even if the strain possesses the tdh or trh gene, it does not necessarily expresses these proteins. Finally, TDH- and TRH-negative strains may also possess other virulence factors such as type III secretion systems (TTSSs), which inject bacterial effector proteins into host cells (Park et al., 2004). Therefore, detection of V. parahaemolyticus bacterial cells is recommended over detection of their toxins for food or environmental samples. In Japan, the official test method for food samples require the detection of total V. parahaemolyticus rather than only pathogenic strains (Hara-Kudo and Kumagai, 2014). Moreover, V. parahaemolyticus, like coliform, is used as an indicator strain of the sanitary quality of foods. If V. parahaemolyticus is detected in boiled octopus or crab, for instance, this indicates that contamination of V. parahaemolyticus occurred from the environment or cooking equipment such as a cutting board after boiling them.

The major method for detecting V. parahaemolyticus is via culture: enrichment in culture broth, plating on selective agar, and identification of suspected colonies. V. parahaemolyticus are usually isolated on TCBS (thiosulfate citrate bile sucrose) agar. It is difficult to distinguish colonies between V. parahaemolyticus and other sucrose non-fermenting spp. such as V. mimicus and V. vulnificus because they all form green colonies like V. parahaemolyticus. To identify isolated colonies, biochemical tests that take extra time are required. Thus, the culture method is time-consuming, laborious, and inefficient. Nevertheless, contamination of V. parahaemolyticus in foods must be detected as quickly as possible because raw seafood, a major source of V. parahaemolyticus infection, is shipped immediately after production due to its short expiration date. Many rapid detection methods have been developed including conventional polymerase chain reaction (PCR) (Kim et al., 1999), quantitative real-time PCR (Takahashi et al., 2005), loop-mediated isothermal amplification (Nemoto et al., 2011), and immunodot blotting assay (Sakata et al., 2012) (Prompamorn et al., 2013). These methods detect V. parahaemolyticus rapidly, but they all require well trained personnel and expensive equipment such as a thermal cycler that it is impractical for an on-site test.

To facilitate a rapid detection assay with practical on-site testing capabilities, herein, we generated novel monoclonal antibodies (mAbs) for specific detection of V. parahaemolyticus, and then evaluated their characteristics. We developed an immunochromatographic (IC) strip using the anti-V. parahaemolyticus mAbs engineered in this study. Moreover, we analyzed the epitope recognized by our mAbs.

Section snippets

Bacterial strains

Bacterial strains used in this study were obtained from the following sources: American Type Culture Collection (ATCC; Manassas, VA, USA); National Collection of Type Cultures (NCTC; a Culture Collection of Public Health England, London, UK); Research Institute for Microbial Diseases (RIMD; Osaka University, Osaka, Japan); Japan Collection of Microorganisms (JCM; RIKEN BioResource Center, Tsukuba, Japan). Other strains were isolated from the environment and clinical sources.

Preparation of monoclonal antibodies against V. parahaemolyticus

Production of mAbs

Production and characterization of mAb detection for V. parahaemolyticus

Hybridoma clones were obtained by fusing splenocytes from mice immunized with heat-killed V. parahaemolyticus with P3U1 myeloma cells. Out of forty 96-well plates, the clones reacted against V. parahaemolyticus and not other strains of Vibrio spp. as screened by antigen-coated ELISA. Culture supernatants of screened hybridoma were further analyzed the reactivity to 12 serogroups of V. parahaemolyticus by antigen-coated ELISA, and obtained clones were categorized into four groups (Table 2).

Discussion

In this study, we generated V. parahaemolyticus-specific mAbs and developed an IC strip that can produce a test result within 20 min via a simple procedure. This detects any serogroups (O1 to O12) and isolates (clinical, food, and environmental strains) of V. parahaemolyticus, regardless of the presence of tdh or trh. The sensitivity of the IC strip was affected by NaCl concentration in alkaline peptone water, and its sensitivity was highest in 1% NaCl among 1%–3% conditions tested (data not

Acknowledgments

We would like to thank Professor Emeritus Sumio Shinoda, Collaborative Research Center for Infectious Diseases in India, Okayama University, for guidance our work and kindly providing most of the Vibrio spp. strains isolated from environment or clinical used in this study. We also thank Professor Seiichi Kondo and Professor Yasunori Isshiki, School of Pharmaceutical Sciences, Josai University, for technical advice about V. parahaemolyticus treatment.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

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