Identification of a Novel Haemophilus parasuis-Specific B Cell Epitope Using Monoclonal Antibody against the OppA Protein

Mitchell Ho,Nan Zheng,Xi Li,Zheng Chai,Fucheng Jiang,Zhuo Wang,Fang Fu,Xiangling Wang,Xueyun Zhang

doi:10.1371/journal.pone.0084516

Mitchell Ho, Nan Zheng + Show 7 more

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https://doi.org/10.1371/journal.pone.0084516

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Abstract

Monoclonal antibody (MAb) 1B3 against Haemophilus parasuis (H. parasuis) was generated by fusing SP2/0 murine myeloma cells and spleen cells from BALB/c mice immunized with the whole-bacterial-cell suspension of H. parasuis HS80 (serotype 5). The MAb 1B3 showed strong reactivity with 15 serotype reference strains of H. parasuis using Dot blot and Western blot analysis. Immunoprecipitation and protein spectral analysis indicated that MAb 1B3 recognized by Oligopeptide permease A (OppA) belongs to the ATP binding cassette transporter family. In addition, a linear B-cell epitope recognized by MAb 1B3 was identified by the screening of a phage-displayed 12-mer random peptide library. Sequence analysis showed that MAb 1B3 was recognized by phages-displaying peptides with the consensus motif KTPSEXR (X means variable amino acids). Its amino acid sequence matched 469KTPAEAR475 of H. parasuis OppA protein. A series of progressively truncated peptides were synthesized to define the minimal region that was required for MAb 1B3 binding. The epitope was highly conserved in OppA protein sequences from the isolated H. parasuis strains, which was confirmed by alignment analysis. Furthermore, the minimal linear epitope was highly specific among 75 different bacterial strains as shown in sequence alignments. These results indicated MAb 1B3 might be potentially used to develop serological diagnostic tools for H. parasuis.

Highlights

Haemophilus parasuis (H. parasuis) is a Gram-negative, nonhemolytic, NAD-dependent bacterium belonging to the Pasteurellaceae family
The hybridoma 1B3 was generated by the fusion of murine myeloma SP2/0 cells with splenocytes from mice immunized with H. parasuis
The other bacteria such as S. enterica, S. aureus, enterotoxigenic Escherichia coli (ETEC) and A. pleuropneumoniae were used as the negative controls

Summary

Introduction

Haemophilus parasuis (H. parasuis) is a Gram-negative, nonhemolytic, NAD-dependent bacterium belonging to the Pasteurellaceae family. This bacterium is the causative agent of Glasser’s disease. H. parasuis has become an important pathogen in the pig industry all over the world [1,2]. The identification of H. parasuis has traditionally been accomplished by culture isolation and biochemical analysis [3]. Antibodies with a high affinity and specificity for bacterial protein could be used to detect the pathogens by immunological methods. With such high quality antibodies in conjunction with the advent of new technologies, cultural enrichment may be necessary for the detection

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