Evidence obtained with monoclonal antibodies that O antigen is the major antigen responsible for the cross-reactivities between serotypes 4 and 7 of Actinobacillus (Haemophilus) pleuropneumoniae

Abstract

Monoclonal antibodies (MAbs) against Actinobacillus (Haemophilus) pleuropneumoniae serotype 4 (reference strain M62 and field isolate F6) were produced and characterized. Three hybridoma clones were raised against strain M62, and 13 were raised against strain F6. The predominant antibody class was immunoglobulin M (IgM), although IgG2a and IgG2b were also obtained. Three of the MAbs produced to field isolate F6 (5C5, 1E10, and 5H7) did not recognize the reference strain of serotype 4, another (6F7) was reactive with both reference strains of serotypes 4 and 7, and the remaining 12 MAbs reacted only with the reference strain of the homologous serotype. All epitopes recognized by MAbs, except for one (6F7), were sensitive to periodic acid oxidation, and all of them were resistant to boiling in the presence of sodium dodecyl sulfate and reducing conditions, as evidenced by immunodot. Enhanced chemioluminiscence-immunoblot assays revealed that 10 MAbs (3E12, 5B8, 7C3, 6F7, 7F5, 7E6, 5G4, 4F1, 7E10, and 4B8) recognized a ladder-like banding pattern, which is in accordance with the O side chain antigen of lipopolysaccharide (LPS), while the remaining 6 MAbs (5C5, 5H7, 1E10, 6D11, 6B4, and 5E4) blotted with high-molecular-weight regions composed of a single banding pattern. The suitability of MAbs for serotyping of 78 field isolates was also examined. A high correlation was found between the results previously established by indirect hemagglutination with polyclonal sera and those obtained by enzyme-linked immunosorbent assay with MAbs. According to the different immunoreactivity of MAbs, three groups were established: group I (MAbs 3E12, 5B8, 7C3, 6F7, and 7F5), group II (MAbs 7E6, 5G4, 4F1, 7E10, and 4B8), and group III (MAbs 5C5, 5H7, and 1E10). MAbs 6D11, 6B4, and 5E4 could not be included in any of the described above. At least six different immunodominant epitopes on the O antigen of the A. pleuropneumoniae serotype 4 LPS were identified. Finally, the implications of the effect of the O antigen of LPS in cross-reactions between serotypes 4 and 7 are clearly evidenced.