Development of an indirect ELISA for detection of anti-Mycoplasma hyopneumoniae IgG in naturally infected pathogen-induced convalescent sera

Abstract

Background: Immunization of pigs with an inactivated Mycoplasma hyopneumoniae vaccine (bacterin) generates hyperimmune serum that contains high concentrations of anti-M. hyopneumoniae IgG. Commercially available IgG-ELISA kits cannot distinguish between anti-M. hyopneumoniae IgG in inactivated bacterin-induced hyperimmune sera and convalescent sera resulting from natural M. hyopneumoniae infection. Establishment of an ELISA to detect anti-M. hyopneumoniae IgG in convalescent sera will facilitate the evaluation of the M. hyopneumoniae status of pig farms.

Results: In this study, we expressed and purified recombinant Mhp366-N protein, which contains an epitope recognized by M. hyopneumoniae convalescent sera but not hyperimmune sera, for use as a coating antigen. For the M. hyopneumoniae convalescent serum IgG-ELISA, the optimal antigen concentration, blocking buffer, blocking time, dilution of serum, incubation time with serum, secondary antibody dilution, secondary antibody incubation time and colorimetric reaction time were 0.25 µg/mL, 2.5 % skim milk, 1 h, 1:500, 0.5 h, 1:10,000, 1 h and 15 min, respectively. Validation of the M. hyopneumoniae convalescent serum IgG-ELISA showed a cut-off value of 0.323, the intra-assay CV ranged from 3.27 to 7.26 %, the inter-assay CV ranged from 3.46 to 5.93 %, and the assay was able to differentiate convalescent sera from antibodies to 7 other porcine respiratory pathogens. The convalescent serum IgG-ELISA detected no anti-M. hyopneumoniae IgG in hyperimmune serum samples while a commercial IgG-ELISA identified 95/145 of these sera as positive. The accuracy of the M. hyopneumoniae convalescent serum IgG-ELISA was comparable to the sIgA-ELISA but better than the commercial IgG-ELISA.

Conclusions: The convalescent serum IgG-ELISA is a reproducible, sensitive, and specific indirect ELISA to detect anti-M. hyopneumoniae IgG in naturally infected pathogen-induced convalescent sera. This ELISA could be used to carry out large scale surveillance of M. hyopneumoniae infection in pig farms regardless of vaccination status.

Keywords: Convalescent sera; IgG; Indirect ELISA; Mhp366; Mycoplasma hyopneumoniae.

Fig. 1

Expression and purification of the Mhp366-N protein. SDS-PAGE (a) and Western blotting (b) showing Mhp366-N in recombinant bacteria. Soluble (Lane 2, Fig. 1 a and b) and insoluble forms (Lane 3, Fig. 1 a and b) of Mhp366-N protein were expressed using IPTG induction (Lane 1, Fig. 1 a). No Mhp366-N was detected in E. coli BL21(DE3) containing the pET-28a(+) empty vector (Lane 1, Fig. 1 b). c Mhp366-N protein was purified by Ni affinity chromatography. Lane 1: loading material; Lane 2: flow through; Lane 3–5: affinity purification with a linear imidazole gradient, 0.1 M (Lane 3), 0.2 M (Lane 4), and 0.5 M (Lane 5); Lane 6–9: isolation of target protein

Fig. 2

Optimization of the working conditions of the M. hyopneumoniae convalescent serum IgG-ELISA. Optimal coating antigen concentration was 0.25 µg/mL in coating buffer (a). The optimal blocking buffer was 2.5 % skim milk dissolved in PBS (b), and the optimal incubation time for the blocking step was 1 h (c). The optimal dilution of serum and secondary antibody were 1:500 (d) and 1:10,000 (f) diluted in blocking buffer. The optimal incubation times for serum and secondary antibody were 0.5 h (e) and 1 h (g). The optimal colorimetric reaction time was 15 min (h)

Fig. 3

Specificity and sensitivity of the M. hyopneumoniae convalescent serum IgG-ELISA. a The ELISA detected no cross reactions with sera containing antibodies against 7 porcine respiratory pathogens, including M. hyorhinis (Mhr), A. pleuropneumoniae (App), S. suis serotype 2 (SS2), classical swine fever virus (CSFV), porcine reproductive and respiratory syndrome virus (PRRSV), porcine circovirus type 2 (PCV2) and pseudorabies virus gB protein (gB-PRV). b Five sera gave positive results at dilutions of 1:100, 1:500, 1:1,000 and 1:2,000, 1 serum gave a positive result at a dilution of 1:4,000, and 5 sera gave negative results at dilutions of 1:8,000 or more. Convalescent serum could be diluted up to 2,000 times for this assay