Development of a blocking ELISA for detection of Mycoplasma hyopneumoniae infection based on a monoclonal antibody against protein P65

Abstract

Mycoplasma hyopneumoniae causes porcine enzootic pneumonia, an economically important disease of swine. A more sensitive and reliable method for detection of serum antibodies is needed for epidemiological investigations and to evaluate the effect of immunization. We expressed the M. hyopneumoniae protein P65 in Escherichia coli and produced a monoclonal antibody (mAb) that bound specifically to recombinant P65. Using this mAb, a blocking enzyme linked immunosorbent assay (ELISA) was developed. The blocking ELISA had similar specificity to and sensitivity with the commercial ELISA produced by IDEXX. Thus, this blocking ELISA is a useful test for serological confirmation of M. hyopneumoniae infection.

Fig. 1.

Western blot of recombinant P65, M. hyopneumoniae whole cell proteins, M. hyorhinis whole cell proteins and E. coli containing the empty expression vector probed with the anti-P65 mAb. Lane M, molecular mass standards (Pierce Pre-stained Protein Molecular Weight Markers); lane 1, recombinant P65; lane 2, M. hyopneumoniae whole cell proteins; lane 3, M. hyorhinis whole cell proteins; and lane 4, E. coli containing the pET-32a (+) vector.

Fig. 2.

Receiver-operating characteristic (ROC) curve for anti-P65 blocking ELISA. A. Receiver-operating characteristic (ROC) curve for anti-P65 blocking ELISA. B. The OD₄₅₀ value of positive and negative samples detected by anti-P65 blocking ELISA.

Fig. 3.

Specificity analysis of the blocking-ELISA. PI value of antisera against M. hyopneumoniae, M. hyorhinis, Haemophilus parasuis (HPS), pseudorabies virus (PRV), porcine circovirus type 2 (PCV2), porcine reproductive and respiratory syndrome virus (PRRSV), classical swine fever virus (CSFV) and foot-and-mouth disease virus (FMD) (n=6).