Mapping Of Epitopes Of Mycoplsama Hyopneumoniae P97 C-terminal Protein And Initial Study Of Antigen Capture ELISA For Mycoplsama Hyopneumoniae

Immunobloting assay was made with P97 C-Terminal protein expressed in 10 partially overlaping or consecutive fragments and the monoclonal antibody(A3C9 and B4D5), to map primary epitopes of the C-Terminal protein. In order to further study the epitopes structure of P97 C-Terminal protein, Pepscan was used to exactly identify the minimum epitopes of the C-Terminal protein. Finally, two minimum epitopes F905PMAFSY911 (905～911aa)and G991TPNQGKKAE1000 (991～1 000aa)were identified. Compared with different Mhp strains, the epitopes were tatally homologous in them, so they are conservative species-specific epitopes. The results of growth inhibition assay showed that two strains of monoclonal hybridoma cells which could stably secrete specific antibody(A3C9 and B4D5)were able to inhibit the growth of M.hyopneumoniae cells in vitro, especially B4D5 antibody. The assay also showed that Both of peptides of the two epitopes have better reactionogenicity and immunogenicity ,to be candidate peptides of epitopes vaccine.Also, An antigen capture enzyme-linked immunosorbent assay(AC-ELISA)to detect Mycoplasma hyopneumoniae antigen has been developed. Its means features included a HRP-labeling MAb AF11 and a capture MAb A3C9(A3C9 and AF11 reacted with different peptides of epitopes).The optimal working condition was listed as below, The capture MAb was diluted to 1:800(0.559μg/well), the Mhp standard antigen was diluted to 1:16 and the HRP-labeled AF11 was diluted to 1:400(1.178μg/well), the reaction time for HRP-labled MAb and capture MAb were all 60mins in the assay. The pH 9.6(0.05 mol/L)Na2CO3-NaHCO3 buffer and 5% gelatin from cold water fish skin were separately used as coating solution and blocking buffer. The result of the specific test demonstrated the two MAbs had better specificity for Mhp J strain. In the assay there were no significant cross-reactivity with other species(Y-goat, PG2, PG1, 27399, 87001 and BST-7).CVs of intra-plate and inter-plate was lower than 10%. Our test was equally sensitive to Mhp and had an immunologic sensitivity of 6.4×104 colony-forming units per milliliter. In comparison with culture-amplified antigen detection assays, the AC-ELISA has no significant difference(χ2 =3,P>0.05). The coincidence of AC-ELISA with culture-amplified antigen detection assays and PCR were separatly 50% and 75%.The study is not only important for further understanding the structure and function of Mhp P97 C-Terminal protein, but also useful for designing the diagnostic reagent and vaccine. It is a valuable clinical significance that the antigen capture ELISA for detection of Mycoplasma hyopneumoniae .