Preparation Of Monoclonal Antibodies Against The Domain 4 Of Pyolysin And The Establishment Of A Double Antibody Sandwich ELISA For Quantitative Detection Of Pyolysin

Trueperella pyogenes(T.pyogenes)is a Gram-positive,irregular,non-motile,non-spore-form Brevibacterium.It is considered to be the most common opportunistic pathogen bacteria that is widely present on the surface of domestic mucous membranes,it can cause multiple organ suppurative infections in the host.Pyolysin(PLO)is one of the major virulence factors of T.pyogenes.As an exotoxin,it has the biological properties of lysing cells.Similar to other members of the Family of cholesterol-dependent cytolysin(CDCs),the PLO protein molecule has four domains.The role of the domain 4 is to identify and combine cholesterol on the cell membrane.In this study,five monoclonal antibodies(mAbs)against PLO D4 were prepared,four of them(2G3,3C8,3G10,4C9)have strong anti-hemolytic activity,and 3C7 has lower anti-hemolytic activity.Antigen epitope mapping by Western-blot,The results showed that the antigenic epitope against 3C7 was located at amino acid(AA)519-534 of the immature PLO(PLO 519-534AA).The antigenic epitope for 4 mAbs with strong anti-hemolytic activity is located at amino acid 487-502 of the immature PLO(PLO 487-502AA).This epitope contains a complete undecapeptide(PLO 491-501AA),it was confirmed that the undecapeptide has an important effect on the pore-forming activity of PLO.Western blot experiments showed that the above 5 mAbs could all react with PLO obtained by crude purification of T.pyogenes cultures.It shows that 5 mAbs can be used for the detection of wild type PLO(wtPLO).Since the titers of 2G3 ELISA reaction was significantly higher than that of the other 4 mAbs,then,we used 2G3 as a capture antibody to established a sandwich ELISA method and optimized with the HRP-labeled anti-PLO monoclonal antibody(HRP-AP-1A3)that has been stored in our laboratory.According to this method,PLO content of T.pyogenes HLJ-0912 and T.pyogenes HLJ-1005 strains secreted in culture medium was detected in different culture times.The PLO content in cultures was detected when the strain HLJ-0912 was grown to 10-14 h.In summary,five mAbs were successfully prepared in this study,and proved that the mAb against undecapeptide can effectively inhibit the hemolytic activity of PLO.At the same time,finished the preliminary application of these mAbs prepared in this study,quantification of wtPLO secreted by T.pyogenes HLJ-0912 strain using sandwich technology ELISA.Provides the basis for the virulence analysis of T.pyogenes.