A Preliminary Study On The Origin And Protective Properties Of Nontreponemal Antibodies

Background Nontreponemal antibodies have been widely utilized as the diagnostic markers in clinical practice,however,the origin of these antibodies and their protective properties in the process of Treponema Pallidum(Tp)are still unknown.Aims To investigate the origin of nontreponemal antibodies by conducting a series of animal experiments;To identify the location of cardiolipin antigen in membrane;To screen potential proteins that combine with cardiolipin antigen;To evaluate the protective properties of nontreponemal antibodies.Methods(1)Rabbits were administered with active Tp and inactive Tp respectively,and treponemal and nontreponemal antibodies in serum were tested.To be more detailed,the inactivation of Tp includes formalin treatment,heat treatment and ultrasonication.Rabbits with cardiolipin antigen injection were served as controls;(2)Rabbits were administered Tp which has been subjected to different treatments to damage the outer membrane,and treponemal and nontreponemal antibodies in serum were tested.The treatments placed to Tp include Triton X-100,repeated centrifugation,and sodium citrate.Rabbits administered with heat treated Tp were served as controls;(3)Ion Mobility Mass Spectrometry(IMMS)was employed to screen the proteins that can be combined with cardiolipin antigen;(4)After the induction of the secretion of nontreponemal antibodies in rabbits,Rabbits was challenged with Tp and to evaluate the impact of nontreponemal antibodies in Tp infection process and protective properties.Results(1)Inactive Tp can induce rabbit to secret nontreponemal antibodies in low titer,by contrast,active Tp can result in the high and steady production of nontreponemal antibodies in a shorter time(P<0.001);Cardiolipin antigen alone cannot lead to the production of anticardiolipin antibody(ACA),but combined with BAS protein,cardiolipin antigen can induce the production of ACA.(2)In the attempt of removing outer membrane of Tp by using TritonX-100 and repeated centrifugation,it was found that Tp still has some parts of outer membrane after above-mentioned treatments,and low titers of nontreponemal antibodies can be observed in those immunized rabbits.Sodium citrate,however,is capable of removing outer membrane completely,and rabbits cannot produce nontreponemal antibodies after injection of sodium citrate treated Tp.(3)In total,23 potential proteins that can be combined with cardiolipin antigen were identified by using IMMS,most of them were ATP-binding cassette proteins,GTP-binding proteins and microtubule associated proteins.(4)Rabbits with high production of nontreponemal antibodies by immunization were administered with Tp,and the intensity of inflammatory infiltration in skin lesions was elevated and the advent of ulcer was delayed(P<0.01).However,no significant differences were observed in the blood Tp DNA load and lymph node infection experiment when comparing with controls(P>0.05).Conclusions(1)ACA was found in Tp,which can induce host to produce nontreponemal antibodies.Cardiolipin antigen can only present immunogenicity if combined with proteins;The high production of nontreponemal antibodies after Tp infection can be attribute to the joint work of cardiolipin antigen from Tp and ACA from host,the latter one may have the principle role.(2)Cardiolipin antigen is located in the outer membrane of Tp.(3)23 potential proteins that can combine with cardiolipin were identified by using IMMS.(4)Nontreponemal antibodies can delay the advent of ulcer to some extent during Tp infection,however,they do not have protective properties.In brief,the source of nontreponemal antibodies and its protective properties during Tp infection were elucidated through this study,and it is of great significance for correctly understanding and applying the clinical value of nontreponemal antibodies.