Preparation Of Mycobacterium Tuberculosis MPT64 Monoclonal Antibody And Its Preliminary Application

Background and ObjectTuberculosis（TB）caused by Mycobacterium tuberculosis（Mtb）infection is a widespread chronic infectious disease.MPT64 is one of the secreted antigens of Mtb and is also the Mtb virulence factor.MPT64 regulates host immune response and induces protective effect against Mtb infection.Therefore,MPT64 is one of the candidate antigens for TB diagnostic and vaccine research.In this study,the purpose of this study was to prepare anti-MPT64 monoclonal antibody（mAb）and establish a double-antibody sandwich ELISA.Based on the above mAbs,the regulatory effect of MPT64 on host innate immunity and the role of MPT64 in host anti-Mtb infection were investigated.This study provides a tool for the rapid diagnosis of MPT64 in TB,and clarifies the mechanism of action of MPT64 in host anti-Mtb infection.Methods and Results1.Preparation of Mycobacterium tuberculosis MPT64 monoclonal antibodyProkaryotic expression of MPT64 recombinant protein and affinity chromatography purification,subcutaneous immunized mice,splenocytes were fused with hybridoma cells,and 14 mAbs were obtained by initial screening and monoclonalization.ELISA and Western blot identification showed that each mAb could specifically recognize the recombinant MPT64 protein,the isoform was Ig G,and the titer was 1:80～1:2 560.Coupled ELISA screening obtained C8H9,F5E2 and H2F4 paired antibodies with A5B2strain mAbs,respectively.Only H2F4 and A5B2 strains of mAb ascites were successfully prepared,and purified by affinity chromatography.The mAb potencies of the purified H2F4 and A5B2 strains were 1:102 400 and 1:51 200,respectively,and the results of SDS-PAGE and Western blot showed that the two mAbs had high purity and could specifically identify the natural MPT64 in Mtb bacteria,and did not have non-specific reactions with BCG,Mycobacterium smegmatis and Staphylococcus aureus.2.Establishment of MPT64 double antibody sandwich ELISABiotin-labeled A5B2 mAb detection antibody was prepared,H2F4 mAb was used as the capture antibody of MPT64 double antibody sandwich ELISA,and the concentration of capture antibody and detection antibody was optimized by checkerboard titration to1.25μg/m L and 2.5μg/m L,respectively.Taking the conventional conditions as the initial reaction conditions,the reaction conditions of each link were optimized in turn,and the optimal conditions for the system were as follows:CBS（p H 9.6）coated with H2F4 mAb（1.25μg/m L）100μL,allowed to stand overnight at 4℃,washed;2%BSA closed at room temperature for 1 h,washed;The samples to be measured were incubated at room temperature for 2 h and washed;Incubate 100μL of Biotin-A5B2 mAb（2.5μg/m L）at room temperature for 2 h and wash;Avidin-HRP（0.5μg/m L）was incubated at room temperature for 1 h and washed;TMB chromogenesis,2 M H₂SO₄ termination,microplate reader detection OD₄₅₀.The key parameters of the established ELISA system were analyzed,and the results showed that the lower limit of detection of MPT64 was 39 ng/m L,the linear range was39～1 250 ng/m L（R²=0.982 3）,the coefficient of variation was less than 9.5%,the recovery rate was 89.177～104.390%,and the specificity of MPT64 reaction was good,and there was no cross-reaction with BCG,unrelated bacteria,and media components.Further detection of the supernatant cultured at different growth stages of Mtb H37Ra strain showed that the MPT64 content was proportional to the number of Mtb viable bacteria,and it was detected positive at the earliest 12th day of culture at a concentration of 4.09 ng/m L.3.Regulation of host innate immunity by MPT64 and its role in anti-Mtb infectionMice were injected with high and low doses of MPT64 mAb through the tail vein and intraperitoneal cavity,and then infected with Mtb H37Ra nasal drops to detect the number of organs and the content of MPT64 antibodies in mice.The results showed that at least 2 w of mAbs could exist for intravenous and intraperitoneal high-dose injections,and the mAbs injected at low intraperitoneal doses had been metabolically cleared.The results showed that there was no significant difference in the number of bacteria between the treatment groups,and the number of lung bacteria in the low-dose intravenous mAb group increased slightly,and the number of lung bacteria in the high-dose intravenous mAb group decreased.Anti-MPT64 mAb pretreated bone marrow-derived macrophages（BMDM）and then infected with Mtb H37Ra,and found that mAb pretreatment 6 h earlier did not affect the intracellular survival of Mtb,and 0 h earlier pretreatment of mAb promoted intracellular survival of Mtb.MPT64 protein stimulated BMDM and MH-S macrophages in vitro,and real-time quantitative PCR assays showed that the transcription levels of pro-inflammatory factors IL-1β,IL-6,TNF-α,i NOS and key proteins NLRP3,ASC and Caspase-1 were up-regulated.In addition,the results of BMDM cell infection model showed that the inflammatory response induced by MPT64significantly inhibited the intracellular viability of Mtb infection in the early stage（1～3days）,and the effect disappeared in NLRP3^-/-BMDM,indicating that MPT64 promoted the clearance of Mtb infection by the host and was related to NLRP3 activation.ConclusionsIn this study,14 anti-MPT64 mAbs were prepared,paired antibodies were obtained,and an MPT64 double-antibody sandwich ELISA detection system was established,which was used to quantitatively analyze MPT64 in Mtb culture medium to assist in the analysis of Mtb content.It was preliminarily confirmed that MPT64 could induce the activation of NLRP3 inflammasomes and promote the clearance effect of macrophages on Mtb infection,but this effect was not obvious in mice.