Preparation and specificity analysis of monoclonal antibody against 6 kD early secretory antigenic target of Mycobacterium tuberculosis
NING Huanhuan1 REN Rui1 KANG Jian1 BAI Lu2 LU Yanzhi1 XIE Yanling2 ZHANG Fanglin1 BAI Yinlan1
1.Department of Microbiology and Pathogen Biology, School of Preclinical Medicine, Air Force Medical University, Shaanxi Province, Xi’an 710032, China; 2.School of Life Sciences, Yan’an University, Shaanxi Province, Yan’an 716000, China
Abstract：Objective To prepare a monoclonal antibody (mAb) against 6 kD early secretory antigenic target (ESAT-6) of Mycobacterium tuberculosis and to analyze its specificity. Methods ESAT-6 was purified by affinity chromatography and immunized mice with the protein. Splenocytes from ESAT-6 immunized mice were fused with myeloma cells, and hybridoma cell lines secreting mAb were screened. mAb was purified from ascites. The subclass and relative affinity of mAb were assayed by enzyme-linked immunosorbent assay. The specificity of the mAb was analyzed by Western blot. Results The hybridoma cell line secreting ESAT-6 antibody was obtained and mAb was purified. The subclass of ESAT-6 mAb was IgG1 with a relative affinity of 3.9×10-5. The mAb specifically recognizes ESAT-6 in Mtb. Conclusion The preparation of mAb against ESAT-6 lays a foundation for the application of ESAT-6 in the diagnosis and treatment of tuberculosis and its mechanism.
[1] Sia JK，Rengarajan J. Immunology of Mycobacterium tuberculosis Infections [J]. Microbiol Spectr，2019，7（4）：10.
[2] Gloria LL，Bastos ML，Santos Júnior BD，et al. A simple protocol for tuberculin skin test reading certification [J]. Cad Saude Publica，2021，37（8）：e00027321.
[3] Abebe F. Immunological basis of early clearance of Mycobacterium tuberculosis infection： the role of natural killer cells [J]. Clin Exp Immunol，2021，204（1）：32-40.
[4] Ahmed A，Rakshit S，Adiga V，et al. A century of BCG： Impact on tuberculosis control and beyond [J]. 2021，301（1）：98-121.
[5] Sathkumara HD，Muruganandah V，Cooper MM，et al. Mucosal delivery of ESX-1-expressing BCG strains provides superior immunity against tuberculosis in murine type 2 diabetes [J]. Proc Natl Acad Sci U S A，2020，117（34）：20848-20859.
[6] Vazquez Reyes S，Ray S，Aguilera J，et al. Development of an In Vitro Membrane Model to Study the Function of EsxAB Heterodimer and Establish the Role of EsxB in Membrane Permeabilizing Activity of Mycobacterium tuberculosis [J]. Pathogens，2020，9（12）：1015.
[7] Hamada Y，Cirillo DM，Matteelli A，et al. Tests for tuberculosis infection： landscape analysis [J]. Eur Respir J，2021， 58（5）：2100167.
[8] Lu Y，Kang J，Ning H，et al. Immunological characteristics of Mycobacterium tuberculosis subunit vaccines immunized through different routes [J]. Microb Pathog，2018，125：84- 92.
[9] 谢燕玲，宁唤唤，梁璇，等.结核分枝杆菌分泌蛋白MPT64单克隆抗体的制备及其特异性[J].中国人兽共患病学报，2022，38（5）：387-393.
[10] Miotto P，Goletti D，Petrone L. Making IGRA testing easier： First performance report of QIAreach QFT for tuberculosis infection diagnosis [J]. Pulmonology，2022，28（1）：4-5.
[11] Pathan AA，Wilkinson KA，Klenerman P，et al. Direct ex vivo analysis of antigen-specific IFN-gamma-secreting CD4T cells in Mycobacterium tuberculosis-infected individuals： associations with clinical disease state and effect of treatment [J]. J Immunol，2001，167（9）：5217-5225.
[12] Dirix V，Dauby N，Hites M，et al. Optimal Detection of Latent Mycobacterium tuberculosis Infection by Combined Heparin-Binding Hemagglutinin（HBHA）and Early Secreted Antigenic Target 6（ESAT-6）Whole-Blood Interferon Gamma Release Assays [J]. J Clin Microbiol，2022，60（5）：e0244321.
[13] Miller MA，Gortazar C，Roos EO，et al. Serological reactivity to MPB83 and CFP10/ESAT-6 antigens in three suid hosts of Mycobacterium bovis infection[J]. Vet Microbiol，2019， 235：285-288.
[14] Wu X，Wang Y，Weng T，et al. Preparation of immunochromatographic strips for rapid detection of early secreted protein ESAT-6 and culture filtrate protein CFP-10 from Mycobacterium tuberculosis [J]. Medicine（Baltimore），2017， 96（51）：e9350.
[15] Diouani MF，Ouerghi O，Refai A，et al. Detection of ESAT-6 by a label free miniature immuno-electrochemical biosensor as a diagnostic tool for tuberculosis [J]. Mater Sci Eng C Mater Biol Appl，2017，74：465-470.
[16] Omar RA，Verma N，Arora PK. Development of ESAT-6 Based Immunosensor for the Detection of Mycobacterium tuberculosis [J]. Front Immunol，2021，12：653853.
[17] Xie J，Mu Z，Yan B，et al. An electrochemical aptasensor for Mycobacterium tuberculosis ESAT-6 antigen detection using bimetallic organic framework [J]. Mikrochim Acta，2021， 188（11）：404.
[18] Poulakis N，Gritzapis AD，Ploussi M，et al. Intracellular ESAT-6： A new biomarker for Mycobacterium tuberculosis infection[J]. Cytometry B Clin Cytom，2016，90（3）：312- 314.
[19] Ning H，Zhang W，Kang J，et al. Subunit Vaccine ESAT-6：c-di-AMP Delivered by Intranasal Route Elicits Immune Responses and Protects Against Mycobacterium tuberculosis Infection [J]. Front Cell Infect Microbiol，2021，11：647220.
[20] Palanisamy GS，Smith EE，Shanley CA，et al. Disseminated disease severity as a measure of virulence of Mycobacterium tuberculosis in the guinea pig model [J]. Tuberculosis（Edinb），2008，88（4）：295-306.
[21] Solans L，Aguiló N，Samper S，et al. A specific polymorphism in Mycobacterium tuberculosis H37Rv causes differential ESAT-6 expression and identifies WhiB6 as a novel ESX-1 component [J]. Infect Immun，2014，82（8）：3446- 3456.
[22] Peng X，Jiang G，Liu W，et al. Characterization of differential pore-forming activities of ESAT-6 proteins from Mycobacterium tuberculosis and Mycobacterium smegmatis [J]. FEBS Lett，2016，590（4）：509-519.
[23] 姚航平，卢洪洲.抗结核杆菌ESAT-6单克隆抗体TBEA8及应用：中国，CN101987872B [P]. 2011-11-09.
[24] 解晓莉.结核分枝杆菌ESAT-6蛋白单克隆抗体的研制与鉴定[D].江苏：扬州大学，2014.
[25] 刘树玲，杨秀旭，李浩，等.结核分枝杆菌ESAT-6抗原鼠单克隆抗体的制备及初步鉴定[J].生物技术通讯，2011，22（2）：258-260.
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