title: Novel monoclonal antibodies to ESAT-6 and CFP-10 antigens for ELISA-based diagnosis of pleural tuberculosis.
abstract: OBJECTIVE
  To elucidate the potential of monoclonal antibodies (mAbs) of culture filtrate protein 10 (CFP-10) and early secretory antigenic target 6 (ESAT-6) in tuberculosis (TB) diagnosis.
  
  
  METHODS
  We generated and characterised monoclonal and polyclonal antibodies against Mycobacterium tuberculosis-specific antigens ESAT-6 and CFP-10 by immunising BALB/c mice with an ESAT-6/CFP-10 fusion protein. Stable hybridoma cell lines were established and mAbs were specifically identified by immunoblotting and immunoprecipitation. The mouse mAbs were used to coat plates, and biotin-labelled polyclonal antibodies were used to detect the antigens. One hundred and seventy-three samples of sputum culture supernatants and pleural effusion aspirates have been tested.
  
  
  RESULTS
  The ESAT-6 enzyme-linked immunosorbent assay (ELISA) detected the culture supernatants and pleural effusion specimens that were positive for M. tuberculosis, but failed to identify M. tuberculosis-positive specimens in the non-M. tuberculosis culture supernatants or control specimens. This yielded a sensitivity of 95.4% and a specificity of 100% for the ESAT-6-specific ELISA. The CFP-10 ELISA presented less satisfactory sensitivity and specificity, of respectively 81.6% and 92.2%. Results showed positive detection rates of ESAT-6 and CFP-10 of 86.8% (33/38) and 76.3% (29/38) for the diagnosis of tuberculous pleural effusion in patients bacteriologically negative for M. tuberculosis culture.
  
  
  CONCLUSIONS
  The ESAT-6 and CFP-10 ELISAs incorporating mAbs generated in this study serve as potential tools in the laboratory diagnosis of TB.
authors.affiliation: State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, Zhejiang, China.
authors.forename: T-T
authors.initials: TT
authors.lastname: Feng
authors.forename: C-M
authors.initials: CM
authors.lastname: Shou
authors.forename: L
authors.initials: L
authors.lastname: Shen
authors.forename: Y
authors.initials: Y
authors.lastname: Qian
authors.forename: Z-G
authors.initials: ZG
authors.lastname: Wu
authors.forename: J
authors.initials: J
authors.lastname: Fan
authors.forename: Y-Z
authors.initials: YZ
authors.lastname: Zhang
authors.forename: Y-W
authors.initials: YW
authors.lastname: Tang
authors.forename: N-P
authors.initials: NP
authors.lastname: Wu
authors.forename: H-Z
authors.initials: HZ
authors.lastname: Lu
authors.forename: H-P
authors.initials: HP
authors.lastname: Yao
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal; D000942:Antigens, Bacterial / Q000276:immunology*; D001426:Bacterial Proteins / Q000276:immunology*; D017931:DNA Primers; D030561:Databases, Nucleic Acid; D004195:Disease Models, Animal; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D006801:Humans; D015151:Immunoblotting; D047468:Immunoprecipitation; D051379:Mice; D008807:Mice, Inbred BALB C; D009169:Mycobacterium tuberculosis / Q000276:immunology* / Q000302:isolation & purification; D012680:Sensitivity and Specificity; D013183:Sputum / Q000382:microbiology; D014373:Tuberculin / Q000276:immunology; D014396:Tuberculosis, Pleural / Q000175:diagnosis*
chemical_list: D000911:Antibodies, Monoclonal; D000942:Antigens, Bacterial; D001426:Bacterial Proteins; C117940:CFP-10 protein, Mycobacterium tuberculosis; D017931:DNA Primers; C093276:ESAT-6 protein, Mycobacterium tuberculosis; D014373:Tuberculin