title: [Preparation and characterization of reference samples of Mycobacterium tuberculosis culture filtrate protein-10 for time-resolved fluoroimmunoassay].
abstract: OBJECTIVE
  To prepare reference samples of Mycobacterium tuberculosis culture filtrate protein-10 (CFP-10) and CFP10-streptavidin fusion proteins (CFP10/SA) for time-resolved fluoroimmunoassay (TRFIA).
  
  
  METHODS
  The CFP10 gene was amplified by PCR from Mycobacterium tuberculosis strain H37Rv and cloned into pET24b, pET24b-streptavidin (SA) or pET21a-SA expression vectors. The recombinant proteins CFP10, CFP10-SA and SA-CFP10 were expressed in Rosetta cells, purified via nickel affinity chromatography and refolded by dialysis. The sensitivity and stability of the resultant proteins as reference samples were evaluated by double-antibody sandwich TRFIA.
  
  
  RESULTS
  CFP10-SA and SA-CFP10 fusion proteins were expressed as inclusion bodies, whereas CFP10 was expressed in a soluble form. The resultant purity of the 3 recombinant proteins all exceeded 95%. TRFIA results showed that CFP-SA fusion protein possessed the best sensitivity (0.02 µg/L) and stability.
  
  
  CONCLUSIONS
  The reference samples of CFP10 for TRFIA detection have been successfully prepared and can be used in the development of a diagnostic kit for Mycobacterium tuberculosis.
authors.affiliation: School of Biotechnology, Southern Medical University, Guangzhou 510515, China. guo.fang86@163.com
authors.forename: Fang-fang
authors.initials: FF
authors.lastname: Guo
authors.forename: Li-lin
authors.initials: LL
authors.lastname: Zou
authors.forename: Ying-song
authors.initials: YS
authors.lastname: Wu
authors.forename: Zhi-ming
authors.initials: ZM
authors.lastname: Hu
authors.forename: Jin-long
authors.initials: JL
authors.lastname: Li
authors.forename: Jian-xin
authors.initials: JX
authors.lastname: Lv
authors.forename: Ji-min
authors.initials: JM
authors.lastname: Gao
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics / Q000592:standards*; D015200:Fluoroimmunoassay / Q000379:methods*; D005784:Gene Amplification; D009169:Mycobacterium tuberculosis / Q000302:isolation & purification*; D012015:Reference Standards
chemical_list: D001426:Bacterial Proteins; C117940:CFP-10 protein, Mycobacterium tuberculosis