title: Use of recombinant purified protein derivative (PPD) antigens as specific skin test for tuberculosis.
abstract: OBJECTIVE
  Purified protein derivative (PPD) is currently the only available skin test reagent used worldwide for the diagnosis of tuberculosis (TB). The aim of this study was to develop a Mycobacterium tuberculosis specific skin test reagent, without false positive results due to Bacillus Calmette-Guerin (BCG) vaccination using recombinant antigens.
  
  
  METHODS
  Proteins in PPD IC-65 were analyzed by tandem mass spectrometry and compared to proteins in M. tuberculosis culture filtrate; 54 proteins were found in common. Top candidates MPT64, ESAT 6, and CFP 10 were overexpressed in Escherichia coli expression strains and purified as recombinant proteins. To formulate optimal immunodiagnostic PPD cocktails, the antigens were evaluated by skin testing guinea pigs sensitized with M. tuberculosis H37Rv and BCG.
  
  
  RESULTS
  For single antigens and a cocktail mixture of these antigens, best results were obtained using 3 μg/0.1 ml, equivalent to 105 TU (tuberculin units). Each animal was simultaneously tested with PPD IC-65, 2 TU/0.1 ml, as reference. Reactivity of the multi-antigen cocktail was greater than that of any single antigen. The skin test results were between 34.3 and 76.6 per cent the level of reactivity compared to that of the reference when single antigens were tested and 124 per cent the level of reactivity compared to the reference for the multi-antigen cocktail.
  
  
  CONCLUSIONS
  Our results showed that this specific cocktail could represent a potential candidate for a new skin diagnostic test for TB.
authors.affiliation: Mycobacterial Antigens Laboratory & Enzymology & Applied Microbiology Laboratory, National Institute for Research & Development in Microbiology & Immunology, Cantacuzino, Romania. henriette_stavri@hotmail.com
authors.forename: Henriette
authors.initials: H
authors.lastname: Stavri
authors.forename: Nadia
authors.initials: N
authors.lastname: Bucurenci
authors.forename: Irina
authors.initials: I
authors.lastname: Ulea
authors.forename: Adriana
authors.initials: A
authors.lastname: Costache
authors.forename: Loredana
authors.initials: L
authors.lastname: Popa
authors.forename: Mircea Ioan
authors.initials: MI
authors.lastname: Popa
mesh_terms: D000818:Animals; D000942:Antigens, Bacterial / Q000276:immunology*; D006168:Guinea Pigs; D009169:Mycobacterium tuberculosis / Q000276:immunology; D011994:Recombinant Proteins / Q000276:immunology; D014373:Tuberculin / Q000276:immunology*; D014374:Tuberculin Test; D014376:Tuberculosis / Q000175:diagnosis*
chemical_list: D000942:Antigens, Bacterial; D011994:Recombinant Proteins; D014373:Tuberculin