title: A new ELISA method for serological diagnosis of Legionella pneumophila: use of five purified proteins, FLA, MOMP, MIP, IP, and PILE, as diagnostic antigen.
abstract: BACKGROUND
  Legionella pneumophila plays an important role in human infection. Commercial ELISA kits commonly used, which take Legionella pneumophila whole-cell protein as the coating antigen, often have cross-reactivity among serogroups or species. In this study, five Legionella pneumophila proteins FLA, MOMP, MIP, IP, and PILE were purified and further applied in serological diagnosis of Legionella pneumophila infections compared with R & D Legionella ELISA kits.
  
  
  METHODS
  The five recombinant plasmids pET-fla, pET-momp, pET-mip, pET-ip, and pET-pile were transformed into E. coli BL21 and then induced them with IPTG. The expression products were analyzed by SDS-PAGE and purified by affinity chromatography. Indirect enzyme-linked immunosorbent assays (ELISAs) were established with the five purified proteins FLA, MOMP, MIP, IP, and PILE altogether as the coating antigen and tested for the presence of IgG, IgM, and IgA antibody independently from 50 positive sera and 40 negative sera, compared with R & D IgG, IgM, and IgA Legionella ELISA kits.
  
  
  RESULTS
  The FLA protein about 42 kDa in size, the MOMP protein about 45 kDa, the MIP protein about 40 kDa, the IP protein about 46 kDa, and the PILE protein about 35.7 kDa were separately expressed and purified. Compared with R & D IgG, IgM, and IgA Legionella ELISA kit, the outcome of indirect ELISAs set up with the five purified proteins showed that for IgG the sensitivity was 90.4%, the specificity was 97.4%, the area under ROC curve was 0.939, the kappa value was 0.865, the 95% confidence interval was 0.883 - 0.995. For IgM the sensitivity was 91.8%, the specificity was 95.1%, the area under ROC curve was 0.935, the kappa value was 0.866, the 95% confidence interval was 0.876 - 0.994. For IgA the sensitivity was 93.6%, the specificity was 95.3%, the area under ROC curve was 0.945, the kappa value was 0.889, the 95% confidence interval was 0.890 - 0.999.
  
  
  CONCLUSIONS
  The proteins FLA, MOMP, MIP, IP, and PILE were successfully expressed and purified, and they seemed to be suitable coating antigens for the serological diagnosis of Legionella pneumophila.
authors.forename: Hui
authors.initials: H
authors.lastname: Sun
authors.forename: Haifeng
authors.initials: H
authors.lastname: Ma
authors.forename: Li
authors.initials: L
authors.lastname: Liu
authors.forename: Xiuqin
authors.initials: X
authors.lastname: Cao
authors.forename: Zhiwei
authors.initials: Z
authors.lastname: Yang
mesh_terms: D000907:Antibodies, Bacterial / Q000276:immunology*; D000942:Antigens, Bacterial / Q000276:immunology; D001426:Bacterial Proteins / Q000097:blood* / Q000276:immunology; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D006801:Humans; D007070:Immunoglobulin A / Q000276:immunology; D007074:Immunoglobulin G / Q000276:immunology; D007075:Immunoglobulin M / Q000276:immunology; D016952:Legionella pneumophila; D007877:Legionnaires' Disease / Q000097:blood* / Q000175:diagnosis*; D019696:Peptidylprolyl Isomerase / Q000276:immunology; D010957:Plasmids / Q000378:metabolism; D018272:Porins / Q000276:immunology; D012372:ROC Curve; D011994:Recombinant Proteins / Q000276:immunology; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
chemical_list: D000907:Antibodies, Bacterial; D000942:Antigens, Bacterial; D001426:Bacterial Proteins; C000591932:FLA protein, Legionella pneumophila; D007070:Immunoglobulin A; D007074:Immunoglobulin G; D007075:Immunoglobulin M; C000591933:PILE protein, Legionella pneumophila; D018272:Porins; D011994:Recombinant Proteins; C072453:major outer membrane protein, Legionella pneumophila; C512922:Mip protein, Legionella pneumophila; D019696:Peptidylprolyl Isomerase