The calculation of cut-off values as well as the interpretation of the results of the Ani Labsystems kits were performed according to the manufacturer’s instructions. == Authors’ contributions == HN performed experiments, analysed the data, and wrote the manuscript. logistic regression analysis. We showed that combination of rAtpD and rP1-C discriminated maximally between the patients infected withM. pneumoniae(children and adults) and the Rabbit polyclonal to USP37 healthy subjects for the IgM class, performing better than the single recombinant antigens or the commercial whole-cell extract. == Conclusion == These results suggest that AtpD can be used as an antigen for the immunodiagnosis of early and acuteM. pneumoniaeinfection in association with adhesin P1, providing an excellent starting point for the development of point-of-care diagnostic assays. == Background == Mycoplasma pneumoniaeis a cell wall-less bacterium belonging to theMollicutesclass, which invades the human host respiratory epithelium by adhering with a tip-like attachment organelle. Several proteins, including the major surface adhesins P1, P30, P116 and proteins HMW1 to HMW3, as well as proteins A, B and C, interact to constitute this tip-like attachment organelle [1-5].M. pneumoniaecauses atypical pneumonia and other respiratory tract infections (RTIs) such as tracheobronchitis, and is responsible for up to 20% of all cases of LY341495 community-acquired pneumonia, especially among school-aged children and young adults [6,7]. M. pneumoniaeis intrinsically resistant to beta-lactams antibiotics usually given as the first-line treatment of RTIs. Macrolides and related antibiotics represent the treatment of choice forM. pneumoniaerespiratory infections. Consequently, an early and specific diagnosis is necessary to give the patient the correct antibiotic treatment. Serology, including the complement fixation test (CFT) and different enzyme-linked immunosorbent assays (ELISA), is the most common laboratory method utilized for the diagnosis ofM. pneumoniaeinfection although culture methods and PCR are also performed. The CFT may have limited value because it also steps antibodies derived from earlier infections and antibodies toM. pneumoniaeglycolipid antigens; thus, it can react with antigens of different origins [7]. Previous studies comparing the CFT to the PCR detection ofM. pneumoniae, however found good sensitivity and specificity for the CFT [8,9]. Many ELISA-based assays, using protein extracts, membrane preparations, glycolipid extracts or whole cell lysates have been developed for the detection ofM. pneumoniaeinfection [8]. In particular, good sensitivity has been observed for assays with P1 adhesin-enriched extracts [8,10,11]. In a study by Beersmaet al.[8], 12 commercial serologic assays forM. pneumoniaespecific immunoglobulins M and LY341495 G and the CFT were evaluated with PCR used as the “gold standard”. The IgM assay that showed the best sensitivity and specificity were from your Ani Labsystems (77% and 92%, respectively) corresponding to P1-enriched extracts. Other studies have reported the superiority of assays using the P1-enriched antigen on the basis of a comparison of the strength of the immune response [9,11,12]. Nonetheless in the same studies, high IgG seroprevalence has been observed in the control sera ranging from 36% (Virotech assay) to 93% (Ani Labsystems assay). The variability of LY341495 the ELISA results observed in these studies suggests the need for improved sensitivity and specificity among commercialised serological assays used to detectM. pneumoniaeinfection [8]. Recently, many studies have reported great desire for using a recombinant protein corresponding to the C-terminal portion of the P1 adhesin, which has been described as the immunodominant antigen inM. pneumoniae[2,13-17]. Antigenic properties of recombinant proteins P116 and P30 have also been shown [15,18,19]. A combination of frequently acknowledged antigens could be useful for diagnostic purposes. Thus, the identification of antigenicM. pneumoniaeRTI-related proteins appears to be a prerequisite for the development of serological test kits based on recombinant antigens. In this study, we used serologic proteome analysis ofM. pneumoniaeM129 total extracts to simultaneously identify candidate antigens inducing an antibody response [20]. We focused on the ATP synthase beta subunit (AtpD) ofM. pneumoniaeas it was likely to generate an antibody response inM. pneumoniae-infected children and adults at an early stage of contamination. TheatpDgene (mpn598) contains an open reading frame of 1 1,428 nucleotides and encodes a protein of 475 amino acids, with a calculated molecular weight of 52,486 Da [21-23]. It was cloned and expressed inE. colito obtain recombinant protein. We then compared.