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Enzyme-linked immunosorbent assay (ELISA) was used to confirm the proteomics results. Interleukin-25 (IL-25) level was measured with an IL-25 ELISA kit according to the instructions (BioSource, Nivelles, Belgium). Optical density at 450?nm was measured using a spectrophotometer. All samples were assayed in duplicates. Related results were presented as the mean?��?standard deviation (SD) from three independent experiments. Mean values were statistically compared using ANOVA tests or Student��s t-test. Statistical significance was defined as p?[http://www.selleckchem.com/products/Staurosporine.html see more] a comparative proteomic analysis of cell-free sputum from TB patients and healthy controls was performed. Pooling of the samples facilitated the initial identification of differentially regulated proteins in the patients with active pulmonary tuberculosis and healthy controls. Pooling also reduced the experimental variations in the data and minimized the data files subjected to computer-intensive comparative analysis. Many published research findings were from pooled samples [8,11]. Our pretest data indicated that the profiles for the pooled samples could generally mirror the [http://www.selleckchem.com/products/gsk126.html GSK126] protein profiles of individuals. So, in our study, pooled samples were used for proteomics analysis as described in other studies [12,13]. Representative 2D gel images are shown in Fig.?(a,b). A mean of 900 protein spots per gel were recognized and the volume of all spots was evaluated. Of 185 differentially expressed protein spots, 150 were decreased and 35 were increased in the TB sample compared with the controls. Compared with the controls, levels of proteins in the TB sample were increased on average by 2.2- to 6-fold, but decreased on average by 2.1- to 100-fold. All differentially expressed proteins are summarized in Table?. The result of matrix-assisted laser desorption/ionization-time [http://en.wikipedia.org/wiki/Transducin Transducin] of flight/time of flight-mass spectrometry MALDI-TOF/TOF-MS analysis of IL-25 is shown in Fig.? as an example. Out of the 62 proteins, 15 were up-regulated, whereas 47 were down-regulated in the TB samples compared with the controls. Among the 62 identified proteins, 55 are from humans and seven are from bacteria, respectively. Because proteomics results were derived from the pooled sputum samples, to validate our proteomics results, ELISA analysis was further performed for the detection of individual samples from all the cases. To confirm the proteomic results, IL-25 was selected and subjected to analysis. The data showed that IL-25 had an increased abundance in the TB group compared with the controls (Fig.?). The result was consistent with the proteomic data.