Therefore a rapid test with accurate diagnostic performance is greatly needed for TB diagnosis [11]. A serology blood test, which is easy to perform, low in cost, and easy with testing large amount of samples, represents a promising method for TB diagnosis [12] and has attracted great interests from investigators. perform, low in cost, and easy with testing large amount of samples, represents a promising method for TB diagnosis [12] and has attracted great interests from investigators. In the past few years, encouraging progress has been Rabbit Polyclonal to SFRS7 made in TB serodiagnosis by using specific antigens. A number ofMtbantigens have been investigated for their use in TB diagnosis [12C16] and several promising antigens have been identified [17]. However, Abiraterone (CB-7598) Abiraterone (CB-7598) currently no single antigen is able to achieve sufficiently high sensitivity and specificity simultaneously. The strategy of combining multiple antigens has been shown to significantly enhance diagnostic performance, but none of the combination tests has been reported with satisfactory accuracy for S? TB diagnosis [18, 19]. Consequently, efforts are still desired to identify novel and more effective antigens for TB diagnosis, especially for S? TB cases. TheMtbregions of difference (RD) encode numerous specific antigens and some of them have been extensively studied for TB diagnosis, such as ESAT6 (Rv3875), CFP10 (Rv3874), CFP-21, and MPT-64 [12, 20]. However, they have not been thoroughly investigated as TB-specific antigens. In the present study, our aim was to identify TB-specific antigens and screen combinational antigens with high accuracy for TB diagnosis. We cloned five immunodominant antigens encoded in theMtbRD1 and overexpressed the protein fragments. The IgG levels of the five antigens in different TB populations (S+/C+, S?/+, and S?/C?) were then assessed with indirect ELISA. The accuracy of the tests using the five antigens individually and in combination was evaluated for TB diagnosis. 2. Methods 2.1. Abiraterone (CB-7598) Study Population and Serum Collection The study participants, including 298 active PTB patients and 94 healthy individuals, were selected consecutively from November 2012 to December 2013 in Beijing Chaoyang District Centre for Disease Control and Prevention. The active PTB was diagnosed based on clinical symptoms, including coughing, fever, coughing up of blood, and pulmonary fibrocavity infiltrates on chest radiograph. For the suspected TB cases, sputum smear and culture test was performed as reported previously [21]. Final TB diagnosis was based on the result of sputum smear and culture test as well as symptomatic improvement after anti-TB therapy. No patient was identified with HIV-1 infection. The patients were further divided into three groups: (1) smear-positive for acid-fast bacilli and culture-positive pulmonary TB (S+/C+ group, = 117), (2) smear-negative and culture-positive pulmonary TB (S?/C+ group, = 80), and (3) smear-negative and culture-negative group (S?/C? group, = 101). Healthy controls were recruited from routine checkup population who showed no clinical symptoms of TB with no prior history of TB infection. In addition, TST and chest radiograph were performed on the healthy controls to exclude potential TB infection. Before any anti-TB treatment was given to the patients, 3?mL of fasting peripheral venous blood was drawn and collected from each patient. Within 4?hr of blood collection, the samples were centrifuged at 1,200?g for 10?min at 4C to spin down the blood cells. Then the supernatant was transferred into a new ice cold centrifuge tube and centrifuged as the above. The supernatant was transferred into an RNase/DNase-free tube and stored at ?80C until use. 2.2. Cloning, Expression, and Purification of the Recombinant Antigens The BioSun Version 3.0 software (Developed by the Center of Computational Biology, Beijing Institute of Basic Medical Sciences) was used for B-cell epitope prediction as described in our previous report [17]. The coding sequences of five immunodominant antigens (Rv3871, Rv3874, Rv3875, Rv3876, and Rv3879) were selected and amplified by PCR fromM. tuberculosisH37Rv genomic DNA with specific end nuclease restriction sites (Xho I and Xba I). The gene fragments were then inserted into the prokaryotic expression plasmid pBVIL1 and overexpressed inEscherichia coliHB101 as reported previously [17]. The recombinant proteins were purified by ion exchange and gel filtration, and the protein concentration was determined by the Bradford method (Pierce, Rockford, IL). The purified proteins were aliquoted and stored under ?80C. 2.3. Indirect ELISA Microplates were coated with individual antigens (Rv3871, Rv3874, Rv3875, Rv3876, and Rv3879), respectively, at 5?indicates the least number of antigens defined for positive detection. In clinical practice, a diagnostic test with ~90% or higher specificity is usually required. Thus, we made an effort to further test various combinations of RD1 antigens for TB diagnosis, aiming to minimize the number of antigens used in the test while improving the diagnostic performance, especially test specificity..