Serodiagnosis of tuberculosis requires detection of antibodies against multiple antigens of Mycobacterium tuberculosis, because antibody profiles differ among the patients. Using fusion proteins with epitopes from two or more antigens would facilitate in the detection of multiple antibodies. For subsequent development of serodiagnostic assay, total seven recombinant antigens were expressed in E. coli. These included three native antigens FbpC1, HSP, CHP, and four fusion proteins tn1FbpC1-tnPstS1, tn2FbpC1-tnPstS1, tnHSP-tn1FbpC1 and HSPXFbpC1. Two other native antigens PstS1 and HSPX, produced in our lab by fellow researchers, were also included in this study for comparative purposes. Fusion construct tn2FbpC1-tnPstS1 was produced by linking FbpC1 fragment of 97-111 amino acid residues, comprising of the two epitopes, to the N-terminus of truncated PstS1. Fusion construct tn1FbpC1-tnPstS1 was produced by linking FbpC1 fragment of 34-111 amino acid residues, which comprised of an N-terminal flanking region in addition to the two epitopes, to the N-terminus of truncated PstS1. The truncated 1-99 amino acid peptide from HSP, comprising of its epitope and the flanking N-terminal region, was linked to the FbpC1 fragment of 34-111 amino acid residues. Also, the ORFs of HSPX and FbpC1 were linked in tandem to produce HSPX-FbpC1 fusion protein. Upon induction of E. coli cells harboring the recombinant plasmids, the antigenic proteins were expressed and analyzed by SDS-PAGE. The levels of expression as determined densitometrically using Syngene GeneTools were found to be 39%, 36%, 34%, 35%, 30%, 32% and 36% of the total cell proteins for FbpC1, HSP, CHP, tn1FbpC1-tnPstS1, tn2FbpC1-tnPstS1, tnHSP-tn1FbpC1 and HSPX-FbpC1, respectively. CHP, tn1FbpC1- tnPstS1 and tn2FbpC1-tnPstS1 were expressed in the cells as insoluble inclusion bodies, whereas HSP, tnHSP-tn1FbpC1 and HSPX-FbpC1, was recovered in the soluble fraction of E. coli cell lysate. FbpC1 was insoluble when expressed at 37 ºC, but was recovered in soluble fraction when E. coli cells were grown in M9NG medium at 18 ºC. CHP, inclusion bodies were solubilised with 2M urea, at high pH, whereas, tn1FbpC1-tnPstS1 and tn2FbpC1-tnPstS1 inclusion bodies were solubilised with 8 M urea, and then were refolded through dialysis. All the antigens were purified through Ni-affinity chromatography and anion exchange chromatography. After purification, percentage recoveries of FbpC1, HSP, CHP, tn1FbpC1-tnPstS1, tn2FbpC1-tnPstS1, tnHSPtn1FbpC1 and HSPX-FbpC1 were 25%, 25%, 20%, 30%, 33%, 29% and 35% respectively. Their yields were 17.5, 16.2, 12.24, 18.9, 17.8, 16.7 and 22.68 mg per litre per OD600, respectively. Rabbits were immunized with the purified native antigens and polyclonal antisera was prepared. All the fusion antigens were found to be reactive with the antisera produced against the corresponding antigens. For ELISA, various parameters like coating concentration of each antigen, coating buffer, coating time and temperature, dilution of plasma sample, dilution of secondary antibody, and the TMB reaction time were optimized for each antigen by using the negative and positive control plasma samples. Variations of only one parameter were analyzed at a time, and N/P ratio was calculated for each variation using the formula �/� = %&''( *+,-.//0. 123 45& (&6''478& 92(432: ;:''<='' %&''( *+,-.//0. 123 45& ;2<7478& 92(432: ;:''<='' . The condition which gave smallest value of N/P ratio was selected as optimum. ELISA analysis of the plasma samples of active pulmonary TB patients against FbpC1, PstS1, HSP, HSPX, CHP, tn1FbpC1-tnPstS1, tn2FbpC1-tnPstS1, tnHSP-tn1FbpC1 and HSPX-FbpC1 showed sensitivities of 60%, 36.6%, 34.4%, 31.1%, 12.7%, 60%, 72.2%, 67.7% and 55.5%, respectively. Sensitivity of tn2FbpC1-tnPstS1 (72.2%) was nearly the same as the expected combined value for the two individual antigens (72.7%). However, the sensitivity of tn1FbpC1- tnPstS1, was only 60%. tnHSP-tn1FbpC1 showed 67.7% sensitivity which is slightly less than the expected combined value for the two individual antigens (75%), but still significantly higher than that of each of the individual antigen. Sensitivity of HSPXFbpC1 (55.5%) was quite less than the expected combined sensitivity of the individual HSPX and FbpC1 (72.2%). The area under ROC curve was used to determine the overall ability of the test to discriminate between patients and healthy individuals. ROC curve for individual proteins had the AUC values of 0.9496, 0.8169, 0.9097, 0.7892 and 0.5254 for FbpC1, PstS1, HSP, HSPX and CHP while the AUC values for fusion proteins tn1FbpC1-tnPstS1, tn2FbpC1-tnPstS1, tnHSP-tn1FbpC1 and HSPX-FbpC1 were 0.9467, 0.9585, 0.9092, and 0.9049, respectively. The fusion protein tn2FbpC1-tnPstS1 had the highest AUC value, indicating its superior diagnostic capability. ELISA analysis of the plasma samples of lymphatic TB patients against FbpC1, PstS1, HSP, HSPX, CHP, tn1FbpC1-tnPstS1, tn2FbpC1-tnPstS1, tnHSP-tn1FbpC1 and HSPXFbpC1 showed sensitivities of 38.33%, 16.66%, 13.33%, 11.66%, 6.66%, 40%, 46.66%, 40% and 25%, respectively. ELISA analysis of the plasma samples of pleural TB patients against FbpC1, PstS1, HSP, HSPX, CHP, tn1FbpC1-tnPstS1, tn2FbpC1-tnPstS1, tnHSPtn1FbpC1 and HSPX-FbpC1 showed sensitivities of 40%, 20%, 18%, 18%, 6%, 38%, 48%, 42% and 24%, respectively. Comparative modeling of the fusion antigens was performed through MODELLER using the crystal structures of FbpC1 and PstS1, and predicted structures of HSP and HSPX. Differential antigen–antibody contact regions of the fusion antigens were analyzed through solvent accessibility analysis using CPORT. Data for secondary structure analysis by CD spectrometry was in reasonable agreement with the X-ray crystallographic data of the native proteins and the predicted structure of the fusion proteins. Comparative molecular modeling suggests that the epitopes of the constituent proteins are better exposed in tn2FbpC1-tnPstS1 as compared to those in tn1FbpC1-tnPstS1. Therefore, removal of the N-terminal non-epitopic region of FbpC1 from 34-96 amino acids seems to have unmasked at least some of the epitopes, resulting in greater sensitivity. Molecular modeling of HSPX-FbpC1 fusion molecule indicated that B-cell epitopic region of HSPX had lost its native conformation and formed a random coil. Solvent accessible surface area analysis also showed that the epitopes of FbpC1 in HSPX-FbpC1 seemed to be relatively less accessible. Perhaps these could be the reasons for the decreased sensitivity of HSPX-FbpC1 fusion molecule. The high level of sensitivity of tn2FbpC1-tnPstS1 and tnHSP-tn1FbpC1, not reported before shows that these fusion proteins have great potential for use in serodiagnosis of tuberculosis.