Streptavidin gold was trapped by the biotin in the control line. was 94% when the kit was tested using known TB positive sloth CGB bear sera samples. 47.4% of the in-contact sloth bears turned seropositive using the rapid POC TB diagnostic kit. Seropositivity in other wild animals was 25% when the sera samples were tested using the kit. A point of care TB sero-diagnostic kit with the combination of proteins was developed and the kit was validated using the sera samples of wild animals. Electronic supplementary material The online version of this article (10.1007/s12088-017-0688-7) contains supplementary material, which is available to authorized users. complex. Wild animals can mount an antibody response to environmental Mycobacterium resulting in false positivity in TB diagnosis [27]. The present study was aimed at developing a Rapid TB antibody detection kit using recombinant fusion protein of ESAT-6::CFP-10 along with purified protein derivatives (PPDs) of for the improved diagnostic sensitivity and also with PPDs of for assessing the diagnostic specificity. To the best of our knowledge, this is the first report on the development and evaluation of a point of care kit using the above combination of antigens for the diagnosis of TB. Materials and Methods maslinic acid Immunochemicals and Reagents Purified protein derivatives of (BoPPD) and sub species (AvPPD) were procured from Prionics AG (Wagistrasse, Schlieren-Zurich, Switzerland). Wild animal sera samples were sourced from various zoos, rescue centers and temple elephants around India. Bovine positive and negative reference sera were from Animal and Plant Health Agency (APHA), UK. Cloning and Expression of Recombinant ESAT-6::CFP-10 Fusion Protein Coding sequence of ESAT-6 and CFP-10 of was synthesized as fusion gene construct (GenScript, USA). The gene construct was cloned into prokaryotic expression maslinic acid vector pET28a (Novagen) and the plasmid clone was used to transform chemically competent BL21 DE3 cells (Invitrogen). The protein expression was induced in the clone using 1?mM IPTG for overnight at 25?C. The HIS6 tagged ESAT-6::CFP10 fusion protein was purified from the soluble fraction of the bacterial lysate using NiCNTA agarose (immobilized metal affinity chromatography). Briefly, a 5?ml NiCNTA agarose column was equilibrated with 10 column volumes of tris buffered saline (TBS) and the soluble fraction of the bacterial lysate was passed through the column and the column was washed with 20 column volumes of TBS containing 50?mM imidazole and the recombinant protein was eluted using 500?mM imidazole. The pooled protein fractions were dialyzed against PBS (pH 7.4) and purity of the protein was assessed using SDS-PAGE. The protein was identified in a western blot using anti-His antibody. LPS Removal from the Purified TB Antigens LPS from recombinant fusion protein ESAT-6::CFP-10 was removed using Triton X100 as per the procedure [22]. Briefly, Triton X ?100 was added to the protein sample to a final concentration of 1% (v/v) and incubated at 4?C for 1?h with continuous mixing. The sample was centrifuged at 7500?rpm for 10?min at 30?C and the upper miceller phase was collected without disturbing the LPS rich middle and lower phase. Triton X ?100 was added again to the upper phase to a final concentration of 0.5% (v/v) and the remaining steps were repeated as mentioned above. maslinic acid Then, the recombinant protein was analysed in SDS-PAGE and western blot. Characterization of Recombinant Protein The ESAT-6::CFP-10 fusion protein was characterized using reference culture positive and culture negative sera samples in immuno-blot. Identity of the protein was further established by LCCMS/MS analysis of the trypsin-treated protein sample. Lateral Flow Assay (LFA) Design.