[Google Scholar] 22. ELISA commercial kit experienced a sensitivity of 97% and specificity of 95%. Conclusion: Our data show that rEgAgB8/2 is an appropriate source of antigen for the serological diagnosis of human hydatid cyst. Co-expression of the rEgAgB/2 along with other subunits of AgB may enhance the performances of these antigens for the serodiagnosis of human CE. have been evaluated in different serodiagnosis assays of human hydatid cyst[8,13]. Among them, antigen 5 (Ag5) and antigen B (AgB) are frequently used antigens. AgB, known as the main protein responsible for metabolic adaptation INH6 and survival of the parasite, is the prominent antigen of hydatid cyst fluid. The antigen is usually a lipoprotein of 120-160 kDa, consisted of a group of about 8-kDa subunits. Under reducing conditions in SDS-PAGE, it dissociates into 8, 16, and 24 kDa subunits[17,18]. The 8-kDa subunit, the smallest one, has offered the best overall performance in the diagnosis of CE and has been extensively used in synthetic, recombinant, or native forms in different serological assays[10,12,14,19-21]. Molecular studies have exhibited that AgB is usually encoded by a multigene family that is variably expressed, with at least five major gene clusters named EgAgB1-5[18]. The putative protein isoforms encoded by the five EgAgB genes differ 44C81% in amino acid sequence[18]. A comparison between the AgB8/1 and AgB8/2 nucleotide sequences has showed 53.5% identity among exons and 50% identity between introns of these two INH6 subunits[22]. Accordingly, the immunodiagnostic performances of different subunits of AgB (EgAgB8/1-5) seem to be different. Formerly, it has been demonstrated that this recombinant EgAgB8/2 (rEgAgB8/2) has a higher diagnostic value in comparison with the rEgAgB8/1 or native AgB for the INH6 serodiagnosis of human CE[22]. In our previous study, we successfully cloned and expressed the EgAgB8/1 subunit of AgB and evaluated its diagnostic efficacy in CE. A sensitivity and specificity of 93% and 92% was found for the Serpinf2 EgAgB8/1 subunit[14]. In the current study, we focused on other subunits of the antigen where the EgAgB8/2 was expressed in host, and its overall performance for serological diagnosis of human CE was evaluated using an ELISA system. MATERIALS AND METHODS Construction, optimization, and cloning of AgB8/2 Construction, optimization, and cloning of rEgAgB8/2 were performed as previously explained with some modifications[14]. Briefly, the DNA sequence of AgB8/2 was extracted from NCBI GenBank database with the accession quantity of “type”:”entrez-nucleotide”,”attrs”:”text”:”DQ835667.1″,”term_id”:”111052781″,”term_text”:”DQ835667.1″DQ835667.1. The target sequence was optimized based on codon usage and the optimized gene fragment was synthesized (Biomatik, Ontario, Canada) and cloned in pBluescript II SK (+) vector. The fragment was subsequently excised by DH5. The recombinant plasmid was extracted using QIAprep Spin Miniprep Kit (Qiagen, Hilden, Germany). The extracted plasmid was transformed into BL21 (DE3)pLysS cells for expression under the T7 promoter. The expression of AgB8/2 was induced by 0.5 mM IPTG (Invitrogen, USA) at 37 C for four hours. Protein purification The cells were harvested by centrifugation (1800 g at 4 C for 10 min), and the resultant pellet was re-suspended in a lysis buffer (Triton-X100, PBS [pH 8], and 0.5 M EDTA) and sonicated (5 30 s) on ice. The resultant cell lysate was centrifuged at 15,000 g at 4 C for 30 min and incubated at -20 C overnight. After that, the obvious supernatant was collected, and the recombinant protein was purified by affinity chromatography, based on its glutathione S-transferase (GST)-tag, using immunoaffinity column (Sigma-Aldrich, Germany). The bound protein was eluted with elution buffer (reduced glutathione, 50 mM Tris, pH 8). Finally, desalination of purified protein was done using a dialysis membrane in PBS (pH 7.5)[14]. SDS-PAGE and Western blotting SDS-PAGE of the recombinant and the native protein (native AgB purified from hydatid cyst fluids as explained previously)[21] were carried out on 18% (w/v) polyacrylamide gel made up of 0.1% SDS as documented before[14]. Patients and control sera Hydatid cyst patients sera were obtained from 30 pathologically-confirmed CE patients. In addition, 32 samples of nonce patients, including subjects affected by giardiasis (n = 3), hymenolepiasis (n = 3), toxocariasis (n = 3), toxoplasmosis (n = 3), fascioliasis (n = 3), malaria (n = 2), cryptosporidiosis (n = 2), trichostrongyliasis (n = 1), patients with autoimmune diseases (n = 9; including three with vitiligo, three with Beh?ets disease, and three with systemic lupus erythematous) as well as patients with FUO (fever of unknown origin;.