Wuchereria bancrofti protein WbSXP-1 was identified and established as a potential candidate for the diagnosis of lymphatic filariasis. For the economic production of rWbSXP-1, osmotically (salt) inducible Escherichia coli GJ1158 was preferred. Cultivation and expression was optimized in 3 L airlift bioreactor (ALB) and was successfully extended to 30 L ALB. Purification of rWbSXP-1 his-tag protein was optimized in technical scale using FPLC and the maximal recovery of rWbSXP-1 with significant level of purity was achieved using the combination of IMAC and gel filtration. Quality criteria for immuno-reactivity of purified rWbSXP-1 were established for diagnostic applications. Enhancement of sensitivity in rapid diagnostic format was optimized to effectively detect weak to strong antibody reactivity in individuals exposed to lymphatic filariasis. Performance of the rapid format during field evaluation was successful. The accelerated stability assessment of the rapid format satisfied the requirements of WHO-cGMP norms. This investigation presents a successful technical scale production and purification of rWbSXP-1 considering the future industrial application and an enhanced rapid flow through antibody assay for the diagnosis of human lymphatic filariasis.
Prior studies have demonstrated that transglutaminase (TGase) from the human filarial parasite Brugia malayi is critical for the growth and development of the larval stages. In this report, we describe the cloning and partial characterization of a cDNA encoding the B. malayi TGase (BmTGase). Using RT-PCR and RACE-PCR, the cDNA was amplified from adult worm mRNA. BmTGase is 1,881 bp long and codes for a protein with a predicted molecular mass of 54 kDa. Amino acid sequence analysis of BmTGase revealed significant homology to the protein disulfide isomerase (PDI), particularly, to the PDI-related protein ERp60, a PDI isoform found in the lumen of endoplasmic reticulum. The activity of recombinant B. malayi TGase enzyme (rBmTG) was found to be calcium-dependent and could be inhibited by EDTA. ELISA studies showed that approximately 88% of 48 sera from healthy Indian patients living in a bancroftian filariasis endemic area were reactive with rBmTG. In contrast, only 33% of sera from patients with clinical filariasis were reactive to rBmTG. Non-endemic sera were uniformly non-reactive. Additional studies are needed to elucidate the role, if any, of B. malayi TGase in protective immunity to filariasis.
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