Production and Easy One-Step Purification of Bluetongue Recombinant VP7 from Infected Sf9 Supernatant for an Immunoenzymatic Assay (ELISA).

Bluetongue (BT) is non-contagious, vector-borne viral disease of domestic and wild ruminants, transmitted by midges (Culicoides spp.) and is caused by Bluetongue virus (BTV). BTV is the type species of the Orbivirus genus within the Reoviridae family and possesses a genome consisting of 10 double-stranded RNA segments encoding 7 structural and 4 nonstructural proteins. Viral Protein 7 (VP7) is the major sera group-specific protein and is a good antigen candidate for immunoenzymatic assays for the BT diagnosis. In our work, BTV-2 recombinant VP7 (BTV-2 recVP7), expressed in Spodoptera frugiperda (Sf9) cells using a baculovirus system, was produced and purified by affinity chromatography from the supernatant of infected cell culture. The use of the supernatant allowed us to obtain a high quantity of recombinant protein with high purity level by an easy one-step procedure, rather than the multistep purification from the pellet. RecVP7-BTV2 was detected using a MAb anti-BTV in Western blot and it was used to develop an immunoenzymatic assay.

Development of a rapid lateral flow immunochromatography assay for the detection of group-specific antibodies against VP7 protein of bluetongue virus in multiple species.

Bluetongue virus (BTV), the causative agent of bluetongue disease infects many domestic and wild ruminants. In the present study, colloidal gold nanoparticle-based lateral flow immunochromatography assay (LFIA) was developed to detect the group-specific antibodies to BTV in serum samples of sheep, goats, cattle, and camel. The recombinant VP7 protein of BTV conjugated to colloidal gold nanoparticles (GNPs) was used as a detector reagent. Recombinant streptococcal protein G and monoclonal antibody to BTV group-specific antigen were immobilized as the test and the control line, respectively on a nitrocellulose membrane. The protein G could capture the specific antibodies to BTV present in the serum of multiple ruminant species susceptible to BTV in a common test format and could eliminate the requirement of multiple anti-species antibodies. Upon addition of serum sample, GNP-rVP7 protein-serum complex migrated laterally onto the strip via capillary action and results were analyzed based on appearance of red colour band at test and control line. Serum samples (n = 481) of sheep, goats, cattle, and camel segregated as positive and negative by the commercial competitive-ELISA (c-ELISA) kit were tested in the fabricated LFIA strips to analyze the performance of the assay. In comparison with c-ELISA, the relative diagnostic sensitivity (DSn) of 95.2% with 91.6-97.6 (95%)) confidence interval and relative diagnostic specificity (DSp) of 99.6% 97.8-100.0 (95%) confidence interval were obtained for the optimized LFIA. The agreement between the LFIA and the c-ELISA was excellent as indicated by the kappa coefficient value of 0.949 (SE = 0.0142) with 0.9219 to 0.9779 (95%) confidence interval. The recombinant protein G based LFIA is a sensitive, specific, rapid, one-step test that can be used in the field or poorly equipped laboratories for serological diagnosis and serosurveillance of bluetongue in multiple susceptible species.

Evaluation of thermo-stability of bluetongue virus recombinant VP7 antigen in indirect ELISA.

This study shows the thermo-stability of lyophilized and purified recombinant VP7 bluetongue virus (BTV) protein in the presence of two sugar stabilizers (trehalose and mannitol) at different temperature. Truncated VP7 protein purified by nickel affinity column was lyophilized in the presence of trehalose and mannitol at 60 mM final concentration and then exposed to different temperature like 4, 25, 37 and 45 °C for various periods like 5 months, 7 weeks, 7 days and 48 h, respectively. After thermal treatment, the reactivity of the protein was evaluated in indirect ELISA. At 4 and 25 °C, the protein was stable up to 5 months and 7 weeks, respectively, irrespective of stabilizers used. At 37 °C, it was stable up to 3 days with both the stabilizers, after which it lost its stability and reactivity. At 45 °C, the protein was stable up to 30 and 24 h with trehalose and mannitol stabilizers, respectively. Both stabilizers found suitable for stability of the protein. However, trehalose appeared to have better stabilizing effect, particularly at higher temperatures than the mannitol. Trehalose could be used as stabilizer for freeze-drying the recombinant VP7 protein if an indirect ELISA kit based on the purified rVP7 protein is supplied to different laboratories of the country for detection of BTV antibody in sheep.