Y were compared. OVCAR 3 cells (ovarian carcinoma) were used as negative

Y were compared. OVCAR 3 cells (ovarian carcinoma) were used as negative control cells for binding of VLP (data not shown).Cell CultureHuh 7 and Huh7.5 cells [28] were maintained in Dulbecco’s PD168393 modified Eagle get 3PO Medium (DMEM, Sigma) supplemented with 10 fetal bovine serum at 37uC under 5 CO2. Sf21 cells were maintained in TC100 insect cell Medium (Sigma) with 10 fetal bovine serum at 26uC.Generation of HCV-LPsThe sequence encoding core-E1-E2 for genotype 3a from cDNA corresponding to RNA isolated from patient blood has been cloned in pGEMT Easy vector (Acc. No. core: GU172376 and E1E2: GU172375). The core-E1-E2 region was subsequently subcloned in pFastBac HTb at 12926553 BamHI-EcoRI site (2.256 kb). Similarly, the core-E1-E2 of genotype 1b was amplified from replicon Con 1FL (Acc. No. AJ238799) [29] and cloned into pFastBac HTc in frame. After the generation of bacmid, integration of DNA specific for core-E1-E2 into the baculoviral genome was confirmed by PCR amplification using M13F and E2R primers for genotype 3a or core F and M13R primers for genotype 1b. The recombinant baculoviruses were rescued from the bacmid and the viruses were amplified in Sf 21 cells. Time course expression of the core-E1-E2 protein in insect cells by recombinant baculovirus was tested 24, 48, 56 and 72 h of post infection at 10 moi. Wild type baculovirus infection cell extracts were used as controls.Immunization of Mice and Establishment of HybridomaPurified VLP (30 mg for each mouse) emulsified with Freund’s adjuvant was administered subcutaneously to 6? weeks old female BALB/c mice three boosters (15 mg for each mouse) at interval of three weeks. After a month, the mice were finally injected intraperitoneally with 100 mg of the antigen in saline and four days later the animals were sacrificed. The spleens were excised, and the splenocytes were fused with Sp2/0 mouse myeloma cells using polyethylene glycol 4000 (Merck). Hybridoma were selected on HAT (Hypoxanthine-aminopterin-thymidine medium) supplemented by IMDM subsequently. Hybridoma obtained were tested for specific antibody production using ELISA and subcloned to obtain single cells. Monoclonal antibodies (mAbs) were purified from culture supernatant by affinity chromatography on a protein A-Sepharose column by following standard procedures [31].Purification of HCV-LPsSf21 cells were infected with recombinant baculovirus at a moi of 5?0, and cells were harvested 72 h post infection. Cell pellets 1516647 were washed with phosphate buffered saline (PBS: 50 mM phosphate buffer pH 7.2 containing 150 mM NaCl) thrice and were resuspended using a tissue homogenizer in a lysis buffer (50 mM Tris, 50 mM NaCl, 0.5 mM EDTA, 1 mM PMSF, 0.1 NP40 and 0.25 protease inhibitors). The lysate was centrifuged at 15006g for 15 min at 4uC and the supernatant was pelleted over a 30 sucrose cushion. The pellet was resuspended in 20 mM Tris and 150 mM NaCl which was then applied on a 20 to 60 sucrose gradient in SW41 rotor (Beckman). After 22 h of ultracentrifugation at 30,000 rpm at 4uC, fractions (1 ml) were collected and tested for E1 and E2 by enzyme-linked immunosorbent assay (ELISA) and western blotting. Anti E1 2 polyclonal antibody raised in rabbit was used for the above assays. Fractions containing HCV-LPs were diluted with 10 mMImmunoassays. (i) ELISAMicrotiter ELISA plates (Nunc) were coated overnight with antigen (HCV-LP) (5 mg/ml in PBS) followed by blocking of unoccupied sites with 0.5 gelatin in PBS. The plates were incub.Y were compared. OVCAR 3 cells (ovarian carcinoma) were used as negative control cells for binding of VLP (data not shown).Cell CultureHuh 7 and Huh7.5 cells [28] were maintained in Dulbecco’s modified Eagle medium (DMEM, Sigma) supplemented with 10 fetal bovine serum at 37uC under 5 CO2. Sf21 cells were maintained in TC100 insect cell Medium (Sigma) with 10 fetal bovine serum at 26uC.Generation of HCV-LPsThe sequence encoding core-E1-E2 for genotype 3a from cDNA corresponding to RNA isolated from patient blood has been cloned in pGEMT Easy vector (Acc. No. core: GU172376 and E1E2: GU172375). The core-E1-E2 region was subsequently subcloned in pFastBac HTb at 12926553 BamHI-EcoRI site (2.256 kb). Similarly, the core-E1-E2 of genotype 1b was amplified from replicon Con 1FL (Acc. No. AJ238799) [29] and cloned into pFastBac HTc in frame. After the generation of bacmid, integration of DNA specific for core-E1-E2 into the baculoviral genome was confirmed by PCR amplification using M13F and E2R primers for genotype 3a or core F and M13R primers for genotype 1b. The recombinant baculoviruses were rescued from the bacmid and the viruses were amplified in Sf 21 cells. Time course expression of the core-E1-E2 protein in insect cells by recombinant baculovirus was tested 24, 48, 56 and 72 h of post infection at 10 moi. Wild type baculovirus infection cell extracts were used as controls.Immunization of Mice and Establishment of HybridomaPurified VLP (30 mg for each mouse) emulsified with Freund’s adjuvant was administered subcutaneously to 6? weeks old female BALB/c mice three boosters (15 mg for each mouse) at interval of three weeks. After a month, the mice were finally injected intraperitoneally with 100 mg of the antigen in saline and four days later the animals were sacrificed. The spleens were excised, and the splenocytes were fused with Sp2/0 mouse myeloma cells using polyethylene glycol 4000 (Merck). Hybridoma were selected on HAT (Hypoxanthine-aminopterin-thymidine medium) supplemented by IMDM subsequently. Hybridoma obtained were tested for specific antibody production using ELISA and subcloned to obtain single cells. Monoclonal antibodies (mAbs) were purified from culture supernatant by affinity chromatography on a protein A-Sepharose column by following standard procedures [31].Purification of HCV-LPsSf21 cells were infected with recombinant baculovirus at a moi of 5?0, and cells were harvested 72 h post infection. Cell pellets 1516647 were washed with phosphate buffered saline (PBS: 50 mM phosphate buffer pH 7.2 containing 150 mM NaCl) thrice and were resuspended using a tissue homogenizer in a lysis buffer (50 mM Tris, 50 mM NaCl, 0.5 mM EDTA, 1 mM PMSF, 0.1 NP40 and 0.25 protease inhibitors). The lysate was centrifuged at 15006g for 15 min at 4uC and the supernatant was pelleted over a 30 sucrose cushion. The pellet was resuspended in 20 mM Tris and 150 mM NaCl which was then applied on a 20 to 60 sucrose gradient in SW41 rotor (Beckman). After 22 h of ultracentrifugation at 30,000 rpm at 4uC, fractions (1 ml) were collected and tested for E1 and E2 by enzyme-linked immunosorbent assay (ELISA) and western blotting. Anti E1 2 polyclonal antibody raised in rabbit was used for the above assays. Fractions containing HCV-LPs were diluted with 10 mMImmunoassays. (i) ELISAMicrotiter ELISA plates (Nunc) were coated overnight with antigen (HCV-LP) (5 mg/ml in PBS) followed by blocking of unoccupied sites with 0.5 gelatin in PBS. The plates were incub.