West Nile Virus, NS2b/NS3 Protease (WNV)

NS3 and NS2b are non-structural viral proteins that play an essential role in viral replication and are therefore a potential target for treatment and prevention of West Nile Virus disease. NS3 is a trypsin-like serine protease that requires NS2b as a cofactor. Hybridoma: Produced from a hybridoma resulting from the fusion of a mouse myeloma with B cells obtained from a mouse immunized with purified, E. coli-derived, recombinant West Nile Virus NS3 Protease complex (rwnvNS3/NS2b; aa 1-184 of NS3; Accession # NP_776018, and aa 49-96 of NS2b; Accession # NP_776017). Applications: Western Blot, Neutralization, Immunoprecipitation, Direct ELISA Recommended Dilution: Western Blot: This antibody can be used at 1-2ug/mL with the appropriate secondary reagents to detect west nile virus NS3/NS2b complex. Using a colorimetric detection system, the detection limit for rwnvNS3/NS2b complex is approximately 5 ng/lane under non-reducing and reducing conditions. Chemiluminescent detection will increase sensitivity by 5 to 50 fold. Neutralization of west nile virus NS3 bioactivity: This antibody has been used to inhibit/neutralize the enzymatic activity of rwnvNS3/NS2b. The antibody was preincubated with the enzyme at different molar ratios at room temperature for 30 min. The enzyme was then assayed with a peptide substrate, Ac-REKR-AFC. 50% of the proteolytic activity was inhibited by the antibody at approximately 13ug/mL (IC50) under conditions in which the enzyme was present at 10ug/mL and the substrate concentration was 200uM. Considering the molecular masses of the enzyme (27kD) and the antibody (150kD), IC50 was achieved at approximately 1:4 molar ratio of the antibody to the enzyme. Immunoprecipitation: This antibody was used at a concentration of 25ug/mL to immunoprecipitate rwnvNS3/NS2b from conditioned cell culture medium. The recovered rwnvNS3/NS2b can be detected in Western blots using a polyclonal antibody. Direct ELISA: This antibody can be used at 0.5-1.0ug/mL with the appropriate secondary reagents to detect west nile virus NS3/NS2b complex. The detection limit for rwnvNS3/NS2b is approximately 1 ng/well. Optimal dilutions to be determined by the researcher. Storage and Stability: Lyophilized powder may be stored at 4 degrees C for short-term only. Reconstitute to nominal volume by adding sterile 40-50% glycerol and store at -20 degrees C. Reconstituted product is stable for 12 months at -20 degrees C. For maximum recovery of product, centrifuge the original vial after thawing and prior to removing the cap. Further dilutions can be made in assay buffer.