Abstract
Antigen Used for Immunization
Ovine fetal turbinate (OFTu) cells were grown in 10 T150 tissue culture flasks and, when 90% confluent, were infected with NA1/11 (5 MOI). When cytopathic effect was evident (3–5 days) and the majority of cells displayed a rounded morphology, the media was centrifuged (1500 rpm, 4°C, 10 min). The supernatant was subjected to sucrose gradient ultracentrifugation to separate the viral particles from other matter. The viral particles were removed and heat-inactivated for 90 min at 96°C, sonicated, and stored at −80°C. A protein assay reagent (Bio-Rad, Hercules, CA) was utilized to determine the protein concentration of the purified virus.
Method of Immunization
Ten 8-week-old female BALB/c mice were immunized via intraperitoneal (i.p.) injection with 50 μg of viral protein and bentonite (Fisher Scientific, Waltham, MA). Boosting of mice started 4 weeks after initial immunization, consisting of i.p. injection of 50 μg viral proteins four times per week in 2-week intervals.
Parental Cell Line Used for Fusion
Splenocytes were fused with SP2/0 myeloma cells.
Selection and Cloning Procedure
The fused cells were re-suspended in RPMI 1640 medium (Hyclone, Logan, UT) supplemented with 20% FBS, 10 U/mL IL-6 (Roche, Mannheim, Germany), OPI media supplement (Sigma-Aldrich, St Louis, MO), and HAT media supplement (Sigma-Aldrich). They were then plated into 96-well tissue culture plates (1.2×105 cells/well) in a volume of 200 μL. After incubation for 7 to 10 days (37°C, 5.0% CO2), the culture medium in each well was analyzed by indirect ELISA to detect the presence of MAb against viral protein. Hybridomas reacting to orf virus protein were further purified and tested.
Heavy and Light Chains of Immunoglobulin
IgG (IgG1).
Specificity
Mass spectrometry was conducted after IP to confirm specific binding to ORFV086 protein.
Specific Antigen Identified
Orf virus (NA1/11) proteins were purified by sucrose gradient ultracentrifugation, immunoprecipitated with purified MAb, and finally analyzed by mass spectrometry.