Five female Balb/c mice (6 weeks old) weighing between 22 and 25 g were purchased from Charles River Laboratories, Inc. Wilmington, MA, USA. The mice were fed with standard commercial diet and housed in a clean facility at the Kansas State University. Animal care and protocols were approved by Institutional Animal Care and Use Committee (IACUC#3517) at Kansas State University. All animal experiments were done under strict adherence to the IACUC protocols.
Generation of monoclonal antibody to C-strain E2 protein
Expression and purification of C-strain E2 protein using a baculovirus expression system were performed as previously described . The purified C-strain E2 protein was concentrated using Amicon Ultra Centrifugal Filters 30,000 NMWL (Millipore, Billerica, USA) and measured using BCA assay kit (Pierce, USA) according to the manufacture’s recommendations.
For mAb production, 50 μl (1 μg/μl) purified E2 protein plus equal volume of 2% Alhydrogel (Invitrogen, CA, USA) was used as an immunogen to inject each of the Balb/c mice via intraperitoneal injection. Three booster immunizations with same dose were conducted at 2 week intervals. Three days after the final booster injection, one mouse with the highest anti-E2 antibody titer was humanely euthanized using carbon dioxide (CO2) in euthanasia chambers and sprayed with 70% ethanol. Spleen cells were collected and fused with the mouse myeloma partner SP2/0-Ag14 (ATCC, MD, USA) by using polyethylene glycol 1500 (Boehringer Mannheim, IN, USA) at a ratio of 10:1. The hybridoma cells were maintained in RPMI1640 medium (Gibco, NY, USA) with 20% fetal bovine serum (FBS, Hyclone, UT, USA). Supernatants from growing hybridomas were screened by an ELISA for reactivity to E2 protein as previously described . The positive hybridoma clones were subcloned three times by limiting dilution until monoclones were obtained. Characterization of these monoclonal antibodies will be published elsewhere. One mAb, designated 6B211 was used in this study. Its isotype was classified with an antibody-isotyping kit (Roche Diagnostics Corporation, IN, USA). The reaction of 6B211 with native and β-mercaptoethanol treated C-strain E2 protein was analyzed by western blot as we described previously .
Indirect fluorescent antibody assay (IFA) test
The reactivity of mAb with different BVDVs was tested by IFA as described previously . Briefly, MDBK cells (bovine kidney cells, ATCC, CCL22) grown in 96-well plate were infected with BVDVs (BVDV-32 strain, genotype 1; BVDV-0427 strain, genotype 1; BVDV-AV6 strain, genotype 1; BVDV-125 strain, genotype 2) at a multiplicity of infection (MOI) of 0.1 for 3 days. Cells were fixed in cold acetone and washed two times with PBST. Supernatant of hybridomas (1:50 diluted) was added and plates were incubated at 37 °C for 1 h (hr). Plates were washed three times with PBST and Alexa Fluor 488 goat anti-mouse IgG (H + L) (Life Technologies, MA, USA) was added at 1:200 dilution to each well and incubated at 37 °C for 1 h. Finally, the plate was washed three times with PBST and examined under a fluorescence microscope.
Neutralizing antibody test
Purified mAbs 6B211 (1 mg/ml) and WH303 (1 mg/ml, Animal and Plant Health Laboratories Agency, Wey Bridge, United Kingdom) were first diluted five-fold and then serially diluted two-fold. The diluted samples (in duplicate) were incubated with 100 TCID50 (50% tissue culture infective dose) of CSFV C-strain in DMEM with 10% FBS for 1 h at 37 °C. Residual virus infectivity was determined by adding 1.0 × 104 ST cells to each well with serum-virus mixture in 96-well plate and incubated at 37 °C for 3 days. The cells were subjected to immunofluorescence staining with E2-specific mAb WH303 and Alexa Fluor 488 goat anti-mouse IgG (H + L) (Life Technologies, MA, USA). Neutralizing antibody titers (NAT) were expressed as the reciprocal of the highest dilution that caused 50% neutralization.
Competitive enzyme-linked immunosorbent assay (cELISA)
The 6B211 was purified by HiTrap™ Protein G column (GE Healthcare Life Sciences, PA, USA) followed by conjugating with Horseradish Peroxidase (HRP) using EZ-Link™ Plus Activated Peroxidase (Thermo Scientific, NJ, USA) according to the manufacturer’s instruction. The HRP-6B211 was dialyzed with Slide-A-Lyzer Dialysis Cassettes (Thermo Scientific, NJ, USA) against PBS and stored in Pierce™ Peroxidase Conjugate Stabilizer (Thermo Scientific, NJ, USA).
The systematic checkerboard procedure was used to optimize the concentration of capture antigen and HRP-6B211. The optimal dilution of serum and blocking solution were determined experimentally. The established cELISA was performed in Corning® 96 Well Clear Flat Bottom Polystyrene High Bind Microplate (Corning, NY, USA). Briefly, plates were coated overnight with C-strain E2 (0.625 μg/ml, 100 μl/well) in PBS (without calcium and magnesium, pH 7.4, Thermo Scientific, NJ, USA) at 4 °C. After washing three times with PBST, the plates were blocked with blocking buffer by incubating at 37 °C for 1 h; after washing, 50 μl of diluted serum samples and 50 μl of diluted HRP-6B211 were added to each well and mixed well by pipetting. The plates were incubated at 37 °C for 1 h. After washing five times, 100 μl of room-temperature TMB Stabilized Chromogen (Invitrogen, CA, USA) were added and incubated at room temperature (RT) for 10 min; after adding 100 μl/well of 2 N Sulfuric Acid (Ricca Chemical Company, TX, USA), the absorbance at 450 nm were obtained using SpectraMAX microplate reader (Molecular Devices, CA, USA). The OD450 of the samples were converted to a percent inhibition (PI) value using the following formulation: PI (%) = (OD450 value of negative controls − OD450 value of sample)/OD450 value of negative controls × 100%.
The cut-off value that served as the threshold to separate VNT positive sera from VNT negative sera was determined by testing negative sera of unvaccinated pigs and VNT positive sera of C-strain or C-strain E2 subunit vaccinated pigs (21–56 DPV).
Serial derived serum samples after vaccination with C-strain from 0 to 56 DPV at every 7 days were used for testing the kinetics of antibody titers of pigs (n = 2) at different time intervals post-vaccination.
Reproducibility and statistical analysis of the cELISA
Inter-assay and intra-assay reproducibility for the established cELISA was evaluated by testing CSFV antibody negative (n = 20) serum samples and C-strain VNT positive pig serum samples (n = 20). For the intra-assay reproducibility, each serum sample (in duplicate) was detected by the same batch of pre-coated ELISA plates. For the inter-assay reproducibility, each serum sample was detected by three batches of pre-coated ELISA plates. Sensitivity and specificity analysis were carried out by the web-based MedCalc statistical software (https://www.medcalc.org/calc/diagnostic_test.php). Statistical analysis of reproducibility was carried out by calculate the mean PI value and coefficient of variation (CV) of replications of each test. Statistical analysis of the degree of agreement (Kappa value) and correlation between the established cELISA and VNT were carried out by McNemar’s test and Pearson correlation coefficient analysis in SPSS Statistics for Windows, version 25.0 (IBM Crop, NY, USA). Differences were considered statistically significant when p < 0.05. Scatter plots was generated using the same program.