A neutralizing monoclonal antibody-based competitive ELISA for classical swine fever C-strain post–vaccination monitoring

Abstract

Background: Virus neutralization test (VNT) is widely used for serological survey of classical swine fever (CSF) and efficacy evaluation of CSF vaccines. However, VNT is a time consuming procedure that requires cell culture and live virus manipulation. C-strain CSF vaccine is the most frequently used vaccine for CSF control and prevention. In this study, we presented a neutralizing monoclonal antibody (mAb) based competitive enzyme-linked immunosorbent assay (cELISA) with the emphasis on the replacement of VNT for C-strain post–vaccination monitoring. Results: One monoclonal antibody (6B211) which has potent neutralizing activity against C-strain was generated. A novel cELISA was established and optimized based on the strategy that 6B211 can compete with C-strain induced neutralizing antibodies in pig serum to bind capture antigen C-strain E2. By testing C-strain VNT negative pig sera (n=445) and C-strain VNT positive pig sera (n=70), the 6B211 based cELSIA showed 100% sensitivity (95% confidence interval: 94.87 to 100%) and 100% specificity (95% confidence interval: 100 to 100%). The C-strain antibody can be detected in pigs as early as 7 days post vaccination with the cELISA. By testing pig sera (n=139) in parallel, the cELISA showed excellent agreement (Kappa=0.957) with VNT. The inhibition rate of serum samples in the cELISA is highly correlated with their titers in VNT (r 2 =0.903, p<0.001). In addition, intra- and inter-assays of the cELISA exhibited acceptable repeatability with low coefficient of variations (CVs). Conclusions: This novel cELISA demonstrated excellent agreement and high level correlation with VNT. It is a reliable tool for sero-monitoring of C-strain vaccination campaign because it is a rapid, simple, safe and cost effective assay that can be used to monitor vaccination-induced immune response at the population level.

A Novel Competitive ELISA for Specifically Measuring and Differentiating Immune Responses to Classical Swine Fever C-Strain Vaccine in Pigs

“…Generation of monoclonal antibodies against C-strain E rns were performed as we previously described [21]. Briefly, 50 µL (1 µg/µL) purified E rns protein plus equal volume of Alhydrogel 2% (InvivoGen, San Diego, CA, USA) was used as an immunogen to inject each of five female Balb/c mice (purchased from Charles River Laboratories, Inc. Wilmington, MA, USA) via intraperitoneal injection for mAb production.…”

“…Optimization of the concentration of capture antigen and HRP-1504, the dilution of serum, and the blocking solution was performed as we described previously [21]. Briefly, Corning ® 96 Well Clear Flat Bottom Polystyrene High Bind Microplates (Corning, NY, USA) were coated overnight with C-strain E rns (0.31 µg/mL, 100 µL/well) in PBS (without calcium and magnesium, pH7.4, Thermo Scientific, Bridgewater, NJ, USA) at 4 • C. After blocking, 50 µL of diluted serum samples and 50 µL of diluted HRP-1504 (0.625 µg/mL) were added to each well and mixed well by pipetting.…”

Establishment of a Blocking ELISA Detection Method for Against African Swine Fever Virus p30 Antibody

“…Following extensive washing, reaction was developed by adding chromogenic substrate solution (TMB) (Beyotime Biotechnology Co., Ltd., Shanghai, China) for 10 min and stopped with Stop Solution for TMB Substrate (Beyotime Biotechnology Co., Ltd., Shanghai, China). The plates were read at 630 nm, and the raw data were transformed to an Excel sheet and consequently the percent of inhibition (PI value) of each test sample was calculated using the formula: PI (%) = [(OD 630 value of negative controls -OD 630 value of sample)/OD 630 value of negative controls] × 100%, as described by Wang et al (35).…”

Recent Advances in the Diagnosis of Classical Swine Fever and Future Perspectives

“…The C-strain antibody can be tested in pigs as early as 7 days post vaccination with the cELISA. This cELISA is a reliable, rapid, simple, safe, and cost-effective tool for sero-monitoring of C-strain vaccination at a population level [ 56 ]. In addition, another group developed a high-throughput VNT by using the recombinant CSFV possessing a small report tag and luciferase system.…”

“…A cELISA was developed in our group based on the strategy that the C-strain-specific mAb will compete with the C-strain vaccine-induced antibodies in pig serum to bind the capture antigen (C-strain E rns ) [unpublished data]. Different from the CSFV neutralizing monoclonal anti-E2 antibody based cELISA for sero-monitoring of C-strain vaccination at a population level [ 56 ], this novel anti-E rns mAb-based cELISA is a valuable tool for measuring and differentiating immune responses to C-strain vaccination and/or infection in pigs. The data about the establishment and validation of this C-strain specific cELISA will be published separately at a later date.…”