• Development of an ELISA kit to detect and evaluate Bovine Viral Diarrhea Virus antigen in the critical stages of vaccine production

PECORA A; OSTACHUK A; LEVY S; ESPINOZA A; DUS SANTOS MARIA JOSE; WIGDOROVITZ A

Melbourne, Australia

Simposio; 13th International Symposium of the World Association of Veterinary Laboratory Diagnosticians; 2007

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Development of a ELISA kit to detect and evaluate Bovine Viral Diarrhea Virus antigen in the critical stages of vaccine production A. Pecora1, A. Ostachuk1, S. Levy2, A. Espinoza2, M. J. Dus Santos1* and A. Wigdorovitz1 (1) Instituto de Virología, CICVyA, INTA-Castelar, Buenos Aires, Argentina (2) Biogénesis-Bago, Argentina * mdussantos@cnia.inta.gov.ar IntroductionBVDV is the causal agent of a worldwide spread disease. It infects bovines of all ages, causing reproduction problems and altering biological products of high commercial value, resulting in considerable industrial losses. Persistently infected cattle disseminate large amounts of virus into the environment representing a mechanism by which the virus persists. Due to worldwide distribution of BVDV, disease control is based on the segregation of persistently infected cattle and efficient vaccination of the herd. According to that, the aim of vaccination against BVDV is to prevent transplacental transmission of the virus. Production of a BVDV vaccine usually presents the difficulty of obtaining enough antigen in order to induce high levels of neutralizing antibodies The objective of this work was to standardize an enzyme-linked immunosorbent assay (ELISA) to detect and evaluate BVDV antigen in the critical stages of vaccine production. Material & methodsELISA procedure U-bottom polystyrene microplates (Maxisorp, NUNC) were coated with an anti-E2 mouse monoclonal antibody (1:100 dilution)  Samples were added at the corresponding dilution and incubated 1h at 37°C. As primary antibody, sera from rabbit immunized with a truncated version of E2 (1:2000) were used. Peroxidase-conjugated goat anti-rabbit IgG (1:2000) were used as secondary antibody. The reaction was visualized with ABTS (Sigma) and absorbance was measured at 405 nm in a Multiskan EX (Labsystyem) reader. Daily calibration curves using four concentrations (104 ng/ml, 26 ng/ml, 6.5 ng/ml and 1.6 ng/ml) (each level in duplicate) were prepared with purified tE2, and used to compute E2 levels. Standard curves A stable CHO-K1 cell line expressing a truncated version of BVDV E2 glycoprotein (tE2) was used for construction of standard curves. E2t was purified by Immobilized Metal Affinity Chromatography. Purity and protein concentrations were determined by bicinconic method and SDS-PAGE and Coomasie blue staining using a BSA standard curve followed by densitometry analysis using Image J software. ResultsA sandwich ELISA kit for the detection of BVDV was developed. In order to quantify E2 in the sample tested, a standard curve was performed using purified tE2 as antigen. 8 concentrations of purified E2t were prepared for using as standards. An aliquot of each standard was tested 12 times. A linear dose-response curve was obtained (from 417 to 0.4 ng/ml of purified E2t) with a coefficient of correlation R≥0.93. Four concentrations of purified tE2 (104 ng/ml, 26 ng/ml, 6.5 ng/ml and 1.6 ng/ml) were prepared for use as QC samples for evaluation of intra and inter-assay precision and accuracy. Excellent levels of repeatability and reproducibility were obtained. The limit of detection of E2 in the supernatant of the stable cell line was 0.15 ng E2/ul. The test also allows detecting E2 glycoprotein in BVDV cell culture production. For that purpose BVDV samples were treated with 0.25% Triton X114 (Sigma-Aldrich).  The assay could detect up to 10 4,5 TCID50/ml BVDV, Singer strain. Different BVDV productions and experimental and commercial vaccines were assayed. The ELISA was able to detect and quantify E2 in live BVDV, inactive BVDV and in the formulated vaccine. Discussions & conclusionsResults obtained indicate that the assay developed is a reliable tool to monitor the most relevant BVDV antigen in the process of vaccine formulation. Preliminary results showed that the ELISA is able to detect BVDV in serum from persistently infected animals. Further analysis of a larger amount of samples should be performed in order to assess the feasibility of using this assay for diagnosis of PI cattle. ReferencesInsert text