• Validation of an ELISA for quantification of Bovine Viral Diarrhea virus antigen in the critical stages of vaccine production

ANDREA PECORA; MARIA SOL PEREZ-AGUIRREBURUALDE; SUSANA LEVY; DIEGO BOCHOEYER; MARIA JOSE DUS SANTOS; ANDRES WIGDOROVITZ

MADRID

Simposio; World Association of Veterinary Laboratory Diagnosticians-14th International Symposium; 2009

WAVLD

Introduction Bovine Viral Diarrhea Virus (BVDV) is the causal agent of a worldwide spread disease. It infects bovines of all ages, causing reproductive problems and altering biological products of high commercial value, resulting in considerable industrial losses (1,3). Production of a BVDV inactivated vaccine usually presents the difficulty of obtaining enough quantity and quality of BVDV antigenic proteins. Evaluation of vaccines requires the immunization of cattle in order to determine the neutralizing antibody titers induced by the vaccine. An alternative to the use of cattle for testing vaccines is the in vitro measurement of key viral antigens. This report describes the validation of a sandwich-type, indirect ELISA, which is able to detect and quantify BVDV E2 glycoprotein in live and inactivated BVDV.   Material & Methods ELISA procedure: U-bottom polystyrene microplates (Maxisorp, NUNC) were coated with an anti-E2 mouse monoclonal antibody (6) (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. Recombinant antigen preparation: A stable CHO-K1 cell line expressing a truncated version of BVDV E2 glycoprotein (tE2) was used as antigen. The standard curve was derived from CHO-tE2 cells.  The supernatants were four fold diluted in blocking buffer and the resulting samples comprised the following concentrations: 100 ng/ml, 50 ng/ml, 25 ng/ml, 12.5 ng/ml and 6.25 ng/ml. E2t was purified by Immobilized Metal Affinity Chromatography. Purified proteins were stored in aliquots of 10 ml at –70°C. Purified tE2 was used as quality control (QC). Stability of tE2 in  crude supernatant and after purification was assayed by incubating the preparations at 37ºC, 4ºC, -20ºC and -70ºC for 1, 8 and 21 days. Stability after three freeze-thaw procedures was also assessed by determining % recovery in the ELISA. Optimization of working conditions: The optimal antigen concentration and antibodies dilution were determined through standard checkboard titration procedures. Optimal blocking buffer and exposure time were also determined. Limit of detection, correlation and limit of quantification: LOD was estimated by interpolating the mean of 42 negative values plus 3 standard deviations in a standard titration curve. Eight replicates in 14 (purified tE2) and 9 (tE2 in crude supernatant) independent assays were performed in order to determine working range, lower limit of quantification and absolute correlation. Precision: Nine dilutions of purified E2t and CHO-tE2 supernatant were prepared. Each sample was assayed in 8 replicates within the plate. Variation between replicates, intra-assay variation and inter-assay variation were calculated. Inter-laboratory assay: Fifteen randomly coded samples including dilutions of QC samples, dilutions of tE2 in crude supernatant, dilutions of BVDV and dilutions of the negative controls and capture,  primary and developing antibodies were sent to 4 laboratories. The laboratories performed 8 assays, each every 7-10 days. Coefficient of variation for intra- and inter- laboratory assays were calculated.   Results Antigen preparation: tE2 in crude supernatant showed to be stable at 37ºC, 4ºC, -20ºC and -70ºC in the conditions assayed and was not affected after 3 freeze-thaw procedures. For this reason, tE2 in crude supernatant was an optimal reagent for daily calibration curves.  Optimization of ELISA procedure: The optimal dilution of capture and first antibodies and the conjugate were set at 1:400, 1:2000 and 1: 4000, respectively. It was established that PBS-T 1% skimmed milk resulted the best blocking buffer. Finally, it was shown that the optimal exposure time was 30 min. Validation parameters and the CV are summarized in the following table: Validation parameters Result Correlation (tE2 concentration vs absorbance) R= 0.944 p<0.001 Repeatability intra-plate inter-plate   13.5 % 15.1 %