DEVELOPMENT OF AN IN PROCESS CONTROL FILTRATION-ASSISTED CHEMILUMINOMETRIC IMMUNOASSAY TO QUANTIFY FOOT AND MOUTH DISEASE VIRUS (FMDV) NON CAPSID PROTEINS IN VACCINE ANTIGEN BATCHES

CAPOZZO, ALEJANDRA; MARTINEZ, MANUEL; LAVORIA, MARÍA DE LOS ÁNGELES Y SCHIELEN, WIM

Viena

Otro; OPEN SESSION OF THE EuFMD STANDING TECHNICAL COMMITTEE; 2010

FAO

Introduction: In many countries, Foot and Mouth Disease (FMD) is controlled by vaccination and surveillance against viral Non Capsid Proteins (NCP); therefore vaccines are required not to induce antibodies against NCP. Vaccine purity is evaluated by repeated inoculation of naïve cattle, an expensive and time consuming protocol that raises several animal welfare concerns. We have developed an in vitro in process control test applying the technology of filtration-assisted chemiluminometric immunoassay (FAL-ELISA), to detect and quantify NCP in vaccine antigen batches. Materials and methods: Samples were filtered through PVDF-filter microplates pre-coated with a monoclonal antibody against NCP. Filtration removes all unbound components in the sample and captured NCP are detected by anti-NCP conjugate followed by incubation with the substrate, luminol/peroxide. Limit of detection was estimated by serial dilution of recombinant NCP and NCP-spiked vaccine antigen batches (VABs). Positive and negative samples were confirmed by serology and Western Blot. Results: The assay showed to be highly specific. Analytical detection limit was 2 ng for purified NCP and 4ng for vaccine antigen batches spiked with NCP per well. Vaccine components did not interfere with the antibody and substrate reactions in the assay. FAL-ELISA could detect a three-fold dilution of a proven serology-positive VAB. Discussion: We have developed an in vitro in process control filtration-assisted chemiluminometric immunoassay (FAL-ELISA), sensitive enough to detect and quantify NCP in any formulation regardless its volume and composition. FAL-ELISA is an alternative for the in vivo tests. This development observes the objective to Replace, Reduce and Refine the use of animals for quality control of immunobiologicals.