DEVELOPING A COMMERCIAL MULTI-ANTIGEN CONFIRMATORY ELISA TO DETECT ANTIBODIES TO AFRICAN SWINE FEVER VIRUS UNDER BIOSAFETY LEVEL 2 CONDITIONS

African Swine Fever (ASF) is a rapidly emerging disease of domesticated pigs caused by a highly virulent strain of ASFV. In 2021, ASFV was detected in close proximity to the U.S., in the Dominican Republic and Haiti, placing the U.S. on high alert for ASFV and ramping up surveillance and disease management efforts. ASF serological diagnostic tests and reagents have significant market demand to support domestic biosecurity to prevent ASF entry to the U.S. and for international disease management where ASF is endemic, emerging, or in close proximity. Currently, the immunoperoxidase test (IPT) or indirect fluorescent antibody test (IFAT) using ASFV infected cells, and immunoblotting are the only confirmatory serological methods available and recommended for ASF. However, enzyme linked immune-sorbent assay (ELISA) is an ideal format for surveillance due to its speed, low-cost, and technical accessibility (training, equipment, and biological safety). The goal of this project is to deliver new diagnostic reagents and assays for a confirmatory ASF ELISA. Currently available commercial ASF ELISA have sensitivity issues to detect certain ASFV strains, such as Estonia and Georgia. Many diagnostic targets have their strengths and weaknesses, but none can provide comparable performance as tests that use ASF-infect cells or cell lysates (IPT, IFAT, immunoblotting). Thus, to improve ELISA performance, a mixture of ASFV antigens must be used. We previously developed ASF ELISAs, and confirmed the strengths of p30, p54, and p72 antigens. In this project, we aim to develop a confirmatory ELISA using additional antigens to detect multiple ASF antibodies. Following reagent and ELISA development, we intend to validate the ELISA and pursue USDA-licensure. The ASF ELISA will be useful to maintain and prove ASF free status. In the event of an outbreak, this will support rapid recovery to regain market access.Technical ObjectivesObjective 1. Production of twelve different immunogenic ASFV proteinsWe will use baculovirus expression systems to express recombinant ASF antigens. We will employ 6x his-tag on each recombinant antigen to facilitate purification by immobilized metal affinity chromatography (IMAC). Each purified antigen will be screened for ASF antibody detection.Objective 2. Screening antigens by iELISA: benchmark >95% sensitivity and specificity2.1. Screen antigens by iELISA (a) antigen concentrations, microtiter plates and blocking buffers, (b) sample and reagent diluents, and (c) plates and reagent preservatives buffers. We will screen twelve ASFV proteins that were previously characterized to be antigenic or immunogenic but have not yet been used to develop diagnostic tests. Antigens will be evaluated by their ability to detect ASF antibodies from ASF-infected animals.2.2. Validate antigens by iELISA and select top four candidates. We will select four antigens based on their sensitivity to detect ASFV antibodies, from multiple ASFV strains, early stages of infection, and ASFV vaccinated pigs. Then, we will use four new antigens, three well-characterized antigens (p30, p54, and p72), and a negative control antigen to develop an eight-antigen confirmation test. Following initial screening, we will work with the Canadian Food Inspection Agency, National Center for Foreign Animal Disease (NCFAD), collaborating with the OIE-recognized ASF Reference laboratory, Headed by Dr. Aruna Ambagala. Dr. Ambagala has an extensive sample collection for validating ASF diagnostics. His lab will perform validation experiments to evaluate the performance of each antigen identified in the ELISA screen.2.3. Initiate mAb development for top four antigen candidates. Four validated antigens, based on their sensitivity to detect ASFV antibodies, will be used to initiate mAb development. The mAb reagents can be used in research and diagnostics, but as a long-term process, mAb development and validation will not be conducted in the scope of our Phase I project. However, new mAb may provide valuable tools and positive controls for ELISA improvement and commercialization during a Phase II project.Objective 3. Develop confirmation test: benchmark >99.9% sensitivity and >99.75% specificity?3.1. Optimize compatibility of eight iELISA conditions. During validation we will develop several ELISA reagents (buffers, diluents, etc.) for each of the antigens individually. In this objective, they will be evaluated for their compatibility with the other antigens to determine if reformulation will be necessary. Objective 2 will be conducted in such a way that we will have reference data to identify compatible formulas and avoid incompatible conditions. 3.2. Validate iELISA confirmation test. Following development, we will work with Dr. Ambagala (NCFAD) to validate performance of the iELISA confirmation test, using the same strategies, sample sets, and comparison as performed for single-antigen iELISA screening.