Location: Foreign Arthropod Borne Animal Disease Research
Project Number: 3022-32000-025-007-S
Project Type: Non-Assistance Cooperative Agreement
Start Date: Sep 1, 2021
End Date: Aug 31, 2024
Objective:
The overarching objective of this agreement is to develop reagents and new technologies for detection and surveillance of transboundary zoonotic arboviruses that are recognized as emerging animal disease threats. The area of focus will be directed toward diagnostic assays for Japanese encephalitis and Rift Valley fever viruses. Specific objectives include: 1) develop and express new recombinant viral proteins, 2) develop and validate new enzyme-linked immunosorbent assays (ELISAs), and 3) generate new monoclonal antibodies (mAbs).
Approach:
This aim of this project is to develop new sensitive and specific enzyme-linked immunosorbent assays (ELISAs) for the detection and surveillance of the transboundary arboviral disease threats Japanese encephalitis virus (JEV) and Rift Valley fever virus (RVFV). Assays to be developed will include indirect (iELISA) and blocking (bELISA) ELISAs. Objective 1: The approach will entail first designing at least two recombinant JEV proteins and two RVFV proteins. Protein expression and purification protocols will then be optimized to maximize antigen purity and yield. Objective 2: These recombinant proteins will then be used for the development of new iELISAs and/or bELISAs. Antigen coating for ELISA plates will be optimized. To minimize non-specific antigen-antibody interactions, optimal blocking buffers will also be determined. Sample diluents and antibody conjugate diluents will be evaluated and adjusted to reduce background and increase assay sensitivity. Preservatives for plates and reagents will be tested and the stability of each will be evaluated. Validation experiments of the optimized assays will be performed using negative and positive serum samples. Development of the iELISA will precede the bELISA because the bELISA requires monoclonal antibody development and validation. Objective 3: Following validation of antigen specificity through the iELISA, new monoclonal antibodies will be developed. Recombinant protein antigens will be used for mouse injections. Test-bleeds from each mouse will be screened to select which animals are producing the most specific antibodies. The selected animals will be used to generate hybridoma clones to produce monoclonal antibodies (mAbs). Each hybridoma clone will be screened to select clones that produce the most specific mAbs. Following clone selection, sub-clones will be generated, which will undergo a final screening for specificity prior to scaling up production of mAbs.
