The proposed research aims to meet the urgent requirement for immunological reagents with which SARS serodiagnostic tests can be developed. Validated, reliable tests are urgently needed to provide public health laboratories, clinical centers, hospital and private reference laboratories with the means to test patients for SARS infection. For this purpose, immunoreactive antigens which can serve as potential targets for SARS antibody detection will be identified, and antibodies will be generated to SARS antigens with which antigen capture assays can be developed. These objectives will be pursued with a dual strategy comprising epitope scanning via overlapping synthetic peptides and recombinant antigen production. The target antigen will be the SARS virus spike glycoprotein, a potent immunogen in other coronaviruses. Immunoreactive epitopes will be sought based on sequence analysis, predictive algorithms, iterative peptide synthesis and analogy with known immunoreactive epitopes within the spike protein of other coronaviruses. Candidate immunoreactive epitopes will be evaluated against sera from diagnosed SARS patients. Monoclonal and polyclonal antibodies will be generated to identified immunoreactive SARS antigens for use in development of antigen capture assays. The overall goal of the proposed project is to develop the key reagents and resources needed for the subsequent development of diagnostic immunoassays for SARS infection. The project thus represents an exploratory effort, given the current state of knowledge of SARS, consistent with the R21 grant mechanism.
Four specific aims are proposed in this project: ? ? (1) Identification of antigenic sequences within the SARS genome, in particular within the S (spike) protein. ? ? (2) Evaluation of the immunoreactivity of recombinant or synthetic antigens with SARS patient sera by IgG and IgM ELISA. ? ? (3) Determination of specificity of potential SARS antigens sera from human blood donors, patients with non-SARS respiratory infections and with other potentially cross-reactive conditions. ? ? (4) Generation of monoclonal and polyclonal antibodies to specific SARS antigens, for use in antigen capture assays. ? ? ?
