I. BROAD,LONG-TERM OBJECTIVES: The current objective is to develop inexpensive point of care diagnostics using new monoclonals against SARS antigens. The same antibodies may have therapeutic utility as well. II.
SPECIFIC AIMS : (a)To develop high affinity MAbs against SARS associated envelope (E), membrane glycoprotein (M), the nucleocapsid protein (NP) or spike proteins (SP) useful in homosandwich(repeat epitopes as in whole virus) or heterosandwich (as in shed monomeric antigens) assay;(b) To develop bispecific antibodies with specificity to NP (or SG) and HRPO;(c) To optimize an ultrasensitive molecular velcro homosandwich assay of the whole SARS virus on microplates(d) To develop an immunoswab or dipstick assay for samples collected from throat or sputum for screening applications. III. RESEARCH DESIGN: The various SARS antigens will be used to immunize mice by short-term and long-term protocols with s.c., i.p.,and i.v. injections. Immunized splenocytes and myeloma cells will be fused to obtain antigen specific hybridomas. The hybridoma supernatants from different clones will be tested for their ability to detect antigens in homosandwich and heterosandwich formats with recombinant antigen. We will also develop quadromas to generate bispecific monoclonal antibodies (bsMAb) with one paratope capable of binding to SARS antigen and the other to an enzymatic marker like peroxidase. We will explore different ELISA formats for detection of whole virus, for NP or other shed antigens. The immunoswab assay will be optimized using the commercially available nasopharyngeal swab with a flexible aluminum shaft for direct detection of SARS virus or its shed antigen using bsMAbs. Alternatively, we will also test the utility of our Mabs labeled with collidal gold to develop a prototype immunoswab assay that could potentially be used for rapid screening of suspected individuals. The simplicity of the proposed immunoswab assay is its utility in screening at airports and high risk communities and health care workers.
