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. ? ?

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project--Cooperative Agreements (U01)
Project #
1U01AI061233-01
Application #
6818409
Study Section
Special Emphasis Panel (ZAI1-LR-M (M1))
Program Officer
Cassels, Frederick J
Project Start
2005-03-15
Project End
2010-02-28
Budget Start
2005-03-15
Budget End
2006-02-28
Support Year
1
Fiscal Year
2005
Total Cost
$241,074
Indirect Cost
Name
University of Alberta
Department
Type
DUNS #
208095844
City
Edmonton
State
AB
Country
Canada
Zip Code
T6 2-E1
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Raghuwanshi, Dharmendra; Mishra, Vivek; Das, Dipankar et al. (2012) Dendritic cell targeted chitosan nanoparticles for nasal DNA immunization against SARS CoV nucleocapsid protein. Mol Pharm 9:946-56
Palaniyappan, A; Das, D; Kammila, S et al. (2012) Diagnostics of severe acute respiratory syndrome-associated coronavirus (SARS-CoV) nucleocapsid antigen using chicken immunoglobulin Y. Poult Sci 91:636-42
Das, Dipankar; Kammila, Sriram; Suresh, Mavanur R (2010) Development, characterization, and application of monoclonal antibodies against severe acute respiratory syndrome coronavirus nucleocapsid protein. Clin Vaccine Immunol 17:2033-6
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Suresh, Mavanur R; Bhatnagar, Pravin K; Das, Dipankar (2008) Molecular targets for diagnostics and therapeutics of severe acute respiratory syndrome (SARS-CoV). J Pharm Pharm Sci 11:1s-13s
Bhatnagar, Pravin K; Das, Dipankar; Suresh, Mavanur R (2008) Sequential affinity purification of peroxidase tagged bispecific anti-SARS-CoV antibodies on phenylboronic acid agarose. J Chromatogr B Analyt Technol Biomed Life Sci 863:235-41
Kammila, Sriram; Das, Dipankar; Bhatnagar, Pravin K et al. (2008) A rapid point of care immunoswab assay for SARS-CoV detection. J Virol Methods 152:77-84
Das, Dipankar; Suresh, Mavanur R (2006) Copious production of SARS-CoV nucleocapsid protein employing codon optimized synthetic gene. J Virol Methods 137:343-6