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United States Department of Agriculture

Agricultural Research Service

Title: Immunoassays Utilizing Atomic Force Microscopy As a Readout Method: a Platform for the Detection of Viral Pathogens

Authors
item Kwarta, Karen - IOWA STATE UNIVERSITY
item Driskell, Jeremy - IOWA STATE UNIVERSITY
item Neill, John
item Ridpath, Julia
item Porter, Marc - IOWA STATE UNIVERSITY

Submitted to: Pittsburgh Conference
Publication Type: Abstract Only
Publication Acceptance Date: November 15, 2004
Publication Date: February 27, 2005
Citation: Kwarta, K., Driskell, J., Neill, J.D., Ridpath, J.F., Porter, M. 2005. Immunoassays utilizing atomic force microscopy as a readout method: a platform for the detection of viral pathogens [abstract]. The Pittsburgh Conference (PITTCON). Paper No. 1830-2.

Technical Abstract: High throughput, differential detection of viral pathogens is central to combating bio-terrorism threats and maintaining a safe food supply. To address this need, we have explored the development of a virus detection method that couples the binding specificity of antibody-antigen interactions and the ultrahigh resolution imaging capabilities of atomic force microscopy (AFM) as a direct readout method. The designed platform can be adapted for the detection of multiple antigens in the same sample. In addition to detecting binding of specific antibodies, this technology allows the direct visualization of bound viruses. Thus the size and shape of bound viruses may be observed. This presentation demonstrates this capability by the detection of feline calicivirus (FCV) captured directly from serum by immobilized anti-FCV monoclonal antibodies (mAbs) and quantified by TappingMode AFM. Specificity was demonstrated by linear relationship between viral concentration and detection and by competition with free antibodies. Single binding events (i.e. the binding of one viral particle) could be detected. Thus, volume size is the only limit to sensitivity. In an effort to reduce the overall analysis time, binding kinetics were examined at two different temperatures (25 °C and 37 °C). Results show that the exposure time can be reduced to 1 h at 37 °C while maintaining sensitivity. This method was also used as a diagnostic screening tool to identify the most effective anti-FCV mAb and to determine binding efficiencies for different strains of FCV. The ability of different chemical fixatives to enhance the stability of the antibody-antigen interaction under stringent rinsing conditions will also be discussed.

Last Modified: 9/2/2014
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