Submitted to: Journal of Virological Methods
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 8/29/2006
Publication Date: 12/1/2006
Citation: Driskell, J.D., Kwarta, K.M., Lipert, R.J., Vorwald, A.C., Neill, J.D., Ridpath, J.F., Porter, M.D. 2006. Control of antigen mass transfer via capture substrate rotation: an absolute method for the determination of viral pathogen concentration and reduction of heterogeneous immunoassay incubation times. Journal of Virological Methods. 138(1-2):160-169. Interpretive Summary: Many diagnostic tests in use today use antibodies as the method to detect the presence of specific pathogens. These tests are well characterized and are done under very defined conditions. However, many of the incubation periods used in the tests are long and thus make the assay a long process to determine whether a pathogen is present or not. Recently, the use of surface enhanced Raman spectroscopy (SERS) was used as the readout technology in the detection of specific antibody-antigen. The use of SERS also decreases the labor involved in sample preparations. Feline calicivirus and porcine parvovirus were used as model systems in the work demonstrating the utility of this technology. The work described here furthers these studies with the finding that rotation of the sample substrate greatly increases virus binding and decreases the time of incubation necessary to detect specific antigen-antibody interaction. It was also possible to use the rotation method to accurately quantitate the actual number of viral particles present, something that previously was not possible to do. These findings will be useful not only for SERS but can also be utilized in other assays where antibody binding is and integral part of the assay.
Technical Abstract: Immunosorbent assays are commonly employed as diagnostic tests in human healthcare and veterinary medicine and are strongly relevant to the methodologies for bioterrorism detection. However, immunoassays often require long incubation times, limiting sample throughput. As an approach to overcome this weakness, this paper examines the use of rotating capture substrates to increase the flux of antigen to the surface, thereby reducing the incubation time. To assess the capability of this approach, porcine parvovirus (PPV) was selectively extracted from a sample solution exposed to a gold substrate modified with a covalently immobilized layer of anti-PPV monoclonal antibodies. The captured PPV were then directly imaged and quantified by atomic force microscopy. Substrate rotation rates were systematically varied to control the flux of PPV to the capture surface. Analysis of the experimental results, in combination with established theory for rotation-induced flux, allows an accurate determination of PPV concentration. Furthermore, the relationship between the quantal concentration units of 2 50% tissue culture infective dose (TCID50) and quantitative concentration units of viruses/mL can be calculated. The benefits of substrate rotation are demonstrated by comparing dose-response curves, established by exposing the capture platform to the sample solutions for 10 min at 25ºC, for an immunoassay performed under stagnant conditions to one performed with substrate rotation at 800 rpm. The limit of detection (LOD) improved to 3.4 x 10**4 TCID50/mL (~80 fM) by rotating the capture substrate at 800 rpm from an LOD of 3.2 x 10**5 TCID50/mL (~800 fM) under stagnant conditions. The potential to broadly apply this technique to heterogeneous immunoassays is also briefly discussed.