Immunoassay performance: recognition elements, equilibrium and mass transfer considerations

This dissertation explores the importance of mass transfer, equilibrium, and recognition elements on performance of immunoassay platforms. Atomic Force Microscopy (AFM) and Surface-Enhanced Raman Scattering (SERS) are employed as readout methods. AFM was utilized to evaluate the effectiveness of monoclonal antibodies used in a heterogeneous immunoassay for porcine parvovirus (PPV) and feline calicivirus (FCV). These results were employed to develop immunoassay protocols for PPV and FCV, and to assess the effect of elevated temperatures on the rate of mass transfer, and therefore the accumulation of PPV and FCV. Theoretical accumulation rates were compared to experimental observations as monitored by AFM. The effect of sample volume and capture area on limits of detection was examined for PPV utilizing SERS as a readout method. A model of the equilibrium for heterogeneous immunoassays revealed that the use of small capture substrate areas and large sample volumes would prevent depletion of the analyte solution, and would provide the highest surface concentration of analyte. Experimental findings for the detection of PPV upheld this prediction. Finally, the use of aptamers as alternatives to antibodies in a SERS-based detection platform for thrombin was examined. The conditions for aptamer immobilization and thrombin extraction were evaluated, the limit of detection for the aptamer-based assay was compared to that for an antibody-based assay, and possible underpinnings for the differences in performance discussed