Two sphere immunoassay on a microfluidic device

Clinical measurements of biologically important proteins and peptides are routinely carried out in the laboratory setting using immunoassays. However, the most recent trend in medicine is the advance of personalized medicine. The goal is to tailor the diagnosis, care and the drugs to the individual. To make this a reality, fast and inexpensive, analytical tools need to be developed that are capable of measuring multiple biological markers at one time in small volumes. When an assay can use very small volumes less invasive methods for obtaining samples such as blood can be used (finger prick). The measurement of more than one biomarker from the same sample using the traditional ELISA method requires a separate immunoassay for each analyte. This is expensive, requires large amount of sample and is time consuming. A faster and more economical way is by developing a multiplexing assay where not one but several analytes are measured at one time. The immunoassay we developed uses microspheres (capture spheres) conjugated with monoclonal antibodies specific to the targeted analyte. These microspheres capture the antigen and form a microsphere-antibody-antigen complex, which are then bound by a secondary monoclonal antibody, recognizing a different exposed epitope on the antigen. This secondary antibody is conjugated to a second microsphere (detection sphere). The capture and detection spheres are loaded with fluorescent dyes, each with a different wavelength, allowing for easy differentiation. Detection of the capture sphere-antigen-detection sphere complexes can accomplished by three different formats, 96 well plate, Flow cytometer and Microfluidic devices. In this Thesis we will discuss the development of this fast and sensitive aminoassay using these three formats. We will show that the assay can be performed in less then 15 minutes with sensitivity comparable to, or better than that obtained with the gold standard ELISA assay. Also we will calculate the theoretical limits of this assay and compare them to a commercial single bead assa