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Utilization of Bradford Assay to Aid in Development of a Novel Point-of-Use Immunobiosensor

Immunodiagnostics play a critical role in disease diagnosis and monitoring. Further advancement of immunoassays will extend the reach of medical technology to allow testing in the field with inexpensive and disposable point-of-use biosensors that respond to target molecule(s) (antigens) in test solutions by a change in one or more of the biosensor’s properties. An immunobiosensor that utilizes a biochemically-enhanced paper test strip is being researched and developed in our laboratory. The work presented here does not focus on the design or use of the immunobiosensor, but rather on development of an analytical tool/assay method to better understand the immunobiosensor’s test strip composition during its manufacturing process and its functional behavior during an assay. The objective of the work described here was to develop and apply a modified Bradford assay to enable determination of how much antibody is crosslinked to the paper strip during manufacture via measurement of the depletion of the applied antibody from the applied solution. Additionally, this modified Bradford assay allowed for the quantification of immobilized-antibody capture (specific binding) of antigen in an “active test strip” during testing, and for the quantification of nonspecifically bound antigen in a “reference test strip” during testing to enable determination of the antibody’s specific antigen-binding activity (and percent loss of activity). This method was also useful for determining rinse times at various steps during test strip manufacturing required to remove free, unbound protein that is present in the pores of the paper strip and for determining exposure times needed for various solution/protein treatments. In our study it was found that more than 80% of the applied antibody was bound to the paper strip during the crosslinking step with different antibodies experiencing similar crosslinking efficiencies. The activity of the crosslinked antibody in the test strip was also quite good, retaining 22% of the supplier’s reported antibody’s specific antigen-binding capacity, much greater than typically found in ELISA.