(190f) Characterization of Antibody Immobilization Methods Using the QCM-D

Biosensors sensors which incorporate biomolecules such as proteins, cells or DNA to detect the target molecule (analyte) find applications in numerous fields such as medical diagnostics, environmental applications and food analysis. The high specificity, sensitivity and rapid detection times have warranted great interest in the field of biosensors. Biosensors which utilize proteins such as antibodies or enzymes to sense the target molecules are usually immobilized onto substrates. But, the reliability of antibody based biosensors has been a major issue as immobilization of the antibodies on substrates tends to make them inactive, as a result of which the antibody loses its ?biosensing' ability. The reasons for inactivity could be due to the denaturation of the antibodies on the surface or because of the unfavorable orientation of the surface antibodies wherein the antibody is unable to bind to the antigen.

In this work, we examine some of the most commonly used techniques to immobilize antibodies and provide a quantitative means to evaluate the different immobilization chemistries. We have studied the immobilization of Mouse IgG antibody via three different immobilization schemes and its interaction with Antimouse IgG antibody. Mouse IgG Antibodies were immobilized by covalent attachment to the substrate (randomly oriented), attachment via Protein A which specifically binds to the Fc region of the antibody (oriented) and finally, attachment by Biotinlyated antibody to a Streptavidin covered substrate (oriented). Equilibrium adsorption studies were conducted for each of the immobilization chemistries using the quartz crystal microbalance (QCM), a surface acoustic wave based sensor. The QCM can measure extremely low masses (ng/cm2) with very high precision by accurately measuring the decrease in the resonant frequency of the quartz crystal when mass is deposited on it. The frequency of resonance of the quartz crystal decreases linearly with the mass adsorbed (Saurberey's equation) and so, a very accurate and sensitive measurement of mass adsorbed can be made. The equilibrium adsorption isotherm between the Antibody and Antigen follows the conventional Langmuir Adsorption model, from which we can obtain useful information about the association equilibrium constant (Ka) and the surface concentration (Ab0) of active antibody. From the results of our work, we can conclusively say that the surface concentration of active antibodies is higher for the oriented immobilization of antibodies as compared to the randomly oriented antibody, which makes it more suitable for application in biosensors.