Analytical interpretation of surface plasmon resonance spectroscopy using representative antibody-antigen systems

Surface plasmon resonance spectroscopy (SPRS) is an evanescent wave spectroscopy traditionally employed to examine the intrinsic properties of thin metal films (silver or gold). In dielectric film(s), the index of refraction n, the imaginary index k and the thickness d are properties that are obtainable in SPRS. The presentation of the SPRS spectrum is reflectivity (0--1) verses internal angle referenced to the normal of the metal film. Our interest in SPRS is in the examination, both quantitatively and qualitatively, of antigen (Ag) binding to antibody (Ab) immobilized on the metal surface. This is manifest by an angular shift of the SPR spectrum. In this work we have explored liquid phase SPRS for the quantitation of two medically significant antigens. Using a Protein G-Ab based biosensor, the first of these antigens evaluated was human epidermal growth factor receptor 2 (HER-2), an important tumor marker for diagnosis of breast cancer. The second biomarker was prostate specific antigen (PSA), an equally important tumor marker in the diagnosis of prostate cancer. In our work, we have found several instances of negative SPR shifts. We envision this as a "net" shift composed of both positive and negative components affected by the configuration of the Ab. We have detected total PSA levels at 9ng/mL and complexed PSA at 16ng/mL. HER-2 measurements were strictly qualitative. SPR studies were also conducted with bovine serum albumin (BSA) to further evaluate biosensor behavior and quantification schemes using numerical methods of integration. BSA experiments were performed using a static electric field to study this effect on mass transport limitations. BSA was detected at 3.7ng/mL. Finally, bulk layer refractive index effects were evaluated both theoretically and experimentally for simple bulk buffer systems in contact with the metal surface.