Abstract 4899: Development of a quantitative measurement of p53 – p53 antibody interactions

Abstract

Abstract We are developing methods to quantify antibody interactions that includes a quartz crystal microbalance (QCM) system to measure, on a molecular basis, the interaction of p53 and anti-p53 antibodies. Previously, as a model system, we developed a reference device consisting of p53 protein (human wild type), characterized by mass spectroscopy and immobilized at various concentrations on a glass slide. The device is designed as a reference control to be used with immunohistochemical (IHC) assays that incorporate commercially available anti-p53 antibodies and probes. In the current study, we compared three different monoclonal antibodies (clones: BP53.12, PAb 1801 and DO-1) for their ability to detect p53 in HT1080 cells. Both visible microscopy (diaminobenzidine probe) and flow cytometry (AlexaFluor 488 probe) showed significant increases in staining intensity compared to controls without primary antibody. However, quantification of these observations requires direct measurement of the concentration of the p53 and the concentrations and specificities of the antibodies. For this the p53 protein is covalently immobilized on glass coated quartz crystals and the resonance frequency shift is followed in-situ. The functionalized sensor is then incubated with the anti-p53 antibody, which provides a direct gravimetric measure of antibody-antigen binding. This allows the comparison of the surface immobilized protein concentrations with those previously obtained by fluorescence measurement. The p53 functionalized QCM system offers an independent measure of surface immobilized protein concentration and is essential in the development of our IHC calibration prototypes. These results also provide a benchmark for comparing systems that may be used in developing molecular diagnostic assays such as aspiration biopsy analysis and the detection of biomarker proteins in blood and urine. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4899. doi:10.1158/1538-7445.AM2011-4899