Standards for immunohistochemical imaging: a protein reference device for biomarker quantitation.

Author(s): Atha DH,  Manne U,  Grizzle WE,  Wagner PD,  Srivastava S,  Reipa V

Journal: J Histochem Cytochem

Date: 2010 Nov

Major Program(s) or Research Group(s): CBRG

PubMed ID: 20805583

PMC ID: PMC2958133

Abstract: We are developing a reference device to be used in the validation of immunohistochemical imaging of biomarkers by microscopy. The prototype device consists of p53 protein immobilized at various concentrations on a glass slide. The device is designed as a reference control to be used with assays that incorporate commercially available anti-p53 antibodies. p53 protein was characterized by mass spectrometry and covalently immobilized through amide linkage to the (3-aminopropyl)trietoxysilane-modified glass surface. This procedure is reproducible and provides a chemically stable product in high yield. The surface-bound protein was shown to be immunoreactive by its specific interaction with anti-p53 antibody (Ab) and detection by absorbance and fluorescence spectroscopy. Also, comparison was made with microscopic images of Ab-stained tissue samples, known to stain positive for p53. Further development will be required to establish accurate surface protein concentrations in the range required for specific clinical applications.