Standards for immunohistochemical imaging: a protein reference device for biomarker quantitation.
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.
Atha
Donald H
DH
Biochemical Science Division, National Institute of Standards and Technology, Bldg. 227, Room A243, Mail Stop 8311, 100 Bureau Drive, Gaithersburg, MD 20899-8311, USA. donald.atha@nist.gov
Manne
Upender
U
Grizzle
William E
WE
Wagner
Paul D
PD
Srivastava
Sudhir
S
Reipa
Vytas
V
D000595:Amino Acid Sequence; D015415:Biomarkers / Q000378:metabolism; D005898:Glass / Q000737:chemistry; D006801:Humans; D055767:Immobilized Proteins / Q000737:chemistry / Q000276:immunology / Q000378:metabolism; D007150:Immunohistochemistry / Q000295:instrumentation* / Q000592:standards*; D013058:Mass Spectrometry; D008853:Microscopy; D057054:Molecular Imaging / Q000295:instrumentation* / Q000592:standards*; D011786:Quality Control; D012015:Reference Standards; D015203:Reproducibility of Results; D013050:Spectrometry, Fluorescence; D016159:Tumor Suppressor Protein p53 / Q000737:chemistry / Q000276:immunology / Q000378:metabolism
D015415:Biomarkers; D055767:Immobilized Proteins; D016159:Tumor Suppressor Protein p53
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