Biochemical Science Division, National Institute of Standards and Technology, Bldg. 227, Room A243, Mail Stop 8311, 100 Bureau Drive, Gaithersburg, MD 20899-8311, USA.
J Histochem Cytochem. 2010 Nov;58(11):1005-14. doi: 10.1369/jhc.2010.956342. Epub 2010 Aug 30.
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.
我们正在开发一种参考设备，用于通过显微镜验证生物标志物的免疫组织化学成像。该原型设备由固定在载玻片上的不同浓度的 p53 蛋白组成。该设备设计为与包含市售抗 p53 抗体的测定一起使用的参考对照。通过质谱法对 p53 蛋白进行了表征，并通过酰胺键共价固定在（3-氨丙基）三乙氧基硅烷修饰的玻璃表面上。该过程具有重现性，并以高产率提供化学稳定的产物。通过其与抗 p53 抗体（Ab）的特异性相互作用及其通过吸光度和荧光光谱的检测，显示表面结合的蛋白质具有免疫反应性。此外，还与已知 p53 染色阳性的 Ab 染色组织样本的显微镜图像进行了比较。需要进一步开发以确定特定临床应用所需范围内的准确表面蛋白浓度。
