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

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.

Figure 1

Mass spectrum of p53 used for immobilization on a glass slide. Three primary sequences, indicated by arrows, are shown from trypsin-digested fragments that uniquely identify p53. G, glycine; L, leucine; I, isoleucine; F, phenylalanine; P, proline; S, serine; T, threonine; Y, tyrosine; N, asparagine; Q, glutamine; D, aspartic acid; E, glutamic acid; K, lysine; R, arginine; H, histidine.

Figure 2

Diagram of protein immobilization chemistry. The p53 protein was immobilized through amide linkage to the (3-aminopropyl)trietoxysilane-modified glass surface using previously described methods (Weetall 1993). Diagram modified from Weetal with permission. Appl Biochem Biotechnol 41:157–188, 1993.

Figure 3

Absorbance spectra of local slide areas using the chromogenic DAB system. Effect of different treatments: p53 + p53 antibody (Ab)—covalently immobilized p53, incubated with anti-p53 Ab; BSA + p53 Ab—covalently immobilized BSA, incubated with anti-p53 Ab; p53—covalently immobilized p53, no Ab; BSA—covalently immobilized BSA, no Ab.

Figure 4

Fluorescent spectra of immobilized p53/p53 Ab/IgG–biotin using different streptavidin-conjugated fluorescent probes (fluorescent Cy3; quantum dot 605; Si nanocrystal; and Blank, no added probe). Dashed lines show BSA controls. Excitation wavelengths for fluorescent Cy3, quantum dot 605, and Si nanocrystal probes were 530, 450, and 280 nm, respectively.

Figure 5

Images of microscope slides containing immobilized p53: (A) chromogenic DAB probe, transillumination; (B) fluorescent Cy3 probe, laser illumination; (C) fluorescent Cy3 probe, laser illumination with filter. Immobilized p53 is located in center of slide; immobilized control BSA is located at both ends of slide and is not visible.

Figure 6

Microscopic images of p53 visualized using chromogenic probe: comparison of colon tissue sections and an immobilized p53 reference. Sequential slide sections taken from a biopsied colon tissue sample were prepared and stained using the Ventana chromogenic DAB system and compared with immobilized p53 reference slides using the same chromogenic system. (A) Tissue treated with anti-p53 Ab; (B) tissue, control (no primary Ab); (C) immobilized p53 reference treated with anti-p53 Ab; (D) immobilized p53 reference, control (no primary Ab). Bar = 50 μm.