Quantitation of HER2 and telomerase biomarkers in solid tumors with IgY antibodies and nanocrystal detection

Abstract

In an effort to improve affinity biomarker validation in fixed patient tissue specimens, we have developed a novel quantum dotbased bioimaging system that utilizes chicken IgY antibody for high sensitivity and specificity relative quantitation of cancer proteins. Monospecific, polyclonal IgYs were generated against human HER2 and telomerase, and analytically validated for specificity by western blot and immunohistochemistry on tumor and normal cells and for relative affinity by layered peptide array (LPA). IgYs bound desired targets in cell lines and fixed tissues and showed greater affinity than commercial mammalian antibodies for both HER2 and telomerase proteins. In tissue microarray experiments, HER2 quantitation with IgY antibody and quantum dot imaging correlated well with chromogenic in situ hybridization (CISH), whereas telomerase quantitation suggested a trend toward correlation with prostate cancer Gleason Grade and differentiation. Although patient numbers were small, these findings demonstrate the feasibility of relative quantitation of cancer biomarkers with IgY and quantum dot fluorophores, and show promise for rigorous clinical validation in large patient cohorts. ' 2008 Wiley-Liss, Inc.Key words: IgY antibody; cancer biomarker; HER2; telomerase; quantum dots Few new early cancer biomarkers have surfaced in recent years. 1 Among novel proteomic biomarkers for early cancer detection, some have proven controversial. 2,3 Analytical and clinical validation of cancer biomarkers has suffered from bias in the design, conduct and interpretation of such research, 4 incompletely validated imaging, 5 and lack of affinity standards 6 and antibodies that did not, in fact, detect correct targets. 7 Here, we describe a novel approach to cell-based bioimaging with relative quantitation for biomarker validation. We report characterization of two new IgY antibodies for quantitation of model cancer biomarker systems, HER2 and telomerase, 8 and explore analytical improvements, including low cross-reactivity IgY-isotype chicken polyclonal antibodies raised against recombinant polypeptides; digital quantification of antibody signals with streptavidin-conjugated semiconductor nanocrystals to obviate photobleaching of organic fluorescent dyes; complete z-plane fluorescence image capture using 3D-deconvolution microscopy; high-throughput, automated, robotic slide processing; and quantitative, massively parallel, high-throughput analysis of peptide antigen-antibody interactions by layered peptide array (LPA) technology. 9

Immunohistochemical determination of the appropriate anti-hTERT antibodies for in situ detection of telomerase activity in frozen sections of head and neck squamous cell carcinomas and tumor margin tissues

“…That is why Ab 8 (code ab23699), Ab 9 (code 600-401-252), Ab 10 (code NB 100-297) and Ab 11 (code NB 100-317) are also well suited for the detection of hTERT expression and field cancerization. Considering the evidence given by Wu et al (23,106) and our results for immunoabsorption, the following conclusion can be drawn: antibodies Ab 4 and Ab 9 to Ab 11 are particularly well suited for identifying field cancerization and for proof of MRD. We were unable to demonstrate absorption of these antibodies with the nucleolin peptide ab25315 (Fig.…”

“…Manufacturers of the NCL-L-hTERT antibody should feel prompted by the critical publications of Wu et al to re-examine the antigen specificity of their antibody. Xiao et al (106) conclude from their studies on culture cells and tissues fixated in formalin that chicken IgG antibodies can have definite advantages over the commonly used mammal IgG antibodies and exhibit less non-specific staining. Irrespective of the afore-mentioned objections, many authors continue to use the monoclonal NCL-L-hTERT for immunohistochemical proof of hTERT (63,66,(107)(108)(109)(110)(111)(112).…”

Cell Lines as Candidate Reference Materials for Quality Control of ERBB2 Amplification and Expression Assays in Breast Cancer

“…Chicken anti-HER2 IgY antibody (YXPB-IgY-hHER2/p) was prepared as described previously (13 ). Mouse anti-HER2 IgG monoclonal antibody (CB11) was purchased from Ventana Medical Systems.…”

Biosynthesis of Quantum Dots and Their Potential Applications in Biology and Biomedicine

“…In [88], using QDs with maximum emission wavelength 605 nm to detect CA 125 in ovarian cancer specimens of different types (fixed cells, tissue sections, and tumor xenografts) with high specificity and sensitivity is reported. Several studies on the detection of human epidermal growth factor receptor (HER 2) for breast cancer diagnosis with QDs have been completed [89]. HER 2 on breast cancer cell membrane is found to be overexpressed in approximately 25-30 % breast cancer and plays an important role in breast cancer prognosis and treatment selection.…”