Profiling of cytokine expression by biotin‐labeled‐based protein arrays

Abstract

Global analysis of protein expression holds great promise in basic research and patient care. Previously we demonstrated that multiple cytokines could be detected simultaneously using an enzyme-linked immunosorbent assay protein array system with high sensitivity and specificity. In this paper, we described a biotin-labeled-based protein array system to detect multiple cytokines simultaneously from biological samples. In this new approach, proteins from a variety of biological sources are labeled with biotin. The biotin-labeled proteins are then incubated with antibody chips. Targeted proteins are captured by the array antibodies spotted on the antibody chips. The presence of targeted proteins is detected using Cy3- or Cy5-conjugated streptavidin and signals are imaged by laser scanner. The system also can be easily adapted to a two-color binding assay, allowing measurement of the levels of proteins in a test sample with respect to a reference sample at the same chip. To demonstrate its potential applications, we applied this technology to profile human cytokines, chemokines, growth factors, angiogenic factors and proteases in estrogen receptor (ER)+ and ER- cells. These results suggest that biotin-labeled-based antibody chip technology can provide a practical and powerful means of profiling hundreds or thousands of proteins for research and clinical purposes.

Glomerular endothelium in kidneys with congenital nephrotic syndrome of the Finnish type (NPHS1)

“…The experiments were performed according to manufacturer's instructions and analysed as previously described [18]. The results were expressed as relative signal intensities so that positive control spots included in each membrane were given an intensity value of 100.…”

Antibody array-based technologies for cancer protein profiling and functional proteomic analyses using serum and tissue specimens

“…In neoplastic diseases, they have been evaluated to a lower extent, although the implementation of cytokine antibody arrays is increasing in many aspects of cancer research, such as the discovery of biomarker candidates, molecular mechanisms of cancer development, preclinical studies, and the effects of cancer compounds. Studies linking clinical material (serum) and in vitro systems have revealed the potential of cytokine profiling using antibody arrays for characterizing hematological neoplasias [8][9][10], or in serum of patients with breast cancer [30,31]. Cytokine profiles can support differentiation between cancer patients from control subjects and also stratify patients with leukemia based on clinical outcome.…”

“…Several reports have also compared the reproducibility and differences among the several technologies available for multiplexing cytokine measurements, including not only planar antibody arrays but also bead-based technologies [13][14][15]. Cytokine profiles of cell lysates have also been analyzed by means of cytokine arrays and compared to those obtained on body fluids and tissue extracts [30]. Commercially available cytokine arrays were applied to conditioned media of cancer cells to dissect functional cytokine-secreted signatures associated to the overexpression of critical breast cancer target genes in breast cancer cells.…”

Changes in glomerular mesangium in kidneys with congenital nephrotic syndrome of the Finnish type

“…The experiments were performed according to the manufacturer's instructions and analyzed as previously described [25].…”