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Assay Tutorials : Dec 1, 2011 (Vol. 31, No. 21)

Analysis of Multiple Angiogenesis Analytes

New Panel Reveals More Information about Protein Interactions and Signaling Pathways
  • Wen-Rong Lie, Ph.D.
  • ,
  • Debra MacIvor, Ph.D.
  • ,
  • David Hayes, Ph.D.

Angiogenesis, the formation of new blood vessels, is a tightly controlled process with a key role in normal growth, development, wound healing, and transplantation rejection. When angiogenic pathways are disrupted, insufficient or excessive angiogenesis results in diseases such as coronary artery disease, ischemic chronic wound healing, autoimmune disease, macular degeneration, and cancer.

Angiogenic signaling in tumors is similar to normal angiogenesis, mediated by soluble growth factors, membrane-bound receptors, and cell-cell and cell-matrix interactions. Such signaling regulates cell migration, which is vital to angiogenesis. However, there are multiple differences between tumor angiogenesis and normal blood vessel formation.

Tumor endothelial cells proliferate faster than nontumor endothelial cells. Tumor vasculature differs from normal vasculature in morphology, enhanced leakiness, and structural abnormalities. Finally, tumor vessels are often less efficient at transporting oxygen to and removing waste products from all of the tumor tissues, resulting in frequent tumor cell necrosis.

The study of individual biomarkers, whether intracellular or circulating, is often inadequate to fully characterize the angiogenesis process in both normal and diseased states. Multiplexing of analytes offers significant advantages when studying complex pathways and biomarker interactions such as that found in angiogenesis.

This tutorial will describe the use of xMAP® bead-based, multiplexing technology (Luminex) with the MILLIPLEX® MAP Human Angiogenesis/Growth Factor Panel 1 (EMD Millipore). The panel includes 17 analytes that can be “plexed” together to study the role of circulating angiogenic biomarkers in a variety of diseases, as well as normal growth and development.

xMAP Multiplex Platform

Traditional “singleplex” protein-detection methods can be impractical for efficient exploration of complex cell-signaling pathways and protein-protein interactions such as those found in angiogenesis. The xMAP multiplex platform enables researchers to differentiate dozens of analytes per sample, significantly reducing the time, labor, and costs associated with methods such as ELISA and Western blotting, which are only semi-quantitative and offer limited throughput.

The xMAP platform consists of fluorescently dye-labeled magnetic microspheres, flow cytometry-based (LX200™ or FLEXMAP 3D®) or CCD camera-based (MAGPIX®) instruments, and software for data acquisition and analysis. With the xMAP platform, sandwich assays are performed on a bead (Figure 1).

Each bead contains two red dyes; 10 different concentrations of the dye yield 100 different possible combinations. Since the beads are in solution rather than fixed to a plate, up to 50 different beads with different capture antibodies can be used with one sample yielding results for up to 50 different proteins. When the beads pass through the reader, or in the case of MAGPIX camera, flow into the chamber, the ratio of the two dyes indicates the bead number.

Streptavidin-phycoerythrin, which fluoresces green, is used as the common detection reagent binding to biotinylated antibodies. The detection system reads red and green thus providing identification of the analyte and quantitation of the amount bound to the bead.

MILLIPLEX® MAP Angiogenesis Panel

The MILLIPLEX MAP Angiogenesis panel contains 17 analytes reported to be involved in the process of angiogenesis including cytokines and growth factors, as well as proteins requested by researchers. The configurable panel represents pro-angiogenic and anti-angiogenic factors, allowing researchers to study the process of angiogenesis in both normal and diseased states including cancer and cardiovascular disease. Inclusion of VEGF-A, C, and D and other factors allow the study of lympho- and vasculogenesis, the formation of the embryonic circulatory system.

Figure 2 shows the standard curves developed for a serial dilution of the 17 analytes. Premixed standards for the analytes were prepared as a 1:3 dilution. Data is reported as the average of duplicate wells and shown as median fluorescence intensity at each concentration. The data was fitted using a five-parameter fit, from which sample values were interpolated. The average values from duplicate wells were used to generate the values shown in Figure 3.

A collection of serum samples obtained commercially (Bioreclamation) were run using the MILLIPLEX MAP Human Angiogenesis/Growth Factor Panel protocol. The concentration value from duplicate wells was obtained by interpolation of the standard curve (shown in Figure 2) for each of the 17 analytes.

The mean value observed from healthy control (n=8), pregnancy (n=5), and various tumor types (n= 35) is displayed with blue columns representing control samples; red columns, pregnancy; and green columns, various tumor types. The insert shows two analytes (leptin and angiopoietin-2), which have higher values in serum than the other 15 members of this panel.

This new angiogenesis/growth factor panel allows researchers to study multiple analytes in one sample, yielding more information about complex protein interactions and signaling pathways than traditional methods deliver. Fully configurable, researchers can use the entire panel, or select those analytes of most interest.