Tuesday, January 25, 2011

Cell Line Biomarker Characterization using a 22-plexed Human panel and a MAGPIX® Reader

Diagnostic biomarkers are a key element in cancer research. Research has focused primarily on intracellular biomarkers such as HER2 or BRAC1 that identify specific tumors, provide genetic or phenotypic information that can clarify the process of oncogenesis. Recently more attention is being placed on identifying soluble extracellular circulating biomarkers, which can provide information on the body response to cancer, as well as the relationship between a tumor cell and its environment. Because cancer is a series of different disease states, the study of individual biomarkers is usually inadequate to study the complex relationship between a tumor and its environment. Only with a large panel of different biomarkers can one discern the autocrine and paracrine interactions of the tumor and its host. While some biomarkers are tumor specific, such as PSA, others such as IL-8, are found in tumors of many different origins.

Recently, a new paradigm of xMAP Reader has been provided by Luminex which does not utilize flow cytometry principles for detection. The MAGPIX® Reader System has been developed by Luminex exclusively for MagPlex® microspheres. This technology allows for multiplex analyte determinations on biological samples. By using multiplexed bead sets several analytes can be quantitated simultaneously, saving time, money and precious sample. We are using a panel of known tumor biomarkers to characterize tumor cell lines of known lineage under different conditions to obtain a better understanding of the biology specific to different tumor types.

Figure 1. Standard Curves for the EMD-Millipore 22-Plex human Circulating Cancer Biomarkers using ELx50 Microplate strip Washer.
Figure 1. Standard Curves for the EMD-Millipore 22-Plex human Circulating Cancer Biomarkers using ELx50 Microplate strip Washer.

A key element in the ability to multiplex numerous samples is to adequately remove unbound materials while retaining the beads. Here we have used the ELx50 Microplate strip washer to automate the wash steps required to run the 22-Plex human Circulating Biomarkers panel from EMD-Millipore. As demonstrated in Figure 1, individual analyte calibration curves can be generated that will allow concentration determinations for 22 different analytes on each sample.
Figure 2. HepG2 Cell line Biomarker characterization.
Figure 2. HepG2 Cell line Biomarker characterization.

When dilutions (1:1, 1:3, and 1:6) of conditioned media supernatant from HepG2 cells are analyzed for the secretion of biomarkers a discrete pattern of expression is observed. HepG2 cells demonstrate measurable amounts of AFP, CYFRA21-1, IL-8, MIF, OPN, sFas, and VEGF that correlates with the concentration of the input material.

Determined concentrations of HepG2 cell supernatant.
Table 1. Determined concentrations of HepG2 cell supernatant
By interpolating the standard curves depicted In Figure 1 one can calculate levels of secreted biomarkers (Table 1). Significant amounts of specific cancer biomarkers are secreted by this human hepatocarcinoma cell line. Using this as a model we will be able to test other cell lines, including primary cell lines, under various conditions and treatments.

By, BioTek Instruments

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