Michael Steffey, Sr. Associate Scientist, In Vitro Operations


February 21, 2017

In recent years many researchers have focused on the analysis of circulating soluble cancer biomarkers as an indicator of the host immune response to oncogenesis.1,2,3 Multiple biomarkers can be quantified in order to fully characterize changes in homeostasis brought on by tumor growth and metastasis. The ability to multiplex tumor-type specific antigens and general immune response biomarkers in a small plasma or serum sample can provide the researcher a broad picture of test agent efficacy.

MI Bioresearch’s (MI) use of Luminex® technology can quantify multiple cytokines, chemokines, and growth factors from a single well using as little as 12.5µl of plasma or serum. The technology uses fluorescently-coded magnetic microspheres coated with specific capture antibodies to bind the biomarker of interest. We then mix the sample with the biotinylated detection antibody to form a sandwich complex similar to an ELISA. A streptavidin-phycoerythrin reporter is added in the final step and instrument analysis follows. The instrument uses fluidics/optics/data analysis similar to that of a red/green laser flow cytometer and can process 96-well plates containing standards and samples in high throughput mode.

Fig. 1: Bio-Plex Pro™ Rat Cytokine 24-plex Assay:

Fig. 1: Bio-Plex Pro™ Rat Cytokine 24-plex Assay

Standards are plated in duplicate and the curves are analyzed using a four parameter nonlinear regression curve fit. The data shown (Figure 1) is from the Bio-Plex Pro™ Rat Cytokine panel but the number and types of analytes can be customized to suit the needs of the investigator.

In Figure 2, rat plasma samples were diluted four-fold in the supplied diluent and 50ul of the sample was assayed in triplicate. These samples show elevated levels of M1 macrophage inflammatory markers IL-1β, IL-6, TNF-α, IL-18, and MCP-1 and diminished levels of VEGF, a reported immunosuppressive cytokine.3 Different cancer models can display contrasting circulating cytokine profiles and studies can be designed to monitor changes in the particular profile desired. Panels can be created for mouse, rat, and human samples.

Fig. 2: Rat plasma samples analyzed for 24 different cytokines using the Bio-Plex Pro™ Rat Cytokine 24-plex Assay:

Fig. 2: Rat plasma samples analyzed for 24 different cytokines using the Bio-Plex Pro™ Rat Cytokine 24-plex Assay

MI multiplexes key biomarkers of tumor growth in small samples of plasma or serum providing the researcher with a practical but robust method for quantifying test agent efficacy in studies involving multiple cohorts. MI and an investigator can together customize the analyte panels to answer specific study questions and couple the in vitro analysis with MI’s in vivo imaging and pharmacology capabilities to generate complete preclinical oncology data packages.

Contact us today, to arrange your next Luminex® study helping to quantify your biomarkers and finding your test agent’s efficacy.

1N. Henry, and D. Hayes. Cancer biomarkers. Molecular Oncology 6 (2012) 140-146
2N. Goossens, S. Nakagawa, X. Sun, and Y. Hoshida. Cancer biomarker discovery and validation. Transl Cancer Res. 2015 June; 4(3): 256–269
3B. Burkholder et al. Tumor-induced perturbations of cytokines and immune cell networks. Biochimica et Biophysica Acta 1845 (2014) 182-201