Microarrays, cytokines

Antibody arrays are used in detection and expression profiling of e.g. cytokines (Figure 3)2 4 and allergens (www.vbc-genomics.com). Microarrays containing antigens are developed for detection of autoantibodies in rheumatoid diseases5. 

A single cell suspension of LNC is prepared under aseptic conditions and cultured for various periods of time. The production by LNC of type lcytokines (such as IFN-y and IL-12) and type 2 cytokines (such as IL-4, IL-5, IL-10 and IL-13) is measured using cytokine specific ELIS As or cytokine microarrays. It is possible to measure the spontaneous production of IL-5, IL-10, IL-12, IL-13 and IFN-y by LNC without restimulation in vitro. However, IL-4 appears to be produced in comparatively small amounts and to induce detectable levels of this cytokine an additional stimulus is required concanavalin A (con A), a T lymphocyte mitogen. Culture of LNC derived from mice treated with chemical respiratory sensitizer with con A will stimulate the production of detectable levels of IL-4. Treatment with the same mitogen of LNC derived from naive or vehicle-treated mice fails to induce measurable IL-4 secretion [86],... 

The basal immunological mechanisms are complex but can now be analyzed with refined and more powerful laboratory methods, such as the microarray technique. With the help of this technique, additional information besides that concerning IgE and cytokine levels about the association between adaptive and innate immune response as well as other cellular responses can be gathered (6-8). 

A number of commercial antibody-based microarrays for multiplexed cytokines analysis are now available (Beckman Coulter BD Biosciences Panom-ics Pierce S S Zyomyx and others). Cytokines are essentially biomarkers of cell injury, inflammation, and apoptosis. They are released by cells in culture in response to drug action (Turtinen et al., 2004) or are elevated in serum in various disease states. Moreover, numerous cytokines are involved in cellular response and many serve as dual effectors (Asao and Fu, 2000). As a result, anticytokine microarrays are being evaluated in drug discovery for off-target toxicity testing to replace standard ELISA plate formats. 

Huang etal. (2002) prepared an antibody array for the simultaneous detection of 43 cytokines. They were able to verify the down-regulation of MCP-1 cytokine in transfected cells (human glioblastoma cells transfected with cx43 expression vector) relative to control cells. The antibody array is an emerging technology. In at least one study based upon the use of a commercial membrane format, the cytokine microarray failed to accurately determine cytokine levels in bacterial and lipopolysaccharide (LPS)-stimu-lated whole human blood (Copeland, 2004). 

Membranes cast upon glass slides also fall into the 3D surface category. Most notable are the nitrocellulose-coated FAST slides offered by Schleicher Schuell (S S) BioScience. A cytokine micro-ELISA product xmder the trade name Provision has been introduced in single-slide and 96-well spacing (64 usable wells) formats. It is called FAST Quant (Harvey, 2003). Historically, nitrocellulose membrane has been used for the sequestering of both proteins and nucleic acids. The adaptation by S S of this microporous (0.2-p pores) material cast into a microarray format has been relatively straightforward. 

Song, Y, Boyer, D., Leung, L, Moheb, S., Gu, J., Doan, A.T., Khan, A., Flores, 1., Yee, F.S., and Wong, J.R., A microarray system A novel multiplexed assay platform for cytokine profiling, 95th Annual Meeting, American Association for Gancer Research, Orlando, FL, March 27-31, 2004. 

Other early work includes that of Moody et al. (2001) who spotted anticytokine monoclonals onto the bottom of polystyrene microtiter plates (Max-isorp, Nalge Nunc) and measured cytokine levels in stimulated peripheral blood mononuclear cells. Finally, although not strictly a microarray, the microwell array system developed by Michael Snyder s group at Yale University to measure kinase activity is a simple and elegant approach (Zhu et al., 2000). The "protein chip" is comprised of microwells fabricated in a flexible elastomer of PDMS [poly(dimethylsiloxane)] substrate by a molding process. 

Matson, R.S., Milton, R.G., Gress, M.D., and Rampal, J.B., Microarray-based cytokine immunosorbent assay. Oak Ridge Gonference, 2001, Poster 20. 

While in most instances the RCA microarray immunoassay results were comparable to published performance reports on commercial ELISA (Quan-tikine, R D Systems), there were notable exceptions in which ELISA appeared to out-perform the microarray by 10- to 50-fold sensitivity. Such differences may be due to variations in binding affinities exhibited by the particular capture antibodies employed in the assay. In studies involving lipopolysaccharide (LPS)-induced secretion of cytokines from human... 

In reality, these forays represent miniaturization of the standard sandwich ELISA to attain higher throughput assays by multiplexing a limited number (<50) of analytes, e.g., cytokine panels. Even at these low densities, quantification problems arise in part due to a lack of robustness in the printing process and also in the selection and stability of monoclonal antibodies that must be highly specific and of high binding affinity to be useful for microarrays. 

Genomic tools 31 This method takes advantage of select genomic (microarray-or PCR-based approaches) and proteomic (antibody array analysis) tools to identify the receptors for growth factors, hormones, cytokines, and other components of cell signaling pathways expressed by a culture of interest. 

Many assays that have been bead-captured and gel-based have been converted to 96-well polystyrene microtiter plates to standardize the format for automation and higher throughput. In many ways, plate formats are a bridge to the higher-density microarray formats and remain quite useful until full conversion to microarray or microfluidic formats can be made. Microtiter plate assays or activated microbeads (i.e., Luminex) for panels of cytokines are widely available because of their use in clinical medicine. 

Antibody arrays immobilized on glass surfaces mimic DNA microarrays in format and spot size. The biggest challenge in protein profiling using antibody microarrays is selection of validated antibodies that are useful in the desired sample environment. Many of the initial reports used antibody arrays assayed for cytokines because serum presents a relatively simple sample assay environment compared to tissue and also because there are numerous validated antibodies available for this clinically important set of proteins. Tissue and cell lysates present more complex assay environments with more opportunities for antibody cross-reactivity and other interferences which erode the biological meaningfulness of the data. 

---