Tuesday June 29, 2004
We are interested in studying the in vivo and ex vivo response to different immunomodulators and are using the RayBiotech cytokine antibody array as a rapid means to get a snapshot of the response. This technique allows for the determination of the type of immune response as defined by the cytokines that are produced, e.g. acute phase, Th1, Th2 type responses. The cytokine protein array system from RayBiotech Inc. allows simultaneous detection of multiple cytokines, chemokines, and growth factors in one experiment. The assay is easy, simple (some optimization required with regard to the appropriate dilution of the sample), sensitive, specific, requires a small sample volume, cost effective, requires minimal expertise and no prior sample preparation is needed. This is in contrast to the alternative methods which are limited to the measurement of individual cytokines. For example, setting up ELISA assays to measure different cytokines would be a cumbersome approach. They are also expensive, labor and time intensive and require an ELISA reader.
Different array systems are available from the company that allow for the detection of up to 120 cytokines, chemokines and growth factors from tissue culture supernatant, serum, plasma, cell lysate or certain tissue lysates. In addition to the different human or mouse cytokine/chemokine combinations, the company also provides an opportunity to get a custom array with your choice of cytokines, but of course this involves extra cost.
The assay is basically a sandwich dot blot. The procedure is simple and straightforward with three basic steps. (1) Incubate the sample (Serum, Tissue culture supernatant etc.) with the array membrane to allow cytokine binding to the immobilized antibodies on the array; (2) Detect the captured cytokines with a mixture of biotin-labeled anti-cytokine antibodies, creating an antibody "sandwich" around the bound cytokines, (3) Visualize active cytokines using streptavidin-HRP. The kit includes a chemiluminescent detection system that can be documented with X-ray film. The intensities of the signals can be quantitated by densitometry. After preparing a digital image of the result, the optical densities of the individual spots are quantified with the TotalLabtm analysis (Nonlinear Dynamics, Co.). An internal control on each membrane can be used to normalize the results within the membrane for comparison of different membranes. The system includes all the reagents required to do the assay.
I have used both mouse and human array system with serum and tissue culture supernatants as my samples and have gotten wonderful results with both the systems. It indicates the levels of the different kind of cytokines being stimulated. The assay allows for easy screening of the different immunomodulating peptides of interest. Results can be obtained within one day after an overnight incubation with sample. The only problems that we have had with the system are that we prefer using the ECL plus chemiluminescent system from Amersham Biosciences instead of the one included in the kit. Also, the internal positive controls on the membrane give a very high signal compared to the expected levels from samples. When the expression of a cytokine of interest is low, requiring a longer exposure time, the internal positive controls become overexposed preventing quantitation and they shadow the neighboring cytokine dots. This problem has been addressed by RayBioTech but the amount of the positive controls are still high, in my opinion.
Overall, my experience with cytokine antibody array system from Raybiotech has been very good. We are using this system regularly for our studies. It is the right choice for fast screening of multiple cytokine, chemokine and growth factor expression.
Neena Goel, Ph.D.
Research Assistant Professor
Department of Microbiology/Immunology
Northeastern Ohio Universities College of Medicine