Sanne Hoeks

Chapter 6 90 METHODS TO ANALYZE CYTOKINES Cytokines can be measured at various levels. mRNA can be detected by real time PCR 44 and intracellular proteins can be detected by flowcytometric assays of permeabilized cells. 45 Due to the range and location of cytokine activity, there are a plethora of methods by which to measure cytokine levels. Irrespective of the chosen methodology, several factors are key to creating the optimal protocol by which to analyze cytokines as biomarkers. Parameters should include (a) a high specificity, affinity and sensitivity (b) make use of a simple protocol (c) be reproducible and reliable (d) be time and cost effective and (e) allow the detection of multiple cytokines within a small sample volume. 46, 47 This review focuses on the measurement of cytokines at a protein level describing various techniques that can be used for their detection. Pro’s and con’s of the various methodologies are summarized in Table 1. Bioassays Bioassays look at cytokines and their activity within a system by focusing on their biological activity and using this as a read out. In a bioassay the activity of a sample is tested on a sensitive cell line and the results of this activity are compared to a standard cytokine preparation. This assay is highly sensitive and permits the detection of bioactive molecules however, it is also semi-quantitative, low in specificity, shows a narrow analytical range, is time consuming and requires a large sample size. 46 In addition to these factors, cell lines respond to different molecules present within a sample thus, any changes observed may be the result of one or multiple compounds. 48 This combination of disadvantageous factors leads to the innovation of more specific and sensitive assays by which to measure cytokines directly. Protein Microarrays Protein microarrays analyze the interaction, function and activity of proteins on a large scale. 49 This sensitive, high-throughput method allows large numbers of proteins to be measured rapidly, economically and in parallel. 50 The protein chip used in this procedure is comprised of a support surface to which a range of capture proteins are bound. Fluorescently labeled probe molecules are then added to the array and upon interaction with the bound capture protein, a fluorescent signal is released and read by a laser scanner. 51 Analytical (capture) microarrays use antibodies, aptamers or affibodies bound to the chip surface in order to bind the specific and desired protein within a complex protein solution, commonly cell lysate. The subsequent protein interactions provide information on the expression levels, binding affinity and or specificity of the proteins within the solution permitting comparisons of protein expressions between various solutions. The protein microarray system is applicable for biomarker detection by allowing protein expression profiling however, some challenges do exist for this procedure. Downfalls come in the form of difficulties manufacturing chips with stable proteins holding the necessary primary or