Cytokine assays are an assessment of the preparation and treatment of blood and tissue samples related to cytokines. Cytokine assays are essential in disease diagnosing and monitoring because cytokines are pivotal players in the progression or regression of a pathological process and are biomarkers for many disease. Cytokines assays can be described as a serious of methods and protocols, including bioassays, protein microarrays, high-performance liquid chromatography (HPLC), sandwich enzyme-linked immunosorbent assay (ELISA), Meso Scale Discovery (MSD) electrochemiluminescence and bead based multiplex immunoassays (MIA).
Bioassays is a kind of a cytokine assays of looking 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 cytokine 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. 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. This combination of disadvantageous factors leads to the innovation of more specific and sensitive assays by which to measure cytokines directly.
Protein microarrays analyze the interaction, function and activity of cytokines on a large scale. This sensitive, high-throughput
method allows large numbers of cytokines to be measured rapidly, economically and in parallel. The protein chip used in this assay 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. Analytical (capture) microarrays use antibodies, aptamers or affibodies bound to the chip surface in order to bind the specific and desired cytokine 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 cytokine assay of 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 tertiary structure as these are vital in their interactive ability and biological activity. Protein array shelf life is relatively short due to protein denaturation and difficulties still exist in finding and isolating capture molecules for the wide range of proteins within the human genome. A complex balance between quantifying amounts of bound protein, maintaining sensitivity and reducing background noise is difficult to obtain especially due to the low affinity or low specificity of capture agents. Most poignant however, is the inability of this cytokine assay to provide a complete view of the proteome with abundant proteins overpowering the detection of less abundant proteins whose levels are also key in therapeutic analysis.
Unlike protein microarrays, high-performance liquid chromatography (HPLC) does not identify cytokines using protein–protein
interactions but rather, as compounds with specific weights, hydrophobicity, protonating abilities, ligand affinity and ion exchange. In short, HPLC is a kind of cytokine assay using the specific chemistry of each compound as a method of identification and separation. This method allows the quantification and purification of compounds by loading a sample onto a separation column containing solid particles under pressure. The sample is then separated into individual compounds according to their interaction with the column particles. The separation is in itself influenced by the liquid solvent condition and the chemical interactions between sample and solvent. HPLC has successfully been used to purify and separate cytokines such as IL-1 derived from various cells such as macrophage and epidermal cells. However, even with the ability to achieve better separation than ordinary liquid chromatography, HPLC is a less than optimal method of analysis. Some disadvantages of this process include a high cost and complexity, the coelution of compounds with similar structure and polarity, the irreversible absorption of compounds which then remain undetected and the low sensitivity of the apparatus to certain compounds as a result of the speed of the process.
It is known that several parameters must be met in order for a assay to be optimal for cytokine biomarker discovery. As seen in bioassays, protein microarrays and HPLC, parameters are often either suboptimal or conflicting within an cytokine assay. Brining each of these factors close to or within optimal range will, however, give way to the perfect assay. There are two types of sandwich antibody assays, those that are plate-based and those that are bead-based. Plate-based assays such as Sandwich Enzyme-linked immunosorbent assay (ELISA) and Meso Scale Discovery electrochemiluminescence (MSD) as well as bead-based assays such as multiplex immunoassays (MIA) are currently on the forefront of achieving the parameter goals required for cytokine biomarker discovery. In essence, these sandwich assays work with the principle of sandwiching a cytokine between two specific antibodies that intern bind to two none competing epitopes of that cytokine. For bead based analysis, the antibodies are either coated to a solid carrier (bead), acting as the capture antibody and or, in the case of the second antibody, bound to a labeled reporter. In plate-based assays however, the capture antibodies are bound in distinct positions within the wells of 96 well plate.
The ELISA procedure in cytokine assay encompasses the detection of an analyte within a liquid sample in a liquid environment within a reaction chamber. In lieu with heterogeneity of the assay, the desired component is separated from the analytical mixture by binding to an immobilized solid phase, usually the bottom of a transparent plate. Following this binding, substrate is added which is enzymatically converted, resulting in an optical change (colored or fluorescent) that allows the quantitative and qualitative measurement of the desired compound. The ELISA protocol of cytokine assay allows for high specificity and sensitivity as well as a wide analytical range and reproducibility, all of which are dependent on the type of biological fluid and cytokine being measured. Downfalls in this assay, however, include the inability to distinguish between bioactive and inactive compounds, varying binding affinity of antibodies as a result of differences in the internal structure of recombinant proteins used to generate these antibodies, large sample volumes, high reagent costs, a narrow dynamic range and the fact this assay only permits the measurement of one cytokine at a time in a specified sample volume. In order to overcome the inability to detect multiple cytokines simultaneously, the ELISA protocol was advanced to include a sequential ELISA analysis and ELISPOT assays however, these assays are time consuming, laborious and limited in their ability to detect a spectrum of cytokines. Taking into account the complex interaction between multiple cytokines during a disease process, an assay had to be developed that combated one of the major pitfalls of the ELISA protocol, that of an inability to sufficiently and accurately measure multiple cytokines.
The principle of MSD is based on a reaction in which an electron transfer in electrochemically generated intermediates causes these molecules to enter an excited state. Once excited these molecules can emit a photon of light when re-entering a lower energy level. Initially, capture antibodies are coated onto the surface of a plate. Samples are then incubated on the plate followed by the addition of an electrochemiluminescent tagged antibody. Analysis of this plate reveals fluorescent regions in which specific interactions have occurred between the antibodies and analyte, allowing both a quantitative and qualitative analysis of the desired compound. This cytokine assay is highly sensitive, has low background, does not incorporate washing steps and most importantly, allows the detection of multiple analytes at the same time. What remains unknown however is, how this assay will perform in various sample matrices.
In bead-based multiplex immunoarrays, identifiable bead sets are stably coated with desired and specific capture antibodies. These beads are then incubated with a small sample volume allowing the capture of the analyte that binds specifically to the capture antibody. Following this, labeled detection antibodies bind to the analyte- capture antibody-bead complex to make a four membersolid phase sandwich that when passed through the detection system allows the identification and quantification of the desired compound. In comparison to ELISA, These cytokine multiplex assays in general are more sensitive, show a broad analytical and dynamic range – measuring a few pg/ml, are highly specific, are rapid, require smaller sample volumes and allow the simultaneous measurement of up to 500 different proteins. Even in the presence of all these advantageous features however, these multiplex assays, similar to other antibody based technologies, are affected by the presence of heterophilic and auto- antibodies. These antibodies cause false positive and false negative signals by binding to either the capture antibody, detection antibody or to the antigen. In order to combat this phenomenon three methods can be undertaken. The first involves heterophillic/auto- antibody blockage with animal serum (ineffective when antibody titers are high- as seen in a diseased state). The second is through the use of internal assay markers that allow the interfering antibodies to be monitored. These internal markers can clearly indicate when heterophillic or auto-antibodies are influencing data and can thus allow the exclusion of unfit samples during assay analysis. The third and most preferable method is the removal of these antibodies by incubating samples with either protein-L, or with antibodies cocktail such as HeteroBlock.
• Keustermans G C E, et al. Cytokine assays: an assessment of the preparation and treatment of blood and tissue samples[J]. Methods, 2013, 61(1): 10-17.