Sanne Hoeks

Cytokine assays 93 6 Enzyme-linked immunosorbent assay The ELISA procedure 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 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. 58, 59 Downfalls in this procedure, 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 the protocol only permits the measurement of one cytokine at a time in a specified sample volume. 59 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. 46 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. At present, some of the most common multiplex assays used are the bead-based Luminex multi-analyte profiling (xMAP) technology [Luminex, Austin Texas, USA] and the cytometric bead array (BD Biosciences, San Diego California, USA) 58 or, the plate-based MSD array (Meso Scale Discovery, Gaithersburg, USA). 57 Meso scale discovery 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. 60 Initially, capture antibodies are coated onto the surface of a plate. Samples are then incubated on the plate followed by the addition of an electrochemiluminescence 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 method 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. 57