Non-specific binding controls – wells that contain no analyte but that include all other assay components necessary to generate a signal these may be subtracted from all data points to correct for background, a process often referred to as blanking.Wavelength correction – this is typically built into a spectrophotometer and addresses changes in background absorption that can occur due to factors such as the type of plasticware or the presence of condensation.Other types of control worth considering include: They provide an indication of non-specific binding within the ELISA and, along with positive controls, are used in defining the standard curve. Negative controls lack one or more of the assay components necessary to detect the target analyte. They are used to generate a concentration curve against which the analyte of interest can be quantified, making them fundamental to the interpretation of ELISA data. Like positive controls, standards are known quantities of the target analyte. They typically comprise a known amount of the target analyte and produce a maximal assay signal that can be used in defining the standard curve. Positive controls function to confirm that the ELISA is performing as expected. It is good practice to use the same batch of control/standard material across all plates in an assay to avoid any intraplate variability, and to perform comparative studies every time a new lot is brought into use. For this reason, they should always be included on every plate and should ideally be run in duplicate or triplicate. Incubation time/temperature for signal developmentĪdditionally, it is important to identify suitable controls and standards and to determine how best to store and handle these.Ĭontrols and standards are essential to any immunoassay but are of particular importance to ELISA where they are used to quantify the analyte of interest.Incubation time/temperature for directly-conjugated detection antibody.The concentration of conjugated detection antibody.Sample dilution (dilution factor and choice of diluent).Incubation time/temperature for blocking.Choice and concentration of blocking solution.Incubation time/temperature for coating.In the example workflow described in Figure 10, factors requiring optimization are: Checkerboard titration experiment to simultaneously optimize the concentrations of both coating or primary and detection antibodies. By setting up multiple plates in parallel to compare different blocking solutions, incubation times, washing solutions and other factors, optimal assay conditions can rapidly be determined. For instance, using the example workflow depicted in Figure 10 (Part 2), the coating or primary antibody might be titrated across the 96 well microplate and the detection (secondary) antibody down the microplate while all the other variables are kept constant. Although a lesser degree of optimization is necessary when using pre-configured ELISA kits or matched antibody pairs, it is still essential that researchers establish optimal settings for their own unique study.Ī popular approach to ELISA optimization is to set up a checkerboard assay whereby the effect of several different variables can be compared in parallel. Where an ELISA is designed from scratch, each step must be thoroughly optimized to achieve the maximal assay window (the difference between full signal and background).