To detect antigen A, purified antibody specific for antigen A is linked chemically to an enzyme. The samples to be tested are coated onto the surface of plastic wells, to which they bind nonspecifically; residual sticky sites on the plastic are blocked by adding irrelevant proteins (not shown). The labeled antibody is then added to the wells under conditions that prevent nonspecific binding, so that only binding to antigen A causes the labeled antibody to be retained on the surface. Unbound labeled antibody is removed from all wells by washing, and bound antibody is detected by an enzyme-dependent color-change reaction. This assay allows arrays of wells known as microtiter plates to be read in fiberoptic multichannel spectrometers, greatly speeding the assay. Modifications of this basic assay allow antibody or antigen in unknown samples to be measured as shown in Figs. A.6 and A.25.
is added to the well and allowed to bind to the unlabeled antigen under conditions in which nonspecific adsorption is blocked, and any unbound antibody and other proteins are washed away. Antibody binding in RIA is measured directly in terms of the amount of radioactivity retained by the coated wells, whereas in ELISA the binding is detected by a reaction that converts a substrate into a reaction product of a different color (Fig. A.5). The color change can be read directly in the reaction tray, making data collection very easy and providing a quantitative measurement of reaction product concentration; furthermore, ELISA also avoids the hazards of radioactivity. This makes ELISA the preferred method for most direct-binding assays. In a variation of this assay, labeled antiimmunoglobulin antibodies can also be used in RIA or ELISA as a second layer, following the binding of unlabeled antibody to unlabeled antigen-coated plates. The use of such a second layer amplifies the signal, because at least two molecules of the labeled anti-immunoglobulin antibody are able to bind to each unlabeled antibody. As mentioned above, RIA and ELISA can also be carried out in reverse when the goal is to determine the amount of antibody in a solution; in this case, unlabeled antibody is adhered to the plates, labeled antigen is added, and the amount of labeled antigen bound after washing is measured.
A modification of ELISA known as a capture or sandwich ELISA (or more generally as an antigencapture assay) is commonly used to detect secreted products such as cytokines. Rather than the antigen being directly attached to a plastic plate, antigen-specific antibodies are bound to the plate. These are able to bind antigen with high affinity, and thus concentrate it on the surface of the plate, even with antigens that are present in very low concentrations in the initial mixture. A separate labeled antibody that recognizes a different epitope from that recognized by the immobilized first antibody is then used to detect the bound antigen.
Another variant of the antigen-capture assay, often referred to as a multiplex assay, has been developed to allow quantitation of multiple antigens in a single sample. This technique is often utilized to examine the levels of multiple cytokines in clinical serum samples, or in sera from experimental animals, cases in which it is not feasible to assess each cytokine of interest individually. For this type of assay, small microspheres are differentially labeled with fluorescent dyes that can be distinguished based on their distinct emission spectra. Microspheres labeled with a given fluorescent dye are conjugated to antibodies specific for one antigen, for instance, a single cytokine. The microspheres— up to 100 different microspheres with unique identifiers—are added to the sample to capture the antigen. Bound antigen is then detected using a second antibody that binds the antigen at a distinct site. This second antibody is conjugated to a different fluorescent dye, and the magnitude of its fluorescence is a measure of the quantity of bound antigen. The machine that performs this multiplex analysis, the Luminex® analyzer, then measures the amount of fluorescence associated with each differentially labeled microsphere.