Immunoassays ELISA - Proteins Analytical Techniques

Immunoassays ELISA

Enzyme-linked immunosorbent assay (ELISA) pro-vides a means to quantitatively measure extremely small amounts of proteins in biological fluids and serves as a tool for analyzing specific proteins during purification. This procedure takes advantage of the observation that plastic surfaces are able to adsorb low but detectable amounts of proteins. This is a solid phase assay. Therefore, antibodies against a certain desired protein are allowed to adsorb to the surface of microtitration plates. Each plate may contain up to 96 wells so that multiple samples can be assayed. After incubating the antibodies in the wells of the plate for a specific period of time, excess antibody is removed and residual protein binding sites on the plastic are blocked by incubation with an inert protein. Several microtitration plates can be prepared at one time since the antibodies coating the plates retain their binding capacity for an extended period. During the ELISA, sample solution containing the protein of interest is incubated in the wells and the protein (Ag) is captured by the antibodies coating the well surface. Excess sample is removed and other antibodies which now have an enzyme (E) linked to them are added to react with the bound antigen.

The format described above is called a sandwich assay since the antigen of interest is located between the antibody on the titer well surface and the antibody containing the linked enzyme. Figure 10 illustrates anumber of formats that can be used in an ELISA. A suitable substrate is added and the enzyme linked to the antibody–antigen–antibody well complex con-verts this compound to a colored product. The amount of product obtained is proportional to the enzyme adsorbed in the well of the plate. A standard curve can be prepared if known concentrations of antigen are tested in this system, and the amount of antigen in unknown samples can be estimated from this standard curve. A number of enzymes can be used in ELISAs. However, the most common ones are HRP and AP. A variety of substrates for each enzyme are available which yield colored products when catalyzed by the linked enzyme. Absorbance of the colored product solutions is measured on plate readers, instruments which rapidly measure the absorbance in all 96 wells of the microtitration plate, and data processing can be automated for rapid throughput of information. Note that detection approaches partly parallel those discussed in the section on Blotting. The above ELISA format is only one of many different methods. For example, the microtitration wells may be coated directly with the antigen rather than having a specific antibody attached to the surface. Quantitation is made by comparison with known quantities of antigen used to coat individual wells.

Another approach, this time subsequent to the binding of antigen either directly to the surface or to an antibody on the surface, is to use an antibody specific to the antibody binding the protein antigen,that is, a secondary antibody. This latter, secondary, antibody contains the linked enzyme used for detec-tion. As already discussed in the section on blotting, the advantage to this approach is that such antibodies can be obtained in high purity and with the desired enzyme linked to them from commercial sources. Thus, a single source of enzyme-linked antibody can be used in assays for different protein antigens. Should a sandwich assay be used, then antibodies from different species need to be used for each side of the sandwich. A possible scenario is that rabbit antibodies are used to coat the microtitration wells; mouse antibodies, possibly a monoclonal antibody, are used to complex with the antigen and then a goat anti-mouse immunoglobulin containing linked HRP or AP is used for detection purposes.

As with immunoblots discussed above, strepta-vidin or avidin can be used in these assays if biotin is covalently linked to the antibodies and enzymes (Fig. 10).

If a radioactive label is used in place of the enzyme in the above procedure, then the assay is a solid phase radioimmunoassay (RIA). Assays are moving away from the use of radioisotopes, because of problems with safety and disposal of radioactive waste and since non-radioactive assays have compar-able sensitivities.