Immunoblotting, dot-blotting, and ELISPOT assays: methods and applications.

Immunoblotting (or Western blotting) involves the transfer of proteins that have been separated by electrophoresis or isoelectric focusing (IEF) from the electrophoresis gel to a membrane, to which they become bound. The bound molecules are then detected by a specific probe, usually an antibody. The advantage of immunoblotting is that it combines the resolution obtained by electrophoretic separation of mixtures of proteins in a gel with the specificity of antibodies used as probes to identify individual antigens. Proteins can be separated by any of the electrophoretic techniques available by using agarose, polyacrylamide in the presence or absence of sodium dodecyl sulfate (SDS), isoelectric focusing (IEF), or two-dimensional polyacrylamide gel electrophoresis (2-D PAGE). Direct overlay of the gel with antibody (immunofixation) has been used to identify antigens of interest, but such methods suffer from the disadvantages of prolonged incubation times, resulting in diffusion of the bands, and consequent loss of resolution. Immunofixation is also primarily limited to agarose gel systems, since antibody molecules cannot readily penetrate polyacrylamide gels, owing to the gels’ small pore size. Because of these difficulties, the idea of transferring electrophoretically separated proteins from a gel to a membrane, where they are readily accessible to high relative molecular mass (M,) probes, such as antibody molecules, opened up a new vista for ,electrophoretic analysis of proteins. The method of transfer is termed blotting, since the pattern of bands on the membrane is an exact replica of the pattern in the original gel. The Southern-blotting method for analysis of DNA was named after Ed Southern; hence, analysis of RNA molecules by a similar technique was called Northern blotting, and analysis of proteins by transfer to a membrane and detection by antibody became known as Western blotting or immunoblotting. Dot-blotting, slot-blotting, and line-blotting are variants of this in which antigens are applied directly to a membrane without prior separation and identified in the same way. The amount of antigen present in the mixture can be measured quantitatively. ELISPOT assays use similar principles to measure the number of cells secreting specific antibody or other proteins. Many adaptations have been developed and applied to a wide variety of fields of research and clinical applications. In this chapter I shall discuss the basic techniques and principles of immunoblotting, dot-blotting, and the ELISPOT assay, and describe a selection of some of their