Immunochemical Detection of Antigens After Electrotransfer Immunoblotting

4 Immunochemical Detection of Antigens After Electrotransfer (Immunoblotting) 

Macromolecules blotted onto membranes are detected very specifically and sensitively by antibodies. The prerequisite is the existence of epitope(s) on the surface of blotted antigens after denaturation during SDS-PAGE. Very often, even fragile epitopes (which are 

Immunochemical detection is possible in one step, if the detecting antibody carries the signal-forming principle. But also cascades of steps are used to identify the first-bound antibody and by it the antigen immobilized by blotting. Antibodies form these cascades, i.e., if the first bound antibody is from species A, e.g., rabbit, a second antibody from other species, e.g., goat, directed against the primary, is used. 

A second possibility is the modification (conjugation) of an antibody by a label, e.g., biotin, which is detected later on by a specific receptor, e.g., (strept)avidin. In each case the last part of such a cascade has to carry a measurable label. Such labels are enzymes, fluorescent dyes, colloids, radioactive isotopes, paramagnetic substances, and others. 

As indicator enzymes horseradish peroxidase (HRP or HRPO), alkaline phosphatase (AP), or /i-galactosidase, are favored, since they are relatively robust, have a high product-forming rate, are easy to purify, and are cheap. The most used colloids are from gold, silver, and iron, and iodine isotopes are mostly taken as radioactive labels in immunoassays. 

---