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Abstract

Immunoassay is a biochemical method that identifies and quantifies unknown analytes (protein, lipid, nucleic acid etc.) in solution (serum, urine etc.) using antibody-antigen reactions. There are many different formats and variations of an immunoassay, but the key point is still specific antibody-antigen recognition. What is an antigen and an antibody? Why can an antibody bind to an antigen?An antigen is a molecule that is recognized by the immune system, particularly by antibodies. Proteins, polysaccharides, pesticides, antibiotics, toxins, and hormones all can be antigens. But not all antigens can stimulate the immune system to generate antibodies. An antigen that can elicit an immune response, and particularly antibody synthesis is called an immunogen. Proteins with molecular weights higher than 5-10 kDa are immunogens, while small peptides, pesticides, antibiotics, or hormones are not. These low-molecular-weight substances are called haptens and must be chemically coupled to larger carrier molecules, such as bovine serum albumin or keyhole limpet hemocyanin, in order to elicit specific antibody formation.An antibody is a Y-shaped large protein (160 kDa) bearing two combining sites (paratopes) that bind to a limited surface (epitope) of the antigen specifically. Epitopes are mostly located on the hydrophilic part of the antigen, and their size is 10-15 amino acid residues that can be contiguous on the protein chain (linear epitope) or discontinuous yet spatially close on the protein chain.The antibody-antigen reaction is a typical reversible bimolecular reaction having rate constants for the forward and backward reactions that are dependent on the concentration of the antigen (Ag) and antibody (Ab), affinity for the antigen as defined by the association constant of the antibody for its antigen, temperature, pH, and other environmental conditions. Immunoassays are based on the binding and complex of an antigen to an antibody and people use some physical or chemical means to measure and quantify the antigen-antibody complex.