Precipitation of Immunoglobulins and Antigen

INTERACTIONS AND PRECIPITATION OF IMMUNOGLOBULINS AND ANTIGEN

B lymphocytes are essential mediators of adaptive immune response. Plasma cells, the activated form of B cells after specific-antigen stimulation immunsely produce and secrete proteins, antibodies, which are soluble gycloproteins that bind specifically and non-covalently to their unique and individual antigens. Antibody specificity is determined by the variable region, composed of the combination of light and heavy chains. The binding of soluble antigen to antibody forms an immune complex. This specific interaction enables antibodies to neutralize, opsonize and inactivate toxic substances and pathogens. However, these complexes vary in size depending on both the antigen and antibody concentrations. When sufficient antibody is available to crosslink, due to the duplicate binding sites on an antibody molecule, from two arms, large immune complexes are subsequently formed. These complexes are cleared by the reticuloendothelial system of cells which bear Fc receptors and complement receptors. Moreover, small immune complexes, which form when antigen is in excess, are deposited in and damage small blood vessels. (1)

The direct measurement of antibody binding to antigen is the principle used most commonly in the majority of quantitative serological assays. Moreover, a majority of assays rely on the ability of antibody binding to alter the physical state of the antigen in interacts with. It is these secondary interactions that are usually detected. These secondary reactions can be detected in a variety of ways, through agglutination, complement fixation, but more specifically as seein by the amount of formed precipitate. This precipitate is formed when sufficent amounts of antibody are mixed with soluble macromolecular antigens. This visible precipitate is composed of large aggregates of antigen crosslinked by antibodies. This precipitate reaction is usually refered to as a precipitin reaction. (1)

However, the amount of precipitate and consequently its' visibility, directly depends on the quantities of both antigen and antibody and on the ratio between them. Increasing the amount of antigen added to a fixed amount of serum containing antibody, the subsequent formation of precipitate increases up to a given maximum (for given specific amount of antibody) and then declines. Adding small amounts of antigen to excess amounts of antibody, causes the formation of antigen-antibody complexes in which each molecule of antigen is bound extensively by antibody and crosslinked to adjacent molecules of antigen. Moreover, adding large amounts of antigen, only small antigen-antibody complexes are able to form and are fairly soluble in the antigen excess zone. Between these two zones, extremes, all of the antigen and antibody is found in the precipitate, forming a zone of equivalence and an insoluble precipirtate.

At the zone of equivalence large lattices of antigen and antibody complexes are formed by crosslinking. (1) The precipitin reaction is influenced by the number of binding sites each antibody has for a given antigen and by the maximum number of antibody binding sites found on an antigen molecule. These quantities are refered to as the valence of each antibody and the valence of the antigen, respectively. The valence of an antibody molecule depends upon its' structural classification (e.g. IgM versus IgG). In order to form a precipitate, the valence of both the antibody and the antigen must be at least two or more. The site on an antigen which binds an unique antibody molecule is called an antigenic determinant (epitope). (1)

The precipitin reaction has become instrumental in numerous assays to detect and quantify components found in serum and other biological fluids. Numerous different assays are all based on the precipitin reaction, but each is able to provide unique information distinct from the other assays due to differences in their procedures. (1)

 

References:

1. Janeway, C.A., Travers, P., Walport, M., and Capra, J.D. 1999. Immunobiology: The immune system in health and disease. Garland Publishing, 4th ed., New York, USA.

2. Delves, P., and Roitt, I. 1999. Encyclopedia of Immunology. Academic Press Inc., 2nd ed., San Diego, USA.

3. Cruse, J. and Lewis, R. 1995. Illustrated Dictionary of Immunology. CRC Press Inc., USA.

4. Bryant, N. 1986. Laboratory Immunology and Serology. B. Venable, W.B. Saunders Company, 1st ed., Philadelphia, USA.