Introduction to Precipitation Reaction
It is a type of antigen–antibody reaction, in which the antigen occurs in a soluble form. When a soluble antigen reacts with its specific antibody, at an optimum temperature and PH in the presence of electrolyte antigen antibody complex forms insoluble precipitate. This reaction is called precipitation reaction. A lattice is formed between the antigens and antibodies; in certain cases, it is visible as an insoluble precipitate. Antibodies that aggregate soluble antigens are called precipitins.
The interaction of antibody with soluble antigen may cause formation of insoluble lattice that will precipitate out of solution. Formation of an antigen–antibody lattice depends on the valency of both the antibody and antigen. The antibody must be bivalent; a precipitate will not form with monovalent Fab fragments. Antigen must be bivalent or polyvalent; that is it must have at least two copies of same epitope or different epitopes that react with different antibodies present in polyclonal sera. Antigen and antibody must be in appropriate concentration relative to each other.
- Antigen access: Too much antigen prevents efficient crosslinking/lattice formation.
- Antibody access: Too much antibody prevents efficient crosslinking/lattice formation.
- Equivalent Antigen and Antibody: Maximum amount of lattice (Precipitate) is formed
Antigen and antibody reaction occurs optimally only when the proportion of the antigen and antibody in the reaction mixture is equivalent. On either side of the equivalence zone, precipitation is actually prevented because of an excess of either antigen or antibody. The zone of antibody excess is known as the prozone phenomenon and the zone of antigen excess is known as postzone phenomenon.
In the prozone phenomenon, there is too much antibody for efficient lattice formation. This is because antigen combines with only few antibodies and no cross-linkage is formed. In postzone phenomenon, small aggregates are surrounded by excess antigen and again no lattice network is formed. Thus, for precipitation reactions to be detectable, they must be run in the zone of equivalence.
When instead of sedimenting, the precipitate remains suspended as floccules, the reaction is known as flocculation.
Precipitation reactions are based on the interaction of antibodies and antigens. They are based on two soluble reactants that come together to make one insoluble product, the precipitate. These reactions depend on the formation of lattices (cross-links) when antigen and antibody exist in optimal proportions. Excess of either component reduces lattice formation and subsequent precipitation. Precipitation reactions differ from agglutination reactions in the size and solubility of the antigen. Antigens are soluble molecules and larger in size in precipitation reactions. There are several precipitation methods applied in clinical laboratory for the diagnosis of disease. These can be performed in semi-solid media such as agar or agarose, or non-gel support media such as cellulose acetate.
Applications of precipitation reaction
- Detection of unknown antibody to diagnose infection e.g. VDRL test for syphilis.
- Standardisation of toxins and antitoxins.
- Identification of Bacteria e.g. Lancified grouping of streptococci.
- Identification of bacterial component e.g Ascoli’s thermoprecipitin test for Bacillus anthracis