Precipitation test shows the following features:
· It is a type of antigen–antibody reaction, in which the antigen occurs in a soluble form.
· It is a test in which antibody interacts with the soluble antigen in the presence of electrolyte at a specified pH and temperature to produce a precipitate. 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.
· When instead of sedimenting, the precipitate remains sus-pended as floccules, the reaction is known as flocculation.
· 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.
· The antigen must be either bivalent or polyvalent; that is, it must have at least two copies of the same epitope, or have different epitopes that react with different antibodies present in polyclonal antisera.
◗ Prozone phenomenon
Antigen and antibody reaction occurs optimally only when the proportion of the antigen and antibody in the reaction mixture is equivalent (zone of equivalence) (Fig. 14-1). On either side of the equivalence zone, precipitation is actually prevented because of an excess of either antigen or anti-body. The zone of antibody excess is known as the prozonephenomenon and the zone of antigen excess is known as postzone phenomenon.
Marrack in 1934 proposed the lattice hypothesis to explain the prozone phenomenon. Marrack’s hypothesis is based on the assumptions that each antibody molecule must have at least two binding sites, and antigen must be multivalent. In the zone of equivalence where optimum precipitation occurs, the number of multivalent sites of antigen and antibody are approximately equal. In this zone, precipitation occurs as a result of random, reversible reactions whereby each anti-body binds to more than one antigen and vice versa, form-ing a stable network or lattice. As they combine, it results in a multimolecular lattice that increases in size until it precipi-tates out of solution.
The prozone and postzone phenomena are taken into con-sideration in the interpretation of serological tests, because false negative reactions can occur in either of these conditions. A false negative reaction suspected to be due to prozone phe-nomenon can be rectified by diluting out the antibody and performing the test. In the postzone phenomenon, excess anti-gen may obscure the presence of small amount of antibodies. Typically, such a test is repeated with an additional patient specimen taken about a week later. This would give time for the further production of antibodies. If the test is negative on this occasion, it is unlikely that the patient has that particular antibody.