The precipitin reaction is a phase-change phenomenon observed in antibody-antigen reactions. Kraus (1), its discoverer, demonstrated formation of a precipitate when spent bacterial culture broth was mixed with a specific antiserum. Marrack and Smith (2) showed that recovery of precipitate was largely independent of solution conditions or the presence of nonspecific proteins, so the precipitin reaction was not a simple phase change. Marrack (3) proposed correctly that the precipitate was an extended lattice in which multivalent antibody molecules form links with two or more antigen molecules, which, in turn, can form multiple links with other antibody molecules.
Since lattice formation requires multiple intermolecular links per monomer, the precipitin reaction is very sensitive to the ratio of antibody and antigen. A precipitin curve, shown in Figure 1, refers to the rise, peak, and fall in amount of precipitate recovered as a fixed amount of serum is titrated with antigen. The maximum amount of precipitate forms when antigen and antibody are present in similar molar amounts (the equivalence zone). At a low antigen:antibody ratio, few antibody molecules will bind more than one antigen molecule, hence the multiple intermolecular links necessary for a lattice again do not form (antibody excess zone). At a high antigen:antibody ratio, all antibody combining sites will be saturated with antigen, and few antigen molecules will be shared between two antibodies (antigen excess zone). The zones are defined empirically as those sets of reactions whose supernatants give additional precipitate upon adding a small increment of antibody (in the antigen excess zone) or antigen (in the antibody excess zone). In the equivalence zone, little antibody or antigen remains in the supernatant, and no additional precipitate can be induced. With some antigens and antisera, complete antibody precipitation occurs over an extended range of antigen additions, while excess antigen is still observed in the supernatant. In such cases, the term "inhibition zone" refers to the region of the antigen excess zone in which the absolute recovery of precipitate declines.
Figure 1. Precipitin curve. An antibody is titrated with antigen, and the resulting precipitates are isolated and quantitated.
Heidelberger and Kendall (4, 5) analyzed the composition of precipitates and discerned a general relation that formed the basis of a quantitative assay of antigen or antibody preparations of unknown concentration. In this method, a precipitin curve is obtained, and the experimental points in the equivalence and antibody excess zones are identified as described above. (Points in the antigen excess zone are not used.) These authors found for many antibody-antigen systems that a straight line results if the ratio of antibody to antigen in the precipitate is plotted against the amount of antigen added, illustrated in Figure 2. (A frequent simplifying assumption is that all antigen added is precipitated.) The linearity of the relation allowed easy interpolation; hence the antigen content of an unknown could be determined by comparing the antibody:antigen ratio in the precipitate from the unknown to the results of precipitin reactions from standards of known concentration.
Figure 2. Precipitin curve analyzed by the method of Heidelberger and Kendall (4). Data from the equivalence and antibody excess zones in Figure 1 are replotted as the ratio of antibody to antigen found in each precipitate versus the quantity of antigen added to initiate the precipitation.
Although the precipitin curve technique illustrated in the figures is no longer used for analysis, the precipitin reaction itself has been incorporated into many immunological techniques (see Immunoelectrophoresis). One of the most visually elegant applications is the Ouchterlony double-diffusion assay (see Immunoassays), in which one can infer properties such as molecular weight and cross-reactivity of an antigen or antiserum preparation from the shape of a precipitin line on an agar plate (6). A turbidimetric assay based on the precipitin reaction is widely used in clinical laboratories. In this type of immunoassay, antibody that is free in solution reacts with microscopic antigen-coated latex beads (or free antigen reacts with antibody-coated beads) to farm large cross-linked aggregates that are easily and sensitively detected by light scattering (7).
