Precipitation in fluids

As mentioned, the phenomenon of immunoprecipitation appears when a solution of antigenic material is mixed with immune serum containing corresponding antibodies. The reaction is registered by a gradually increasing turbidity, changing to the appearance of precipitating or flocculating aggregates. A common procedure for immunoprecipitation analyses is to use a series of test tubes containing a constant amount of one of the reactants, and adding the corresponding reactant in serial, stepwise dilutions. A rough estimation of the grade of precipitation can be made by the naked eye or, more precisely, by tur-bidimetric or nephelometric measurements. The most adequate measurement of the intensity of the precipitation reaction is made by weighing the precipitate or by chemical analyses, e.g. N-determination, or by physical measurements when, for instance, dye- or isotope-labeled materials are involved in the reaction. Moreover, such measurements make it possible to determine the antigen:antibody ratios in the precipitated material. By extensive experimental work along these lines, basic information on immunoprecipitation and its characteristics has been obtained. In this connection, monospecific anti-

Test of supemalants

Test of supemalants

Eïcess At) Equiva- Excess Ag lence

Figure 1 Schematic representation of a precipitation curve when a constant amount of antibody was tested against increasing amounts of corresponding antigen (ovalbumin-rabbit antiovalbumin). The amounts of precipitated material (O—O). the antibody:antigen (Ab:Ag) ratio in the precipitates (•—•) and the presence of unprecipitated antigen or antibody in the super-natants are indicated.

Eïcess At) Equiva- Excess Ag lence

Figure 1 Schematic representation of a precipitation curve when a constant amount of antibody was tested against increasing amounts of corresponding antigen (ovalbumin-rabbit antiovalbumin). The amounts of precipitated material (O—O). the antibody:antigen (Ab:Ag) ratio in the precipitates (•—•) and the presence of unprecipitated antigen or antibody in the super-natants are indicated.

bodies, usually of the immunoglobulin G (IgG) class, and corresponding multivalent antigen have commonly served as a suitable model system.

The most important characteristics of the precipitation reaction may be summarized as follows. There are two stages to the reaction, the first being the very rapid binding of antibody to antigen, which occurs within seconds. The binding is governed by the degree of structural complementarity which provides serological specificity. Physically, van der Waal forces, hydrogen bonds, hydrophobic bonds and electrostatic forces are involved. The union is firm but reversible and at dissociation each reactant can be recovered unaltered. The strength of binding is measured by the grade of avidity of the antiserum, high avidity implying greater firmness. The second stage of the precipitation reaction develops more slowly, lasting minutes to hours, the primary antigen-antibody complexes forming larger secondary complexes in a lattice formation which end up as visible precipitating aggregates. This aggregation is electrolyte dependent. A prerequisite for the formation of registcrable aggregates is that the molecules of the participating reactants have a valency of at least 2 (e.g. bivalent IgG) and a multivalent antigen. An important observation is that in the im-munoprecipitation reaction, antigen and antibody molecules combine in varying proportions depending on the ratio of reactants when they are initially mixed. This phenomenon of multiple proportions is in contrast to what happens in a common, pure chemical precipitation reaction where the reactants mostly combine in a nonvariable proportion.

The variation in the amount and composition of antigen-antibody aggregates in an immunoprecipi-tation system assayed by serial dilution of one of the reactants is represented by the precipitation curve in Figure 1. The amount and the antigen:antibody ratio of the precipitates are indicated, as well as the presence or absence of free antigen or antibody in the supernatants. The general characteristics of the reaction are, in short, as follows. At a certain antigen:

antibody ratio, maximal precipitation takes place, with neither free antigen nor antibody in the supernatant (zone of equivalence, a quantitatively balanced system). Where the antigen:antibody ratio differs from that of equivalence - zones of either excess antigen or antibody - the conditions are less favorable for the creation of an extended lattice formation. Consequently, at either side of the zone of equivalence, smaller amounts of precipitate are formed, and where the reactants are greatly out of balance a zone of complete inhibition may be observed. It should be noted that this phenomenon occurs even though primary antigen-antibody binding has taken place. Whether there is total or partial solubility of antigen-antibody complexes in the regions around the zone of equivalence depends on the species origin of the immune serum and the nature of the antigen. So, for instance, horse immune sera reacting with the corresponding protein antigens usually give a more sharply defined point of maximum precipitation, and complete inhibition occurs at excess of antigen as well as of antibody,

Antigen Diluent Aril body

Tube flocculation test

Time

Antigen consfant concentration

(min)

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