The formation of a precipitate when antibody and soluble antigen are mixed is one of the most striking phenomena of serological reactions; some of the older and many of the more recent studies of it have yielded results of great interest and importance.
The visible result of the chemical interaction of antigen and antibody. Such precipitate was first described in 1897, and it was later noted that the optimal ratio of antigen to antibody concentrations resulted in the visible precipitate. Not all antibodies will result in precipitation, yet they may participate in agglutination reactions or add onto particulate antigens, and evidence for their occurrence together with precipitating antibody can be obtained for most sera. Precipitins may be noted qualitatively or be quantified by noting the end-point dilution (titer) of serum required to give a precipitate at the threshold of visiblity, or the amount of antibody may be determined in milligrams or micrograms by analysis of the precipitate with correction for the antigen contained therein.
Amount of Precipitate: Zones
From Boyd ()
Either the amount of antiserum or the amount of antigen can be varied; it is usual to keep the concentration of one reagent constant, and add different amounts of the other. Since the precipitates obtained are now usually analyzed by some modification of the micro-Kjeldahl technic developed by Parnas and Wagner, it is advisable to adjust the amounts of both reagents so as to obtain the appropriate amount of nitrogen (or of any other constituent being estimated in the precipitate). From the results so obtained we can calculate back to the basis of constant amounts of serum or of antigen. A series of such determinations made with different proportions of reagents gives a system of data which if plotted in three dismensions will yield a surface which shows how the composition of the precipitate depends on the concentrations of the two reagents; two-dismensional graphs can be made by showing appropriate sectiosn of this.
Most of the precipitating antisera that have been studied quantitatively so far were obtained from the horse or the rabbit. (Pennell and Huddleson used goat serum). In the horse there seems to be a striking difference in the behavior of antibodies against proteins (such as antitoxins) and antibodies against bacterial polysaccharides; in the rabbit this difference is not found, but both kinds of antibodies behave more or less alike, resembling in their precipitating behavior the anti-carbohydrate antibody of the horse. It should be noted, however, that this similarity does not extend to their solubility in water, or their molecular weights.
It is generally found that if more than a certain relative amount of antibody is added, no precipitate is produced, soluble compounds being formed, whereas carbohydrates are precipitated even with large excess of antibody. In the language of our discussion of agglutination, horse anti-protein sera exhibit a prezone. Rabbit antibodies, whether against protein or carbohydrate, do not show a prezone with excess antibody. It may be suspected that the difference is at least partly due to a difference in solubility of the various antibodies, for it will be recalled that the anti-carbohydrate antibodies in the horse, and most antibodies of the rabbit, are predominantly found in the euglobulin (least soluble) fraction of the serum proteins, while the anti-protein antibody in the horse is mostly of pseudoglobulin (more soluble) character.
All precipitating systems exhibit the phenomenon of the postzone which is unfortunately usually called the prezone, (since in precipitin reactions it is usually the antigen which is diluted, so that the strongest antigen solutions come first); for this reason and for others, it seems best to use Heidelberger's term "inhibition zone." We may define this as the zone in which there is insufficient antibody to precipitate the antigen present. The antibody in this zone forms soluble compounds with the antigen.
The actual amount of precipitate obtained from a given amount of antiserum depends in the first place on the amount of precipitating antibody present, and in the second place on the amount of antigen added. As increasing amounts of antigen are added, the amount of precipitate increases up to a maximum, then declines as the inhibition zone is reached.
The Ouchterlony Test
In this test, antibody and possible antigens are placed in wells in agar plates and allowed to diffuse toward one another. The antibody is placed in a center well and antigens (specific or nonspecific) are placed in surrounding wells. When an antibody and its specific antigen meet one another and are at the proper concentrations, the precipitate will form a visible white line between the two wells. This line is called a precipitin line.
Multiple lines formed in the Ouchterlony test (double-diffusion precipitation) when rabbit antiserum (centre well) reacts in agar gel with four different antigen preparations (peripheral wells). Non-identity and partial identity of antigens are shown respectively by crossing over and spur formation between precipitin lines.()