Antigen-antibody precipitation

Oudin, J. (1980) Immunochemical analysis by antigen-antibody precipitation in gels. Methods Enzymol. 70,166-198. 

Now, while it is possible, by lowering the surface tension of the liquid medium to 50 dyn/cm by means of solutes of low molecular weight, to dissociate antigen-antibody precipitates (of the pure VDW type) (6), whole mammalian serum (with a surface tension < 50 dyn/cm) definitely has no such dissociating power. Upon some reflection this is not as anomalous as it might seem. Table I shows that it is mainly because of the presence of proteins (of molecular weight > 20,000) that blood (or plasma) has a surface tension y < 50 (and not y 70), and it cannot be expected that molecules with dimensions of the order of 100 A will effect a separation by purely physical means between other proteins of the same approximate size that are only about 2 A apart at their site of interaction (13). 

In 1967, Ritchie reported a direct technique for measurement of albumin and immunoglobulins by the development of antigen-antibody precipitates in liquid media. In this method turbidity produced by the antigen-antibody complex was determined by measuring absorbance at 420 nm with a spectrophotometer (Rl). 

Immunochemical Analysis by Antigen-Antibody Precipitation in Gels... 

The author s aims are a) to classify as rationally as possible the techniques of immunochemical analysis by antigen-antibody precipitation in gels (,b) to state the more or less general principles and laws which hold true for all or part of the techniques (c) to help the reader in understanding the bases of the different techniques in a way that does not require any special knowledge of physics and (d) to give a few examples to illustrate that this understanding of the principles and laws, plus an analysis of the reactions may raise, and eventually solve, problems of appreciable importance. 

Approximately 2 years after antigen-antibody precipitation in gels was first proposed for immunochemical analysis, a number of techniques began to appear in the literature. No attempt is made to describe or enumerate all these techniques or their variations, which are described elsewhere. ... 

In contrast to the techniques described, several procedures use antigen-antibody precipitation in gels as a means of immunochemical analysis with an electric field as the motor of the reagents. 

This number is equal to that of the precipitation zones, provided that what one is counting is actually antigen-antibody precipitation zones and not something else (see discussion of sources of error). The number of antigens determined is a minimum number because the concentration of the antibodies against one antigen (or of an antigen) may be below the threshold detectable value and because a precipitation zone may be hidden by others, especially when they are numerous. 

With standardized reactions between a class-specific antiserum and its immunoglobulin, it is possible to measure the level of that immunoglobulin in serum, etc. Turbidimetry of antigen-antibody precipitates is not recommended as too many variables influence the flocculence of the... 

Assuming that antigen-antibody precipitates have fattice-like structures (see Fig. 33.4 in the text), draw simple sketches showing possible arrangements of antigen and antibody molecules in a precipitate in which the ratio of antibodies to antigens is (a) 1.14 and (b) 2.83. 

Figure 20. The free runs to the left are rabbit antihemocyanin from three individual Sandy Lop rabbits. The antigen antibody precipitates were prepared at equivalence, washed twice with normal salive, dissolved in 8 Af urea and applied on the gel. The pattern to the right is obtained from pooled mouse antihemocyanin prepared in the same way. The electrofocusing was performed on 5% polyacrylamide gel with 2% Ampholine carrier ampholytes, pH range 3-10, 6 M urea. (Awdeh et dl., 39).

II, and III, obtained with two antigens (VI and X) and three antisera (C, E, and F), that the antigen-antibody precipitate is either dissolved slightly or carried away mechanically by the saline or borate buffer solutions with which it is washed. The loss in this way is, however, small, amounting to about 5 to 15% for eight or ten extra washings with 10-ml. portions of solution. 

Fig. 1.—Effect of added hapten (in amounts given) on amount of antigen-antibody precipitate (Table VIII).

Fig. 3.—Calculated effect of vaiiaticm of scdubility of antigen-antibody precipitate on amount of precipitate all curves for initial antibody concentration B >= 25 and...

Fig. 5.—Calculated effect of addition of hapten on amount of antigen-antibody precipitate, for Btotai = 25, JC = Vs. and X - r - 1.

Fig. 7.—Calculated dependence of amount of antigen-antibody precipitate on hapten-antibody bond-strength constant K, for Atotai - Bwtai = 25, K = Vz. s = 1.

Experiments on the Danysz phenomenon (73) and other related experiments indicate that a long time—many days—is needed for equilibrium to be approached for reactions involving change in composition of antigen-antibody precipitates. 

The second of oiu arguments for the multivalence of antibody molecules is based on the solubility of antigen-antibody precipitates in... 

It has been found by experiment that substances of the type RX, containing two different haptenic groups, do not form precipitates with either anti-R serum or anti-X serum Alone, but do form precipitates with a mixture of the two specific antisera. This provides proof of the effective bivalence of the dihaptenic precipitating antigen, and thus furnishes further evidence for e framework theory of antigen-antibody precipitation. In these experiments tiie anti-R serum and anti-X serum were made by injecting rabbits with sheep serum coupled with diazotized p-arsanilic add and diazotized p-aminobenzoic acid, respectively, and the RX substances used were l-amino-2-/>-(p-azophenylazo)-phenylarsonic acid-3,6-disul-fonic add 7-p-(p-azophenylazo)-benzoic acid-8-hy-droxynaphthalene and l,8-dihydroxy-2-/>-azo-phenylarsonic acid-3,6-disulfonic acid-7-p-(p-azo-phenylazo)-benzoic acid-naphthalene. 

An experimental test of the framework theory of antigen-antibody precipitation. Science 98 (1943) 263-264. (Linus Pauling, David Pressman, and Dan H. Campbell). 

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