A Quantitative Study of the Precipitin Reaction Between Type Iii Pneumococcus Polysaccharide and Purified Homologous Antibody

Abstract

Of all the reactions of immunity the precipitin test is perhaps the most dramatic and striking. While other immune reactions are more delicate, the precipitin test is among the most specific and least subject to errors and technical difficulties. Attempts at its quantitative interpretation and explanation (1, 2) have been hampered either by the difficulty of finding suitable analytical methodsf or by the failure to separate the reacting substances from closely related, non-specific materials with which they are normally associated.With the aid of recent work it has been found possible to avoid these difficulties to some extent. The isolation of bacterial polysaccharides which precipitate antisera specifically (3) and possess the properties of hap tens (4) has not only afforded one of the components of a precipitin reaction in a state of comparative purity, but has greatly simplified the analytical problem. Since many of these polysaccharidescontain no nitrogen, and antibodies presumably are nitrogenous, the latter may be determined in the presence of any amount of the specific carbohydrate. Moreover, Felton's method for the separation of pneumococcus antibodies from horse serum (5) not only permits the isolation of a high proportion of the precipitin, freed from at least 90 per cent of the serum proteins and much of the serum lipoid, but is also applicable on a sufficiently large scale to furnish the amounts of antibody solution needed to make quantitative work possible. It is realized that antibody solutions of this type do not contain pure antibodies--indeed, only 40 to 50 per cent of the nitrogen is specifically precipitable--but since so small a proportion of the original serum protein remains with the antibody a far-reaching purification actually has been effected. It should thus be possible with the aid of antibodies purified by Felton's method to obtain data of a preliminary character which should point toward the mechanism of the reaction. The present paper is concerned with such data obtained in a quantitative study of the precipitin reaction between the soluble specific substance of Type III pneumococcus and Type III pneumococcus antibody solution. EXPERIMENTAL Materials and Methods.--a. Solutions of Soluble Specific Substance, Type IllPneumococcus.--The soluble specific substance of Type III pneumococcus (6)* used was kinky supplied by Drs. O. T. Avery and W. F. Goebel of The Rockefeller Institute for Medical Research. It was ash-free, contained 0.04 per cent of nitrogen, and showed [aid = -32 °. A weighed amount of anhydrous substance was suspended in 0.9 per cent saline, dissolved with the aid of 0.1 normal sodium hydroxide, and the solution was diluted with saline, adjusted to pH 7.6 and made up to volume with saline to yield a 1 per cent solution. This was sterilized in the autoclave and used as a stock solution for making up other dilutions. These were prepared with sterile saline under aseptic precautions, and were kept in the icebox.b. Type III Pneumococcus Antibody Solution.--The antibody solu...

Quantitative Studies on the Precipitin Reaction

“…12) result in a spatial configuration which is altered when these charges are reduced by high salt concentrations, with a consequent diminution in specificity, or reactivity with corresponding spatially orientated groupings in the antibody molecule. At any rate there is much reason to ascribe an ionic mechanism to this effect, as was originally done in the case of the S IH-antibody reaction itself (10). Salt effects similar to the above have also been noted by Hammarsten and collaborators (13), in the reaction between proteins and nucleic acids.…”

The Results of some Quantitative Experiments on the Serum Precipitation Reaction

“…In this case acid in excess is adsorbable by the resulting compound, and is not traceable in the supernatant fluid in equivalent amount. Heidelberger and Kendall (1929), working with a non-protein antigen from pneumococcus and homologous antisera, have suggested that, in this case, the reaction between antigen and antibody is following a modification of the laws of mass action. They state that more than one type of complex is formed: one part of antibody being capable of reacting with one part of antigen to yield one type of complex, whilst one part of antibody may react with two parts of antigen to form a second type of complex, and so on.…”

Studies in Agglutination

“…This antibody solution is almost entirely free of serum protein other than serum globulin, 90 per cent being removed in its preparation (9). This finding is further evidence, although indirect, in favor of the importance, in the production of the inhibition zone, of modified agglutinin (antibody globulin).…”

“…In support of such a suggested explanation we have observed one serum (pneumococcus, Type I, horse) in which an inhibition zone appeared when incubation was at 56 ° but not at 37 ° . It is of interest in this connection that Heidelberger and Kendall (9) in studying the precipitin reaction have observed flocculation at icebox temperatures in mixtures which showed no precipitation at room temperature. Whenever natural inhibition zone serum is available further investigation will be carried forward.…”