In the first paper of this series (1) 1 it was shown that the precipitin reaction might be considered the resultant of a series of competing bimolecular reactions, the quantitative outcome of which depended on the relative proportions in which the components were mixed. It was thus found possible to express the entire course of the precipitin reaction between a specific polysaccharide and its homologous antibody by simple equations derived from the mass law. In the second paper of the series (2) it was shown that. these considerations were equally applicable to an antigen-antibody system in which the antigen was R-salt-azo-biphenyl-azo-crystalline egg albumin. This antigen, a deep red dye, could be determined with accuracy in precipitates over the entire range of the reaction, thus permitting the separate quantitative estimation of the amounts of antigen and antibody nitrogen precipitated.In the present communication the information obtained with the aid of the specific polysaccharide and the protein dye is applied to a system involving a colorless antigen, crystalline egg albumin, and its homologous antibody. The antigen used has the advantages of homogeneity, known molecular weight, and of having been studied quantitatively in respect to its behavior in the precipitin reaction by a number * The work reported in this communication was carried out under the Harkness Research Fund of the Presbyterian Hospital.1 A statement was omitted in this paper that the sera used were absorbed with pneumococcus protein and "C" substance before purification of the antibody. 697 on

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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...

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