Many studies on prophylactic immunization against pneumococcal pneumonia have been made, using a number of different antigenic preparations. Almost all investigators have concluded that immunization exerts a beneficial effect. In most of the studies, however, certain variables have clouded interpretation of the results. Among the variables, the following appear to be of greatest moment: differences in the composition of the immunized and control groups; uncertainty as to whether the specific pneumococcal types included in the immunizing preparation were the same as those currently causing pneumonia; failure to determine whether the observed decline in cases in the immunized group was due to a decrease in cases caused by the pneumococcal types included in the vaccine; inadequate control of the antigenicity of the preparations used.The subject of antipneumococcal immunization has been reviewed recently by Heffron (1) and references will be made in the present paper to certain aspects only.The studies of Lister and Ordman (2) among native laborers in South Mrican mines between 1930 and 1934 suggest that immunization with a polyvalent pneumococcal vaccine reduces the incidence of pneumonia caused by the same pneumococcal types.

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

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...

1. A method is given for the concentration and purification of the soluble specific substance of the pneumococcus. 2. The material obtained by this method is shown to consist mainly of a carbohydrate which appears to be a polysaccharide built up of glucose molecules. 3. Whether the soluble specific substance is actually the polysaccharide, or occurs merely associated with it, is still undecided, although the evidence points in the direction of the former possibility.

1. Highly purified rabbit Type III pneumococcus anticarbohydrate proved to be homogeneous in the ultracentrifuge and its sedimentation constant, 7.0·10–13, did not differ from that of the principal component of normal rabbit globulin or of immune rabbit globulin containing up to 50 per cent of anti-egg albumin. The molecular weight of antibody in the rabbit is therefore probably very close to that of the principal normal globulin component, namely, 150,000. 2. Highly purified horse Type I pneumococcus anticarbohydrate, on the other hand, was only homogeneous in the ultracentrifuge when prepared from sera stored without preservative. Its sedimentation constant, 18.4·10–13, coincided with that of the principal globulin component in most of the Felton solutions and purified antibody solutions studied. The molecular weight of pneumococcus anticarbohydrate in the horse is probably three to four times that of the principal normal globulin component. 3. The significance of the differences between pneumococcus anticarbohydrate formed in the rabbit and in the horse is discussed. 4. Results are given of ultracentrifuge studies on the molecular species in solutions of egg albumin-anti-egg albumin specific precipitates dissolved in excess egg albumin. The implications of the results are discussed.

1. The evidence presented indicates that Mg++, or other cation such as Ca++, Ni++, or Co++, is essential for the hemolytic action of C'. Ca++, Ni++, and Co++ are less effective than Mg++. The hemolytic system usually does not contain sufficient Mg++ for optimal hemolytic activity so that a marked enhancement can be obtained by addition of extra Mg++. 2. The enhancing action of tissue fluids can be ascribed to their contribution of Mg++. 3. Substances which bind Mg++ and Ca++ are anticomplementary when added to the usual hemolytic system which contains only a small quantity of Mg++. This type of anticomplementary effect can be overcome by addition of extra Mg++. 4. Ca++ may also be essential to the lytic process but its action is much less pronounced than that of Mg++.

hydrolysis of the collagen, although the organized skin structure inhibited diffusion of the enzyme and greatly decreased the speed of the reaction.Specimens of collagen tanned with quinone, gallotannic acid, copper sulfate and formaldehyde were all hydrolyzed by trypsin while chrome tanned collagen was not.In 1917 Dochez and Averyl showed that there was contained in filtrates from pneumococcus cultures and in the body fluids of experimentally infected animals and of patients suffering from pneumonia, a soluble substance which reacts specifically in antipneumococcus serum of the homologous type. This substance, which was found to be thermostable, precipitable by alcohol o r acetone, non-dialyzable, and not digested by trypsin, is now being subjected to a more intensive chemical study.Eight-day, autolyzed cultures of Type I1 Pneumococcus in phosphate broth were concentrated to 1/15 volume and precipitated with 1.2 volumes of alcohol. The precipitate, centrifuged at high speed, yields a compact middle layer containing the specific soluble substance. By repeated fractionation with alcohol or acetone, first in neutral, then in dilute acetic acid solution, followed by repeated fractional precipitation with ammonium sulfate and final dialysis, about 1 gm. of a highly purified preparation was obtained for each 75 liters of culture used.In its present state of purity the specific soluble substance is amorphous and yields a viscous solution in water. A 1 per cent.solution gives no biuret test, yields no precipitate with phospho-1

In the first paper of this series (1) it was concluded as a first approximation that under a standard set of conditions the entire course of the precipitin reaction between the specific polysaccharide of Type III pneumococcus and homologous purified antibody could be quantitatively accounted for by three simple equations. The mass law was believed to hold for these equations, the more so as the reactions were found to be reversible. Studies of the theoretical factors involved have since been continued under more varied conditions, and the present paper describes experiments which have necessitated modification of the conclusions originally drawn. EXPEI~r&rENTALThe quantitative predpitin determinations were made as in previous papers (2-4), except that the technique was modified as described below in order to study the effect of varying a given set of conditions. In general, precipitates were analyzed, rather than superuatants, as had been done in (I). Much of the serum used was obtained through the kindness of Dr. William H. Park, to whom the writers again wish to express their gratitude. Unless otherwise stated, antibody solutions were prepared according to Felton (6).