A QUANTITATIVE THEORY OF THE PRECIPITIN REACTION : VI. THE REACTION BETWEEN MAMMALIAN THYROGLOBULINS AND ANTIBODIES TO HOMOLOGOUS AND HETEROLOGOUS PREPARATIONS

1. Quantitative data for both homologous and heterologous precipitin reactions of human, hog, beef, and sheep thyroglobulins show that these reactions have the same mechanism as other instances of the precipitin reaction and may be expressed quantitatively by the same equations derived from the law of mass action. 2. It is shown that all of the added antigen is precipitated in the region of antibody excess and in the equivalence zone, so that in these portions of the reaction range the composition of the specific precipitate may be calculated from the nitrogen precipitated and the amount of antigen nitrogen added. 3. The thyroglobulin-antibody reaction is characterized by low antibody N to antigen N ratios, as would be expected with an antigen of high molecular weight. Molecular ratios varying from 60:1 to 1:1 were calculated for the extremes of the reaction range, indicating a very large number of immunologically reactive groupings on the thyroglobulin molecule. 4. Failure of thyroxine or diiodotyrosine to inhibit specific precipitation was confirmed, but it is shown that this need not mean that these substances do not occur in thyroglobulin, as has been claimed

THE PRECIPITIN REACTION BETWEEN TYPE III PNEUMOCOCCUS POLYSACCHARIDE AND HOMOLOGOUS ANTIBODY : II. CONDITIONS FOR QUANTITATIVE PRECIPITATION OF ANTIBODY IN HORSE SERA

The experiments recorded above show that in the case of antipneumococcus horse serum or purified antibody the arbitrary immunological procedure (37° for 2 hours, overnight in the ice box) does not permit either the establishment of a true equilibrium or the precipitation of the maximum amount of antibody nitrogen. Analyses of such horse sera for antibody content should therefore be carried out at 0° and the determinations should be allowed to stand in the cold for at least 24 hours in order to insure the completion of the reaction. It is believed that the similarity of the nitrogen: S III ratios in the specific precipitate, whether obtained from whole serum or from purified antibody, and the failure of added serum to influence the amount of nitrogen precipitated show that the absolute chemical method for the estimation of antibody actually measures antibody and not antibody plus a more or less indefinite amount of non-specific protein. An objection to the use of the method is thus shown to be unfounded

PREDICTED AND UNPREDICTED CROSS-REACTIONS OF AN ACETYLPHOSPHOGALACTAN OF SPOROBOLOMYCES YEAST

The teichoic acid of streptococcal Group N, with end groups of galactose phosphate, had been shown to cross-react with antipneumococcal sera of types VI, XIV, XVI, and XXVII. End groups of D-galactose-1-phosphate in the phosphogalactans of Sporobolomyces yeasts made it predictable that these galactans would precipitate the same antipneumococcal sera and also antisera to streptococcal Group N. The predictions were verified, and other unpredicted reactions were found. Precipitation of much of the antibody in an antipneumoccal type XVIII horse serum was shown to be due to O-acetyl-D-galactose residues in the phosphogalactan, in accord with earlier information that an O-acetyl sugar was a principal determinant of S XVIII. The new results identify this sugar as D-galactose. Since it is linked 1,3- in S XVIII, the O-acetyl group in the Sporobolomyces galactan is probably also on a 1,3-linked residue. Another major cross-reaction in anti-S. paratyphi A serum characterizes the galactose residues in the "O" polysaccharide of the bacillus as members of the D-series probably linked in tandem 1,6-, 1,6-; 1,6-, 1,3-; 1,3-, 1,6-; or 1,3-, 1,3-. Reactions of periodate-oxidized-reduced products confirm the conclusions stated above. Quantitative data are given

A QUANTITATIVE THEORY OF THE PRECIPITIN REACTION : II. A STUDY OF AN AZOPROTEIN-ANTIBODY SYSTEM

1. A quantitative theory of the precipitin reaction based on the laws of classical chemistry has been tested on an azoprotein-antiprotein system and found to apply. 2. With its aid relationships may be deduced which permit the calculation of the behavior of an antidye serum over its entire range after a few quantitative chemical analyses have been made for antigen and antibody in the precipitate. 3. An empirical relation is also presented which further reduces the number of analyses necessary. 4. A study of supernatants in the inhibition zone has shown that the entire amount of dissolved antigen-antibody compound present is precipitated when supernatants are analyzed for antigen by the precipitin method

SPECIFIC AND NON-SPECIFIC POLYSACCHARIDES OF TYPE IV PNEUMOCOCCUS

1. Three nitrogen-containing polysaccharides have been isolated from autolyzed cultures of Type IV pneumococcus: (1) a type-specific carbohydrate differing markedly from those of Type I, II, and III pneumococcus, and representing a type of substance hitherto not observed among specific polysaccharides, (2) a chemically similar carbohydrate without specific function, and (3) the "C" substance, or species-specific polysaccharide of Tillett, Goebel, and Avery. 2. The chemical differences between the specific polysaccharides of Pneumococcus are discussed, and the relationship of the new examples to chitin is pointed out and its bearing indicated on the unsettled controversy as to whether or not chitin occurs in bacteria. 3. The data of Tillett, Goebel, and Avery on the "C" substance have been extended

STUDIES ON THE PRECIPITIN REACTION : PRECIPITATING HAPTENS; SPECIES DIFFERENCES IN ANTIBODIES

1. Partial hydrolysis products of the specific polysaccharide of Type III pneumococcus ranging from 550 to 1,800 in formula weight can be quantitatively freed from unhydrolyzed polysaccharide. 2. The fractions yield specific precipitates with Type III antipneumococcus horse serum but fail to precipitate homologous rabbit antisera, giving rise only to specific inhibition. The aldobionic acid, the structural unit of S III, does not precipitate antisera. 3. A possible explanation and a possible application of the findings are pointed out

A QUANTITATIVE THEORY OF THE PRECIPITIN REACTION : III. THE REACTION BETWEEN CRYSTALLINE EGG ALBUMIN AND ITS HOMOLOGOUS ANTIBODY

Page 702, at the bottom of the 3rd column of Table II, for 0.36, read 0.26. This change necessitates the following correction also. Page 707, 3rd line from the top, for the entire amount, read much. Page 715, 9th line from the top, for actually occurs, read occurs to a great extent. 12th line from the top, for was 0.36 mg., and precisely this amount was, read was 0.36 mg. without deducting about 0.1 mg. for solubility, whereas 0.26 mg. was

THE PRECIPITIN REACTION BETWEEN TYPE III PNEUMOCOCCUS POLYSACCHARIDE AND HOMOLOGOUS ANTIBODY : III. A QUANTITATIVE STUDY AND A THEORY OF THE REACTION MECHANISM

The precipitin reaction between the specific polysaccharide of Type III pneumococcus and homologous antibody formed in the horse can be accounted for quantitatively by assuming the chemical combination of the components in a bimolecular reaction, followed by a series of competing bimolecular reactions which depend upon the relative proportions of the components. These reactions would lead to the formation of larger and larger aggregates until precipitation ultimately occurred. The mathematical formulation of this theory on the basis of the mass law is described. The derived expressions are shown to be in accord with the experimental findings and the constants used in these expressions are shown to have definite significance. In spite of the wide variation in the properties of individual sera these expressions permit the complete description of the behavior of an unknown serum with S III without an unduly burdensome number of analyses. The quantitative theory presented has been found applicable to other instances of the precipitin reaction, as will be shown in subsequent papers

QUANTITATIVE STUDIES ON ANTIBODY PURIFICATION : I. THE DISSOCIATION OF PRECIPITATES FORMED BY PNEUMOCOCCUS SPECIFIC POLYSACCHARIDES AND HOMOLOGOUS ANTIBODIES

1. Quantitative data are given on the effect of changes in hydrogen ion concentration and of salt solutions of high concentration on certain immune precipitates obtained at lower salt concentration. 2. Advantage is taken of the shift in reaction equilibrium brought about by the salt in the case of pneumococcus carbohydrate-anti-carbohydrate precipitates to enable the preparation, in a single step from unconcentrated serum, of antibody solutions in which up to 93 per cent of the total nitrogen is immune nitrogen. The method permits successive absorptions of a serum to be made with the same specific precipitate. 3. A modification of Felton's alkaline earth hydroxide dissociation procedure is proposed which yields highly purified antibody with precipitates which have been subjected to several successive salt dissociations

A QUANTITATIVE STUDY OF THE PRECIPITIN REACTION BETWEEN TYPE III PNEUMOCOCCUS POLYSACCHARIDE AND PURIFIED HOMOLOGOUS ANTIBODY

1. A quantitative study of the reaction between the soluble specific substance of Type III pneumococcus and its homologous antibody has been made. 2. The entire reaction, from excess of antibody, to excess of specific substance with its accompanying inhibition zone effect, may be expressed by three mass-law equations. 3. The significance of these findings is discussed