A QUANTITATIVE THEORY OF THE PRECIPITIN REACTION : IV. THE REACTION OF PNEUMOCOCCUS SPECIFIC POLYSACCHARIDES WITH HOMOLOGOUS RABBIT ANTISERA

1. The reaction between the specific polysaccharide of Type III pneumococcus and homologous antibody in rabbit sera is quantitatively accounted for by expressions similar to those derived from the mass law for the corresponding horse sera. Preliminary data are also given for the Type I reaction. 2. Differences and similarities of the reaction with antibodies produced by the two animals are discussed. 3. Calculations are made of the equivalent composition of the specific precipitate at various reference points in the reaction range. 4. Certain theoretical and practical implications of the findings are pointed out.     

A QUANTITATIVE THEORY OF THE PRECIPITIN REACTION : V. THE REACTION BETWEEN CRYSTALLINE HORSE SERUM ALBUMIN AND ANTIBODY FORMED IN THE RABBIT

1. The reaction between crystalline horse serum albumin and homologous antibody in rabbit sera is quantitatively accounted for by expressions similar to those derived from the law of mass action for other immune precipitating systems. 2. The reaction of an azo dye prepared from crystalline serum albumin by coupling with diazotized R-salt-azo benzidine was also studied with homologous antibody and anti-serum albumin. 3. Quantitative data obtained on cross reactions with the two antigens differ markedly from data on the corresponding reactions in the egg albumin system and indicate that tyrosine and perhaps histidine, while important in determining the serological specificity of egg albumin, have little connection with the specificity of serum albumin. 4. Calculations are made of the equivalent composition of the specific precipitate at various reference points in the reaction range.     

A QUANTITATIVE STUDY OF THE CROSS REACTION OF TYPES III AND VIII PNEUMOCOCCI IN HORSE AND RABBIT ANTISERA

1. A preparation of the specific polysaccharide of Type VIII pneumococcus is described in which the use of heat, strong acid, and alkali were avoided. 2. Quantitative estimations are given of the homologous and cross reacting precipitin and agglutinin in Types III and VIII antisera produced in the rabbit and in the horse. Quantitative data are also given on the mechanism of the Type VIII precipitin reaction and the cross reaction between the Type III polysaccharide and Type VIII antipneumococcus horse serum. 3. The significance of the data is discussed.     

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.     

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.     

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.     

A FURTHER STUDY OF THE CROSS REACTION BETWEEN THE SPECIFIC POLYSACCHARIDES OF TYPES III AND VIII PNEUMOCOCCI IN HORSE ANTISERA

1. The cross reaction of the specific polysaccharide of Type VIII pneumococcus with Type III antipneumococcus horse serum has been studied quantitatively and found similar to the S III-anti-S VIII reaction. 2. Contrasted with the general similarity of the two-segment reaction curves were distinct qualitative and quantitative differences in the course and character of the reciprocal reactions with respect to each segment. 3. These differences could be interpreted in terms of the known chemical differences between the specific polysaccharides of the two types. A minimum molecular weight of 62,000 was calculated for S III and 140,000 for S VIII. 4. It was also found possible to fractionate the Type VIII antibody into portions characteristic of each segment of the cross reaction curve. At least three different kinds of Type III and Type VIII anticarbohydrates were identified.     

QUANTITATIVE STUDIES ON THE PRECIPITIN REACTION : EFFECT OF SALTS ON THE REACTION

1. A quantitative study has been made of the effect on the precipitin reaction between the specific polysaccharide of Type III pneumococcus and the homologous antibody of salt concentrations ranging from O.1 M to 1.79 M, including the effect of ions of higher valence. 2. Within these limits, observed decreases in precipitated antibody with increasing salt concentration appear to be due to a decrease in the amount of antibody combined with the S III, rather than to an increase in solubility of the S III-antibody compounds. 3. The egg albumin-antibody reaction is far less sensitive to changes in salt concentration than is the S III-antibody reaction.     

QUANTITATIVE STUDIES ON THE PRECIPITIN REACTION : THE DETERMINATION OF SMALL AMOUNTS OF A SPECIFIC POLYSACCHARIDE

A method, based on the precipitin reaction, is given for the micro determination of the specific polysaccharide of Type III pneumococcus. As little as 0.01 mg. of S III can be determined. The method should be applicable to any specific polysaccharide upon standardization of an homologous antibody solution or antiserum in the region of excess antibody.     

THE PRECIPITIN REACTION OF ANTIPNEUMOCOCCUS SERA : I. THE PRECIPITIN INDEX.

The mechanism of the precipitin reaction between antipneumococcus sera and the type-specific soluble carbohydrate is investigated. The sensitivity of the reaction is found to be generally constant when expressed by the product of the concentrations of the two reacting substances. Precipitin index (P.I.) is defined as one-millionth of the reciprocal value of this product. See PDF for Structure Zonal phenomena and their bearing on the absolute concentration and the equivalent weight of the antibody are discussed. The greater tendency towards exhibition of a post-zone in Type III is connected with the lower acid equivalent of the homologous specific carbohydrate. The influence of the addition of normal serum and the influence of slight changes in pH are studied. The addition of normal serum as well as the increase in pH promote the pro-zone, whereas decrease in pH promotes the post-zone. The sensitivity of the reaction is accordingly influenced by the pH in different ways depending on the range of concentration. The precipitin index allows the recognition and elimination of zonal irregularities. Thus it offers a method for the standardization of pneumonia antibody.     

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.     

THE PRECIPITIN REACTION OF ANTIPNEUMOCOCCUS SERA : III. THE RATIO OF PRECIPITIN TO PROTECTIVE ANTIBODY IN TYPE II

The ratio precipitin/protective antibody is given for several fresh antipneumococcus horse sera (Type II). The application of the precipitin test here dealt with and that of similar ones, based on the conception of a parallelism between precipitin and protective antibody, is limited to unrefined horse sera.     

THE PRECIPITIN REACTION OF ANTIPNEUMOCOCCUS SERA : II. THE RATIO OF PRECIPITIN TO PROTECTIVE ANTIBODY

The precipitin indices for a number of monovalent and polyvalent antipneumococcus sera were determined under known conditions, and found to vary as did the number of protective units. The ratio precipitin index/protective units in monovalent sera was found to lie between 2.8 and 4.8 for Type I and to be about ten times greater for Type III. Lower values were found in polyvalent horses and when mixing heterologous monovalent sera with each other. The influence of the duration of treatment upon the quotient was studied. Several refined and concentrated preparations showed a relative increase in precipitin activity.     

SPECIFICITY OF THE PRECIPITIN REACTION IN TOBACCO MOSAIC DISEASE

1. Leaf extracts of Sudan grass, Hippeastrum equestre Herb., lily, and Abutilon striatum Dicks. (A. Thompsoni hort.), each affected with its respective mosaic disease, and peach affected with yellows disease, were tested for their ability to precipitate antiserum for virus extract of tobacco mosaic disease. No precipitate occurred. 2. Nicotiana glutinosa L., N. rustica L., and Martynia louisiana Mill. were added to the list of hosts of tobacco mosaic virus which have been tested with antiserum for the same virus in N. tabacum L. var. Turkish. The object was to determine the presence or absence of material reacting with the specific precipitins such as that already demonstrated in extracts of tomato, pepper, and petunia affected with the same virus. The presence of specific substances was demonstrated in every case. 3. The viruses of ringspot and cucumber mosaic diseases were multiplied in Turkish tobacco and leaf extracts of the affected plants were used in turn as antigens in precipitin tests with antiserum for tobacco mosaic virus extract of Turkish tobacco. A slight precipitation resulted in the tubes containing undiluted antiserum and virus extract such as occurs when juice from normal tobacco is used with undiluted antiserum. No precipitate was demonstrable that was specific for virus extracts of tobacco affected with either ringspot or cucumber mosaic disease. 4. The results favor the interpretation that the specific antigenic substance in virus extract of tobacco mosaic disease is foreign antigenic material, possibly virus itself, not altered host protein.     

ANTIPROTEINS IN HORSE SERA : III. ANTIBODIES TO RABBIT SERUM ALBUMIN AND THEIR REACTION WITH ANTIGEN

1. Two horses were injected subcutaneously with alum-precipitated rabbit serum albumin. 2. The resulting antibody resembled diphtheria antitoxin and anti-egg albumin in the horse in giving a sharp zone of flocculation with antigen, in being water-soluble, in reactivity toward an anti-antibody rabbit serum, and in its electrophoretic properties. 3. The effect of continued immunization, and of variation in volume and temperature on the reactivity of the antibody are discussed. 4. Intravenous injection of the same antigen into horses did not give rise to detectable amounts of antibody of the same type.     

STUDIES ON RABBIT LYMPHOCYTES IN VITRO : VI. THE INDUCTION OF BLAST TRANSFORMATION WITH SHEEP ANTISERA TO RABBIT IGA AND IGM

Sheep antisera specific for the three major immunoglobulin groups of the rabbit, i.e. IgG (γ-chain), IgA (α-chain), and IgM (µ-chain), are each able to induce blast transformation of the peripheral lymphocytes of the rabbit when added to in vitro cultures. The per cent of lymphocytes transformed with each antiserum indicate that one-fourth of the peripheral lymphocytes carry or have the capacity to synthesize molecules of all three of the major immunoglobulin groups, and that the remaining three-fourths carry or have the capacity to synthesize only two (IgG and IgM). The data do not permit direct conclusions concerning the ability of a single cell to produce molecules belonging to more than one immunoglobulin group at the same time, or the ability of a given cell to make a transition from the synthesis of molecules of one immunoglobulin group to those of another group.     

QUANTITATIVE STUDIES ON THE PRECIPITIN REACTION : ANTIBODY PRODUCTION IN RABBITS INJECTED WITH AN AZO PROTEIN

1. The preparation is described of a deep red protein dye, R-salt-azo-benzidine-azo-crystalline egg albumen, which contains no more than traces of protein with the original egg albumen specificity. 2. Based on previous publications of the writers, a quantitative method is given for the micro estimation of precipitin in the antisera to the dye. The method gives the actual weight of precipitin and may be applied to the determination of the maximum amount of precipitable antibody in any antiserum. 3. Data are given (1) on the influence of the period between the final injection and the bleeding on the precipitin content of rabbit antisera to the azo protein; (2) on the magnitude of the antibody response following the injection of multiple doses of the antigen varying within wide limits; (3) on the variations in the precipitin content of the sera of rabbits given successive courses of antigen injections; and (4) on the stability of antisera stored in the cold. 4. Four antisera were obtained in which over 100 times as much precipitin was recovered as the amount of antigen injected. This supplements the growing mass of evidence against the theory that specific antigen fragments are actually incorporated into the antibody molecule.     

EXPERIMENTAL SYPHILIS IN THE RABBIT : VII. AFFECTIONS OF THE EYES.

From the study of a number of instances of eye infection in the rabbit, it was found that a variety of affections might occur following scrotal or testicular inoculations of Treponema pallidum. Those observed included ciliary injection, conjunctivitis, keratitis, and iritis which might occur separately or in combination with one another, except that keratitis and iritis were always accompanied by a reaction in the ciliary vessels and usually by a conjunctivitis. Several forms of each of these affections were described, and while some of them were regarded as presenting a very characteristic picture, it was recognized that the conditions present in other cases were not sufficiently distinctive to permit of a clinical diagnosis. With a few exceptions, however, the pathology of the lesions was sufficient to identify them as processes of a syphilitic nature. It was also found that this group of lesions usually arose from a common focus of infection which was located in the episcleral tissues immediately surrounding the cornea. From this point, the infection tended to spread to the conjunctiva and the cornea, or toward the canal of Schlemm and the spaces of Fontana and thence to the ciliary body, the iris, and the choroid. The localization of the lesion and the mode of extension were held to be responsible for the combination of manifestations usually observed. From an analysis of the circumstances under which affections of the eyes occurred, it was found that the great majority of them occupied a definite position in the scheme of tissue reactions, being the only generalized lesions developed or the last type of lesion to appear. These facts, together with the unusual frequency of relapse in these affections, were believed to indicate that a low degree of protection was conferred upon these tissues by reactions taking place elsewhere and that the protection afforded by the local reaction was of a feeble character. This deduction was in part confirmed by the fact that it was found to be possible to increase or decrease the incidence of eye lesions by the use of experimental means which varied the scheme of reaction in animals inoculated with a given strain of Treponema pallidum.     

PRECIPITIN RESPONSE IN THE BLOOD OF RABBITS FOLLOWING SUBARACHNOID INJECTIONS OF HORSE SERUM

1. Rabbits which have received a single dose of normal horse serum in the subarachnoid space produce precipitins in the blood in greater abundance, of higher titer, and persisting longer than those in control rabbits which have received a similar injection intravenously. 2. Repeated subarachnoid injections of normal horse serum in rabbits induce precipitins in the blood early. These may appear in high titer as soon as 1 week after the initial injection, whereas in rabbits similarly treated intravenously no precipitins are found at this time. They may appear a few days afterward and reach a high titer. 3. No anaphylactic manifestations occurred in rabbits treated repeatedly with subarachnoid injections of normal horse serum when the precipitin content of the blood was high. 4. Anaphylactins, as determined by passive transfer of anaphylaxis, were demonstrated in sera with high precipitin content. 5. These experiments may explain clinical evidences of anaphylaxis, observed when an initial intravenous injection of horse serum followed a series of intraspinal injections of such serum.