Horse Antiserum Research Articles

A Ring Precipitin Test for Estimating the Concentration of Antibody in Small Amounts of Immune Serum

Summary and Conclusions A ring precipitin method has been described for estimating the concentration of antibody in small amounts (0.03 to 0.21 cc. is sufficient for a preliminary titration and for duplicate checks) of immune serum. The test depends on allowing an optimal amount of antigen to diffuse into increasing dilutions of the antiserum in such a way that the smallest detectable concentration of antibody is precipitated in the form of a visible ring on the upper surface of the gelatinized antigen. Data are presented on the control of variables and on the quantitative aspects of the procedure in the titration of anti-horse serum, as checked by the “optimal proportions” method of Dean and Webb. Preliminary information on the titration of other antigen-antibody systems has been given.

The Reputed Antigenic Relationship between Organisms of the Brucella Group on the One Hand, and of the Pasteurella, Pfeifferella and Proteus Groups on the Other

1. Provided that due precautions are taken to ascertain the normal level of agglutinins in a given host, and that antigenically smooth strains are used, there is every reason to believe that the occurrence of agglutinins in a titre above the normal range of variation is due to infection—latent, active or past—with the specific organism in question, or in a few instances with an organism, usually of the same genus, sharing a similar antigen.2. This conclusion may have to be modified for the occurrence of agglutinins toProteusOX19 in human typhus sera, since the exact relationship of this organism toRickettsia prowazekiis still doubtful. If, however, future work shows the truth of Felix's (1933) contention that serological types ofProteusX correspond to serological types of typhus virus (Rickettsia), even this apparent exception will fall within the general rule.3. The examination of sixty-four horse and fifty-three cattle sera, and the performance of numerous cross-agglutination and cross-absorption experiments with rabbit and horse antisera, lend no support to the suggestion made by certain workers of the existence of an antigenic relationship between Brucella strains on the one hand, and strains of Pfeifferella, Pasteurella andProteusX on the other.

The Dissimilarity of the Results of Precipitin Titrations Performed with a Constant Amount of Antiserum and with a Constant Amount of Antigen

1. Antisera, made against crystalline egg-albumin, have been examined by titrating a constant amount of antiserum against falling amounts of the antigen (the usual Dean and Webb method), and also by the reverse procedure in which the amount of antigen was constant and that of antiserum varied. The ratios by the two methods were never found to agree; nevertheless, in the majority of cases the relationship which the ratio by one method bore to the ratio by the other method was of the same order, and it is possible that this relationship is constant for crystalline egg-albumin and its antisera.2. The conclusions reached in my previous work (1931) require modification, because in those experiments insufficiently extensive ranges were used and as a result it was found that when varying amounts of anti-horse serum were titrated against a fixed amount of antigen, particulation was quickest in the tube containing most antiserum.3. Titrations by the reverse method over a fairly considerable range have provided evidence that the proportions of crystalline egg-albumin and a given homologous antiserum favourable for the most rapid particulation are constant despite absolute quantitative changes. The proportions in which these reagents particulate most rapidly are similarly constant in titrations by the usual Dean and Webb method.4. Why the two methods of titration give different results I am unable to explain, but the point of optimal particulation found by the Dean and Webb method probably coincides with the point of neutrality of antigen and antibody. Since in the Ramon (1923) method of titrating diphtheria toxin and antitoxin the reverse method, with constant antigen and varying antiserum, is used, the work reported here suggests that the determination of the relationship to each other and to animal values of the results of titrations of toxin and antitoxin by both methods might yield helpful information.5. Some points of practical importance in the performance of optimal proportions titrations are appended.

Observations on Serological Reactions with Albumose Preparations.

In continuation of the experiments briefly reported in this journal, the following observations were made. Immune sera prepared with azoheteroalbumose or azoprotoalbu-mose did not distinguish between the azocompounds of heteroal-bumose and protoalbumose of the same origin. There was a distinct specificity, however, with some immune sera in tests with azoalbumoses derived from different material such as horse serum, eggwhite or Witte peptone. The reactions just mentioned were inhibited by azoalbumoses, particularly those of the same origin as the material used for immunization. Since in most of the experiments described, crude preparations were used, the question arises whether the precipitin reactions with the azoalbumose preparations are due to the albumoses themselves or to the presence of other substances, such as metaprotein or plas-tein. Evidence against the latter assumption is the fact that reactions were also obtained with azocompounds of purified hetero- and protoalbumose from Witte peptone and that the reactions with azometaprotein and azoplastein were rather weaker than those given by the azoalbumoses. By immunization with a crude preparation of heteroalbumose (not coupled with diazobenzene) made by peptic digestion of horse serum, immune sera were obtained which gave precipitin reactions of moderate strength with the substance used for immunization and furthermore with metaprotein, prepared by short peptic digestion of horse serum, and with unchanged horse serum. No reactions took place with heteroalbumoses made from egg white or Witte peptone. On the other hand, in conformity with previous work, a common anti-horse serum precipitin reacted only with horse serum but not at all with the metaprotein and the heteroalbumose from horse serum.

EXPERIMENTAL STUDY OF A HORSE ANTIPOLIOMYELITIC SERUM

Through the kindness of Dr. W. H. Park we have been enabled to study a horse antipoliomyelitic serum. This preparation has been supplied us in three forms: citrated blood plasma, serum, and globulin concentrate. We have tested these preparations in vitro and in vivo for inactivating or neutralizing or, to use perhaps a better term, antiviral effects against a constant, potent, filtrate virus of poliomyelitis. The preparations exhibited these effects when combined in vitro. Their action in this respect appears to be greater and more constant than that found by Stewart and Haselbauer for the Pettit antipoliomyelitic horse serum. On the other hand, in vivo tests carried out by us were less successful. In comparison with the constancy of action, under given conditions, of convalescent monkey and human sera, the antipoliomyelitic horse serum displayed striking irregularity, and certain preparations were devoid of protective power. The precise nature of the inactivating substances in the horse antiserum and their relation to the corresponding substances in convalescent sera have still, to be determined. As far as one absorption test carried out by us indicates, precipitin does not play a major rôle in the inactivating process. When an active globulin concentrate was filtered through Berkefeld candles, it lost its in vitro inactivating power. This is not true of convalescent sera in the native state. No tests have, however, been made with globulin concentrates from such sera. The experiments described in this paper raise the question whether, therapeutically considered, the antipoliomyelitic horse serum should be regarded as an exact equivalent of, and hence employed as a perfect substitute for, convalescent serum. This question can only be answered by further experiment and observation.

The Results of some Quantitative Experiments on the Serum Precipitation Reaction

The work described in this paper is based, very largely, on the work of Dean and Webb (1926), and the methods used in the various experiments are similar to those described by them. The results are discussed in two parts. The first is concerned with the mixture of different specimens of anti-horse serum, and with the relationship of the antibody in one specimen to the antibody in another. The second part deals with the nature of the reaction between antigen and antibody in the serum precipitation reaction. The two parts are dependent on each other, and the subject of the second suggested itself whilst the work on the first was in progress. The order in which the work is described is mostly that in which it was done. Nevertheless, conclusions arrived at in one part are, in one or two cases, of importance in the consideration of the other part, and vice versa. A question as to the possibility of strengthening the ratio of an antiserum by the addition of another antiserum led to the earlier experiments.

A Note upon the Serum-Prophylaxis of Measles with special reference to two Horse anti-sera, by E. H. R. HARRIES, M.D., D.P.H., Medical Superin tendent, City Hospitals, University Teacher of Fevers, Birmingham. (Member.)

A Note upon the Serum-Prophylaxis of Measles with special reference to two Horse anti-sera, by E. H. R. HARRIES, M.D., D.P.H., Medical Superin tendent, City Hospitals, University Teacher of Fevers, Birmingham. (Member.)

ANAPHYLACTIC SHOCK CAUSED BY ANTIBODY IN ANIMALS SENSITIZED BY ANTIGEN—REVERSED PASSIVE ANAPHYLAXIS

Anaphylactic shock occurs (in rabbits) when the usual procedure for the production of passive anaphylaxis is reversed; that is, when an animal previously treated with antigen receives the corresponding antiserum by way of the circulating blood. This susceptibility to the action of anti-horse serum produced by injection of antigen reaches maximum intensity after an interval of 4 hours presumably required to permit penetration of the antigen in sufficient concentration into the tissues. Desensitization to the action of a shock-producing dose of anti-horse serum can be brought about by repeated small doses of the same antiserum. Anaphylactic shock and local anaphylaxis manifested by the acute inflammation of an immunized animal when injected with the antigen used for immunization (Arthus phenomenon) occur under analogous conditions; that is, when antigen and antibody meet within the tissues. The peculiar characters of these reactions are dependent upon the site of entry of the irritating agent, which is the vascular system in one instance and tissue spaces in the other, and upon the concentration of antigen and antibody within susceptible tissues. Meeting of antigen and antibody within susceptible tissues is sufficient to explain the phenomena of local and general anaphylaxis so that it is unnecessary to assume the sudden formation of a toxic substance (anaphylatoxin).

Acute Inflammation Caused by Antibody in an Animal Previously Treated with Antigen

Abstract Normal rabbits may be sensitized to the local action of horse serum by injection of serum obtained from an animal immunized by repeated injections of the antigen. Horse serum injected into the dermis of this passively sensitized animal causes acute inflammation (Arthus phenomenon). This passive transfer of sensitization shows that the blood serum of the immunized animal contains an antibody which introduced into a normal animal renders it sensitive. Furthermore, simultaneous injection of horse serum and anti-horse serum from rabbit causes acute inflammation which fails to occur when anti-horse serum is replaced by normal serum. These observations suggest the possibility that acute inflammation will occur whenever antigen and antibody come into contact within the tissues. The attempt has been made to determine whether a normal rabbit which has received horse serum will react with acute inflammation when serum from an immunized or “sensitized” animal is injected into its tissues.

Concentration of the protective bodies in anti-pneumococcus serum by means of specific precipitation

The addition of a water-clear extract of pneumococcus to the homologous antiserum produces a voluminous precipitate which carries down with it the agglutinins and practically the totality of the protective bodies against pneumococcus infection in animals. This precipitate when washed and resuspended in saline solution to the original volume of serum protects as well as the whole serum. The protein content of such solutions has varied from 0.09 to 0.34 per cent. as contrasted with about 6 per cent, in the original serum. The solution of this precipitate is not necessary to insure protection, and when produced by dilute alkali (NaOH) frequently destroys the immune bodies.This concentration of the immune bodies differs from the method described by Dehne and Hamburger1 for fixing tetanus antitoxin by means of antihorse serum and similar results with diphtheria antitoxin described by Weill,—Halle and Lemaire2 in that the immune serum serves as a precipitin and not as precipitogen. By this reversal of reaction ...