Production Of Antitoxin Research Articles

ANTITOXIN PRODUCTION IN CASES OF DIPHTHERIA NOT TREATED WITH SERUM

ANTITOXIN PRODUCTION IN CASES OF DIPHTHERIA NOT TREATED WITH SERUM

The Production of Diphtheric Antitoxin in Tissue-Cultures

Abstract It has been known for a long time that antibodies can be produced by tissues cultivated in vitro. Carrel and Ingebrigtsen (1) cultivated lymph-node and bone-marrow tissues of the guinea pig in homologous plasma to which goat's erythrocytes were added, and reported the development of hemolysin. Lüdke (2) cultivated splenic tissue from rabbits who had been injected with ox or goat-erythrocytes, dysenteric or typhoid vaccine, and found hemolysins and agglutinins. Schilf (3) found bacteriolysins to vibrio cholerae, and Meyer and Loewenthal (4) proved that agglutinins are present in cultures of spleen, lymph-node, etc. R. Kimura (5) found agglutinins for B. coli, and precipitins for chicken and rabbit-sera. Many of the subsequent studies on the subject of antibody-production in tissue-cultures have been made in our Institute, and relate to hemolysin (6), precipitin (6), bacterial agglutinin (6, 7, 10), hemagglutinin (8), bactericidin (7), anaphylaxis antibody (8), virulicidal substance (9) and complement-fixation antibody (10).

Pneumococcus Toxin and Antitoxin

A number of years ago one of us, while working with the scarlet fever skin test, attempted to demonstrate the presence of in the filtrates of broth cultures of the pneumococcus. Although some skin reactions were observed, no satisfactory evidence of the was obtained. In 1935 we1 reported a method of producing a concentrated and purified scarlet fever toxin. It occurred to us that the presence of a in low concentration in pneumococcus culture filtrates might be demonstrated by making use of similar concentration and purification. It had been shown by Parker2 that anaerobically produced autolysates from broth cultures of pneumococci caused, in guinea pigs, hemorrhagic edema of the lungs and necrosis of the skin. Parker3 also demonstrated that antiautolysate serums could be produced in rabbits and horses by the repeated injection of autolysate. While the present study was in progress, Coca4 reported his work on toxins produced in anaerobic cultures of pneumococci. He concluded that under ordinary atmospheric conditions of bacterial cultivation the pneumococcus produces a toxin which lacks the property of stimulating antitoxin production in human beings. The specific purpose of this study was to produce a pneumococcus in a concentrated and purified form from pneumococci grown aerobically in ordinary veal infusion broth with about 0.1% human blood added.

A study of the pneumococcus toxins

Summary 1.A toxin has been demonstrated in filtrates of pneumococcus cultures the injection of which in young children caused a rise in temperature which reached 105°F. in the more susceptible children. 2.All of 34 children under 3 years of age, excepting two who previously suffered either bronchopneumonia or a severe bronchitis, were found susceptible to the type I toxin. 3.All of 31 persons suffering from or convalescent from pneumococcus pneumonia were found immune to the type I toxin (skin test). 4.The toxin seems to be type-specific. 5.The toxin is not the type-specific polysaccharide. 6.Cultures grown in the absence of artificially supplied CO 2 have been found to contain toxin which, however, seemed not to be capable of stimulating antitoxin production in human beings. 7.Two injections in children were followed, in the majority of instances, by prompt antitoxin production (one or two weeks). 8.Serum from two pneumonia convalescents neutralized the pneumococcus type I toxin.

A Study of the Pneumococcus Toxins

Summary A toxin has been demonstrated in filtrates of pneumococcus cultures the injection of which in young children caused a rise in temperature which reached 105°F. in the more susceptible children. All of 34 children under 3 years of age, excepting two who previously suffered either bronchopneumonia or a severe bronchitis, were found susceptible to the type I toxin. All of 31 persons suffering from or convalescent from pneumococcus pneumonia were found immune to the type I toxin (skin test). The toxin seems to be type-specific. The toxin is not the type-specific polysaccharide. Cultures grown in the absence of artificially supplied CO2 have been found to contain toxin which, however, seemed not to be capable of stimulating antitoxin production in human beings. Two injections in children were followed, in the majority of instances, by prompt antitoxin production (one or two weeks). Serum from two pneumonia convalescents neutralized the pneumococcus type I toxin.

The Individual as a Factor in Antidiphtheria Immunity

Summary The experiments dealt with the influence of different methods of immunization upon the antitoxic response of groups of guinea pigs. The points investigated included the condition of the antigen, the total amount of antigen, the division and spacing of doses and the route of injection. The adequacy of the different methods for group immunization was analyzed on the basis of the percentage of individuals made Schick negative. The total amount of antigen influenced the immunity response but to different extents in different zones of dosage: its influence was masked in the zone of “hyperimmunization,” but within the zone “adequate for group immunization” it was manifested in speed of response, and within the zone “inadequate for group immunization” both in speed of response and in percentage of a group finally made immune. However, the amount of antigen could be counterbalanced by other factors: to some extent by the route of injection but especially by the division and spacing of injections. The results are discussed in terms of the expression: A = f(C × S) in which A represents antitoxin production, C, the capacity to respond to specific stimulus and S, the stimulus of the specific antigen. In the present paper this formula was used in the analysis of the development of immunity; in later papers it will be modified for the analysis of immunity possessed long after the last injection of antigen.

Hemolytic Streptococcus Toxins and Antitoxins

Abstract Veldee stated that the use of a toxin prepared from the NY-5 strain of the hemolytic streptococcus in his rabbit ear method of testing was preferable to the use of toxins from other sources for determining the neutralizing value of antitoxins for therapeutic use. We have followed this suggestion for some time, employing a NY-5 toxin for test purposes and also for the production of antitoxin in horses. In May, 1932 we discovered that, although our antitoxin gave satisfactory neutralization values when tested against our own toxin, it failed to do so on titration with standard toxin obtained from the National Institute of Health. This was decidedly at variance with our earlier experience, for previously our antitoxins had neutralized both our own and National Institute toxins equally well. This finding led us to obtain transplants of the NY-5 strains from the National Institute of Health and from the Bureau of Laboratories of the New York State Department of Health.

IS THERE A SCARLET FEVER TOXOID?

In early studies of diphtheria toxin, it was observed that on standing the toxin undergoes spontaneous change resulting in loss of toxicity. But this loss of toxicity is not accompanied by corresponding loss in ability to neutralize diphtheria antitoxin. In elaborating his theory of immunity, Ehrlich employed the term to designate this nontoxic substance which is still able to bind antitoxin. The French prefer the term anatoxin for this substance. The change from toxin to toxoid, which takes place spontaneously in solutions of diphtheria toxin, may be hastened by addition of certain chemicals. Ramon used formaldehyde to accelerate and complete the change from diphtheria toxin to toxoid and utilized the formolized product for active immunization. He found the same method useful in obtaining toxoid from tetanus toxin. Toxoids that have lost their toxicity but retain the ability to neutralize and stimulate the production of antitoxin are satisfactory agents for

Prof. Georges Dreyer, C.B.E., F.R.S

GEORGES DREYER was bom on July 4, 1873 at Shanghai, where his father, Capt. G. H. N. Dreyer of the Danish Royal Navy, was stationed at the time. He was educated in Copenhagen, where he qualified in medicine in 1898 after a very brilliant career as a student. While serving as a medical officer in the Navy, he found time to do some work with Salomonsen in the Pathological Department of the University, and continued this later when he was house physician at the fever hospital. For a time he was responsible for the production of diphtheria antitoxin at the newly established Serum Institute, and he received his M.D. degree in 1900 for a thesis on diphtheria toxin and antitoxin. Dreyer became a Privatdocent in the University of Copenhagen, and carried on an astounding amount of research on a wide variety of subjectstyphoid agglutination, the effect of light on bacteria and protozoa, the action of enzymes, and other subjects. An interest in mathe matics had been-early stimulated by his father, and in all his varied research he attempted to obtain accurate quantitative results. He found existing methods unsatisfactory for exact quantitative work, and developed new technique in many branches of biological research. He travelled extensively in Scandinavia, Germany, France and England, and came into contact with the leading workers in many fields. An excellent linguist, he spoke Danish, French, German and English fluently.

Production of Antitoxins by Means of Bacteriophage

Production of Antitoxins by Means of Bacteriophage

Index

Abstract A comparison of the toxicity of various meningococcus preparations 269 A hypothetical mechanism of antibody formation 423 Active immunization of white mice by a non-polysaccharide and probably nonprotein derivative of the pneumococcus 405 Agglutination and precipitation, Specific, II. Velocity of the reactions 153 Agglutination reactions in rheumatoid arthritis. II. The nature and significance of agglutination reactions with streptococcus hemolyticus 205 Agglutinin responses to typhoid-paratyphoid vaccination, The 81 Agglutinogens M and N of Landsteiner and Levine, Heredity of the, III. Medicolegal application for the determination of non-paternity 63 Alum on horses used for the production of diphtheria antitoxin, The effect of. 261 Anaphylaxis in the rabbit, Studies in, I. A study of the factors concerned in the establishment of maximal hypersensitiveness in rabbits to egg white and horse serum 101 Anaphylaxis in the rabbit, Studies in, II. A study of the effect of histamine, barium chloride and epinephrine of unstriped muscle of the guinea pig and rabbit 125

The Effect of Alum on Horses Used for the Production of Diphtheria Antitoxin

Summary and Conclusions Three groups of horses were immunized for the production of diphtheria antitoxin. Group 1 received toxoid containing 0.5 per cent potassium alum, followed by diphtheria toxin containing alum. Group 2 received diphtheria toxoid, followed by diphtheria toxin—both without alum. Group 3 received diphtheria toxin-antitoxin, followed by diphtheria toxin—both without alum. As a result of the marked intolerance of the horses of group 1 toward alum antigens, they received less than threetenths of the antigen dosage given to the horses of Group 2. At trial bleedings after three months the average antitoxin titers per cubic centimeter for the horses of groups 1 and 2 were practically the same, namely, 360 units and 350 units, respectively; while the average titer of group 3 was 580 units. Based on the relative antigen doses and the subsequent antitoxin titers, the antigenic effectiveness of immunization with alum-treated toxoid and toxin was about three times as great as that of toxoid and toxin without addition of alum. A cumulative toxic effect was observed in the horses of group 1 after repeated doses of alum combined with diphtheria antigens. During a period of several weeks after injections containing alum had been discontinued, most of the horses in this group gradually declined and two-thirds of them died with symptoms of alum poisoning. The disastrous late after-effects upon horses of repeated injections of alum along with diphtheria antigens completely overshadow any increase in immunizing power attributable to the addition of the alum. It appears improbable, however, that the addition of a small amount of alum to the two or three doses of toxoid used for human immunization would exert any harmful effect.

Anaphylaxis to Diphtheria Toxin

Abstract Previously reported experiments (1, 2, 3) on anaphylaxis apparently due to the toxin or toxoid antigen were made upon actively sensitized (antitoxic immune) animals. The results had indicated that while other diphtheria antigens participated to some extent, the major part of the anaphylaxis could be assigned to the toxin or toxoid antigen, provided one used both for the immunization and for the intravenous test minimal amounts of unheated filtrate in which toxin or toxoid was the dominant antigen, and selected for intravenous test the individual guinea pigs that had been “earliest” in antitoxin production. Passive sensitization possesses certain advantages over active sensitization in the distinction of reactions due to toxin-antitoxin from reactions due to other diphtheria antigen-antibody combinations, for with the passive method it is possible to examine each of the sensitizing sera for antibodies other than antitoxin and, when desired, to remove the other antibodies by absorption of the antisera before using them for sensitization.

Method for Increasing and Prolonging the Production of Antitoxins in Horses

Abstract When horses are immunized by the injection of increasing doses of diphtheria toxin or anatoxin, the serum of the animals comes to contain increasing quantities of antitoxin. At the end of a period varying with different factors referring to the antigen or the animal, the antitoxic power of the serum reaches a certain degree which appears to be the highest degree which it is possible to attain under any conditions. As soon as this maximum is reached, therefore, it is considered preferable, by certain laboratories, to bleed the animals completely. In other laboratories, the practice is to allow a period of about eight days to elapse following the last injection of the antigen and then bleed the animal in two fractions of 6 to 8 liters each, done a few days apart. This double bleeding is repeated every month.

Comparative Studies of Infusion and Infusion-Free Diphtheria Toxin in Antitoxin Production and in Standardization by the Flocculation, Subcutaneous, and Intracutaneous Tests

Summary The practical application to routine and experimental purposes of diphtheria toxin produced with an infusion-free peptone medium is reported. The procedures followed in the routine preparation of the medium and toxin are given in some detail. The marked reduction in cost of production of the toxin as compared with that of meat-infusion toxin, and the greater uniformity in the potency obtained are pointed out. Results of comparative studies of the flocculation-reaction test are recorded. The infusion-free toxin was found to be at least as satisfactory as the infusion toxin, in these tests. The micrometer-syringe method proved less accurate than the “pipette method” in which larger volumes of toxin are measured for the test. A comparison of the flocculation-reaction test with the subcutaneous and the intracutaneous animal method for determining diphtheria antitoxic values showed closer agreement with the subcutaneous test. Variation was less than 10 per cent with the subcutaneous and nearly 20 per cent with the intracutaneous method. In titrations of sera from certain horses, especially during the early stages of immunization, wide variations were noted between the results of the flocculation and subcutaneous tests on the one hand and those of the intracutaneous test on the other. The flocculation reaction was found to be a satisfactory method for the approximate estimation of the unit content of concentrated antitoxins. The flocculation test was used to determine the time at which optimum antitoxic titer might be expected in the serum of horses immunized against the infusion-free toxin by different methods. The value of the toxin as an antigen was also studied in a series of twelve horses. The results demonstrated that the infusion-free toxin is equal to, if not better than, meat-infusion toxins in the routine immunization of horses for diphtheria antitoxin production. Three horses given injections of purified (ultrafiltered) infusion-free peptone toxin appeared to respond as well as horses receiving other toxins and possibly with less disturbance of the normal metabolic processes. Further studies are in progress. The chief points which are brought out by this investigation may be enumerated as follows: Diphtheria toxin of uniformly high potency has been produced in infusion-free peptone medium on a routine basis for a period of nearly two years.The infusion-free toxin compares in all respects very favorably with routine infusion toxin for the immunization of horses in the production of diphtheria antitoxin.This toxin has also been used extensively in comparative flocculation-reaction tests and found in all respects to compare favorably with the infusion toxin.The flocculation-reaction test for standardization of diphtheria antitoxin proved more accurate when relatively large volumes were measured by pipettes than when the micrometer-syringe method was used.Closer agreement was found between the flocculation-reaction-test and the subcutaneous animal method for the determination of antitoxic content than between the flocculation and the intracutaneous animal test, or between the subcutaneous and the intracutaneous test.The flocculation-reaction test was satisfactory for preliminary estimations of the antitoxin content of the concentrated as well as of the untreated serum.Besides its other advantages, the great saving in production cost as compared with the usual meat-infusion toxin makes the infusion-free peptone toxin an important economic factor where large amounts of toxin are required for the production of diphtheria antitoxin, for standardization, and possibly also, for the active immunization of persons with toxoid.

Studies of the Flocculation-Reaction Time in the Course of Immunization and the Quantitative and Qualitative Changes of the Proteins

Summary A consistent increase in the flocculation-reaction time has been found during the immunization period of horses used for diphtheria antitoxin production. It is suggested that the speed of reaction is an expression of the interfacial state of the antitoxic-protein-molecule complex. A study was made of various quantitative and qualitative characteristics of antitoxic sera as related to the flocculation reaction. Nine horses were immunized with infusion-free toxin, by both a slow and a rapid immunization technic. Three horses were immunized with purified infusion-free toxin and three horses with a routine toxin containing meat infusion. During the immunization period the horses were followed for any biochemical changes in order to determine, if possible, the existence of any relationship between the flocculation-reaction time and quantitative differences of one or more of the physicochemical constants of sera. A technic was developed for determination of the surface tension of plasma in order to exclude the errors present when serum is used.

Hypersensitiveness to Diphtheria Bacterial Products

Summary This paper reports further experiments on guinea pigs actively sensitized against diphtheria toxoid filtrate. Five points are made. First, that the anaphylactic reaction of the actively sensitized guinea pigs is inhibited by incubation of the test dose of antigen (toxin or toxoid filtrate) in mixture with antitoxic horse globulin prior to its intravenous injection. Second, that actively sensitized guinea pigs are made refractory by intravenous injection of the antitoxic horse globulin or serum shortly before the test injection of the toxin or toxoid filtrate antigen. Third, that guinea pigs anaphylactically reactive to toxoid filtrate are made refractory by subcutaneous injections of toxoid filtrate ten and seven days before the time of intravenous test, but not by similar injections of diphtheria bacterial vaccine. Fourth, that the anaphylactic reaction is inhibited if filtrate absorbed with antitoxic rabbit serum is used as the intravenous antigen, but not if filtrate similarly absorbed with antibacterial rabbit serum is used. Fifth, that the capacity of toxoid filtrate either to invoke antitoxic immunity or to actively sensitize, is greatly reduced, and under certain quantitative conditions entirely lost, if filtrate absorbed with antitoxic rabbit serum is used as immunizing or sensitizing material, while the same filtrate similarly treated with antibacterial rabbit serum retains the capacity both to invoke antitoxin production and to sensitize against the toxoid filtrate.

Hypersensitiveness to Diphtheria Bacterial Products

Summary This paper reports the anaphylactic reactions of guinea pigs immunized with toxoid and subsequently injected intravenously either with phenolized filtrate containing the toxin antigen or with formolized filtrate containing the toxoid antigen. It was proved that the acute death of the antitoxic immune animals represented true anaphylactic reactions specific to bacterial products contained in diphtheria filtrate. While it was recognized that other constituents participate to some extent, the evidence as a whole indicated that the toxin or toxoid antigen is the principal agent of the anaphylactic reaction of antitoxic immune guinea pigs to unheated filtrates containing a relatively large proportion of toxin or toxoid. The complications due to the substances other than toxin or toxoid are least prominent if one uses both for the immunization and for the intravenous test, minimal amounts of unheated filtrate in which toxoid is the dominant antigen, and finally selects for intravenous test the individual guinea pigs that had been “earliest” in antitoxin production. As might be expected, the complications become more prominent if either the immunization or the intravenous anaphylaxis test is made with diphtheria filtrate in which toxin or toxoid is not the dominant antigen (e.g., filtrates of weakly toxicogenic strains or heated filtrates of toxicogenic strains). Most of the experiments were made with toxoid-immunized guinea pigs, but after a proper rest period animals immunized with toxin-antitoxin gave anaphylactic reactions to intravenous injection of formolized toxoid-containing filtrates.

An Analysis of the Influence of Irradiation by means of a Mercury Vapour Lamp upon the Health and Fertility of a Breeding Stock of Guinea-pigs and upon the Health of their Offspring during the First Six Weeks of Life

The maintenance of a constant supply of healthy guinea-pigs is an important part of the work of laboratories which are engaged in the production of diphtheria and tetanus antitoxin. The subacute infective processes to which malnutrition predisposes enhance the effect of the test dose of toxin, with the result that irregular deaths among the animals under test render difficult the titration of the antitoxin. For this reason it is desirable that breeding stocks should be kept in suitable animal houses under the best hygienic conditions possible. This policy has been followed in the Serum Department of the Institute for many years; there have been comparatively few introductions of stock from outside sources, and within recent years special attention has been devoted to diet.

SERUM DISEASE AND SERUM ACCIDENTS

This report was prepared at the request of the Council on Pharmacy and Chemistry. The Council considered the report, approved it, and authorized its publication. W. A.Puckner, Secretary. A little more than forty years ago, Hericourt and Richet established the principle of passive immunization. They demonstrated that the blood of an animal immunized by repeated injections of a micro-organism contains something which protects an otherwise untreated animal against infection by the organism used to immunize the first animal. The discovery of this important principle of passive immunization prepared the way for the production of diphtheria antitoxin in 1890 by von Behring and for all the other therapeutic serums and antitoxins which have been offered to the medical men of the present generations. The prophylactic or therapeutic value of a few of these serums is beyond question; of some, the most that can be said is that they are probably