Mixture Of Toxin Research Articles

The flocculation of tetanus toxin and toxoid. I.

1. The flocculation of tetanus toxins and toxoids produced in a modified Tarozzi broth was made the subject of an exhaustive study. 2. At least three flocculation zones were found. 3. The lowest flocculation value always proved to agree with the true point of equivalence of the antitoxin-binding (A.B.V.). 4. When mixtures of toxin and antitoxin were injected into mice, only about half the total quantity of antitoxin that can be bound (as appears from A.B.V. or Lf) appeared to be required for the neutralization of the toxic effect. The quantity of toxin thus found was indicated as M.T.V. So this M.T.V. is approximately 1/2 × A.B.V. or Lf. 5. During the incubation of tetanus cultures all flocculation values (3 or 4) increase steadily. 6. The d.l.m. also increases regularly, but much more quickly, hence the ratio m.l.d./Lf increases very considerably during incubation. 7. At the same time the ratio M.T.V./Lf remains constant, i.e. about 1/2. 8. The possible theoretical explanation of the different problems was discussed.

The combination of antitoxin with toxin and toxoid.

1. Non-avid tetanus antitoxin appears to have a higher titre in relation to an avid standard if titrations are made against test toxins with a high toxoid content. The least avid serum tested appeared to be 2·87 times as potent when titrations were made against a toxin grown for 35 days than when a 2-day growth was used. Using the same test doses avid sera did not show this discrepancy.2. A mixture of short-growth toxin and formol toxoid reacted similarly to the long-growth toxin.3. Some non-avid tetanus antitoxic sera showed evidence of difference in goodness of fit with two test toxins; one serum was 2·49 times the titre of another when tested against one toxin and only 1·37 times when tested against another toxin.4. Diphtheria toxoid has less affinity than toxin for antitoxin; this difference is greater with formol toxoids of poor immunizing efficiency; the difference is further accentuated if non-avid anti-toxin is used.5. The Danysz effect may be very small if non-avid antitoxin is used.6. In the presence of excess antitoxin, toxin or formol toxoid can combine with 3, 4 or more times its equivalent as judged byin vivotitrations for free antitoxin.7. The serum ratio, i.e.in vivotitre/in vitrotitre of high or low ratio antitoxin in multiple combinations is nearer unity than that of the original antitoxin.

Antigens in Scarlatinal Erythrogenic Toxin Demonstrable by the Oudin Technic

Summary Dick toxin contains a mixture of antigens for which specific antibodies exist in the sera of horses immunized for the production of scarlatinal antitoxin. At least eight distinct precipitin systems are represented in a mixture of commercial antitoxin and standard erythrogenic toxin employed in this laboratory. Several methods of identifying individual precipitin systems, based on the Oudin technic, have been examined: Specific adsorption of one antibody from the serum gives the most unequivocal results. Replacement of the antigen layer on agar in which zones have been established is a reliable method, but produces some unexplained effects. Agreement between the slopes of lines representing the relation between concentration of antigen and rate of migration should be used only as supportive evidence when dealing with ill-defined preparations. At least one antigen present in Dick toxin, antigen A, is derived from the heart-infusion broth used in toxin production. The erythrogenic toxin proper is represented by the fourth zone (D) in tubes containing our standard toxin and antitoxin.

Studies of the Experimental Myocarditis in Rabbits to Diphtheria Toxin (I)

Rabbits were sensitized with the mixture of diphtheria toxin or anatoxin and rabbit-heart-muscle-phosphatide. After reinjection with the same antigen, their organs, especially heart were investigated histologically. Great degenerative change of myocardium and some pericapillaritis in epicardium were found, and electrocardiogram showed myocardial damage. Without heart-muscle-phosphatide, not so severely changed.

Reactivation of Overneutralized Mixtures of Influenza Virus and Antibody

Abstract Introduction. The detection of influenza carriers may be missed because the virus in nose and throat washings is overneutralized by antibody in the mucous as the virus is liberated from the parasitized cells. The purpose of this study was to determine if overneutralized influenza virus could be reactivated. Numerous investigators have used diverse methods for the purpose of reactivating neutral mixtures of toxins, phages, viruses and bacteria and their specific antibodies. Apparently-neutral mixtures of toxin and antitoxin have been made toxic by simple dilution (1–4). This same “dilution phenomenon” has been observed where apparently-neutral phage-antiphage (5, 6), viral-antiviral (7–19) and bacterial-antibacterial (20) mixtures were employed. Separation of the virus from its antibody by cataphoresis (11, 21) and centrifugation (20, 22–24) has been reported. Adsorption of viruses on Chamberland L2 filter powder, blood clot, euglobulin precipitate, and kaolin, as well as papaindigestion of antibody also have been used to accomplish the same end (9, 10, 12, 16, 25).

Inability of Penicillin to Neutralize Dick and Schick Toxins

Summary and Conclusions1. When mixtures of penicillin and Dick toxin were injected intracutaneously into normal subjects along with parallel mixtures of Dick toxin and isotonic salt solution, no significant difference was observed in the number of positive Dick tests in the 2 series. Incubation of the mixtures, previous to testing, did not influence the outcome.2. Similar results were obtained with Schick toxin.3. Penicillin injected into an area of rash of patients with scarlet fever did not cause blanching.4. Dick positive patients receiving penicillin for an unrelated disease did not become Dick negative after a week of penicillin therapy.5. It is concluded that penicillin has no neutralizing effect upon Dick or Schick toxin.We wish to thank Dr. John H. Hanks for his interest and advice.

Titration of Tetanal Toxins and Toxoids by Flocculation

Abstract In 1926 G. Ramon and P. Descombey (1) described the phenomenon of flocculation which takes place in mixtures of tetanal toxin and tetanal antitoxic serum. In the same communication Ramon and Descombey reported that with certain sera “abnormal” flocculations may occur. Other workers have also reported, in the case of tetanus, similar “abnormal” flocculations, that is flocculations which occur in zones which do not correspond to true toxin-antitoxin neutralization. In order to overcome the difficulty presented by false zones of flocculation a number of solutions have been proposed. In 1937 Ramon (2) reported that he had prepared a serum which flocculated in a single zone. This was accomplished by rapidly immunizing horses with the filtrate from broth cultures of tetanus which had been incubated for only a few days. Prévot (3) claimed that true and false flocculation could be distinguished by a method based on graphic representation.

Effect of Alkyl-Dimethyl-Benzyl-Ammonium Chlorides (Zephiran) Upon Tetanus Toxin

Summary(1) Alkyl-dimethyl-benzylammonium chlorides (Zephiran chloride) in a concentration of 1 : 2,000 inactivates tetanus toxin. This effect takes place almost immediately. Zephiran chloride in a dilution of 1:20,000 fails to do so. (2) Human serum mixed with Zephiran chloride prior to the addition of tetanus toxin markedly interferes with the inactivation of toxin by this compound. Addition of serum to a mixture of 2kphiran chloride and tetanus toxin does not reactivate the toxin.

ARTIFICIAL ANTIBODIES

In vitro synthesis of type specific antipneumococcus precipitins and agglutinins has been reported recently by Pauling and Campbell¹of the Department of Chemistry, California Institute of Technology. Substances simulating specific antibodies formed outside the animal body were demonstrated thirty years ago by Russian immunologists. Ostromyschlenski and Petroff,²for example, incubated a mixture of diphtheria toxin and normal horse serum and obtained an end product with all the therapeutic properties of diphtheria antitoxin. By incubating diphtheria toxin with different protein fractions of normal horse serum, Kryshanowski³prepared two artificial diphtheria antitoxins of different therapeutic value. By somewhat different technics, Sdrawosmisslow⁴and Kimmelstiel⁵incubated diphtheria toxin with commercial trypsin and reported the successful test tube synthesis of a "toxin trypsinate" with the therapeutic properties of an antidiphtheritic serum. In anticipation of future commercial value, artificial antitoxin made by incubating diphtheria toxin with normal serum plus pancreatin was

STUDIES ON THE MOLECULAR WEIGHT OF DIPHTHERIA TOXIN, ANTITOXIN, AND THEIR REACTION PRODUCTS

Purified diphtheria antitoxic horse pseudoglobulin has been prepared which is homogeneous by sedimentation, diffusion, and electrophoresis. Immunologically, however, the preparation contains only 43.5 per cent antitoxin specifically precipitable by toxin. The inactive pseudoglobulin remaining after specific precipitation was found to have the same physical and chemical properties as the original antitoxic pseudoglobulin. Although the molecular weight of antitoxin is the same as that of the normal horse serum globulins, the electrophoretic mobility does differ from those normally present. The molecular weight of diphtheria toxin is 70,000 and of antitoxin is 150,000. From ultracentrifuge studies on the two reactants and on mixtures of toxin and antitoxin in the soluble inhibition zones, the average molecular composition of the specific floccules at certain reference points throughout the equivalence zone and the maximum "valence" of toxin and antitoxin with respect to each other have been calculated. The significance of the results has been discussed in relation to antigen-antibody reactions in general and a possible explanation for the exceptional behavior of the toxin-antitoxin reaction in the region of excess antitoxin has been suggested.

Studies on Serum Fractions

Summary The supernatant fluid of the indicating mixture of antipneumococcal horse serum and pneumococcal SSS showed a smaller ratio to the precipitin prepared with the specific precipitates of pneumococcal SSS (P.V.Ab.) than the original serum, inindicating a decrease of the B-fraction; its ratio to the precipitin prepared with the floccules of diphtheric toxoid-antitoxin (P.V.At.) was the same as that of the original serum. The supernatant fluid of indicating mixtures of diphtheric toxin and its antitoxin showed a smaller ratio to the precipitin prepared with the floccules of diphtheric toxoid-antitoxin (P.V.At.) than did the original serum, indicating a decrease of the A-fraction. This decrease of A-fraction (a fraction reacting with P.V.At.) was shown to be less marked in a combined diphtheria- and tetanus-antitoxic horse serum, of which diphtheric antitoxin constituted only a small part of its A-fraction, than the decrease of the same fraction in antidiphtheric horse sera. These observations indicate that diphtheric antitoxin or pneumococcal antibody constitutes only a part of the fraction reacting with P.V.At. or P.V.Ab. respectively.

Staphylococcus Toxin Combined with Lens Extract as a Desensitizing Agent in Individuals with a Cutaneous Sensitivity to Lens Extract

Lens extract, when combined with staphylococcus toxin and injected intracutaneously, first sensitizes and then desensitizes the rabbit to lens extract. Injection of lens extract alone does not produce this effect. Because of this synergistic effect of the toxin on lens extract, a mixture of lens extract and staphylococcus toxin was used to treat two patients with intraocular inflammation associated with cutaneous sensitivity to lens extract. Both patients lost the cutaneous sensitivity to lens extract after treatment and it is probable, but has not been proved, that the ocular tissues were also desensitized. From the Wilmer Ophthalmological Institute, Johns Hopkins Hospital and University.

ACTIVE IMMUNIZATION OF GUINEA PIGS WITH THE VIRUS OF EQUINE ENCEPHALOMYELITIS

ACTIVE IMMUNIZATION OF GUINEA PIGS WITH THE VIRUS OF EQUINE ENCEPHALOMYELITIS

Vitamin C and Diphtheria Toxin

SummaryVitamin C under certain conditions increased the resistance of guinea pigs to injection of 1 M.L.D. of a standardized diphtheria toxin. Injections of mixtures of toxin and vitamin C if the solutions had been in contact for 1 hour at room temperature before injection were less toxic. The guinea pigs that had survived these injections 5 to 7 weeks showed but slight if any immunity when tested by the injection of 2 M.L.D.'s of the toxin. Vitamin C caused no destruction of the antitoxic properties of antitoxin or of the slightly toxic toxin antitoxin mixture. Only clinical work can determine whether the increased resistance to diphtheria toxin shown by guinea pigs treated with vitamin C can be applied to human beings. While we were engaged in these experiments Dr. C. G. King told us that he and Dr. Bessey2 were working in the same field and had also found a relationship between vitamin C and diphtheria toxin.We wish to thank Mr. Andrew Mackey for his aid in carrying out the experiments.

Hemolytic Streptococcus Toxins and Antitoxins

Summary Identical mixtures of scarlet fever streptococcus toxin and antitoxin have been injected subcutaneously in the ear and intracutaneously in the body of rabbits selected as reactive to this toxin. The reactivity of the two injection sites is the same for such mixtures. The larger area made available by the use of the body skin may, therefore, be expected to yield results comparable with those obtained by Veldee's method. For titration of toxins and the residual in formalinized toxins, the ear reactions have proved easier to evaluate than skin reactions.

Meningococcus Toxin and Antitoxin

Summary Live cultures, whole killed cultures, washed killed cultures, washings of live cultures, washings of killed cultures and toxin (meningococcus bouillon filtrate) have more or less of a similar effect when injected intracisternally in 250-gram guinea pigs. Experimental meningococcus antitoxin has a neutralizing effect against meningococcus toxin, the washings of killed cultures and to some degree against washed cultures, when mixed with these substances before injection, but shows no evidence of neutralizing live cultures or killed cultures when the mixtures are injected intracisternally. Antimeningococcic serum has evidenced some neutralizing effect against meningococcus toxin when the mixture of toxin and serum was injected intracisternally. Exposure for two hours at incubator temperature has no appreciable deleterious effect on the toxin or mixtures of toxin and antitoxin.

Manson Medal for Tropical Medical Research

THE Manson medal for tropical medical research, given triennially by the Royal Society of Tropical Medicine and Hygiene, has this year been awarded to Dr. Theobald Smith. In association with Kilborne in 1893, Theobald Smith showed that red-water fever in cattle is transmitted by ticks, and at the same time demonstrated the passage of infection through the ova of one generation of ticks to the next. This discovery proved that a protozoal parasite of mammals could be disseminated by the bite of a blood-sucking arthropod. Probably because Theobald Smith was dealing with a cattle rather than a human disease, this great event in the history of medicine received scant recognition, though it preceded the better-known work of Bruce on the transmission of sleeping sickness and that of Ross on malaria. Indeed, Theobald Smith was the first to transfer the insect transmission theory of protozoal disease from the realms of hypothesis to those of established fact. In 1904, Theobald Smith reported anaphylaetic symptoms in dogs and rabbits, and, in a letter to Ehrlich, wrote the original description of the classical anaphylactie shock in guinea-pigs often known as the Theobald Smith phenomenon. In 1907 he proved that it was possible to immunise guinea-pigs actively by the injections of a balanced mixture of diphtheria toxin and antitoxin, and two years later suggested further investigations of the method with the view of its ultimate application to human beings, thus anticipating Von Behring and Park's work by several years. The previous recipients of the Manson medal have been Sir David Bruce (1933), Senator Ettore Machiafava (1926), and Sir Ronald Ross (1929).

Factors Influencing the Antigenic Power of Toxin-Antitoxin Mixture

Abstract The ability of mixtures of diphtheria toxin and antitoxin to bring about, when injected subcutaneously, active immunity to poisoning by diphtheria toxin as well as power to resist diphtheritic infection, is now firmly established by the published studies of numerous workers in this country and abroad. It is recognized that certain mixtures have a greater antigenic power than others, but the factors which give rise to these differences in activity are not well understood. We have had the opportunity to study the influence of several factors on the relative efficiency of such mixtures, having devoted attention especially to differences between mixtures containing greater or less excess of toxin; the influence on antigenic value of the character of the toxin entering into the mixture; and the effectiveness of one large dose as compared to that of several small doses.

A Comparison of Methods for Determination of Potency as Observed on a Group of Horses Immunized against Diphtheria

Summary The potencies of antidiphtheritic sera obtained on trial bleedings during the course of immunization of a group of 20 horses were obtained by three different methods: The official test of the Hygienic Laboratory, based on prolongation of life of guinea pigs when injected subcutaneously with the trial serum along with an L + dose of standardized toxin. The so-called intracutaneous method, based upon the production of erythema at the site of intradermal injection into a guinea pig of a mixture of trial serum and standardized toxin. Flocculation in vitro when the trial serum is mixed with standardized toxin. The intracutaneous test yields results in closer agreement with those obtained by the official test than does the flocculation test. The last gives consistently lower values. The substantial accuracy of the intracutaneous test, as well as its economy of time and materials, makes it probably the method of choice for estimation of antidiphtheritic potency on trial bleedings.

DIPHTHERIA IMMUNIZATION

At the present time, toxin-antitoxin is undoubtedly the most popular agent in America for producing an active immunity against diphtheria. The original preparation, first recommended by von Behring 1 in 1913, was an underneutralized mixture of toxin and antitoxin, so combined that tests made on guinea-pigs showed a slight excess of toxin. Von Behring worked on the hypothesis that the slight excess of toxin had an immunizing action. This theory, however, was disproved by Park, 2 who showed the immunizing effect of fully neutralized mixtures of toxin-antitoxin, and also by Loewenstein and Bousson, 3 who used over-neutralized mixtures. The latter investigators showed, further, that immunity produced by the use of toxin-antitoxin mixtures develops later than the immunity induced by the use of free toxin. They concluded that a dissociation of the combination of toxin and antitoxin took place in the body, and that the toxin which was gradually liberated stimulated