Injection Of Antitoxin Research Articles

Neutralizing chimeric heavy-chain antibody targeting the L-HN domain of Clostridium botulinum neurotoxin type F.

Botulinum toxin (BoNT) from Clostridium botulinum is the most toxic biotoxin known and is also an important bioterrorism agent. After poisoning, the only effective treatment is injection of antitoxin. However, neutralizing nanoantibodies are safer and more effective, representing a promising therapeutic approach. Therefore, it is important to obtain effective neutralizing nanoantibodies. Hence, the present study aimed to construct a phage antibody library by immunizing a camel and screening specific clones that bind to the L-HN domain of BoNT/F and constructing chimeric heavy-chain antibodies by fusing them with a human Fc fragment. The antibodies' affinity and in vivo neutralizing activities were evaluated. The results showed that 2µg of F20 antibody could completely neutralize 20 × the median lethal dose (LD50) of BoNT/F in vitro. Injection of 5mg/kg F20 at 1h, 2h, 3h, and 4h into mice after BoNT/F challenge resulted in complete survival in vivo. Overall, the antibody might be a candidate for the development of new drugs to treat botulism.

Characterization of Botulinum Neurotoxin Type A Neutralizing Monoclonal Antibodies and Influence of Their Half-Lives on Therapeutic Activity

Botulinum toxins, i.e. BoNT/A to/G, include the most toxic substances known. Since botulism is a potentially fatal neuroparalytic disease with possible use as a biowarfare weapon (Centers for Disease Control and Prevention category A bioterrorism agent), intensive efforts are being made to develop vaccines or neutralizing antibodies. The use of active fragments from non-human immunoglobulins (F(ab')2, Fab', scFv), chemically modified or not, may avoid side effects, but also largely modify the in vivo half-life and effectiveness of these reagents. We evaluated the neutralizing activity of several monoclonal anti-BoNT/A antibodies (mAbs). F(ab')2 fragments, native or treated with polyethyleneglycol (PEG), were prepared from selected mAbs to determine their half-life and neutralizing activity as compared with the initial mAbs. We compared the protective efficiency of the different biochemical forms of anti-toxin mAbs providing the same neutralizing activity. Among fourteen tested mAbs, twelve exhibited neutralizing activity. Fragments from two of the best mAbs (TA12 and TA17), recognizing different epitopes, were produced. These two mAbs neutralized the A1 subtype of the toxin more efficiently than the A2 or A3 subtypes. Since mAb TA12 and its fragments both exhibited the greatest neutralizing activity, they were further evaluated in the therapeutic experiments. These showed that, in a mouse model, a 2- to 4-h interval between toxin and antitoxin injection allows the treatment to remain effective, but also suggested an absence of correlation between the half-life of the antitoxins and the length of time before treatment after botulinum toxin A contamination. These experiments demonstrate that PEG treatment has a strong impact on the half-life of the fragments, without affecting the effectiveness of neutralization, which was maintained after preparation of the fragments. These reagents may be useful for rapid treatment after botulinum toxin A contamination.

A unique experiment in neurological surgery: intracerebral injection of antitoxin for tetanus

The discipline of neurological surgery was considered primarily "hopeless" and, at best, experimental in the late 19th century. Harvey Cushing's efforts during his initial uncharted voyage through the surgery of the human cranium were rudimentary and exploratory. A direct review of his available patient records from early in his career provides the opportunity to look back at Cushing as a physician-scientist, uncovering work that demonstrates that he was at the forefront of neurosurgical intervention in avenues that have been largely overlooked. The authors present Cushing's notes pertaining to a case of tetanus. This case represents the intersection of neurosurgery and tetanus treatment in Dr. Cushing's intracerebral injection of antitoxin to treat generalized tetanus.

Survival analysis for respiratory failure in patients with food-borne botulism

Introduction. Botulism is a rare presynaptic neuromuscular junction disorder caused by potent toxins produced by the anaerobic, spore-forming, Gram-positive bacterium Clostridium botulinum. Food-borne botulism is caused by the ingestion of foods contaminated with botulinum toxin. In March 2006, there was a large outbreak of food-borne botulism associated with the ingestion of home-canned bamboo shoots in Thailand. The survival analyses for respiratory failure in these patients were studied and are reported here. Methods. A prospective observational cohort study was conducted on this outbreak. The primary outcome of interest was the time to respiratory failure. The secondary outcome was the time to weaning off ventilator. The prognostic factors associated with respiratory failure and weaning off ventilator are presented. Results. A total of 91 in-patients with baseline clinical characteristics were included. Most cases first presented with gastrointestinal symptoms followed by neurological symptoms, the most striking of which being difficulty in swallowing. Common clinical features included ptosis, ophthalmoplegia, proximal muscle weakness, pupillary abnormality, and respiratory failure. Forty-two patients developed respiratory failure requiring mechanical ventilation and the median duration on ventilator was 14 days. The median length of hospital stay for all patients was 13.5 days. Difficulty in breathing, moderate to severe ptosis, and dilated and fixed pupils were associated with respiratory failure. Among patients who were on ventilators, a short incubation period and pupillary abnormality were associated with a longer period of mechanical ventilation. All patients had antitoxin injection and there was no mortality in this outbreak. Conclusion. The history of difficult breathing and the findings of moderate to severe ptosis and pupillary abnormality were associated with severe illness and respiratory failure. A long incubation time was associated with a better prognosis. Although botulism is a potentially fatal disease, there was no mortality in this outbreak. All patients had antitoxin injection and good intensive care that resulted in good clinical outcomes.

Neurosurgical treatment for tetanus*

Soon after the discovery of the tetanus antitoxin, animal experiments led to the conclusion that intracerebral injection of the antitoxin would be useful in the treatment of tetanus. The French surgeon Quenu carried out the first injection and gave a description of the neurosurgical technique recquired. The method was applied in other European countries until it was declared to be less efficient than simple subcutaneous or intravenous injections. Outside Europe, intracerebral injections of tetanus antitoxin were reported and photographed in Montevideo, Uruguay, in 1901.

The mechanism of action of tetanus toxin: effect on synaptic processes and some particular features of toxin binding by the nervous tissue.

1. In experiments involving injection of tetanus toxin into the rat diaphragm it was shown that the toxin, by acting on the presynaptic apparatus, disturbs the neuromuscular transmission. 2. Electron microscopic investigation does not reveal any visible ultrastructural changes in neuromuscular and axosomatic synapses of spinal motoneurons in tetanus intoxication. However, synaptic vesicles accumulate in the axon terminals. 3. Neuraminidase splits off the sialic acid and liberates the toxin from the protagon-tetanus toxin complex. 4. Toxin neutralized by antitoxin is still able to react with protagon. 5. Toxin bound to protagon can be neutralized by antitoxin; when neutralized, the toxin is retained in complex with protagon. Partial neutralization of the toxicity of the protagon-toxin complex is effected by small amounts of antitoxin; complete neutralization of residual toxicity requires comparatively greater amounts of antitoxin. 6. Fragments of antitoxin [F(ab′)2 and Fab′] are more effective for complete neutralization of toxin bound on protagon than the initial JgG-antitoxin. 7. Injections of antitoxin into the cisterna magna during the incubation period and at earlier stages of experimental tetanus ascendens and descendens are more effective than intramuscular, intravenous or intracarotid injections. At later stages of intoxication all ways of antitoxin injection used are ineffective. 8. The tetanus toxin molecule has at least three groupings; one of them is responsible for the binding on its receptor in brain substance, the other for the interaction with antitoxin and the third for tis specific toxic effect.

Immunology as scholarly discipline.

PERSPECTIVES IN BIOLOGY AND MEDICINE Volume 16 · Number 1 · Autumn 1912 IMMUNOLOGY AS SCHOLARLY DISCIPLINE* SIR MACFARLANE BURNETt It is now nearly fifty years since I became interested in immunology . Believe it or not, in 1923 I sketched out some ideas on how Semon's mnemic theory of memory, now forgotten, might be applied to antibody function. I showed them to my chief, C. H. Kellaway, who gave me John Hunter's advice to Jenner: "Don't think. Do the experiments." Well, I have done many experiments, but have never really stopped thinking about immunology since then. If I seem to be unduly enthusiastic or optimistic about immunology, I am certain that this is one audience that will forgive me, and when a heresy or two creep in, as they will, I hope that you will be tolerant also of them. The year 1923 was in many ways a good time to come into a hospital laboratory to begin a career in what was then called medical research. In Australia that tended to be a synonym for medical bacteriology and the related serological work. In those days there were still plenty of cases of typhoid fever in the wards, and my first publication was on H and O type agglutination in typhoid. That was the beginning of a continuing opportunity to watch at firsthand the development of immunology from where it stood after the great days of von Behring, Ehrlich, and Bordet at the turn of the century. I want to say a little about some of those who brought immunology to its present prosperous state, to look at how it has changed its pattern with the years, and to speculate in broad terms where it may be going next. I still find it impossible not to dream about the future of immunology, and, after all, I have seen one or two of my earlier * Keynote address, First International Congress of Immunology, Washington, D.C., August 1, 1971. t School of Microbiology, University of Melbourne, Parkville, Victoria 3052, Australia. Perspectives in Biology and Medicine · Autumn 1972 | 1 dreams come true. Let me start with an outline of immunology around 1923-1925. In the first place it was concerned almost wholly with immunity against microorganismal infection. The fact that men with faces pocked by smallpox or milkmaids who had had the cowpox were immune (i.e., did not contract the disease on exposure to smallpox cases) remained the prototype situation, much as it had been in Jenner's time. The major current interests were in elucidating the nature of immunity to diphtheria and the process of recovery from pneumococcal pneumonia. The only significant activity outside the field of infectious disease, as such, was concerned with the occasional harmful effects of the increasing numbers of injections that medical treatment now called for. Iatrogenic disease probably first became significant in the form of serum sickness following injections of antitoxin and the reactions to incompatible or contaminated blood transfusions . It was already evident that the body resented the entry of foreign material, whether this was serum protein from a horse or red cells from a donor of incompatible blood group. Without any specific formulation of a doctrine beyond Ehrlich's phrase "horror autotoxicus ," the capacity of the organism to differentiate between self-components proper to the body and nonself-components was being tacitly accepted. Landsteiner, of course, was the leading spirit in sorting out the human blood groups. His recognition of what, in the last analysis , must have been chemical differences between cell surface components probably played a major part in bringing Landsteiner to the study of the immunological specificity of chemically defined haptens. From then onward the central themes of immunology were chemically oriented. Of course its medical applications were broadened and elaborated, but, after Landsteiner, immunology was no longer simply something that might be of value for preventing or curing infections. It had become, and has remained, primarily a field for scholarly enquiry . Antibody had been defined as a soluble protein, and when Tiselius developed the technique of quantitative electrophoresis it became gamma globulin. Thereafter the history of antibody studies has been essentially determined by the general progress of protein chemistry and...

Response of monkeys to immunization with cholera toxoid, toxin, and vaccine: reversion of cholera toxoid.

Rhesus monkeys have a severe, local reaction to a 50-ag subcutaneous dose of formalinized, purified cholera toxoid. The reaction was delayed, reached a maximum seven to nine days after injection, and was characterized by local edema and erythema, with prominent cellular infiltration in a later, accentuated phase of the reaction. The reaction, which was similar histologically to that produced by an injection of cholera toxin, was prevented by the injection of antitoxin with the toxoid. Also, no reaction to toxoid was seen either in monkeys passively immunized with antitoxin given intravenously or in actively immunized monkeys that had circulating endogenous antitoxin at the time of booster injection. It was concluded that reversion of cholera toxoid to toxin in vivo caused the reaction. A sustained rise in titers of antitoxin in the serum was produced by the toxoid. A marked polymorphonuclear cellular response to conventional cholera vaccine with formation of local microabscess was also observed.

台湾におけるジフテリア菌に関する研究

In order to analyse the mechanisms of host-parasite relationships in diphtherial infections, 4 strains of Corynebacterium diphtheriae isolated in Taiwan were first compared for their virulence by intradermal test in cortisone-treated and control (non-treated) groups of rabbits. It was found that #74 strain showed much weaker reaction as compared with other strains, i.e. #103, #78 and #69. This difference was apparent even after dilution of the inoculum to 10-1 or 10-2. In the dose-response curve, in which the amount of living bacilli (viable count) was taken into account, such difference in virulence was clearly observed. Therefore, the difference in virulence is not due to the difference in the number of viable bacilli present in each inoculum, but rather to the inherent ability of the individual bacillus itself to survive and produce toxin in the host.In the cortisone-treated groups of rabbits and guinea pigs, the skin reaction was generally markedly weaker than in control groups. The erythema at the site of injection was almost absent, and if present, it was observed to form a ring-form halo around the outer edge of induration. The induration was also less in size and thickness, and ulceration was rarely observed in the cortisone-treated groups.After intradermal injection of diphtheria bacilli, when the animals were not treated with a single injection of antitoxin to prevent premature death, the mortality of the infected rabbits and guinea pigs was not significantly different between the cortisone-treated and control groups. However, the positive rates of re-isolation of the diphtheria bacilli from regional lymph nodes and spleens of rabbits were significantly higher in cortisone treated than in control groups. Thus, the factors of parasite, i.e. inherent potency of the bacilli to survive and multiply in the host and toxin-producing ability, as well as the host factors such as amount of corticosterioids excreted, etc. seem to be the important determinants of diphtherial infection in man.

SIMULTANEOUS ACTIVE AND PASSIVE IMMUNIZATION OF GUINEA-PIGS AGAINST TETANUS.

1. Simultaneous active and passive immunization of guinea-pigs using tetanus toxoid adsorbed on to aluminium hydroxide produced a higher antitoxin response than simultaneous immunization using plain toxoid. The response was also better than that produced by plain toxoid alone.2. A 24 hr. interval between the injection of plain toxoid and the subsequent injection of antitoxin gave no better results than when toxoid and antitoxin were given at the same time.3. The response to simultaneous immunization, using adsorbed toxoid, of guinea-pigs sensitized to horse serum, was superior in the first 6 weeks to that of guinea-pigs which had not been sensitized.4. The suggestion that simultaneous active and passive immunization of man should be performed with adsorbed toxoid rather than plain toxoid is supported by these results.

Immunization against tetanus

1. A study of the various aspects of passive tetanus immunization and combined active and passive immunization in different animal species has been undertaken.2. The criteria on which conclusions have been based consisted of titration of antitoxin serum levels and mouse and guinea-pig protection tests using a challenge of 20 M.L.D. of tetanus toxin.3. A study of the rate of elimination of heterologous tetanus antitoxin in normal and sensitized animals showed that the latter eliminated antitoxin more rapidly from circulation after subcutaneous injection but no difference could be detected following intravenous administration. More important is the profound loss of serum antitoxin in both animal groups from the seventh day onwards.4. The rate of elimination in humans receiving 1500 units of horse antitoxin subcutaneously was approximately the same in normal subjects and those who had been previously exposed to horse serum.5. The interference of an active primary immune response by the simultaneous injection of antitoxin in rabbits was consistently more marked with fluid toxoid than with adsorbed toxoid. Thus, combined active and passive immunization of persons who have never been actively immunized, is not recommended, especially with fluid toxoid.6. Administration of adsorbed toxoid 7 days or more after passive immunization with heterologous antitoxin does not interfere with the primary immune response.7. Comparison of the immunizing potency in rabbits of fluid and adsorbed toxoids showed that the latter was far superior.8. Homologous antitoxin was demonstrated in man and animals to be immunologically superior to the heterologous type in every respect. It persisted unchanged in a therapeutically active concentration for at least 3 weeks in humans and its protective action in guinea-pigs was at least 100 times greater than heterologous antitoxin. Since homologous antitoxin can be used in low doses it will not interfere with the primary immune response in combined immunization.9. A recommended prophylactic dose of homologous antitoxin for man would be 200–250 units.

Guillain‐Barré syndrome resulting from tetanus antitoxin injection

Guillain‐Barré syndrome resulting from tetanus antitoxin injection

The relationship of toxin and antitoxin injection site to tetanus development in the rat.

The pattern of development of symptoms was studied following injection of 3 MLD (minimum lethal dose) of tetanus toxin in each of the following sites in the rat: vein, muscle, endoneurium, epineurium, spinal cord, subarachnoid space, and subdural space. Similar observations were made when 3 MPD (minimum protective dose) of antitoxin were injected into each of the above sites a few minutes after the intramuscular injection of toxin. Local tetanus followed intramuscular, endoneurial, and epineurial injection of the toxin; blood-borne tetanus also appeared in the latter instance. Tetanus dolorosus followed intraspinal injection of toxin. Both dorsal tetanus and blood-borne tetanus developed after subdural administration of toxin. Blood-borne tetanus, alone, appeared following intravenous and subarachnoid injection of toxin. In no case did injection of antitoxin at the various sites listed above prevent local tetanus from developing after intramuscular injection of toxin. Of the various sites injected with antitoxin, the intramuscular, intraspinal, and intravenous were the most effective. It is suggested that the endoneurial tissue spaces serve as a conduit for tetanus toxin from the muscle to the CNS. The perilemma of the peripheral nerve trunk may act as a selectively permeable membrane which permits tetanus toxin to pass from the epineurium to the endoneurium. Outward diffusion of toxin from the endoneurial spaces is apparently markedly reduced. The antitoxin probably is prevented from permeating this barrier in either direction. The importance of absorption of tetanus toxin by the lymphatic and blood circulation is discussed.

Priapism: An Unusual Complication of Tetanus Antitoxin Injection

Priapism: An Unusual Complication of Tetanus Antitoxin Injection

Quantitative Studies on the Neutralization of Pathogenic Agents in Tissues by Circulating Antibodies

Summary and Conclusions The investigations reported in the present paper are part of the more general problem of studying immunity reactions with the aid of the methods of the direct and indirect test or, more specifically, by determining the Ai, Ad and Ai/Ad values. These terms have been defined and discussed in the first part of the paper. In particular, the present communication is concerned with the experimental analysis of previously reported observations. It had been found that the intracerebral or rather intraventricular Ai values were very different for individual tetanal toxins and thus revealed hitherto unknown qualitative differences between the toxins. In the present paper, it has been shown that similar differences exist between the intramuscular Ai values of tetanal toxins. On the basis of considerations which have been discussed in the first part of the paper and need not be repeated, the assumption was tentatively made that the differences in the Ai values might be due to differences in the rapidity with which toxins either enter the nerve endings or else are neutralized by antitoxin at the site of injection. In other words, the differences in the Ai values were explained by differences in the relative avidities of the toxins for nerve tissue and antitoxin. The validity of this explanation was tested by experiments in which the antitoxin was injected at various intervals after the toxin. It was found that an interval of 3 hours between the injection of toxin and the subsequent injection of antitoxin increased the antitoxin requirements 16 times for toxin 64 (Ai = 0.0031 ml) and 260 times for toxin 641B (Ai = 0.2 ml). This result shows that after a certain lapse of time the toxin with the high Ai value is more difficult to dislodge from the tissue than the toxin with the low Ai value. This observation can hardly be explained otherwise than by assumming that toxins with a high Ai value have either a greater affinity for nerve tissue or a lower avidity for antitoxin. After eliminating differences in the Lf values of the individual toxins, the avidities of the toxins for antitoxin were determined by measuring the velocity of flocculation in the Ramon test. These “reduced” flocculation rates were very similar for 5 tetanal toxins. Moreover, the small differences were in no way related to the Ai values. It follows that the differences in the Ai values of tetanal toxins cannot be explained by differences in their avidities for antitoxin. In connection with the above described experiments, this result leaves only the alternative that the differences in the Ai values of the toxins are due to differences in the avidities for nerve tissues.

Production of Anemia by Tetanus Toxin

SummarySmall sublethal doses of tetanus toxin produce a typical secondary anemia—low red cell count, reduced hemoglobin index, increased sedimentation rate. Injection of antitoxin produced a prompt return to normal, the red cell count rose rapidly as a result of large numbers of reticulocytes pouring into the system.The severe anemia following the administration of toxin and the prompt recovery following that of antitoxin may serve as a simple early diagnostic test of possible tetanus intoxication in wounded.

IMMUNITY TO TETANUS INDUCED BY COMBINED ALUM-PRECIPITATED DIPHTHERIA AND TETANUS TOXOIDS

To the pediatrician the problem of prophylaxis against tetanus is of particular importance. One half of the deaths from tetanus occur among children under 15 years of age. Tetanus may develop after receipt of a superficial scratch and even in persons lacking objective evidence of an injury for which no medical attention was sought. The routine injection of antitoxin in all cases of injury, a procedure carried out in the accident rooms of many hospitals and elsewhere, is obviously a dangerous practice when applied to allergic persons, especially those hypersensitive to horse dander, horse serum or both. The frequency of anaphylactic (allergic, hyperergic) shock among hypersensitive persons following the injection of serum is much higher than the reported incidence of 1 instance per 50,000 injections of serum in unselected cases. Numerous examples illustrating the inadequacy of passive immunization of soldiers against tetanus were furnished during the World War of 1914-1918.

Investigations on the Pathogenesis of Tetanus III

Summary and Conclusions In this and previous communications, investigations on the pathogenesis of tetanus have been reported. The theories of Courmont and Doyon, Meyer and Ransom and Abel have been subjected to experimental tests. We believe to have established the following facts: Tetanal toxin does not reach the C-N-S directly by way of the circulation. This follows from an observation referred to as the R-(route) phenomenon. It was found that the amount of circulating antitoxin which protects animals from death is determined by the route of introduction of the toxin. The same test-dose of toxin, when injected by the intracutaneous or intramuscular routes, requires up to 80 times more antitoxin than after intravenous injection. This result is obviously incompatible with the assumption that the toxin reaches the C-N-S from the tissue after absorption into the circulation.Tetanus cannot be explained by an action of the toxin on the sensory nerve-endings (Courmont and Doyon). Section of the posterior spinal nerve-roots does not influence the R-phenomenon or local tetanus.Tetanus cannot be explained by the activity of a secondary toxic substance formed in the muscle (Courmont and Doyon) or in the C-N-S (Firor, Lamont and Shumacker). Transverse lesions of the dorsal cord, followed by intravenous injection of antitoxin and intramuscular injection of toxin into one hind leg, confines tetanus to the lower extremities and considerably prolongs the survival-time of the experimental animals. This shows that generalized tetanus cannot be explained by a vascular transport of a secondary toxic substance to higher levels of the C-N-S.Tetanal toxin reaches the C-N-S by way of the motor nerves. The R-phenomenon disappears after section of the peripheral nerves or anterior roots.Tetanal toxin spreads within the C-N-S. As mentioned under the third conclusion, after intravenous injection of antitoxin, intramuscular injection of toxin into one hind leg and transverse lesion of the cord, tetanus remains confined to the lower extremities. This shows that interruption of the neural pathways within the cord prevents the passage of the toxin to higher levels of the C-N-S. Moreover, the appearance of tetanus in both hind legs proves the migration of the toxin from one side of the cord to the other.Local tetanus is of central origin. Under optimal experimental conditions antitoxin is 80 times more potent by the intraventricular than the intravenous route in preventing local tetanus in the hind leg. Since the possibility could be excluded that the intraventricularly injected antitoxin reaches the myoneural junction by way of the nerve this experiment shows conclusively that local tetanus is due to an action of the toxin on the C-N-S.Our experiments therefore confirm the nerve-carriage theory of Meyer and Ransom in all essential points.Our results provide conclusive evidence for the existence of the blood-C-N-S barrier. Since tetanal toxin is extremely toxic when injected into the lumbar cord its inability to reach the C-N-S by way of the circulation can be explained only by an impermeability of the capillaries of the C-N-S to the toxin. This, in connection with the electro-negative charge of tetanal toxin, confirms the rule of Friedemann and Elkeles according to which the blood-C-N-S barrier is impermeable to electro-negative substances.It is now established that an inanimate substance can be transmitted within the nerve to the central nervous system. The implications of this fact for the pathogenesis of neurotropic virus diseases are discussed.

A Study of Natural and Acquired Immunity to Staphylococcal Toxin in Monkeys

Summary and Conclusions The natural antistaphylolysin (alpha) of the serum of a series of 65 monkeys (M. irus and M. mulatta) was found to vary from 0 to 25 international units; the largest number had from 4 to 5. Individual monkeys, like rabbits and human beings, varied in their response to parenteral introduction of staphylotoxin. The maximal titers reached depend upon the route of injection, as well as upon their natural levels. Animals with high initial titers (10 units or more per cubic centimeter) develop antibodies more rapidly and reach a much higher level than those with low values. High titers, up to 250 units per cubic centimeter, resulted from repeated injection of small doses of toxin, either intracutaneously or intrabronchially, the rapidity of antibody-production being in the order given. Intranasal instillation, with or without ether-anesthesia, yielded low titers. Neither after active nor passive immunization could any definite relationship be established between the titers of circulating antibody (alpha-antistaphylolysin) and the intensity of cutaneous reactions to staphylotoxin. As is true with the Schick test, there was no fixed level at which individual animals passed from the negative to the positive group or vice versa. After injection of antitoxin, skin reactions remained negative at a time when the serum titers had returned to approximately their original levels.

Quantitative Studies on the Neutralization of Pathogenic Agents in Tissues by Circulating Antibodies

Summary The neutralization of diphtheric toxin in the skin by intravenously injected antitoxin follows the law of multiple proportions provided that the antitoxin is given before the toxin. If the antitoxin is injected only two minutes after the toxin, neutralization no longer follows the law of multiple proportions, larger amounts of toxin requiring more antitoxin than calculated. From the above experimental observations it follows that when the injection of antitoxin precedes that of the toxin the latter is neutralized before combining with the tissue. The endpoint of titration is the same whether the antitoxin is injected immediately, 1, 2, and sometimes even 4 or 6 hours before the toxin is injected intracutaneously. The distribution of antitoxin between blood and skin, therefore, must reach equilibrium immediately after its intravenous injection. This rapid exchange of antibodies between blood and tissue is explained by the assumption that the cutaneous reaction will be negative when the toxin is neutralized in a molecular zone of fluid bordering on the tissue structures and that within this area the exchange takes place in no measurable time. The result of the cutaneous test under these experimental conditions, therefore, is independent of the velocity of the toxin-tissue reaction. The application of these conclusions to experiments with viruses is discussed.