Units Of Antitoxin Research Articles

Quantitation of commercial equine tetanus antitoxin by competitive enzyme-linked immunosorbent assay

In the USA, the potency of commercially prepared equine tetanus antitoxin is determined by the method outlined in the Code of Federal Regulations, Title 9, Part 113.451. In the current test, commercial equine tetanus antitoxin is tested by a toxin neutralization test in guinea pigs. The in vivo test measures antitoxin content through effectiveness of protection of guinea pigs injected with diluted mixtures of antitoxin and a standard toxin. A competitive enzyme-linked immunosorbent assay, designed as an in vitro alternative to the in vivo test, measures antitoxin content based on a competitive reaction between standard or unknown serum and murine monoclonal antibody specific for tetanus toxin. The monoclonal antibody used in the assay delayed death in mouse passive protection studies and reacted with the C fragment of tetanus toxin. No cross-reaction was observed when the antibody was tested with the toxins of Clostridium chauvoei, C. novyi, C. perfringens, or C. sordellii. The in vitro test will measure the antitoxin content of serum samples containing 100–1500 units of antitoxin. Tetanus antitoxin titers obtained by the competitive enzyme-linked immunosorbent assay compared favorably with the toxin neutralization test conducted in guinea pigs. The in vitro assay serves as a feasible alternative to the in vivo test because it can be completed in less time, is reproducible, and eliminates the use of test animals.

Quantitation of commercial equine tetanus antitoxin by competitive enzyme-linked immunosorbent assay.

In the USA, the potency of commercially prepared equine tetanus antitoxin is determined by the method outlined in the Code of Federal Regulations, Title 9, Part 113.451. In the current test, commercial equine tetanus antitoxin is tested by a toxin neutralization test in guinea pigs. The in vivo test measures antitoxin content through effectiveness of protection of guinea pigs injected with diluted mixtures of antitoxin and a standard toxin. A competitive enzyme-linked immunosorbent assay, designed as an in vitro alternative to the in vivo test, measures antitoxin content based on a competitive reaction between standard or unknown serum and murine monoclonal antibody specific for tetanus toxin. The monoclonal antibody used in the assay delayed death in mouse passive protection studies and reacted with the C fragment of tetanus toxin. No cross-reaction was observed when the antibody was tested with the toxins of Clostridium chauvoei, C. novyi, C. perfringens, or C. sordellii. The in vitro test will measure the antitoxin content of serum samples containing 100-1500 units of antitoxin. Tetanus antitoxin titers obtained by the competitive enzyme-linked immunosorbent assay compared favorably with the toxin neutralization test conducted in guinea pigs. The in vitro assay serves as a feasible alternative to the in vivo test because it can be completed in less time, is reproducible, and eliminates the use of test animals.

Suppression of the immune response to diphtheria toxoid in murine schistosomiasis

Studies in humans and experimental animals indicate that infection with schistosomes results in impaired immune response to a variety of antigens. Since artificial immunization against a number of infections is frequently attempted in populations in which schistosomiasis is endemic, we have attempted to determine whether this impairment could be demonstrated in response to a commonly used immunogen. We have compared the serum antitoxin response to diphtheria toxoid (DTd) in normal, uninfected mice with that in mice bearing schistosome infections of different duration. Animals were infected with 100 Schistosoma mansoni cercariae 16, 12, 8, 4 and 2 weeks prior to, on the same day as, and 4 weeks after the first of three 1 μg doses of DTd given at 3 week intervals. The antitoxin level of each mouse was taken as the mean of two sera obtained 1 and 2 weeks after the 3rd dose of DTd. The mean antitoxin level in uninfected-immunized control mice was 0.0457 Antitoxin Units (AU) ml −1. 71% of mice in this group developed antitoxin levels ≥0.01 AU ml −1. Only 28% of the infected-immunized mice achieved antitoxin levels ≥0.01 AU ml −1. In infected-immunized mice with infections of all durations, both the percentage of mice with ≥0.01 AU ml −1 (0 to 50%) and mean antitoxin levels (0.003 to 0.016 AU ml −1) were lower than in uninfected-immunized mice. Antitoxin levels in animals infected 16, 12, 8 and 2 weeks prior to, simultaneously with, and 4 weeks after the first dose of DTd were significantly lower ( P=<0.05) than those of controls. Mice infected 4 weeks prior to the first dose of DTd had antitoxin levels 64% below controls but this difference was not significant. If similar immunosuppression occurs in human schistosomiasis, these findings may have implications for childhood immunization programs in areas where schistosomiasis is endemic.

Measurement of tetanus antitoxin II. Toxin neutralization

Existing methods for titration of tetanus antitoxin fail to provide an accurate and sensitive technique for conducting assays on small volumes of sera. The need for such a technique is real since in those parts of the world where tetanus is a problem, the collection of adequate serum samples is usually also a problem. The toxin neutralization method described permits titration of tetanus antitoxin in twofold dilution steps from as little as 0·001 AU/ml. If the titration includes the lower limit of the range of antitoxin units to be tested—which differs according to the toxin test dose—the volume of serum required is 0·6 ml. For most antisera, however, the toxin test dose and range of serum dilutions for testing can be chosen, on the basis of titres determined by indirect haemagglutination, so that dilutions from an initial serum dilution of 1:2 will suffice which means that only 0·3 ml of serum is requited. By restricting the range of serum dilutions for testing, prior titration of tetanus antitoxin by indirect haemagglutination also avoids the need for large numbers of mice that the titration of a twofold series by toxin neutralization would otherwise entail. The method is simple and economical and the results obtained are accurate and reproducible provided that no significant variation occurs in the body weight of the mice.

Characterisation and neutralisation of Aeromonas hydrophila enterotoxin in the rabbit ileal-loop model.

Cell-free culture filtrates and crude enterotoxin preparations from six strains of Aeromonas hydrophila caused the accumulation of fluid in rabbit ileal loops. This activity was due to a non-dialysable, heat and acid-labile antigenic protein and was lost when culture filtrates and crude enterotoxin preparations were heated at 60 degrees C for 20 min. or 56 degrees C for 30 min. respectively. Maximum activity was observed at pH 8.0-10.0; there was a gradual loss at lower pH and activity was abolished in culture filtrates held at pH 3.0 and crude enterotoxin preparations held at pH 4.0. Titration of the crude enterotoxin preparations in rabbit ileal loops showed that the ED50 (equivalent to 1 unit of toxin) was contained in 25 microgram of protein; a logarithmic plot of the neutralisation coefficients against antiserum concentrations showed that one unit of antitoxin was contained in 42 x 10(-4) ml of the antiserum.

IgE Synthesis in Man

Abstract Specific IgE and IgG antibodies were measured in 39 adolescent and adult humans preselected to have less than 0.125 antitoxin units (AU) ml of tetanus antitoxin (IgG) irrespective of previous immunization history. Mean antitoxin values (IgG) measured by sheep red cell hemagglutination (SRBC) increased by 2 logs to 1.49 AU/ml sera for tetanus and by 1 log to 0.025 AU/ml sera for diphtheria. Specific tetanus IgE was noted to increase after immunization from a mean 2.0 ± 0.2% counts bound to 3.1 ± 0.6% counts bound, but was not statistically significant. Diphtheria-specific IgE rose significantly (p &amp;lt; 0.05) from a mean 1.5 ± 0.2% counts bound to a mean 4.0 ± 1.2% counts bound in atopic individuals and from a mean 1.2 ± 0.0% counts bound to a mean 2.2 ± 0.5% counts bound in nonatopics. These data document the development of tetanus and diphtheria IgE antibodies after booster immunization.

Toxoid of Clostridium botulinum type F: purification and immunogenicity studies.

Toxin from Clostridium botulinum type F was recovered from dialysis cultures and partially purifed by: (i) ammonium sulfate and ethanol precipitation; (ii) O-(diethylaminoethyl)-cellulose chromatography; or (iii) diethylaminoethyl-cellulose chromatography followed by O-(carboxymethyl)-cellulose chromatography. Toxin purities as reflected by specific activity were 1.83 X 10(6), 9.8 X 10(6), and 2.0 X 10(7) mouse 50% lethal doses (LD50)/mg of N, respectively, for toxins purified by the three methods. The toxins were converted to toxoids by incubation at 35 C in the presence of 0.3 to 0.45% formalin for 21 to 35 days. Toxoids were immunogenic in guinea pigs, as demonstrated by serum antitoxin response and the immunized animals' resistance to challenge by type F botulinal toxin. The immune response to type F toxoids was lower when toxoids of serotypes A, B, C, D, and E were combined with the type F toxoid than when the type F toxoid only was administered. The toxoid prepared from the most highly purified toxin (method [iii]) conferred the highest immunity in guinea pigs at a given dose level. A relation between serum antitoxin level and resistance to challenge was observed. At least 50% of the groups of guinea pigs with 0.015 antitoxin units or more per ml survived challenge by 10(5) mouse LD50 of type F botulinal toxin. A dose of 3.75 mug of N of the most highly purified type F toxoid in combination with the other five serotypes of botulinal toxoid invoked an immune response in guinea pigs comparable to that considered adequate for the other toxoids.

Problems in the use of the in vitro toxigenicity test for Corynebacterium diphtheriae.

A method for the in vitro toxigenicity test for C. diphtheriae that has given accurate results when compared with tests in vivo is reported in detail. The major factors, such as the antitoxin, serum, and basal ingredients, that are known to cause erroneous results in the in vitro test are discussed. Antitoxins obtained from five sources were found to produce non-specific lines of precipitation with known nontoxigenic strains when used at the previously advocated concentration of 500 antitoxin units (AU) per ml. At a concentration of 100 AU per ml. of the only antitoxin currently commercially prepared in the United States, all 115 strains tested gave the correct reaction.

DURATION OF IMMUNITY AFTER ACTIVE IMMUNISATION AGAINST TETANUS

The serum-tetanus-antitoxin concentration was measured in healthy adults whose immunisation histories were fully known. Of ninety persons who had had a basic immunisation course of spaced injections of plain tetanus toxoid, twelve (13.3%) had less than 0.01 unit of tetanus antitoxin per ml. of serum 2-4 years later. Within 2-4 years of immunisation with adsorbed toxoid only one of eighty persons (1.25%) had less than 0.01 unit of antitoxin per ml. of serum, and the mean serum-tetanus-antitoxin of this group was about four times greater than in those who had had plain toxoid. It is suggested that people who have had a basic immunisation course of plain toxoid should be given a booster within 3 years of the last injection, preferably with adsorbed tetanus toxoid.

Studies on diphtheria immune globulin prepared from outdated human blood.

Summary. Approximately one per cent of 8,481 units of outdated plasma tested had 4 or more units of diphtheria antitoxin per ml. Two lots of diphtheria immune globulin (human) were prepared from outdated plasma rich in diphtheria antitoxin. Plasma of this potency was satisfactory to prepare diphtheria immune globulin having 80 or 90 units of antitoxin per ml. When diphtheria immune globulin (human) containing 250 units of antitoxin was injected into human volunteers, serum antitoxin levels of at least 0.01 units per ml were reached in two to seven days and lasted for 21 to 36 days.

Studies on Diphtheria Immune Globulin Prepared from Outdated Human Blood

Approximately one per cent of 8,481 units of outdated plasma tested had 4 or more units of diphtheria antitoxin per ml. Two lots of diphtheria immune globulin (human) were prepared from outdated plasma rich in diphtheria antitoxin. Plasma of this potency was satisfactory to prepare diphtheria immune globulin having 80 or 90 units of antitoxin per ml. When diphtheria immune globulin (human) containing 250 units of antitoxin was injected into human volunteers, serum antitoxin levels of at least 0.01 units per ml were reached in two to seven days and lasted for 21 to 36 days.

Tissue culture method for toxigenicity testing of Corynebacterium diphtheriae.

A method for toxigenicity testing of Corynebacterium diphtheriae in tissue cultures was developed. Results were obtained by comparing destruction of the monkey kidney or, preferably, rabbit kidney monolayer by 0.1 ml of the C. diphtheriae culture in Elek's broth containing 20% rabbit serum with the appearance after the addition of 0.2 ml of a mixture of the C. diphtheriae culture and diphtheria antitoxin. The mixture of C. diphtheriae broth culture and 10 antitoxin units per ml was incubated for 1 hr at room temperature before it was added to the tissue cultures which were then incubated as long as 5 days; most results, however, were read in 72 hr. Elek's broth medium was superior to heart infusion broth for toxin production by C. diphtheriae. Addition of 20% rabbit serum improved toxin production in either broth. Numerous toxigenic and atoxigenic C. diphtheriae cultures were tested for toxigenicity in primary rabbit and monkey kidney tissue cultures. If properly controlled, this in vitro method appeared to have an advantage over the in vitro agar gel method; its results were comparable with the rabbit intradermal test. With the wider use of tissue cultures in most laboratories, we believe that the tissue culture method for toxigenicity would be more economical and easier to perform than the animal intradermal method.

Tissue Culture Method for Toxigenicity Testing of Corynebacterium diphtheriae

A method for toxigenicity testing of Corynebacterium diphtheriae in tissue cultures was developed. Results were obtained by comparing destruction of the monkey kidney or, preferably, rabbit kidney monolayer by 0.1 ml of the C. diphtheriae culture in Elek's broth containing 20% rabbit serum with the appearance after the addition of 0.2 ml of a mixture of the C. diphtheriae culture and diphtheria antitoxin. The mixture of C. diphtheriae broth culture and 10 antitoxin units per ml was incubated for 1 hr at room temperature before it was added to the tissue cultures which were then incubated as long as 5 days; most results, however, were read in 72 hr. Elek's broth medium was superior to heart infusion broth for toxin production by C. diphtheriae. Addition of 20% rabbit serum improved toxin production in either broth. Numerous toxigenic and atoxigenic C. diphtheriae cultures were tested for toxigenicity in primary rabbit and monkey kidney tissue cultures. If properly controlled, this in vitro method appeared to have an advantage over the in vitro agar gel method; its results were comparable with the rabbit intradermal test. With the wider use of tissue cultures in most laboratories, we believe that the tissue culture method for toxigenicity would be more economical and easier to perform than the animal intradermal method.

Immunological Reactivity of Trypsinized Clostridium botulinum Type E Toxin.

SummaryThe number of LD50's of C. botulinum type E toxin necessary to produce a toxin-antitoxin precipitin line in the Ouchterlony gel-diffusion test is much greater for trypsin-activated than for non-activated toxin. For the same lethal challenge dose, less antitoxin is needed to protect mice against toxin after trypsinization than before the toxin is activated. Antitoxin units required for animal protection are related to the amount of toxin which is activated rather than to mouse toxicity obtained by trypsinization. The data indicate that tryptic activation of type E toxin results in appearance of more toxic groupings without a concomitant increase in the number of immunologically reactive sites.

PREVENTION OF TETANUS BY ANTITOXIN OF BOVINE ORIGIN

SIXTY-NINE non-immune patients requiring passive tetanus immunisation received a dose of 1500 units of antitoxin prepared from bovine serum purified by ethanol fractionation. Although more than a third of the patients had reacted adversely to a previous dose of equine antitoxin, not one systemic allergic reaction was reported after the dose of bovine antitoxin. Three factors are considered to have contributed to this virtual absence of reactions—the change to another species, the lesser reactivity of bovine serum, and the removal of reactive components by ethanol fractionation. But the use of the bovine species and the application of ethanol fractionation to all heterologous antisera is not a practical proposition at present.

Tetanus after antitoxin prophylaxis: Report of six cases

1. 1. Six cases of tetanus after administration of 1,500 units of antitoxin at the time of injury are reported. 2. 2. Factors possibly contributing to the development of tetanus in these patients including age, type of wound, incubation period, antitoxin sensitivity and initial wound care are analyzed. 3. 3. Evidence is presented supporting administration of minimal doses of 5,000 units of tetanus antitoxin for all nonsensitive patients requiring prophylaxis. 4. 4. Puncture wounds of the extremities are again emphasized as particularly “tetanus prone”.

Disappearance of human diphtheria antitoxin from human passive transfer skin sites.

SummaryThis paper has described further experiences with a passive transfer technic which depends upon the ability of intradermal antitoxin to neutralize Schick toxin introduced simultaneously or later at the same sites. Partial, total or no inhibition of toxic reactions indicates the amount of antitoxin remaining at the skin sites. The amount of antitoxin which has disappeared may be calculated by difference, assuming that 1 Lf toxin = 1 Unit of antitoxin. On the basis of this test 50% of antitoxin introduced at skin sites was generally found to disappear in 10 to 14 hours. No difference in disappearance from the skin was observed between precipitating antitoxin, non-precipitating antitoxin and skin sensitizing antitoxin.

X irradiation and toxin neutralization by antitoxin.

A small area of the skin of normal rabbits was exposed to 1000 r and, after a given interval, 15 units of horse serum diphtheria antitoxin were injected subcutaneously in that area and simultaneously a lethal (25 MLD) dose of homologous toxin in another area. As controls, non-irradiated rabbits of similar weight were injected with the same quantities of antitoxin and of toxin at the same time as the irradiated rabbits and under identical conditions. Five intervals following the irradiation were employed for the injection of the antitoxin and the toxin: 1 to 3 hours, 24 hours, 7 days, 14 days, and 90 days. It was found that, of the irradiated rabbits, 55% survived the toxin when the antitoxin was injected 1 to 3 hours after the exposure, 100% survived when the antitoxin was injected 24 hours after, 47% survived when the antitoxin was injected 7 days after, 27% survived when the antitoxin was injected 14 days after and none of the rabbits survived when the antitoxin was injected 90 days after. Of the non-irradiated controls, an average of 20% survived the toxin. The results indicated that a restricted amount of antitoxin injected subcutaneously in an irradiated area up tomore » 7 days after exposure to 1000 r was more effective in the neutralization of a lethal dose of toxin than the same amount injected into a corresponding area of non-irradiated rabbits and the most marked neutralization occurred 24 hours after the exposure. By 14 days after the exposure, the neutralization of the toxin reached the control level, but 90 days after, no neutralization of the toxin was observed. The basis for the non-neutralization of the toxin in any of the rabbits was apparently not an insufficiency of the subcutaneouslyinjected antitoxin, which consisted of 15 units, since 5 units injected intravenously were found to be ample for neutralization of the same dose of toxin. The non-protection was due most likely to the localization of the proteins of the antitoxin, consisting of 0.75 mg, in the area of injection, preventing thereby the antitoxic fraction from reaching the toxin in time for neutralization. When the 15 units of antitoxin were injected in the irradiated area 1 to 3 hours after exposure to 1000 r, 45% of the rabbits showed localization of the protein. None of the rabbits showed localization when injected 24 hours after, 53% showed localization when injected 7 days after, 73% showed localization when injected 14 days after and 100% showed localization of the 0.75 mg protein when injected 90 days after the exposure to 1000 r. With regard to the controls, an average of 80% of the non-irradiated rabbits localized this fraction of a milligram of protein in the subcutaneously injected area. Localization in these experiments was undoubtedly temporary in nature, preceding proteolysis. But the length of time of localization was apparently sufficient to so retain the antitoxic fraction in the injected area as to prevent it from reaching and neutralizing the toxin. (auth)« less

Tetanus antitoxin tittration by haemagglutination at a low level of test.

An investigation has been made of methods of estimating the antitoxin content of small samples of human sera by haemagglutination procedures.The direct agglutination test appears to be satisfactory when dealing with groups of sera, but was unreliable for testing individual sera.The discrepancies observed in the direct agglutination test were greatly reduced by adopting an agglutination inhibition test at the LA/100 level of test.The difference in agglutinating capacity of both avid and non-avid sera perin vivounit of antitoxin did not appear to be related to avidity.

A Method for Purifying Diphtheria Toxoid and Combining it with Protamine

Discussion and Summary The use of protamine offers a means of separating bacillary protein and therefore removes a major protein component of crude diphtheria toxoid. No specific and constant formula, in terms of quantity of protamine or with respect to ionic strength can be given which will apply equally well to crude toxoids made in broths of varying composition. It is necessary to determine optimal conditions for each lot. It is desirable to employ toxoids that contain only minimal amounts of protein derivatives precipitated by trichloracetic acid at pH 4.0. Using such preparations, this step precipitates toxoid of high purity, once the bacillary protein is removed. On further treatment with Norite, preparations containing 2000 to 2100 Lf/mgN were obtained. Several additional steps have been employed between the removal of bacillary protein and the addition of trichloracetic acid when the crude toxoid contained larger amounts of protein derivatives originating in the production broth. The chemical steps employed in preparing toxoid of this degree of purity do not appear to have resulted in noticeable changes in the stability of the toxoid, since flocculation has occurred fairly promptly with solutions in phosphate buffer held at 2 C for approximately 2 years and solutions held at 37 C for 4 weeks showed only 10 to 20 per cent loss in Lf. Precipitation of purified toxoid by protamine, and adsorption of the complex on a small quantity of aluminum hydroxide, gave a product which, when suspended in phosphate buffer and warmed at 37 C for 2 weeks, lost only 5 to 10 per cent of its Lf value; when warmed for 4 weeks the loss was 20 per cent. The absence of change in the toxoid molecule as a result of the procedures used is further indicated by the fact that guinea pigs have produced 4 units of antitoxin following injection of the protamine-precipitated product. The high degree of purity of the toxoid obtained is reflected in the absence of disturbing reactions following injection of the protamine-toxoid into human adults. It seems possible that even the occasional individual who is sensitive to toxoid as such will experience less discomfort if given the protamine-precipitated material, since this would be expected to reduce the rate of release of toxoid at the site of the injection. Some data are presented dealing with electrophoretic, and sedimentation-measurements of the purified toxoid.