The use of the Toxin Binding Inhibition (ToBI) test for the estimation of the potency of the diphtheria component of vaccines

The Toxin Binding Inhibition (ToBI) test, previously developed for the estimation of diphtheria and tetanus antitoxin in human sera, was adapted for the estimation of the potency of diphtheria components in vaccines. Data are presented to show that antitoxin titres of individual sera of mice obtained by the ToBI test are in good agreement with those obtained in the Vero cell test. In addition, diphtheria potency and 95% confidence interval of twelve batches of vaccine in different compositions were estimated by the ToBI test and the results were compared with those obtained in Vero cells. A significant correlation could be demonstrated. It is concluded from this study that the ToBI test is a valuable model in the potency assay of diphtheria toxoids, based on antitoxin induction in mice.     

The use of the in vitro toxin binding inhibition (ToBI) test for the estimation of the potency of tetanus toxoid

The in vitro toxin binding inhibition (ToBI) test was used to determine antitoxin responses in mice immunized with tetanus toxoid. The ToBI test showed good correlation with the in vivo toxin neutralization (TN) test in titration of sera of mice immunized with various doses of DPT-Polio, DT-Polio and a tetanus reference preparation. Estimates of potency of tetanus toxoid obtained in mice by ToBI test correlated significantly with those obtained in mice by the lethal challenge test. In addition, potency values of the European reference preparation, succeedingly estimated by ToBI test and lethal challenge test in a single group of guinea-pigs, showed good correlation. From the study it is concluded that the ToBI test is a promising alternative to the toxic challenge procedure in the potency assay of tetanus toxoid vaccines. A substantial refinement and reduction in the use of animals can be achieved. Additional savings can be made by combining diphtheria and tetanus potency testing.     

Immunogenicity test of tetanus component in adsorbed vaccines by toxin binding inhibition test

Samples from 20 lots of diphtheria-tetanus (adult use dT) vaccine and from 20 lots of diphtheria-tetanus-pertussis (DTP) vaccine were used to standardize and validate the in vitro toxin binding inhibition (ToBI) test for the immunogenicity test of the tetanus component. The levels of tetanus antitoxin obtained by ToBI test were compared to those obtained using the toxin neutralization (TN) test in mice routinely employed to perform the quality control of the tetanus component in adsorbed vaccines. The results ranged from 1.8 to 3.5 IU/ml for dT and 2 to 4 IU/ml for DTP by ToBI test and 1.4 to 3 IU/ml for dT and 1.8 to 3.5 IU/ml for DTP by TN in mice. These results were significantly correlated. From this study, it is concluded that the ToBI test is an alternative to the in vivo neutralization procedure in the immunogenicity test of the tetanus component in adsorbed vaccines. A substantial refinement and a reduction in use of animals can be achieved.     

Potency control of diphtheria component in adsorbed vaccines by in vitro neutralization tests.

Samples from 20 lots of dT vaccine and from 20 lots of DTP vaccine were used to standardize and validate the Vero cell and the toxin binding inhibition (ToBI) tests for the potency control of diphtheria component. For the Vero cell method, violet crystal solution was used to stain the cells and estimate the endpoint of diluted diphtheria antitoxin. Diphtheria anatoxin was used for performing the ToBI test instead of toxin. The results obtained by both in vitro tests were similar to those obtained by in vivo toxin neutralization test in guinea pigs. The various analysis and the chi(2) test applied to evaluate the reproducibility and homogeneity, respectively, among in vitro tests and in vivo toxin neutralization test did not detect statistical significant difference for both analysed vaccines. An excellent correlation among in vitro tests and in vivo neutralization test was observed by Spearman's correlation coefficient.     

Transplacental antibodies. Part II: Maternal antibodies against the toxins of C. diphtheriae and C. tetani

Examinations of 297 sera for diphtheria antitoxin and 160 sera for tetanus antitoxin were carried out in 1981. All sera were obtained from the cord blood of mothers between 15 and 34 years of age. The mothers were divided into four age groups each of which was further subdivided into the primipara and multipara subgroups. The aim was to assess the age-specific variations in response to active immunization against diphtheria and tetanus. The protective level of diphtheria antitoxin (at least 0.01 I.U./ml) was recorded in the serum of 96.3% of examinees and the rates of seropositivity were found to fall with increasing age. The protective level of tetanus antitoxin (at least 0.1 I.U./ml) was found in the serum of 95.2% of mothers. The serologic response encountered in groups of older mothers was a clear-cut demonstration that the country-wide mass immunization against tetanus carried out between 1974 and 1975 was highly effective and fully justified. The variations in the diphtheria and tetanus antitoxin levels found in the primipara and multipara subgroups were not statistically significant.     

Microtissue Culture Test for the Titration of Low Concentrations of Diphtheria Antitoxin in Minimal Amounts of Human Sera

A microtissue culture method for the assay of low concentrations of diphtheria antitoxin in human sera has been developed, using a monkey kidney cell (VERO) culture technique. Results obtained with sera from nonvaccinated children and with immune sera from children vaccinated with three and four injections of diphtheria pertussis tetanus vaccine were in agreement with antitoxin levels considered necessary to denote immunity to diphtheria. The use of microplates and organic buffer for culturing the animal cells improved the stability of the tissue culture system. The described method is sensitive, economical, and applicable for the titration of antitoxin in human sera particularly from infants and children from whom a minimum amount of serum is available.     

Diphtheria and tetanus immunity among blood donors in Toronto

OBJECTIVE: To determine the diphtheria and tetanus antitoxin levels among blood donors in Toronto. DESIGN: Cross-sectional seroprevalence study. SETTING: Two fixed-site blood-donation clinics in Toronto from September to November 1994. PARTICIPANTS: Blood donors 20 years of age or older were eligible to participate; of the 781 eligible donors, 710 (90.9%) participated in the study. MAIN OUTCOME MEASURES: Diphtheria and tetanus antitoxin levels and factors associated with disease susceptibility, such as vaccination history, country of birth, age and sex. A diphtheria antitoxin level lower than 0.01 lU/mL and a tetanus antitoxin level lower than 0.15 lU/mL were considered nonprotective. RESULTS: Among the participants, 147 (20.7%) had a diphtheria antitoxin level in the nonprotective range, and 124 (17.5%) had a tetanus antitoxin level that was nonprotective. Increasing age and lack of written vaccination records were associated with susceptibility to the 2 diseases. Birth outside Canada was significantly related to tetanus susceptibility. CONCLUSION: Adults over 50 years of age who did not know their vaccination history were the least likely to be protected against diphtheria and tetanus. The greatest benefit of any immunization strategy would be gained by targeting this group.     

Use of the toxin-binding inhibition test for estimation of tetanus antitoxin in serum

The usefulness of the toxin binding inhibition test (ToBI) for the titration of tetanus antibody in human sera was assessed. Sera from 80 healthy people with different vaccination histories that had been previously tested by the in vivo toxin neutralization (TN) test were retested by the ToBI test. The lowest tetanus antibody titre which could be detected by using 0.1 Lf/ml tetanus toxin was 0.01 IU/ml. Comparison between the estimates obtained by the ToBI test and those obtained by the TN test (r = 0.93 and r = 0.7 for titration low titre sera) showed good correlation. No overestimation of antibody content was seen in titrating low titre sera by the ToBI test. It is concluded that the ToBI-test is a reliable alternative to toxin neutralization test in mice.     

A comparison of enzyme-linked immunosorbent assay (ELISA) with the toxin neutralization test in mice as a method for the estimation of tetanus antitoxin in human sera

An enzyme-linked immunosorbent assay (ELISA) has been developed for the measurement of tetanus antitoxin in human sera as an alternative to the toxin neutralization test in mice, the currently accepted method of assay. The ELISA was found to be simple and quick to perform and required only small amounts of materials. In addition, the assay was found to give reproducible estimates of antitoxin levels and to measure antitoxin at levels as low as 0.01 IU per ml, a sensitivity similar to that of the neutralization test. Furthermore, a comparison of the results of the ELISA and the neutralization test involving 80 human sera, including sera with both high and low antitoxin levels, showed close agreement in antitoxin levels obtained by the two methods. It was concluded that ELISA was an acceptable alternative to the toxin neutralization test in mice for the measurement of tetanus antitoxin levels in human sera.     

The titration of tetanus antitoxin III. A comparative evaluation of indirect haemagglutination and toxin neutralization titres of human sera

The tetanus antitoxin titres of 174 serum samples from healthy adults were determined by a standardization indirect haemagglutination test (IHA) and the conventional toxin neutralization (TN) test. The serum samples were titrated by the IHA test using glutaraldehyde-fixed and toxoid sensitized sheep erythrocytes before and after the treatment of the sera with 2-mercaptoethanol (2-ME). The IHA method has been found to be very sensitive and specific for the estimation of tetanus antitoxin in human sera. The IHA titres before the treatment of the sera with 2-ME were generally about four times higher than the TN titres and the correlation coefficient between these titres was 0.94. The IHA titres after the treatment of the sera with 2-ME were in good agreement with the TN titres and there was no statistically significant differences between the titres by the two methods. The tetanus antitoxin titres of 50% of the sera were below the minimum protective titres of tetanus antitoxin (0.01 IU/ml). In 19.5% of the sera the antitoxin level (IU/ml) ranged from 0.01 to 0.1, in 20.1% from 0.1 to 1.0 and in 10.4% from 1.0 to 10.0.     

Dynamic changes of horse serum T-globulin immunization with snake venoms, tetanus and diphtheria toxoids

In course of immunizing horses with snake venoms, tetanus and diphtheria toxoids, a new serum component, T-globulin, was formed and migrated between the beta- and gamma-globulins. The T-globulin content was parallel with the antibody titre after the middle course of immunization. There were many components in snake antivenin and T-globulin was composed of most of those components. The components of diphtheria T-globulin were the same as those of crude antitoxin and tetanus T-globulin except one precipitin.     

Persistence of antibody after accelerated immunisation with diphtheria/tetanus/pertussis vaccine.

OBJECTIVE--To determine the persistence of antibody to diphtheria, tetanus, and pertussis in children receiving an accelerated schedule of primary immunisation. DESIGN--Controlled study of antibody testing of blood samples from children immunised according to various schedules: three doses of triple vaccine completed at 8-13 calendar months, 6-7 calendar months, before 6 calendar months, or three doses followed by diphtheria/tetanus before age 2. SETTING--Plymouth Health Authority. SUBJECTS--129 children aged 4 years who had received three doses of diphtheria/tetanus/pertussis vaccine with or without a diphtheria/tetanus booster. MAIN OUTCOME MEASURES--Diphtheria and tetanus antitoxin concentrations and antibody titres to pertussis toxin, filamentous haemagglutinin, and agglutinogens 2 and 3. RESULTS--All children had protective concentrations of antitoxin to diphtheria and tetanus (greater than or equal to 0.01 IU/ml). There was no evidence of a significant difference in diphtheria or tetanus antitoxin concentrations and pertussis antibody titres in children immunised with an accelerated course (third dose of triple vaccine before 6 months) compared with those who received a longer course (third dose at 8-13 months) with no booster (geometric mean antitoxin concentration 0.411 (95% confidence interval 0.273 to 0.618) v 0.426 (0.294 to 0.616) for diphtheria and 0.358 (0.231 to 0.556) v 0.299 (0.197 to 0.453) for tetanus; geometric mean antibody titres 903 (500 to 1631) v 1386 (848 to 2266) for pertussis filamentous haemagglutinin, 179 (130 to 248) v 232 (167 to 322) for pertussis toxin, and 2002 (1276 to 3142) v 3591 (2220 to 5809) for agglutinogens 2 and 3). CONCLUSION--Immunisation with three doses of triple vaccine at monthly intervals completed before 6 months of age probably provides adequate protection against diphtheria, tetanus, and whooping cough which will persist until the age of the preschool booster.     

Immunity of children to diphtheria,tetanus, and poliomyelitis.

A survey of titres of diphtheria and tetanus antitoxins and of antibodies to polioviruses in the sera of 291 schoolchildren aged 15, 11, and 7 years showed that high immunisation rates can evoke protective concentrations of tetanus antitoxin in 98% of children and protective levels of the antibodies to diphtheria and all three types of poliomyelitis in 85% of children. Reinforcing immunisation at school entry appeared to be necessary to maintain adequate titres of diphtheria antitoxin in children up to 15 years of age, not essential to maintain adequate titres of tetanus antitoxin, and to have little effect on the titres of antibodies to poliomyelitis.     

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.     

Evaluation of the immunological effectiveness of the planned vaccinal prophylaxis of diphtheria and tetanus in Sverdlovsk and Sverdlovsk Province

The level and intensity of antitoxic immunity to diphtheria and tetanus in children and adolescents were determined. The presence of tetanus antitoxin in titers exceeding the protective level in 96.3-98.5% of the examined children and adolescents is indicative of a high actual coverage by immunization. Protective titers against diphtheria were lower. There was no essential difference in the levels of protection in children immunized according to the vaccination schedule and in those immunized with some deviations from this schedule. A considerable part of newborns and children aged 3 months had antibodies to diphtheria and tetanus antitoxins. After the third booster immunization changes in antidiphtheria immunity characteristics occurred only in 2.5% of the vaccines and no changes in antitetanus immunity characteristics were observed.     

The reduction of animal usage by the application of indirect haemagglutination in the potency testing of diphtheria toxoid in combined vaccines

Eight Adsorbed Diphtheria-Tetanus vaccines and 13 Diphtheria-Tetanus-Pertussis vaccines made by four different manufacturers were tested for the potency of the diphtheria components in guinea-pigs by the method of British Pharmacopoeia (1973). Two-hundred-and-ten guinea-pig sera consisting of ten sera related to each vaccine sample thus obtained were titrated for diphtheria antitoxin by indirect haemagglutination (IHA) and the conventional toxin neutralization (TN) tests. Statistical analysis of the results showed a good correlation between the titres obtained with the two tests. The potencies of the diphtheria components of various vaccines calculated from the antitoxin content of the respective guinea-pig sera titrated by the IHA test correlated significantly with the potencies obtained from the antitoxin content titrated by the routinely used TN test. The use of IHA in place of the TN test thus offers as an alternative that permits a reduction in animal usage.     

Titration of tetanus antitoxin by passive hemagglutination. I. Titration of guinea-pig antitoxins at various periods of immunization.

An improved technique for passive hemagglutination (HA) for titration of tetanus antitoxin was described. The use of highly purified tetanus toxoid and of improved diluent increased the specificity and reproducibility of the test. Several hundreds of specimens of guinea-pig serum taken at various stages of immunization were titrated by HA and toxin neutralization (NT) in mice. The ratio of HA to NT titers varied significantly depending on the immunization stage; higher at early stages and lower at later stages. The high HA/NT ratio was not due to the IgM antitoxin, which is very rare in guinea pigs. The variation in discrepancy between HA and NT titers decreased considerably by grouping the serum specimens with respect to the stage of immunization. Thus, it is possible to predict the in vivo titer of a tetanus antitoxin accurately enough for clinical study. The HA test may be useful as an alternative method for titrating tetanus antitoxin in the field trials. Moreover, it can be used for the study of characteristics of antitoxins.     

Significance of circulating toxin and antitoxin in unimmunized tetanus cases of neonates and infants

The level of circulating tetanus toxin, antitoxin and their individual influence on the outcome of tetanus cases were determined in unimmunized 125 neonatal and 39 infant cases of tetanus. PHA (passive haemagglutination) test showed 40% positive cases for toxin while its absence in the remaining cases indicated of either toxin fixation to the central nervous system (CNS) or it got neutralized by antitoxin. TN (toxin neutralization) and PHA test carried out in 46 sera samples revealed a strong positive correlation (r = 0.9) showing that 35/46 (76%) and 38/46 (82.6%) samples were positive for antitoxin, respectively. 25.4% of the neonate and infant cases and 34% of the control group had a protective serum tetanus antitoxin level. 42.5% of the paired sera from unimmunized mothers and their neonates showing nonprotective antitoxin levels suggested that a high level of antitoxin is needed for transplacental transfer, although transfer may not play a decisive role in the resistance against the disease. The presence of toxin or antitoxin in the clinical cases did not affect the outcome of the disease, although in neonates, presence of toxin was found to be a bad prognostic sign. This study explicitly advocates for the need to improve the vaccination coverage strategy.     

Duration of immunity in children who received a primary complex of inoculations with ADT-M anatoxin and an assessment of postponed secondary revaccination against diphtheria and tetanus (the results of an epidemiologic trial)

The prolonged observations of the immunological effectiveness of adsorbed diphtheria-tetanus toxoid with reduced antigen content in children who had received the primary course of immunization with this preparation showed that the preparation induced the development of prolonged and intensive immunity to both infections. In 2-3 years after the first booster immunization the protective level of diphtheria antitoxin was registered in 89.9% and that of tetanus antitoxin, in 99% of children. 6 years later the level of immunity remained practically unchanged: the titers of diphtheria antitoxin above the protection level were determined in 92% and those of tetanus antitoxin, in 97% of children. These data made it possible to increase intervals between booster immunizations to 6-7 years in children of this category. The results of the epidemiological trial made to find out the possibility of a change in the timing of the second booster immunization confirmed the expediency of postponing booster immunization from 6 and 11 years to 9 and 16 years of age.     

The immunization of children with combined diphtheria and tetanus vaccine in Sweden

Two groups derived from 97 children three-four months of age were vaccinated with diphtheria and tetanus vaccines containing either a routinely prepared diphtheria toxoid or a more purified preparation. Two injections were given with an interval of one month and a third injection was given one year after the first. Prior to the third injection no child was without protection against diphtheria, i.e. had an antitoxin titre less than 0.01 IU ml-1. After the third injection 95 and 94% of the children vaccinated with the routinely and more purified diphtheria toxoids, respectively, had diphtheria antitoxin titres greater than 1 IU ml-1 (estimated to provide protection for at least ten years). Systemic reactions such as fever and malaise occurred in five children. Local reactions greater than 10 cm were observed in three children and reactions greater than 5 but less than or equal to 10 cm were seen in 14% of the children. The routinely prepared combined diphtheria and tetanus vaccine, DT, produced very good immunity against diphtheria with moderate side effects. The use of a more purified diphtheria toxoid in the combined vaccine produced the same immunity and side effects.