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.     

Combined estimation of tetanus and diphtheria antitoxin in human sera by the in vitro Toxin-Binding Inhibition (ToBI) test

The use of the principle of inhibition of toxin binding to an antitoxin coated immunoassay plate as described in a previous paper for tetanus antitoxin titration, was adapted for the estimation of diphtheria antitoxin in human sera. With a few modifications, a Toxin-Binding Inhibition (ToBI) test was developed which could be used for a combined estimation of both tetanus and diphtheria antitoxin levels. The application of streptavidin-biotinylated peroxidase complex when using small serum samples (less than 50 microliters) is discussed. Antitoxin titres (both diphtheria and tetanus) of 0.002 IU ml-1 were detectable by the ToBI test, this being far below the level considered to be protective in man. Sera from 140 adults with different vaccination histories were titrated for both tetanus and diphtheria antitoxin. Good correlations were found between the estimates obtained by the ToBI test and those obtained by the toxin-neutralization (TN) test in mice (tetanus antitoxin) and those obtained in the in vitro neutralization test in VERO cells (diphtheria antitoxin). It is concluded that the ToBI test is a simple and reliable alternative to the functional models currently in use for the estimation of diphtheria and tetanus antitoxin levels. In addition, the ToBI test eliminates the need for laboratory-animal or cell-culture facilities and can be performed with small quantities of serum as required in field trials.     

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.     

Immunity to diphtheria, six to 15 years after a basic three-dose immunization schedule

The results of a study of the immunity to diphtheria of 283 girls (9-18 years of age) vaccinated at the age of two years with three doses of vaccine, are reported. The rabbit skin test was used to determine the titre of serum diphtheria antitoxin. 55.8% of the subjects were found to be protected (titre greater than or equal to 0.1 IU/ml), 38.9% were only relatively immune (titre greater than or equal to 0.01- less than 0.01 IU/ml), and 5.3% were unprotected (titre less than 0.01 IU/ml). The antitoxin titres showed a tendency to decrease with time. Even so, 6-15 years after vaccination, the percentages of protected and partially protected subjects were still high (95%).     

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.     

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.     

The titration of tetanus antitoxin IV. Studies on the sensitivity and reproducibility of the toxin neutralization test

The quality of tetanus toxin affected the sensitivity of the toxin neutralization (TN) test greatly. By using purified toxin a minimum level of 0.001 IU/ml of tetanus antitoxin could be detected whereas with crude toxin a level of 0.025 IU/ml only could be detected. The TN test described in this report permitted titration of tetanus antitoxin in twofold dilution steps from levels as low as 0.001 IU/ml using 0.6 ml of serum only at the L+/5000 level of purified tetanus toxin. Treatment of the sera with 2-mercaptoethanol (2-ME) did not affect the TN titres showing that the TN test detects the neutralizing antibodies (IgG) which are not affected by 2-ME. The TN test was found to be a highly sensitive and reproducible test.     

The immunization of adults against diphtheria in Sweden

Two hundred and three women who disclaimed vaccination against diphtheria were divided into four groups and injected with either 2.0 or 6.25 Lf of a routine diphtheria toxoid or of a more purified preparation. One hundred and twenty-six of these women who did not show a secondary antibody response were given a second and a third injection one month and one year, respectively, after the first injection. Prebooster (third injection) antitoxin titres of greater than or equal to 0.01 IU ml-1 (the minimum level for protection) were found in 22 and 37% of those who received 2.0 and 6.25 Lf, respectively. Postbooster titres of greater than or equal to 1.0 IU ml-1 (calculated to give a protection of at least ten years of duration) were found in 23 and 58% of those who received 2.0 and 6.25 Lf, respectively. The rate of untoward reactions was low. Fever of short duration occurred in five women. Four out of the five women received 6.25 Lf of the more purified diphtheria toxoid and one 2 Lf of the routine toxoid. Local reactions greater than 10 cm were observed in three women. All received the higher dose, 6.25 Lf of diphtheria toxoid. Local reactions greater than 5 but less than or equal to 10 cm occurred in up to 13% (6.25 Lf of diphtheria toxoid). No significant difference between the groups of women vaccinated with routine or more purified toxoid was found. It was concluded that the diphtheria toxoids in the two doses of 2 Lf and 6.25 Lf did not induce a satisfactory immune response. To induce adequate protection the dose of diphtheria vaccine needs to be the same for adults and children, i.e. 12.5 Lf.     

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.     

Comparative study of resistance to tetanus and indices of the passive hemagglutination reaction in experimental animals

The article presents the results of the study of resistance to tetanus in 450 guinea pigs immunized against tetanus in a single injection and having antitoxin titers in their blood, as determined in the passive hemagglutination test, from less than or equal to 0.01 IU/ml to 1.6 IU/ml and more. The degree of protection in the immunized animals was determined by their challenge with Clostridium tetani spores in DCL and LD50.     

ELISA for the routine determination of antitoxic immunity to tetanus

Serum samples from 727 persons with different vaccination histories were assessed for tetanus antitoxin content in an enzyme linked immunosorbent assay (ELISA) and tested for tetanus toxin neutralization activity in mice in order to compare the results obtained by the two methods. Neutralizing antibody activities in sera from individuals previously completely vaccinated correlated well with results obtained by ELISA and the accuracy increased with increasing antitoxin concentration in serum. This correlation was observed in sera from persons vaccinated recently as well as in sera from persons vaccinated many years ago. In sera from persons with an incomplete vaccination history ELISA was found to be an unreliable tool for the prediction of in vivo results. Many of these sera had antitoxin levels by ELISA far above the in vivo values, probably due to the presence of non specific or low avidity antitoxin which is detected in ELISA. The lowest ELISA value reliably predictive of protective antibody activity in serum irrespective of vaccination history was found to be 0.16 IU/ml. It was concluded that ELISA is useful for larger population studies as an initial test, but sera with an antitoxin content below 0.16 IU/ml should also be assessed in a neutralization system.     

The titration of tetanus antitoxin V. Effect of formalization method for the fixation of sheep erythrocytes on the indirect haemagglutination test

Two methods of fixation of sheep erythrocytes with formaldehyde for the titration of tetanus antitoxin by the indirect haemagglutination (IHA) test have been compared. The cells fixed with 3% formaldehyde at 4-8 degrees C for 24 h (formaldehyde (I) fixed cells) were less sensitive than the cells fixed with 3% formaldehyde at 4-8 degrees C for 24 h and subsequently treated with 40% formaldehyde at 4-8 degrees C for a further 24 h (formaldehyde (II) fixed cells). The correlation between the toxin neutralization (TN) and IHA titres using formaldehyde (I) fixed cells was better than that obtained with formaldehyde (II) fixed cells. There was no statistically significant difference between TN and IHA titres after treatment of the sera with 2-Mercaptoethanol using formaldehyde (I) fixed cells. Formaldehyde (I) fixed cells can be used for two months with adequate sensitivity to detect the minimum protective level of tetanus antitoxin in the sera.     

Neonatal tetanus despite protective serum antitoxin concentration

Using the ELISA technique to estimate serum antibodies against tetanus toxin, seven neonates with clinical tetanus were found to have antibody levels 4-13 times higher than the presumed minimum protective level of 0.01 IU/ml. All but one of their mothers had been vaccinated with tetanus toxoid in pregnancy. In two other neonates, whose mothers had received multiple booster doses of toxoid during pregnancy, the anti-toxin concentrations were 100- and 400-times the presumed protective level. Therefore the toxin dose may overwhelm the pre-existing anti-toxin level and produce disease. Furthermore, multiple booster injections of tetanus toxoid may not only enhance serum anti-toxin titres, but could also lead to an ineffective immune response.     

The titration of tetanus antitoxin II. A comparative evaluation of the indirect haemagglutination and toxin neutralization tests

Serum samples from 77 guinea pigs immunized against tetanus have been titrated for tetanus antitoxin by a standardized indirect haemagglutination (IHA) test and the conventional toxin neutralization (TN) test. These sera were titrated before and after treatment of the sera with 2-mercaptoethanol (2-ME) by the IHA test using unfixed sheep erythrocytes and erythrocytes fixed with glutaraldehyde, formaldehyde and pyruvic aldehyde. The titres of these sera obtained by IHA using unfixed and glutaraldehyde-fixed sheep erythrocytes before treatment of the sera with 2-ME were two to six times higher than the TN titres, whereas the IHA-titres using formaldehyde- and pyruvic aldehyde-fixed sheep erythrocytes were 10 times higher than the TN titres in some of the sera. There was no statistically significant difference between TN and IHA titres using unfixed and glutaraldehyde-fixed sheep erythrocytes after the treatment of the sera with 2-ME.     

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.     

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.     

Relationship between the routes of antitoxin administration and the effectiveness of treating experimental tetanus

Experiments were conducted on 100 rabbits with asscending, hematogenic and cerebral tetanus caused by the administration of tetanus toxin (1 Dcl). The therapeutic efficacy of "Diaferm-3" antitoxin was compared depending on the route--intracysternal or intralumbar--of its administration (400 IU/kg). Intracysternal antitoxin administration proved to be thrice as effective as the intralumbar one (31.4 and 10.2% of the sick animals recovered, respectively). The latter route was effective only in animals with the ascending intoxication, this apparently being connected with the site of entrance of the toxin into the central nervous system by the peripheral nerves of the hind limbs.     

酶联免疫破伤风抗体定量试剂的研制及应用

制备一种精确的破伤风抗体定量试剂,用于人源破伤风抗体的定量及人群破伤风抗体水平的测定。以人源破伤风免疫球蛋白国家标准品建立定量反应曲线,经系统优化后建立双抗原夹心法定量检测系统。定量反应曲线显示,抗体浓度在10~120m IU/m l之间,相关系数r=0.9993。精密度(CV)≤7%。实际应用验证,未经(TTC)免疫的献血员中,具有保护性抗体水平(0.01 IU/m l)的比例只占12.2%。经3针(TTC)免疫后,抗体水平均大于0.01 IU/m l,经动物体内中和试验法(NT)定量的3批破伤风免疫球蛋白,用制备的酶联免疫破伤风抗体定量试剂测定,其回收率分别为109%、98%和93%。结论,该试剂可用于破伤风抗体的精确定量。     

A comparison of the indirect haemagglutination test with the toxin neutralization test for the estimation of diphtheria antitoxin in mouse sera

Serum samples from 42 groups of mice immunized for different immunization periods with various doses of Adsorbed Diphtheria-Tetanus Vaccine, Adsorbed Diphtheria-Tetanus and Pertussis Vaccine and a standard diphtheria toxoid were assayed for their diphtheria antitoxin content by indirect haemagglutination (IHA) and by toxin neutralization (TN) tests. A very good correlation of 0.91 was obtained between the results of the two methods. There was no statistically significant difference between the IHA and the TN titres obtained. Adsorption with sheep red cells and treatment of the sera with 2-mercaptoethanol had no effect on the IHA titres. The minimum level of antitoxin detectable by the IHA test was 0.00039 IU ml-1. IHA proved to be a sensitive, specific and reproducible method which can be used reliably for the assay of diphtheria antitoxin in mouse sera.     

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.