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.     

Determination of the antigenic activity of diphtheria anatoxin in liquid and sorbed preparations using an erythrocyte diphtheria antigen (anatoxin) diagnostic agent

On the basis of the experimentally established dependence of the degree of binding of diphtheria toxoid with standard diphtheria antitoxin on the duration of their joint incubation with the maximum binding occurring in 3-4 hours of incubation at 37 degrees C, the method of the in vitro determination of the antigenic activity of diphtheria toxoid in liquid and adsorbed preparations is proposed. The method is based on the principle of double binding with the use of diphtheria antigenic (toxoid) erythrocyte diagnosticum. The antigenic activity of diphtheria toxoid, evaluated by the degree of its maximum binding with diphtheria antitoxin, correlated with its antitoxin-binding activity in animal experiments and did not correlate with its flocculating activity. The antigenic activity of diphtheria toxoid in adsorbed preparations, evaluated by their maximum binding with standard diphtheria antitoxin, was shown to be closely related to the immunogenic potency of these vaccines in animals.     

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.     

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.     

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.     

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.     

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.     

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.     

Vero细胞法检测白喉抗毒素的研究

参照Ｍｉｙａｍｕｒａ等报道,建立了微量细胞培养检测白喉抗毒素的方法。以家兔皮肤试验为参照,Ｖｅｒｏ细胞培养法敏感度为９８．１１％,特异度为８４．００％,符合率为９６．２０％,相关系数ｒ＝０．９３,在白喉血清抗毒素测定中值得推广     

应用Vero细胞检定法和家兔皮内中和法检测吸附白喉类毒素的效价

Ｖｅｒｏ细胞法测定ＤＴＰ疫苗中白喉类毒素的效价与家兔皮内中和法（简称ＮＴ法）所得结果两法之间有一种平行关系,并且有很好的相关性,ｒ＝０．９３。但Ｖｅｒｏ细胞法测得平均值低于ＮＴ法,二者之间的平均比率为０．２８７,Ｓ＝０．０８９。本试验结果与１９９５部颁规程ＮＴ法０．２５ＩＵ／ｍｌ相比,确定了Ｖｅｒｏ细胞法以０．０７ＩＵ／ｍｌ作为检定吸附ＤＴＰ效价的最低要求。     

Antibody status to poliomyelitis,measles, rubella,diphtheria and tetanus,Ontario, 1969-70: deficiencies discovered and remedies required.

A serologic survey was made in 15 health unit areas, testing some 5000 individuals in the age groups 4 to 6, 11 to 13, 15 to 17 and 23 to 45 years. Two types of serious deficiency were found. Only 65% of children 4 to 6 years old had antibodies to all three types of poliovirus, the antibodies being due almost entirely to immunization with Salk vaccine. Even in children who had had six or more doses only 74% had antibodies to the three types. The high percentage of students 11 to 13 and 15 to 17 years old with poliovirus antibodies can be attributed largely to natural infection and to Sabin vaccine in the mass campaign of 1962, as well as to Salk vaccine. In children who had received Sabin vaccine as well as Salk vaccine a very high level of immunity was found. The immunity of the school-age population will decline to an insufficient level unless Sabin vaccine is used after immunization with Salk vaccine. Of children 4 to 6 years old 18% had no diphtheria antitoxin and 6% had no tetanus antitoxin. Even in those who had had six or more doses of the antigens 5% had no diphtheria antitoxin and 1 to 2% had no tetanus antitoxin. This apparently refractory state is probably due to the use of unadsorbed toxoids, and it is clear that adsorbed toxoids should be used. In the adults, diphtheria antitoxin was found in only 55% and tetanus antitoxin in only 38%.     

Pathogenesis of diphtheria carrier state from the immunological point of view.

Results of a comparative investigation of diphtheria antitoxin and type-specific antibacterial antibodes in 264 carriers of diphtherial bacteria, 41 diphtheria patients and 263 non-infected subjects are presented. A high level of antitoxin did not prevent the development of toxigenic-strain carrier state. A basically similar immunological antibacterial response was observed in patients with manifest forms of diphtheria and in carriers of toxigenic strains; such a response could not as yet be detected in carriers of non-toxigenic strains. It has been suggested that the infectious process in the toxigenic-strain carrier state is due to factors of the virulence responsible for infectivity and invasiveness of the diphtherial microbe. The toxin plays no pathogenic role in carrier state.     

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 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.     

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.     

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.     

Fatal diphtheria in an older woman

A previously healthy 68-year-old woman presented with fever and sore throat. Her condition was initially diagnosed as necrotizing streptococcal tonsillitis and was treated with penicillin G, given intravenously. A swab of her throat taken for culture at the time of admission yielded Corynebacterium diphtheriae 48 hours later. At that time an electrocardiogram showed new T-wave inversion — evidence of diphtheritic myocarditis. She was immediately given 60 000 units of equine diphtheria antitoxin (following a test dose), but later that day she began choking, became apneic and died. The patient had not received any immunizing agents as a child, and no antitoxin was detected in a blood sample obtained prior to administration of the antitoxin. Her death re-emphasizes the seriousness of diphtheria, an infection to which many elderly people are susceptible.     

Determination of the toxigenicity of Corynebacterium diphtheriae by using paper indicator disks

The method for the determination of the toxigenicity of C. diphtheriae by means of discs impregnated with diphtheria antitoxin is proposed. The discs can be prepared long before use and stored for a year. The determination of toxigenicity with these discs is not inferior in its sensitivity to the routine procedure of the immunoprecipitation test. The disc method decreases the consumption of unavailable diphtheria antitoxin by 10 times, that of culture media by 2-2.5 times: besides, it is less labor consuming.     

IgA and IgA diphtheria antitoxin responses from human tonsil lymphocytes.

Human tonsil lymphocytes were stimulated with diphtheria toxoid and then cultured in a Marbrook culture system so that antibodies could be measured in the culture supernatant. Specific antibodies were measured with excess radiolabeled antigen and antisera specific for each immunoglobulin class. Good IgG and IgA diphtheria antitoxin responses have been obtained and responding culture supernatants were shown to neutralize toxin. The relationship between antitoxin response in vitro and immunization of donors with toxoid was investigated. It was found that at least two immunizations after the age of 6 months were necessary to prime the tonsils for an in vitro antibody response. The IgG and IgA in culture supernatants were demonstrated by immunodiffusion and were measured by radioimmunoassay. By sucrose density gradient ultracentrifugation, it was shown that 40% of the IgA produced in the cultures was greater than 7S. Evidence was obtained that neither the IgA nor the specific IgA antitoxin bears secretory piece. It appears that human lymphocytes from tonsils produce polymer IgA in vitro without secretory piece.     

Diphtheria-neutralizing antibody levels in healthy adults from Rio de Janeiro, Brazil

In Brazil, until 2004, the immunization policy against diphtheria involved childhood vaccination with no official routine booster dose administered after 15 years of age. This study assessed functional antibody levels against diphtheria among blood donors. A total of 140 blood samples were collected, and diphtheria antitoxin levels were evaluated by Vero cell neutralization test. The mean age of the population was 34 years old (range: 18-61 years); 37.8% females and 62.2% males. Overall, 30.7% (95%, CI: 23.4-38.7) individuals presented neutralizing antitoxin antibody titers < 0.01 IU/ml; 42.1% (95%, CI: 34.1-50.4) showed values between 0.01-0.09 IU/ml and, 27.1% (95%, CI: 20.2-34.9) had (3) 0.1 IU/ml. In the subgroup of individuals with history of diphtheria immunization during childhood (85%), a number of 28.5% showed unprotective levels of circulating neutralizing antibody (< 0.01 IU/ml). Despite the continuous progress of immunization programs directed to Brazilian population, currently healthy adults remain susceptible to diphtheria.