Caprylate/chromatography process to produce highly purified tetanus immune globulin from human plasma.

While tetanus toxoid vaccination has reduced the incidence of tetanus in the developed world, this disease remains a substantial health problem in developing nations. Tetanus immune globulin (TIG) is used along with vaccination for prevention of infection after major or contaminated wounds if vaccination status cannot be verified or for active tetanus infection. These studies describe the characterisation of a TIG produced by a caprylate/chromatography process. The TIG potency and presence of plasma protein impurities were analysed at early/late steps in the manufacturing process by chromatography, immunoassay, coagulation and potency tests. The caprylate/chromatography process has been previously shown to effectively eliminate or inactivate potentially transmissible agents from plasma-derived products. In this study, the caprylate/chromatography process was shown to effectively concentrate TIG activity and efficiently remove pro-coagulation factors, naturally present in plasma. This TIG drug product builds on the long-term evidence of the safety and efficacy of TIG by providing a product with higher purity and low pro-coagulant protein impurities.

Detection of Tetanus Antibody Applying a Cu-Zn-In-S/ZnS Quantum Dot-Based Lateral Flow Immunoassay.

Lateral flow test strip (LFTS) enables rapid, portable, and low-cost point-of-care testing (POCT) diagnosis. Quantum dots (QDs), which are fluorescent semiconductor nanocrystals with distinctive and unique photophysical properties, have become promising candidates to serve as labels for LFTS with improved sensitivity. Here, by using QDs as a signal reporter, we report a fluorescent LFTS for detection of tetanus antibody. This LFTS possess a high sensitivity for tetanus antibody, with a detection limit of 0.001 IU/mL. This assay was also applied for detection of tetanus antibody in human serum. More importantly, these strips can retain their specificity and sensitivity for at least 4 months when they are stored at 4 °C.

Tetanus antitoxin potency assessment by surface plasmon resonance and ToBI test.

Potency testing of tetanus antitoxin must be performed in vivo, in a very painful, stressful and prone to high variability assay. It is, therefore, mandatory to find alternatives to this kind of potency assessment. Immunochemical tests as ELISA or ToBI test are already available but usually results in a poor correlation to the in vivo protection. Considering research and development of mono and oligoclonal antibodies against tetanus and the improvement of equine polyclonal antitoxin production and control, we developed an alternative instrumental test for tetanus antitoxin by using surface plasmon resonance. Tetanus antitoxin from hyperimmune equine sera (16 batches) were tested and the results indicated excellent concordance and correlation to the in vivo test (Lin's ρ = 0.9). This innovative approach should now be improved in order to extend it to oligoclonal and monoclonal human antibodies aiming to replace mice for the potency assessment of tetanus antitoxin especially during research and development steps.

Determination of low tetanus or diphtheria antitoxin titers in sera by a toxin neutralization assay and a modified toxin-binding inhibition test

A method for the screening of tetanus and diphtheria antibodies in serum using anatoxin (inactivated toxin) instead of toxin was developed as an alternative to the in vivo toxin neutralization assay based on the toxin-binding inhibition test (TOBI test). In this study, the serum titers (values between 1.0 and 19.5 IU) measured by a modified TOBI test (Modi-TOBI test) and toxin neutralization assays were correlated (P < 0.0001). Titers of tetanus or diphtheria antibodies were evaluated in serum samples from guinea pigs immunized with tetanus toxoid, diphtheria-tetanus or triple vaccine. For the Modi-TOBI test, after blocking the microtiter plates, standard tetanus or diphtheria antitoxin and different concentrations of guinea pig sera were incubated with the respective anatoxin. Twelve hours later, these samples were transferred to a plate previously coated with tetanus or diphtheria antitoxin to bind the remaining anatoxin. The anatoxin was then detected using a peroxidase-labeled tetanus or diphtheria antitoxin. Serum titers were calculated using a linear regression plot of the results for the corresponding standard antitoxin. For the toxin neutralization assay, L+/10/50 doses of either toxin combined with different concentrations of serum samples were inoculated into mice for anti-tetanus detection, or in guinea pigs for anti-diphtheria detection. Both assays were suitable for determining wide ranges of antitoxin levels. The linear regression plots showed high correlation coefficients for tetanus (r² = 0.95, P < 0.0001) and for diphtheria (r² = 0.93, P < 0.0001) between the in vitro and the in vivo assays. The standardized method is appropriate for evaluating titers of neutralizing antibodies, thus permitting the in vitro control of serum antitoxin levels.

Measurement of tetanus antitoxin in oral fluid: a tool to conduct serosurveys.

BACKGROUND: Serosurveys that measure tetanus antitoxin can complement immunization coverage surveys to allow evaluation of immunization services in developing countries. Measurement of IgG tetanus antitoxin in oral fluid was investigated as a practical and noninvasive alternative to and correlate of serum antibodies. METHODS: Serum and oral fluid were collected from Malian infants, toddlers and adults (males without a history of tetanus vaccination). Specific IgG tetanus antitoxin was measured by enzyme-linked immunosorbent assay in serum (S-ELISA) and oral fluid (OF-ELISA). RESULTS: One hundred forty-two pairs of serum and oral fluid samples were collected from infants, 35 pairs from toddlers and 35 pairs from adults. IgG tetanus antitoxin titers measured by OF-ELISA were 100-fold lower than those measured by S-ELISA but they correlated strongly (r = 0.90, P < 0.001). All 35 toddlers who had received 2 or 3 doses of diphtheria-tetanus-pertussis (DTP) vaccine (100%) had serum tetanus antitoxin levels >or=0.15 IU/mL and 28 of 35 (80%) had oral fluid values >or=0.0015 IU/mL. Among adults lacking a history of tetanus immunization, only 6 of 35 (17.1%) had serum titers >or=0.15 IU/mL and 4 of 35 (11%) had oral fluid titers >or=0.0015 IU/mL in oral fluid. CONCLUSIONS: IgG tetanus antitoxin in oral fluid correlates well with levels in serum. OF-ELISA values >or=0.0015 IU/mL constitute protection against tetanus and in subjects >12 months of age imply multiple prior contacts with immunization services. IgG tetanus antitoxin measured by OF-ELISA provides a logistically practical alternative for performing seroprevalence surveys.

Seroprevalence of tetanus antibody in Turkish population and effectiveness of single-dose tetanus toxoid.

The study presented here was performed to determine the rates of immunity to tetanus in a cross section of the Turkish population and to assess the impact of a single-dose tetanus toxoid in previously unvaccinated individuals. Among 18-year-old participants who received their last tetanus booster 4 years prior to the study and 22-year-old participants who received the booster 8 years previously, certain durable protective antibody (>1 IU/ml) was detected in 100%. However, only 3.3% of 20-year-old participants who did not know whether they had been vaccinated as children or young adults had a protective antibody level. After administering a single dose of tetanus vaccine to this group, the rate of protective antibody increased from 3.3% to 96.7%. For the prevention of tetanus, the optimal policy should focus on improving rates of complete primary immunization in adults and children and giving a single booster every 10 years, or at least at age 40 or 50 years.

Immunity against diphtheria and tetanus in German blood donors.

After the recent diphtheria epidemics in Eastern Europe in the early 1990s, we re-evaluated the diphtheria and tetanus immunity of 321 German blood donors (192 men and 129 women). The mean antitoxin levels of all blood donors in this study, measured by commercial ELISA, revealed a questionable protection (0.1-1.0 IU/ml) against diphtheria. In 1994, 66.4% were without immunity against diphtheria (55.0% in 1997/98), 32.1% (41.5% in 1997/98) showed questionable protection and only 1.5% (3.5% in 1997/98) had protective antitoxin levels. The evaluation of tetanus immunity revealed only 0.5% (1.1% in 1997/98) of the subjects with no protection and 9.1% (8.5% in 1997/98) with questionable protection. For this reason, we conclude that the diphtheria epidemics only lead to an insufficient improvement of the immunization status in a healthy German population.

Improved ELISA for determination of anti-diphtheria and/or anti-tetanus antitoxin antibodies in sera.

Double-antigen ELISAs for detection and quantification of anti-tetanus or anti-diphtheria antibodies in serum have been developed. The assays showed good correlations with established toxin neutralizing assays and were functionally specific for IgG antibodies. The double-antigen set-up allows specific antibodies to bind to antigen-coated microtitre wells with one arm and the free arm to bind to biotin-labelled antigen. The amount of antibodies able to bind labelled antigen was assessed by adding enzyme-conjugated streptavidin and colour substrate followed by measurement of the colour using an ELISA reader. The double-antigen principle makes it possible to compare samples of different species on the same plate, permitting the direct use of existing international references of animal or human origin. The double-antigen ELISAs showed a detection limit of 0.00002 IU/ml for both antibodies and were suitable for quantifying antibodies in blood samples collected on filter paper as well as in serum. The assays required no special equipment compared to traditional ELISA.

Adjuvanticity of pGPL-Mc and LRS in the immune responses of monkeys to oral immunization with diphtheria and tetanus toxoids.

Experiments were carried out to examine the adjuvanticity of polar glycopeptidolipids of Mycobacterium chelonae (pGPL-Mc) or the London rocket seed (LRS) when combined with diphtheria and tetanus toxoids in an oral immunization of the African green monkey. The results showed that none of the monkeys receiving diphtheria and tetanus toxoids combined with 25 mg/kg of pGPL-Mc showed an increase in the the level of diphtheria antitoxin (DA) on the third and sixth weeks following the first and the second immunizations. One monkey from this group responded with increased seroneutralizing antibodies 3 weeks after the third feeding. On the other hand, one monkey, 3 weeks after the first immunization, and three monkeys, 3 weeks after the second and third oral vaccinations, showed an increase in specific anti-diphtheria antibody responses when the toxoids were combined with 25 mg/kg of LRS. The anti-diphtheria antitoxin responses of monkeys receiving diphtheria and tetanus toxoids combined with 50 mg/kg of pGPL-Mc or 50 mg/kg of LRS were significantly enhanced compared to the groups administered 25 mg/kg of the two adjuvants. The increase was observed in four out of five pGPL-Mc administered and in three out of five LRS-receiving monkeys. The results show that pGPL-Mc induced the highest titres of anti-diphtheria antitoxin compared to LRS, whereas the level of anti-diphtheria antitoxin titre of the two monkeys receiving the toxoids alone was less than 0.1 i.u./ml of serum throughout the experiment. According to the statistical analyses, no significant differences were recorded between the diphtheria antitoxin responses of monkeys following the first, second or third administration of LRS-adjuvated diphtheria and tetanus toxoids. However, a significant difference (P < or = 0.05) was observed in the diphtheria antitoxin response between the first and the second immunization of monkeys administered with toxoids adjuvated with 50 mg/kg of pGPL-Mc. The tetanus antitoxin responses of all monkeys were less than 0.1 i.u. of antitoxin per millilitre of serum throughout the study, which is considered not to be protective. However, we have recorded an anti-tetanus antitoxin titre of more than 0.2 i.u./ml of serum in one monkey that received diphtheria and tetanus toxoids combined with 50 mg/kg of pGPL-Mc.

Antibody response to Haemophilus influenzae type b capsular polysaccharide conjugated to tetanus toxoid in preterm infants.

OBJECTIVE: To evaluate the antibody response to a Haemophilus influenzae type b capsular polysaccharide (HibCP) tetanus toxoid (TT) conjugate vaccine (HibCP-TT) in preterm infants. SUBJECTS: Thirty-five healthy preterm infants with gestational ages (GA) from 27 to 36 weeks and birth weights from 920 to 2550 g. Controls were 37 term infants. METHODS: All infants were immunized with HibCP-TT at 2, 4 and 12 months of age. Antibodies to HibCP and TT were determined at each immunization and 1 month after the second and third. RESULTS: After two doses of HibCP-TT the preterm infants with GAs < or = 30 weeks (n = 8; mean GA, 29.5 weeks) had a significantly lower HibCP antibody response than the preterm infants with GAs > 30 weeks (n = 23; mean GA, 34.2 weeks) (P = 0.004), who for their part had a response not significantly different from that of the term infants. After the third dose there were no significant differences among the groups. The response to the TT part of the vaccine showed the same pattern. CONCLUSION: Although the most immature infants may show an inadequate antibody response to the initial immunizations, many preterm infants can benefit from vaccination with HibCP-TT when starting immunization at the same chronologic age as term infants.

[Effects of drugs on the results of passive hemagglutination test]. / Vliianie lekarstvennykh sredstv na rezul'tat reaktsii priamoi gemaggliutinatsii.

Study on the effects of some drugs on the results of passive hemagglutination test used for the detection of antitetanus antibodies in the blood serum showed that some drugs had a pronounced dose-dependent effect on antibody titers in experiments in vitro with standard antitoxic serum. The possibility of such errors should not be disregarded when assessing the results of the test.

Latex agglutination test for detection of tetanus antitoxins.

A rapid and easy method of slide agglutination test for the detection of human tetanus antitoxins was developed in this study. Testing reagents were prepared from carboxylated polystyrene latex particles with tetanus toxin by soluble carbodiimide. The test was performed on a glass slide with a drop of test sample and a drop of testing reagent. The agglutination reaction was usually completed within five minutes. Sensitivity of this test for tetanus antitoxins can be reached at 0.125 IU/ml. Therefore, the latex agglutination test can be used to determine the immune status of a patient in an emergency.

[Evaluation of antitoxin and antibacterial activity of human preparations of normal immunoglobulin. I. Level of tetanus antitoxin in preparations of IMIG and IVIG]. / Ocena aktywnosci antytoksycznej i antybakteryjnej preparatów ludzkiej normalnej immunologlobuliny. I. Poziom antytoksyn tezcowych w preparatach IMIG i IVIG.

The level of anti-tetanus antibodies were measured in 791 lots of IMIG and 330 lots of IVIG of human normal gamma globulin. The antibody levels varied 15-80 IU/ml of IMIG lots and 5-20 IU/ml of IVIG lots. Every lot of human normal gamma globulin (IMIG or IVIG) having high anti-tetanus activity may be an acceptable alternative to specific tetanus immune globulin.

[Effectiveness of tetanus vaccination in an adult population 10 years after the last dose]. / Efectividad de la vacunación antitetánica a los 10 años de la última dosis en una población adulta.

OBJECTIVE: To check if antitetanus immunity after a correct vaccination lasts the anticipated 10 years. DESIGN: Sero-epidemiological crossover study. Determination of tetanus antitoxin by the ELISA method. SETTING: The town of Barrado in Cáceres. PATIENTS AND OTHER PARTICIPANTS: 136 adult men and women (average age 54.6 +/- 15.9 years; range, 24 to 87) who had received their most recent vaccination during a 1981-1982 campaign. 71.32% received three doses. MEASUREMENTS AND MAIN RESULTS: 82.35% +/- 6.40 of the sera analysed (percentage +/- C.I. 95%) maintained adequate levels of protection (> or = 0.01 Ui/ml). 100% of those under 35, revaccinated in 1980-81 because they were ELISA negative in spite of earlier school vaccination, were protected. The average concentrations did not differ significantly by gender and age-groups, although a tendency to decrease with age was noted. CONCLUSIONS: Anti-tetanus vaccination in the individuals studied provided lasting protection, detectable for 10 years in 76-89% of cases. This level of effectiveness lends support to the criterion of administering back-up doses every 10 years.

Prevalence of hyperimmunization against tetanus in Italians born after the introduction of mandatory vaccination of children with tetanus toxoid in 1968.

Systematic mandatory immunization of children against tetanus was started in Italy in 1968. Prevalence of tetanus hyperimmunization (> 5 IU/ml) was assessed among 214 immune subjects born after 1968 and found to be 17.3%. This figure is significantly higher (p < 0.01) than the 10.8% found in a previous study of subjects born before 1968. This increase is statistically significant in the Center (p < 0.01) and in the South (p < 0.05) of Italy, but not in the North. Hyperimmunization is not associated with family size (odds ratio 2.16; C. I. 95% = 0.5-7.6) or the father's years of education (odds ratio 1.83; C. I. 95% = 0.6-5.3). No difference was found between urban and rural areas of residence. Indiscriminate administration of booster doses of tetanus vaccine in hyperimmune subjects in some areas could result in unnecessary vaccinations, which can cause hypersensitivity reactions.

[Preventive tetanus immunization and avoidance of side effects of booster immunization]. / Tetanusimpfschutz und Vermeidung von Nebenreaktionen bei Auffrischimpfungen.

Prevalence of antibodies against tetanus toxoid (tetanus antitoxin titre) was measured in 5,235 males aged 17-60 years, 3,069 (59%) aged 20-22 years. Taking as criterion a threshold value of 0.1 IU/ml, 5,071 (97%) had adequate tetanus immunity. 1,301 of 4,355 aged 21-30 years (30%) actually had antibody concentrations above 6.3 IU/ml. Booster injections are especially contraindicated in this latter group because of the danger of side effects with further toxoid doses. Thoughtless routine booster immunization should therefore be avoided.

[The kinetics of vaccine antibodies against tetanus toxoid, diphtheria toxoid, measles virus, poliomyelitis virus and pneumococcus after allogeneic and autologous bone marrow transplantation and revaccination. 3: The kinetics of vaccine antibodies against tetanus toxoid and diphtheria toxoid after allogeneic and autologous bone marrow transplantation and combined revaccination against diphtheria and tetanus]. / Untersuchungen zur Kinetik der Impfantikörper gegen Tetanustoxoid, Diphtherietoxoid, Masern-Virus, Poliomyelitis-Virus und Pneumokokken nach allogener und autologer Knochenmarktransplantation und Wiederholungsimpfung. Teil 3: Kinetik der Impfantikörper gegen Tetanustoxoid und Diphtherietoxoid nach allogener und autologer Knochenmarktransplantation und kombinierter Wiederholungsimpfung gegen Diphtherie und Tetanus.

The 3rd part of the paper deals with the results of a combined revaccination against diphtheria and tetanus in a group of 25 children after allogeneic bone marrow transplantation (BMT) with and without graft versus host disease (GvHD) and after autologous transplantation. It can be shown that in the allogeneic transplanted groups with and without GvHD it is possible to build up a tetanus and diphtheria antitoxin titre in a safe protective cause by a 2nd basic immunisation consisting of 3 single vaccinations starting about 9 to 12 months later. For autologous transplanted children only 1 to 2 vaccinations at a later term than for the allogeneic transplanted children may possibly be sufficient.