Antitoxin Titers Research Articles

Measurement of Tetanus Antitoxin in Oral Fluid

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. 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). 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. 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.

Nasal immunization studies using liposomes loaded with tetanus toxoid and CpG-ODN

To increase the systemic and mucosal immune responses against the nasally administered tetanus toxoid, liposomes as a drug delivery system and CpG-ODN as an adjuvant were evaluated. Rabbits were nasally immunized with entrapped tetanus toxoid (TT) and CpG-ODN in neutral liposomes and systemic and mucosal immune responses were determined. Liposomes containing TT and CpG-ODN were prepared by dehydration–rehydration method. The volume mean diameter of liposomes was 2.3 ± 0.6 μm. Encapsulation efficiency of TT and CpG-ODN was determined as 54.0 ± 8.8 and 60.1 ± 7.4, respectively. The leakage of the encapsulated TT from liposomes reached 7.38% after 3 months. Encapsulated TT kept its intact structure, and its immunoreactivity was also completely preserved, as shown by SDS–PAGE and ELISA methods. The highest serum IgG and antitoxin titers were observed in groups immunized with solution formulations ( P < 0.001). However the highest mucosal sIgA titers were achieved by liposomes encapsulated with TT. CpG-ODN as an adjuvant was able to increase the serum IgG and antitoxin titers when co-administered with TT solution ( P < 0.05) or co-encapsulated with TT in liposomes ( P < 0.01), but failed to increase the sIgA titers in nasal lavages. No hemolysis occurred on incubation of liposomes and human RBCs. Also after nasal administration of plain liposomes to human volunteers, no local irritation was seen. Intranasal administration of liposomes encapsulated with vaccines showed to be an effective way for inducing the mucosal immune responses.

Induction of systemic and mucosal immune responses by intranasal administration of alginate microspheres encapsulated with tetanus toxoid and CpG-ODN

In the induction of systemic and mucosal immunity, particulate antigens are more effective than soluble antigens; possibly because they are more efficiently endocytosed by mucosal-associated lymphoid tissue (MALT) M cells. In this study, we determined the systemic and mucosal immune responses in rabbits following intranasal immunization with encapsulated tetanus toxoid (TT) and CpG-ODN in alginate microspheres. The microspheres were less than 4 μm in diameter. Encapsulation efficiency of TT and CpG-ODN was determined as 47.7 ± 6.6 and 34.2 ± 7.4, respectively. Release of TT and CpG-ODN in a simulated model with nasal cavity was 14.2 ± 3.06 and 36.7 ± 2.4% after 4 h. Encapsulated TT preserved its intact structure, but its immunoreactivity was decreased to about 91 ± 5%. The highest serum IgG and antitoxin, and nasal lavage IgA titers were observed in groups immunized with microsphere formulations. CpG-ODN as an adjuvant could increase the serum IgG and antitoxin titers when co-administered with TT solution, but its co-encapsulation with TT in alginate microspheres failed to potentiate the systemic immune response while induced high IgA titers in nasal lavages. No hemolysis was occurred on incubation of alginate microspheres and human RBCs. Also after nasal administration of plain microspheres to human volunteers, no local irritation was observed. Intranasal administration of microspheres encapsulated with vaccines showed to be an effective way for inducing a variety of immune responses and that a strong systemic IgG and mucosal IgA responses can be induced in rabbits with intranasal administration of alginate microspheres encapsulated with TT.

Protective Effect of Botulinum C/D Mosaic Toxoid against Avian Botulism

Avian botulism is a paralytic disease caused by a toxin produced by Clostridium botulinum type C. Since type C isolates from cases of avian botulism produced a neurotoxin consisting of a mosaic form of parts of type C and D neurotoxins, we examined the antitoxin titers in the convalescent sera of botulism-affected birds which belonged to family Anatidae. ELISA using the C/D mosaic neurotoxin as an antigen revealed that the antibody was detected in the sera at 2 weeks, but not at 5 weeks after the onset, suggesting that the antibody only appeared for a short period in the convalescent phase. However, we failed to detect the antibody titers with anti-chicken IgG instead of anti-duck IgG. We therefore examine the immunological properties of IgG among different families and species. The results revealed that different species of IgG in the same family exhibited strong cross-reactivity. Ducks immunized once with the toxoid together with a commercial oil-adjuvanted vaccine were found to develop sufficient antibody to protect against a challenge with a lethal toxin dose. The ELISA titers did not correspond to the neutralization titers in the sera of immunized ducks at the early stage during immunization. These findings suggest that the neutralizing titer was more useful than the ELISA titer for evaluating the protection against the toxin, but the ELISA technique may be applicable for detecting the occurrence of botulism.

Kinetics of Local and Systemic Immune Responses after Vaginal Immunization with Recombinant Cholera Toxin B Subunit in Humans

Vaginal vaccination seems to be the best strategy for inducing specific immunoglobulin A (IgA) and IgG antibody responses in the female genital tract. The relative efficiencies of one, two, and three vaginal doses of recombinant cholera toxin B subunit (CTB) in generating mucosal and systemic immune responses in healthy women were evaluated, and the kinetics of the immune responses were monitored for responding volunteers for up to 12 months after the last vaccination. A single dose of CTB failed to generate CTB-specific IgA antibody responses in cervical secretions. Two vaccinations induced significant increases in IgA antitoxin titers in seven of nine volunteers, and four volunteers also developed IgG antitoxin responses. The magnitudes of the responses were 20-fold for IgA antitoxin and 7.1-fold for IgG antitoxin. A third vaccination did not significantly increase the antitoxin responses, although the frequency of IgG responses was slightly higher than that after the second vaccination. In serum, CTB-specific antibodies were observed already after a single vaccination. However, two vaccinations were required to induce marked IgA as well as IgG antitoxin titer increases in the majority of volunteers. The postvaccination levels of antitoxin antibodies in serum were comparable after two and three vaccinations. At 12 months after vaccination, significantly elevated IgA and IgG antitoxin levels in cervical secretions could still be detected in approximately half of the volunteers who had initially responded to the vaccine. Antitoxin titer increases in serum were found in most of the vaccinees at follow-up.

Immunité antidiphtérique de la population algérienne : Étude séroépidémiologique

Objective. – The antidiphtheria immunity of the Algerian population is not well known. Thus, a study was carried out on 1755 blood samples, in 1998 to better assess this vaccinal status. Three epidemics occurred in Algeria, in the years 1993, 1994, 1995, and then immunization sensitizing campaigns were introduced in September 1995. Material and method. – The procedure that was used was based on the immunoenzymatic method, the reagents of which were ready for use. The interpretation of our results and vaccinal policy deduction were performed according to the VIROTECH Diphtheria ELISA technique which offers five classes in antitoxin titers: I. <0.1 IU/ml basic immunization immediately. II. 0.1–1.0 IU/ml booster immunization immediately. III. 1.0–1.4 IU/ml booster vaccination 5 years later. IV. 1.5–2.0 IU/ml booster vaccination 7 years later. V. >2.0 IU/ml booster vaccination 10 years later. Results. – In our series, 17.03% of the samples had a titer <0.1 IU/ml considered as insufficiently protective, an immunization was therefore recommended. 59.82% had titers ranging between 0.1 and 1 IU/ml, 5.98% had titers ranging between 1 and 1.4 IU/ml, 3.36% had titers ranging between 1.5 and 2 IU/ml, and 13.79% had titers >2 IU/ml.

Evaluation of diphtheria convalescent patients to serve as donors for the production of anti-diphtheria immunoglobulin preparations

Aims: The study was conducted to evaluate the possibility of selecting convalescent diphtheria patients to serve in emergency situations as donors for the production of anti-diphtheria immunoglobulin. To select suitable donors, the criterion of an antitoxin titer ≥3.0 IU/ml was used. In addition, the effects of treatment and the effect of immunization with diphtheria toxoid on the level of anti-diphtheria toxin antibodies were evaluated. Scope: Three groups of diphtheria patients were included in the study. The first group ( n=23) consisted of patients who had a basic antibiotic treatment, with or without serotherapy using horse antitoxin and/or human immunoglobulin. The second group ( n=12) comprised patients examined immediately after the onset of disease. The immunological history of this group was not known. The third group ( n=20) included patients with a known immunization history, treated only with antibiotics but having received a booster immunization with diphtheria toxoid. Antitoxin titers were measured using the toxin binding inhibition (ToBI) assay. Conclusions: In the first group, 47.8% (11/23) of the patients had a diphtheria antibody titer ≥3.0 IU/ml. For most of them, however, the antibody titers could have resulted from treatment with exogenous antibodies from horse antitoxin or human immunoglobulin (18/23). Only two of the 11 high-titer subjects had received antibiotics only. Among the second group, only two (16.76%) of the patients had an antibody titer of ≥3.0 IU/ml. In the third group 50% (10/20) of the patients showed an antibody titer of ≥3.0 IU/ml prior to vaccination, and therefore could be directly considered as donors. Three weeks after booster vaccination, 70% (14/20) had an antibody titer of ≥3.0 IU/ml and 1 year after booster vaccination, 28.6% (2/7) of the subjects still had titers of ≥3.0 IU/ml. In 40% of these patients, a decrease was observed 3–4 weeks after the booster dose. It was concluded that convalescent diphtheria patients could be considered as donors in an emergency situation, since approximately half of them showed antitoxin titers of ≥3.0 IU/ml.

Phase II safety and immunogenicity study of type F botulinum toxoid in adult volunteers

An aluminum hydroxide ( alum)-adsorbed, purified, botulinum F toxoid (Bot F) vaccine was manufactured to be administered as a stand-alone monovalent vaccine or to be added to the current botulinum pentavalent toxoid vaccine to make a hexavalent vaccine. We conducted a phase II trial of the Bot F vaccine over 3 years in 144 healthy adult volunteers to identify one of three vaccination schedules that was safe and maximally immunogenic for adult volunteers. We vaccinated 116 volunteers 1–3 times with Bot vaccine, and 28 volunteers 1–3 times with a licensed, alum-adsorbed hepatitis B vaccine (Engerix-B) as a reaction control group. After 1 year, 42 Bot volunteers with low, mouse anti-toxin titers (<0.10 IU/ml) received a booster injection and were followed for an additional year. The Bot vaccine inoculated three times over 28–42 days was generally well tolerated and safe, whether injected by the subcutaneous (s.c.) or intramuscular (i.m.) route, although it caused significantly more local reactions than did the HBV vaccine. Two vaccination schedules of three primary injections over 42 days (days 0, 14 and 42 or days 0, 21 and 42) provided significantly better protective immunity (anti-toxin levels >0.02 IU/ml) than did vaccinations given over 28 days (days 0, 7 and 28). Vaccine reactogenicity and immunogenicity were similar over 42 days whether administered subcutaneously or intramuscularly. However, even the most immunogenic schedule left 7–16% of volunteers unprotected at day 56 and 33–42% of vaccinees unprotected at 1 year. The booster dose administered at 1 year induced high levels of protective serum anti-toxin in all persons assayed which persisted for at least one additional year. A more potent vaccine formulation will be required to protect more individuals after primary immunization.

Tetanus antitoxin seroprevalence in patients with Behçet's disease.

Anti-tetanus antibody status was evaluated by enzyme-linked immunosorbent assay in 82 patients with Behçet's disease and 79 healthy individuals. 76 patients with Behçet's disease (92.7%) and 74 healthy controls (93.7%) had protective antibody titres against tetanus, with geometric mean levels of 1.0194 +/- 1.2755 and 1.3899 +/- 1.6533 IU/ml, respectively. The difference between the 2 groups was not statistically significant. There was a significant inverse correlation between antitoxin titres and age in patient and control groups. These findings imply that patients with Behçet's disease have intact overall immunity against tetanus.

Tetanus immunisation in geriatric patients with accidental wounds: how much is needed?

In the industrialised world, the elderly carry the highest risk for tetanus. This prospective serological study was performed to evaluate the reliability of tetanus immunisation histories and the antibody response to tetanus vaccinations in the elderly. 40 individuals >65 years with a bleeding trauma were included in the study. Their tetanus vaccination histories were investigated, and accordingly a single booster (group A, n = 7), or a three dose basic immunisation against tetanus (group B, n = 33) was provided. In addition, tetanus antitoxin levels were determined. Age varied between 67 and 95 years. Inconsistencies regarding the vaccination history were found between patients and their physicians in 30% (12/40), between patient statements and vaccination documents in 57.1% (8/14) and between physicians' records and vaccination documents in 35.7% (5/14). Antitoxin titres >0.15 IU/ ml were considered protective, giving a seroprevalence of 92.5%. Sensitivities and negative predictive values for tetanus immunity were 30.6% and 10.7% based on patient histories and 2.8% and 7.9% based on physicians' records. In group A, after the single booster immunisation the median titre rose from 1.2 to 14.2 IU/ml, in group B from 0.9 to 5.3 IU/ml after the first, and to 9.6 IU/ml after the third tetanus toxoid dose (p <0.001 using Friedman's test). In Switzerland, elderly patients with a tetanus prone wound provide an unreliable vaccination history but their seroprotection against tetanus is high. A single booster for secondary immunisation is therefore sufficient. It is not necessary to take the largely unreliable vaccination history into account.

Tetanus immunisation in geriatric patients with accidental wounds: how much is needed?

In the industrialised world, the elderly carry the highest risk for tetanus. This prospective serological study was performed to evaluate the reliability of tetanus immunisation histories and the antibody response to tetanus vaccinations in the elderly. 40 individuals >65 years with a bleeding trauma were included in the study. Their tetanus vaccination histories were investigated, and accordingly a single booster (group A, n = 7), or a three dose basic immunisation against tetanus (group B, n = 33) was provided. In addition, tetanus antitoxin levels were determined. Age varied between 67 and 95 years. Inconsistencies regarding the vaccination history were found between patients and their physicians in 30% (12/40), between patient statements and vaccination documents in 57.1% (8/14) and between physicians' records and vaccination documents in 35.7% (5/14). Antitoxin titres >0.15 IU/ ml were considered protective, giving a seroprevalence of 92.5%. Sensitivities and negative predictive values for tetanus immunity were 30.6% and 10.7% based on patient histories and 2.8% and 7.9% based on physicians' records. In group A, after the single booster immunisation the median titre rose from 1.2 to 14.2 IU/ml, in group B from 0.9 to 5.3 IU/ml after the first, and to 9.6 IU/ml after the third tetanus toxoid dose (p <0.001 using Friedman's test). In Switzerland, elderly patients with a tetanus prone wound provide an unreliable vaccination history but their seroprotection against tetanus is high. A single booster for secondary immunisation is therefore sufficient. It is not necessary to take the largely unreliable vaccination history into account.

Frequent nasal administrations of recombinant cholera toxin B subunit (rCTB)-containing tetanus and diphtheria toxoid vaccines induced antigen-specific serum and mucosal immune responses in the presence of anti-rCTB antibodies

Vaccination via a mucosal route is a very attractive means for immunization, because both local and systemic immune responses are inducible and vaccines can be administered easily and safely from infants to elderly persons. For developing widely applicable mucosal vaccines using recombinant cholera toxin B subunit (rCTB) as a safe adjuvant, we examined whether frequent nasal administrations of rCTB-containing same and different vaccines could induce antigen-specific immune responses without induction of systemic tolerance and suppression by pre-existing anti-rCTB immunity. Ten repetitive nasal administrations to mice of tetanus toxoid (TT) + rCTB or diphtheria toxoid (DT) + rCTB raised and maintained high levels of antigen- and rCTB-specific serum IgG including high levels of tetanus/diphtheria antitoxin titres and raised nasal, salivary, lung, vaginal and fecal secreted IgA, suggesting that the regimen did not induce systemic tolerance to TT/DT and rCTB. Mice successively received repetitive five doses of TT as the first antigen and subsequent five doses of DT as the second antigen, and vice versa, raised serum IgG to the second antigen at various levels including low but sufficient protective levels of antitoxin titres and induced mucosal IgA in the lungs, the vaginas and feces, but hardly in the nasal secretions and salivas. After an interval of 22 weeks between the dosage of the first and second antigens, mice induced serum IgG to the second antigen at high levels and mucosal IgA in all sites. In conclusion, anti-TT and -DT serum and mucosal antibody responses induced by repeated intranasal immunization using rCTB adjuvant lasted for a long period, and for improving the effectivity of vaccination, different rCTB-containing vaccines should be administered at appropriate intervals.

Tetanus toxoid coverage as an indicator of serological protection against neonatal tetanus.

A Multiple-Indicator Cluster Survey (MICS) was conducted at mid-decade in more than 60 developing countries to measure progress towards the year 2000 World Summit for Children goals. These goals included the protection of at least 90% of children against neonatal tetanus through the immunization of their mothers, as measured by tetanus toxoid (TT) coverage. In the Central African Republic (CAR), serological testing was added to the MICS to understand better the relationship between survey estimates of TT coverage and the prevalence of serological protection. In the CAR MICS, mothers of children younger than one year of age gave verbal histories of the TT vaccinations they had received, using the MICS TT questionnaire. A subsample of mothers was tested for tetanus antitoxin, using a double-antigen enzyme-linked immunoadsorbent assay (ELISA). Seropositivity was defined as a titre of > or =0.01 IU/ml, and TT coverage was defined as the proportion of mothers protected at delivery, according to their history of TT vaccinations. Among the 222 mothers in the subsample, weighted TT coverage was 74.4% (95% Confidence Interval (CI); 67.0% - 81.7%) and tetanus antitoxin seroprevalence was 88.7% (95% CI; 83.2% - 94.2%). The weighted median antitoxin titre was 0.35 IU/ml. Tetanus toxoid coverage in the CAR was lower than the prevalence of serological protection against neonatal tetanus. If this relationship holds for other countries, TT coverage estimates from the MICS may underestimate the extent to which the year 2000 goal for protecting children against neonatal tetanus was reached. We also showed that a high level of serological protection had been achieved in a country facing major public health challenges and resource constraints.

Induction and distribution of intestinal immune responses after administration of recombinant cholera toxin B subunit in the ileal pouches of colectomized patients.

The induction and dissemination of mucosal immune responses to recombinant cholera toxin B subunit (rCTB) administered into the ileal pouches of patients, who had been colectomized because of ulcerative colitis, was analyzed. Biopsies from the duodenum and ileal pouch were collected, along with peripheral blood and ileostomy fluids. Two immunizations induced strong CTB-specific immunoglobulin A (IgA) antibody-secreting cell (ASC) responses in the duodenum in five of five patients, whereas weaker and less-frequent ASC responses were noted in the ileal pouch. Intestine-derived CTB-specific IgA ASCs were found in peripheral blood in three of the five patients. The vaccination also induced significant IgA antitoxin titer rises in ileostomy fluid in all of the patients. Increased production of gamma interferon in cell cultures from the ileal pouch was found in four of five patients after the vaccination. These results clearly indicate that rCTB administered into the distal ileum is capable of inducing B-cell responses in the "entire" small intestine and that homing of immunocompetent cells occurs preferentially to the duodenum.

Novel use of anaerobically induced promoter, dmsA, for controlled expression of fragment C of tetanus toxin in live attenuated Salmonella enterica serovar Typhi strain CVD 908- htrA

The anaerobically induced promoter dmsA (P dmsA ) was adapted to optimize in vivo expression of foreign antigens in attenuated Salmonella enterica serovar Typhi live vector vaccines CVD 908- htrA. P dmsA from Escherichia coli and two derivatives, P dmsA2 and P dmsA3 were cloned into a plasmid driving the expression of a gene encoding tetanus toxin fragment C. Expression of fragment C varied from a low level induced by pTET dmsA, to moderate and high levels induced, respectively, by pTET dmsA2 and pTET dmsA3. Mice were immunized intranasally with CVD 908- htrA harboring pTET dmsA2 or pTET dmsA3, and the serum antitoxin response was compared to that elicited by CVD 908- htrA(pTET nir15) (P nir15 is a benchmark anaerobically activated promoter). S. Typhi carrying pTET dmsA2 elicited modest tetanus antitoxin titers while S. Typhi harboring pTET dmsA3 generated elevated titers (GMT=55 384) that were higher than elicited by pTET nir15 (GMT=4354) ( P=0.007). Mice immunized with CVD 908- htrA carrying pTET dmsA3 and pTET nir15 survived tetanus toxin challenge. P dmsA derivatives are attractive promoters for in vivo expression of foreign genes in attenuated live vector vaccines.

Evidence of increased carriage of Corynebacterium spp. in healthy individuals with low antibody titres against diphtheria toxoid.

This study evaluated whether a correlation exists between carriage of corynebacteria and the lack of immunity to diphtheria toxoid. Samples of both nasal and pharyngeal secretions were taken from 500 apparently healthy subjects of both sexes and of all ages and inoculated onto Tinsdale's medium. A serum sample was also taken for ELISA test to determine the titre of diphtheria toxin antibodies. None of the subjects carried Corynebacterium diphtheriae. Ninety-three strains of Corynebacterium spp. were isolated from 93 subjects and 86 of these were classified to species or group level by biochemical tests. C. xerosis was the most common (25.8%) followed by C. pseudodiphthericum (16.1%), C. jeikeium and C. striatum (both 10.8%), and C. urealyticum (9.7%). Three other species accounted for approximately 20% of strains and seven were unclassified as biochemically atypical corynebacteria. Non-protective antibodies to diphtheria toxin were found in 80 of the 93 subjects and a strong statistical association was demonstrated between carriage of corynebacteria and non-protective levels of anti-toxin antibodies. The remaining 13 subjects had protective levels of antitoxin antibodies. In contrast, only 45 of the 407 non-colonized subjects had non-protective antitoxin titres. The prevalence of carriage increased with age among males as did the percentage of non-protected subjects. The prevalence of female carriers of corynebacteria was significantly lower. Serum samples from 12 subjects with different antibody titres to diphtheria toxoid reacted to varying degrees with whole-cell lysates of a number of species of corynebacteria. The results suggest that a causal relationship may exist between nasopharyngeal carriage of corynebacteria and a low anti-diphtheria toxin immune response.

Adequate immune response to tetanus toxoid and failure of vitamin A and E supplementation to enhance antibody response in healthy children

The effects of vitamin A and vitamin E supplementation on the IgG response to tetanus toxoid after primary immunization were evaluated in a prospective, randomized controlled clinical trial involving 89 healthy infants with normal serum vitamin A and E levels at 2 months of age. Before the first dose of DPT vaccine, the infants were randomly enrolled into four different study groups [Group I (n=24): 30,000 IU vitamin A for 3 days just after each three doses of primary vaccination, Group II (n=21): 150 mg oral vitamin E for only 1 day after the injections for primary immunization, Group III (n=21): vitamins A and E together in the same order, Group IV (n=23) no vitamin after DPT vaccines]. Serum tetanus antitoxin (IgG) titres were measured three times; initially at 2 months of age before the first dose of DPT, secondly at 5 months of age 1 month after primary immunization and thirdly at 16–18 months of age before the booster dose of DPT. Before the first dose of the DPT vaccine, 1 month after the third DPT injection and at 16–18 months before the booster dose of DPT, there was no significant difference in serum tetanus antitoxin levels between these four groups. A significant increase was observed in all the groups when serum tetanus antitoxin levels before (2 months) and after (5 months) primary immunization were compared. In addition, serum antibody levels against tetanus significantly decreased in the four groups before booster vaccination. Before the beginning of primary immunization, 15 infants (16.8%) had serum tetanus antitoxins (IgG) below protective level. After three doses of DPT, all the infants had protective antitoxin levels. At 16–18 months of age before booster dose, four infants (10%) also had serum tetanus antitoxins (IgG) below the protective level. No side-effects were observed except bulging fontanelle in two infants in Group I.

Induction of systemic and mucosal antibody responses in mice immunized intranasally with aluminium-non-adsorbed diphtheria toxoid together with recombinant cholera toxin B subunit as an adjuvant

Nasal mucosal immunization is very attractive for vaccination to prevent various bacterial and viral infectious diseases because of induction of systemic and mucosal immune responses. The aim of the present study was to investigate the possibility of changing the immunization procedure of diphtheria toxoid (D t) from intramuscular or subcutaneous injection to intranasal administration. Intranasal immunization with aluminium-non-adsorbed diphtheria toxoid (nD t) together with recombinant cholera toxin B subunit (rCTB, 10 μg) induced, at a concentration of 5 Lf, high levels of serum D t-specific IgG antibody responses and high or moderate levels of the specific IgA antibody responses in all mice and only a slight level of the specific IgE antibody responses in some mice. Furthermore, sufficiently high diphtheria antitoxin titres more than 0.1 international units (IU) ml −1 were obtained from mice which showed high levels of serum D t-specific IgG antibody responses. Under the same experimental conditions, induction of significant levels of mucosal D t-specific IgA antibody responses occurred in the nasal cavity, the lung, the saliva and vaginal secretions and the small and large intestines of all mice, although there were different titres between individual mice. Similar results were also obtained with rCTB-specific serum IgG and IgA and mucosal IgA antibody responses; serum rCTB-specific IgE antibody titres were not detected. These results show that intranasal administration of nD t with rCTB must be a very useful means for vaccination against diphtheria.

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.