Toxin-neutralizing Titers Research Articles

Hyperimmune Targeting Staphylococcal Toxins Effectively Protect Against USA 300 MRSA Infection in Mouse Bacteremia and Pneumonia Models.

Staphylococcus aureus has been acquiring multiple drug resistance and has evolved into superbugs such as Methicillin/Vancomycin-resistant S. aureus (MRSA/VRSA) and, consequently, is a major cause of nosocomial and community infections associated with high morbidity and mortality for which no FDA-approved vaccines or biotherapeutics are available. Previous efforts targeting the surface-associated antigens have failed in clinical testing. Here, we generated hyperimmune products from sera in rabbits against six major S. aureus toxins targeted by an experimental vaccine (IBT-V02) and demonstrated significant efficacy for an anti-virulence passive immunization strategy. Extensive in vitro binding and neutralizing titers were analyzed against six extracellular toxins from individual animal sera. All IBT-V02 immunized animals elicited the maximum immune response upon the first boost dose against all pore-forming vaccine components, while for superantigen (SAgs) components of the vaccine, second and third doses of a boost were needed to reach a plateau in binding and toxin neutralizing titers. Importantly, both anti-staphylococcus hyperimmune products consisting of full-length IgG (IBT-V02-IgG) purified from the pooled sera and de-speciated F(ab’)2 (IBT-V02-F(ab’)2) retained the binding and neutralizing titers against IBT-V02 target toxins. F(ab’)2 also exhibited cross-neutralization titers against three leukotoxins (HlgAB, HlgCB, and LukED) and four SAgs (SEC1, SED, SEK, and SEQ) which were not part of IBT-V02. F(ab’)2 also neutralized toxins in bacterial culture supernatant from major clinical strains of S. aureus. In vivo efficacy data generated in bacteremia and pneumonia models using USA300 S. aureus strain demonstrated dose-dependent protection by F(ab’)2. These efficacy data confirmed the staphylococcal toxins as viable targets and support the further development effort of hyperimmune products as a potential adjunctive therapy for emergency uses against life-threatening S. aureus infections.

Development of Protective Immunity in New Zealand White Rabbits Challenged with Bacillus anthracis Spores and Treated with Antibiotics and Obiltoxaximab, a Monoclonal Antibody against Protective Antigen.

ABSTRACTThe recommended management of inhalational anthrax, a high-priority bioterrorist threat, includes antibiotics and antitoxins. Obiltoxaximab, a chimeric monoclonal antibody against anthrax protective antigen (PA), is licensed under the U.S. Food and Drug Administration's (FDA's) Animal Rule for the treatment of inhalational anthrax. Because of spore latency, disease reemergence after treatment cessation is a concern, and there is a need to understand the development of endogenous protective immune responses following antitoxin-containing anthrax treatment regimens. Here, acquired protective immunity was examined in New Zealand White (NZW) rabbits challenged with a targeted lethal dose of Bacillus anthracis spores and treated with antibiotics, obiltoxaximab, or a combination of both. Survivors of the primary challenge were rechallenged 9 months later and monitored for survival. Survival rates after primary and rechallenge for controls and animals treated with obiltoxaximab, levofloxacin, or a combination of both were 0, 65, 100, and 95%, and 0, 100, 95, and 89%, respectively. All surviving immune animals had circulating antibodies to PA and serum toxin-neutralizing titers prior to rechallenge. Following rechallenge, systemic bacteremia and toxemia were not detected in most animals, and the levels of circulating anti-PA IgG titers increased starting at 5 days postrechallenge. We conclude that treatment with obiltoxaximab, alone or combined with antibiotics, significantly improves the survival of rabbits that received a lethal inhalation B. anthracis spore challenge dose and does not interfere with the development of immunity. Survivors of primary challenge are protected against reexposure, have rare incidents of systemic bacteremia and toxemia, and have evidence of an anamnestic response.

Systemic antibody responses induced by a two-component Clostridium difficile toxoid vaccine protect against C. difficile-associated disease in hamsters

Clostridium difficile infection (CDI) has been identified as the leading cause of nosocomial diarrhoea and pseudomembranous colitis associated with antibiotic therapy. Recent epidemiological changes as well as increases in the number of outbreaks of strains associated with increased virulence and higher mortality rates underscore the importance of identifying alternatives to antibiotics to manage this important disease. Animal studies have clearly demonstrated the roles that toxins A and B play in gut inflammation as well as diarrhoea; therefore it is not surprising that serum anti-toxin A and B IgG are associated with protection against recurrent CDI. In humans, strong humoral toxin-specific immune responses elicited by natural C. difficile infection is associated with recovery and lack of disease recurrence, whereas insufficient humoral responses are associated with recurrent CDI. The first generation of C. difficile vaccine that contained inactivated toxin A and B was found to be completely protective against death and diarrhoea in the hamster C. difficile challenge model. When tested in young healthy volunteers in Phase I clinical trials, this investigational vaccine was shown to be safe and immunogenic. Moreover, in a separate study this vaccine was able to prevent further relapses in three out of three patients who had previously suffered from chronic relapsing C. difficile-associated diarrhoea. Herein we examined the immunogenicity and protective activity of a next-generation Sanofi Pasteur two-component highly purified toxoid vaccine in a C. difficile hamster model. This model is widely recognized as a stringent and relevant choice for the evaluation of novel treatment strategies against C. difficile and was used in preclinical testing of the first-generation vaccine candidate. Intramuscular (i.m.) immunizations with increasing doses of this adjuvanted toxoid vaccine protected hamsters from mortality and disease symptoms in a dose-dependent manner. ELISA measurements of pre-challenge sera showed that the median anti-toxin A and anti-toxin B IgG titres in the group of surviving animals were significantly higher than the median values in the group of animals that did not survive challenge. Assessment of the neutralizing activity of these sera revealed a statistically significant difference between the levels of both toxin A and toxin B neutralizing titres in protected versus unprotected animals as the median anti-toxin A and anti-toxin B neutralizing titres from surviving animals were higher than the median values from animals that succumbed to challenge. Statistically significant correlations between the toxin-specific binding titres and toxin neutralizing titres were seen for both toxin A and toxin B responses. The role of circulating anti-toxin antibodies in immunity against disease was evaluated by passive transfer of immune sera against C. difficile toxoids to naïve hamsters. Passively immunized animals were protected against morbidity and mortality associated with C. difficile challenge. Taken together, these results indicate the ability of i.m. immunization with inactivated toxins A and B to induce robust dose-dependent anti-toxin A and anti-toxin B IgG responses, the principal role of circulating anti-toxin antibody in immunity against disease and that antibody toxin binding and neutralization titres can serve as correlates of protection in the hamster challenge model of C. difficile.

Serological Correlate of Protection in Guinea Pigs for a Recombinant Protective Antigen Anthrax Vaccine Produced from Bacillus brevis

ObjectiveRecombinant protective antigen (rPA) is the active pharmaceutical ingredient of a second generation anthrax vaccine undergoing clinical trials both in Korea and the USA. By using the rPA produced from Bacillus brevis pNU212 expression system, correlations of serological immune response to anthrax protection efficacy were analyzed in a guinea pig model. MethodsSerological responses of rPA anthrax vaccine were investigated in guinea pigs that were given single or two injections (interval of 4 weeks) of various amounts of rPA combined with aluminum hydroxide adjuvant. Guinea pigs were subsequently challenged by the intramuscular injection with 30 half-lethal doses (30LD50) of virulent Bacillus anthracis spores. Serum antibody titers were determined by anti-PA IgG ELISA and the ability of antibodies to neutralize the cytotoxicity of lethal toxin on J774A.1 cell was measured through the toxin neutralizing antibody (TNA) assay. ResultsTo examine correlations between survival rate and antibody titers, correlation between neutralizing antibody titers and the extent of protection was determined. Toxin neutralization titers of at least 1176 were sufficient to confer protection against a dose of 30LD50 of virulent anthrax spores of the H9401 strain. Such consistency in the correlation was not observed from those antibody titers determined by ELISA. ConclusionNeutralizing-antibody titers can be used as a surrogate marker.

Development and evaluation of an ovine antibody-based platform for treatment of Clostridium difficile infection.

Treatment of Clostridium difficile is a major problem as a hospital-associated infection which can cause severe, recurrent diarrhea. The currently available antibiotics are not effective in all cases and alternative treatments are required. In the present study, an ovine antibody-based platform for passive immunotherapy of C. difficile infection is described. Antibodies with high toxin-neutralizing titers were generated against C. difficile toxins A and B and were shown to neutralize three sequence variants of these toxins (toxinotypes) which are prevalent in human C. difficile infection. Passive immunization of hamsters with a mixture of toxin A and B antibodies protected them from a challenge with C. difficile spores in a dose-dependent manner. Antibodies to both toxins A and B were required for protection. The administration of toxin A and B antibodies up to 24 h postchallenge was found to reduce significantly the onset of C. difficile infection compared to nonimmunized controls. Protection from infection was also demonstrated with key disease isolates (ribotypes 027 and 078), which are members of the hypervirulent C. difficile clade. The ribotype 027 and 078 strains also have the capacity to produce an active binary toxin and these data suggest that neutralization of this toxin is unnecessary for the management of infection induced by these strains. In summary, the data suggest that ovine toxin A and B antibodies may be effective in the treatment of C. difficile infection; their potential use for the management of severe, fulminant cases is discussed.

Effective induction of protective systemic immunity with nasally administered vaccines adjuvanted with IL-1

IL-1α and IL-1β were evaluated for their ability to provide adjuvant activity for the induction of serum antibody responses when nasally administered with protein antigens in mice and rabbits. In mice, intranasal (i.n.) immunization with pneumococcal surface protein A (PspA) or tetanus toxoid (TT) combined with IL-1β induced protective immunity that was equivalent to that induced by parenteral immunization. Nasal immunization of awake (i.e., not anesthetized) rabbits with IL-1-adjuvanted vaccines induced highly variable serum antibody responses and was not as effective as parenteral immunization for the induction of antigen-specific serum IgG. However, i.n. immunization of deeply anesthetized rabbits with rPA + IL-1α consistently induced rPA-specific serum IgG ELISA titers that were not significantly different than those induced by intramuscular (IM) immunization with rPA + alum although lethal toxin-neutralizing titers induced by nasal immunization were lower than those induced by IM immunization. Gamma scintigraphy demonstrated that the enhanced immunogenicity of nasal immunization in anesthetized rabbits correlated with an increased nasal retention of i.n. delivered non-permeable radio-labeled colloidal particles. Our results demonstrate that, in mice, IL-1 is an effective adjuvant for nasally administered vaccines for the induction of protective systemic immunity and that in non-rodent species, effective induction of systemic immunity with nasally administered vaccines may require formulations that ensure adequate retention of the vaccine within the nasal cavity.

A Heterologous Helper T-Cell Epitope Enhances the Immunogenicity of a Multiple-Antigenic-Peptide Vaccine Targeting the Cryptic Loop-Neutralizing Determinant ofBacillus anthracisProtective Antigen

We previously showed that a multiple antigenic peptide (MAP) displaying amino acids (aa) 305 to 319 from the 2beta2-2beta3 loop of protective antigen (PA) can elicit high-titered antibody that neutralizes lethal toxin (LeTx) in vitro and that this loop-neutralizing determinant (LND) specificity is absent in PA-immune rabbits. Some immune rabbits were, however, nonresponders to the MAP. We hypothesized that the immunogen elicited suboptimal major histocompatibility complex (MHC) class II-restricted T-cell help and that introduction of a functional helper T-cell epitope would increase MHC-restricted responsiveness and the magnitude and affinity of the antibody responses. In the current study, we characterized the T- and B-cell responses to LND peptides in mice, then designed second-generation MAP immunogens for eliciting LND-specific immunity, and tested them in rabbits. The 305-319 sequence was devoid of helper T-cell epitopes in three strains of mice; however, a T-B peptide comprising aa 305 to 319, colinearly synthesized with the P30 helper epitope of tetanus toxin, elicited robust LeTx-neutralizing immunity in mice. T-B MAPs displaying B-cell epitopes 304 to 319 (MAP304) or 305 to 319 (MAP305) elicited high-titer, durable antibody responses in rabbits which exhibited potent neutralization of LeTx in vitro. All MAP304-immune rabbits demonstrated neutralization titers exceeding that of hyperimmune sera of rabbits immunized with PA in Freund's adjuvant, with peak neutralization titers 23-, 6-, and 3-fold higher than that of the PA antiserum. Overall, immunization with MAPs containing the P30 epitope elicited higher antibody and toxin neutralization titers and peptide-specific affinity than immunization with an LND MAP lacking a helper epitope. P30-containing MAP304 represents a promising LND-specific vaccine for anthrax.

Detoxified Lethal Toxin as a Potential Mucosal Vaccine against Anthrax

The nontoxic mutant lethal factor (mLF; which has the E687C substitution) and functional protective antigen (PA63) of Bacillus anthracis were evaluated for their use as mucosal vaccines against anthrax in A/J mice. Intranasal vaccination of three doses of 30 microg of mLF or 60 microg of PA63 elicited significant serum and mucosal antibody responses, with anthrax lethal toxin-neutralizing titers of 40 and 60 in immune sera, respectively. However, only 30% and 60% of the vaccinated animals in the two groups could survive a challenge with 100 times the 50% lethal dose of B. anthracis Sterne spores, respectively. In contrast, vaccination with three doses of the combination of 30 microg of mLF and 60 microg of PA63, the detoxified lethal toxin, elicited antibody responses against LF and PA significantly higher than those elicited after vaccination with mLF or PA63 individually by use of the same dose and schedule. Vaccination with the detoxified lethal toxin resulted in significantly higher lethal toxin-neutralizing antibody titers in sera (titer, 90). Animals vaccinated with three doses of the detoxified lethal toxin were completely protected against the spore challenge. The data suggest that mLF and PA63 have a mutual enhancement effect for evoking systemic and mucosal immune responses and that the detoxified lethal toxin can be used as an efficient mucosal vaccine against anthrax.

Protection against Shiga Toxin-ProducingEscherichia coliInfection by Transcutaneous Immunization with Shiga Toxin Subunit B

Enterohemorrhagic Escherichia coli (EHEC) strains are important human food-borne pathogens. EHEC strains elaborate potent Shiga toxins (Stx1, and/or Stx2) implicated in the development of hemorrhagic colitis (HC) or hemolytic-uremic syndrome (HUS). In this report, we evaluated the immunogenicity and protective efficacy of Stx1 subunit B (StxB1) administered by transcutaneous immunization (TCI). Three groups of Dutch Belted rabbits received patches containing StxB1, StxB1 in combination with Escherichia coli heat-labile enterotoxin (LT), or LT alone. An additional group of naïve rabbits served as controls. The protective efficacy following TCI with StxB1 was assessed by challenging rabbits with a virulent Stx1-producing strain, RDEC-H19A, capable of inducing HC and HUS in rabbits. Antibodies specific to StxB1 from serum and bile samples were determined by enzyme-linked immunosorbent assay and toxin neutralization test. Rabbits immunized with StxB1 demonstrated improved weight gain and reduced Stx-induced histopathology. Rabbits receiving StxB or StxB1/LT showed a significant increase in serum immunoglobulin G titers specific to StxB1 as well as toxin neutralization titers. These data demonstrated that the StxB delivered by TCI could induce significant systemic immune responses. Thus, Stx subunit B vaccine delivered by a patch for a high-risk population may be a practical approach to prevent (and/or reduce) Stx-induced pathology.

Adenovirus-based Prime-boost Immunization for Rapid Vaccination Against Anthrax

Prime-boost vaccination using plasmid DNA and replication-defective adenovirus vectors has emerged as a highly effective strategy for vaccinating against viral pathogens. However, its ability to provide protection against bacterial disease has never been assessed. Here we evaluate prime-boost vaccination approaches for immunizing against anthrax. We show that mice primed with DNA and boosted with an adenovirus vector, both expressing domain four of Bacillus anthracis protective antigen (PA), have higher antibody and toxin-neutralizing titers than mice immunized with either single modality alone. DNA-primed/adenovirus-boosted mice also had significantly higher antibody and toxin-neutralizing titers than mice immunized with Anthrax Vaccine Adsorbed. High levels of antigen-specific interferon-gamma-secreting cells were present in vaccinated mice indicating that a cell-mediated immune response had also been stimulated. Both DNA-primed/adenovirus-boosted and adenovirus-primed/adenovirus-boosted mice were fully protected from Sterne strain spore challenge. We also show that a single injection with an adenovirus vector-expressing domain four of PA can provide partial protection from spore challenge 2 weeks after immunization and full protection 3 weeks after immunization. These results demonstrate that adenovirus-based prime-boost vaccination can provide rapid protection from anthrax and that this approach may be an effective strategy for immunizing against bacterial as well as viral pathogens.

Multicomponent anthrax toxin display and delivery using bacteriophage T4

We describe a multicomponent antigen display and delivery system using bacteriophage T4. Two dispensable outer capsid proteins, Hoc (highly antigenic outer capsid protein, 155 copies) and Soc (small outer capsid protein, 810 copies), decorate phage T4 capsid. These proteins bind to the symmetrically localized capsid sites, which appear following prohead assembly and expansion. We hypothesized that multiple antigens fused to Hoc can be displayed on the same capsid and such particles can elicit broad immunological responses. Anthrax toxin proteins, protective antigen (PA), lethal factor (LF), and edema factor (EF), and their functional domains, were fused to Hoc with an N-terminal hexa-histidine tag and the recombinant proteins were over-expressed in E. coli and purified. Using a defined in vitro assembly system, the anthrax-Hoc fusion proteins were efficiently displayed on T4 capsid, either individually or in combinations. All of the 155 Hoc binding sites can be occupied by one antigen, or they can be split among two or more antigens by varying their molar ratio in the binding reaction. Immunization of mice with T4 phage carrying PA, LF, and EF elicited strong antigen-specific antibodies against all antigens as well as lethal toxin neutralization titers. The triple antigen T4 phage elicited stronger PA-specific immune responses than the phage displaying PA alone. These features offer novel avenues to develop customized multicomponent vaccines against anthrax and other pathogenic diseases.

Comparative Analysis of Tetanus Antitoxin Titers of Sera from Immunized Mice and Guinea Pigs Determined by Toxin Neutralization Test and Enzyme-linked Immunosorbent Assay

Abstract. Enzyme-linked immunosorbent assay (ELISA) and toxin neutralization (TN) test were comparatively evaluated for determination of antibodies to tetanus toxin in sera of immunized guinea pigs and mice at different intervals. Tetanus specific IgG antibodies by ELISA were found in sera of guinea pigs at ninth day after inoculation and in mice sera at seventh day after inoculation when there were no TN antibodies. Two weeks after injection of mice with aluminium phosphate adsorbed tetanus toxoid, anti-tetanus toxin IgG levels determined by ELISA were 35 to 40 times higher than the TN titers. At 4 weeks, ELISA IgG antibody levels of mice and guinea pig sera were still statistically significantly higher than TN titers but with a good correlation between ELISA and TN titers (correlation coefficient of mice sera 0·89). Mice sera at 2 weeks after two doses of tetanus toxoid (interval between doses 30 days) and 8 weeks after a single injection showed similar ELISA and TN titers (P > 0·1). Guinea pig sera at 6 weeks after a single injection showed similar TN and ELISA titers (P > 0·4). During the course of immunization in mice and guinea pigs, low avidity and low affinity IgG antibodies start appearing early which are detected by ELISA but not by TN test. Therefore ELISA results on sera before 6 weeks of immunization determined against a hyper-immune reference serum do not give a true picture of TN levels.

Comparative study of a whole-cell pertussis vaccine and a recombinant acellular pertussis vaccine

The safety and immunogenicity of an acellular pertussis vaccine containing the genetically detoxified pertussis toxin PT-9K/129G, filamentous hemagglutinin, and pertactin, together with diphtheria and tetanus toxoids, were compared with those of a whole-cell pertussis component-diphtheria-tetanus vaccine. Four hundred eighty infants were enrolled into this prospective, multicenter, double-blind study. Each infant was randomly given three doses of one of the two vaccines at 2, 4, and 6 months of age. Both local and systemic adverse reactions, reported within 48 hours and 7 days of each injection, were less frequent after the acellular vaccine than after the whole-cell vaccine. The enzyme-linked immunosorbent assay titers to pertussis toxin, filamentous hemagglutinin, and pertactin, as well as the pertussis toxin-neutralizing titer measured by the Chinese hamster ovary cell assay, were significantly higher after the acellular vaccine was given. Both vaccines induced adequate levels of anti-diphtheria and anti-tetanus antibodies. We conclude that the recombinant acellular pertussis vaccine produces fewer reactions than the whole-cell vaccine and provides a high antibody response against the antigens of Bordetella pertussis involved in bacterial adhesion and systemic toxic effects.

Neutralizing antibodies in 6-year-old children after five doses of tetanus toxoid

It has recently been suggested that multiple boosters of tetanus toxoid may enhance serum antitoxin titres but may not necessarily lead to an effective immune response. Tetanus antitoxin titres by haemagglutination inhibition and mouse toxin neutralization tests were determined in sera of 64 children, 5 and 6 years old. Primary vaccination against tetanus was given as four doses of diphtheria pertussis-tetanus (DPT) vaccine beginning in the second or third month of life, and a booster dose given to schoolchildren at 6 years of age. In our area more than 90% of children receive five doses of tetanus toxoid before their seventh birthday. The children were given 0.5 ml of DPT or DT containing 10 Lf ml −1 tetanus toxoid at each injection. The haemagglutination titres and the toxin neutralization titres were much higher in 6-year-old than in 5-year-old children. We concluded that the fifth dose is an effective booster in 6-year-old children.

Acellular pertussis vaccine composed of genetically inactivated pertussis toxin: Safety and immunogenicity in 12- to 24- and 2- to 4-month-old children

To determine whether a nontoxic derivative of pertussis toxin obtained by recombinant DNA technology, PT-9K/129G, is a good candidate for a new pertussis vaccine, we examined the safety and the immunogenicity in children of a vaccine containing 15 micrograms of PT-9K/129G protein and 0.5 mg of aluminum hydroxide per dose. Fifty-three children 12 to 24 months of age and 21 infants aged 2 to 4 months were injected with two and three doses, respectively. The vaccine did not induce significant local or systemic reactions and elicited an increase of antibody titer in more than 98% of the children. The geometric mean of the toxin-neutralizing titers increased after each dose and was 85 units in children given two doses and 196 units in those given three doses. Two children who had detectable antibody levels before the first immunization had a high response (greater than 320 units) to the first vaccine dose. The findings suggest that PT-9K/129G is a promising antigen to be included in the development of acellular pertussis vaccines.

Immunization of pigs with a purified Shiga-like toxin II variant toxoid

Passive transfer of neutralizing antibodies and active immunization with a toxoid of purified Shiga-like toxin II variant (SLT-IIv, edema disease toxin) were used to protect pigs against challenge with SLT-IIv. Six 6-week-old pigs were passively immunized by intraperitoneal administration of an immunoglobulin preparation from porcine antiserum against purified SLT-IIv. Six 6-week-old pigs and twelve 2-week-old pigs were actively immunized by two intramuscular injections of 25 μg of SLT-IIv toxoid given 2 weeks apart. The 24 immunized pigs and an equal number of age-matched unimmunized control pigs were all challenged by intravenous injection of purified SLT-IIv (6 ng/kg body weight). The six passively immunized pigs acquired neutralizing SLT-IIv antibody titers of 1280 or 2560 and the 18 actively immunized pigs mounted a serum immune response with toxin neutralizing titers ranging from 128 to 10 240. All 24 immunized pigs survived the challenge but nine pigs with the lowest titers showed mild clinical signs of edema disease. All 24 control pigs died within 30 hours of challenge.

Production of a Shiga-like cytotoxin by Campylobacter

Cell lysates and culture supernatants of 36 Campylobacter isolates from patients with enteritis were tested for cytotoxic activity on HeLa cells. Cytotoxic activity was considered Shiga-like if neutralized by monoclonal antibody to the B subunit of Shiga-like toxin I of Escherichia coli and rabbit anti-Shiga toxin. Fifteen of the Campylobacter isolates produced no detectable cytotoxin, 10 produced a non-neutralizable cytotoxin, and 11 produced low levels of a cell-associated SLT. However, under low stringency conditions no hybridization was observed between a DNA fragment containing cloned SLT-I genes and restriction enzymedigested total DNA from a Campylobacter strain that produced low levels of a Shiga-like toxin I. The Shiga-like toxin neutralizing titers in sera from 15 patients with C. jejuni infections, 5 patients infected with S. sonnei, and 20 healthy persons were then determined. No rise in neutralizing titer between acute and convalescent sera of patients with C. jejuni infection or S. sonnei infection was observed, although 27% of C. jejuni-infected patients, 40% of S. sonnei-infected patients, and 30% of the healthy controls had neutralizing activity in their sera. These data indicate that low levels of Shiga-like toxin are produced by some Campylobacter isolates but that SLT is genetically distinct from the SLT-I toxin produced at high levels by certain E. coli. The findings also suggest that exposure to SLTs is common in the adult population but not as a consequence of infection with C. jejuni or S. sonnei.

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.

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 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.