ELISA for the routine determination of antitoxic immunity to tetanus

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

Modification of the ELISA for the estimation of tetanus antitoxin in human sera

The use of indirect ELISA for the quantitation of tetanus toxin neutralizing antibodies in human sera is limited by marked overestimations in low titered sera. The reasons for the discrepancy between the results obtained by ELISA and by in vivo assay and modifications of the ELISA to overcome the problem were investigated. Catching ELISA and indirect ELISA using trays coated with the contaminant proteins in toxoid preparations indicated that antibodies to contaminants were only partly responsible for the discrepancy and the introduction of these modifications did not solve the problem. In ELISA competition experiments with toxin neutralizing monoclonal antibodies, the human immunoglobulins irrelevant in toxin neutralization, but detectable in indirect ELISA, were found to be difficult to inhibit in their binding to the solid antigen phase. These might represent antitoxins bound bivalently to the solid phase but with affinities in monovalent binding insufficient for toxin neutralization or other coupled antibodies due to conformational changes of the antigen. A competition ELISA with toxin in solution was therefore developed to assess selectively the antitoxin capable of binding the antigen in solution and by this approach the in vivo activities of even low titered sera were accurately predicted. This antigen competition ELISA may be easily introduced into routine tetanus serology and the principle may also be of value for the in vitro detection of functional antibodies to other antigens.     

The use of the in vitro toxin binding inhibition (ToBI) test for the estimation of the potency of tetanus toxoid

The in vitro toxin binding inhibition (ToBI) test was used to determine antitoxin responses in mice immunized with tetanus toxoid. The ToBI test showed good correlation with the in vivo toxin neutralization (TN) test in titration of sera of mice immunized with various doses of DPT-Polio, DT-Polio and a tetanus reference preparation. Estimates of potency of tetanus toxoid obtained in mice by ToBI test correlated significantly with those obtained in mice by the lethal challenge test. In addition, potency values of the European reference preparation, succeedingly estimated by ToBI test and lethal challenge test in a single group of guinea-pigs, showed good correlation. From the study it is concluded that the ToBI test is a promising alternative to the toxic challenge procedure in the potency assay of tetanus toxoid vaccines. A substantial refinement and reduction in the use of animals can be achieved. Additional savings can be made by combining diphtheria and tetanus potency testing.     

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.     

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.     

Use of the toxin-binding inhibition test for estimation of tetanus antitoxin in serum

The usefulness of the toxin binding inhibition test (ToBI) for the titration of tetanus antibody in human sera was assessed. Sera from 80 healthy people with different vaccination histories that had been previously tested by the in vivo toxin neutralization (TN) test were retested by the ToBI test. The lowest tetanus antibody titre which could be detected by using 0.1 Lf/ml tetanus toxin was 0.01 IU/ml. Comparison between the estimates obtained by the ToBI test and those obtained by the TN test (r = 0.93 and r = 0.7 for titration low titre sera) showed good correlation. No overestimation of antibody content was seen in titrating low titre sera by the ToBI test. It is concluded that the ToBI-test is a reliable alternative to toxin neutralization test in mice.     

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.     

An alternative to the toxin neutralization assay in mice for the potency testing of the Clostridium tetani, Clostridium septicum, Clostridium novyi Type B and Clostridium perfringens Type D epsilon components of multivalent sheep vaccines

Potency testing of veterinary vaccines containing clostridial antigens currently requires the vaccination of laboratory rabbits followed by the determination of specific antitoxin concentration in the rabbit sera by toxin neutralization test in mice. ELISAs are described as an alternative method to toxin neutralization for the determination of Clostridium tetani, Clostridium septicum, Clostridium novyi Type B and Clostridium perfringens Type D epsilon antitoxins. The assays were found to be rapid, specific and economical and showed good correlation with the toxin neutralization test.     

The toxin binding inhibition test as a reliable in vitro alternative to the toxin neutralization test in mice for the estimation of tetanus antitoxin in human sera

A method for the screening of human sera for tetanus antibodies has been developed and evaluated. The toxin binding inhibition test (ToBI-test) is based on inhibition of the binding of tetanus toxin to an antitoxin-coated immunoassay microtitre plate by tetanus antibodies. Serum samples from 191 healthy adults with different vaccination histories have been titrated for tetanus antibodies by the toxin neutralization (TN) test in mice, by toxoid-ELISA and by the ToBI-test. In every respect, the ToBI-test proved to be the best in vitro alternative to the TN-test in mice. Comparisons showed a higher degree of correlation between the ToBI-test and the TN-test than between the toxoid-ELISA and the TN-test. Furthermore, no overestimation of antibody content was seen in titrating low titre sera by the ToBI-test. In contrast, several false positive results were seen when using the toxoid-ELISA. It is concluded that the ToBI-test is a reliable and precise alternative to the TN-test and can be performed under simple laboratory conditions in a short time.     

Significance of circulating toxin and antitoxin in unimmunized tetanus cases of neonates and infants

The level of circulating tetanus toxin, antitoxin and their individual influence on the outcome of tetanus cases were determined in unimmunized 125 neonatal and 39 infant cases of tetanus. PHA (passive haemagglutination) test showed 40% positive cases for toxin while its absence in the remaining cases indicated of either toxin fixation to the central nervous system (CNS) or it got neutralized by antitoxin. TN (toxin neutralization) and PHA test carried out in 46 sera samples revealed a strong positive correlation (r = 0.9) showing that 35/46 (76%) and 38/46 (82.6%) samples were positive for antitoxin, respectively. 25.4% of the neonate and infant cases and 34% of the control group had a protective serum tetanus antitoxin level. 42.5% of the paired sera from unimmunized mothers and their neonates showing nonprotective antitoxin levels suggested that a high level of antitoxin is needed for transplacental transfer, although transfer may not play a decisive role in the resistance against the disease. The presence of toxin or antitoxin in the clinical cases did not affect the outcome of the disease, although in neonates, presence of toxin was found to be a bad prognostic sign. This study explicitly advocates for the need to improve the vaccination coverage strategy.     

The titration of tetanus antitoxin III. A comparative evaluation of indirect haemagglutination and toxin neutralization titres of human sera

The tetanus antitoxin titres of 174 serum samples from healthy adults were determined by a standardization indirect haemagglutination test (IHA) and the conventional toxin neutralization (TN) test. The serum samples were titrated by the IHA test using glutaraldehyde-fixed and toxoid sensitized sheep erythrocytes before and after the treatment of the sera with 2-mercaptoethanol (2-ME). The IHA method has been found to be very sensitive and specific for the estimation of tetanus antitoxin in human sera. The IHA titres before the treatment of the sera with 2-ME were generally about four times higher than the TN titres and the correlation coefficient between these titres was 0.94. The IHA titres after the treatment of the sera with 2-ME were in good agreement with the TN titres and there was no statistically significant differences between the titres by the two methods. The tetanus antitoxin titres of 50% of the sera were below the minimum protective titres of tetanus antitoxin (0.01 IU/ml). In 19.5% of the sera the antitoxin level (IU/ml) ranged from 0.01 to 0.1, in 20.1% from 0.1 to 1.0 and in 10.4% from 1.0 to 10.0.     

The use of an immunoenzymatic assay for the estimation of tetanus antitoxin in human sera: a comparison with seroneutralization and indirect haemagglutination

The ability of an enzyme-linked immunosorbent assay (ELISA) to detect tetanus antitoxin in human sera has been evaluated in comparison with the in vivo seroneutralization test. The results of this study, carried out on 171 serum samples, show that ELISA is a sensitive and specific in vitro test for immunity to tetanus in man; it reveals the minimum protective level of 0.01 IU/ml and is well correlated with seroneutralization. A comparison has also been made with indirect haemagglutination. Differences in specificity in low titered sera, although not statistically significant, have been observed. Reported data suggest that the ELISA may be used for the estimation of tetanus antitoxin in sero-epidemiological surveys and for clinical purposes with reliability equal--and perhaps superior--to that of IHA.     

Studies on the antibody composition and neutralizing activity of tetanus antitoxin sera from various species of animals in relation to the antigenic substructure of the tetanus toxin molecule

On the basis of the antigenic substructure of tetanus neurotoxin, the antitoxin compositions of horse, rabbit and human tetanus antitoxin sera, in terms of their contents of antibodies against four antigenic determinant groups (alpha, beta-1, beta-2 and the "topographic" determinant group gamma) so far known for the toxin were studied by quantitative precipitation reactions using purified toxin, complementary fragments alpha, beta and fragment beta-1 (a subfragment of fragment beta) of the toxin. The antitoxin antibody composition varied slightly depending on the antiserum preparation. In addition, different patterns of antitoxin antibody composition and toxin-neutralizing ability, characteristic of horse, rabbit and man were found: horse antitoxin sera contained all four kinds of antibodies and horse anti-gamma showed low toxin-neutralizing ability, while human antisera lacked anti-alpha and had anti-gamma with high neutralizing activity but contained anti-beta-1 with no detectable neutralizing activity. Rabbit sera showed an intermediate pattern between those of horse and human sera. In all antisera, antibodies against determinants on the isolated fragment beta account for approximately 80-50 percent of the total precipitable antibodies and anti-beta-2 antibody was invariably present. Immunodiffusion analyses showed that the antitoxin compositions of mouse and guinea pig antisera resembled those of human antisera. In mice, fragment beta was almost as efficient as whole toxin toxoid in eliciting a protective immune response on an equal weight basis, whereas fragments beta-1 and alpha were both relatively poor antigens.     

Immunogenicity test of tetanus component in adsorbed vaccines by toxin binding inhibition test

Samples from 20 lots of diphtheria-tetanus (adult use dT) vaccine and from 20 lots of diphtheria-tetanus-pertussis (DTP) vaccine were used to standardize and validate the in vitro toxin binding inhibition (ToBI) test for the immunogenicity test of the tetanus component. The levels of tetanus antitoxin obtained by ToBI test were compared to those obtained using the toxin neutralization (TN) test in mice routinely employed to perform the quality control of the tetanus component in adsorbed vaccines. The results ranged from 1.8 to 3.5 IU/ml for dT and 2 to 4 IU/ml for DTP by ToBI test and 1.4 to 3 IU/ml for dT and 1.8 to 3.5 IU/ml for DTP by TN in mice. These results were significantly correlated. From this study, it is concluded that the ToBI test is an alternative to the in vivo neutralization procedure in the immunogenicity test of the tetanus component in adsorbed vaccines. A substantial refinement and a reduction in use of animals can be achieved.     

Titration of Cholera Antitoxin in Human Sera by Microhemagglutination with Formalinized Erythrocytes

Microtiter hemagglutination tests employing formalinized sheep erythrocytes sensitized with either crude or purified cholera toxin were used to assay the cholera antitoxin content of human sera. Comparable results were obtained with either crude or purified toxin-sensitized cells with the exception of two sera that gave unusually high hemagglutination titers with the crude toxin. Sera from 13 convalescent cholera patients showed a high degree of correlation between antitoxin levels as determined in vitro by the hemagglutination test and in vivo by the skin permeability factor neutralization test. Fourfold or greater rises in antitoxin levels between acute and convalescent sera were detected in 9 of 15 patients with bacteriologically proven cholera. No significant increases in titer were observed in 14 cases of noncholera diarrhea. Cholera antitoxin was detected by hemagglutination in only 1 of 33 sera, obtained from eight countries, containing vibriocidal antibodies. Formalinized sheep erythrocytes sensitized with toxin and stored at 4 C in the presence of 1:10,000 thimerosal were stable and sensitive for at least 6 months (the longest time tested).     

Comparative evaluation of the methods of determining perfringens A antitoxin

The methods for determining the level of type A Perfringens antitoxin in human blood sera were examined and compared. The ratios for correlating the data obtained in the toxin neutralization test (NT) in vivo, in the passive hemagglutination test (PHT), and as a result of the enzyme-labeled immunosorbent assay (ELISA) with regard to the antitoxin level measured in the NT in vitro were equal to 0.88, 0.64 and 0.39, respectively. The sensitivity of the NT in vivo and in vitro was 0.25 IU/ml, that of the PHT 0.01-0.005 IU/ml, and that of the ELISA 0.01-0.02 IU/ml Perfringens antitoxin. To perform the NT, not less than 1 ml blood serum is required, while for the PHT and ELISA, 0.1-0.05 ml. Provided hyghly purified anatoxin is used for preparing the erythrocyte diagnosticum Perfringens, and polysterene plates are sensitized in performing the ELISA, all the reactions are specific. While titrating human blood sera containing type A Perfringens antitoxin, use in the PHT may be made of type A Perfringens rabbit antiserum as reference.     

Titration of tetanus antitoxin by passive hemagglutination. I. Titration of guinea-pig antitoxins at various periods of immunization.

An improved technique for passive hemagglutination (HA) for titration of tetanus antitoxin was described. The use of highly purified tetanus toxoid and of improved diluent increased the specificity and reproducibility of the test. Several hundreds of specimens of guinea-pig serum taken at various stages of immunization were titrated by HA and toxin neutralization (NT) in mice. The ratio of HA to NT titers varied significantly depending on the immunization stage; higher at early stages and lower at later stages. The high HA/NT ratio was not due to the IgM antitoxin, which is very rare in guinea pigs. The variation in discrepancy between HA and NT titers decreased considerably by grouping the serum specimens with respect to the stage of immunization. Thus, it is possible to predict the in vivo titer of a tetanus antitoxin accurately enough for clinical study. The HA test may be useful as an alternative method for titrating tetanus antitoxin in the field trials. Moreover, it can be used for the study of characteristics of antitoxins.     

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.     

Evaluation of a guinea pig model to assess interference in the immunogenicity of different components of a combination vaccine comprising diphtheria, tetanus and acellular pertussis (DTaP) vaccine and haemophilus influenzae type b capsular polysaccharide conjugate vaccine.

A guinea pig model to assess the immunogenicity of a combination vaccine containing diphtheria, tetanus and acellular pertussis (DTaP) vaccine and Haemophilus influenzae type b (Hib) capsular polysaccharide conjugated to tetanus toxoid (HibT) was evaluated comparatively with the mouse immunogenicity test to study the effect of combining these antigens on the immunogenicity of various components. The immunogenicity test in mice was performed by subcutaneous injection of groups of 10 animals twice at an interval of four weeks with 1/10 of a single human dose of various formulations of combination vaccines, DTaP or HibT vaccine. The animals were bled at 4 and 6 weeks and IgG or total antibodies to various components were determined by ELISA or RIA. The guinea pig immunogenicity model included groups of animals injected subcutaneously twice at an interval of six weeks with 1.5 times the single human dose of various formulations. The animals were bled at 4, 6 and 8 weeks and serum samples were tested for antibodies to various components by ELISA, RIA and/or neutralization tests. Additionally, potency of tetanus and diphtheria components was assessed as per the US Food and Drug Administration's regulations. Aluminium phosphate (AIPO(4)) adsorbed HibT vaccine or HibT as a combination with AIPO(4)adsorbed DTaP vaccine showed significant increases in IgG antibodies to tetanus toxin in mice as well increased tetanus antitoxin levels in guinea pigs as compared to soluble HibT vaccine. In general, combining DTaP and HibT vaccines did not affect the antibody levels to tetanus and diphtheria toxoids whereas DTaP-HibT combination vaccine elicited significantly lower IgG antibodies to pertussis toxin and filamentous haemagglutinin than DTaP vaccine alone, particularly after first injection. Mice showed similar Hib antibody responses for the combination and HibT alone whereas guinea pigs consistently showed lower anamnestic responses to Hib for combination formulations than for HibT alone. Reducing the amount of HibT and/or tetanus toxoid in the combination formulations reduced this suppression of Hib antibody response in guinea pigs. Suppression of Hib antibody response in combination vaccines has also been reported from recent clinical trials. Based on the results from this study, it appears that the guinea pig model may be able to predict the human response to various components of combination vaccines.     

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.