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 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.     

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.     

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.     

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.     

The titration of tetanus antitoxin V. Effect of formalization method for the fixation of sheep erythrocytes on the indirect haemagglutination test

Two methods of fixation of sheep erythrocytes with formaldehyde for the titration of tetanus antitoxin by the indirect haemagglutination (IHA) test have been compared. The cells fixed with 3% formaldehyde at 4-8 degrees C for 24 h (formaldehyde (I) fixed cells) were less sensitive than the cells fixed with 3% formaldehyde at 4-8 degrees C for 24 h and subsequently treated with 40% formaldehyde at 4-8 degrees C for a further 24 h (formaldehyde (II) fixed cells). The correlation between the toxin neutralization (TN) and IHA titres using formaldehyde (I) fixed cells was better than that obtained with formaldehyde (II) fixed cells. There was no statistically significant difference between TN and IHA titres after treatment of the sera with 2-Mercaptoethanol using formaldehyde (I) fixed cells. Formaldehyde (I) fixed cells can be used for two months with adequate sensitivity to detect the minimum protective level of tetanus antitoxin in the sera.     

Vero细胞法检测白喉抗毒素的研究

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

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.     

Potency control of diphtheria component in adsorbed vaccines by in vitro neutralization tests.

Samples from 20 lots of dT vaccine and from 20 lots of DTP vaccine were used to standardize and validate the Vero cell and the toxin binding inhibition (ToBI) tests for the potency control of diphtheria component. For the Vero cell method, violet crystal solution was used to stain the cells and estimate the endpoint of diluted diphtheria antitoxin. Diphtheria anatoxin was used for performing the ToBI test instead of toxin. The results obtained by both in vitro tests were similar to those obtained by in vivo toxin neutralization test in guinea pigs. The various analysis and the chi(2) test applied to evaluate the reproducibility and homogeneity, respectively, among in vitro tests and in vivo toxin neutralization test did not detect statistical significant difference for both analysed vaccines. An excellent correlation among in vitro tests and in vivo neutralization test was observed by Spearman's correlation coefficient.     

Microtissue Culture Test for the Titration of Low Concentrations of Diphtheria Antitoxin in Minimal Amounts of Human Sera

A microtissue culture method for the assay of low concentrations of diphtheria antitoxin in human sera has been developed, using a monkey kidney cell (VERO) culture technique. Results obtained with sera from nonvaccinated children and with immune sera from children vaccinated with three and four injections of diphtheria pertussis tetanus vaccine were in agreement with antitoxin levels considered necessary to denote immunity to diphtheria. The use of microplates and organic buffer for culturing the animal cells improved the stability of the tissue culture system. The described method is sensitive, economical, and applicable for the titration of antitoxin in human sera particularly from infants and children from whom a minimum amount of serum is available.     

An investigation of a mouse model to estimate the potency of the diphtheria component in vaccines

A mouse model to estimate the potency of the diphtheria toxoid components in vaccines using Vero cells to detect the neutralizing antibodies in the sera from immunized mice is described. The results obtained with this mouse model correlated significantly with those obtained in the lethal challenge test in guinea-pigs. For this reason it is suggested that the potency test in guinea-pigs be replaced by this mouse model because a considerable reduction in the number of animals used and costs can be achieved by the introduction of this mouse test for the routine control of the potency of the diphtheria component of vaccines.     

Transplacental antibodies. Part II: Maternal antibodies against the toxins of C. diphtheriae and C. tetani

Examinations of 297 sera for diphtheria antitoxin and 160 sera for tetanus antitoxin were carried out in 1981. All sera were obtained from the cord blood of mothers between 15 and 34 years of age. The mothers were divided into four age groups each of which was further subdivided into the primipara and multipara subgroups. The aim was to assess the age-specific variations in response to active immunization against diphtheria and tetanus. The protective level of diphtheria antitoxin (at least 0.01 I.U./ml) was recorded in the serum of 96.3% of examinees and the rates of seropositivity were found to fall with increasing age. The protective level of tetanus antitoxin (at least 0.1 I.U./ml) was found in the serum of 95.2% of mothers. The serologic response encountered in groups of older mothers was a clear-cut demonstration that the country-wide mass immunization against tetanus carried out between 1974 and 1975 was highly effective and fully justified. The variations in the diphtheria and tetanus antitoxin levels found in the primipara and multipara subgroups were not statistically significant.     

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).     

Multicorrelation analyses of toxic and antigenic components of DPT and DP vaccines produced in Japan

The combined diphtheria-pertussis-tetanus and combined diphtheria-pertussis vaccines, of which the contents of endotoxin, LPF and HSF were reported in the previous paper, have been tested for the respective potencies of pertussis, diphtheria and tetanus components. In order to obtain some information concerning possible adjuvanticities of the toxic components of pertussis cells, relationship between respective toxic activities and the potencies of the respective antigenic components were tested by applying the multiple regression analyses. The results showed that the effects of the toxic components were fairly complicated. Within the ranges of contents of the respective toxic components of the vaccines used in the present experiment, LPF appeared to enhance all the three potencies as the content increased, while the effects of endotoxin and HSF were either enhancing or suppressive depending on the antigenic components.     

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.     

Serological Diagnosis of Human Melioidosis with Indirect Hemagglutination and Complement Fixation Tests

An indirect hemagglutination (IHA) test and a complement fixation (CF) test were evaluated from test results on sera from 212 human melioidosis patients of which 119 were culturally proved cases. Significant antibody titers (IHA titers of 1:40 or greater and CF titers of 1:4 or greater) were demonstrated with either test in all except five patients. IHA and CF titers ranged as high as 1:20,480 and 1:1,024, respectively. Antibodies were usually demonstrated by both tests 1 week after onset of disease. Transient seronegative reactions during the course of disease were seen in sera of approximately 19% of the patients with either IHA and CF but rarely with both tests. High titers in either test were obtained by the third week of disease and reached maximum levels in 4 to 5 months. Titers usually were detectable for 9 or more months. Antibodies were detected by IHA and CF tests in 80 to 100% of the sera obtained at various time intervals from 9 months to 2 or more years after disease onset. Antibody persistence occurred in patients who had a short disease course, as well as in patients with prolonged, complicated infections. The IHA test had excellent specificity when evaluated with normal human sera and diverse antimicrobial sera from hyperimmunized rabbits and human patients. The CF antigen appeared to contain common antigens with some but not all types of Pseudomonas aeruginosa. The specificity of the CF antigen could be enhanced without appreciable effect on its sensitivity by use of a titer of 1:8 in lieu of 1:4 as a criterion for a significant reaction. Either test could be used advantageously for the laboratory diagnosis of melioidosis.     

Effect of strain differences on the potency testing of rabies vaccines in mice

Antigenic differences between several strains of rabies virus, namely CVS, SAD and Flury (LEP) strains, were studied in cross-challenge experiments or cross-neutralization tests performed on sera of mice immunized with vaccines containing each strain. A typical wild fox virus strain was also included as challenge virus. The strain differences affected the relative potencies of the three vaccines in the European Pharmacopoeia mouse protection test for veterinary rabies vaccines, in that higher antigenic values were obtained when the vaccine strain was homologous to the challenge virus. This observation was confirmed by neutralizing antibody induction in mice.     

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.     

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.     

The development of an enzyme-linked immunosorbent assay for measuring the potency of vaccines containing Clostridium chauvoei antigens

Current standards (British Pharmacopeia (Veterinary) 1985) for vaccines containing Clostridium chauvoei require a potency test based on a challenge assay in guinea-pigs. Animal welfare and cost considerations favour the development of alternatives. Most veterinary clostridial vaccines are multi component, requiring assays in rabbits to test the potency of components other than C. chauvoei. We describe the application of an ELISA to measure the response to C. chauvoei vaccines in rabbits. The antigen is a sonicated extract of C. chauvoei strain CH4, intended to include a mixture of cellular and soluble antigens. The rabbit response to more than 70 vaccines containing C. chauvoei has been assessed against a reference serum which has been assigned an arbitrary potency of 100 units ml-1. The antibody titres of rabbit sera have been compared with the results of guinea-pig challenge potency tests on the same vaccines. The pass level in the guinea-pig potency test is equivalent to a rabbit ELISA titre of 50 units ml-1.