Toxin Binding Inhibition Test Research Articles

ELISA and modified toxin-binding inhibition test for quality control of the clostridial vaccine processes

This study aimed to assess and standardize the ELISA and modified ToBI test in vitro methods in order to verify the potency of epsilon toxicoid in comparison with the in vivo TCP method. The following epsilon toxoids were used: NIBSC standard from batches 375/07, 532/08, 551/08, 373/07 and 378/07. These were evaluated using a TCP test, ELISA and ToBI tests. The results indicate that the correlation ratio between the dilutions of standard NIBSC toxicoid and absorbance values of 89.44% obtained with the ELISA method support the use of the curve to evaluate epsilon toxoids. However, it was observed that the absorbance values were similar for all toxoids, thus presenting no significant difference between higher and lower concentration toxoids. For the ToBI test, the correlation ratio of 96.76, obtained in the curve pattern, demonstrates the effectiveness of the curve to be used in the epsilon toxoid evaluation. The correlation ratio between the titration degrees of toxoids obtained through TCP and ToBI tests was higher than 90%. It is concluded that the type of ELISA test used does present discriminative power for toxoids with different concentrations, which does not support the use of this technique for such a purpose. The ToBI test can be used as a screening method for it is sensitive and effective to detect epsilon toxicoid produced by C. perfringens type D.

Equine Influenza and Equine Herpesvirus Vaccination: Evaluation of Immune Response and Safety of Concurrent Administration of Equip® FT and Equip® EHV 1,4

AimsThe concurrent administration of EHV1,4 and EIV vaccines is not currently recommended. This study aimed to evaluate safety and immunogenicity of concurrent administration of Equip® FT and Equip® EHV1,4 (treatment group T04) as compared with vaccination with Equip® FT (treatment group T02) or Equip® EHV1,4 (treatment group T03) alone.MethodsEach treatment group included 10 ponies vaccinated on study Days 0 and 42. Immune responses to vaccine antigens were measured on Day 56. Tetanus antibody titres were determined with the toxin binding inhibition test (ToBI). Immunity to equine influenza virus (EIV) induced by vaccination was measured by single radial haemolysis (SRH; antibody response) and IFN gamma assay (a marker of cell‐mediated immunity; CMI). Immune responses to Herpesvirus 1,4 were measured by complement fixation (CF) and IFN gamma assay.ResultsConcurrent administration of EIV and EHV1,4 vaccines was well tolerated. Concurrent administration was proven to be efficacious against tetanus with similar serological immune response of treatment group T04 as compared with T02. Mean SRH results for ponies that received both vaccines were above 85 mm2 for A/eq/Borlange/91 and A/eq/Kentucky/98 antigens, a threshold associated with clinical protection against closely related EIV strains. The highest levels of IFN gamma response were measured in group T02. Although concurrent vaccination seemed to have an effect on both equine herpesvirus (EHV) serology and EHV CMI, EHV results obtained during this study were not conclusive and may have been influenced by a natural EHV‐4 infection occurring prior to study start.Conclusion and practical relevanceConcurrent administration of Equip® FT and Equip® EHV1,4 was well tolerated and induced mean serum antibody levels consistent with clinical protection for tetanus and EIV. However, it was not possible to gather relevant information about the EHV immune response under the conditions of this study.Ethical animal researchThe study was conducted under the Animal Health Trust Home Office Project License and with approval of the Zaventem Ethics Review Assessment team (Zoetis). Sources of funding: The study was sponsored by Pfizer/Zoetis Animal Health. Competing interests: Equip® FT and Equip® EHV1,4 are owned by Zoetis, who sponsored this study. Romain Paillot reports no conflict of interest. All other authors are employed by the study sponsor.

Equine Influenza and Tetanus Immunisation: Evaluation of Antibody Response and Safety of Equip® FT and Equip® F Administration after Reduction of the Primary Course Interval to 3 Weeks

AimsA 6‐week interval is currently recommended during the primary course of the equine influenza (EI) vaccines Equip® FT or Equip® F. This study aimed to evaluate immunogenicity and safety of a 3‐week accelerated interval in order to improve flexibility and to reduce the period of susceptibility to EI virus (EIV) infection.MethodsThree groups of 10 ponies were respectively vaccinated with the vaccine Equip® FT according to the recommended 6‐week V1–V2 interval (T02), Equip® F or Equip® FT according to an experimental accelerated 3‐week interval (T03 and T04, respectively). These groups were boosted (V3) with Equip® F 5 months after V2. Ten ponies in a control group were injected with saline (T01). The antibody response to the vaccine antigens (tetanus toxoid and EIV) were measured 2 weeks after V2 and V3 with the toxin binding inhibition test (ToBI) and single radial haemolysis (SRH), respectively. All animal work received ethical approval.ResultsThe accelerated schedule of immunisation was well tolerated. At both time points, in all vaccinated groups, SRH least squares means for A/eq/Borlange/91 and A/eq/Kentucky/98 antigens were well above 85 mm2, a level described previously to provide clinical protection against homologous EIV strains. Non inferiority of SRH least square means were demonstrated at 2 weeks post V2 for both antigens for T03 and T04, and for A/eq/Kentucky/98 antigen in group T04 after V3. For tetanus,ToBI least squares means were 86.3 iu/mI for T02 and 45.9 iu/ml for T04 after V2 and 2.6 iu/ml for T02 and 2.2 iu/ml for T04 after V3. A level of 0.02 iu/ml has been described as being protective in various publications.Conclusion and practical relevanceA 3‐week accelerated primary course interval was well tolerated and serology results suggested good immunogenicity.Ethical animal researchThe study was conducted under the Animal Health Trust Home Office Project License and with approval of the Zaventem Ethics Review Assessment team (Zoetis). Sources of funding: The study was sponsored by Pfizer/Zoetis Animal Health. Competing interests: Equip® FT is owned by Zoetis, who sponsored this study. Romain Paillot reports no conflict of interest. All other authors are employed by the study sponsor.

Modified toxin-binding inhibition (ToBI) test for epsilon antitoxin determination in serum of immunized rabbits

The aim of the present study was to evaluate and standardize the ToBI test in vitro as a substitute for the serum neutralization test in mice for quality control of clostridial vaccines. The ToBI test in vitro was used to evaluate 40 serum samples of known antibody content, obtained from rabbits immunized against clostridiosis with experimental vaccine. The correlation between epsilon antitoxin titers in rabbit sera, determined by the ToBI test and serum neutralization in mice, ranged from 0.222% to 0.452% in polyvalent vaccines and from 0.154% to 0.387% in monovalent vaccines. Interplate coefficients of variation were not significant, reaching 0.350% in polyvalent vaccines and 0.400% in monovalent vaccines, indicating high homogeneity. In conclusion, the ToBI test in vitro is suitable for assessing the potency of clostridial vaccines and may be used as an alternative method able to replace current in vivo tests.

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

A method for the screening of tetanus and diphtheria antibodies in serum using anatoxin (inactivated toxin) instead of toxin was developed as an alternative to the in vivo toxin neutralization assay based on the toxin-binding inhibition test (TOBI test). In this study, the serum titers (values between 1.0 and 19.5 IU) measured by a modified TOBI test (Modi-TOBI test) and toxin neutralization assays were correlated (P < 0.0001). Titers of tetanus or diphtheria antibodies were evaluated in serum samples from guinea pigs immunized with tetanus toxoid, diphtheria-tetanus or triple vaccine. For the Modi-TOBI test, after blocking the microtiter plates, standard tetanus or diphtheria antitoxin and different concentrations of guinea pig sera were incubated with the respective anatoxin. Twelve hours later, these samples were transferred to a plate previously coated with tetanus or diphtheria antitoxin to bind the remaining anatoxin. The anatoxin was then detected using a peroxidase-labeled tetanus or diphtheria antitoxin. Serum titers were calculated using a linear regression plot of the results for the corresponding standard antitoxin. For the toxin neutralization assay, L+/10/50 doses of either toxin combined with different concentrations of serum samples were inoculated into mice for anti-tetanus detection, or in guinea pigs for anti-diphtheria detection. Both assays were suitable for determining wide ranges of antitoxin levels. The linear regression plots showed high correlation coefficients for tetanus (r(2) = 0.95, P < 0.0001) and for diphtheria (r(2) = 0.93, P < 0.0001) between the in vitro and the in vivo assays. The standardized method is appropriate for evaluating titers of neutralizing antibodies, thus permitting the in vitro control of serum antitoxin levels.

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.

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 χ2 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.Copyright 2002 The International Association for Biologicals. Published by Elsevier Science Ltd. All rights reserved .

European Sero-Epidemiology Network: standardisation of the results of diphtheria antitoxin assays

A European Sero-Epidemiological Network (ESEN) was established with the aim to co-ordinate and harmonise serological surveillance of immunity to communicable diseases in Europe. In this study the inter-laboratory standardisation of diphtheria toxin antibody measurements is reported. A standard panel of 162 sera was tested by the participating laboratories using an in vitro assay of their choice: VERO cell toxin neutralisation assay (NT), double-antigen delayed time-resolved fluorescence immuno-assay (DA-DELFIA), double-antigen enzyme-linked immunosorbent assay (DAE), toxin binding inhibition test (ToBI) and an indirect enzyme-linked immunosorbent assay (ELISA). The results were standardised using regression against the NT. The variations due to inter-laboratory and inter-assay variation, which would otherwise make it difficult directly to compare the main serum bank results by the different laboratories and the various assays were successfully minimised by the standardisation. The regression equations obtained will be used to transform the respective local results of testing the main serum bank into the reference test unitages. This study also gave the opportunity to compare the various assays within and between laboratories. This demonstrated a very high correlation between DA-DELFIA, DAE, ToBI and the NT. The ELISA showed a good correlation, too, however sera below some 0.1 IU/ml were overestimated.

Development, validation and acceptance of alternative methods in the quality control of vaccines: A case report

Information about levels of protection against tetanus is needed both for the assessment of immune status and for the estimation of the potency of batches of tetanus toxoid. Originally, levels of protective antibodies in human serum samples were titrated in an in vivo toxin neutralization (TN) test. Potency testing was based either on an indirect protection test or a direct challenge test. In the former test, rabbits or guinea pigs are immunized and bled, followed by titration of serum samples in a TN test. In the latter test, used in the quality control of tetanus toxoid for human use, protective immunity is induced by vaccination in guinea pigs or mice and subsequently challenging them directly with tetanus toxin. In the mid-1980s, an in vitro model was developed at the National Institute of Public Health and Environmental Protection (RIVM) for estimating levels of tetanus antitoxin in serum samples. This model, the toxin-binding inhibition (ToBI) test, was validated recently for both diagnostic testing and potency testing. Although accurate estimation of antitoxin levels is more important for diagnostic testing than for potency testing, the ToBI test has been adopted for determining the immune status but not for potency testing. One major reason is that there are no official guidelines for titration of human serum samples. By contrast, potency testing is performed in accordance with the monographs of national and international pharmacopoeias, which complicates acceptance for technical and bureaucratic reasons. This paper focuses on the validation studies performed at the RIVM. In particular, attention is paid to the various problems encountered. Suggestions for the role of the European Centre for Validation of Alternative Methods (ECVAM) ∗ ∗ The superscript ECVAM in the text refers to a recommendation to the European Centre for the Validation of Alternative Methods (ECVAM). These recommendations are summarized at the end of this paper. in the quality control of vaccines are also presented.

Interlaboratory Validation of in vitro Serological Assay Systems to Assess the Potency of Tetanus Toxoid in Vaccines for Veterinary Use

Abstract. An interlaboratory validation study was carried out in seven laboratories to evaluate the suitability of in vitro serological assay systems for the assessment of the potency of tetanus toxoid in single and multicomponent vaccines for veterinary use. Nine commercial vaccines and one experimental tetanus toxoid preparation were selected for immunization purposes according to Method A of the European Pharmacopoeia. Levels of tetanus antibodies in guinea-pig and rabbit serum samples were estimated by indirect ELISA, toxin binding inhibition (ToBI) test, passive haemagglutination (HA) test and by the prescribed standard toxin neutralization (TN) test in mice. Estimates of potency obtained by in vitro tests and by TN test were in good agreement for the various vaccines tested and for antibody levels of individual serum samples in the range 2·6 IU/ml to 266 IU/ml. Significant (P < 0·05) intralaboratory variation occurred less frequently for ELISA and ToBI test than for HA test. The frequency of significant (P < 0·05) interlaboratory variation was acceptable for the ELISA and the ToBI test but greater variation was observed for the HA test. It is concluded that the ELISA and ToBI tests are suitable in vitro assay systems for assessing the potency of tetanus toxoid in batches of single and multicomponent vaccines for veterinary use. Rigid standardization of the HA test is essential before this test can be used for the same quality control purpose.

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.

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 (<50 μl) 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 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.