Binding Inhibition Test Research Articles

Induction of systemic, mucosal, and cellular immunity against SARS-CoV-2 in mice vaccinated by trans-airway with a S1 protein combined with a pulmonary surfactant-derived adjuvant SF-10.

There is a need for vaccines that can induce effective systemic, respiratory mucosal, and cellular immunity to control the COVID-19 pandemic. We reported previously that a synthetic mucosal adjuvant SF-10 derived from human pulmonary surfactant works as an efficient antigen delivery vehicle to antigen presenting cells in the respiratory and gastrointestinal tracts and promotes induction of influenza virus antigen-specific serum IgG, mucosal IgA, and cellular immunity. The aim of the present study was to determine the effectiveness of a new administration route of trans-airway (TA) vaccine comprising recombinant SARS-CoV-2 spike protein 1 (S1) combined with SF-10 (S1-SF-10 vaccine) on systemic, local, and cellular immunity in mice, compared with intramuscular injection (IM) of S1 with a potent adjuvant AddaS03™ (S1-AddaS03™ vaccine). S1-SF-10-TA vaccine induced S1-specific IgG and IgA in serum and lung mucosae. These IgG and IgA induced by S1-SF-10-TA showed significant protective immunity in a receptor binding inhibition test of S1 and angiotensin converting enzyme 2, a receptor of SARS-CoV-2, which were more potent and faster achievement than S1-AddaS03™-IM. Enzyme-linked immunospot assay showed high numbers of S1-specific IgA and IgG secreting cells (ASCs) and S1-responsive IFN-γ, IL-4, IL-17A cytokine secreting cells (CSCs) in the spleen and lungs. S1-AddaS03™-IM induced IgG ASCs and IL-4 CSCs in spleen higher than S1-SF-10-TA, but the numbers of ASCs and CSCs in lungs were low and hardly detected. Based on the need for effective systemic, respiratory, and cellular immunity, the S1-SF-10-TA vaccine seems promising mucosal vaccine against respiratory infection of SARS-CoV-2.

Screening of natural extracts with anti-norovirus effects and analysis of this mechanism in grape seed extract.

Norovirus (NoV）is a major causative virus of viral gastroenteritis and requires a general disinfection method because it is resistant to common disinfectants such as ethanol and chlorhexidine. This study aimed to find natural extracts as candidates for versatile disinfectant ingredients. The antiviral effect of natural extracts against NoV can be evaluated using the feline calicivirus (FCV）-inactivation test and NoV virus-like particle (NoV-VLP）-binding inhibition test. In this study, screening of natural extracts with anti- NoV effects was performed using these two methods. Of the 63 natural extracts examined, 14 were found to have high FCV-inactivation and NoV-VLP-binding inhibitory effects. In addition, we evaluated the NoV-VLPbinding inhibitory effect of grape seed extract（GSE）containing proanthocyanidins under multiple concentration conditions and treatment times and determined that the binding inhibitory effect of GSE was concentration- and time-dependent. Electron microscopy showed that GSE-treated NoV-VLPs aggregated, distorted, and swelled, suggesting that GSE directly interacts with NoV particles. The results suggest that some natural extracts containing GSE can be used as components of disinfectants against NoV.

Automated detection of neutralizing SARS-CoV-2 antibodies in minutes using a competitive chemiluminescence immunoassay

The SARS-CoV-2 pandemic has shown the importance of rapid and comprehensive diagnostic tools. While there are numerous rapid antigen tests available, rapid serological assays for the detection of neutralizing antibodies are and will be needed to determine not only the amount of antibodies formed after infection or vaccination but also their neutralizing potential, preventing the cell entry of SARS-CoV-2. Current active-virus neutralization assays require biosafety level 3 facilities, while virus-free surrogate assays are more versatile in applications, but still take typically several hours until results are available. To overcome these disadvantages, we developed a competitive chemiluminescence immunoassay that enables the detection of neutralizing SARS-CoV-2 antibodies within 7 min. The neutralizing antibodies bind to the viral receptor binding domain (RBD) and inhibit the binding to the human angiotensin-converting enzyme 2 (ACE2) receptor. This competitive binding inhibition test was characterized with a set of 80 samples, which could all be classified correctly. The assay results favorably compare to those obtained with a more time-intensive ELISA-based neutralization test and a commercial surrogate neutralization assay. Our test could further be used to detect individuals with a high total IgG antibody titer, but only a low neutralizing titer, as well as for monitoring neutralizing antibodies after vaccinations. This effective performance in SARS-CoV-2 seromonitoring delineates the potential for the test to be adapted to other diseases in the future.Graphical

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.

Galβ1‐6Gal, antigenic epitope which accounts for serological cross‐reaction in diagnosis ofEchinococcus multilocularisinfection

Parasitic helminths express various antigenic carbohydrates, which often account for serological cross-reactions. In serodiagnosis, it is essential to inspect cross-reactivity between the target parasite and other parasites in order to assess diagnostic performance. Our previous study showed that the Galbeta1-6Gal sequence was a common epitope between Echinococcus multilocularis (Em) and E. granulosus (Eg). Furthermore, compounds with this sequence from Fasciola hepatica (Fh) reportedly were recognized by sera with Eg infection. Our aim is to investigate whether this sequence is one of the widely common epitopes in many kinds of parasites. For various parasites, sera with Fh infection cross-reacted at the highest frequency (71.4%) against Em antigen. In patients with other parasitic infections, sera showed cross-reactions against Fh antigen bound to Em antigen with a high frequency (23.7%). Binding inhibition tests with commercial Galbeta1-6Gal disaccharide showed that Galbeta1-6Gal was the common epitope between not only Em, Eg and Fh, but also between various other parasites. Furthermore, the presence of the Galbeta1-6Gal epitope in Em antigen was confirmed by immunoblot testing with the specific antibody for this sequence. This study showed that the Galbeta1-6Gal sequence is one of the antigenic epitopes that accounts for serological cross-reactivity between Em and various other parasites.

On-line determination of 3,5,6-trichloro-2-pyridinol in human urine samples by surface plasmon resonance immunosensing

An immunochemical method for the analysis of 3,5,6-trichloro-2-pyridinol (TCP), a major urinary metabolite of chlorpyrifos, is developed using a surface plasmon resonance (SPR)-based biosensor. The stability of the assay was assessed by covalently linking the analyte derivative to a thin, gold-modified sensor surface. For optimization of analyte derivative immobilization, sensor chips were activated via alkanethiol monolayers with terminal amine or carboxyl groups. Binding inhibition tests were performed in untreated urine samples and compared to those obtained in distilled water and PBS was used as control. In all cases, similar detection limits, at the micrograms per litre level (0.1-0.24 microg L(-1)), were attained for TCP assays independently of the dilution buffer. Reproducibility of measurements was studied throughout more than 130 regeneration cycles, which allowed the repeated use of the same immunosensor surface without significant variation of the SPR signal. All measurements were developed in real-time in only 10 min, using a SPR portable system. The device could be applied as a valuable analytical method to both environmental screening and clinic diagnostics.

Development of a TIRF-based biosensor for sensitive detection of progesterone in bovine milk

A total internal reflectance fluorescence (TIRF)-based biosensor for progesterone in bovine milk was developed and tested by measuring the progesterone level in daily milk samples for 25 days, covering a whole estrus cycle. The detection is based on total internal reflectance fluorescence. The assay has been designed as a binding-inhibition test with a progesterone derivative covalently immobilized on the sensor surface and a monoclonal anti-progesterone antibody as biological recognition element. First an existing progesterone assay was optimized by reducing the assay time per measurement, resulting in an assay time of about 5 min and reaching a limit of detection (LOD) of 0.04 ng mL −1 and a quantification limit (LOQ) of 0.34 ng mL −1. After calibration the assay was tested by measuring the progesterone level in daily milk samples over several weeks. An estrus cycle of a cow could be measured. As results become available within minutes without any preparation or pre-concentration of the milk samples the fully automated TIRF-based biosensor for progesterone can be used in-line in the milking parlor and thus could be an important tool for reproductive management of dairy cattle detecting heat and predicting pregnancy, which are critical parameters in milk production.

Real-time detection of chlorpyrifos at part per trillion levels in ground, surface and drinking water samples by a portable surface plasmon resonance immunosensor

Continuous monitoring of the organophosphate pesticide chlorpyrifos in real water samples has been performed using a portable surface plasmon resonance (SPR) immunosensor. The immunoassay was based on a binding inhibition test by using the covalent immobilization of an analyte derivative onto the gold-coated sensing surface. Sensor reusability was assured through the formation of alkanethiol self-assembled monolayers (SAMs). In this format, the biosensor surface remained stable over 190 measurements including regeneration cycles. The reproducibility and precision of SPR measurements were evaluated by performing independent immunoassays for three replicates of each sample. The batch-assay variability was also estimated using three different gold-coated sensor surfaces. The assay provided a highly sensitive detection of chlorpyrifos at nanogram per litre levels (55 ng L −1) in laboratory buffer. No significant matrix effects were found for ground, river and drinking water samples, where the limits of detection ranged from 45 to 64 ng L −1 and the recovery values were between 80 and 120%. The validation of chlorpyrifos immunoassay was performed using conventional chromatographic methods (GC–MS). The correlation between both techniques was proved by the good correlation coefficient ( r 2 = 0.995) obtained. Chlorpyrifos immunoassays were developed using the β-SPR sensor system commercialised by the company SENSIA, S.L. (Spain). Rapid analysis of samples, without previous extraction or clean-up, was accomplished in only 20 min, including the regeneration cycle. The high sensitivity, low-time of response and portability of the β-SPR biosensor platform, would allow its application as a field analytical method for environmental waters.

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.

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.

A new method for determining anti-B cell antibodies and their specificity using flow cytometry

We describe a new flow cytometric B cell crossmatch method with improved sensitivity and specificity. It is based on the coating of B cell surface immunoglobulins with an unconjugated polyvalent anti-human immunoglobulin antibody. The method also provides a means for determining the specificity of anti-B cell antibodies and, potentially, for anti-HLA class II antibody specificity differentiation (DR, DQ or DP) in a binding inhibition test using mouse monoclonal antibodies directed against human HLA class II antigens.

A sensitive enzyme-linked immunosorbent assay for human interleukin-8

In order to quantify human interleukin-8 (IL-8), which is chemotactic for T cells and basophils as well as neutrophils, we developed an enzyme-linked immunosorbent assay (ELISA). Since binding inhibition tests indicated that three monoclonal antibodies (mAbs; BS-1, WS-4, WS-6) blocked the binding of 125I-labelled IL-8 to neutrophils, we tested an ELISA using these mAbs as primary antibodies, rabbit anti-IL-8 Ab as the secondary antibody, and alkaline phosphatase-labelled goat anti-rabbit Ab as the conjugate. Among the three mAbs tested, WS-4 was the most sensitive with a detection limit of 16 pg/ml. Several other cytokines, including monocyte chemotactic and activating factor (MCAF), which is structurally related to IL-8, showed no cross-reactivity in this system, indicating that this ELISA is specific for IL-8. The coefficients of variation for the intra- and interassays were below 10%. Furthermore, this ELISA also detected natural IL-8 (including both 72 and 77 amino acid forms) produced by cultured human cells and cell lines stimulated with IL-1, suggesting that this system will be useful in the detection of natural IL-8 in various body fluids.

Detection of cancer-associated antigen in feces using monoclonal antibodies in the diagnosis of colon carcinoma

Monoclonal antibodies against colon and pancreatic cancer, CL-2, CL-3, PS-9, PS-10, were used to detect the associated antigens in feces of patients with gastrointestinal carcinoma and non-cancer diseases. Binding inhibition test by SABC-ELISA method were performed for the measurement of the antigen level. Results showed that the associated antigen detected in feces of patients with colon cancer were significantly higher than that of noncancer disease or normal subjects. The positive rates were 61.1% as detected with CL-2; 53.4% with CL-3; 55.0%, PS-9; and 53.3% PS-10 in cancer patients while that in normal subjects were 7%; 9%; 8%; and 8% respectively. When ‘cocktail’ of CL-2, PS-9 and PS-10 were used, the positive rates were 92.5% in colon cancer and 14% in normal subjects. In seven out of the sixty patients with colon cancer studied who were graded as Dukes A, the results were all positive. The results seem superior to the serologic detection and may provide a promising new approach in the early diagnosis of colon cancer.

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

The in vitro toxin binding inhibition (ToBl) test was used to determine antitoxin responses in mice immunized with tetanus toxoid. The ToBl 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 ToBl 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 ToBl test and lethal challenge test in a single group of guinea-pigs, showed good correlation. From the study it is concluded that the ToBl 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.

Production of Monoclonal Antibodies against a New Carcinoma-associated Marker in View of Developing a Serological Test

By immunizing a mouse with human metastatic breast tumor cells from patient effusions and infiltrated lymph nodes, a monoclonal antibody (MLuC2), which identifies a new carcinoma-associated marker, was raised. The reactivity of this reagent was studied by immunohistochemistry on live and fixed cells from tumor cell lines and on frozen sections from surgical specimens. Besides reacting with 73% of breast carcinomas, MLuC2 also reacted with 93% of non-small cell lung carcinoma (NSCLC) and with a few normal tissues. The MLuC2-recognized molecule (CaMLuC2), whose MW was 90 KDa according to immunoblotting experiments, was found to be detectable in the serum and could therefore be of particular interest for serological diagnostic applications. Since the CaMLuC2 epitope was not polyexpressed on the bearing molecule, we produced a new generation of MAbs in order to define epitopes coexpressed with CaMLuC2 on the same 90 kDa molecule, and which are therefore suitable to develop a double-determinant immunoradiometric assay (DDIRMA) for the detection of this marker in the sera of lung carcinoma patients. Different analyses by immunohistochemistry, binding inhibition tests and DDIRMA, proved that the two new reagents developed, MLuC8 and MLuC9, recognize the same or closely related epitopes, which are however different from CaMLuC2, but which are all present on the same molecule. Preliminary immunoradiometric tests performed on sera from lung cancer and control patients showed a good specificity but a low sensitivity. In fact, only 42% of the 28 tested sera samples from NSCLC patients scored positive despite the fact that more than 90% of the NSCLC expressed the relevant antigen.

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 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.