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.     

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.     

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.     

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 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.     

A comparison of enzyme-linked immunosorbent assay (ELISA) with the toxin neutralization test in mice as a method for the estimation of tetanus antitoxin in human sera

An enzyme-linked immunosorbent assay (ELISA) has been developed for the measurement of tetanus antitoxin in human sera as an alternative to the toxin neutralization test in mice, the currently accepted method of assay. The ELISA was found to be simple and quick to perform and required only small amounts of materials. In addition, the assay was found to give reproducible estimates of antitoxin levels and to measure antitoxin at levels as low as 0.01 IU per ml, a sensitivity similar to that of the neutralization test. Furthermore, a comparison of the results of the ELISA and the neutralization test involving 80 human sera, including sera with both high and low antitoxin levels, showed close agreement in antitoxin levels obtained by the two methods. It was concluded that ELISA was an acceptable alternative to the toxin neutralization test in mice for the measurement of tetanus antitoxin levels in human sera.     

Murine red blood cells as efficient carriers of three bacterial antigens for the production of specific and neutralizing antibodies.

Three bacterial toxoids, CRM 197 (mutagenized diphtheria toxin), tetanus toxoid (formaldehyde-treated tetanus toxin), and PT-9K/129G (double mutant of pertussin toxin) were encapsulated within red blood cells (RBCs) of B6D2F1 and Balb/C mice according to a mild procedure based on hypotonic dialysis-isotonic resealing that yielded undamaged RBCs. The toxoid-loaded RBCs were injected intravenously in order to immunize animals and their effects were compared to those of identical amounts (30-95 micrograms per mouse subdivided into multiple injections) of the corresponding free toxoids injected intravenously in saline. Sera from treated mice were collected and tested for titers of specific antibodies against each of the three antigens and also for titers of neutralizing antibodies, i.e., affording protection from toxic effects induced by the corresponding native toxins. In all experiments, significant seroconversion was observed with both immunization systems. Titers of both specific and neutralizing antibodies against CRM 197 and tetanus toxoid were several-fold higher upon immunization with the RBC-encapsulated toxoids, than with the free toxoids. These differences were not due to qualitatively different recognition patterns of antigenic determinants by the two types of sera. Conversely, intravenous immunization with pertussis toxoid either as RBC-encapsulated or as free antigen elicited a comparably high production of specific and of neutralizing antibodies. These data demonstrate that properly engineered RBCs behave as natural carriers and possibly adjuvants for antigens of vaccinal interest.     

Methods for analysis of biologically functional antibodies to the HIV-1 gp120 principal neutralizing domain.

The humoral response to HIV-1 infection has been demonstrated by a variety of immunoassays utilizing viral proteins. While several assays detect HIV-1 infection with high sensitivity and great specificity, little progress has been made to develop immunoassays correlative with disease progression and viral transmission. Antibodies toward the V3 domain of HIV-1 envelope can prevent virus infection and block virus-mediated cell fusion in vitro. Such properties may be critical to the course of the disease. Furthermore, understanding the role of neutralizing antibodies against HIV-1 during infection in humans and generating biologically relevant neutralizing antibodies are paramount to developing an efficacious AIDS vaccine. In this study we explored peptide binding and neutralization assays and their relation to predicting disease progression and viral transmission. Biologically relevant polyclonal and monoclonal neutralizing antibodies that were derived from natural HIV-1 infection of humans, experimental infections of chimpanzees, and viral envelope protein peptide immunizations were characterized. Comparison of V3-specific monoclonal antibodies by antigen-limited ELISA and a quantitative HIV-1 neutralization assay demonstrated a less than optimal predictive relationship between binding and neutralization potency. On the other hand, polyclonal sera from goats immunized with V3-specific peptides derived from three different HIV-1 strains, as well as sera from other HIV-1-infected individuals demonstrated correlation between binding affinity and neutralization.     

猪和牛轮状病毒抗原和抗体的检测

应用ELISA直接双抗体夹心法检查轮状病毒抗原,24份仔猪和29份犊牛的腹泻粪样,分别有12和16份阳性。用病毒RNA电泳分析检查阳性粪样,各出现两种病毒RNA电泳型,用中和试验检查17份成年牛和16份成年猪血清,分别有16和15份病毒抗体阳性。将其与ELISA间接法和结合法进行了比较。     

Quantitation of poliovirus antigens in inactivated viral vaccines by enzyme-linked immunosorbent assay using animal sera and monoclonal antibodies

Recent advances in methods for the manufacture of inactivated poliovirus vaccines have resulted in increased vaccine immunogenicity. In conjunction with this capability it is important to have available highly sensitive and quantitative potency assays. The potential suitability of enzyme-linked immunoassay (ELISA) was evaluated using animal sera with neutralizing antibodies or neutralizing monoclonal antibodies for antigen detection in potency tests. The monoclonal antibodies developed, which bound D antigen but not C antigen, were neutralizing unless relatively weakly reactive. Those that bound C antigen only were non-neutralizing. Those that bound both C and D antigens were sometimes neutralizing. D-specific and D/C-specific neutralizing monoclonal antibodies against type-2 poliovirus protected mice on passive immunization against paralytic disease and death from the MEF strain virus. Potency measurements by ELISA using either D-specific neutralizing monoclonal antibodies or type-specific goat sera for antigen detection were sensitive and precise. Tests using C-specific monoclonal antibodies for antigen detection indicated that increased C antigen content may result in falsely elevated reactivities of animal sera with some vaccines. Monoclonal antibodies may be useful ELISA reagents for IPV potency testing.     

Increasing the potency of neutralizing single-domain antibodies by functionalization with a CD11b/CD18 binding domain

Recombinant single domain antibodies (nanobodies) constitute an attractive alternative for the production of neutralizing therapeutic agents. Their small size warrants rapid bioavailability and fast penetration to sites of toxin uptake, but also rapid renal clearance, which negatively affects their performance. In this work, we present a new strategy to drastically improve the neutralizing potency of single domain antibodies based on their fusion to a second nanobody specific for the complement receptor CD11b/CD18 (Mac-1). These bispecific antibodies retain a small size (?30 kDa), but acquire effector functions that promote the elimination of the toxin-immunocomplexes. The principle was demonstrated in a mouse model of lethal toxicity with tetanus toxin. Three anti-tetanus toxin nanobodies were selected and characterized in terms of overlapping epitopes and inhibition of toxin binding to neuron gangliosides. Bispecific constructs of the most promising monodomain antibodies were built using anti Mac-1, CD45 and MHC II nanobodies. When co-administered with the toxin, all bispecific antibodies showed higher toxin-neutralizing capacity than the monomeric ones, but only their fusion to the anti-endocytic receptor Mac-1 nanobody allowed the mice to survive a 10-fold lethal dose. In a model of delayed neutralization of the toxin, the anti- Mac-1 bispecific antibodies outperformed a sheep anti-toxin polyclonal IgG that had shown similar neutralization potency in the co-administration experiments. This strategy should have widespread application in the development of nanobody-based neutralizing therapeutics, which can be produced economically and more safely than conventional antisera.     

Determination of Tetanus Toxin and Toxoid by ELISA Using Monoclonal Antibodies

The procedure for obtaining monoclonal antibodies TT-1, TT-2, and TT-3 against tetanus toxin/toxoid is described. It is shown that the commercial DTP vaccine and tetanus toxoid conjugated with a low-molecular-weight hapten can both be used as immunogens. Monoclonal antibodies TT-1 and TT-2 neutralized tetanus toxin in vivo. The monoclonal antibodies obtained were used to design and compare several schemes of quantitative determination of tetanus toxoid and toxin by ELISA. A more sensitive competitive ELISA allowed the detection of as much as 0.01 EC/ml toxoid and 50 LD₅₀/ml toxin.     

Sublingual immunization with an engineered Bacillus subtilis strain expressing tetanus toxin fragment C induces systemic and mucosal immune responses in piglets

Sublingual (SL) and intranasal (IN) administration of a Bacillus subtilis-based tetanus vaccine was tested in piglets, which more closely mimic the human immune system than mice. Piglets were immunized by the SL, IN or oral routes with vaccine expressing tetanus toxin fragment C, or commercial tetanus vaccine given by intramuscular injection as a control. Tetanus toxoid specific ELISA and passive neutralization tests were used to measure IgG and IgA levels in serum and mucosal secretions, and assess protective serum antibodies, respectively. The nature of the immune response was explored by MHC Class II, TGF-β1 expression, and ELISA assays for multiple cytokines. SL or IN immunization of piglets induced neutralizing tetanus toxoid specific serum antibody and local salivary and vaginal IgA responses. Standard tetanus vaccine resulted in systemic antibodies, whereas oral administration of the Bacillus-based vaccine was ineffective. Further analyses indicated a balanced Th1/Th2 response to SL or IN immunization. CONCLUSION: This study demonstrates for the first time that SL or IN administration is effective for inducing both systemic and mucosal responses in a piglet model, indicating that SL or IN delivery of a B. subtilis-based tetanus vaccine can be a simple, non-invasive, low cost strategy to induce immunity to tetanus.     

Determination of tetanus toxin and toxoid by ELISA using monoclonal antibodies

The procedure of obtaining monoclonal antibodies TT-1, TT-2, and TT-3 against tetanus toxin/toxoid is described. It is shown that both commercial DTP vaccine and tetanus toxoid conjugated with a low-molecular-weight hapten can be used an immunogens. Monoclonal antibodies TT-1 and TT-2 neutralized tetanus toxin in vivo. The monoclonal antibodies obtained were used to design and compare several schemes of quantitative determination of tetanus toxoid and toxin by ELISA. A more sensitive competitive ELISA allowed detecting as much as 0.01 EC/ml toxoid and 50 LD50/ml toxin.     

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.     

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.     

Increasing the potency of neutralizing single-domain antibodies by functionalization with a CD11b/CD18 binding domain

Recombinant single domain antibodies (nanobodies) constitute an attractive alternative for the production of neutralizing therapeutic agents. Their small size warrants rapid bioavailability and fast penetration to sites of toxin uptake, but also rapid renal clearance, which negatively affects their performance. In this work, we present a new strategy to drastically improve the neutralizing potency of single domain antibodies based on their fusion to a second nanobody specific for the complement receptor CD11b/CD18 (Mac-1). These bispecific antibodies retain a small size (˜30 kDa), but acquire effector functions that promote the elimination of the toxin-immunocomplexes. The principle was demonstrated in a mouse model of lethal toxicity with tetanus toxin. Three anti-tetanus toxin nanobodies were selected and characterized in terms of overlapping epitopes and inhibition of toxin binding to neuron gangliosides. Bispecific constructs of the most promising monodomain antibodies were built using anti Mac-1, CD45 and MHC II nanobodies. When co-administered with the toxin, all bispecific antibodies showed higher toxin-neutralizing capacity than the monomeric ones, but only their fusion to the anti-endocytic receptor Mac-1 nanobody allowed the mice to survive a 10-fold lethal dose. In a model of delayed neutralization of the toxin, the anti- Mac-1 bispecific antibodies outperformed a sheep anti-toxin polyclonal IgG that had shown similar neutralization potency in the co-administration experiments. This strategy should have widespread application in the development of nanobody-based neutralizing therapeutics, which can be produced economically and more safely than conventional antisera.     

Acellular pertussis vaccines: neutralization by immune sera of the lethality of pertussis toxin and viable Bordetella pertussis for chick embryos.

Vaccines containing acellular pertussis components, either separate or combined with other microbial antigens, were evaluated for specific immune responses in guinea-pigs and mice. The capacity of sera to protect chick embryos from the lethal effect of pertussis toxin was independent of the Chinese hamster ovary cell clumping neutralization titre and the antigen binding ELISA anti-toxin titre. Direct correlations did not exist between ELISA titres to Pt, FHA, fimbria or 69 kDa and capacity to prevent killing of embryos by different strains of Bordetella pertussis. With the exception of one combination vaccine product, addition of foreign microbial antigens to acellular pertussis vaccines did not significantly alter capacity of the sera to protect embryos against toxin or bacteria.     

Tandem copies of a human rotavirus VP8 epitope can induce specific neutralizing antibodies in BALB/c mice

The VP8 subunit protein of human rotavirus (HRV) plays an important role in viral infectivity and neutralization. Recombinant peptide antigens displaying the amino acid sequence M(1)ASLIYRQLL(10), a linear neutralization epitope on the VP8 protein, were constructed and examined for their ability to generate anti-peptide antibodies and HRV-neutralizing antibodies in BALB/c mice. Peptide antigen constructs were expressed in E. coli as fusion proteins with thioredoxin and a universal tetanus toxin T-cell epitope (P2), in order to enhance the anti-peptide immune response. The peptide antigen containing three tandem copies of the VP8 epitope induced significantly higher levels of anti-peptide antibody than only a single copy of the epitope, or the peptide co-administered with the carrier protein and T-cell epitope. Furthermore, the peptide antigen containing three copies of the peptide produced significantly higher virus-neutralization titres, higher than VP8, indicating that a peptide antigen displaying repeating copies of the amino acid region 1-10 of VP8 is a more potent inducer of HRV-neutralizing antibodies than VP8 alone, and may be useful for the production of specific neutralizing antibodies for passive immunotherapy of HRV infection.     

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.