Toxin Neutralization Test In Mice Research Articles

Development of an ELISA for measuring the activity of tetanus toxoid in vaccines and comparison with the toxin neutralization test in mice

An enzyme-linked immunosorbent assay (ELISA) has been developed to measure anti-tetanus toxoid antibody levels in immunized guinea-pig sera as a useful alternative to the currently used toxin neutralization test (TNT) in determining the activity of the tetanus toxoid in vaccines. The ELISA was found to measure antibody levels as low as 5.8 × 10 −5 IU/ml. Furthermore, a comparison of the results from ELISA and TNT involving 132 different commercial vaccines showed a very good correlation ( r=0.94, p<0.001) between antibody levels measured by both methods. The results suggest that the proposed ELISA is a reliable, simple and economical alternative to the TNT in mice for assessing the activity of tetanus toxoids in vaccines.

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

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

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

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

Monoclonal antibodies against tetanus toxin and toxoid.

Monoclonal antibodies against tetanus toxin and its toxoid were produced by immunizing mice with toxoid or toxin. They were measured by an enzyme-linked immunosorbent assay (ELISA), by a toxin neutralization test in mice (in vivo prevention test), and by their ability to prevent binding of 125I-toxin to brain membranes or gangliosides (in vitro prevention test). Six monoclonal antibodies obtained by immunization with toxoid (anti-toxoid 1-6) were investigated in more detail. They belonged to IgG class 1. Three of them (anti-toxoid 1, 2 and 3) recognized both toxoid and toxin as well as fragment B and the light chain of toxin, but not fragment C. Two other antibodies (anti-toxoid 4 and 5) were directed against toxoid only. Neither of them prevented toxin action in vitro or in vivo. Anti-toxoid 6 recognized toxin, toxoid and fragment C, but not light chain, and prevented toxin action in vitro and in vivo. Immunization against toxin was initiated with a toxin-antitoxin complex and boosted with toxin. We studied six antibodies in more detail, all of IgG type 2. Their KD against 125I-tetanus toxin varied from 10(-9) to 10(-10) M. Anti-toxin 2 recognized toxin, toxoid, light chain and fragment B, but not fragment C. The others reacted with toxin, toxoid and fragment C, but not with light chain or fragment B. All of them prevented toxin action in vitro and in vivo. As calculated from the maximal extinction achieved in the ELISA, tetanus toxin combined with a maximum of two different antibody molecules from our set. Gel filtration data indicate that tetanus toxin reacts with monoclonal antibodies one by one. Compared with polyclonal antiserum, monoclonal antibodies yield flatter slopes in both in vitro and in vivo prevention tests. Thus, they cannot substitute for the polyclonal antibodies in clinical situations, and cannot be calibrated in international units.

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.

Quantification of tetanus antitoxin by toxin neutralization test in mice. A comparison between lethal and paralytic techniques

Tetanus antitoxin may be assayed by toxin neutralization (TN) tests in mice using either the death of mice or early symptoms of tetanic paralysis as the titration endpoint. The present investigation presents a statistical comparison of the two techniques. The correlation between the two techniques is very good. The paralytic technique reduces animal suffering considerably, but the inspection of the animals needs to be carried out more carefully and the method is therefore more time consuming than the lethal technique.

Quantification of Tetanus Antitoxin in Human Sera

We described recently an improved counter-immunoelectrophoretic method for quantification of tetanus antitoxin. The toxin neutralization test in mice is considered to correlate well with protection in humans. In the present study, the correlation coefficient between the two methods was 0.89. Sera containing more than 7.0 I.U./ml could be quantified directly by counter-immunoelectrophoresis, while sera containing less tetanus antitoxin had to be concentrated prior to quantification. The passive haemagglutination test was also compared with the toxin neutralization test in mice. The correlation coefficient between the two methods was 0.76.

Quantification of tetanus antitoxin in human sera. I. Counter-immunoelectrophoresis.

An extensive sero-epidemiological survey of immunity to tetanus is to be performed in Norway during 1978 and 1979 and thus a simple and reliable method for screening sera for content of tetanus antitoxin is needed. An improved counter-immunoelectrophoretic method for quantification of tetanus antitoxin is described. The toxin neutralization test in mice is considered to correlate well with protection in humans. Counter-immunoelectrophoresis has the advantage of using tetanus toxoid instead of toxin as well as being more rapid and less expensive. Specific antibodies of the IgG, IgM, and IgA classes could be read simultaneously for many sera. This may be of importance, since it is reported in the literature that only antibody of the IgG class is capable of neutralizing tetanus toxin in mice. Counter-immunoelectrophoresis is limited by being less sensitive than the toxin neutralization test in mice.

Immunologic Relationships Among the Spotted Fever Group of Rickettsias Determined by Toxin Neutralization Tests in Mice with Convalescent Animal Serums

Summary Tests of certain variables inherent in the spotted fever toxin neutralization test in mice revealed that a) spotted fever group toxins remained stable for a period of at least 3 hr at either room temperature (26°C) or at 2°C, b) serum antitoxin titers were maximum 15 min after mixing antiserum with toxin and they did not change significantly during 2 hr further incubation at room temperature, c) a relationship existed between the serum neutralization titer and the concentration of toxin neutralized: a 2-fold increase in quantity of toxin reduced the serum titer 4-fold. Three strains of Rickettsia rickettsii associated with spotted fever disease in man behaved alike in reciprocal cross neutralization tests. A strain of R. rickettsii from the rabbit tick Haemaphysalis leporis-palustris, showed a significant serologic difference from one but not from the other two of these strains of R. rickettsii. The rickettsias of boutonneuse fever, South African tick bite fever, Kenya tick typhus and Indian tick typhus, were indistinguishable from one another by the mouse toxin test. These studies with Indian tick typhus confirm recent information concerning its serologic relationship with this group of rickettsias. R. rickettsii and Rickettsia conorii were clearly differentiated from one another by the mouse toxin test. Neither of these two rickettsias produced more than a slight degree of cross neutralization with the other. The rickettsia of Siberian tick typhus could be distinguished by the mouse toxin technique from R. rickettsii and R. conorii as well as from other spotted fever group strains studied. However, a relatively high one-way cross neutralization occurred between the Siberian agent and certain strains of R. rickettsii and the rickettsia from the tick Amblyomma maculatum, but not R. conorii. Two strains of Siberian tick typhus rickettsias, one from the tick Dermacentor nuttalli, and the other of human origin, gave complete cross neutralization with one another and were essentially alike in their cross reactions with other members of the spotted fever group. The maculatum rickettsia exhibited distinct serologic differences from all other spotted fever strains studied. This rickettsia did, however, give notably high nonreciprocal cross reactions with R. conorii strains and with the rickettsia of Siberian tick typhus. Guinea pig antisera against Rickettsia australis and Rickettsia akari showed little or no cross neutralization of toxins of 10 other strains of spotted fever group rickettsias.