Mouse Neutralization Test Research Articles

Antigenic variation of wild and vaccine rabies strains of Egypt.

Nineteen street rabies virus strains, isolated in Egypt from humans (two), dogs (nine), cats (two), farm animals (two), gerbils (three), and a jackal were antigenically analyzed. The Pasteur strain used for the preparation of human rabies vaccine, the Flury high and low egg passage stains (HEP, LEP) used for animal vaccines, and the challenge virus standard (CVS) strain were also assayed. All were examined by the indirect fluorescent antibody test, using a panel of 20 monoclonal antibodies against the nucleocapsid of rabies and rabies-related viruses. The rabies isolates demonstrated patterns of reactivity with the antinucleocapsid panel different from those of the Pasteur, HEP, and CVS strains. Representative human, dog, and rodent isolates were analyzed by neutralization tests in mice, with a second panel of 19 monoclonal antibodies against rabies and Mokola envelope glycoproteins. With this panel, the isolates demonstrated patterns of reactivity different from the vaccine strains. These data indicate antigenic variation between wild virus and vaccine strains.

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.

Reactogenicity and immunogenicity of the newly developed Purified Chick Embryo Cell (PCEC)-rabies vaccine in man

Purified Chick Embryo Cell (PCEC) rabies vaccine was given to 88 healthy adults according to six different vaccination schedules. Local side effects were reported on reactivity forms after 16.4% of PCECV injections, general symptoms were recorded after 15.1% of the 292 doses administered. IgE antibodies specific for chicken proteins determined by the Radio Allergo Sorbens Test (RAST) could not be shown before and after the vaccinations. With no exception, all 88 vaccinees developed high titres of complement-fixing and neutralizing antibodies as determined by the Rapid Fluorescent Focus Inhibition Test and Serum Mouse Neutralization Test. For the first time, induction of serum interferon by PCEC rabies vaccine has been shown in man. In rabies vaccination, PCEC vaccine seems to be as effective as Human Diploid Cell Strain (HDCS) vaccine.

Human rabies immunoglobulin assayed by the rapid fluorescent focus inhibition test suppresses active rabies immunization

The rabies antibody content of each of ten lots of human rabies immunoglobulin was titrated by both the mouse neutralization test and the rapid fluorescent focus inhibition test. The two tests did not give comparable results, the antibody titres obtained by the mouse neutralization test being 1·4–9·6 times higher than those obtained by the rapid fluorescent focus inhibition test. This titre difference was associated with a consistently lower antibody response in human volunteers who had received post-exposure rabies vaccine treatment which included the administration of RIG assayed by the RFFIT.

The mouse neutralization test in comparison with the rapid fluorescent focus inhibition test: Differences in the results in rabies antibody determinations

Sixteen lots of rabies immune globulin (RIG) and six lots of rabies immune horse serum (RIS) from different producers were examined for rabies antibody by the standard mouse neutralization test (MNT) and the rapid fluorescent focus inhibition test (RFFIT). An equine rabies standard serum was assayed in parallel. In comparisons of RIS with this standard the MNT and RFFIT gave comparable results. In comparisons of RIG the antibody values in the MNT was two to ten times higher than that in the RFFIT in 15 out of 16 lots. The MNT and RFFIT are thus not fully comparable when measuring rabies antibodies in RIG. The choice of the titration method is obviously important in the measurement of the antibody concentration in RIG or RIS in IU against an equine rabies reference preparation. The described differences could have consequences for the use of RIG.

Microneutralization test in PK(15) cells for assay of antibodies to louping ill virus.

A microneutralization test in PK(15) cells was developed to measure the neutralizing antibody response of a group of ponies experimentally challenged with louping ill virus. Viral cytopathic effect was maximal after 6 days of incubation, at which point titration endpoints were clear-cut and readily determinable. The assay compared favorably with the mouse neutralization test for accuracy and ease of performance.

Rabies neutralizing antibodies determination by the modified counterimmunoelectrophoresis test and the rapid fluorescent focus inhibition test

The correlation between the modified counterimmunoelectrophoresis and the rapid fluorescent focus inhibition tests for detecting rabies virus antibodies in human sera was studied, using as reference the standard mouse neutralization test. Statistical analyses showed good correlation among the three serological tests. There were no statistically significant differences in titers obtained by serum neutralization and rapid fluorescent focus inhibition tests and the estimated titers based on the results of the modified counterimmunoelectrophoresis test.

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.

The isolation and preliminary characterization of a rhabdovirus in Australia related to bovine ephemeral fever virus

CSIRO 368 virus was isolated from blood collected in the Northern Territory from a healthy cow and electron microscope studies showed that the isolate had rhabdovirus morphology. Fluorescent antibody studies and complement fixation tests related the virus to bovine ephemeral fever (BEF) virus. Neutralization tests in both suckling mice and Vero cells showed that the virus was not BEF virus. Antibodies to CSIRO 368 virus were found in cattle sera from northern and eastern Australia and Papua New Guinea. Antibodies were found in 16 out of 45 buffalo, some of which also had antibodies to BEF virus. In contrast, none of the 419 deer tested had antibodies to CSIRO 368 virus, although 142 of the same deer had antibodies to BEF virus. No antibodies to CSIRO 368 virus were detected in 16 goats, 54 horses, 10 pigs, 31 sheep, 25 kangaroos, or 14 human beings. Both CSIRO 368 and BEF viruses were found to be sensitive to ether and chloroform, but were not affected by the DNA inhibitor 5-bromo-2′-deoxyuridine, showing that they probably had the same type of nucleic acid — namely RNA. CSIRO 368 was also shown to grow to higher titres in BHK21 cells than in Vero cells. Temperature sensitivity studies at −20, 4 and 37°C showed that the presence of foetal calf serum increased the survival time markedly at −20°C, but only slightly at 4 and 37°C. The virus survived the longest at −20°C in the presence of foetal calf serum.

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.

Sensitivity of the biotin-avidin-peroxidase (BAP) technique for rabies virus neutralizing antibody assay and the measurement of neutralizing antibodies of vaccinated human sera.

The biotin-avidin-peroxidase (BAP) technique for measuring serum neutralizing (NT) antibody against rabies virus in human sera was established. When the focus reduction rates with serial serum dilutions were plotted on a probit chart, a linear regression line was drawn. The slopes (b-value) of the regression lines were identical irrespective of the different test samples. The sensitivity of the BAP test for detecting NT antibody was highest when compared with that of the rapid fluorescent focus inhibition test (RFFIT) and mouse neutralization test. In addition, Iow level-antibody titer (< I : 5) which could not be detected by mouse neutralization test was detected by the BAP test and the RFFIT. Serum NT antibody response was easily followed by the BAP test with individuals who received 3 doses of preexposure preventive rabies vaccine. Most of the vaccinees produced a detectable amount ofserum NT antibody by a single dose ofvaccine and after the second vaccination, all vaccinees produced high level of the antibody and the titers increased continuously until day 2 1 , however, the titers on day 78, two months after the last vaccination, showed a tendency ofdecline. INTRODUCTION NT antibody to rabies virus has been mostly measured by mouse neutralization test (Atanasiu, 1973) and rapid fluorescent focus inhibition test (RFFIT) (Smith et al., 1 973). The former method requires a large number of mice and at least 2 weeks of long period until the final results can be obtained. The latter method is much more superior to mouse neutralization test for detecting antibody in every respect of the sensitivity, reproducibility and rapidity. However, this method has a disadvantage that the fluorescent staining is unstable and the stained cells easily fade out and has a difficulty in testing many specimens collectively. In order to resolve this problem, we previously studied the biotin-avidin-peroxidase (BAP) technique for measurement of rabies virus infectivity and the NT antibody to rabies virus (Shichijo et al., 1 982). The BAP technique provides such advantages that the specimens can be examined under an ordinary microscope and can be stored for 1 Department of Microbiology, Medical College of Oita, Oita 87(~9 1 , Japan 2 The Chemo-SeroTherapeutic Research Institute, 668 Shimizu Machi, Kumamoto 860, Japan

Comparaison de quatre techniques de titrages sérologiques des anticorps contre le virus de la rage chez le chien

Four different serological techniques were used for the determination of antibody levels against rabies virus in 55 vaccinated street dogs: mouse neutralization test (reference), rapid fluorescent focus inhibition test, plaque reduction test and immunoenzymatic test (protein A). The results obtained with each of the last three methods were compared with those obtained with the reference test: correlation coefficients were, respectively, 0.810, 0.812 and 0.682, each being significantly correlated with the others. Therefore the three techniques may each be recommended as an alternative to the mouse neutralization test for routine titrations; the immunoenzymatic method, which titrates not only neutralizing antibodies being, nevertheless, better used as a screening test.

Antibody responses to human diploid cell vaccine for rabies with and without human rabies immune globulin.

One hundred one volunteers with no exposure to rabies were given human diploid cell vaccine (HDCV) for rabies with or without 20 international units of human rabies immune globulin (HRIG)/kg of body weight to evaluate schedules for therapy with HDCV and HRIG after exposure. All of the volunteers who received three or more doses of HDCV alone or four or more doses of HDCV with HRIG developed high titers of neutralizing antibodies by day 35, which persisted for at least 60 days. By day 7, of the 61 volunteers given HRIG and HDCV, 53% had neutralizing antibodies by a mouse neutralization test and 67% had neutralizing antibodies by a rapid fluorescent focus inhibition test. Similar antibody levels were found in volunteers given HRIG alone, a finding which suggests that low or undetectable early titers after administration of HDCV and HRIG were due to inadequate HRIG dosage rather than any interaction between the passive antibody (HRIG) and the vaccine antigen. These results suggest that trials with 30 or 40 international units of HRIG/kg in combination with HDCV are warranted.

Antigenic variants of rabies virus

Monoclonal antibodies directed either against the nucleocapsid or the glycoprotein of rabies virus were reacted by indirect immunofluorescence and mouse neutralization tests with vaccine strains and rabies field isolates of various origin. Major antigenic determinants allow the grouping of viruses and minor ones the differentiation of virus strains. A protection study showed the existence in nature of certain antigenic variants against which conventional vaccines do not fully protect. The isolation of two such variants from ‘imported’ bats in the Federal Republic of Germany is reported here for the first time.

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.

Antibody development in garter snakes (Thamnophis spp.) experimentally infected with western equine encephalitis virus.

Garter snakes (Thamnophis spp.) have been considered to possibly play an important role in the ecology of western equine encephalitis (WEE) virus. Serological tests (hemagglutination-inhibition, complement-fixation, neutralization test in mice, and plaque neutralization) to detect antibody in these reptiles following laboratory exposure t this virus have, in our experience, been unsatisfactory. A new test, the snake globulin precipitation (SGP) test, has been developed and we consider it to be reliable in detecting antibody in WEE virus-infected garter snakes. Antibody has been detected in these snakes over 4.5 years following inoculation with WEE virus. The SGP test should be a valuable tool in obtaining further information regarding the possible role of these cold-blooded vertebrates in the ecology of this important arbovirus.

Rift Valley fever virus infection in man in Nigeria.

Recently, Rift Valley fever virus (RVFV) disease has assumed public health significance as associated with human fatalities on a significant scale both in the North, Central and South of Africa. In West Africa, little information is available about RVFV disease. However, in Nigeria, four strains of the virus have been isolated from sheep, culicoides, and mosquitoes; also, RVFV antibodies were demonstrated in some domestic animal sera. The West African dwarf sheep, which is indigenous to Nigeria, is highly suspectible to experimental RVFV infection. No information is available about RVFV disease in man. Human blood and serum samples collected from different parts of Nigeria were examined for RVFV and its specific antibodies using mouse inoculation technique, complement fixation (CF), and neutralization (N) tests. In addition, a limited number of hospital records of suspected RVFV cases were examined. No RVFV was isolated from 19,026 samples examined for virus content. Of the 2,223 sera tested, 146 or 6.6% were positive for RVFV antibodies by the CF test. There was a good correlation between the CF and N tests, in RVFV serodiagnosis. One hundred forty-four of the 146 CF-positive sera were positive in N tests; in addition, all of the 50 CF-negative sera were negative in N tests for RVFV antibodies. From the limited hospital records examined, it appears that RVFV infection in Nigeria presents as an influenza-like illness. On one occasion, there was ocular complication following a febrile illness, associated with the development of specific RVFV antibodies. With less than 10% of the surveyed population immune to RVFV, the potentials for a severe eqizootic of RVFV disease in Nigeria appear great. Further studies on such factors as climate and vector population, as well as the reservoir and host range of RVFV, are in progress.

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.

Rabies neutralizing antibody determination by the interference inhibition test (IIT) and the mouse neutralization test (MNT)

The CVS-11 strain of rabies virus was shown to interfere with plaque formation by vesicular stomatitis virus (VSV) in a continuous line of monkey kidney cells (V3). Interference was inhibited by rabies antibody and an interference inhibition test (IIT) was devised to titrate rabies neutralizing antibody in 94 human sera. The sera were also tested by the mouse neutralization test (MNT) and the results obtained by either method were comparable. The IIT was highly reproducible and since only routine tissue culture facilities are required for the test, the technique is recommended as an alternative to the MNT and the rapid fluorescent focus inhibition test (RFFIT).