Comparison of Three Serological Tests for Titration of Rabies in Immunized Individuals
S. Simani PhD
WHO-Collaborating Center for Reference and Research on Rabies, Pasteur Institute of Iran
Background-Several immunological assays have
been applied to measure the titer of rabies antibodies.
The titer of these antibodies is a critical point for
post-exposure treatment of people who are injured by
rabid animals. This study was designed to evaluate three
serological tests for titration of anti-rabies antibodies
as the previous studies were reported to be
Methods-The mouse neutralization test (MNT), the rapid fluorescent focus inhibition test (RFFIT), and the enzyme linked immuno-sorbent assay (ELISA) were used to measure the titer of rabies antibodies amongst sixty-one vet students at Tehran University. The students were vaccinated according to the pre-exposure anti-rabies regimen, on days 0, 7, and 28. Blood samples were collected two weeks after the last day of vaccination, and the sera were titered using the techniques of MNT, RFFIT and ELISA. Statistical analysis of the results was done on the average titers of each technique, using the analysis of variance test.
Results-The titers obtained with the MNT and RFFIT techniques were comparable but differed from the results obtained by the ELISA.
Conclusion-The ELISA should not be substituted for MNT, but the RFFIT can be used as an alternative test for MNT to determine the rabies anti-bodies level.
Key Words · Rabies · MNT · ELISA · RFFIT · antibody titers.
Rabies is one of the oldest and most devastating viral diseases afflicting humans. It is usually transmitted through the saliva via bites, and only a very rapid immunization protocol administered immediately after encounter is effective in preventing the onset of symptoms and disease. After having become symptomatic, prevention of full onset of the disease is impossible. The causative agent is a neurotropic virus belonging to the Rhabdoviridae family. The rabies viral particles are bullet-shaped, consisting of an internal helical structure which is the nucleocapsid, and an outer bilayer lipoprotein membrane. Glycoprotein projections on the surface of the outer membrane induce neutralizing antibodies.12,15
The best post-exposure treatment after attacks by a rabid animal or one suspected of being rabid, consists of cleansing the bite wound by flushing with soap and water, and starting an immunization protocol consisting of 5 doses of vaccine administered on days, 0,3,7,14,30 as soon as possible. The wounded areas should not be closed by suturing.3,10
In 1881 Louis Pasteur published the first report on the successful treatment of a nine year old boy who was severely bitten by a rabid dog) by vaccination with the rabbit spinal cord vaccine.3 Vaccination remained the sole treatment of exposed individuals until 1954. Since then simultaneous administration of vaccine and anti-rabies serum to the victims, bitten in the head and face region was introduced by Pasteur Institute of Iran.1,2 Such victims can not be treated by vaccination as Louis Pasteur recommended.
To treat the suspected victims, the WHO has recommended the usage of homologous or heterologous antirabies serum at the dosages of 20 IU/kg and 40 IU/kg body weight, respectively.8 Such treatment requires laboratory techniques for measurement of effective antirabies levels in the sera used for treatment. It is also necessary to measure the titer of rabies antibodies to evaluate the effectiveness of vaccination in establishing immunization. Three procedures are available to measure rabies antibody levels, the MNT, RFFIT, and ELISA. Using these techniques, inconsistent results have been reported by others.4,5,9,14,16 The MNT has been accepted as a standard method for determination of rabies antibody titer by WHO.11 We have compared these techniques to find the more sensitive method and consequently use it as routine test in our laboratory.
Materials and Methods
The sera used in this study were obtained from 55 veterinary students. They were collected before vaccination and also two weeks after the last injection of the standard pre-exposure anti-rabies vaccine. Vaccination against rabies is mandatory for vet students in Iran due to the risk of exposure to rabies. Every student received a total of 3 doses of anti-rabies vaccine, on days 0, 7 and 28 by intramuscular injections. The vaccines were supplied by the Pasteur-Merieux, lot K829, and prepared from rabies virus infected Vero Cell Lines inactivated with beta-propiolacton. All serum samples were tested by the tree methods as follows:
The Mouse Neutralization Test (MNT):
The challenge virus strain (CVS) used in this study was titrated at 10-7 as described by Kaplan.11 A constant dose of titrated virus was added to the sera which serially diluted at 1/5.
A reference serum of known titer was included in all assays. The virus/serum mixtures at dilutions of 1/5, 1/25, 1/125, 1/625, 1/3125 were then inoculated intracerebrally to the groups of 8 young adult mice. Totally 40 mice were inoculated with the virus/serum mixture. The percentage of mortality was determined and the serum dilution which protects 50% of the animals was calculated by the Reed and Muench method.11,13
The Rapid Fluorescent Focus Inhibition Test (RFFIT)
The BHK cells were grown and used in the RFFIT according to the standard methods.11 The sera to be examined were diluted at 1/3 in the 96 wells microplates. Diluted sera were then mixed with a constant dose of challenge virus that cause infection in 80% of the cells as described.13 The sera/virus mixture were incubated at 37° C for an hour. After incubation, susceptible cells were added to the serum/virus mixtures, and after a further 24 hours of incubation, the cell monolayer was acetone fixed and stained with a fluorescent antibody in order to detect the presence of non-neutralized virus (fluorescent foci). Calculations were done by the Reed and Muench method.7,11,13
The Enzyme Linked Immuno Sorbent Assay (ELISA)
This assay was performed to detect rabies virus anti-glycoprotein antibodies in the sera using Platelia Rage kit as recommended by the supplier (Diagnostic Pasteur 72200 France). Various dilutions of serum samples, a positive control serum, and a negative control were added to wells of microplates coated with the rabies virus surface glycoprotein. The plate was incubated and washed appropriately, and then staphylococcus Protein A conjugated with Peroxidase was added. After a further incubation and washing, O-phenyl-diamine (OPD) and H2O2 was added and color was developed in the dark. The plate was read at 492 nm in an automated plate reader. Titers of serum samples were determined using standard curves based on the absorbances obtained with the positive control serum.17
The average titers of each technique were compared using statistical methods.
Results and Discussion
This study was performed on 55 vet students of whom 90.9% were males and the remaining were females. None of the three titration techniques detected anti-rabies antibodies in the pre-vaccination sera. The titer of rabies antibodies were measured after vaccination.
It was found that in many vaccinated students, the titer by ELISA was very low, while it was high when it was measured by other methods (MNT and RFFIT).
The mean±SD coefficient of variation of titers of antibody was 36.3±24.4 (67%) for MNT and 34.5±20.8 for RFFIT which were quite similar and comparable. ELISA showed lower mean of titer (14.5±13.6) and much greater coefficient of variation (94%).
Table 1 is two-by-two comparisons of the 3 techniques. It shows that despite differences in some subjects, the results of MNT and RFFIT are almost comparable.
Using the STATA statistical software program, demonstrated that the mean titers of anti-rabies antibodies obtained by MNT, RFFIT and ELISA were similar, but the mean titers from ELISA were less than MNT, RFFIT and ELISA were significantly different (P<0.0001, DF=2,162). Further statistical analysis by Scheffe test demonstrated that the mean titers of rabies antibodies obtained by, MNT and RFFIT were similar, but the mean titers from ELISA were less than MNT and RFFIT. Similar findings were also reported by others.4,9 However a few studies suggested that these three techniques had produced the same results.14,16
While according to the kit supplier (Diagnostic Pasteur. 72200, France), the ELISA indirectly evaluates the reaction of the viral glycoproteins with the rabies antibodies, the MNT and RFFIT directly measure the reaction of whole viral antigens. This is the advantage of these techniques over ELISA. It is clear from the table that the mean titer obtained by ELISA is lower than those of MNT and RFFIT. This may be due to the fact that staphylococcus protein A which is used in ELISA does not recognize the human IgG3 subclass of antibodies.6 It might be possible to obtain higher titer of rabies antibody in ELISA if anti-human antibody IgG is used instead of staphylococcus protein A as suggested.6,17
Since the time required to perform RFFIT is shorter than MNT (24 hours versus 21 days), it is suggested that the RFFIT could be substituted for the standard MNT.
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17 Turner M, Owen M. Immunoglobulins, In: Roitte I, Brostoff J, Male D. eds. Immunology. Washington DC: ASM Press, 1993.
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