| dc.description.abstract | Rabies is world-wide in distribution. Despite significant
scientific advances in its prevention and control, the disease
has been spreading in most parts of the world where it
continues to persist as a major public health problem. The
disease is endemic in Kenya where the dog plays a major role
in its transmission to man.
In view of the almost 100% mortality rate of human rabies,
prevention becomes essential. Also in animals, rabies is a
disease approaching a 100% case fatality rate, but it has been
known since the days of Pasteur that some animals survive
both natural and experimental infection, and that a healthy
and infective carrier state may occur. Since efficient
vaccines are now available, vaccination may be performed on a
large scale in relevant animals and in high-risk human
populations.
The emergence of highly efficient vaccines has to a large
extent obviated the need to confirm adequate immune responses
of vaccinated individuals by serum antibody determinations.
Nevertheless, a rapid, specific and reproducible assay for the
detection and accurate quantitation of antibodies to rabies
virus would be highly desirable for the following purposes :
(i) evaluation of potency of vaccines,
(ii) evaluation of individual immune responses,
(iii) standardization of hyperimmune antiserum for
therapeutic/prophylactic use,
(iv) determination of efficacy of large-scale vaccination
programmes,
(v) aid in the diagnosis of rabies in suspected
non-vaccinated cases,
(vi) comparative studies of rhabdoviruses.
The results obtained by the mouse neutralization test
(MNT) and the rapid fluorescent focus inhibition test (RFFlT)
have been recognized as true reflections of the protective
potency of a serum, and can be expressed as international
units per millilitre (I.U./ml) in comparison with an
internationally recognized reference serum. Both tests suffer
from poor reproducibility and demands for highly skilled
man-power. The absolute requirements for live infective
challenge virus, a large number of animals or tissue culture
capability as well as special facilities and equipment render
these two neutralization tests unsuitable for routine or
large-scale use in most countries. Although promising, most
of the replacement tests have been found to be inadequate
largely because of poor correlation with the MNT and the
RFFlT.
In this study, an inhibition enzyme immunoassay (INH-EIA)
for the detection and quantitation of rabies antibodies was
developed and evaluated. In this system, the interaction of
specific enzyme-labelled antibodies with their antigen is
inhibited by non-labelled antibodies of the same specificity.
The antibody titre is expressed as that dilution of a serum
sample which gives 50% inhibition. This can subsequently be
converted to equivalents of I.U./ml calculated on the basis
of an antirabies reference serum whose potency has been
determined by the MNT or the.RFFIT.
The INH-EIA was carried out by coating microtitre plates
with a predetermined dilution of a rabies virus preparation.
The IgG fraction of absorbed serum from a goat immunized with
human diploid cell rabies vaccine from InstitutMerieux,
France, was conjugated with horseradish peroxidase and shown
to be specific for rabies virus components. It was shown
that the conjugate possessed antibody activities to both the
glycoprotein and the ribonucleoprotein components of the
virus when compared with known antiglycoprotein and
antiribonucleoprotein sera obtained from other laboratories.
No inhibition was observed when dilutions of tissue
culture homogenates and known negative sera from 7 different
animal species were assayed.
A high ionic strength salt (1M KCl) buffer containing 2%
polyethylene glycol 6000 was used as test serum diluent. The
composition of the diluent would enhance antigen-antibody
interactions and might consolidate these reactions, thus
presumably allowing the estimation of even low affinity
antibodies. The INH-EIA was performed sequentially allowing
antibodies of the test serum to interact with antigen prior to
the addition of conjugate. It is expected that such an assay
would be capable of detecting both low and high affinity
antibodies, and also low levels of antibody. The ability of
the INH-EIA to detect low levels of antibody was evident when
low but definite antibody activity (equivalent to 0.25 - 1.95
I.U./ml) was detected in 5 human sera obtained 7 days after
primary intradermal vaccination with human diploid cell rabies
vaccine. The assay gave a mean titre equivalent to 0.06
I.U./ml (range 0.04 - 0.08 I.U./ml) in 14 sera from
non-vaccinated humans. The ability of the INH-EIA to detect
low levels of antibodies should render the test suitable for
the intra-vitam diagnosis of rabies. This became evident when
a serum sample from a previously unvaccinated rabid goat, bled
one week before death, gave an antibody titre corresponding
to 2.0 I.U./ml, i.e. ten times the highest titre obtained in
serum samples from non-vaccinated humans and animals.
The reproducibility of the INH-EIA was assessed by
performing 15 complete titrations of each of two antirabies
sera obtained from the World Health Organization (WHO) and
the Institut Herieux. The WhO serum which had a potency of
10 I.U./ml as determined by the MNT, gave a mean titre
correspondng to 8.7 I.U./ml (range 7.2 - 9.9, coefficient of
variation + 10%) calculated on the basis of the Institut
Merieux serum. The Institut Merieux serum gave a mean titre
corresponding to 174 I.U./ml (range 151.6 - 205.4 I.U./ml,
coeff. var. + 10%) when the WHO serum was used as a standard.
The Institut Merieux serum had been titrated against an
international standard by using the RFFIT. A mean titre
equivalent to 194 I.U./ml (range 183 - 209, coeff. var. + 6%)
was found. Thus, excellent agreement was found between the
INH-EIA and both the MNT ana the RFFIT.
In the INH-EIA, duplicate single serum dilutions of 1:2
could be used to accurately quantitate rabies antibodies as
long as the inhibition was within 20-75%. This procedure
proved valuable in the screening of large numbers of serum
samples from any animal species within a short time.
The highest antibody titre obtained in sera from
non-vaccinated persons and animals was 0.2 I.U./ml.
Consequently, this titre was chosen as a cut-off point for
the differentiation of rabies antibody positive and negative
sera. Some of the sera from man and different animal species
gave antibody titres above the equivalent of 0.2 I.U./ml.
This was notable with sera obtained from goats, cattle,
elephants and hyenas. Since no antirabies vaccination is
carried out in these animals in Kenya, it is surmised that
the animals may have been exposed to the rabies virus or to
one of the rabies-related viruses.
It is concluded that INH-EIA is a highly reproducible,
sensitive and specific method for the detection and
quantitation of antibodies to rabies virus and correlates
well with the internationally accepted MNT and RFFIT. The
method allows the screening at a single serum dilution of
large numbers of specimens from different animal species. It
is therefore likely that the method will find considerable
application in seroepidemiological studies of rabies.
The INH-EIA appears to be an attractive substitute for
both the hNT and the RFFIT because of its high sensitivity and
specificity, high reproducibility, ease of performance and
interpretation, rapidity and low cost. Further investigations
are needed to confirm whether the antibody titres obtained in
the INH-EIA represent true reflections of various levels of
protective immunity. | en |
