Note: Descriptions are shown in the official language in which they were submitted.
W O96/17249 PCT/GB95/02766
SPONGI~O.~. EN~FPHALOPATHY DETECTION r~l~ODS
The present invention relates to methods for the detection of
spongiform encephalopathies in animals, and in particular for the
detection of bovine spongiform encephalopathy (BSE) in cattle.
.
Spongiform encephalopathies are a group of diseases which include
scrapie in sheep and Creutzfeldt-Jakob disease (CJD) in humans.
BSE is a notifiable fatal neurodegenerative disease found in cattle.
BSE is of major importance to the British farming industry.
Currently cases of BSE are identified by clinical manifestations in
the animal. Cases are confirmed by post-mortem analysis of brain
tissue, for instance by histopathology, by detection of scrapie
associated fibrils or proteinase K resistant protein.
These methods have the disadvantage that they necessitate the
slaughter of potentially-infected animals which may turn out to be
disease-free. Alternatively, clinical signs may be absent or
go undetected, thus leaving infected animals in the herd.
Harrington et ~1 (New England Journal of Medicine (1986) Vol 315, No
5, pp 279-283) used high resolution two dimensional polyacrylamide
gel electrophoresis (2DPAGE) to discover the presence of 4 abnormal
proteins in the cerobrospinal fluid of human patients suffering from
CJD. However, the precise identity of these proteins was not
ascertained.
Thus there exists a need for a pre-mortem test for spongiform
encephalopathies which can be used when diagnosing
potentially-infected animals.
The present invention has now provided a method for detecting
spongiform encephalopathies in animals which addresses some, and in
preferred forms all, of these problems.
W O96/17249 PCT/GB95/02766
According to one aspect of the present invention there is provided
method for detecting the presence of spongiform encephalopathy in an
animal comprising determining the presence and/or amount of agent in
a body fluid of the animal which cross-reacts with antibody raised
against apoliprotein E, and relating the result of this determination
to the infection status of the animal.
~referably the result of the determination is compared with a control
value, and the relationship between the two is correlated with the
infection status of the animal.
Preferably the method is used to detect BSE.
Apolipoprotein E is a cholesterol transporting protein produced in
the peripheral and central nervous system. Its presence in either
multiple- or single-forms has been categorised in cerebrospinal fluid
(CSF) and serum.
Thus the discovery that spongiform encephalopathy infection in an
animal can be correlated with the presence of, or an increase in the
concentration of, an agent or agents in the body fluids of that
animal, forms the basis for the methods of the current invention.
The agent or agents are cross reactive with anti-apolipoprotein E,
have a molecular weight of around 34-38 kDa, and have a pI of around
5.4 - 5.7. This is consistent with their identification as
apolipoprotein E, and the term 'Apo E agent' as used hereinafter is
intended to embrace any agent which has these properties (including
apolipoprotein E itself and isoforms or multiple-forms thereof).
It should be noted that there is no requirement to accurately
quantify the Apo E agent concentration because spongiform
encephalopathy may be detected by comparison with a control.
The control value may be derived from the Apo E agent concentration
in a different animal (for which the infection status is known) and
which is analysed in parallel with the test animal. Alternatively,
W 096/17249 PCT/GB95/02766
the control value may come from the same animal, or be a known
standard.
The control value may be determined using the same method used for
the test animal, or may be derived by a different analytical method.
The results from the 'control' animal may be used to derive a
standard positive- or negative-control value, or to calibrate the
test animal result.
Preferably the body fluid analysed in the method is CSF since
authentic apolipoprotein E is the major apolipoprotein found in this
fluid. Additionally, the proximity of the CSF to brain means that
neurological disorders which produce alterations in the protein
composition of the brain may be manifested in the CSF. Methods for
extracting samples of CSF are well known to those skilled in the art.
The invention embraces any method for analysing the concentration of
Apo E agent in a body fluid of an animal which is currently comprised
in the art, and any methods which may later become available.
Preferably the presence and/or amount of Apo E agent in a body fluid
of the animal is derived by the use of PAGE or 2DPAGE to separate out
the Apo E agent from other agents in the body fluid, and then
stAining the gel and making densitometry measurements in the region
of the gel of interest in order to determine the presence and/or
amount of the Apo E agent.
Preferably the identity of the Apo E agent ls confirmed by use of
immunogenic material, for instance antibody raised against Apo E
agent or a synthetic peptide based on a sequence thereof. Suitable
immunogen-based techniques for identifying the presence of
cross-reactive agents are well known to those skilled in the art eg.
ELISA or Western Blotting.
In alternative embodiments of the invention, these immunogenic
techniques may be used both to identify the Apo E agent, and
W 096/17249 PCT/GB95/02766
also to estimate its concentration.
Thus the invention makes available methods for detecting the presence
of spongiform encephalopathy in a test animal which address many, and
in preferred forms all, of the problems of the prior art. The
balance between test certainty and ease of use will be dependent on
the precise method of Apo E agent analysis chosen for use in the
methods of the current invention. However, the pre-mortem diagnosis
of BSE in cattle opens up the possibility of mass-testing in herds,
thereby reducing the likelihood of slaughtering uninfected ~ni~ls or
'missing' infected ones.
The methods of the present invention will now be described, by way of
illustration only, through reference to the following example and
figures. Other embodiments falling within the scope of the invention
will occur to those skilled in the art in the light of this.
FXAMP!.F. - IDENTIFICATION OF BSE IN CATT!F
Sample preparation: CSF samples were collected from BSE-positive
cattle and BSE-negative cattle. In each case the diagnosis was
confirmed by post-mortem histopathology and electron microscopy. CSF
samples were taken by cisternamagna puncture after death and
concentrated 10-15 fold. Volumes of CSF cont~ining 40 ,ug total
protein were mixed in a 4:1 ratio with denaturing solution (lg sodium
dodecyl sulphate (SDS) and 0.232g dithiothreitol in lOml water) and
heated at 95C for 5 minutes. Samples were then pulse centrifuged.
Electrophoresis: The prepared samples were 2D electrophoresed using
a Millipore Investigator 2D electrophoresis system according to the
method in the instruction manual. First dimensional
iso-electric-focussing was carried out in 26 cm threaded glass tubes
with 1 mm inner diameter in a pH gradient of 3-10 for 18000 volt
hours after pre-focussing the gels for 1 hour to 1500 V. Second
dimension SDS-PAGE was carried out using 1 mm thick large format gels
(23 cm x 23 cm) with 12.5% acrylamide and no stacking gel.
W O96/17249 PCT/GB95/02766
St~ining and Image analysis: The 2D gels were silver stained
according to the Millipore manual. Gels were scanned with an
0 i ~ia scanner XRS and analysed using Bioimage software and
Investigator Database programme (Millipore) using a sunSPARC station
computer.
Confirmation of the identity of Apo E agent: The 2D gels were
electroblotted onto Immobilon-P membranes overnight at 30V using a
Bio-Rad Trans-Blot cell. The blots were blocked using Tween 80 for l
hour and then incubated for 90 minutes with sheep antiserum
cont~ining polyclonal antibody raised against authentic
apolipoprotein E. Bound sheep antibodies were detected using rabbit
anti-sheep IgG and a horseradish peroxidase detection system. A
number of agents in the region of interest (approximate molecular
weight of 34-38 kDa, and a pI of around 5.4 - 5.7) were found to have
cross reacted with anti-apolipoprotein E antibody.
Comparison of BSE-negative and BSE-positive cattle: A comparison of
the stained gels from typical BSE-positive and -negative samples is
shown in Fig l(a) and Fig l(b). As can be seen the number and
intensity of the silver stained spots in the region corresponding to
agents having an approximate molecular weight of 34-38 kDa, and a pI
of around 5.4 - 5.7 (labelled 'Apo E') is higher in the BSE-positive
sample.
A comparison of the mean optical density of those silver-stained
spots on the gels which were also found to cross react with
anti-apolipoprotein E antibody is found below:
W O96/17249 PCT/GB95/02766
~g~ No BSE-negative BSE-Dositive
1 0.13 0.47
2 0.42 o.84
3 0.45 o.64
4 0.45 0.99
o.46 1.02
6 0.41 o.69
7 o.87 1.12
By comparing the two sets of optical density readings it can be seen
that each agent is present in consistently higher amounts in the
BSE-positive (n=31) animals than in the BSE-negative (n=27) Anir~lc,
thus indicating that the presence and/or amount of these agents can
be used to detect the likely presence of BSE in potentially infected
Ani r~l !:; .
