Title: Diagnostic assay for
transmissible spongiform encephalopathies
United States Patent: 7,368,247
Issued: May 6, 2008
Denis Francois (Geneva, CH), Sanchez; Jean-Charles (Geneva, CH),
Zimmermann; Catherine Gabrielle (Geneva, CH), Guillaume; Elisabeth (Annemasse,
Assignee: Universite De
Geneve (Geneva, CH)
Appl. No.: 10/238,557
Filed: September 10, 2002
Heart and brain fatty acid binding
proteins (H-FABP, B-FABP) are markers for TSEs, especially CJD. The
invention provides a diagnostic assay for either of these markers,
preferably by enzyme immunoassay using a specific antibody thereto. Since
H-FABP is also a marker for acute myocardial infarction (AMI), to
distinguish CJD from AMI requires an assay specific to AMI, e.g. using
troponin-1 or CK-MB as a marker, also to be carried out.
Description of the
SUMMARY OF THE INVENTION
It has now surprisingly been found that two fatty acid binding proteins (FABP),
known as heart (H-FABP) and brain (B-FABP), are markers for TSEs. Thus, the
invention provides a diagnostic assay for a TSE or the possibility thereof
in a sample of body fluid taken from a subject suspected of suffering from
the TSE, which comprises determining the concentration of heart or brain
fatty acid binding protein (H-FABP or B-FABP) in the sample. The method is
especially applicable to the diagnosis of CJD, especially new variant CJD,
in human patients, and to BSE in ruminant animals such as cattle.
Conveniently the method is carried out using an antibody to H-FABP or B-FABP,
whereby the extent of the reaction between the antibody and the FABP in the
sample is assayed and related to the concentration of FABP in the sample.
The concentration thus determined is used to make or assist in making a
The present invention enables an assay of high sensitivity, specificity and
predictive accuracy for CJD to be carried out. "Sensitivity" is defined as
the percentage of true positives given by the assay on samples taken from
patients in whom clinical examination has confirmed CJD. "Specificity" means
the percentage of true negatives given by the assay on control samples, i.e.
from patients in whom clinical examination has not revealed CJD. "Predictive
accuracy" means the ratio of true positives to total positives (true+false)
expressed as a percentage.
H-FABP is a known marker of acute myocardial infarction (AMI), see Ishii, J.
et al., "Serum concentrations of myoglobin Vs human heart-type cytoplasmic
fatty-acid binding protein in early detection of acute myocardial
infarction", Clinical Chemistry 1997;43 1372-1378. Therefore, in order to
use an assay for H-FABP for the diagnosis of CJD in humans to better
advantage, it is desirable to perform another kind of assay for AMI (one in
which the marker is not a FABP) in order to eliminate from the diagnosis for
CJD those patients who are positive in the AMI assay.
Thus, in a particular embodiment, the invention provides a method which
comprises determining the concentration of H-FABP in a first assay, as
defined above, whereby a positive result indicates either a CJD or acute
myocardial infarction, and which further comprises carrying out a second
diagnostic assay, for acute myocardial infarction (AMI) only, whereby a
positive result in the H-FABP assay and a negative result in the assay for
AMI indicates that the patient might be suffering from CJD. Assays using
Troponin-I and Creatine Kinase-MB (CK-MB) as early biochemical markers of
acute myocardial infarction (AMI) are well known and suitable for the above
A similar H-FABP and also a brain-specific fatty acid binding protein (B-FABP)
have been found in the brain of mice, see Pu, L. et al., Molecular and
Cellular Biochemistry 198, 69-78 (1999). Brain H-FABP (not to be confused
with B-FABP) is believed to differ from heart H-FABP by a single amino acid
substitution. However, B-FABP differs considerably. Sellner, P. A. et al.,
"Development role of fatty acid binding proteins in mouse brain" Dev. Brain
Res. 89, 33-46 (1995), estimated the DNA homology at 69%, while A. Schreiber
et al., "Recombinant human heart-type fatty acid binding protein as standard
in immunochemical assays", Clin. Chem. Lab. Mod. 36(5), 283-288 (1998),
mention 0.64% amino acid sequence homology and that a monoclonal antibody to
human H-FABP is cross-reactive with human B-FABP to the extent of only 1.7%.
Now that the present inventors have found that H-FABP is a marker for CJD,
it is a very reasonable prediction that B-FABP will also be. Since B-FABP is
specific to brain tissue and does not appear to react significantly with a
monoclonal antibody to H-FABP, it will not give positives for AMI, making a
separate assay for AMI unnecessary.
DESCRIPTION OF PREFERRED EMBODIMENTS
For the method of assay, the sample can be taken from any convenient body
fluid of the subject, but preferably plasma or serum (rather than whole
blood). Cerebrospinal fluid (CSF) is another useful fluid, particularly when
testing animals such as cattle.
The method is considered applicable to all types of TSE, including those
referred to above, and to any human or animal suffering or suspected of
suffering therefrom. Particularly, the invention is applicable to all types
of CJD in humans, including new variant, sporadic and genetic (familial).
Further, it is applicable to BSE in cattle and BSE-like disease in other
animals, e.g. deer.
The marker, H-FABP or B-FABP, is preferably measured by an immunoassay,
using a specific antibody to H-FABP and measuring the extent of the antigen
(H-FABP or B-FABP)/antibody interaction. For the diagnosis of human
patients, the antibody is preferably anti-human H-FABP or B-FABP. Similarly,
if the subject is an animal the antibody is preferably anti- to the H-FABP
or B-FABP of the same animal variety, e.g. anti-bovine H-FABP or B-FABP if
the patient is bovine. However, there is some cross reactivity of the
antibodies between species, often enabling a heterologous antibody to be
used: for example anti-rat/mouse H-FABP can be used to detect BSE in cattle.
It may be a monoclonal antibody (conveniently mouse) or an engineered
antibody. Preferably a mouse anti-human, anti-bovine etc. monoclonal
antibody is used. Antibodies to H-FABP are known, e.g. 66E2 and 67D3
described by Roos, W. et al., "Monoclonal antibodies to human heart type
fatty acid-binding protein", J. Immunol. Methods 183 149-153 (1995).
Antibody 66E2 is commercially available. Also, the usual Kohler-Milstein
method may be used to raise H-FABP or B-FABP antibodies. The source of
protein for this purpose can be the naturally derived or recombinant
DNA-prepared protein. Recombinant human H-FABP and B-FABP have been
described by Schreiber, A. supra and Shimizu, F. et al., "Isolation and
expression of a cDNA for human brain fatty acid binding protein (B-FABP)",
Biochim Biophys. Acta 1354, 24-28 (1997), respectively. Less preferably, the
antibody may be polyclonal.
Any known method of immunoassay may be used. A sandwich assay is preferred.
In this method, a first antibody to the FABP is bound to the solid phase
such as a well of a plastics microtitre plate, and incubated with the sample
and with a labelled second antibody specific to the H-FABP or B-FABP to be
detected. Alternatively, an antibody capture assay could be used here, the
test sample is allowed to bind to a solid phase, and the anti-FABP antibody
is then added and allowed to bind. After washing away unbound material, the
presence or amount of antibody bound to the solid phase is determined using
a labelled second antibody, anti- to the first.
In another embodiment, a competition assay could be performed between the
sample and a labelled FABP or a peptide derived therefrom, these two
antigens being in competition for a limited amount of anti-FABP antibody
bound to a solid support. The labelled FABP or peptide could be
pre-incubated with the antibody on the solid phase, whereby the FABP in the
sample displaces part of the FABP or peptide thereof bound to the antibody.
In yet another embodiment, the two antigens are allowed to compete in a
single co-incubation with the antibody. After removal of unbound antigen
from the support by washing, the amount of label attached to the support is
determined and the amount of protein in the sample is measured by reference
to standard titration curves established previously.
The label is preferably an enzyme. The substrate for the enzyme may be
colour-forming, fluorescent or chemiluminescent.
It is highly preferable to use an amplified form of assay, whereby an
enhanced "signal" is produced from a relatively low level of protein to be
detected. One particular form of amplified immunoassay is enhanced
chemiluminescent (ECL) assay. Here, the antibody is preferably labelled with
horseradish peroxidase, which participates in a chemiluminescent reaction
with luminol, a peroxide substrate and a compound which enhances the
intensity and duration of the emitted light, typically 4-iodophenol or
Another preferred form of amplified immunoassay is immuno-PCR. In this
technique, the antibody is covalently linked to a molecule of arbitrary DNA
comprising PCR primers, whereby the DNA with the antibody attached to it is
amplified by the polymerase chain reaction. See Hendrickson, E. R. at al.,
Nucleic Acids Research 23, 522-529 (1995) or Sano, T. et al., in "Molecular
Biology and Biotechnology" ed. Robert A. Meyers, VCH Publishers, Inc.
(1995), pages 458-460. The signal is read out as before.
In a particularly preferred procedure, an enzyme-linked immunosorbent assay
(ELISA) was developed to detect H-FABP in serum. Since H-FABP is a marker
for AMI as well, Troponin-I or CK-MB levels were assayed in order to exclude
any heart damage. As described in the Example, these assays were assessed in
serial plasma and CSF samples, from patients lacking AMI and CJD, patients
with AMI, patients with dementia and patients with confirmed CJD through
autopsy. The sensitivity, specificity and predictive accuracy for H-FABP in
CJD above a suitable cut-off level were all 100%. Thus, H-FABP detection
combined with the Troponin-I or CK-MB assay provides a useful serum marker
of CJD diagnosis or brain damage.
The use of a rapid microparticle-enhanced turbidimetric immunoassay,
developed for H-FABP in the case of AMI, Robers, M. et al., "Development of
a rapid microparticle-enhanced turbidimetric immunoassay for plasma fatty
acid-binding protein, an early marker of acute myocardial infarction", Clin.
Chem. 44, 1564-1567 (1998), should drastically decrease the time of the
assay. Thus, the full automation in a widely used clinical chemistry
analyser such as the "COBAS" MIRA Plus system from Hoffmann-La Roche or the
"AXSYM" system from Abbott laboratories should be possible and applied for
routine clinical diagnosis of CJD.
The H-FABP or B-FABP can be measured by other means than immunoassay. For
example, the sample can be subjected to 1 or 2-DE gel electrophoresis and
the amount of the FABP estimated by donsitometric scanning of the gel or of
a blot therefrom.
The assay of the invention can be used together with one or more other
pre-mortem assays for the TSE, including specifically those assays described
above. Such combined procedures are particularly useful in diagnosing BSE in
ruminant animals such as cattle.
Claim 1 of 9 Claims
1. A method of identifying a subject
suffering from a transmissible spongiform encephalopathy selected from the
group consisting of Creutzfeldt-Jakob disease (CJD) and Bovine Spongiform
encephalopathy (BSE) in a test subject comprising: (i) performing an
immunoassay for determining a level of brain-derived heart fatty acid
binding protein in a body fluid sample derived from said test subject
suspected of suffering from a transmissible spongiform encephalopathy
selected from the group consisting of CJD and BSE; and (ii) comparing the
level of brain-derived heart fatty acid binding protein in the body fluid
sample derived from said test subject to the level of brain-derived heart
fatty acid binding protein in a body fluid sample derived from a control
subject wherein the control subject does not exhibit a transmissible
spongiform encephalopathy selected from the group consisting of CJD and
BSE; wherein an elevated level of brain-derived heart fatty acid binding
protein in the body fluid sample derived from said test subject compared
to the level of brain-derived heart fatty acid binding protein in the body
fluid sample derived from the control subject, in combination with
clinical evaluation of acute myocardial infarction in the subject and
assaying for a marker of acute myocardial infarction in the subject and
obtaining a non-elevated level of the marker in comparison with a normal
control which is characteristic of the subject having no acute myocardial
infarction, identifies that the test subject suffers from the
transmissible spongiform encephalopathy selected from the group consisting
of CJD and BSE.
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