EPREUVE DIAGNOSTIQUE POUR LA SCLEROSE EN PLAQUES

DIAGNOSTIC TEST FOR MULTIPLE SCLEROSIS

Abrégé anglais

2083583 9115765 PCTABS00007
A test for the diagnosis of multiple sclerosis by identifying an
abnormally high level of a metabolic protein having a molecular
weight of approximately 67,000 Daltons in blood plasma. A
treatment method employing introduction into the patient of a quantity of
purified like or related protein from related plasma.

Revendications

WO 91/15765 PCT/US91/02163


WE CLAIM:

1. A method of testing a suspected multiple
sclerosis patient for multiple sclerosis, comprising of
identifying the presence of a protein having a molecular
weight of about 67,000 Daltons in the blood plasma of
said patient.

2. The method of claim 1 including sampling
said patient's blood for identification of the presence
of said protein when said patient is in a fasting state.

3. The method of claim 2 wherein said patient
has been fasting at least eight hours.

4. The method of claim 1 wherein said protein
whose presence in said plasma is to be identified is one
whose concentration in the blood is responsive to food
intake by the patient.

5. The method of claim 1, including performing
gel electrophoresis of a blood sample from said suspected
multiple sclerosis patient and analyzing a gel prepared
from said blood sample to detect said protein in said
blood sample.

6. A method of treating a multiple sclerosis
patient comprising introducing into said patient purified
metabolic plasma proteins from the plasma of a normal
person.

Description

WO 91/1576~ PCI'/1,3S91/02D63
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DIAGNOSTIC TEST FOR MULTIPLE SCLEROSIS

TECHNICAL FIELD
The present invention relates to a test and
treatment for multiple sclerosis.

BACKGROUND ART
The suggestion that the circulatory system
might be involved in the genesis of multiple sclerosis
(MS) was first reviewed and amplified in 1948. This
theory is based on the presence o~ abnormal capillary
circulation in the nailbed and coldness of one or another
extremity, with or without neurological involvement. It
is based on early histopathological changes with thrombi
or changes of platelets in the micro vascular action at
or near sclerotic patches or areas of impaired blood~
brain barrier, and on frequent subcutaneous hemorrhages
in MS patients.
Blood plasma of NS infected persons was
observPd in the early 1950's to di~fer from no~m2l in
two-dimensional paper chromatography. Indirectly, this
was supported by studies with the red cell mobility test
in MS. These revealed that the electrophoretic mobility
of red cells was derived not from the red cells them-
selves but from the plasma. The incubation of MS red
cells in normal plasma returned their slow mobility to
normal. Conversely, incubation of normal red cells in MS
plasma reduced their mobility to the level observed in
MS. Finally, it was observed that infusion of normal
blood plasma into MS patients returned their slow red
cell mobility to normal. This indicated that the
abnormality was due to one or more unknown plasma
components.

DISCLOSURE OF THE INVENTION
The present invention recognizes a distinct and
reproducible difference between the plasma of MS patients




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and controls, consisting of the detectable presence in
fasting MS patients of a protein not found in fasting
control subjects. The protein has an apparent molecular
weight of around 67,000 Daltons, and has been tentatively
identified as a plasma~derived transfer protein. Thus,
MS can be diagnosed by testing for the presence of such a
protein in the blood plasma of fasting subjects.
The protein thus associated with MS exhibits
many of the characteristics of the previously isolated
phospholipid transfer protein (LTP II), [ref: Albers, et
al. Isolation and characterization of a phospholipid
transfer protein (LTP II) from human plasma, J. of Lipid
Research, Vol 29, 1988, pp 1593-1602], although it has
not been positively identified as such.
Furthermore, MS patients could be treated by
introducing a purified form of this or a related protein
obtained from normal plasma into patients with MS. This
treatment has not yet been clinically tested and proven~
but appears promising as a result of the changes seen in
blood samples obtained from ~S patients after such
laboratory treatment. This protein is the only
abnormality yet discovered in the plasma of MS patients/
and it is thought that it may in fact be the factor which
is altered to achieve the improvement seen to result from
clinical infusion of normal plasma into MS patientsO
It is therefore a principal object of the
present invention to provide a method for definitely
diagnosing Multiple Sclerosis.
It is another object of the present invention
to provide a treatment for MS patients.
The foregoing and other objectives, features,
and advantages of the invention will be more readily
understood upon consideration of the following detailed
description of the invention, taken in conjunction with
the accompanying drawings.




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WO91/15765 PCT/~S91/02163
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BRIEF DESCRIPTION OF THE DRAWINGS
FIG. l is a view of a densitometer readout of a
polyacrylamide gel of albumin deficient plasma from an MS
patient.
FIG. 2 is a representation of a polyacrylamide
gel of albumin, albumin-deficient plasma samples from MS
patients and controls, and molecular weight markers.

DETAILED DESCRIPTION OF THE INVENTION
In accordance with the present invention
multipIe sclerosis can be diagnosed definitely and at an
earlier stage than has previously been possible, by
- detecting the presence of a protein which is not present
in detectable quantities in fasting persons without MS.
The validity of this diagnosis was studied
using blood from 63 subjects. The samples used in this
study were matched for sex, age and diet for a total of
31 controls and 32 MS patients ranging in age from 23-63
years. Thirty percent in both groups were female and 80%
were on a low fat diet. The MS patient diet consisted of
20 grams of saturated fat per day or less; the controls
consumed up to 40 grams of saturated fat per day.
Sodium Dodecyl Sulfate Polyacrylamide Gel
Electrophoresis (SDS-PAGE) was used to 6eparate the
proteins of plasma samples from the subjects. Albumin
and other proteins were removed from the plasma samples
prior to electrophoresis and a scanning densitometer was
employed to read and evaluate the gels.
Readouts from the scanning densitometer showed
three differences between plasma from controls and that
from the MS patients. These differences were observed in
the portions of the plasma whose molecular weights were
in the range of 20,000-35,000 Daltons, and at approxi-
mately 67,000 and l00,000 Daltons. In the 20,000-35,000
Dalton range the difference consisted mainly of slight
differences in the peaks of low molecular-weight proteins




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WOgl/15765 PCT/US91/0211~
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in MS samples. However, they did not follow any set
pattern, and their significance is therefore doubtfulO
The peak near lO0,000 Daltons ~howed a slighk
decrease (5-10%~ in some MS patients when compared to the
- 5 controls; however, this decrease was only apparent in 67%
of the cases, and therefore is not conclusive.
The peak at approximately 67,000 Daltons was
the only consistent difference noted in the plasma of MS
patients. This peak is consistently a doublet in MS
patients (see FIG. l, peaks lO and 12), and is always a
singlet in the controls. In FIG. l peak lO represents
residual albumin in a plasma sample from a fasting MS
patient, and peak 12 represents the protein observed in
fasting MS patients but not in fasting subjects without
MS.
FIG. 2, a representation of a polyacrylamide
gel, shows a band 14 of an increased protein mass in
albumin-deficient MS plasma samples A, B, C, D when
compared to control samples E, F, G. By comparison to
molecular weight markers 16, the protein present in
samples A, B, C , D appears to have a molecular weight
around 67,000 Daltons. The plasma of MS patients
responds to food intake by exhibiting increasing amounts
of this protein. This protein was tentatively identified
as a plasma derived lipid transfer protein.
From the foregoing, it will be seen that a
definite and reliable assay can be employed for the
clinical diagnosis of MS, to detect the disease during or
perhaps be~ore the onset of symptoms. The easiest and
most reliable form of this assay would be one of several
well known antibody detection procedures. The assay
could take several forms, such as an Enzyme Linked
Immunoabsorbance Assay ~ELISA), immunodiffusion,
immunoelectrophoresis or other protein detection systems.




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WO91/15765 PCT/US9R/0~63
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EXAMPLE
Albumin was removed from the blood of a fasting
(greater than eight hours) subject with an Affi-Gel ~lu2
column from Pharmacia Biochemicals, Uppsala, Sweden, (1O5
x lO cm) according to the manufacturers directions. The
plasma proteins were then separated by ~odium dodecyl
sulfate polyacrylamide gel electrophoresis (SDS-PAGE),
with a 7.5% separating gel. Molecular weight markers
used during electrophoresis were prestained SDS-7B
obtained through Sigma Chemical Company. The resultant
gels were stained with coammassie blue, and destained in
10% acetic acid and 15~ methanol overnight. After
destaining, the gels were preserved by air drying between
sheets of biomembrane obtained from Biodesign of New York
in accordance with their protocol. This procedure was
performed with no heat added, in order to minimize
distortion of the gels. Gels were read and analyzed on a
Hoeffer densitometer GS365 with supporting software,
looking for the doublet peak at 67,000 Daltons indicative
of multiple sclerosis. The indicative doublet peak was
noted in the case of each MS patient and was absent in
each non-MS control sample.

The presence of the MS-associated protein
indicates an abnormality in the plasma. Because the
protein found in elevated quantities is metabolically
related, presence of the protein in MS patients in a
fasting state would indicate an abnormality in the
metabolism of MS patients, and could be indicative of an
abnormality in the protein. This is believed to be true
because it has been shown that patien~s on a low fat diet
have fewer and less severe exacerbations. Therefore
infusion of a complementary metabolic protein ~rom normal
plasma could stabilize the patient's metabolism.
The terms and expressions which have been
employed in the foregoing specification as used therein
as terms of description and not of limitation, and there



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WO91/15765 PCT/US~1/02163

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is no intention, in the use of such terms and
expressions, of excluding equivalents of the features
shown and described or portions thereof, it being
recognized that the scope of the invention is defined and
limited only by the claims which follow.




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