CA2083583A1 - Diagnostic test for multiple sclerosis - Google Patents

Abstract

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.

Description

WO 91/1576~ PCI'/1,3S91/02D63 ~ 2~;3S~8~
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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 ~ 3 ~ ~35~3 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 . :, ,, . : :..... . ~. ,: ,. . , : .

WOgl/15765 PCT/US91/0211~

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., -, ~ ", 't 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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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 :~ . . - ,"'.1: .,.. : . . . . .: ..
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WO91/15765 PCT/US~1/02163 2~'8`~5 8 3 6 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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Claims (6)

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.