GB2224837A - Immunoassay of body fluids - Google Patents

Abstract

A method of detecting and/or monitoring a demyelination process comprises testing a body fluid to assay for P2 protein and/or fragments thereof. The method uses preferably a polyclonal antibody raised against P2 protein. The method may be particularly useful in monitoring demyelination processes liable to occur in multiple sclerosis and viral diseases of the nervous system.

Description

IMMUNOASSAY OF BODY FLUIDS This invention relates to the immunoassay of body fluids and, in particular, to the diagnosis and monitoring of demyelination processes in diseases of the nervous system by in vitro testing of body fluids.

It has been known for many years that, in multiple sclerosis (disseminated sclerosis) and certain other diseases of the nervous system, the normal myelin sheath around the nerves is broken down. Myelin is a complex material comprising various proteins and lipids. There is a protein in myelin, called myelin basic protein (MBP), which is known to be released upon breakdown of the myelin. It has been suggested (Journal of Neurology, Neurosurgery, and Psychiatry 1988; 51 : 1334-1337) that cerebrospinal fluid MBP levels are probably an indication of the degree of demyelination in multiple sclerosis (MS) patients and its quantitation might be used to monitor disease activity.

However, MBP is in practice a very difficult material to measure with any accuracy and hence its potential for use in connection with monitoring MS and other demyelination diseases is very limited and not considered to be at all satisfactory.

I have now found that there is another breakdown product of myelin which can better be used to monitor demyelination processes, and which can further be used for diagnostic purposes.

According to the present invention, there is provided a method of testing a body fluid to detect and/or monitor a demyelination process, wherein the body fluid is assayed for P2 protein and/or fragments thereof. The assay will normally use antibody raised against P2.

P2 protein is a natural constituent of myelin in human beings and other mammals. Upon demyelination, the protein is released (together with other substances) either in relatively whole form or partly fragmented, and reference herein to the P2 protein released during demyelination includes reference both to any whole protein which may be present as well as any fragments of P2 protein. Thus, the assay of the invention is made on whole P2 or on fragments thereof, or on both.

There are various diseases of the central nervous system in which demyelination occurs. Among these is MS.

Hitherto, there has been no absolute clinical test for establishing the presence of MS: diagnosis has necessarily been dependent, in part at least, upon subjective assessment by the patient and doctor, and tests of a non-specific nature. Furthermore, there has been no objective reliable test for monitoring the activity of demyelination in MS. As a result, it has been difficult to assess whether drug or other therapy has been effective or not in the treatment or amelioration of the condition.

The method of the present invention is particularly useful in the diagnosis and monitoring of MS.

In particular, whilst in normal healthy individuals and in the majority of neurological diseases, P2 protein is undetectable in spinal fluid, in MS patients and particularly when the disease is active, the level of P2 protein in spinal fluid is grossly elevated. I do not know of any other disease state in which this is so. Thus, the method of the present invention can be used for the diagnosis of MS. It can also be used to monitor demyelination in MS patients for any purpose, especially to monitor the activity of the disease such as in relation to specific drug or other treatments.

The detection of P2 protein in a body fluid (preferably spinal fluid but other fluids such as serum may be used), and measurement of the amount of P2 therein, is preferably effected by an immunoassay procedure. As will be clear to those skilled in the art, many of the known immunoassay techniques can be used for an assay of this type. I prefer currently to use an ELISA technique (i.e. an enzyme-linked immunosorbent assay) and an example thereof will be described hereafter in detail. However, other label assays can be used including radioimmunoassays, fluoroimmunoassays and other enzyme-label procedures.

I have found that polyclonal antibody to P2, such as that raised in rabbits to the P2 of myelin (or the associated oligodendrocyte cells), is satisfactory in the ELISA technique on spinal fluid for the purposes of the present invention. Monoclonal antibody, made by the hybridoma technique using mouse cells, can also be used but is not at present preferred.

Whilst in the case of diagnosis of MS, a definite positive detection of the presence of a significant amount (as opposed to trace) of P2 protein in spinal fluid is normally adequate for a firm diagnosis, it is nevertheless preferred of course to obtain also some measure of the quantity of P2 protein. To this end, whatever immunoassay technique is used, reference will be made to standard results (i.e. to results obtained using accurately known quantities of P2 protein in the assay mixture).

Whilst the method of the invention is especially useful to diagnose and monitor MS, it can also with advantage be used to monitor the activity of demyelinating processes in diseases of the nervous system due to viruses.

The invention includes an immunoassay kit for carrying out the method of the invention, which kit includes antibody to P2 and other necessary means for effecting the immunoassay utilising the said antibody.

The method of the invention will now be described, by way of example only, with particular reference to diagnosis of MS.

Example The method of the invention was carried out by the ELISA technique on samples of spinal fluid obtained from various patients suffering from MS. Essentially, the technique consisted in coating wells of microtitre plates with serial dilutions of spinal fluid samples, contacting the coatings with rabbit anti-P2 protein antibody, then contacting the coatings with goat anti-rabbit-IgG antibody linked to an enzyme; and finally measuring the amount of enzyme so bound to the coating by contacting the coatings with a colourgenerating substrate. Measurement of the colour so formed gave an indication of the amount of P2 protein in the spinal fluid sample.

Details of the materials used and the method steps employed are as follows: Materials a. Coating buffer: For 1 litre 1.59 g Na2C03 2.93 g NaHC03 + 1 litre distilled water b. 'Sterilin' 96 well transparent microtitre plates c. Phosphate buffered saline + Tween (PBS-T) (0.1%) (Tween is a trademark.) 1.07g Na2HP04 0.39g NaH2PO4.2H20 8.5 g NaCl + 1 litre distilled water + 1 ml Tween 20 d. Substrate: p-nitrophenyl phosphate 1 mg/ml in diethanolamine buffer, pH 9.8 For 20 ml: 1.95 ml diethanolamine 16 ml H20 2 mg MgC12 + 0.1M HC1 to adjust pH to 9.8 + 1 mg/ml (i.e. 20 mg) p-nitrophenyl phosphate e. 3 M NaOH: 1.20 g NaOH + 10 ml H20 f. Polyclonal antibody to P2 protein, raised in rabbits g. Polyclonal IgG antibody to rabbit anti-P2 protein antibody, raised in goats, and then labelled with alkaline phosphatase h. P2 protein (for controls) i. Gelatine in PBS (dilution 0.01%) Method 1. The microtitre plate wells are first coated with various dilutions of spinal fluid (CSF) sample and control antigen (P2 protein) such that each well receives 100 ul. For this purpose, doubling dilutions of CSF in coating buffer are made up ranging from 1/20 to 1/40960. Similarly, doubling dilutions of control antigen in coating buffer were made up ranging from 1/200 to 1/409600. After incubation for 1 hour at room temperature and then overnight at 40C (the plates being covered with plastics film prior to incubation), the coated wells were washed three times with PBS-T.

2. A solution of gelatine in PBS is now placed in each well (100 ul per well) to block non-specific binding sites.

After covering the wells with plastics film and incubating for three hours at room temperature, the wells are washed three times with PBS-T.

3. Next, the polyclonal anti-P2 protein antibody is added to the wells at a dilution of 1/320 in PBS-T, using 1OOul per well. The wells are covered with plastics film and incubated at room temperature for 3 hours, after which they are washed three times with PBS-T. At this stage, those wells having P2 protein in their coatings will have antibody bound thereto.

4. There is then added to the wells a solution in PBS-T of the goat (anti-rabbit IgG) antibody-alkaline phosphatase conjugate at a dilution of 1/1000 and in an amount of lOOul per well. The wells are covered with plastics film and then incubated for one hour at room temperature and overnight at 4"C, after which the wells are washed three times with PBS T. At this stage, enzyme-labelled antibody will be bound to the anti-P2 protein antibody in the wells.

5. Finally, the enzyme substrate reagent is added to the wells (lOOul per well) and the wells stored in the dark at room temperature for 1 hour to allow the colour to develop.

At the end of the 1 hour period, the enzyme/substrate reaction is quenched using 50ul/well of 3M sodium hydroxide solution. Each well is then subjected to colorimetric assay by measuring its absorbance at 405nm in a spectrophotometer.

The absorbances obtained (for the CSF samples and antigen controls) are plotted against dilution.

6. As will be understood by those skilled in the art, in addition to wells having CSF and (as controls) P2 protein therein, there are also other wells which did not have any 0SF or P2 protein therein, but in which various combinations of reagents are placed for control purposes, i.e. to indicate any cross-reactions etc.

Results In the accompanying single figure drawing, I have shown the optical absorbance results plotted against a function of the sample dilution (actually -loglo dilution). It will be seen that there are three curves labelled, respectively, "high", "low" and "undetectable". Each curve is a composite of at least 10 individual curves from at least 10 different samples. I have not shown a curve for standard results (using P2), but such a curve is very close to the "high" curve.

When 0SF samples are analysed for P2 by the method of the invention, I have found that the shape of the absorbance/dilution curve obtained is closely similar to one of the three curves shown in the drawing. That is to say, there is seldom any difficulty in saying that the curve for any particular sample is of the "high", "low" or "undetectable" curve shape. 0SF samples giving curves of the "high" shape are only obtained from MS patients, and if the sample gives a curve of this shape, a positive diagnosis of MS can be made.

On the other hand, over 90% of samples which give curves similar in shape to the "undetectable" curve are from patients who do not have MS. Only 50% of patients whose 0SF samples give a curve of the "low shape" are diagnosed as MS.

Results to date based on assay of a large number of 0SF samples show that all those giving a curve of the high shape are from MS patients.

Claims (15)

CLAIMS:

1. A method of testing a body fluid to detect and/or monitor a demyelination process, wherein the body fluid is assayed for P2 protein and/or fragments thereof.

2. A method as claimed in claim 1, wherein the body fluid is assayed using antibody raised against P2 protein.

3. A method as claimed in claim 2, wherein the body fluid is assayed by an enzyme-linked immunosorbent assay.

4. A method as claimed in either claim 2 or claim 3, wherein the antibody is a polyclonal antibody.

5. A method as claimed in claim 4, wherein the polyclonal antibody is raised in rabbits.

6. A method as claimed in either claim 2 or claim 3, wherein the antibody is a monoclonal antibody.

7. A method as claimed in claim 6, wherein the monoclonal antibody is made by a hybridoma technique using mouse cells.

8. A method as claimed in any one of the preceding claims, comprising the preliminary step of preparing a set of standard results by assay of known quantities of P2 protein in an assay mixture, the method then being carried out on a body fluid and the results thereof compared with said standard results to give a quantified test result.

9. A method as claimed in any one of the preceding claims, wherein the demyelination process to be detected and/or monitored is that liable to occur in multiple sclerosis.

10. A method as claimed in any one of claims 1 to 8, wherein the demyelination process to be detected and/or monitored is that liable to occur in a vital disease of the nervous system.

11. A method of testing a body fluid to detect and/or monitor a demyelination process substantially as described herein and with reference to the Example.

12. An immunoassay kit for detecting and/or monitoring a demyelination process comprising antibody to P2 protein.

13. An immunoassay kit as claimed in claim 12, wherein said antibody is a polyclonal antibody.

14. An immunoassay kit as claimed in claim 13, wherein the polyclonal antibody is raised in rabbits.

15. An immunoassay kit substantially as described herein.