JPH021558A - Method of diagnosing multiple sclerosis and substance thereof - Google Patents

Abstract

PURPOSE: To diagnose the presence of multiple sclerosis quickly, simply and accurately using an antibody produced from an antigen substance composed of homogenized substance of lymphocyte of a multiple sclerosis patient. CONSTITUTION: An antigen substance specifically related with the lymphocyte of a patient diagnosed crinically as multiple sclerosis is obtained. The antigen accelerates quantitative formation of a specific antibody even for an immunologically active different kind of animal. A sample containing a specific antibody thus produced causes immunological reaction on the lymphocyte of a multiple sclerosis patient regardless of the state of patient at the time of sampling lymphocyte. When this antibody is employed in various immunological operations including immunoassay, fluorescent antibody assay, radiation immunoassay, etc., a multiple sclerosis patient can be diagnosed quickly, simply and accurately.

Description

DETAILED DESCRIPTION OF THE INVENTION BACKGROUND OF THE INVENTION The present invention relates generally to the diagnosis of multiple sclerosis symptoms in humans, and more particularly to novel antigens, antibody preparations, and novel methods useful for the diagnosis of multiple sclerosis. The present invention relates to epidemiological reagents containing antigens and antibodies.

multiple sclerosis
), often called infectious sclerosis, is morphologically characterized by patches of demyelination in the brain and spinal cord;
Clinically, it is a slow-progressing disease of the central nervous system characterized by many signs and symptoms that ebb and flow.

The etiology of multiple sclerosis is essentially unknown, and the disease may be caused by autoimmune mechanisms, slow viral infections, toxins such as metals, metabolic factors such as myelin division factors, and abnormal blood clots. This is thought to be due to vascular lesions caused by the mechanism.

Among the various manifestations of multiple sclerosis symptoms are sensory disturbances (especially vision), spastic limb weakness, cerebellar ataxia, nystagmus, bladder dysfunction, irritability, and two or more of these. There are more combinations than that.

Diagnosis of the condition is practically impossible due to the overlap of the above symptoms of multiple sclerosis and similar symptoms of other diseases. Diagnosis of a medical condition is often made on the premise of a ``classic'' medical history, consisting of remissions and worsening of various symptoms over many years, while systematically excluding the co-occurrence of other diseases that produce similar symptoms.

Sources of symptoms include, for example, intracranial lesions, cerebral vascular disorders, acoustic neuromas, cerebellar tumors, gliomas of the brainstem, lung tumors, amyotrophic lateral sclerosis, syphilis, pernicious anemia, cervical columnar arthritis. Collateral testing, which involves substantial cost and often discomfort to the patient, is performed to rule out disc rupture, platysma and hereditary ataxias.

Substantial efforts have been directed toward developing diagnostic methods and materials useful for early diagnosis of multiple sclerosis. A positive result on the colloidal test for cerebral spinal fluid is considered to support a positive diagnosis, but is not conclusive. The same applies to tests for elevated gamma globulin in the cerebral spinal fluid (which tends to confirm the diagnosis in advanced stages of the disease, but is not helpful in early diagnosis). . Similarly, active demyelination, which is often associated with this disease, is indicated by elevated cerebrospinal fluid basal protein assays, but test levels decline rapidly once the acute exacerbation ends. An inconclusive correlation exists between the presence of pathology and elevated levels of measles antibodies in serum and cerebral spinal fluid. Some investigators have also proposed that electron microscopy of lymphocytes for certain distinct morphological changes may provide an effective basis for the diagnosis of multiple sclerosis.

Recent advances in the field are reviewed in Angers et al., April 9, 1979.
Chemical and Engineering News CC
hemicaJ & Engineering New
s)” on page 22. Briefly summarized, Angers et al. used essentially non-specific blood extracts (so-called "multiple sclerosis-related substances °') from the blood of multiple sclerosis patients whose disease is reversing or in progress. Inhibition of leukocyte aggregation (Leuko-cyte adhere)
nce 1nhibitionHLAI) test. This LAI test involves measuring the decrease in the ability of test sample red blood cells to adhere to a glass surface after incubation in MSRM. This rather complicated, time-consuming and expensive operation is said to have a confirmation accuracy of about 90% for multiple sclerosis patients and about 95% for non-multiple sclerosis patients.

Thus, there is a very substantial need in the art for diagnostic methods and materials that quickly, simply, and accurately determine the presence of multiple sclerosis symptoms. Desirably, such methods should be highly specific for multiple sclerosis (ie, do not give false positive results in the case of other central nervous system diseases). Furthermore, such methods should be able to establish the presence of a disease state in its early stages, should be able to be used regardless of whether the disease is worsening or remitting, and should not require painful or dangerous removal of patient tissue samples.
Furthermore, it should preferably consist of standardized laboratory techniques that do not require expensive or difficult-to-operate equipment.

BRIEF SUMMARY According to the main features of the present invention, antibodies are provided that specifically immunologically react with antigenic substances associated with lymphocytes of patients suffering from multiple sclerosis. This antibody will be used to develop diagnostic reagents suitable for use in standardized immunological evaluations applied to lymphocytes from patients suspected of suffering from the disease.

Other features and advantages of the invention will be apparent to those skilled in the art upon consideration of the following detailed description.

DETAILED DESCRIPTION The basis for the practice of the present invention is, at least in part, to have a specific association with the blood (primarily lymphocytes) of patients clinically diagnosed as suffering from multiple sclerosis. It lies in the discovery of antigenic substances.

Furthermore, it has been found that this antigen can promote the quantitative formation of specific antibodies even in immunologically active xenospecies animals (eg rabbits). Samples containing specific antibodies generated in this manner are used to detect lymphocytes from multiple sclerosis patients, essentially independent of the patient's remission or deterioration status or stage of disease progression at the time of lymphocyte sampling. was found to immunologically react with Importantly, this antibody formulation is essentially immunologically unresponsive in non-MS patients, even those with central nervous system diseases associated with MS and often misdiagnosed. That's true.

The antibodies of the present invention are suitably used in various immunological procedures, including immunodiffusion assays, fluorescent antibody assays, radiation immunoassays, etc., each of which involves the development of reagents suitable for such uses. Dew.

The examples set forth below illustrate the practice of the invention according to certain currently preferred operations. More specifically, the examples address the preparation of lymphocytes for use either as antigen inocula or as patient test samples, the development of specific antibodies, and the use of antibodies in immunodiffusion assays.

Example 1 Lymphocytes used as antigen inoculum for developing antibodies and in diagnostic tests according to the invention are prepared by the procedures exemplified below. 10m1
A sample of venous blood is collected in a degassed tube and mixed thoroughly with, for example, 0.5 ml of 0.1 M sodium citrate.

Lymphocytes are prepared using lymphocyte preparation medium (Lymphocyte
It is separated from the blood using Pre-eparation Medium (Bionetics), centrifuged at constant speed, and the lymphocyte layer is removed. The cells are homogenized, the cell membranes are disrupted, and the homogenized lymphocytes are stored (at -80°C) until needed. The antigenic components of such lymphocyte preparations are extremely stable;
Immunologically “intact” (i.e.
ntact) and can withstand at least four "freeze-thaw" cycles (from 80°C to 22°C).

Example 2 Antibodies of the present invention are obtained by the following exemplary procedure. Example 1. 6 ml of homogenized lymphocytes obtained from one or more patients clinically diagnosed with multiple sclerosis were mixed with 4 ml of Freund's complete supplement (Fr.
eund's Complete Adjuvant) and mixed into a stable suspension. Each rabbit receives four 0.5 ml intramuscular injections of the lymphocyte/supplementary fluid suspension (two animals each) and 1 ml of homogenized lymphocytes without the addition of supplementary fluid is administered into the peripheral ear vein.

Intramuscular injections are repeated 7, 14, and 21 days after the initial inoculation. Two weeks after the last injection, the rabbits are sacrificed and blood is removed by cardiac puncture.

Serum is separated from whole blood and subjected to purification to remove non-specific antibodies and, in particular, those produced in the animal in response to human lymphocyte composition other than multiple sclerosis antigenic components. This separation is achieved, for example, by continuous adsorption contact with lymphocyte homogenates from non-diseased patients (controls). With 4 consecutive contacts per hour, e.g.
1 ml aliquot of control lymphocyte homogenate at 7°C.
4 ml of serum sample treated with
’. The antibodies thus obtained are quite stable. Preliminary electrophoretic analysis indicates that the specific multiple sclerosis antibody appears to be microglobulin.

Example 3 Diagnostic testing of a patient's lymphocyte sample is performed by the procedures exemplified below. A commercially available immunodiffusion medium (Clinical Science) was used, with one central well-like depression and six depressions radiating outward at uniform distances from the center depression. The gel medium used was high quality agar in 0.1N barbidol buffer.

A recess volume of about 0.12 m1 is suitable. Example 2
Approximately 0.15 ml of antibody (serum) is placed in the central well. Approximately 0.12 ml of lymphocyte homogenate obtained according to Example 1° from a clinically diagnosed multiple sclerosis patient is added to three peripheral wells. Each of the remaining three cells had approximately 0.1
2ml of lymphocyte homogenate is added. The medium is incubated at room temperature for 12 to 18 hours and then submerged in a 0.85% saline soak for 4 hours at room temperature. The medium is visually checked for the presence or absence of a precipitin zone between the center and peripheral wells. A band appearing between the central depression and the test sample depression and an identical band between the central depression and the multiple sclerosis control is indicative of multiple sclerosis in the patient. The absence of a band is taken to indicate the absence of a medical condition.

Example 4゜Example 3. The feasibility of the exemplary operation is demonstrated by the results of the following two series of tests. Table 1 relates to the results of 50 diagnostic tests performed on lymphocyte homogenates obtained from residents of the Erie, Pennsylvania area. As described, lymphocytes are present in patients with non-multiple sclerosis (°“normal°°) and clinically diagnosed multiple sclerosis (°“
patients with clinically diagnosed central nervous system pathologies other than multiple sclerosis ("Multiple Sclerosis"). Immunodiffusion test data are displayed as “°positive゛°” for a positive multiple sclerosis result and “negative” when there is no indicator precipitin zone. Test/Clinical Diagnosis Correlation is “°
It is indicated by +゛, and -゛ means the absence of such a correlation.

Sample Test Number Result 1 Positive 2 Negative 3 Positive 4 Positive 5 Negative 6 Negative 7 Positive 8 Negative 9 Positive 10 Negative 11 Negative 12 Positive 13 Negative Table 1 Multi-hardness 10 normal 10 normal Shiduo-hard 10 disease 10-hard 10-hard normal 10-hard normal Hard Normal Polyhard - (a) 34 Positive Polyhard 135
Negative multi-hard 36 positive multi-hard 137
Positive Multihard 138 Negative Multihard 39 Positive ゛ Multihard 140
Negative normal 141 Negative hard 42 Negative normal 143
Positive multihard 144 Positive multihard
145 positive hard 146
Positive multi-hard 147 positive
Morbidity 48 Negative Normal 149
Negative normal 150 Negative normal
10(a) The clinical diagnosis later changed to multiple sclerosis.

Table 2 below relates to the results of 33 clinical tests conducted on lymphocyte homogenates obtained from residents of the Kalamazoo, Michigan area. Again, the sample is normal,
Obtained from multiple sclerosis and other central nervous system patients.

Table Clinical Diagnosis Normal Normal Diabetes + SLE (a) + Morbidity Normal + (b) Normal + (c) Epilepsy, sickle cell normal - (b) SLE Test/Clinical Diagnostic correlation + + Negative Multi-hard negative Normal Ten-negative Multi-hard negative Morbidity Ten-positive SLE Negative Morbidity Ten-negative Normal Ten-negative Multi-hard negative SLE + Negative a-Year Ten negative SLE + Negative Normal Ten negative Parkinson+ Negative Morbidity Parkinson+ Negative Normal 10 Negative Normal 10 Negative Normal 10 Positive Polyhard 10 Systemic facial fixed erythema (b) Son of multiple sclerosis patient (c) Wife of multiple sclerosis patient In carrying out the present invention according to the above disclosure, It is anticipated that numerous modifications and changes will occur to those skilled in the art.

As described above, the antibodies of the present invention are expected to be useful in providing a variety of immunological diagnostic reagents. In addition to its usefulness in immunodiffusion assays, antibody-containing serum can be used in immunologically inert particles (
used to sensitize stabilized red blood cells, latex beads, etc.). Isolation and purification techniques well known in the art allow the active ingredient to be released in a more concentrated state when used in labeled form as a reagent in fluorescent antibody and radioimmunoassay clinical techniques. applied in antibody preparation to ensure the enrichment of specific antibodies from serum antibodies, together with antibody components that are likely to be present in substantially smaller concentrations in the serum of multiple sclerosis patients. It is also envisaged that it will be possible to isolate and activate specific lymphocyte antigens associated with It is also usefully used as a clinical reagent component, especially in competitive type immunoassays.

Such purified antigens are useful as therapeutic agents in the treatment of multiple sclerosis by a therapeutic technique commonly known as induction therapy. According to such techniques, low doses of disease-causing antigenic substances are administered with the aim of evoking a generalized systemic response in the patient.

Claims (1)

[Claims] A specific antibody that is capable of immunologically reacting with lymphocyte components that have a specific association with multiple sclerosis symptoms and that constitutes a clinical reagent consisting of antibodies formed by using lymphocytes from multiple sclerosis patients as antigens. An antigenic substance suitable as an inoculum for preparing target multiple sclerosis antibodies, the antigenic substance consisting of a homogenized product of lymphocytes of a patient suffering from multiple sclerosis.