JPH03211461A - Method for deciding multiple sclerosis - Google Patents

Abstract

PURPOSE:To allow the monitoring of the disease state of the multiple sclerosis and the decision of the measure for medicine administrating and a treatment effect by taking a spinal fluid from a patient in the disease state of the multiple sclerosis and measuring the concn. of the human tumor necrossis factors in the spinal fluid. CONSTITUTION:The spinal fluid taken by lumber puncture from the patient in the disease state of the multiple sclerosis is rapidly frozen and preserved. The spinal fluid is thawed just before the quantitative measurement of the human tumor necrossis factor TNF-alpha and this spinal fluid specimen is subjected to the quantitative measurement of the TNF by an enzyme immunoassay method for the TNF-alpha by a two antibody method using a fluorescent substrate. The TNF-alpha exists at a high value in the spinal fluid of the multiple sclerosis patient than in the spinal fluid of other nervous diseases and further the TNF-alpha increases in the advanced stage as compared to the lysis stage and the monitoring of the diseases state of the multiple sclerosis is possible. This method is helpful for deciding the measure of the medicine administration and the therapeutic effect of the medicine.

Description

DETAILED DESCRIPTION OF THE INVENTION a. Industrial application field The present invention relates to human tumor necrosis factor
sis F actor-alpha This is referred to as “TN
By specifically measuring the amount of TNF-α in human cerebrospinal fluid using a quantitative measurement method for TNF-α (sometimes abbreviated as “F-α”), we hope to help diagnose multiple sclerosis and determine its pathological condition. It concerns a method for doing so.

b0 Background of the invention Multiple sclerosis (■ultiple 5 clerosis)
) is a typical demyelinating disease that frequently occurs in the age group of 15 to 50 years. Clinical symptoms are diverse and result from multiple lesions in the central nervous system. It is often accompanied by motor paralysis such as numbness in the limbs and visual impairment, but is not limited to these. The incidence rate in Japan is low at 3 to 4 per 100,000 population, but in Europe and America it is ten times higher.
Genetic factors are thought to be involved in the onset of this disease. However, the cause of the incident at the central office is unknown. In the focal area of the central office, intermembrane formation occurs, infiltration of cells such as macrophages, and destruction of miniphosphorus are observed. For this reason, MRI ←mag
eneticrecovance imaaino)
It is possible to perform a diagnosis by the central office using the . However, because there is no fundamental treatment available, once the disease develops, the disease often continues to progress, with repeated periods of remission and exacerbation. There is still no specific and simple testing method that can monitor the onset and progression of disease, and there is a strong need for its development.

On the other hand, TNF-α is a type of cytotoxic protein produced by macrophages, also called cachectin, and is a protein with extremely diverse biological activities.

Its activity broadly affects the activation of the body's immune system.

TNF-α production is induced by the action of endotoxins and bacterial cells on macrophages 1), but is regulated by cytodynamics such as interleukin 1 and gamma inkferon 2).

TNF-α promotes the specific expression of class I major histocompatibility antigen 3), induces the production of granulocyte-macrophage colony-stimulating factor 4) and IL-15), and decreases riboprotein lipase activity 6). It has many activities such as damaging tumor cells 7) and vascular endothelial cells 8) and acting as an endogenous fever factor 9).

1) [E, A, Carswell et al., Pr.
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Acad, sci, USA, 72.36
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] 3) ET, Coff1n et al., ProcJ
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Sc+, USA, 83. 446 (1986) ]
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1. 201 (1988)] 5) E Nawroth et al., J. EX.
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)] 6) [8, Beutler et al., Nature 3
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76, 1113 (1986)] 9) [D tnarello et al., J. Ex.
p, Med, 163°1433 (1986)
] TNF-α, which has such diverse activities, may play a central role in the body's defense mechanism against infection and the operation of the immune system in various diseases.
There has been a great deal of interest in measuring its amount in body fluids such as blood.

It has already been reported that the amount of TNF-α in body fluids such as blood increases in certain pathological conditions. Furukawa et al. found that serum TNF-α levels were increased in Kawasaki disease 10), and Eutler et al. reported that TNF-α is a mediator of endotoxic shock 11). In addition, S cuderi et al.
, Maury et al. 13)
, Waage et al. 14) and Alkwill et al. 15) reported that blood levels of TNF-α are increased in meningitis 14) and malignant tumors 15).

lo) [S, Furukawa et al.
, CIin, I m1un.

Ismnopathol, 48. 247 (1988
) ] 11) [B, Beutler et al., N
ature, 320. 584 <1986> ] 12) P. 5cuderi et al., The Lan
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[)ember 13. (198B) t),
1364] 13) [P, J, Maury et al., J.
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987) ] 14) [A, Waage et al., The L
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(1987) ii, 1229] Considering the biological activity of TNF-α, the present inventors investigated the possibility of involvement of TNF-α in multiple sclerosis. In severe lesions, macrophage infiltration is observed, symptoms of inflammation are observed, and there are many facts suggesting that the immune system is involved in the onset of the disease, so it is accumulated in the diseased area. We have come to believe that there is a high possibility that TNF-α exists in the cerebrospinal fluid.

C3 Objective of the Invention As described above, the present invention provides a method for determining multiple sclerosis and a method for determining its severity.

d0 Structure of the Invention Therefore, the present inventors collected cerebrospinal fluid from patients with various pathological conditions of multiple sclerosis, and conducted a highly sensitive quantitative measurement of TNF-α by collecting cerebrospinal fluid from patients with other neurological diseases. When we conducted a study, we found that TNF-α was present in the cerebrospinal fluid of multiple sclerosis patients at higher levels than in other neurological diseases, and that TNF increased during exacerbation compared to during remission. We have discovered that it is possible to monitor changes in , leading to the present invention.

Specifically, the present invention includes the following inventions.

(1) A method for determining multiple sclerosis by measuring human tumor necrosis factor in human cerebrospinal fluid.

(2) A method for determining the severity of multiple sclerosis by measuring human tumor necrosis factor in human cerebrospinal fluid.

The present invention will be explained in more detail below.

<Preparation of cerebrospinal fluid specimen> cerebrospinal fluid collected by conventional lumbar puncture may be used as is for measurement. If it takes time to measure, it may be frozen as soon as possible after collection, at -20°C or lower; if storage will be for a long time, it may be stored desirably at -70°C or lower, and used for measurement after thawing. It is preferable not to repeatedly freeze and thaw the specimen.

<Measurement of TNF-α> Any method can be used to measure TNF-α as long as it can specifically and sensitively quantify TNF-α. However, 1
A method that can quantitatively measure TNF at a concentration of ~100 H/d is essential for determining the disease state of the central government.
Desirable for measuring NF-α. Furthermore, when using a bioassay, it is desirable to confirm that the activity is due to TNF-α by a neutralization experiment using an anti-TNF antibody.

Preferably, the patent application filed earlier (Japanese Patent Application No. 1996-14
No. 6657: Filed on June 12, 1999: Title of invention “T
It is preferable to measure by the method described in ``Methods for Measuring NF-α, Kits, and Diagnostic Methods'', but any method may be used as long as it has sensitivity and specificity equivalent to or higher than this method.

<Determination of Multiple Sclerosis> The determination by the central government can be aided by measuring the concentration of TNF-α in the cerebrospinal fluid.

In other words, the central office has high concentrations of TN compared to other neurological diseases.
F-α is detected, and the higher the TNF-α concentration, the worse the disease state of the disease tends to be.

e0 Effects of the Invention According to the present invention, it is possible to monitor the pathological condition of multiple sclerosis, and it can be used as a guideline for administering drugs such as immunosuppressants and anti-inflammatory drugs used by the central government, and to help determine the therapeutic effects of the same drugs. Now you can.

f. Examples Hereinafter, the present invention will be described in detail with reference to Examples, but the present invention is not limited to the following Examples.

(TNF- in the cerebrospinal fluid of patients with multiple sclerosis and other neurological diseases)
Measurement of α concentration) 26 patients with multiple sclerosis, including 18 patients with relapsing type (exacerbation stage 1)
Cerebrospinal fluid was collected from 2 patients with remission, 6 with chronic progressive disease, 8 with chronic progressive disease, and 12 with other neurological diseases (7 with polyneuropathy, 5 with cerebrovascular disease) and promptly cryopreserved.

Immediately before the quantitative measurement of TNF-α, it was melted, and the previously filed patent application (Japanese Patent Application No. 1-146657 was published on June 12, 1999).
Quantitative measurement of TNF-α was carried out according to the method described in the patent application titled “Method for measuring TNF-α, kit, and diagnostic method”).

That is, using 50 μm of cerebrospinal fluid specimen, TNF-α was quantitatively measured by an enzyme immunoassay method (sensitivity limit: 3.4 pg/d) for TNF-α using a two-antibody method using a fluorescent substrate. The results are summarized in the table below.

<Measurement of the amount of TNF-α in the cerebrospinal fluid of patients> According to the above table, the TNF-α concentration in the cerebrospinal fluid of patients with multiple sclerosis is significantly higher than that of other neurological diseases, with a risk rate of less than 1%. It was also found that in the relapsing type, TNF-α was significantly increased during the exacerbation phase compared to the remitting type, with a risk rate of less than 0.1%.

Therefore, the measurement of TNF-α in cerebrospinal fluid according to the present invention is extremely effective for determining multiple sclerosis, determining the severity, and monitoring the disease state.

Claims (1)

[Claims] 1. A method for determining multiple sclerosis by measuring human tumor necrosis factor in human cerebrospinal fluid. 2. A method for determining the severity of multiple sclerosis by measuring human tumor necrosis factor in human cerebrospinal fluid.