JP3104197B2 - Method for measuring complement receptor expression level - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for measuring the amount of complement receptor expression, and more particularly, to the expression of complement receptor expression for use in a method for detecting Kawasaki disease based on the degree of activation of polymorphonuclear leukocytes. How to know the degree of.

[0002]

2. Description of the Related Art Kawasaki disease is one of the intractable febrile diseases presenting with inflammatory symptoms accompanied by systemic acute rash such as erosion of oral mucosa, edema of limbs, swelling of lymph nodes, etc. observed in infants. The disease, sometimes sometimes exhibit serious symptoms such as mergers and fatal coronary artery aneurysms of meningitis, is a dangerous disease life-threatening, the cause has not yet been clarified, therefore the test
Intellectual method is also not only to determine Overall, etc. The above symptoms and laboratory values, rapid detection was difficult.

[0003] Furthermore, it is said that Kawasaki disease was apt to be misdiagnosed as another disease such as polyarteritis nodosa based on its symptoms, and that it was not uncommon for the patient to receive a wrong treatment and become life-threatening. It is believed that accurate methods of detecting the disease are needed.

[0004]

As described above, a rapid and accurate method for detecting Kawasaki disease is desired, but no effective method has been found yet.

[0005] By the way, "means for solving the problem"
As described in detail in the section, the present inventors have found that the expression level of complement receptor of polymorphonuclear leukocytes is higher in Kawasaki disease patients than in healthy subjects and patients with other diseases. As a method of quantifying the complement receptor, there is known a method of measuring the amount of active oxygen generated when polymorphonuclear leukocytes recognize complement-fixed zymosan as a foreign substance and phagocytose.

[0006] However, the expression level of the complement receptor varies among individuals and individual cells, and also varies depending on the state of cells. According to the above method, the absolute amount of the complement receptor can be measured, but it is not possible to know how much the receptor is expressed with respect to the ability of the cell.

It is an object of the present invention to provide a method for determining the degree of complement receptor expression and a method for rapidly and accurately detecting Kawasaki disease using the same.

[0008]

Means for Solving the Problems As a result of intensive studies to solve the above problems, the present inventor has found that the activation of granulocytes, especially polymorphonuclear leukocytes, in blood of Kawasaki disease patients is healthy. In addition, it was found that the complement receptor of polymorphonuclear leukocytes expressed a remarkable difference with respect to the maximum expression level (the expression level when a sufficient amount of cell chemotactic factor was adsorbed). Depending on whether it is possible to know the relative expression level of the complement receptor per cell,
The present invention has been completed.

[0009] That is, the present invention relates to a method of cell chemotaxis in which the amount of active oxygen generated in a reaction system in which blood or a blood component containing polymorphonuclear leukocytes and zymosan to which human complement is bound is mixed. This is a method for measuring the expression level of complement receptor expression in polymorphonuclear leukocytes, characterized by using, as an index, a value divided by the amount of active oxygen generated when a sex factor is added. Further, the present invention is characterized in that the amount of active oxygen generated is measured in a reaction system obtained by mixing blood or blood components containing polymorphonuclear leukocytes collected from a detection target with zymosan bound to human complement. Provide a method for detecting Kawasaki disease.

[0010] In the present specification, the term "complement receptor expression level" refers to the degree of expression of a complement receptor that is already expressed in the state in which a cell chemotactic factor is not acted, unless otherwise specified. Hereinafter, the present invention will be described in detail.

<1> Principle of the present invention The method for measuring the expression level of the complement receptor of the present invention is based on the fact that the expression level of the complement receptor varies among individuals, individual cells, and even the state of cells. By using as an index the degree of expression of the complement receptor of polymorphonuclear leukocytes relative to the maximum expression level (the expression level when a sufficient amount of cell chemotactic factor is adsorbed), It is to know the relative expression level of complement receptor.

In addition, the method for detecting Kawasaki disease of the present invention has found that the complement receptor expression level of polymorphonuclear leukocytes is higher in Kawasaki disease patients than in healthy subjects and patients with other diseases. Things.

[0013] The complement receptor is expressed on the cell surface of polymorphonuclear leukocytes. The expression level of this complement receptor varies depending on the state of the cell. When polymorphonuclear leukocytes and a cell chemotactic factor coexist, the cell chemotactic factor is adsorbed on the cell chemotactic factor receptor on the cell surface, and complementation is performed. The expression level of body receptors increases. It is known that most of the blood cells exhibiting such a reaction are polymorphonuclear leukocytes (Robert C. Allen et.al Current Opinionic Infe
ctions Diseases 1992, 5,389-398).

When polymorphonuclear leukocytes coexist with complement-fixing zymosan, complement receptors on the cell surface of polymorphonuclear leukocytes recognize complement-binding zymosan as a foreign substance and phagocytose it. As described above, polymorphonuclear leukocytes to which a cell chemotactic factor has been acted have an increased complement receptor expression level, and thus have an increased effect of phagocytosing complement-fixed zymosan.

At this time, when the cell chemotactic factor is added in an excessive amount to the number of cell chemotactic factor receptors of the polynuclear leukocytes, the phagocytic ability in the blood is maximized. The phagocytic ability at this time (hereinafter, also simply referred to as "maximal phagocytic ability") is divided by the phagocytic ability of polymorphonuclear leukocytes that do not act on cell chemotactic factors (hereinafter, also referred to as "basal phagocytic ability"). The amount of the complement receptor already expressed in the state where the chemotactic factor does not act can be shown as a ratio to the maximum expression level. Hereinafter, this value is referred to as a CL-R value. This CL-R value is nothing but the complement receptor expression level aimed at by the present invention.

The present inventor has found that this CL-R value differs depending on various diseases, and is particularly high in patients with Kawasaki disease, especially in other diseases. If basal phagocytic ability is high to some extent, this value may be able to detect Kawasaki disease, but in consideration of accuracy and versatility, CL-
It is preferable to use the R value as an index.

The phagocytic ability of polymorphonuclear leukocytes can be measured by quantifying the amount of active oxygen generated since zymosan phagocytoses. Further, the amount of generated active oxygen can be measured as luminescence intensity using an active oxygen amplifier or the like. As described above, by measuring the expression level of the complement receptor as chemiluminescence mediated by active oxygen, it is possible to perform highly sensitive and accurate measurement as compared with a conventional radioimmunoassay or the like.

<2> Method for Measuring the Expression of Complement Receptor of the Present Invention and Method for Detecting Kawasaki Disease In the method of the present invention, the phagocytic ability of polymorphonuclear leukocytes is measured, for example, by measuring the blood or blood components of a patient or their blood components. Diluent, complement-fixed zymosan, and active oxygen amplifier
The determination is performed by quantifying the amount of active oxygen generated in the mixed reaction system using an appropriate buffer or the like. At this time, when a cell chemotactic factor is added to the reaction system, the maximum phagocytic ability can be measured. Hereinafter, these reactions, reagents, materials, and the like used for the measurement and the measurement method will be described.

(1) Reagents and Materials Used in the Method of the Present Invention First, the reagents and materials used in the present invention will be described. The polymorphonuclear leukocytes used in the method of the present invention are collected from the blood of the detection subject. In addition, among blood cells, most of the phagocytosis is enhanced by the cell chemotactic factor is polymorphonuclear leukocytes, so blood may be used as it is, or a blood fraction containing polymorphonuclear leukocytes may be used. . In the present invention,
It is preferable to use blood or a diluent thereof because it is simple. The amount of blood may be, for example, about 100 μm obtained by diluting 100 times.

It is preferable to add a blood coagulation inhibitor such as heparin or EDTA (sodium ethylenediaminetetraacetate) so that blood collected from the detection subject does not coagulate.

The cell chemotactic factor used in the present invention includes:
For example, C5a, fMLP, LTB4, PAF and the like can be mentioned. Among them, C5a and fMLP are generally well-known and commercially available. The amount of these added at the time of measurement may be an excess amount with respect to the cell chemotactic factor receptor on the surface of the polymorphonuclear leukocyte, and may be 10 picomoles to 200 picomoles.
It is about picomolar.

The complement-fixing zymosan used in the present invention includes C ₃ b, C ₄ b, iC
Complement component such as ₃ b is obtained by zymosan bond. These can be easily obtained, for example, by adding zymosan to the serum from which the immunoglobulin fraction has been removed, reacting at 37 ° C. for about 30 minutes, and then washing with PBS. In addition, what is marketed can be used for zymosan. The amount added to the measurement system is 10 ⁷ to 1 in excess of the complement receptor.
0 ⁸ is appropriate.

When the phagocytic ability of polymorphonuclear leukocytes to zymosan is measured as the amount of active oxygen generated, an active oxygen amplifier is used together with the above blood or blood fraction, cell chemotactic factor and complement-fixing zymosan. Coexist. The active oxygen amplifier refers to a substance that emits light by being oxidized and excited by active oxygen, and examples thereof include luminol, luciferin, and resigenin. The appropriate amount of luminol added to the measurement system is 10 ^-4 to 10 ^-5 mol.

As means for measuring the luminous intensity,
A commercially available chemiluminescence meter may be used, for example, Biol-Mate LB953 (Berthold).

The above reagents may be purchased or adjusted individually, but commercially available kits such as AXIS
A kit (manufactured by Exos Emis (U.S.A.)) may be used. This kit contains at least C ₃ as a complement
b, complement-fixed zymosan containing iC ₃ b, and luminol and lucigenin as active oxygen amplifiers.

Since the active oxygen generated in the reaction system has extremely high reactivity, it is preferable to measure the emission intensity immediately.

(2) Basic phagocytic ability, maximum phagocytic ability and C
Method of calculating LR value In the method of the present invention, the phagocytic ability of polymorphonuclear leukocytes is measured by measuring the blood or blood components of a patient or a diluent thereof, complement-fixed zymosan, and an active oxygen amplifying agent. The determination is performed by quantifying the amount of active oxygen generated in a reaction system mixed using a buffer or the like. The value obtained without adding a cell chemotactic factor to this reaction system corresponds to basal phagocytic ability, and the value obtained when a cell chemotactic factor is added corresponds to maximum phagocytic ability.

In the above-mentioned measuring system, it is preferable to measure the background of the amount of luminescence, that is, the value measured without adding the complement-fixed zymosan, and subtract this from the value obtained above.

On the other hand, the CL-R value is calculated from these values by the following equation.

[0030]

## EQU00001 ## CL-R value = Basic phagocytic ability / Maximum phagocytic ability (1)

[0031]

Next, the action of the method of the present invention is described as
A description will be given together with specific measurement examples of the maximum phagocytic ability and the CL-R value.

Prepare four test tubes A, B, C and D,
Added Hank's solution containing active oxygen amplification agent 600μl in A, adding 10 ⁸ active oxygen increases additive medium and complement binding zymosan 600μl in B, the C 600
μl active oxygen amplifier added medium and cell chemotactic factor 1
0-100 pmol was added, keep adding active oxygen increases additive medium and complement binding zymosan 10 ⁸ and a cell chemotactic factor from 10 to 100 pmol of 600μl in D.

Separately, blood is collected and heparin or EDTA
A blood diluent is prepared by diluting an anticoagulant such as the above with a blood dilution medium 100 times. 100 μl of the blood diluent is added to A, and the luminescence intensity CL- (A) is measured immediately.

To B, 100 μl of a blood diluent is added, and the luminescence intensity CL- (B) is measured immediately. 100μ for C
1 of the blood diluent is added, and the luminescence intensity CL- (C) is measured immediately.

To D , 100 μl of the blood diluent is added, and the luminescence intensity CL- (D) is measured immediately. These CL-
From (A), CL- (B), CL- (C), and CL- (D), the basal phagocytic ability and the maximum phagocytic ability are determined by the following equations.

[0036]

## EQU00002 ## Basal phagocytic ability = [CL- (B)]-[CL- (A)] (2) Maximum phagocytic ability = [CL- (D)]-[CL- (C)]. (3) Further, the CL-R value is calculated from the above values according to the above equation (1).

<4> Measurement Example CL-R of blood of healthy subjects and various diseased patients by the above method
Table 1 shows examples of the measured values. In this measurement, the AXIS kit described above was used, and 20 cells were used as the cell chemotactic factor.
Picomolar C5a (manufactured by Exos Emis) was used, EDTA was used as an anticoagulant, and 10 ^-5 mol of luminol was used as an active oxygen amplifier.

[0038]

[Table 1]

From these results, it can be seen that the CL-R value changes depending on various diseases, the most characteristic of which is that this value is more prominent in Kawasaki disease patients than in patients with other diseases and healthy subjects. It is expensive. With this knowledge,
It turns out that Kawasaki disease can be determined by measuring the phagocytic ability, particularly the CL-R value.

Further, by measuring the amount of generated active oxygen as chemiluminescence having high detection sensitivity, it is possible to accurately measure the CL-R value. Conventionally, many studies on the relationship between Kawasaki disease and complement have been performed using radioimmunoassay, but differences in complement levels between Kawasaki disease and other diseases or between Kawasaki disease and healthy individuals have been found. Had not been.

Therefore, to clarify the features of the present invention, the expression level of the complement receptor is defined as the amount of active oxygen generated when polymorphonuclear leukocytes take up complement-fixed zymosan, and the amount of chemiluminescence is determined. Is used to measure the sensitivity and quantitatively, and the extent to which it is expressed relative to the maximum expression level of the complement receptor (the expression level when a sufficient amount of cell chemotactic factor is adsorbed). The purpose is to know the relative expression level of the complement receptor per cell by using it as an index.
In addition, the present inventors have found a causal relationship that polymorphonuclear leukocytes of Kawasaki disease patients have high expression levels of complement receptors, and use this finding and a method for measuring the expression levels of complement receptors for the detection of Kawasaki disease .

[0042]

Embodiments of the present invention will be described below.

[0043]

Example 1 A 1-year-old boy suspected of Kawasaki disease due to fever and the temperature did not decrease was measured and detected for the amount of complement receptor expression by the method of the present invention. 20 picomoles of C5a as cell chemotactic factor and 10 ^-5 as active oxygen amplifier
When the CL-R value was measured in the same manner as described above using a molar luminol and an AXIS kit, the value was 0.48, which was significantly higher. The diagnosis of Kawasaki disease was made based on the comprehensive judgment of the detailed and biochemical tests performed at the same time.

[0044]

[Example 2] For a one-year-old boy who had fever for 10 days,
Measurement and detection of complement receptor expression level by the method of the present invention
I know . AX using C5a as a cell chemotactic factor
When the CL-R value was measured using an IS kit, the result was 0.09.
Was low. At the same time, the patient was diagnosed with acute nonlymphocytic leukemia based on a detailed examination and a biochemical examination.

[0045]

Example 3 A 4-year-old girl with swelling of lymph nodes accompanied by fever, suspected of having polyarteritis nodosa, was measured and detected for the amount of complement receptor expression by the method of the present invention. When C5a was used as a cell chemotactic factor and the CL-R value was measured using an AXIS kit, it was significantly higher at 0.32. At the same time, comprehensive evaluation of the detailed and biochemical tests revealed Kawasaki disease.

As described above, it is clear that Kawasaki disease can be detected by the detection method of the present invention.

[0047]

According to the present invention, the expression level of the complement receptor can be measured not as an absolute amount but as a relative amount indicating the degree of expression relative to the ability of the cell. Further, the detection method of the present invention enables accurate and simple detection of Kawasaki disease.

Claims (4)

(57) [Claims] 1. An amount of active oxygen generated in a reaction system in which blood or a blood component containing polymorphonuclear leukocytes and zymosan to which human complement is bound is mixed, and a cell chemotactic factor is further added to the reaction system. A method for measuring the expression level of complement receptor expression of polymorphonuclear leukocytes, characterized in that a value obtained by dividing by the amount of active oxygen generated upon addition is used as an index. 2. The method according to claim 1, wherein an active oxygen amplifier is added to the reaction system, and the amount of active oxygen generated is measured by chemiluminescence. 3. A method of measuring the amount of active oxygen generated in a reaction system in which blood or blood components containing polymorphonuclear leukocytes collected from a detection target and zymosan to which human complement is bound are mixed. Kawasaki disease detection method . 4. The method according to claim 3, wherein a value obtained by dividing the amount of active oxygen by the amount of active oxygen generated when a cell chemotactic factor is further added to the reaction system is used as an index. For detecting Kawasaki disease.