JP3045172B2 - Chemical method for measuring human IL-6 using IL-6 receptor and kit therefor - Google Patents

Description

The present invention relates to a method for chemically measuring human IL-6 and a kit therefor.

[Conventional technology]

Interleukin-6 (BSF2 / hereinafter abbreviated as IL-6)
Is a protein having various important physiological activities and widely involved in cell proliferation and differentiation. Furthermore, it has been reported that abnormal production of IL-6 may be a causative factor of various autoimmune diseases (see Kishimoto, Blood, 74, pl, 1989).

Knowing the physiological concentration of IL-6, which exerts various physiological activities in vivo, is expected as a new diagnostic marker for various diseases. As a method for measuring the biological activity of IL-6, a highly sensitive method capable of measuring 100 fg / ml of IL-6 has been reported (T. Matsuda et al., Eur. J. Immunol., 18, p951, 1).
988). However, this method has the disadvantage that a large number of analytes cannot be measured quickly and easily. On the other hand, in the immunochemical measurement of IL-6 reported so far, antibodies obtained by immunizing various animals with recombinant (recombinant) IL-6 are used. Therefore, in the blood
Report that IL-6 is present by binding to α2-macroglobulin (T. Matsuda et al., J. Immunol., 142, p148).
As described above, there is a risk that the concentration of IL-6 in a biological sample expected to exist in a form different from that of recombinant IL-6 cannot be measured with high accuracy.

[Problems to be solved by the invention]

Therefore, the present invention provides a biologically active substance, that is, IL
An object of the present invention is to provide a chemical assay method for IL-6 which can be applied to any form of IL-6 as long as it has a property of binding to the -6 receptor, and a kit for use in the method.

[Means for solving the problem]

The present inventors have conducted various studies in order to solve the above-mentioned problems. As a result, by using IL-6 receptor, several hundreds of animals can be obtained without using antibodies obtained by immunizing animals with recombinant IL-6.
We have obtained a new finding that human IL-6 at a concentration of pg / ml can be measured, and based on this, completed the present invention.

Accordingly, the present invention provides for contacting a solid support having human IL-6 receptor immobilized thereon with labeled human IL-6 and an analyte, and binding the labeled support to the solid support via the human IL-6 receptor. Measuring the amount of human IL-6 or the amount of labeled IL-6 not bound to the solid support, and determining the presence or amount of IL-6 in the sample based on the measured value. The present invention provides a method for detecting or measuring IL-6 in a test sample.

The present invention further comprises binding the human IL-6 in the assay sample to the solid support via the IL-6 receptor by contacting the solid support on which the human IL-6 receptor is immobilized with the analyte, and IL-6 attached to a solid support
And a method for detecting or measuring human IL-6.

The present invention also provides a kit for performing the first method, wherein the kit comprises a solid support on which a human IL-6 receptor is immobilized and labeled human IL-6. .

The present invention further provides a kit for performing the second method, wherein the kit comprises a solid support on which a human IL-6 receptor is immobilized and an anti-human IL-6 antibody.

[Specific description of the invention]

1. Chemical measurement method of IL-6 The chemical measurement method of IL-6 is based on the physiological concentration of IL-6 contained in human serum, urine, other joint fluid, etc., and culture when human cells are cultured. It is intended to accurately and rapidly measure the IL-6 concentration in the supernatant.

In the measurement method of the present invention, IL- immobilized on a solid support is used.
Various methods using a specific binding reaction between IL-6 receptor and IL-6 are included. In a first embodiment, the IL-6 receptor immobilized on a solid support has a labeled IL
IL-6 competitively binds to IL-6 in an analysis sample, and labeled IL bound to a solid support via an IL-6 receptor
-6 amount or labeled IL remaining in the reaction solution without binding
-6 was measured, and IL-6 in the sample was determined based on the measured value.
Is determined (or concentration). According to a second aspect, the IL-6 receptor immobilized on a solid support contains
IL-6 is bound, and the amount of the bound IL-6 is measured by a conventional method.

In either embodiment, the IL-6 receptor is first immobilized on a solid support. As the solid support, a microtiter plate, various types of beads such as plastics such as polystyrene and polypropylene, inorganic materials such as metal ceramics, and the like, which are commonly used in immunoassays, can be used. When immobilizing the IL-6 receptor on a solid support, the IL-6 receptor may be directly immobilized, or a substance that binds to the IL-6 receptor, for example, an antibody against the IL-6 receptor is first immobilized, and then the IL-6 receptor is immobilized. The -6 receptor may be bound. IL
Immobilization of an antibody against the -6 receptor or the IL-6 receptor can be performed according to a conventional method. For example, these proteins may be dissolved in a PBS solution at a concentration of, for example, 2 μg / ml, added to the wells of the plate 100 μ at a time, and allowed to stand overnight.

Next, according to the first aspect, the immobilized IL-6
The receptor is brought into contact with a mixture of the analyte and the labeled IL-6 at a fixed ratio. Examples of the label of IL-6 include ¹²⁵ I and ³⁵ S. The amount of labeled IL-6 bound to the IL-6 receptor or the amount of unlabeled IL-6 not bound to the IL-6 receptor is competitive because the IL-6 and the labeled IL-6 in the assay sample competitively bind to the IL-6 receptor. It is a feature of the present invention to measure the concentration of IL-6 in the analysis sample by measuring the amount of IL-6. As a method for measuring the above labeled IL-6, ¹²⁵ I
In the case of labeled IL-6, after the binding reaction between IL-6 and IL-6 receptor for a certain period of time, the support is washed with a normal solution, and the radioactivity of the support is measured with a γ counter. ^In the case of 35S-labeled IL-6, it can be measured in the same manner.

In the second embodiment, the solid support on which the IL-6 receptor is immobilized is incubated with the analyte, and the IL-6 present in the sample is allowed to bind to the IL-6 receptor on the solid support. Next, the amount of IL-6 bound to the solid support is measured according to a conventional method. This measurement can be performed by various methods. For example, a rabbit-derived antibody against IL-6 is bound to IL-6 bound to a solid support, and then
A labeled sheep-derived antibody to rabbit immunoglobulin is bound to a rabbit-derived antibody IL-6 antibody bound to a solid support. As a substance for labeling a sheep-derived antibody, for example, enzymes such as alkaline phosphatase, horseradish peroxidase, β-D-galactosidase, and bition can be used. Known detection methods can be used. As a method for detecting these labeling substances, for example, any means known in immunoassays can be used.

2. IL-6 receptor IL-6 receptor is a protein on the cell membrane that specifically binds to IL-6 in vivo and is involved in IL-6 signal transduction. The IL-6 receptor suitable for the present invention may be purified from human-derived cells or the like by a biochemical method, or may be produced by genetic engineering. The protein is
Amino acid sequence portions other than the amino acid sequence portion that contributes to binding to IL-6 may be changed by substitution, deletion, or insertion. Examples of such an IL-6 receptor include a soluble IL-6 receptor detached from a cell membrane, that is, an extracellular portion of the IL-6 receptor. In addition,
For the IL-6 receptor, the primary structure of amino acid, DNA, etc. are disclosed in detail in the specification of Japanese Patent Application No. 1-9774.
It is disclosed in detail in the specification of 271862.

3. Assay Kit A kit for performing the method of the first aspect of the present invention comprises a solid support on which IL-6 is immobilized by an appropriate method, and labeled IL-6. The kit may further include a buffer for diluting the sample.

Further, a kit for carrying out the method of the second embodiment comprises a solid support on which IL-6 is immobilized by an appropriate method and a rabbit,
Includes anti-IL-6 antibodies derived from animals such as rats. The kit can further include a labeled second antibody to the animal antibody. However, it is not necessary to include this second antibody in the kit because it is easily available separately as a universal labeled antibody. The kit may further include a buffer for diluting a sample, a reagent for detecting a label, and the like.

〔Example〕

EXAMPLES Hereinafter, the present invention will be described in more detail with reference to Examples, but the present invention is not limited to these Examples.

Example 1. Chemical assay of human IL-6 using beads 500 mg of SPA beads (Amersham) suspended in a TBS buffer to which an anti-mouse immunoglobulin antibody has been bound, and a mouse-derived anti-human IL-6 receptor MT18 which is a monoclonal antibody (Y. Hirata et al., J. Immunol., 143, p2900, 1989)
20 μg) and rotated at 2 ° C. overnight. The next day, after washing with PBS to remove unbound MT18, the beads were suspended in 10 ml of PBS. Next, 20 μg of soluble IL-6
Receptor (see Japanese Patent Application No. 271865) and 2
It was left at ℃ for 4 hours. Subsequently, the assay buffer (PB
The beads were washed with (S / 0.1% BSA) to remove unbound soluble IL-6 receptor, and suspended in 10 ml of the same buffer. 50
μl of ¹²⁵ I-IL-6 (30000 cpm), 50 μl of known concentration
IL-6 solution, 100 μl of the above-treated beads, 100 μl
Were mixed and the light generated from the SPA beads due to ¹²⁵ I-IL-6 binding was measured in a β-scintillation counter.

FIG. 1 shows that the bound ¹²⁵ I-IL-6 decreases as the IL-6 concentration in the sample increases, that is, the IL-6 concentration in the sample can be measured by the present method.

Example 2. Chemical assay of human IL-6 using plate (method without using anti-IL-6 antibody) PBS containing 1 µg / ml of the above MT18 antibody was added to a 96-well microtiter plate at 100 µl / well. In addition, it was left at 4 ° C. overnight. After washing, add 100 μl of 1% BSA and add
Left at room temperature for hours. After washing with BPS / 0.05% Tween 20, the culture supernatant of CHO cells secreting the IL-6 receptor was added, and the mixture was allowed to stand at room temperature for 2 hours. PBS / 0.05% Tween20
After washing, a mixture of 50 μl of ¹²⁵ I-IL-6 (30000 cpm) and 50 μl of a known concentration of IL-6 solution was added to each well, and the mixture was allowed to stand at room temperature for 2 hours. PBS / 0.05% Tween2
After washing with 0, the radioactivity of each well was measured with a γ-counter.

FIG. 2 shows that as the IL-6 concentration in the sample increases, the residual radioactivity of the plate decreases, that is, the IL-6 concentration in the sample can be measured by this method.

Example 3. Chemical assay of human IL-6 using a plate (method using anti-IL-6 antibody) PBS containing 2 µg / ml of the above MT18 antibody was added to a 96-well microtiter plate at 100 µl per well. , Left overnight at 4 ° C. After washing with PBS / 0.05% Tween 20, 100μ
Was added and left at room temperature for 2 hours. PBS / 0.05
After washing with% Tween 20, the culture supernatant of CHO cells secreting IL-6 receptor was added, and the mixture was allowed to stand at room temperature for 2 hours. After washing with PBS / 0.05% Tween 20, IL-
6 solutions were added and left at room temperature for 2 hours. After washing, 5μg /
100 ml of rabbit-derived anti-IL-6 polyclonal antibody was added per well, and the mixture was allowed to stand at room temperature for 2 hours. PBS / 0.05
After washing with 20% Tween20, 100 μl of an anti-rabbit immunoglobulin antibody (Cosmo Bio) derived from sheep conjugated to alkaline phosphatase diluted 1000 times was added to each well.
Left at room temperature for 2 hours. After washing, 1 mg / ml alkaline phosphatase substrate (Cosmo Bio) was added to each well at 10 mg / ml.
After adding 0 μ and leaving the mixture at 37 ° C. for about 30 minutes, the color development at 405 nm was measured.

FIG. 3 shows that the color develops strongly with an increase in the IL-6 concentration in the sample, that is, the IL-6 concentration in the sample can be measured by this method.

〔The invention's effect〕

The chemical assay method for IL-6 provided by the present invention can be used for any form of IL-6 with high sensitivity and speed, as long as it has biological activity, that is, as long as it has the property of binding to the IL-6 receptor. This enabled multiple samples to be measured simultaneously. This method is useful for examining the physiological concentration of IL-6 in each disease, elucidating the role of IL-6 in a disease, and examining the effects of other drugs. In addition, this method
It is also possible to use a substance that inhibits the binding between IL-6 and the IL-6 receptor in a high-sensitivity and rapid search,
It is also useful for developing inhibitors of IL-6.

[Brief description of the drawings]

FIG. 1 shows that the amount of ¹²⁵ I-IL-6 bound to SPA beads when a sample containing no IL-6 was measured by the method described in Example 1 was 100, and that recombinant IL-6 derived from E. coli was used at various concentrations. The figure shows the amount of ¹²⁵ I-IL-6 bound to SPA beads when the contained sample was measured by the same method. FIG. 2 shows the amount of ¹²⁵ I-IL-6 remaining on the plate when a sample containing no IL-6 was measured by the method described in Example 2.
100 indicates the amount of ¹²⁵ I-IL-6 remaining on the plate when a sample containing recombinant E. coli-derived recombinant IL-6 at various concentrations was measured by the same method. FIG. 3 shows the color development of a plate when samples containing various concentrations of recombinant IL-6 derived from Escherichia coli were measured by the method described in Example 3.

Continuation of the front page (56) References JP-A-1-3198 (JP, A) JP-A-59-108959 (JP, A) JP-A-63-40858 (JP, A) JP-A-2-103465 (JP, A) , A) Science Vol. 241, No. 5867 (1988), p. 825-828 J CLIN INVEST, Vol. 84, no. 6 (1989) p. 2008-2011 (58) Fields surveyed (Int. Cl. ⁷ , DB name) G01N 33/53 G01N 33/566

Claims (5)

(57) [Claims] (1) contacting a solid support on which human IL-6 receptor is immobilized with labeled human IL-6 and an analyte;
Then, the amount of labeled human IL-6 bound to the solid support via the human IL-6 receptor or the amount of labeled IL-6 not bound to the solid support was measured. A method for detecting or measuring IL-6 in a sample, wherein the method determines the presence or absence or the amount of IL-6 in the sample. (2) contacting a human IL-6 receptor-immobilized solid support with an analytical sample to bind human IL-6 in the analytical sample to the solid support via the IL-6 receptor; A method for detecting or measuring human IL-6, comprising measuring IL-6 bound to a solid support. 3. The method for measuring IL-6 bound to a solid support by using an anti-IL
The method according to claim 2, wherein the method is performed using an antibody -6. 4. A kit for performing the method according to claim 1, comprising a solid support on which a human IL-6 receptor is immobilized and labeled human IL-6. 5. A kit for carrying out the method according to claim 2 or 3, comprising a solid support on which a human IL-6 receptor is immobilized and an anti-human IL-6 antibody. .