JPH0599922A - Immunochemical measuring method for human gp130 and kit therefor - Google Patents

Abstract

PURPOSE:To perform quick measurement by mixing a first material having affinity for human gp130, which is bonded to a solid supporting body, and a second material having affinity for the human gp130, which is bonded to a label material at the same time or later. CONSTITUTION:As a first material having affinity for human gp130, which is bonded to a solid supporting body, the composite body of monoclonal antibody or polyclonal antibody for the human gp130, a human interleukin 6(IL6) and a human IL-6 receptor and the like are used. As a detectable label material, which is bonded to a second material, the label materials such as, e.g. fluorescence material, light emitting material and light absorbing material, which are used in an ordinary immunoassay, are used. The measurement is performed with an optical detecting apparatus. The first material having the affinity for the human gp130 is mixed with a sample. The second material having the affinity for the human gp130, which is bonded to the detectable label material, is mixed at the same time or later.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to an immunochemical assay method for human gp130 and a kit therefor.

[0002]

2. Description of the Related Art Interleukin-6 (hereinafter abbreviated as IL-6) binds to IL-6 receptor on target cells,
Furthermore, by associating with gp130, which is a protein on the cell membrane, it induces various important physiological activities and is widely involved in cell proliferation and differentiation (Hirano et al., Im.
munology Today, 11, p443, 1990). Recently IL-
A thrombocytosis effect of 6 was discovered (Ishihashi et al., Blood, 74, p.
1241, 1989), and is expected as a new drug. In addition, it has been reported that abnormal production of IL-6 is a causative factor of various autoimmune diseases, and human IL-6 antibody is expected as a new drug that inhibits the action of IL-6 (B. Klein et al., Eur. Cytokine Net., 1, p193,
See 1990).

Recently, soluble IL-6 receptor was found in blood and urine, and the genetically engineered soluble IL-6 receptor bound to IL-6 and then associated with gp130 on the cell membrane. However, it was found that the IL-6 signal was transmitted (Yasukawa et al., J. Biochem. 108, p673, 1).
990).

As described above, in order to study the physiological activity of IL-6 in more detail, it is necessary to know not only the IL-6 concentration in the living body but also the gp130 concentration. Further, in order to develop IL-6 or an inhibitor of IL-6 as a medicine, it is necessary to measure gp130 in a biological sample.

[0005]

The structure and manufacturing method of IL-6 and the IL-6 receptor are known (IL
-6; JP-A-63-157996, JP-A-1-503354, IL-
6 receptor; JP-A-2-288898 and JP-A-3-133390
No.) and its measuring method (IL-
6; JP-A-3-135766, IL-6 receptor; Japanese Patent Application No. 2-
No. 128150).

On the other hand, with respect to human gp130, its structure (Japanese Patent Application No. 2-140069) and its antibody (Japanese Patent Application No.
No. 15090) is known, but its measuring method is not known.

[0007]

[Means for Solving the Problems] The present inventors have conducted extensive studies to provide a simple and accurate immunochemical assay method for human gp130 and a kit therefor, and carried out a monoclonal antibody against gp130 and a polyclonal antibody. The present invention, which is a immunochemical assay method for gp130 by appropriately combining a null antibody or a complex of IL-6 and IL-6 receptor and a kit therefor, has been completed.

That is, the present invention relates to a solid support,
Human gp130, wherein a sample is mixed with a first substance having an affinity for human gp130, and simultaneously or thereafter is mixed with a second substance having an affinity for human gp130 bound to a detectable labeling substance. Is an immunochemical assay. The present invention also includes a solid support having a first substance having an affinity for human gp130 bound thereto and a second substance having an affinity for human gp130 bound with a detectable labeling substance, Is an immunochemical assay kit for human gp130, which is stored in a container that provides a reaction space. Hereinafter, the present invention will be described in detail.

As the solid support used in the present invention, those used in ordinary immunoassay can be used. Specific examples thereof include a plastic support having a surface excellent in protein adsorption ability such as polystyrene and polypropylene, and an inorganic support such as a metal ceramic. The shape of the solid support is not particularly limited and, for example, in addition to spherical or disk-shaped ones, the wall surface itself of the plate (microtiter plate) having irregularities can be used as the solid support. Further, the size of the solid support is not limited.

In the present invention, as will be described later, the first substance having an affinity for human gp130 finally bound to a solid support, human gp130, and human bound to a detectable labeling substance forming a complex consisting of a second substance having an affinity for gp130,
At this time, it is necessary to separate the so-called free second substance that does not form the complex from the solid support. In order to easily carry out this separation operation, the solid support should be a solid support that can be easily separated from the solution phase, that is, a solid support consisting of a substance having a certain size and having a larger specific gravity than the solution For example, it is preferable to use a solid support containing a magnetic substance so that it can be separated from the solution by utilizing magnetic force.

The first substance having an affinity for human gp130 bound to a solid support is human gp1.
Examples thereof include a monoclonal antibody or polyclonal antibody against 30, a complex of human IL-6 and human IL-6 receptor, and the like.

The human IL-6 receptor used for this complex is gp1 by forming a complex with IL-6.
As long as it can bind to 30, it is possible that, for example, the soluble human IL-6 receptor in which the transmembrane region and the intracellular region of the cell are removed and the molecular weight of the soluble human IL-6 receptor is reduced due to steric hindrance. Is less and preferable. The complex can be easily prepared by simply mixing human IL-6 and human IL-6 receptor.

The antibody having an affinity for human gp130 and the complex of human IL-6 and human IL-6 receptor described above can be prepared according to the description of the above-mentioned documents and the like.

On the other hand, the second substance having an affinity for human gp130 bound to a detectable labeling substance is, like the first substance, a monoclonal antibody or polyclonal antibody against human gp130, human IL. -6 and human IL
It may be selected from a complex of -6 receptors. However, a combination of human IL-6 and human IL-6 receptor is used as either one, and a combination in which the recognition sites of human gp130 of the first substance and the second substance are different is selected.

In the present invention, a combination of two types of monoclonal antibodies having different recognition sites or IL-6 and IL
A combination of a −6 receptor complex and a monoclonal antibody having a different recognition site from the complex is particularly preferable for highly accurate measurement.

As the detectable labeling substance to be bound to the second substance, for example, a labeling substance such as a fluorescent substance, a luminescent substance, a light absorbing substance, a chemiluminescent substance, a radioisotope and the like used in a usual immunoassay can be used. .. Among these labeling substances, fluorescent substances, luminescent substances, light-absorbing substances, chemiluminescent substances and the like can be measured by ordinary optical detectors and are therefore preferable as the labeling substances. In addition, the enzyme used in the enzyme immunoassay is
Since the signal actually measured can be amplified, it can be exemplified as a preferable labeling substance.

The binding of the first substance to the solid support can be carried out by a conventional method. For example, the antibody dissolved in a PBS solution may be added to the wells of the plate and allowed to stand overnight. .. For immobilizing the complex of human IL-6 and human IL-6 receptor, after binding an antibody against IL-6 or an antibody against IL-6 receptor as described above, human IL-6 or human IL-6 -6 Receptor may be mixed and allowed to stand overnight.

In the present invention, human gp13, such as urine, serum, plasma, or a liquid containing crushed substances such as cell membranes, collected from human,
What can be expected to have 0 can be used as a sample.
In addition to the above, for example, a cell culture solution in the case of producing human gp130 using Escherichia coli or animal cells by a genetic engineering method can be used as a sample.

In the measuring method of the present invention, a complex consisting of the first substance, gp130 in the sample and the second substance bound to the labeling substance is first formed on the solid support. For this,
The sample may be first mixed with the solid support bound to the first substance, and then the second substance bound to the detectable labeling substance may be mixed thereto, or the sample, the solid support bound to the first substance from the beginning. The body and the second substance bound to the detectable labeling substance may be mixed at once. When the sample is mixed with the solid support bound to the first substance and then the second substance bound to the detectable labeling substance is mixed, the second substance bound to the detectable labeling substance is mixed. An operation of separating the solid support from the liquid phase may be performed before.

After forming the complex on the solid support as described above, the second substance bound to the free (non-complexed) label in the liquid phase is separated. When the solid support is a solid phase, this operation may be performed, for example, by suctioning and removing the liquid phase and washing the solid support with an appropriate buffer solution. When the solid support contains a magnetic substance, magnetic force may be applied to obtain the solid support, and the solid support may be washed with an appropriate buffer solution.

After that, the amount of the labeling substance in the complex remaining on the solid support was measured, and compared with the value when the same operation was performed for the known gp130, gp13 in the sample was compared.
The density (quantity) of 0 can be known.

However, when the second substance bound to the known amount of the labeling substance is used in the present invention, the second substance bound to the free labeling substance which does not form a complex on the solid support is used. Gp13 in the sample can also be measured
You can know the zero amount. In this case, the labeled amount in the separated liquid phase may be measured.

The immunochemical assay kit for human gp130 provided by the present invention is a kit for carrying out the assay method described so far. Therefore, this kit comprises at least a solid support bound to a first substance having an affinity for human gp130 and a second substance having an affinity for human gp130 bound to a detectable labeling substance. Has been. However, in order to simplify the transportation and control the quality of the reagent, the solid support bound to the first substance having an affinity for human gp130 and the human gp130 bound to the detectable labeling substance are It is preferable that the second substance having an affinity is housed in a container that provides a reaction space, and further contains an appropriate stabilizer, etc., and is sealed with an inert gas or the like in a dry state. Those which have been described are particularly preferable. Thus, in a kit containing a solid support bound to a first substance and a second substance bound to a detectable labeling substance in a container, a sample is placed in this container,
If the separation operation as described above is performed after leaving it for a suitable time,
After that, the gp130 can be measured only by measuring the labeling substance, and the kit is suitable for a quick and easy measurement of the gp130.

[0024]

INDUSTRIAL APPLICABILITY According to the immunochemical assay method for human gp130 of the present invention and the kit for the same, human gp in a sample is
130 quantities can be measured quickly and easily.

This means that, for example, a study of a complex ontogenetic mechanism and immune mechanism consisting of IL-6, IL-6 receptor and gp130, a therapeutic drug comprising IL-6 and IL-6 receptor or their diagnosis. It supports the development of medicines. In addition, naturally, early diagnosis of diseases related to gp130 and the like are also possible.

Further, according to the present invention, monitoring can be performed when establishing a method for genetically engineering a large amount of gp130, which is produced only in an extremely small amount in the natural state.

[0027]

EXAMPLES Examples will be shown below to explain the present invention in more detail, but the present invention is not limited to these examples.

Example 1. Production of rat polyclonal antibody against gp130 40 μg of CHO cell-derived soluble gp130 (Japanese Patent Application No. 3-217924) dissolved in PBS per rat for 2 weeks
Subcutaneous immunization was performed once, a total of three times, and one week later, whole blood was collected and the antibody was purified by a usual method.

Example 2. Immunochemical assay for gp130 using complex 2 μg / ml anti-gp130 monoclonal antibody AM64
(T. Taga et al., Cell, 63, p1149, 1990) containing 100 µl of coating buffer (0.05 M sodium carbonate, pH 9.6) per well of a 96-well microtiter plate and overnight at 4 ° C. I left it. 100 per well after washing
μl of 1% BSA-PBS was added and left at room temperature for 2 hours. After washing, 100 μl of a sample (CHO cell culture supernatant, etc.) was added and left at room temperature for 2 hours. After washing, IL-6 and IL-
100 μl of 1% BSA-PBS containing 5 μg / ml of each of 6 receptors was added to each well, and the mixture was allowed to stand at room temperature for 2 hours. After washing, add 100 μl of 5 μg / ml guinea pig-derived anti-human IL-6 receptor polyclonal antibody per well,
It was left overnight at 4 ° C. After washing, 100 μl / well of 1000 times diluted alkaline phosphatase-labeled rabbit-derived anti-guinea pig immunoglobulin antibody (Cosmo Bio)
In addition, it was left for 2 hours at room temperature. After washing, 1 mg / ml alkaline phosphatase substrate (Sigma) was added to 100 wells per well.
μl was added, and the mixture was allowed to stand at 37 ° C. for about 30 minutes, and then color development at 405 nm was measured. Soluble g derived from CHO cells purified as a sample
Figure 1 shows the results measured by this method using p130.

From FIG. 1, a monoclonal antibody against gp130 was used as the first substance, and human IL- was used as the second substance.
According to the measurement method of the present example using the complex of 6 and human IL-6 receptor, it can be seen that gp130 in the concentration range of 2.4 ng / ml to 156 ng / ml can be measured. Moreover, it can be seen that in this range, the signal increase is almost linear, and a highly sensitive measurement is realized.

Example 3. Immunochemical assay for gp130 without complex The procedure was the same as in Example 2 until the plate was coated with the antibody and the sample was added. This was allowed to stand for 2 hours at room temperature and then washed, and 1% BSA-containing 5 μg / ml of the rat-derived anti-gp130 polyclonal antibody obtained in Example 1.
100 μl of PBS was added to each well, and the plate was left at room temperature for 2 hours. After washing, 100 μl of a 1000-fold diluted alkaline phosphatase-labeled goat-derived anti-rat immunoglobulin antibody (Cosmo Bio) was added to each well, and the mixture was allowed to stand at room temperature for 2 hours. After washing, add 100 µl of 1 mg / ml alkaline phosphatase substrate (Sigma) to each well, and add about 30 at 37 ℃.
After leaving for a minute, color development at 405 nm was measured. Fig. 2 shows the result of measurement by this method using purified CHO cell-derived soluble gp130 as a sample.

From FIG. 2, a monoclonal antibody against gp130 was used as the first substance, and gp130 was used as the second substance.
According to the assay method of this example using a polyclonal antibody against gp1, gp1 in the concentration range of 2.4 ng / ml to 156 ng / ml was used.
It turns out that 30 can be measured.

Example 4. Immunochemical measurement of gp130 in human serum By the method described in Example 2, the concentration of soluble gp130 in serum was measured for 10 healthy persons (denoted as A to J). Table 1 shows the measured results.

[0034] From this table, it can be seen that the concentration of soluble gp130 in the serum of healthy persons is 22.0 ng / ml on average, which is within the range that can be sufficiently measured by the measuring method shown in Example 2 or Example 3. .. This means that gp in serum is measured by the assay method of the present invention.
130 shows that it is possible to know diseases that affect the concentration.

[Brief description of drawings]

FIG. 1 is a graph showing the measurement results of the method shown in Example 2,
The color development of the plate for each soluble gp130 concentration is shown. Open circles show the results when IL-6 and soluble IL-6 receptor were used at 5 μg / ml, and black circles show soluble I-6.
The results obtained when only the L-6 receptor was used at 5 μg / ml are shown.

FIG. 2 shows the color development of the plate for each soluble gp130 concentration as measured by the method described in Example 3.

Claims (4)

[Claims] 1. A human gp bound to a solid support and a sample with a first substance having an affinity for human gp130, and simultaneously or subsequently bound to a detectable labeling substance.
Mixing a second substance having an affinity for 130,
Immunochemical assay for human gp130. 2. A human g that recognizes different sites between a first substance and a second substance having an affinity for human gp130.
An antibody against p130.
The immunochemical assay method described in. 3. The method according to claim 1, wherein either the first or second substance having an affinity for human gp130 is a complex of IL-6 and IL-6 receptor. Immunochemical assay. 4. A solid support having a first substance having an affinity for human gp130 bound thereto and a second substance having an affinity for human gp130 bound with a detectable labeling substance, which are Is contained in a container that provides a reaction space, and an immunochemical assay kit for human gp130 is characterized.