JPH03251763A - Method and kit for measuring cytokinin - Google Patents

Abstract

PURPOSE:To measure the concn. of a trace of cytokinin in blood of a healthy person with high sensitivity by using an anti-cytokin antibody subjected to affinity purification to immobilize the same on a solid phase and measuring cytokinin by chemoluminescence method or a bioluminescence method. CONSTITUTION:A column is packed with a substance having strong biological affinity to an objective component and a liquid mixture containing the objective component is allowed to flow in the column and a predetermined chemical agent is injected in the column to recover the objective component which is, in turn, subjected to affinity purification to obtain the anti-cytokin antibody immobilized on a solid phase. After cytokinin being an antigen is bonded to the anti-cytokinin antibody, a labelled antibody to which a label substance bonded is bonded to cytokin bonded to the antibody on the solid phase. Next, the label substance of the labelled antibody is subjected to luminescence if necessary by other reagent. A measuring limit is lowered by the use of the antibody subjected to affinity purification treatment. The quantity of light in luminescence reaction is measured to make it possible to indirectly measure the concn. of cytokinin. As the solid phase to be used, polyethylene beads are designated.

Description

DETAILED DESCRIPTION OF THE INVENTION A. Industrial Field of Application The present invention relates to a method for measuring cytokines and a kit for the measurement, and more particularly to a method for measuring minute amounts of cytokines with high sensitivity and a method for measuring the same. This relates to a kit for use.

B1 Summary of the Invention The present invention involves binding an affinity-purified anti-cytokine antibody immobilized on a solid phase and an antibody labeled with a labeling substance to a cytokine using an antigen-antibody reaction, and then chemically converting the labeling substance. By measuring by a luminescence method such as a luminescence method or a bioluminescence method, and measuring the concentration of the cytokine corresponding to the concentration, it is possible to measure extremely small amounts of cytokines.

C9 Prior Art Cytokines are physiologically active substances derived from lymphocytes or non-lymphocytes and have various biological activities such as cell division, proliferation, and suppression.

Cytokines include various factors, for example,
Colony stimulating factor (C3F) and interleukin (IL)
) etc. are known. C3F includes granulocyte colony-stimulating factor (G-C3F), which differentiates and proliferates granulocytes, macrophage colony-stimulating factor (M-C3F), which differentiates and proliferates macrophages, and granulocytes/macrophages that form both granulocytes and macrophages. Colony stimulating factor (0M
C3F), multi-colony stimulating factor that proliferates granulocytes, macrophages, and their precursor cells, pluripotent bone marrow stem cells.

In addition, IL includes ILl, whose main function is to induce the production of T cell growth factors from helper T cells, IL-2, which has the function of proliferating T cells, and IL, which is a growth factor for mast cells and bloodstream stem cells. -3. IL-4 is a factor that specifically produces 1gG-type and IgE-type antibodies, IL-5 is a factor that induces the proliferation and differentiation of B cells that produce antibodies, and IL-5 is a factor that induces the proliferation and differentiation of B cells that produce antibodies. Examples include IL-6, which is a factor that induces differentiation and promotes antibody production.

Cytokines exist in extremely small amounts in vivo, but they are factors that deeply influence immune regulation. Therefore, it is known that the concentration of cytokines in the living body increases or decreases depending on changes in the living body, although within a very small amount. Therefore, by measuring the concentration of cytokines, it is possible to accurately understand the condition of the living body, and it is expected to confirm the effectiveness of disease prevention and treatment.

Measurement of cytokines is also useful for determining the dosage in advance when administering cytokines as drugs into living organisms.

D6 Problems to be Solved by the Invention In light of the above points, it is important to clarify the clinical significance of cytokines, and in doing so, it is necessary to measure cytokine levels in the blood of healthy people.

For example, for G-C3F, a measurement kit based on a combination of an anti-G-C3F antibody that has not been subjected to affinity purification and a colorimetric method is commercially available, and the detection limit is 1,000.
It is possible to measure up to a sensitivity of pg/xg.

However, the concentration of cytokines in the blood of healthy people is
It is said to be several pg/il, and at present there is no highly sensitive measurement method that can measure cytokines in healthy people, so the clinical significance of cytokines is still unknown and practical clinical application has not been possible.

Means for Solving the E0 Problem The present invention provides an anti-cytokine antibody that is measured by a luminescence method such as a chemiluminescence method or a bioluminescence method, and that is affinity-purified to an anti-cytokine antibody immobilized on a solid phase used in the measurement. Furthermore, preferably, by preparing an anti-cytokine antibody immobilized on a solid phase using a solid phase immersion solution concentration in a specific range; antibodies and
This method provides a highly sensitive method for measuring cytokines by performing an antigen-antibody reaction with cytokines in a phosphate buffer with shaking, and then reacting with an anti-cytokine antibody labeled with a labeling substance.

Further, the present invention provides a measurement kit for carrying out the above-described method for highly sensitive measurement of cytokines.

That is, it is a measurement kit consisting of an anti-cytokine antibody that has been subjected to affinity purification treatment for immobilization on a solid phase, and an anti-cytokine antibody labeled with a labeling substance that enables luminescence detection. In addition, if necessary, cytokines as standard antigens at various concentrations for creating a calibration curve, substrates for enzymes required when enzymes are used as labeling substances, and buffers necessary for antigen-antibody reactions can be added to the kit. . Beads, plates, and tubes can also be attached as solid phases. In addition, the capacity and concentration of the constituent components of the measurement kit of the present invention can be determined as appropriate depending on the purpose.

F1 effect (1) Affinity purification of antibodies involves filling a column with a substance that has a strong affinity (biological affinity) for the target component, pouring a mixture containing the target component, and removing the target component remaining in the column. In this method, a chemical that has the role of removing the substance is poured into the next process, and the target component is recovered.

By affinity purification, only those anti-cytokine antibodies that specifically bind to cytokines can be extracted and used. This increases the affinity with the antigen and enables highly sensitive measurements using luminescent methods. be able to.

(2) When obtaining an anti-cytokine antibody immobilized on a solid phase, the solid phase is immersed in a buffer solution with an affinity-purified anti-cytokine antibody at a concentration of 0.3 to 10 (μg/jIσ).

This is clear from FIG. 2 of Example 2 later,
Outside the above concentration range, the detection limit of measurement using the luminescence method increases, resulting in several pg/m (1 or less, especially 5 pg/jI).
This is because detection below e becomes difficult.

In addition, methods for immobilizing antibodies on solid phase include physical adsorption,
Alternatively, conventional methods such as chemical bonding methods such as glutaraldehyde method and polylysine method can be used.

The solid phase used here is commonly used beads,
A plate or tube can be used, and its material can be plastic such as polystyrene or polyethylene, glass, or others.

(3) Measurement of cytokines according to the present invention is carried out by antigen-antibody reaction and luminescence method as follows.

■ First, a cytokine, which is an antigen, is bound to an anti-cytokine antibody (hereinafter referred to as a solid-phase antibody) immobilized on a solid phase, and then an anti-cytokine antibody labeled with a labeling substance is added to the cytokine bound to this solid-phase antibody. (Hereinafter referred to as labeled antibody.)
combine.

(2) Next, the labeled antibody bound to the cytokine is made to emit light using a labeling substance and, if necessary, other reagents. Although the labeled antibody does not need to be an affinity-purified antibody, it is preferable to use an affinity-purified antibody in order to further lower the measurement limit.

■Measure the amount of light generated by the luminescence reaction in (■), and indirectly measure the concentration of cytokines.

The reaction between the solid-phase antibody and the cytokine is preferably carried out in a phosphate buffer and under shaking conditions. This is because, as shown in Examples below, it is possible to detect lower concentrations than when using other buffers, such as boric acid buffers.

Moreover, uniform and sufficient specific binding can be obtained by shaking.

(4) The above luminescence method includes a chemiluminescence method and a bioluminescence method, and the concentration of the cytokine is measured by measuring the amount of light generated according to the following reaction formula.

(2) What is directly measured by the luminescence method is a labeled substance, and the labeled substance refers to a substance that is labeled with an anti-cytokine antibody. Also itself 1O-1811012/112
It is preferable to use a compound that can be detected to a certain degree and that does not reduce its activity when labeled with an anti-cytokine antibody.

(2) When using bioluminescence as a detection means, the labeling substance is an enzyme such as firefly luciferase that directly emits light upon reaction with a substrate.

In addition, when using the chemiluminescence method, the labeling substance is a chemiluminescent substance such as acridinium ester, isoluminol derivative, dioxysecn derivative, or glucose oxidase (hereinafter referred to as Oxidizing enzymes such as C0D) can be used.

Substrate 10 Enzyme → Hydrogen peroxide In addition, as a catalyst, those used in ordinary chemiluminescence methods such as potassium ferricyanide and peroxidase can be used.

G. Examples Next, examples will be described using cytokines as examples, but the present invention is not limited to these examples in any way.

Example 1 (A) Affinity purification treatment of anti-G-C3F antibody CNB r-Sepharose 4B (manufactured by Pharmacia) coupled with G-C3F was used as a packing material,
A solution of G-C3F antiserum is poured into the column, and the anti-G-C3F IgG remaining in the column is collected.
C3F IgG to anti-G CSF F(ab')
2 and anti-G-C3FFab' were obtained.

(B) Preparation of solid phase antibody ■ Anti-G CS affinity purified in (A) above
Polystyrene beads (φ-6, 5 mm) were added as a solid phase in a borate buffer (pH 8,5) containing FF(ab')2 or non-affinity purified anti-G-C8F(ab')2. Soaked and stored overnight.

■ Next, the solid phase in ■ is washed with borate buffer containing bovine serum albumin (BSA), and affinity-purified anti-G-C3F (ab') or non-affinity-purified anti-G-C5FF ( A solid phase antibody of ab')2 was obtained.

(C) Preparation of enzyme-labeled antibody Preparation was carried out by the maleimide method using COD as a labeling substance. That is, ■ 0,1 mol/l phosphate buffer containing maleimidized COD (
A dimethylformamide solution containing a crosslinking agent is added to the solution (pH 7.0) to introduce a maleimide group into the COD.

■(i) Phosphate buffer (0.1 mo (1/(1, f) H6,0) with β-mercaptoethylamine 1,1 mg, EDTA5jI
0,1 mo+2/(l phosphate buffer (pH 6,0) containing m6Q/12 was added and stirred at a temperature of 37°C.

(ii) Next, desalting was performed using a PD-10 column (manufactured by Pharmacia) filled with Sephadex G-25.

■ Preparation of COD-labeled anti-G-CS 'F antibody - (i)
0,1 m containing 31 g of maleimidized COD of ■
o(!/0 phosphate buffer (pH 6,0) with ■ anti-G-
0,1 mo1 containing C3F Fab'5.4*y
2/ Add 1 phosphate buffer, (ii) Add 0.1mol 12/12 phosphate buffer (pH 6,0). Stir at 30°C and leave at 4°C.

(iii) Add the mixed solution of (ii) above to TSK G
It was purified using a 3000SW (manufactured by Tosoh) column to obtain a COD-labeled anti-G-C3F antibody.

(D) Solid-phase antibody obtained in (B) above and C obtained in (C)
After performing an antigen-antibody reaction using the OD-labeled antibody according to the following procedure, the detection limit of G-C3F was measured by a chemiluminescence method.

(2) The solid phase antibody obtained in (B) above was added to the G-C3F solution.

(2) A phosphate buffer (pH 7.0) was further added to perform an antigen-antibody reaction between the solid phase antibody and G-C3F.

(2) Next, the solid phase was washed, and the COD-labeled antibody obtained in (C) was added to perform an antigen-antibody reaction with G-C8F bound to the solid phase at room temperature.

■ Furthermore, wash the solid phase from ■ and transfer it to another test tube.
Add 5 mol/1 glucose and add C of the labeled enzyme.
Reacted with OD.

■ Sampling was performed after adding 0.15N HCA' to the solution of ■■, and 2
/A'luminol solution and 6X 10"3mo7/l
Add KsFe(CN)s solution of
The detection limit of F was measured.

■ The results are shown in Table 1 and Figure 1. For comparison, Table 1 also shows the measurement results by the colorimetric method. in this case,
゛As a labeling enzyme, horseradish peroxidase (H
RP) was used.

Table 1 ("F-test") From Table 1, measurement by the chemiluminescent method was more sensitive than measurement by the colorimetric method, and could be measured down to low concentrations.

In addition, anti-G-C3 with affinity treatment applied to the solid-phase antibody
When the F antibody was used, it was possible to measure even lower concentrations than when the solid phase antibody was not subjected to affinity treatment.

Example 2 In the purification of the solid phase antibody in Example 1 (B), the relationship between the concentration of the anti-G-C3F antibody immersion solution in which the solid phase was immersed and the detection limit was investigated.

■ Affinity-purified goat anti-G-C3FF (
ab') = 0, 1 mo (1/ρ boric acid buffer 1 l
1 (pH 8,5) to the following concentrations.

Concentration; 0.05.0.1.0.5.1.5.10.2
Polystyrene beads (φ-6, 5zm) were immersed in solutions with concentrations of 5 (μg/mc) 1 and 2 and left overnight.

■ Solid phase of 0.11Ilo1 containing 0.1% BSA
Solid-phase antibodies for each immersion solution concentration were obtained by washing with 2/Q borate buffer (pH 8,5).

■ Next, use COD as the labeled enzyme, affinity-purified goat anti-G-CSF Fab' and non-affinity-purified goat anti-GC3F Fab as antibodies.
An enzyme-labeled antibody was obtained using the method shown in Example 1 (C).

(2) The detection limit of G-C3F was measured by the method of Example 1 (D) using the solid-phase antibodies and enzyme-labeled antibodies of (2) and (2). The results are shown in Figure 2.

The antigen-antibody reactions of the solid-phase antibody, enzyme-labeled antibody, and G-C3F were performed in a phosphate buffer (pH 7.0) under shaking.

From Figure 2, the concentration of the immersion liquid is 0.3 μg/x (1 to 10 μg/x).
g/x (number pg/j when there is I 12 or less, special 5
It is possible to measure C3F at a concentration of p g/xQ or less.

Example 3 In the antigen-antibody reaction of Example 1 (D), the solid phase antibody and G
The detection limits of G-C3F by the chemiluminescence method were compared when the -C8F reaction was carried out in a phosphate buffer under shaking conditions and when the reaction was carried out using a borate buffer.

Solid phase antibody affinity goat anti-G CS F F (
ab') labeled antibody COD-labeled affinity goat anti-G
-C3F Fab' (hereinafter in the margin) Table 2 From Table 2, if the antigen-antibody reaction is performed in a phosphate buffer under shaking conditions, the detection limit can be further lowered and measurements can be made to low concentrations.

Example 4 6.5■φ polystyrene balls with 0.3μy/x(1
~10 μ9/112 affinity purified anti-G-C3F
Solid-phase antibody prepared by immersion in antibody solution, solution of COD-labeled anti-G-C8F antibody prepared by maleimide method, standard antigen (1,0,5,0,10,25°50p9/Iris)
A G-CSF measurement kit consisting of the following was created.

Example 5 (A) Affinity purification treatment of anti-IL-2 antibody CNB
IL-2 in r-Sepharose 4B (manufactured by Pharmacia)
A solution of an anti-IL-2 IgG fraction (manufactured by Collaborative) is poured into the column using a coupled material as a packing material, and the anti-IL-2 IgG remaining in the column is collected. IL-2F(ab')
t and anti-IL-2 Fab'.

(B) Preparation of solid phase antibody ■ Anti-IL 2 affinity purified in (A) above
Borate buffer containing F(ab')2 (pH 8,5)
Inside, polystyrene beads (φ6, 5 x
Soak R) and set aside overnight.

(2) Next, the solid phase in (2) is washed with a borate buffer containing bovine serum albumin (BSA) to obtain an affinity-purified anti-IL-2F (ab') solid phase antibody.

(C) Preparation of enzyme-labeled antibody Preparation is performed by the maleimide method using COD as a labeling substance. That is, (1) A dimethylformamide solution containing a crosslinking agent is added to a 0.1 mo+2/Q phosphate buffer (pH 7.0) containing maleimide-formed COD to introduce a maleimide group into the COD.

■(i) Phosphate buffer (0.1 m
β-mercaptoethylamine 1.1 mg, EDTA 5xmo (2/12) in 0.1 mo (1/(l) phosphate buffer (pH 6,0)).
0) and stirred at a temperature of 37°C.

(ii) Next, desalting is performed using a PD-10 column (manufactured by Pharmacia) filled with Sephadex G-25.

■Preparation of COD-labeled anti-IL-2 antibody (1)
1 mo12/ (l phosphate buffer (pH 6,0)
0,1 containing anti-IL-2 Fab' 5.4m9
Add mo12/(1 phosphate buffer), (ii) Add 0.1 moC/12 phosphate buffer (
pH 6,0). Stirring was carried out at a temperature of 30°C,
Leave it at ℃.

(iii) Add the mixed solution of (11) above to TSK G
Purification is performed using a 3000SW (manufactured by Tosoh) column to obtain a COD-labeled anti-IL-2 antibody.

(D) Solid-phase antibody obtained in (B) above and C obtained in (C)
After performing an antigen-antibody reaction using the OD-labeled antibody according to the following procedure, the detection limit of IL-2 is measured by chemiluminescence method.

(2) Add the solid phase antibody obtained in (B) above to the IL-2 solution.

(2) A phosphate buffer (pH 7.0) is further added to perform an antigen-antibody reaction between the solid-phase antibody and IL-2.

(2) Next, the solid phase is washed, and the COD-labeled antibody obtained in (C) is added to perform an antigen-antibody reaction with IL-2 bound to the solid phase at room temperature.

■ Next, wash the solid phase from ■ and transfer it to another test tube.
Mo12/12 glucose is added and reacted with the labeling enzyme COD.

■ Sampling was performed after adding 0.15N HCl2 to the solution of ■■.
Q-luminol solution and 6x 10-'mo(2/
1! KJe(CN)a solution was added, and the luminescence amount after 163-45S was measured with a luminometer (manufactured by Meidensha). L-
Measure the detection limit of 2.

Using this system, the detection limit is 1 to 10 (T) g/x12)
IL-2 can be measured.

Example 6 6.5xxφ polystyrene balls with 0.3μ9/x(
1-10 μ9/yt (l of affinity purified anti-IL-
Solid-phase antibody prepared by immersing in 2 antibody solution, COD-labeled anti-IL-2 antibody solution prepared by maleimide method, standard antigen (1,0,5,0,10,25,50py/zff
) can be prepared as an IL-2 measurement kit.

■0 Effects of the invention The present invention uses affinity-purified solid-phase antibodies,
Since we decided to measure the concentration of cytokines using a luminescence method using antigen-antibody reactions, the measurement limit can be lowered compared to conventional methods, and it is possible to detect minute amounts of cytokines on the order of several pg/mQ. It is.

[Brief explanation of drawings]

FIG. 1 is a graph showing the calibration curve of G-C3F, and FIG. 2 is a graph showing the detection limit of G-CSF with respect to the solid phase immersion solution concentration.

Claims (6)

[Claims] (1) After binding an anti-cytokine antibody immobilized on a solid phase and an anti-cytokine antibody labeled with a labeling substance to a cytokine using an antigen-antibody reaction, the labeling substance is measured by a detection means, and the labeled substance is measured by a detection means. In the method of measuring the concentration of a cytokine in response to the concentration of a labeling substance, the anti-cytokine antibody immobilized on the solid phase is affinity purified, and the detection means is a luminescence method. Method for measuring characteristic cytokines. (2) The anti-cytokine antibody immobilized on the solid phase has 0.
The method for measuring cytokines according to claim 1, characterized in that the method is prepared by immersing a solid phase in a buffer solution of affinity-purified anti-cytokine antibodies of 3 μg/ml to 10 μg/ml. (3) The method for measuring cytokines according to claim (1), wherein the detection means is a chemiluminescence method or a bioluminescence method. (4) the cytokine and the affinity-purified anti-cytokine antibody immobilized on a solid phase in a phosphate buffer;
and a claim characterized in that the reaction is carried out in a shaking state (
1) Method for measuring cytokines as described in section 1). (5) Affinity-purified anti-cytokine antibody for immobilization on a solid phase, anti-cytokine antibody labeled with a labeling substance that enables luminescence detection, cytokine as a standard antigen as required, substrate for labeled enzyme, and buffer Cytokine measurement kit consisting of liquid, etc. (6) The cytokine measurement kit according to claim (5), wherein the luminescence detection is based on a chemiluminescence method or a bioluminescence method.