JP2000266748A - MOUSE IgE MEASUREMENT KIT USING ANTI-MOUSE IgE ANTIBODY AND MEASURING METHOD - Google Patents

Abstract

PROBLEM TO BE SOLVED: To carry out easily operable measurement with excellently high precision and sensitiveness by comprising an antibody recognizing at least a part of mouse immunoglobulin E(IgE) and performing an antigen-antibody reaction in the presence of serum containing no mouse IgE. SOLUTION: A measurement kit comprises various solid phase carrier, a mouse IgE standard, a detection reagent, non-mouse IgE-containing serum, and a reaction medium or a sample diluting solution and the like in addition to a various antibody recognizing at least a part of mouse IgE. This kit and a method thereof are used for measuring mouse IgE. In this case, any sample including blood, plasma, serum, and IgE may be used as an objective sample. As the measurement method, an agglutination method, a sandwich method, a competitive method, and a solid phase direct method and the like are available. In the agglutination method, an antibody is bound to the surface of a particle or a blood cell, and a degree of agglutination is measured as an index. In any of the measurement methods, a combination of a label enzyme - a coloring substrate can be substituted by a combination of a label enzyme - bioluminescent/chemical luminescent substrate, a label enzyme - a fluorescent substrate or the like.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a measurement kit for measuring mouse IgE in a target sample, including an antibody recognizing mouse immunoglobulin E (IgE), and a mouse immunoglobulin E (IgE) recognition kit. The present invention relates to a measurement method for measuring mouse IgE in a target sample using an antibody to be tested.
The results obtained by measuring mouse IgE by the measurement method of the present invention provide useful information for the pathology of various allergic diseases, therapeutic effects by drugs, and prediction and diagnosis in animal model experiments using mice.

[0002]

2. Description of the Related Art When a foreign substance enters a living body, an immune reaction occurs in an attempt to eliminate the foreign substance. The case where this immune response causes various diseases to the living body is called allergy, and is currently classified into five types from type I to type V according to the mechanism of action of this allergic reaction. In recent years, the number of patients with various allergic diseases represented by atopic dermatitis, especially infants, has become a social problem, but many of these allergic diseases produce excessive allergen-responsive IgE This is called an immediate (type I) allergy that develops.

[0003] In general, the blood IgE concentration is kept at a very small level of about several ng / mL in a state where no allergy has developed.
IgE concentrations increase tens to thousands of times. IgE produced in large quantities binds to receptors on mast cells and is cross-linked on the cell membrane by allergens that have entered the body, causing inflammatory factors such as histamine to be released from mast cells into the blood from mast cells and causing allergic diseases It is believed that.

[0004] With the goal of treating this type I allergy as the ultimate goal, studies for elucidating the mechanism of regulating IgE production have been actively conducted, and drugs for suppressing the inflammatory reaction caused by chemical mediators such as histamine and leukotriene released from mast cells, Drugs that inhibit the binding of IgE to the receptor are being developed.

[0005] Therefore, quantifying the amount of IgE in the blood is an important diagnostic criterion for type I allergic diseases, particularly atopic diseases. Also, parasitic infections, severe liver damage,
It is also known to be elevated in eczema and IgE myeloma, and quantifying the amount of IgE in blood is useful for discriminating these diseases.

[0006] Human blood IgE can be measured by an ELISA system or an RIA system combining antibodies against human IgE, and is performed by a sandwich method or a competition method. In addition, a method for measuring IgE involved in the development of allergy utilizing the binding to IgE receptors (FcεRI and FcεRII) present on mast cells and basophils has also been developed (Japanese Patent Application Laid-Open No. 7-072150). 5-218920), JP-A-8-101
194 (Japanese Patent Application No. 6-237590).

[0007]

Problems to be Solved by the Invention As an animal model,
Generally, mice are used, and reagents for measuring mouse IgE are already commercially available, but the measurement sensitivity is at most 10 ng.
/ ml, and 100 μl of a sample sample diluted 25 times or more was required. Therefore, the present inventors, the operation is simple, measurement accuracy and measurement sensitivity, excellent reproducibility,
We have been working hard to develop a mouse IgE measurement system (kit) that is intended to be widely and generally used in the future.

As a result, by using an antibody that recognizes mouse IgE and performing an antigen-antibody reaction in the presence of mouse IgE-free serum, it is possible to reduce the amount of antibody by about 500
The present invention relates to a measurement kit and a measurement method which are simple, excellent in measurement accuracy, measurement sensitivity and reproducibility for measuring mouse IgE in a target sample based on the finding that a measurement sensitivity about twice as high can be obtained, and the present invention. Was completed.

[Means for Solving the Problems]

That is, the present invention includes an antibody that recognizes at least a part of mouse IgE, and performs an antigen-antibody reaction on mouse IgE.
The present invention relates to a measurement kit for measuring mouse IgE in a target sample, which is performed in the presence of non-containing serum. Furthermore, the present invention uses an antibody that recognizes at least a part of mouse IgE, and performs an antigen-antibody reaction in the presence of mouse IgE-free serum.
The present invention relates to a measuring method for measuring the This method is preferably performed using the above-described measurement kit of the present invention. The antigen-antibody reaction in the assay method of the present invention is characterized in that it is performed in the presence of mouse IgE-free serum. here,
“Mouse IgE-free serum” means a serum that does not substantially contain mouse IgE (only contains mouse IgE that is lower than the detection sensitivity of the measurement method of the present invention). The final concentration of the mouse IgE-free serum in the antigen-antibody reaction solution can be appropriately determined by those skilled in the art, and is preferably 5% by volume to 90% by volume, more preferably 10% by volume, in order to obtain high measurement sensitivity.
% By volume to 50% by volume. Such mouse IgE-free serum can be obtained by passing serum of warm-blooded animals such as rabbits, guinea pigs, rats, mice, goats, sheep, horses, pigs, chickens, etc. It can be prepared by a method known to those skilled in the art such as absorption. Mouse IgE-free serum may be added directly to the system, for example, during an antigen-antibody reaction, or an antibody or mouse.
The target sample (sample) containing IgE or the diluted solution for diluting the labeled antibody may be contained in advance.

[0010]

BEST MODE FOR CARRYING OUT THE INVENTION In the present invention, mouse IgE refers to all IgE present in mouse blood. That is, in blood, there are those that bind to IgE receptors (FcεRI and FcεRII) on mast cells and basophils as free IgE, and those that do not bind to these receptors, but any of them may be used. Alternatively, a mixture thereof may be used. Alternatively, IgE released in a culture supernatant obtained by artificially culturing cells capable of producing mouse IgE such as mouse B cells and mouse myeloma cells may be used. Furthermore, a recombinant mouse prepared by genetic engineering using B cell mRNA etc.
IgE may be used.

[0011] The measurement method of the present invention is characterized by including an antibody that recognizes at least a part of mouse IgE. This antibody will be described below. The antibody used in the present invention may be any antibody that can recognize the above-mentioned mouse IgE as an antigen. Therefore, the antibody used in the present invention may be a polyclonal antibody or a monoclonal antibody. When a polyclonal antibody is used as the immobilized antibody, it is preferable to use a column specifically immobilized with purified mouse IgE to suppress nonspecific reaction of the antibody. . Further, a chimeric antibody or the like produced by genetic engineering may be used. Furthermore,
F (ab ′) ₂ , Fab ′, fragments obtained by removing the Fc portion of an antibody such as Fab, which are obtained by allowing a proteinase such as pepsin or papain to act on the above antibody, are also included in the antibody used in the present invention. . These fragments contain the Fc of the antibody.
Since there is no portion, non-specific binding to a solid phase such as a microtiter plate is low, and it is preferable as a labeled antibody to be labeled with a chemical substance such as an enzyme in the present invention. In order to obtain high measurement sensitivity, it is particularly preferable to use F (ab ') ₂ as a labeled antibody. For example, in a measurement method in which a two-stage antigen-antibody reaction is performed, such as a solid phase sandwich method, the immobilized antibody (primary antibody) used for coating the carrier is a polyclonal antibody or a monoclonal antibody, or a labeled secondary antibody. For example, two or more types of antibodies can be used, such as a fragment obtained by removing the Fc portion derived from a polyclonal antibody.

The antibody used in the present invention can be prepared by any method known to those skilled in the art, such as by genetic engineering using B-cell mRNA or the like, or by immunizing an animal. The following method is generally used. First, an animal is immunized using mouse IgE or a part thereof as an immunogen. The immunogen used here may be any as long as a desired antibody recognizing the above-mentioned mouse IgE can be obtained, and the whole or part of the mouse IgE may be used. It may be artificially produced.

When mouse IgE to be used as an immunogen is obtained from nature, various chromatographic methods such as salting out, dialysis, affinity chromatography, ion exchange chromatography, and hydrophobic chromatography are used from the mouse serum or plasma fraction containing IgE. And can be purified. In addition, it is also possible to culture appropriate cells such as mouse myeloma cells that produce IgE and purify them from the culture supernatant.

To artificially produce mouse IgE, a DNA encoding the amino acid sequence of the desired mouse IgE is prepared,
An appropriate host cell may be transformed with the DNA, and the DNA may be recovered from a culture supernatant of the transformed host cell.

An immunized animal useful for immunizing an animal using the mouse IgE obtained as described above as an immunogen is a rabbit,
They are warm-blooded animals such as guinea pigs, rats, sheep, goats, pigs and chickens. The method of immunizing these animals may be a method commonly used in the art. still,
When using two kinds of anti-mouse IgE antibodies,
Those derived from different immunogens can also be used.

Next, an antibody is obtained from the immunized animal. This method may also be performed by a method conventionally known in the art. An example is as follows.

When the target antibody is a polyclonal antibody, the antiserum obtained from the immunized animal is applied to an existing protein G column, and the column-adsorbed fraction is eluted with an acidic buffer such as a glycine hydrochloride buffer to elute IgG. After collecting the fractions, the target solvent is replaced by dialysis or the like. Next, it is determined whether or not this fraction has anti-mouse IgE activity. As a specific method of determination, according to a known enzyme immunoassay,
A method of reacting an antibody with an antigen adsorbed on a plate and reacting it with a secondary antibody labeled with an enzyme, and measuring the enzyme activity by a color reaction of a substrate by the action of the enzyme, or a known method using an antigen labeled with a radioisotope And the like, but various known methods can be used.

When preparing a monoclonal antibody, spleen cells of an immunized animal are fused with a parent cell for cell fusion such as a myeloma cell line, and a suitable one is selected from the obtained fused cells (hybridomas). Then, the fused cells are cultured in vitro or in vivo, and a monoclonal antibody having higher specificity is collected from the culture mixture.

The antibody obtained as described above is purified if necessary. The purification method may be any combination of known methods such as salting out, dialysis, ion exchange chromatography, and affinity chromatography.

In the measurement method of the present invention, the antibody obtained as described above is used. The antibody may be, for example, a mouse IgE, which is a test substance in a target sample, as an immobilized antibody and / or a labeled antibody. It is preferably used to trap the test substance mouse IgE in the target sample by an antigen-antibody reaction with the antibody. The labeled antibody and the mouse IgE standard can be provided in various forms such as a solution or freeze-dried. Immobilized antibodies are available in beads, microtiter plates, test tubes,
It is prepared by binding an antibody that recognizes at least a part of mouse IgE to the surface of a solid support such as a nitrocellulose membrane or a nylon membrane by a method known to those skilled in the art. The measurement kit of the present invention contains various reagents, materials, instruments, and the like according to the measurement method and principle described below, in addition to various antibodies that recognize at least a part of mouse IgE. For example, various solid-phased carriers (in the case where the measurer prepares the solid-phased antibody for measurement), mouse IgE standard, detection reagent, mouse IgE-free serum, reaction medium or diluted solution for sample And others known to those skilled in the art.

As described above, the measurement kit and the measurement method of the present invention aim to measure mouse IgE in a sample. In this case, the target sample is blood, plasma,
Serum and culture supernatant of IgE-producing cells are preferred, but not limited thereto, as long as the solution contains mouse IgE. The principle of detecting a test substance in the measurement kit and the measurement method of the present invention is not limited to a specific one. Examples of the measuring method include an aggregation method, a sandwich method, a competitive method, and a solid phase direct method.

In the agglutination method, an antibody is bound to the surface of particles, for example, latex particles or erythrocytes, so that the aggregation of particles occurs when mouse IgE is present, and mouse IgE is measured using the degree of aggregation of the particles as an index. In addition, in this agglutination method, generally used particles such as gelatin, microbeads, and carbon particles can be used in addition to latex and red blood cells.

In the sandwich method, the solid phase direct method, and the competitive method, labeled antibodies and antigens are used, and enzyme immunoassay (EIA), radioimmunoassay (RIA), chemiluminescence immunoassay (chemiluminescence immunoassay), The measurement can be performed based on principles such as fluoroimmunoassay (fluorescence immunoassay) and time-resolved fluorescence immunoassay (TR-FIA).

Hereinafter, a solid-phase sandwich method based on the principle of EIA will be described as a preferred example of the present invention. In the solid phase sandwich method using EIA, first, an antibody that recognizes mouse IgE, which is labeled with an enzyme such as peroxidase, alkaline phosphatase, or β-galactosidase, is prepared. Further, an antibody recognizing mouse IgE is adsorbed on the solid phase to be used. Next, the antibody non-adsorbed surface of the solid phase is subjected to a blocking treatment with a protein that does not affect the reaction system, for example, BSA (bovine serum albumin). Sample (sample)
Alternatively, the standard is added to the solid phase, and the antigen-antibody reaction in the first step is performed. After the reaction, the solid phase is washed, and the above-mentioned enzyme-labeled antibody is added, and mouse IgE reacted with the immobilized antibody is subjected to the antigen-antibody reaction in the second step.
Note that a sample (sample) or standard and an enzyme-labeled antibody can be added simultaneously. After removing the excess enzyme-labeled antibody by a washing operation, a chromogenic substrate corresponding to the enzyme to be used, for example, 1,2-phenyldiamine and H ₂ O ₂ , P-nitrophenyl phosphate, 2-nitrophenyl-β-D -Galactoside, 3,3 ', 5,5'-tetramethylbenzidine and the like are added and reacted with the enzyme. Since the color development of the substrate depends on the amount of the enzyme and thus the amount of mouse IgE substance in the sample, mouse IgE can be quantified by measuring the amount of the end product of color development.

In the solid phase direct method, a sample (sample) is directly adsorbed on a solid phase, and the mouse IgE non-adsorbed surface of the solid phase is blocked with a protein that does not affect the reaction system, such as BSA (bovine serum albumin). Treated, then mouse IgE
Is added and allowed to react. Later,
The same operation as in the solid phase sandwich method is performed to determine the presence or absence or quantification of mouse IgE in the sample.

In the solid phase competition method, a certain amount of mouse IgE recognized by the antibody to be used is directly adsorbed on a solid phase, and then subjected to a blocking treatment, whereupon an enzyme-labeled antibody recognizing mouse IgE and a sample (sample) And. After reacting for a certain period of time, washing is performed to remove substances not bound to the solid phase, and a chromogenic substrate is added to react with the enzyme. After the reaction, mouse IgE in the sample is quantified by measuring the degree of inhibition of binding of the enzyme-labeled antibody to solid-phase mouse IgE due to the addition of the sample. The antibody was first adsorbed to the solid phase, enzyme-labeled mouse IgE was added at the same time as the sample (sample), and the addition of the sample was measured to determine the degree of inhibition of the binding of the labeled substance to the immobilized antibody. Mouse IgE in the sample) may be quantified.

As a method other than the above, the antigen-antibody reaction is performed not in the solid phase but in the liquid phase, and thereafter, the mouse IgE bound to the labeled antibody is insolubilized by a coagulation sedimentation method using an antibody or a physicochemical method. There is also a method of quantification. Also,
Instead of labeling an antibody recognizing mouse IgE, it is also possible to obtain a secondary antibody recognizing the antibody, label the antibody, perform an antigen-antibody reaction, and measure mouse IgE.

In any of the sandwich method, the solid-phase direct method, and the competitive method, the combination of the labeling enzyme and the chromogenic substrate is used.
It is possible to change to a combination of a labeling enzyme-bioluminescent substrate or a chemiluminescent substrate, a labeling enzyme-fluorescent substrate and the like.
In this case, a typical combination of the enzyme-luminescent substrate is
There are alkaline phosphatase-AMPPD, horseradish peroxidase luminol, luciferase luciferin, etc., and a typical combination of enzyme-fluorescent substrate is alkaline phosphatase-umbelliferyl phosphate, horseradish peroxidase-p-hydroxyphenylpropionic acid, etc. is there.

Further, in the above three kinds of measurement methods, an antibody or an antigen directly or indirectly labeled with a radioactive substance, a chemiluminescent substance or a fluorescent substance is used in place of the enzyme, and the radioactivity, luminescence and fluorescence intensity are used. By measuring
It is also possible to measure mouse IgE in a sample (sample). As the radioactive substance, ¹²⁵ I, ¹³¹ I and the like are generally used, and a typical chemiluminescent substance is acridinium ester. When measuring fluorescence intensity, as a more sensitive method, a chelating agent is directly or indirectly bound to an antibody or antigen, and the intensity of fluorescence emitted from the chelating agent after irradiation with excitation light is time-resolved. A method (time-resolved fluorescence immunoassay) of measuring mouse IgE in a sample by performing the above measurement is also useful.
Europium is a typical rare earth metal.

Other reaction conditions and the like in each measurement method of the present invention can be appropriately determined by those skilled in the art according to the purpose, the type of the target sample, the measurement principle, and the like.

As shown in the following examples, the detection system and the measurement method using the measurement kit of the present invention are very stable in measuring IgE in mouse serum. Further, the target sample required for the measurement is only a small amount of only a few μl, and has high sensitivity such that mouse IgE of 0.5 ng / ml or more can be detected. The measurement kit and the measurement method of the present invention described above provide useful information for the pathology of various allergic diseases, therapeutic effects by drugs, and prediction and diagnosis in animal model experiments using mice.

[0032]

EXAMPLES Hereinafter, the present invention will be described more specifically with reference to examples, but these are shown as examples of the present invention, and the present invention is not limited thereto. The abbreviations used in the description are those commonly used in the art.

[0033]Production of mouse IgE-free serum  A mouse IgE-free rat serum was prepared as follows. (1) Preparation of anti-mouse IgE antibody-immobilized resin Anti-mouse IgE antibody purified from goat anti-mouse IgE antiserum
N-hydroxysuccinimide activated resin (AmershamPhar
maciaBiotech, HiTrap NHS-activated)
As described in the instruction manual. (2) Absorption operation of mouse IgE Rat serum was applied to anti-mouse IgE antibody-immobilized resin at 1 mm / min.
Collect the fractions that passed through at the linear flow rate,
Mouse IgE in rat serum was measured by the
Was. The serum IgE concentration in serum should be below the measurement limit.
The above operation was repeated.

[0034]Horseradish peroxidase-labeled goat F (a
b ') Preparation of ₂ anti-mouse IgE polyclonal antibodies  (1) Goat F (ab ')_TwoPreparation of anti-mouse IgE The IgG fraction obtained from goat anti-mouse IgE antiserum was
Dialyze overnight against sodium acid buffer (pH 4.5). Same buffer
To a concentration of 2 mg / ml, and dissolved in the same buffer.
Tapepsin was added to a final concentration of 4%. Keep at 37 ° C
Incubate in the incubator for 24 hours
Enzymatic digestion was performed. After enzyme digestion, 0.2M NaHCo_ThreeEquilibrate with
Separation with a Superose 12 column (Pharmacia)
(ab ’)_TwoFractions were collected. F (ab ') recovered_TwoFraction 2mg
/ ml and stored at -80 ° C until use. (2) Periodate-activated horseradish peroxidase
Goat F (ab ')_TwoCoupling with anti-mouse IgE. Periodic acid-activated horseradish prepared according to known methods.
Peroxidase (4 mg / ml, 0.2 M NaHCO_ThreeDissolved in)
Goat F (ab ') prepared in (1)_TwoAnti-mouse IgE (2mg / ml)
Mix in equal amounts and incubate at room temperature, protected from light for 6 hours.
I did it. After that, the total amount of protein subjected to coupling
Add an equal volume of sodium borohydride and release at 4 ° C overnight
Was placed. Dialysis overnight against 20 mM Tris-HCl, 150 mM NaCl (pH 7.4)
After dialysis, dilute to a concentration that can be used in ELISA.
Used in the measurement system.

Solid phase sandwich based on the principle of ELISA
A measurement system for mouse IgE by the solid phase sandwich method based on the principle of measurement ELISA by the ELISA method was prepared as follows.

[0036]

Embodiment 1A Monoclonal antibody-enzyme-labeled polyc
Lonal antibody system  (1) Coating of carrier with anti-mouse IgE antibody Anti-mouse IgE rat monoclonal antibody (Crystal Chem I
nc.) in carbonate buffer (pH 9.6) to 2 μg / ml.
Dissolved. Put this in a microtiter plate (Nunc
100μ on Micro Module Plate, Maxsorp-F8)
The mixture was dispensed in 1 l increments and left at 4 ° C. overnight. (2) Blocking of microtiter plate Each well was washed with 300 μl of a washing solution (150 mM NaCl, 0.05% Tween20).
After washing three times with 20 mM Tris-HCl buffer (pH 7.4) containing
Blocking solution (0.1% BSA (Oriental Yeast), 15
20 mM Tris-HCl buffer containing 0 mM NaCl, 0.05% Tween20
(pH 7.4)), and left at 25 ° C for 2 hours. (3) Measurement of mouse IgE After removing the blocking solution from each well, dilute each well.
Solution (30% mouse IgE-free rat serum, 0.1% BSA, 150
20 mM Tris-HCl buffer containing mM NaCl and 0.05% Tween 20 (p
H7.4)) was dispensed in an amount of 95 μl. Add 5 μl of sample serum here
And left at 25 ° C. for 4 hours. As standard mouse IgE,
Using mouse myeloma cell-derived purification (100 ng / ml),
This was serially diluted two-fold with a diluting solution, and 5 μl was similarly added to each well.
In addition, it was left at 25 ° C. for 4 hours. In addition, the measurement range in this system
For samples other than those in the box,
Measured. After washing each well 5 times with 300 μl of washing solution,
Horseradish peroxidase-labeled goat anti-mouse IgE polyc
Dilute the lonal antibody with the diluent, add 100 μl each, and
For 2 hours. Next, wash each well 5 times with 300 μl of washing solution
After washing, TMB solution (3,3 ', 5,5'tetramethylbenzidine)
Was added thereto in an amount of 100 μl each, and reacted at 25 ° C. under light shielding for 30 minutes. So
Thereafter, 100 μl of 1N sulfuric acid was added to stop the reaction.
Measure the absorbance of each well using a microplate reader.
The measurement was performed at a main wavelength of 450 nm and a sub wavelength of 630 nm. Figure 1 shows the standard mouse
Dilution linearity of serially diluted 2-fold IgE (100 ng / ml)
Is shown. It shows good dilution linearity.

[0037]

Embodiment 2B polyclonal antibody-enzyme-labeled polyc
Lonal antibody system  (1) Coating of anti-mouse IgE antibody on carrier Anti-mouse IgE rat polyclonal antibody was added to carbonate buffer
(PH 9.6) so as to be 10 μg / ml. this
The microtiter plate (Nunc
Plate, Maxsorp-F8) at 4 ° C.
Left overnight. (2) Blocking of microtiter plate Each well was washed with 300 μl of a washing solution (150 mM NaCl, 0.05% Tween20).
After washing three times with 20 mM Tris-HCl buffer (pH 7.4) containing
Blocking solution (0.1% BSA (Oriental Yeast), 15
20 mM Tris-HCl buffer containing 0 mM NaCl, 0.05% Tween20
(pH 7.4)), and left at 25 ° C for 2 hours. (3) Measurement of mouse IgE After removing the blocking solution from each well, dilute each well.
Solution (mouse 30% IgE-free rat serum, 0.1% BSA, 150%
20 mM Tris-HCl buffer containing mM NaCl and 0.05% Tween 20 (p
H7.4)) was dispensed in an amount of 95 μl. Add 5 μl of sample serum here
And left at 25 ° C. for 4 hours. As standard mouse IgE,
Using mouse myeloma cell-derived purified IgE (100 ng / ml),
This was serially diluted 2-fold with a diluting solution, and 5 wells were similarly added to each well.
μl was added and left at 25 ° C. for 4 hours. In addition, measurement in this system
For samples outside the specified range, dilute in advance with the sample dilution solution.
And then measured. Wash each well 5 times with 300 μl wash solution
Later, horseradish peroxidase-labeled goat anti-mouse IgE
Dilute the polyclonal antibody with the diluent and add 100 μl each.
And left at 25 ° C. for 2 hours. Next, wash each well with 300
After washing 5 times with μl, the TMB solution (3,3 ', 5,5'
100 μl each and react for 30 minutes in the dark at 25 ° C.
I let it. Then, add 100 μl of 1 N sulfuric acid to stop the reaction.
Stopped. Microplate reader for absorbance of each well
The measurement was carried out at 450 nm in the main wavelength and 630 nm in the sub wavelength using a laser beam. FIG.
Diluted two-fold serial dilutions of standard mouse IgE (100 ng / ml).
Shows linearity. It shows good dilution linearity.

[0038]

Embodiment 3C polyclonal antibody-enzyme labeled F (a
b ') ₂ polyclonal antibody system  (1) Coating of anti-mouse IgE antibody on carrier Anti-mouse IgE rat polyclonal antibody was added to carbonate buffer
(PH 9.6) to give a concentration of 2 μg / ml. this
The microtiter plate (Nunc
Plate, Maxsorp-F8) at 4 ° C.
Left overnight. (2) Blocking of microtiter plate Each well was washed with 300 μl of a washing solution (150 mM NaCl, 0.05% Tween20).
After washing 5 times with 20 mM Tris-HCl buffer (pH 7.4) containing
Blocking solution (0.1% BSA (Oriental Yeast), 15
20 mM Tris-HCl buffer containing 0 mM NaCl, 0.05% Tween20
(pH 7.4)), and left at 25 ° C for 2 hours. (3) Measurement of mouse IgE After removing the blocking solution from each well, dilute each well.
Solution (30% mouse IgE-free rat serum, 0.1% BSA, 150%
20 mM Tris-HCl buffer containing mM NaCl and 0.05% Tween 20 (p
H7.4)) was dispensed in an amount of 95 μl. Add 5 μl of sample serum here
And left at 4 ° C. for 16 hours. As standard mouse IgE,
Use purified IgE (200 ng / ml) derived from usmyeloma cells.
This was serially diluted two-fold with a diluting solution, and 5 μl was similarly added to each well.
In addition, it was left at 4 ° C. for 16 hours. In addition, the measurement range in this system
For samples other than those in the box,
Measured. After washing each well 5 times with 300 μl of washing solution,
Horseradish peroxidase-labeled goat F (ab ')_TwoAnti mouse Ig
E Dilute the polyclonal antibody with the diluent and add 100 μl
And left at 25 ° C. for 2 hours. Next, wash each well with 300
After washing 5 times with μl, the TMB solution (3,3 ', 5,5'
100 μl each and react for 30 minutes in the dark at 25 ° C.
I let it. Then, add 100 μl of 1 N sulfuric acid to stop the reaction.
Stopped. Microplate reader for absorbance of each well
The measurement was carried out at 450 nm in the main wavelength and 630 nm in the sub wavelength using a laser beam. FIG.
Dilution of serial two-fold dilutions of standard mouse IgE (200 ng / ml)
Shows linearity. It shows good dilution linearity. Actual
When measuring the amount of IgE in the sample, use the mouse IgE standard
The amount of IgE in the sample was converted as a quasi-curve.

[0039]

Example 4 D monoclonal antibody-enzyme-labeled F (a
b ') ₂ polyclonal antibody system (1) Coating of anti-mouse IgE antibody on carrier Anti-mouse IgE rat monoclonal antibody (Crystal Chem I)
nc.) was dissolved in a carbonate buffer (pH 9.6) to a concentration of 1 μg / ml. Put this in a microtiter plate (Nunc
100μ on Micro Module Plate, Maxsorp-F8)
The mixture was dispensed in 1 l increments and left at 4 ° C. overnight. (2) Blocking of microtiter plate Each well was washed with 300 μl of a washing solution (150 mM NaCl, 0.05% Tween20).
After washing 5 times with 20 mM Tris-HCl buffer (pH 7.4) containing
Blocking solution (0.1% BSA (Oriental Yeast), 15
20 mM Tris-HCl buffer containing 0 mM NaCl, 0.05% Tween20
(pH 7.4)) 300 μl was added, and the mixture was left at 25 ° C. for 2 hours. (3) Measurement of mouse IgE After removing the blocking solution in each well, a dilution solution (30% mouse IgE-free rat serum, 0.1% BSA, 150%) was added to each well.
20 mM Tris-HCl buffer containing mM NaCl and 0.05% Tween 20 (p
H7.4)) was dispensed in an amount of 95 μl. Here, 5 μl of the sample serum was added, and left at 4 ° C. for 16 hours. As standard mouse IgE, purified mouse myeloma cell-derived IgE (32 ng / ml) was serially diluted 2-fold with a diluting solution, and 5 μl was similarly added to each well and left at 4 ° C. for 16 hours. In addition, a sample outside the measurement range in this system was measured after diluting with a sample diluting solution in advance. After washing each well five times with 300 μl of a washing solution, horseradish peroxidase-labeled goat F (ab ′) ₂ anti-mouse IgE
The polyclonal antibody was diluted with a diluent, added in 100 μl portions, and left at 25 ° C. for 2 hours. Next, wash each well with 300
After washing 5 times with μl, 100 μl of a TMB solution (3,3 ′, 5,5 ′ tetramethylbenzidine) was added thereto, and the reaction was carried out at 25 ° C. under light shielding for 30 minutes. Thereafter, 100 μl of 1 N sulfuric acid was added to stop the reaction. The absorbance of each well was measured at a primary wavelength of 450 nm and a secondary wavelength of 630 nm using a microplate reader. FIG.
Shows the dilution linearity of a two-fold serial dilution of standard mouse IgE (32 ng / ml). It shows good dilution linearity. When actually measuring the amount of IgE in the sample, this was used as a mouse IgE standard curve to convert the amount of IgE in the sample. By using the above-described measurement kit of the present invention, mouse IgE of 0.5 ng / ml or more could be detected using 5 μl of mouse serum.

[0040]

Example 5 Basic Performance Test of Mouse IgE Measurement Kit (1) Dilution Test of Serum Sample According to the test method of Example 4, a dilution test of a mouse serum sample was actually performed. Two sera of two sera with unknown IgE amounts were serially diluted with a sample diluent, and the IgE concentration of the sera was calculated based on a standard curve obtained from simultaneously measured standard mouse IgE. As shown in the results of FIG. 5, good linearity substantially passing through the origin is obtained. (2) Addition / recovery test A mouse IgE addition / recovery test was performed according to the test method described above. Standard mouse IgE was added to three serum samples, and the recovery rate of the added IgE amount was determined from the measured values. As shown in Table 1, good results were obtained with an addition recovery rate of 84.8% to 126.8% (average value 107.2%).

[0041]

[Table 1]

(3) Simultaneous reproducibility test A simultaneous reproducibility test was performed according to the test method described above. As shown in Table 2, the CV value is 10% or less, indicating good simultaneous reproducibility.

[0043]

[Table 2]

(4) Daily Difference Reproducibility Test A daily difference reproducibility test was performed according to the test method described above. As shown in Table 3, the CV value is 10% or less, indicating good reproducibility of daily difference.

[0045]

[Table 3]

The results of (3) and (4) show that this detection system is very stable in measuring IgE in mouse serum. In addition, when using this measurement system, only 5 μl of mouse serum can be used.
Mouse IgE of 5 ng / ml or more was detectable.

[Brief description of the drawings]

FIG. 1 shows a mouse myeloma-derived standard in a monoclonal antibody-enzyme-labeled polyclonal antibody system.
IgE (100 ng / ml) was diluted by 2-fold serial dilution, and the absorbance was measured at a measurement wavelength of 450 nm and a sub-wavelength of 630 nm by the method of the example of the present invention (solid-phase sandwich method based on the principle of ELISA). FIG.

FIG. 2 shows a polyclonal antibody-enzyme-labeled polyclonal antibody system using a mouse myeloma-derived standard.
IgE (100 ng / ml) was diluted by 2-fold serial dilution, and the absorbance was measured at a measurement wavelength of 450 nm and a sub-wavelength of 630 nm by the method of the example of the present invention (solid-phase sandwich method based on the principle of ELISA). FIG.

FIG. 3 shows that in a polyclonal antibody-enzyme-labeled polyclonal F (ab ′) ₂ antibody system, mouse myeloma-derived standard IgE (200 ng / ml) was diluted by two-fold serial dilution.
5 is a graph showing the results of measuring the absorbance at a measurement wavelength of 450 nm and a sub-wavelength of 630 nm by the method of the example of the present invention (solid-phase sandwich method based on the principle of ELISA).

FIG. 4 shows monoclonal antibody-enzyme-labeled F (a
b ′) In a _two- polyclonal antibody system, mouse myeloma-derived standard IgE (32 ng / ml) was diluted by 2-fold serial dilution, and the method of the example of the present invention (solid-phase sandwich method based on the principle of ELISA) was used. It is a graph which shows the result of having measured the light absorbency in measurement wavelength 450nm and subwavelength 630nm.

FIG. 5 shows that a mouse serum sample was applied to a measurement system at 0.0625 μm.
l, 0.125 μl, 0.25 μl using the method of Example 4 of the invention
5 is a graph in which the absorbance at a measurement wavelength of 450 nm and a sub-wavelength of 630 nm was measured by (solid-phase sandwich method based on the principle of ELISA), and the concentration was calculated from FIG. 4.

Claims (15)

[Claims] 1. A mouse IgE in a target sample comprising an antibody that recognizes at least a part of mouse IgE, wherein the antigen-antibody reaction is performed in the presence of mouse IgE-free serum.
Measurement kit for measuring. 2. The measurement kit according to claim 1, wherein two kinds of combinations of a polyclonal antibody and a monoclonal antibody are used as the antibody. 3. The measurement kit according to claim 1, wherein the polyclonal antibody is in the form of F (ab ′) ₂ or Fab ′. 4. The assay kit according to claim 1, wherein the serum is a serum derived from rabbit, guinea pig, rat, mouse, goat, sheep, horse, pig, or chicken. 5. The measurement kit according to claim 1, wherein an enzyme immunoassay is used as a measurement method. 6. The measurement kit according to claim 1, wherein a radioimmunoassay is used as a measurement method. 7. The measurement kit according to claim 1, wherein a fluoroimmunoassay is used as a measurement method. 8. The measurement kit according to claim 1, wherein a chemiluminescence immunoassay is used as a measurement method. 9. The measurement kit according to claim 1, wherein a time-resolved fluorescence immunoassay is used as the measurement method. 10. The measurement kit according to claim 1, wherein an immunoassay using the aggregation of particles as an index is used as a measurement method. 11. The measurement kit according to claim 10, wherein said particles are latex particles. 12. A mouse IgE in a target sample, wherein an antigen-antibody reaction is performed in the presence of a mouse IgE-free serum using an antibody that recognizes at least a part of mouse IgE.
A measuring method for measuring. 13. The method according to claim 12, wherein the target sample is selected from plasma, serum, or cell culture supernatant. 14. The method according to claim 12, wherein the final concentration of the mouse IgE-free serum in the antigen-antibody reaction solution is 5% by volume to 90% by volume. 15. The method according to claim 12, wherein mouse IgE of 0.5 ng / ml or more contained in the target sample can be detected.