JPH08101194A - Measuring method for ige in blood - Google Patents

Abstract

PURPOSE: To check existence of IgE or determine the quantity of existing IgE by bringing a diluted blood or blood component into contact with soluble α-chain of a high affinity IgE receptor (FcεRI). CONSTITUTION: A receptor peculiar to IgE having a high affinity is FcεRI solely, and the part committed to the bond with IgE is α-chain. Therefore, by bringing a solid carrier to which anti-IgE antibody is fixed, into contact with a test specimen or its diluted matter, if IgE exists in the test specimen, the IgE is bonded with the anti-IgE antibody and fixed to the solid carrier. Then, by bringing this solid carrier into contact with the soluble α-chain of FcεRI, the α-chain is coupled with the fixed IgE. The existence of IgE in the test specimen or its quantity is determined from existence or quantity of the soluble α-chain of the thus bonded FcεRI.

Description

Detailed Description of the Invention

[0001]

This invention uses FcεRI,
It relates to a method of measuring IgE in human blood.

[0002]

2. Description of the Related Art In recent years, an increase in allergic diseases has been reported all over the world, and even in Japan, it was reported to be several percent in the 1955's, but now it is 10-20%, and it is 30 at the end of this century. It is estimated to reach%. An allergic reaction is an immune reaction that damages its own tissues.
It is classified into four types from type IV to type IV, but most of the allergic diseases mentioned here refer to type I allergy (immediate type allergy).

In type I allergy, when an allergen (allergen) invades the body, it binds to an IgE antibody specific to the antigen. This IgE antibody has previously bound to the high affinity IgE receptor (FcεRI) on mast cells or basophils at the Fc portion, and the binding of allergen and IgE antibody causes receptor aggregation and degranulation, resulting in histamine and The release of chemical mediators such as leukotoluene induces a general allergic reaction.

The IgE antibody that induces this allergic reaction is in the order of 150 U to 300 U / ml or less in normal human serum, but 10 ² to 10 ⁴ times as much IgE antibody in the serum of atopic patients, which is one of allergic diseases. It is included. In addition, it is generally known that IgE in blood is generally elevated in type I allergic diseases. Therefore, it is important to measure the total IgE amount in the patient's blood or the antigen-specific IgE amount in allergic diseases. Plays an important role in diagnosis.

Currently, the method for measuring IgE in blood is IgE.
There is a method using a specific antibody (monoclonal antibody or polyclonal antibody), such as RIA system or E
Measurement with a LISA system is possible. However, the method using these antibodies against IgE is the same as I existing in blood.
It cannot be determined if the gE antibody is functional. In other words, as long as the antigen-antibody reaction is used, IgE that does not have the ability to bind to receptors on mast cells and basophils is also detected, and even those that have been degraded and those that have been denatured are detected. Will end up. That is, IgE that is not involved in the allergic reaction is also detected in vivo.

[0006] In the case of an antibody-based detection system, the specificity and affinity of the antibody obtained by the type of IgE used for immunization, whether monoclonal antibody or polyclonal antibody, or the difference in immune reaction of immunized animals. It is possible that the sexes are different. In this case, if the specificity differs depending on the antibody used, a certain specific IgE may be detected, and it is possible that not all IgE can be detected. In particular, when constructing a sandwich system by ELISA, it is necessary to detect with a combination of two different antibodies, so this risk is doubled, and different results may be obtained with the combination of antibodies used.

[0007] In addition, there are factors in blood that inhibit the specific antigen-antibody reaction, and it is expected that the measurement system will be affected. Examples of this factor include rheumatoid factor and Na
F, bilirubin, hemoglobin, chyle and the like can be mentioned. Furthermore, heparin, sodium oxalate, EDTA, sodium citrate, etc., which are blood coagulants for blood collection, are also considered as factors that inhibit the antigen-antibody reaction.

[0008]

Related Art The present inventors succeeded in expressing a soluble FcεRI molecule in baculovirus and insect cells, and were able to obtain a large amount of soluble FcεRI molecule. It has also been reported that an IgE detection system using the FcεRI molecule can be constructed (Japanese Patent Application No. 5-201199).
However, the measurement of IgE in human blood was not performed.

[0009]

Accordingly, the present invention provides a method for selectively and specifically detecting and quantifying IgE having an ability of inducing an allergic reaction existing in vivo, particularly in blood. Is.

[0010]

As a result of studies to solve the above problems, the present inventors have found that the α chain and I chain of the FcεRI molecule are
Established a method for selectively and accurately detecting and quantifying IgE, which has the ability to induce allergic reaction in blood in vivo by utilizing the specific and high affinity binding of gE molecule did.

Therefore, the present invention provides a method for detecting or measuring IgE in a test sample which is blood or a blood component, wherein the high affinity IgE receptor (FcεRI) is soluble after the blood or the blood component is diluted as desired. A method is provided, which comprises contacting with an α chain, measuring the presence or absence or the amount of binding between IgE and a soluble α chain of FcεRI, and determining the presence or amount of IgE from the result.

In one embodiment of the above method, anti-Ig
If IgE is present in the test sample by contacting the immobilized carrier on which the E antibody is immobilized with the test sample or a dilution thereof, the IgE is immobilized on the solid carrier by binding with the anti-IgE antibody. This solid support is FcεR
By contacting with the soluble α chain of I, the FcεR
The soluble α chain of I is bound to the immobilized IgE, and the presence or amount of the soluble α chain of FcεRI bound in this way determines the presence or amount of IgE in the test sample.

In another aspect of the invention, FcεRI
By contacting the solid carrier having the soluble α-chain immobilized with the test sample or a dilution thereof with IgE in the test sample.
If present, the IgE is immobilized on a solid support by binding to the soluble α-chain of FcεRI, and then the solid support is contacted with an anti-IgE antibody, whereby the IgE
The IgE antibody is immobilized by binding the antibody to the immobilized IgE, and the presence or quantity of IgE in the test sample is determined from the presence or quantity of the immobilized IgE. In the present invention, when referring to blood or blood components, these mean whole blood, or components separated from blood such as serum and plasma, bone marrow fluid, lymph fluid, urine, saliva, and other various body fluid products derived from blood. To do.

[0014]

[Detailed description] FcεRI is a mast cell, basophil,
It is expressed only on skin dendritic cells and has high affinity (K
_a= 10^TenM^-1) And specifically bind IgE
Is a glycoprotein molecule. As a molecule that binds to IgE
For B cells, T cells, macrophages, etc.
Expressing low-affinity IgE receptor (FcεRII:
K _a= 10⁷M^-1) Exists. I with low affinity
FcγRII and FcγRIII, which are gG receptors, also aggregate
Affinity comparable to that of aggregated IgE for IgG
Has been shown to bind by sex, but has a high affinity
FcεRI is the only IgE-specific receptor.

FcεRI is one molecule of α chain and one molecule of β
Chain, consisting of a tetramer of two γ-chains, and it has been clarified that the part of the tetramer involved in IgE binding is the α-chain (Kinet, JP (1990). , Curr.O
pinion Immunol. 2, p499-505). This α chain is a membrane protein that penetrates the cell membrane and is known to have a sugar chain in vivo.

The extracellular domain of this α chain has two immunoglobulin homologous domains and is considered to be an immunoglobulin superfamily. It has been suggested that the second domain of these two domains has an essential site for binding to IgE, but since the binding activity cannot be achieved by only one domain, the binding site is composed of two domains. It is considered to be determined by the three-dimensional structure.

When this FcεRI α chain was expressed in CHO cells with the transmembrane region removed, the α chain became soluble and was secreted extracellularly. This solubilized α chain had a high affinity for IgE, and even when the sugar chain was removed, it showed a similar high affinity (Blank, U., C., and K).
inet, JP (1991) J. Biol. Chem. 266, p2639-2646).
The present inventors have succeeded in efficiently producing this soluble FcεRI in a large amount in a system of recombinant baculovirus and insect cells (Japanese Patent Application No. 5-201991). In addition, IgE has been successfully detected by using this soluble FcεRI.

Fc used for detecting IgE in blood according to the present invention
The εRIα chain can also be produced using animal cells such as yeast recombinant and CHO. Recombinantly produced F
By using the cεRIα chain, a soluble FcεRIα chain can be stably obtained with good reproducibility.

Soluble FcεRIα chain and IgE in the present invention
Compared with the detection method using an antibody, the first characteristic of the measurement method using specific binding of FcεR
Since the IgA chain is used for detection, only IgE having a function involved in an allergic disease is detected, which enables more disease-related diagnosis. Secondly, soluble Fc
The specific binding between εRIα chain and IgE has a high affinity, and this affinity is higher than the affinity of the antibody generally used for the antigen-antibody reaction, and the detection sensitivity and detection time It may be advantageous for shortening.

Thirdly, although this is common with the first feature, the binding of the anti-IgE antibody produced in other animals such as mouse, rabbit or goat and IgE by the antigen-antibody reaction cannot occur in the human body. But soluble FcεR
The binding between the Iα chain and IgE occurs in the human body. This makes it possible to eliminate the influence of substances in serum that act inhibitory to the antigen-antibody reaction when detecting IgE in serum, which is carried out in the present invention.

As a method for measuring IgE using the soluble FcεRIα chain and IgE of the present invention, an immunological measuring method utilizing the specific reaction of various antigen / antibody conventionally used can be considered. The detailed method of this immunological assay is described in detail in "New application examples of immunoassay and its application to the development of diagnostic reagents and therapeutic agents" (Management Education Publishing). In this case, the FcεRIα chain in the present invention can be replaced with a specific antibody for all immunoassays.

As a method for measuring the total amount of IgE in blood, a sandwich system can be considered. For example, soluble Fc
εRI α chain is known as a carrier generally used in enzyme-linked immunosorbent detection methods (eg, ELISA, EIA etc.) such as microplates, beads, nitrocellulose membranes, nylon membranes, radioimmunodetection methods (eg RIA etc.), etc. Combine by the method of.

The coupling method should be selected as efficiently as possible. Samples (serum etc.) on these carriers
To contact. In this case, serum or the like can be directly contacted, but generally, it is diluted with an appropriate sample diluent and contacted to prevent nonspecific binding of nonfunctional IgE to the carrier, Only functional IgE capable of binding the soluble FcεRIα chain should bind to the soluble FcεRIα chain.

Then, the unbound fraction is washed by a commonly used method, and specific for IgE labeled with an enzyme (for example, horseradish peroxidase, β-D-galactosidase, alkaline phosphatase), biotin, radioisotope and the like. Soluble Fc by reacting with a specifically bound antibody
Bind to IgE bound to εRIα chain. The soluble FcεRI is obtained by washing the unbound fraction and detecting the labeled enzyme by a commonly used method.
IgE having a function of binding to the α chain can be detected.

On the contrary, an antibody which specifically binds to IgE is detected by an enzyme immunological method such as microplate, beads, nitrocellulose membrane, nylon membrane (for example, ELISA, E).
IA, etc.), radioimmunoassay (eg, RIA, etc.), etc., and a commonly used carrier is bound by a known method. The binding method should be selected as efficiently as possible. A sample (serum or the like) is brought into contact with these carriers.
In this case, serum or the like can be contacted as it is, but generally, it is diluted with an appropriate sample diluent and contacted,
It should prevent binding of substances that cause non-specific reactions and should bind IgE in all analytes.

Next, the unbound fraction is washed by a commonly used method, and soluble FcεRIα labeled with an enzyme (for example, horseradish peroxidase, β-D-galactosidase, alkaline phosphatase), biotin, radioactive isotope and the like. I reacted with the chain and bound to the antibody
Bind to gE. By washing the unbound fraction by a commonly used method and detecting a labeled enzyme or the like, IgE having a function of binding to the soluble FcεRIα chain can be detected.

It is necessary that the antibody used in either of the above methods does not inhibit the binding of soluble FcεRIα chain to IgE. That is, it is known that the soluble FcεRIα chain binds to the CH3 domain of IgE, and it is important to select an antibody that does not inhibit this binding. Further, it is preferable that this antibody is capable of binding IgE bound to the soluble FcεRIα chain, and is capable of binding all IgE and not detecting specific IgE.

As another method for detecting total IgE in blood, a competitive method represented by the RIA method can be considered. This method is characterized in that the molecule that binds IgE is soluble in F
cεRIα chain only, another like sandwich system
The point is not to use one antibody. The detailed method is described in the patent of the present inventors (Japanese Patent Application No. 5-218920). Or for more various methods, "New application examples of immunoassays and application to the development of diagnostic reagents and therapeutic agents"
(P33-p42, Management Education Publishing).

By using the soluble FcεRIα chain of the present invention, it is possible to measure not only the total IgE in blood but also the specific IgE amount of each antigen causing allergies. As this antigen, an antigen existing in nature may be purified, or a gene may be isolated and an antigen may be obtained by gene recombination with the gene. This detailed method is also described in the patent of the present inventors (Japanese Patent Application No. 5-218920).

[0030] For more detailed method, "New application examples of immunoassay and application to development of diagnostic reagents and therapeutic agents" (p3-p)
60). According to the present invention, it becomes possible to measure the total amount of IgE involved in allergy in blood or the amount of IgE for each antigen. It is considered that the correlation between the conventional method and the method according to the present invention is better as a whole according to the examples of the present invention, but when the individual specimens are examined, there are some cases where the measured values deviate. These facts indicate that the measurement method of the present invention is IgE.
This is considered to be the result of specifically measuring IgE and selectively measuring IgE involved in allergy. By using this measurement method, Ig closely related to pathological conditions and symptoms
It is thought that E can be measured. Furthermore, Ig by antigen
By measuring the amount of E, the main causative substance of allergy can be identified, which is effective as a guideline for treatment. In the case of the discrepancy between the conventional measurement method and this measurement method, IgE not conventionally involved in allergy is measured, or conversely, a specific I
It is considered that there was an example in which gE was not measured.
This IgE measurement method using the FcεRIα chain provides more effective information for diagnostic treatment than the conventional method.

[0031]

According to the present invention, it becomes possible to selectively detect only IgE involved in allergic diseases in blood. Further, by using the soluble FcεRIα chain, the influence of substances that inhibit the antigen-antibody reaction in serum can be eliminated as much as possible, and it has become possible to construct a detection system having a form close to that in the living body. This is useful for diagnosing allergic diseases and various diseases such as parasitic infection, severe liver damage, eczema, IgE myeloma and the like.

Furthermore, the IgE detection system of the present invention is also effective in the treatment of allergies, its determination, and elucidation of the pathological condition.

[0033]

The present invention will be described in more detail with reference to the following examples, but the present invention is not limited to the examples.

Example 1. Coating of carrier with anti-IgE antibody
Measurement of IgE in blood by anti-IgE antibody coating method (1) Coating of carrier with anti-human IgE antibody Anti-human IgE mouse monoclonal antibody (SIGMA) 0.05M sodium phosphate buffer containing 0.25M NaCl It was dissolved in (pH 8.0) at 1.25 μg / ml. This is a microtiter plate (Nunc,
Microwell module plate, Maxisorp
-F8) was dispensed in 100 μl aliquots and left overnight at 4 ° C.

(2) Blocking of microtiter plate Each well was added with 330 μl of washing solution (2.7 mM KCl, 1
After washing twice with 0.1 M phosphate buffer (pH 7.4) containing 37 mM NaCl and 0.05% Tween 20, a blocking solution (0.5% (w / v) sodium caseinate (Wako Pure Chemical Industries) was included) Phosphate buffer (2.7 mM KC
1, 0.1 M phosphate buffer containing 137 mM NaCl (pH
7.4)) 300 μl was added and left at room temperature for 2 hours.

(3) Measurement of IgE Each well was washed 3 times with 330 μl of the washing solution, and then each well was diluted with a specimen dilution solution (0.5 M NaCl, 1% bovine serum albumin, 0.05% Tween 20 and 0.1 M phosphoric acid). 100 μl of buffer solution (pH 7.0) was dispensed. 10 μl of sample serum was added thereto and left at room temperature for 2 hours. Standard I
As gE, serum-derived standard IgE (2000 U / ml)
Was measured. The sample was diluted with 2-fold serial dilution, 10 μl was similarly added to each well, and the mixture was allowed to stand at room temperature for 2 hours. For samples outside the measurement range in this system, use 10% diluted sample.
The measurement was made by diluting by a factor of 2 or 100.

Each well was washed 3 times with 330 μl of the washing solution, and then the biotin-labeled soluble FcεRIα chain fragment (0.1 mg
/ Ml) is a labeling diluent (0.15 M NaCl, 1% bovine serum albumin in 0.1 M phosphate buffer (pH 7.
4)) was diluted 1000 times, 100 μl was added to each well, and the mixture was left for 1 hour. In addition, wash each well with 33
After washing 3 times with 0 μl, horseradish peroxidase-labeled avidin (1 mg / ml; Vector) was added to 50 ml with a labeling diluent.
It was diluted to 00 times, 100 μl of each was added, and the mixture was left at room temperature for 1 hour.

Next, each well was washed four times with 330 μl of the washing solution, and OPD tablets (Wako Pure Chemical Industries) were dissolved (0.1 M sodium phosphate buffer (pH 5.0), 0.009% H ₂ O).
₂ ) After dissolving in 50 ml, 100 μl of each was added, and the mixture was allowed to react for 30 minutes at room temperature in the dark. Then add 2N sulfuric acid to 100μ
1 was added to stop the reaction. The absorbance at 492 nm of each well was measured with a microtiter plate reader. FIG.
The standard curve when the serum-derived standard IgE (2000 U / ml) was serially diluted 2-fold is shown in FIG. Good dilution linearity is obtained. When actually measuring the amount of IgE in the sample, the amount of IgE in the sample was converted from this standard curve.

(4) Simultaneous reproducibility test Simultaneous reproducibility test was conducted according to the above test method. Specimen is serum containing 2030 IU / ml IgE with specimen diluent 2
Double serial dilution, 2030, 1015, 508, 128,
Alternatively, n = 8 was performed for those containing 0 IU / ml of IgE, only the sample diluent, and 3 samples of unknown IgE amount.
As shown in the results in Table 1, the CV value is 6.37% at the maximum, which shows good simultaneous reproducibility. This indicates that the detection system using this soluble FcεRIα is a very stable system for measuring IgE in serum.

[0040]

[Table 1]

Example 2. Carrier of soluble FcεRIα fragment
Of IgE in blood by coating on soluble FcεRI
α Fragment Coating Method (1) Coating of Soluble FcεRIα Fragment on Carrier Soluble FcεRIα fragment was dissolved in 0.05M sodium phosphate buffer (pH 9.6) at 2 μg / ml. Microtiter plate (Nunc, Microwell module plate, Maxisorp-F)
100 μl was dispensed into 8) and left at 4 ° C. overnight.

(2) Blocking of microtiter plate Each well was added with 330 μl of washing solution (2.7 mM KCl, 1
After washing twice with 0.1 M phosphate buffer (pH 7.4) containing 37 mM NaCl and 0.05% Tween 20, a blocking solution (0.5% (w / v) sodium caseinate (Wako Pure Chemical Industries) was included) Phosphate buffer (2.7 mM KC
1, 0.1 M phosphate buffer containing 137 mM NaCl (pH
7.4)) 300 μl was added and left at room temperature for 2 hours.

(3) Measurement of IgE After washing each well with 330 μl of the washing solution three times, a sample dilution solution (0.5 M NaCl, 1% bovine serum albumin in 0.1 M phosphate buffer (pH 7.0) was added to each well. )) 1
00 μl was dispensed. 10 μl of sample serum was added thereto and left at room temperature for 2 hours. As the standard IgE, serum-derived standard I
gE (2000 U / ml) was measured. 2 with sample dilution solution
After serially diluting, 10 μl was added to each well and left at room temperature for 2 hours. In addition, samples outside the measurement range in this system were diluted 10 times or 100 times with a sample diluent and measured.

Each well was washed 3 times with 330 μl of washing solution, and then horseradish peroxidase-labeled goat anti-human IgE was added.
A polyclonal antibody (Cappel) was added with a labeled diluent (0.1
0.1M containing 5M NaCl, 1% bovine serum albumin
Dilute 2000 times with phosphate buffer (pH 7.4)) and
Each 100 μl was added and left at room temperature for 1 hour. Next, after washing each well 4 times with 330 μl of washing solution, OPD tablets (Wako Pure Chemical Industries) were dissolved in 50 ml of dissolution solution (0.01% H ₂ O ₂ , 0.1M sodium phosphate buffer (pH 5.0)). After 10
0 μl of each was added, and the mixture was allowed to react at room temperature for 20 minutes while protected from light.

Thereafter, 100 μl of 2N sulfuric acid was added to stop the reaction. The absorbance at 492 nm of each well was measured with a microtiter plate reader. FIG. 2 shows the dilution linearity of 2-fold serial dilution of serum-derived standard IgE (1000 U / ml). It shows good dilution linearity.
When actually measuring the amount of IgE in the sample, the amount of IgE in the sample was converted from this standard curve.

Example 3. Conventional method, anti-IgE antibody coating method
(Example 1) and soluble FcεRIα fragment coating method (actual)
Measurement of IgE in serum of allergic patients in Example 2) The amount of IgE in serum of allergic patients or normal persons was determined by the conventional method (hereinafter, Ergia method), anti-IgE antibody coating method (Example 1, hereinafter, FcR method), soluble FcεRIα. It was measured by three methods, a fragment coating method (Example 2: hereinafter FcR coating method).

The conventional method is Elgia-IgE (International Reagents Co., Ltd.)
Was measured by an automatic measuring device (Elsia Auto) according to the attached measuring method. The FcR method is the method of Example 1,
The R coating method was measured by the method of Example 2. As the standard IgE, the serum-derived IgE attached to Ergia IgE was used, and the measurement was performed in each system to draw a standard straight line. Approximately 90 cases of serum of allergic patients or normal persons were measured by the three methods, but samples exceeding the measurement range were diluted with a sample diluent and remeasured. Tables 2 and 3 show the measured values of the three methods.

[0048]

[Table 2]

[0049]

[Table 3]

FIG. 3 shows the correlation between the measured values of the Ergia method and the FcR method, FIG. 4 shows the correlation between the measured values of the Ersia method and the FcR coat method, and FIG. 5 shows the measured values of the FcR method and FcR coat method. The correlation of the measured values was shown. The correlations of the respective measured values are relatively well correlated, and the correlation coefficients are 0.949, 0.
It is 981, 0.983. However, in some samples, there are cases in which the number of units deviates in each measurement method.

In particular, there are few examples in which the measured values of the FcR method and the FcR coat method are different from each other.
In the two methods using the RIα fragment (Figs. 3 and 4), there are some examples of deviation. This is because the method for measuring IgE using the soluble FcεRIα fragment of the present invention binds FcεRI to functional I.
This is because only gE is detected. It is also considered that the influence of substances that inhibit the antigen-antibody reaction in blood can be excluded.

Example 4. RIA method, Ergia method, FcR
Of IgE in serum by the method and FcR coat method RI which is one of detection systems of IgE using antigen-antibody reaction
The measurement results of the A method and the measurements of the Ergia method, FcR method, and FcR coat method were compared. The RIA method was measured using Pharmacia IgE rear (hereinafter RIST method: Pharmacia Biosystems). About 130 specimens already measured by RIST method, Ersia method, FcR method, F
It was measured by the cR coating method. The results are shown in Tables 4 and 5.

[0053]

[Table 4]

[0054]

[Table 5]

Ig in four methods as in Example 3
The E measurements are relatively correlated. However, No.
Examples such as 85 and No. 112 are seen. It is considered that these are examples in which the usefulness of the measurement system using the soluble FcεRIα fragment was strongly shown.

Reference Example 1. Biotin of soluble FcεRIα fragment
Soluble F secreted and purified by expression in a chin-labeled baculovirus expression system
Biotinylation of the cεRIα chain was performed as follows. The purified soluble FcεRIα chain was prepared at 0.2 mg / ml in a 0.1 M sodium hydrogen carbonate and 0.2 M sodium chloride solution. 1.7 mg / ml in 2.9 ml of this solution
30 μl of NHS-LC-Biotin (Pierce)
The reaction was continued for 2 hours at room temperature with gentle stirring. Further, 0.2 M glycine solution (pH 8.0) was added, and the mixture was gently stirred at room temperature for 1 hour. This solution was dialyzed against 0.2 M sodium chloride, 20 mM Tris-HCl buffer (pH 7.6) to remove the reaction reagent, and biotin-labeled soluble FcεR
Iα chain was obtained.

Reference Example 2. Soluble human FcεRI α chain inheritance
Preparation of Plasmid Having Child Fragment Plasmid BS-having Human FcεRI α Chain Gene Fragment
From SK (+) herα, DNA synthesizer (8700DNA Synt
The target soluble human FcεRI α chain gene fragment was prepared by a PCR reaction using a primer DNA synthesized using hesizer, MilliGen / Biosearch). As the primers, NTV primer and PAS primer were prepared. The respective sequences are shown below. NTV primer; 5'-AAAAACTCGAGATGGCTCCTGCCATGGAATCCCCTACT (SEQ ID NO: 1) PAS primer; 5'-AAAAAGGATCCTTAAGCTTTTATTACAGTAATGTTGA (SEQ ID NO: 2)

The PCR reaction was carried out under the following conditions. 1
00 μl of reaction solution [1 ng plasmid BS-SK (+) h
erα DNA, 1 μM NTV primer, 1 μM P
AS primer, 10 mM Tris-HCl (pH 8.
3), 50 mM KCl, 1.5 mM MgCl ₂ , 0.0
01% gelatin, 200 μM dATP, 20
0 μM dCTP, 200 μM dGTP, 200 μM
dTTP, 2.5U AmpliTaq DNA polymerase (Takara Shuzo)] was prepared and the DNA Thermal Cycler (PerkinElmer Ce
30 cycles of 94 ° C. for 1 minute, 50 ° C. for 2 minutes, and 72 ° C. for 3 minutes.

A 20 μl portion of the reaction solution was applied to 0.8% agarose and electrophoresed. About 0.6 separated
The kb DNA fragment was transferred from agarose using a GeneClean kit (BIO101) in 16 μl of TE solution [10 mM.
Tris-HCl (pH 7.5), 1 mM EDTA]. There, 10X reaction buffer [100 mM Tr
is-HCl (pH 7.5), 1.5M NaCl, 70
mM MgCl ₂ ], 2 μl was added, and BamHI, XhoI
1 μl of each was added and reacted at 37 ° C. for 1 hour.

After heat inactivating the enzyme by treating the reaction solution at 65 ° C. for 10 minutes, a 5 μl portion of the enzyme was added to BamH.
Cloning vector pT cut with I and XhoI
3T7U19X (Japanese Patent Application No. 3-106600) with 10 ng DNA Ligation Kit (Takara Shuzo Co., Ltd.) Solution A 3
The mixture was mixed with 0 μl and 6 μl of solution B and reacted at 16 ° C. for 1 hour to ligate both DNAs.

E. coli XL1-Blue (Stratagene) was added to Hanahan using 10 μl of the obtained DNA solution.
Method (DNA cloning: A practical approach (ed.D.
M.Glover), vol.1, p109-, IRC press (1885)] and plated on an L-amp plate containing X-Gal, and selected white colonies for ampicillin resistance and β-galactosidase deficiency. Colonies were selected.

Recombinant phages were prepared by culturing the selected colonies in L-amp medium and infecting them with Herpape phage VCSM13. Single-stranded DNA was prepared from the prepared recombinant, and the prepared DNA was about 0.8 μg.
Using the DNA Sequencing System / Taq polymerase (Applied Biosystems) according to the conditions recommended by the manufacturer, and the reaction product is the DNA sequencer Model 370A.
(Version 1.30, Applied Biosystems) was used to analyze and determine the nucleotide sequence.

Approximately 1 μg of recombinant plasmid DNA prepared by the alkaline lysis method was subjected to Cycling sequence using M13RPI primer (Applied Biosystems).
The reaction is performed according to the method recommended by the manufacturer by the encing method (Applied Biosystems Technical Manual), and the reaction product is DNA sequencer Model 370A (version 1.
30, Applied Biosystems) and analyzed to determine the nucleotide sequence.

Of the sequences determined by both methods, the structural gene part is not different from that of the previously reported human FcεRIα chain gene, and there is an XhoI site introduced by the NTV primer before the translation initiation codon. , And when the 25th valine of the encoded amino acid sequence is the 1st position, there may be a translation termination codon TAA and a BamHI site introduced by the PAS primer immediately after the codon encoding the alanine at the 172nd position. The confirmed recombinant plasmid pNTV2 was obtained.

Reference Example 3. Human soluble FcεRIα chain inheritance
Isolation of daughter fragment Total RNA Sepa from KU812 cells (4 × 10 ⁷ cells)
RNA was extracted using a rator kit (Clontech) according to the method recommended by the manufacturer. MR from extracted RNA
PolyA RNA was purified using NA Separator Kit (Clontech) according to the method recommended by the manufacturer. 40
It was possible to obtain μg of polyA RNA. Using 5 μg of this, double-stranded cDNA was synthesized using a Time Saver cDNA synthesis kit (Pharmacia) according to the method recommended by the manufacturer.

10 μl of a part of the obtained cDNA solution (150 μl) was used to prepare the above-mentioned NTV primer, P
Using AS primer, 100 μl of reaction solution (1 μM
NTV primer, 1 μM PAS primer, 10 mM
Tris-HCl (pH 8.3), 50 mM KCl,
1.5 mM MgCl ₂ , 0.001% gelati
n, 200 μM dATP, 200 μM dCTP, 2
00 μM dGTP, 200 μM dTTP, 2.5U
AmpliTaq DNA polymerase (Takara Shuzo)) was prepared and DN
Using A Thermal Cycler, 94 ℃ for 1 minute, 50 ℃
The reaction was carried out for 30 cycles of 2 minutes at 72 ° C and 3 minutes at 72 ° C.

A 20 μl portion of the reaction solution was applied to 0.8% agarose and electrophoresed. About 0.6 separated
The kb DNA fragment was transferred from agarose using the GeneClean kit in 16 μl of TE solution [10 mM Tris-
HCl (pH 7.5), 1 mM EDTA]. 10X reaction buffer [100 mM Tris-HCl]
(PH 7.5), 1.5M NaCl, 70 mM MgCl
₂ ], and add 1 x each of XhoI and BamHI
μl was added and the mixture was reacted at 37 ° C. for 1 hour.

After heat inactivating the enzyme by treating the reaction solution at 65 ° C. for 10 minutes, a portion of 5 μl of the enzyme was deactivated by Xho.
Cloning vector pBl cut with I and BamHI
uescript II KS (+), DNA with 10 ng
A ligation kit, solution A (30 μl) and solution B (6 μl) were mixed and reacted at 16 ° C. for 1 hour to ligate both DNAs.
E. coli XL1-b using 10 μl of the obtained DNA solution
transforming lue according to the method of Hanahan,
An L-amp plate containing X-Gal was plated and white colonies were selected to select ampicillin-resistant β-galactosidase-deficient colonies.

Plasmid DNA prepared from colonies
Dy primer cycling sequencing kit (Applied Biosystems Co., Ltd.) was used to react 1.5 μg with M13RP1 or -21M13 primer according to the conditions recommended by the manufacturer, and the reaction product was reacted with DNA sequencer Mo.
The nucleotide sequence of the inserted fragment was determined by analysis using del 373A.

The inserted fragment has the same sequence as the reported sequence, has an XhoI site immediately before the translation initiation codon ATG, and has an alanine codon present at position 172 when valine at position 24 is at position 1. It was confirmed that the translation stop codon and the BamHI site were inserted immediately below.
It is clear that the plasmid thus obtained can be used in place of pNTV2 in Reference Example 2.

Reference Example 4. Expression of soluble human FcεRIα chain
Construction of current plasmid Plasmid pN having soluble human FcεRI α chain gene
1 μg of TV2 in 10 μl of reaction solution [10 mM Tri
s-HCl (pH 7.5), 100 mM NaCl, 7 mM
MgCl ₂ , 12U XhoI, 10U BamHI]
At 37 ° C. for 1 hour.

After completion of the reaction, the DNA was extracted with phenol / chloroform and then recovered by the ethanol precipitation method. This DNA fragment was made blunt-ended by a method recommended by the manufacturer using a DNA blunting kit [Takara Shuzo]. The reaction solution was subjected to 0.8% agarose electrophoresis, and the separated fragment of about 0.6 kb was separated from the agarose piece by G
Harvested in 10 μl TE solution using the eneClean kit.

On the other hand, pAcYM1 which is a baculovirus expression vector (Matsuura, Y. et al., J. Gen. Virol. 68 , p123
3-1255) 1 μg in 10 μl of reaction solution [10 mM Tri
s-HCl (pH 7.5), 150 mM NaCl, 7 mM
MgCl ₂ , 10 U BamHI] at 37 ° C.
Allowed to react for hours. The reaction solution was subjected to 0.8% agarose electrophoresis, and the separated fragment of about 10 kb was recovered from a small piece of agarose in 10 μl of TE solution using the GeneClean kit.

2 μl of a solution containing this recovered fragment and p
3 μl of the solution containing the fragments recovered from NTV2 and 25 μl of DNA ligation kit A solution and 5 μl of solution B respectively
It was mixed with 1 and reacted at 16 ° C. for 1 hour to ligate both DNAs. E. coli XL1-blue was transformed with 10 μl of each of the obtained DNA solutions according to the method of Hanahan, plated on L-amp plates, and ampicillin-resistant colonies were selected. Colony plasmid D
The baculovirus expression vector pAcYM1-NTV was selected by examining the restriction enzyme sites of NA and selecting a transformant having a plasmid to which each fragment was ligated.
I got

Reference Example 5. Soluble human FcεRI α chain expression
Isolation of Recombinant Virus Sf-9 cell line, which is a night-throwing moth-intestine cell line, and pAcYM1-NTV, a baculovirus expression vector,
and 1 μg of natural baculovirus DNA were infected by the calcium phosphate precipitation method. The method of infection was according to the method of Matsuura (Journal of Experimental Medicine, p281-284, 1990). Plaques not synthesizing polyhedrin were selected by post-infection plaque assay. Recombinant virus was isolated by performing this plaque assay three times.

MOI (multiplicity
of infection) = 1 to infect Sf-9 cells for 10
72 in Grace's medium (Gibco) containing% FCS
By culturing for a long time and analyzing a part of the culture supernatant by Western blotting, soluble human FcεR
Ac-NTV, a recombinant virus expressing Iα chain, was isolated. MOI (multiplicity of infe
ction) = 1 to infect Sf-9 cells and 10% FC
About 1 × 10 ⁸ pfu / ml was obtained by culturing for 72 hours in Grace medium (Gibco) containing S and collecting the culture supernatant.
The recombinant virus was recovered.

Reference Example 6. The purified recombinant virus expression and expression products MOI (multiplicity of infectio
n) = 5 and infected Sf-9 cells (4 × 10 ⁸ cells) with Grace medium (Gibco) containing 10% FCS.
It was cultured in 400 ml of medium for 72 hours, and the culture supernatant was collected. Ammonium sulfate was added to the collected supernatant to a saturated concentration of 60%, and the mixture was kept at 4 ° C. for 2 hours. The precipitate was collected by centrifugation at 10,000 rpm for 30 minutes.

The collected precipitate was added to TBS (20 mM Tri
After dissolving it in s-HCl [pH 7.5], 150 mM NaCl), it was dialyzed against the same solution at 4 ° C. overnight. The solution after dialysis was collected, applied to a Sephacryl S200 column (2.6 cm × 60 cm) equilibrated with TBS, and separated by TBS.
Fractions having a molecular weight of around 30,000 to 40,000 were collected.

The anti-FcεRIα monoclonal antibody column was prepared as follows. BrCN-activ
Aerated Sepharose 4B (Pharmacia) was swollen in 1 mM hydrochloric acid at room temperature for 15 minutes. The swollen gel was washed and swollen repeatedly on a glass filter with 1 mM hydrochloric acid, and then washed with a coupling buffer solution (0.5 M sodium chloride, 0.1 M sodium bicarbonate (pH 8.3)).

Immediately, 2 ml of this gel was added to 2 ml (5 mg / ml) of a purified anti-FcεRIα monoclonal antibody solution dissolved in a coupling buffer, and the mixture was gently stirred at room temperature for 2 hours. The supernatant was removed and 0.2 M glycine hydrochloric acid (pH 8.0) was added.
The gel was suspended in and the mixture was gently stirred at room temperature for 2 hours. Next, this was repeatedly washed with a coupling buffer solution and a 0.1 M acetate buffer solution containing 4.0 M sodium chloride (pH 4.0) to obtain an anti-FcεRIα monoclonal antibody column.

Anti-FcεR of the fractions obtained by gel filtration
Purification with an Iα monoclonal antibody column was performed as follows. Anti-FcεRIα equilibrated with 20 mM Tris-HCl buffer (pH 7.6) containing 0.5 M sodium chloride
The FcεRIα-containing fraction obtained by gel filtration of the culture supernatant was applied to the monoclonal antibody column, and the column was thoroughly washed with the buffer used for equilibration.

Next, 3M sodium thiocyanate and 0.
20 mM Tris-HCl buffer containing 5 M sodium chloride (pH
Elution from the column was performed at 7.6), and the eluted fraction was immediately dialyzed against 20 mM Tris-HCl buffer pH (7.6) containing 0.1 M sodium chloride to obtain soluble FcεR.
A purified Iα fragment was obtained.

Reference Example 7. Purity test of purified sample Purity test of purified sample was carried out as follows using SDS-polyacrylamide gel electrophoresis. Concentrate the purified sample and use the sample buffer (2% sodium dodecyl sulfate,
5% 2-mercaptoethanol, 7% glycerol,
62.5 mM containing 0.005% bromphenol blue
It was suspended in Tris-HCl buffer (pH 6.8)) and boiled at 100 ° C. for 5 minutes.

This sample was electrophoresed using a concentration gradient slab gel for SDS-polyacrylamide gel electrophoresis (manufactured by Daiichi Pure Chemicals Co., Ltd.) having a separation gel concentration of 10-20%, and a silver staining kit (Daiichi Pure Chemicals) The protein was detected by staining the gel with Yakuhin Co., Ltd.). As a result, the molecular weight is 3
A single band spread around 0k to 35k was detected.

[0085]

[Sequence list]

 SEQ ID NO: 1 Sequence Length: 38 Sequence Type: Nucleic Acid Number of Strands: Single Strand Topology: Linear Sequence Type: Synthetic DNA Sequence AAAAACTCGA GATGGCTCCT GCCATGGAAT CCCCTACT 38

SEQ ID NO: 2 Sequence Length: 37 Sequence Type: Nucleic Acid Number of Strands: Single Strand Topology: Linear Sequence Type: Synthetic DNA Sequence AAAAAGGATC CTTAAGCTTT TATTACAGTA ATGTTGA 37

[Brief description of drawings]

FIG. 1 is a serum-derived standard IgE (2000 U / m 2).
2 is a graph showing the results of measuring the absorbance at 492 nm by diluting l) with 2-fold serial dilution and using the method of Example 1 of the present invention (anti-IgE antibody coating method).

FIG. 2 is a serum-derived standard IgE (1000 U / m 2).
l) is diluted by 2-fold serial dilution, and 492 is diluted by the method of Example 2 of the present invention (soluble FcεRIα fragment coating method).
It is a graph which shows the result of having measured the light absorbency in nm.

FIG. 3 is a graph showing the correlation between the value measured by the conventional Ergia method and the value measured by the FcR method of the present invention regarding the amount of IgE in human serum.

FIG. 4 is a graph showing the correlation between the IgE amount in human serum measured by the conventional Ergia method and the measured value by the FcR coat method of the present invention.

FIG. 5 is a graph showing the correlation between the amount of IgE in human serum measured by the FcR method of the present invention and the measured value by the FcR coat method of the present invention.

 ─────────────────────────────────────────────────── ─── Continuation of front page (72) Inventor Mitsuaki Yanagida 1-3-1 Nishitsurugaoka, Oi-cho, Iruma-gun, Saitama Tonen Corporation Research Institute (72) Kenjiro Yamaguchi Nishitsuruga, Oi-cho, Iruma-gun, Saitama Oka 1-3-1 Tonen Co., Ltd. Research Institute (72) Inventor Shintaro Yagi Nishitsurugaoka, Oi-cho, Iruma-gun, Saitama Prefecture 1-3-3 Tonen Co. Research Institute (72) Inventor Fukuya Toyoji Saitama 1-3-1 Nishitsurugaoka, Oi-cho, Iruma-gun Tonen Corporation Research Institute (72) Inventor Chiyasu Ra, 2-14-13 Hanazono, Hanamigawa-ku, Chiba-shi, Chiba

Claims (4)

[Claims] 1. I in a test sample which is blood or a blood component
In the method for detecting or measuring gE, blood or a blood component is optionally diluted and then contacted with a soluble α chain of a high affinity IgE receptor (FcεRI) to determine whether or not IgE binds to the soluble α chain of FcεRI. A method comprising measuring the amount of binding and determining the presence or amount of IgE from the result. 2. The method of claim 1, wherein the blood or blood component is whole blood or serum or plasma. 3. A solid is prepared by contacting a fixed carrier on which an anti-IgE antibody is immobilized with a test sample or a dilution thereof, if IgE is present in the test sample, the IgE is bound to the anti-IgE antibody. The soluble α-chain of FcεRI is bound to the immobilized IgE by immobilizing it on a carrier and then contacting the solid α-carrier with the soluble α-chain of FcεRI, thus binding FcεRI.
The presence or absence or amount of IgE in the test sample is determined from the presence or absence or amount of the soluble α chain of Escherichia coli.
The method described in. 4. If IgE is present in the test sample by contacting the test sample or a dilution thereof with a solid support on which the soluble α chain of FcεRI is immobilized, the IgE is added to the F sample.
It is immobilized on a solid support by binding to the soluble α-chain of cεRI, which is then contacted with an anti-IgE antibody, whereby the IgE antibody is immobilized on said immobilized IgE.
The presence or amount of IgE in a test sample is determined from the presence or amount of the immobilized IgE by immobilizing the IgE antibody by binding with IgE antibody.
The method described in.