JP2000266746A - Specific ige antibody measuring method - Google Patents

Abstract

PROBLEM TO BE SOLVED: To measure a specific IgE antibody in a blood serum using a normal plate for ELISA without receiving interference due to competition of a coexisting specific IgG antibody. SOLUTION: After pretreating a blood serum with a ligand specifically adsorbing an IgG antibody and removing the specific IgG antibody to a degree not causing a competition with a specific IgE antibody, the specific IgE antibody is measured by a normal ELISA method. A ligand fixed to an insoluble carrier is preferable as the ligand specifically adsorbing the IgG antibody.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a method for measuring a specific IgE antibody. According to the present invention, a conventional ELIS can be used without interference from competition of co-existing specific IgG antibodies.
Specific IgE antibodies in serum against any antigen immobilized on the plate for A can be measured, which is useful for more detailed analysis of antigens causing allergy.

[0002]

2. Description of the Related Art Since the discovery of IgE antibodies, which are antibodies that play a decisive role in allergic reactions, many studies have been reported on their measurement methods, and specific IgE antibodies have been reported.
The clinical importance of measuring antibodies is also widely recognized.

[0003] In the field of clinical testing for allergies, specific
The CAP-RAST system is widely used for measuring IgE antibodies. The value measured with the caplast system is the RAST for each antigen (allergen).
It has become the de facto standard as a unit (Ua / ml). However, it is difficult to measure specific IgE antibodies using antigens that are not commercially supplied for caplast systems. In order to analyze an antigen that causes allergy, that is, an allergen in more detail, it is necessary to develop a method that can easily measure a specific IgE antibody using any antigen.

In order to measure a specific antibody using an arbitrary antigen, an ordinary 96-well microplate was used.
It is recommended to use ELISA method. However, specific in serum
IgE measurement was performed using a standard 96-well microplate.
LISA is not easy. The reason is that IgE in serum
Antibodies are usually present at very low concentrations, typically tens of thousands of times lower than IgG antibodies, and the serum contains co-specific IgG antibodies with the same specificity as the specific IgE antibody to be measured. This causes a problem of competition with the specific IgG antibody.

[0005] For example, consider a case where a specific IgE antibody in serum against an antigen immobilized on a plate is measured by ELISA using a usual 96-well microplate. In a 96-well microplate, the immobilized density of the antigen, ie, the amount of the immobilized antigen per unit area, is not so high. Also, since the concentration of specific IgE antibody is measured at a low concentration, the dilution ratio of the serum cannot be too high. for that reason,
The concentration of the coexisting specific IgG antibody becomes considerably high, and the amount of the specific antibody combining the IgG antibody and the IgE antibody becomes excessive with respect to the amount of the immobilized antigen. In such a state, the immobilized antigen is mostly occupied by the predominant specific IgG antibody, which makes it difficult for the specific IgE antibody to bind to the immobilized antigen (Zeiss et a
l., J. Allergy Clini. Immunol., 67, 105 (1981)).
In addition, when the serum is diluted at a high magnification so that the amount of the specific antibody is smaller than the amount of the immobilized antigen, the concentration of the specific IgE antibody becomes very low, and the detection often becomes difficult.

[0006] In order to avoid such problems caused by competition with specific IgG antibodies and to accurately measure specific IgE antibodies in serum, the following methodologies have been considered.

[0007] By increasing the amount of immobilized antigen bound, a method has been considered in which measurement can be performed in a state where the amount of immobilized antigen is larger than the amount of specific antibody even when a serum with a low dilution ratio is used. This is a method using a cellulose polymer which is a sponge-like substance to which an antigen is bound at high density (Ceska et al., J. Allergy Clin. Immunol., 49, 1 (1
972)). This method is currently applied to the caplast system, which is widely used in the field of clinical testing for allergies, and its data is commonly known as the RAST unit (Bousquet et al., J. Allergy Clin. Im
munol., 85, 1039 (1990)). However, since it is not easy to prepare a sponge-like substance on which an antigen is immobilized, it is difficult to perform measurement using any antigen that is not commercially supplied for a cap-last system. In addition, the standard ELIS
A dedicated device different from A is required.

[0008] A method using an anti-IgE secondary antibody labeled with acetylcholinesterase, an enzyme having extremely high molecular activity, has been reported (Wal et al., Food & Agricultu).
ralImmunology, 7, 175 (1995); Grassi et al., J. Imm
unological Methods, 123, 193 (1989)). The diluted antiserum was used to reduce the amount of the specific antibody to the amount of the immobilized antigen, and as a result, specific IgE bound to the immobilized antigen was used.
This method is capable of detecting a specific IgE antibody with an enzyme having extremely high molecular activity even when the amount of the antibody is extremely small. However, acetylcholinesterase is not used in normal ELIS
Anti-IgE antibodies labeled with that enzyme, which is not the enzyme commonly used in A, are currently not available. Anti-IgE antibodies that can be practically used without cross-reactivity problems are limited to some extent, and the production of a new acetylcholinesterase-labeled anti-IgE antibody requires a lot of time and labor. In addition, since a very small amount of a substance is to be detected, how to suppress the influence of a non-specific signal becomes a major problem.

A method called Capture ELISA has been reported. This is a method in which a specific IgE antibody is detected by selecting an IgE antibody in the antiserum with an immobilized anti-IgE antibody and reacting with a labeled antigen (Plebani et a
l., J. Immunol. Methods. 90, 241 (1986); Sakaguchi
et al., J. Immunological Methods, 116, 181 (1989);
Olivieri et al., J. Immunol.Methods, 157, 65 (199
3)). In this method, first, only antibodies of the IgE class are captured, so that competition with IgG antibodies does not occur. But,
It is necessary to prepare a label for each antigen to be examined,
Modification of the antigen by labeling can be problematic. In addition, competition between specific IgE antibodies and specific IgG antibodies can be avoided, but the problem arises that competition between antigen-specific IgE antibodies and other IgE antibodies that are not specific for the antigen, i.e., non-specific IgE antibodies. However, antiserum with high non-specific IgE antibody concentrations, often found in the sera of allergic patients, may interfere with the detection of specific IgE antibodies. In such a case, it is necessary to perform the measurement under the condition that the total amount of IgE antibody in the serum is smaller than the amount of immobilized anti-IgE antibody.

[0010] A method has been reported in which IgG antibodies are reduced by ammonium sulfate precipitation and then specific IgE antibodies are measured by ordinary ELISA (Haba et al., J. Immunological Methods, 85, 3).
9 (1985)). However, in this method, conditions under which ammonium sulfate salting out can be performed are limited, and the treatment is difficult with a small amount of antiserum.

As described above, several measurement methods have been considered, and any of these methods is specific for any antigen.
It is difficult to easily measure IgE antibodies using a standard ELISA plate.

[0012]

DISCLOSURE OF THE INVENTION The present invention has been made to solve the above-mentioned problems, and is intended to solve the problem of the coexisting specific IgG antibody using a conventional ELISA plate and a conventional ELISA method. Specific IgE in serum for any antigen immobilized on the plate without interference from competition
It is an object to provide a method for easily measuring an antibody.

[0013]

SUMMARY OF THE INVENTION The present invention relates to a method for measuring a specific IgE antibody in serum without being interfered by competition of a specific IgG antibody coexisting therewith. The serum to be measured is treated with a ligand that specifically adsorbs the IgG antibody, and the specific IgG antibody is removed to such an extent that competition with the specific IgE antibody does not occur.
Measure antibody. By using this method, the usual
Specific Ig for any antigen using ELISA plate
E antibody can be measured easily and is useful for more detailed analysis of antigens that cause allergy.

[0014]

BEST MODE FOR CARRYING OUT THE INVENTION The amount of antigen that can be immobilized on a normal 96-well microplate for ELISA is at most several hundred nanograms per square centimeter. Very low compared to the antigen amount of tens of micrograms per square centimeter. Therefore, in the present invention, in order to measure the specific IgE antibody concentration for any antigen immobilized on the plate, the specific IgG antibody coexisting in the serum is removed in advance to the extent that competition does not occur, and then the normal ELISA is performed. Specific IgE antibody is measured by the method.

As a method for adsorbing and removing the IgG antibody, protein A and protein G of Staphylococcus aureus cell wall protein, which are ligands having a property of selectively binding to the IgG antibody, can be used. However, for the purpose of measuring specific IgE antibodies, it is desirable to use protein G. The reason is that
First, protein G is more likely to be a mammalian Ig than protein A.
High affinity for G antibody (Akerst
rom and Bjorck, J. Biol. Chem., 261, 10240 (198
6)). In addition, protein A may show some binding to specific IgE antibodies, whereas protein G does not show such binding (Peng et al., Int.
Arch. Allergy Immunol., 104, 204 (1994)).

Considering that IgG antibodies including specific IgG antibodies bound to protein G are removed from serum, it is desirable that protein G is immobilized on an insolubilizing carrier such as beads. In the actual removal treatment, after serum and immobilized protein G are mixed and treated, IgG antibodies bound to the immobilized protein G can be removed from the serum by centrifugation.

In the present invention, an IgG antibody is
It must be removed to the extent that it does not compete with the E antibody. The amount of specific IgG antibodies in human serum involved in competition with specific IgE antibodies may vary depending on the individual sera, but the total IgG antibody combined with non-specific IgG antibodies is usually 8 to 16 mg. / ml is known. Therefore, in a mixed solution obtained by mixing the immobilized protein G and the serum, the adsorption capacity of the immobilized protein G at a certain dilution ratio of the serum increases the IgG present in the mixed solution.
If the adsorption capacity of immobilized protein G is about 16 mg / ml or more when converted to a serum stock solution based on the total amount of antibodies, in other words, based on the dilution ratio of serum, it is considered that most of the IgG antibodies have been removed. You can think.

However, the dilution ratio of the serum at this time also needs to be within a certain range in which a specific IgE antibody can be detected. When the adsorption capacity of the immobilized protein G is small, a large amount of the immobilized protein G is required, and the serum is diluted to a high magnification, which may make it difficult to detect a specific IgE antibody. Therefore, in order to efficiently remove IgG antibodies and to achieve a serum dilution ratio that allows detection of trace amounts of specific IgE antibodies, it is desirable that the protein G adsorption capacity be as large as possible.

The immobilized protein G can be prepared using various carriers. Also, various types of immobilized protein G are currently commercially available, and can be obtained and used. For example, Protein G with high adsorption capacity immobilized by Pierce (UltraLink (TM) Immo
bilized Protein G, UltraLink (TM) Immobilized Prot
ein G Plus, etc.) are beads in which protein G is immobilized on a carrier (UltraLink (TM) Biosupport Medium) provided by the company and has a surface area of about 26 mg / ml (UltraLin
k (TM) Immobilized Protein G) or about 40mg / ml (U
ltraLink (TM) Immobilized Protein G Plus)
It has the ability to adsorb IgG antibodies and is easily separated by centrifugation. Therefore, the dilution ratio of serum does not become too high when mixed with immobilized protein G, and a trace amount of specific IgE antibody can be detected.

As described above, Ig containing specific IgG antibody
The serum from which the G antibody has been removed can be purified using a standard ELISA plate on which any antigen is immobilized and a standard ELISA method.
Specific IgE antibodies can be measured without competition with G antibodies. In addition, it is possible to confirm that competition with the specific IgG antibody has not occurred by confirming whether or not the serum dilution ratio and the detection intensity of the specific IgE antibody show a correlation.

By using the measuring method of the present invention, the EL
ISA can measure specific IgE antibodies against any antigen.For example, in addition to the RAST unit, which is widely used as a common measure in the field of allergy clinical tests, allergen analysis based on individual cases Can be performed in more detail.

[0022]

Example 1 Serum Protein G Treatment: Serum collected from a milk allergic patient was treated with phosphate buffered saline (PBS).
Two-fold dilutions and two-fold dilutions of immobilized protein G gel (Pierce) with PBS were mixed in equal amounts and allowed to react at room temperature for 1 hour with shaking. After the reaction, the mixed solution was centrifuged to remove the immobilized protein G. Measurement of specific IgE antibody by ELISA: The serum protein-specific IgE antibody in the serum of a milk-allergic patient treated with protein G as described above was measured by ELISA. 96
50 μl of a 10 μg / ml skim milk solution dissolved in 0.1 M carbonate buffer (pH 8.7) was dispensed into each well of a well microplate (Nunc) and immobilized at 5 ° C. for 16 hours or more. 0.05% T as blocking operation to reduce non-specific adsorption
Dissolved in phosphate buffered saline containing ween20 (PBS-T)
Dispense 0.2 ml of 1% BSA solution (PBS-TB) into each well,
It was left at room temperature for 1 hour. After washing each well three times with PBS-T, 50 μl of serum from a patient with milk allergy was added to each well and reacted at room temperature for 2 hours. Wash each well with PBS-T
After washing twice, 50 μl of horseradish peroxidase-conjugated anti-human IgE antibody (Seikagaku) diluted 1000-fold with PBS-TB was dispensed to each well, and reacted at room temperature for 2 hours. After each well was washed three times with PBS-T, 0.1 ml of a fluorescent peroxidase substrate (Pierce) was added to each well and reacted for 30 minutes to 1 hour. Thereafter, 0.1 ml of a reaction stop solution is added to each well, and the fluorescence intensity at an excitation wavelength of 320 nm and an emission wavelength of 440 nm is measured by cytoflow 4000.
(Japan Perceptive). Figure 1 shows the results
Shown in When protein G treatment was not performed, there was no correlation between the fluorescence intensity of the specific IgE antibody and the dilution ratio in a region where the serum dilution ratio was low, indicating that competition with the specific IgG antibody occurred. On the other hand, in the case of protein G treatment, a good correlation between the fluorescence intensity and the dilution ratio was observed even in a region where the serum dilution ratio was low, and it was found that competition with a specific IgG antibody could be avoided.

[0023]

[Test Example 1] It was confirmed whether or not protein G treatment had an effect on IgE antibodies using a human myeloma IgE antibody (Seikagaku Corporation). 96-well microplate (Nu
nc) was dissolved in 0.1 M carbonate buffer (pH 8.7) in each well.
50 μl of 10 μg / ml anti-human IgE antibody (Seikagaku Corporation)
The solution was dispensed at a time and immobilized at 5 ° C. for 16 hours or more. As a blocking operation to reduce non-specific adsorption, 0.2 ml of a 1% BSA solution (PBS-TB) dissolved in phosphate buffered saline (PBS-T) containing 0.05% Tween 20 was dispensed into each well, and For 1 hour. After washing each well three times with PBS-T, 50 μl of human myeloma IgE antibody is added to each well and incubated at room temperature.
Allowed to react for hours. After washing each well three times with PBS-T, 50 μl of horseradish peroxidase-conjugated anti-human IgE antibody (Seikagaku Corporation) diluted 1000-fold with PBS-TB is dispensed to each well and reacted at room temperature for 2 hours. I let it. After washing each well three times with PBS-T, 0.1 ml of a fluorescent peroxidase substrate (Pierce) is added to each well.
The reaction was performed for minutes to 1 hour. Then, add 0.1 ml of the reaction stop solution to each well, and excite at 320 nm and emission wavelength.
The fluorescence intensity at 440 nm was measured with a Cytoflow 4000 (Nippon Perceptive). As a result, as shown in FIG. 2, it was confirmed that protein G treatment had no negative effect on the detection of human myeloma IgE antibody.

[0024]

[Test Example 2] The protein G treatment performed in Example 1 resulted in the Ig in serum of the milk allergic patient in Example 1.
The effect on the E antibody was confirmed by measuring the total IgE antibody. How to make a human myeloma
The test was performed in the same manner as in Test Example 1 except that the serum of an allergic patient was used instead of the IgE antibody. As a result, as shown in FIG. 3, it was confirmed that the protein G treatment had no negative effect on the detection of IgE antibodies in the serum of allergic patients.

[0025]

According to the method of the present invention, coexisting specific
A specific IgE antibody in the serum of an allergic patient against any antigen immobilized on an ordinary ELISA plate can be simply and accurately measured without being disturbed by IgG antibody competition. The value measured by this method is useful for a more detailed analysis of an antigen causing allergy together with a RAST unit widely used in the clinical field.

[Brief description of the drawings]

FIG. 1 shows the effect of protein G treatment on the measurement of specific IgE antibodies in serum of milk allergy patients in Example 1.

FIG. 2 shows the effect of protein G treatment on human myeloma IgE antibody in Test Example 1.

FIG. 3 shows the effect of protein G treatment on total IgE antibodies in serum of milk allergy patients in Test Example 2.

Claims (3)

[Claims] 1. Using a ligand that specifically adsorbs an IgG antibody, a specific IgG antibody coexisting with a specific IgE antibody having the same specificity is previously removed, and then the specific antibody is detected by ELISA.
Specific Ig in serum characterized by measuring IgE antibody
E How to measure antibodies. 2. The method according to claim 1, wherein the ligand that specifically adsorbs the IgG antibody is a ligand immobilized on an insolubilized carrier. 3. The method according to claim 1, wherein the ligand that specifically adsorbs the IgG antibody is protein G.