JP2000065832A - Filter type biological specific reaction measurement carrier and measurement using it - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an accurate analysis result without requiring any pretreatment of a sample by using a porous filter made of a plurality of specific kinds of organic polymers and provided with a specific pore size. SOLUTION: A measurement carrier is constructed of a filter type organic polymer having a pore size of about 1-100 μm. The organic polymer is one of polymers selected from polyamide, polyacrylate, acrylic resin, methacrylic resin and the like or a mixture of two or more kinds of polymers selected from them. The surface of the filter type organic polymer is treated by hydrolysis or corona discharge of the fibrous surface or application of an active substance. A first substance specifically reacting with a measured substance is arranged in the lower part of the filter type biological specific reaction measuring carrier, and a biological specific reaction measuring apparatus provided with an absorbing layer absorbing liquid penetrating through the measuring carrier is used. The measured substance is antigen specific human IgG or human IgE.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to an antigen-antibody reaction,
The present invention relates to a method for measuring a substance involved in a specific reaction based on a biological specific reaction such as DNA-DNA, DNA-RNA, or RAN-RNA hybridization, and a carrier for measuring a filter-like biological specific reaction used in the measurement method. .

[0002]

2. Description of the Related Art A substance that specifically reacts with a substance to be measured is
For example, as a method of immobilizing the substance on a porous matrix such as a membrane or a filter and analyzing the substance to be measured using the same, for example, Methods in Enzymology;
646-656 (1981), a method for measuring IgE is reported. In addition, Tokushu Sho 60-500
In Japanese Patent No. 384, as a porous matrix,
It is reported that a filter made of glass, metal, polysaccharide, or the like can be used.

[0003]

Cellulose and its derivatives, which are widely used as porous matrices in immunochromatography and the like, have a dense structure and are difficult to pass through liquids containing highly viscous liquids and particles, and have been used particularly as specimens. There is a problem that the serum becomes clogged, resulting in an incorrect analysis result. To avoid this,
Before applying serum etc., dilute serum to reduce viscosity,
There is a problem in terms of operability and simplicity, such as the necessity of performing a pre-filter filtration or a centrifugal separation operation to remove particles and the like.

[0004] In order to reduce the pretreatment operation of such a sample, there are some cases where a glass fiber filter having a coarser liquid permeability than a membrane is used as a porous matrix. However, since glass has a negative charge, a substance in a sample is likely to be non-specifically bound by ionic bonding or the like, and this non-specifically bound substance often results in incorrect analysis.

[0005] The present invention provides a filter-like carrier for measuring a biologically specific reaction, which can avoid a sample pretreatment and can obtain a correct test result. The present invention further provides a measuring method using the carrier for measuring a biologically specific reaction.

[0006]

Means for Solving the Problems In view of the above circumstances, the present inventors have conducted intensive studies and as a result, have found that in an analytical method using a substance which specifically reacts with a substance to be measured as a solid phase, a specific type of organic polymer is used. By using a porous filter having a specific pore size, even if immersed in a liquid, without impairing the physical properties of the filter due to swelling of the fiber, etc.
The present inventors have found that complicated sample pretreatment is unnecessary, non-specific reactions due to substances in the sample are suppressed, and correct analysis results are obtained, and the present invention has been completed.

That is, the present invention has the following configuration. (1) A filter-like carrier for measuring a biological specific reaction, comprising a filter-like organic polymer having a pore diameter of 1 to 100 µm. (2) The organic polymer is polyamide, polyamide imide,
Polyarylate, polyimide, polyetherimide, polyetheretherketone, polyethylene, polyethylene oxide, polyester, polycarbonate, polystyrene, polysulfone, polyethersulfone, polyparamethylstyrene, polyallylamine, polyvinyl alcohol, polyvinyl ether, polyvinyl butyral, polyvinyl Formal, polyphenylene ether,
Polyphenylene sulfide, polybutylene terephthalate, polypropylene, polymethylpentene, acrylic resin, methacrylic resin, epoxy resin, xylene resin,
Guanamine resin, diallyl phthalate resin, vinyl ester resin, phenol resin, unsaturated polyester resin,
At least one selected from the group consisting of furan resin, polyimide, poly-p-hydroxybenzoic acid, polyurethane, maleic resin, melamine resin and urea resin resin
The carrier according to (1), which is a species or a mixture of two or more species. (3) The filter-like organic polymer is subjected to hydrolysis of the fiber surface, corona discharge, plasma treatment, UV-ozone treatment,
The carrier for measuring a biological specific reaction in a filter according to (1) or (2), wherein at least one type of surface treatment selected from the group consisting of application of an active substance is applied. (4) The first substance which specifically reacts with the substance to be measured is placed on the lower part of the carrier for measurement of a specific reaction in the form of a filter according to any of (1) to (3), Using a biologically specific reaction measuring instrument provided with an absorption layer for absorbing the liquid that has passed through the specific reaction measuring carrier, a sample containing the substance to be measured, and a detectable signal are generated from the top of the porous filter of the instrument. Sequentially supplying a second substance that specifically reacts with the analyte to which the substance is bound, and calculating the amount of the analyte in the sample by measuring a signal derived from the signal generating substance. Measurement method to be performed. (5) The substance to be measured is an antigen-specific human IgG (4)
The measurement method described. (6) The filter-like substance under the carrier for measuring a biologically specific reaction according to any one of (1) to (3), wherein a fourth substance specifically reacting with the third substance is immobilized. The first substance that specifically reacts with the substance to be measured from the top of the porous filter using a biologically specific reaction measurement instrument provided with an absorption layer that absorbs the liquid that has passed through the carrier for measurement of biologically specific reaction. And a mixed solution obtained by adding the target substance to the conjugate of the third substance and reacting the same, and a second substance that specifically reacts with the target substance to which the detectable signal-generating substance is bound, is sequentially supplied. A measurement method comprising calculating the amount of a substance to be measured in a sample by measuring a signal derived from a signal generating substance. (7) The method according to (6), wherein the third substance is biotin, and the fourth substance is one selected from the group consisting of avidin, streptavidin and an anti-biotin antibody. (8) The substance to be measured is an antigen-specific human IgG (6)
Or the measuring method according to (7). (9) The analyte is antigen-specific human IgE (6)
Or the measuring method according to (7).

[0008]

BEST MODE FOR CARRYING OUT THE INVENTION In the present invention, an organic polymer used as a carrier has a feature that swelling due to water absorption is small. Specifically, for example, polyamide, polyamideimide, polyarylate, polyimide, polyetherimide, polyetheretherketone, polyethylene, polyethylene oxide, polyester, polycarbonate, polystyrene, polysulfone, polyethersulfone, polyparamethylstyrene, polyallylamine , Polyvinyl alcohol, polyvinyl ether, polyvinyl butyral, polyvinyl formal, polyphenylene ether, polyphenylene sulfide, polybutylene terephthalate, polypropylene, polymethylpentene, acrylic resin, thermoplastic resin such as methacrylic resin, epoxy resin, xylene resin, guanamine resin,
Examples include diallyl phthalate resin, vinyl ester resin, phenol resin, unsaturated polyester resin, furan resin, polyimide, poly-p-hydroxybenzoic acid, polyurethane, maleic acid resin, melamine resin, and urea resin resin. Of these, polyamide, polystyrene and polypropylene are particularly preferred. These may be used alone or as a mixture of two or more.

The shape of the porous filter in the present invention is generally a so-called surface filter or depth filter, but a depth filter is preferable in order to maintain excellent liquid permeability. The pore size of the porous filter is preferably 1 to 100 μm, more preferably 1 to 10 μm.

[0010] The above-mentioned porous filter, antibody, antigen, D
In order to enhance the binding to biological substances such as NA, RNA and sugar, it is possible to modify the surface by applying an appropriate surface treatment.

The method of performing the above-mentioned surface treatment is not particularly limited, but introduction of a functional group by limited hydrolysis of the resin or fiber surface, corona discharge, plasma treatment, UV-ozone treatment, carbodiimide A general method includes a method of applying an active substance such as (polycarbodiimide) to the surface.

The absorbent layer used in the present invention is not particularly limited as long as it can absorb and contain an aqueous liquid, but is made of cellulose or cellulose derivative fiber from the viewpoint of the aqueous liquid absorption rate. Desirably, it is a laminate.

The backflow prevention layer is not always necessary in the present invention, but is preferably provided when it is necessary to disconnect the liquid between the porous filter and the absorption layer. As the backflow preventing layer, for example, those having high hydrophobicity and high porosity such as a polyolefin nonwoven fabric sheet and a wave are suitable.

The measuring method of the present invention is based on a biological specific reaction. Examples of biological specific reactions include:
(1) antigen-antibody reaction, (2) DNA-DNA, RNA
-DNA, RNA-RNA hybridization,
(3) sugar-lectin reaction, (4) ligand-receptor reaction, (5) avidin-biotin reaction and the like. One of them becomes the first substance or the second substance,
The other is the substance to be measured. Not only can these be used alone, but also several reactions can be combined.

When a biologically specific substance is bound to a porous filter, it may be bound in a state of being formed into a porous filter, or may be formed into a filter after the binding. A non-covalent bond such as a hydrophobic bond or an ionic bond can be used as a bonding method, but it is preferable to form a covalent bond in order to prevent the substance from being detached from the carrier. As a method of covalent bonding, a glutaraldehyde method using a carboxyl group, an amino group or an SH group, a carbodiimide method and a maleimide method are generally used.

Examples of the substance that generates a detectable signal used in the present invention include peroxidase, β-D-galactosidase, alkaline phosphatase, malate dehydrogenase, glucose-6-phosphate dehydrogenase, and glucose. Enzymes such as oxidase and invertase, radioisotopes such as I ¹²⁵ , fluorescein isothiocyanate, tetramethyl rhodamine,
Fluorescent substances such as tetramethyl-damine isothiocyanate, dimethylaminonaphthalene-5-sulfonium chloride, fluoram, europium chelate, luminescent substances such as acridinium ester and acridinium sulfonamide, colloidal gold, colloidal selenium, and colored latex And other substances.

The method for measuring the signal-generating substance may be performed according to a known method. For example, when the signal-generating substance is an enzyme, the enzyme is reacted with a substrate, and then the reflection absorbance, fluorescence intensity and luminescence of the reaction product are measured. The strength or the like may be measured. When the signal generating substance is a radioisotope, the radiation dose may be measured. When the signal generating substance is a fluorescent substance, the fluorescence intensity may be measured directly. When the signal generating substance is a luminescent substance, the luminescence intensity may be measured directly.

The washing solution used in the present invention is not particularly limited, but usually a buffered physiological saline, distilled water or the like is used. If necessary, a surfactant or the like can be added.

In the present invention, the substance to be measured is
That is, although the substance to be measured is not particularly limited, hepatitis A virus, hepatitis B virus, hepatitis C virus (hereinafter referred to as HCV), hepatitis D virus, hepatitis E virus, and F type Hepatitis virus, hepatitis G virus, HIV, HTLV-I, herpes virus, rotavirus, toxoplasma, rubella, chlamydia, spirocheta, Helicobacter pylori, treponema.
Infectious disease related markers such as Pali dam, CEA, CA19
-9, CA125, AFP, ferritin, DUPAN-
II, tumor markers such as fecal occult blood, other CRP, AS
O, RF, apolipoproteins, β ₂ -microglobulin, total IgE, specific IgE and the like.

Hereinafter, the present invention will be described in detail with reference to specific examples.

Example 1 Measurement of HCV Antibody (Preparation of Reaction Apparatus) A nylon filter having a pore size of 1, 5, or 10 μm was slowly immersed in distilled water to uniformly wet it. Thereafter, the inside of the dot blot apparatus was slightly depressurized with a peristaltic pump while being sandwiched between dot blot apparatuses (Biodot manufactured by Bio-Rad). 100 μl of buffered saline (pH 7.2) from the injection port of the dot blot apparatus.
L, 100 μL of HCV antigen peptide solution, 100 μL of buffered physiological saline (pH 7.2), and 100 μL of buffered physiological saline containing an antigen stabilizer. Supplied. Thereafter, the nylon filter was removed from the dot blot apparatus, excess water was removed on a filter paper, dried at room temperature, sealed with a desiccant until use, and stored in a cool and dark place. At the time of use, the portion of the nylon filter to which the antigen was bound was cut off, set on the opening side of the container having an opening, and further laminated with an absorbent layer made of cellulose, and then fixed with a bottom plate so that the laminate did not fall off. A reaction device was used.

(Preparation of Labeled Antibody) An anti-human IgG monoclonal antibody (manufactured by Kappel) and horseradish peroxidase (manufactured by Toyobo Co., Ltd.) were bound according to the method of Nakane, and a column was used to bind the anti-human IgG monoclonal antibody and horseradish peroxidase. The conjugate was purified and stored in a cool place until use.

(Performance of Analysis) Buffered saline (pH 7.2) 50 was taken through the opening of the reaction apparatus prepared as described above.
μL was supplied, and the filter was hydrated. afterwards,
Specimen 50μ with anti-HCV antibody without pretreatment
L, 50 μL of a buffered physiological saline (pH 7.2) containing 0.05% Tween 20, and 20 μL of a solution prepared by diluting the labeled antibody are sequentially supplied after the immediately preceding solution is completely absorbed. did. 0.05% Tween
100 μm of buffered saline (pH 7.2) containing n20
L was supplied twice after the solution supplied immediately before was completely absorbed, and the excess labeled antibody remaining on the filter was washed and removed. 50 μL of a 3,3 ′, 5,5′-tetramethylbenzidine solution was supplied as a substrate for horseradish-derived peroxidase, and after reacting for 1 minute, the reflection absorbance at 670 nm was read.

In terms of sensitivity, the nylon filter having a small pore size was good, but the anti-HCV anti-HCV which was not pretreated in any of the nylon filters having any pore size was good.
The measurement of the antibody was possible. Table 1 shows the results.

[0025]

[Table 1]

Comparative Example 1 Using a nylon filter having a pore diameter of 0.2 μm or 0.45 μm, preparation of a reaction device, preparation of a labeled antibody, and analysis were performed in the same manner as in Example 1. With these nylon filters having a pore diameter of 1 μm or less, the filter was clogged when a sample containing an anti-HCV antibody was applied, so that subsequent reagents could not be applied and analysis could not be performed to the end. .

From the comparison between Example 1 and Comparative Example 1, it was found that, in order to perform analysis without causing clogging even when serum not subjected to pretreatment such as passing through a prefilter was used, 1 μm was used.
This indicates that a porous filter having a pore size of m or more is required.

Example 2 Measurement of Anti-Treponemal Antibody (Preparation of Reaction Apparatus) Nylon fiber, polyethylene fiber,
Polyester fiber, polypropylene fiber, and polystyrene fiber were cut to a length of 3 mm and immersed in a methanol solution of polycarbodiimide. The reaction was carried out with stirring at 40 ° C. for 3 hours to form a polycarbodiimide layer on the fiber surface. After washing with methanol, it was dried. Then, the surface-treated fiber was added to the Treponema pallidum extract purified solution, and reacted at room temperature for 3 hours to give Treponema pallidum fibers.
The pallidum extract purified was combined.

[0029] The fibers after binding are buffered in physiological saline (pH
After washing three times in 7.2), the cells were suspended in a buffered physiological saline (pH 7.2) containing a stabilizer. The fiber suspension was transferred onto a glass filter and aspirated slowly to prepare a filter to which the purified Treponema pallidum extract was bound. The pore diameter at this time was about 3 to 30 μm. The filter was sealed with a desiccant until use and stored in a cool place. At the time of use, the filter is cut to an appropriate size, set on the opening side of the container having an opening, and after further laminating the absorbent layer made of cellulose, the laminate is fixed with a bottom plate so as not to fall, and the reaction device and did.

(Preparation of Labeled Antibody) An anti-human IgG monoclonal antibody degraded with pepsin derived from swine stomach to produce Fab ′ and bovine small intestine-derived alkaline phosphatase (manufactured by Boehringer) into which a maleimide group was introduced were mixed, and then incubated at 4 ° C. for 20 hours The reaction was performed. The alkaline phosphatase-Fab 'complex was purified and concentrated by a column, and stored in a cool place until use.

(Performance of Analysis) Buffered saline (pH 7.2) 50 was taken through the opening of the reaction apparatus prepared as described above.
μL was supplied, and the filter was hydrated. afterwards,
50 μL of a sample containing an anti-Treponemal pallidum antibody,
Buffered saline containing 0.05% Tween 20 (p
H7.2) 50 μL, and 20 μL of the prepared diluted solution of the labeled antibody were sequentially supplied after the immediately preceding solution was completely absorbed. Furthermore, 0.05% Tween20
100 μL of buffered saline (pH 7.2) containing
The solution was supplied after the solution supplied immediately before was completely absorbed, and the excess labeled antibody remaining on the filter was washed and removed. 4-substrate as alkaline phosphatase substrate
Supply 50 μL of methyl umbelliferyl phosphate solution,
After reacting for 1 minute, the upper surface of the filter was irradiated with light having an excitation wavelength of 360 nm, and the fluorescence was measured at 450 nm. All the filters were able to quantitatively measure the Treponema pallidum antibody without causing clogging. Table 2 shows the results.

[0032]

[Table 2]

Comparative Example 2 (Preparation of Reaction Apparatus) A glass fiber filter having a pore diameter of 3 μm was slowly immersed in distilled water to uniformly wet it. After that, the inside of the dot blot apparatus was slightly depressurized with a peristaltic pump while being sandwiched between commercially available dot blot apparatuses. Buffered saline (pH
7.2) 100 μL, Treponema pallidum extract purified product solution 100 μL, buffered saline (pH 7.2) 1
00 μL, buffered saline containing a stabilizer (pH 7.
2) 100 μL was supplied sequentially after the liquid supplied immediately before was aspirated. Thereafter, the glass fiber filter was removed from the dot blot apparatus, excess water was removed on a filter paper, dried under reduced pressure, sealed with a desiccant until use, and stored in a cool dark place. When used, Treponema
The part where the purified paridum extract was bound was cut off, set on the opening side of the container having an opening, and further laminated with an absorbent layer made of cellulose. .

(Analysis) Preparation and analysis of a labeled antibody were carried out in the same manner as in Example 2. Similarly to the filter of Example 2, the glass fiber filter was not clogged with the sample, and the analysis could be performed to the end. However, human IgG non-specifically remained on the glass fiber filter, and the T. pallidum-specific I
The measurement of gG was difficult.

From the comparison between Example 2 and Comparative Example 2, when a glass fiber filter is used as a carrier, human Ig
Since G remains non-specifically, antibody measurement cannot be performed.
By using nylon, polyethylene, polyester, polypropylene, and polystyrene as filter materials, human I
gG hardly remained on the filter, indicating that it was useful for antibody measurement. The results are shown in Table 2.

Example 3 Measurement of Specific IgE (Preparation of Reaction Apparatus) A filter in which polystyrene fiber and polypropylene fiber were mixed was subjected to corona discharge treatment. Anti-biotin polyclonal antibody was added to a 0.1 M carbonate buffer (p
H9.6), the filter was immersed in a solution having a concentration of 10 μg / mL, and incubated at 40 ° C. for 3 hours to allow physical adsorption. After incubation, 0.05
% Tween 20 in buffered saline (pH 7.
The plate was sufficiently washed in 2), and finally was immersed in a buffered physiological saline (pH 7.2) containing a stabilizer, and then dried under reduced pressure. The filter was sealed with a desiccant until use and stored in a cool place. At the time of use, the above filter is cut into an appropriate size, set on the opening side of a container having an opening, and further, after laminating an absorbent layer made of cellulose, fixing with a bottom plate so that the laminated material does not fall, the reaction is performed. It was an instrument.

(Preparation of labeled antibody) An anti-human IgE monoclonal antibody (manufactured by Kappel) and horseradish peroxidase were bound in the same manner as in Example 1, and a conjugate of the anti-human IgE monoclonal antibody and horseradish peroxidase using a column. Was purified and stored in a cool place until use.

(Preparation of Biotin-House Dust Conjugate)
A commercially available biotinylation reagent (manufactured by Nacalai Tesque) and house dust were mixed and incubated at room temperature for 3 hours. Thereafter, the resultant was purified with a Sephadex G-25 column (manufactured by Pharmacia Biotech) to prepare a biotin-house dust conjugate.

(Performance of Analysis) Buffered saline (pH 7.2) 50 was passed through the opening of the reaction apparatus prepared as described above.
μL was supplied, and the filter was hydrated. afterwards,
A solution obtained by mixing 50 μL of an allergic patient sample and 50 μL of a biotin-allergen conjugate and allowing to stand for 10 minutes, and 20 μL of a diluted solution of the prepared labeled antibody are sequentially supplied after the immediately preceding solution is completely absorbed. did. Buffered saline containing 0.05% Tween 20 (p
H7.2) 100 μL was supplied twice after the solution supplied immediately before was completely absorbed, and the excess labeled antibody remaining on the filter was washed and removed. 50 μL of a 3,3 ′, 5,5′-tetramethylbenzidine solution was supplied as a substrate for horseradish-derived peroxidase, and after reacting for 1 minute, the reflection absorbance at 670 nm was read.

FIG. 1 shows the measurement results. High values of reflection absorbance were obtained in patients with house dust allergy. On the other hand, in the negative specimen, there was no example in which human IgE non-specifically remained on the filter.

[0041]

As described above, the carrier for measuring a filter-like biologically specific reaction and the measuring method using the same according to the present invention are highly accurate and free from the effects of non-specific reactions by a simple operation. Obtaining the analysis result is realized.

[Brief description of the drawings]

FIG. 1 is a view showing the result of analyzing a sample of an allergic patient by the method of the present invention.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) G01N 33/545 G01N 33/545 Z

Claims (9)

[Claims] 1. A filter-like carrier for measuring a biologically specific reaction, comprising a filter-like organic polymer having a pore size of 1 to 100 μm. 2. An organic polymer comprising polyamide, polyamide imide, polyarylate, polyimide, polyether imide, polyether ether ketone, polyethylene, polyethylene oxide, polyester, polycarbonate, polystyrene, polysulfone, polyethersulfone, polyparamethylstyrene, Polyallylamine, polyvinyl alcohol, polyvinyl ether, polyvinyl butyral, polyvinyl formal, polyphenylene ether, polyphenylene sulfide, polybutylene terephthalate, polypropylene, polymethylpentene, acrylic resin, methacrylic resin, epoxy resin, xylene resin, guanamine resin, diallyl phthalate resin, Vinyl ester resin, phenol resin, unsaturated polyester resin, furan resin, The filter-like biological material according to claim 1, which is at least one kind or a mixture of two or more kinds selected from the group consisting of polyimide, poly-p-hydroxybenzoic acid, polyurethane, maleic acid resin, melamine resin and urea resin resin. Carrier for specific reaction measurement. 3. The method according to claim 1, wherein the filter-like organic polymer has been subjected to at least one type of surface treatment selected from the group consisting of hydrolysis of the fiber surface, corona discharge, plasma treatment, UV-ozone treatment, and application of an active substance. Item 4. The carrier according to Item 1 or 2, for measuring a biological specific reaction. 4. The filter-like biological substance according to any one of claims 1 to 3, wherein the first substance that specifically reacts with the substance to be measured is provided on the lower part of the carrier for measurement of a filter-like biological specific reaction. Using a biological specific reaction measurement instrument provided with an absorption layer that absorbs the liquid that has passed through the specific reaction measurement carrier, from the top of the porous filter of the instrument, a sample containing the substance to be measured,
And sequentially supplying a second substance that specifically reacts with the analyte to which the detectable signal-generating substance is bound, and measuring the signal derived from the signal-generating substance to reduce the amount of the analyte in the sample. A measuring method characterized by calculating. 5. The method according to claim 4, wherein the substance to be measured is an antigen-specific human IgG. 6. A filter-like biological specific reaction measuring carrier according to claim 1, wherein a fourth substance specifically reacting with the third substance is immobilized. The first substance that specifically reacts with the substance to be measured from the top of the porous filter using a biologically specific reaction measurement instrument provided with an absorption layer that absorbs the liquid that has passed through the carrier for measurement of biologically specific reaction. And a mixed solution obtained by adding and reacting the target substance to the conjugate of the third substance, and a second substance that specifically reacts with the target substance to which the detectable signal-generating substance is bound, are sequentially supplied. A measurement method comprising calculating the amount of a substance to be measured in a sample by measuring a signal derived from a signal generating substance. 7. The method according to claim 6, wherein the third substance is biotin, and the fourth substance is one selected from the group consisting of avidin, streptavidin and an anti-biotin antibody. 8. The method according to claim 6, wherein the substance to be measured is an antigen-specific human IgG. 9. The method according to claim 6, wherein the substance to be measured is antigen-specific human IgE.