JPH026753A - Enzyme immunoassay - Google Patents

Abstract

PURPOSE:To directly measure haptene without using a high-polymer compd. having the same ligand as the ligand of the haptene by using an enzyme- antibody conjugate and a high-polymer material on which enzyme can act. CONSTITUTION:The antibody constituting the enzyme-antibody conjugate which is the conjugate of the antibody reacting with the haptene and the enzyme is the fragment of Fab, etc., and reacts with the antigen determinant of the haptene. The high-polymer material on which said enzyme acts includes the substrate of enzyme, for example, starch powder, amylose, etc. The object to be measured is the haptene having the antigen determinant contained in the specimen. The specimen is, for example, blood (plasma, serum), urine, etc. and the measurement can usually be made with the specimen which is held intact without requiring a special pretreatment. The haptene has >=1 or 2 antigen determinants. The enzyme activity can be measured by bringing the haptene in the specimen into reaction with the enzyme-antibody conjugate of the antibody reacting with the haptene into reaction and acting the high-polymer material on the conjugate.

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The present invention relates to an extremely useful and simple enzyme immunoassay method for measuring haptens that are important for treatment or diagnosis in the medical field.

[Conventional technology]

Enzyme immunoassay is conventionally known as a method for quantifying biochemical substances in a specimen. This method required an operation to separate the free labeled substance from the measurement system. In order to solve this complexity, a so-called homogeneous enzyme immunoassay method that does not require separation operations has been developed. In particular, JP-A-61-8004
Similar to the present invention, the enzyme immunoassay method described in No. 9 is an invention that uses hapten in a sample as the measurement target, and uses a polymer compound having the same ligand as the hapten for an enzyme-antibody conjugate in the sample. The hapten is measured by performing a competitive reaction with the hapten and measuring the enzyme activity using a polymeric substance on which the enzyme acts as a substrate.

[Problem to be solved by the invention]

The method described in JP-A-61-80049 provides those skilled in the art with a much simpler method than conventional enzyme immunoassays. However, in measuring haptens, it is essential to use a polymer compound having the same ligand as the hapten, so the compound must be obtained, and there has been a desire to develop a simpler method.

[Failure to solve the problem]

As a result of intensive research aimed at overcoming the conventional drawbacks, the present inventors have completed the present invention by discovering a method that allows direct measurement of hapten without using a polymer compound having the same ligand as hapten.

The present invention is an enzyme immunoassay method for measuring haptens in a specimen using an enzyme-antibody conjugate, which is a conjugate of an enzyme and an antibody that reacts with haptens, and a polymeric substance on which the enzyme can act.

That is, the present invention is an enzyme immunoassay method in which enzyme activity is measured by reacting a hapten in a sample with an enzyme-antibody conjugate of an enzyme and an antibody that reacts with the hapten, and allowing a polymeric substance to act on the conjugate. be.

The measurement target of the enzyme immunoassay of the present invention is a hapten having an antigenic determinant contained in a specimen. The type of peripheral body is not limited, and examples thereof include blood (plasma, serum), lymph fluid, urine, and the like. In the case of plasma, serum, urine, etc., measurements can be performed on the specimen as it is, usually without requiring any special pretreatment.

Haptens have one or more antigenic determinants, and examples include drugs such as digoxin, theophylline, phenoharbicool, phenihein, penicillin, amikacin, prostaglandin, testosterone, progesterone, and thyroxine. I can list hormones such as The term "hapten" used in the present invention refers to a low molecular weight compound having a molecular weight of about 10,000 or less, as described above.

The antibody constituting the enzyme-antibody conjugate used in the present invention reacts with the antigenic determinant of a hapten. The antibody may be a fragment such as F(ab')z, Fab, Fab', etc.

As a method for producing antibodies, a conjugate of a hapten or its derivative and a protein is administered to warm-blooded animals such as rabbits, goats, horses, guinea pigs, and chickens at 1 g of 0.3 to 2 IT per 1 kg of body weight.
It is formed in the animal's body by injecting it several times subcutaneously in the back, into the Fundopat, thigh muscle, etc. together with an adjuvant. The antibody may be used as it is, or it may be used after being purified by a known method for collecting antibodies, ie, immunoglobulins, from serum.

On the other hand, this antibody can also be obtained as a monoclonal antibody. In that case, one of the antigens mentioned above is injected several times into the peritoneal cavity of a mouse along with an adjuvant, and pancreatic cells are taken out and fused with mouse myeloma cells using polyethylene glycol or the like. Then, by cloning those fused cells that produce the antibody, they are grown as monoclonal cells, and by growing them in the peritoneal cavity of a mouse, a single antibody, that is, a monoclonal antibody, can be produced in large quantities.

On the other hand, the enzymes constituting the enzyme-antibody conjugate include, for example, amylase, dextranase, cellulase, collagenase, mannase, protease, elastase, lipase, and glucoamylase. Among these enzymes, amylase, dextranase, cellulase, mannase, and glucoamylase are preferably used because their activities can be easily measured.

Enzyme substrates can be cited as polymeric substances on which these enzymes act. Examples include starch, amylose, amylopectin, dextran, etc. For details, see Enzyme Handbook J (Asakura Shoten,
1982), edited by the Japanese Biochemical Society [Biochemistry Handbook 1 (Maruzen, 1980), so refer to it and select as appropriate.

Furthermore, the polymeric substance may have a functional group or compound that can be detected directly or indirectly.

The method of binding the enzyme and antibody may be determined by taking into consideration the functional groups of both. As the functional group, an amino group, an imidazole group, a phenyl group, etc. can be used. For example, methods for bonding between amino groups include a diisocyanate method,
Many methods are known, including the glutaraldehyde method, difluorobenzene method, and benzoquinone method. Further, as methods for bonding between an amino group and a carboxyl group, the carbodiimide method, the Woodward reagent method, etc. are known. A periodic acid oxidation method (Nakane method) is known as a method for crosslinking chains. A method of using a thiol group for crosslinking is, for example, by reacting cysteine with the carboxyl group on the other side by the carbodiimide method to introduce a thiol group, and then bonding the two using a thiol group-reactive divalent crosslinking reagent. There are known ways to do this. Similarly, diazotization methods, alkylation methods, and the like are known as methods that utilize phenyl groups. The bonding method is not limited to these examples, and in addition, for example, r
Method in Immunology and
An appropriate method can be selected and used from among the methods described at the bottom of ``Immunochemistry'' or ``Enzyme Immunoassay Method'' (Igakushoin, published in 1978) edited by Ishikawa, Kawai, and Miyai. The binding ratio of enzyme and antibody is 1:
Needless to say, the ratio is not limited to 1, and any ratio can be taken depending on the purpose. After the reaction, the resulting enzyme-antibody conjugate can be purified by an appropriate combination of gel filtration, ion exchange chromatography, affinity chromatography, etc., and, if necessary, dried by freeze-drying or the like.

In carrying out the present invention, although it is not necessarily necessary nor does it define the present invention, if the sample contains an enzyme of the same type as the enzyme of the enzyme-antibody conjugate,
It is preferable to bring into contact an enzyme inhibitor that inhibits the enzyme in the specimen to a greater degree than the activity of the enzyme bound to the conjugate.

It goes without saying that it is most desirable for this enzyme inhibitor to be one that completely deactivates the enzyme contained in the sample and does not inhibit the enzyme bound to the conjugate at all; It is sufficient that the blank value is not increased, and the enzyme activity may be restored by deactivating the enzyme inhibitor after the measurement. Furthermore, in a free state, it may be inactivated by an enzyme inhibitor. Known enzyme inhibitors with such specificity may be used as this enzyme inhibitor, but it is also possible to administer the enzyme contained in the sample to a warm-blooded animal to obtain its antibodies. It can also be used as an enzyme inhibitor. The method for obtaining antibodies can be the same as the method for obtaining antibodies against haptens described above.

To specifically explain the implementation of the present invention, for example, a certain amount of an aqueous liquid sample such as plasma, serum, lymph, urine, etc. is mixed with an enzyme-antibody conjugate, and after incubation for a certain period of time, a polymer The substance is added and incubated at a constant temperature, preferably at a substantially constant temperature around 37°C, for a certain period of time, an enzyme reaction stop solution is added, and the color development in the reaction solution is controlled by visible or ultraviolet light. The content of hapten in a liquid sample can be determined by measuring using light having a maximum absorption wavelength or a wavelength near it, and using a calibration curve prepared in advance and based on the principle of colorimetric measurement. Alternatively, the hapten content in the liquid sample can be determined by measuring the intensity of fluorescence in the reaction solution and using a calibration curve prepared in advance. By keeping the amount of liquid sample used, incubation time, and temperature constant, quantitative analysis of haptens can be performed with high precision.

In carrying out the present invention, a buffer solution is usually used as in this type of analysis method, and examples of buffer solutions that can be used include glycerophosphoric acid, acetic acid, maleic acid, phosphoric acid, MES, MOPS, etc. , P I PES, B
15-Tris and the like. In the present invention, the enzyme activity varies depending on the buffer solution used, but if it is set below a specific pl+, the amount of hapten increases and the enzyme activity increases;
If the value is set to + or more, the amount of hapten increases and the enzyme activity decreases, and the measurer can select as appropriate depending on what kind of measurement result he or she desires during the measurement.

The present invention can be easily carried out as described above, but as explained above, this method is not limited to the so-called solution method, and can also be incorporated into a normal multilayer analytical element. It is possible.

When the enzyme-antibody conjugate used in the enzyme immunoassay method of the present invention and the polymer substance on which the enzyme can act are incorporated into a dry analytical element, it is possible to use a layer structure similar to that of various known dry analytical elements. can. In addition to the immunoreaction reagent layer described below, the dry immunoassay element has a multilayer structure including a support, a porous development layer, a detection layer, a light shielding layer, an adhesive layer, a filtration layer, a water absorption layer, an undercoat layer, and other layers. May have. As such analysis elements, U.S. Patent Nos. 3,992,158 and 4,042
, No. 335 and Japanese Patent Application Laid-Open No. 55-164356.

When using a light-transparent water-impermeable support, the dry immunoassay element of the present invention can practically have the following configuration.

Of course, the present invention is not limited to this.

(1) A reagent layer on a support, and a developing layer on the second layer.

(2) A support having a detection layer, an immunoreaction reagent layer, and a development layer in this order.

(3) A support having an immunoreaction reagent layer, a light reflection layer, and a development layer in this order.

(4) A detection layer, an immunoreaction reagent layer, a light reflection layer on the support,
It has development layers in this order.

(5) A detection layer, a light reflection layer, an immunoreaction reagent layer on the support,
It has development layers in this order.

(6) A support having a detection layer and an immunoreaction reagent-containing development layer in this order.

(7) Support: A support having a detection layer, a light reflection layer, and a development layer containing an immune reaction reagent in this order.

A water-absorbing layer containing a hydrophilic polymer as a main component may be provided between the support and the immunoreactive reagent layer or the detection layer. Moreover, the immunoreaction reagent layer and the detection layer may be porous layers. A second reagent layer containing a reagent other than the immunoreactive reagent (eg, a coloring reagent composition) can also be provided.

As the support, polymer sheets 1 to 1 which are light-transparent (transparent), water-impermeable, and have a smooth surface are preferred.

Examples of preferred supports include polyethylene terephthalate and polystyrene. On the surface of the support, a detection layer,
In order to firmly adhere a hydrophilic layer such as a water absorption layer, an immunoreactive reagent layer, or a second reagent layer, an undercoat layer made of a hydrophilic polymer such as gelatin is provided, or an undercoat layer made of a hydrophilic polymer such as gelatin is provided, or a treatment such as ultraviolet irradiation treatment or glow discharge treatment is applied. It is preferable to perform a hydrophilic treatment. Note that a light-reflective or opaque support may also be used.

Hydrophilic polymers used in the detection layer or water absorption layer include, for example, gelatin and derivatives thereof (e.g. phthalated gelatin), cellulose derivatives (e.g. hydroxymethylcellulose), agarose, acrylamide copolymers, methacrylamide copolymers, acrylamide or methacrylamide. Copolymers with various vinyl monomers, etc. can be used.

As the material constituting the porous spreading layer, for example, filter paper, nonwoven fabric, woven fabric (for example, plain woven fabric), knitted fabric (for example, tricots) [fabric], glass fiber filter paper, etc. can be used. Among these, woven fabrics, knitted fabrics, etc. are preferable as the spreading layer. Fabrics and the like may be subjected to glow discharge treatment as described in JP-A No. 57-66359. For the spreading layer, in order to adjust the spreading area, spreading speed, etc.,
No. 222770, Japanese Patent Application No. 122875, No. 61-1980, No. 61
It may contain a hydrophilic polymer or a surfactant as described in No. 122876 and No. 61-143754.

The immunoreaction reagent layer is a water-permeable layer containing an enzyme-antibody conjugate, which is a conjugate of an enzyme and an antibody that reacts with Habten, and a polymeric substance on which the enzyme can act. Preferred embodiments include an embodiment in which the above two components are contained in a porous spreading layer (an embodiment in which the developing layer also serves as an immunoreaction reagent layer);
There are embodiments in which the components are contained in a porous layer (different from the spreading layer). When the immunoreaction reagent layer is a porous layer, the same porous material as the spreading layer can be used. It is preferable that the immunoreaction reagent layer or the spreading layer contain the aforementioned pl+buffer and a surfactant (preferably a nonionic surfactant).

An adhesive layer for adhering and laminating the developing layer may be provided on layers such as a reagent layer, a light reflection layer, a filtration layer, a water absorption layer, and a detection layer. The adhesive layer is preferably comprised of a hydrophilic polymer that is capable of adhering the porous layer when swollen with water, such as gelatin, seratin derivatives, polyacrylamide, starch, and the like.

The light-reflecting layer is used to measure the reflectance of detectable changes (color change, color development, etc.) occurring in the detection layer, reagent layer, etc. from the light-transmitting support side. liquid color,
In particular, when the sample is whole blood, it blocks the red color of hemoglobin and also functions as a background layer. The light reflective layer is
It is preferably a water-permeable layer in which light-reflecting fine particles such as titanium dioxide and barium sulfate are dispersed using a hydrophilic polymer as a binder. The binder is preferably composed of gelatin, gelatin derivatives, polyacrylamide F, # powder, etc.

In the dry immunoassay element, light-reflecting particles such as titanium dioxide may be contained in the developing layer, reagent layer, detection layer, etc., instead of or at the same time as providing the light-reflecting layer.

Dry immunoassay elements can be prepared by known methods described in the aforementioned patent specifications.

Dry immunoassay elements are cut into small pieces, such as squares with a side of about 15 mm to about 3 ONIl, or circles of approximately the same size, and are cut into small pieces such as squares or circles of approximately the same size. Kaisho 58-32350
It is preferable from various viewpoints such as production, packaging, transportation, storage, and measurement operations to use it as an immunoslide in a slide frame as described in Japanese Patent Publication No. 58-501144. Depending on the purpose of use, it can be used in the form of a long tape and stored in a cassette or magazine, or small pieces can be attached or stored in a card with an opening.

The dry immunoassay element can analyze a hapten, which is an analyte, in a liquid sample by the operations described in the above-mentioned patent specifications. For example, an aqueous liquid sample droplet such as whole blood, plasma, serum, lymph fluid, urine, etc. in a range of about 5 μr to about 30 μe, preferably about 8 μ2 to about 15 μe is spotted on the spreading layer.
inkling at a substantially constant temperature in the range of about 20°C to about 40°C, preferably around 37°C, for a period of time ranging from 10 minutes to 10 minutes, to achieve color development within the element. Alternatively, the discoloration can be measured by reflection photometry from a light-transmitting support using light at or near the maximum absorption wavelength of visible light or ultraviolet light, and then measured in a liquid volume using the principle of colorimetric measurement using a pre-prepared calibration curve. The content of hapten can be determined.

Alternatively, the hapten content in the liquid sample can be determined by measuring the intensity of fluorescence within the element and using a calibration curve prepared in advance. By keeping the amount of liquid sample applied, incubation time, and temperature constant, quantitative analysis of haptens can be performed with high precision. In embodiments using a light-reflective or opaque support, color development or discoloration within the analytical element is measured by reflection from the outermost layer side opposite to the support.

The measurement operation is according to JP-A-60-125543 and JP-A-60-125543.
220862, JP-A No. 61-294367, JP-A No. 58-161867, etc., can perform highly accurate quantitative analysis with extremely easy operation.

Hereinafter, the present invention will be explained in more detail with reference to Examples.

[Example] Example 1 ■Preparation of CHM-forming amylase 5 mg of Bacillus subtilis amylase was added to 1 ml of 0.1M glycerophosphate at pH 6.3. Container, CHM
52mg/mg, DMF solution 100pl! was added and left to react at room temperature for 1 hour. This reaction solution was put into a Sephadex G-25 column, and 0.1M of p) 17.0 was added.
Gel filtration was performed by flowing glycerophosphoric acid, and a fraction that passed through was collected.

■Preparation of anti-theophylline mouse IgG F (ab”)2 0.1M containing anti-theophylline mouse IgG10■
30 papain in 2 ml of acetate buffer (pt15.5)
0 μg was added and stirred at 37°C for 18 hours. 0. I
The pH was adjusted to 6°0 by adding N-NaOH. This reaction solution was preliminarily mixed with 0.1M phosphate buffered 1mM EDTAi solution (p
The mixture was placed in an AcA-44 gel column buffered with 1+6.3) and eluted with the above phosphate buffer.

Collect the peaks eluted around the molecular weight of about 100,000 and
Concentrate in females and add the desired anti-theophylline mouse IgG F (
ab')z was obtained.

■α-amylase anti-theophylline mouse IgG Fa
b" Preparation of conjugate anti-theophylline mouse TgGF (ab
) z 6 mg in 0.1 M phosphate buffer 1 mM
DTA? 10 mg per ml (pH 6,0)
/ml of 2-mercaptoethylamine and stirred for minutes. This reaction solution was gel-filtered through a Sephadex G-25 column buffered in advance with 0.1 M glycerophosphate buffer (pH 7, 0) to remove unreacted 2-mercaptomethylamine, and to remove HS-Fab'. Obtained.

To this was added 1 mg of CHM-modified α-amylase prepared in ①, and the mixture was reacted at 37°C for 90 minutes. Next, this reaction solution was mixed with 20 cocoon glycerophosphate buffered 51 calcium chloride solution (
A fraction with a molecular weight of 200,000 or more was collected by gel filtration using an AcA-34 gel column buffered with pH 7.0, and concentrated to obtain the desired enzyme-antibody conjugate.

■Measurement concentration of theophylline: 0 to 20 μg/kai theophylline solution (1% BSA
Contains) 50 μp of the enzyme-antibody conjugate solution prepared in ■
μe was added and allowed to react for 20 minutes. Next, use Daiichi Chemical Co., Ltd.'s Neo-Amylase Test [No.
After adding 1 mal of the suspension in mR of 0.1M acetate buffer (pH 5,5) and reacting for 30 minutes, 0.5ml of 0.5N-Na0t+ was added thereto. Add 300
The mixture was centrifuged at 0 rpm for 1 minute, and the absorbance of the supernatant at a wavelength of 620 nm was measured. The relationship between the theophylline concentration and absorbance thus obtained is shown in FIG.

Example 2 ■Anti-phenobarbicur mouse IgG F(ab')z
Preparation of anti-phenoharbital mouse containing TgGiOmg.
1 g of 1M acetate buffer (pH 15,5) 2 mflVhapaiy300t was added, and the mixture was stirred at 37°C for 18 hours.

0. This reaction solution, whose pH was adjusted to 6.0 by adding IN-Na00, was placed in an AcA-44 gel column that had been buffered in advance with a 0.1M phosphate buffered 1mM EDTA solution (pH 6,3), and the above phosphate buffer solution was added. The peak portion eluted with a molecular weight of around 100,000 was collected and concentrated to 1 mρ,
The desired anti-phenoharbicur mouse IgG F (ab'
) got z.

■α-amylase anti-phenoharbital mouse JgG
Preparation of Fab conjugate Anti-phenobarbicur mouse IgG prepared in ① (
1 m of 0.1 M phosphate buffer containing 6 mg of ab')z
MEDTA? Liquid (pH 6,0) 10 per mft
mg/mA of 2-mercaptoethylamine and stirred for minutes. This reaction solution was gel-filtered through a Sephadex G-25 column buffered in advance with 0.1 M glycerophosphate buffer (pH 7, 0) to remove unreacted 2-mercaptomethylamine, and to remove HS-Fab'. Obtained.

To this was added 1+ng of CHM-modified α-amylase prepared in the same manner as in Example 1, and the mixture was reacted at 37°C for 90 minutes.

Next, this reaction solution was buffered with 20 mM glycerophosphate buffered 5 mM calcium chloride solution (pH 7.0).
The fractions having a molecular weight of 200,000 or more were collected by gel filtration using a No. 34 gel column and concentrated to obtain the desired enzyme-antibody conjugate.

(2) Measuring concentration of phenoparbital: 0 to 1000 μg 50 μe of the enzyme-antibody conjugate solution prepared in (2) was added to 50 μe of a 7 ml phenobarbital solution (containing 1% BSA) and allowed to react for 20 minutes. next,
Daiichi Chemical Co., Ltd.'s neo-amylase test "Daiichi" 1
4 d of 0.1 M acetate buffer (pl+ 6,
After adding 1 d of the suspension suspended in 5) and reacting for 30 minutes, 0.5 h of 0,5 N-NaOH was added thereto, and the mixture was centrifuged at 3000 rpm for 1 minute to obtain a supernatant with a wavelength of 620 nm.
The absorbance at m was measured.

The relationship between the phenobarbicur concentration and absorbance thus obtained is shown in FIG.

Example 3 A crosslinking agent-containing water-absorbing layer was coated with an aqueous solution on a colorless transparent polyethylene terephthalate (PET) sheet (support) with a thickness of 180 μm on which a ceratin undercoat layer was provided so as to have the following coating amount. It was coated using a wafer and dried.

Alkali-processed gelatin 6.6 mg/rd nonylphenoxy polyglycidol (contains 10 glycidyl units on average) 330 mg/rd
Y? Bis[(hinylsulfonylmethylcarbonyl)amino]methane 380mg/on top of the crosslinking agent-containing water absorption layer, a detection layer was applied using an aqueous dispersion to the following coating amount and dried. .

Acid-treated gelatin] Og/I polymer aqueous latex (1) 3 g/rrr (solid content 10%) Nonylphenoxy polyglycidol (contains 10 glycidyl units on average) 2 g/rrr
A light shielding layer was applied onto the detection layer using an aqueous dispersion in the following coating amount so that the dry layer thickness was 7 μm, and was dried.

Alkali-treated gelatin 2.9 g/n (Rutile type titanium dioxide fine particles 13 g/r'
l (nonylphenoxy polyglycidol (contains 10 glycidyl units on average) 400mg
/r+i On top of the light shielding layer, dry layer thickness 5 with the following coating amount
The adhesive layer was applied using an aqueous dispersion so that the adhesive layer was adjusted to pm and dried.

Alkali-processed gelatin 6.7g/m Nonylphenoxy polyglycidol (contains 10 glycidyl units on average) 600mg/m
n? Next, water was almost uniformly supplied to the surface of the adhesive layer at a rate of 30 g/rrr to moisten it, and on top of this, a 36 gauge knitted PET spun yarn equivalent to 50 denier was knitted to a thickness of about 250 μm.
(Tricks) The fabrics were laminated and adhered almost uniformly by applying light pressure to form a porous spread layer.

Next, an aqueous dispersion of the substrate and immunoreaction reagent composition was applied onto the developing layer and dried so as to have the following coating amount.

Diamil-L (product name) 3.5 g /
α-amylase anti-theophylline mouse IgG Fab
'Bound (Example 1) 6.2mg/rr
rnonylphenoxypolyethoxyethanol (average 40
500 mg/n (contains oxy-modified tyrene units)
A multilayer immunoslide for theophylline analysis was completed by placing it in the frame of the slide described in No. 2350.

Performance evaluation experiment A 50ml solution containing a known amount of theophylline at pH 7.0 was added to the developing layer of the multilayer immunoslide for theophylline analysis described above.
10 µe of Mglyptaphosphate buffer solution was added dropwise. 37°
After reacting at C for 20 minutes, the reflective optical density at 540 nm was measured from the support side. The results are shown in Figure 3.

From the calibration curve shown in FIG. 3, it is clear that the dry immunoassay element for theophylline analysis of the present invention can quantify theophylline with high accuracy.

〔Effect of the invention〕

In the present invention, haptens can be measured using only an enzyme-antibody conjugate, which is a conjugate of an enzyme and an antibody that reacts with hapten, and a polymeric substance on which the enzyme can act.

[Brief explanation of the drawing]

Figures 1 and 2 show the relationship between the concentration and absorbance of theophylline and phenobarbital. FIG. 3 is a diagram showing a calibration curve of the dry immunoassay element for theophylline analysis of Example 3. Patent applicant: Fujirebio Co., Ltd. Representative: Fuji Photo Film Co., Ltd. Person: Patent attorney China/China Political situation: 1 person ^4- Funeral map Theophylline concentration (, yg/ml) Figure

Claims (2)

[Claims] (1) Enzyme immunoassay method for measuring hapten in a sample using an enzyme-antibody conjugate, which is a conjugate of an enzyme and an antibody that reacts with hapten, and a polymeric substance on which the enzyme can act. (2) Claim (1) wherein the enzyme is amylase, dextranase, cellulase, mannase or glucoamylase.
) The analysis method described in