JPH02176465A - Ligand measuring method - Google Patents

Abstract

PURPOSE:To enhance the measuring accuracy of ligand and to simplify operation by combining a solid phase material having a molecular sieve effect and macromolecular substrate. CONSTITUTION:Ligand is incorporated in a specimen. An antigen determinant for the ligand or an antibody for the antigen determinant is fixed in the inside. Gel-state solid-phase material having molecular sieve effect can make a bonded material enter into the inside. The ligand is brought into contact with the bonded material of the antibody for the antigen determinant of the ligand or the antigen determinant, and reaction is made to occur. Furthermore, the gel- state solid-phase material is separated or the macromolecular substrate of enzyme which cannot enter into the inside of the gel-state solid-phase material is added into reacted material, and the enzyme reaction is made to occur. Then the activity of the enzyme is measured.

Description

[Detailed Description of the Invention] [Industrial Application Field] Analysis of trace components derived from drugs or various diseases contained in body fluids such as serum and urine is extremely meaningful for diagnosing diseases and determining the progress of treatment. It is used in daily clinical tests. The present invention relates to a method for measuring this trace component.

[Prior Art] Body fluids such as blood contain a wide variety of components, some of which may include substances with similar molecular weights, substances with similar physiological activities, or substances with similar structures. many. Therefore, this analytical method is required to have high specificity and to be able to quantify down to minute amounts.

Various methods have been developed for detecting these trace components of blood, but enzyme immunoassay methods have been gaining acclaim due to their superior sensitivity, specificity, and short processing time. However, in the case of conventional enzyme immunoassay methods, the sensitivity is not yet sufficient and it is not easy to determine accurate concentrations.

Therefore, the present inventors conducted studies to develop an analysis method with even higher sensitivity, and developed a method that utilizes a combination of an antibody against a ligand to be measured and an antibody against an enzyme. In this method, a ligand to be measured and an enzyme are subjected to a competitive reaction with respect to the bound substance, and the activity of the enzyme is then measured, thereby quantifying the ligand (Japanese Patent Laid-Open Publication No. 164960/1982). In this case, a method of further competitively reacting an antibody that reacts with the antibody against the ligand or an antibody that reacts with the antibody against the enzyme (Japanese Patent Application Laid-Open No. 178360/1983), or a method of making a compound of the ligand and a polymer compound, or A method for competitively reacting polymers (Japanese Unexamined Patent Publication No. 178361/1982) was also developed.

[Problem that the invention seeks to solve]

These methods developed by the present inventors have improved sensitivity and simplified operation, but the sensitivity is limited to lng/IR.
It was about 1.

[Means for solving the problem] Therefore, as a result of various studies in order to develop a method that can measure ligands with even higher sensitivity, we found that by combining a solid phase with a molecular sieving effect and a polymer substrate, we were able to achieve even higher sensitivity. It was discovered that the ligand could be measured, and based on this knowledge, the present invention was completed.

That is, the present invention provides a method for measuring a ligand contained in a specimen, in which the antigenic determinant of the ligand or an antibody against the antigenic determinant is immobilized inside the ligand, and the conjugate described below is also immobilized inside the ligand. A gel-like solid phase having a molecular sieving effect that allows entry into the ligand, and an antibody against an antigenic determinant of the ligand or a conjugate of an antigenic determinant and an enzyme are brought into contact with each other to cause a reaction, and further, the gel-like solid phase is separated, Alternatively, it relates to a method for measuring a ligand, characterized in that a polymeric substrate for the enzyme that cannot penetrate into the interior of the gel-like solid phase is added to the reactant to cause an enzymatic reaction, and the enzyme activity is measured.・It is something to do.

The object to be measured in the present invention is a substance containing an antigenic determinant contained in a specimen. The type of specimen is not limited, but includes, for example, serum and urine. In the case of serum, urine, etc., no special pretreatment is usually required and measurements can be performed as they are.

Ligands have one or more antigenic determinants, such as hormones derived from various endocrine glands, immunoglobulin, albumin, plasma proteins such as ferritin, H.
Antigens include viruses such as B antigen, bacteria, α-fetoprotein, carcinoembryonic antigen, and other antigens present in various organs, blood, and urine.

The antibody immobilized on the gel solid phase must react with the antibody. This antibody has F(ab')z.

Fragments such as Pab' and Fab are also included.

As a method for producing antibodies, a ligand or a conjugate of a ligand and a protein is administered to warm-blooded animals such as rabbits, goats, horses, guinea pigs, and chickens at a dose of 0.3 to 2 μg per 1 kg of body weight once to several times subcutaneously on the back or on the foot. It is injected into the bud, thigh muscle, etc. together with an adjuvant to form within the animal's body. Since this antibody is a mixture of antibodies that recognize various antigenic determinants, it is used after being separated. Affinity chromatography is preferably used as a separation method; for example, the ligand is decomposed with an enzyme or chemical reagent, a gel is filtered, the gel is separated using ion exchange chromatography, etc., and each antigen fraction is insolubilized to create an affinity column. ,
This column can be used to separate the antibody mixture described above. In addition, commercially available products of this antibody also exist. In the method of the present invention, antibodies do not need to be separated into single antibodies; it is sufficient to divide them into at least two groups.

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 into the peritoneal cavity of a mouse several times together with an adjuvant, and lung 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 grown in the peritoneal cavity of a mouse, thereby making it possible to produce a single antibody, that is, a monoclonal antibody in large quantities.

The gel-like solid phase allows antibodies or antigens to enter the interior and immobilize them, and also allows a conjugate of antibodies or antigens and enzymes, which will be described later, to enter the interior thereof. As such a solid phase, various gels commonly used in chromatography for separating proteins can be used. Examples include dextran gel, polyacrylamide gel, silica gel, etc. The pore size of the gel to be used can be determined depending on the size of the ligand, etc. For example, if the molecular weight of the Gantt is 50,000, and the molecular weight of the antibody-enzyme conjugate is about 10,000 to 200,000, the gel will penetrate. The lower limit of the molecular weight of the enzyme substrate that inhibits is 100
It should be around 10,000 to 2,000,000.

As a method for immobilizing antibodies or antigens on a gel-like solid phase, for -a, a method of activating CNBr or a method of converting the antibody or antigen into an activated ester after carboxylation is used. Alternatively, it is also possible to epoxidize with dicrycidyl ether or the like and use this epoxy to carry out condensation. In addition, an appropriate method can be selected from known enzyme immobilization methods.

During this immobilization, the antibody or antigen enters not only the surface of the gel-like solid phase but also the interior of the solid phase, and due to the difference in area, most of the antibody or antigen is immobilized inside.

The antigen bound to the enzyme may be the antigen itself or a ligand. Alternatively, it is preferable that the antibody bound to the enzyme reacts with a different antigenic determinant from that with which the gel-like solid phase antibody reacts. This antibody is IgG, rgM or IgA,
It may be a fragment of F(ab')z or Fab, or it may be chemically modified such as DNP, acetylation, biotinylation, or nitration. This antibody is not limited to one type,
There may be two or more types.

Antibodies that recognize these different antigenic determinants can be easily obtained by the method for producing monoclonal antibodies using the cell fusion method described above. Alternatively, the above-mentioned warm-blooded animals may be used to produce an antibody group and then isolated. In that case, it is not necessary to separate the antibodies into a single antibody (for example, the antibodies may be divided into two groups, one of which is bound to the above-mentioned enzyme, and the other is immobilized on a gel-like solid phase). Also,
This antibody does not need to be completely separated, and the other antibody may be mixed in to the extent that the measurement is not inhibited.

The enzymes constituting the conjugate can act on polymeric substrates, and among them, enzymes whose activity can be easily measured are preferred. Such enzymes include, for example, amylase, dextranase, cellulase, glucoamylase, collagenase, mannase, protease, elastase, lipase, peroxidase, β-galactosidase, and the like.

The enzyme substrate itself may be a low-molecular substance, or it may be made into a polymer by binding a high-molecular compound.

The method of binding the enzyme to the antibody or antigen may be determined by considering the functional groups of both. As the functional group, an amino group, a carboxyl group, a hydroxyl group, a thiol group, an imidazole group, a phenyl group, etc. can be used. For example, when bonding between amino groups, a diisocyanate method, a glutaraldehyde method, a difluorobenzene method can be used. , benzoquinone method, etc. are known. In addition, as a method for bonding between an amino group and a carboxyl group, in addition to the method of converting the carboxyl group into a succinimide ester, the carbodiimide method and the Woodward reagent method are known. There is also a periodic acid oxidation method (Nakane method). When using a thiol group, for example, the carboxyl group on the other side is esterified with succinimide, this is reacted with cysteine to introduce a thiol group, and both are bonded using a thiol group-reactive divalent cross-linking reagent. can do. Methods that utilize phenyl groups include diazotization and alkylation. The binding method is not limited to these examples, and in addition, for example, rMe
thod in Immunology and Im
Methods described in books such as "munochemistry" or "enzyme immunoassay" can be appropriately selected and used. The coupling ratio is not limited to 1:l,
It goes without saying that any ratio can be used depending on the purpose. After the reaction, the gel is purified by an appropriate combination of ion exchange chromatography, affinity chromatography, etc., and if necessary, dried by freeze-drying or the like.

The glue gunt to be measured is brought into contact in an aqueous solution with an antibody against the antigenic determinant, a gel solid phase on which the antigen is immobilized, and a conjugate of an antibody and an enzyme against the antigen or antigenic determinant. At this time, the temperature of the solution is approximately 20 to 45°C, and the pH is generally approximately 4 to 9. In order to keep the pH constant, a buffer such as a phosphate buffer or an acetate buffer may be used if necessary. In this case, the appropriate amounts of the gel solid phase and the bound substance vary depending on the type of solid phase, the type of ligand, the contact conditions, etc., and are therefore preferably determined by testing in advance. The order in which the ligand is brought into contact with the gel solid phase and the conjugate is not critical (either may come first or both may be done simultaneously).

On the other hand, if the enzyme of the conjugate is amylase or the like and the same type of enzyme is contained in the sample, the degree of inhibition of the enzyme in the sample is determined by the activity of the enzyme bound to the conjugate. It is preferable to contact the enzyme inhibitor to a degree that inhibits the enzyme.

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. The blank value may not be increased in the measurement, and the enzyme activity may be recovered by deactivating the enzyme inhibitor after the measurement. On the other hand, the effect of the enzyme inhibitor is a problem, and the enzyme is in a state bound to an antibody, and in a free state, it may be inactivated by the 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 may be the same as the method for obtaining antibodies against the aforementioned ligands. The enzyme inhibitor may be added at any time as long as it can substantially prevent separation of the polymer substrate, which will be described later, by the enzyme in the sample, and is usually added before the addition of the polymer substrate. However, since the inhibitory effect of the enzyme inhibitor is generally much faster than the rate of decomposition of the substrate by the enzyme, the enzyme inhibitor may be added at the same time as the polymeric substrate or after some delay.

The conjugate reacted with the ligand is brought into contact with a polymer substrate and reacted.

The bound substance to be brought into contact with the polymer substrate may be separated from the reactant, but usually it may remain contained in the reactant.

It goes without saying that this polymeric substrate is a substrate for the enzyme of the conjugate, but in addition, it must be large enough to prevent it from entering the interior of the gel-like solid phase. For example, if the gel-like solid phase used is one that blocks molecules with a molecular weight of about 1 million to 2 million, a polymer substrate with a molecular weight of 2 million or more is used. Examples of polymeric substrates include insoluble or soluble starch for α-amylase, dextran for dextranase, cellulose for cellulase, and collagen for collagenase.
In the case of mannase, mannan, in the case of protease, protein, in the case of elastase, elastin, and in the case of lipase, various oils and fats can be mentioned.

This polymeric substrate may be soluble or insoluble. Furthermore, even when the substrate itself is low molecular weight, it can be used after being polymerized to a predetermined molecular weight by binding or polymerizing an insoluble or water-soluble polymer compound. For example, when peroxidase is used as the enzyme, dextran or the like can be bound to the leuco dye as a substrate to give it a predetermined molecular weight.

Enzyme reaction conditions may be determined appropriately depending on the enzyme used.

After the enzymatic reaction, determine the enzyme activity. Enzyme activity can be determined by tracking the increase in decomposed products due to this enzymatic reaction, the decrease in polymeric substances that are raw materials, and other changes in the system due to the enzymatic reaction.

[Effect]

The method of the present invention utilizes the molecular sieving effect of a gel-like solid phase on a polymer substrate. That is, when an antibody is immobilized on a gel-like solid phase, the immobilized antibody reacts with the antigenic determinant of the ligand that reacts with the antibody, and the ligand is bound to the antibody. Then, the antibody portion of the antibody-enzyme conjugate reacts with the antigenic determinant of the ligand with which it reacts, thereby binding the ligand and the conjugate. As a result, the conjugate becomes bound to the gel-like solid phase via the ligand. Alternatively, each antigenic determinant of Gant that binds to a ligand and an enzyme reacts competitively with an antibody immobilized on a gel-like solid phase and binds thereto. As a result, the ligand-enzyme conjugate becomes bound to the gel-like solid phase in inverse proportion to the amount of ligand.

When a ligand is immobilized on a gel-like solid phase, each antigenic determinant of the ligand and the immobilized ligand competitively reacts with the antibody portion of the antibody-enzyme conjugate that reacts with it, and binds to it. do. As a result, the antibody-enzyme conjugate becomes bound to the gel-like solid phase in inverse proportion to the amount of ligand.

Since most of the antibodies or ligands immobilized on the gel-like solid phase are inside the solid phase, the bound substance is also bound inside the solid phase. On the other hand, since the enzyme substrate of the conjugate is a polymer and cannot enter the solid phase, this substrate can undergo an enzymatic reaction only outside the solid phase. Therefore, the amount of ligand determines the amount of bound substance bound within the solid phase, and as a result, the amount of enzyme outside the solid phase that can react with the substrate is determined, so by measuring this enzymatic reaction, the amount of ligand can be determined. be able to.

[Example] Example 1 ■ Anti-human α-fetoprotein mouse IgG(ab
') Preparation of z-immobilized gel-like solid phase 5 g of CNBr-activated Sepharose 4B (manufactured by Pharmacia) was dissolved in 5 g of 1 mM hydrochloric acid aqueous solution.
Wash with 0.00 rrdl and 0.1M NaHCO30.5
MNaC1'll liquid pH 8,0)? Anti-human α-fetoprotein mouse IgG(ab′)z(2
20 rtdl of the solution (containing 0 mg) were added and the suspension was stirred at room temperature in an end-over-end mixer overnight.

This Cefbroth 4B gel was thoroughly washed with PBS (pH 7,0), and 0.1M ethanolamine solution (pH 8.0) was washed with PBS (pH 7.0).
, 0) and left at 37°C for 1 hour. P this
Wash with BS (pH 7.0) and add 50 d of PBS (pH 7.0).
,0) suspended.

■ Preparation of CHM β-amylase Dissolve 1 mg of β-amylase in 1 ml of 0.1M phosphate buffer, pH 7.0, and add CIMS 1 mg/mf.
100μ2 of D liver solution was added and left to react at room temperature for 1 hour.

This reaction was placed on a Bio GefP-2 column and the pH
The gel was filtered by flowing 0.1 M phosphate buffer solution of 7.0, and the fraction that passed through was collected.

■ Anti-human α-fetoprotein goat IgG (ab
”) Preparation of 8 containing anti-human α-fetoprotein goat IgG10
．． 300 μg of pepsin was added to 2 ml of 1 M acetate buffer (pt(4,0)), and the mixture was stirred at 37°C for 18 hours.

The pH was adjusted to 6.0 by adding 0, I N NaOH, and the reaction solution was preliminarily buffered with 0.1M phosphate buffer and 1 mM EDTA.
Sephacryl S-30 buffered solution (pH 6,3)
0 gel column and eluted with the above phosphoric acid solid solution.

Collect the peaks eluted around the molecular weight of about 100,000 and
m1 to obtain the target anti-human α-fetoprotein goat IgG(ab')x.

■ Preparation of β-amylase-anti-human α-fetoprotein goat IgG Fab' conjugate Anti-human α-fetoprotein goat IgG prepared in ■
(ab')z 3 mg in 0.1 M phosphate buffer 1
mM EDTA solution (pH 6,0) 10 m in L d
g/d of 2-mercabutethylmian hydrochloride aqueous solution 10
0 μl was added and stirred at 37°C for 90 minutes. This reaction solution was applied to a gel on a Sephadex G-25 column buffered in advance with 0 and 1 M phosphate buffer (pl+7.0) to remove unreacted 2-mercaptomethylamine, and the tls
-Fab" was obtained. To this was added 1 mg of CHM-modified β-amylase prepared in
It was left overnight at °C. Next, this reaction solution was filtered through a glycel CPG column buffered with 20mM'J acid-buffered saline (pH 7.0) to collect a fraction with a molecular weight of 170,000 or more, which was condensed. The desired conjugate was obtained.

■ Add the anti-human α prepared in ■ to 50μ2 of an α-fetoprotein solution (containing 8% PEG) with a measured concentration of human α-fetoprotein from 0 to 640 ng/ml.
- Fetoprotein Mouse IgG (monoclonal antibody) immobilized gel solid phase (100 ul) and 50 μ2 of the conjugate solution prepared in (2) were added and reacted for 20 minutes. next,
Starch with a molecular weight of 20 million or more 50 mg/glucosidase, β-glucosidase 2 U/d, glucose oxidase 2 U
/ml, POD IU/ml, 8n+M phenol, 2.5mM 4-aminoantipyrine and 0.1
Add a solution 1- of pll, 0 containing M phosphoric acid to 37°
After reacting at C for 30 minutes, the absorbance of the supernatant at a wavelength of 505 nm was measured. The relationship between α-fetoprotein concentration and absorbance thus obtained is shown in FIG.

Example 2 ■ Preparation of anti-human ferritin mouse monoclonal IgG-binding Sepharose 4B CNBr-activated Sepharose 4B (manufactured by Pharmacia)
5g was thoroughly washed with 1mM HCffi aqueous solution, and 0. I
MNaHCOt O, 5M NaCl solution (pH 8, o
) Anti-human ferritin mouse monoclonal I dissolved in
15 ml of gG (containing 20 ml) solution was added and the suspension was stirred overnight in an end-over-end mixer at room temperature.

This Sepharose 4B gel was dissolved in PBS (pl+7.0).
After washing with , it was suspended in 0.1 M glycine buffer (pH 8,0) and left at 37°C for 1 hour. This is expressed as PBS (ρ1
17.0) and 50 ml of PBS (pH 7,
0>.

■ Preparation of CIIMized glucoamylase 5 mg of glucoamylase was added to pl+ containing 10 mM 0-zenatroline.
Dissolve in 1d of 0.1M glycerophosphate buffer of 6.3,
4-(maleimidomethylclohexane-1-carboxylic acid) succinimide ester (CHMS) 2 rN/l
100μ of Ir1 dimethylformamide (DMF) solution
2 was added and left to react at room temperature for 1 hour. This reaction solution was put into a Sephadex G-25 column, pH 6,
Gel filtration was performed by flowing 0.1 M phosphate buffer (No. 3), and the through-through fraction was collected.

■ Anti-human ferritin mouse monoclonal IgG (
Preparation of ab')z anti-human ferritin mouse monoclonal IgG 10m
g in 0.1 M acetate buffered 3 mM EDTA solution (pH 5
, 5) Add 300 μg of papain to 2 ml and incubate at 37°C for 1 hour.
Stirred for 8 hours. 0. I NNaOH was added to adjust the pH to 6.0, and the reaction solution was previously buffered with 0.1M phosphate buffer 1.
d It was placed in a Sephacryl S-300 gel column buffered with EDTA solution (pH 6,3) and eluted with the above phosphate buffer. The peak portion eluted around a molecular weight of approximately 100,000 was collected and concentrated to 1 ml to obtain the desired anti-human ferritin mouse monoclonal IgG(ab')z.

■ Preparation of glucoamylase-anti-human ferritin mouse monoclonal IgG Pab' conjugate Anti-human ferritin mouse monoclonal IgG (ab') prepared in ■
0.1M phosphate buffer 1mM E containing z6 flag
Add 100 μ2 of a 10 mg/- 2-mercaptoethylamine hydrochloride aqueous solution to 1 m of TA solution (pH 6,0),
Stirred at 37°C for 90 minutes. This reaction solution was prepared in advance at 0.1
Gel filtration was performed on a Sephadex G-25 column buffered with M phosphate buffer (p! 47.0) to remove unreacted 2-mercaptomethylamine, yielding 15-Fab'. Add to this CHM-modified glucoamylase 1 prepared in
mg was added and reacted at 37°C for 90 minutes. Next, this reaction solution was gel-filtered through a Sephacryl S-300 column buffered with O, lM acetate buffered 5mM calcium chloride solution (pH 6,0) to collect fractions with a molecular weight of 200,000 or more. The desired conjugate was obtained.

■ Preparation of glucoamylase substrate Dissolve 5 g of soluble starch in 50 d of INNaOH aqueous solution, add surfactant Emulgen 8101 d,
Furthermore, benzene 252 relatives and epichlorohydrin 2r
trl was added and the mixture was reacted at 60°C for 6 hours without stirring. The formed precipitate was washed with methanol and then with water,
The suspension was suspended in water, heated at 100°C for 1 hour, and used as a substrate.

■Measurement of ferritin in human serum Dilute human serum containing human ferritin with goat serum.
n dilution series were prepared. Take 100μ2 of each in a small test tube, and add Streptomyces viritosporus Nα297-82FERM-P5405 to the conjugate prepared in ①.
amylase inhibitor produced by 100μg/r
50 μ2 of a solution containing ul and an anti-human ferritin monoclonal antibody with different recognition sites were combined with the conjugate antibody.■
100 μl of the solid gel phase prepared above was added and heated at 37° C. for 30 minutes.

Next, 50 mg/si of the starch prepared in ■, 2 U/ml of glucose oxidase, 1 peroxidase
U/if, po6. containing 8mM phenol, 2.5mM 4-aminoantipyrine and 0.1M acetic acid. Solution 1 of s
After adding tnQ and reacting at 37°C for 30 minutes, the absorbance of the supernatant at a wavelength of 505 nm was measured. The relationship between the ferritin concentration and absorbance thus obtained is shown in FIG.

Example 3 ■ Preparation of gel-like solid phase immobilized with anti-human l-18E mouse 1gG 5g of CNBr-activated Sepharose 4B gel was added to 1mMl1
1 aqueous solution, and 0.1 MNallCOz-0.
, 5M NaCe solution (pt(8,0) containing anti-lemon gE mouse IgG (containing 20 mg)?8 solution 15z++1
was added and the suspension was stirred overnight in an end-over-end mixer at room temperature. Next, this Sepharose 4B gel was washed with PBS (pH 7,0), suspended in 0.1M ethanolamine aqueous solution (pH 8,0), and heated at 37°C for 1 hour.
I left it for a while. Wash this with PBS (PH7,0),
Suspended in PBS (pH 7, 0>50d).

■ Anti-human IgE mouse monoclonal IgG Fab'
- Preparation of peroxidase conjugate CIIM-conjugated per metabiodase was prepared in the same manner as in Example 1 (2), using peroxidase instead of β-amylase.

Anti-human IgE mouse monoclonal IgG (ab“
) 0.1M phosphate buffer containing 23mg 1mM EDTA
2 of 10rng/a1 in solution (pl+6.0)l ml
-100μ2 of an aqueous mercaptoethylamine hydrochloride solution was added, and the mixture was stirred at 37°C for 90 minutes. This reaction solution was preliminarily
．． The gel was passed through a Sephadex G-25 column buffered with 1 M phosphate buffer (pH 7.0) to remove unreacted 2-
Mercaptoethylamine was removed and )Is-Fab'
I got it. To this was added the previously prepared CIIM-modified permetase-dase 5■ and allowed to react at 37°C for 90 minutes. Next, this reaction solution was gel-filtered through a Sephacryl S-300 column buffered with 0.1M acetate buffer and 5mM calcium chloride solution (pH 6.0) to collect a fraction with a molecular weight of approximately 100,000, which was concentrated. The desired conjugate was obtained.

■ Preparation of peroxidase substrate 5 g of dextran with a molecular weight of 5 million to 40 million
The suspension was suspended in 0 ml of pyridine, 5 g of succinic anhydride and 200 mg of 4-dimethylaminopyridine were added thereto, and the mixture was stirred at room temperature overnight. After washing this dextran three times with ethanol and once with dimethylformamide,
tyrl dimethylformamide and add 5
g of 1-ethyl-3-(3dimethylaminopropyl)carbodiimide hydrochloride and 2.5 g of N-hydroxysuccinimide were added and left at room temperature for 1 hour. To the supernatant obtained by centrifugation were added 10 ml of a 50 mg/m luminol solution in dimethylformamide and 10 tnR of 0.2 M sodium carbonate buffer (pH 8,0), followed by stirring at room temperature for 2 hours.

The dextran was precipitated and separated by adding excess methanol. After repeating a purification operation in which the obtained precipitate was dissolved in dimethylformamide and precipitated with methanol, the precipitate was dried under reduced pressure.

■ Measurement of human rgE 0-5100ng/ml human IgE solution 1oan
90 μl of the gel-like solid phase prepared in step ① and 10 μl of the bound product prepared in step ① were added to the mixture, and the mixture was allowed to react at 37°C for 30 minutes.

Next, 300μ of the 50μ/- aqueous solution of the substrate prepared in
1 was added and stirred. Add 311111 H20□ to this supernatant.
The luminescence intensity was measured 1 second after adding 100 μ2 of the aqueous solution. The relationship between the concentration of HiI-1gH and the luminescence intensity thus obtained is shown in FIG.

Example 4 ■ Anti-theophylline mouse IgG(ab')z
Preparation of immobilized gel-like solid phase 5 g of Sephadex G-200 gel was suspended in a 0.3N aqueous sodium hydroxide solution containing 2 ml/ml of sodium borohydride. After adding 251d of 1,4-butanediol diglycidyl ether and shaking at 37"C for 3 hours, the gel was washed with sufficient water on a glass filter. The gel thus obtained was .Anti-theophylline mouse I dissolved in 1M carbonate buffer (pH 9.5)
The suspension was suspended in a 20-ml solution of gGF(ab')z (containing 20 ml) and stirred overnight at room temperature using an end-over-end mixer. This gel was thoroughly washed with PBS (pH 7.0), suspended in 0.1 M ethanolamine solution (pH 8.0), and left at 37°C for 1 hour. This is PBS (p
Wash with 50 d of PBS (pH 7,0).
suspended in.

■ Preparation of theophylline amylase Theophylline 5■ and N-hydroxysuccinimide 2.
5 rag was dissolved in 500 ul of dimethylformamide, 5 µ of water-soluble carbodiimide (EDC) was added, and the mixture was stirred for 2 hours at room temperature.Next, 2 µ of α-amylase derived from Bacillus subtilis was dissolved in 0.1 M glycerophosphate buffer (pH
8,0) ld, and 250μ2 of the above dimethylformamide solution was added. After being left at room temperature for 3 hours, 0.1
Gel filtration was performed using Sephadex G-25 equilibrated with M glycerophosphoric acid solution (pH 7.0) to remove unreacted theophylline.

■Measurement of thiophorine 100μ anti-theophylline mouse IgG gel prepared in ■
Solution containing theophylline in l (0-10077g/d)
and 50 μl of the theophylline amylase conjugate prepared in ■! , (1 μg/all) and 2
It was left at room temperature for 0 minutes. Next, a 20IIIM glycerophosphate buffer containing 1% amylose with a molecular weight of 4 million or more, α-gelcomidase 3U/d, glucose oxidase 2U/d, Poo (peroxidase) 2U/d, thiaminoantipyrine 3I1M, and phenol 8IIIM. 800 μl of the supernatant was added and reacted at 37°C for 30 minutes, and the color development of the supernatant was measured by absorbance at 50511I11.

The table shows the concentration and absorbance.

Concentration Absorbance 1100u/d O, 86050μg/d
0.80 25 u g/mi O,706,25 μg
/In10.55 1.5 ug/1uft 0.45 [Effects of the Invention] The method of the present invention can measure various ligands with high sensitivity. Furthermore, the operation is extremely simple since separation and washing operations after the reaction are not required.

[Brief explanation of the drawing]

Figures 1 to 3 all show the results obtained by measuring the relationship between the concentration of various ligands and absorbance or luminescence intensity using the method of the present invention. Patent applicant: Fujirebio Co., Ltd. Figure 1 α-fetoprotein diagram Ferritin rJ /mi diagram Human gE nil/ml

Claims (1)

[Claims] In a method for measuring a ligand contained in a specimen, a molecular sieving effect that allows the ligand to be immobilized inside the antigenic determinant of the ligand or an antibody against the antigenic determinant, and to allow the conjugate described below to enter the inside thereof. and an antibody against the antigenic determinant of the ligand or a conjugate of the antigenic determinant and the enzyme to react, and further, the gelled solid phase is separated or left untreated. A method for measuring a ligand, which comprises adding a polymeric substrate for the enzyme that cannot penetrate into the interior of the gel-like solid phase to the reactant, causing an enzymatic reaction, and measuring enzyme activity.