JPH0421820B2 - - Google Patents

Description

[Detailed Description of the Invention] (Industrial Application Field) Analysis of trace components derived from drugs or various diseases contained in liquids such as serum and urine is extremely meaningful for diagnosing diseases and determining the progress of treatment. , is used in daily clinical tests. The present invention relates to a method for measuring low molecular weight components among these trace components.

(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. Furthermore, since it is used as a daily test, it is desirable that it be simple and routine.

Various methods have been developed for detecting these trace components of blood, but enzyme immunoassay is the preferred method due to its excellent sensitivity, specificity, and ability to process large amounts of samples in a short time. However, in the case of conventional enzyme immunoassay methods, the sensitivity is still not sufficient, and the washing operation is complicated and the tube needs to be changed, making it difficult to determine accurate concentrations. It wasn't.

Therefore, the present inventors conducted studies to develop an analysis method with higher sensitivity and fewer complicated operations, and developed a method that utilizes a combination of an antibody against the antigenic determinant-containing substance to be measured and an antibody against an enzyme. developed. This method is a method for quantifying antigenic determinant-containing substances by competitively reacting an enzyme with the antigenic determinant-containing substance to be measured against this bound substance, and then measuring the activity of this enzyme
-164960). At that time, there is a method in which an antibody that reacts with the antibody against the antigenic determinant-containing substance or an antibody that reacts with the antibody against the enzyme is further subjected to a competitive reaction (Japanese Patent Application No. 58-51494 (Japanese Unexamined Patent Publication No. 58-59)
178360)) or a method of competitively reacting a conjugate of an antigenic determinant-containing substance with a polymer compound or a polymer of an antigenic determinant-containing substance (Japanese Patent Application No. 58-51495 (Japanese Unexamined Patent Publication No. 59-178361)) ) was also developed.
After further investigation, they developed a number of methods for measuring antigenic determinant-containing substances that are closely related to this technology and filed patent applications. A method in which the antibody is reacted with a conjugate of an enzyme capable of acting on a water-insoluble polymeric substance, and then the enzymatic activity of the conjugate is measured (Japanese Patent Application No. 58-231241, JP-A No. 60-123767)
It was hot.

(Problems to be Solved by the Invention) This method, like other methods, can not only measure antigenic determinant-containing substances with high specificity and extremely high sensitivity, but also has simpler operations than other methods. Although it had the advantage of not consuming much of the antigenic determinant-containing substance because it was only used for antibody production, it had the problem of low measurement sensitivity for low-molecular antigenic determinant-containing substances. Ta.

(Means for Solving the Problems) The present invention provides a method for measuring antigenic determinant-containing substances that solves the above-mentioned problems. The present invention is characterized in that a new combination of an antigenic determinant-containing substance and a polymer compound, which share at least one antigenic determinant with each other, is newly introduced to cause a competitive reaction.

That is, the present invention comprises a substance 1 containing an antigenic determinant to be measured having a molecular weight of 20,000 or less, a substance 2 containing an antigenic determinant that shares at least one antigenic determinant with this substance 1 and a molecular weight of 100,000 Daltons. The conjugate with the above water-soluble polymer compound is brought into contact with the conjugate of the antibody and amylase that reacts with this common antigenic determinant in an aqueous solution, and then the conjugate of the antibody and amylase is reacted with the conjugate of the antibody and amylase. The present invention relates to a method for measuring a substance containing an antigenic determinant, which comprises bringing an insoluble starch into contact with an enzyme to cause an enzymatic reaction, and measuring amylase activity.

The object to be measured in the method of 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.

Antigenic determinant-containing substance 1 (hereinafter referred to as ligand 1) is a substance having one or more antigenic determinants and a molecular weight of 20,000 or less; examples include digoxin, theophylline, phenobarbital, phenytoin, and penicillin. , drugs such as amikacin, and hormones such as prostaglandin, testosterone, progesterone, and thyroxine.

Antigenic determinant-containing substance 2 (hereinafter referred to as ligand 2) must have at least one antigenic determinant in common with ligand 1. One or more of the antigenic determinants of Ligand 2 may be common to Ligand 1, and all of them may be common. Thus, Ligand 2 may be the same as Ligand 1.

The polymer compound bound to the ligand 2 has a molecular weight of 100,000 Daltons or more and is water-soluble. Examples of the polymer compound include soluble dextran, carboxymethylated dextran, aminated dextran, polysaccharides such as amylose and derivatives thereof, proteins such as gelatin, hemocyanin, and ferritin, and polyethylene glycol. These need only meet predetermined conditions when bound to the ligand 2. For example, even if it is a relatively low-molecular substance such as bovine serum albumin, it can be made into a polymer by self-polymerizing it. It may also be a digitized version.

The ligand 2 itself may be polymerized by polymerization. The polymerization method may be appropriately selected from among the methods for bonding the ligand 2 and the polymer compound described below. For example, polymerization may be performed using a divalent crosslinking agent such as carbodiimide or glutaraldehyde.

The method of bonding the ligand 2 and the polymer compound may be determined by considering the functional groups of both. The functional group is
Amino groups, carboxyl groups, hydroxyl groups, thiol groups, imidazole groups, phenyl groups, etc. can be used. For example, when bonding between amino groups, diisocyanate method, glutaraldehyde method, difluorobenzene method, benzoquinone method, etc. Many 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 an iodic acid oxidation method (Nakane method). When using a thiol group, for example, the carboxyl group on the other side is converted into a succinimide ester, and this is reacted with cysteine to introduce a thiol group.
A thiol group-reactive divalent crosslinking reagent can be used to link both. Methods using phenyl groups include diazotization and alkylation.
The binding method is not limited to these examples, and in addition, for example, "Method in Immunology"
The binding ratio that can be used 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, purification is performed by an appropriate combination of gel filtration, ion exchange chromatography, affinity chromatography, etc., and if necessary, drying is performed by freeze-drying or the like.

The antibodies making up the conjugate must react with the common antigenic determinant of Ligand 1 and Ligand 2. This antibody contains F(ab') ₂ , Fab',
Fragments such as Fab are also included.

As a method for producing antibodies, Ligand 1 or Ligand 2, or a conjugate of either of these ligands and a protein, is administered to mixed-breed animals such as rabbits, goats, horses, guinea pigs, and chickens at a dose of 0.3 to 2 mg per 1 kg of body weight.
- Inject subcutaneously into the back, foot pads, thigh muscles, etc. together with an adjuvant several times to allow it to form within the animal's body. The antibody may be used directly as a serum, or after being purified by a known method for collecting antibodies, ie, immunoglobulins, from the serum.

On the other hand, this replacement 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 spleen 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. can.

The binding method between amylase and antibody may be determined by considering the functional groups of both. The functional group can be an amino group, a carboxyl group, a hydroxyl group, a thiol group, an imidazole group, a phenyl group, etc., and the bonding method can be selected as appropriate from the bonding method of the ligand 2 and the polymer compound described above. good. After the reaction, purification is performed by an appropriate combination of gel filtration, ion exchange chromatography, affinity chromatography, etc., and if necessary, drying is performed by freeze-drying or the like.

A combination of Ligand 1 and Ligand 2 contained in the sample and a polymer compound (hereinafter referred to as polymerized ligand 2)
That's what it means. ) is brought into contact with the above antibody-amylase conjugate in a solution. At that time, the temperature of the solution is
A temperature of about 20 to 45°C and a pH of about 4 to 8.5 are usually appropriate. To keep the pH constant, if necessary,
Buffers such as phosphate buffer and acetate buffer may also be used. In this case, the appropriate amount of the antibody-amylase conjugate varies depending on the type of antibody, the type of ligand, the contact conditions, etc., and is therefore preferably determined by testing in advance. Ligand 1 and polymerized ligand 2 for the antibody-amylase conjugate
The order in which they are contacted is not critical; they may be contacted first or at the same time.

The antibody-amylase conjugate reacted with Ligand 1 and polymerized Ligand 2 is brought into contact with water-insoluble starch to react.

The conjugate of antibody and amylase that is brought into contact with water-insoluble starch may be separated from the reaction product, but usually it may remain contained in the reaction product. Even if starch itself is soluble, it can be used after being bound to an insoluble carrier or made insoluble by polymerization.

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

On the other hand, if the sample contains amylase of the same type as the amylase of the conjugate, the amylase in the sample inhibits the amylase to a greater extent than the amylase bound to the conjugate. It is preferable to bring the inhibitor into contact with the inhibitor.

It goes without saying that it is most preferable for this amylase inhibitor to be one that completely deactivates amylase contained in the sample and does not inhibit amylase bound to the conjugate at all. The blank value may not be increased in the measurement, and the amylase activity may be recovered by deactivating the amylase inhibitor after the measurement. On the other hand, the action of amylase inhibitors is a problem, and amylase is in a state bound to an antibody, and in a free state, it may be inactivated by amylase inhibitors. A known amylase inhibitor with such specificity may be used as this amylase inhibitor, but it is also possible to administer amylase contained in a sample to a warm-blooded animal to obtain antibodies, and then use this as an antibody. It can also be used as an amylase inhibitor. The method for obtaining antibodies may be the same as the method for obtaining antibodies against the aforementioned ligands. The amylase inhibitor may be added at any time as long as it can substantially prevent the decomposition of water-insoluble starch, which will be described later, by amylase in the sample, and it is usually added before the addition of this starch. However, since the inhibitory effect of an amylase inhibitor is generally much faster than the decomposition rate of starch, which is a substrate, by amylase, the amylase inhibitor may be added at the same time as the starch or after some delay.

After the enzymatic reaction, amylase activity is determined. Amylase activity is caused by an increase in decomposed products due to this enzymatic reaction,
It is sufficient to track the decrease in starch, which is a raw material, and other changes in the system due to enzymatic reactions.

(Function) The method of the present invention utilizes the fact that ligand 1 causes steric hindrance to the subsequent enzymatic reaction by binding to the antibody portion of the conjugate. Since the starch subjected to the enzymatic reaction is insoluble, most of the contact with the amylase moiety of the conjugate occurs between solid and liquid, resulting in significant steric hindrance due to polymerization of amylase. In order to confirm this, the present inventors investigated using an α-amylase system, and found that in the case of pentaose, there was almost no decrease in amylase activity due to the polymerization of amylase, whereas in the case of insoluble starch, In some cases, amylase activity was significantly reduced.

The present invention is characterized by newly introducing polymerized ligand 2 into such a system. That is,
By causing this polymerized ligand 2 to react competitively with ligand 1 against the antibody, the polymerized ligand 2 reacts with the antibody that did not react with ligand 1, and this has a large effect on the subsequent enzymatic reaction. Causes steric hindrance. Therefore, a large difference in steric hindrance between ligand 1 and polymerized ligand 2 appears,
Even if Ligand 1 is a small molecule, it can be measured with high sensitivity.

(Example) Preparation of CHM amylase 5 mg of Bacillus subtilis amylase was added to PH
Dissolve in 1 ml of 0.1M phosphate buffer from 6.3,
100μ of a DMF solution containing 2mg/ml of CHMS was added and left to react at room temperature for 1 hour. This reaction solution was put into a column of Sephadex G-25, and the pH was adjusted to 6.3.
Gel filtration was carried out by flowing 0.1M phosphate buffer, and the flow-through fraction was collected.

Preparation of anti-digoxin rabbit IgGF (ab') ₂ Add 10 mg of anti-digoxin rabbit IgG to 2 ml of 0.1 M acetate buffer (PH4.0) and add 300 μg of pepsin.
Stirred at ℃ for 18 hours. Add 0.1NNaOH to pH
was adjusted to 6.0, and the reaction solution was placed in a Sephacryl S-300 gel column buffered in advance with a 0.1M phosphate buffered 1mM MEDTA solution (PH6.3), and eluted with the above phosphate buffer. The peak portion eluted around the molecular weight of approximately 100,000 was collected and concentrated to 1 ml.
The desired anti-digoxin rabbit IgGF (ab′) ₂ was obtained.

α-amylase-anti-digoxin rabbit
Preparation of IgGFab′ conjugate Anti-digoxin rabbit IgGF prepared by
(ab′) ₂ 0.1M phosphate buffer containing 6mg 1mMEDTA
Add 100μ of a 10mg/ml 2-mercaptoethylamine hydrochloride aqueous solution to 1ml of the solution (PH6.0),
Stirred at 37°C for 90 minutes. Prepare this reaction solution in advance.
Unreacted 2-mercaptomethylamine was removed by gel filtration with a Sephadex G-25 column buffered with 0.1M phosphate buffer (PH6.3), and HS
−Fab′ was obtained. CHM-ized α prepared with this
- 2 mg of amylase was added and reacted at 37°C for 90 minutes. Next, add this reaction solution to 0.1M acetate buffer (5mM).
The gel was filtered through a Sephacryl S-300 column buffered with a calcium chloride solution (PH6.0) to collect fractions with a molecular weight of 200,000 or more, which were concentrated to obtain the target conjugate.

Preparation of polymerized digoxin 10 mg of digoxin was suspended in 500μ of ethanol, and 500μ of a 0.2M sodium metaperiodate aqueous solution was added thereto. This mixture was stirred at room temperature for 1 hour, and then a 1M aqueous ethylene glycol solution was added.
1μ was added. Hemocyanin 10mg after 5 minutes
was added and stirred at room temperature for 3 hours. 0.5mg of this
of NaBH ₄ was added and stirred for an additional 18 hours at 4°C. This reaction solution was dialyzed three times and then passed through Sephadex G-50 equilibrated with 0.02M phosphate buffered saline pH 7.0 to remove unreacted digoxin. The void fractions were pooled to obtain the desired polymerized digoxin.

Measurement of digoxin Add 50μ of a conjugate solution prepared by adding 50μ of a solution containing 0 to 12.5ng of digoxin to serum.
Polymerized digoxin 50μ made with
(100 ng/ml) and 50 µ of human amylase inhibitor (1.0 mg/ml) were added and allowed to react for 20 minutes. Add 1.0 ml of blue starch suspension to the reaction solution and further react at 37°C for 20 minutes.
The reaction was stopped by adding 1 ml of 0.5N NaOH. After stirring, the mixture was centrifuged at 3500 rpm for 2 minutes, and the absorbance of the resulting supernatant at 620 nm was measured.

A calibration curve showing the relationship between the obtained absorbance and the concentration of digoxin is shown in FIG.

(Effects of the Invention) The method of the present invention can measure low-molecular ligands with high specificity and extremely high sensitivity. In addition, the operation is simple, and the ligand can be quantified easily and inexpensively. The method of the present invention can be used to measure any type of low-molecular ligand.

[Brief explanation of drawings]

FIG. 1 shows the relationship between the amount of digoxin and the absorbance measured by the method of the present invention.

Claims (1)

[Claims] 1 An antigenic determinant-containing substance 1 to be measured with a molecular weight of 20,000 or less, and a water-soluble antigenic determinant-containing substance 2 with a molecular weight of 100,000 daltons or more that shares at least one antigenic determinant with this antigenic determinant-containing substance 1 A conjugate with a polymer compound is brought into contact with a conjugate of an antibody that reacts with this common antigenic determinant and amylase in an aqueous solution to react, and then a water-insoluble starch is added to the conjugate of the antibody and amylase. 1. A method for measuring a substance containing an antigenic determinant, which comprises contacting the two to cause an enzymatic reaction and measuring amylase activity.