JPH0340832B2 - - Google Patents

Description

[Detailed Description of the Invention] (Field of Industrial Application) 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. , 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. 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 antigenic determinant-containing substance to be measured with an enzyme against this bound substance, and then measuring the activity of this enzyme.
-38975, Japanese Unexamined Patent Publication No. 164960/1983).
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. 1983-
51494, Japanese Patent Application Laid-open No. 59-178360), or a method of competitively reacting a combination 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, 178361) was also developed. After further investigation, they developed a series of methods for measuring substances containing antigenic determinants that are closely related to this technique, and filed patent applications. Among them, there is a method in which a conjugate of the antibody and an enzyme capable of acting on a water-insoluble polymer substance is applied to a substance containing an antigenic determinant to be measured, and then the enzymatic activity of the conjugate is measured (patent application No. 58-231241, Japanese Patent Publication No. 1983
-123767) was received. Further, the antigenic determinant-containing substance to be measured is brought into contact with a combination of an antibody and amylase against one antigenic determinant of the antigenic determinant-containing substance and an antibody against another antigenic determinant of this antigenic determinant-containing substance. Subsequently, a method for measuring the amylase activity, etc. of the conjugate was developed (Japanese Patent Application No. 143801/1983, Japanese Patent Application Laid-open No. 23970/1983).

(Problems to be Solved by the Invention) Compared to the former method, the latter method was superior in that the measurement sensitivity was one to two orders of magnitude higher;
The latter method requires the addition of an amylase inhibitor that specifically acts on amylase in the sample, since the sample such as serum contains amylase.

(Means for Solving the Problems) The present invention extends this latter invention by using, in place of amylase, an enzyme that can act on water-insoluble polymeric substances. This method is based on the knowledge that substances containing antigenic determinants can be measured with high sensitivity. Among these enzymes, if an enzyme not included in the sample is used, there is an advantage that the measurement can be performed without using any particular enzyme inhibitor.

That is, the present invention provides a method for measuring a substance containing two or more antigenic determinants contained in a specimen, in which the antigenic determinant-containing substance is coated with an antibody against one antigenic determinant of the antigenic determinant-containing substance and water. A conjugate with an enzyme capable of acting on an insoluble polymeric substance and an antibody against other antigenic determinants are brought into contact with each other to react, and the conjugate is further brought into contact with the polymeric substance to cause an enzymatic reaction. The present invention relates to a method for measuring a substance containing an antigenic determinant, which is characterized by measuring activity.

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. For serum, urine, etc., no special pretreatment is usually required;
Measurements can be made as is.

Substances containing antigenic determinants (hereinafter referred to as ligands)
has two or more antigenic determinants, such as hormones derived from various endocrine glands, plasma proteins such as immunoglobulin, albumin, and ferritin, viruses such as HB antigen, bacteria, α
- Antigens present in various organs, blood, and urine, such as fetoprotein and carcinoembryonic antigen.

The antibodies making up the conjugate must be reactive with the ligand. This antibody has F
Also included are fragments such as (ab′) ₂ , Fab′, and Fab.

As a method for producing antibodies, a ligand or a conjugate of a ligand and a protein is administered per kilogram of body weight to warm-blooded animals such as rabbits, goats, horses, guinea pigs, and chickens.
0.3 to 2 mg once to several times subcutaneously on the back, foot pads,
It is injected into the 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, affinity chromatography is performed in which the ligand is decomposed with enzymes or chemical reagents, separated by gel filtration, ion exchange chromatography, etc., and each antigen fraction is insolubilized. A column can be constructed and used to separate the antibody mixture described above. Also,
This antibody is also commercially available. In the method of the present invention, antibodies do not need to be separated into single antibodies,
It is sufficient to divide it 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 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 enzymes constituting the conjugate can act on water-insoluble polymer compounds, and among them, enzymes whose activity can be easily measured are preferred. Such enzymes include, for example, amylase, dextranase, cellulase, collagenase, mannase,
protease, elastase, lipase, etc.

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, 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 an 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 the two 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 (Method in Immunology and
"Immunochemistry" or "Enzyme immunoassay"
You can select and use the methods as appropriate from among the methods described in books such as . It goes without saying that the coupling ratio is not limited to 1:1 and can be any ratio depending on the purpose. After the reaction, purification is performed using an appropriate combination of gel filtration, ion exchange chromatography, affinity chromatography, etc., and if necessary, drying is performed using freeze-drying or the like.

The antibody that is allowed to act on the ligand together with the conjugate antibody reacts with a different antigenic determinant from the antigenic determinant that the conjugate antibody reacts with. This antibody is IgG, IgM or IgA, and F
It may be a fragment such as (ab') ₂ or Fab, or it may be one that has been chemically modified, such as DNP, acetylation, biotinylation, or nitration. This antibody is not limited to one type, and may include 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. Also,
The above-mentioned warm-blooded animals may be used to produce antibodies, which may then be 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 may be bound to the above-mentioned enzyme, and the other may be used for this antibody. Furthermore, this antibody does not need to be completely separated, and the other antibody may be mixed to the extent that the measurement is not inhibited.

It may be preferable to bind a water-soluble polymer to this antibody in order to increase sensitivity. Water-soluble polymers have a molecular weight of 1000 or more, and include proteins such as albumin and hemocyanin, polysaccharides, polyethylene glycols, and polynucleotides. The binding method may be appropriately selected from among the methods described above for binding antibodies to enzymes.

Similarly, this antibody may be further reacted with an antibody against this antibody to form a polymer. The second antibody is, for example, rabbit IgG against goat IgG, and can be obtained using the first antibody or a combination of the first antibody and a ligand as an antigen according to the method for obtaining antibodies described above. The second antibody may be brought into contact with the first antibody before or after the first antibody is brought into contact with the ligand, but it is operationally convenient to add it at the same time.

The ligand to be measured is brought into contact with a conjugate of an antibody to the one antigenic determinant and an enzyme and an antibody to another antigenic determinant in a solution. that time,
The temperature of the solution is about 20-45℃, and the pH is usually 4-45℃.
Approximately 8.5 is appropriate. To keep the pH constant,
If necessary, a buffer such as a phosphate buffer or an acetate buffer may be used. In this case, the appropriate amounts of the conjugate and antibody vary depending on the type thereof, the type of ligand, the contact conditions, etc., and are therefore preferably determined by testing in advance. The order in which the ligand is contacted with the conjugate and the antibody is not critical; either may come first or both may be brought into contact simultaneously.

On the other hand, if the sample contains an enzyme of the same type as the enzyme of the conjugate, the degree of inhibition of the enzyme in this sample is greater than the degree of inhibition of the activity of the enzyme bound to the conjugate. It is preferable to bring the inhibitor into contact with the inhibitor.

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 the decomposition of a water-insoluble polymeric substance, which will be described later, by the enzyme in the sample, and is usually added before the addition of this polymeric substance.
However, since the inhibitory effect of an enzyme inhibitor is generally much faster than the rate of decomposition of a substrate by the enzyme, the enzyme inhibitor may be added at the same time as the polymeric substance or after some delay.

The conjugate reacted with the ligand is brought into contact with a polymeric substance and reacted.

The bond to be fused with the polymeric substance may be separated from the reactant, but usually it may remain contained in the reactant.

This polymeric substance is one in which the enzyme in the conjugate can undergo an enzymatic reaction, and is usually a substrate, but it is insoluble in water. Examples of polymeric substances include insoluble starch for α-amylase, cellulose for cellulase, collagen for collagenase, and mannan for mannase.
Examples include insoluble proteins in the case of protease, elastin in the case of elastase, and various oils and fats in the case of lipase. Even if this polymer substance itself is soluble, it can also be used after being bound to an insoluble carrier or made insoluble by polymerization.

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

After the enzymatic reaction, determine the enzyme activity. Enzyme activity is
What is necessary is to track the increase in decomposed products due to this enzymatic reaction, the decrease in the polymeric material that is the raw material, and other changes in the system due to the enzymatic reaction.

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

A feature of the present invention is that a new antibody that recognizes an antigenic determinant different from that of the antibody of the conjugate is introduced into such a system. In other words, one antigenic determinant portion is bound to the antibody portion of the conjugate, and this antibody binds to the other antigenic determinant portion of the ligand, thereby making the conjugate larger and further increasing its steric hindrance. . This greatly increases the sensitivity of ligand measurement.

(Example) Example 1 Preparation of cellulase substrate Paper was cut into pieces of 20 cm x 20 cm and immersed in a previously prepared reactive blue solution (5 g reactive blue, 5 g Na ₂ CO ₃ distilled water 200 ml). did. The mixture was heated to 60°C and continued to be heated for 3 days with occasional stirring. The paper was thoroughly washed with distilled water to remove excess dye. Next, dry it in a constant temperature dryer and cut it into 1cm x 5
The desired substrate was obtained by cutting into cm-sized pieces.

Preparation of cellulase-anti-human IgG mouse IgG conjugate Dissolve 10 mg of cellulase in 2 ml of 0.1 M phosphate buffer at pH 6.0 and prepare a dimethyl sulfoxide solution of 4-(maleimidomethylcyclohexane-1-carboxylic acid) succinimide ester (CHMS) ( 2mg/ml) was added thereto, and the mixture was left at room temperature for 1 hour. This reaction solution was gel-filtered using Sephadex G-25 to remove unreacted CHMS. This CHMS-modified cellulase was concentrated to 1 ml.

Meanwhile, 10 ml of anti-human IgG mouse IgG was added at 5 mA.
200 μl of a dioxane solution of 9 mg/ml S-acetylmercaptosuccinic anhydride (SAMS) dissolved in 2 ml of 0.1 M phosphate buffer, pH 7.5 containing EDTA.
added. Then, after leaving it at 37℃ for 1 hour, 1N
200μ of hydroxylamine aqueous solution (PH7.5) was added. After 30 minutes, the reaction solution was gel-filtered through Sephadex G-25 to remove unreacted SAMS. This HS
-Anti-human IgG Mouse IgG solution was added to 1 ml of the above CHM-modified celltase and left at 37°C for 2 hours.
This reaction solution was gel-filtered with Sephacryl S-300 to obtain the desired cellulase-anti-human IgG mouse IgG conjugate.

Measurement of human IgG Add 50μ of a standard solution containing human IgG to 50μ of a solution containing a cellulase-anti-human IgG mouse IgG conjugate.
Then, 50μ (0.1 mg/ml) of anti-human IgG mouse IgG that recognizes an antigenic determinant other than the antibody of the conjugate was added, and the mixture was left at 37°C for 30 minutes. This has a PH5.0
1 ml of 0.1M acetate buffer was added, and then one sheet of the blue cellulose paper prepared above was added. After 1 hour, the absorbance of the reaction solution was measured at a wavelength of 650 nm.
Figure 1 shows the amount of human IgG in the standard solution and the absorbance. In the figure, white circles indicate the case where anti-human IgG mouse IgG was added, and black circles indicate the case where it was not added.

Example 2 Preparation of insolubilized blue dextran 2 g of Blue Dextran 2000 (product of Pharmacia) was dissolved in 25 ml of 0.6N NaOH aqueous solution.
To this were added 1 g of cellulose powder (product of Pharmacia) and 30 mg of NaBH ₄ . 8 ml of diglycidyl ether was added with stirring, and the mixture was stirred overnight at room temperature. After the reaction, the resulting lumps were crushed with a spatula and thoroughly washed with distilled water. The insolubilized blue dextran was fractionated by centrifugation. Add 1g of this insolubilized blue dextran to 0.1M acetate buffer.
Suspended in 50ml.

Preparation of CHM-modified dextranase Dissolve 1 mg of dextranase in 1 ml of 0.1M phosphate buffer (PH7.0), and dissolve 1 mg of CHMS/ml in DMF.
100μ of the solution was added and left to react at room temperature for 1 hour. This reaction solution was put into a column of Bio Gel P-2, and gel filtration was performed by flowing 0.1M phosphate buffer at pH 7.0, and the flow-through fraction was collected.

Anti-human α-fetoprotein goat IgG F
Preparation of (ab′) ₂ 10mg of anti-human α-fetoprotein goat IgG
300μ of pepsin in 2ml of 0.1M acetate buffer (PH4.0)
g was added thereto, and the mixture was stirred at 37°C for 18 hours. 0.1N
The pH was adjusted to 6.0 by adding NaOH, and the reaction solution was preliminarily mixed with 0.1M phosphate buffered 1mM EDTA solution (PH
6.3) and eluted with the above phosphate buffer.
The peak portion eluted at a molecular weight of approximately 100,000 was collected and concentrated to 1 ml to obtain the target anti-human α-fetoprotein goat IgG F(ab') ₂ .

Preparation of dextranase-anti-human α-fetoprotein goat IgG Fab′ conjugate Antigen human α-fetoprotein goat IgG F(ab′) ₂ containing 3 mg in 0.1 M phosphate buffer 1
10 mg/ml 2-mercaptoethylamine hydrochloride aqueous solution in 1 ml of mM EDTA solution (PH6.0)
100μ was added and stirred at 37°C for 90 minutes. This reaction solution was gel-filtered through a Sephadex G-25 column buffered in advance with 0.1M phosphate buffer (PH7.0) to remove unreacted 2-mercaptomethylamine, yielding HS-Fab'. Add 1 mg of CHM-modified dextranase prepared above, and cool at 37°C.
After reacting for 90 minutes, the mixture was left at 4°C overnight. Next, this reaction solution was mixed with 20mM phosphate buffered saline (PH
7.0) and collected fractions with a molecular weight of 170,000 or more, which were concentrated to obtain the target conjugate.

Measurement of human α-fetoprotein Add 50μ of an α-fetoprotein solution (containing 8% PEG) with a concentration of 0 to 1 mg, and 50μ of the conjugate solution prepared in and 50μ of anti-human α-fetoprotein mouse IgG (monoclonal antibody). (500μ
g/ml) and allowed to react for 20 minutes. Add 1.0 ml of insolubilized blue dextran suspension to the reaction solution and further react at 37°C for 30 minutes.
The reaction was stopped by adding 1 ml of 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.

FIG. 2 shows a calibration curve showing the relationship between the obtained absorbance and the concentration of human α-fetoprotein.
The white circle in the figure is anti-human α-fetoprotein mouse.
The case where IgG was added is shown, while the black circle shows the case where it was not added.

(Effects of the Invention) The method of the present invention can measure a ligand in a specimen with high specificity and extremely high sensitivity. The method of the present invention has been disclosed in earlier patent applications (Japanese Patent Application No. 59-27710, Japanese Patent Application Laid-open No. 60-1989).
171461), the sensitivity can be further improved by one to two orders of magnitude. 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 ligand. The reagent used in the method of the present invention has the advantage that a trace amount is sufficient because the ligand is not used directly and the ligand is only used for producing the antibody. Therefore, the method of the present invention is particularly effective when the same ligand as the target to be measured is difficult to obtain or expensive.

[Brief explanation of the drawing]

Figure 1 shows the results of comparing the relationship between human IgG concentration and absorbance when cellulase was used as the enzyme and when antibodies against other antigenic determinants were added (white circles) and when antibodies against other antigenic determinants were not added (black circles). FIG. 2 shows the results of a similar comparison for human α-fetoprotein using dextranase as the enzyme.

Claims (1)

[Scope of Claims] 1. A method for measuring a substance containing two or more antigenic determinants contained in a specimen, wherein the antigenic determinant-containing substance is coated with an antibody against one antigenic determinant of the antigenic determinant-containing substance. A conjugate with an enzyme capable of acting on a water-insoluble polymeric substance is brought into contact with an antibody against another antigenic determinant, and the conjugate is brought into contact with the polymeric substance to cause an enzymatic reaction. , a method for measuring an antigenic determinant-containing substance, characterized by measuring enzyme activity. 2. The measuring method according to claim 1, wherein the antibody against the other antigenic determinant is bound to a water-soluble polymer. 3. The measuring method according to claim 1 or 2, characterized in that the antibody directed against this antibody is further brought into contact with an antibody directed against another antigenic determinant.