JPS6180050A - Measurement of substance possessing antigen determining group by utilizing enzyme - Google Patents

Abstract

PURPOSE:To measure a substance possessing an antigen determining group with high sensitivity, by using enzyme capable of acting on a water-insoluble high-molecular substance. CONSTITUTION:A ligand being a substance possessing two or more of antigen determining groups contained in a specimen is contacted and reacted with a combined substance of the antigen to one antigen determining group of said ligand and enzyme capable of acting on a water-insoluble high-molecular substance, and the antigen to the other antigen determining group. Further, the aforementioned combined substance is contacted with the aforementioned high- molecular substance to perform enzymatic reaction and enzymatic activity is measured to determine the substance possessing the antigen determining groups. The enzyme is one capable of acting on the water-insoluble high- molecular compound and the bonding method of the antigen may be determined with due regard to both functional groups. The antibody acting on the ligand is one reacted with the antigen determining group different from the antigen determining group reacted with the antigen of the combined substance.

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, in order to be used as a daily test, it is desirable to make it simple and routine.Although methods for detecting these trace components in consanguineous relatives have been developed for a long time, they lack sensitivity, specificity, and the shortness of time required for large amounts of specimens. Enzyme-linked immunosorbent assays, which have advantages such as time processing, are currently on the rise. However, in the case of the conventional enzyme immunoassay method, the sensitivity is still not sufficient, and the washing operation is complicated and dusty.
It was not easy to obtain an accurate concentration of gold because it sometimes required replacing the cheap.

Therefore, the inventors of the present invention conducted studies to develop an analysis method with higher sensitivity and fewer complicated operations.
We have developed a method that utilizes a conjugate of an antibody against a target antigenic determinant-containing substance and an antibody against an enzyme. In this method, the antigenic determinant-containing substance to be measured is competitively reacted with the antigenic determinant-containing substance and the enzyme, and the activity of the enzyme is then measured, thereby quantifying the total amount of the antigenic determinant-containing substance (Japanese Patent Laid-Open No. 59-1111). Public bulletin). 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), or a method in which the antigenic determinant-containing substance and the polymer compound are further reacted competitively. A method of competitively reacting a conjugate or a polymer of antigenic determinant-containing substances (Patent application 1982-
No. 51495) 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 58-231241). 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 this antigenic determinant-containing substance and an antibody against another antigenic determinant of this antigenic determinant-containing substance. There was also a method (Japanese Patent Application No. 143801/1982) in which the amylase activity of the conjugate was subsequently measured.

(Problem to be solved by the invention) Compared to the former method, the latter method is superior in that the measurement sensitivity is one or two orders of magnitude higher; It was necessary to add amylase inhibitor y-, which acts specifically on amylase in the sample because of its inclusion.

(Means for Solving the Problems) The present invention extends the 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 θ11 can be determined 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 the total 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. In the case of blood and urine, no special pretreatment is usually required and the measurement can be performed as is.

Antigenic determinant-containing substances (hereinafter referred to as antigens) are substances that have two or more antigenic determinants, such as hormones derived from various endocrine glands, immunoglobulins, albumin, plasma proteins such as ferritin, etc. , viruses such as HB antigen, bacteria, α-fetoprotein, carcinoembryonic antigen, and other antigens present in various organs, blood, and urine.

The antibodies making up the conjugate must be reactive with the ligand. This antibody has F(ab')2.

Fragments such as Fab' and Fab are also included.

A method for producing antibodies involves administering a ligand or a conjugate of a ligand and a protein to a warm-blooded animal such as a rabbit, goat, horse, guinea pig, or chicken weighing 1 kg. l) 0.3-2
M9 is injected subcutaneously into the back, foot pad, thigh muscle, etc. together with anopant one to several times to form in the animal's body. Since this antibody is a mixture of antibodies that recognize various antigenic determinants, it can be used by separating it. It is best to use affinity chromatography as a separation method. For example, the ligand is decomposed with enzymes or chemical reagents, separated by gel filtration, ion exchange chromatography, etc., and an affinity column is used in which each antigen is insolubilized. The antibody mixture can be separated by running around the entire column. 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 annunvant, and the lung cells HD are extracted and fused with mouse myeloma cells using polyethylene glycol or the like. Then, the cells that produce the antibody from among these fused cells are grown as monoclonal cells by whole cloning, and by growing them in the peritoneal cavity of mice, single antibodies, that is, monoclonal antibodies, can be produced in large quantities. .

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, gakistranase, cellulase, collagenase, mannase, protease, elastase, lipase, and the like.

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 amine 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, the Neusonanate method, glutaraldehyde method, difluorobenzene method is used. , benzoquinone method, etc. are known. In addition, as a method for completely bonding between an amino group and a carboxyl group, in addition to the method of converting the carboxyl group into a sacunnimide ester, the carboimide method and the Woodward reagent method are known. There is also a periodic acid oxidation method (Nakane method) for crosslinking. When using a thiol group, for example, a thiol group is introduced by esterifying the carboxyl group on the other side with cysteine, and then a thiol group-reactive divalent cross-linking reagent is used to connect both sides. can be combined. Methods that utilize phenyl groups include noazotization and alkylation. The coupling method is not limited to these examples, and in addition, for example, r Method
in Immunology and Immunoc
It is possible to use an appropriate method selected from among the methods described in books such as hemistry J or "enzyme immunoassay". 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 carried out by an appropriate combination of gel filtration, ion exchange chromatography, affinity chromatography, etc., and if necessary, drying is carried out by freeze-drying or the like.

The antibody that acts on Gant, together with the antibody of this conjugate, reacts with an antigenic determinant different from the antigenic determinant with which the antibody of the conjugate reacts. This antibody is IgG,
IgM or IgA, F (a b')z
It may be a fragment such as rFab, or it may be chemically modified, such as DNP, acetylation, biotinylation, or nitration. This antibody is not limited to one type, and it is better to have two or more types.Antibodies that recognize different antigenic determinants can be easily obtained by the above-mentioned method for producing monoclonal antibodies using the cell fusion method. . 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 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, such as proteins such as albumin and hemocyanin, and polysaccharides.

These include kalide, polyethylene glycol, and polynucleotide. The binding method may be appropriately selected from among the methods described above for binding antibodies to enzymes.

Similarly, this antibody may be made into a polymer by further reacting the entire antibody against this antibody. 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. This second
Although the antibody may be brought into contact with the first antibody before or after the first antibody is brought into contact with the ligand, it is operationally convenient to add the antibody at the same time.

A conjugate of an antibody and an enzyme against the one antigenic determinant and an antibody against another antigenic determinant are brought into contact with the glue gunt to be measured in a solution. At that time, @the temperature of the liquid is 20~4
Appropriate temperature is about 5°C and pH is usually about 4 to 8.5. In order to keep - 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 conjugate and antibody vary depending on the type of the compound, the type of the 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 come into contact with the antibody at the same time.

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. A known enzyme inhibitor with such specificity may be used as this enzyme inhibitor, but it is also possible to obtain antibodies against the enzyme contained in the sample by administering it to a whole warm-blooded animal. 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 ligands described above. 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 in the case of α-amylase, cellulose in the case of cellulase, collagen 9 in the case of collagenase, mannan in the case of mannase, and insoluble protein in the case of protease. , elastin in the case of nilastase, and iE-
In the case of oil, various oils and fats can be mentioned. Even if this polymer substance itself is soluble, it can 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 can be determined by tracking the increase in decomposed products due to this enzymatic reaction, the decrease in the raw material polymer 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.

In order to confirm this, the present inventors conducted an investigation using an α-amylase system, and found that in the case of pentaose, there was almost no decrease in enzyme activity due to the polymerization of the enzyme. In this case, the enzyme activity decreased significantly.

A feature of the present invention is that a new antibody that recognizes all antigenic determinants, which is different from the antibody of the conjugate, is introduced into such a system. That is, one antigenic determinant portion binds to the antibody portion of the conjugate, or this antibody binds to another antigenic determinant portion of the Gantt, thereby making the conjugate larger and further increasing its steric hindrance. This greatly improves the measurement sensitivity of the holder.

(Example) Example 1 ■ Preparation of cellulase substrate 2 Cut paper into a size of 20 (7) water 200
me). The mixture was heated to 60° C. and continued to be heated for 3 days with occasional stirring. The P paper was thoroughly washed with distilled water to remove excess dye. Subsequently, it was dried in a constant temperature dryer and cut into a size of 1 m x 5 crn to obtain the desired substrate.

■ Preparation of cellulase-anti-human IgG mouse IgG conjugate Dissolve cellulase 10m9'1p86.0 in 2 ml of O, lM phosphate buffer and 1-(maleimidomethylcyclohexane-1-caranoic acid) succinimide ester (
CHMS ) in trimethyl sulfoxide solution (zmpAJ
)200μ! was added and left at room temperature for 1 hour. This reaction solution was filtered using Sephadex G-25 to remove unreacted CHMS. This CHMS-modified cellulase was concentrated to 11 nl.

On the other hand, anti-human IgG-7 mouse IgG 10'9'
Fl: 0.1 at pH 7,5 with 5 mA EDTA
M! Dissolve in 2 ml of J acid buffer g m;)/r
ug of S-acetylmercaptosuccinic anhydride (SAM
S ) nooxane solution 200μ! added. Then 3
After standing at 7°C for 1 hour, it was added to 200μ of a 1N hydroxylamine aqueous solution (pH 7.5). After 30 minutes, the reaction solution was filtered through Safedex G-25 to remove unreacted SAMS.
was excluded. This H8-antihuman) IgG mouse IgG@ solution was added to 1 ml of the CHM celltase described above, and the mixture was heated at 37°C for 2 hours.
I left it for a while. This reaction solution was filtered through heptafacrylic S-300, and the target cellulase-antibiotic IgG mouse I
A gG conjugate was obtained.

■ Measurement of human IgG Cellulase-antihuman) IgG Mouse IgG conjugated solution containing 50μ! 2) Add standard solution 50a containing IgGi and anti-human IgG mouse IgG 50 tJ (0.1 m (//me)) that recognizes an antigenic determinant different from the bound antibody and leave it at 37°C for 30 minutes. pH 5.0 to 0
．． A total of 1 me of 1M acetate buffer was added, and then a total of 1 sheet of blue cellulose P paper prepared in ① was added. After 1 hour, the absorbance of the reaction solution was measured at a wavelength of 650 nm. FIG. 1 shows the amount of human JgG in the standard solution and the absorbance.

In the figure, white circles indicate the case where anti-human IgG mouse IgG1 was added, and black circles indicate the case where it was not added.

Example 2 ■ Insolubility ratio Preparation of blue dextran Blue dextran 2000 (Pharmacia product)
2 ji f 0.6 N NaOH aqueous solution 25rnl
1 g of this cellulose powder (product of Pharmacia) and 9f of NaBH430rn were added. While stirring, 5 rnl of noglycinolether was added, and the mixture was stirred overnight at room temperature. After the reaction, the resulting lumps were crushed with a sterilizer and thoroughly washed with distilled water. The insolubilized blue dextran was fractionated by centrifugation. 1g of this insolubilized blue dextran
was suspended in 0.1 M acetate buffer Q 5 Q ntl.

■ Preparation of CHM-forming dextranase Dextranase 1 m9 e 0.1M at pH 7.3
Dissolve in 1 ml of phosphate buffer, CHMS l mgf
100 μA of a DMF solution of il was added and left to react at room temperature for 1 hour. This reaction solution f Bio Gel P
-2 column and 0,1 M of Pi-t 7.0.
! Gel filtration was carried out by flowing J-acid buffer, and the flow-through fraction was collected.

■ Anti-human α-fetoprotein/goat IgG (a
b') Preparation of 2 Anti-human α-fetoprotein goat ■gG 10 m9 was sifted into 2 ml of 0.1 M acetate buffer (pH 4.0).
300 μgt of fufu was added and stirred at 37° C. for 18 hours. 0
．． 1 NNaQH was added to adjust the pH to 6.0, and the reaction solution was pre-buffered with 0.1M phosphate buffered 1mM EDTA solution (pH 6.3).Sephacryl S-300r
and eluted with the above phosphate buffer. Collect 1 ml of gold from the peak eluting at a molecular weight of around 100,000.
Concentrate K and target anti-human α-fetoprotein goat ■g
GF (a b') 2 was obtained.

■ Preparation of dextranase-anti-human α-fetoprotein goat IgG Fal)' conjugate Anti-human α-fetoprotein goat IgCr F(ab')23 prepared in ■
1ffl of 0.1M phosphate buffer containing 1mM EDTA (pH 6, o) containing 9e 1 OrrQ/m in 1me
2-Mercazotoethylamine hydrochloride water soluble gioooμ
Ai was added, and the mixture was stirred at 37°C for 90 minutes. This reaction solution was filtered through a Cefazo-X G-25 column buffered in advance with 0.1M phosphoric acid buffer (pH 7.0) to remove unreacted 2-mercaptomethylamine gold. H8Fa
b' was obtained. To this was added CHM-formed r-chitranase 1 da prepared in ①, and after reacting at 37°C for 90 minutes, it was left at 4°C overnight. Next, this reaction solution was mixed with glycel CPG buffered with 20 mM' acid-buffered saline (pH 7,0).
A fraction with a molecular weight of 170,000 or more was collected by gel filtration through a column, and this fraction was concentrated to obtain the target conjugate.

■ Measurement of human α-fetoprotein Concentration 1i 0-1 m9 α-fetoprotein solution (8
%PEG content) 50μ! In, conjugate solution 5 prepared in ■
0 μjl and U anti-human α-fetoprotein mouse IgG
(Monoclonal antibody) 50 ttA (5004/m
e) was added and reacted for 20 minutes. Insolubilized blue dextran suspension in reaction solution 1. Q m, e f plus 3
Further reaction was carried out at 7°C for 30 minutes, and 0.5 NNaOH1
ml k was added to stop the reaction. After stirring this, 35
Centrifugation was performed at 00 rpm for 2 minutes, and the resulting supernatant was centrifuged at 620 n.
The total absorbance at m was measured.

A calibration curve showing the relationship between the obtained absorbance and the concentration of human α-fetoprotein is shown in FIG. The white circle in the figure is anti-human α
-Fetoprotein Mouse IgG'i is added, while black circles are not added.

(Effects of the Invention) The method of the present invention allows the measurement of a concentration in a sample with high specificity and extremely high sensitivity. The method of the present invention was developed in the earlier application (
Compared to the method disclosed in Japanese Patent Application No. 59-27710), the sensitivity can be further improved by one to two orders of magnitude. In addition, it is easy to operate, and it is possible to quantify the amount of water at a low cost and easily. The method of the present invention can be used to measure any type of throat.

The method of the present invention also has the advantage that the reagent used in the method of the present invention does not require direct use of the Gantt, and only a trace amount of the ligand is needed for the production of 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 a comparison between when cellulase was used as the enzyme and an antibody against an antigenic determinant was added (white circles) and when it was not added (black circles), where the relationship between human IgG concentration and absorbance was km. FIG. 2 shows the results of a similar comparison of human α-fetoprotein using dextranase as the enzyme. Patent Applicant Fujirebio Co., Ltd. Agent Patent Attorney 1) Masahiro Naka 1 0j560.6252-5 10 40 160 64
0 2560 IgG 7ag/ml Figure 2 J-Fetophage Dtin

Claims (3)

[Claims] (1) In a method for measuring a substance containing two or more antigenic determinants contained in a sample, the antigenic determinant-containing substance is combined with an antibody against one antigenic determinant of the antigenic determinant-containing substance and a water-insoluble antibody. A conjugate with an enzyme capable of acting on a polymer substance and an antibody against another antigenic determinant are brought into contact and reacted, and further, the conjugate is brought into contact with the polymer substance to cause an enzymatic reaction, and the enzymatic activity is determined. A method for measuring an antigenic determinant-containing substance, characterized by: (2) The measuring method according to claim 1, wherein the antibody against another 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.