JPS613065A - Measurement of antigen - Google Patents

Abstract

PURPOSE:To enable the measurement of an antigen handily with a very high sensitivity, by adding a polymer of a specified enzyme substrate and an enzyme for cutting the polymer off at the end thereof anew. CONSTITUTION:In the measurement of an antigen (e.g. digoxine) contained in serum or urine, by the enzyme-immunological method, first, a bond of an antigen to be measured, a substance (ligand) having an antigen determinant common to the antigen and a polymer (e.g. polymer of amino acid or the like) is brought into contact with an antigen reacting to the common antigen determinant. Then, an enzyme (e.g. esterase) is made to act on the bond in order to cut the coupling between the bound ligand and the polymer while another enzyme (e.g. carboxypeptidase A) to cut the polymer at the end thereof. The resulting composed products are measured by colorimetiric assay or the like. In this method, as a plurality of signalling substances are generated from a 1-molecule bond, the antigen can be measured with a high sensitivity. In addition, since reagents necessary for the reaction can be applied simultaneously, this method is simple to operate and also excellent as clinical analysis.

Description

[Detailed Description of the Invention] (Industrial Application Field) The present invention uses enzyme immunoassay to measure antigens contained in serum, urine, etc. when kept, such as trace components derived from drugs or various diseases. It is about the method.

(Prior art) Various methods are known for enzyme immunoassay, one of which involves binding an enzyme substrate to the same antigen as the measurement target, and reacting this with an antibody and an enzyme that breaks down this bond. There is a known method for measuring degradation products as antigens (
C1in Chem,, 23/8.

pp. 1402-1408 (1977)).

(Problems to be Solved by the Invention) This method has a problem in that the sensitivity is low because the amount of decomposed products is small.

(Another means to solve the problem) The present inventors have developed a polymer of a specific enzyme substrate and this
-) We have discovered that antigens can be measured easily and with very high sensitivity by newly adding an enzyme that cleaves polymers from the ends, and based on this knowledge, we have completed the present invention.

That is, in the present invention, a combination of an antigen to be measured, a substance having a common antigenic determinant with the antigen, and a polymer is brought into contact with an antibody that reacts with the common antigenic determinant, and further The present invention relates to a method for measuring an antigen, which comprises using an enzyme that cleaves between a substance having an antigenic determinant and a polymer, and an enzyme that cleaves the polymer from its terminal.

The antigen to be measured is not particularly limited, but antigens with a volume of 10,000 or less are particularly suitable for the method of the present invention. Examples of such antigens include drugs such as nocoxin and theophylline, steroid hormones, and incilin. High-altitude antigens, such as ferritin and α-fethoflotine, can be degraded with protease or the like, or reduced in molecular weight as synthetic peptide antigens, and if necessary, the antigenic determinant portion can be separated and measured. Therefore, polymer antigens such as HD antigens, cancer antigens such as CA 19-9, viral antigens such as 1-IB virus, HB, and herpes virus, plasma proteins such as fibrin degradation products, and immunoglobulins can also be used in the method of the present invention. can be measured.

The substance to be bound to the polymer described below (hereinafter referred to as a ligand) also has a common antigenic determinant with the antigen to be measured. At least one antigenic determinant may be common, or all antigenic determinants may be common. Therefore, the ligand may be the same as the antigen to be measured.

The polymer is a polymer of amino acids, sugars, ethylene oxide, vinyl alcohol, phosphoric acid, nucleotides, etc.

Examples of amino acid polymers include L-tyrosine polymers, L-
tricytophane polymer, L-phenylalanine polymer,
These include L-lysine polymers, L-glutamic acid polymers, and natural peptides made of various amino acids. Examples of sugar polymers include α-1,6-linked glucose polymers, β-galacto/depolymers, and dextran. The ethylene oxide polymer is polyethylene glycol, and the vinyl alcohol polymer is polyvinyl alcohol. Phosphoric acid polymers include polyphosphoric acid and hexametaphosphoric acid. Polymers of nucleotides include polyadenine, polyguania, polytin, polythymine, and natural polynucleotides composed of various nucleotides.

The degree of polymerization of these polymers is not less than dimer, and is not particularly limited, but it is usually preferable that about 10 to 10,000 molecules are polymerized.

The method of binding the ligand to the polymer varies depending on the type of enzyme used to cleave this bond.

When using esterase, ψrie combines a carboxyl group and a hydroxyl group. When the ligand side is a carboxyl group, if the ligand is pozotide, the carboxyl end, glutamic acid residue, aspartic acid residue, etc. may be used. When the ligand is other and does not have a carboxyl group, a hydroxyl group, a nitrogen group, an amine group, an aldehyde group, etc. may be converted into a carboxyl group by a known method and used. If the polymer has a hydroxyl group, it may be used as the hydroxyl group on the polymer side.If the polymer does not have a hydroxyl group, a halogen, an aldehyde group, a carboxyl group, a carbonyl group, an amide group, etc. can be added using a known method. This can be used instead of a hydroxyl group. The same applies when the ligand side is a hydroxyl group and the polymer side is a carboxyl group. As a method for bonding carboxyl groups and hydroxyl groups, an activated ester method, an acid chloride method, or a method using an anhydrating agent such as carboimide may be used.

However, when using bezotidase, these functional groups are introduced into the ligand or polymer side as necessary to obtain a combination of amine groups and carboxyl groups, and then these are bonded using known bezotide synthesis methods. That's fine.

In addition, in this case, when using an amino acid polymer,
This polymer can also be formed by bonding amino acids one by one to a ligand, and then bonding amino acids one after another to this using the R peptide bonding method.

When using other enzymes, appropriate functional groups may be selected depending on the enzyme and bonded using known methods.

Antibodies that react with common antigenic determinants can be produced according to known methods. That is, antigen ligands or conjugates of these and proteins are administered to warm-blooded animals such as rabbits, goats, horses, guinea pigs, and chickens at doses of 0.3 to 2 m9 per 1 kg of body weight.
is injected subcutaneously into the back, foot pads, thigh muscles, etc. together with an adjuvant one to several times to form in the animal's body.

For this antibody, serum may be used as it is, or it may be used after being purified by a known method for collecting antibodies, ie, immunoglobulin, from serum.

On the other hand, this antibody can also be obtained as a monoclonal antibody. In that case, one of the antigens mentioned above is injected into the peritoneal cavity of the mouse together with an associate several times, and the 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.

This antibody has F(ab'), + Fab', F
Fragments with ab are also included.

The order in which this antibody is brought into contact with the antigen to be measured and the above-mentioned conjugate is not critical, and either may come first, but unless there is a particular purpose, both may be added together.

The pH and temperature of the solution that causes this antigen-antibody reaction should be kept under conditions that facilitate the reaction, usually pH 4.5 to 105.
The temperature should be about 20 to 45°C. The reaction time is usually sufficient to allow a sufficient antigen-antibody reaction, and may be about 5 to 30 minutes.

The enzyme that cleaves between the ligand of the conjugate and the polymer is selected appropriately depending on the type of bond, and for example, esterase, peptidase, lipase, cholinesterase, galactosidase, glucondase, etc. can be used.

Depending on the type of polymer, the enzyme that cleaves the polymer from the terminal is carboxy-! ! Butidase A1 caldexybezotidase B1 glucoamylase, polyethylene glycol oxidase, polyvinyl alcohol oxidase, polyphosphatase, echinonucleotidase, etc. may be used.

These enzymes may be added after the above-mentioned antigen-antibody reaction is completed, or all reagents may be added at once to allow the antigen-antibody reaction and the enzyme reaction to occur in parallel. Each enzymatic reaction may be performed simultaneously or separately. When carried out separately, either enzyme reaction may occur first.

In order to speed up the enzymatic reaction, it is preferable to use as much amount of each enzyme as possible. It is not necessarily the case that each enzymatic reaction is preferably carried out under conditions in which the enzyme is likely to act; therefore, appropriate temperature, pH, etc. are set depending on each enzyme.

Appropriate methods are selected to measure the decomposed products depending on the type of polymer; for example, in the case of amino acids, colorimetric determination using ninhydrin, enzymatic method using H2O2 produced by oxidation with amino acid okindase, or It can be quantified by chemical methods such as colorimetric determination or fluorescence analysis. In addition, when thironne is released, it can also be measured using a coupled enzyme system using dirosinase.

When the polymer is sugar, for example, if the decomposition product is glucose, H2C is produced by the action of glucose okindase. may be chemiluminescent, or the quinone imine dye produced by oxidative condensation using phenol, no-ξ-ogindase and 4-aminoantipyrine may be colorimetrically determined. In addition, measurement can also be performed using the 02 electrode.

When the polymer is polyethylene glycol, 2,5-nochlorophenolindophenol may be oxidized by the action of polyethylene glycol oxidase, and the resulting dye may be determined colorimetrically. In the case of polyvinyl alcohol, it may be oxidized with polyvinyl alcohol oxidase and raw sil H2O2 may be determined by a conventional method.

In the case of polyphosphoric acid, it can be quantified by the molybdate method or enzymatic measurement method.

The methods for measuring decomposition products are not limited to these.
The method may be appropriately selected from among various known methods.

(Function) In the method of the present invention, the antigen to be measured and the ligand in the bound substance are caused to react competitively with the antibody. In this case, since the antigen to be measured preferentially binds to the antibody, the amount of the bound compound that reacts with the antibody depends on the amount of antigen in the sample. The conjugate that has reacted with the antibody becomes difficult to react with the enzyme due to steric hindrance, and the conjugate that has not reacted with the antibody is preferentially degraded by the enzyme.

(Effects of the Invention) Since the method of the present invention generates a plurality of gunar substances from one molecule of conjugate, antigens can be detected at 1l11 with high sensitivity.
Can be determined. Since the reagents necessary for the reaction can be added all at once, it is easy to operate and is an excellent clinical analysis method.

(Implementation ψ11) Carboxytheophyllin 7100 ~ was dissolved in DMF 2 me, 100 μl of thionyl chloride was added at 0° C., and the mixture was reacted at 4° C. for 3 hours and then at room temperature for 1 hour. child'! Add 50 m of monomethoxy polyethyl glycol 4000 to 1.
9 and reacted at room temperature for 16 hours. The reaction solution was applied to a Sephadex G-25 column and developed with 20mM 'J acid buffered saline solution pH 6.5 to remove unreacted theophylline to obtain a theophylline-polyethylene glycol conjugate.

To 50 μl of serum sample containing 50 μg of theophylline O, 1001 Al portions of the solution containing the above conjugate 250 tt9 was added, and further 100 μl of a solution containing anti-theophylline mouse IgG1 was added, followed by reaction at 37° C. for 20 minutes. Next, Carpigysis Esthesis 2000UA/X, t
? Liethylene glycol oxidase °4000UAl
and nochlorophenol indophenol 0.01mM
Add 3 ml of a solution containing and react at 37°C for 30 minutes,
The absorbance of the reaction solution at 580 nm was measured. The results obtained are shown in the drawings.

Next, various serums were measured in the same manner as above, and the theophylline concentration was determined using the curve in the drawing as a calibration curve. The results are shown in the table below. The same table also shows the results of measuring the same serum using a conventional method, Radioimmunoa, Sei (RIA).

Theophylline concentration (μ!17m(') A 2.1 1.
6B 4.5 4
．． 3C10,110,6 D I8,5 16.
9E 1.2 1
．． 0F 23.1 2
2.5G 31 2
9.4

[Brief explanation of the drawing]

The drawing shows an example of the relationship between theophylline concentration in serum and absorbance. Patent applicant Fujirebio Co., Ltd. Representative Patent attorney 1) Masahiro Naka 0 +
, 4 16 64 256 +000pg/ml

Claims (2)

[Claims] (1) A combination of an antigen to be measured and a substance and a polymer having a common antigenic determinant with the antigen is brought into contact with an antibody that reacts with the common antigenic determinant, and further the antigenic determinant of the combination is A method for measuring an antigen, which comprises causing an enzyme to cleave a bond between a substance having a substance and a polymer, and an enzyme to cleave the polymer from the end to act. (2) The method for measuring an antigen according to claim 1, wherein the polymer is a dimer.