JPS59164960A - Assay of antigenic determinant-containing substance - Google Patents

Abstract

PURPOSE:To enable simplified quantitative determination of an antigenic determinant-containing substance (referred to as ligand) contained in a specimen with a high sensitivity, by measuring the activity of an enzyme after the ligand is made to react with an enzyme or a conjugate between the enzyme and a high molecular compound and a conjugate between an antibody against the ligand and one against the enzyme. CONSTITUTION:A ligand (hormone, immunoglobulin, HB antigen or the like) in a specimen such as serum or urine is made to react with a mixture of an enzyme such as hexonase or a conjugate between the enzyme and a high molecular compound (protein such as soluble dexitran or gelatin) and a conjugate between an antibody against the ligand and one against the enzyme (or a conjugate between the both antibodies and the high molecular compound) in a solution. After the reaction, the enzyme is allowed to react with a substrate to measure the rate of the changes in absorbance at a specified wavelength due to a reaction product and a color-forming reagent. a calibration curve obtained by using a specimen with a known concentration is used to enable simplified determination of the concentration of a ligand in a specimen with a high accuracy without requiring any pretreatment thereof.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for measuring trace components derived from various diseases.

Microanalysis of body fluid components such as serum and urine has become a powerful means for diagnosing diseases and determining the progress of treatment. Therefore, various methods for analyzing body fluid components have been developed, and among these, immunological analysis methods are widely used in daily tests because of their excellent sensitivity and specificity.

Immunological analysis methods that utilize the extremely high affinity between antigens and antibodies include radioimmunoassays that use radioactive isotopes as labeling substances, enzyme immunoassays that use enzymes, and the like. However, because radioimmunoassay uses radioactive isotopes, it has various problems, such as use in limited facilities, disposal of waste liquid, and short shelf life. Therefore, enzyme immunoassay is attracting attention, but it is inferior to radioimmunoassay in terms of operability and sensitivity.

As a result of various studies in order to develop an analysis method with higher sensitivity and fewer complicated operations, the present inventors discovered that the combination of an antibody against an antigenic determinant-containing substance, which is the object of measurement, and an antibody against an enzyme was used for measurement. It was discovered that when an enzyme is brought into contact with the target antigenic determinant-containing substance, the enzyme activity changes depending on the amount of the antigenic determinant-containing substance, and this reaction can be used to easily prepare antigenic determinant-containing substances. We have now completed a completely new method that allows measurements to be performed quickly and with high sensitivity.

That is, the present invention combines an antigenic determinant-containing substance contained in a specimen with an enzyme or a conjugate of an enzyme and a polymer compound into a conjugate of an antibody to the antigenic determinant-containing substance and an antibody to the enzyme in a solution. Or it relates to a method for measuring an antigenic determinant-containing substance, which comprises bringing an antibody against the antigenic determinant-containing substance into contact with a combination of an antibody against the enzyme and a polymer compound, and then measuring the activity of the enzyme. be.

Hereinafter, the content of the present invention will be explained in detail.

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 substances (hereinafter referred to as "ligands") are those having one or more antigenic determinants, such as hormones derived from various endocrine glands, plasma proteins such as immunoglobulin, albumin, and ferritin; These include antigens that exist in various organs, blood, and urine, such as viruses such as HB antigen, bacteria, α-fetoprotein, and carcinoembryonic antigen.

The ligand is preferably one that has a large effect on the enzymatic activity that is subsequently measured when it binds to the antibody conjugate described below.
In this respect, those having a molecular weight of 100,000 Daltons or more are particularly suitable for the method of the present invention.

The enzyme may be any enzyme from which the antibody can be obtained.

Most enzymes 1 can be used in the method of the present invention because they form antibodies in animals when administered to them. Even enzymes derived from animals are no exception, as antibodies can usually be obtained by administering them to a foreign animal. For enzymes, it is more convenient to use a simple method for measuring activity. Examples of b-memes include glucose-6-phosphate dehydrogenase, hexokinase, α-amylase, malate dehydroke9ase, alkaline phostaphatase, peroxidase, β-galactosidase, creatine kinase, reconuclease, It can suppress benicidase, etc.

If the activity of the enzyme does not change significantly even after reacting with the antibody conjugate described below, it is preferable to bind the enzyme to a polymer compound in advance to form a polymer before use.

The polymer compound preferably has a molecular weight of 100,000 Daltons or more and is water-soluble. Examples of polymer compounds include:
Examples include soluble dextran, carboxymethylated dextran, reduced dextran, polysaccharides such as amylose and derivatives thereof, proteins such as gelatin, hemocyanin, and ferritin, and polyethylene glycol. If these IDs are combined with an enzyme and meet certain conditions, for example, even if it is a soft and low molecular weight substance such as bovine serum albumin, it can be self-polymerized. It may also be made into a polymer.

Polymerization may be performed on the antibody conjugate described below in addition to the enzyme, or both the enzyme and the antibody conjugate may be polymerized.

The method of binding the enzyme and the polymer compound may be determined by taking into consideration the functional groups of both. As functional groups, amino groups, carboxyl groups, hydroxyl groups, thiol groups, imidazole groups, phenyl groups, etc. can be used. For example, when bonding between amine groups, diisocyanate method, glutaraldehyde method, difluorobenzene method can be used. A number of methods are known, including the benzoquinone method and the benzoquinone method. In addition, as a method for bonding between an amine group and a carboxyl group, there are known methods of converting a carboxyl group into a succinimide ester, such as the carbodiimide method and the Woodward 9 reagent method. There is also a periodic acid oxidation method (Nakane method) that cross-links the chains. If all thiol groups are to be used, 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 combined using a thiol group-reactive divalent cross-linking reagent. Can be combined. Methods that utilize phenyl groups include diazotization and alkylation. The bonding method is not limited to these examples, and in addition, for example, r M
method in Immunochemist, ry
It is possible to use an appropriate method selected from among the methods described at the bottom of the page, such as J or "Enzyme-antibody assay". 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 dull filtration, ion-exchange chromatography, affinity chromatography, etc., and drying is performed, if necessary, by freeze-drying.

Antibodies against ligands (hereinafter referred to as anti-ligand antibodies).

) and antibodies against the enzyme (hereinafter referred to as anti-enzyme antibodies) can both be obtained according to known methods for obtaining antibodies. For example, administer 1 kg of ligand or enzyme to warm-blooded animals such as rabbits, goats, horses, guinea pigs, and chickens.
Antibodies may be formed in the animal's body by injecting it together with an adipant once or several times into the back, foot pads, thigh muscles, etc., about 90.3 to 2 cm. This antibody is IgG,
Not only IgMIgA, etc., but also proteolytic enzymes such as pepsin can reduce F (ab') 2, F a b '% F
It may also be used after being decomposed into a, b, etc.

Anti-enzyme antibodies can completely inhibit enzyme activity, partially inhibit it, or not inhibit it at all by reacting with the enzyme, but any of these can be used in the method of the present invention. can.

These antibodies, whether or not they are the aforementioned fragments, can be purified as appropriate by known methods for obtaining IgG'i from serum, such as imitation precipitation, ion exchange chromatography, filtration, affinity chromatography, etc. then use it.

On the other hand, these antibodies can also be obtained as monoclonal antibodies. In that case, the aforementioned ligand or enzyme is injected into the peritoneal cavity of the mouse together with an adipant several times, and the spleen cells are removed and fused with mouse myeloma cells using polyethylene glycol or the like. and,
Among these fused cells, those that produce the antibody can be grown as monoclonal cells by cloning, and the resulting monoclonal cells can be grown in the peritoneal cavity of a mouse to produce a total amount of monoclonal antibodies.

As a method for binding an anti-ligand antibody and an anti-enzyme antibody, all of the methods for binding proteins to each other among the aforementioned methods for binding an enzyme and a polymer compound can be used. For example, a glutaraldehyde method, a periodic acid oxidation method, a maleimide method, a diisocyanate method, a benzoquinone method, a cardediimide method, etc. can be used. In addition, 5PD, which combines NH2 and SH groups,
P method, method using lectins such as protein A that binds to sugar chains of IgG, two types of F in the presence of a reducing agent.
A method for recombining the SH groups in (a b')2 can also be used.

The conjugate is not limited to one unit each of anti-ligand antibody and anti-enzyme antibody, but may also be one in which several units of each are bound, or one in which they are further bound to form a polymer.

In that case, it goes without saying that the ratio may be changed as appropriate.

In some cases, it may be better to bind this antibody conjugate to a polymer compound and polymerize it, as with the enzyme described above. In that case, the polymer compound may be appropriately selected from those mentioned above, and the bonding method may be the same as described above. This polymerization may be performed on one or both antibodies before performing the binding between the antibodies, or may be performed after performing the binding between the antibodies.

The antibody conjugate and its polymerized product are purified by an appropriate combination of dull filtration, ion exchange chromatography using a cation exchange resin, anion exchange resin, affinity chromatography, etc., and then freeze-dried if necessary. ″.

A ligand contained in a specimen and an enzyme or a polymeric product thereof are brought into contact with the antibody conjugate or a polymeric product thereof in a solution. In this case, the appropriate temperature of the solution is about 20 to 45°C, and the voice 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. Appropriate amounts of enzymes or their polymers and antibody conjugates or their polymers vary depending on their type, the type of ligand, the contact conditions, etc., and are preferably determined by testing in advance. The contact time between the antibody conjugate, the ligand, and the enzyme is usually such that sufficient reaction can occur; for example, at 37° C., about 20 to 60 minutes is appropriate.

The order in which the antibody conjugate is contacted with the ligand and the enzyme is not critical, and either may come first or may be done simultaneously.

After these contacts are made, the enzyme activity is measured and the amount of ligand in the sample is calculated. Enzyme activity may be measured according to known methods. For example, when glucose-6-phosphate dehydrogenase is used as the enzyme, glucose-6-'J
When hexokinase is used, glucose and ATP may be added to the system to react. , N.A.D.
It can be determined by adding a substrate solution containing P and glucose-6-1 non-acid dehydrogenase and measuring the reaction mixture and the amount of I'i-NADPH produced.

The method of the present invention can measure ligands with high specificity and extremely high sensitivity. In addition, the operation is simple, and it is possible to quantify the entire amount of the ligand at low cost and easily.

Practical examples are shown below.

Practical example 1 1) Preparation of a conjugate between anti-human α-fetoglotin goat Fab' and anti-hexokinase guinea pig Fab' Human α-fetoprotein (AFP)' by a known method
The anti-AFP goat antiserum obtained by immunizing goats was purified using an AFP-Sepharose 4B affinity column,
A dried product of anti-AFP goat-specific IgG was obtained from Kozue.

To this #H anti-AFP goat-specific IgG 20f, 0.1" of Penocin (Shikuma d) was added, and the pH was adjusted to 0.1 at pH 4.2.
Digestion was performed overnight at 37°C in IJum buffer.

This digested product was adjusted to pH 7.8 by adding IN NaOHf, passed through a Cefazo, Kusu G-1000 column, and the absorbance of the effluent was measured at 280 nm. A fraction containing

Anti-AFP Goat F (abs2'5 RTK 1 mM
FJ (6,0) containing EDTA was dissolved in 2 ml of 0.1 M IJ awakening buffer, and this solution was
- Add 0.2- of mercaptoethylamine solution to 37°C.
The reaction was extended for 90 minutes. Sephadex G- for reaction f'
25 (1 cm x 40 crn), and collect the flow-through fractions and use anti-AFP-goat Fab'. N,N'-(1,2phenylene)bismaleimide 4Ivt-Dissolved in acetone 0.5M, pi(
The mixture was added to the anti-AFP-Yamate Fab' fraction described above and reacted at 30°C for 20 minutes. This reaction product was passed through Sephadex G = 25 (1 crn x 40 cm) and subjected to dull filtration, the flow-through fractions were collected, and the PEG
-20,000 to obtain an anti-AFP goat Fab'-maleimide conjugate. On the other hand, anti-dish guinea pig-specific IgG i obtained by injecting cyhexokinase (HK) i into guinea pigs by a known method.
Anti-HK guinea pig specific IgG L:D+'r was purified using an HK-Sepharose 4B affinity column.
1 product was obtained. This 4M equipment 201 is the anti-AFP
Treated similarly to goat-specific IgG, anti-HK guinea pig F
A fraction containing (ab')29.8m9 was obtained.

anti-dish guinea pig) F(ab')25■ with the anti-A
2- by the same operation as FP goat F (a b')2
Reduction with mercaptoethylamine and Sephadex G-
The mixture was filtered through a filter using a filter with a filter of 25 to obtain anti-dish guinea pig Fab'i.

The obtained anti-dish guinea pig) Fab'iab' anti-AFP goat Fab'-maleimide conjugate was mixed with -t of the mixture.
-6.8 and allowed to react at 4°C for 24 hours. The reaction solution was concentrated using PEG-20000ffi and filtered through a Sephadex G-100 column (1crnX100cr11) to collect fractions corresponding to F(ab')2.
Anti-AFP goat Fab' - anti-f (K guinea pig) Fab'
conjugate binding total 89011) polymerized hexokinase (H
K) Preparation of rabbit serum albumin 50mfl (202mA
and prepared to pi-1'i6.0. To this solution was added 11 g of 1-ethyl-3(3-dimethylaminopropyl)carbodiimide hydrochloride (IDC).
The reaction was carried out at room temperature for about 1 hour while maintaining the pH at 6.0. After centrifuging the reaction solution, the supernatant solution was filtered using Sephacryl S-300 (10
The mixture was filtered through a filter (100 x 100 mm), and the clear fractions were collected. This fraction was concentrated using PEG-20000 to obtain polymerized rabbit serum albumin.

This polymerized rabbit serum albumin 10 rtu;l f
It was dissolved in HzO1rrtl and set to 'pi(6.0).

HK2■ and EDC1011'19 were added to this solution, and the mixture was reacted at room temperature for about 30 minutes while maintaining the pH at 6.0. After centrifuging the reaction solution, the supernatant was purified by gel filtration through a Sepharose 6B column, and fractions having HK activity were collected to obtain polymerized HK.

111) Quantification of human α-7 ethoprotein (AFP) 1
Test tube arrays containing 10 μl of each dilution were prepared by serially diluting AFP f 2 n at 0 μl/rrte. Each dilution contains 40 till k of anti-AFP goat Fab'-anti-HK guinea pig Fab' conjugate.
Add 20mM nonaphosphate buffered saline solution of pH 7.0 of No. 13, and further determine the specific activity (ΔOD
340 nm/min) bath solution containing polymerized HKi of 0.06.

20 μm of the solution was added and left at room temperature for 30 minutes.

In each test tube, add 0.67 MD-
Glucose, 16.5mM ATP, 6.8mM
NADP, 13.3221M MgC42, and 3
pli 8.0 containing 00 IU/IILlG6PDH
50mM of When the mixture was added to J-Su hydrochloric acid buffer solution t1 and mixed well, the change in absorbance in 0D54D was measured, and the results shown in Table 1 were obtained.

Table 1 Qn, 17 20.0 68 17.5 135 16.3 270 15.2 540 12.4 1080 10.5 Measurement was performed in the same manner as above using 5oμA' each for 5 human dish serum samples, Using the results in Table 1 as a calibration curve, A
The concentration of FP was determined. On the other hand, in parallel, a conventional radioimmunoassay (RIA method) was used to detect the AF of the same serum.
P concentration was measured.

The results obtained are shown in Table 2.

Table 2 AFP concentration A 200nl 192nliB 5
23 545 C421401 D 823 811 E 495 502 Example 2 1) Anti-human IgG guinea pig IgG (αHIgG) and anti-glucose 6-phosphate dehydrogenase guinea pig) IgG (α
Preparation of conjugate with G6PDHIgG) αH1gG51W was dissolved in 1vLl of 0.1M phosphate buffer at pH 6.3, and 2nVrnl of 4-maleimidomethylcyclohexane-1-carboxylic acid succinimide ester (CHMS) in dioxane bath solution was added to this. 100μ7t
- The blade was opened and left at room temperature for 1 hour. This solution was placed on a column of Sephadex G-25 (1y++
Glu filtration was performed with a phosphate buffer to remove unreacted CBMS, and the resulting 4-maleimidomethylcyclohexane-
conjugate of 1-carboxylic acid and αHIgG (CHM-formed αH
The total solution of IgG) was concentrated to 1 volume.

Dissolve 5 ml of αG6PDHIgG in 0.1 M phosphate buffer of 7.5 containing 5 mM EDTA, add 100 μl of a dimethyl sulfoxide solution of 9 myALA' S-acetylmercagutosuccinic anhydride, and add 3
It was heated at 7°C for 1 hour. Subsequently, 110 μm of a 1M hydroxylamine solution having a pH of 7.5 was added, and the mixture was allowed to react at 37° C. for 30 minutes. Sephadex G
-25 was used, and the unreacted S
- Acetylmercagutosuccinic acid was removed.

The thus obtained SH-conjugated αG6PDHIgG'i 1
The mixture was concentrated to mJ, and the above-mentioned concentrated solution of CHM-modified αHIgG lIn1 was added thereto, and the mixture was allowed to react overnight at 4°C. This reaction solution was mixed with Sephacryl-8-30,0 (1 crn
αHIgG and αG6PD
A 1:1 conjugate with HIgG was obtained.

11) Polymerization of the conjugate The above conjugate was dialyzed against 0.1M carbonate buffer at pH 8.0. The protein concentration of the dialysis residual solution was set to 'r 5 m9/ml, and pre-synthesized carboxydextran (
MW = l Q 7) Succinimide ester 20~
The mixture was stirred at 37°C for 2 hours using a knife.

Next, the target α-f (IgG-αG6PD
A HIgG-dextran conjugate (G-H-D conjugate) was obtained.

111) Quantification of human IgG Add 50 μl of each human IgG bath solution to human IgG at various concentrations
After adding 50μ7 of the conjugate and heating at 37°C for 1 hour, 100μlk of glucose-6-phosphate dehydrogenase was added.

The reaction was carried out at 37°C for 20 minutes, and the reaction solution contained pH 8 containing 5mM glucose-6-phosphate, 6.8rnMNADP, 1M glycylglycine and 20mM MgCZ2.
, 5 and Substrate Bath Solution 1, OTLl', and the rate of increase in absorbance at a wavelength of 340 nm at 30°C was determined, and the results shown in Part 3 were obtained.

Table 3 Human IgG ΔA340nm/
rr1inOμIIo, o s.

5 0.070 25 0.06250
0.050 200 0.035800
Regarding 4 samples of 0.028 human serum,
Measurement was carried out in the same manner as above using 50 μl'fr of each sample, and the concentration of IgG was determined using the results of the third coating as a calibration curve. On the other hand, in parallel to this, all IgG concentrations in the same serum were measured using the conventional 5RID method.

The obtained results are shown in Table 4.

4th coat IgG concentration serum Present invention method 5RID method F 12.2 mg/TL111.8 pack G
11.8 12.0 H8, 98, 2 I 14.3 13.5 Procedural amendment (voluntary) 1. Indication of case Japanese Patent Application No. 1983-38975 2. Name of invention 3. Amendment of method for measuring substances containing antigenic determinants Relationship with the case involving the person who filed the patent application Name of patent applicant Fujirebio Co., Ltd. 4 Agent residence 2-1-3 Kyobashi, Chuo-ku, Tokyo 104 The statement in the 6th amendment to the statement of contents is amended as follows.

15 pages 19 lines 0.67 0.1〃 20 lines
16.5 0.56, 8 0.2 16 pages 1 line 300 3 19 pages 15 lines 50.5 〃 16 lines 6.8 1.3 10.1

Claims (1)

[Claims] An antigenic determinant-containing substance contained in a sample and an enzyme or a conjugate of an enzyme and a polymer compound are mixed in a solution with a conjugate of an antibody to the antigenic determinant-containing substance and an antibody to the enzyme or the antigenic determinant. A method for measuring a substance containing an antigenic determinant, which comprises bringing into contact a bond of an antibody against the substance with a polymer compound and an antibody against the enzyme, and then measuring the activity of the enzyme.