JPS59178360A - Measurement of antigen determinant-containing substance - Google Patents

Abstract

PURPOSE:To achieve a highly sensitive and handy measurement by bringing a conjugate between an antigen determinant-containing substance (ligand) contained in a specimen and an enzyme and a second antibody with respect to an antibody of a ligand into contact with a conjugate between an antibody of the ligand and an antibody of the enzyme in a solution. CONSTITUTION:A ligand contained in a specimen, an enzyme or a high molecular compound thereof and the second antibody of an anti-ligand antibody or the second antibody of an anti-enzyme antibody are brought into contact with said antibody conjugate or a high molecular compound thereof in a solution. Here, the temperature of the solution is appropriately about 20-45 deg. and pH about 4- 8.5 normally. It is preferable in securing a moderate change in the enzyme activity that the component element should be converted into a high molecule to a certain extent and for the purpose, the second antibody is used. After the contact therebetween, the enzyme activity is measured to calculate the quantity of the ligand in the specimen. The measurement of the enzyme activity can be done by a widely-accepted method.

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 immunotherapy is attracting attention,
Radioimmunoassays were lacking in terms of operability and sensitivity.

The present inventors have conducted various studies to develop a measurement method that does not have the drawbacks mentioned above. It has been found that when a substance containing an antigenic determinant is brought into contact with an enzyme that reacts with an antibody against the enzyme, the enzyme activity changes depending on the amount of the substance containing an antigenic determinant. Utilizing this reaction, we have completed a completely new method that enables the simple measurement of antigenic determinant-containing substances with high sensitivity and without the above-mentioned separation procedure. After that, the present inventors continued their research and newly added a second antibody whose antigen is an antibody against the above-mentioned antigenic determinant-containing substance or a second antibody whose antigen is an antibody against the above-mentioned enzyme to the reaction system. For example, the inventors discovered that enzyme activity changes more sensitively depending on the amount of a substance containing an antigenic determinant, and based on this finding, the present invention was completed.

That is, the present invention provides an antigenic determinant-containing substance contained in a specimen, an enzyme or a combination of an enzyme and a polymer compound, and a second antibody against the antibody of the antigenic determinant-containing substance (or a second antibody against the antibody of the enzyme). 2. The antibody is brought into contact with a conjugate of an antibody of the antigenic determinant-containing substance and an antibody of the enzyme, or a conjugate of an antibody of the antigenic determinant-containing substance, an antibody of the enzyme, and a polymer compound in a solution. The present invention relates to a method for measuring a substance containing an antigenic determinant, which comprises subsequently measuring the activity of the enzyme.

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.

The antigenic determinant-containing substance (hereinafter referred to as a ligand) 1 has one or more antigenic determinants, for example,
Hormones derived from various endocrine glands, immunoglobulins,
These include plasma proteins such as albumin and ferritin, viruses such as HB antigen, bacteria, antigens present in various organs, blood, and urine such as α-7 ethoprotein 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 can be used in the method of the present invention because they form antibodies in the animal body when administered to the animal body. Even enzymes derived from animals are no exception, as antibodies can usually be obtained by administering them to a foreign animal. For concentration, an easy method of measuring activity is preferred. Examples of enzymes include glucose-6-phosphate dehydrogenase, Hegin kinase, alpha-amylase, malate dehydrogenase, alkaline bosphatase,
It can target benicilidase, β-galactosigase, taleatinginase, ribonuclease, benicilidase, etc.

If the activity of the enzyme does not change much even if the enzyme is reacted with a conjugate of an antibody described below, it is preferable to bind oxygen to a polymer compound in advance to form a polymer before use. It is appropriate that the polymeric compound has a molecular weight of 100,000 gluton or more and is water bathable. Examples of polymeric compounds include soluble dextran, carboxymethylated dextran, aminated dextran, polysaccharides such as amylose, derivatives thereof, proteins such as gelatin, hemocyanin, ferritin, and polyethylene glycol. These only need to meet certain conditions when bound to an enzyme. 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 be something that has been done.

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 the functional group, an amine group, a carboxyl group, a hydroxyl group, a thiol group, an imiguzole group, a phenyl group, etc. can be used. For example, when bonding between amine groups, the soifocyanate method, the glutaraldehyde method, Many methods are known, including the difluorobenzene method and the benzoquinone method. In addition, as a method for bonding between an amino group and a carboxyl group, methods such as the carboximide method and the Woodward reagent method, which convert the carboxyl group into a succinimide ester, are known, and the amine group and the sugar chain are cross-linked. There is also a periodic acid oxidation method (Nakane method). When using a thiol group, for example, the carboxyl group on the other side is converted into a zacunnimide ester, and this is reacted with cysteine to introduce a thiol group, and then both are linked using a thiol group-reactive divalent cross-linking reagent. Can be combined. Methods that utilize phenyl groups include diazotization and algylation. The bonding method is not limited to these examples, and in addition, for example, [Me
thod in Imrr+unochemistry
It is possible to use an appropriate method selected from methods described in books 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 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.

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

), an antibody against the enzyme (hereinafter referred to as anti-enzyme antibody), and a second antibody thereof can all be obtained according to known methods for obtaining antibodies. For example, when injecting a liganded V-enzyme into warm-blooded animals such as rabbits, goats, horses, guinea pigs, and chickens, the injection rate should be 03/kg body weight.
~2mg, and when injecting an anti-ligand antibody or anti-enzyme antibody, inject about 3~2mg of O6 per 10 body weight once or several times subcutaneously in the back, 7) i4 into the throat, thigh muscle, etc. together with adipant. Antibodies may be formed in the body of the animal using the method. This antibody is IgG%
Not only IgA etc. but also proteolytic enzymes such as pepsin
(ab') 2 , Fab', Fab, etc. may be decomposed and used.

The anti-enzyme antibody and its second antibody may completely inhibit enzyme activity, partially inhibit it, or not inhibit it at all by reacting with the enzyme; It can be used in any method. These antibodies, whether or not they are 7 fragments, can be purified as appropriate by known methods for obtaining IgG from serum, such as ammonium sulfate precipitation, ion exchange chromatography, dull filtration, and affinity chromatography. used from

On the other hand, these antibodies are treated as monoclonal antibodies (
You can also sip it. In that case Id, i' to the mouse
Inject Gant or Enzyme along with Aju Gant several times into the cavity, etc., and remove the bulge cells and fuse them with mouse myeloma cells using polyethylene glycol or the like. Then, among these fused cells, those that produce the antibody are grown as monoclonal cells by cloning, and the resulting monoclonal cells are grown in the peritoneal cavity of a mouse, thereby making it possible to produce a large 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, in addition to OC, which can use the glutaraldehyde method, periodic acid oxidation method, maleimide method, diisocyanate method, benzoquinone method, and carboimide method, S
PDP method, protein A that binds to sugar chains of IgG
A method using a lectin such as , a method of recombining the SH groups of two types of F(ab') 2 in the presence of a reducing agent, etc. 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 arbitrary depending on the purpose.

In some cases, it may be better to bind this antibody conjugate to a single polymeric compound and polymerize it, similar to 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, if necessary, freeze-dried.

The ligand contained in the sample, the enzyme or its polymer, and the second antibody of the anti-synthetic antibody or the second antibody of the anti-enzyme antibody.
The antibody is brought into contact with the antibody conjugate or polymerized product thereof in a solution. At that time, the temperature of the solution is 16.20-45
℃ and the pH is usually about 4 to 8.5. In order to keep I))-1 constant, any buffer such as phosphate buffer or acetate buffer may be used as necessary. Enzyme or polymerized product thereof, second antibody of anti-ligand antibody or second antibody of anti-enzyme antibody and antibody conjugate or polymerized product thereof Alternatively, it is better to determine the second antibody by testing in advance as it varies depending on the conditions at the time of contact.The second antibody may be added only one of the second antibody of the anti-ligand antibody or the second antibody of the anti-enzyme antibody. Both may be added.

The amount of second antibody added is the amount necessary to appropriately change the enzyme activity, and this also varies depending on the enzyme, antibody conjugate, ligand type, and the conditions at the time of contact. It is best to determine this through testing in advance.

The contact time between the antibody conjugate, the enzyme, and the second antibody is usually such that sufficient reaction occurs; for example, at 37°C, about 20 to 60 minutes is appropriate. The order in which the binding substance is contacted with the ligand, enzyme, and second antibody is not critical; any one may come first.
Or they may be done simultaneously.

In the case of the method of the present invention, it is preferable that each component be polymerized to some extent in order to ensure an appropriate change in enzyme activity, and for this purpose a second antibody is used. By adding this second antibody, an antibody-enzyme matrix is formed, and as the amount of ligand increases, secondary steric hindrance becomes stronger and therefore the matrix becomes more concentrated.
It is thought that the enzyme activity appears to be low. This effect is even greater when combined with polymerization of enzymes or conjugates.

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, if glucose-6-phosphate dehydrogenase is used as the enzyme (in the case of Q, the reaction system in which the above contact is carried out contains glucose-6-phosphorus, dihydrogen, and NADP+), Then, apply anti-[j+ and generate NADPH at a wavelength of 340 nm.
It can be determined from the increase in absorbance of .

When using -xokinase, gluconis, ATP'INADP+ and glucose-6 are added to the reaction system.
- Adding a substrate solution containing phosphate dehydrogenase to react,
Again, it may be determined by measuring the amount of NADPH produced.

The method of the present invention can measure ligands with high specificity and extremely high sensitivity. And, the operation force is 1 unit,
It is possible to quantify the ligand cheaply and easily.

The practical value of the method of the present invention is further enhanced by C which increases the sensitivity of the method of the photoinvention.

Examples are shown below.

Example 1 1) Anti-human IgG guinea pig IgG (α-h IgG
) and anti-hexokinase guinea pig) IgG (α-HK
IgG) binding and preparation of proboscis α-hI) g05m9 to 0.1M phosphorus'tW slow↑y7,
3 liquid (Pi-16,3) dissolved in 1 ml, 2 m9/m
i-no-4-(71/(midomethi/lz Schif0hexane-1-carboxylic acid)sacunnimide ester (CHMS
) was added thereto, and the mixture was reacted at room temperature for 1 hour. The reaction solution was put into a column of Sephadex G-25, and 0.1M of pH 6.5 containing 1mM EDTA was added.
'Glup filtration was performed with J acid buffer to remove unreacted CHM.
After removing S, the resulting conjugate of 4-maleimidomethylcyclohexane-1-carboxylic acid and α-h IgG (CH
The solution of M-alpha-h IgG ) was concentrated by 1rtllK.

α-! (Dissolve K Igo 5 m9 in 0.1 M IJ acid buffer, pH 7.5, containing 5 mM EDTA, and add 100 μl of a dimethyl sulfoxide solution of 9 mg/ml S-acetylnucleanhydride tosuccinic anhydride to this.
was added and heated at 37°C for 1 hour. Next, pi-17
, 5 was added, and the mixture was allowed to react at 37° C. for 30 minutes. This reaction solution was filtered using Cefazo and Kusu G-25, and unreacted S
- Acetylmercaptosuccinic acid was removed. This S R
-α-I (KIgG was condensed to 1 ml, and the above-mentioned C1 (M-α-h IgG was concentrated to 1 ml).
k was added, and the reaction was allowed to proceed overnight at 4°C. This reaction solution was filtered through heptaphryl S-30Q to obtain α-I (KIgG and α-I).
- A fraction of bound hIgG (containing 4 mg) was obtained.

11) Quantification of human IgG Add the above α-)IK IgG to each 50μ of the human) IgG bath solution of various types of sputum.
- α-hIgG conjugate 50 conjugates total 50 μl and α ~ Mormoy F IgG - 1/100 dilution of IgG serum 1
After adding 00 μl, the mixture was heated at 25° C. for 1 hour.

Add 25 μl (containing 0.04 μg) of hexokinase to this, react at 25°C for 30 minutes, and then add 0.1 μl to the reaction solution.
M glucose, 0.5 mM ATP, 0.2
mMNADP) 3 TJ/ml IJ/ :l
-S6-phosphate dehydrogenase and 13.3 mM MgCt
Add 3 ml of Lys buffered heart solution (50 mM) containing pH 2.0 as the substrate solution and adjust the temperature at wavelength 34.0 at 25°C. O
When the increase in absorbance at nmK was determined, the results shown in the table below were obtained.

Human IgG (ΔA
7m1n)X100O40nm O,6ttjJ/ml l 9.52
．． 0 17.05.0
14.120.0
11.3100
8.5 1 each for 5 human serum samples
Measurement was carried out in the same manner as above using 50 till f of each 1:000 diluted serum. Then, using the results in the above table as a calibration curve,
The acidity of gG was determined. Meanwhile, in parallel, the IgG concentration of the same serum was measured using the conventional 5RID method.

The results obtained are shown in the table below.

IgG concentration 88.4169/ml 9.1- m9/mlB
16.1 17.2C11,510,3 D 12.1 11.8E
7. s8.1 Example 2 1) Anti-glucose-6-phosphadephydrodanase mouse IgG ((χ-G6PDHIgG)) Co., Ltd.] was used. This G 6 P
A 1'9/rnl solution of DH is mixed with Freund's complete anupant to make an emulsion, and its Q,
lvrl into the peritoneal cavity of BALB/C mice over 8 years old.
Every week ((I noted it 3 times. Then, after another week, I took 50 tt & / 0.1 ml of G 6 P in the coccyx style.
DH solution was injected, and 3 days later, 4 lactobacilli were produced.

Grind the spleen to separate the spleen and visceral cells, and use Po'J ethylene glycol 150 (1-) to remove mouse myeloma.
The cells were fused with 3U1.

The obtained fused cells were dispensed into 961-quel plates,
I (cultured in AT medium. Cells in each well were incubated with G 6 P
D) ELISA using a plate with I immobilized on a solid phase
Mice I that have reactivity to G 6 PDH as determined by
We found 5 wells that seemed to contain gG.

These 5 wells of cells were diluted and cloned using the limiting dilution method, and examined using an inhibition assay applying the gLIsA method.
Two cell lines that appear to recognize different antigenic determinants of 6 PDH were obtained.

This cell line was cultured in 10% FC8-RPMI medium.
The proliferated cells were injected into the peritoneal cavity of B'ALB/C mice that had been previously injected with pristane, and about 10 ml of ascites was collected 2 weeks later.

This ascites was salted out with ammonium sulfate saturated with 45% salt, and the resulting precipitate was separated. This precipitate was mixed with a small amount of phosphate buffer pi (7
0 and equilibrated with the same buffer.
The IgG fraction was collected by Glu filtration using a 00 column.

Equal amounts of each IgG obtained from the two cell lines that recognize different antigenic determinants thus obtained were mixed and α-G 6
PDT (as IgG).

! :) Anti-human α-fetoprotein mouse IgG (
Preparation of human α-fetoprotein (α-AFP IgG) by the same procedure as above.
Two types of mouse monoclonal antibodies against AFP were obtained. Each antibody was purified to IgG, and the two types were mixed and used as α-AFP IgG.

111) α-AFP IgG and α-G6 PDI
(Preparation of conjugate with IgG Dissolve Dextran T500 (manufactured by Pharmacia, average molecular weight 500,000) 5'Om9 in 17 n-l of water, and add 0.1 M periodic acid to this solution.) +) Rum aqueous solution 0. 2N
was added and reacted overnight at 4°C. Add 0.15 μl of ethylene glycol to this, react for 5 minutes, and then add 1 mM
The mixture was filtered through a Sephadex G-25 column equilibrated with acetic acid buffer (PH5, 0), and the flow-through fractions were collected. This fraction contains α-AFP IgG and α-G 6
A solution of 20 Da of PDT (mixture with IgG) dissolved in 10 mM carbonate buffer (pH 9.5) was added, and the pH was adjusted to 9.5.
After adjusting the temperature, the mixture was allowed to react at room temperature for 2 hours.

0.4 borohydride) IJium aqueous solution 0.5 m
The reaction mixture was further reacted at 4° C. for 2 hours, and the reaction mixture was dialyzed against 20 mM chloride buffer (pH 7.0). The dialysate was filtered through a heptafacrylic S-300 column to fractionate the high molecular weight fraction, and AFP IgG and α-G6P were separated.
A fraction bound to DHIgG was obtained.

iV) Quantification of α-fetoprotein (AFP) Add the above α-AF to 50 μl of each AFP solution at various concentrations.
PIgG-α-G 6 PDH IgG binding binding surface fraction 5
After adding 100 μl of rabbit anti-mouse IgG IgG fraction containing G 6 PDH at 25°C.
The mixture was heated for 30 minutes to react.

Q, 5 nh was added to the reaction solution as a substrate for G 6 PDH.
Contains Mglufus-6-phosphate, 0.5mM NADP and 20mM MgC12 tr 0. When adding IM glycyldarisine buffer (P) 18.5) and determining the increase in absorbance at a wavelength of 340 nm at 25°C,
The results shown in the table below were obtained.

O beating 36.5 50 28.1 100 22.3 200 19.8 400 16.9 800 15.1 Measure the 5 human serum samples in the same manner as above using 50 μl each, and calibrate the results in the table above. AFP used for lines
The concentration of was determined. In addition, in parallel with this, the conventional method RI
The concentration of AFP in the same serum was measured using Method A.

The results obtained are shown in the table below.

AFP concentration A 200? '[91877Ll? 8 6
0 53 C320334 +) 530 '551 E 105 112 Patent applicant: Fuji Organ Pharmaceutical Co., Ltd.

Claims (1)

[Claims] An antigenic determinant-containing substance contained in a specimen, an enzyme or a combination of an enzyme and a polymer compound, and a second antibody to the antibody of the antigenic determinant-containing substance or a second antibody to the antibody of the enzyme are mixed in a solution. contact with a conjugate of an antibody of the antigenic determinant-containing substance and an antibody of the enzyme, or a conjugate of an antibody of the antigenic determinant-containing substance, an antibody of the enzyme, and a polymer compound, and then increase the activity of the enzyme. A method for measuring an antigenic determinant-containing substance, characterized by: