JPH02222837A - Novel measurement of antigen - Google Patents

Abstract

PURPOSE:To enable realization of a high-sensitivity measurement of an antigen by bonding a functional group or a label to the antigen to obtain a decorated antigen for use. CONSTITUTION:In a process (A), a functional group or a label is bonded to an antigen to be measured in a liquid to be inspected to form a decorated antigen. In a process (B), the decorated antigen is bonded to a carrier through an antibody against the antigen and then, the carrier is separated from the liquid to be inspected. In a process (C), the decorated antigen is dissociated from the carrier in a process (a) and a decorated antigen-antibody composite composed of the decorated antigen and the antibody against the antigen is dissociated from the carrier in a process (b). In a process (D), the decorated antigen or the decorated antigen-antibody composite as mentioned in the process (C) are measured.

Description

DETAILED DESCRIPTION OF THE INVENTION (Industrial Field of Application) The present invention relates to a method for measuring an antigen, and particularly to a method for measuring an antigen that can measure even low-molecular-weight antigens with high sensitivity.

[Prior art]

The measurement of biological components, especially antigenic substances in human body fluids, is of great clinical importance. Measurement of antigenic substances is widely used for endocrine testing by measuring hormones, cancer diagnosis, drug quantification, etc.

Previously, the measurement of trace amounts of antigenic substances as described above was carried out using RIA, E
This is done by immunological measurements such as IA and FIA.

Immunological assay methods are broadly classified into competitive methods and Sanderutsch methods.

The immunoassay method was a competitive method by Yallo et al.
It was started by IA. The competitive method is a measurement method that utilizes the inhibition of the binding of a labeled antigen to an antibody by the antigen to be measured. If the amount of labeled antigen and antibody added is increased, inhibition by a small amount of antigen cannot be detected, and if the amount of labeled antigen and antibody added is decreased, there is a limit because the rate of binding of the labeled antigen to the antibody decreases. Therefore, the measurement limit is generally about 1 fIIol (10-ISs+ol)
It is.

On the other hand, the sandwich method is a measurement method in which the antigen to be measured is bound to a carrier bound to an antibody and a labeled antibody in a sandwich manner. After using a large amount of both the antigen-binding antibody on the carrier and the labeled antibody, the carrier can be washed to remove excess labeled antibody, so the measurement limit is 1.
00 to 1000 times lower concentration, anal (10-”
It is possible to measure up to the mol) level. Recently, T.S.
Protein hormones such as H, hCC; etc. can be tested by ELISA, IRM
It is common to measure by the sand inch method called A.

However, there are limitations to the antigens that can be measured by the Sand-Deutsch method. In other words, an antigen that can be measured by the Sandwich method must have an antigen site that can bind to an antibody at at least two sites simultaneously, and low-molecular-weight antigens cannot be measured by the Sandwich method.

In addition, even for antigens that can theoretically be measured by the sandwich method, it may be difficult to obtain antibodies against two antigenic sites, or even if antibodies against two antigenic sites can be obtained, the Antibody titers can only be obtained at one antigen site, and highly sensitive measurements may not be possible.

(Intelligence B to be Solved by the Invention) Under such circumstances, the purpose of the present invention is to provide a highly sensitive antigen measurement method that utilizes the characteristics of the sandwich method using only an antibody against one antigen site. There is a particular thing. The object of the present invention is to provide a method that can measure vF with high sensitivity similar to the Sandwich method even for low molecular antigens such as peptides, steroids, and drugs that cannot be measured using the conventional Sandwich method.

[Means for Solving the Problems] The present inventor obtained a modified antigen by binding a functional group or a label to an antigen, and by using this modified antigen, the conventional The authors discovered that it is possible to directly measure the antigen in a sample solution, similar to the sandwich method, and that it is possible to measure antigens with only one antibody antigen site with higher sensitivity than the competitive method. I came up with an invention.

The present invention involves the following steps (A), (B), (C), and (D): Step (A): A functional group or a label is bonded to the antigen to be measured in the test liquid to form a modified antigen. The process of forming.

Step (B): A step of binding the modified antigen to the carrier via an antibody against the antigen, and then separating the carrier and the test liquid.

Step (C): the next (a) or (b) step.

(a) A step of dissociating the modified antigen from the carrier.

(Step of dissociating a modified antigen-antibody complex composed of a modified antigen and an antibody against the antigen from the bl carrier.

Step (D): A step of measuring the modified antigen or modified antigen-antibody complex described in (C).

This is a method for measuring an antigenic substance, which is characterized in that it includes the following.

Hereinafter, the present invention will be explained in accordance with the order of the steps.

About A This is a step of obtaining a modified antigen in a test liquid.

Examples of the test liquid include body fluids such as serum, plasma, Ua, saliva, and urine, and buffer solutions containing antigens to be measured. As the antigen to be measured, all substances containing an antigen site as an antigen, substantially all antigenic substances that can be measured by conventional immunoassay methods, can be measured.

Examples include thyroid-stimulating hormone (TSH), which is classified as a protein hormone, and placental gonadotropin (hcG).
), fibrin degradation products (FDP) classified as plasma proteins
, C-reactive protein (CRP), α-fetoprotein (AFP), which is classified as an oncofetal protein, oncofetal protein (C
EA), angiotensin 11, which is classified as a habten.
Vasopressin, somatostatin, atrial natriuretic hormone, endothelin, luteinizing hormone-releasing hormone (LH-RH), peptides such as casinin, progesterone, testosterone, steroids such as cortisol, total thyroxine (T4), triiodothyronine (
T3), catecholamines, digoxin, etc.
Low molecular weight antigens with a molecular weight of 2,000 or less can also be measured. Desirable antigens in the present invention are antigens classified as habten.

Particularly desirable antigens are peptides having amino groups in the molecule.

Any functional group can be used as long as there is a substance that specifically binds to the functional group and there is substantially no substance that specifically binds to the functional group or the functional group in the test liquid. Good examples include dinitrophenyl group, trinitrophenyl group, fluorescein group, and biotinyl.

The label may be any label used in immunoassays (enzymes, radioactive substances, luminescent substances, etc.).
Examples include fluorescent substances and metal compounds.

For example, enzymes include peroxidase, β-D-galactosidase, alkaline phosphatase, etc., radioactive substances include iodine, hydrogen, etc., fluorescent substances include fluorescein isothiocyanate, and luminescent substances include acridinium salts.

As the label that is directly bound to the antigen, radioactive substances, luminescent substances, fluorescent substances, etc. are preferable.

Examples of methods for forming a modified antigen by bonding a functional group or label to the antigen to be measured include a method in which a functional group or label is bonded via a chemical bond using a reactive group of the antigen. Ru.

Regarding reactive groups possessed by antigens, for example, proteinaceous hormones generally have -NH, groups. Furthermore, since Habten binds to proteins to create antibodies, it has a reactive group for binding. Such reactive groups are subjected to chemical bonding.

Such groups include -NH, -3H, -COO
Examples include H group and -CR2 group.

When a functional group or a label is bonded through a chemical bond using such a reactive group of an antigen, a functional group or a label compound having a reactive group for the reactive group of the antigen or to which such a reactive group is bonded is used. It will be done. A reactive group for a reactive group on an antigen refers to a reactive group that reacts substantially quantitatively with a reactive group on an antigen.For example, when an antigen has an -NHI group as a reactive group, an N-hydroxysuccinimide group, an acid anhydride group, etc. When the compound or antigen has a -SH group as a reactive group, it is a maleimide group, a pyridyl disulfide group, etc.

Examples of compounds for introducing such functional groups include N-sulfosuccinimidyl-6-(biotinamide)
Examples include hexanoate, 3-(N-maleimido-propionyl)biotin, N-succinimidyl-2,4-dinitrophenyl-5-aminocaproate, N6-pyothinyl-L-lysine hydrazide, p-diazobenzoylbiocytin, and the like.

Examples of compounds for introducing labels include N-succinimidyl-3-(4-hydroxy-5-(ltJ)iodophenyl)propionate, N-hydroxysuccinimide acridinium ester, europium (E u34
) Chelate compounds and the like.

The amount of the compound to be added to the test solution to introduce a functional group or label should be determined based on the amount of the compound to be added to the test solution, in addition to the antigen to be measured. Because of the presence of compounds, it is only necessary to add a sufficient amount to ensure that the reactive groups of the antigen in the test solution can react efficiently.Additionally, adding more than necessary will increase the background and reduce the measurement sensitivity. decreases.

The reaction temperature is 0 to 55° C., preferably 4 to 40° C., at which denaturation of the antigen and protein in the test solution does not occur. The reaction time is 10 minutes to 48 hours, preferably 30 minutes to 12 hours.

After the reaction is completed, in order to eliminate the reactivity of the unreacted functional group or the compound for introducing the label, it is preferable to add a substance that reacts with the reactive group of the compound. Examples of such a substance include N-sulfonate. In the case of succinimidyl-6-(biotinamide) hexanoate, Lm glycine etc.
-(N-maleimido-propionyl)biotin includes 2-mercaptoethanol and the like.

B is a step in which the modified antigen is specifically bound to a carrier using an antibody against the antigen, and the modified antigen is then separated from the test liquid.

The first method for binding a modified antigen to a carrier via an antibody against the antigen is to bind an antibody against the antigen to a carrier and then react with the modified antigen. The second method involves reacting a modified antigen with an antibody against the antigen,
This method involves binding to a carrier via an antibody.

In the second method, a functional group is bound to an antibody against the antigen, and the modified antigen and the antibody are reacted to form a modified antigen-antibody complex. The functional group used here is selected to be different from the functional group used in step (A). A substance that specifically binds to the functional group is bound to a carrier,
This is a method of reacting a modified antigen-antibody complex.

Examples of substances that specifically bind to a functional group include a specific antibody for habten, and avidin and streptavidin for biotin.

Antibodies against the antigen to be measured can be obtained by known methods. That is, polyclonal or monoclonal antibodies are obtained by immunizing an animal with the antigen or the antigenic site of the antigen. When the antigen or the antigenic site of the antigen is habten, it is combined with a protein and immunized to obtain antibodies.

As the carrier, any carrier that can be used in immunoassays may be used; examples thereof include polystyrene, polyacrylic, Teflon, paper, glass, agarose, and the like. Moreover, there is no particular restriction on the shape.

An example of a method for binding an antibody to an antigen or a substance that specifically binds to a functional group to a carrier is a method used for binding an antibody to a carrier in the Sand-Deutsch method.

After binding the modified antigen to the carrier, the carrier and the test liquid are separated.

The separation method may be any method conventionally used in immunoassays using carriers.

It is preferable to wash the carrier before proceeding from step (B) to step (C) described below.

Washing may be carried out under conditions commonly used in the Sandersch method.

Regarding C, this step is a step in which the modified antigen to be measured bound to the carrier via the antibody against the antigen is separated from the modified contaminants bound non-specifically.

Dissociation of the modified antigen from the carrier can be achieved using acids, alkalis,
This can be done by treating with a highly concentrated inorganic salt or the like.

In general, when dissociating with an acid, the pH is set to pl+5 or less, preferably PH 0.5 to 3.5; when dissociating with an alkali, the pH is set to 9 or higher; when dissociating with a high concentration inorganic salt, the pH is set to 2M or higher. This is done by adjusting the salt concentration. The treatment is generally carried out at a temperature of 0 to 45°C for 10 minutes to several tens of hours.

When the second method is used in step (B), a modified antigen-antibody complex consisting of a modified antigen and an antibody against the antigen is produced from a carrier by adding a substance having the same site as the functional group bound to the antibody. Can be dissociated.

For example, when the functional group is dinitrophenyl, dinitrophenyl amino acid (4L dinitrophenyl lysine) is used, and when the functional group is biotinyl, biotin is used.

Furthermore, when a functional group is bonded to the antibody via a -8-3- bond, the modified antigen-antibody complex can be dissociated with a reagent that cleaves the -5-S- bond.

Regarding D, this step is a step of measuring the modified antigen or the modified antigen-antibody complex composed of the modified antigen and an antibody against the antigen dissociated in step (C), and is measured by a known method including the immune complex transfer method. It can be done.

For example, if the dissociated modified antigen is an antigen bound to a functional group, a carrier bound to a substance that specifically binds to the functional group and an antibody against the antigen bound to a label ( This is a method of measuring a modified antigen using a labeled antibody) using the Sand-Deutsch method.

Measurement by the sandwich method may be performed by any of the following methods: reacting with a labeled antibody and then reacting with a carrier, reacting with a carrier and then reacting with a labeled antibody, or reacting a carrier and a labeled antibody simultaneously.

The second method is to bind a modified antigen to a carrier via an antibody against the antigen in the same manner as in step (B), and perform measurement focusing on the functional group or label of the modified antigen.

As a method for measuring by focusing on the functional group, measurement is performed by the Sand-Deutsch method using a carrier and a label in which a label is bound to a substance that specifically binds to the functional group. Examples of labels include conjugates of avidin or streptavidin and enzymes.

In methods other than the first method, a reaction is carried out in which a modified antigen bound to a functional group is reacted with a label in which a 4I-responsive substance is bound to a substance that specifically binds to the functional group, thereby converting the modified antigen to a modified antigen bound to a label. It is also possible to perform it before step (D).

〔Example〕

The present invention will be explained below with reference to examples, but the present invention is not limited to these.

Example 1 1 to 0.1 M sodium chloride and 1 g/j! 0.0 containing bovine serum albumin. OIM sodium phosphate buffer, pH 1.0, was prepared and designated as buffer A.

Angiotensin! - Bovine serum albumin conjugate was prepared by known methods using glutaraldehyde.

Angiotensin! - A rabbit anti-angiotensin antiserum was prepared by immunizing a rabbit with the bovine serum albumin conjugate by a known method using a complete solo indage band.

IG F ab'Fab's IgG was obtained by salting out with sodium sulfate and DEAE cellulose, F(ab')* was obtained by pepsin digestion of IgG, and Fab' was obtained by reduction of F(ab') using known methods. [Ishikawa et al., Journal of Immunoassay (J, Ls+5unoasaay) Vol. 4, No. 20
9 (1983)).

Angiotensin I (0.5 g) was added to activated CH-Cefbroz 4B (0.15 g) according to the Pharmacia manual.
) was insolubilized.

Rabbit anti-angiotensin 1 Fab' was prepared by a known method using N-succinimidyl-6-maleimidohexanoate as a cross-linking agent [Hashida et al., Journal of Applied Biochemistry (J.

Appl, Biochem, ) Volume 6, Page 56 (1
984)) Labeled with peroxidase.

Rabbit anti-angiotensin I Fab "-peroxidase was prepared using an angiotensin I-Sepharose 4B column and eluted at pH 2.5 using a known method (11-h, R
Uan et al., Clinical Kimi Power Acta (CIin,
Chis, Acta, Volume 147, Page 167 (1)
985)).

The biotinyl non-specific rabbit IgG of biotinyl Sagi IG was prepared by a known reaction between maleimide non-specific rabbit IgG and N-biotinyl-2-mercaptoethylamine [Kono et al., Journal Clinical
Laboratory Analysis (J, Cl1n, Lab
, Anal, Vol. 2, p. 19 (1988)).

Isako: 2 navy blue IuJ Shi14 rabbit anti-angiotensin II IgGtl solution (oral, 1 g/
l) or biotinyl non-specific rabbit IgG solution (0.1 g
A polystyrene ball [diameter 3.2 mm (Precidine Plastic Ball Co., Ltd., Chicago)] was coated using a known method [Ishikawa et al., Scandinavian Journal of Immunology (Scand.

J. Ismunol, Volume 8 (Supplement 7), Page 43 (
Rabbit anti-angiotensin I or biotinyl non-specific rabbit [gG] insolubilized polystyrene balls were prepared by physical adsorption (1978).

Streptavidin solution (0.1 g/j) was added to a biotinyl non-specific rabbit IgG insolubilized polystyrene ball at 30°C.
The mixture was reacted for 4 hours to prepare streptavidin-insolubilized polystyrene balls.

1 d of blood was collected from a healthy individual into an ice-cold test tube containing 10.5 μm of disodium ethylenediaminetetraacetate (EDTA) and centrifuged at 4°C to prepare plasma. Before the measurement, plasma was pre-injected with 4-8 hydroxyquinoline, an angiotensin I-converting enzyme inhibitor.
．． After incubation for 20 minutes under water cooling with an equal volume of 16+vM phosphate buffer, pH 4,6, O containing 4g MEDTA was added.
, l M sodium carbonate buffer, pH 9.1 at 12.5
Diluted twice.

■! Dissolve 0.56 ml of Angiotensin I manufactured by Okazayo in 0.5 m of distilled water, and add 0.165 M of 8-hydroxyquinoline, 4
Standard solutions of various concentrations were prepared by diluting with 0,1 M sodium carbonate buffer, ρ 8.5, containing mMEDTA and 1 g/It bovine serum albumin.

50μ2 of angioncin'-plasma or standard solution was added to 88-H sulfosuccinimidyl-6-(biotinamido)hexanoate (sul
fosuccinimidyl-6-(biotina
aido) hexan.

ate) in dimethylformamide solution containing 3μ2 and 30
Incubated at °C for 11 hours. Then, add 1M glycine-sodium hydroxide, pH 8, 5, 7 μl and 30°C.
Incubate for 1 hour and add 10exM sodium phosphate W street solution, pH 6,0,90ul containing 1 g/l bovine serum albumin, 0.1 M sodium chloride, Ig/2 sodium azide mMEDTA! was added.

150μ2 of the above reaction solution and rabbit anti-angiotensin■
Incubate with insolubilized polystyrene balls for 20"C1 night. After incubation, remove the reaction solution and add O,L
After washing twice with 10mM sodium phosphate buffer containing M sodium chloride, p) 17.0.2d, buffer Al00μ! Add IM salt #20〃l and heat at 30℃ for 1
Incubated for hours.

Remove the polystyrene balls and add 1M sodium phosphate buffer, p117.0.10μ! and 0.9M sodium hydroxide 20μ! It was neutralized. Then, buffer A20 containing affinity-purified heron anti-angiotensin IFab'-peroxidase (50 or ■ ol)! Ij! and incubate at 20°C for 3 hours and at 4°C - night,
Two more streptavidin-insolubilized polystyrene balls were added and incubated at 20°C for 4 hours with shaking.

The reaction solution was removed, and the polystyrene ball was washed in the same manner as above. Then, the peroxidase activity bound to the polystyrene balls was reduced to 3-(4-hydroxyphenyl).
Probionin quacid as a substrate, 30°C, l#r
Fluorescence optical measurements were carried out using the known method of BI [Kanaso et al.
Analytical Letters Volume 16 (B19), No. 15
Page 09 (19B3)).

The fluorescence intensity was measured using 0.2/1 quinine dissolved in 0.05 M sulfuric acid as a standard.

The measurement results of the standard solution are shown in Figure 1. The measurement limit is 1:1
g (10 anal), and it was possible to measure up to 6.5 ng/R using 2 still plasma.

Two types of plasma (angiotensin I concentration 56 and 1102n
/ 1) Three concentrations of angiotensin! (65～
As a result of measuring the spike recovery rate using plasma to which 6,400) was added, the spike recovery rate was 85 to 104%.

As a result of measuring the measurement circle variation coefficient at 13 concentrations ranging from 56 to 5.558 ng/l, the measurement circle variation coefficient was 1.8 to 1.
0% (n=5).

The measurement limit is 80 times lower concentration than conventional competitive methods.

Example 2 Buffer, IgG, F (ab'), , Fab'
Preparation of Kashinin-Sepharose 4B, preparation of affinity-purified Cyaninin Fab''-peroxidase, and preparation of biotinyl non-specific rabbit 1gG steel and protein-insolubilized solid phase were carried out in the same manner as in Example 1. Ta.

The kashinin-bovine serum albumin conjugate of rabbit kashinin was combined with S-acetylmercaptosuccinic anhydride and m-maleimidobenzoyl-
It was prepared by a known method using N-hydroxysuccinimide ester.

Rabbits were immunized with the Kashinin-bovine serum albumin conjugate by a known method using complete and incomplete Freund's adjuvant to prepare rabbit anti-Kashinin antiserum.

7 days of blood was collected from a rat in a water-cooled test tube containing 10.5μ1g of disodium ethylenediaminetetraacetate (EDTA) and centrifuged at 4'C to prepare plasma. Before measurement, 2 mL of 0.1 M sodium phosphate buffer, pH 7.5, containing 4 mM EDTA was added.
Diluted 5 times.

■Dissolve Kashinin 1.0■ manufactured by Junkimushisei in distilled water O45, d, and make 4mM
0,1 containing EDTA and 1g/l bovine serum albumin
Standard solutions of various concentrations were prepared by diluting with M sodium phosphate buffer, pH 7.5.

50μ2 of Zuohuango Ihuo Teiding plasma or standard solution was mixed with 53-1 sulfosuccinimidyl-6-(biotinamide) hexanoate (suI).
fosuccinlmldyl-6-(bio tj
The mixture was incubated with 3μ2 of a dimethylformamide solution containing naa+1do) hexanoa to) for 1 hour at 30°C. It was then incubated with 1M glycine-sodium hydroxide, PH8.
Sodium chloride, Ig/l sodium azide mM
105M sodium phosphate buffer containing EDTA, pH
6.0.90ttl was added.

150 .mu.i of the above reaction solution was incubated with rabbit anti-casinin insolubilized polystyrene balls at 20.degree. C. for 1 night.

After incubation, remove the reaction solution and add 10-M sodium phosphate buffer containing 0.1 M sodium chloride, pH 7.
After washing twice with ,0,2W1, buffer A100μ
2 and 20μ2 of eye salt M2 were added and incubated at 30°C for 1 hour.

The polystyrene balls were removed and neutralized with 20 μl of 1M sodium phosphate buffer, pH 7.0, 1077ff and 0.9M sodium hydroxide. Thereafter, affinity-purified Eurasian antisynin Fab'-peroxidase (5
Buffer A containing 0 fmol) 20 μj! and 20℃
, incubate for 3 hours and 4°C 1-night, then incubate for 2 more hours.
Streptavidin-insolubilized polystyrene balls were added and incubated under shaking for 20"C for 14 hours.

The reaction solution was removed, and the polystyrene ball was washed in the same manner as above. Thereafter, the peroxidase activity bound to the polystyrene balls was measured optically by a known method using 3-(4-hydroxyphenyl)propionic acid as a substrate at 30° C. for 11 hours. (Kankan et al., Analytical Letters [Vol. 16 (B19), No. 150]
9 (1983)).

Fluorescence intensity was determined by 0.2IRg dissolved in 0.05 M sulfuric acid.
#! Measurements were made using quinine as a standard.

The measurement results of the standard solution are shown in FIG. Measurement limit is 130f
g (100asol), 21! It was possible to measure up to 65 ng/f using plasma.

Three concentrations of Kashinin (650-19,500ng/
As a result of measuring the speech power a recovery rate using plasma to which 1) was added, the addition recovery rate was 101 to 119%. 840
As a result of measuring the measurement circle variation coefficient at six concentrations of ~26.700 ng/Il, the measurement circle variation coefficient was 7.9-13% (
n=5).

Comparative Example I 11 hills 0.15? I sodium chloride, 0.1% gelatin and 0
．． A 0.05 M sodium phosphate buffer containing 0.02% sodium azide, pH 7.0, was prepared and designated as buffer B.

Rabbit Kashinin ′ The same antiserum as in Example 2 was used.

5r-tyrosine The N-terminally tyrosinated kashinin of kashinin was labeled by a known method using Na"' I and chloramine T, and +zsl-
Tyrosinated kashinin was prepared.

The specific radioactivity of 1tsl-tyrosinated kashinin is 11μC4
/μg.

I quasi-kaihuangai I Kashinin 0.5■ Q, I M borate buffer, pH 8
, 5, 1d and diluted with buffer B to prepare standard solutions of various concentrations.

A solution of rabbit anti-kashinin antiserum prepared by diluting Kashinin's standard solution 100/7ε with buffer B 200 uffi, +15
1-Tyrosinated Kashinin Buffer IBt8i 100ul
- (10,000 cps) and 8200 μl of buffer at 4° C. for 18 hours.

Then 0.25% Dextran T-70 and 2.5%
200μ of buffer B suspension containing activated carbon (Noritz) A)
2 was added and left at 4°C for 15 minutes, then heated to 4°C1300
Centrifuged at 0 rpm for 20 minutes. The radioactivity of the supernatant was
Measured using a counter.

The measurement results of the standard solution are shown in FIG. Measurement limit is 100p
g (75fs+ol).

The measurement limit of Example 2 according to the present invention is 750 times lower than that of Comparative Example 1 using a conventional competitive method.

Example 3 As a common buffer, 0.01 M sodium phosphate buffer containing 0.1 M NaCl, Ig/l bovine serum albumin, pH 7.0, (buffer A) and 0.1 M
NaC1,Ig/j! Bovine serum albumin and 1 M
0.1 M sodium phosphate buffer containing EDTA,
A pH of 1.0 (buffer C) was used.

The anti-arginine vasopressin antiserum of the heron nimbasishin is arginine-vasopressin (
A conjugate of Peptide Institinito 1nc, + Osaka) and bovine thyroglobulin was subcutaneously administered 8 times at 2-3 week intervals to sensitize male New Sealant white rabbits.

Arginine vasopressin (5 g) was prepared using the carbodiimide method [Skowski et al., Journal of Laboratory Medicine].
・And Clinical Medicine (J, Lab,
Cl1n, Med, ), vol. 80, 134-14
4 (1972)), ■-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (Calbiochem Behring, U.S., California, U.S. 8)
using 50I1 g of bovine thyroglobulin (Sigma Chemical Co., St. Louis MO, USA).
was combined with The conjugate was administered as a 0.4 μ/d saline solution emulsified with an equal volume of Freund's complete adjuvant (Miles Laboratories, Inc., Elkhart, Indiana). The dose of conjugate was 0°2/4 rabbits. Thirteen days after the final sensitization, blood was collected and the antiserum was stored at -20°C.

IgG, F (ab')z and Fab were prepared in the same manner as in Example 1, and their amounts were determined from the absorbance at 280n-.

Arginine Vasopressin Syn-Sepharose 4B Book 1 Arginine Vasopressin (0,61■) was prepared according to the Pharmacia manual and activated CH-Sepharose 4B (
0.17g, Pharmacia Fine Chemicals A
B, Uppsala, Sweden).

Anti-arginine vasopressin Fab' was prepared by a known method [Shide et al., Journal of Applied Biochemistry (cited above)].
Horseradish peroxidase (Grade I, RZ
=3.0. Boehringer Mannheim GmbH, Mannheim, FRG).

Rabbit anti-vasopressin Fab'-peroxidase was affinity-purified using an arginine vasopressin-Sepha 0-S 4B column and elution at pH 2.5 using a known method (Rouen et al., Clinical Biochemistry Acta, supra).

In the same manner as in Example I, polystyrene balls (3, 2
m diameter, Immuno Chemical, Okayama 1 Japan) were coated with rabbit anti-arginine vasopressin IgG (0.1 g/j). Preparation of biotinyl non-specific rabbit IgG, biotinyl non-specific rabbit IgG polystyrene balls and streptavidin-insolubilized polystyrene balls. was based on Example 1. These protein-insolubilized polystyrene balls were
It was stored at 4°C in II buffer A containing g/l NaN.

Samples and luginine vasopressin urine samples are from 22 to
Sampled from the first morning excrement of a 35-year-old healthy person.
Diluted 20 times with buffer C.

Arginine vasopressin (0°61■) is 0.5d
of 0.2 M acetic acid and diluted with 1N solution C.

Luginin Bapre Shin's Biotinylation of samples was performed by two different methods.

■ Direct biotinylation □ Add 100 μl of diluted arginine vasopressin solution or diluted urine to 10 μl of 66M
MN-hydroxysuccinimide biotin (Zy*ed
Lab, Inc. + San Francisco.

California) / dimethyl sulfoxide (DMS)
O) Incubate with solution for 1 hour at 20°C. After incubation, the reaction solution was incubated with 10 μl of 1 M glycine-NaOH buffer, pH 7.0, for 30 min at 20° C., followed by 30 μl containing 5 g/2 NaN. of Buffer C was added.

■ Indirect biotinylation □ 100μ2 of diluted arginine vasopressin solution or diluted urine with 5μ2 of 63M
N-succinimidyl-6-maleimidohexanoate (
Dojindo, Kumamoto) / dimethyl oxide solution for 20'
C, incubated for 1 hour. After incubation, the reaction solution was incubated with 5μ2 of 99mM reduced glutathione (Dojindo, Kumamoto) in 10.1M sodium phosphate buffer, pH 7.0, at 20°C for 1 hour, and then with 5μ2 of 138mM N-hydroxysaccharide. It was incubated with cinimidobiotin/DMSO solution at 20°C for 1 hour. Finally, 5μ2 of 2M glycine-
NaOH buffer, PII 7.0 was added and incubation was carried out at 20°C for 30 minutes. After that, 5g/j! 30μ2 of buffer C containing NaN was added. The outline is as shown in Part 2 below.

Margin below C] The biotinylated test solution (150 μl) was incubated with two anti-arginine vasopressin IgG insolubilized polystyrene balls at 4° C. overnight.

Then, the polystyrene ball was 2d of 0.1M NaC2
10mM sodium phosphate buffer, pH 7.0, was added and removed for 9 minutes, washed twice, and incubated at 4°C for 1 hour with a mixture of buffer Al 001Ij2 containing 1mM EDTA and 20u2 of 1M hydrochloric acid.

After removing the polystyrene ball, add 10 μl to the remaining solution (7)
LM sodium phosphate IL buffer, pH 7.0 and 20tI
j! of LM NaOH was added to neutralize. The neutralized mixture was purified with an affinity-purified anti-arginine vasopressin Fab'-peroxidase conjugate (200f-ol
) and non-specific rabbit F (ab'L (0,1■
) with 20 .mu.l of u1 buffer A containing 4.degree. C. overnight. Thereafter, two streptavidin-insolubilized polystyrene balls were added and incubated at 4'C for 5 hours. After removing the 0 reaction solution, the polystyrene balls were washed twice as described above, and the peroxidase activity bound to the polystyrene balls was determined in Example 1. Similarly, using 3-(4-hydroxyphenyl)propionic acid as the substrate, 30
'C, Measured for 60 minutes. The fluorescence intensity was 0.2 μ/l quinine 150-M sulfuric acid solution as standard, and the excitation wavelength was 320 nm.
(At an emission wavelength of 4050-
10, Shimadzu Corporation, Kyoto).

The detection limit for arginine vasopressin is the minimum that produces a significant (knee test, p<0.001. N=5) increase in activity relative to nonspecifically adsorbed peroxidase activity (background) in the absence of arginine vasopressin. of arginine vasopressin.

■ When directly biotinylated, the detection limit for arginine vasopressin is 54 fg (50aa*o!/1u
be) (Figure 4, white circles).

■ In the case of indirect biotinylation, the detection limit of arginine vasopressin is 11 fg (10 amol/1 ub
e) (Fig. 4, black circles).

This is a competitive radioimmunoassay [Morton et al., Journal of Endocrinology (J, E
ndocrl, nol, ), 65S, 411-4
24 (1975)), [Grenzer et al.
Acta Hndocrinol
oglca), vol. 106, pp. 317-329 (198
4)) Or competitive enzyme immunoassay [Uno et al.

Experimentia +3
8, pp. 786-787 (1982)), which is 23 to 400 times lower. The measurement range of urinary arginine vasopressin was 2-2.000 ng/l in a 5 μl urine sample.

Nine concentrations of arginine vasopressin (6,6-63
．． When two concentrations (20 and 80 ng/j) of arginine vasopressin were added to a urine sample (5 μN) containing 5 ng/2), the recovery rate of arginine vasopressin added to the urine sample was 86.4 ± 7. .6% (average believer SD, 77-101%; n-18).

6.6-117.3ng/j! Measurement accuracy was determined for urine samples with and without arginine vasopressin at 14 different concentrations. The measurement circle variation coefficient is 3.0~
It was 12.6% (n-5).

In the case of indirect biotinylation, steric inhibition that may occur due to biotin binding in close proximity to the epitope site of the peptide could be avoided, and the detection limit was 5 times lower than that of the direct method. This shows that habten having an amino group can also be measured with high sensitivity by the method of the present invention.

〔effect〕

As explained above, the present invention makes it possible to measure antigens with high sensitivity by utilizing the characteristics of the sandwich method using only antibodies against fall antigen sites, and also to measure antigens that could not be measured using the conventional sandwich method. Even in low molecular antigens,
Like the Sand Inch method, it can provide measurements with higher sensitivity than conventional competing methods.

[Brief explanation of the drawing]

FIGS. 1, 2, and 4 are calibration curves of an example of antigen measurement according to the method of the present invention. FIG. 3 is a sample of a comparative example of antigen measurement using a conventional method. Figure 1 Anzo large tesojino 1 Reika [wrapping L/+ Yunie] Figure 2 Power - 5 mouthfuls of nnisoji! (αynoL/7 child knees Figure 3

Claims (1)

[Scope of Claims] 1. A method for measuring an antigenic substance, which includes the following steps (A), (B), (C), and (D). Step (A): A step of binding a functional group or label to the antigen to be measured in the test liquid to form a modified antigen. Step (B): A step of binding the modified antigen to the carrier via an antibody against the antigen, and then separating the carrier and the test liquid. Step (C): the next (a) or (b) step. (a) A step of dissociating the modified antigen from the carrier. (b) A step of dissociating a modified antigen-antibody complex composed of a modified antigen and an antibody against the antigen from the carrier. Step (D): A step of measuring the modified antigen or modified antigen-antibody complex described in (C).