JP2606722B2 - Ultrasensitive antigenic substance measurement method - Google Patents

Description

The present invention relates to an ultrasensitive method for measuring an antigenic substance.

[Conventional technology]

Measurement of biological components, particularly antigenic substances in human body fluids, is of great clinical importance. The measurement of antigen is widely used for tests such as endocrine test by measuring hormone, diagnosis of cancer, diagnosis of infectious disease and the like.

Heretofore, the trace amount measurement of an antigenic substance as described above has been performed by immunological measurement. In recent years, a method using a carrier has been widely used in immunoassays. ELISA
, A sandwich-type assay such as the IRAM method, and a competitive-type assay such as the second antibody solid phase method.

Conventionally, a sandwich-type antigen measurement method traps an antigen in a test solution on a carrier in which antibodies are insolubilized,
There is a method of measuring this using a labeled antibody against the antigen.

[Problems to be solved by the invention]

In such a measurement method, in any case, the measurement sensitivity is regulated by the background derived from the label component non-specifically bound to the carrier, and there is a limit to high-sensitivity measurement.

The object of the present inventor is to provide a highly sensitive measuring method which has never been available under such circumstances.

[Means for solving the problem]

In general, the present invention relates to a method for measuring an antigenic substance, and comprises the following steps (A), (B), (C) and (D): Step (A): Or (b) process.

(A) After forming a complex composed of the antigen substance to be measured in the test liquid and one or more active ingredients,
A step of binding the complex to a carrier. Here, (1) one or more functional groups are bound to at least one active ingredient, and the active ingredient can bind to the antigenic substance to be measured, and the functional group bound to the active ingredient Can bind to a reactive group bound to the carrier, and (2) a reactive group is bound to the carrier, and the reactive group can bind to a functional group bound to the active ingredient.

(B) a reaction for forming a complex composed of the antigen substance to be measured in the test solution and one or more active ingredients, and a reaction between a functional group of the active ingredient and a reactive group of the carrier. A step performed on a carrier to form a complex of the complex and the carrier. Here, (1) one or more functional groups are bound to at least one active ingredient, and the active ingredient can bind to the antigenic substance to be measured, and the functional group bound to the active ingredient Can bind to a reactive group bound to the carrier, and (2) a reactive group is bound to the carrier, and the reactive group can bind to a functional group bound to the active ingredient.

Step (B): a step of dissociating the complex from the carrier after washing the carrier to which the complex is bound.

Step (C): a step of washing the carrier after binding the complex to another carrier. Here, the carrier has a reactive group capable of binding to the complex.

Step (D): a step of measuring the complex on the carrier according to (C).

It is characterized by including.

 Hereinafter, the present invention will be described in the order of steps.

Step (A) As the test liquid, for example, serum, plasma, cerebrospinal fluid, saliva,
Examples include body fluids such as urine and buffers. As the antigenic substance to be measured, all substances having an antigenic site as an antigen, and virtually all substances which can be measured by a conventional immunological assay can be measured. Examples include enzymes such as γ-glutamyl transpeptidase (γ-GTP), alkaline phosphatase, glycosyltransferase, thyroid stimulating hormone (TS
H), luteinizing hormone (LH), placental gonadotropin (hCG), insulin, secretin, growth hormone (G
H), proteinaceous hormones, fibrin degradation products (FD
P), C-reactive protein (CRP), α ₁ - acid glycoprotein _{(α 1 -AGP), α 1} - antitrypsin (alpha ₁ -A
T), α ₂ - plasmin inhibitor (α ₂ -PI), β
₂ - microglobulin (beta ₂ -MG), plasma proteins such as immunoglobulins, alpha-fetoprotein (AFP), carcinoembryonic protein (CEA), oncofetal protein such as fetal ferritin, lymphocytes, viral microorganisms And haptens such as digoxin, thyroxine, triiodotyrosine, cortisol, and prostaglandin.
These antigens can be measured not only in a free state but also in a state of being bound to an immune complex or a binding protein in a test solution.

 The active ingredient is 1 or 1 and 2 below.

1. Antibodies. The antibody referred to here may be any of a monoclonal antibody and a polyclonal antibody obtained by immunizing the antigen to be measured or a substance having the same antigen recognition site as the antigen by a known method.

2. A component that causes an antigen-antibody reaction with the above antibody.

(For example, antigens, anti-antibodies, etc.) These active ingredients are usually one of the functional groups involved in binding the complex to the carrier in step (A) and / or step (C).
Species or a combination of two or more species may be used.

Here, as the functional group, for example, hapten such as dinitrophenyl group or trinitrophenyl group, biotin,
Antibodies or antigens other than the antigen to be measured and the corresponding antibody bound via —S—S— bonds, the hapten or the biotin, and the like.

Preferably, the functional group is a component in the test solution which does not inhibit the binding to the carrier in the step (A), does not easily dissociate in the washing in the step (B), and facilitates dissociation during dissociation. The functional group involved in the bonding in the step (C) is preferably a functional group capable of efficiently bonding to another carrier from the solution dissociated in the dissociating step (B).

The measurement of the complex in the step (D) may be performed by a conventional method, and a preferable method is a method using a label.

When a label is used, it is preferable to bind the label to at least one of the active ingredients, and the time of the label introduction is, as in the conventional method of labeling, before the measurement of the complex in step (D). It may be at any time before or during the steps (A) to (D).

The label may be any substance used for measurement in immunological measurement, such as enzymes, radioactive substances, luminescent substances,
Examples include a fluorescent substance and a metal compound.

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

These functional groups and labels may be bound to the active ingredient via a carrier that does not affect the steps (A) to (D). Such intervention is particularly preferred when the active ingredient is a small molecule. Examples of the carrier include non-specific rabbit IgG, bovine serum albumin, dextran and the like.

As a method for binding a label, any binding method for binding a label to an antibody or an antigen in a conventional immunoassay may be used.

The formation of a complex composed of an antigen and an active ingredient is performed by adding one or more active ingredients to a test solution. 2
Preferably, more than one active ingredient is used, and the active ingredient that binds the functional group and the active ingredient that binds the label are used separately.

The formation of the complex is performed under the conditions used for a normal antigen-antibody reaction. Generally 0 to 45 ° C, several hours to several tens of hours,
Preferably, it is formed at 20 to 37 ° C. for 1 to 6 hours. The complex thus formed is bound to a carrier.

As the carrier, all those used in conventional immunological assay methods can be used. For example, polystyrene,
Polyacryl, Teflon, paper, glass, agarose and the like can be mentioned. Further, the shape may be any shape.

The carrier must have a reactive group for binding the complex formed in step (A) or for forming the complex on the carrier.

Any reactive group that binds to the carrier is used as the reactive group, as long as it binds to an antigen to be measured, an active ingredient, a functional group, a label, or an immunologically active site newly generated by complex formation. .

As the reactive group, a reactive group capable of easily dissociating the complex in step (B) is preferable.

Examples of such a reactive group include ordinary ones corresponding to the functional groups. For example, 1) when the functional group is a hapten such as a dinitrophenyl group or a trinitrophenyl group, an antibody against these is used.

2) When the functional group is biotin, avidin or streptavidin can be used.

3) When the functional group is an antigen via an -S-S- bond or an antibody, the corresponding antibody or antigen can be mentioned.

The binding of the reactive group to the carrier is performed by a known method for preparing a carrier in an immunological assay.

For the binding of the complex to the carrier, conditions usually used for the immunological measurement using the carrier are employed.

It is desirable to simultaneously add the active ingredient and the carrier to the test solution to form the complex on the carrier, because the process can be simplified.

Regarding Step (B) The washing is carried out under conditions usually used for immunological measurement using a carrier.

The dissociation of the complex is preferably performed without decomposing the complex. When the binding between the complex and the carrier is due to the antigen-antibody reaction,
By making the binding constant of the antigen-antibody reaction smaller than the binding constant of the complex formation, the complex can be dissociated with an acid, an alkali, a high-concentration inorganic salt, or the like.

A more preferred method of dissociating the complex from the carrier without decomposing it is to add a substance having the same reaction site as the functional group involved in the binding between the complex and the carrier.

For example, when the functional group is dinitrophenyl, a dinitrophenyl amino acid (eg, dinitrophenyl lysine),
When the functional group is biotin, it is biotin, and when the functional group is an antigen, antibody, hapten, biotin or the like bonded via -SS-,-
A reagent that cleaves SS is used.

Step (C) The carrier used in step (A) is used.

Any reactive group that binds to the carrier is used as the reactive group, as long as it binds to an antigen to be measured, an active ingredient, a functional group, a label, or an immunologically active site newly generated by complex formation. .

Preferred reactive groups are, in addition to the reactive groups shown in step (A), a component to be measured, an active component, a label or
A reactive group that binds to an immunoactive site newly generated by complex formation is exemplified.

The component to be measured in the complex and the reactive group that binds to the active component in the antigen-antibody reaction are particularly preferable because the introduction of one functional group can be omitted.

When selecting the same reactive group as the reactive group used in the step (A), it is necessary to separate the complex from the solution dissociated in the step (B). It is preferable to use a reactive group different from that in the step (A). In this case, the dissociating step (B) and the step of binding to the carrier (C) can be performed simultaneously.

For the binding of the complex to the carrier, the conditions usually used for the above-mentioned immunoassay using the carrier are employed as in the step (A).

The above steps (B) and (C) may be repeated as necessary.

Step (D) In order to measure the complex on the carrier, there is a method of measuring the label introduced into the active ingredient described in the step (A).

As described above, according to the present invention, an antigenic substance that can be measured by a conventional immunological assay can be measured with higher sensitivity than before.

When performing the antigen measurement, it is possible to use a combination of complexes according to the purpose as described below.

1. At the time of the sandwich type measurement, antibodies obtained from two kinds of animals capable of sandwiching are used as active ingredients.
A carrier having a functional group bound to one side and a label bound to the other to form a complex, bound to the carrier by the functional group in step (A), and an anti-antibody bound to the functional group-bound antibody in step (C) Is used to reduce the non-specific binding of the labeled antibody to the carrier, and the label in the complex on the carrier is measured in step (D).

2. When two kinds of antibodies obtained from the same kind of animal are used as active ingredients, two kinds of functional groups are bound to one side and a label is bound to the other side to form a complex, and the functional group is formed in step (A). By using a carrier capable of binding the other functional group in step (C), non-specific binding of the labeled antibody to the carrier is reduced, and the step (D) The label of the complex is measured.

3. In the case of competitive measurement, an antigen and an antibody are used as active ingredients. A label is bound to the antigen, a functional group is bound to the antibody, and a complex is formed. In step (A), the carrier is bound to the carrier by a functional group, and in step (C), the carrier to which the anti-antibody to the antibody is bound is bound. This reduces non-specific binding of the labeled antigen to the carrier, and measures the label in the complex on the carrier in step (D).

Hereinafter, the present invention will be described with reference to Examples, but the present invention is not limited thereto.

〔Example〕

Example 1 Buffer 0.01 M sodium phosphate buffer containing 0.1 M NaCl, 1 mM MgCl ₂ , 0.1% bovine serum albumin and 0.1% NaN ₃ , pH 7.
0 was prepared and used as buffer A.

hTSH standard product The standard product of hTSH kit "Daiichi" (Daiichi Radioisotope, Tokyo) was used.

Preparation of IgG, F (ab ') ₂ , Fab' IgG was prepared by salting out with sodium sulfate and DEAE cellulose, and F (ab ') ₂ was prepared by digesting IgG with pepsin to produce Fab'.
By reduction of F (ab ') _2, each known method [Ishikawa et al., Journal of Immunoassay (J.Immunoas
say) Volume 4, page 209 (1983)].

Dinitrophenyl-monoclonal mouse (anti-hTSHβ-
Subunit) Preparation 1. mercapto succinyl of IgG ₁ - monoclonal mouse (anti hT
Preparation of SHβ-subunit) IgG ₁ Monoclonal mouse (anti-hTSHβ-subunit) Ig
A thiol group was introduced into G ₁ , (Mallink Clot, St. Louis, Mo.) by a known method using S-acetylmercaptosuccinic anhydride (Ishikawa et al., Journal of Immunoassay, supra).
The number of thiol groups introduced per molecule was 10.5.

2.Preparation of maleimide-dinitrophenyl-L-lysine 0.1 mM sodium phosphate buffer containing 5.5 mM dinitrophenyl-L-lysine, 5 mM EDTA, pH 7.0, 0.9 ml and 5 mM N
0.10 ml of N, N-dimethylformamide containing succinimidyl-6-maleimidohexanoate (supra)
The reaction was performed at 30 ° C. for 30 minutes.

3. Dinitrophenyl-monoclonal mouse (anti-hTSHβ
- subunit) Preparation mercapto succinyl of IgG ₁ - monoclonal mouse (anti hT
(SHβ-subunit) IgG ₁ 0.1 mg sodium phosphate buffer solution containing 0.1 mg of 5 mM EDTA, pH 6.0, 4.5 ml, and maleimide-dinitrophenyl-L-lysine solution 0.5 ml
The reaction was performed at 0 ° C. for 30 minutes. The reaction solution was treated with Sephadex G-25.
Gel filtration was performed using a column. Column size is 1.0 × 30c
m, eluent used was 0.1 M sodium phosphate buffer, pH 7.0. The number of monoclonal mouse (anti hTSHβ- subunit) IgG ₁ 1 introduced dinitrophenyl groups per molecule was 7.2 units.

Preparation of dinitrophenyl-bovine serum albumin Bovine serum albumin (fraction V, Armor,
(Kanky Key, Illinois) in the same manner as above.

The number of dinitrophenyl groups introduced per bovine serum albumin molecule was 7.0.

Preparation of Protein-Sepharose 4B hCG (2.5 mg), dinitrophenyl-bovine serum albulin (10 mg) and non-specific mouse IgG (20 mg) were prepared according to the Pharmacia's instructions, CNBr-activated Sepharose 4B (1 mg).
g).

Affinity purification of IgG Rabbit (anti-hCG) IgG (Dakopatz, Glostrup,
Rabbit (anti-hCG) F (a)
b ') ₂ , rabbit (anti-dinitrophenyl-bovine serum albumin) IgG (Miles, Elkurt, IN) and rabbit (anti-mouse IgG) IgG (National Institute of Medical Biology, Nagoya) are hCG and dinitrophenyl-bovine, respectively. Serum albumin and non-specific mouse IgG insolubilized Sepharose 4
A known method using a B column and eluting at pH 2.5 (Kono et al.,
Journal of Biochemistry (J. Biochem.)
100, p. 1247 (1986)].

Measurement of β-D-galactosidase activity β-D-galactosidase activity was determined using 4-methylumbelliferyl β-D-galactoside as a substrate.
After reaction at 16 ° C. for 16 hours, fluorescence was measured by a known method (Imakawa et al., Anals Clin. Biochem., Vol. 21, p. 310 (1984)). And a 0.1 M glycine-NaOH buffer in which 10 ^-8 M 4-methylumbelliferone was dissolved, pH 10.3, as a standard.

Preparation of affinity-purified rabbit anti- (hCG) Fab'-β-D-galactosidase Affinity-purified rabbit anti- (hCG) Fab '
-O-phenylenedimaleimide as a crosslinking agent in a known manner [Ishikawa et al., Scandinavian Journal of
Immunology (Scand.J.Immunol.) Vol. 8 (Supplement 7) No. 43
Page (1978)] labeled with β-D-galactosidase. β-
Fab'2.1 pieces bound to one molecule of D-galactosidase.

Buffer A containing 0.8 mg of Fab'-β-D-galactidase
0.5 ml was passed through buffer A non-specific mouse serum protein-Sepharose 4B column. The amount of the labeled antibody was calculated from the enzyme activity.

Preparation of affinity-purified rabbit (anti-dinitrophenyl-bovine serum albumin) IgG, affinity-purified rabbit (anti-mouse IgG) IgG-insolubilized solid phase Affinity-purified rabbit (anti-dinitrophenyl-bovine serum albumin) IgG solution (0.1 g / 1) or affinity Using a purified rabbit (anti-mouse IgG) IgG solution (0.1 g / 1), a polystyrene ball [3.2 mm in diameter (Precision
Plastic Ball Co., Chicago)] on the surface in a known manner [Ishikawa et al., Scandinavian Journal of
Immunology (supra)] insolubilized by physical adsorption.

Measurement of hTSH Affinity purified rabbit anti- (hCG) Fab'-β-D-
To 0.01 ml of buffer A containing 200 fmol of galactosidase, 0.01 ml of buffer A containing 0.1 mg of non-specific mouse IgG was added, and the mixture was added at 20 ° C.
Left for hours.

Separately, dinitrophenyl-monoclonal mouse (anti-hT
SHβ- subunit) buffer A0.02m containing IgG _1, 150fmol
0.02 ml of buffer A containing 0.1 mg of non-specific rabbit F (ab ') _{2 in} l
Was added and left at 20 ° C. for 2 hours.

Thereafter, the two solutions were mixed and reacted at 20 ° C. overnight with 0.09 ml of buffer A in which various concentrations of hTSH standard were dissolved. After the reaction, two affinity-purified rabbit (anti-dinitrophenyl-bovine serum albumin) IgG insolubilized polystyrene balls were added, and the mixture was reacted at 20 ° C. for 4 hours.

After removing the reaction solution, the polystyrene ball is
And washed twice.

1 mM dinitrophenyl-L-lysine, non-specific rabbit F
0.15 ml of buffer A containing 0.1 mg of (ab ') _{2 and two} affinity-purified rabbit (anti-mouse IgG) IgG-insolubilized polystyrene balls were added and reacted at 20 ° C. for 4 hours.

After washing the affinity-purified rabbit (anti-mouse IgG) IgG-insolubilized polystyrene ball in the same manner as described above, the β-D-galactosidase activity bound to the polystyrene ball was increased by 30%.
The reaction was measured at 16 ° C. for 16 hours. The results are shown in FIG. 1 together with Comparative Example 1 described later. That is, FIG. 1 shows the results of the present invention and the conventional example in the relationship between the hTSH concentration (nU / tube, horizontal axis) and the fluorescence intensity (vertical axis) corresponding to specifically bound β-D-galactosidase. It is a graph shown.

Comparative Example-1 Buffer, hTSH standard, β-D-galactosidase activity measurement, affinity purified rabbit (anti-hCG) Fab'-β
Purification of -D- galactosidase, monoclonal mouse (anti hTSHβ- subunit) Preparation of IgG ₁ insolubilized polystyrene balls were in accordance with Example 1.

Measurement of hTSH Monoclonal mouse anti- (hTSHβ-subunit) Ig
G ₁ insolubilized polystyrene balls including standard hTSH buffer A0.1
The mixture was reacted with 5 ml at 20 ° C. overnight.

After removing the reaction solution, the polystyrene balls were washed twice with 2 ml of buffer A, and then affinity-purified rabbits (anti-hC
G) 0.15 ml of buffer A containing 200 fmol of Fab'-β-D-galactosidase and 0.1 mg of non-specific rabbit F (ab ') ₂ and 20
It was made to react at 4 degreeC for 4 hours.

After washing the polystyrene ball at the same time as described above, the β-D-galactosidase activity bound to the polystyrene ball was measured.
It was measured after reacting at 30 ° C. for 1 hour. The results are shown in FIG.

Example 2 Buffer, Preparation of IgG, F (ab ') ₂ , Fab', Preparation of Protein-Sepharose 4B, Affinity Purification of IgG, Affinity Purification (anti-dinitrophenyl-bovine serum albumin) IgG insolubilized solid phase, Preparation of the affinity-purified rabbit (anti-mouse IgG) IgG insolubilized solid phase followed the method of Example-1.

hGH standard product The standard product of hGH RIA kit (Dynabot, Tokyo) was used.

Dinitrophenyl-monoclonal mouse (anti-hGH) IgG
Preparation was performed according to the method of Example-1. Monoclonal mouse (anti-hGH) introduced dinitrophenyl group ₁ per molecule IgG was 5.7 units.

Preparation of rabbit (anti-hGH) Fab'-β-D-galactosidase The method of Example 1 was followed. 4.1 Fab's bound to one molecule of β-D-galactosidase.

Measurement of hGH 0.1 mg of non-specific mouse IgG was added to 0.01 ml of buffer A containing 200 fmol of purified rabbit anti- (hGH) Fab'-β-D-galactosidase.
Was added and the mixture was allowed to stand at 20 ° C. for 2 hours.

Separately, dinitrophenyl-monoclonal mice (anti-hG
Non Rabbit F in buffer A0.02ml containing H) IgG ₁ 150fmol
(Ab ') ₂ Add 0.02 ml of buffer A containing 0.1 mg, and add
Left for hours.

Thereafter, the two solutions were mixed and reacted overnight at 20 ° C. with 0.09 ml of buffer A in which various concentrations of hGH standard were dissolved. Two affinity-purified rabbit (anti-dinitrophenyl-bovine serum albumin) IgG insolubilized polystyrene balls were added to the reaction solution, and the mixture was reacted at 20 ° C. for 4 hours.

After removing the reaction solution, the polystyrene ball was placed in buffer A2.
Washed twice with ml.

1 mM dinitrophenyl-L-lysine, non-specific rabbit F
0.15 ml of buffer A containing 0.1 mg of (ab ') _{2 and two} affinity-purified rabbit (anti-mouse IgG) IgG-insolubilized polystyrene balls were added and reacted at 20 ° C for 4 hours.

The affinity-purified rabbit (anti-mouse IgG) IgG-insolubilized polystyrene balls were washed as described above, and the β-D-galactosidase activity bound to the polystyrene balls was reduced by 30%.
The reaction was measured at 16 ° C. for 16 hours. The results are shown in Comparative Examples below.
2 is shown in FIG. That is, FIG. 2 shows the results of the present invention and the conventional example in the relationship between the hGH concentration (pg / tube, horizontal axis) and the fluorescence intensity (vertical axis) corresponding to specifically bound β-D-galactosidase. It is a graph shown.

Comparative Example-2 The buffer, the activity measurement of β-D-galactosidase, and the preparation of a solid phase insolubilized with a monoclonal mouse (anti-hGH) IgG were performed in accordance with Example 1.

The hGH standard and purified rabbit (anti-hGH) Fab'-β-D-galactosidase were prepared according to the method of Example-2.

Measurement of hGH 0.15 ml of buffer A containing standard hGH was reacted with a monoclonal mouse (anti-hGH) IgG ₁ insolubilized polystyrene ball at 20 ° C. overnight.

After removing the reaction solution, the polystyrene balls were washed twice with 2 ml of buffer A, and the rabbit (anti-hGH) Fab'-β-D-
200 fmol of galactosidase and non-specific rabbit F (ab ') ₂₀ .
The mixture was reacted with 0.15 ml of buffer A containing 1 mg for 4 hours at 20 ° C.

The polystyrene ball was washed as described above, and the β-D-galactosidase activity bound to the polystyrene ball was measured.
It was measured after reacting at 30 ° C. for 1 hour.

 The results are shown in FIG.

〔The invention's effect〕

As described above, the method of the present invention can measure an antigen with ultra-high sensitivity, and thus contributes to the diagnosis of various hormone secretion abnormalities such as thyroid disease and hypopituitarism, and various infectious diseases.

[Brief description of the drawings]

1 and 2 are graphs showing β-D-galactosidase activity of the present invention and the conventional example.

Claims (4)

(57) [Claims] 1. The following (A), (B), (C) and (D)
A method for measuring an antigenic substance, comprising the steps of: Step (A): The next step (a) or (b). (A) After forming a complex composed of the antigen substance to be measured in the test liquid and one or more active ingredients,
A step of binding the complex to a carrier. Here, (1) one or more functional groups are bound to at least one active ingredient, and the active ingredient can bind to the antigenic substance to be measured, and the functional group bound to the active ingredient Can bind to a reactive group bound to the carrier, and (2) a reactive group is bound to the carrier, and the reactive group can bind to a functional group bound to the active ingredient. (B) a reaction for forming a complex composed of the antigen substance to be measured in the test solution and one or more active ingredients, and a reaction between a functional group of the active ingredient and a reactive group of the carrier. A step performed on a carrier to form a complex of the complex and the carrier. Here, (1) one or more functional groups are bound to at least one active ingredient, and the active ingredient can bind to the antigenic substance to be measured, and the functional group bound to the active ingredient Can bind to a reactive group bound to the carrier, and (2) a reactive group is bound to the carrier, and the reactive group can bind to a functional group bound to the active ingredient. Step (B): a step of dissociating the complex from the carrier after washing the carrier to which the complex is bound. Step (C): a step of washing the carrier after binding the complex to another carrier. Here, the carrier has a reactive group capable of binding to the complex. Step (D): a step of measuring the complex on the carrier according to (C). 2. The method according to claim 1, wherein at least one active ingredient is labeled. 3. In the step (B), after washing the carrier to which the complex is bound, the complex is dissociated from the carrier by adding a substance having the same reactive site as the functional group in the step (A). The method according to claim 1 or 2, wherein the method is a step. 4. The method according to claim 1, wherein the reactive group in the step (C) is
4. A reactive group different from the above.
The method according to any one of claims 1 to 4.