JPH05203652A - Antibody enzyme immunoassay - Google Patents

Abstract

PURPOSE:To enable analysis with high sensitivity and high reproducibility without causing deterioration of S/N by bringing a two-head antibody into contact with a ligand, and measuring the activity of the antibody enzyme of the two- head antibody being coupled to the ligand, the two-head antibody consisting of a moiety of an antibody for the ligand and a moiety of the antibody enzyme which can generate signals. CONSTITUTION:Antigens (ligand L) 12 in a sample are coupled to a solid-phase antibody 10 (first immunoreaction). Those antigens which are not yet coupled are removed (washing). A two-head antibody(Bs-Ab) comprising a moiety 14a of the ligand specific antibody and a moiety 14b of an enzyme antibody is added and coupled to the antigens 12 being coupled to the solid-phase antibody 10 (second immunoreaction). Those two-head antibodies 14 which are not yet coupled to the antigens 12 are removed (washing). An enzyme substrate (S) is added to detect a product (Prod) so as to measure the amount of the coupled two-head antibodies (enzyme reaction).

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an immunoassay method using an antibody enzyme, and more particularly to an antibody enzyme immunoassay method using a double-headed antibody.

[0002]

BACKGROUND OF THE INVENTION Analysis of blood, urine, etc. is very useful for diagnosing pathological conditions and determining the course of treatment, and plays an important role in the field of clinical examination. As a method of analyzing such a trace component (ligand), a method of immunologically measuring using an antibody against the trace component has been widely applied (for example, "Immunology Illustrated" translated by Tomio Tada, Nankodo,
1990, p327-339). The immune reaction is a specific and high-affinity reaction between an antigen and an antibody, and it is possible to quantify the antigen to be measured by analyzing the equilibrium state between the antibody and the antigen using a labeled antigen or a labeled antibody. it can. An enzyme immunoassay method (enzyme immunoassay; EI) uses an enzyme as the label and utilizes chemical amplification by the enzyme.
A). The enzyme immunoassay method has been actively used in recent years as a simple and highly sensitive analysis method. For details, see, for example, Eiji Ishikawa and Akio Tsuji, "Enzyme Immunoassay Method" (Kyoritsu Publishing,
1987).

At present, the most commonly used enzyme immunoassay method for antigen measurement is the so-called sandwich method. The sandwich method is a method for measuring a multivalent antigen having two or more antigenic determinant sites, and is used for measuring different antigenic determinant sites.
Use different types of antibodies. A typical sandwich EIA method can be performed by the following steps, for example. 1. The antigen in the sample is bound to the immobilized antibody (first antibody) (first immune reaction). 2. Remove unbound antigen (wash). 3. An enzyme-labeled antibody (a second antibody different from the immobilized antibody) is added and allowed to bind to the antigen on the solid phase (second immune reaction). 4. Unbound enzyme-labeled antibody is removed (washing). 5. Add an enzyme substrate and measure the amount of enzyme-labeled antibody bound (enzyme reaction). In such a method, it is necessary to label the second antibody with an enzyme to prepare an enzyme-labeled antibody, and the antibody and the enzyme were chemically bound mainly by using a crosslinking reagent (for example, Eiji Ishikawa, Tadashi Kawai, Kiyoshi Miyai "Enzyme Immunoassay" (Medical Shoin, 1987)
Year)).

In the enzyme immunoassay, the performance of the enzyme-labeled antibody used has a great influence on the measurement sensitivity and reproducibility. However, an enzyme-labeled antibody obtained by chemically bonding an antibody and an enzyme has a problem in this performance.

Glutaraldehyde, which is a typical cross-linking reagent, binds and cross-links with amino groups of antibodies and enzymes, but the binding is random and reacts indiscriminately with amino groups of antibodies and enzymes to cause antibody activity and enzyme activity. Spoil. In addition, the labeling efficiency is low because only the enzyme or the antibody polymerizes. Moreover, as a result of random reaction, homopolymers and heteropolymers increase, and once these polymers are formed, the enzyme-labeled antibody (enzyme: antibody = 1: 1) is the ideal complex. Since it is difficult to separate the EIA method, the sensitivity and reproducibility of the EIA method are reduced.

The periodate method is a method in which the sugar chain of an enzyme is oxidized to form an aldehyde group, and the aldehyde group and the amino group of the antibody are reacted and bonded. in this way,
Although the decrease in the enzyme activity is small, the decrease in the antibody activity is inevitable because the antibody has no selectivity for the amino group. In addition, it cannot be applied to enzymes that do not have sugar chains.

In the maleimide method, a maleimide group introduced into an enzyme is bound / crosslinked with a thiol group at the hinge portion of an IgG antibody. According to this method, the enzyme can be selectively bound to the hinge portion distant from the antibody active site, so that the decrease in antibody activity is small. However, since the enzyme has no selectivity for amino groups, if the enzyme has two or more amino groups, the enzyme activity will decrease to some extent. Further, if two or more molecules of antibody are bound to one molecule of enzyme, the concentration of the labeled enzyme per molecule of antibody will be low, and the increase in sensitivity cannot be expected.

There are various other enzyme labeling methods by chemical bonding, but all of the enzyme-labeled antibodies obtained have the same and slightly different drawbacks as the above. In addition, the enzyme-labeled antibody by these chemical bonds is a combination of molecules having a large molecular weight, that is, an antibody and an enzyme, so that a defect that non-specific adsorption to a solid phase such as a microtiter plate or beads is likely to occur easily occurs. .. Therefore, there are problems that the sensitivity as expected cannot be obtained and the reproducibility is poor.

In addition, instead of using a chemically bound enzyme-antibody complex as in the prior art, we immunologically bind an enzyme to a two-headed antibody (BS antibody; Bi-specific antibody). We also developed a method of analysis and examined its performance (Japanese Patent Application No. 3-113774). A marked improvement in sensitivity and reproducibility was observed by the method using this two-headed antibody. However, in this method, a large amount of enzyme must be added to the reaction system, so that a new problem has arisen that the S / N ratio is lowered due to nonspecific adsorption of the added enzyme.

[0010]

SUMMARY OF THE INVENTION The present invention has been made in view of the above-mentioned circumstances, and an assay with high reduction in antibody activity and high sensitivity and good reproducibility can be carried out without reduction in S / N ratio. It is an object of the present invention to provide an immunoassay capable of performing the same.

[0011]

The object of the present invention is to contact a ligand with a two-headed antibody comprising a half-body of an antibody against a ligand and a half-body of an antibody enzyme capable of generating a detectable signal, It was achieved by an antibody-enzyme immunoassay characterized by measuring the activity of the antibody enzyme of the two-headed antibody bound (or not bound) to the ligand.

[0012]

In the present invention, instead of using a chemically bound enzyme-antibody complex as in the conventional case, a double-headed antibody (B
S-antibody; Bi-specific antibody) is used for immunoassay. This is problematic by using a two-headed antibody consisting of a half-body of an antibody enzyme capable of generating a detectable signal and a half-body of a specific antibody having antigen specificity for a test substance (ligand). It is based on the finding by the inventor that the decrease in sensitivity is not seen.

The two-headed antibody has a dimer structure similar to that of a normal antibody and is not chemically modified, so that the antibody activity is not impaired. Moreover, the enzyme activity of the antibody enzyme is not impaired. In addition, since it is not necessary to add an enzyme or the like other than the two-headed antibody, the S / N ratio does not decrease due to nonspecific adsorption of the added enzyme.

[0014]

Detailed Description of the Structure of the Invention Antibody Enzyme The antibody enzyme in the present invention refers to an antibody capable of converting its specific binding pair into another product. In other words, it is an antibody that retains enzymatic activity (or catalytic activity). The antibody enzyme (Antibody Enzyme) is also called a catalytic antibody (catalytic Antibody), Abzyme and the like.

In general, an antibody is characterized as a protein that recognizes a specific molecule (antigen) and specifically binds to it. On the other hand, the enzyme, which is the same protein, has the function of not only specifically binding to a specific molecule (enzyme substrate) but also catalyzing the chemical reaction of the molecule. That is, both enzymes and antibodies are in-vivo proteins capable of forming a binding pair with a specific substance, but antibodies can be distinguished from enzymes in that they have no ability to catalyze chemical reactions. However, since it is possible to obtain antibodies specific to almost all molecules in principle, on the basis of the property shared by antibodies and enzymes, that is, the property of specifically binding, Attempts have been made to obtain antibodies that catalyze chemical reactions. However, in the early studies, it was not possible to find an antibody having a catalytic effect, because it produced an antibody against the substrate (Biochemistry, 5 ,
2836 (1966), FEBS Letter, 100 , 137 (1979)). On the other hand, PG Schulz et al. And RA Lerner et al. Produced an antibody against an analog of the transition state of the reaction based on L. Pauling's idea that the active center of the enzyme has a structure complementary to the transition state of the reaction. And found that this antibody has catalytic activity (Science, 234 , 1570 (1986), Science, 2
34 , 1566 (1986)). Since then, many researchers have produced antibodies with such catalytic activity, and the idea of antibody enzyme (catalytic antibody, Abzyme) has been widely accepted.

A transition state analog is a stable analog that is similar in shape and charge to a substance (intermediate) in a transition state in a certain enzymatic reaction. Such a transition state analog acts as an antigen and is an antibody. The antibody that induces
It binds to substances in the transition state of the reaction process, stabilizes them, and further functions as a catalyst. For example, in the hydrolysis reaction of a carboxylic acid ester, the reactant ester generally has a planar molecular structure having no charge. Hydrolysis begins with the attack of water molecules, passes through a charged tetrahedral intermediate (transition state), and then rapidly decomposes into carboxylic acids and alcohols. In this intermediate, the bond direction between atoms also changes, and the distance between atoms is about 1.2.
Doubles. Due to such an unstable characteristic, it cannot be isolated and its antibody cannot be obtained (see the following chemical formula 1).

[0017]

[Chemical 1]

However, when the central carbon atom (C) of this tetrahedral structure is replaced with a phosphorus atom (P), a stable compound called a phosphoric acid ester having a similar configuration is obtained. In addition, the bond distance between phosphorus and oxygen is about 20% longer than the normal bond between carbon and oxygen, and is close to the bond distance in the actual transition state. In fact, phosphoric acid esters having such properties are known to inhibit certain hydrolases. By immunizing with such a transition state analog as an antigen, an antibody having this enzyme activity, that is, an antibody enzyme can be obtained.

The antibody enzyme used as one of the half isomers of the two-headed antibody in the present invention refers to an antibody having such an enzyme activity. The present invention uses antibody enzymes as an alternative to conventional labeling enzymes. Therefore, an antibody enzyme as used herein is one that is capable of producing a product or degradation product capable of producing a detectable signal from a substrate. For example, there is a substrate that uses a substance whose product generated by the ester decomposition in the above formula 1 is a dye.
As the antibody enzyme, rather than using a polyclonal antibody as it is obtained by immunizing with a transition state analog as it is, a monoclonal antibody is prepared and one having high catalytic activity is selected, which is used in the present invention. It is preferable to use it as a material for the two-headed antibody.

The double-headed antibody immunoglobulin (antibody) is not one type and is classified into several classes based on its chemical structure, but its basic structure is the structure found in IgG. That is, an L chain having a molecular weight of about 25,000 and an H chain having a molecular weight of about 50,000 form an S-S bond (disulfide bond) to form one unit (half-mer), and these equivalent half-mers further form an S-S bond of the H-chain. In 2
Quantify to form one IgG molecule. The antigen-binding site (Fab site) exists in each halfmer. That is,
IgG has one antigen specificity, but has the ability to bind to two antigen molecules (homologous bivalent). The concept itself of making an antibody having two different antigenic specificities (a so-called two-headed antibody having a different divalent) by chemically treating the same divalent IgG has already existed, and various methods have been developed. ing. The two-headed antibody used in the present invention can be produced by these known techniques, and is composed of a half-body of an antibody enzyme capable of generating a detectable signal and a half-body of a specific antibody against an analyte (ligand). To be done.

For example, according to the method of Nisonoff et al., Two types of I
After digesting gG with pepsin to remove the Fc portion, each was reduced to obtain a half-body Fab ′, which was reoxidized to prepare a two-headed antibody (Arch. Biochem. Biophy.
s., 90 , 460-462, (1961)). In this method, in addition to the heterodivalent antibody to which the different antibody half-dimers Fab 'are bound, the homologous divalent antibody to which the half-dimer Fab' having the same antigen specificity is bound (self-associated) is also formed. Therefore, in this case, the heterobivalent antibody is separated and purified from the homobivalent antibody. Or Bren
According to the method of Nan et al., one of the half-dimers Fab 'may be temporarily protected at the SH group with nitrobenzoic acid (Science, 22.
9 , 81, (1985)). By this, a heterodivalent two-headed antibody can be selectively prepared from two kinds of monoclonal antibodies. Bren
The method of Okumura et al., which is a simplified version of the method of Nan et al., may be followed (Japanese Patent Laid-Open No. 2-76899).

In each of the above-mentioned two-headed antibody, F (ab ') ₂ with the Fc portion removed was used. However, the Fc portion was not removed and the F (ab') ₂ was reduced to obtain a halfmer. It may be oxidized and bound to the body. However, the Fc portion is likely to be nonspecifically adsorbed on the solid phase. Therefore, for high sensitivity measurement, I
It is preferable to use Fab 'obtained by digesting gG with pepsin to remove the Fc portion and further reducing it to form a double-headed antibody.

The two-headed antibody can also be produced by fusing two types of hybridomas to produce a heterohybridoma. It can also be prepared by fusing a hybridoma and spleen cells. A method for producing a monoclonal antibody, a method for purifying the same, and a method for obtaining an F (ab ') ₂ fragment can be obtained by the methods described in various publications (for example, "Tomonoyama Sakuji et al.," Monoclonal Antibody Experimental Manual). ] Kodansha, 1988
Year).

Analysis Method The antibody enzyme immunoassay method of the present invention can be specifically performed as follows. (See Figure 1). 1) The antibody (ligand L) 12 in the sample is immobilized onto the immobilized antibody 10
(First immune reaction). 2) The unbound antigen 12 is removed (washing). 3) A two-headed antibody (BS-Ab) 14 consisting of a half-body 14a of a ligand-specific antibody and a half-body 14b of an enzyme antibody is added to bind to the antigen 12 bound to the immobilized antigen 10 (second
Immune reaction). 4) The two antibodies that have not bound to the antigen 12 are removed (washing). 5) An enzyme substrate (S) is added, and the amount of bound two-headed antibody is measured by detecting the product (Prod) (enzyme reaction).

In the above, the present invention is applied to the sandwich method to measure the antibody enzyme activity of the two-headed antibody bound to the solid phase. However, for the free two-headed antibody not bound to the solid phase. The activity of the bound antibody enzyme may be measured. Further, the method for carrying out the present invention is not limited to such a sandwich method. For example, a fixed amount of solid-phase antigen and an antigen (ligand) in a sample compete with a fixed amount of two-headed antibody, and the amount of the two-headed antibody bound to the solid-phase is determined by measuring the enzyme activity of the antibody. Good. Alternatively, the two-headed antibody may be bound to the solid-phase antigen in advance, and the amount of the two-headed antibody on the solid-phase reduced by competition with the antigen (ligand) in the sample added later may be measured ( So-called replacement method).

[0026]

[Synthesis Example 1] Synthesis of hapten for antibody enzyme Assuming a reaction for producing p-nitrophenol (yellow: absorption at 400 nm) from substrate 1 according to the following reaction formula, an enzyme antibody which catalyzes this was prepared.

[0027]

[Chemical 2]

First, a compound having the following structural formula was synthesized as a transition state analog (hapten) of the above reaction.

[0029]

[Chemical 3]

The outline of the reaction synthesis route is shown in FIG. First, compound 1 (17 g; 0.1 mol) and compound 2 (20 g;
0.1 mol) was mixed and heated at 160 ° C. for 7 hours. After cooling, the low boiling point portion was distilled off to obtain 13 g of the target compound 3 as a colorless oil. Compound 3 (6.5 g; 0.
(026 mol) is dissolved in 10 mL of ethanol and 12N hydrochloric acid is added to 80
mL was added. The mixture was heated to 160 ° C. for 2.5 hours. After cooling, the solvent was distilled off under reduced pressure to obtain 4.4 g of the target compound 4 as a solid. Compound 4 (5.1 g; 0.028 mol) was dissolved in thionyl chloride (40 mL) and stirred at room temperature for 4 hours (compound 5 was produced by this reaction). After distilling off excess thionyl chloride under reduced pressure, the residue was dissolved in 40 mL of chloroform, and p-nitrophenol (12 g; 0.086 mol) was dissolved therein.
And 40 mL of a chloroform solution of triethylamine (8.7 g; 0.086 mol) were added dropwise. After the dropping, the mixture was stirred for 2 hours at room temperature and then subjected to usual post-treatment, and the organic layer was distilled off to obtain crystals. The crystals were recrystallized from a mixed solvent of ethyl acetate / hexane to obtain 7.2 g of the target compound 6. Compound 6 (3.0 g; 0.005 mol) was added to 200 mL of 0.2N NaOH solution, and the mixture was heated with stirring at 100 ° C. for 1.5 hours. After acidification with hydrochloric acid, nitrophenol was removed by repeated extraction with ether. The aqueous layer was collected and concentrated with an evaporator to deposit white crystals. By recrystallizing from ethanol, 900 mg of the desired compound 7 (transition state analog) was obtained.

[0031]

[Synthesis Example 2] Synthesis of hapten-KLH complex The compound 7 obtained in Synthesis Example 1 was used as a hapten to bind to a carrier protein keyhole limped hemocyanin (KLH) to synthesize an hapten-KLH for immunization. Hapten (Compound 7: 5 mg), N-hydroxysuccinide (4 mg
), Dicyclohexylcarbodiimide (7.4 mg) and pyrrolidinopyridine (0.4 mg) were dissolved in 2 mL of methylene chloride and reacted at room temperature for 1 day. After removing the generated urea body, 1 mL of an aqueous KLH solution (6 mg / mL) was gently added, and the mixture was reacted at room temperature for 3 hours. Then, it was dialyzed against water for 2 days to synthesize a hapten-KLH complex. A hapten-BSA complex for measuring antibody titer was synthesized by a completely similar method.

[0032]

[Synthesis Example 3] Synthesis of Substrate Am. Chem. Soc. , 109 , 2174-2
Substrate 1 having the following structure was synthesized by the method described in 176 (1987).

[0033]

[Chemical 4]

[0034]

Example (1-1) Preparation of antibody enzyme The hapten-KLH complex synthesized in Synthesis Example 2 was dissolved in a phosphate buffer solution (10 mM, pH7.4, containing 0.9% NaCl) to give 40 μg.
/ mL solution. Balb / C mice were immunized with 500 μL of a mixture of this with an equal amount of Freund's complete adjuvant (for booster immunization, mixed with Freund's incomplete adjuvant). Boosts were given 4 times every 3 weeks. After stopping immunization for 2 months, 2 every 2 weeks
After the immunization was performed and the antibody titer was confirmed, the spleen of the immunized mouse was taken out, and the spleen cells and myeloma cells (SP2) were cell-fused according to a conventional method using polyethylene glycol. The hapten-BSA complex-immobilized microtiter plate was used for screening by the ELISA method, and 93 types of hapten-binding antibody-producing cells were selected.
The cells were intraperitoneally injected into Balb / C mice. The antibody-containing ascites produced in the abdominal cavity of the mouse was collected and the IgG fraction was collected by the ammonium sulfate precipitation method. The obtained IgG fraction was purified with a protein A column (MAPS-2 kit; manufactured by Bio-Rad).

(1-2) Evaluation of catalytic ability of antibody enzyme From the antibodies having a hapten-binding ability selected in Example 1-1, an antibody having a catalytic ability was selected by the following method. Substrate 1 (0.12mg / mL; 10mM Tris-HCl buffer solution; pH8.5) 0.2mL of Synthetic Example 3 and purified antibody (2mg / ml) 0.1mL were mixed, and substrate 1 was decomposed to form paranitro. The absorption of phenol was followed at 400 nm. From 93 types of antibodies, an antibody (antibody enzyme) capable of degrading a substrate was found to be 3 clones (antibody enzyme 4A1, 5H2, 1G2) (Fig. 3).
reference).

For the antibody enzyme 4A1 having the highest catalytic ability, purified IgG of 10 mg / mL (acetate buffer; pH 4.2)
Then, 0.5 mg of pepsin (manufactured by Sigma) was added to 1 mL of the mixture, and the mixture was reacted at 37 ° C. for 20 hours. After reaction, Superdex-2
Gel filtered through 00 column (Pharmacia) to obtain F (ab ') ₂
Fractions were obtained.

(2) Preparation of anti-CRP antibody Commercially available CRP (C-reactive protein) was added to a phosphate buffer solution (10 mM,
pH 7.4, containing 0.9% NaCl) to give a 400 μg / mL solution. This was mixed with an equal volume of Freund's complete adjuvant (for booster immunization, mixed with physiological saline) 50
Balb / C mice were immunized with 0 μL. Booster immunizations were given 5 times every 2 weeks. After confirming the antibody titer, the spleen of the immunized mouse was taken out, and the spleen cells and myeloma cells (SP
2) and were fused with each other according to a conventional method using polyethylene glycol. The antibody-producing fused cells obtained by screening by the ELISA method were intraperitoneally injected into Balb / C mice. The anti-CRP antibody-containing ascites produced in the mouse abdominal cavity was collected, and the IgG fraction was collected by the ammonium sulfate precipitation method. The IgG fraction thus obtained was separated from the protein A column (MA
PS-2 kit; manufactured by Bio-Rad). Make purified IgG 10 mg / mL (acetate buffer; pH 4.2) and add 1 mL
0.5mg of pepsin (Sigma) was added to
Reacted for hours. After the reaction, gel filtration was carried out using a Superdex-200 column (manufactured by Pharmacia) to obtain an F (ab ') ₂ fraction.

(3) Preparation of double-headed antibody Fraction of F (ab ′) ₂ fraction of antibody enzyme 4A1 prepared in (1-2)
To 0.45 mL of 3 mg / mL solution (phosphate buffer solution; pH 6), 0.05 mL of 0.5 M 2-mercaptoamine was added, and the mixture was reacted at 30 ° C for 90 minutes,
Reduced. After the reaction, gel filtration was performed using a Sephadex G-25 column to obtain a half-body Fab ′ of the antibody enzyme. on the other hand,
3 mg / mL of F (ab ') ₂ fraction of anti-CRP antibody prepared in (2)
To 0.45 mL of the solution (phosphate buffer solution; pH 6), 0.05 mL of 5 mM dithiothreitol was added, and the mixture was allowed to react at 30 ° C. for 30 minutes for reduction. After the reaction, 0.05mL of dithiobisnitrobenzoic acid (50mM)
The reaction was stopped by addition, and the reduced thiol group was masked. Gel filtration was performed on this Sephadex G-25 column to obtain a half-meric anti-CRP-Fab '. The antibody enzyme half-body Fab ′ thus obtained and an anti-CRP antibody half-body Fab ′ were mixed in equal amounts and allowed to stand at room temperature for 10 hours to cause association. Bound F (ab ') ₂ (2 antibodies)
Was purified by gel filtration on a Superdex-200 column (Pharmacia).

(4) Comparative Example As a comparative example, an antibody enzyme-labeled antibody by chemical bond was used. That is, (1-1) the antibody enzyme 4A1 monomer,
The anti-CPP antibody monomer of (2) was combined with the glutaraldehyde two-step method to prepare an anti-CRP-antibody enzyme complex.

(5) Preparation of anti-CRP-immobilized plate 50 μL of anti-CRP IgG (50 μg / mL, PBS solution) prepared in (2) was placed in each well of a 96-well microtiter test plate (Nunc). Sensitized overnight at 4 ° C.
Then, wash each well with PBS and add 300 μL of 3% BS.
A PBS solution containing A was added to each well to block nonspecific adsorption sites. In the sandwich method of the present Example, the first antibody to be immobilized and the second antibody, which is a half-body of the two-headed antibody, bind to different antigenic determinants of one antigen, and originally different antigens. Use one with specificity. However, since CRP is a pentamer, the same antibody as the anti-CRP antibody used for the two-headed antibody was immobilized in this example. However, as the immobilized anti-CRP antibody, intact IgG that was not digested with pepsin was used.

(6) Quantification of CRP CRP was diluted stepwise with a PBS solution, 50 μL of each was placed in each well of the anti-CRP antibody-immobilized plate prepared in (5), and reacted at 37 ° C. for 2 hours ( First immune reaction).
After that, the unbound CRP was removed by washing three times with a PBS solution. 2 head antibody (10 μg /
100 μL of PBS solution containing (mL) was added, and the mixture was reacted at 37 ° C. for 60 minutes (second immunoreaction). Then 3 with PBS solution
Washed twice. Then, 200 μL of Tris-HCl buffer solution (pH 8.5) containing 0.08 mg / mL of the substrate 1 of Synthesis Example 3 was added, and the mixture was stirred at room temperature.
Leave for 20 minutes. Then, measure the 400 nm absorbance of each well with a microplate reader (Corona),
A calibration curve was created.

In the comparative example, instead of the two-headed antibody, (4)
The PBS solution containing the anti-CRP-antibody enzyme complex (12 μg / mL) prepared in 1. was used. The reaction is the same as for the two-headed antibody. As shown in FIG. 4, the example (-○-) was about 10 times more sensitive than the comparative example (-●-).
This means that the antibody enzyme not only has enzymatic activity as a labeling reagent, but it is also possible to use this two-headed antibody in combination with the half-body of the anti-ligand antibody as a half-mer for more sensitive immunoassay. It shows that

[Brief description of drawings]

FIG. 1 is a schematic diagram illustrating an immunoassay method of the present invention.

FIG. 2 is a diagram showing a synthetic route of a hapten (transition state analog) used for producing an antibody enzyme in an example of the present invention.

FIG. 3 shows the catalytic activity of 36 clones of hapten-binding antibody.

FIG. 4 is a diagram showing a calibration curve showing the results of Examples and Comparative Examples.

[Explanation of symbols]

 10 Immobilized antibody, 12 Antigen (ligand) 14 Two-headed antibody 14a Half-dimer of anti-ligand specific antibody 14b Half-dimer of enzyme antibody

Continuation of the front page (72) Inventor Yukio Sudo 3-1146 Izumisui, Asaka-shi, Saitama Fuji Shashin Film Co., Ltd.

Claims (2)

[Claims] 1. A double-headed antibody that is bound to a ligand by contacting a double-headed antibody consisting of a half-body of an antibody against the ligand and a half-body of an antibody enzyme capable of generating a detectable signal to the ligand. An antibody enzyme immunoassay characterized by measuring the activity of an enzyme. 2. A two-headed antibody comprising a half-body of an antibody against the ligand and a half-body of an antibody enzyme capable of generating a detectable signal, which is brought into contact with the ligand and is not bound to the ligand. An antibody-enzyme immunoassay method characterized by measuring the antibody enzyme activity of.