JPH0466870A - Novel labeling body and immunoassay using the same - Google Patents

Abstract

PURPOSE:To make a highly sensitive immunoassay possible by preparing and using a labeling body trough biotin-avidin or biotin-streptoavidin system. CONSTITUTION:A labeled body is prepared by reacting a biotin binding body with avidin or streptoavidin labeled body. Upon this, the biotin binding body is obtained by combining a biotin derivative with an immunity-reacting substance of antigen, etc. in a buffer solution. The labeling body of avidin, etc. is obtained as follows for example in the case where the labeling substance is enzyme, avidin labeling enzyme of beta-galactosidase; a maleimid radical is derived into avidin, etc. by reacting avidin with N-succinimidil-6-maleimid hexanoate and then beta-galactosidase is reacted. A highly sensitive result can be obtained by making the immunity measurement using the labeling body prepared in such a way.

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The present invention relates to a novel label and an immunoassay using the same. More specifically, the present invention relates to an immunoreactive substance labeled through a bond between biotin and avidin or a bond between biotin and streptavidin, and an immunoassay method for a target substance using the immunoreactive substance.

[Conventional technology]

Conventionally, quantitative measurement of a target substance in a sample by immunoassay involves directly binding a label to an immunoreactive substance that can form an immune complex with the target substance, and then binding the label to a solid phase. An immunoassay method is widely used in which an immune complex of the target substance is formed on a solid phase in the form of a sandwich with another immunoreactive substance, and then the labeled substance bound to the solid phase is measured. There is.

For example, if the labeled substance is an enzyme and the target substance in the sample is an antibody, a sandwich-like immune complex with the antibody, which is the target substance, is formed on the solid phase by the enzyme-labeled anti-antibody and the antigen bound to the solid phase. However, a commonly used method is to measure the activity of an enzyme bound to a solid phase. In addition, if the target substance in the sample is an antigen, an enzyme-labeled antibody and an antibody bound to a solid phase can be used to
A commonly used method is to form a sandwich-like immune complex with a target substance, an antigen, on a solid phase and measure the activity of an enzyme bound to the solid phase.

However, the biggest problem in measuring target substances such as antigens and antibodies is the nonspecific binding of the label to the solid phase. There are two major causes of non-specific binding of the label to the solid phase: direct non-specific binding of the label to the solid phase and non-specific binding of a substance in the sample that can bind to the label. Separated. Several methods have been published for increasing measurement sensitivity and reliability of measurement values by reducing the influence of this non-specific binding as much as possible. For example, in order to reduce the direct non-specific binding of enzyme labels to the solid phase, various methods for producing enzyme labels have been studied. 45 (19B?)]. In addition, non-specific binding of substances capable of binding to the labeled substance in the sample to the solid phase is an important problem when the target substance is an antibody. A method has been reported in which a sandwich-like immune complex is formed on a solid phase with an antigen bound to a solid phase and an antibody as a target substance, and the labeled substance bound to the solid phase is measured (Endo et al., Cl1n ,
Chim, Acta, 103.6 Fu77 (19
80)).

Recently, as an improved method, instead of using a solid phase directly bound to an antigen, an antigen bound to a hapten such as a dinitrophenyl group is used to form a sandwich-like immune complex of the target substance, an antibody, and a labeled antigen. A method has been reported in which the sandwich-like immune complex is bound to a solid phase bound to an anti-hapten antibody such as an anti-dinitrophenyl group antibody, and the label bound to the solid phase is measured. In this method, the reaction between an antigen and a specific antibody is carried out in a solvent, and the formed sandwich-like immune complex can be bound to a solid phase using a bond with high binding capacity, allowing for highly sensitive antibody measurement in a short time. It became possible (Japanese Patent Application Laid-Open No. 1-312464).

Furthermore, a method for reducing non-specific binding of a label to a solid phase has been developed by the present inventor. That is, this is a method in which a sandwich-like immune complex formed on a solid phase as described above and consisting of a target substance, a labeled substance, and an immunoreactive substance in a specimen is eluted from the solid phase, and then the labeled substance is measured. for example,
In the measurement of antibodies, even higher sensitivity can be achieved by adding a hapten to the solid phase bound to the sandwich-shaped immune complex, allowing the sandwich-shaped immune complex to be eluted from the solid phase, and measuring the enzyme activity in the eluate. Measurement has become possible (Japanese Patent Application No. 1-17873).

In addition, by binding this eluate to a solid phase bound to an anti-antibody, antibody class-specific measurements (1 mmune C
complex transfer method) has also become possible (Japanese Unexamined Patent Publication No. 2-28558).

As described above, measuring antigens, antibodies, etc. using labeled substances is a useful method, and various improved methods have been proposed.

For example, various enzyme labeling methods have been developed as methods for producing enzyme-labeled substances such as antibodies and antigen proteins and peptides. The development of enzyme labeling methods for antibodies and antigens is described in Ishikawa et al. (Proteins, Nucleic Acids, Enzymes, supra). In other words, the glutaraldehyde method, which utilizes the amino groups of antibodies and enzymes, has been used in the past. In recent years, new bifunctional crosslinking agents have been developed, and by using one amino group and the other thiol group instead of amino groups, polymer formation has been suppressed and measurement sensitivity has been improved.

However, the production of enzyme-labeled products using conventional methods involves direct binding of biologically active substances such as antigens and antibodies to enzymes, so the reaction is delicate and there are problems with reproducibility and production. In reality, problems have been pointed out, such as the need for a certain amount of sample or more, which limits the scope of its applicability. That is, in boat fishing and enzyme labeling of antibodies, the optimal conditions differ not only depending on the antibody class but also on individual antibodies. In particular, antigens for antibodies that are important for clinical diagnosis are unstable, require consideration of special conditions for solubilization, or when amino groups and thiol groups coexist in one antigen. The bifunctional crosslinking agent has problems such as the formation of antigen polymers. Therefore, it was necessary to consider the labeling method and labeling conditions for each antigen. Furthermore, it has been pointed out that in order to react an antigen with a large molecular weight with an enzyme, it is necessary to increase the concentration of both, and it has also been pointed out that even slight changes in conditions affect the properties of the labeled substance, resulting in poor reproducibility. Furthermore, in some cases, current direct labeling techniques may not yield labels.

For example, antigens required for viral antibodies such as HTLV-1, HCV, HBs, and HIV, and receptors such as acetylcholine receptors and TSH receptors required for antibody measurement.Scepters are membrane proteins that bind to enzymes. Conditions containing large amounts of detergent are required to dissolve the antigen to possible concentrations. However, many enzymes lose their activity under these conditions. In addition, to solve this problem, there is a method of using an epitope peptide, which is the recognition part of an antibody, as an antigen, but in this case, an amino acid involved in the reaction is added to the recognition part between an enzyme label such as lysine or cysteine. However, there are limitations to using conventional direct enzyme labeling methods.

In addition, recently, labeled substances in which antigens and antibodies are directly labeled with fluorescent substances or luminescent substances have been used, but in this case as well, there were limitations to the use of direct labeling methods for peptides containing lysine or cysteine in the recognition site. .

For these reasons, there is a demand in the art for the development of an enzyme labeling method that is simpler and has a wider range of applications, and a biotin labeling method has been developed to meet these demands.

For example, by creating a sandwich-like complex with an antigen in a specimen using a biotin-labeled antibody and a solid phase bound to the antibody, and then reacting with an enzyme, a fluorescent substance, etc. bound to avidin or streptavidin, This is a method for measuring the amount of antigen bound to a solid phase (M, Wilke
r et al., Analytical Biochemist
y, 171.1-32.1988).

[Problem to be solved by the invention]

Regarding the advantages and disadvantages of using this biotin-labeled substance, M.
, Wilker et al. (Analytical Bioch.
emistry.

(mentioned above).

The advantage of this method is that biotin is a stable compound, has a carboxyl group, easily reacts with proteins, bebutide, etc.
It is easy to introduce functional groups such as groups, and by using this compound, biotin can be easily and simply introduced into proteins, peptides, etc. Compounds into which various functional groups have been introduced are already commercially available.

On the other hand, avidin and streptavidin-labeled substances, which are labeled substances bound to avidin and streptavidin, which have the ability to bind biotin, are common reagents, and substances conjugated with avidin, etc. for various labels are already commercially available. It can be easily synthesized and obtained. The binding strength of biotin, avidin, and streptavidin is high, and they can be quantitatively bound even in dilute solutions.

However, a drawback of this method using a biotin label is that the label bound to avidin or streptavidin tends to bind nonspecifically to the solid phase, so that measurement sensitivity is not improved. That is, when using a biotin-labeled substance,
Even though it is easy to label with avidin etc. during the measurement operation, there is a problem with measurement sensitivity, and various efforts have been made to solve this problem, but so far no solution has been achieved. do not have.

Therefore, an object of the present invention is to provide a novel label useful for immunoassays.

Another object of the present invention is to provide a highly sensitive immunoassay method using a labeled substance.

[Means to solve the problem]

Under these circumstances, the present inventors have conducted extensive research.

First, a biotin-conjugate of an immunoreactive substance such as an antigen is combined with an avidin-labeled substance or a streptavidin-labeled substance to prepare a labeled substance via a biotin-avidin or biotin-streptavidin system, and then It was discovered that sandwich-type immunoassays can be performed using the isolated and purified labeled product in the same manner as with conventional labeled products, leading to the present invention. That is, the present invention provides: (1) a label represented by the general formula (a) or (b) through the binding of biotin and avidin or the binding of biotin and streptavidin; (a): X-biotin-avidin-label; Thing (b):
X-biotin-streptavidin-labeled product (wherein,
represents an immunoreactive substance selected from the group consisting of antigen, antibody, and anti-antibody. ), (2) An object characterized by having a step of forming a sandwich-like immune complex containing a target substance in a specimen on a solid phase using the label and solid phase substance described in (1) above. It relates to immunoassay of substances.

In the label of the present invention, X represents an immunoreactive substance selected from the group consisting of antigens, antibodies, and anti-antibodies, and is capable of forming an immune complex with the target substance in the specimen. Therefore,
For example, if the target substance is an antibody, X is the antigen or anti-antibody,
When the target substance is an antigen, X represents an antibody.

Here, the antigen refers to a substance that can induce an antigen-antibody reaction or an immune response, and includes proteins, polysaccharides, complexes thereof, complexes with lipids, etc., and bebutides containing antigenic determinants of these antigens. It also represents. The peptides also include synthetic peptides synthesized by conventional methods, such as synthetic peptides of antigenic determinants.

Here, the antibody includes (Fab) 2 and Fab', which are part of the antibody.

Here, the label is not particularly limited as long as it can be used in immunoassays, and usually includes enzymes, luminescent substances, fluorescent substances, metal compounds, and radioactive substances. for example,
The enzymes are β-D-galactosidase, peroxidase, alkaline phosphatase, the fluorescent substance is fluorescein isothiocyanate, the metal compound is europium complex, the luminescent substance is acridium salt, and the radioactive substance is 3) (, 14C, +2Si, +31).
1.32p etc. are used.

The label of the present invention can be easily produced as follows. That is, it can be obtained by first binding biotin to an immunoreactive substance such as an antigen or antibody, then reacting the biotin conjugate with an avidin or streptavidin label, and purifying it using a column or the like as necessary. can.

The production of biotin conjugates can be easily carried out according to existing known techniques (Bayer, B, A, et al.

Methods in Bnzymology, 62
．． '308-315 (1979)).

That is, a biotin derivative, for example, a succinimide derivative, and an immunoreactive substance such as an antigen are mixed in a buffer solution, or a photoreaction is performed. After the reaction, unreacted biotin derivatives are removed using a column or the like, if necessary.

Furthermore, it may be desirable to insert a spacer between an immunoreactive substance such as an antigen and biotin.

For example, an antigen has a glutathione group.
It can also be obtained by introducing e) and then reacting with a biotin derivative.

In addition, various biotin derivatives, such as MPB (3-(
N-maleimido-propionyl) biocytin), B
Thiol group, amino group, aldehyde or carbodiimide activated carboxyl group using NH3 (biotin N-hydroxysuccinimide ester), BCH2 (N6-biotinylated monolysine hydrazide) or DBB (p-diazobenzoyl biocytin), respectively. Alternatively, biotin can be attached to a phenol or imidazole group.

Furthermore, when the immunoreactive substance is a peptide consisting of 100 or less amino acids, biotin can be introduced at a desired position without changing the immunoactivity of the peptide using known peptide synthesis methods.

That is, the functional group of the amino acid side chain is semi-permanently
Biotinylation at any position can be performed by the conventional method of organic peptide synthesis, in which the amino acid is blocked with a temporary protecting group, the N-terminal amino group is replaced with a temporary protecting group, and amino acids are sequentially added. .

Selection of protecting groups and removal conditions can be made appropriately by those skilled in the art by applying the techniques shown at the bottom. , co-authored by Michinori Waki, published by Maruzen, pp. 143-17.
3).

For example, during peptide synthesis, Nε-2-chlorobenzyloxycarbonyl-lysine and 5-4-methylbenzyl-cysteine are used to protect the ε-amino group of lysine and the β-thiol group of cysteine. One example is a method in which peptide synthesis is performed by protecting the α-amino group with a BOC (Nα-1-butyroxycarbonyl) group. After peptide synthesis, the N-terminal BOC is cleaved with the mild acid TFA (tetrafluoroacetic acid) to free only the N-terminal α-amino group, and biotin is attached, followed by Nε-2-talolopenzyloxy group. By cleaving the carbonyl group and 4-methylbenzyl group with a stronger acid HF (hydrogen fluoride), a peptide biotinylated only at the N-terminus can be obtained.

In addition, side chain functional groups include glutamic acid, aspartic acid α and β-carboxyl groups, serine, and threonine β-carboxyl groups.
-hydroxyl group, aromatic hydroxyl group of tyrosine, etc., but the above method is particularly useful when these are blocked by existing peptide biotinylation methods,
In some cases, this is a necessary method.

Surface antigens such as HIV (human immunodeficiency virus) and HCV (hepatitis C virus), which have been attracting attention in recent years, also have peptide chains containing such active functional groups. It is a suitable material for labeled objects.

If the immunoreactive substance is an antibody or an antigen with a molecular weight of 50,000 or more,
The number of biotin molecules that bind to one molecule of antibody or antigen is usually 1 to 10 molecules, preferably 1 to 4 molecules.

When the immunoreactive substance is a part of an antigen or antibody with a molecular weight of 5,000 or more and less than 50,000, the number of biotin molecules bound to one antibody molecule is usually 1 to 5 molecules, preferably 1 to 2 molecules.

When the immunoreactive substance is an antigen with a molecular weight of less than 5.000 or a peptide containing an antigenic determinant of the antigen, the number of biotin molecules bound to one antibody molecule is usually one to two molecules, preferably one molecule. .

Avidin or streptavidin labels can also be easily produced using known techniques (Bayer et al., supra). For example, when the label is an enzyme, the avidin-labeled enzyme for β-galactosidase is a combination of avidin and N-succinimidyl-6-maleimidohexanoate (N-succinimidyl-6-maleimidohexanoate).
minidyl-6-maleimidohexan.

A maleimide group is introduced into avidin or streptavidin by reacting with β-ate), and then β-
It can be obtained by reacting with galactosidase.

The number of molecules of avidin or streptavidin that can be bound to one molecule of enzyme is usually one to four molecules, preferably one to two molecules.

When the label is a luminescent or fluorescent substance, it can be obtained by mixing a reactant of the luminescent or fluorescent substance, such as a reactant with succinimidyl, and avidin or streptavidin.

The number of luminescent substances or fluorescent substances that bind to one molecule of avidin or streptavidin is 1 to 30 molecules, usually 2 to 10 molecules.

The reaction between the biotin conjugate and the avidin or streptavidin label is obtained by mixing solutions of both. For example, in a buffer solution with a pH of 6 to 9, usually at 0 to 40°C.
, preferably at 4 to 37°C, usually for 10 minutes to 48 hours, preferably for 10 minutes to 8 hours. The mixing ratio of biotin conjugate and avidin or streptavidin label is:
The molar ratio is usually 0. 1 to 100, preferably 0.3 to 10.

If the immunoreactive substance is a low molecule and it is easy to separate the generated label and the unreacted biotin conjugate, the biotin conjugate can be added in excess.

If the immunoreactive substance is a polymer and it is easy to separate the generated labeled product from the unreacted avidin or streptavidin labeled product, an excess amount of the avidin or streptavidin labeled product can be added.

In the immunoassay method of the present invention, a sandwich-like immune complex containing a target substance in a specimen is bound to a solid phase using a labeled substance and a solid-phase substance obtained by the method described above, and then the labeled substance is bonded to a solid phase. The amount of the target substance is determined by measuring the amount of the target substance. Here, the solid roughening substance refers to a substance that can bind a target substance to a solid phase and form a sandwich-like immune complex on the solid phase. Usually, when the target substance is an antigen, it represents an antibody, and when the target substance is an antibody, it represents an antigen or an anti-antibody. The immobilized substance is used by binding to a solid phase or by labeling one of the binding pairs.

Next, the immunoassay method of the present invention will be explained.

This method is classified into the following two methods and four types of embodiments are exemplified.

The first method is to measure a label in a sandwich-like immune complex formed on a solid phase.

The second method is a method in which a sandwich-like immune complex formed on a solid phase is eluted from the solid phase, and then the labeled substance is measured.

First, in the first embodiment, which is an example of the first method, a sandwich-like immune complex with a target substance in a sample, which is a substance to be measured, is formed on a solid phase using a label and a solid-phase substance bound to a solid phase. This method measures the label formed on the solid phase and bound to the solid phase.

As a measurement method, a sample containing the target substance is reacted with a solid-phase substance, the sample is aspirated, the solid phase is washed, and then the solid phase and the label are reacted. Aspirate unreacted labeled substance,
After washing the solid phase, the label bound to the solid phase is measured.

Alternatively, a method may be used in which a sample containing a labeled body, a solid-phase substance, and a target substance are simultaneously reacted, the reaction solution is aspirated, the solid phase is washed, and then the labeled substance bound to the solid phase is measured. The conditions for measurement can be the same as those for the known Sand-Germany immunoassay method.

The immobilized substance in this first embodiment may be produced by any known method of binding proteins to a solid phase.

For example, agarose, polystyrene, polyacrylic, Teflon, paper, glass, etc. are commonly used as the solid phase.
Polystyrene is preferred.

The antigen can be bound to the solid phase by physical adsorption. Furthermore, when the antigen is a hapten, the antigen is bound to a suitable carrier protein and then bound to a solid phase. Examples of carrier proteins include albumin and non-specific immunoglobulin. In addition, the method for producing the solid phase bound to antibodies is the method by Ishikawa et al. (Scan
d, J, Immun.

19.8.43 (1978)).

In the second embodiment, which is an example of the first method, in the first embodiment, the antigen, etc. in the homophasing substance that forms an immune complex with the target substance in the specimen is directly bound to the solid phase. In the second embodiment, a sandwich-like immune complex is formed on a solid phase via an immobilized substance having two binding pairs. The term "binding pair" as used herein refers to a pair of substances that can be strongly and selectively bound together through physical bonding. Examples include haptens and anti-habten antibodies. Binding pairs of the invention do not include biotin-avidin or streptavidin binding pairs. Furthermore, binding pairs that affect the reaction between the target substance to be measured and the label cannot be used.

Preferred binding pairs include a dinitrophenyl group and an anti-dinitrophenyl group antibody.

It is desirable that one of the binding pairs (for example, the dinitrophenyl group in the above example) labeled on the immobilized substance has a low molecular weight. That is, in a binding pair of a hapten and an anti-habten antibody, it is desirable to label the hapten with an antigen or the like.

One such desirable bond pair is a dinitrophenyl group.

An antigen or antibody bound to one of the binding pairs can be produced by a known method. For example, Hashida et al., J.A.
ppl, Biochem, 6.56 (1984
) can be easily obtained using N-succinimidyl-6-maleimidohexanoate as a crosslinking agent.

The other member of the binding pair (eg, the anti-dinitonophenyl group antibody in the example above) is an anti-hapten antibody when one is a hapten. The other such desirable binding pair includes anti-dinitrophenyl group antibodies.

The method for producing the solid phase to which the other member of the binding pair is bound may be any known method for binding a protein to a solid phase.

The measurement method is to add a labeled immobilized substance to a sample containing the target substance, and then, after the reaction, add a reaction solution to the surface phase bound by the other member of the binding pair, and perform the reaction. After aspirating the reaction solution and washing the surface phase, the labeled substance bound to the solid phase is measured, or the labeled substance, the immobilized substance labeled with one of the binding pairs, and the solid phase bound with the other of the binding pair. is simultaneously added to a sample containing the target substance, and after the reaction, the reaction solution is aspirated, the solid phase is washed, and the labeled substance bound to the solid phase is measured. The latter has fewer steps and is preferable.

The measurement conditions can be the same as the known Sand-Germany immunoassay method.

In the present invention, in both the first aspect and the second aspect, results with higher sensitivity can be obtained compared to the conventional method.
This embodiment is preferable because it provides more sensitive results.

The third aspect, which is an example of the second method, is a method in which the sandwiched immune complex bound to the solid phase in the first or second aspect is eluted, and then the labeled substance in the eluate is measured.

When the first embodiment is adopted as a method for forming a sandwich-like immune complex containing the target substance in a sample using a labeled substance, when binding an antigen etc. to a solid phase, the sandwich-like immune complex is formed. They must be bound through a bond that can be eluted without dissociation.

A preferred method is to bind the sandwiched immune complex to a solid phase in a second manner, and after washing, add a compound containing or to the binding pair, and elute the sandwiched immune complex from the solid phase. .

As a specific example of one of the binding pair or a compound containing it, one of the binding pair has a dinitrophenyl group-labeled antigen as the labeled antigen, and the other side of the binding pair has a solid phase bound with an anti-dinitrophenyl group antibody as the bound surface phase. When used, dinitrophenol-lysine may be mentioned.

For elution, add one of the binding pairs or the compound containing it to 0.1
Add a buffer solution containing ~10mM and pH 6.0 to 9.0, usually 0 to 40°C, preferably 20 to 37°C, usually 10 minutes to
It is desirable to carry out the treatment for 48 hours, preferably for 1 to 6 hours.

A fourth embodiment, which is an example of the second method, is a method of binding the immune complexes eluted in the third embodiment to a new solid phase. The new solid phase refers to a solid phase that has the ability to bind sandwich-like immune complexes but does not have the ability to bind labels. When the target substance is an antibody, a desirable solid phase includes a solid phase bound to an anti-antibody. In particular, as an anti-antibody, I
By selecting antibodies that can identify antibody classes such as gG, A, and M, it is possible to measure antibodies of each class of antibodies in a sample.

As a measurement method, in the third embodiment, when a new solid phase is added at the same time as one of the binding pairs or a compound containing it, the sandwich-like immune complex can be eluted from the solid phase and simultaneously bound to the new solid phase, This is the preferred method. after that,
In this method, the reaction solution is aspirated, the new solid phase is washed, and then the labeled substance bound to the new solid phase is measured.

Measurement of the labeled substance is carried out in the same manner as in ordinary immunoassays, and can be carried out by a known method.

To reduce the effects of nonspecific adsorption of the label to the solid phase,
The second method is preferred, and the fourth aspect is particularly desirable.

[Margin below]

[Examples] Hereinafter, the present invention will be explained in more detail with reference to Examples and Reference Examples, but the present invention is not limited to these Examples.

Preparation of standard solution of rabbit anti-anginatensin III IgG: Angiotensin ■ was combined with bovine serum albumin by a known method using glutaraldehyde.

This angiotensin - bovine serum concentration of rubumin is 6 to 9.
White rabbits were immunized with New Sealant with complete Freund's adjuvant at weekly intervals to obtain rabbit anti-angiotensin antiserum [Tanaka et al., Bioche
m, Biophs, Res, [:ommun,, 1
60, 40-45 (1989)).

From this antiserum, salting out with sodium sulfate and DEAE
Using cellulose, rgG is purified and I per unit volume is
gG was measured.

Pab”-peroxidase was created using the obtained total IgG [Tanaka et al., Biochem, Biophs,
Res.

Commun 9, supra], this label was adsorbed onto an angiotensin I-Sepharose 4B column. Of the peroxidase activity added to the column, 5.1% was adsorbed. Anti-anginatensin IIgG accounted for 5.1% of the total 1gG of antiserum.

This anti-anginatensin IIgG was diluted with rabbit serum to prepare standard solutions of rabbit anti-anginatensin IIgG at various concentrations.

Buffer: Buffer A: 0. IM! A thorium buffer solution of sodium chloride acid was prepared and designated as buffer solution A.

Buffer B: 5mM [l! A 0.1M sodium phosphate buffer containing DTA, pH 6.0, was prepared and designated as buffer B.

Buffer C: 0.1M sodium chloride, 0.1g/I!
Bovine serum albumin (Fraction V1 Armor, Kankakee, IL), 10mM containing 1.0mM magnesium chloride and 1.0g/β sodium azide
Prepare sodium phosphate buffer, pH 7.0, and add buffer C.
And so.

Buffer D: 1.0g/12 bovine serum albumin,
10mM sodium phosphate buffer containing 1.0mM magnesium chloride and 1,0g/Il sodium azide;
The pH was adjusted to 7.0 and used as a buffer solution.

β-D-galactosidase activity measurement: β-D-galactosidase activity is measured by 4-methylumbelliferyl β-
After reacting with D-galactoside as a substrate at 30°C for 150 minutes, fluorescence optical measurements were performed using a known method [Imayo et al., A
nn, CI in, Biochem.

, 21.310-317 (1984): ]. Fluorescence intensity was measured using O in which 10-8M 4-methylumbelliferone was dissolved, 1M glycine-NaOH buffer, pH 10.3.
was measured as a standard.

Example 1 1. Preparation of maleimide-avidin 03 ml of buffer solution AO in which 3 mg of avidin (avidin D, Vector Laboratories, California) was dissolved.
30 μl of a solution of 3.3 mM N-succinimidyl-6-7 reimidohexanoate (Isotope Kagaku Kenkyusho, Kumamoto) in N,N-dimethylformamide was added and reacted at 30° C. for 30 minutes. The reaction solution was purified using a Sephadex G-25 column (1, OX 30 cm) using buffer B as an eluent.

The amount of avidin has an extinction coefficient of 1.4 g-' at 280 nm;
1 itar, cm"", and the molecular weight was calculated as 68,000. 1.0 maleimide groups were bound per molecule of avidin.

2. Preparation of β-D-galactosidase-avidin β-D
- Maleimide-avidin 0
, 5 mg (7.4% mol) of buffer B O,
54- was added and reacted at 4°C for 20 hours. The reaction solution was purified using an Ultrogel AcA22 column (1.5 x 45 cm) using buffer C as an eluent.

1.2 avidins were bound per molecule of β-D-galactosidase.

3. Preparation of maleimide angiotensin I Mix 0.13 ml of distilled water in which 1.5 mg (1,2, Umol) of angiotensin 1 was dissolved and 1.2 ml of 0.1 M sodium phosphate buffer (pH 7,5). 55 to the mixture
0.13-mM N-succinimidyl-6-maleimidohexanoate in N,N-dimethylformamide
was added and reacted at 30°C for 300 minutes. The reaction solution was purified using a Sephadex G-10 column (1, OX 45) using buffer B as an eluent.

4. Preparation of glutathione-angiotensin I Maleimido-angiotensin I 0.69 mg (0.46μ
Buffer B containing 3.1 mM glutathione (0.2 ml) was added to 82 ml of buffer in which 3.1 mM glutathione was dissolved. Add 30℃, 3
It reacted for 00 minutes. After the reaction, 820 μl of a buffer containing 0.1M N-ethylmaleimide was added, and the reaction was carried out at 30° C. for 300 minutes.

5. Preparation of biotiny-glutathione-angiotensin ■ 4. Glutathione-angiotensin ■ solution 2.2 m
l! Add 130μl of 1.0M sodium hydroxide to
After adjusting the pH to 7.2, 0.2 rnl of a 34 mM biotin-N-hydroxysuccinimide solution in N,N-dimethylformamide was added, and the mixture was reacted at 30°C for 300 minutes. Sephadex G-10 column (1, OX 4
5 cm ) using buffer A as the eluent.

6. Preparation of β-D-galactosidase-avidin-biotinyl-glutathione-angiotensin■ β-D-galactosidase-avidin 0.22 mg (0
, 36 nmol) was dissolved in 1.5 ml of buffer C, and 73 μg of biotinyl-glutathione-angiotensin I was dissolved.
Buffer A0.3rd in which (3.6 nmol) was dissolved was added and reacted at 30°C for 3 hours. The reaction solution was purified using an Ultrogel AcA44 column (1, OX45Cm) using buffer C as an eluent.

Preparation 1 of Dinitrophenyl-Bovine Serum Albumin A thiol group is introduced into bovine serum albumin by a known method using S-acetylmercaptosuccinic anhydride, and a maleimide group is introduced into dinitrophenyl-lysine. reacted with [Kono et al., J, Cl1n, L
ab, Anal, 2.209-214 (1988
) ).

The amount of bovine serum albumin is determined by adjusting the extinction coefficient at 280 nm to 0.
63 g-', 1 iter, cm-', molecular weight 66
, 200. The amount of dinitrophenyl group is 3
The extinction coefficient at 60 nm is 17.400 mod', 1i
Calculated as ter, cm-'.

Absorbance measurements at 280 nm and 360 nm revealed that 5.5 dinitrophenyl groups were bound per molecule of bovine serum albumin.

2. Preparation of mercaptoacetyl-dinitrophenyl-bovine serum albumin Buffer A in which 3.7 mg of dinitrophenyl-bovine serum albumin was dissolved 1. Orrtg, 3JmMN-
N,N of succinimidyl-8-acetylthioacetate
-Add 0.1- of dimethylformamide solution, 30°C,
The reaction was carried out for 300 minutes. After the reaction, add 1M Tris-HCl buffer (
pH7,0) 50μβ, 0.1M B[1TA(f)
) 17.0) Add 50μβ and 75μ of 1M hydroxylamine solution (pH 7,0), react at 30°C for 15 minutes, and then remove the reaction solution using a Sephadex G-25 column (1, OX 30cm). Buffer B was used as the eluent for purification. There were 2.3 thiol groups bound per molecule of dinitrophenyl-bovine serum albumin.

3. Maleimide-avidin was prepared according to the previous section.

4. Preparation of dinitrophenyl-bovine serum albumin-avidin mercaptoacetyl-dinitrophenyl-bovine serum albumin 1.0 mg (15 nmol) was dissolved in buffer BO.
Add buffer solution 81.1d in which 5 nmol) was dissolved, and heat at 4°C.
, reacted for 200 hours. Transfer the reaction solution to Ultrogel AcA44
column (1,5x 100 cm) with buffer A
was purified as an eluate.

Absorbance measurements at 280 nm and 360 nm revealed that 1.1 avidin was bound per molecule of bovine serum albumin.

5. Preparation of dinitrophenyl-bovine serum albumin-avidin-biotinyl-glutathione-angiotensin I Dinitrophenyl-bovine serum albumin-avidin 48
Buffer A in which μg (0.36 nmol) was dissolved 1.
7 in, biotinyl-glutathione-angiotensin I 73,! Buffer A in which j g (36 nmol) was dissolved 0.3 ill! was added and reacted at 30°C for 3 hours. The reaction solution was purified using an Ultrogel AcA44 column (1, Ox 45 cm ) using buffer C as an eluent.

Rabbit (anti-dinitrophenyl-bovine serum albumin)
IgG was adsorbed onto a dinitrophenyl-bovine serum albumin-Sepharose 4B column and eluted at pH 2.5. This affinity-purified rabbit (anti-dinitrophenyl-bovine serum albumin) I8GI7) 0.1 g/A solution was applied to the surface of 3.2 mm polystyrene beads using a known method [Ishikawa et al., 5cand, J. Immu.
nol, Vol. 8 (Supplement 7), p. 43 (197 g)] to make it insolubilized by physical adsorption.

Measurement of rabbit anti-anginatensin IIgG Standard solution of rabbit anti-anginatensin IIgG 20μβ 0.33M
Dinitrophenyl-bovine serum albumin-avidin-biotinyl-glutathione-engiotensin I (100 fmo) dissolved in buffer D130 μβ containing NaCl
l) and β-D-galactosidase-avidin-biotinyl-glutathione-angiotensin I (100f
rnol) at 20° C. for 3 hours with shaking. After incubation, the reaction solution was affinity-purified with rabbit (anti-dinitrophenyl-bovine serum albumin) IgG insolubilized solid phase (
2 pieces) at 20℃ for 3 hours with shaking, then 4
The mixture was allowed to stand overnight at ℃. Then, affinity-purified rabbit (anti-dinitrophenyl-bovine serum albumin) Ig
G-insolubilized solid phase was added to buffer D2 containing 0.1 M NaCl.
After washing twice with ml, the bound β-D-galactosidase activity was measured.

The results are shown in Figure 1.

Example ■ Dinitrophenyl-bovine serum albumin-avidin-biotinyl-glutathione-angiotensin 11β-D
- Galactosidase-avidin-biotinyl-glutathione-angiotensin and affinity purified rabbit (anti-dinitrophenyl-bovine serum albumin) Ig
The G-insolubilized solid phase was prepared by the method of Example I.

Measurement of rabbit anti-anginatensin IIgG 20μl of standard solution of rabbit anti-anginatensin IIgG is 0.33M
Dinitrophenyl-bovine serum albumin-avidin-biotinyl-glutathione-angiotensin I (100 fmo
l) and β-D-galactosidase, bidin, biotinylated glutathione, and angiotensin I (100f
mol) at 20°C for 3 hours with shaking. After incubation, the reaction solution was affinity-purified with rabbit (anti-dinitrophenyl-bovine serum albumin) IgG insolubilized solid phase (2
Incubate at 20℃ for 3 hours with shaking, then at 4℃
It was left undisturbed overnight. Then, affinity purified rabbit (anti-dinitrophenyl-bovine serum albumin) IgG
The insolubilized solid phase was added to buffer D2 containing 0.1 M NaCl.
- was washed twice. After washing, affinity-purified rabbit (anti-dinitrophenyl-bovine serum albumin) IgG
The insolubilized solid phase was treated with 1.0 mM dinitrophenyl-L-
Buffer D15 containing IJ Gin and 0.3 M NaC]
Incubate at 20°C with 0 μA for 1 hour with shaking. After incubation, the affinity-purified rabbit (anti-dinitrophenyl-bovine serum rubumin) IgG insolubilized phase was removed, and the β-D-galactosidase activity contained in the solution was measured.

The results are shown in Figure 1.

Example ■ Dinitrophenyl - bovine serum rubumin - biotin - biotinylated glutathione - angiotensin ■, β-D
- Galactosidase-Avidin-Biotinyl-Glutathione-Angiotensin 1 and Affinity Purified Rabbit (Anti-dinitrophenyl-Bovine Serum Rubumin) IgG
The insolubilized phase was prepared by the method of Example I.

Affinity purification (anti-rabbit IgG) Preparation of IgG insolubilized solid phase (anti-rabbit IgG) After adsorbing IgG to a rabbit IgG-Sepharose 4B column, it was eluted at pH 2.5. This affinity purified rabbit (anti-rabbit IgG) Ig
Using a 0.1 g/β solution of G, a known method [Ishikawa et al., 5cand.

J. Immunolo, supra] and insolubilized by physical adsorption.

Measurement of rabbit anti-anginatensin IIgG 20 μm standard solution of rabbit anti-anginatensin IIgG was diluted with 0.33 M
Dinitrophenyl-bovine serum albumin-avidin-biotinyl-glutathione-angiotensin I (100 fm) dissolved in 130 μA of buffer D containing NaCl.
ol) and β-D-galactosidase-avidin-biotinyl-glutathione-angiotensin I (100
fmol) at 20°C for 3 hours with shaking. After incubation, the reaction solution was affinity-purified with rabbit (anti-dinitrophenyl-bovine serum albumin) IgG insolubilized solid phase (
2 pieces) at 20℃ for 3 hours with shaking, then 4
The mixture was allowed to stand overnight at ℃. Then, affinity-purified rabbit (anti-dinitrophenyl-bovine serum albumin) Ig
G insolubilized solid phase to 0. Buffer D containing I M NaCl
Washed twice with 2d. After washing, affinity purified rabbit (anti-dinitrophenyl-bovine serum albumin) Ig
G insolubilized solid phase with 1.0 mM dinitrophenyl-L-
Buffer D15 containing IJ lysine and 0.3 M NaCl
0 μl and affinity purified (anti-rabbit IgG) Ig
Incubate with G insolubilized solid phase (2 pieces) at 20°C for 1 hour while shaking. After incubation, the affinity-purified rabbit (anti-dinitrophenyl-bovine serum albumin) IgG insolubilized solid phase was removed, and the mixture was further incubated at 20° C. for 2 hours with shaking.

Affinity purification (anti-rabbit IgG) IgG insolubilized solid phase Buffer D2m12 containing I M NaCl
After washing twice with , the bound β-D-galactosidase activity was measured.

The results are shown in Figure 1.

Example (2) Affinity-purified rabbit (anti-dinitrophenyl-bovine serum albumin) IgG insolubilized solid phase was prepared by the method of Example (2). Affinity purification (anti-rabbit IgG)
The IgG insolubilized solid phase was prepared by the method of Example ①.

Preparation of Roux-Angiotensin I 1, Preparation of Biotinylated-Angiotensin I Angiotensin I 1. Mix 0.18-mL of distilled water in which Omg (0.77 μmol) was dissolved and 1.6-mL of buffer A, and add 0.18rnf of 44 mM biotin-N-hydroxysuccinimide in N,N-dimethylformamide to the mixture.
! was added and reacted at 30°C for 30 minutes. Sephadex G-10 column (1, OX 45cm) from the reaction solution
was used for purification using buffer A as the eluent.

2. Preparation of β-D-galactosidase avidin-biotinyl-angiotensin I Add biotiny-angiotensin to buffer C1.5 mff in which 0.22 mg (0.36 nmol) of β-D-galactosidase avidin prepared in Example ① was dissolved. I
Buffer A in which 5.5 μg (3.6 nmol) was dissolved
0.3- was added and reacted at 30°C for 3 hours. From the reaction solution) Logel AcA44 column (1, OX 45cm
), using buffer C as the eluent.

3 ml of buffer A0, in which 5.5 μg (3.6 nmol) of biotinylated angiotensin I was dissolved in 7 mf of buffer A L, in which 48 μg (0.36 nmol) of dinitrophenyl-bovine serum albumin-avidin prepared in Example I was dissolved. was added and reacted at 30°C for 30 minutes. From the reaction solution, remove Ultrogel AcA44 column (1,
OX 45 cm ) using buffer C as the eluent.

Measurement of rabbit anti-anginatensin IIgG 20μβ standard solution of rabbit anti-anginatensin IIgG was mixed with 0.33M
Dinitrophenyl-bovine serum albumin-ahisin-biotinylated angiotensin I (100 fmol) and β dissolved in buffer D130μj2 containing NaCl
-D-galactosidase-avidin-biotinylated-angiotensin I (100 fmol) and incubated at 20°C for 3 hours with shaking. After incubation, the reaction solution was incubated with affinity-purified rabbit (anti-dinitrophenyl-bovine serum albumin) IgG insolubilized solid phase (2 pieces) at 20°C for 3 hours with shaking, and then allowed to stand at 4°C overnight. after that,
Affinity-purified rabbit (anti-dinitrophenyl-bovine serum albumin) IgG insolubilized solid phase at 0. I M
Washed twice with buffer D2rnl containing NaCl. After washing, add 1.0 m of affinity-purified rabbit (anti-dinitrophenyl-bovine serum albumin) IgG insolubilized solid phase.
M dinitrophenyl-lysine and 0.3 M Na
Affinity purification with 150 μl of buffer D containing C1 (
anti-rabbit IgG) with IgG insolubilized solid phase (2 pieces)
Incubate at 0°C for 1 hour with shaking. After incubation, the affinity-purified rabbit (anti-dinitrophenyl-bovine serum albumin) IgG insolubilized solid phase was removed, and further incubated at 20°C for 2
The mixture was kept warm under shaking for an hour. Affinity purification (anti-rabbit I
gG) After washing the IgG insolubilized surface twice with buffer D2- containing 0.1 M NaCl, bound β-D-galactosidase activity was measured.

The results are shown in Figure 2.

Example 1. Synthesis of biotinyl-HIV epitope peptide (A) Boc-Arg(Tos)-Arg(Tos)
-Gln-Leu-Leu-Gly-11e-Trp
(For) -G Iy-Cys (MBzl) -3
et (Bzl)-G 1y-Lys(CIZ)-Le
u-1ie-Cys (MBzl)-0CH2-(=)
- Peptides labeled as RBSIN are manufactured by Applied Biosystems, Inc. in the United States using the peptide solid phase synthesizer (AB).
I430A) according to the manufacturer's instructions. Briefly, Boc-Cy with the first carboxy-terminal amino acid attached to a solid-phase chloromethylated resin.
s (MBzl)-0CL-@;? -RBSIN (0
,5 mmole/g) 1.0 g as starting material,
The peptide chain was extended by sequentially attaching amino acids from the carboxy terminus. Among the raw amino acids used, Lys, Ser, Cys, Trp, which have functional groups in their side chains,
Each Arg was protected as follows.

Lys(CIZ): N2-2-Chlorobenz
Yloxycarbonyl.

5er (Bzl): 0-Benzyl.

Cys(MBzl): S-4-Nethy]benz
yl.

Trp(For): N1′″-Forn+yl.

Arg(Tos): N'-Tosyl, and the α-amino group is a Boc group (Nα-t-Butyloxycarb
onyl).

Trifluoroacetic acid (TPA) was used to cleave the Bo group at each step during the synthesis, and dicyclohexylcarbodiimide (DCCD) was used to form a peptide bond. The finally synthesized protected peptide resin was taken out from the apparatus and dried to obtain 1.99 g of solid.

(B) Biotinyl-Arg (Tos>-Arg
(Tos)-Gin-Leu-LeuGly-11a-
Trp (For) -G 1y-Cys (MBzl
) -3er (Bzl) -Gly-Lys (CI
Z) -Leu-11e-Cys (MEtz 1)
-4 solids synthesized with DC82 RBS I N (A)
0% TFA/CH2Cl□ and (i, 2 N HCI/
The Boc- group is cleaved by treatment with Dioxane. The acid and solvent are removed under reduced pressure, and the resin is thoroughly washed with CH2Cl2 and dried on a glass filter. This and 1-Hydroxybenzotriazole
O, 28g 1 biotin 0.5g dimethylformamide (DMF) 50rd! 0.37 ml of water-soluble carbodiimide was added under cooling and stirring. added. After reacting overnight at room temperature and confirming that the ninhydrin reaction was negative, the resin was placed on a glass filter, thoroughly washed with CH2Cl2, and dried to obtain 2.0 g of biotinylated protected peptide resin.

(C) Biotinyl-8rg-Arg-Gln
-Leu-Leu-Glylle-Trp-Gly-C
ys-3er-Gly-Lys-Leu=11e-Cy
Biotinylated protected peptide resin 1 synthesized in s(B),
0.3 g (0.25 mmol) of p-cresol.
33-butanedithiol 1. Om! ! The resin and all the protecting groups were cleaved by treatment with hydrogen fluoride (HF) 5.4-containing at -5°C for 60 minutes. After distilling off excess hydrogen fluoride under reduced pressure, additives are removed by washing with ether. The residue was dissolved in 5% acetic acid, applied to a Dowex 1X2 (acetate) column (10 g), and the flow-through fraction was lyophilized to obtain 330 mg of powder. This one is 0.
The mixture was dissolved in 200 i of 0.05% ammonia water, and a 0.01M potassium ferricyanide solution was added dropwise, and the end point was the point at which the yellow color of the reaction solution did not fade. The peptide cyclized by oxidizing cysteine was analyzed by reverse-phase HPLC at 10
~60% CH3[:N-820 (containing 0.1% TF8) was purified by gradient. The 30-45% gradient concentration elution fraction was purified again using the same HPLC system.
Finally, 32 mg of the target product was obtained. Amino acid analysis confirmed that all the constituent amino acid residues had been introduced into this product as theoretically expected. Furthermore, since it was completely adsorbed on the avidin column, it was considered that it was biotinylated.

2. Preparation of β-D-galactosidase-avidin-biotene-HIV epitope peptide 0.15 mg of β-D-galactosidase avidin prepared in Example 1
Biotinyl-HIV epitope peptide 5, OAt g in buffer C1,0- in which (0,25 nmol) was dissolved
5.0 μA of buffer A in which (2.5 nmol) was dissolved was added, and the mixture was incubated at 30° C. for 2 hours. The reaction solution was transferred to a column of Ultrogel AcA 44 (1, Ox 45cm
) to purify and isolate β-D-galactosidase-avidin-biotin-HIV epitope peptide (0.09 mg).

Reference example (anti-rabbit IgG) Preparation of Fab'-peroxidase (anti-rabbit IgG) Fab' was prepared using a known method [Hashida et al., J. Appl. Bioch
Em., 6.56-63 (1984)].

Angiotensin I - Preparation of bovine serum albumin insolubilized solid phase 1, Mercaptoacetyl - Preparation of bovine serum albumin Buffer A0 in which 2.0 mg of bovine serum albumin was dissolved
．． 40 μl of a solution of 8.8 mM N-succinimidyl-8-acetylthioacetate in N,N-dimethylformamide was added to 4 ml, and the mixture was reacted at 30° C. for 300 minutes. After the reaction, the reaction solution was adjusted to 1.0M)! J Soot-acid buffer (pH 7゜0
) 40 μβ, 0.1 M BDTA (pH 7,0) 4
0μβ and 1.0M hydroxylamine hydrochloride solution (p
H7,0) was reacted with 60 μp at 30° C. for 15 minutes. Sephadex G-25 column (1,0
x 30 cm ) using buffer B as the eluent. There were 6.0 thiol groups bound per molecule of bovine serum albumin.

2. Angiotensin ■ - Preparation of bovine serum albumin Mercaptoacetyl - Bovine serum albumin 1.5 mg
Example ■
0.34 mg of maleimide-avidin (23
Add 83 ml of buffer solution in which 0 nmol) was dissolved, and
t, reacted for 300 minutes. Ultrogel AcA from the reaction solution
44 columns (1,5x 45 cm) with 0.1
0.1 M sodium phosphate buffer (p
H7,5) was purified as an eluent.

Due to the decrease in thiol groups, 6.0 angiotensin ■ were bound per molecule of bovine serum albumin.

3. Angiotensin I - Preparation of solid phase insolubilized with bovine serum albumin Angiotensin ■ - 0°1 g of bovine serum albumin/
1 solution onto the surface of polystyrene beads in 3.2 mm using a known method [Ishikawa et al., 5cand, J.

Immunol, supra] was insolubilized by physical adsorption.

Measurement of rabbit anti-anginatensin IIgG :33
130μ of 10mM IJ buffer containing M NaCl and 1.0g/41' bovine serum fulbumin
β was mixed. The mixed solution was kept at 37° C. with shaking for 3 hours together with an angiotensin ①-bovine serum albumin insolubilized solid phase. Thereafter, the angiotensin I bovine serum albumin-insolubilized solid phase was washed twice with 2 d of 10 mM sodium phosphate buffer (pH 7,0) containing 0.1 M NaCl. After washing, the insolubilized solid phase of giotensin I-bovine serum T rubumin was dissolved in 0.3 M NaCl and 1.0 g/
10mM containing j2 bovine serum albumin! Sodium J phosphate buffer pH 7.0) Dissolved in 150 μp (
anti-rabbit IgG) F-b'-peroxidase (50
ng) at 37°C for 3 hours with shaking. After incubation, the angiotensin I-bovine serum albumin insolubilized solid phase was washed in the same manner, and the bound peroxidase activity was measured.

Peroxidase activity was measured optically by fluorescence using 3-(4-hydroxyphenyl)propionic acid as a hydrogen donor. The fluorescence intensity was measured using 1 mg/β quinine dissolved in 50 mM sulfuric acid as a standard.

The results are shown in Figure 2.

[Brief explanation of drawings]

FIG. 1 shows the measurement results of Example I, (1) and (2). FIG. 2 shows the measurement results of Example (2) and Reference Example. In both figures, the horizontal axis represents the serum concentration of rabbit anti-angiotensin IIgG, which is the measurement target, and the vertical axis represents the fluorescence intensity representing the amount of the detected label.

Claims (3)

[Claims] (1) A labeled substance represented by the general formula (a) or (b) through the binding of biotin and avidin or the binding of biotin and streptavidin. (a): X-biotin-avidin-labeled product (b): X-biotin-streptavidin-labeled product (
In the formula, X represents an immunoreactive substance selected from the group consisting of antigen, antibody, and anti-antibody. ) (2) The labeled substance according to claim (1), wherein the immunoreactive substance according to claim (1) is a synthetic peptide. (3) A step of forming a sandwich-like immune complex containing the target substance in the specimen on a solid phase using the label and solid phase substance according to claim (1) or (2). An immunoassay method for the target substance.