JPH0829419A - Antigenic substance detecting method - Google Patents

Abstract

PURPOSE:To provide an antigenic substance detecting method by which excellent detecting sensitivity can be exhibited. CONSTITUTION:A first antibody component 11 and a second antibody component 12 are joined together, and a linker bivalent antibody 1 is perpared. Next, an antigenic substance 2 is specifically joined to the first antibody component 11 through a first hapten 21. An alkali phosphatase 7 labeled by a second hapten 72 is specifically joined to the second antibody component 12. Next, a fluorescent material 3 being the substrate is reacted with the alkali phosphatase 7, and a signal is generated, and this is detected.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for detecting an antigenic substance such as nucleic acid and protein, and more particularly to a method for binding an antigenic substance to a fluorescent substance via an antibody.

[0002]

2. Description of the Related Art As a method for detecting a nucleic acid DNA having a specific base sequence, as shown in FIG.
91, probe gene 9 labeled with biotin 93
2, then, an excess of streptavidin 94 is bound to the probe gene 92 by an antigen-antibody reaction, and further, a biotinylated enzyme 95 is bound to the remaining active site of the bound streptavidin 94, and the enzyme activity is There is a way to detect. This detection method is generally performed in a solution, a filter, or a living tissue.

[0003]

However, in the above-described conventional detection method, as shown in the right part of FIG. 7, biotin 93 (B) on the probe gene 92 is present at all active sites of streptavidin 94 (S). Bind to each other, and biotinylated enzyme 95 (E) becomes streptavidin 9
There is a risk that it will not bind to 4.

In this case, the detection sensitivity deteriorates and the nucleic acid D
It is difficult to accurately detect NA. Also, the method of detecting a protein having a specific amino acid sequence has the same problem as described above.

In view of the above conventional problems, the present invention aims to provide a method for detecting an antigenic substance which exhibits excellent detection sensitivity.

[0006]

According to the present invention, a bivalent antibody linker is prepared by previously binding a first antibody component that specifically binds to an antigenic substance and a second antibody component that specifically binds to a signal substance. Next, an antigenic substance is bound to the first antibody component of the divalent antibody linker, a signal substance is bound to the second antibody component of the divalent antibody linker, and then a signal is generated from the signal substance. Then, the method for detecting an antigenic substance is characterized by detecting the signal.

What is most noticeable in the present invention is a bivalent antibody linker comprising a first antibody component that specifically binds to an antigenic substance and a second antibody component that specifically binds to a signal substance. Is to use.

The bivalent antibody linker has a bivalent active site composed of the first antibody component and the second antibody component. The first antibody component and the second antibody component are sites that specifically bind to the antigenic substance and the signal substance, respectively.

The first antibody component is specifically bound to the antigenic substance via, for example, the first hapten. Examples of the first hapten include biotin (biotin), dioxigenin (digoxigenin),
dinitrophenol (DNP) (dinitrophenol), trinitrophenol (TNP)
(Trinitrophenol), fluorescein
(Fluorescein), tetramethylrhod
amine B isothiocyanate (TR
ITC) (tetramethylrhodamine B isothiocyanate), rhodamine (rhodamine), Tex
as Red (Texas Red), Lucifer
Yellow (Lucifer Yellow), 4,4-di
Fluoro-5,7-dimethyl-4-bor
a-3a, 4a-diaza-s-indacene-
3-propionyl (BODIPY) (4,4-difluoro-5,7-dimethyl-4-bora-3a, 4a-
Diaza-s-indacene-3-propionyl), 7-n
itrobenz-2-oxa-1,3-diazol
-4-yl (NBD) (7-nitrobenz-2-oxa-1,3-diazol-4-yl), (o-11- (5
-Dimethylaminonaphthalene
-1-sulfonyl) amino) undecyl
(Dansyl), coumarin, coumarin, DNA, RNA, virus or the like can be used.

For example, when the first hapten is biotin, an anti-biotin antibody or streptavidin can be used as the first antibody component. Streptavidin is a substance that specifically binds to biotin similarly to the anti-biotin antibody, and therefore acts as an antibody of biotin. When the first hapten is digoxigenin, an anti-digoxigenin antibody can be used as the first antibody component. When the first hapten is DNP, an anti-DNP antibody can be used as the first antibody component.

The second antibody component is specifically bound to the signal substance via the second hapten, for example. Examples of the second hapten include biotin (biotin), dioxigenin (digoxigenin),
dinitrophenol (DNP) (dinitrophenol), trinitrophenol (TNP)
(Trinitrophenol), fluorescein
(Fluorescein), tetramethylrhod
amine B isothiocyanate (TR
ITC) (tetramethylrhodamine B isothiocyanate), rhodamine (rhodamine), Tex
as Red (Texas Red), Lucifer
Yellow (Lucifer Yellow), 4,4-di
Fluoro-5,7-dimethyl-4-bor
a-3a, 4a-diaza-s-indacene-
3-propionyl (BODIPY) (4,4-difluoro-5,7-dimethyl-4-bora-3a, 4a-
Diaza-s-indacene-3-propionyl), 7-n
itrobenz-2-oxa-1,3-diazol
-4-yl (NBD) (7-nitrobenz-2-oxa-1,3-diazol-4-yl), (o-11- (5
-Dimethylaminonaphthalene
-1-sulfonyl) amino) undecyl
(Dansyl), coumarin, coumarin, DNA, RNA, virus or the like can be used.

For example, when the second hapten is digoxigenin, an anti-digoxigenin antibody can be used as the second antibody component. The second hapten is DNP
In this case, an antibody DNP antibody can be used as the second antibody component. When the second hapten is biotin, an anti-biotin antibody or streptavidin can be used as the second antibody component.

The first antibody component and the second antibody component may be of the same kind, but preferably of different kinds. This allows the antigenic substance to be detected with high sensitivity without binding of the antigenic substance to both the first antibody component and the second antibody component.

The first antibody component and the second antibody component form a bivalent antibody linker by chemical bonding. The first antibody component and the second antibody component are bound by, for example, a thiol group and a maleimide group. As a method for forming the above-mentioned bivalent antibody linker, for example, there is a method in which the inactive sites of the first antibody component and the second antibody component are cleaved or modified, and the cleaved or modified sites are bound by a chemical reaction.

Specifically, for example, the inactive site of the first antibody component is maleimidated, and the inactive site of the second antibody component is cleaved by an enzymatic reaction such as pepsin to expose a thiol group at its end. Then, the first antibody component and the second antibody component can be combined as shown in FIGS. In the figure, "Fab" means the first antibody component or the second antibody component at the amino group end, and "Fab '" means the first antibody component or the second antibody component at the thiol group end.

The signal substance is, for example, a liposome containing a fluorescent substance or an enzyme having a substance capable of becoming a fluorescent substance as a substrate. The fluorescent substance contained in the liposome is a covalent fluorescent substance bound to the liposome by a covalent bond, a non-covalent fluorescent substance encapsulated in the liposome, or the like. A liposome is a kind of artificial lipid membrane,
It is a closed vesicle composed of a bilayer membrane. A large number of fluorescent substances can be incorporated into the liposome, and the fluorescence intensity can be increased.

Further, the fluorescent substance generally has a property that the fluorescence intensity increases several tens of times in a hydrophobic atmosphere. Moreover, the fluorescent substance is well soluble in an aqueous solution. Therefore, the fluorescent substance is
In particular, the fluorescence intensity is amplified 50 to 200 times by being incorporated into the liposome having both hydrophilicity and hydrophobicity. Therefore, the detection sensitivity of the analyte is improved.

The liposome can incorporate a hydrophilic substance into the aqueous phase of the membrane and a hydrophobic substance into the molecular membrane. Further, various substances can be directly bound to the liposome. Therefore, substances that are difficult to be used can be easily incorporated into the liposome, the selection range of fluorescent substances is widened, and it can be applied to the multicolor fluorescence detection method.
Examples of liposomes include those formed from 3000 to 4000 phospholipids.

The fluorescent substance contained in the liposome is, for example, a covalent fluorescent substance covalently bound to the liposome or a non-covalent fluorescent substance encapsulated in the liposome. Since the above-mentioned covalently binding fluorescent substance is covalently bound to a molecule (for example, phospholipid) constituting the liposome, it can be bound to the liposome to the same extent as the number of reactive groups of the number of molecules. On the other hand, when the number of covalently bound fluorescent substances bound to one liposome is less than 10, the fluorescence intensity of the fluorescent aggregate is weakened, and the detection sensitivity of the analyte may be lowered.

Examples of the covalently binding fluorescent substance include compounds having a coumarin skeleton, compounds having a naphthalene skeleton, compounds having a fluorescein skeleton, compounds having a perylene skeleton, compounds having a pyrene skeleton,
There are compounds having an anthracene skeleton, compounds having a rhodamine skeleton, and the like.

As the compound having a coumarin skeleton, 4
-Methylumbelliferyl phosphocholine, 7-hydroxycoumarin-3-carboxylic acid-octadecyl ester and the like can be used. As the compound having a naphthalene skeleton, NBD-labeled phosphatidylethanolamine, dansyl-labeled phosphatidylethanolamine, or the like can be used.

As the compound having a fluorescein skeleton, fluorescein-labeled phosphatidylethanolamine or the like can be used. As the compound having a perylene skeleton, 1-hexadecanoyl-2- (3-perylenedodecanoyl) -sn-glycero-3-phosphocholine or the like can be used.

As the compound having a pyrene skeleton, 1,
2-bis- (1-pyrenedecanoyl) -sn-glycero-3-phosphocholine, 12- (1-pyrendecanoyl) phosphocholine and the like can be used. As the compound having an anthracene skeleton, 12- (9-anthroyloxy) -stearic acid or the like can be used. As the compound having a rhodamine skeleton, rhodamine-labeled phosphatidylethanolamine or the like can be used.

The non-covalently bound fluorescent substance is not bound to the molecules constituting the liposome, but is incorporated in the aqueous phase of the liposome membrane or in the hydrophobic molecular membrane. 1
The number of non-covalently bound fluorescent substances incorporated in each liposome is preferably 10 to 10,000. When it is less than 10, the fluorescence intensity of the fluorescent aggregate is weak and the detection sensitivity of the analyte may be lowered. On the other hand, 1000
When it exceeds 0, the water solubility of the liposome is lowered and the size thereof is increased, so that the steric hindrance may be considerably increased.

As the above non-covalently binding fluorescent substance, 4-
Methylumbelliferone, 7-hydroxy-4-biphenyl-umbelliferone, 3-hydroxy-2-naphthoic acid 2-phenylanilide, 3-hydroxy-2-naphthoic acid-2,4-dimethylanilide, 6-bromo- 2-
Hydroxy-3-naphthoic acid 2-methylanilide, 3-
Hydroxy-2-anthranoic acid 2-methylanilide, pyrene, fluorescein, perylene, rhodamine, Texas red, or a DNA-binding fluorescent substance may be used. As a DNA-binding fluorescent substance, Hoechst3
3258 (made by Hoechst Japan), DAPI (SI
GMA), ethidium bromide and the like can be used.

Examples of the enzyme having as a substrate a substance that can be a fluorescent substance include alkaline phosphatase, acid phosphatase, peroxidase, esterase, glucosidase, and phospholipase. Alkaline phosphatase is 3-hydroxy-N-
2'-biphenyl-2-naphthalene
carboxamide phosphate est
er (HNPP) and Fast Red TR are used as substrates.

Examples of the above-mentioned antigenic substance include substances having antigenicity such as tissue cells, viruses, nucleic acids, and proteins.

[0028]

In the present invention, an antigenic substance is detected by using a bivalent antibody linker. The bivalent antibody linker is a first antibody component that specifically binds to an antigenic substance,
A second antibody component that specifically binds to the signal substance. Therefore, the bivalent antibody linker has active sites for the antigenic substance and the signal substance separately. Therefore, when the bivalent antibody linker binds to the antigenic substance, the active site that always binds to the signal substance remains.

Therefore, when the antigenic substance directly binds to the first antibody component, the same number of signal substances as the number of the antigenic substance and the antigenic substance binds to the first antibody component via the first hapten. In this case, the same number of signal substances as the number of the first hapten can be bound with certainty. Therefore,
Unlike the conventional method, the antigenic substance binds to all the active sites of the antibody, and the active site of the antibody is not deactivated.
Therefore, excellent detection sensitivity is exhibited.

As described above, according to the present invention, it is possible to provide a method for detecting an antigenic substance that exhibits excellent detection sensitivity.

[0031]

【Example】

Example 1 Regarding a method for detecting an antigenic substance according to an example of the present invention,
This will be described with reference to FIGS. In this example, a human peripheral blood lymphocyte chromosome to which a biotin-labeled probe gene is complementarily bound is detected as the antigenic substance 2. As shown in FIG. 1, the outline of the detection method is as follows. First, an anti-biotin antibody (anti-B) as the first antibody component 11 and an anti-digoxigenin antibody (anti-D) as the second antibody component 12 are combined, Prepare the bivalent antibody linker 1. Next, biotin (B) as the first hapten 21 was added to the first antibody component 11.
The human peripheral blood lymphocyte chromosome 29 is specifically bound via the probe gene 20 labeled with.

The anti-digoxigenin antibody as the second antibody component 12 includes alkaline phosphatase 7 labeled with digoxigenin (D) as the second hapten 72.
Is specifically bound. Next, the alkaline phosphatase 7 is reacted with the fluorescent substance 3 which is its substrate to generate a signal, which is detected.

The above detection method will be described in detail below. (1) Preparation of bivalent antibody linker Modification of anti-digoxigenin antibody Anti-digoxigenin antibody (Boehringer) 2 mg
And phosphate-sodium hydroxide buffer (0.1M.
7) Dissolve in 300 μl, and add SMCC (Succ
inimidyl-4- (N-maleimideme
thyl) -cyclohexane-1-carbo
xylate / DMF, 5mg / 250μl) 10μ
l, and let stand at 30 ° C. for 30 minutes.

Then, after centrifuging this, the supernatant was separated with Seph.
adex G-25 (column size: 1.0 x 15c
m: 0.1 M phosphoric acid-sodium hydroxide, pH 6) for desalting by gel filtration. Eluted components of the polymer were collected and concentrated to 200 μl by centrifugal ultrafiltration to obtain a maleimidated anti-digoxigenin antibody solution.

Modification of anti-biotin antibody 3 mg of anti-biotin antibody (manufactured by Vector) was dialyzed against 0.1 M acetic acid-NaOH buffer (pH 4.5), 0.2 mg of pepsin (manufactured by Sigma) was added, and the mixture was incubated at 37 ° C. It was left standing for 16 hours. The precipitate was removed by centrifugation, and the supernatant was gel-filtered (Ultrogel AcA44, column size:
1.0 × 45 cm, buffer: 0.1 M phosphoric acid-sodium hydroxide pH: 7).

The elution fraction of the anti-biotin antibody protein was collected and ammonium sulfate was added at a concentration of 0.66 g / ml (wt%) to precipitate the anti-biotin antibody (salting out). After centrifugation, the precipitate was added to a phosphate-sodium hydroxide buffer solution (0.
1 mol pH: 6) Dissolve in 0.4 ml, add 0.1 g / ml sodium azide 4 μl for preservative, and add F
(Ab ') ₂ fragment solution.

The F (ab ') ₂ fragment solution was added with 0.
1M mercaptoethylamine hydrochloride (0.1M phosphoric acid-sodium hydroxide buffer pH: 6,5 mM ED
50 μl of TA) was added, and the mixture was allowed to stand at 37 ° C. for 90 minutes. After gel filtration, the variable part simple substance (Fab ') fractions were collected and concentrated to 0.4 ml by salting out to obtain a thiolated anti-biotin antibody solution.

In the binding reaction between the anti-digoxigenin antibody and the anti-biotin antibody, a maleimidated anti-digoxigenin antibody solution and a thiolated anti-biotin antibody solution were mixed and incubated at 30 ° C. for 1 hour, followed by gel filtration (Ultrogel Ac
A44, 0.1M phosphoric acid-sodium hydroxide buffer,
0.1 M NaCl: pH 7) was performed to separate the divalent antibody linker (see FIG. 3) in which the anti-digoxigenin antibody and the anti-biotin antibody were bound at a ratio of 1: 1.

(2) DNA using a bivalent antibody linker
Detection of human peripheral blood lymphocytes was denatured at 75 ° C. for 3 minutes with a denaturing solution, and then in situ hybridization was performed using the human c-myc gene labeled with biotin by nick translation as a probe DNA. Next, this is 50% formami
Excess probe DN after washing with de, 2XSSC
A was removed.

Next, blocking treatment is performed with a skim milk powder aqueous solution to bind the anti-biotin antibody active site of the bivalent antibody linker to biotin of the probe DNA, and
Digoxigenin-labeled alkaline phosphatase was bound to the active site of the anti-digoxigenin antibody.

Next, a fluorescent substance which is a substrate of alkaline phosphatase, namely 3-hydroxy-N-2'-b.
iphenyl-2-naphthalenecarb
oxamide phosphate ester (H
NPP) and Fast Red TR (manufactured by Sigma) were reacted at room temperature four times for 30 minutes. Chromosomes were counterstained with Hoechst-quinacrine. Slides were mounted with mounting solution. Next, when the chromosome was observed using a fluorescence microscope, as shown in Fig. 2, a strong red fluorescence signal (white portion in the photograph) on chromosome 8 could be detected, and at the same time, the fluorescence staining pattern of the chromosome. Was detected.

On the other hand, as a comparative example (general method), streptavidin was used in place of the bivalent antibody linker of this example for detection, but no specific fluorescent signal could be detected. From this, it is clear that the detection method of this example was able to detect human peripheral lymphocyte chromosomes with higher sensitivity than ever before.

Example 2 This example is a method for detecting alkaline phosphatase as an antigenic substance, and uses a fluorescent substance encapsulated in liposomes as a signal substance. Further, an anti-digoxigenin antibody is used as the first antibody component that specifically binds to the antigenic substance, and an anti-biotin antibody is used as the second antibody component that specifically binds to the signal substance. Furthermore, in this example, the alkaline phosphatase used as the signal substance in Example 1 is used as the antigenic substance.

The outline of the above detection method will be described with reference to FIG. 5. First, the fluorescent substance 30 was covalently bonded to the liposome 39 and labeled with biotin as the second hapten 22. Rhodamine was used as the fluorescent substance 30. Rhodamine is a covalently bound fluorescent substance that is covalently bound to the liposome 39.

Next, on a slide glass, the anti-alkaline phosphatase antibody 40 was immobilized on the beads 4 to bind alkaline phosphatase as the antigenic substance 25, and the anti-alkaline phosphatase as the first hapten 71 was labeled with digoxigenin (D). Alkaline phosphatase antibody 70 was reacted.

Next, the above-mentioned digoxigenin was used in Example 1.
The anti-digoxigenin antibody of the bivalent antibody linker 1 of 1. was bound as the first antibody component 110. Further, the anti-biotin antibody of this bivalent antibody linker 1 was used as the second antibody component 120 and bound to the above-mentioned liposome 39. Then, the fluorescent substance 30 covalently bonded to the liposome 39 was detected.

This will be described in detail below. Preparation of liposome and encapsulation of fluorescent substance in liposome Lecithin (1 μmol), cholesterol (0.65 μm)
mol), rhodamine-DHPE (0.25 μmol)
And biotin-X-DHPE (0.1 μmol) were dissolved in 5 ml of chloroform as a solvent, placed in an eggplant-shaped flask, stirred well, and then the solvent was removed by an evaporator.

Again, 3 ml of chloroform as a solvent was put in this eggplant-shaped flask, and after stirring, the solvent was removed by a rotary evaporator. This was repeated 3-5 times. Then, a thin film liposome was formed on the wall surface of the flask, and this was transferred to a vacuum desiccator and vacuum sucked with a vacuum pump for one day to completely remove the solvent. A fluorescent substance (rhodamine) is bound to the surface of the prepared liposome by a covalent bond.

Then, after replacing the inside of the flask with nitrogen, 5 ml of a buffer (0.1 mol / liter phosphate buffer, 5 mM EDTA, pH: 6) was added to about 6
Immerse it in a warm water bath at 0 ° C and use a bath-type sonicator for 20
Sonicated for minutes. Then, a small-diameter liposome was prepared while the thin film peeled off from the wall of the eggplant flask.
0.2 μm pores for uniform liposome size
The liposomes were extruded by filtering with a polytetrafluoroethylene (PTFE) membrane filter (1) to adjust the diameter to 0.2 μm or less.

Detection of Alkaline Phosphatase Using Fluorescent Substance-Containing Liposomes POLYBEAD, CARBOX manufactured by Polyscience
A trace amount of alkaline phosphatase was detected on a slide glass using YLATEMICROSPHERES (beads).

That is, an alkaline phosphatase antibody (A
The solution containing P) was reacted. At this time, the concentration of alkaline phosphatase was adjusted so that one AP was bound to one bead. Next, after reacting with anti-alkaline phosphatase antibody labeled with digoxigenin, excess anti-alkaline phosphatase antibody was added to TRIS.
buffer (100 mM Tris-HCl, 1
It was washed with 50 mM NaCl; pH 7.5). Then, the divalent antibody linker prepared in Example 1 was reacted, and then the unreacted divalent antibody linker antibody was reacted with TRI.
Washed well with S buffer. Then, the liposomes were reacted for 30 minutes.

The detection was carried out by observing with a fluorescence microscope (magnification: 1000 times). As a result, as shown in FIG. 6, fluorescent bright spots (white areas) judged to be bound to the dispersed beads could be detected. Also, one liposome appeared to have the same bright spot size. From this, it can be seen that the bivalent antibody linker was able to bind one liposome containing a fluorescent substance and one alkaline phosphatase, and was able to detect one alkaline phosphatase.

Therefore, a substance that connects with the antigenic substance,
That is, it can be said that a reaction system capable of detecting one antigenic substance could be established by using the bivalent antibody linker of the present invention and one liposome. Therefore, it can be said that the detection method of the present invention is an ultra-sensitive detection system in the clinical field.

Further, in the present example, the analyte alkaline phosphatase was immobilized on the beads for detection, but when alkaline phosphatase immobilized on a filter such as a membrane or alkaline phosphatase present in living tissue was detected. Also, there is no steric hindrance due to liposomes, and detection can be performed with high sensitivity.

[Brief description of drawings]

FIG. 1 is an explanatory view showing a method for detecting a human peripheral blood lymphocyte chromosome in which a biotin-labeled probe gene is complementarily bound in Example 1.

FIG. 2 is an explanatory view with a fluorescence micrograph (magnification: 1000 times) showing a detection result of a fluorescent staining pattern of a chromosome formed on a slide (substrate) in the detection method of Example 1.

FIG. 3 is an explanatory view showing the binding state of the first antibody component and the second antibody component of the bivalent antibody linker of the present invention.

FIG. 4 is an explanatory view showing another binding state of the first antibody component and the second antibody component of the bivalent antibody linker of the present invention.

FIG. 5 is an explanatory diagram showing a method for detecting alkaline phosphatase of Example 2.

6 is a fluorescence micrograph showing the morphology of alkaline phosphatase in the detection method of Example 2 (magnification: 1000).
2 times) explanatory diagram.

FIG. 7 is an explanatory diagram showing a conventional method for detecting an antigenic substance.

[Explanation of symbols]

 1. ． ． Bivalent antibody linker, 11, 110. ． ． First antibody component, 12, 120. ． ． Second antibody component, 2, 25. ． ． Antigenic substance, 21, 71. ． ． First hapten, 22, 72. ． ． Second hapten, 3,30. ． ． A fluorescent substance, 39. ． ． Liposomes, 4. ． ． Beads, 40,70. ． ． Anti-alkaline phosphatase antibody, 7. ． ． Alkaline phosphatase,

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Kyoko Kondo 2-1, 1-1 Asahi-cho, Kariya city, Aichi Prefecture Aisin Seiki Co., Ltd.

Claims (12)

[Claims] 1. A bivalent antibody linker is prepared by previously binding a first antibody component that specifically binds to an antigenic substance and a second antibody component that specifically binds to a signal substance, and then:
An antigenic substance is bound to the first antibody component of the divalent antibody linker, a signal substance is bound to the second antibody component of the divalent antibody linker, and then a signal is generated from the signal substance to produce the signal. A method for detecting an antigenic substance, which comprises detecting 2. The first antibody component according to claim 1, wherein the first antibody component is bound via the first hapten bound to the antigenic substance,
A method for detecting an antigenic substance, which is characterized by being specifically bound to the antigenic substance. 3. The antigenic substance according to claim 1 or 2, wherein the second antibody component is specifically bound to a signal substance via the second hapten bound to the signal substance. Detection method. 4. The first hapten according to claim 2 or 3, wherein the first hapten is biotin, digoxigenin, dinitrophenol, trinitrophenol, fluorescein, tetramethylrhodamine B isothiocyanate, rhodamine, Texas red, lucifer yellow, 4,4-.
Difluoro-5,7-dimethyl-4-bora-3a, 4a
-Diaza-s-indacene-3-propionyl, 7-nitrobenz-2-oxa-1,3-diazol-4-yl, dansyl, coumarin, DNA, RNA, or a virus. A method for detecting an antigenic substance. 5. The method according to any one of claims 2 to 4,
The second hapten is biotin, digoxigenin, dinitrophenol, trinitrophenol, fluorescein, tetramethylrhodamine B isothiocyanate, rhodamine, Texas red, lucifer yellow, 4,4-difluoro-5,7-dimethyl-4-bora-. 3a, 4a-diaza-s-indacene-3-propionyl, 7-nitrobenz-2-oxa-1,3-diazol-4-yl, dansyl, coumarin, DNA, RN
A method for detecting an antigenic substance, which is one of A and virus. 6. The method according to any one of claims 1 to 5,
A method for detecting an antigenic substance, wherein the first antibody component and the second antibody component are bound by a thiol group and a maleimide group. 7. The method according to any one of claims 1 to 6,
The method for detecting an antigenic substance, wherein the signal substance is a liposome containing a fluorescent substance, or an enzyme having a substance capable of becoming a fluorescent substance as a substrate. 8. The fluorescent substance contained in the liposome according to claim 7, is a covalent fluorescent substance covalently bound to the liposome or a non-covalent fluorescent substance encapsulated in the liposome. A method for detecting an antigenic substance characterized by being present. 9. The covalently binding fluorescent substance according to claim 8, wherein the covalent bond fluorescent compound is a compound having a coumarin skeleton, a compound having a naphthalene skeleton, a compound having a fluorescein skeleton,
A method for detecting an antigenic substance, which is one or more selected from the group consisting of a compound having a perylene skeleton, a compound having a pyrene skeleton, a compound having an anthracene skeleton, and a compound having a rhodamine skeleton. 10. The non-covalently binding fluorescent substance according to claim 8, wherein 4-methylumbelliferone, 7-hydroxy-4-biphenyl-umbelliferone, 3-hydroxy-2-naphthoic acid 2-phenylanilide is used. , 3-Hydroxy-2-naphthoic acid-2,4-dimethylanilide, 6-bromo-2-hydroxy-3-naphthoic acid 2-
Methylanilide, 3-hydroxy-2-anthranoic acid 2-methylanilide, pyrene, fluorescein,
A method for detecting an antigenic substance, which is one or more selected from the group consisting of perylene, rhodamine, Texas red, and a DNA-binding fluorescent substance. 11. The antigen according to claim 7, wherein the enzyme using the substance that can be a fluorescent substance as a substrate is any one of alkaline phosphatase, acid phosphatase, peroxidase, esterase, glucosidase, and phospholipase. Method for detecting volatile substances. 12. The method for detecting an antigenic substance according to claim 1, wherein the antigenic substance is any one of tissue cells, viruses, nucleic acids, or proteins.