HK1167891A

HK1167891A - Method for detecting substance in biological sample

Info

Publication number: HK1167891A
Application number: HK12108515.5A
Authority: HK
Inventors: Takakura Yoshimitsu; Oka Naomi; Kondo Kazuhiro
Original assignee: Japan Tobacco Inc.
Priority date: 2009-03-02
Filing date: 2010-03-02
Publication date: 2012-12-14

Description

Method for detecting substance in biological sample

Technical Field

The present invention relates to a method for qualitatively and/or quantitatively detecting a substance in a biological sample. The method of the present invention enables detection of substances which are present in a small amount in a biological sample and are generally difficult to detect.

Background

Using eggs to which a substance to be detected is specifically bound by hydrophobic binding, covalent binding, or the like Method for detecting substance carrying body of white matter

In order to detect a substance in a biological sample, a method using a substance that specifically binds to the substance (a substance to be detected) has been widely used. As a substance that specifically binds to a substance to be detected, an antibody, another protein, or the like is often used. These substances can be immobilized on a carrier such as a microplate, a bead or a sensor chip. As a general method for immobilization, hydrophobic binding, covalent binding, and the like are known.

"hydrophobic binding" is to bind a carrier to a specific protein by utilizing the interaction between the hydrophobic surface of the carrier and the hydrophobic portion of the protein that specifically binds to a substance to be detected (hereinafter, also referred to as "specific protein"), and is simple and requires no special reagent. However, the binding force is generally weak, and when the protein is used for ELISA (enzyme linked immunosorbent assay), the protein is often detached from the carrier by a washing operation after the binding. When a specific protein is bound to a carrier by hydrophobic binding, many proteins may lose their functions completely or partially.

"covalent bonding" is a strong bonding force by utilizing the interaction between a functional group (for example, an amino group) in a specific protein and a functional group (for example, a carboxyl group) provided on the surface of the carrier. However, when a specific protein is bound to a carrier by covalent binding, the function of many proteins is completely or partially lost, as in the case of hydrophobic binding.

In addition to hydrophobic bonding and covalent bonding, the following methods are known: a method in which a plurality of histidines are fused to the ends of a protein, and the fusion protein having the histidine tag is bound to a substrate or the like, for example, a protein chip, whose surface is provided with nickel. However, the interaction between the histidine tag and the nickel ion is not so strong, and it is also known that there is nonspecific binding between the nickel ion and various biomolecules.

In general, it is an important issue to reduce non-specific binding that causes a background signal in a specific binding assay system using a solid phase to which a protein that specifically binds to a substance to be detected is bound by hydrophobic binding or covalent binding as described above. As a method for solving this problem, the following methods are proposed: for example, a method in which an extract solution of a bacterial component is contained in a reagent for detection (Japanese patent laid-open No. Sho 59-99257); a method of adding a culture component of a host cell into a sample, the host cell being introduced with a vector (vector) that is the same as a vector for producing a recombinant protein that specifically binds to a substance to be detected and does not contain a vector encoding a gene for the protein (Japanese patent laid-open No. 8-43392); a method in which an aqueous extract solution derived from cells that are the same as the cells producing the recombinant protein that specifically binds to the substance to be detected and do not contain the protein is subjected to heat treatment, and then the water-soluble fraction is added to a sample (Japanese patent application laid-open No. 2004-301646). By these methods, a certain effect of suppressing nonspecific binding can be observed.

Substance detection method using avidin-biotin binding

Avidin is a glycoprotein derived from egg white, and binds very strongly to biotin (vitamin H). Avidin interacts with biotin as one of the strongest non-covalent bindings (Green (1975) Adv Protein Chem 29: 85-133). On the other hand, streptavidin is an avidin-like protein derived from actinomycetes, and binds strongly to biotin. Up to now, the (strept) avidin-biotin interaction has been widely used in the fields of molecular biology and biochemistry, such as detection of antigens and antibodies, because of its strength of action (Green (1990) Methods Enzymol 184: 51-67).

A method of binding a protein to a carrier by utilizing the biotin-binding property of avidin or streptavidin has been proposed. That is, a method of binding (strept) avidin to a substrate such as a microplate by covalent binding or hydrophobic binding, and further binding biotinylated protein to the substrate to immobilize the protein.

Further, the following technique is also reported in japanese patent laid-open No. 4-236353: avidin is first bound to the substrate to which biotin is bound by avidin-biotin binding, and then biotinylated desired protein is bound thereto by binding to another biotin pocket (biotin pocket) of avidin, thereby obtaining immobilization in the order of substrate-biotin-avidin-biotin-desired protein. The detection of the substance to be detected can be performed using the plate immobilized by such a method.

However, even in the analysis using avidin-biotin bonding, there is a problem that it is necessary to deal with a background signal, as in the case of the method using a solid phase to which a protein specific to a substance to be detected is bonded by hydrophobic bonding, covalent bonding, or the like. In order to solve the above problems, in addition to the above non-specific inhibition method, the following methods are proposed: a method in which a solid phase to which deactivated (strept) avidin is bound is brought into contact with a sample, and then, the solid phase to which active (strept) avidin is bound is brought into contact (Japanese patent application laid-open No. H8-114590); a method in which an avidin-bonded solid phase is bonded to a biotinylated substance and then contacted with a conjugate between polyethylene glycol and biotin (Japanese patent application laid-open No. 11-211727); a method of contacting with a biotin-containing solution (Japanese patent laid-open publication No. 2002-48794), and the like. However, it cannot be said that a sufficient practical effect is obtained.

Fusion proteins using avidin or streptavidin have been produced for the purpose of using as a protein marker, a diagnostic marker, and a cell-specific targeting factor (Airenne et al (1999) Biomol Eng 16: 87-92). Among these fusion proteins, in particular, fusion proteins between avidin or streptavidin and antibodies such as scFv, Fab fragments, and IgG have been studied for their application to specific targets of drugs for cancer cells and the like. Furthermore, the assumption of a column for immobilizing scFv by avidin-biotin binding using a fusion protein of streptavidin and scFv was described (Kiprivov et al (1995) Hum antibody Hybrid 6: 93-101, Dubel et al (1995) J immunological Methods 178: 201-209). However, no example of detecting a substance to be detected in a biological sample by immobilization of a biotin-binding protein has been known. Although there is an example in which streptavidin fusion protein is immobilized on a biotinylated plate and ELISA is performed using an antibody using the fused protein as an antigen (WO2002/046395), the report merely shows the possibility that the streptavidin fusion protein can be immobilized on a support without losing the activity.

Documents of the prior art

Patent document

Patent document 1: japanese patent laid-open No. Sho 59-99257

Patent document 2: japanese Kokai publication Hei-8-43392

Patent document 3: japanese patent laid-open No. 2004-301646

Patent document 4: japanese unexamined patent publication No. 8-114590

Patent document 5: japanese patent laid-open No. H11-211727

Patent document 6: japanese laid-open patent publication 2002-48794

Patent document 7: WO2002/046395

Patent document 8: WO02/072817

Non-patent document

Non-patent document 1: green (1975) Adv Protein Chem 29: 85-133

Non-patent document 2: green (1990) Methods Enzymol 184: 51-67

Non-patent document 3: airenne et al (1999) Biomol Eng 16: 87-92

Non-patent document 4: kiprivov et al (1995) Hum antibody Hybrid 6: 93-101

Non-patent document 5: dubel et al (1995) J Immunol Methods 178: 201-209

Non-patent document 6: takakura et al (2009) FEBS J276: 1383-1397

Disclosure of Invention

Problems to be solved by the invention

The object of the present invention is to provide a method for qualitatively and/or quantitatively detecting a substance in a biological sample.

The present invention has an object to provide a method for qualitatively and/or quantitatively detecting/measuring a substance present in a biological sample in a trace amount while suppressing a background signal.

Means for solving the problems

As a result of intensive and diligent research efforts by the present inventors, the following system was developed: this system utilizes the binding between biotin-binding proteins and fixes a protein that specifically binds to a substance to be detected and a fusion protein of the biotin-binding protein to a support. As described above, in this system, it was found that processing of a background signal is a large problem particularly in order to detect a substance present in a biological sample in a trace amount, and thus the present invention was conceived in order to reduce the background signal. Specifically, when a cell disruption extract and a biotin-binding protein are added to a biological sample, the effect of reducing nonspecific binding is significant. Alternatively, a cell disruption extract prepared from cells to which a biotin-binding protein has been bound by genetic engineering techniques may be added instead of adding the biotin-binding protein, whereby the same effects can be obtained.

Further, the present inventors found that: in a system in which a fusion protein of a protein that specifically binds to a detection substance and a biotin-binding protein is immobilized on a support, the biotin-binding protein is brought into contact (blocking) with the support before addition of a biological sample, whereby a substance at a lower concentration can be stably measured while suppressing a background signal.

The present invention is based on the above knowledge and provides the following detection methods: a detection method in which a substance for specifically detecting a substance to be detected by avidin-biotin binding is immobilized on a support, which method reduces non-specific binding and has higher sensitivity.

The present invention includes the following embodiments, but is not limited thereto.

[ embodiment 1]

A method of detecting a substance in a biological sample, comprising:

1) preparing a fusion protein of a protein that specifically binds to a substance to be detected and a biotin-binding protein, and a carrier to which biotin is bound;

2) binding the carrier prepared in step 1) to the fusion protein by binding biotin-binding protein to each other, thereby producing a carrier to which the fusion protein is bound;

3) mixing (a) a biological sample, and

(b-i) a cell disruption extract prepared from the same host cell as used for expressing the fusion protein of step 1), and a biotin-binding protein, or

(b-ii) a cell disruption extract solution prepared from a cell in which a biotin-binding protein has been expressed by genetic engineering techniques in the same cell as the host cell used for expression of the fusion protein in step 1);

mixing and adding the mixture to the carrier combined with the fusion protein prepared in the step 2); then, the user can use the device to perform the operation,

4) and detecting the substance to be detected bound to the protein, which is a protein that specifically binds to the substance to be detected in the fusion protein.

[ embodiment 2]

A method of detecting a substance in a biological sample, comprising:

3) blocking the carrier by bringing the carrier to which the fusion protein is bound, which is produced in the step 2), into contact with a biotin-binding protein;

4) after the blocking step of step 3), a biological sample is added to the carrier to which the fusion protein has been bound, and then,

5) and detecting the substance to be detected bound to the protein, which is a protein that specifically binds to the substance to be detected in the fusion protein.

[ embodiment 3]

A method of detecting a substance in a biological sample, comprising:

4) after the blocking step of step 3), (a) a biological sample, and

mixing and adding the mixture into a carrier combined with the fusion protein; then, the user can use the device to perform the operation,

[ embodiment 4]

The method according to embodiment 1 or 3, wherein a cell disruption extract solution extracted from cells containing an arbitrary carrier is added as the cell disruption extract solution in step 3(b-i) of embodiment 1 or step 4(b-i) of embodiment 3.

[ embodiment 5]

The method according to any one of embodiments 1 to 4, wherein the biotin-binding protein is tamavidin or a mutant thereof.

[ embodiment 6]

The method according to any one of embodiments 1 to 5, wherein the biological sample is selected from the group consisting of blood, serum, cerebrospinal fluid, saliva, sweat, urine, tears, lymph, and breast milk.

[ embodiment 7]

A carrier for detecting a substance in a biological sample, which is bound to a fusion protein of a biotin-binding protein and a protein that specifically binds to a substance to be detected, by binding between biotin and the biotin-binding protein, the carrier for detecting a substance in a biological sample being prepared by:

3) the carrier having the fusion protein bound thereto prepared in step 2) is brought into contact with the biotin-binding protein to block the carrier.

[ embodiment 8]

A kit for detecting a substance in a biological sample, comprising:

A) a carrier which is bound to a fusion protein of a biotin-binding protein and a protein specifically binding to a substance to be detected by binding between biotin and biotin-binding protein, and

a reagent for diluting a biological sample comprising the following B-i) or B-ii);

b-i) a cell disruption extract prepared from the same cells as the host cells used for expression of the fusion protein of A), or

B-ii) a cell disruption extract prepared from a cell in which a biotin-binding protein is expressed by a genetic engineering technique in the same species as the host cell used for expressing the fusion protein of A); and/or

C) A blocking agent comprising a biotin-binding protein.

ADVANTAGEOUS EFFECTS OF INVENTION

The method of the present invention can suppress a background signal and stably perform detection with higher sensitivity in the detection of a substance to be detected in a biological sample. The method of the present invention enables detection of substances which are present in a biological sample in a trace amount and are generally difficult to detect.

Drawings

FIG. 1 is a schematic diagram showing the blocking of biotin-binding protein in FIG. 1. The white circles indicate biotin, the black ovals indicate biotin-binding proteins, and the white ovals indicate proteins that specifically bind to the substance to be detected. FIG. 1A: immobilization with fusion protein without blocking by biotin-binding protein; FIG. 1B: no blocking by biotin-binding protein, no immobilization of fusion protein; FIG. 1C: the biotin-binding protein is used for blocking and the fusion protein is immobilized; FIG. 1D: the blocking and fusion protein-free immobilization was performed by a biotin-binding protein.

FIG. 2 is a view showing the results of detection of a fusion protein of SITH-1 and TM2 expressed in Escherichia coli by immunoblotting (Western blotting). As a control, Escherichia coli introduced with only the vector was used.

FIG. 3 is a graph showing the effect of adding an extract obtained by disrupting Escherichia coli to serum on nonspecific binding and the effect of blocking with tamavidin 2. FIG. 3-1 shows the results when blocking was performed (only) by BSA. That is, the result obtained by subtracting the measurement value of the biotinylated plate (0.5% BSA blocking) which had not been subjected to any immobilization from the measurement value of the SITH-1-TM2 fusion protein-immobilized plate (0.5% BSA blocking) is shown. FIG. 3-2 shows the results when blocking was performed with BSA and TM 2. That is, the results obtained by subtracting the measurement value of biotinylated plate (50. mu.g/ml TM 2/0.5% BSA blocking) without any immobilization from the measurement value of the SITH-1-TM2 fusion protein immobilized plate (50. mu.g/ml TM 2/0.5% BSA blocking) are shown.

FIG. 4 column 2 of FIG. 4 shows the results of detection of the fusion protein of Harpin and TM2 expressed in E.coli by immunoblotting. As a control, Escherichia coli introduced with the vector alone was used (column 1).

Detailed Description

I. Detection method of the present invention (embodiment 1)

The detection method (embodiment 1) of the present invention is a method for detecting a substance in a biological sample, and includes:

3) mixing (a) a biological sample, and

Detection substance in biological sample

The present invention relates to a method for detecting a substance in a biological sample.

The biological sample in the present invention is not particularly limited as long as it may contain a substance to be detected. The sample may be a sample containing cells, tissues or fragments thereof collected from a living body, for example, a body fluid, more preferably blood, serum, cerebrospinal fluid, saliva, sweat, urine, tears, lymph fluid, breast milk, or the like.

Such body fluids are diluted and used as necessary. The dilution ratio is usually about 2 to 10000 times, preferably about 100 to 1000 times, but is not limited thereto. The solution for dilution may be any buffer solution, and may contain a suitable blocking agent. As the blocking agent, a substance having a high effect of inhibiting nonspecific binding is preferred, and blocking agents known to those skilled in the art, such as BSA or casein, can be used. In embodiment 2 of the present invention, a biotin-binding protein is used as a blocking agent. For which case it will be described below.

The substance to be detected of the present invention is not particularly limited as long as it is a substance to be detected or measured in a biological sample, and proteins such as antibodies and antigens, fragments thereof, peptides, nucleic acids, saccharides, glycolipids, and the like can be preferably used.

The present invention also enables measurement of substances in biological samples at low concentrations that are difficult to detect or accurately quantify by conventional methods. For example, when the substance to be detected is, for example, an antibody in serum, and further when the antibody titer is low (for example, when the serum is diluted 1000-fold, it is difficult to detect, but when the serum is diluted 100-fold, the antibody titer is low), it is necessary to suppress the dilution rate of the serum, which results in an increase in nonspecific binding derived from components in serum.

Examples of the substance to be detected of the present invention include, but are not limited to: an antibody to a Small protein (Small protein encoded by the intermediate Transcript of HHV-6) (SITH-1) encoded by the latent infection intermediate stage Transcript of HHV-6. Other examples of the additives include: antibodies to antigens other than those described above of herpes virus, and antibodies to virus-associated antigens derived from cytomegalovirus, hepatitis virus, HIV virus, HTLV virus, measles virus, influenza virus, and the like; or an antigen associated with a bacterium derived from Helicobacter pylori (Helicobacter pylori) or the like; or an antibody to a fungal-associated antigen, and the like.

Based on the contents described in PCT/JP2008/67300 and U.S. provisional application No.61/102441 SITH-1

(1) SITH-1 protein and nucleic acid

The structure and function of the SITH-1 protein, nucleic acid are as disclosed in PCT/JP2008/67300, the entire contents of which are incorporated herein.

SITH-1 is a factor involved in latent infection with herpes virus, and more specifically, a protein specifically expressed in latent infection with herpes virus. Here, "specifically express when the herpesvirus is latently infected" means that when the herpesvirus is latently infected (non-proliferative infection) in a host infected with the herpesvirus, a gene or a gene product derived from the herpesvirus is specifically expressed.

Examples of the proteins and nucleic acids of SITH-1 include (a) proteins represented by SEQ ID NO: 1, and a nucleic acid encoding the protein.

Consisting of SEQ ID NO: 1, which is a protein specifically expressed in latent infection with human herpesvirus-6 (HHV-6), as shown in the following reference examples. The SITH-1 protein is a polypeptide having the sequence of SEQ ID NO: 1, a protein consisting of 159 amino acids and having a molecular weight of about 17.5 kDa.

The SITH-1 protein is encoded by a nucleic acid of the SITH-1 gene. The cDNA of the SITH-1 gene is shown as SEQ ID NO: 3, (1.79 kbp), the nucleotide sequence from position 954 to 956 being a start codon (Kozak ATG), and the nucleotide sequence from position 1431 to 1433 being a stop codon (TAA). Thus, in the context of the nucleic acid sequence as set forth in SEQ ID NO: 3, the SITH-1 nucleic acid has a nucleotide sequence from positions 954 to 1430 as an Open Reading Frame (ORF), and the ORF has a size of 477 base pairs (about 0.48 kbp). In the cDNA of SITH-1, the base sequence representing the ORF region is as shown in SEQ ID NO: 2, respectively. In addition, the nucleotide sequence of SEQ ID NO: 2 contains 3 bases of a stop codon.

The SITH-1 nucleic acids are normally expressed in the cytoplasm of cells latently infected with HHV-6, but are not expressed in cells infected with proliferation. The nucleic acid encoding the SITH-1 protein is encoded by a DNA having a complementary strand relationship with a gene specific for HHV-6 latent infection reported so far (H6LT), and the expression of which is enhanced in the intermediate stage of the latent infection with HHV-6. From these facts, it can be said that: the SITH-1 protein is a protein specifically expressed upon latent infection with HHV-6.

The SITH-1 protein binds to CAML (calcium-modulating cyclophilin ligand), an accession # U18242, which is a host protein, and increases the calcium concentration in glial cells. CAMLs are known to be found in the brain and lymphocytes in host organisms, and are proteins that increase intracellular calcium concentration. Furthermore, it is considered that the increase in intracellular calcium concentration caused by the expression of the SITH-1 protein activates all signal transduction in the latently infected cell, contributing to the efficient reactivation of HHV-6.

HHV-6 is known to be latently infected in glial cells in the brain, and it can be considered that: during latent infection or during the intermediate stage of latent infection state with high activity, HHV-6 increases the calcium concentration in glial cells when SITH-1 is expressed. It is considered that an increase in intracellular calcium concentration in brain cells is largely related to mental disorders such as mood disorders (japanese journal of research 2003).

The SITH-1 protein has: and (3) a function of binding to a host protein, namely CAML, maintaining the activity and increasing the intracellular calcium concentration. In addition, mental disorders can be induced by expressing the protein in glial cells in the brain, which are considered to be the most potent cells expressing the SITH-1 protein. It is thus assumed that: the SITH-1 protein is expressed in latent infection or early reactivation of herpes virus, and has a function of causing mental disorder in a host.

(2) Antibodies against SITH-1

An antibody against SITH-1 can be obtained as a polyclonal antibody or a monoclonal antibody by a known method using the SITH-1 protein or a mutant thereof, or a partial peptide thereof as an antigen. Known methods include, for example: methods described in the literature ("Antibodies: A Laboratory, ColdSpring Harbor Laboratory, New York (1988)"), monoclonal antibody hybridoma and ELISA, lecture (1991) "by Kawasaki et al. The antibody thus obtained can be used for detection/measurement of the SITH-1 protein.

The term "antibody" means an immunoglobulin (IgA, IgD, IgE, IgG, IgM and their Fab fragments, F (ab')₂Fragments, Fc fragments), mention may be made, as examples: polyclonal antibodies, monoclonal antibodies, single chain antibodies, anti-idiotype antibodies, and humanized antibodies, but are not limited to these antibodies.

The phrase "antibody recognizing the SITH-1 protein" is intended to include intact molecules and antibody fragments (e.g., Fab and F (ab')₂Fragments). Fab and F (ab')₂And other fragments of the SITH-1 antibody. Such fragments are typically produced by papain (to produce Fab fragments) or pepsin (to produce F (ab')₂Fragment) produced by cleavage of proteolysis.

It is considered that in a patient suffering from a mood disorder or an individual who may suffer from a mood disorder, the expression level of the SITH-1 protein increases, and as a result, the SITH-1 antibody titer also increases. For the purposes of the present invention, in one embodiment, a patient with a mood disorder or an individual likely to have a mood disorder can be identified by detecting the SITH-1 antibody in a biological sample.

Fusion protein having bound thereto a protein that specifically binds to a substance to be detected and a biotin-binding protein Carrier for proteins (Steps 1) and 2 of embodiment 1))

The detection method of the present invention utilizes a carrier that binds to a fusion protein of a biotin-binding protein and a protein that specifically binds to a substance to be detected, through the binding between biotin and biotin-binding proteins.

The carrier of the present invention can be produced by a method comprising the steps of:

2) the carrier prepared in step 1) is bound to the fusion protein by binding of biotin-binding proteins, thereby producing a carrier to which the fusion protein is bound.

Biotin-bonded carrier

"Biotin" refers to D- [ (+) -cis-hexahydro-2-oxo-1H-thieno- (3, 4) -imidazole-4-pentanoic acid]General name of (a). Is a water-soluble vitamin classified in vitamin B group, also called vitaminB₇(vitamin B)₇) Or sometimes referred to as vitamin H, coenzyme R. Biotin binds very strongly to avidin, one of the glycoproteins contained in egg white, and thus its absorption is hindered. For this reason, biotin deficiency is caused by a large intake of raw egg white.

"Biotin" in the present specification includes, in addition to the above-mentioned Biotin, Biotin analogues such as iminobiotin (iminobiotin) (Hofmann et al (1980) Proc Natl Acad Sci USA 77: 4666-.

Systems using biotin-avidin (biotin-binding protein) complexes are widely used in the fields of tissue immunology, DNA analysis, clinical examination, and the like. The method for binding the protein of the present invention to the carrier is as follows: the carrier is bound with a fusion protein in which a desired protein is bound to a biotin-binding protein by biotin-avidin binding. The method of the present invention can act very efficiently without impairing the function of the protein, as compared with the conventional binding using biotin-avidin binding.

The material constituting the solid support includes: cellulose, teflon (registered trademark), nitrocellulose, agarose, dextran, chitosan, polystyrene, polyacrylamide, polyester, polycarbonate, polyamide, polypropylene, nylon, polyvinylidene fluoride, latex, silica, glass fiber, gold, platinum, silver, copper, iron, stainless steel, ferrite (ferrite), silicon wafer, polyethylene, polyethyleneimine, polylactic acid, resin, polysaccharide, protein (albumin, etc.), carbon, or a combination thereof, but is not limited thereto. In addition, a material having a certain strength, a stable composition, and less nonspecific binding is preferable.

The shape of the solid support includes, but is not limited to, beads, magnetic beads, thin films, microtubes, filter membranes, plates, microwell plates, carbon nanotubes, sensor chips, and the like. As is known in the art, pits, grooves, filter bottoms, etc. may be provided on flat solid support such as membranes, plates, etc.

In one embodiment of the invention, the beads may have a sphere diameter in the range of about 25nm to about 1 mm. In a preferred embodiment, the beads have a diameter in the range of about 50nm to about 10 μm. The size of the beads may be selected according to the particular application. Since some bacterial spores have a size on the order of about 1 μm, it is preferred that the beads used to capture the spores involved have a diameter greater than 1 μm.

Without being limited thereto, for example, in the case where high detection sensitivity is desired, such beads as described above can be preferably used as the solid support from the viewpoints of the frequency of contact between the substance to be detected and the substance that specifically binds thereto and the ease of washing operation.

Examples of the method of binding biotin to the carrier include a method using a biotinylation reagent. As the biotinylation reagent, for example: EZ-Link (registered trademark) sulfo-NHS-Biotin manufactured by PIERCE (linker Length, reactive group in sequence in parentheses) ((registered trademark))Primary amine), EZ-Link (registered trademark) sulfo-NHS-LC-Biotin ((C)Primary amine), EZ-Link (registered trademark) sulfo-NHS-LCLC-biotin (Primary amine), EZ-Link (registered trademark) PFP-biotin (CAmine), EZ-Link (registered trademark) Maleimide-PEO₂Biotin(s) (Mercapto group), EZ-Link (registered trademark) Biotin-PEO₂Amine (A), (B), (C) and (C)Carboxyl group), EZ-Link (registered trademark) Biotin-PEO₃LC amines (Carboxy), EZ-Link (registered trademark) Biotin-hydrazide (R)Aldehyde group), EZ-Link (registered trademark) biotin-LC-hydrazide (Aldehyde group), EZ-Link (registered trademark) NHS-iminobiotin ((II)Primary amine), and the like, but not limited to the above.

The biotinylation reagent can bind biotin to a desired carrier such as a microplate, a microbead, or a sensor chip by a known method. For example, there is a method using a carrier having various functional groups such as amino group, carboxyl group, mercapto group, tosyl group, epoxy group, maleimide group, and active ester (for example, magnetic beads, Sepharose beads, agarose beads, latex beads, microtiter plates, etc.). In this case, for example, in the case of using a biotinylation reagent containing an NHS ester, the biotinylation reagent containing an NHS ester may be dissolved using an organic solvent such as DMSO (dimethlysulfoxide) or phosphate buffer at pH7 to 9, and biotin may be bound by adding to an immobilization support having an amino group. For example, when a biotinylation reagent containing an amino group is used, biotin may be bound by converting the carboxyl group of the immobilization support into an active ester using carbodiimide such as EDC (1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride), and then adding a biotinylation reagent dissolved in a buffer solution at a pH around 5. In addition, in the biotinylated immobilization carrier, it is preferable that unreacted functional groups are deactivated and then blocked with BSA or the like.

In addition, biotinylated commercial supports can be used. As the biotinylated microplate, for example: reacti-Bind^TMThe biotin-coated polystyrene plate (manufactured by PIERCE) is not limited thereto. Examples of biotinylated microbeads that can be used include, but are not limited to, BioMag biotin (manufactured by Polysciences), nanobag (registered trademark) -D biotin, nanomag (registered trademark) -silica biotin (manufactured by corefrnt), nanobag (registered trademark) -silica biotin (manufactured by Upstate), polystyrene-based microbeads, beamlyte (registered trademark) biotin beads (manufactured by Upstate), biotin agarose, 2-iminobiotin-agarose (manufactured by Sigma), or highly crosslinked agarose (manufactured by Biosearch Technologies).

As for the length of the linker connecting the carrier to biotin, it is preferably at least longer thanMore preferablyThe above is more preferableAbove, it is more preferable thatThe above.

Protein capable of binding specifically to substance to be detected

The protein that specifically binds to the substance to be detected (hereinafter, also referred to as "specific protein") is not particularly limited. In one embodiment of the present invention, for example, in complementary binding between an antigen and a ligand such as an antibody or a hormone and a receptor, or complementary binding between a lectin and a sugar or a nucleic acid, one of them can be selectively analyzed from a sample to be tested by utilizing the ability of forming a specific complex with the other, but the present invention is not limited to these.

More specifically, examples of the protein include: antibodies, antigenic proteins, lectins, peptides, or protein a, protein G, protein L, receptors, enzyme proteins, and the like. The antibody may be IgG, or an antibody fragment containing an antigen binding site such as scFv or Fab, and the antigen protein may be a protein derived from viruses such as hepatitis a/c virus, HIV, influenza, and herpes virus, a protein derived from bacteria such as helicobacter pylori, a tumor marker such as CEA and PSA, or a sex hormone. Lectins are sugar-binding proteins, and include: monosaccharide-specific lectins such as mannose-specific lectins, GalNAc-specific lectins, GlcNAc-specific lectins, fucose-specific lectins, sialic acid-specific lectins, and oligosaccharide-specific lectins. Further, DNA/RNA binding proteins and the like can be exemplified. Examples of the peptide include peptides composed of 2 to 100 amino acids, preferably 4 to 50 amino acids, and more preferably 6 to 30 amino acids, but are not limited thereto.

In addition, although not limited thereto, the SITH-1 protein is exemplified as a protein that specifically binds to a substance to be detected in the present invention.

Biotin-binding protein

The present invention comprises immobilizing a protein that specifically binds to a substance to be detected on a support by utilizing the binding between biotin-binding proteins. In the present invention, "binding between biotin-binding proteins" is sometimes referred to as "avidin-biotin binding".

The biotin-binding protein may be any protein capable of binding to avidin, streptavidin, neutravidin (neutravidin), AVR protein (biochem. J., (2002), 363: 609-617), Bradavidin (J.biol. chem., (2005), 280: 13250-13255), Rhizavidin (biochem. J., (2007), 405: 397-405), or tamavidin (WO 02/072)817) And biotin-binding proteins such as mutants thereof, and any of these can be preferably used. Preferably at least biotin has a dissociation constant (KD) of 10^-6The following, more preferably 10^-8Hereinafter, more preferably 10^-10The following. Note that the biotin-binding protein added to the sample to be tested and the biotin-binding protein for blocking the carrier are as described below.

As the biotin-binding protein, tamavidin highly expressed in Escherichia coli and mutants thereof can be preferably used in particular. Tamavidin is a biotin-binding protein found in Pleurotus cornucopiae (Pleurotus conopoidea), a basidiomycete used as an edible mushroom (WO02/072817, Takakura et al (2009) FEBS J276: 1383-. Examples of mutants of tamavidin include high-binding ability and low-non-specific binding tamavidin (PCT/JP 2009/64302).

"tamavidin" according to the present invention means tamavidin 1, tamavidin 2, or a mutant thereof. In particular, typically, the tamavidin of the invention may be a polypeptide comprising SEQ ID NO: 5 or SEQ ID NO: 7, or may be a protein consisting of an amino acid sequence comprising SEQ ID NO: 4 or SEQ ID NO: 6 in the sequence listing. Alternatively, the tamavidin of the invention may be a polypeptide comprising SEQ ID NO: 5 or SEQ ID NO: 7, or may also be a mutant of a protein consisting of an amino acid sequence comprising SEQ ID NO: 4 or SEQ ID NO: 6, a protein having the same biotin-binding activity as tamavidin 1 or 2, or a protein having high binding ability/low non-specific binding activity. In the present specification, tamavidin 1, tamavidin 2 and their mutants are sometimes collectively referred to as "tamavidin" for short.

The mutant of Tamavidin 1 or 2 may be a protein having the same biotin-binding activity as Tamavidin 1 or 2, comprising an amino acid sequence set forth in SEQ ID NO: 5 or 7, which has an amino acid sequence having 1 or more amino acids deleted, substituted, inserted and/or added. Substitutions may be conservative substitutions, meaning that a particular amino acid residue is replaced with a residue having similar physicochemical characteristics. Non-limiting examples of conservative substitutions include: substitution of an aliphatic group-containing amino acid residue such as mutual substitution of Ile, Val, Leu or Ala, and substitution of a polar residue such as mutual substitution of Lys and Arg, Glu and Asp, or Gln and Asn.

Mutants obtained by deletion, substitution, insertion and/or addition of amino acids can be produced by the following methods: in the DNA encoding the wild-type protein, this is achieved, for example, by site-directed mutagenesis as a well-known technique (for example, see Nucleic Acid Research, Vol.10, No.20, p.6487-6500, 1982, the entire contents of which are incorporated herein by reference). In the present specification, "1 or more amino acids" are preferably amino acids to the extent that they can be deleted, substituted, inserted and/or added by site-directed mutagenesis. In addition, "1 or more amino acids" in the present specification may be 1 or several amino acids, as the case may be. Without being limited thereto, "1 or more amino acids" means amino acids within 50, preferably within 40, within 30, within 20, within 10, within 8, within 5, within 3. the mutant of tamavidin 1 or 2 may also be a mutant comprising a sequence identical to SEQ id no: 5 or 7 has an amino acid sequence having at least 60% or more, preferably 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more, more preferably 99.3% or more amino acid identity, and has the same biotin-binding activity as tamavidin 1 or 2, or a protein having high binding ability/low non-specific binding activity.

The% identity of two amino acid sequences can be determined by visual inspection and mathematical calculation. Alternatively, the percent identity of two protein sequences can be determined by comparison of the sequence information using the GAP computer program available from the University of Wisconsin Genetics Computer Group (UWGCG) based on the algorithm of Needleman, S.B. and Wunsch, C.D. (J.mol.Bol., 48: 443-. Preferred default parameters for the GAP program include: (1) scoring matrices (scoring matrix), blosum62 as described in Henikoff, S. and Henikoff, J.G (Proc. Natl. Acad. Sci. USA, 89: 10915-; (2) a gap penalty of 12 points; (3) a gap length penalty of 4 points; and (4) no penalty for end gaps.

Other procedures for sequence comparison used by those skilled in the art may also be used. Regarding percent identity, for example: sequence information can be compared and determined using the BLAST program described in Altschul et al (nucleic acids res., 25, p.3389-3402, 1997). This program can be used on the network at the National Center for Biotechnology Information (NCBI) or DNA Data Bank of Japan (DDBJ) websites. This website describes in detail various conditions (parameters) for identity search using the BLAST program, and can change the partial settings as appropriate, but the search is usually performed using default values. The% identity between two amino acid sequences can also be determined by a program such as genetic information processing software GENETYX Ver.7 (manufactured by GENETYX), or by the FASTA algorithm. At this time, the search may be performed using a default value.

The% identity of two nucleic acid sequences can be determined by visual inspection and mathematical calculation, and furthermore, the comparison is more preferably performed using a computer program to compare the sequence information. Representative preferred computer programs are the Wisconsin software package of the genetics computer group (GCG; Madison, Wisconsin), the "GAP" program version 10.0 (Devereux et al, 1984, Nucl. acids Res., 12: 387). Using this "GAP" program, it is possible to compare not only two nucleic acid sequences but also two amino acid sequences and to compare nucleic acid sequences with amino acid sequences.

The "biotin-binding protein" constituting the fusion protein bound to the carrier is used to bind the fusion protein to the carrier by binding biotin-binding protein, thereby producing a carrier to which the fusion protein is bound. Thus, although not limited thereto, it is preferable that the biotin binding activity of the fusion protein formed using a mutant of tamavidin 1 or tamavidin 2 is not significantly reduced as compared with the fusion protein formed using a wild-type of any of them.

Thus, without limitation, mutants of tamavidin 1 are preferably of the following sequence: SEQ ID NO: 5, N14, S18, Y34, S36, S78, W82, W98, W110 and D118 are not modified. Note that, for example, Y34 represents SEQ ID NO: 5 amino acid sequence 34. Alternatively, in the case of modifying these amino acids, the amino acids are preferably modified to have similar properties or structures, and preferably, when asparagine (N14) is used, glutamine (Q) and aspartic acid (D) may be modified, and aspartic acid is preferably modified; when serine (S18, S36, S78), it may be modified to threonine (T) or tyrosine (Y), preferably to threonine; when tyrosine (Y34), it may be modified to serine (S), threonine (T) or phenylalanine (F), preferably to phenylalanine; when tryptophan (W82, W98, W110), it may be modified to phenylalanine (F); when aspartic acid (D118), glutamic acid (E), asparagine (N) may be modified, and asparagine is preferred.

Furthermore, the tamavidin 2 mutant is preferably of the following sequence: SEQ ID NO: 7 (W69, W80, W96, W108) is not modified. Alternatively, in the case of modifying these amino acids, it is preferable to modify them to amino acids similar in nature or structure, such as phenylalanine (F). Furthermore, it is expected that amino acid residues believed to directly interact with biotin (N14, S18, Y34, S36, S76, T78, D116) are also unmodified. Alternatively, in the case of modifying these amino acids, it is preferable to modify the amino acids so that the binding to biotin can be maintained, and it is preferable to modify the amino acids individually so that the amino acids are, for example, asparagine (N14), glutamine (Q), aspartic acid (D), preferably aspartic acid; when aspartic acid (D40), asparagine (N) may be modified; when serine (S18, S36, S76), it may be modified to threonine (T) or tyrosine (Y), preferably to threonine; when tyrosine (Y34), it may be modified to serine (S), threonine (T) or phenylalanine (F), preferably to phenylalanine; in the case of threonine (T78), it may be modified to serine (S), tyrosine (Y), preferably to serine; when aspartic acid (D116), glutamic acid (E), asparagine (N) may be modified, and asparagine is preferably modified.

In the present invention, preferred tamavidin modifications comprise the following (PCT/JP 2009/64302).

In a nucleic acid comprising SEQ ID NO: 7, or an amino acid sequence having 1 to a plurality of amino acid mutations in the sequence, or an amino acid sequence having 80% or more identity to the sequence, and showing a biotin-binding activity, wherein 1 or more residues selected from the group consisting of:

1) SEQ ID NO: 7, an arginine residue at position 104;

2) SEQ ID NO: 7, lysine residue at position 141;

3) SEQ ID NO: 7, a lysine residue at position 26; and

4) SEQ ID NO: 7, lysine residue at position 73.

More preferably a modified biotin-binding protein selected from the group consisting of:

in SEQ ID NO: 7, a modified biotin-binding protein in which the arginine residue at position 104 is substituted with a glutamic acid residue and the lysine residue at position 141 is substituted with a glutamic acid residue (R104E-K141E);

in SEQ ID NO: 7 in which the aspartic acid residue at position 40 is substituted with an asparagine residue and the arginine residue at position 104 is substituted with a glutamic acid residue (D40N-R104E);

in SEQ ID NO: 7 in which the aspartic acid residue at position 40 is substituted with an asparagine residue and the lysine residue at position 141 is substituted with a glutamic acid residue (D40N-K141E); and

in SEQ ID NO: 7, a modified biotin-binding protein wherein the aspartic acid residue at position 40 is substituted with an asparagine residue, the arginine residue at position 104 is substituted with a glutamic acid residue, and the lysine residue at position 141 is substituted with a glutamic acid residue (D40N-R104E-K141E).

Fusion protein of protein specifically binding to substance to be detected and biotin-binding protein

The present invention fixes a fusion protein of a protein that specifically binds to a substance to be detected and a biotin-binding protein (hereinafter, also referred to as "biotin-binding protein fusion protein" or "fusion protein") on a support.

The method for preparing the biotin-binding protein fusion protein is not particularly limited, and for example, expression can be performed by a known genetic engineering technique. For example, a fusion protein can be obtained by expressing a fusion protein gene encoding a biotin-binding protein and a desired protein using an expression system such as E.coli. For high expression in E.coli, tamavidin or a mutant thereof is preferably used.

In the biotin-binding protein fusion protein, the biotin-binding protein may be directly bound to a desired protein, or may be bound via a linker, preferably an amino acid linker. The length of the linker may be at least 1 amino acid or more, preferably 5 amino acids or more, and more preferably 6 amino acids or more. In order to further improve the binding force between biotin and tamavidin bound to the carrier, the binding force is preferably 10 amino acids or more, more preferably 12 amino acids or more, 15 amino acids or more, 18 amino acids or more, and still more preferably 25 amino acids or more. Furthermore, it is speculated that such linkers may also enhance the activity of tamavidin fusion proteins. The amino acid constituting the linker is not particularly limited, and preferably is composed of a repeating neutral amino acid such as glycine, serine, or alanine. For example, the following may be mentioned but not limited to: GGGGS, GGSGG, GASAG, GSGAA, GSGSGSA, GGGGSG, GGGSGGS, GGSGGGGS, AAAAGSGAA, GGGGSGGGGSGGS, GGGGSGGGGSGGGGSGGGGSG GGGS (SEQ ID NO: 8-18), etc.

Further, the biotin-binding protein may be bound to either the N-terminal side or the C-terminal side of the desired protein. In addition, when expressing a desired protein, a leader sequence for targeting the periplasmic space can be used in the case where the expression is more suitable for the periplasmic space than in the cytoplasm of E.coli, for example. Such leader sequences include, but are not limited to, the following PelB (Lei et al (1987) J Bacteriol 169: 4379-4383), OmpA (Gentry-Weeks et al (1992) J Bacteriol 174: 7729-7742), and the like.

When the biotin-binding protein fusion protein is obtained from the soluble fraction, the crude protein extract may be contacted with a biotinylated carrier without purification, whereby the fusion protein is bound to the biotinylated carrier and then sufficiently washed, whereby purification of the fusion protein and immobilization on the carrier can be achieved at once. Alternatively, purification may be carried out using a column to which biotin analogue such as iminobiotin (Hofmann et al (1980) ProcNatl Acad Sci USA 77: 4666-4668) is bound, followed by binding to a biotinylated carrier.

Alternatively, a purification tag may be further added to the N-terminus or C-terminus of the biotin-binding protein fusion protein. Such labels are considered to be, but not limited to, the following: c-myc epitope tags (Munro and Pelham (1986) Cell 46: 291-300), histidine tags (Hochuli et al (1988) Bio/Technol 6: 1321-1325, Smith et al (1988) J Biol Chem 263: 7211-7215), Halo tags (Los and Wood (2007) Methods Mol Biol 356: 195-208), Flag tags (Einhauer and Jungbauer (2001) J Biochem Biophys Methods 49: 455-465), and the like, or combinations thereof.

When the fusion protein is obtained from the insoluble fraction, a well-known method can be used: proteins are solubilized using chaotropic salts (chaotropic salt) such as urea and guanidine hydrochloride, and after solubilization, folding of the proteins is promoted (refolding) by using dialysis or the like while removing the chaotropic salts slowly (Sano and Cantor (1991) Bio/Technology 9: 1378-1381, Sano et al (1992) Proc Natl Acad Sci USA 89: 1534-1538).

Alternatively, when the desired Protein is expressed as an insoluble fraction in E.coli, for example, a Protein which binds to maltose (Bach et al (2001) J Mol Biol 312: 79-93), thioredoxin (Jurado et al (2006) J Mol Biol 357: 49-61), glutathione S transferase (Tudyka and Skerra (1997) Protein Sci 6: 2180-: 99-105, or further fusion of a partner to a fusion protein to prepare a 3-plex fusion protein. Maltose binding protein, thioredoxin, and glutathione S transferase can be used as a purification tag.

The expression system of the fusion protein may be expressed by other known expression systems such as insect cells, plant cells, mammalian cells, yeast cells, Bacillus subtilis cells, and cell-free expression systems. In particular, when a protein to be fused (e.g., a lectin or the like) is expressed by a plant cell, it is also preferable to express the fusion protein by using an expression system of the plant cell. The person skilled in the art can select an appropriate expression system in consideration of the properties of the fusion partner protein.

Conjugation of fusion proteins to Supports

In the present invention, a biotin-bonded carrier or biotin-binding protein fusion protein is prepared, and the carrier and the protein are brought into contact with each other to bind to each other by avidin-biotin binding.

Without being limited thereto, a crude extract of cell disruption containing a biotin-binding protein fusion protein is prepared so that the total protein concentration is 0.1mg/ml to 5mg/ml, preferably 0.2mg/ml to 2 mg/ml. The extract is contacted with the carrier to which biotin is bonded at 10 to 40 ℃, preferably 20 to 30 ℃ for 5 minutes to 2 hours, preferably 30 minutes to 1 hour. Alternatively, the biotin-binding protein fusion protein purified to a concentration of 0.1. mu.g/ml to 5. mu.g/ml is brought into contact with a carrier to which biotin is bound.

Adding a biological sample to the carrier (step 3 of embodiment 1)

The method according to embodiment 1 of the present invention, after preparing a carrier to which a fusion protein is bound, includes, as step 3):

mixing (a) a biological sample, and

(b-ii) a cell disruption extract liquid prepared from a cell in which a biotin-binding protein has been expressed by genetic engineering techniques in the same cell as the host cell used for expression of the fusion protein in step 1).

Mixing and adding the mixture to the carrier to which the fusion protein is bound prepared in the step 2).

In general, in a detection method using a carrier, the following methods are known for reducing non-specific binding that causes a background signal: a method in which a bacterial component extract is contained in a detection reagent (Japanese patent application laid-open No. 59-99257); a method of adding a culture component of a host cell into a sample, the host cell being introduced with a vector that is the same as a vector for producing a recombinant protein that specifically binds to a substance to be detected and does not contain a gene encoding the protein (Japanese patent application laid-open No. 8-43392); a method in which an aqueous extract solution derived from cells that are the same as the cells producing the recombinant protein that specifically binds to the substance to be detected and do not contain the protein is subjected to heat treatment, and then the water-soluble fraction is added to a sample (Japanese patent application laid-open No. 2004-301646).

The present inventors have attempted to use the above-described method in a fusion protein system, but have failed to obtain sufficient effects. However, as a result of intensive studies, a method that can obtain more significant effects has been devised. Specifically, it was found that when the sample to be tested is brought into contact with the carrier, it is preferable to allow both the cell disruption extract and the biotin-binding protein to be present at the same time.

In one embodiment, the biological sample is admixed with the addition of: a cell disruption extract prepared from the same host cell as used for expressing the fusion protein, and a biotin-binding protein. When the biotin-binding protein and the cell disruption extract are added to the sample to be tested, either one of them may be added first, or both of them may be added at the same time. Alternatively, the biotin-binding protein and the cell disruption extract are mixed and then added to the sample to be tested.

In another embodiment, a cell disruption extract prepared from cells in which a biotin-binding protein is expressed by genetic engineering techniques in the same kind of cells as the host cells for expressing the fusion protein may be added to the sample. Specifically, there may be used: the cell disruption extract is obtained by recombining the biotin-binding protein into an expression vector and expressing the recombinant protein in the cell.

When the sample to be tested is serum or the like, the serum is usually diluted 10-10000 times, preferably 100-1000 times, more preferably 100-500 times, with the cell disruption extract.

The cells derived from the cell disruption extract solution may be Escherichia coli cells, yeast cells, mammalian cells, insect cells, plant cells, etc., and are not particularly limited, but cells of the same type as the host cells used for expression of the fusion protein are preferred. For example, in the case where the fused cells are prepared using E.coli, it is preferable that the cell disruption extract is also prepared using E.coli. When the fusion protein is expressed by a cell-free system, the cell disruption extract used may be used as it is, or the cell disruption extract may be suspended in a desired buffer solution.

More preferably, the cell is a cell of biological origin, preferably of a different species than the organism from which the biological sample is derived. For example, when the biological sample is a human-derived sample, the cell disruption extract is preferably prepared from cells derived from a non-human organism. However, for example, when a fusion protein is expressed using mammalian cells, a cultured cell line prepared from a specific cancer cell or the like is generally used. In this case, since the content of human cells in a human cultured cell line is substantially different from that of human cells in a living body (for example) derived from a biological sample, the effect of the present invention can be obtained even when a cell disruption extract solution of a human cultured cell line is used.

The cells used for preparing the cell disruption extract may contain any carrier, and preferably contain an empty carrier. The empty vector may be a vector of the same type as the vector used for expressing the protein or fusion protein thereof to which the substance to be detected specifically binds, and which does not contain a gene encoding the above-mentioned protein, or may be any vector in which the empty vector further contains any nucleic acid, for example. The vector used for expressing the protein or the fusion protein thereof to which the substance to be detected specifically binds may be any unrelated known vector.

The extract solution obtained by disrupting cells is not particularly limited as long as it is a component derived from cells, and for example, a protein component, a saccharide component, a lipid component, or a mixture thereof can be used. Preferably, soluble extracts of the cells can be used.

The method for preparing the extract solution for cell disruption is not particularly limited, and various methods can be used. In general, it can be prepared as follows: the soluble component can be obtained by disrupting or solubilizing cells cultured in an appropriate medium by a physical method such as ultrasonic waves, a chemical method using a surfactant or the like, an enzymatic treatment or the like, and then subjecting the cells to centrifugation, filtration or the like. In order to prolong the shelf life, it is preferable to add a protease inhibitor to a clarified liquid by centrifugation, filtration, or the like, or to perform a heat treatment such as an autoclaving treatment to inhibit or inactivate various enzymes derived from cells, or the like. The concentration of the cell disruption extract added may be changed depending on the intensity of the non-specific reaction to be generated, and a concentration sufficient for absorbing the non-specific reaction may be appropriately set.

As an example of a specific method for preparing a cell disruption extract solution, although not limited thereto, for example, in the case of Escherichia coli cells, Escherichia coli (which may contain a vector and may contain a gene encoding a biotin-binding protein) may be inoculated into LB medium containing an antibiotic, shake-cultured at 25 ℃ to 37 ℃ to give an OD600 of 0.25 to 1, preferably 0.4 to 0.6, then IPTG of 0.1mM to 5mM, preferably 0.5mM to 1mM, is added, and further shake-cultured at 25 ℃ to 37 ℃ for 2 hours to 24 hours, preferably 4 hours to 16 hours. The culture solution was centrifuged to recover the cells, the cells were suspended in a desired buffer solution and then disrupted, and the disrupted solution was centrifuged to recover the supernatant as a crude extract of Escherichia coli.

The biological sample and the cell disruption extract can be added to the carrier by any method. However, the biological sample must be contacted with the cell disruption extract before the biological sample is contacted with the carrier. That is, the biological sample and the cell disruption extract may be brought into sufficient contact with each other, and it is not always necessary to add the components derived from the cell disruption extract to the carrier together with the biological sample. For example, a carrier to which a cell disruption extract component is bound can be prepared, and the treated biological sample can be used therein. Specifically, the biological sample may be passed through a column for cell disruption extract liquid components.

When a crude cell disruption extract is mixed with a biological sample, the sample is reacted with a crude cell disruption extract prepared from a desired buffer solution (which may contain BSA, casein, a commercially available blocking agent, or the like) at a total protein concentration of 0.05mg/ml to 5mg/ml, preferably 0.5mg/ml to 5mg/ml, at 10 ℃ to 30 ℃, preferably 20 ℃ to 30 ℃ for 30 minutes to 4 hours, preferably 1 hour to 2 hours, although not limited thereto. When the biological sample is serum, the biological sample is not limited to serum, and may be diluted 100 to 1000 times by the crude cell disruption extract.

Adding biotin-binding protein to test sample

As a feature of the present invention, a cell disruption extract solution prepared from cells of the same species as the host cells for expressing the fusion protein and a biotin-binding protein are mixed with a biological sample, and the mixture is added to a support (step b-i), or a cell disruption extract solution prepared from cells of the same species as the host cells for expressing the fusion protein, in which the biotin-binding protein has been expressed by genetic engineering techniques, is mixed with a sample to be tested, and the mixture is added to a support (step b-ii).

In the method of the present invention, the addition of biotin-binding protein to a sample to be tested can ultimately suppress the background signal.

Such a biotin-binding protein may be the same as or different from the biotin-binding protein constituting the fusion protein. In addition, it may be a wild type or a mutant, and the biotin-binding ability may be the same as, or higher than, or lower than that of the wild type.

In addition, as an embodiment of addition, biotin-binding protein (may be naturally derived or may be a protein expressed by genetic engineering) may be added as it is, or may be dissolved in an appropriate liquid and then added. Further, the following embodiments are also possible: for example, a mixture of a sample and a cell disruption extract solution is treated (for example, by a column) with a carrier having a biotin-binding protein immobilized thereon without directly adding the biotin-binding protein to the sample. (Process b-i).

When the biotin-binding protein is added to the crude cell disruption extract, the concentration of the biotin-binding protein added is, but not limited to, 1. mu.g/ml to 500. mu.g/ml, preferably 10. mu.g/ml to 100. mu.g/ml, in terms of the final concentration. When expressing the biotin-binding protein in the cell by genetic engineering, the concentration of the biotin-binding protein may be the same, but the present invention is not limited thereto.

Alternatively, a cell extract containing a biotin-binding protein obtained by introducing a gene encoding the biotin-binding protein into a host cell and expressing the gene, and disrupting the host cell (step b-ii) can be used. In this case, the biotin-binding protein can be expressed in a desired host by a method known to those skilled in the art, but when a fusion protein of a protein that specifically binds to a substance to be detected and the biotin-binding protein is expressed by genetic engineering, it is preferably the same as the host.

When the host is Escherichia coli, a gene encoding a biotin-binding protein is incorporated into an expression vector, and the expression vector is introduced into Escherichia coli, and the Escherichia coli is cultured while inducing protein expression. The induction conditions such as the expression vector, the host E.coli strain, the medium components, the IPTG concentration, and the culture temperature can be appropriately selected.

Method for detecting substance to be detected (step 4 of embodiment 1)

The detection method of the present invention detects a substance to be detected that binds to a protein that specifically binds to the substance to be detected in a fusion protein.

The method for detecting the substance to be detected can be appropriately selected by those skilled in the art based on the properties of the desired protein. Preferred methods include: enzyme-linked immunosorbent assay methods (including ELISA, Sandwich ELISA), immunoassay methods such as Radioimmunoassay (RIA), nucleic acid hybridization assay methods, surface plasmon resonance assay methods, and the like. A substance that is immobilized by avidin-biotin binding and specifically binds to/interacts with a substance to be detected is reacted with a sample to be detected, and then the substance to be detected is detected.

In the immunoassay, for example, when the substance to be measured is an antibody, the antigen is immobilized, and the antibody present in the sample to be detected is reacted with the antigen, and the detection is performed by a method known to those skilled in the art. For example, when the sample to be tested is a human-derived material, a human antibody that binds to an antigen is detected using an anti-human antibody. In this case, the amount of the antibody can be indirectly measured and quantified by measuring the final amount of fluorescence, enzyme activity, or radioactivity by labeling the anti-human antibody with fluorescence, enzyme, or radioisotope in advance. In addition, when the substance to be measured is an antigen, an antibody against a certain portion (epitope) of the antigen is made into a solid phase, and then it is reacted with the antigen present in the sample to be tested. Then, it is further reacted with an antibody against other epitopes of the antigen. The amount of the antigen can be indirectly measured by labeling the secondary antibody against the other epitope in advance as described above. Alternatively, in the nucleic acid hybridization assay, avidin-biotin binding is used to immobilize nucleic acid having several tens to several hundreds or several thousands of bases in a sequence region complementary to the nucleic acid to be assayed. The sample is reacted with a sample containing a nucleic acid labeled with a fluorescent or radioactive isotope in advance, and the amount of fluorescence or radioactivity is measured.

The labeling may be carried out by any method known to those skilled in the art, and commercially available anti-human antibodies labeled with fluorescence or enzyme may be used. As the fluorescent label, for example, a label such as fluorescein and rhodamine, or a fluorescent protein such as GFP can be used. Examples of the enzyme label include peroxidase, alkaline phosphatase, luciferase, and glucose oxidase, but are not limited to these enzymes. Substrates for measurement by these enzymes are commercially available, and in the case of peroxidase, for example, TBA or substrates for chemiluminescence can be used. Examples of the radioactive isotope include: iodine (I)¹²⁵I、¹²¹I) Carbon (C)¹⁴C) Sulfur (S), (S)³⁵S), and tritium (³H) In the case of nucleic acids, phosphorus (B) may be mentioned³²P), and the like.

The amount of antigen, antibody present in a biological sample (sample) can be readily calculated by comparison with the amount present in a standard preparation, such as a standard sample of a healthy person or a standard sample of a typical patient in the case of a clinical sample, using, for example, a linear regression computer algorithm. Such assays for detecting antigens, antibodies, for example, with regard to ELISA, such as Iacobelli et al, BreastCancer Research and Treatment 11: 19-30 (1988).

Alternatively, for example, when the amount of a substance to be detected, for example, an antibody, in a biological sample is small (when the antibody titer is low), or when a biological sample such as serum is not specifically bound to itself in a large amount, the influence of background signals due to non-specific binding increases. Therefore, by appropriately subtracting the background signal from the measurement value, the substance to be detected can be more accurately measured. The person skilled in the art can appropriately judge the subtracted background according to the experimental system.

For example, as an example of such an embodiment, there is a "human/monkey anti-type I and type II collagen IgG antibody measurement kit" (manufactured by Chondrex corporation) for detecting an antibody against collagen present in serum. In this kit, a background value (a measurement value obtained from only the secondary antibody without adding serum) is subtracted from the measurement value of the sample.

In addition, in the case where there is a large amount of nonspecific binding in a biological sample itself such as serum, in order to subtract the background due to such nonspecific reaction, as described in example 2, an embodiment is also effective in which the measurement value of the region in which the SITH-1 antigen is not immobilized (but the blocking operation using BSA or the like is performed in the same manner as the region in which the SITH-1 antigen is immobilized), and the measurement value of the serum (biological sample) containing an anti-SITH-1 antibody (the substance to be detected in the biological sample) is added, is subtracted from the measurement value of the carrier (plate) in which the fusion protein of the SITH-1 antigen (the protein that specifically binds to the substance to be detected) and tamavidin is immobilized. Alternatively, as described in example 2 or example 3, the measurement value of the carrier on which tamavidin is immobilized (bound) can be subtracted from the measurement value of the carrier (plate or magnetic bead) on which the fusion protein of the SITH-1 antigen and tamavidin is immobilized, thereby calculating the measurement value.

As a further example of the embodiment for subtracting the specific nonspecific reactions from the sample, there may be a case where the measurement value of the wells to which collagen is not immobilized is subtracted from the measurement value of the collagen antibodies in serum using the wells of a microtiter plate to which collagen is immobilized, as in the above-described "human/monkey anti-type I and type II collagen IgG antibody measurement kit". In addition, as a kit according to a similar embodiment, there is "virus antibody EIA 'Shengyan' herpes IgM" for detecting an anti-herpes simplex virus IgM-type antibody in serum or plasma (manufactured by Denka Shengyan Co., Ltd.).

Further, the following embodiments are preferred: the measured value can be obtained more accurately by subtracting the measured value of the region of an arbitrary protein (for example, GFP or the like in the case where the organism of origin is a mammal, although not limited thereto) which is not an antibody possessed by the organism of origin (for example, SITH-1, the organism of origin is a human) to which the sample is immobilized. The method of solid-phase immobilization is not particularly limited, and it is preferable to prepare a fusion protein with a biotin-binding protein and immobilize the fusion protein on a biotinylated carrier by biotin-avidin binding.

The calculation method described above can be appropriately designed and selected by those skilled in the art according to the properties of the biological sample, the characteristics of the antibody used, and the like.

The detection method of the present invention is preferably capable of specifically detecting an antibody having a low antibody titer in serum.

The effect of embodiment 1 of the present invention can be understood by, for example, the following embodiments: the results of the TM2/pTrc99A/BL21 cell disruption extract (black triangles) of FIG. 3-1 of example 2 were compared with those of PBS (white squares) or pTrc99A/BL21 cell disruption extract (black circles). Further, the S/N ratio of the crude extract of BL21 introduced with TM2/pTrc99A in Table 3 of example 3 was compared with the S/N ratio of the disrupted extract of BL21 introduced with PBS or pTrc 99A.

Detection method of the present invention (embodiment 2)

The detection method (embodiment 2) of the present invention is a method for detecting a substance in a biological sample, and includes:

In the method of embodiment 2 of the present invention, the method of producing the carrier to which the fusion protein is bound in steps 1 and 2 is the same as in embodiment 1. The same applies to the last step of detecting a substance to be detected that binds to a protein that specifically binds to a substance to be detected in a fusion protein (step 4 in embodiment 1, step 5 in embodiment 2).

The method of embodiment 2 is characterized in that the carrier blocking step (step 3 of embodiment 2) is performed before the biological sample is added to the carrier.

In the present invention, the carrier to which the fusion protein is bound can be further contacted with the biotin-binding protein before the addition of the biological sample to the carrier, thereby more effectively suppressing the background signal. Without being bound by theory, it is believed that this is because the biotin moiety in a free state (free) which is not bound to the fusion protein is bound to the biotin-binding protein on the surface of the carrier (FIG. 1).

The biotin-binding protein to be contacted with the conjugate carrier may be the same as or different from the biotin-binding protein constituting the fusion protein. Further, the mutant may be a wild type or a mutant. The biotin-binding ability may be equal to or higher or lower than that of the wild type, and is preferably equal to or lower than that of the wild type. For example, the biotin-binding activity of the protein is lower than that of the wild-type protein, and the dissociation constant (KD) of the protein to biotin is less than 10^-5(M) in the presence of a protein. For the preparation of such biotin-binding proteins, there are methods of chemical modification (US-A-5051356), and the most preferred is A method of modifying the amino acid sequence (Qureshi et al (U.S.)2002)J.Biol.Chem.、276、46422-46428)。

In the present invention, the method for contacting the biotin-binding protein with the conjugate carrier is not particularly limited. That is, the biotin-binding protein may be added as it is, or may be dissolved in an appropriate liquid and brought into contact with the carrier. In addition, a blocking agent known to those skilled in the art, such as BSA or casein, may be used together.

Without being limited thereto, biotin-binding protein is added to a desired buffer (e.g., TBS or PBS containing Tween-20 or Triton at a concentration of 0.02% to 1%, preferably 0.1 to 0.5%) at a final concentration of 1. mu.g/ml to 500. mu.g/ml, preferably 10. mu.g/ml to 100. mu.g/ml. Further, BSA or casein may be added thereto at a final concentration of 1. mu.g/ml to 50. mu.g/ml, preferably 5. mu.g/ml to 10. mu.g/ml. The blocking solution is contacted with a carrier which binds to the biotin-binding protein fusion protein by avidin-biotin binding at 10 to 40 ℃, preferably 20 to 30 ℃ for 10 minutes to 2 hours, preferably 30 minutes to 1 hour.

The effects of embodiment 2 of the present invention can be understood by, for example, the following embodiments: in example 2, the values of pTrc99A/BL21 cell disruption extract (black circles) in FIGS. 3-1 and 3-2 were compared with each other when the antibody was 0. When the antibody is 0, the fluorescence should be 0, assuming that there is no nonspecific binding, since there is no antibody. Here, comparing the black circles in the case of antibody 0 in fig. 3-1 and 3-2, it is considered that the fluorescence value decreased almost in half when the carrier was blocked with TM2, and the nonspecific binding decreased greatly. In fig. 3-2 in which the carrier was sealed by TM2, the entire graph was moved downward as compared with fig. 3-1. In addition, the results of cell disruption of the extract solution by pTrc99A/BL21 in Table 2 of example 2 can be understood by comparing the value of "blocking by BSA" with the value of "blocking by BSA and TM 2".

Detection method of the present invention (embodiment 3)

The detection method (embodiment 3) of the present invention is a method for detecting a substance in a biological sample, and includes:

a method of detecting a substance in a biological sample, comprising:

4) after the blocking step of step 3), (a) a biological sample, and

Embodiment 3 is a combination of embodiment 1 and embodiment 2. Specifically, the cell disruption extract and the biotin-binding protein of embodiment 1 were added, and the carrier was blocked by the biotin-binding protein of embodiment 2.

The effect of embodiment 3 of the present invention can be understood by, for example, the following embodiments: the results of the TM2/pTrc99A/BL21 cell disruption extract (black triangles) of FIG. 3-2 in example 2 were compared with other results, for example, the results of PBS (white squares), of FIGS. 3-1 and 3-2. In example 4, the S/N ratio of the crude extract of BL21 introduced into TM2/pTrc99A of Table 5 was compared with the S/N ratio of the crushed extract of BL21 introduced into PBS, E.coli (BL21) or pTrc 99A.

Support bound with fusion protein

Further, the present invention provides a carrier for detecting a substance in a biological sample. That is, the carrier of the present invention is a carrier that binds to a fusion protein of a biotin-binding protein and a protein that specifically binds to a substance to be detected by binding between biotin and biotin-binding proteins, and is produced by the following method:

Thus, the carrier of the present invention is preferably bound to a fusion protein of a protein that specifically binds to a substance to be detected and a biotin-binding protein by avidin-biotin binding, and unreacted biotin on the carrier is preferably bound to the biotin-binding protein by avidin-biotin binding.

VII. kit

Further, the present invention provides a kit for detecting a substance in a biological sample. The kit of the present invention comprises:

A) a carrier bound to a fusion protein of a biotin-binding protein and a protein specifically binding to a substance to be detected by binding between biotin and biotin-binding protein, and

a reagent for diluting a biological sample comprising B-i) or B-ii) and/or;

C) A blocking agent comprising a biotin-binding protein.

The reagent for diluting the biological sample may be the cell disruption extract (and biotin-binding protein) itself, or a reagent for further diluting the cell disruption extract together with the biological sample, and may include an appropriate buffer solution, a commercially available solvent such as a cell diluent or a serum diluent.

Examples

The present invention will be specifically described below with reference to examples, which are not intended to limit the technical scope of the present invention. The present invention can be easily modified and changed by those skilled in the art in light of the description of the present specification, and these are included in the technical scope of the present invention.

In examples 1 and 2, the fusion protein of the SITH-1 protein and tamavidin 2 derived from human herpesvirus 6(HHV-6) was expressed using E.coli, and the crude extract of E.coli obtained by ultrasonication was allowed to react directly with a biotinylated microplate, and the fusion protein was immobilized by tamavidin-biotin binding. The SITH-1 plate thus obtained was reacted with human serum (containing rabbit anti-SITH-1 antibody) diluted with a crude extract of Escherichia coli, and the amount of anti-SITH-1 antibody contained in human serum was measured by using, as a model clinical sample, a commercially available human serum to which rabbit anti-SITH-1 antibody (antiserum) diluted in stages was added.

Example 1 construction of a vector for expressing a fusion protein of the SITH-1 Gene and tamavidin 2

In this example, a gene encoding a fusion protein in which tamavidin 2 (hereinafter, referred to as TM2) was placed on the C-terminal side of SITH-1 via a linker was designed.

1-1. design of primers

To construct the SITHl-TM2 fusion gene, first, primers for fusing the two genes, SITH-1 and TM2, mediated by a linker (5 xlinker: GGGGSGGGGSGGGGSGGGGSGGGGS) (SEQ ID NO: 18) were designed.

Namely, the following primers were designed: a primer (SITH1C-5xlink-TM2N-F) (SEQ ID NO: 19) comprising a DNA sequence coding for the C-terminal site of SITH-1 on the 5 'side, a linker at the center, and the N-terminal site of TM2 on the 3' side, and a primer (SITH1C-5xlink-TM2N-R) (SEQ ID NO: 20) comprising a DNA sequence coding for the N-terminal site of TM2 on the 5 'side, a linker, and the C-terminal site of SITH-1 on the 3' side.

Next, a primer (SITH15 ' EcoRI-F (SEQ ID NO: 21)) comprising the 5 ' portion of the N-terminal region of SITH-1 and provided upstream thereof with an EcoRI restriction endonuclease cleavage site (CCATGG), and a primer (TM2CtermBam) (SEQ ID NO: 22) consisting of the 3 ' portion of the gene encoding TM2 and downstream thereof with a BamH I restriction endonuclease cleavage site (GGATCC) sequence were designed. The primers used to construct the fusion gene of SITH-1 and TM2 are summarized in Table 1.

[ Table 1]

Primer for constructing fusion gene of SITH-1 and TM2

Restriction enzyme recognition sites are underlined. Linker sequences are represented in lower case font.

1-2.PCR

To construct the SITH1-TM2 fusion gene, two-stage PCR was performed.

The first stage of PCR was performed by the following amplification: the site of SITH-1 was amplified using the FLAG expression vector (SIGMA) plasmid in which the SITH-1 gene (ORF) (SEQ ID NO: 2) was recombined as a template, and primers SITH 15' EcoRI-F and SITH1C-5xlink-TM2N-R, and the site of TM2 was amplified using the vector pTrc99A plasmid (WO02/072817) in which the TM2 gene was recombined as a template, and primers SITH1C-5xlink-TM2N-F and TM2 CtermBam.

The PCR reaction conditions are as follows: to 20. mu.l of the reaction solution, 500ng of template DNA, 2. mu.l of 10 XExTaq buffer (TaKaRa Co.), 1.6. mu.l of 2.5mM dNTP, 20 picomoles of each primer, and 0.1. mu.l of 5U/. mu.l Ex Taq were added, and the reaction was carried out at 96 ℃ for 3 minutes 1 time using a GeneAmp PCR System 9600 (PERKINELMER); 95 ℃, 1 minute, 60 ℃, 1 minute, 72 ℃, 2 minutes, 20 times; 72 ℃ for 6 minutes, 1 time. As a result, 579bp of PCR product was obtained in the SITH-1 part, and 528bp of PCR product was obtained in the TM2 part. These PCR products were fractionated by agarose gel electrophoresis in TAE buffer. Gels of the respective PCR products were excised, and the products were collected by QIAEX II gel extraction kit (QIAGEN). The extraction method is carried out according to the instruction of the kit.

Using the above two PCR products as templates, second-stage PCR was performed using primers SITH 15' EcoRI-F and TM2 CtermBam. The reaction conditions are as follows: to 20. mu.l of the reaction solution were added 500ng each of the template DNA, 2. mu.l of 10 XPyrobest buffer (TaKaRa Co.), 1.6. mu.l of 2.5mM dNTPs, 20 picomoles each of the primers, and 0.1. mu.l of 5U/. mu.l Pyrobest DNA polymerase, and the mixture was subjected to 96 ℃ for 3 minutes and 1 time using a GeneAmp PCR System 9600(PERKIN ELMER); 95 ℃, 1 minute, 60 ℃, 1 minute, 72 ℃, 2 minutes, 20 times; 72 ℃ for 6 minutes, 1 time. As a result, a 990bp PCR product was obtained.

1-3 cloning

The SITH1-TM2 fusion gene obtained by PCR was cloned into the vector pCR4BluntTOPO (manufactured by Invitrogen).

Specifically, first, ligation (ligation) was performed according to the instructions attached to the vector kit. The DNA was introduced into E.coli TB1 by electroporation according to the usual method (Sambrook et al 1989, Molecular Cloning, A laboratory manual, 2)^ndedition) to extract plasmid DNA. For the plasmid for which the presence of the insert was confirmed, the nucleotide sequence was sequenced using M13 primer (TaKaRa Co.), ABI PRISM fluorescent sequencer (Model 310 Genetic Analyzer, Perkin Elmer Co.), and compared with the designed gene sequence, thereby confirming the absence of mutation. The plasmid having the recombinant fusion gene was double digested with EcoRI and BamHI, subjected to agarose gel electrophoresis and purification in the same manner as described above, and the DNA fragment was recovered. This fragment was ligated with E.coli-use expression vector pTrc99A (produced by Pharmacia) previously digested with EcoRI and BamHI using a ligation kit (produced by TaKaRa). The ligation product was transformed into E.coli TB1, and plasmid DNA was extracted and further transformed into E.coli BL 21. To obtainThe E.coli colony of (1) was used as a template, and the presence or absence of the inserted gene was confirmed by PCR amplification of the inserted gene site using SITH 15' EcoRI-F and TM2 CtermBam.

As described above, the expression vector for the fusion protein of SITH-1 and TM2, SITH1-TM2/pTrc99A, was completed. The base sequence of SITH1-TM2 in the coding expression vector SITH1-TM2/pTrc99A is shown as SEQ ID NO: 23, and the coded amino acid sequence is shown as SEQ ID NO: as shown at 24.

With regard to SITH1-TM2, on the 5' side, two amino acid residues (base numbers 4-9 of SEQ ID NO: 23, amino acid residue numbers 2-3 of SEQ ID NO: 24) were added after the translation-initiating methionine, since recombination was made into pTrc99A using the EcoRI site. Next, the sequences of SITH-1 (base numbers 10-483 of SEQ ID NO: 23, amino acid residues 4-161 of SEQ ID NO: 24), 5xlinker (base numbers 484-558 of SEQ ID NO: 23, amino acid residues 162-186 of SEQ ID NO: 24), and TM2 (sequence excluding Met) (base numbers 559-981 of SEQ ID NO: 23, amino acid residues 187-326 of SEQ ID NO: 24) were continued.

1-4. coli expression

Coli BL21 having SITH1-TM2/pTrc99A introduced thereinto was inoculated into 50ml of LB medium containing the antibiotic ampicillin (final concentration: 100. mu.g/ml), and cultured with shaking at 30 ℃ until the absorbance at OD600 reached 0.5. Then, 1mM IPTG was added, followed by shaking culture at 30 ℃ for 5 hours. The cells were collected by centrifugation of 50ml of the culture. The cells were suspended in 3ml of 0.1MHEPES/KOH (pH7.4) and disrupted by ultrasonication. The disruption solution was centrifuged (15000rpm), and the supernatant was used as a crude extract of Escherichia coli. In order to confirm the expression of the TM2 fusion SITH-1 protein, the proteins contained in the crude extract were fractionated by SDS-PAGE and analyzed by immunoblotting.

The rabbit anti-SITH-1 antibody (antiserum) prepared by purifying SITH-1 expressed in E.coli and immunizing rabbits) and the alkaline phosphatase-labeled anti-rabbit IgG antibody (manufactured by BIO RAD) were each diluted 1000-fold to detect SITH-1. The results are shown in FIG. 2. A band (band) around 35kDa was detected in E.coli expressing the TM2 fusion SITH-1. This size is essentially consistent with the molecular weight of 34.8kDa for the TM2 fusion SITH-1. Further, Escherichia coli BL21(Takakura et al (2009) FEBS J.276: 1383-1397) into which pTrc99A and TM2/pTrc99A were introduced was cultured in the presence of IPTG in the same manner as described above, and then a crude extract of Escherichia coli was prepared.

Example 2 detection of anti-SITH-1 antibodies in the Presence of human serum by ELISA

The crude extract of Escherichia coli expressing the TM2 fusion SITH-1 obtained in example 1 was diluted with 0.1M HEPES/KOH (pH7.4) to give 2mg/ml of total soluble protein, and 100. mu.l of the extract was added to each well of a biotin plate (manufactured by Sumitomo Bakelite). After standing at room temperature for 1 hour, the TM2 fusion SITH-1 protein was immobilized on a biotin plate by tamavidin-biotin binding. Then, each well of the plate was washed 3 times with TBS Buffer (TBST) containing 0.1% Tween20, and 250. mu.l of 5. mu.g/ml BSA/TBST solution or 50. mu.g/ml purified TM 2/5. mu.g/ml BSA/TBST solution was added to each well, allowed to stand at room temperature for 1 hour, and blocked. Then, each well was washed 3 times with TBST.

Next, Human Serum (Human Serum pool, manufactured by Cosmo-Bio Inc.) was diluted 100-fold with PBS or the crude extract of E.coli introduced with pTrc99A containing 5mg/ml of total soluble protein prepared in examples 1 to 4 or the crude extract of E.coli introduced with TM2/pTrc99A containing 5mg/ml of total soluble protein to obtain a solution, and rabbit anti-SITH-1 antibody (antiserum) was added to the solution, wherein the rabbit anti-SITH-1 antibody (antiserum) was diluted stepwise to 0.002, 0.001, 0.0005 or 0.00025 in volume ratio, and 100. mu.l of each well of the TM2 fusion SITH-1-immobilized plate was added thereto, followed by reaction at room temperature for 1 hour.

The antibody titer in the serum of the rabbit anti-SITH-1 antibody (antiserum) was estimated to be about 50-fold higher than that in the serum of a typical depression (mood disorder) patient. Therefore, a rabbit anti-SITH-1 antibody (antiserum) was mixed with a commercially available (healthy human) human serum in an amount of 1/50 by volume, and this was considered as a model sample of a typical serum for a depression patient. Accordingly, the above-mentioned antibody dilution rate is considered to be equivalent to the case where the serum of a patient suffering from depression is diluted about 10 to 80 times.

As a control, the following two regions were set, respectively: for the biotin plate not subjected to any immobilization, a region blocked with only the above-mentioned BSA and a region blocked with both of the above-mentioned blocking operations with BSA and TM2 were used. When the blocking operation is performed using a solution containing tamavidin, since a part of tamavidin in the blocking solution specifically binds to biotin on the plate, it is considered that the same state as that of the system using the tamavidin-immobilized plate is obtained. Then, 100. mu.l of each of the mixtures obtained by adding the rabbit anti-SITH-1 antibody diluted stepwise to 100-fold human serum diluted with PBS solution, crude extract of Escherichia coli introduced with pTrc99A or TM2/pTrc99A (total soluble protein 5mg/ml) was added thereto, and the mixture was reacted at room temperature for 1 hour. The rabbit anti-SITH-1 antibody was reacted with TM2 fusion SITH-1 in the presence of human serum, and then washed 3 times with TBST.

Then, in order to detect the SITH-1 immobilized on the plate by tamavidin, the rabbit anti-SITH-1 antibody after reaction/binding, and the human IgG in the serum considered to be non-specifically bound in each well, 100. mu.l each of the mixed solutions of horseradish peroxidase-labeled goat anti-rabbit IgG antibody and peroxidase-labeled goat anti-human IgG antibody diluted 5000 times with TBST was added, and the mixture was allowed to stand at room temperature for 1 hour. Then, the peroxidase activity was measured by washing 3 times with TBST. The activity was determined by the following method: 100. mu.l of SuperSignal ELISA PicoChemimetric Substrate (PIERCE) was added to each well, and the mixture was allowed to stand at room temperature for 5 minutes, and then the luminescence amount was measured by a microplate reader Infinite M200 (manufactured by TECAN).

As data, the measurement of the luminescence amount in each concentration region of the rabbit anti-SITH-1 antibody and in each control region (region not immobilized with TM2 fusion SITH-1, subjected to blocking with BSA or BSA and TM2, and treated with human serum containing anti-SITH-1 antibody at each concentration) was performed at the same time, and the value of the control region was subtracted from the value of the luminescence amount in the region immobilized with TM2 fusion SITH-1. And this was used as a detection amount of the anti-SITH-1 antibody contained in the serum.

The results of the effect of the addition of the E.coli disrupted extract to the serum on nonspecific binding and the effect of the blocking with TM2 on nonspecific binding are shown in FIG. 3. In FIG. 3, the region in which the serum was diluted 100-fold with PBS is represented by white squares, the region in which the serum was diluted 100-fold with the E.coli disrupted extract having only the expression vector is represented by black circles, and the region in which the serum was diluted 100-fold with the E.coli disrupted extract expressing TM2 is represented by black triangles. The ordinate (fluorescence amount) of each graph represents the amount of the anti-SITH-1 antibody detected, and the abscissa represents the dilution ratio of the anti-SITH-1 antibody diluted in stages.

FIG. 3-1 shows: the amount of the antibody against SITH-1 in the serum was measured when the human serum was diluted with PBS (white square), crude E.coli extract (black circle) introduced with pTrc99A or E.coli extract (black triangle) introduced with TM2/pTrc99A without blocking with biotin-binding protein (TM 2).

Fig. 3-2 shows: when the serum was blocked with biotin-binding protein (TM2), the amount of the antibody to SITH-1 in the serum was detected by diluting the serum with PBS (white square), crude E.coli extract (black circle) introduced with pTrc99A, or crude E.coli extract (black triangle) introduced with TM2/pTrc 99A.

In order to compare the blocking effect and the effect of each serum dilution, the S/N ratio was calculated by the following method.

S/N ratio of the amount of anti-SITH-1 antibody detected in the region to which anti-SITH-1 antibody was added/the amount of anti-SITH-1 antibody detected in the region to which anti-SITH-1 antibody was not added under each concentration condition

Therefore, a larger S/N ratio indicates a higher detection sensitivity.

The results of calculating the S/N ratio of each region are shown in Table 2.

[ Table 2]

Each region shows the S/N ratio at a dilution ratio of 0 for the anti-SITH-1 antibody (when no anti-SITH-1 antibody is added) and a value of 1

In the region where the serum was diluted with PBS, the anti-SITH-1 antibody contained in the serum was bound to the serum in a large amount without specificity, and a high luminescence amount was also detected in the region where the anti-SITH-1 antibody was not added. On the other hand, in the region to which the crude extract of E.coli introduced with pTrc99A or the crude extract of E.coli introduced with TM2/pTrc99A was added, the fluorescence value in the region to which the SITH-1 antibody was not added was considerably low, and nonspecific binding was drastically reduced. In particular, the non-specific binding was low in the region to which TM2/pTrc99A was added and into which the crude extract of E.coli was introduced (FIGS. 3-1 and 3-2). At this time, it was found that: compared with blocking with BSA alone, blocking with TM2 (BSA + TM2) was quantitative, particularly in the detection of antibodies at low concentrations (dilution rate of 0.0005 or less) (fig. 3-2).

In addition, it was found that the region to which the crude extract of E.coli introduced with pTrc99A was added and the region to which the crude extract of E.coli introduced with TM2/pTrc99A was added were blocked with BSA added with TM2, and the anti-SITH-1 antibody could be detected with higher sensitivity (Table 2). In particular, the S/N ratio was significantly increased in the region to which the crude extract of Escherichia coli introduced with TM2/pTrc99A was added.

The following contents are displayed: the plate on which TM2 fused SITH-1 was immobilized was blocked with a TM 2-containing solution, and then human serum was diluted with a crude extract of TM 2-expressing escherichia coli, whereby nonspecific binding from human serum was reduced, and detection was also possible with good sensitivity, stability, and quantification for very low concentrations of anti-SITH-1 antibody.

Example 3 anti-SITH-1 antibody in the Presence of human serum by ELISA Using magnetic beads Line detection

In this example, when very low concentrations of SITH-1 antibody were detected, the effect of various serum dilutions was examined in a system in which TM2 fusion SITH-1 was immobilized on biotinylated magnetic beads.

3-1 preparation of magnetic beads having immobilized thereon TM2 fused with SITH-1

To 1mL (30mg beads/mL) of magnetic bead Dynabeads M-270 Amine (DynalBiotech Co., Ltd.) was added 1mL of 10mM NHS-Lc-biotin (PIERCE Co., Ltd.). Biotin and magnetic beads were covalently bound by reacting the amino groups with the NHS active ester groups by mixing at room temperature for 30 minutes by inversion. Then, the beads were washed 2 times with 0.1% BSA/0.01% Tween20/PBS solution and finally suspended in PBS buffer. The resulting magnetic beads (30mg beads/mL PBS) were used as biotinylated magnetic beads.

In the same manner as in examples 1 to 4, SITH-1 was fused with TM2 expressed in E.coli, and the crude extract of E.coli prepared was diluted with 0.1M HEPES/KOH (pH7.4) to give a total soluble protein concentration of 5mg/ml, to which biotinylated magnetic beads were added. The TM2 fusion SITH-1 was immobilized to magnetic beads by tamavidin-biotin binding by mixing at room temperature for 1 hour with inversion. Then, the beads were washed 3 times with TBS buffer (TTBS) containing 0.2% Tween 20.

In addition, the crude extract of E.coli (Takakura et al (2009) FEBS J276: 1383-1397) expressing TM2 was diluted with 0.1M HEPES/KOH (pH7.4) so that the total soluble protein concentration was 5mg/ml, and biotinylated magnetic beads were added thereto. TM2 was immobilized by tamavidin-biotin binding by mixing at room temperature for 1 hour with inversion. Washing was performed as described above, and the resultant TM2 immobilized magnetic beads (30mg beads/mL PBS) were then used to subtract out the non-specific binding inherent to the serum sample.

3-2 ELISA assay Using magnetic beads

Human Serum containing the antibody against SITH-1 was prepared by adding the rabbit anti-SITH-1 antibody of example 2 to commercially available Human Serum (Human Serum pool, manufactured by Cosmo Bio Inc.) in an amount of 1/50. On the other hand, human serum to which the SITH-1 antibody had not been added was used as a control.

The crude extract of E.coli expressing the pTrc99A vector product was diluted 1000-fold with PBS, or the crude extract of E.coli expressing the pTrc99A vector product, or the crude extract of E.coli expressing TM2 to obtain a solution, and BSA was further added to make the final concentration 2% (w/v), wherein the crude extract of E.coli expressing the pTrc99A vector product was adjusted with 0.1M HEPES/KOH (pH7.4) to make the total soluble protein concentration 5mg/ml, and the crude extract of E.coli expressing TM2 was adjusted with 0.1M HEPES/KOH (pH7.4) to make the total soluble protein concentration 5 mg/ml. In example 2, human serum was diluted 100-fold, and then rabbit anti-SITH-1 antibody was added to the diluted serum so that the volume ratio was 0.00025 to 0.002. However, in example 3-2, the rabbit anti-SITH-1 antibody was added to human serum at 1/50 (0.02 vol.), and then diluted 1000-fold, so that the dilution rate of the rabbit SITH-1 antibody was 1/50000. Therefore, the above-mentioned antibody dilution rate is at the same level as that in the case of using the serum of a depression patient diluted about 1000 times.

Mu.l of each of the TM2 fusion-SITH-1 immobilized magnetic beads and the TM2 immobilized magnetic beads was added to the diluted serum lmL prepared in this way, and the mixture was mixed by inversion at room temperature for 1 hour to effect reaction. After washing 3 times with TBST, for detection of rabbit anti-SITH-1 antibody bound to SITH-1 antigen immobilized on magnetic beads, horseradish peroxidase-labeled goat anti-rabbit IgG antibody or peroxidase-labeled goat anti-human IgG antibody for detection of human IgG non-specifically bound to each magnetic bead was diluted 5000-fold with TBST containing 2% BSA, respectively, and mixed, 1000 μ l of the mixed solution of peroxidase-labeled secondary antibody was added to each magnetic bead, and the mixture was inverted and mixed at room temperature for 1 hour. Then, the reaction mixture was washed 3 times with TBST, and 100. mu.l of a detection reagent was added thereto for 1 step of ELISAultraTMB (PIERCE), followed by reaction at room temperature for 1 minute. The reaction was stopped by adding 100. mu.l of 2M sulfuric acid, and the degree of color development (absorbance at a wavelength of 450nm, A450) was measured by means of an enzyme reader Infinite M200 (manufactured by TECAN). The measurement was performed 2 times for each treatment area, and the average value was obtained.

In the TM 2-fused SITH-1-immobilized magnetic beads, the a450 values obtained when TM 2-immobilized magnetic beads were used were subtracted from the a450 values obtained when each serum diluent (PBS, crude extract of escherichia coli expressing pTrc99A vector product, crude extract of escherichia coli expressing TM2) was used, and the values were used as data values. The results are shown in Table 3.

[ Table 3] Effect of serum dilutions in detection of SITH-1 antibody in human serum

As shown in Table 3, the value (S) of A450 of the SITH-1 antibody in human serum was the highest when PBS was used as the serum diluent, and the value (N) of A450 of the serum containing no SITH-1 antibody was extremely high, so that the S/N ratio (degree of color development of human serum to which rabbit anti-SITH-1 antibody was added/degree of color development of serum to which rabbit anti-SITH-1 antibody was not added) was the lowest with respect to PBS.

On the other hand, when the crude extract of TM 2-expressing E.coli was used as a serum diluent, the serum containing no SITH-1 antibody exhibited the lowest A450 value, and nonspecific binding was significantly suppressed. In addition, the S/N ratio was the highest when the crude extract of TM2 expressing E.coli was used as a serum diluent, and was higher than 10. The content display comprises the following steps: when using magnetic beads on which TM2 fused SITH-1 was immobilized, non-specific binding could be reduced and anti-SITH-1 antibody could be detected with high sensitivity by mixing a crude extract of E.coli expressing TM2 with human serum.

In the case where the diagnosis of depression (mood disorder) is to be carried out by quantifying the SITH-1 antibody, the human serum used in the present specification to which the rabbit SITH-1 antibody has been added and the human serum to which the rabbit SITH-1 antibody has not been added may be regarded as the serum of a patient suffering from depression (mood disorder) and the serum of a healthy person, respectively. In table 3, when PBS was used as the serum diluent, 0.64 for the depression patient (S) and 0.25 for the healthy person (N), and when the crude extract of BL21 introduced by TM2/pTrc99A was used as the serum diluent, 0.22 for the depression patient (S) and 0.02 for the healthy person (N) were used. In most clinical specimens (sera), it is considered that there are sera with high nonspecific binding even in healthy persons, and there are sera with not significantly high SITH-1 antibody titer in the case of depression. Therefore, as a measurement method for detecting the difference between them, a measurement method capable of reliably detecting specific binding (S) while minimizing non-specific binding (N) is desired. From such a viewpoint, the S/N ratio of the present example is an effective index when used for comparing the effects of the respective serum dilutions and the like.

Example 4 ELISA using a fusion protein of Harpin and tamavidin Detection of anti-HarDin antibodies in the Presence of human serum

In this example, a fusion protein of Harpin (hrpZpss protein, Takakura et al, 2004, Physiol. mol. plant Pathol.64, 83 (89)) and tamavidin 2(TM2) which is a protein derived from plant pathogenic bacteria was expressed using E.coli, and immobilized on a biotinylated microplate by tamavidin-biotin binding, the thus-obtained Harpin plate was blocked with tamavidin 2, and then reacted with serum (serum in which rabbit anti-Harpin antibody and human serum were mixed) diluted with various crude extracts of E.coli, and then goat anti-human IgG antibody and horseradish peroxidase-labeled goat anti-rabbit IgG antibody were labeled with peroxidase, and detection of anti-Harpin antibody was performed.

4-1 construction of vector for expression of fusion protein of Harpin and tamavidin 2 and E.coli expression

A gene encoding a fusion protein in which the sequence of TM2 was ligated to the C-terminal side of Harpin was constructed using PCR. To construct the Harpin-TM2 fusion gene, primers were designed for binding the Harpin, TM2 genes by linker (5 xlinker: GGGGSGGGGSGGGGSGGGGSGGGGS) (SEQ ID NO: 18). The designed primers are shown in Table 4.

[ Table 4] primers for constructing genes encoding Harpin and Tm2 fusion proteins

As shown in Table 4, primers consisting of Harpin C-terminal site on the 5 'side, linker at the center, and TM2N terminal site on the 3' side were designed (HarpinC-5xlink-TM2N-F, Table 4, SEQ ID NO: 27); a primer comprising a DNA sequence encoding in reverse the 5 '-side terminal region of TM2N, the central linker region, and the 3' -side terminal region of Harpin C (HarpinC-5xlink-TM2N-R, Table 4, SEQ ID NO: 26). In addition, a primer HarpinNtermEcoRI-F (comprising an EcoRI cleavage sequence at the 5' end, Table 4, SEQ ID NO: 25) was designed at the N-terminal part of Harpin.

To construct the Harpin-TM2 fusion gene, two-stage PCR was performed. In the first-stage PCR, amplification of the Harpin site was carried out using a plasmid (Takakura et al, 2004, Physiol. mol. plant Pathol.64, 83 (89)) obtained from pCR2.1 vector (Invitrogen) in which Harpin gene (ORF) (SEQ ID NO: 28) (coding for amino acid sequence: SEQ ID NO: 29) was recombined as a template, and using primers HarpinNtermEcoRI-F and HarpinC-5xlink-TM2N-R, and further, amplification of the CtTM 2 site was carried out using a plasmid (WO02/072817) obtained from pTrc99A vector in which TM2 gene was recombined alone as a template, and using primers HarpinC-5xlink-TM2N-F and TM2 ermBam.

The PCR reaction conditions are as follows: to 20. mu.l of the reaction solution were added 100ng of template DNA, 2. mu.l of 10 XExTaq buffer (TaKaRa), 1.6. mu.l of 2.5mM dNTP, 20 picomoles of each primer, and 0.1. mu.l of 5U/. mu.l Ex Taq, and the mixture was subjected to PCR using GeneAmp PCR System 9600(PERKIN ELMER) at 94 ℃, 60 ℃ and 72 ℃ for 25 times for 30 seconds. As a result, a PCR product of about 1kbp was obtained at the Harpin site, and a PCR product of about 0.5kbp was obtained at the TM2 site. These PCR products were fractionated by agarose gel electrophoresis in TAE buffer. Gels of the respective PCR products were cut out, and the products were recovered using QIAEX II gel extraction kit (QIAGEN). The extraction method is according to the instruction of the kit.

The two PCR products were used as templates for the second stage PCR using primers HarpinNtermEcoRI-F and TM2 CtermBam. The reaction conditions are as follows: to 20. mu.l of the reaction solution were added 100ng each of the template DNA, 2. mu.l of 10 XPyrobest buffer (TaKaRa), 1.6. mu.l of 2.5mM dNTP, 20 pmoles each of the primers, and 0.1. mu.l of 5U/. mu.l Pyrobest DNA polymerase, and the mixture was subjected to 94 ℃ for 30 seconds, 60 ℃ for 1 minute, 72 ℃ for 1.5 minutes, 25 times, using a GeneAmp PCR System 9600(PERKIN ELMER). As a result, a PCR product of about 1.5kbp was obtained.

The harpin-TM2 fusion gene obtained by PCR was cloned into the vector pCR4BluntTOPO (manufactured by Invitrogen). After confirming the nucleotide sequence, the plasmid containing the recombinant fusion gene was digested with EcoRI and BamHI, and subjected to agarose gel electrophoresis and purification in the same manner as described above to recover a DNA fragment. This fragment was ligated with an expression vector pTrc99A for E.coli (Pharmacia) previously digested with EcoRI and BamHI using a ligation kit (TaKaRa). The ligation product was transformed into E.coli BL21(DE3), and the nucleotide sequence was confirmed.

As shown above, the harpin-TM2/pTrc99A vector for the expression of the harpin and TM2 fusion proteins was constructed. The base sequence of the coded harpin-TM2 is shown in SEQ ID NO: 30, and the coded amino acid sequence is shown as SEQ ID NO: shown at 31.

Escherichia coli BL21(DE3) introduced with Harpin-TM2/pTrc99A was inoculated into 50ml of LB medium containing the antibiotic ampicillin (final concentration 100. mu.g/ml), and cultured with shaking at 30 ℃ until the absorbance at OD600 reached 0.5. Then, 1mM IPTG was added, and the mixture was further cultured with shaking at 30 ℃ for 5 hours. The cells were collected by centrifugation of 50ml of the culture. The cells were resuspended in 3ml of 0.1MHEPES/KOH (pH7.4), and then disrupted by sonication. The disruption solution was centrifuged (15000rpm), and the supernatant was used as a crude extract of Escherichia coli. In order to confirm the expression of the TM2 fusion harpin protein, the proteins contained in the crude extract were fractionated by SDS-PAGE and analyzed by immunoblotting.

Harpin detection was performed using 1000-fold dilutions of rabbit anti-Harpin antibodies (Takakura et al, 2004, Physiol. mol. plant Pathol.64, 83 (89)) and alkaline phosphatase-labeled anti-rabbit IgG antibodies (manufactured by BIO RAD).

The results are shown in FIG. 4. No specific band was detected in the E.coli extract expressing only the expression vector without the insert (lane 1 of FIG. 4), while a band around 50kDa was detected in E.coli expressing TM2 fusion harpin (lane 2 of FIG. 4). This size corresponds essentially to the molecular weight of 51.4kDa for the TM2 fusion harpin.

4-2 detection of anti-Harpin antibodies in the Presence of human serum by ELISA

The crude extract of TM2 fusion Harpin E.coli was diluted with 0.1M HEPES/KOH (pH7.4) to give a total soluble protein concentration of 1mg/ml, and 100. mu.l was added to each well of a biotin plate (Sumitomo Bakelite). TM2 fusion Harpin was immobilized by tamavidin-biotin binding at room temperature by standing for 1 hour. Then, each well of the plate was washed 3 times with TBS Buffer (TBST) containing 0.1% Tween 20. Mu.l of purified TM2(WO 02/072817)/0.5% BSA/TBST solution (250. mu.l) was added to each well and blocked with TM2 and BSA by standing at room temperature for 1 hour. After blocking, each well was washed 3 times with TBST.

Next, 100-fold dilution of Human Serum (Human Serum pool, manufactured by Cosmo-Bio Inc.) was carried out using PBS, or a crude extract of E.coli expressing 5mg/ml (0.1M HEPES/KOH, pH7.4) of total soluble protein, a crude extract of E.coli expressing pTrc99A vector product expressing 5mg/ml (0.1M HEPES/KOH, pH7.4) of total soluble protein, or a crude extract of E.coli expressing TM2 expressing 5mg/ml (0.1M HEPES/KOH, pH 7.4). To this solution was added 1% BSA, and then 1/500 amounts of rabbit anti-Harpin antibodies, to prepare anti-Harpin antibody-containing sera. Serum to which no Harpin antibody was added was used as a control.

Each 100. mu.l of these sera was added to a plate on which TM2 fusion Harpin was immobilized, allowed to stand at room temperature for 1 hour to react, and then washed 3 times with TBST. In order to detect rabbit anti-Harpin antibodies bound to Harpin antigen, horseradish peroxidase-labeled goat anti-rabbit IgG antibodies and, in order to detect human IgG non-specifically bound to wells, peroxidase-labeled goat anti-human IgG antibodies were diluted 5000-fold respectively with TBST containing 1% BSA and mixed, and 100 μ l each of the peroxidase-labeled secondary antibody mixed solutions was added to each well, and allowed to stand at room temperature for 1 hour to react. Then, the reaction mixture was washed 3 times with TBST, 100. mu.l of a detection reagent was added thereto, and the mixture was reacted at room temperature for 5 minutes in 1 step ELISAultraTMB (PIERCE). The reaction was stopped by adding 100. mu.l of 2M sulfuric acid, and the degree of color development (absorbance at a wavelength of 450nm, A450) was measured by means of an enzyme reader Infinite M200 (manufactured by TECAN). The measurement was performed 3 times for each treatment area, and the average value was obtained. In this example, since a system in which the concentration of the antibody was relatively high and nonspecific binding was small was formed, it was not necessary to subtract the measured value of the region where the antigen was not immobilized.

The results are shown in Table 5.

[ Table 5] Effect of serum dilution on Harpin antibody detection in human serum

As shown in Table 5, the A450 value (S) of harpin antibody in human serum was not significantly different between each serum dilution, and the value (N) of harpin antibody-free serum as a control was decreased in the order of BL21 crude extract introduced into TM2/pTrc99A, BL21 crude extract introduced into pTrc99A, Escherichia coli (BL21) crude extract, and PBS. This shows that: the crude extract of BL21 introduced by TM2/pTrc99A was most effective in suppressing non-specific binding. In addition, the S/N ratio (degree of color development of human serum supplemented with rabbit anti-Harpin antibody/degree of color development of serum not supplemented with rabbit anti-Harpin antibody) was highest when the E.coli extract (crude extract of BL21 introduced with TM2/pTrc 99A) expressing tamavidin 2 was used as a serum diluent.

The following contents show that: by diluting human serum with an extract of Escherichia coli expressing tamavidin 2, nonspecific binding can be suppressed and detection can be performed with high sensitivity.

SEQ ID NO: 1: the amino acid sequence of SITH-1.

SEQ ID NO: 2: the base sequence of the SITH-1 ORF.

SEQ ID NO: 3: the nucleotide sequence of the SITH-1 cDNA.

SEQ ID NO: 4: a base sequence of tamavidin 1.

SEQ ID NO: 5: the amino acid sequence of tamavidin 1.

SEQ ID NO: 6: a base sequence of tamavidin 2.

SEQ ID NO: 7: the amino acid sequence of tamavidin 2.

SEQ ID NO: 8: examples of linker sequences.

SEQ ID NO: 9: examples of linker sequences.

SEQ ID NO: 10: examples of linker sequences.

SEQ ID NO: 11: examples of linker sequences.

SEQ ID NO: 12: examples of linker sequences.

SEQ ID NO: 13: examples of linker sequences.

SEQ ID NO: 14: examples of linker sequences.

SEQ ID NO: 15: examples of linker sequences.

SEQ ID NO: 16: examples of linker sequences.

SEQ ID NO: 17: examples of linker sequences.

SEQ ID NO: 18: examples of linker sequences (used in the examples).

SEQ ID NO: 19: the base sequence of primer SITH1C-5xlink-TM 2N-F.

SEQ ID NO: 20: the base sequence of the primer SITH1C-5xlink-TM 2N-R.

SEQ ID NO: 21: the base sequence of primer SITH 15' EcoRI-F.

SEQ ID NO: 22: base sequence of primer TM2 CtermBam.

SEQ ID NO: 23: the nucleotide sequence of SITH-1-TM 2.

SEQ ID NO: 24: the amino acid sequence of SITH-1-TM 2.

SEQ ID NO: 25: base sequence of primer HarpinNtermEcoRI-F.

SEQ ID NO: 26: the base sequence of primer HarpinC-5xlink-TM 2N-R.

SEQ ID NO: 27: the base sequence of primer HarpinC-5xlink-TM 2N-F.

SEQ ID NO: 28: base sequence of Harpin gene (ORF).

SEQ ID NO: 29: an amino acid sequence encoded by the Harpin gene (ORF).

SEQ ID NO: 30: a nucleotide sequence of harpin-TM 2.

SEQ ID NO: 31: the amino acid sequence of harpin-TM 2.

Claims

1. A method of detecting a substance in a biological sample, comprising:

3) mixing (a) a biological sample, and

2. A method of detecting a substance in a biological sample, comprising:

3. A method of detecting a substance in a biological sample, comprising:

4) after the blocking step of step 3), (a) a biological sample, and

4. The method according to claim 1 or 3, wherein a cell disruption extract solution extracted from cells containing an optional carrier is added as the cell disruption extract solution in the step 3(b-i) of claim 1 or the step 4(b-i) of claim 3.

5. The method according to any one of claims 1 to 4, wherein the biotin-binding protein is tamavidin or a mutant thereof.

6. The method of any one of claims 1 to 5, wherein the biological sample is selected from blood, serum, cerebrospinal fluid, saliva, sweat, urine, tears, lymph and breast milk.

7. A carrier for detecting a substance in a biological sample, which is bound to a fusion protein of a biotin-binding protein and a protein that specifically binds to a substance to be detected, by binding between biotin and the biotin-binding protein, the carrier for detecting a substance in a biological sample being prepared by:

8. A kit for detecting a substance in a biological sample, comprising:

C) A blocking agent comprising a biotin-binding protein.