JPWO2008130053A1 - Fusion protein of protein G and avidins - Google Patents

Abstract

[PROBLEMS] To produce a labeled antibody (an antibody in which a labeling substance is immobilized on an antibody) used in a method for detecting a target antigen, without using a chemical binding method such as a coupling method and sufficiently suppressing nonspecific binding. An anchor material is provided that can be. The fusion protein of the present invention is a fusion protein comprising a protein consisting of protein G or a part thereof and a protein consisting of avidin or a part thereof. The labeled antibody of the present invention comprises an IgG antibody, the fusion protein of the present invention, and a biotinylated labeling substance. [Selection figure] None

Description

  The present invention relates to a fusion protein of protein G and avidins. Specifically, the present invention relates to a recombinant protein having both the binding activity to the Fc region of IgG antibody possessed by protein G and the binding activity to biotin.

An antibody is one of the biological defense factors possessed by higher organisms than cartilaginous fish, and is sometimes called immunoglobulin (Ig). In addition to inactivating by binding to foreign molecules that have entered the body, antibodies have various functions such as promoting phagocytosis by macrophages and the like by aggregation, and activating complement to degrade target proteins.
One of the major features of an antibody is its substrate specificity of “binding only to a specific antigen”. The substrate specificity of the antibody responsible for protecting the body by attacking molecules entering the living body is very high, and also has a strong binding force with the substrate (= antigen substance).
Because it is relatively easy to create and purify antibodies that bind to desired substances by immunizing laboratory animals, methods that use antibodies for searching and detecting substances such as immunostaining, Western blotting, and ELISA Laws were devised one after another and used for research. In addition, a more advanced detection method was devised by the monoclonal antibody production method developed later, and it has come to be used not only for research but also for clinical examination and disease treatment.

Usually, a target antigen detection method using an antibody is performed by labeling a specific substance on the antibody. Labeling substances are added after antigen-antibody reaction, such as substances that enable direct or indirect confirmation of the presence of antibodies such as fluorescent substances and radioactive substances, and enzymes such as horseradish peroxidase (HRP). A substance that makes it possible to confirm the presence of an antibody bound to the target antigen is selected by detecting color development or luminescence accompanying the degradation of the substrate. Further, a method for detecting with higher sensitivity using an oligonucleic acid chain (DNA or the like) as a labeling substance has been developed (Patent Document 1).
Here, various methods for labeling antibodies are known. For example, a diazo coupling method using an azo group (—N ₂ ) contained in an antibody, a method in which a thiol group (—SH) contained in an antibody and a maleimide group of a labeling substance are coupled by a coupling method, etc. Is widely used. All of these methods have advantages such as low cost and easy operation. However, since a hydrolysis reaction is involved in the labeling, the antibody molecule is damaged, or the labeling substance binds to the azo group or thiol group in the reactive group (antigen-binding region) of the antibody, thereby inhibiting or suppressing the antigen-antibody reaction. May be. In addition, the efficiency of labeling may decrease depending on the structure of the antibody and the conditions (temperature, pH, air pressure, etc.) at the time of binding to the labeling substance.

By the way, many methods for binding proteins and chemical substances have been developed so far. As a method often used for binding a labeling substance to an antibody, there is a method using binding of biotin and avidin. The binding between biotin and avidin has a binding force more than 1 million times that of antigen-antibody reaction, and since the binding is substantially irreversible, it is very useful as an anchor that connects substances. .
In some bacteria and viruses, it is known that proteins that bind with high specificity to the Fc region (terminal portion) of antibodies are synthesized. For example, protein A and protein G are well known as such proteins, and these are used when an antibody is bound to a plastic substrate or another substance. In particular, protein G has properties such that it has high affinity for IgG antibodies derived from various animals such as humans, mice and rats, and the binding conditions are very wide. Therefore, when designing an antigen detection method using an antibody, it is often used for labeling the antibody or immobilizing the antibody on a support.

At present, the binding between biotin and avidin and the binding between protein G and an antibody are both widely used and are used for detection of antibody antigen reaction. However, in order to bind avidin or biotin to the antibody, it is necessary to use a chemical bonding method such as the coupling method described above (the same problem as described above). Moreover, since avidin usually exists as a multimer, there is a problem that nonspecific binding occurs between molecules of each other. On the other hand, since protein G also has a binding site for albumin in its molecular structure, it is known that it binds to various components in blood and causes background, for example.
U.S. Pat.No. 5,665,539

Therefore, the problem to be solved by the present invention is that it is not necessary to use a chemical binding method such as a coupling method in producing a labeled antibody (an antibody in which a labeling substance is immobilized on an antibody) used in a method for detecting a target antigen. And it is providing the anchor substance which can fully suppress nonspecific binding.
Furthermore, an object of the present invention is to provide a labeled antibody prepared using the anchor substance, a target antigen detection method using the antibody, a target antigen detection kit containing the antibody, and the like.

  The present inventor has intensively studied to solve the above problems. As a result, the inventors found that the above-mentioned problems can be easily solved by preparing a fusion protein of protein G and avidin as the anchor substance and using this fusion protein for labeling of the antibody, and completed the present invention.

That is, the present invention is as follows.
(1) A fusion protein comprising a protein consisting of protein G or a part thereof and a protein consisting of avidins or a part thereof.
Examples of the fusion protein of the present invention include those in which the avidins are, for example, avidin, streptavidin, or neutravidin.
Examples of the fusion protein of the present invention include those in which a protein consisting of a part of protein G is a protein having a binding activity to the Fc region of an IgG antibody. Specifically, the following (a) or ( What is the protein of b) is mentioned.
(A) a protein consisting of the amino acid sequence shown in SEQ ID NO: 6 (b) consisting of an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 6, and Protein having binding activity with Fc region Examples of the fusion protein of the present invention include a protein in which a part of avidin is a protein having binding activity with biotin. What is the protein of (a) or (b) is mentioned.
(A) a protein comprising the amino acid sequence shown in SEQ ID NO: 8 (b) consisting of an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 8, and Protein with binding activity

(2) The following protein (a) or (b).
(A) a protein comprising the amino acid sequence shown in SEQ ID NO: 10 (b) consisting of an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 10, and Protein with binding activity to Fc region and binding activity to biotin

(3) A gene encoding the protein according to (1) or (2).
(4) A gene comprising the following DNA (a) or (b) and the following DNA (c) or (d):
(A) DNA consisting of the base sequence shown in SEQ ID NO: 5
(B) a DNA that hybridizes under stringent conditions with a DNA consisting of a base sequence complementary to the DNA consisting of the base sequence shown in SEQ ID NO: 5, and has binding activity to the Fc region of an IgG antibody DNA encoding the protein
(C) DNA consisting of the base sequence shown in SEQ ID NO: 7
(D) a DNA that hybridizes under stringent conditions with a DNA comprising a base sequence complementary to the DNA comprising the base sequence shown in SEQ ID NO: 7 and encodes a protein having a binding activity to biotin DNA

(5) A gene comprising the following DNA (a) or (b):
(A) DNA consisting of the base sequence shown in SEQ ID NO: 9
(B) DNA that hybridizes under stringent conditions with DNA consisting of a base sequence complementary to the DNA consisting of the base sequence shown in SEQ ID NO: 9, and binding activity to the Fc region of IgG antibody and biotin DNA encoding a protein having binding activity to

(6) A recombinant vector comprising the gene according to any one of (3) to (5).
(7) A transformant comprising the recombinant vector according to (6).
(8) including a step of culturing the transformant according to (7) above, and a step of collecting a protein having binding activity to the Fc region of IgG antibody and binding activity to biotin from the obtained culture. A method for producing a protein.

(9) A labeled antibody comprising an IgG antibody, the fusion protein according to (1) or (2) above, and a biotinylated labeling substance.
Examples of the labeling substance to be biotinylated in the labeled antibody of the present invention include an oligonucleic acid chain.

(10) An antibody-fusion protein conjugate comprising an IgG antibody and the fusion protein according to (1) or (2) above.
(11) A fusion protein-labeling substance conjugate comprising the fusion protein according to (1) or (2) and a biotinylated labeling substance.
In the fusion protein-labeling substance conjugate of the present invention, examples of the labeling substance to be biotinylated include an oligonucleic acid chain.

(12) An antibody labeling kit comprising at least one selected from the group consisting of the fusion protein according to (1) or (2), the conjugate according to (10), and the conjugate according to (11) .
Examples of the antibody labeling kit of the present invention further include an IgG antibody and / or a biotinylated labeling substance. Here, examples of the labeling substance to be biotinylated include an oligonucleic acid chain.

(13) A method for detecting a target antigen contained in a test sample, comprising the step of bringing a target antigen into contact with the labeled antibody according to (9) to form an antigen-antibody complex, and in the complex Detecting said label.
As the target antigen detection method of the present invention, for example, there are a plurality of types of labeled antigens, and a plurality of types of labeled antibodies that are labeled so that they can be identified as labeled antibodies corresponding to the types of the target antigens. The method using is mentioned. Here, examples of the target antigen include hepatitis C virus antigen.

(14) A target antigen detection kit comprising the labeled antibody according to (9).
Examples of the target antigen detection kit of the present invention include those in which the labeled antibody is a plurality of types of labeled antibodies that are labeled so that they can be identified in accordance with the type of the target antigen. Here, examples of the target antigen include hepatitis C virus antigen.

(15) A target antigen detection kit comprising the conjugate according to (10) and a biotinylated labeling substance, or the conjugate according to (11) and an IgG antibody.
In the target antigen detection kit of the present invention, examples of the target antigen include hepatitis C virus antigen. In the kit, the labeling substance to be biotinylated includes, for example, an oligonucleic acid chain.

(16) An antibody-immobilized support comprising a support having a biotinylated surface, the fusion protein according to (1) or (2), and an IgG antibody.
(17) A method for recovering a target antigen in a test sample, comprising the steps of contacting the test sample with the support according to (16), and recovering the support after the contact, Method.
(18) An antigen collection kit comprising the support according to (16).

(19) A fusion protein-immobilized support comprising the fusion protein according to (1) or (2) and a support having a biotinylated surface.
(20) A method for recovering IgG antibody in a test sample, comprising the steps of contacting the test sample with the support according to (19), and recovering the support after the contact, Method.
(21) An antibody collection kit comprising the support according to (19).
<Cross-reference with related literature>
This application claims the benefit of priority of Japanese Patent Application No. 2007-112779 filed on Apr. 23, 2007, and is incorporated herein by reference.

(a) Schematic diagram showing the synthesis scheme and constitution of the fusion protein of the present invention, (b) The schematic diagram of the production scheme of labeled antibody using the fusion protein of the present invention. Reference numeral 1 denotes a part containing a binding site for IgG antibody Fc region in protein G, and reference numeral 2 denotes a part containing a binding site for biotin in streptavidin. It is the schematic which shows the synthetic scheme of the fusion protein of this invention. It is a figure which shows the information of the one part amino acid sequence of protein G used as a structural component of the fusion protein of this invention, and a base sequence. A region sandwiched between two arrows in the figure corresponds to a part of the protein G. It is a figure which shows the information of the one part amino acid sequence and base sequence of streptavidin used as a structural component of the fusion protein of this invention. A region sandwiched between two arrows corresponds to a part of the streptavidin. It is a photograph which shows the result of the polyacrylamide gel electrophoresis in an Example. It is a graph which shows the detection result of the hEGF antigen in an Example. It is a graph which shows the detection result of the hEGF antigen in an Example. It is a graph which shows the detection result of the hEGF antigen in an Example. It is a graph which shows the detection result of the HCV antigen in an Example. It is a graph which shows the detection result of the HCV antigen in an Example. It is a photograph which shows the result of the polyacrylamide gel electrophoresis in a comparative example. It is a photograph which shows the result of the polyacrylamide gel electrophoresis in a comparative example. It is a graph which shows the detection result of IL-15 antigen in a comparative example.

  Hereinafter, the present invention will be described in detail. The scope of the present invention is not limited to these descriptions, and other than the following examples, the scope of the present invention can be appropriately changed and implemented without departing from the spirit of the present invention.

1. Summary of the Invention As shown in the schematic diagram of FIG. 1 (a), the present inventor has produced a fusion protein in which a protein comprising a part of protein G and a protein comprising a part of streptavidin are linked. FIG. 1 (a) is a schematic diagram showing the configuration of one embodiment of the fusion protein of the present invention, in which the fusion protein comprises a part 1 of protein G (ie, a part containing a binding site for the Fc region of an IgG antibody). , A part 2 of streptavidin (that is, a part containing a binding site for biotin) is included as a constituent component.
FIG. 2 schematically shows the synthesis scheme of the fusion protein of the present invention. Specifically, first, Bo Akerstrom et al. (J. Biol. Chem., Vol. 262, p. 13388-13391, 1987), the results of research on the binding site of protein G to the IgG antibody Fc region, and Strept by Weber. Based on the analysis results of the binding region with biotin using the crystal structure analysis of avidin (Proc. Natl. Acad. Sci. USA, vol.86, p.2190-2194, 1989), protein G And base sequence information encoding each protein of streptavidin. Next, from the obtained base sequence information, a base sequence region that encodes a portion including a substrate (IgG antibody Fc region, biotin) binding site of each protein is selected, and a cDNA fragment having the base sequence is selected by a known gene cloning method. Preparation and isolation were performed. After that, the cDNA fragments isolated by cloning by PCR using the cDNA fragment as a template are joined together (ligation) if necessary, and then inserted into the multicloning site of the expression vector so that a fusion protein can be obtained. did. The recombinant expression vector thus obtained was introduced into Escherichia coli to produce a transformant. The transformant was cultured to express a protein, and the produced fusion protein was recovered and purified.

By using the fusion protein of the present invention, an antibody (IgG antibody) and a biotinylated labeling substance (oligo DNA, HRP enzyme, etc.) can be easily and efficiently bound. That is, without using a conventional coupling method or the like, for example, an antibody and a biotinylated labeling substance are mixed and added to an appropriate solution, and then the fusion protein of the present invention is added. Labeled antibodies can be prepared.
Therefore, when labeling a known substance such as a chemical substance such as HRP enzyme and ALP enzyme, a radioactive substance and an oligonucleic acid chain by binding to an IgG antibody, the labeling is much easier and more efficient than before. Can be made.
In addition, the above-mentioned fusion protein has a site that causes non-specific binding with albumin or the like, which is problematic when protein G is used, and the proteins that are problematic when an avidin protein (streptavidin or the like) is used. Can be prepared in a form that does not include a site that causes non-specific binding. Therefore, compared to the case where a labeled antibody is prepared using wild-type protein G and avidin proteins, non-specific binding at the time of target antigen detection can be significantly suppressed, and the background can be greatly reduced. As a result, the detection sensitivity is greatly improved.

On the other hand, if the fusion protein of the present invention is used, a desired IgG antibody can be easily bound and immobilized on a plate or bead on which biotin is immobilized on the surface, and further, IgG in a test sample such as blood can be immobilized. Since the antibody can be efficiently captured and recovered, it is extremely useful.
As described above, the fusion protein of the present invention can be used in a very useful manner in various applications such as detection and recovery of target antigens and antibodies, and is highly practical.

2. Fusion protein
(1) Fusion protein The fusion protein of the present invention is a fusion protein comprising protein G or a part thereof, and avidin or a part thereof.
Here, the term “fusion protein” refers to an amino acid sequence of one protein and an amino acid sequence of the other protein that are directly linked to each other or indirectly linked via any amino acid sequence that serves as a spacer. The protein consists of a single (series of) amino acid sequence, and usually has the properties (for example, binding activity) of both proteins. In addition, the “fusion protein” as used in the present invention may be a protein consisting only of the above-mentioned directly or indirectly linked amino acid sequences, or a protein consisting of an amino acid sequence partially containing the amino acid sequence. There may be no limitation. Examples of the latter protein include a protein having an amino acid sequence in which any other amino acid sequence is linked to both ends or any one end of the amino acid sequence constituting the former protein. The length of other arbitrary amino acid sequences is not particularly limited, but is preferably, for example, 1 to 300 residues, more preferably 1 to 200 amino acid residues, and still more preferably 1 to 100 amino acid residues. It is a group. Hereinafter, in the present specification, the “fusion protein” is meant to include both the former protein and the latter protein described above.

Further, “a protein consisting of a part thereof” means a protein consisting of an amino acid sequence in a partial region in an amino acid sequence constituting a predetermined protein (protein G and avidins).
In addition, when the fusion protein of the present invention is formed via the amino acid sequence serving as the spacer, the length of the amino acid sequence serving as the spacer is not particularly limited, but is, for example, 1 to 100 residues. More preferably 2 to 50 amino acid residues, still more preferably 2 to 20 amino acid residues, particularly preferably 2 to 10 amino acid residues, and most preferably 2 to 5 amino acid residues. . When the spacer length is in the above range, the three-dimensional structure of each protein constituting the fusion protein does not interfere with each other, and the properties of any protein can be fully exhibited. Moreover, as an amino acid residue used for a spacer, serine, histidine, etc. are preferable, for example, Among them, histidine is more preferable. When these amino acid residues are used as a spacer, the influence on the three-dimensional structure of each protein constituting the fusion protein is reduced, and the properties of any protein can be fully exhibited.

  The fusion protein of the present invention may be a fusion protein containing protein G and avidin, particularly a fusion protein of protein G and avidin, or a fusion containing protein G and a protein comprising a part of avidin. It may be a fusion protein of protein, particularly protein G and a protein consisting of a part of avidin, or a fusion protein comprising a protein consisting of a part of protein G and avidin, particularly a part of protein G It may be a fusion protein of protein and avidin, or a fusion protein comprising a protein consisting of part of protein G and a protein consisting of part of avidin, particularly a protein consisting of part of protein G and avidin It may be a fusion protein with a protein consisting of a part of But not limited to, for example, a fusion protein with a protein consisting of a portion of the protein and avidin consisting part of protein G are preferred. Here, “avidins” as used in the present invention generally means all avidin proteins having specific binding ability to biotin protein, and examples thereof include avidin, streptavidin, and neutravidin, among which streptavidin is preferable. And neutravidin is particularly preferred.

  As the fusion protein of the present invention, for example, a fusion protein comprising a protein consisting of a part of protein G and an avidin or a protein consisting of part thereof, in particular a protein consisting of a part of protein G, and avidin or a part thereof Preferred is a fusion protein comprising a part of the protein, wherein the protein comprising part of the protein G is a protein having a binding activity to the Fc region of an IgG antibody. The protein extracted from the portion having the binding activity in protein G can remarkably reduce non-specific binding with various components (such as albumin) inherent in protein G. A fusion protein specialized in binding ability can be obtained. Therefore, when a labeled antibody prepared using the fusion protein is used in the target antigen detection method (described later), the background during detection can be greatly reduced, and detection sensitivity can be greatly improved. it can.

Here, specifically, the protein comprising a part of protein G is preferably the following protein (a) or (b).
(A) a protein comprising the amino acid sequence shown in SEQ ID NO: 6, particularly a protein comprising the amino acid sequence shown in SEQ ID NO: 6 (b) one or several amino acids are deleted or substituted in the amino acid sequence shown in SEQ ID NO: 6 Or comprising an added amino acid sequence, in particular consisting of an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 6, and binding activity to the Fc region of IgG antibody Protein with

  The information on the amino acid sequence (SEQ ID NO: 2) of wild-type protein G and the base sequence (SEQ ID NO: 1) encoding the amino sequence is registered in a public database. For example, Swiss-Prot (http: / (accessible from /tw.expasy.org/uniprot/) is registered as "entry name: SPG1-STRSG, accession number: P06654" and GenBank (http://www.ncbi.nlm.nih.gov/ Is accessible as "accession number: M13825". The “amino acid sequence shown in SEQ ID NO: 6” referred to in the above (a) and (b) consists of the 228th to 268th amino acid sequence region in the amino acid sequence of wild type protein G (SEQ ID NO: 2). It corresponds to the amino acid sequence (41 amino acid residues in total). Further, in the amino acid sequence of wild-type protein G, the amino acid sequence shown in SEQ ID NO: 6 is an amino acid sequence corresponding to a portion including a binding region with the Fc region of IgG antibody, as described in “Stephern R. et al. , J. Bacteriology, vol. 167, p. 870-880 (1986) ”.

Here, the “amino acid sequence in which one or several amino acids are deleted, substituted or added” is, for example, about 1 to 10 amino acids, preferably about 1 to 5 amino acids deleted, substituted or An added amino acid sequence is preferable.
It is important that the “protein consisting of a deleted, substituted or added amino acid sequence” is a protein that can stably exhibit binding activity to the Fc region of an IgG antibody. Therefore, it is preferable that amino acid residues and the like considered to be important for the binding (substrate binding) with the Fc region of IgG antibody are not mutated (deleted, substituted or added) from the amino acid sequence of wild-type protein G. .
In the present invention, the presence or absence and the degree of binding activity with the Fc region of IgG antibody are determined by, for example, immobilizing purified fusion protein on an immunoplate or the like, adding purified IgG antibody such as goat, and incubating the HRP. It can be measured by sensitizing a labeled anti-goat IgG antibody or the like and detecting the amount of antibody bound to the immobilized protein using various known detection methods (the same applies hereinafter).

  In addition, as the fusion protein of the present invention, for example, a fusion protein containing a protein consisting of a part of avidin and a protein consisting of protein G or a part thereof, particularly a protein consisting of a part of avidin, and protein G Alternatively, preferred is a fusion protein with a protein comprising a part thereof, wherein the protein comprising a part of the avidin is a protein having a binding activity to biotin. Proteins obtained by extracting the portion having the binding activity in avidins significantly reduce non-specific binding caused by self-aggregation activity (activity in which avidin molecules bind to each other) inherently possessed by avidins. Therefore, a fusion protein specialized in the ability to bind to biotin can be obtained. Therefore, when a labeled antibody prepared using the fusion protein is used in the target antigen detection method (described later), the background during detection can be greatly reduced, and detection sensitivity can be greatly improved. it can.

Here, specifically as a protein which consists of a part of avidins, the protein of the following (a) or (b) is mentioned preferably.
(A) a protein comprising the amino acid sequence shown in SEQ ID NO: 8, particularly a protein comprising the amino acid sequence shown in SEQ ID NO: 8 (b) one or several amino acids are deleted or substituted in the amino acid sequence shown in SEQ ID NO: 8 Or a protein comprising an added amino acid sequence, comprising an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 8, and having a binding activity to biotin

  Information on the amino acid sequence (SEQ ID NO: 4) of wild-type streptavidin and the base sequence (SEQ ID NO: 3) encoding the amino sequence is registered in a public database. For example, Swiss-Prot (http: / It is registered as “entry name: SAV STRAV, accession number: P22629” and can be accessed from GenBank (http://www.ncbi.nlm.nih.gov/), which is accessible from /tw.expasy.org/uniprot/. (Accessible) is published as “accession number: X03591”. The “amino acid sequence represented by SEQ ID NO: 8” referred to in the above (a) and (b) consists of the 39th to 183rd amino acid sequence region in the amino acid sequence of wild type streptavidin (SEQ ID NO: 4). It corresponds to the amino acid sequence (total 145 amino acid residues). In addition, in the amino acid sequence of wild-type streptavidin, the amino acid sequence represented by SEQ ID NO: 8 is an amino acid sequence corresponding to a portion including a binding region with biotin, as “Carlos E. et al., Nucleic Acids Res. ., vol. 14, p. 1871-1882 (1986) ”.

  In addition, since avidins usually form a homotetramer and have one biotin-binding domain per subunit, the entire protein has four biotin-binding domains. It is preferable that the biotin-binding domain used in 1 has a single subunit, and for this purpose, it is preferable to use a part of avidin that does not form a tetramer and has only one biotin-binding domain. Until now, monomeric avidin mutants have been actively studied, but have often been unsuccessful (Qureshi, MH, and Wong, SL (2002). Protein Expr Purif 25, 409-415. Laitinen, OH et al., (2003). J Biol Chem 278, 4010-4014.). However, in the present invention, as shown in the Examples, by using a peptide having the amino acid sequence of positions 39 to 183 of streptavidin, a mutant that does not lose the binding activity to biotin could be produced. .

Here, the “amino acid sequence in which one or several amino acids are deleted, substituted or added” is, for example, about 1 to 10 amino acids, preferably about 1 to 5 amino acids deleted or substituted. Or it is preferable that it is the added amino acid sequence.
It is important that the “protein consisting of an amino acid sequence that is deleted, substituted, or added” is a protein that can stably exhibit binding activity to biotin. Therefore, it is preferable that amino acid residues considered to be important for the binding property (substrate binding property) to biotin are not mutated (deleted, substituted or added) from the amino acid sequence of wild-type streptavidin.
In the present invention, the presence / absence and extent of binding activity with biotin are determined by, for example, immobilizing the fusion protein on an immunoplate or the like, then adding biotin-labeled HRP as an aqueous solution at a constant concentration, and then washing the chromogenic substrate. By adding, the amount of biotin bound to the fusion protein can be measured by using various known detection methods (the same applies hereinafter).

More specifically, the fusion protein of the present invention preferably includes the following protein (a) or (b).
(A) a protein comprising the amino acid sequence shown in SEQ ID NO: 10, particularly a protein comprising the amino acid sequence shown in SEQ ID NO: 10 (b) one or several amino acids are deleted or substituted in the amino acid sequence shown in SEQ ID NO: 10 Or comprising an added amino acid sequence, in particular consisting of an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 10, and binding activity to the Fc region of IgG antibody And a protein having binding activity to biotin

  The “amino acid sequence shown in SEQ ID NO: 10” is a series of amino acid sequences partially including the amino acid sequence shown in SEQ ID NO: 6 and the amino acid sequence shown in SEQ ID NO: 8 as components. In the amino acid sequence shown in SEQ ID NO: 10, the 29th to 69th amino acid sequence region is an amino acid sequence (SEQ ID NO: 6) constituting a protein consisting of a part of protein G, and the 76th to 69th amino acids. The 220th amino acid sequence region is an amino acid sequence (SEQ ID NO: 8) constituting a protein comprising a part of avidin (specifically streptavidin), and the 70th to 75th amino acid sequence region is a spacer. An amino acid sequence constituting a part (oligopeptide), which is an amino acid sequence linking the C-terminal of the amino acid sequence shown in SEQ ID NO: 6 and the N-terminal of the amino acid sequence shown in SEQ ID NO: 8. In the amino acid sequence shown in SEQ ID NO: 10, the first to 28th and 221st to 301st amino acid sequence regions correspond to the other amino acid sequences described above.

Here, the “amino acid sequence in which one or several amino acids are deleted, substituted or added” is, for example, about 1 to 10 amino acids, preferably about 1 to 5 amino acids deleted or substituted. Or it is preferable that it is the added amino acid sequence.
It is important that the above-mentioned “protein consisting of a deleted, substituted or added amino acid sequence” is a protein that can stably exhibit both the binding activity to the Fc region of IgG antibody and the binding activity to biotin. It is. Therefore, amino acid residues that are considered to be important for binding to the Fc region of IgG antibodies (substrate binding properties) and amino acid residues that are considered to be important for binding properties to biotin (substrate binding properties) are wild type. It is preferable that the amino acid sequence of protein G or wild type streptavidin is not mutated (deleted, substituted or added).

(2) Recombinant gene The gene of the present invention includes all the genes encoding the various fusion proteins of the present invention described above. Among them, a gene encoding a fusion protein of the amino acid sequence shown in SEQ ID NO: 6 (protein consisting of a part of protein G) and the amino acid sequence shown in SEQ ID NO: 8 (protein consisting of a part of streptavidin) preferable. As such a gene, for example, a gene comprising the following DNA (a) or (b) and the following DNA (c) or (d) is preferably exemplified. The gene may contain a known base sequence (transcription promoter, SD sequence, Kozak sequence, terminator, etc.) necessary for gene expression in addition to the DNA, and is not limited.

(A) DNA consisting of the base sequence shown in SEQ ID NO: 5
(B) a DNA that hybridizes under stringent conditions with a DNA consisting of a base sequence complementary to the DNA consisting of the base sequence shown in SEQ ID NO: 5, and has binding activity to the Fc region of an IgG antibody DNA encoding the protein
(C) DNA consisting of the base sequence shown in SEQ ID NO: 7
(D) a DNA that hybridizes under stringent conditions with a DNA comprising a base sequence complementary to the DNA comprising the base sequence shown in SEQ ID NO: 7 and encodes a protein having a binding activity to biotin DNA

  Here, a gene DNA encoding a protein G or a protein thereof (for example, the DNA of (a) above), and a gene DNA encoding an avidin or a protein thereof (for example of the above (c)) DNA and the like) are based on the nucleotide sequence information of the nucleotide sequence (SEQ ID NO: 1) encoding wild-type protein G and the nucleotide sequence (SEQ ID NO: 3) encoding wild-type streptavidin, respectively. It can be obtained by a known preparation or synthesis method such as a chemical synthesis method (a method using a DNA synthesizer).

  The base sequence shown in SEQ ID NO: 5 is a base sequence (123 base pairs) corresponding to the 1259th to 1381st region of the base sequence shown in SEQ ID NO: 1, and is shown in SEQ ID NO: 7. The base sequence is a base sequence (435 base pairs) corresponding to the 164th to 598th regions of the base sequence shown in SEQ ID NO: 3.

More specifically, the gene encoding the fusion protein of the present invention is preferably a gene containing the following DNA (a ′) or (b ′). The gene may contain a known base sequence (transcription promoter, SD sequence, Kozak sequence, terminator, etc.) necessary for gene expression in addition to the DNA, and is not limited.
(A ′) DNA consisting of the base sequence represented by SEQ ID NO: 9
(B ′) DNA that hybridizes under stringent conditions with DNA consisting of a base sequence complementary to the DNA consisting of the base sequence shown in SEQ ID NO: 9, and binding activity to the Fc region of IgG antibody; DNA encoding a protein with binding activity to biotin

  The “base sequence shown in SEQ ID NO: 9” is a series of base sequences partially including the base sequence shown in SEQ ID NO: 5 and the base sequence shown in SEQ ID NO: 7 as components. In addition, in the base sequence shown in SEQ ID NO: 9, the 85th to 207th base sequence region is a base sequence (SEQ ID NO: 5) encoding a protein comprising a part of protein G, and the 226th to 207th base sequence. The 660th base sequence region is a base sequence (SEQ ID NO: 7) encoding a protein comprising a part of avidin (specifically streptavidin), and the 208th to 225th base sequence regions are spacers. A base sequence encoding a part (oligopeptide), which is a base sequence linking the 3 ′ end of the base sequence shown in SEQ ID NO: 5 and the 5 ′ end of the base sequence shown in SEQ ID NO: 7 . In the base sequence shown in SEQ ID NO: 9, the first to 84th and 661th to 903th base sequence regions correspond to the base sequences encoding the other amino acid sequences described above.

The DNAs of (b), (d), and (b ′) are the DNAs of (a), (c), and (a ′), DNAs that are complementary to them, or fragments thereof, respectively. The obtained product can be obtained from a cDNA library or a genomic library by carrying out a known hybridization method such as colony hybridization, plaque hybridization, or Southern blot using the converted product as a probe. A library prepared by a known method may be used, or a commercially available cDNA library or genomic library may be used, and is not limited. For detailed procedures of the hybridization method, Molecular Cloning, A Laboratory Manual 2nd ed. (Cold Spring Harbor Laboratory Press (1989)) can be referred to as appropriate.
In the implementation of the hybridization method, “stringent conditions” are conditions at the time of washing after hybridization, and the buffer salt concentration is 15 to 330 mM, the temperature is 25 to 70 ° C., preferably the salt concentration is 15 to It means the condition of 150 mM and temperature 55-65 ° C. Specifically, for example, conditions such as 60 mM at 100 mM can be mentioned. Further, in addition to the conditions such as salt concentration and temperature, various conditions such as probe concentration, probe length and reaction time are taken into consideration, and the DNAs of (b), (d) and (b ′) are Conditions for obtaining can be set as appropriate.

  Regarding the DNAs of (b), (d) and (b ′), the hybridizing DNAs are at least 40% relative to the base sequences of the DNAs of (a), (c) and (a ′), respectively. Preferably, the nucleotide sequence has the above homology, more preferably 60% or more, still more preferably 90% or more, even more preferably 95% or more, particularly preferably 98% or more, and most preferably 99% or more. is there.

The DNAs of (b), (d), and (b ′) are not completely identical in nucleotide sequence when compared with, for example, the DNAs of (a), (c), and (a ′). It is particularly preferred that the DNA has a nucleotide sequence such that the amino acid sequences are completely identical, that is, DNA in which a silent mutation is introduced into the DNAs of (a), (c) and (a ′). Mutation-substituted DNAs typified by DNA into which such silent mutations have been introduced include, for example, Molecular Cloning, A Laboratory Manual 2nd ed., Cold Spring Harbor Laboratory Press (1989), Current Protocols in Molecular Biology, John Wiley. & Sons (1987-1997) etc. and can be prepared according to the site-specific displacement induction method. Specifically, it can be prepared using a mutagenesis kit using site-directed mutagenesis by a known method such as the Kunkel method or the Gapped duplex method, and examples of the kit include QuickChange ^TM Site. -Directed Mutagenesis Kit (Stratagene), GeneTailor ^TM Site-Directed Mutagenesis System (Invitrogen), TaKaRa Site-Directed Mutagenesis System (Mutan-K, Mutan-Super Express Km, etc .: Takara Bio) are preferred Can be mentioned. Prepared by performing PCR under appropriate conditions using PCR primers designed to introduce the desired silent mutation or missense mutation and using DNA encoding wild type protein G or wild type streptavidin as a template. You can also The DNA polymerase used for PCR is preferably a highly accurate DNA polymerase, such as Pwo DNA (Polymerase Roche Diagnostics), Pfu DNA polymerase (Promega), Platinum Pfx DNA polymerase (Invitrogen), KOD DNA Polymerase (Toyobo), KOD-plus-polymerase (Toyobo) and the like are preferable. The PCR reaction conditions may be appropriately set depending on the optimum temperature of the DNA polymerase to be used, the length and type of the DNA to be synthesized, etc. For example, in the case of cycle conditions, “90-98 ° C. for 5-30 seconds (thermal denaturation / Dissociation) → 50 to 65 ° C. for 5 to 30 seconds (annealing) → 65 to 80 ° C. for 30 to 1200 seconds (synthesis / elongation) ”is preferably performed under a total of 20 to 200 cycles.

  In the gene of the present invention, codons corresponding to individual amino acids after translation are not particularly limited. Therefore, after transcription, codons generally used in mammals such as humans (preferably frequently used codons) are used. It may also contain a DNA that shows a codon generally used in microorganisms such as Escherichia coli and yeast, plants, etc. (preferably a frequently used codon). May be.

(3) Recombinant Vector In order to express the fusion protein of the present invention, it is first necessary to construct a recombinant vector in which the above-described gene of the present invention is inserted into an appropriate expression vector.
Specifically, two types of genes, a gene encoding a protein containing protein G or a part thereof and a gene encoding a protein containing avidin or a part thereof, are prepared, and both genes are appropriately expressed. A recombinant vector inserted into the vector is constructed. Alternatively, a combination of a gene encoding the fusion protein of the present invention or a part thereof (including the above two genes) prepared in advance using the two types of gene DNAs inserted into an appropriate expression vector. Build a replacement vector.
The type of expression vector is not particularly limited, and for example, a host cell to be used as long as it can hold the inserted gene, such as plasmid DNA, bacteriophage DNA, retrotransposon DNA, retroviral vector, artificial chromosome DNA, etc. A vector suitable for the above can be selected and used as appropriate, but as an expression vector for inserting the two kinds of genes, generally known expression vectors are used as those capable of producing a fusion protein gene. It is preferable. Preferred examples of such an expression vector include pCR2.1 (Invitrogen) and pCR II (Invitrogen).

The gene to be inserted into the expression vector may have a transcription promoter, an SD sequence (when the host is a prokaryotic cell) and a Kozak sequence (when the host is a eukaryotic cell) linked upstream in advance, if necessary. A terminator may be connected downstream, and an enhancer, a splicing signal, a poly A addition signal, a selection marker, and the like may be connected. Each element necessary for gene expression such as the transcription promoter may be included in the inserted gene from the beginning, or may be used when included in the expression vector.
Methods for inserting genes into expression vectors include various methods using known gene recombination techniques, such as methods using restriction enzymes, methods not using restriction enzymes (eg, TA cloning method), and methods using topoisomerase. Can be adopted.

(4) Transformant The fusion protein of the present invention can be expressed by introducing the above recombinant vector into a suitable host to obtain a transformant and culturing it. The “transformant” as used in the present invention means a gene into which a foreign gene has been introduced into the host, for example, a gene into which a foreign gene has been introduced by introducing plasmid DNA or the like into the host (transformation), In addition, it means that any foreign gene is introduced by infecting a host with various viruses and phages (transduction).
The host is not particularly limited as long as it can express the fusion protein of the present invention after the introduction of the recombinant vector. For example, in addition to bacteria and yeast, various animal cells such as humans and mice, And known hosts such as various plant cells.

When bacteria are used as hosts, for example, Escherichia coli (DH5α, etc.), Bacillus subtilis, etc. are used.
When yeast is used as a host, for example, Saccharomyces cerevisiae, Schizosaccharomyces pombe and the like are used.
When animal cells are used as hosts, for example, human fibroblasts, CHO cells, monkey cells COS-7, Vero, mouse L cells, rat GH3, human FL cells and the like are used. Insect cells such as Sf9 cells and Sf21 cells can also be used.
When plant cells are used as hosts, for example, tobacco BY-2 cells are used.
The method for obtaining the transformant is not limited, and can be appropriately selected in consideration of the combination of the host and the expression vector. For example, electroporation, lipofection, heat shock, PEG, Preferred examples include calcium phosphate method, DEAE dextran method, and methods of infecting various viruses such as DNA virus and RNA virus.
In the resulting transformant, the codon type of the gene contained in the recombinant vector may be the same as or different from the codon type of the host actually used, and is not limited.

(5) Production method of fusion protein The fusion protein of the present invention is obtained by culturing the above-described transformant, and collecting a protein having binding activity to the Fc region of IgG antibody and binding activity to biotin from the obtained culture. It can manufacture by the method including the process to do.
Here, “culture” means any of culture supernatant, cultured cells, cultured cells, or disrupted cells or cells. The transformant can be cultured according to a usual method used for host culture. The fusion protein of interest accumulates in the culture.
The medium used for culturing the transformant contains a carbon source, a nitrogen source, inorganic salts, and the like that can be assimilated by the host. Any of the media may be used. For example, when the host is E. coli or the like, general-purpose media such as LB media and TB media can be used.

During culturing, the cells may be cultured under a selective pressure in order to prevent the recombinant vector contained in the transformant from dropping and the gene encoding the target fusion protein from dropping off. That is, when the selectable marker is a drug resistance gene, the corresponding drug can be added to the medium, and when the selectable marker is an auxotrophic complementary gene, the corresponding nutrient factor can be removed from the medium. it can. For example, when culturing human fibroblasts transduced with a vector containing a G418 resistance gene, G418 (G418 sulfate) may be added as needed during the culture.
When cultivating a transformant transformed with an expression vector having an inducible promoter, a suitable inducer may be added to the medium as necessary.
The culture conditions of the transformant are not particularly limited as long as the productivity of the target fusion protein and the growth of the host are not hindered, and are usually 10 ° C to 40 ° C (preferably 20 ° C to 37 ° C). Incubate for 5 to 100 hours. The pH can be adjusted using an inorganic or organic acid, an alkaline solution, or the like. Examples of the culture method include solid culture, stationary culture, shaking culture, and aeration and agitation culture.

After the culture, when the target fusion protein is produced in cells or cells, the protein can be collected by disrupting the cells or cells. As a method for disrupting cells or cells, high-pressure treatment using a French press or homogenizer, ultrasonic treatment, grinding treatment using glass beads, enzyme treatment using lysozyme, cellulase, pectinase, etc., freeze-thawing treatment, hypotonic solution treatment, It is possible to use a lysis inducing treatment with a phage or the like. After crushing, the cells or cell crushing residues (including the cell extract insoluble fraction) can be removed as necessary. Examples of the method for removing the residue include centrifugation and filtration. If necessary, the residue removal efficiency can be increased by using a flocculant or a filter aid. The supernatant obtained after removing the residue is a cell extract soluble fraction and can be a crudely purified protein solution.
In addition, when the target fusion protein is produced in cells or cells, the cells or cells themselves can be recovered by centrifugation, membrane separation, etc., and used without being crushed.
On the other hand, when the target fusion protein is produced outside the cells or cells, the culture solution is used as it is, or the cells or cells are removed by centrifugation or filtration. Thereafter, the target fusion protein can be collected from the culture by extraction with ammonium sulfate precipitation, if necessary.

After collecting the target fusion protein as described above, dialysis, various chromatography (gel filtration, ion exchange chromatography, isoelectric point chromatography, hydrophobic chromatography, affinity chromatography, etc.) as necessary Can also be isolated and purified. In the case of using affinity chromatography, it is preferable to perform affinity column purification using cellulose beads or the like on which IgG antibody is immobilized.
The production yield of the fusion protein obtained by culturing the transformant is, for example, SDS-PAGE (polyacrylamide) in units such as per culture, per wet cell weight or dry weight, per crude enzyme protein, etc. Gel electrophoresis) or the like.
Moreover, the production of the target fusion protein can be carried out using a cell-free protein synthesis system that does not use any living cells, in addition to a protein synthesis system by culturing transformants.

The cell-free protein synthesis system is a system that synthesizes a target protein in an artificial container such as a test tube using a cell extract. Cell-free protein synthesis systems that can be used also include cell-free transcription systems that synthesize RNA using DNA as a template. In this case, the cell extract to be used is preferably derived from the aforementioned host cell. Examples of cell extracts include extracts derived from eukaryotic cells or prokaryotic cells. More specifically, CHO cells, rabbit reticulocytes, mouse L-cells, HeLa cells, wheat germ, budding yeast, Escherichia coli, and the like are extracted. Liquid can be used. These cell extracts may be used after being concentrated or diluted, or may be used as they are, and are not limited. The cell extract can be obtained, for example, by ultrafiltration, dialysis, polyethylene glycol (PEG) precipitation or the like.
Such cell-free protein synthesis can also be performed using a commercially available kit. For example, reagent kits PROTEIOS ^™ (Toyobo), TNT ^™ System (Promega), synthesizer PG-Mate ^™ (Toyobo), RTS (Roche Diagnostics) and the like.
The target fusion protein produced by cell-free protein synthesis can be purified by appropriately selecting means such as chromatography as described above.

3. Labeled antibody
(1) Labeled antibody The labeled antibody of the present invention comprises an IgG antibody, the fusion protein of the present invention (see 2. above), and a biotinylated labeling substance. A biotinylated labeling substance is bound (immobilized) to an IgG antibody via a fusion protein.
The origin of the IgG antibody is not limited, and examples include IgG antibodies derived from mammals such as humans, mice, rats, guinea pigs, rabbits, rabbits, pigs, dogs, cats, monkeys, sheep, cows and horses. Of these, IgG antibodies derived from humans, mice, rats, rabbits and rabbits are preferred.
An IgG antibody having a specific binding ability to a desired target antigen can be used. Examples of target antigens include viral antigens and / or microbial antigens. Here, the types of viruses and microorganisms serving as antigens are not particularly limited, and examples include hepatitis C virus, hepatitis B virus, human immunodeficiency virus, Escherichia coli, tetanus, Staphylococcus aureus, and chlamydia. Among them, hepatitis C virus is preferable.
The IgG antibody may be a polyclonal antibody or a monoclonal antibody, and is not limited, but is preferably a monoclonal antibody.

  The biotinylated labeling substance is various substances that can be used for antibody detection labeling in the antigen detection method using a specific antibody, as long as it is biotinylated (bound with biotin), and is particularly limited. Not done. Examples of biotinylated labeling substances include biotinylated oligonucleic acid chains (oligonucleotide chains, etc.) and various biotinylated chemical substances (enzymes such as HRP and ALP, fluorescent substances, radioactive substances, etc.). Among them, a biotinylated oligonucleic acid chain is preferable. Note that the biotinylation method of the labeling substance is not limited, and a method of introducing biotin by a known chemical or enzymatic treatment (preferably chemical treatment) can be employed.

The oligonucleic acid chain used as a labeling substance is preferably subjected to at least one treatment selected from the following (A) to (I), for example, in order to have a function as a detection label.
(A) Processing to provide a region that can be amplified by the nucleic acid amplification method
(B) Treatment for providing a restriction enzyme cleavage site
(C) Treatment containing radioactive isotopes
(D) Treatment to bind fluorescent dye
(E) Enzyme binding process
(F) Processing to provide a cutting site by light irradiation
(G) Treatment for providing a cutting site with active oxygen
(H) Labeling dendrimer binding process
(I) Treatment for providing at least one base difference in base type

Among these, the treatments (A), (B) and (I) are preferable in terms of easy labeling and high detection sensitivity, and more preferably the treatments (A) and (B). .
In addition, a labeling treatment that combines the treatment of (A) with the treatment of (B), (F), or (G) is also preferable. And a labeling treatment that can be amplified by the treatment (A) using the obtained fragment as a template. When such a labeling treatment is performed, a fragment to be a detection label can be separated and isolated from the antigen-antibody complex to be used as a template, and a more efficient amplification reaction can be carried out. Sensitivity can be improved.

The oligonucleic acid chain subjected to the treatment (A) is an oligonucleic acid chain having a binding region with a primer used in the nucleic acid amplification method. Nucleic acid amplification methods include, for example, nucleic acid amplification methods (for example, PCR method) in which reactions are performed under a plurality of temperature control conditions using a thermal cycler, etc. There is no limitation. Further, the amplification region by the nucleic acid amplification method may be a part or all of the oligonucleic acid chain.
Here, the region that binds to the primer for PCR method is a region that includes two regions serving as groups for designing a primer set composed of a forward primer (F primer) and a reverse primer (R primer), and this It means a region that can be amplified using a primer set, and the specific nucleic acid sequence is not particularly limited. The PCR method is preferably a real-time PCR method from the viewpoint of ease of detection and quantification. In particular, in the case of the TaqMan method, the oligonucleic acid chain having a binding region with a TaqMan probe is used.

The region that binds to the primer for LAMP method consists of 6 regions that serve as a basis for designing a primer set consisting of FIP, BIP, F3 primer, B3 primer (Loop Primer F and / or Loop Primer B as required) It means a region that can be amplified using this primer set, and the specific nucleic acid sequence is not limited.
The region that binds to the primer for the ICAN method is a region that includes two regions that serve as a basis for designing a primer set composed of two chimeric primers (F and R primers), and is amplified using this primer set. It means a region that can be made, and the specific nucleic acid sequence is not limited.

In the present invention, the oligonucleic acid chain that can be used as a labeling substance includes a single-stranded or double-stranded oligonucleotide chain (for example, oligo DNA chain and oligo RNA chain (especially oligo DNA chain)), oligopeptide nucleic acid chain (oligo PNA). (Also referred to as a chain), or a mixed chain thereof is preferable, and an oligonucleotide chain is more preferable. Moreover, the oligonucleic acid chain as used in the field of this invention can also include what contains the oligopeptide chain in one part. The oligonucleic acid chain may be a natural product or a synthetic product, but is usually preferably a synthetic product.
The length of the oligonucleic acid chain used as a label is not particularly limited, but is preferably 100 to 5,000 mer, more preferably 100 to 1,000 mer, and still more preferably 100 to 500 mer. When the length of the oligonucleic acid chain satisfies the above range, the detection sensitivity can be improved and the detection time can be shortened.

  The method for producing the labeled antibody of the present invention is not limited, as long as the above-mentioned IgG antibody, the fusion protein of the present invention (see 2. above), and the biotinylated labeled substance are appropriately mixed and bound to each other. For example, a method based on a procedure in which the fusion protein of the present invention and a biotinylated labeling substance are first bound and an IgG antibody is added to the conjugate and bound is preferable. If necessary, the labeled antibody can be purified and isolated using various types of chromatography (gel filtration, ion exchange chromatography, isoelectric point chromatography, hydrophobic chromatography, affinity chromatography, etc.). .

(2) Antibody-Fusion Protein Conjugate The present invention includes an antibody-fusion protein conjugate comprising an IgG antibody and the fusion protein of the present invention (see 2. above). The details of the IgG antibody are as described above.
The method for producing the antibody-fusion protein conjugate of the present invention is not limited as long as the IgG antibody and the fusion protein of the present invention are appropriately mixed and bound to each other. If necessary, the conjugate can be purified and isolated using the various chromatography described above.
The antibody-fusion protein conjugate of the present invention is intended to bind a desired biotinylated labeled substance to various IgG antibodies, or to bind a desired IgG antibody to a biotinylated support (plate, beads, etc.). In some cases, it can be used effectively.

(3) Fusion protein-labeling substance conjugate In the present invention, a fusion protein-labeling substance conjugate comprising the fusion protein of the present invention (see 2. above) and a biotinylated labeling substance is included. . The details of the biotinylated labeling substance are as described above.
The method for producing the fusion protein-labeling substance conjugate of the present invention is not limited as long as the fusion protein of the present invention and the biotinylated labeling substance are appropriately mixed and bonded together. If necessary, the conjugate can be purified and isolated using the various chromatography described above.
The fusion protein-labeling substance conjugate of the present invention can be used effectively when it is desired to bind a desired biotinylated labeling substance to various IgG antibodies.

(4) Antibody Labeling Kit The antibody labeling kit of the present invention comprises the fusion protein of the present invention (see 2. above), the antibody-fusion protein conjugate of the present invention (see 3. (2) above), and the present invention. A fusion protein-labeling substance conjugate (see 3. (3) above). The kit can be used for the production of the labeled antibody of the present invention described above, and is extremely useful.
The antibody labeling kit of the present invention may contain other components in addition to the components listed above. As other components, for example, either or both of an IgG antibody and a biotinylated labeling substance are preferably exemplified. The details of the IgG antibody and the biotinylated labeling substance are as described above. Furthermore, as other components, for example, various buffers, sterilized water, various reaction containers (Eppendorf tubes, etc.), blocking agents (serum components such as Bovine Serum Albumin (BSA), Skim milk, Goat serum), sodium azide And a preservative such as an experimental operation manual (instruction).

4). Target antigen detection
(1) Method for detecting target antigen The method for detecting a target antigen of the present invention is a method for detecting a target antigen contained in a test sample, which comprises a target antigen and a labeled antibody of the present invention (see 3. (1) above). ) To form an antigen-antibody complex (antigen-antibody complex formation step), and a step of detecting a label in the antigen-antibody complex (label detection step).
As a preferred embodiment of the detection method of the present invention, for example, there are a plurality of types of target antigens contained in the test sample, and the labeled antibodies of the present invention can be identified corresponding to the types of the target antigens. Examples thereof include a method using a plurality of types of labeled antibodies that have been labeled. That is, this method is a method for discriminating and detecting a target antigen contained in a test sample, and it is preferable to perform the discrimination and detection simultaneously (in the same reaction system).
In addition, the detection method of this invention may include other processes other than each process mentioned above. Other steps can be performed using known means and methods.

(1-1) Formation process of antigen-antibody complex
(i) Support In the detection method of the present invention, the target antigen may be fixed to the support or may not be fixed to the support, and is not limited.
The support that can be used in the present invention is not particularly limited as long as it can fix the target antigen and can bring the antibody (antibody solution) into contact with the antigen. Used. For example, a support that can be used in an assay system based on an antigen-antibody reaction is preferable, and specific examples include multi-plastic well plates, plastic beads, latex beads, magnetic beads, plastic tubes, nylon membranes, and nitrocellulose membranes.
Moreover, as the support, as described later, a support in which an antibody capable of specifically binding to the target antigen is immobilized on the support surface can be used. If such a support is used, the target antigen can be immobilized via the antibody. The antibody can be immobilized on the surface of the support using various known methods, but there is a method for efficiently immobilizing using the above-described fusion protein of the present invention (see 2. above). Preferably mentioned. Specifically, the antibody-fusion protein conjugate of the present invention (see 3. (2) above) is brought into contact with a support whose surface has been biotinylated, and the antibody is reacted by a reaction similar to the avidin-biotin binding reaction. Or a method in which a desired IgG antibody is brought into contact with a support to which the fusion protein of the present invention has been contacted after the surface is biotinylated and the fusion protein is bound thereto, and the antibody is immobilized. Can be mentioned.

(ii) Target antigen The type of target antigen to be detected is not particularly limited. For example, the above-described viruses and / or microorganisms (for example, hepatitis C virus), various proteins (including antibody proteins), peptides (oligos) Peptides, polypeptides, etc.), polysaccharides, glycolipids, various nucleic acids (DNA and RNA), and other low-molecular chemical compounds and biological components. As one embodiment of the present invention, a single type of target antigen is targeted for detection, and a plurality of types of labeled antibodies are used to identify to which labeled antibody the target antigen binds more specifically. Also included are assay systems.
Examples of the test sample include biological components (tissue and blood), foods such as meat and vegetables, soil and river water, and combustion waste, but are not limited thereto.
When there are multiple types of target antigens contained in the test sample, the number of types is not particularly limited, but according to the method of the present invention, for example, even if there are 10 types or more, a specific target antigen is clearly identified and detected. In addition, it may be 50 types or more, and may be 100 types or more.

The concentration of the target antigen in the test sample is not limited, but according to the method of the present invention, for example, even if the target antigen is a concentration of ng order or less per 1 μL of the test sample, a specific target antigen is clearly identified. It can be detected, may be pg order or less, and may be fg order or less. In particular, when an IgG antibody labeled with an oligonucleic acid chain that can be amplified by a nucleic acid amplification method is used as the labeled antibody of the present invention, discrimination and detection can be performed with high sensitivity even at a lower target antigen concentration.
In the detection method of the present invention, the target antigen is immobilized on a support and then contacted with the labeled antibody (solution) of the present invention, whereby an antigen-antibody reaction between the target antigen and the labeled antibody is performed. The reaction may be performed without fixing the target antigen to a support or the like. Furthermore, these may be performed in combination.

As a method for immobilizing the target antigen to the support, for example, a method for immobilizing the target antigen directly on the support surface, and an antibody that specifically binds to the target antigen after immobilization on the support surface in advance, A method for immobilizing the target antigen indirectly on the surface of the support by binding the target antigen to the immobilized antibody is not limited. In the case of the latter indirect immobilization method, since a substance that can be a target antigen among a wide variety of substances in a test sample can be selected in advance, detection sensitivity and detection accuracy can be increased. In addition, as an antibody fixed to a support body, an antibody that recognizes a target antigen differently from an antibody added later (labeled antibody or the like) is usually used.
When the target antigen is immobilized on a support, blocking is preferably performed according to a conventional method before adding an antibody (such as a labeled antibody) thereafter.

(iii) Labeled antibody In the detection method of the present invention, the aforementioned labeled antibody of the present invention (see 3. (1) above) is used. Here, when there are a plurality of types of target antigens to be detected, a plurality of types of labeled antibodies that are labeled so that they can be identified corresponding to the types of the antigens are used as the labeled antibodies of the present invention. . The labeling process in the labeled antibody is performed such that a single (one type) labeling process is performed for each IgG antibody capable of specifically recognizing an individual target antigen. By specifying the type, the number of detected target antigens and the type thereof are specified. However, the present invention is not limited to this mode. For example, using a plurality of types of labeled antibodies subjected to a single labeling process (that is, a plurality of types of antibody portions), a plurality of types of target antigens are covered with one type of detection label. Can also be detected automatically. When a plurality of types of labeled antibodies are used, each antibody is preferably a monoclonal antibody, but is not limited.

In the detection method of the present invention, “contacting a target antigen immobilized on a support with a labeled antibody” is not limited to the meaning of directly contacting and binding the target antigen and the labeled antibody. First, the labeled antibody of the present invention that binds a primary antibody that specifically recognizes a target antigen immobilized on a support and then specifically recognizes this primary antibody (or a labeling substance in the primary antibody) It also includes the meaning of indirectly binding the target antigen and the labeled antibody by bringing them into contact with each other. In the case of this indirect binding, a secondary antibody, a tertiary antibody,... N-order antibody may be further bound to the primary antibody. In that case, the labeled antibody of the present invention is n-order. What can specifically bind to the antibody may be used. In addition, it is preferable that the range of said n is 1-11, More preferably, it is 1 or 2.
As a labeled antibody of the present invention, when using a plurality of types of labeled antibodies that are labeled so that they can be identified according to the type of the target antigen, the labeling of the labeling substance for enabling the above-mentioned identification detection Specific modes of processing will be described below. In the following, an example is given in which an oligonucleic acid chain is used as a labeling substance, and a labeled antibody that has been subjected to a labeling treatment in which (A) or (B) described in 3. (1) above or a combination thereof is used. explain. In addition to the cases where these labeling processes are performed, cases where other labeling processes are performed can be understood by referring to known techniques (for example, WO2006 / 049289).

In the case of the labeling treatment (A), for example, the length of the amplified fragment is different from that of the oligonucleic acid chain of another antibody, or the amplified fragment cannot be obtained from the oligonucleic acid chain of the other antibody. It is possible to identify and detect by performing the above or a combination thereof.
Specifically, the lengths of the amplified fragments are different from each other even when the same primer is used. The primer binding position is different, and the width (length) of the amplifiable region is oligonucleic acid. It can be carried out by synthesizing the chains differently. The difference in length of the amplified fragments is not limited, but is preferably 5 mer or more, more preferably 10 mer or more, and even more preferably 50 mer or more, from the viewpoint that discrimination and detection can be performed with good sensitivity.

In the case of the labeling treatment of (B), for example, the length of the fragment obtained after the restriction enzyme treatment is different from the oligonucleic acid chain of another antibody, or the restriction enzyme is derived from the oligonucleic acid chain of the other antibody. Identification or detection can be performed by preventing fragments from being obtained after processing, or by combining them.
Specifically, the lengths of the fragments obtained after the restriction enzyme treatment are different, specifically, the length of the oligonucleic acid chain to be bound to the antibody is substantially the same between the labeled antibodies, but it depends on the restriction enzyme. It can be carried out by synthesizing the cleavage positions so as to be different from each other, or by synthesizing the oligonucleic acid chains to be bound to the antibody so that the lengths of the oligonucleic acid chains are different from each other. The difference in the length of the fragments after the restriction enzyme treatment is not limited, but is preferably 10 mer or more, more preferably 50 mer or more, and still more preferably 100 mer or more from the viewpoint that discrimination and detection can be performed with good sensitivity.
In the case of the labeling process combining (A) and (B), specifically, in the labeling process of (A), restriction enzyme cleavage is performed on the antibody side of the nucleic acid amplifiable region in the oligonucleic acid chain. Except for providing a site, it is substantially the same as the labeling process (A). By performing a nucleic acid amplification method after separating a fragment (template) containing an amplifiable region from a labeled antibody, amplification efficiency can be improved and detection sensitivity can be further enhanced.

(iv) Antigen-antibody reaction When the labeled antibody (solution) of the present invention is brought into contact with a target antigen immobilized (coated) on a support, it is usually subjected to a blocking treatment with a known blocking solution in advance, and a known antigen such as PBS Wash well with the cleaning solution. Then, add an appropriate amount of a solution containing the labeled antibody, perform the binding reaction between the target antigen and the labeled antibody while stirring at room temperature for 30 to 60 minutes, form an antigen-antibody complex, and wash again. Is preferred.
On the other hand, when the labeled antibody (solution) of the present invention is brought into contact with a target antigen that is not immobilized on a support or the like, usually, a test sample containing the target antigen is subjected to appropriate pretreatment, and the target antigen It is preferable to remove or reduce the impurities.

(1-2) Label detection step The detection of the labeled substance in the formed antigen-antibody complex varies depending on the type of the labeled substance in the labeled antibody used. In the following, an example is given in which an oligonucleic acid chain is used as a labeling substance, and a labeled antibody that has been subjected to a labeling treatment in which (A) or (B) described in 3. (1) above or a combination thereof is used. A method for detecting a labeling substance will be described. In addition to the case where these labeling processes are performed, the detection method of the labeling substance when other labeling processes are performed can also be understood by referring to known techniques (for example, WO2006 / 049289). .

In the case of the labeling treatment (A), for example, according to a conventional method, a predetermined primer or the like designed is added, and a fragment obtained by amplifying a specific region by a nucleic acid amplification method such as PCR (for example, about 5 to 30 cycles) is used. obtain. The obtained amplified fragments can be easily detected by turbidity measurement or visual observation, and can be detected by various electrophoresis methods using agarose gel or the like, and a plurality of types can be obtained by comparing the lengths of the amplified fragments. The labeling substance can be identified and detected. In addition, when a fluorescently labeled primer is used, the obtained amplified fragment can be detected by analyzing it with GeneScan software using a DNA sequencer (for example, Applied Biosystems, product name: ABI-3100), If a peak position and its height are identified and compared, a plurality of types of labeling substances can be identified and detected. Furthermore, when a fluorescently labeled primer and probe (such as TaqMan probe) are used, the obtained amplified fragment can also be detected by real-time PCR (for example, product name: MX-3005p real time, manufactured by Stratagene) PCR apparatus; Applied Biosystems, product name: StepOne ^™ Real-Time PCR System, 7300 Real-Time PCR System, etc.).

In the case of the labeling treatment (B), for example, according to a conventional method, a predetermined restriction enzyme solution is added to cleave the oligonucleic acid chain. The obtained fragments can be detected by various electrophoresis methods using an agarose gel or the like, and a plurality of types of labeling substances can be identified and detected by comparing the lengths of the amplified fragments. In the detection, when the fragment concentration after the restriction enzyme treatment is low, such as when the amount of antigen is small, it is preferable to concentrate the reaction solution after the treatment by centrifugation using a spin column or the like, if necessary.
In the case of the labeling treatment combining the above (A) and (B), for example, using a free fragment after restriction enzyme treatment as a template, a predetermined designed primer is added, PCR (for example, about 5 to 30 cycles), etc. A fragment obtained by amplifying a specific region by the nucleic acid amplification method is obtained. The method for detecting the obtained amplified fragment is the same as in the labeling process of (A).

  In the present invention, by using the result obtained in the label detection step as an index, the amount of target antigen (for example, hepatitis C virus amount) in the test sample can be quantified. The said fixed_quantity | quantitative_assay can be suitably selected from the well-known fixed_quantity | quantitative_assay used together with said various detection methods. Specific quantification methods include, for example, measurement of band concentration after electrophoresis using a densitometer, turbidity measurement (monitoring) of an amplification product using an absorptiometer or spectrophotometer, and fluorescence intensity measurement using a spectrofluorometer ( A method of quantifying by using a measurement result such as (monitoring) as an index and comparatively converting it with a control measurement result (a calibration curve or the like) prepared in advance is preferable.

(2) Target antigen detection kit The target antigen detection kit of the present invention comprises the labeled antibody of the present invention (see 3. (1) above). Here, when the kit is a kit for the purpose of identifying and detecting a plurality of types of target antigens, the labeled antibody is labeled with a plurality of types that are labeled so that they can be identified corresponding to the types of target antigens. A labeled antibody is preferred.
The present invention also provides the antibody-fusion protein conjugate of the present invention (see 3. (2) above) and a biotinylated labeling substance, or the fusion protein-labeling substance conjugate of the present invention (see 3. (3) above). And a target antigen detection kit including an IgG antibody.
The target antigen detection kit of the present invention can be used in the above-described method for detecting a target antigen of the present invention, and is extremely useful.

  The target antigen detection kit of the present invention may contain other components in addition to the above components. Other components include, for example, HRP or ALP labeled primary antibody, restriction enzyme, DNA polymerase, PCR primer, dNTP, various buffers, sterile water, Eppendorf tube, phenol chloroform, chloroform, ethanol, nucleic acid coprecipitation agent, various gels (Powder), free radical production / release reagents (HRP and Fe complexes, etc.), serum components such as Bovine Serum Albumin (BSA), Skim milk, Goat serum as blocking agents, and various detergent, fluorescent reagents such as DNA intercalators, light In addition to reactive substances, various plates (including antibody-immobilized plates), various beads (including antibody-immobilized beads), preservatives such as sodium azide, and experimental operation manuals (instructions), various as required Examples include electrophoresis apparatus and laboratory equipment such as PCR.

5). Recovery of antigen or antibody
(1) Antibody-immobilized support The antibody-immobilized support of the present invention is a support comprising a support whose surface is biotinylated, the fusion protein of the present invention (see 2. above), and an IgG antibody. . Specifically, the antibody-immobilized support of the present invention is a support formed by binding an IgG antibody to the biotinylated support surface via the fusion protein of the present invention.
The antibody-immobilized support of the present invention can be used as, for example, a support used in an antigen detection method using a sandwich method. Further, it can be used for capturing and collecting a target antigen in a test sample. Therefore, the present invention includes a method for recovering a target antigen, comprising the steps of bringing a target antigen in a test sample into contact with the antibody-immobilized support, and recovering the support after the contact. is there.
The present invention also includes an antigen collection kit including the above antibody-immobilized support. The kit can contain various known components necessary for antigen recovery from the test sample.

(2) Fusion protein-immobilized support The fusion protein-immobilized support of the present invention is a support comprising the fusion protein of the present invention (see 2. above) and a biotinylated support. . Specifically, the fusion protein-immobilized support of the present invention is a support obtained by binding the fusion protein of the present invention to a biotinylated support surface.
The fusion protein-immobilized support of the present invention can be used for capturing and collecting a target antibody in a test sample. Accordingly, the present invention includes a method for recovering a target antibody, comprising the steps of bringing a target antibody in a test sample into contact with the fusion protein-immobilized support, and recovering the support after the contact. It is.
The present invention also includes an antibody collection kit comprising the above-mentioned fusion protein-immobilized support. The kit can contain various known components necessary for antibody recovery from the test sample.

  Hereinafter, the present invention will be described more specifically with reference to examples. However, the present invention is not limited to these examples.

== Synthesis of fusion protein ==
According to the following procedure, a protein consisting of a part of protein G (a protein including a binding region with IgG antibody Fc region) and a protein consisting of a part of streptavidin (a protein including a binding region with biotin) are constituent components. As a fusion protein was synthesized (see FIG. 2).

1. Public databases Swiss-Prot (accessible from http://tw.expasy.org/uniprot/) and GenBank (accessible from http://www.ncbi.nlm.nih.gov/) From the above, base sequence information and amino acid sequence information on wild type protein G and streptavidin were obtained.
The base sequence information of protein G is shown in SEQ ID NO: 1, and the amino acid sequence is shown in SEQ ID NO: 2. The base sequence information of streptavidin is shown in SEQ ID NO: 3, and the amino acid sequence is shown in SEQ ID NO: 4.

<Protein G>
・ Swiss-Prot:
“Entry name: SPG1-STRSG”, “accession number: P06654”
・ GenBank:
"Accession number: M13825"

<Streptavidin>
・ Swiss-Prot:
“Entry name: SAV STRAV”, “accession number: P22629”
・ GenBank:
"Accession number: X03591"

2. Based on the amino acid sequence information of protein G and streptavidin, an amino acid sequence region necessary for binding to the IgG antibody Fc region in protein G and an amino acid sequence region necessary for binding to biotin in streptavidin are selected. A base sequence encoding an amino acid sequence containing these regions was identified (see the region sandwiched between the arrows in FIGS. 3 and 4).

<Protein G>
The portion containing the binding region with the IgG antibody Fc region:
228th to 268th amino acid sequence region of the amino acid sequence shown in SEQ ID NO: 2 (SEQ ID NO: 6; 41 amino acid residues)
Of the nucleotide sequence shown in SEQ ID NO: 1, the 1259th to 1381st nucleotide sequence region (SEQ ID NO: 5; 123 base pairs)

<Streptavidin>
Portion containing biotin binding region:
39th to 183rd amino acid sequence region of the amino acid sequence shown in SEQ ID NO: 4 (SEQ ID NO: 8; 145 amino acid residues)
Of the base sequence shown in SEQ ID NO: 3, the 164th to 598th base sequence region (SEQ ID NO: 7; 435 base pairs)

3. Based on the base sequence information of protein G and streptavidin, the desired base sequence region was cloned.
Specifically, first, based on the base sequence information on each of protein G and streptavidin specified in 2. above, the same base sequence as the base sequence is obtained by the chemical synthesis method of DNA by the phosphate group site unprotection method. Complementary double stranded DNA with sequence was synthesized. At this time, if necessary, individual double-stranded DNAs synthesized fragmentally were joined with T4 ligase.
Then, each synthesized double-stranded DNA was used as a template, the following forward primer (F primer) and reverse primer (R primer) were designed, and PCR was carried out under the following reaction solution composition and reaction conditions. Double stranded DNA was amplified. In addition, each primer was designed so that the DNA fragment obtained by PCR has a restriction enzyme recognition site at both ends (refer to the base sequence in the lowercase letters).

<Primer for protein G nucleotide sequence>
F primer: 5'-catatg C ACTTACAAATTAATCCTTAA-3 '(SEQ ID NO: 11)
R primer: 5'-gaattc ggatcc TTCACCGTCAACACCGTTG-3 '(SEQ ID NO: 12)

<Streptavidin primer for base sequence>
F primer: 5'-gaattc aagctt GCCGGCATCACCGGCACCTG-3 '(SEQ ID NO: 13)
R primer: 5'-ctgcag CTGCTGAACGGCGTCGAGCG-3 '(SEQ ID NO: 14)

Each PCR was performed under the following reaction solution composition and reaction conditions.
<Reaction solution composition>
Template DNA (1μg / μl): 1μL
TaqDNA polymerase: 0.5unit
F primer (10μM): 5μL
R primer (10μM): 5μL
dNTP (0.2mM each): 8μL
10 × Buffer: 10μL
Sterilized water: appropriate amount (approx. 72μL)
Total: 100μL

<Reaction conditions>
A total of 35 cycles under the cycle condition of “thermal denaturation / dissociation at 95 ° C. for 30 seconds → annealing at 55 ° C. for 30 seconds → synthesis / extension at 72 ° C. for 30 seconds”.

  Whether or not the amplified fragment after PCR has the target nucleotide sequence was determined by first separating the DNA fragment obtained by PCR by agarose gel electrophoresis and confirming its chain length. Subsequently, for each of the chain lengths determined to be correct, sequence analysis is performed individually by a known method to determine whether or not the base sequence is correct (that is, whether or not each base sequence specified in 2 above is included). confirmed.

4). Joining of the two types of DNA fragments The two types of DNA fragments cloned in 3. above were subjected to restriction enzyme treatment using RcoRI according to a conventional method of gene recombination technology, and the treated DNA fragments were obtained by a known method. Collected and purified. Subsequently, the two kinds of DNA fragments after purification were joined by T4 DNA ligase (ligation at each RcoRI site). Using the DNA fragment after conjugation as a template, PCR was carried out using the base sequences shown in SEQ ID NO: 11 and SEQ ID NO: 14 under the same reaction solution composition and reaction conditions as in 3.

5). Insertion into an expression vector The DNA fragment obtained by the PCR described in 4 above is inserted into a multicloning site of an expression vector E. coli protein synthesis plasmid (pCR2.1: Invitrogen) to encode the fusion protein of the present invention. Cloning and selection were performed so that a recombinant vector having the base sequence (SEQ ID NO: 9) was constructed. The base sequence shown in SEQ ID NO: 9 is a base sequence partially including the DNA fragment obtained by the PCR of 4. The amino acid sequence of the fusion protein of the present invention encoded by the base sequence is As shown in SEQ ID NO: 10.

  That is, according to the conventional method of TA cloning using the TA-Cloning reagent attached to the pCR2.1 vector, the DNA fragment obtained by the PCR described in 4 above was introduced into the pCR2.1 vector as an insert. Specifically, the reaction solution after PCR in the above 4 is mixed as it is (or diluted 10-fold with distilled water) at a ratio of 0.5 μl to 2 μl of TA-Cloning reagent, and then at room temperature. The target recombinant vector was constructed by incubating for minutes. The obtained recombinant vector was subjected to the transformation described in 6. below after the above incubation.

6). Recombinant vector introduced into E. coli (production of transformants)
The recombinant vector constructed in 5 above was introduced into E. coli of DH5α species to produce a transformant.
Specifically, first, 2 μl of the solution after the incubation in 4 above was mixed with 50 μl of the competent cell previously dissolved on ice, incubated for 30 minutes on ice, and then heat shocked at 42 ° C. for 1 minute. Incubated again on ice for 30 minutes.
Add 200 μl of TB medium without ampicillin to 50 μl of the transformed Escherichia coli solution, pre-incubate at 200 rpm for 1 hour at 37 ° C., then add equal volume (200 μl) of TB medium with ampicillin (50 μg / ml). 100 μl of this culture solution was applied to an LB plate containing ampicillin (50 μg / ml) and X-gal. Thereafter, the cells were statically cultured overnight at 37 ° C., and only the white ones of the appearing colonies were selected and shake-cultured in 2 ml of TB medium containing ampicillin (50 μg / ml).

After recovering Escherichia coli grown by shaking culture, plasmid DNA contained in Escherichia coli was purified and concentrated based on a conventional alkali prep method.
Since the pCR2.1 vector originally has a sequence such as T7 promoter in the sequence, sequence analysis was performed using the complementary strand of the same sequence to confirm the base sequence of the insert.

7). Production of fusion protein (culture of transformants)
A transformant having a plasmid DNA whose base sequence of the insert was determined to be correct by the sequence analysis of 6. above was obtained at 37 ° C. in 500 ml of TB medium containing ampicillin (50 μg / ml) and IPTG (1 mM). The culture was shaken overnight at 200 rpm.
The bacterial solution after osmotic culture was centrifuged (9,000 rpm, 15 minutes), the supernatant was discarded, and the grown bacterial cells (transformants) were collected. The collected cells were stored frozen at -80 ° C for 1 hour or longer.

8). Recovery and purification of the produced fusion protein The cells cryopreserved in the above 7. were solubilized according to a conventional method. That is, 50 ml of solubilization buffer (50 mM Tris-HCl (pH 7.5), 500 mM NaCl, 20 mM imidazole, 2 M urea, 0.5% TritonX-100) was added to the cells recovered from 500 ml of the bacterial solution after the osmotic culture described in 7 above. Was added and homogenized with a polytron homogenizer. Next, the solubilized bacterial solution was centrifuged at 40,000 G for 2 hours at 4 ° C. in an ultracentrifuge, and the supernatant was collected. Since the protein G-avidin fusion protein is dissolved in the supernatant, 50 μl of cellulose beads (Pierce, product name: AminoLink Plus Coupling Gel) on which IgG antibody is immobilized per 200 ml of the collected supernatant The mixture was mixed and adsorbed at 4 ° C. and 10 rpm for 2 hours. Next, the cellulose beads were collected and washed three times with a washing buffer, and then the collected beads were transferred to the filter column together with the washing solution, and washed again with the washing buffer. Thereafter, the fusion protein bound to the cellulose beads was eluted and purified with a glycine buffer and recovered.
It was confirmed by SDS-PAGE that the purified protein was the target fusion protein. The results are shown in FIG.

== Preparation of oligonucleotide-labeled antibody ==
First, an anti-EGF antibody labeled with a predetermined oligonucleotide was prepared by the following procedure.

1. Preparation of oligonucleotide chain
550mer oligo with 5 'end biotinylated by PCR using PinDNA inserted pcDNA3.1 (Invitrogen) as template DNA and the following primers (5-MUSTagBio, 3-MUSTag515) Nucleotide chains were prepared. Here, 5-MUSTagBio has biotin bound to the 5 ′ end and was used as a 5 ′ primer (F primer). In addition, 3-MUSTag515 was used as a 3 ′ primer (R primer).
On the other hand, as a control, except that 5-MUSTagSS was used instead of 5-MUSTagBio, PCR was performed in the same manner as above to prepare a 550mer oligonucleotide chain with a disulfide group (SS group) introduced at the 5 'end. did. Here, 5-MUSTagSS is the same as 5-MUSTagBio except that a disulfide group (SS group) is bonded to the 5 ′ end instead of biotin (that is, the base sequence is the same), and the 5 ′ primer ( F primer).

<5 'primer>
5-MUSTagBio:
5'-Biotin-CGGAATTCGCGGACGAGGAGAAGCTGCCGCCCGGCTGG-3 '(SEQ ID NO: 15)
5-MUSTagSS:
5'-SS-CGGAATTCGCGGACGAGGAGAAGCTGCCGCCCGGCTGG-3 '(SEQ ID NO: 15)
<3 'primer>
3-MUSTag515:
5'-AGCTTGACGGGGAAAGCCGG-3 '(SEQ ID NO: 16)

The PCR was carried out under the following reaction solution composition and reaction conditions.
<Reaction solution composition>
Template DNA (100μg / μl): 1μL
TaqDNA polymerase: 2.5unit
5 'primer (20μM): 2μL
3 'primer (20μM): 2μL
dNTP (2.5mM each): 8μL
10 × Buffer: 10μL
Sterile water: Appropriate amount (approx. 77μL)
Total: 100μL

<Reaction conditions>
A total of 35 cycles under the cycle condition of “thermal denaturation / dissociation at 95 ° C. for 60 seconds → annealing at 55 ° C. for 60 seconds → synthesis / extension at 72 ° C. for 30 seconds”.

  The amplified product after each PCR was purified into a single oligonucleotide fragment by filtering the supernatant obtained by centrifugation after PCR with MinElute PCR Purification spin column (Qiagen).

2. Production of oligonucleotide-labeled antibodies
(1) Oligonucleotide-labeled antibody of the present invention
(1-1) Binding of biotinylated oligonucleotide chain and fusion protein The biotinylated oligonucleotide chain obtained in 1 above and the fusion protein obtained in the examples were added and mixed in equimolar amounts, respectively. Incubated for 30 minutes at room temperature. As a result, an oligonucleotide chain-fusion protein conjugate (about 66 kDa) in which the biotinylated oligonucleotide chain and the fusion protein were so-called biotin-avidin-bonded was prepared.

(1-2) Binding between oligonucleotide chain-fusion protein conjugate and IgG antibody Oligonucleotide chain-fusion protein conjugate obtained in (1-1) above and anti-hEGF antibody (IgG antibody; about 150 kDa) Were added and mixed in equimolar amounts (100 pmol). Specifically, 6.6 μl of the above conjugate solution of 1 mg / ml and 30 μl of 500 mg / ml anti-hEGF antibody solution were mixed. Here, Anti-hEGF (R & D: Cat # AF236, Lot # AQZ015101) was used as the anti-hEGF antibody. Then, it incubated at room temperature for 30 minutes. As a result, an oligonucleotide-labeled antibody formed by binding the above conjugate with the anti-hEGF antibody was prepared.

(1-3) Purification of labeled antibody The mixture containing the oligonucleotide-labeled antibody obtained in (1-2) above was filtered through a 0.45 µm filter. Fractions were separated by liquid chromatography (gel filtration method using Phamasia SMART System), and only the oligonucleotide-labeled antibody was purified and collected. The collected oligonucleotide-labeled antibody solution was adjusted to a concentration of 2 μg / mL and stored at 4 ° C.

(2) Control oligonucleotide labeled antibody
(2-1) Amination of the fusion protein Protein G (Fr.9: 2.36 mg / ml) and Traut reagent (2 mg / ml) are mixed in the following composition and left at room temperature for 2 hours. Protein G was aminated.

Traut reagent (2mg / ml) 10 mol
Protein G (Fr.9: 2.36mg / ml) 1 mol

(2-2) Deprotection of oligonucleotide chain The SS group-introduced oligonucleotide chain obtained in 1 above was dissolved in 20 mM Tris-HCl (pH 7.4) to prepare a 10 uM oligonucleotide solution. Next, the oligonucleotide solution and 1M DTT solution were mixed at the following ratio and allowed to stand at room temperature for 30 minutes. As a result, a deprotected oligonucleotide chain was prepared in which the SS group of the oligonucleotide chain was reduced to an SH group.

10 μM oligonucleotide solution 45 μl
1M DTT 5μl
50 μl total

Thereafter, the mixed solution was passed through a Sephadex G-50 (500 μl) gel column to remove unnecessary reagents, and the flow-through absorbance was measured.

(2-3) Adapter preparation The aminated protein G obtained in (2-1) above and the deprotected oligonucleotide chain obtained in (2-2) above were mixed at the following ratio, And left for 1 hour. Thereby, an adapter formed by binding protein G and oligonucleotide chain was prepared. In 1 μl of the obtained adapter, 28 ng of protein G and 427 ng of oligonucleotide chain are present.

Aminated protein G 0.82μl (Protein G amount: 1.4μg)
Deprotected oligonucleotide chain 50.0 μl (DNA amount: 21.7 μg)
50.82μl total

(2-4) Binding between adapter and IgG antibody The adapter (protein G to which the oligonucleotide chain was bound) obtained in (2-3) above and an anti-hEGF antibody (IgG antibody; 150 kDa) were each equimolar. (1 mol) was added and mixed. In addition, 1 mol of anti-hEGF antibody was 4.3 μg in terms of IgG amount. Then, it was incubated at room temperature for 1 hour with gentle shaking. Thereafter, in order to remove free anti-hEGF antibody, 50 μl of protein G-immobilized beads (manufactured by BioLabs, product name: Protein G magnetic beads; 1 ml) was added and mixed in the above mixed solution, and magnetic separation was performed. . Since the supernatant after separation was a control oligonucleotide-labeled antibody solution, the supernatant was collected, adjusted to a concentration of 2 μg / mL, and stored at 4 ° C.

== Detection of hEGF antigen using labeled antibody ==
The performance (antigen detection ability) of the oligonucleotide-labeled antibody of the present invention and the control oligonucleotide-labeled antibody obtained in Examples was compared.

1. Preparation of standard dilution series (test sample) of hEGF antigen
Using hEGF recombinant antigen standard solution (hEGF concentration: 3600 fmol / L = 72 pg / mL) and standard diluent, a dilution series of hEGF recombinant antigen solution was prepared. Here, the hEGF recombinant antigen standard solution was prepared by dissolving hEGF recombinant antigen (R & D, product name: Cat # 236-EG) in PBS, and 1% BSA-PBS was used as the standard diluent. used.
Specifically, as shown in the table below, first, an antigen standard solution (72 pg / mL) was added to a standard diluent 208 mL and diluted by a factor of 3.6 to prepare a 20 pg / mL antigen standard solution. Next, this 20 pg / mL antigen standard solution is diluted 5 times with a standard diluent to prepare a 4 pg / mL antigen standard solution, and then the 5-fold dilution operation is similarly repeated 4 times to obtain 0.0064 pg. A dilution series of antigen standard solution of / mL (6.4 fg / mL) was prepared. Moreover, only the standard dilution liquid which does not contain an antigen standard solution was used as Negative Control (NC).

2. HEGF antigen sensitization First, 30 μL / wel of each dilution series of antigen standard solution prepared in 1. was added to each well of an immunoplate on which a primary antibody against hEGF was immobilized. As the above immunoplate, anti-hEGF monoclonal antibody (R & D, product name: Cat # MAB636) is immobilized on an immunomodule plate (product of nunc, product name: CN-468667, 8 stations) by a conventional method. Plates were used.
A plate seal was affixed to the mixed plate, and the mixture was stirred at room temperature (20 to 30 ° C.) for 60 minutes while stirring with a plate mixer to carry out a reaction between the hEGF recombinant antigen and the primary antibody. After completion of the reaction, the liquid in each well was removed with an aspirator.
Thereafter, 400 μL / well of a washing solution (0.05% Tween20-Tris-HCl (pH 7.4) and 0.5 M NaCl) was added to each well and allowed to stand for 20 seconds, and then the washing solution was removed with an aspirator. The same washing operation was further repeated twice (3 times in total).

3. Sensitization of labeled antibody 12.5 mL of labeled antibody diluent (1% BSA-PBS) was added to 50 μL of each oligonucleotide-labeled antibody solution (stock solution) obtained in the Examples, and diluted about 250 times. .
50 μL / well of the diluted oligonucleotide-labeled antibody solution was added to each well after the washing in 2. above. After the addition, the mixture was allowed to stand at room temperature (20 to 30 ° C.) for 60 minutes, and a reaction between hEGF antigen bound to the primary antibody and each oligonucleotide-labeled antibody was performed. After completion of the reaction, the liquid in each well was removed with an aspirator.
Thereafter, 400 μL / well of a washing solution (0.05% Tween20-Tris-HCl (pH 7.4) and 0.5 M NaCl) was added to each well and allowed to stand for 20 seconds, and then the washing solution was removed with an aspirator. The same washing operation was further repeated twice (3 times in total).

4). Amplification process (amplification of oligonucleotide chain)
(1) EcoRI treatment An EcoRI enzyme solution (manufactured by Wako Pure Chemical Industries, Ltd.) was added to each well after the washing in 3. above to cleave the oligonucleotide chain in the oligonucleotide-labeled antibody.
Specifically, an EcoRI enzyme solution was first prepared at the following volume ratio (according to the protocol attached to EcoRI restriction enzyme).

EcoRI (20unit / μL): 0.4
10 x Buffer: 4
Sterile water: 35.6
Total: 40

Next, 30 μL / well of the prepared EcoRI enzyme solution was added to each well. The plate after the addition was allowed to react at 37 ° C. for 30 minutes. After the reaction, the liquid in each well was collected and centrifuged to obtain a supernatant (including the oligonucleotide fragment after EcoRI treatment).

(2) Real-time PCR
The supernatant after centrifugation from each well is used as a template DNA solution, and real-time PCR is performed using the following primers (5-MUSTag-Forw3, 3-MUSTag-GEX) and TaqMan probe (# 1-FAM). went. 5-MUSTag-Forw3 was used as a 5 ′ primer and 3-MUSTag-GEX was used as a 3 ′ primer.

<5 'primer>
5-MUSTag-Forw3:
5'-TGCATCTAGAGGGCCCTATTCTATA-3 '(SEQ ID NO: 17)
<3 'primer>
3-MUSTag-GEX:
5'-GGCAAGCCACGTTTGGTG-3 '(SEQ ID NO: 18)
<TaqMan probe>
# 1-FAM:
5 '-[FAM]-CCTTCTAGTTGCCAGCCATCTGTT-[BHQ] -3' (SEQ ID NO: 19)

The real-time PCR was performed using MX-3005p real-time PCR apparatus (Stratagene) with the following reaction solution composition and reaction conditions.
<Reaction solution composition>
Template DNA (supernatant after centrifugation): 3μL
2 × Buffer: 10μL
(TaqMan Universal PCR master mix; Applied Biosystems)
5 'primer
5- MUSTag-Forw3 (20μM): 0.4μL
3 'primer
3-MUSTag-GEX (20μM): 0.4μL
TaqMan probe
# 1-FAM (2.5μM): 1μL
Reference Dye (Rox II): 0.4μL
Sterile water: appropriate amount (about 4.8μL)
Total: 20μL

<Reaction conditions>
First, heat denaturation at 95 ° C for 10 minutes, followed by "cycle heat denaturation / dissociation at 98 ° C for 15 seconds → annealing / synthesis / extension at 60 ° C for 60 seconds (so-called two-step method)" , Total 40 cycles.

6). Detection process (detection of amplification products)
By measuring the amount of fluorescence that fluctuates with the synthesis reaction of the DNA strand by real-time PCR every minute after the start of the reaction, whether or not the amplified fragment can be detected for each well of each dilution series of hEGF recombinant antigen, and the amount of amplification The change of was observed.

7). Result The detection result of said 6 is shown in FIGS. Specifically, FIG. 6 shows the results of detection using the control oligonucleotide-labeled antibody, and FIG. 7 shows the results of detection using the oligonucleotide-labeled antibody of the present invention. In addition, the results of detection by a conventional ELISA method using a labeled antibody obtained by subjecting the same antibody as the anti-hEGF antibody (IgG antibody) used in the oligonucleotide-labeled antibody of the present invention to HRP according to a conventional method are shown in FIG. This is shown in FIG.
When a control oligonucleotide-labeled antibody was used, no detection result depending on the antigen concentration was observed, and only a detection result with a Δct value of 15 to 16 was obtained at any antigen concentration. In real-time PCR, the Δct value is a value indicating “the number of PCR cycles when the amount of oligonucleotide fragments amplified by PCR exceeds a certain value”. That is, the smaller this value, the greater the amount of oligonucleotide fragments contained in the reaction system. However, as can be seen from FIG. 7, for those with a small amount of antigen or no antigen, a Δct value exceeding 30 was obtained (by preparing a dilution series of oligonucleotide fragments and conducting real-time analysis). However, similar results were obtained (data not shown)). This result means that there are very many oligonucleotide chains on the antigen plate. That is, when the control oligonucleotide-labeled antibody was used, it can be said that a large amount of non-specific adsorption of the labeled antibody occurred on the plate surface. This was due to the non-specificity of the protein G used for the labeled antibody. This is thought to be due to the static adsorptivity. Therefore, when the control oligonucleotide-labeled antibody was used, the background became large and the antigenic substance could not be specifically detected.

  On the other hand, when the detection was performed using the oligonucleotide-labeled antibody of the present invention, there was no background as in the case of using the control oligonucleotide-labeled antibody, and very good results were obtained. When the labeled antibody of the present invention is used, the detection limit is 6.4 fg / mL even though the same antibody as the detection by ELISA (detection limit: 8 pg / mL (FIG. 8)) is used. The sensitivity was improved by 1,250 times compared to ELISA. In addition, compared to the control oligonucleotide labeled antibody, advantages such as “the labeled substance can be bound to the antibody only by mixing operation”, “the operation procedure is small”, “the reaction time is short” are recognized. It was.

== Detection of HCV antigen using labeled antibody ==
1. Preparation of standard dilution series of HCV antigen (test sample) Obtain “Ortho HCV antigen ELISA test” (manufactured by Ortho Clinical Diagnostics), a kit for quantitative detection of hepatitis C virus, and attach to the kit HCV recombinant antigen standard solution (HCV concentration: 3600 fmol / L = 72 pg / mL) and standard dilution solution were used to prepare a dilution series of HCV recombinant antigen solution.
Specifically, as shown in the table below, first, an antigen standard solution (72 pg / mL) was added to a standard diluent 208 mL and diluted by a factor of 3.6 to prepare a 20 pg / mL antigen standard solution. Next, this 20 pg / mL antigen standard solution is diluted 5 times with a standard diluent to prepare a 4 pg / mL antigen standard solution, and then the 5-fold dilution operation is similarly repeated 4 times to obtain 0.0064 pg. A dilution series of antigen standard solution of / mL (6.4 fg / mL) was prepared.

2. HCV patient serum
Three samples of frozen serum from patients who are clearly infected with hepatitis C virus were prepared.
In use, each sample was centrifuged at 15,000 rpm for 15 minutes (4 ° C.) for the purpose of removing impurities, and then only the supernatant was used.
Since the copy number of HCV virus contained in all of these samples was clear, as shown in the following table, 200,000,000 were used using the standard dilution solution attached to the kit for quantitative detection of HCV (the above-mentioned “Ortho HCV antigen ELISA test”) After adjusting to virus / mL, the 5-fold dilution operation was repeated.

3. Primary reaction process (HCV antigen sensitization)
First, 100 μL / well of a reaction solution (supplied with the kit) was added to each well of a multiwell plate (supplied with the kit) on which a primary antibody against HCV was immobilized. Next, the antigen standard solution of each dilution series prepared in 1. and the above-described 2. Each of the patient sera prepared in step 100 was added at 100 μL / well. After the addition, pipetting was performed in each well, and the reaction solution and the antigen standard solution were mixed uniformly.
The mixed plate was stirred at room temperature (20 to 30 ° C.) for 60 minutes with a plate mixer to carry out a reaction between the HCV recombinant antigen and the primary antibody. After completion of the reaction, the liquid in each well was removed with an aspirator.
Thereafter, 400 μL / well of a washing solution (attached to the kit; 10-fold diluted with MilliQ) was added to each well and allowed to stand for 20 seconds, and then the washing solution was removed with an aspirator. The same washing operation was further repeated 5 times (6 times in total).

4). Secondary reaction process (sensitization of HCV secondary antibody)
To 50 μL of an HRP-labeled secondary antibody solution (supplied with the kit) against HCV, 5 mL of an antibody diluent (supplied with the kit) was added and diluted about 100 times.
200 μL / well of the diluted secondary antibody solution was added to each well after the washing in 2. above. After the addition, the mixture was allowed to stand at room temperature (20 to 30 ° C.) for 30 minutes to carry out a reaction between the HCV recombinant antigen and the secondary antibody. After completion of the reaction, the liquid in each well was removed with an aspirator.
Thereafter, the same washing operation as in 2. above was performed for each well the same number of times (6 times in total).

5). Binding step (sensitization of oligonucleotide-labeled antibody)
(1) Preparation of oligonucleotide-labeled antibody First, an anti-HRP antibody labeled with a predetermined oligonucleotide was prepared according to the following procedure.
(1-1) Preparation of oligonucleotide chain
550mer oligo with 5 'end biotinylated by PCR using PinDNA inserted pcDNA3.1 (Invitrogen) as template DNA and the following primers (5-MUSTagBio, 3-MUSTag515) Nucleotide chains were prepared. Here, 5-MUSTagBio has biotin bound to the 5 ′ end and was used as a 5 ′ primer (F primer). In addition, 3-MUSTag515 was used as a 3 ′ primer (R primer).

<5 'primer>
5-MUSTagBio:
5'-Biotin-CGGAATTCGCGGACGAGGAGAAGCTGCCGCCCGGCTGG-3 '(SEQ ID NO: 15)
<3 'primer>
3-MUSTag515:
5'-AGCTTGACGGGGAAAGCCGG-3 '(SEQ ID NO: 16)

The PCR was carried out under the following reaction solution composition and reaction conditions.
<Reaction solution composition>
Template DNA (100μg / μl): 1μL
TaqDNA polymerase: 2.5unit
5 'primer (20μM): 2μL
3 'primer (20μM): 2μL
dNTP (2.5mM each): 8μL
10 × Buffer: 10μL
Sterile water: Appropriate amount (approx. 77μL)
Total: 100μL

<Reaction conditions>
A total of 35 cycles under the cycle condition of “thermal denaturation / dissociation at 95 ° C. for 60 seconds → annealing at 55 ° C. for 60 seconds → synthesis / extension at 72 ° C. for 30 seconds”.

  The amplified product after each PCR was purified into a single biotinylated oligonucleotide fragment by filtering the supernatant obtained by centrifugation after PCR with MinElute PCR Purification spin column (Qiagen).

(1-2) Preparation of oligonucleotide-labeled antibody
(1-2-1) Binding of biotinylated oligonucleotide chain and fusion protein Each was added and mixed, and incubated at room temperature for 30 minutes. As a result, an oligonucleotide chain-fusion protein conjugate (about 66 kDa) in which the biotinylated oligonucleotide chain and the fusion protein were so-called biotin-avidin-bonded was prepared.

(1-2-2) Binding between oligonucleotide chain-fusion protein conjugate and IgG antibody Oligonucleotide chain-fusion protein conjugate obtained in (1-2-1) above and anti-HRP antibody (IgG antibody) And equimolar (100 pmol) each. Specifically, 6.6 μl of the above conjugate solution of 1 mg / ml was mixed with 30 μl of a 500 mg / ml anti-HRP antibody solution. Here, Anti-HRP, Goat-Poly (Gentex. Inc. cat #. GTX72888) was used as an anti-HRP antibody. Then, it incubated at room temperature for 30 minutes. As a result, an oligonucleotide-labeled antibody formed by binding the above conjugate and the anti-HRP antibody was produced.

(1-2-3) Purification of labeled antibody The mixture containing the oligonucleotide labeled antibody obtained in (1-2-2) above was filtered through a 0.45 µm filter. Fractions were separated by liquid chromatography (gel filtration method using Phamasia SMART System), and only the oligonucleotide-labeled antibody was purified and collected. The collected oligonucleotide-labeled antibody solution was adjusted to a concentration of 2 μg / mL and stored at 4 ° C.

(2) Sensitization of oligonucleotide-labeled antibody To 4 μL of the oligonucleotide-labeled antibody solution (stock solution) obtained in (1-2) above, 1 mL of PBS was added and diluted about 250 times.
50 μL / well of the diluted oligonucleotide-labeled antibody solution was added to each well after the washing in 3. above. After the addition, the mixture was allowed to stand at room temperature (20 to 30 ° C.) for 60 minutes, and the HRP labeled with the secondary antibody was reacted with the oligonucleotide-labeled antibody obtained in (1) above. After completion of the reaction, the liquid in each well was removed with an aspirator.
Thereafter, PBS was added to each well at 400 μL / well, allowed to stand for 20 seconds, and then the washing solution was removed with an aspirator. The same washing operation was further repeated twice (total 3 times).

5). Amplification process (amplification of oligonucleotide chain)
(1) EcoRI treatment An EcoRI enzyme solution was added to each well after washing in the above section 4 to cleave the oligonucleotide chain in the oligonucleotide-labeled antibody.
Specifically, an EcoRI enzyme solution was first prepared at the following volume ratio.

EcoRI (20unit / μL): 0.4
10 x Buffer: 4
Sterile water: 35.6
Total: 40

To each well, 40 μL / well of the prepared EcoRI enzyme solution was added. The plate after the addition was allowed to react at 37 ° C. for 30 minutes.
After the reaction, the liquid in each well was collected and centrifuged to obtain a supernatant (including the oligonucleotide fragment after EcoRI treatment).

(2) Real-time PCR
The supernatant after centrifugation from each well is used as a template DNA solution, and real-time PCR is performed using the following primers (5-MUSTag-Forw3, 3-MUSTag-GEX) and TaqMan probe (# 1-FAM). went. 5-MUSTag-Forw3 was used as a 5 ′ primer and 3-MUSTag-GEX was used as a 3 ′ primer.

<5 'primer>
5-MUSTag-Forw3:
5'-TGCATCTAGAGGGCCCTATTCTATA-3 '(SEQ ID NO: 17)
<3 'primer>
3-MUSTag-GEX:
5'-GGCAAGCCACGTTTGGTG-3 '(SEQ ID NO: 18)
<TaqMan probe>
# 1-FAM:
5 '-[FAM]-CCTTCTAGTTGCCAGCCATCTGTT-[BHQ] -3' (SEQ ID NO: 19)

The real-time PCR was performed using MX-3005p real-time PCR apparatus (Stratagene) with the following reaction solution composition and reaction conditions.
<Reaction solution composition>
Template DNA (supernatant after centrifugation): 3μL
2 × Buffer: 10μL
(TaqMan Universal PCR master mix; Applied Biosystems)
5 'primer
5- MUSTag-Forw3 (20μM): 0.4μL
3 'primer
3-MUSTag-GEX (20μM): 0.4μL
TaqMan probe
# 1-FAM (2.5μM): 1μL
Reference Dye (Rox II): 0.4μL
Sterile water: appropriate amount (about 4.8μL)
Total: 20μL

<Reaction conditions>
First, heat denaturation at 95 ° C for 10 minutes, followed by "cycle heat denaturation / dissociation at 98 ° C for 15 seconds → annealing / synthesis / extension at 60 ° C for 60 seconds (so-called two-step method)" , Total 40 cycles.

6). Detection process (detection of amplification products)
By measuring the amount of fluorescence that fluctuates with the synthesis reaction of the DNA strand by real-time PCR every minute after the start of the reaction, whether or not the amplified fragment can be detected for each well of each dilution series of HCV recombinant antigen, and the amount of amplification The change of was observed.

7). Results As described above, the detection method of the present invention was used to detect the antigen using a dilution series of HCV recombinant antigen (72 pg / ml to 0.9 fg / ml) as a test sample. As a result, an antigen concentration of 0.02 pg / ml ( It was possible to detect antigens up to 20 fg / ml) (see FIG. 9).
On the other hand, in the three hepatitis C patient samples, HCV antigen could be detected from any patient with a sensitivity of 8000 copies / mL (see FIG. 10). Usually, it is considered that the detection of 50 fmol / mL, that is, 20,000 to 30,000 copies is the limit in the ELISA method. By using the detection method of the present invention, blood viruses can be detected with higher sensitivity. It was shown that.

Comparative example

<When using a fusion protein with streptavidin capable of forming multimers>
Synthesizing a fusion protein comprising a protein comprising a part of protein G (a protein comprising a binding region with an IgG antibody Fc region) and a protein comprising a part of streptavidin capable of forming a multimer as its constituent components; Simultaneously with studying the synthesis efficiency, an oligonucleotide chain-fusion protein conjugate was prepared with the fusion protein and compared with the fusion protein according to the present invention.

1. Synthesis of fusion protein with streptavidin capable of forming multimers First, a fusion protein having a part of streptavidin capable of forming multimers was synthesized by the same method as in the examples.
A part of the sequence of streptavidin is amino acids 16 to 133. According to US Pat. No. 5328985, a DNA chain having a base sequence encoding a protein having this amino acid sequence (US Pat. No. 5328985; Proc. Natl. Acad Sci., 87: 142 (1990); J. Biol. Chem., 265: 3369 (1990)). This DNA strand is substituted with the region encoding the avidin-derived protein in the recombinant vector producing the fusion protein used in the Examples, and XbaI and BamHI are used to replace one of the streptavidins that can form multimers. A control recombinant vector expressing a fusion protein containing a protein consisting of a part was prepared. And the fusion protein was expressed on the conditions similar to an Example. The results are shown in FIG. In the figure, A and B are different E. coli clones having a recombinant vector. NC is a negative control vector created by self-ligation after blunt-end when the region encoding the avidin-derived protein is cleaved with a restriction enzyme from the recombinant vector used in the Examples. A vector that expressed only a part of G and not the fusion protein was used.
As a result, the fusion protein of the comparative example hardly appeared in the supernatant in which the cells were solubilized, and was contained in a large amount in the cell sediment. This indicates that when the fusion protein of the comparative example is expressed by an expression system using E. coli, the synthesized protein forms an inclusion body in the bacterium and cannot be eluted by the mild solubilization method described in the examples.

Therefore, as shown in US Pat. No. 5,289,985, the cell sediment obtained from the culture solution was solubilized with 7 M guanidine hydrochloride at 37 ° C. for 1 hour, and then 150 mM NaCl-50 mM Tris-HCl (pH 7.5). )-Refolding by dialysis for 24 hours while exchanging several 0.05% Tween 20 buffers. After recovering the dialyzed protein solution, the synthetic protein was recovered by a recovery method using Ni beads and confirmed by electrophoresis. The results are shown in FIG.
As a result, compared to the solubilization treatment according to the example (lane 1), the solubilization treatment with 7M guanidine hydrochloride hardly detected the fusion protein in the cell sediment (lane 2). In addition, as a result of recovering and purifying the fusion protein from the supernatant solubilized with guanidine hydrochloride (lane 4) by a purification method using Ni-beads and performing electrophoresis, the supernatant obtained by the solubilization method of the example ( A much larger amount of synthetic protein was obtained than obtained from lane 3).

  The above results indicate that the selected streptavidin site is different between the example (the present invention) and the comparative example (US Pat. No. 5,289,985). In the example, the streptavidin site capable of forming a monomer is used. In contrast, the comparative example uses a streptavidin site capable of forming a multimer.

2. Preparation of oligonucleotide chain-fusion protein conjugate using fusion protein of comparative example In the same manner as in Examples, an oligonucleotide-labeled antibody was prepared using the fusion protein of comparative example and anti-human IL-15 antibody. Then, in the same manner as in the Examples, IL-15 antigen was detected from serially diluted human IL-15 antigen. The results are shown in FIG.
Based on the results obtained by the RT-PCR method, the ct value was plotted on the vertical axis and the antigen concentration (log10 / unit pg / mL) was plotted on the horizontal axis (FIG. 13A). In FIG. 13A, a solid line indicates an asymptotic line where R ² > 0.95 in the least square method in each graph, that is, a measurement limit. In addition, the graph of FIG. 13A was standardized with negative control values and graphed (FIG. 13B). The detection limit was calculated by the least square method based on the obtained result, and indicated by an arrow in the graph.
As apparent from FIG. 13, both the detection limit and the quantification limit can be detected at a lower concentration by using the fusion protein of the present invention than the fusion protein of the comparative example, and the oligonucleotide label can be detected with higher sensitivity. We were able to create a modified antibody.

  The above results show that the fusion protein of the comparative example was defolded by an extreme treatment with guanidine hydrochloride and then refolded, so that the IgG binding ability, than the fusion protein of the present invention that can be recovered by gentle solubilization treatment, Or it is thought that it is because biotin binding ability is inferior.

3. Conclusion As described above, the fusion protein according to the present invention can be produced in a large amount by gentle solubilization treatment without using a vigorous solubilization method such as guanidine hydrochloride method, by using a streptavidin site capable of forming a monomer. Fusion using a streptavidin site capable of forming a multimer in that the performance of the oligonucleotide chain-fusion protein conjugate synthesized with the resulting fusion protein is improved. It has much better function than protein.

  According to the present invention, it is not necessary to use a chemical binding method such as a coupling method in producing a labeled antibody used for a target antigen detection method, and an anchor substance capable of sufficiently suppressing nonspecific binding. As described above, a fusion protein of protein G and avidins can be provided. In addition, according to the present invention, a labeled antibody prepared using the fusion protein, a target antigen detection method using the antibody, a target antigen detection kit containing the antibody, and the like can be provided.

  The fusion protein of the present invention is extremely useful in that a labeled antibody that can effectively reduce the background at the time of target antigen detection can be produced without impairing the antigen recognition ability of the antibody. In addition, the method for detecting a target antigen using such a labeled antibody can provide much higher detection sensitivity than when using an antibody labeled by a method similar to the conventional method, for example, Detection at an extremely small level is particularly useful in the medical field (for example, detection of hepatitis C virus) that has a great influence on prevention, diagnosis and treatment of diseases.

Claims (32)

  A fusion protein comprising a protein comprising protein G or a part thereof and a protein comprising a part of avidin capable of forming a monomer.   The fusion protein according to claim 1, wherein the avidin is avidin, streptavidin or neutravidin.   The fusion protein according to claim 1, wherein the protein comprising a part of protein G is a protein having binding activity with the Fc region of IgG antibody. The fusion protein according to claim 1, wherein the protein comprising a part of protein G is the following protein (a) or (b).
(A) a protein comprising the amino acid sequence represented by SEQ ID NO: 6 (b) comprising an amino acid sequence in which one or several amino acids have been deleted, substituted or added in the amino acid sequence represented by SEQ ID NO: 6, and Protein with binding activity to Fc region   The fusion protein according to claim 1, wherein the protein comprising a part of avidin capable of forming a monomer is a protein having a binding activity to biotin. The fusion protein according to claim 1, wherein the protein comprising a part of avidin capable of forming a monomer is the following protein (a) or (b).
(A) a protein comprising the amino acid sequence represented by SEQ ID NO: 8 (b) comprising an amino acid sequence in which one or several amino acids have been deleted, substituted or added in the amino acid sequence represented by SEQ ID NO: 8, and Protein with binding activity The following protein (a) or (b).
(A) a protein comprising the amino acid sequence represented by SEQ ID NO: 10 (b) comprising an amino acid sequence in which one or several amino acids have been deleted, substituted or added in the amino acid sequence represented by SEQ ID NO: 10, and Protein with binding activity to Fc region and binding activity to biotin   A gene encoding the protein according to claim 1 or 7. A gene comprising the following DNA (a) or (b) and the following DNA (c) or (d):
(A) DNA consisting of the base sequence shown in SEQ ID NO: 5
(B) a DNA that hybridizes under stringent conditions with a DNA consisting of a base sequence complementary to the DNA consisting of the base sequence shown in SEQ ID NO: 5, and has binding activity to the Fc region of an IgG antibody DNA encoding the protein
(C) DNA consisting of the base sequence shown in SEQ ID NO: 7
(D) a DNA that hybridizes under stringent conditions with a DNA comprising a base sequence complementary to the DNA comprising the base sequence shown in SEQ ID NO: 7 and encodes a protein having a binding activity to biotin DNA A gene comprising the following DNA (a) or (b):
(A) DNA consisting of the base sequence shown in SEQ ID NO: 9
(B) DNA that hybridizes under stringent conditions with DNA consisting of a base sequence complementary to the DNA consisting of the base sequence shown in SEQ ID NO: 9, and binding activity to the Fc region of IgG antibody and biotin DNA encoding a protein having binding activity to   A recombinant vector comprising the gene according to any one of claims 8 to 10.   A transformant comprising the recombinant vector according to claim 11.   A method for producing the protein, comprising: culturing the transformant according to claim 12; and collecting a protein having binding activity to the Fc region of IgG antibody and binding activity to biotin from the obtained culture. .   A labeled antibody comprising an IgG antibody, the fusion protein according to claim 1 or 7, and a biotinylated labeling substance.   The labeled antibody according to claim 14, wherein the labeling substance is an oligonucleic acid chain.   An antibody-fusion protein conjugate comprising an IgG antibody and the fusion protein according to claim 1 or 7.   A fusion protein-labeling substance conjugate comprising the fusion protein according to claim 1 or 7 and a biotinylated labeling substance.   An antibody labeling kit comprising at least one selected from the group consisting of the fusion protein according to claim 1 or 7, the conjugate according to claim 16, and the conjugate according to claim 17.   The kit according to claim 18, further comprising an IgG antibody and / or a biotinylated labeling substance.   A method for detecting a target antigen contained in a test sample, comprising the step of contacting the target antigen with the labeled antibody according to claim 14 to form an antigen-antibody complex, and detecting the label in the complex The method comprising the steps of:   21. The method according to claim 20, wherein a plurality of types of labeled antigens are used, and a plurality of types of labeled antibodies labeled so as to be identified corresponding to the type of the target antigen are used as labeled antibodies.   21. The method of claim 20, wherein the target antigen is a hepatitis C virus antigen.   A kit for detecting a target antigen, comprising the labeled antibody according to claim 14.   24. The kit according to claim 23, wherein the labeled antibodies are a plurality of types of labeled antibodies that are labeled so that they can be identified corresponding to the types of target antigens.   A kit for detecting a target antigen comprising the conjugate according to claim 16 and a biotinylated labeling substance, or the conjugate according to claim 17 and an IgG antibody.   The kit according to claim 23 or 25, wherein the target antigen is a hepatitis C virus antigen.   An antibody-immobilized support comprising a support having a biotinylated surface, the fusion protein according to claim 1 or 7, and an IgG antibody.   A method for recovering a target antigen in a test sample, comprising the steps of bringing the test sample into contact with the support according to claim 27 and recovering the support after the contact.   An antigen collection kit comprising the support according to claim 27.   A fusion protein-immobilized support comprising the fusion protein according to claim 1 or 7 and a support having a biotinylated surface.   A method for recovering IgG antibodies in a test sample, the method comprising the steps of contacting the test sample with the support according to claim 30, and recovering the support after the contact.   An antibody collection kit comprising the support according to claim 30.