# Patent

## Title
Device for capturing target substance

## Abstract

It is intended to provide a target substance-capturing body comprising: a base consisting of a soluble protein; and two or more functional domains capable of binding to different target substances.

## Background
TECHNICAL FIELD
The present invention relates to a target substance-capturing body for capturing a target substance and to a method, a test kit, and so on, for detecting a target substance by use of the target substance-capturing body.
BACKGROUND ART
Biomolecules specifically binding to target substances or low molecular compounds whose target molecules are biomolecules have been expected to be used as candidate substances for pharmaceutical drugs which exert effective physiological activities in vivo on the basis of their specific binding functions to target substances or for target substance-capturing bodies of biosensors.
An example of the biopolymers as described above can include antibodies. The antibody is one of proteins that function in the self-defense mechanisms of animals through which various foreign substances invading their body fluids are detoxicated by the immune systems. In other words, the immune system recognizes a variety of structures on the surface of the foreign substance and produces antibodies specifically binding thereto. As a result, the specific binding of the antibody to the foreign substance detoxicates the foreign substance through the in-vivo immune system. To effectively exert this mechanism, antibodies possess molecular diversity (the number of antibodies with different amino acid sequences for binding to various foreign substances), and the number of antibodies per individual animal is estimated to be 107 to 108. Their specificity in antigen recognition, high antigen-binding ability and molecular diversity account for expectations placed on the use of the antibodies as candidate substances for pharmaceutical drugs or as target substance-capturing bodies.
An antibody has a structure formed by two long and two short polypeptide chains. The long polypeptide chains and the short polypeptide chains are called heavy chains and light chains, respectively. These heavy and light chains individually have variable and constant regions. The light chain is a polypeptide chain composed of two domains, one variable region (VL) and one constant region (CL). The heavy chain is a polypeptide chain composed of four domains, one variable region (VH) and three constant regions (CH1 to CH3).
Each domain of the antibody assumes a tubular structure consisting of approximately 110 amino acids and forms a very stable structure where layers are formed by β-sheets arranged in an antiparallel orientation and are further bound with each other through SS-bond.
The binding of the antibody to various antigen species is known to result from the diversity of amino acid sequences of three complementarity determining region (CDR) retained in each variable region (VH or VL). These three CDRs residing in each of VH and VL are partitioned by framework regions and allow for more highly specific molecular recognition by recognizing the spatial arrangement of a substance to be recognized. The diversity of CDR is generated by DNA reorganization occurring in the antibody gene loci when bone marrow stem cells are differentiated into B lymphocytes, antibody-producing cells. This diversity is known to be produced by causing the DNA reorganization in portions composed of VH, D and JH gene fragments in the heavy chain and in portions composed of Vλ or Vκ gene fragments or Jλ or Jκ gene fragments in the light chain. These genetic recombination processes allow for the molecular diversity of the antibody.
Such antibodies capable of binding to particular substances have conventionally been produced in artificial manners utilizing the antibody production mechanisms in the immune systems of animals as described above and have been used in various industrial fields. One example of the production method thereof includes a method involving immunizing animals (e.g., rabbits, goats and mice) to be immunized with antigen substances of interest together with adjuvants at certain intervals and collecting antibodies produced in their sera. The antibodies thus obtained are a mixture of plural antibodies that recognize various structures present on the surfaces of the antigen substances used in the immunization. The sera containing plural antibodies binding to single antigens as described above are called polyclonal antibodies.
On the other hand, the DNA reorganization occurs independently in each B cell. Therefore, one B cell produces only one type of antibody. To obtain single antibodies, a method involving fusing B cells producing particular antibodies with established tumor cells to produce hybridoma cells has been established. The single antibodies produced from such hybridomas are called monoclonal antibodies.
Antibody fragments Fab, Fab′ and F(ab′)2 obtained by treating the antibodies as described above with a certain kind of proteolytic enzyme are known to have binding ability to the same antigen as those against their parent antibodies and known to be sufficiently available as target substance-capturing bodies.
As described above, such antibodies or antibody fragments are widely available as target substance-capturing bodies adapted for target molecules in biosensors. In this case, the antibodies or antibody fragments are generally immobilized for use on a substrate. A method used for immobilizing the antibodies or antibody fragments are generally selected from physical adsorption and chemical crosslinking methods. In the immobilizing method using physical adsorption, a site of the protein involved in the adsorption cannot be selected arbitrarily when physically adsorbed onto the substrate. Alternatively, in the immobilizing method using chemical bond caused by crosslinking reaction, a functional group of the protein involved in reaction with a crosslinking agent cannot be determined arbitrarily in most cases. Furthermore, when there exist plural reactable functional groups, selectivity among them is exceedingly low. In binding to the substrate through physical adsorption or through chemical bond caused by crosslinking reaction, a site of the protein involved in the binding is generally selected at random. Therefore, if a site directly or indirectly involved in the target substance-binding ability of a protein is identical or overlaps with a site involved in binding onto substrate surface, the target substance-binding ability of the protein might be reduced remarkably.
Moreover, studies have been conducted, which apply Fab and Fab′ fragments containing heavy chain variable regions (VH) and light chain variable regions (VL) that are antibody recognition domains or containing constant regions CH1 and CL for stabilizing them more highly, camel heavy-chain antibody variable regions (VHH), VH and VL. In these studies, a single chain antibody (scFv) is produced in a genetic engineering manner by fusing VH, VL, and so on via amino acids called linkers, and applied as a target substance-capturing body.
For a method for immobilizing such antibody fragments onto a substrate, their features of being able to be produced in a genetic engineering manner have been exploited to study a method involving fusing substrate-affinity peptides or biological compounds (e.g., enzymes) with affinity for compounds immobilized on the substrate into the antibody fragments in a genetic engineering manner. According to this method, such peptides or biological compounds can be selected and fused with the amino terminus (N terminus) or carboxy terminus (C terminus) of the produced antibody fragment molecules so as not to affect their desired antigen-binding ability. Therefore, the antibody fragments bound on the substrate can be expected to be oriented to some extent.
Examples of the substrate-affinity peptides include His tag composed of plural (usually five or more) consecutive histidine residues bonded together. If using a recombinant protein fused with this His-tag, it is possible to arrange desired target substance-capturing bodies on the substrate by applying coating capable of maintaining Ni ions to the substrate surface and utilizing the electrostatic binding between the Ni ions and the His tag.
Anal. Chem. 2004, 76, pp 5713-5720 has disclosed the use of a fusion protein comprising cutinase fused with the N terminus of scFv or the C terminus of VHH against hen egg lysozyme (HEL). This fusion protein is immobilized onto a substrate as follows: at first, SAM layers consisting of triethylene glycol sulfide displaying a suicide substrate for cutinase are formed on a gold substrate and thus the antibody fragment of interest is immobilized onto the substrate via the irreversible binding between the suicide substrate and the cutinase. This document has also disclosed that the antibody fragment immobilized by this method exhibits desired binding ability.
A method for obtaining (producing) the antibodies or antibody fragments capable of being produced in a genetic engineering manner as described above is expected as follows: production methods or systems requiring low cost in total are expected to be adopted in consideration of investment in production facilities using prokaryotes typified by E. coli as hosts, production control during the operation of the production facilities, etc. However, it is difficult to produce, in the prokaryotes, proteins derived from higher organisms including humans as active proteins that maintain desired functions. In many cases, general methods for this purpose have not been established.
A method selected for producing the antibody fragments typified by scFv and Fab in E. coli is a method involving arranging a secretion signal such as pelB at the N terminus and allowing E. coli to secrete the active antibody fragments into the periplasm or a culture supernatant by utilizing the mechanism of inner membrane transport. However, in such a method, antibody fragments cannot be obtained as secreted proteins for some types of antibodies of interest. For example, the desired antibody fragments are sometimes produced as an aggregate of insoluble fractions into the bacterial cell and are not secreted. In this case, the step of solubilizing the obtained aggregate with a denaturant such as guanidine hydrochloride and then refolding the protein structure into an active structure by a dilution or stepwise dialysis method is required and is operationally complicated. Moreover, active protein yields sometimes fall short of acceptable levels.
On the other hand, a method involving fusing the antibody fragments obtained in a genetic engineering manner with secretory proteins to efficiently obtain the antibody fragments as fusion proteins has been known. U.S. Pat. No. 5,969,108 has disclosed a technique for using the antibody fragment as described above as a phage antibody having a structure where the antibody fragment is fused with a coat protein of a phage, particularly a filamentous phage, and expressed and displayed on its surface.
In the production of phages displaying antibodies on their coat protein surfaces, a method involving selecting antibody fragment clones have been utilized as disclosed in the pamphlets of International Publication No. WO088/06630 and International Publication No. WO092/15677 in addition to U.S. Pat. No. 5,969,108 described above.
However, these documents have merely disclosed a method (antibody display method) for fusing an antibody of interest with a phage minor coat protein pIII (hereinafter, pIII) and has not specifically mentioned an antibody display method for other coat proteins. On the other hand, the pamphlet of WO092/15679 has disclosed a specific method for displaying a desired protein on a phage major coat protein pVIII (hereinafter, pVIII).
This document has gained the findings that an antibody protein displayed on pIII causes irreversible reaction with a target substance, and has released a technique for displaying a desired protein on pVIII and effects thereof.
However, all of the documents described above have merely disclosed the technique for displaying an antibody or desired protein on pIII and pVIII.
Descriptions suggesting the immobilization of these antibody fragment-fused phages on a particular substrate or the use of the immobilized phages rendered functional as sensing devices are not found in any of these documents.
On the other hand, Chemistry & Biology, Vol. 11, pp 1081-1091 has disclosed that labeled streptavidin can be detected on a cell.
More specifically, two different peptide chains are displayed on different coat proteins (pIII and pVIII) of a filamentous phage. The peptide chain (RGD-4C: CDCRGDCFC) displayed on the pIII is bound with a target substance (integrin) immobilized on a cell.
As a result, the phage is immobilized on the cell (KS1767) while the streptavidin-binding peptide (R5C2: ANRLCHPQFPCTSHE) is displayed on the pVIII of the phage. According to this document, the labeled streptavidin can thereby be detected on the cell. The document has also disclosed that the phage is bound with a substrate coated with streptavidin to detect the cell.
However, Chemistry & Biology, Vol. 11, pp 1081-1091 has demonstrated that difference in binding with the KS1767 cell is small between the RGD-4/R5C2 peptide-displaying phage and the R5C2-displaying phage. This indicates the low target substance-recognizing (binding) ability of the displayed RGD-4 peptide. Thus, this technique leaves room for improvements from the viewpoint of its use as a biosensing device that specifically detects a particular substance on a substrate.
The most important technique for producing and industrially using an excellent biosensing device is to produce binding molecules (e.g., antibodies) highly specific to target substances at high yields and immobilize the molecules on a substrate or the like with their activities maintained.
DISCLOSURE OF THE INVENTION
A target substance-capturing body of the present invention is characterized by comprising: a base consisting of a soluble protein; and two or more functional domains respectively capable of binding to different target substances.
An device for capturing a target substance of the present invention is an device for capturing a target substance characterized by having the constitution described above and comprising a substrate and the target substance-capturing body in which the two or more functional domains consist of a functional domain capable of binding to a substrate and a functional domain for capturing a target substance different from the substrate, wherein the substrate is bound with the functional domain adapted for the substrate, and the functional domain for capturing the target substance different from the substrate maintains its capturing function.
A detection instrument for detecting a target substance of the present invention is a detection instrument for detecting a target substance to be detected contained in an analyte characterized by comprising: the device of the constitution described above; and detection means for detecting the binding of a target substance to a functional domain for capturing a target substance to be detected comprised in the device.
A kit for detecting a target substance of the present invention is a kit for detecting a target substance to be detected contained in an analyte characterized by comprising: the device of the constitution described above; and detection means for detecting the binding of a target substance to a functional domain for capturing a target substance to be detected comprised in the device.
A method for detecting a target substance of the present invention is a method for detecting a target substance to be detected in an analyte characterized by comprising the steps of: reacting the device of the constitution described above with a target substance to be detected; and detecting the binding of the target substance to be detected to a functional domain of the device.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a diagram schematically showing the constitution of a target substance-capturing body according to the present invention.



BEST MODE FOR CARRYING OUT THE INVENTION
Target Substance-Capturing Body
A target substance-capturing body of the present invention is characterized by comprising a soluble protein and two or more functional domains binding to different target substances. In other words, the target substance-capturing body is a molecule having the functional domains binding to different target substances on the soluble protein used as a base (scaffold). Since the soluble protein is used as a scaffold, the target substance-capturing body has the feature of being easily suspended and solubilized in an aqueous solution. Furthermore, since the target substance-capturing body has the two or more functional domains binding to different target substances, one target substance-capturing body can capture two or more different target substances.
Here, the target substance-capturing body of the present invention will be described with reference to FIG. 1. FIG. 1 is a diagram schematically showing the constitution of the target substance-capturing body of the present invention. In FIG. 1, reference numeral 101 denotes one example of a target substance-capturing body (functional domain-displaying filamentous phage) of the present invention. Reference numerals 102 and 104 denote a pVIII protein and a pIII protein, respectively. These proteins correspond to the soluble proteins. Reference numerals 103 and 105 denote functional domain-fused VIII and functional domain-fused pIII, respectively. They have two functional domains binding to different target substances. Reference numerals 100 and 106 denote one example of a substrate used in the present invention and a target substance, respectively. The substrate 100 and the target substance 106 constitute the different target substances in this example.
Hereinafter, the target substance-capturing body of the present invention and materials preferable for the constitution of a device and so on by use of it will be described in detail.
(Soluble Protein)
The soluble protein used in the present invention functions as a base (scaffold) that holds the functional domains for capturing target substances as described above and also acts as a skeletal structure for obtaining the stable productivity of the target substance-capturing body. Any of previously known proteins may be used as the soluble protein. Particularly preferred is a protein stably present in an atmosphere (e.g., inside of cytoplasm or outside of cytoplasm containing periplasm fractions) in which the functional domains easily assume a structure for stably exerting their functions. Examples of the protein stably present inside of cytoplasm include glutathione-S-transferase and ribosome and β-galactosidase.
For example, the β-galactosidase is an enzyme consisting of a tetramer. Each subunit consists of three regions (N-terminal α-region, middle region and C-terminal ω-region). Even when these regions are separately expressed as their respective fragments, they are known to be spontaneously associated with each other. For example, an α-region-containing defective body/first capturing molecule and an ω-region-containing defective body/second capturing molecule are respectively produced and mixed together for these two molecules to be associated with each other as described above to function as one functional domain.
On the other hand, examples of the protein stably present in periplasm can include periplasm-localized disulfide bond-forming enzymes (DsbA), disulfide bond-isomerizing enzymes (DsbC) and peptidyl prolyl isomerase (PPI). Moreover, they may be fused with the functional domains. The functional domains may be displayed on (fused with) coat proteins of previously known phages, particularly filamentous phages, described below in detail.
The whole soluble protein having a given function may be used as the soluble protein used in the base of the target substance-capturing body of the present invention. Alternatively, not only the whole portion but also a portion of the protein can be used as long as it can maintain solubility and function as the base. When the soluble protein is a protein consisting of a complex of plural units as described above, the protein consisting of all the units may be used in the constitution of the base, or otherwise, each single unit selected therefrom may be used as the soluble protein.
Furthermore, the base may be composed of one soluble protein or plural soluble proteins.
(Filamentous Phage)
Phages capable of supplying the soluble protein to the target substance-capturing body include filamentous phages. Concrete examples thereof include f1, fd, M13, If1, Ike, Xf, Pf1 and Pf3. The fd and M13 phages whose life cycles as well as both genetic and virion structures are known in the art are particularly preferable because of being easily handled in a genetic engineering manner and relatively easily modified for desired properties. When the phage is used as a material for supplying the soluble protein, the phage is constructed as describe below.
It is preferred that the phage should have a structure in which two or more functional molecules of proteins for capturing target substances specifically binding to different target substances are displayed on one or more coat proteins selected from major and minor coat proteins composing the phage surface, that is, the phage shell.
Hereinafter, M13 will be described in detail as one example of the phage preferably used in the present invention with particular emphasis on parts related to the present invention.
(Major Coat Protein: pVIII)
The M13 major coat protein VIII (pVIII) is encoded by a portion called “gene VIII” (gVIII) and consists of 50 amino acids. The pVIII is synthesized as a pVIII precursor with 73 amino acids from the gVIII portion of an E. coli-infecting phagemid with the aid of protein synthetases of the host in the bacterial cell. The N-terminal 23 amino acids of the pVIII precursor are known to be a secretion signal peptide for transporting the synthesized pVIII precursor polypeptide into the cell membrane (inner membrane) After the cleavage of the signal sequence, the resulting pVIII is arranged in the form where its N terminus is embedded in the inner membrane.
(pVIII/Functional Domain Fusion Protein)
The following method is a method for displaying the desired functional domains including antibody fragments on the phage surface by fusing the functional domains with the pVIII so that they can sufficiently exert their functions: for example, a method involving constructing genes for expression on the basis of the technique disclosed in the pamphlet of WO92/15679 and a technique referring to it and expressing proteins onto the phage surface is preferably available. Specifically, a phagemid (recombinant gene) is constructed by functionally combining, if necessary, with a vector portion,
(1) DNA encoding a signal sequence for guiding a protein of interest through the inner membrane to the surface;
(2) DNA encoding a desired functional domain; and
(3) gVIII or DNA encoding N-terminally deleted pVIII
so that desired expression is obtained. This phagemid is used to display a fusion protein between the pVIII and the functional domain on the phage surface or to obtain this fusion protein separated from the phage.
A protein having a previously known gene sequence and having a desired target substance-capturing function as the functional domain can be selected and used as the introduced functional domain. Among others, the preferable functional domain can include antibody fragments. The molecular size of the displayed functional domain is preferably approximately 250 amino acid residues, more preferably approximately 120 amino acid residues. The pVIII used for displaying the functional domain is a small protein with approximately 50 amino acid residues. Therefore, if the displayed functional domain is too large, it is difficult to display the functional domain together with the pVIII on the phage surface because the functional domain does not form the fusion protein with the pVIII and is taken up into the phage.
(Minor Coat Protein pIII)
The pIII is one of phage coat proteins encoded by gene III (gIII). A variety of documents and so on have demonstrated that the desired functional domain is expressed and displayed together with the pIII on the phage surface by inserting DNA encoding the functional domain of interest into the gIII.
(pIII/Functional Domain Fusion Protein)
A fusion protein between the pIII and the functional domain is supplied in the same way as in the pVIII described above by constructing phagemids encoding them and transforming E. coli hosts with the phagemids. However, the necessary infectivity of the phage for the host requires the binding of the N-terminal portion of the pIII with the F pili of the host E. coli. In this case, it is known that phage genome encoding a phage structural protein can be coinfected with a helper phage to thereby display the functional domain (ex. antibody fragment) on the pIII and produce the phage having infectivity. That is to say, it is possible to unevenly make five phage pIII proteins wild-type and fusion-type. In this context, a method for reducing replication efficiency by usually giving a slight defect to the IG (intergenic) region of the helper phage genome is known. When a phage carries phagemid encoding the gene of the antibody fragment to be displayed on pIII, it is known that a phage antibody with a pair of genotype of the phage and phenotype of the antibody fragment displayed on the phage. It is possible to select and produce DNA encoding the antibody fragment/pIII fusion protein in consideration of the function of the expressed antibody fragment with reference to documents such as Science, 1985, 228, 1315-1317.
(Functional Domain)
Two or more different functional domains for capturing target substances are used as the functional domains of the present invention. For example, a functional domain that captures a substrate as a target and a functional domain that captures a target substance to be detected are combined for use as the two or more different functional domains. As a result, a function as a biosensor immobilized on the substrate can be imparted to the target substance-capturing body. The functional domain functions for capturing a target substance, and the preferably used functional domain is capable of being expressed together with the soluble protein in a host and can be designed and produced by use of genetic engineering approaches. For example, the functional domain can be selected and used from proteins such as previously known enzymes and antibodies or functional peptide chains according to its use. Among them, the antibodies, particularly antibody fragments are desirable. The antibodies or antibody fragments have very stable structures, and it is possible to select therefrom those having strong target substance-binding ability. However, the whole antibody molecules are difficult to handle in a genetic engineering manner and still present many problems to be solved for current techniques, particularly in a simple production system using E. coli. In view of such productivity, the preferred embodiment of the present invention is an antibody fragment containing variable regions (VH and VL) or constant regions (CH1 and CL) that are target-binding regions of the antibody molecule or consisting of these regions. Moreover, the functional domain does not have to maintain the whole amino acid sequence or structure of this antibody fragment and may have the minimum amino acids and structure required to possess a desired function. Particularly when the functional domain is fused with pVIII, a low molecular functional domain is desirable as described above. The size of the functional domain used in the present invention is preferably 5 to 300 residues. The functional domain with 5 or more residues is known to have the binding ability to a particular substance. The functional domain with 300 or less residues can be expressed with its solubility maintained.
(Antibody Fragment)
The antibody fragment described in the present invention means a partial region of a monoclonal antibody.
Concrete examples thereof include Fab′, Fab, Fd, Fv (variable fragment of antibody), scFv (single chain Fv) and dsFv (disulfide stabilised Fv). Alternative examples thereof include single domain antibodies (dAb) consisting of variable regions (VH) or light chain variable regions (VL). Furthermore, camel heavy-chain antibody variable regions (VHH), shark antibody-like molecules (IgNAR: immunoglobulin new antigen receptor), and so on may be applied thereto.
In this context, “F(ab′)2” and “Fab′” are fragments that can be obtained by standard methods by treating antibodies with a proteolytic enzyme such as pepsin or papain. That is to say, they are antibody fragments produced as a result of digestion before and after disulfide bond located between two heavy chains (H chains) in the hinge region of the antibody.
The antibody fragment may also be an Fd fragment bound with VH and CH1. Furthermore, the antibody fragment may be an Fv (variable fragment of antibody) fragment or a portion thereof and may be, for example a heavy chain variable region (VH) or light chain variable region (VL) composing Fv or a portion thereof. On the other hand, single chain Fv (scFv) where the carboxy terminus of either of VH or VL is linked with the amino terminus of the other can also be used as a complex where VH and VL are arranged in a single-stranded polypeptide. It is desirable that a linker consisting of one or more amino acids should be provided between VH and VL (not in particular order) forming scFv. It is important to design the residue length of the amino acid linker so as not to provide binding force that prevents the formation of a structure necessary for the binding of VH or VL with an antigen. Specifically, the length of the amino acid linker is generally 5 to 18 residues, and the amino acid linker with 15 residues has been used and studied most frequently. It is possible to obtain these fragments by genetic engineering approaches.
Furthermore, either of VH or VL may be the single domain dAb. However, because most single domain structures are generally unstable, it is preferred that such an unstable single domain dAb should be stabilized by chemical modification such as PEG modification.
(Substrate-Binding Antibody Fragment)
A target of the functional domain, for example the antibody or antibody fragment, displayed on the phage surface may be a substrate that forms a solid phase. For example, our studies have revealed those comprising one or more of SEQ ID NOs: 1 to 57 as antibody fragments showing binding properties to a gold substrate. Concrete examples of VH having SEQ ID NOs: 1 to 57 are shown in SEQ ID NOs: 58 to 74, and concrete examples of VL are shown in SEQ ID NOs: 75 to 77. Amino acid sequences derived from these amino acid sequences with the deletion, substitution or addition of one or several amino acids can be used without problems in the present invention as long as they are sequences that can exert gold-binding properties. One example of the nucleotide sequence of a gold-binding protein is shown below in SEQ ID NOs: 78 to 96.
Furthermore, the gold-binding protein can be constructed as described below.
In other words, it is possible to genetically modify a portion of a site that does not influence binding ability so much in the combination of VH and VL forming gold-binding domains to introduce a cysteine residue into the desired site of the VH and VL so that SS bond can be formed at an interface between the VH and VL. It is also possible to easily form a VH/VL complex by providing two cysteines in the linker.
(Others: Minor Coat Protein)
The introduced binding domains or epitopes can also be displayed as a portion of chimeric minor coat proteins on the filamentous phages. These minor coat proteins are encoded by “genes III, VI, VII and IX”, and each of them is present in approximately 5 copies per virion and is involved in morphogenesis and infection. By contrast, the major coat protein is present in 2500 or more copies per virion. The proteins from the “genes III, VI, VII and IX” are located at the end of the virion.
(Target Substance)
In the present invention, any molecule may be used as a target substance to be captured as long as it is a substance that is capable of serving as an antigen in each approach using antigen-antibody reaction.
The target substances of the present invention are broadly classified into nonbiological substances and biological substances. The nonbiological substances of great industrial value can include:
PCBs as environmental contaminants with varying numbers/positions of substitution of chlorine; dioxins with varying numbers/positions of substitution of chlorine; and endocrine disruptors named so-called environmental hormones (e.g., hexachlorobenzene, pentachlorophenol, 2,4,5-trichloroacetic acid, 2,4-dichlorophenoxyacetic acid, amitrole, atrazine, alachlor, hexachlorocyclohexane, ethylparathion, chlordane, oxychlordane, nonachlor, 1,2-dibromo-3-chloropropane, DDT, kelthane, aldrin, endrin, dieldrin, endosulfan (benzoepin), heptachlor, heptachlor epoxide, malathion, methomyl, methoxychlor, mirex, nitrofen, toxaphene, trifluralin, alkylphenol (5 to 9 carbon atoms), nonylphenol, octylnonylphenol, 4-octylphenol, bisphenol A, di-2-ethylhexyl phthalate, butylbenzyl phthalate, di-n-butyl phthalate, dicyclohexyl phthalate, diethyl phthalate, benzo(a)pyrene, 2,4-dichlorophenol, di-2-ethylhexyl adipate, benzophenone, 4-nitrotoluene, octachlorostyrene, aldicarb, benomyl, kepone (chlordecone), manzeb (mancozeb), maneb, metiram, metribuzin, cypermethrin, esfenvalerate, fenvalerate, permethrin, vinclozolin, zineb, ziram, dipentyl phthalate, dihexyl phthalate and dipropyl phthalate).
Examples of the biological substances include biological substances selected from nucleic acids, proteins, sugar chains, lipids and complexes thereof. To be more specific, the biological substances comprise biomolecules selected from nucleic acid, protein, sugar chains and lipids.
Concretely, the present invention can be applied to any substance as long as it contains a substance selected from DNA, RNA, aptamers, genes, chromosomes, cell membranes, viruses, antigens, antibodies, lectin, hapten, hormones, receptors, enzymes, peptides, sphingoglycolipid and sphingolipid. In addition, bacteria and cells themselves that produce the “biological substances” can be target substances as the “biological substances” intended by the present invention.
Concrete examples of the proteins include so-called disease markers. Examples thereof include: α1-fetoprotein (AFP), an acid glycoprotein produced in hepatic cells for a fetal period and present in fetal blood, which serves as a marker for hepatocellular carcinoma (primary liver cancer), hepatoblastoma, metastatic liver cancer and yolk sac tumor; PIVKA-II, abnormal prothrombin appearing during hepatic parenchymal injury, which is confirmed to specifically appear in hepatocellular carcinoma; BCA225, a glycoprotein that is an antigen immunohistochemically specific for breast cancer, which serves as a marker for advanced primary breast cancer and recurrent/metastatic breast cancer; basic fetoprotein (BFP), a basic fetal protein found in extracts from human fetal serum, intestine and brain tissue, which serves as a marker for ovarian cancer, testicular tumor, prostatic cancer, pancreatic carcinoma, biliary tract carcinoma, hepatocellular carcinoma, renal cancer, lung cancer, gastric cancer, bladder carcinoma and colon cancer; CA15-3, a carbohydrate antigen, which serves as a marker for advanced breast cancer, recurrent breast cancer, primary breast cancer and ovarian cancer; CA19-9, a carbohydrate antigen, which serves as a marker for pancreatic carcinoma, biliary tract carcinoma, gastric cancer, liver cancer, colon cancer and ovarian cancer; CA72-4, a carbohydrate antigen, which serves as a marker for ovarian cancer, breast cancer, colorectal cancer, gastric cancer and pancreatic carcinoma; CA125, a carbohydrate antigen, which serves as a marker for ovarian cancer (particularly, serous cystadenocarcinoma), adenocarcinoma of the uterine body, cancer of the Fallopian tube, adenocarcinoma of the uterine cervix, pancreatic carcinoma, lung cancer and colon cancer; CA130, a glycoprotein, which serves as a marker for epithelial ovarian cancer, cancer of the Fallopian tube, lung cancer, hepatocellular carcinoma and pancreatic carcinoma; CA602, a core protein antigen, which serves as a marker for ovarian cancer (particularly, serous cystadenocarcinoma), adenocarcinoma of the uterine body and adenocarcinoma of the uterine cervix; CA54/61 (CA546), a core carbohydrate-related antigen, which serves as a marker for ovarian cancer (particularly, mucinous cystadenocarcinoma), adenocarcinoma of the uterine cervix and adenocarcinoma of the uterine body; carcinoembryonic antigen (CEA), which has currently been used most widely for assistance in diagnosing cancer as a marker antigen associated with cancer such as colon cancer, gastric cancer, rectal cancer, biliary tract carcinoma, pancreatic carcinoma, lung cancer, breast cancer, uterine cancer and urinary system cancer; DUPAN-2, a carbohydrate antigen, which serves as a marker for pancreatic carcinoma, biliary tract carcinoma, hepatocellular carcinoma, gastric cancer, ovarian cancer and colon cancer; elastase 1, an exocrine pancreatic protease present in the pancreas and specifically hydrolyzing elastic fiber elastin (composing arterial walls, tendons, and the like) in connective tissue, which serves as a marker for pancreatic carcinoma, cystic carcinoma of the pancreas and biliary tract carcinoma; immunosuppressive acidic protein (IAP), a glycoprotein present at high concentrations in the ascites and serum of human patients with cancer, which serves as a marker for lung cancer, leukemia, cancer of the esophagus, pancreatic carcinoma, ovarian cancer, renal cancer, cholangioma, gastric cancer, bladder carcinoma, colon cancer, thyroid carcinoma and malignant lymphoma; NCC-ST-439, a carbohydrate antigen, which serves as a marker for pancreatic carcinoma, biliary tract carcinoma, breast cancer, colon cancer, hepatocellular carcinoma, adenocarcinoma of the lung and gastric cancer; γ-seminoprotein (γ-Sm), a glycoprotein, which serves as a marker for prostatic cancer; prostate-specific antigen (PSA), a glycoprotein extracted from human prostate tissue and present only in prostate tissue, which thus serves as a marker for prostatic cancer; prostatic acid phosphatase (PAP), an enzyme secreted from the prostate and hydrolyzing phosphoric ester at acidic pH, which is used as a tumor marker for prostatic cancer; neuron-specific enolase (NSE), a glycolytic enzyme specifically present in nervous tissue and neuroendocrine cells, which serves as a marker for lung cancer (particularly, small cell carcinoma of the lung), neuroblastoma, nervous system tumor, islet cell cancer, small cell carcinoma of the esophagus, gastric cancer, renal cancer and breast cancer; squamous cell carcinoma-related antigen (SCC antigen), a protein extracted and purified from the hepatic metastatic foci of squamous cell carcinoma of the uterine cervix, which serves as a marker for uterine cancer (cervical squamous cell carcinoma), lung cancer, cancer of the esophagus, head and neck cancer and skin cancer; sialyl Lex-i antigen (SLX), a carbohydrate antigen, which serves as a marker for adenocarcinoma of the lung, cancer of the esophagus, gastric cancer, colon cancer, rectal cancer, pancreatic carcinoma, ovarian cancer and uterine cancer; SPan-1, a carbohydrate antigen, which serves as a marker for pancreatic carcinoma, biliary tract carcinoma, liver cancer, gastric cancer and colon cancer; tissue polypeptide antigen (TPA), a single-stranded polypeptide useful for the speculation, prediction of recurrence, and observation of therapeutic process of advanced cancer particularly in combination with other tumor markers, which serves as a marker for cancer of the esophagus, gastric cancer, colorectal cancer, breast cancer, hepatocellular carcinoma, biliary tract carcinoma, pancreatic carcinoma, lung cancer and uterine cancer; sialyl Tn antigen (STN), a core carbohydrate antigen, which serves as a marker for ovarian cancer, metastatic ovarian cancer, gastric cancer, colon cancer, biliary system cancer, pancreatic carcinoma and lung cancer; cytokeratin (CYFRA) as an effective tumor marker for the detection of non-small cell carcinoma of the lung, particularly squamous cell carcinoma of the lung; pepsinogen (PG), an inactive precursor of two pepsins (PG I and PG II) that are proteases secreted into gastric juice, which serves as a marker for gastric ulcer (particularly gastric ulcer located in the lower part), gastroduodenal ulcer (particularly, recurrent and intractable cases), Brunner's gland adenoma, Zollinger-Ellison syndrome and acute gastritis; C-reactive protein (CRP), an acute phase reactant changed in serum by tissue injury or infection, which shows high values during myocardial necrosis caused by acute myocardial infarction and the like; serum amyloid A protein (SAA), an acute phase reactant changed in serum by tissue injury or infection; myoglobin, a heme protein with a molecular weight of approximately 17500 present mainly in cardiac muscles and skeletal muscles, which serves as a marker for acute myocardial infraction, muscular dystrophy, polymyositis and dermatomyositis; creatine kinase (CK; three isozymes of CK-MM type derived from skeletal muscles, CK-BB type derived from brains and smooth muscles, and CK-MB type derived from cardiac muscles, mitochondrial isozyme and immunoglobulin-linked CK (macro CK)), an enzyme present mainly in the soluble fractions of skeletal muscles and cardiac muscles and migrating into blood by cell injury, which serves as a marker for acute myocardial infraction, hypothyroidism, progressive muscular dystrophy and polymyositis; troponin T, a protein with a molecular weight of 39000 forming a troponin complex with troponin I and troponin C on the thin filaments of striated muscles and participating in the regulation of muscular contraction, which serves as a marker for rhabdomyolysis, myocarditis, myocardial infarction and renal failure; ventricular myosin light chain I, a protein contained in the cells of both skeletal muscles and cardiac muscles, which serves as a marker for acute myocardial infraction, muscular dystrophy and renal failure because a rise in its measurement result means injury and necrosis in skeletal muscles and cardiac muscles; and chromogranin A, thioredoxin and 8-OhdG, which are attracting attention as stress markers in recent years.
(Substrate)
A substrate with any material or shape can be used as the substrate according to the present invention as long as it achieves the object of the present invention. Particularly preferred is a substrate containing gold in at least a portion of its surface.
The material of the substrate used in the present invention may be any material that is capable of forming the structure of the present invention. The material is, for example a material comprising any one or more substances or a complex thereof selected from metals, metal oxides, inorganic semiconductors, organic semiconductors, glasses, ceramics, natural polymers, synthetic polymers and plastics.
The shape of the substrate used in the present invention may be any shape that is capable of forming the structure of the present invention, and is a shape comprising any one or more shapes selected from platelike, particulate, porous, protruded, fibrous, tubular and reticular forms.
Organic polymer compounds can include organic polymer compounds produced by polymerizing polymerizable monomers selected from the group consisting of: styrene-based polymerizable monomers such as styrene, α-methylstyrene, β-methylstyrene, o-methylstyrene, m-methylstyrene, p-methylstyrene, 2,4-dimethylstyrene, p-n-butylstyrene, p-tert-butylstyrene, p-n-hexylstyrene, p-n-octylstyrene, p-n-nonylstyrene, p-n-decylstyrene, p-n-dodecylstyrene, p-methoxystyrene and p-phenylstyrene; acryl-based polymerizable monomers such as methyl acrylate, ethyl acrylate, n-propyl acrylate, iso-propyl acrylate, n-butyl acrylate, iso-butyl acrylate, tert-butyl acrylate, n-amyl acrylate, n-hexyl acrylate, 2-ethylhexyl acrylate, n-octyl acrylate, n-nonyl acrylate, cyclohexyl acrylate, benzyl acrylate, dimethyl phosphate ethyl acrylate, diethyl phosphate ethyl acrylate, dibutyl phosphate ethyl acrylate and 2-benzoyloxyethyl acrylate; methacryl-based polymerizable monomers such as methyl methacrylate, ethyl methacrylate, n-propyl methacrylate, iso-propyl methacrylate, n-butyl methacrylate, iso-butyl methacrylate, tert-butyl methacrylate, n-amyl methacrylate, n-hexyl methacrylate, 2-ethylhexyl methacrylate, n-octyl methacrylate, n-nonyl methacrylate, diethyl phosphate ethyl methacrylate and dibutyl phosphate ethyl methacrylate; and vinyl-based polymerizable monomers such as esters of methylene aliphatic monocarboxylic acids, vinyl esters (e.g., vinyl acetate, vinyl propionate, vinyl butyrate, vinyl benzoate and vinyl formate), vinyl ethers (e.g., vinylmethyl ether, vinylethyl ether and vinylisobutyl ether) and vinyl ketones (e.g., vinyl methyl ketone, vinyl hexyl ketone and vinyl isopropyl ketone).
Examples of inorganic solid matter that can be used include: clay minerals such as kaolinite, bentonite, talc and mica; metal oxides such as alumina, titanium dioxide, zinc oxide, magnetite, ferrite, Nb—Ta complex oxides, WO3, In2O3, MoO3, V2O5 and SnO2; insoluble inorganic salts such as silica gel, hydroxyapatite and calcium phosphate gel; metals such as gold, silver, platinum and copper; semiconductor compounds such as GaAs, GaP, ZnS, CdS and CdSe; and glass and silicon; and complexes thereof.
The substrate include: films consisting of plastics such as polyethylene terephthalate (PET), diacetate, triacetate, cellophane, celluloid, polycarbonate, polyimide, polyvinyl chloride, polyvinylidene chloride, polyacrylate, polyethylene, polypropylene and polyester: and porous polymer membranes consisting of polyvinyl chloride, polyvinyl alcohol, acetylcellulose, polycarbonate, nylon, polypropylene, polyethylene, Teflon, and the like. Alternatively, the substrate can be made into a membrane or sheet form by use of a wooden plate, a glass plate, a silicon substrate, cloth (e.g., cotton, rayon, acryl, silk and polyester) or paper (e.g., high-quality paper, medium-quality paper, art paper, bond paper, recycled paper, baryta paper, cast-coated paper, corrugated cardboard and resin-coated paper). The materials for these membrane or sheet forms may have smooth or uneven surfaces.
Further examples of the substrate include: substrates such as silicon, silica, glass and quartz glass, micro flow passages or holes provided in these substrates by approaches such as photolithography, etching and sandblasting, or those obtained by coating their surfaces with a thin membrane of gold, silver or platinum; substrates such as PDMS (polydimethylsiloxane), PMMA (polymethylmethacrylate), PET (polyethylene terephthalate), PC (polycarbonate) and PS (polystyrene) and micro flow passages or holes provided in these substrates by molding techniques; carbon nanotubes, carbon nanohorn, fullerene, diamond, and aggregates thereof; nanowhisker consisting of alumina, carbon, fullerene, ZnO, or the like; mesoporous thin films, microparticles and monolith structures consisting of SiO2, aluminosilicate, other metallosilicates, TiO2, SnO2, Ta2O5, or the like; microparticles such as gold, silver, copper and platinum; metal oxide particles such as magnetite, ferrite, hematite, gamma-hematite and maghemite; aluminum-silicon mixture membranes and silicon oxide nanostructures obtained by anodically oxidizing them; porous alumina thin membranes, alumina nanohole structures and silicone nanowires.
(Use of Device)
The combination of the functional domain adapted for the substrate that captures the substrate as a target substance and has a substrate-binding property and the functional domain for detection that captures a target substance to be detected contained in a sample (analyte) as a target substance can be used as the functional domains comprised in the target substance-capturing body of the constitution described above.
More specifically, this target substance-capturing body is bonded with the substrate via the functional domain adapted for the substrate to form a device for detection. This device can be used to detect a target substance to be detected (e.g., a variety of nonbiological substances and biological substances described above) by use of the functional domain for detection. Moreover, at least this device and detection means (e.g., optical or electrical measurement instruments and a variety of reagents) capable of detecting the binding of the functional domain for detection with the target substance to be detected can be used to construct a detection instrument or detection kit.
A detecting method can include a method comprising the following steps:
(1) binding the target substance-capturing body to at least a portion of the surface of the substrate via its functional domain adapted for the substrate;
(2) contacting the substrate with an analyte (sample);
(3) washing the substrate; and
(4) detecting a target substance to be detected captured by the functional domain for detection in the target substance-capturing body.
The detection in the step (4) can be performed by a optical detection method (e.g., a method using luminol reaction described in Examples below) using a substance with a detectable label specifically binding to the target substance-capturing body or the target substance to be detected, optical measurement applying enhanced Raman or localized plasmon principles to the use of the substrate having the surface consisting of gold or containing a portion consisting of gold, or electrical measurement utilizing its electrical properties. The specific operation of each of the steps can be performed based on a standard method.
On the other hand, the device for capturing a target substance according to the present invention is preferably available for use in the binding of two kinds of target substances by use of its capturing function and in the immobilization of a target substance onto the substrate by use of the functional domain adapted for the substrate when the target substance-capturing body is immobilized for use on the substrate.
EXAMPLES
Hereinafter, Examples of the present invention will be illustrated.
Example 1
Construction of Plasmid for Expression of pIII Fused with HEL-Binding scFv and Confirmation of its Expression
M13KE (manufactured by NEW ENGLAND BIOLABS.) is used for pIII fusion protein expression. HEL-binding scFv (SEQ ID NO: 97 or 98) is inserted into the multicloning site of the M13KE. This plasmid is inserted into Acc65I/EagI according to the technical bulletin of the company. The resulting plasmid for expression is designated as pM13-HpIII. E. coli is transformed with pM13-GIII by electroporation according to the technical bulletin to express a phage displaying pIII fused with HEL-binding scFv.
The HEL-binding scFv is obtained as a DNA fragment encoding HEL-binding scFv by PCR using, as a template, a plasmid incorporating therein a HEL-binding scFv-encoding gene shown in Journal of Biological chemistry, 2003, 278, pp 8979. The following primers are used in this PCR:









scFv-f




NNNNCCATGCCCGATATCGTCCTGACCCAG
(SEQ ID NO: 112)


 


scFv-r


AGCTACCGCGGAGACGGTGACGAGGGT.
(SEQ ID NO: 113)






The following phage ELISA method is used in the confirmation of the expression:

(1) 80 μL each of serially diluted solutions of VH-displaying phage is dispensed to an amino-modified titer plate immobilizing HEL (manufactured by Seikagaku Corp.) thereon and gently stirred with a shaker for 1 hour;

(2) After the removal of the phage solutions, 90 μL of PBST is dispensed to each well and stirred for 10 minutes, followed by washing and discarding of the supernatant. This procedure is repeated three times;

(3) 75 μl of a solution of HRP-conjugated anti-M13 antibody/PBST (1/5000) is dispensed to each well and gently stirred with a shaker for 1 hour;

(4) The supernatant is discarded. Next, 90 μL of PBST is dispensed to each well and stirred for 10 minutes, followed by washing, and discarding of the supernatant. This washing procedure is repeated three times;

(5) 35 μL each of detection reagents 1 and 2 (Amersham BIOSCIENCE) is dispensed to each well and reacted for 1 minute with gentle stirring; and

(6) The luminescence intensity of luminol is measured.

The same experiment except that M13KO7 (manufactured by NEW ENGLAND BIOLABS.) is used instead of pM13-HpIII is conducted as a comparative experiment. The phage obtained from the pM13-GpIII is confirmed to display gold-binding VH.
Example 2
Construction of Vector for Expression of pVIII Fused with SBA-15-Affinity Peptide
The multicloning site BamHI/SpeI of an f1-based phagemid M13 mp18 (manufactured by NEW ENGLAND BIOLABS.) is used for pVIII fusion protein expression to construct a gene (SEQ ID NO: 101) having sequences in the following order:
(1) promoter sequence, (2) Shine-Dalgarno (SD) sequence, (3) M13-gVIII signal sequence-encoding DNA sequence, (4) SBA-15-affinity peptide-encoding DNA sequence (SEQ ID NO: 99), (5) M13-gVIII, (6) several termination codons, and (7) transcription terminator.
In this Example, the promoter sequence used is a tac promoter. The obtained plasmid is designated as pM13-SpVIII. E. coli is transformed with this plasmid by electroporation according to the technical bulletin of the company to express a phage displaying pVIII fused with SBA-15. The following phage ELISA method is used in the confirmation of the expression:
(A) A solution of 1 mg of SBA-15/PBST and 80 μL of a phage solution are successively added to a 1.5-mL Eppendorf tube and gently stirred with a shaker for 1 hour;
(B) After centrifugation (15000 rpm, 10 min) and the removal of the supernatant solution, 1000 mL of PBST is dispensed again and stirred for 10 minutes, followed by washing and discarding of the supernatant. This procedure is repeated three times;
(C) 500 μl of a solution of HRP-conjugated anti-M13 antibody/PBST (1/5000) is added and gently stirred with a shaker for 1 hour;
(D) After centrifugation (15000 rpm, 10 min), the supernatant is discarded. Next, 1000 μL of PBST is dispensed and stirred for 10 minutes, followed by washing and discarding of the supernatant. This procedure is repeated three times;
(E) 35 μL each of detection reagents 1 and 2 (Amersham BIOSCIENCE) is dispensed to each well and reacted for 1 minute with gentle stirring; and
(F) The luminescence intensity of luminol is measured.
The same experiment except that M13KO7 (manufactured by NEW ENGLAND BIOLABS.) is used instead of pM13-SpVIII is conducted as a comparative experiment. The phage obtained from the pM13-SpVIII is confirmed to display gold-binding VH.
Example 3
Construction of Plasmid for Coexpression of pIII Fused with HEL-Binding scFv/pVIII Fused with SBA-15-Affinity Peptide
The pM13-HpIII obtained in Example 1 and the pM13-SpVIII obtained in Example 2 are used to construct a plasmid for coexpression of gold-binding VH-fused pIII/SBA-15-fused pVIII. The pM13-HpIII and the pM13-SpVIII cleaved with restriction enzymes BspHI/BsmI (both manufactured by NEW ENGLAND BIOLABS.) according to the method of the technical bulletin recommended by the manufacturer. The resulting enzyme reaction solution is subjected to agarose gel electrophoresis. A fragment of around 4.5 kbp obtained from the pM13-HpIII cleavage reaction solution and a fragment of around 0.6 kbp obtained from the pM13-SpVIII reaction solution are collected and purified with a purification kit (manufactured by Promega: trade name Wizard SV Gel and PCR Clean-Up System). Next, the DNA fragments thus obtained are ligated with T4-Ligase (manufactured by Roche) for 2 hours according to the method recommended by the manufacturer. The obtained ligation solution is transformed in the same way as in Example 2 to express and collect a phage. The following method is used in the confirmation of the expression:
(1) A solution of 1 mg of SBA-15/PBST and 80 μL of a phage solution are successively added to a 1.5-mL Eppendorf tube and gently stirred with a shaker for 1 hour;
(2) After centrifugation (15000 rpm, 10 min) and the removal of the supernatant solution, 1000 μL of PBST is dispensed again and stirred for 10 minutes, followed by washing and discarding of the supernatant. This procedure is repeated three times;
(3) Next, 1000 μL of 1 μM HEL is added and gently stirred with a shaker for 1 hour;
(4) After centrifugation (15000 rpm, 10 min) and the removal of the supernatant solution, 1000 μL of PBST is dispensed again and stirred for 10 minutes, followed by washing and discarding of the supernatant. This procedure is repeated three times;
(5) 500 μl of a solution of HRP-conjugated anti-HEL antibody/PBST (1/5000) is added and gently stirred with a shaker for 1 hour;
(6) After centrifugation (15000 rpm, 10 min), the supernatant is discarded. Next, 1000 μL of PBST is dispensed and stirred for 10 minutes, followed by washing and discarding of the supernatant. This procedure is repeated three times;
(7) 35 μL each of detection reagents 1 and 2 (Amersham BIOSCIENCE) is dispensed to each well and reacted for 1 minute with gentle stirring; and
(8) The luminescence intensity of luminol is measured.
The experiments using the phages obtained in Examples 1 and 2 are performed as comparative experiments. The phage obtained in Example 3 is confirmed to have the highest luminescence intensity of luminol.
Example 4
Construction of Vector for Expression of pVIII Fused with Gold-Binding VH
The multicloning site BamH1/SpeI of an f1-based phagemid M13 mp18 (manufactured by NEW ENGLAND BIOLABS.) is used for pVIII fusion protein expression to construct a gene (SEQ ID NO: 102) having sequences in the following order:
(1) promoter sequence, (2) Shine-Dalgarno (SD) sequence, (3) M13-gVIII signal sequence-encoding DNA sequence, (4) gold-binding VH-encoding DNA sequence (SEQ ID NO: 80), (5) M13-gVIII, (6) several termination codons, and (7) transcription terminator.
In this Example, the promoter sequence used is a tac promoter. The obtained plasmid is designated as pM13-GpVIII. The gold-binding property of a phage obtained in the same way as in Example 2 by using the pM13-GpVIII is confirmed by the following method:
(A) 80 μL each of serially diluted solutions of VH-displaying phage is dispensed to a gold-deposited titer plate and gently stirred with a shaker for 1 hour;
(B) After the removal of the phage solutions, 90 μL of PBST is dispensed to each well and stirred for 10 minutes, followed by washing and discarding of the supernatant. This procedure is repeated three times;
(C) 75 μl of a solution of HRP-conjugated anti-M13 antibody/PBST (1/5000) is dispensed to each well and gently stirred with a shaker for 1 hour;
(D) The supernatant is discarded. Next, 90 μL of PBST is dispensed to each well and stirred for 10 minutes, followed by washing and discarding of the supernatant. This washing procedure is repeated three times;
(E) 30 μL each of detection reagents 1 and 2 (Amasham BIOSCIENCE) is dispensed to each well and reacted for 1 minute with gentle stirring; and
(F) The luminescence intensity of luminol is measured.
The same experiment except that M13KO7 (manufactured by NEW ENGLAND BIOLABS.) is used instead of pM13-GVIII is conducted as a comparative experiment. The phage obtained from the pM13-GVIII is confirmed to display gold-binding VH.
Example 5
Construction of Plasmid for Coexpression of pIII Fused with HEL-Binding scFv/pVIII Fused with Gold-Binding VH
The pM13-GpIII obtained in Example 4 and the pM13-SpVIII obtained in Example 2 are used to construct a plasmid for coexpression of gold-binding VH-fused pIII/SBA-15-fused pVIII. The pM1'-GpIII and the pM13-SpVIII are cleaved with restriction enzymes BspHI/BsmI (both manufactured by NEW ENGLAND BIOLABS.) according to the method of the technical bulletin recommended by the manufacturer.
The resulting enzyme reaction solution is subjected to agarose gel electrophoresis. A kbp fragment of the pM13-GpIII cleavage reaction solution and a kbp fragment of the pM13-SpVIII reaction solution are cleaved and purified with a purification kit (manufactured by Promega: trade name Wizard SV Gel and PCR Clean-Up System). Next, the DNA fragments thus obtained are ligated with T4-Ligase (manufactured by Roche) for 2 hours according to the method recommended by the manufacturer. The obtained ligation solution is transformed in the same way as in Example 2 to express and collect a phage.
The following method is used in the confirmation of the expression:
(1) 80 μL each of serially diluted solutions of VH-displaying phage is dispensed to a gold-deposited titer plate and gently stirred with a shaker for 1 hour;
(2) After the removal of the phage solutions, 90 μL of PBST is dispensed to each well and stirred for 10 minutes, followed by washing and discarding of the supernatant. This procedure is repeated three times;
(3) Next, 1000 μL of 1 μM HEL is added and gently stirred with a shaker for 1 hour;
(4) After centrifugation (15000 rpm, 10 min) and the removal of the supernatant solution, 1000 μL of PBST is dispensed again and stirred for 10 minutes, followed by washing and discarding of the supernatant. This procedure is repeated three times;
(5) 500 μl of a solution of HRP-conjugated anti-HEL antibody/PBST (1/5000) is added and gently stirred with a shaker for 1 hour;
(6) After centrifugation (15000 rpm, 10 min), the supernatant is discarded. Next, 1000 μL of PBST is dispensed and stirred for 10 minutes, followed by washing and discarding of the supernatant. This procedure is repeated three times;
(7) 35 μL each of detection reagents 1 and 2 (Amersham BIOSCIENCE) is dispensed to each well and reacted for 1 minute with gentle stirring; and
(8) The luminescence intensity of luminol is measured.
The experiments using the phages obtained in Examples 2 and 4 are performed as comparative experiments. The phage obtained in Example 3 is confirmed to have the highest luminescence intensity of luminol.
Example 6
Construction of DsbA-Fused Gold-Binding VH/HEL-Binding scFv Complex
A target-capturing molecule of the present invention is constructed by the following steps and evaluated:
(1) Construction of DNA encoding gold-binding VH/HEL-binding scFv
(1-1) DNA encoding gold-binding VH (SEQ ID NO: 80), a (GGGGS)3 linker sequence and HEL-binding scFv (SEQ ID NO: 97) is constructed.
(1-2) The DNA is used as a template to perform amplification by PCR using the following primers:










7s4-fw-Nco1:









(SEQ ID NO: 103)











ACATGCCATGGCCCAGGTGCAGTTGGTGGAGTCTG;




and



 



HEL-bk-Hind3:








(SEQ ID NO: 104)











AATGGCAAGCTTGGCCGTGATGATCAGCTTGGTA.







(1-3) The resulting PCR product is cleaved with NcoI/HindIII and inserted into pET-39b (manufactured Novagen). The cleavage with the restriction enzymes (NcoI and HindIII: manufactured by NEB) and the ligation reaction (T4-Ligase: manufactured by Roche) are performed according to the protocols recommended by the manufacturers.

(1-4) 5 μL of the ligation solution thus obtained is added to 50 μL of JM109 competent cells (manufactured by Promega) to perform transformation by heat shock.

(1-5) The cells are then developed onto a plate supplemented with LB/kanamycin (final concentration: 50 μg/mL) and left undisturbed at 37° C. Plasmids where the gene of interest is introduced are screened from the obtained colonies.

(2) Expression of Protein of Interest

(2-1) The plasmids obtained in the step (1) are transformed into BL21 competent cells (manufactured by Promega). This transformation is performed by heat shock in the same way as in the step (1). The cells are then developed onto a plate with LB/kanamycin (final concentration: 50 μg/mL) and left undisturbed overnight at 28° C.

(2-2) The colonies obtained on the plate are poked with a toothpick, then transferred to a solution of 3 mL of LB/kanamycin, and cultured overnight at 28° C.

(2-3) The whole amount of the obtained culture solution is added to 250 mL of 2×YT medium (16 g of triptone, 10 g of enzyme extract and 5 g/L sodium chloride)/kanamycin medium and further cultured for around 8 hours.

(2-4) IPTG is added thereto at a time point of OD600=0.8 to induce the expression of the protein of interest. The overnight culture thereof is performed at 22° C.

(3) Collection of Protein of Interest

(3-1) The culture solution obtained in the step (2) is centrifuged at 6000 rpm for 30 minutes to collect a supernatant. The weight of the supernatant is measured, and the supernatant is stored at 4° C.

(3-2) 60 wt % ammonium sulfate with respect to the weight of the supernatant is added in four portions with stirring, and this stirring is continued for 6 hours.

(3-3) The obtained suspension is centrifuged at 8000 rpm for 20 minutes. After the discarding of the supernatant, the pellet is immersed in 10 mL of Tris buffer (20 mM Tris/500 mM NaCl (pH 7.9 at 4° C.)) and left undisturbed overnight.

(3-4) The solution obtained in the step (3-3) is desalted by dialysis (MWCO: 14000) at 4° C. An external solution in the dialysis is a Tris buffer.

(3-5) The resulting solution is purified with a Ni chelate column. His-Bind (Novagen) is used as a carrier to perform column purification at 4° C. according to the method recommended by the manufacturer.

(3-6) Dialysis for imidazole elimination is performed. A tube and external solution in the dialysis are the same as in the step (3-4).

When the obtained protein solution is confirmed by SDS-PAGE, a single band of approximately 6.5 kDa can be confirmed. This band is confirmed as DsbA-VH (G)-scFv (H) in the same way as in Example 4. This protein is confirmed to be a gold- and HEL-binding protein.
Example 7
Construction of β-Glucosidase-Fused Gold-Binding VH/HEL-Binding scFv Complex
(1) Construction of Vector for expression of Gold-Binding VH/LacΔα-Fused Protein
(1-1) Cloning of LacΔα Fragment
The cloning of a LacΔα fragment can be performed with reference to the method for expression vector production described in Anal. Chem. (2002) 74, pp 2500-2504. The LacΔα is cloned from E. coli DH5α with the following primers:








LacΔα_fw:









(SEQ ID NO: 105)









5′-CCCGGATCCGCGGCCGCCATGACCATGTTACGGAATTCACTGG-3′



 


LacΔα_bk








(SEQ ID NO: 106)









5′-CCCCCCTCGAGTTATTTTTGACACCAGACCAACTGG-3′







(1-2) Insertion into pET-32

The obtained PCR fragment and a vector pET-32 (Novagen) are subjected to restriction enzyme reaction with NotI/XhoI according to the method recommended by the manufacturer. Gel purification is performed in the same way as above. The obtained PCR fragment and DNA fragment (approximately 5.9 kbp) are ligated with T4-Ligase (Roche).

(1-3) Insertion of Gold-Binding VH-Encoding DNA Fragment

The gold-binding VH is cleaved with NcoI/HindIII and inserted into the plasmid obtained in the step (1-2). The gold-binding VH-encoding DNA fragment is amplified by PCR in the same way as in Example 6. Primers used are described below. The obtained PCR fragment and plasmid are cleaved with restriction enzymes NcoI/HindIII and then ligated to construct a plasmid pET-GHΔα encoding trx-gold-binding VH-encoding DNA-LacΔα of interest.










7s4-fw-Nco1_2




ACATGCCATGGCAGGTGCAGTTGGTGGAGTCTG
(SEQ ID NO: 111)


 


HEL-bk-Hind3


AATGGCAAGCTTGGCCGTGATGATCAGCTTGGTA
(SEQ ID NO: 104)






(2) Construction of Vector for Expression of HEL-Binding scFv/LacΔω Fusion Protein

(2-1) Cloning of LacΔω fragment

The cloning of a LacΔω fragment can be performed with reference to the method for expression vector production described in Anal. Chem. (2002) 74, pp 2500-2504 in the same way as above. The LacAo is cloned from E. coli DH5α with the following primers:









LacΔω_fw









(SEQ ID NO: 107)









5′-CCCGGATCCGCGGCCGCCATGACCATGATTACGGATTCACTGG-3′



 


LacΔω_bk








(SEQ ID NO: 108)









5′-CCCCCCTCGAGTTACGGTGCACGGGTGAACTG-3′







(2-2) Insertion into pET-32

The obtained PCR fragment and a vector pET-32 (Novagen) are subjected to restriction enzyme reaction with NotI/XhoI according to the method recommended by the manufacturer. Gel purification is performed in the same way as above. The obtained PCR fragment and DNA fragment (approximately 5.9 kbp) are ligated with T4-Ligase (Roche).

(2-3) Insertion of HEL-Binding scFv-Encoding DNA Fragment

The DNA fragment coding HEL-binding scFv is cleaved with NcoI/HindIII and inserted into the plasmid obtained in the step (2-2). The DNA fragment coding HEL-binding scFv is amplified in the same way as in Example 6. The obtained PCR fragment and plasmid are cleaved with restriction enzymes NcoI/HindIII and then ligated to construct a plasmid pET-HFΔω encoding trx-HEL-binding scFv-encoding DNA-LacΔω of interest.











scFv(HEL)_fw









(SEQ ID NO: 109)











5′-ACATGCCATGGGATATCGTCCTGACCCAGA-3′




 



scFv(HEL)_bk








(SEQ ID NO: 110)











5′-AATGGCAAGCTTCGCGGAGACGGTGACGAGGGT-3′







(3) Fusion Protein Expression

(3-1) Preculture

The plasmids were respectively used to express the proteins of interest in E. coli Origami B (DE3) pLysS (Novagen). Transformation is performed according to the method recommended by the manufacturer. The strains are developed onto a 2×YT plate (2×YT has the same composition as above and is supplemented with 15 g/L agar and further with 50 μg/mL ampicillin, 15 μg/mL kanamycin and 12.5 μg/mL tetracycline as antibiotics). A single colony obtained on the plate is cultured overnight at 37° C. in 5 mL of 2×YT solution (its composition is the same as above except for agar).

(3-2) Main Culture

The obtained culture solution (5 mL) is added to 400 mL of 2×YT medium of the same composition as above. At a time point of OD600=0.8 or more at 37° C., IPTG is added at the final concentration of 10 μM.

The culture is continued in an atmosphere at 16° C. for 12 hours.

(3-3) Collection of Protein

a) Disruption of Bacterial Cells

The culture solution obtained in the step (3-2) is centrifuged at 6000 rpm for 30 minutes, and the supernatant is discarded. The pellet fraction, that is, cell fraction is suspended in 20 mL of phosphate buffer (2.7 mM KCl, 1.8 mM KHPO3, 10 mM Na2HPO3 and 140 mM NaCl). Subsequently, French press is performed under conditions at 4° C. The obtained bacterial cell homogenate solution is centrifuged at 15000 rpm for 10 minutes to obtain a cytoplasm fraction solution.

b) Purification of Fusion Protein

All procedures described below are performed under an atmosphere at 4° C. The gold-binding VH-LacΔα-containing cytoplasm fraction solution and HEL-binding scFv-LacΔω-containing cytoplasm fraction solution thus obtained are mixed. The obtained mixture solution is purified by affinity column chromatography using NP-Sepharose (biosearchtech). The purification method follows the method recommended by the manufacturer. The eluted fraction is dialyzed with a phosphate buffer as an external solution. The external solution is replaced three times at 6-hour intervals.

(4) Confirmation of Double Binding Property of Fusion Protein

The fusion protein obtained in the step (3-3) is examined for its gold- and HEL-binding property in the same way as in Example 6 and confirmed to show a double binding property.

ADVANTAGES OF THE INVENTION
Advantages obtained by the present invention will be described below.
A target substance-capturing body of the present invention is characterized by comprising: a soluble protein used as a base; and two or more functional domains binding to different target substances. Thereby, since the soluble protein serves as a scaffold (base), the feature of being easily suspended or solubilized in an aqueous solution can be imparted to this target substance-capturing body. Furthermore, since the target substance-capturing body is composed of the functional domains respectively binding to different target substances, one molecule can capture two or more different molecules.
The target substance-capturing body of the present invention can be prepared as a protein complex composed of fusion proteins comprising the two or more different functional domains respectively fused with different soluble proteins. Since the target substance-capturing body is composed of such fusion proteins, the solubility of the soluble protein can be utilized effectively.
A soluble protein having a signal peptide or capable of functional addition of a signal peptide is adopted. Thereby, it is possible to produce the target substance-capturing body through a membrane transport process for producing the soluble protein in host cells and secreting it out of the cytoplasm. As a result, post-translational modification necessary to maintain stable protein three-dimensional structures such as disulfide bond formation can be performed in a production step in host bacterial cells.
Moreover, the soluble protein is a protein consisting of two or more constituent units. Furthermore, the two or more functional domains binding to different target substances may respectively be fused with the different constituent units. Thereby, it is possible to ease expression conditions of the fusion protein of the functional domain. Moreover, since plural constituent units derived from an identical protein have self-association ability, they can function as one molecule after association and result in no inconvenience for the capturing molecule.
Moreover, the soluble protein is selected from among phage coat proteins. Thereby, it is possible to stably display the target substance-capturing body on the phage surface without losing the desired functions of the different functional domains. Furthermore, the target substance-capturing body of interest can be obtained by a simple production method.
A functional domain that targets a substrate is included in the two or more functional domains comprised in the target substance-capturing body of the constitution described above. Thereby, it is possible to immobilize this target substance-capturing body in a particular orientation on the substrate. As a result, reduction in capturing ability, which is a problem presented by an immobilization method for conventional target substance-capturing bodies by physical adsorption or chemical crosslinking, can be suppressed.
A device of the present invention comprises the target substance-capturing body immobilized on a substrate, wherein a portion of the surface of the substrate used is composed of gold, and a functional domain having gold-binding ability is held in the target substance-capturing body. Thereby, it is possible to detect the binding between the target substance-capturing body and a target substance to be detected by not only the measurement of the quantity of scattered light but also optical measurement applying enhanced Raman and localized plasmon principles and electrical measurement utilizing its electrical properties. Furthermore, a method for detecting a target substance to be detected by use of the device comprising the target substance-capturing body and the substrate according to the present invention allows for the labeling of a probe or a target substance after reaction without introducing the label thereinto before the reaction as performed previously. As a result, reduction in the functions of labeled substances bound with labeling substances, which is a problem presented by conventional labeling methods, can be prevented. For example, reduction in the binding property between a target substance to be detected contained in a sample and its capturing molecule during reaction between the device and the sample due to the labeling of the target substance before the reaction can be prevented.
Furthermore, according to the method of the present invention, the scope of label selection can be expanded. Accordingly, the optimum label for a target substance can be selected. Thus, the present invention can provide a detection method and detection means capable of performing the reaction of the target substance/device according to the present invention/labeling substance at the same time or at an arbitrary time point.
INDUSTRIAL APPLICABILITY
The present invention provides a target substance-capturing body characterized by comprising a soluble protein and two or more functional domains binding to different target substances. The present invention further provides a device characterized by comprising a substrate bonded with a target substance-capturing body comprising the soluble protein, a functional domain having a substrate-binding property, and one or more functional domains binding to target substances different from the substrate. The present invention makes it possible to provide a technique for producing binding molecules (e.g., antibodies) highly specific to target substances at high yields and immobilizing the molecules with their activities maintained, which has conventionally presented a challenge to the industrial use of a target substance-capturing body.
This application claims priority from Japanese Patent Application No. 2005-192084 filed Jun. 30, 2005, which is hereby incorporated by reference herein.

## Claims

1. A device for capturing a target substance comprising:
a substrate; and
a target substance-capturing body comprising (i) a base comprising a soluble protein and (ii) two or more functional domains respectively capable of binding to different target substances,
wherein the two or more functional domains comprise a functional domain bound to the substrate and a functional domain for capturing a target substance different from the substrate, and
wherein at least a portion of the substrate contains gold, and
wherein the functional domain bound to the substrate is bound to the portion containing the gold and contains the amino acid sequence coded by SEQ ID NO: 80.

2. A detection instrument for detecting a target substance to be detected contained in an analyte comprising:
a device according to claim 1; and
detection means for detecting the binding of a target substance to a functional domain for capturing a target substance to be detected comprised in the device.

3. A kit for detecting a target substance to be detected contained in an analyte comprising:
a device according to claim 1; and
detection means for detecting the binding of a target substance to a functional domain for capturing a target substance to be detected comprised in the device.

4. A method for detecting a target substance to be detected in an analyte characterized by comprising the steps of:
reacting a device according to claim 1 with a target substance to be detected; and
detecting the binding of the target substance to be detected to a functional domain of the device.

5. A detecting method according to claim 4, further comprising the step of preparing the device by reacting the target substance-capturing body with the substrate to bind the target substance-capturing body to the substrate.