JP2009085831A - Capture member and biosensor element - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a capture fixing carrier having a structure for absorbing and diffusing a solvent and capable of selectively and densely fixing only the reagent that will serve as a label for a target matter of detectable reagents. <P>SOLUTION: This capture fixing carrier for detecting the target matter in analyte liquid comprises a capture member for capturing the target matter and a carrier to which the capture member is fixed. The capture member at least contains a capturing molecule, having affinity with respect to the target matter and a carrier material affinity member having affinity with respect to the material constituting the carrier, and the carrier has a structure where the analyte liquid can diffuse into the carrier. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to a capturing member and a biosensor element used in a device for detecting a target substance in a sample liquid.

  A biosensor is a measurement device that utilizes the molecular recognition ability of a living body or a biomolecule. In vivo, examples of combinations of substances having affinity for each other include enzyme-substrate, antigen-antibody, DNA-DNA, and the like. The biosensor uses the principle that one of these combinations can be selectively measured on the other material by fixing or supporting it on a substrate.

  In recent years, biosensors are expected to have a wide range of applications not only in the medical field, but also in the environment and foodstuffs. To expand the range of use, biosensors are small, light, and highly sensitive. Sensors are desired, and as an example, immunochromatographic devices are widely used. FIG. 1 shows an example of an analysis method using an immunochromatography device. In the method shown in FIG. 1, the configuration of the device generally includes a sample inhalation part 8 for inhaling the sample liquid 11, introducing it into the test part, and uniformly infiltrating and diffusing in the test part. A reagent part (not shown) containing a reagent that reacts with the target substance 10 to be detected, a developing part for developing a complex of the target substance 10 and the reagent, a detection part for detecting a detectable substance, an excess sample liquid, an added washing liquid, It consists of each part such as an absorption part 9 that absorbs the developing liquid and prevents backflow. A carrier such as a membrane 7 is used as the development part and the detection part.

  First, a first capture molecule having an affinity for a specific region of a target substance (referred to as an epitope in the case of an antigen-antibody reaction) in the detection region on the surface of the carrier such as the membrane 7 (in the case of an antigen-antibody reaction) Called). On the other hand, the sample inhalation part or the development area is labeled with a detectable reagent (for example, gold colloid), and a second capture molecule having affinity for a specific area of the target substance is applied. As the first capture molecule and the second capture molecule, those having affinity for different regions of the target substance are usually used so as to bind simultaneously to the target substance. Next, the sample liquid is injected into the liquid injection region. When the target substance is contained in the sample liquid, the second capture molecule applied to the sample inhalation part or the development region reacts with the target substance to form a complex. The complex reaches the detection region by diffusing the development region, and is contacted with the first capture molecule immobilized on the carrier, thereby being labeled to the second capture molecule via the target substance. A detectable reagent can be immobilized on a carrier. Then, by measuring the concentration of the detectable reagent immobilized on such a carrier by some method, it is possible to calculate the number and concentration of target target substances.

  In such an immunochromatographic sensor, it is considered that suppressing bleeding of a reagent that becomes a detectable label contributes to improvement in detection sensitivity. Inventions relating to test pieces using a layered inorganic porous material (Patent Documents 1 and 2) are disclosed as techniques for suppressing bleeding of reagents. These are arranged on the carrier surface with a layered inorganic porous material having a high dye adsorbing power in the detection region, and the dye generated by the presence of the target substance is adsorbed on the layered inorganic porous material, thereby suppressing bleeding. We expect improvement.

In addition, when the capturer molecule is immobilized on the carrier, a chemical cross-link that immobilizes the capturer molecule on the carrier by reacting the carrier composed of a material having a functional group or a carrier introduced with a functional group with the capturer molecule. The method is commonly used.
JP 2001-033455 A International Publication No. 03/067258 Pamphlet

  However, since the fixation of the dye or the like in the above invention is nonspecific adsorption fixation, there is a concern that impurities contained in the sample liquid may be fixed, and there is a problem that the signal-noise ratio is deteriorated.

  In addition, when immobilizing a material to be immobilized on a support composed of a material having a functional group by a chemical cross-linking method, the immobilization range of the material to be immobilized is limited unless the region of the functional group to be activated is controlled. There is a problem of becoming larger.

  Accordingly, an object of the present invention is to provide a capturing body immobilization carrier capable of selectively densely immobilizing only a reagent serving as a target substance label among reagents that can be detected in a carrier having a structure that absorbs and diffuses a solvent. Is to provide.

  The capturing body fixing carrier for solving the above-mentioned problems is as follows.

The capturing body fixing carrier of the present invention is a capturing body fixing carrier for detecting a target substance in a sample liquid,
A capturing body member for capturing the target substance;
A carrier on which the capturing body member is fixed,
The capture member is at least
A capture molecule having affinity for the target substance;
A carrier material affinity member having an affinity for the material constituting the carrier,
The carrier has a structure capable of diffusing the sample liquid into the inside of the carrier.

  According to the present invention, the range of immobilization of the first capture molecule (immobilized in the detection region on the surface of the support) on the support in the sandwich method is limited, and diffusion and bleeding of the capture molecule on the support are suppressed. By suppressing, the end of the immobilization region is clarified, and an improvement in the signal-noise ratio can be expected.

《Capturing body member》
In the present invention, the capturing member is a member comprising at least a part of both a capturing molecule and a carrier material affinity member described later.

<< Capture molecule >>
In the present invention, the capture molecule is a substance related to the selective capture of the target substance in the sample liquid. For example, a substance that selectively reacts directly with the target substance in the sample liquid (for example, a receptor or an antibody). , A substance related to the reaction of the target substance (for example, a substance that selectively causes a catalytic action in the reaction of the target substance). The capture molecule may also have a function related to display of the presence or absence and degree of detection, for example, a function of reacting with a substance released by the receptor or a remaining substance to develop a color. Examples of capture molecules used in the present invention include, but are not limited to, enzymes, sugar chains, catalysts, antibodies, antibody fragments, antigens, nucleic acids, color reagents, and the like.

  It should be noted that the detection target of the present invention does not need to be a target substance with which a capture molecule directly reacts, and may be one that can be measured indirectly. For example, measurement can be performed by detecting a target substance that exists specifically in the detection target. Therefore, the detection target is not limited to the biological material, and the size thereof is not limited. However, the target substance may be a biological substance contained in a living organism such as a sugar, protein, amino acid, antibody, antigen or pseudoantigen, vitamin, or nucleic acid, or a related substance or artificially synthesized pseudobiological substance. desirable.

  In addition, the capture component can be used in combination, and as the capture molecule in the present invention, for example, a capture molecule such as a complex enzyme or an antibody-enzyme can be constituted.

《Support material affinity member》
In the present invention, the carrier material affinity member is a material that constitutes a part of the capture member as a portion having a function of fixing the capture member to the carrier, and recognizes the material constituting the carrier, There is no particular limitation as long as it is a substance that specifically adsorbs. As such a material, a protein substrate-binding domain or a material affinity peptide can be preferably used. For example, if the carrier is composed of cellulose fibers, cellulose binding domains can be used. Moreover, when a support | carrier is comprised with a silica gel, a silicon dioxide affinity peptide can be used. These peptides can be prepared by biotechnological techniques if the amino acid sequence is specified. As the cellulose affinity domain, for example, a cellulose binding domain that is a substrate binding domain of cellulase that is a cellulolytic enzyme can be used. Cellulose is roughly classified into soluble cellulose and insoluble cellulose. Insoluble cellulose has crystalline cellulose and non-crystalline cellulose. Therefore, it is known that cellulose-binding domains include domains that bind to crystalline cellulose and domains that bind to amorphous cellulose. It has been. In the present invention, when a cellulose-binding domain is used as the carrier material affinity member, a crystalline cellulose-binding domain is used when the cellulose fiber is rich in crystalline cellulose, and an amorphous cellulose is used when the cellulose fiber is rich in amorphous cellulose. It is preferred to select a binding domain. The cellulose-binding domain used in the present invention is preferably one that does not hydrolyze cellulose serving as a carrier in the cellulose-binding domain itself. On the other hand, non-patent literature (Mehmet Sarikaya, Candan Tamerler, Alex K.-Y. Jin, Klaus Schulten, Francois Baneyx: Molecular biomimetics: nanotechnology through biology. Nature materials 2: 577-585, 2003) It is possible to use the silicon dioxide affinity peptide reported in (1). As a method for selecting a material affinity peptide such as the silicon dioxide affinity peptide, a surface display method for displaying a material affinity peptide on the surface of a cell such as a virus outer shell protein represented by M13 phage, Escherichia coli or yeast. Is known to be available.

  The cellulose-binding domain or the silicon dioxide-affinity peptide, which is a carrier material affinity member in the present invention, has a plurality of repeating structures as long as the capture molecule and its function are not impaired in the capture member in the present invention. It can also be. In addition, the capture molecule and the carrier material affinity member, or the repeated structure of the carrier material affinity member can be directly connected, and the capture molecule and the carrier material affinity member can be freely connected to each other. May be linked by a linker sequence to give The linker sequence is not particularly limited as long as it does not inhibit the functional expression of the capture molecule and the carrier material affinity member. For example, a commonly used linker such as GGGGS composed of glycine (G) and serine (S) An array can be used.

"Affinity"
In the present invention, having affinity means being capable of binding specifically to a certain substance. For example, a cellulose binding domain (CBD) having affinity for cellulose means that CBD has the ability to specifically bind to cellulose. The specific binding force refers to a binding force having an equilibrium dissociation constant (KD) of 1 × e ⁻⁶ M or less.

  The formula for calculating the equilibrium dissociation constant is described below using CBD and cellulose as examples.

1 / [P · C] = KD / [P · C] max × 1 / [P] + 1 / [P · C] max
Free CBD concentration: [P]
Concentration of the region where CBD on cellulose is bound: [C]
Concentration of CBD forming a composite with cellulose: [PC]
Maximum concentration of CBD capable of forming a complex with cellulose: [P · C] max
KD can be calculated by plotting 1 / [P · C] with 1 / [P] in the above equation.

《Configuration of capture body member》
As the constitution of the capturing member in the present invention, there are a case where the capturing molecule and the carrier material affinity member are fused monomers, and a case where they are independent multimers.

  In the case of a monomer, a configuration as a fusion protein in which a capture molecule and a carrier material affinity member are genetically linked can be considered. Specifically, these fusion polypeptides can be obtained using a single chain Fv in which a VH domain, which is an antigen capture domain of an antibody, and a VL domain are linked by a linker as a capture molecule, and using a cellulose-binding domain as a carrier material affinity member. And can be used as a capturing member fixed to a cellulose carrier.

  On the other hand, when the capturing member is used as a multimer, the capturing member is a molecule whose gene sequence is unknown and it is difficult to make the capturing molecule and the carrier material affinity member as one polypeptide chain as described above. It is effective in the case. As a method of constructing such a capture member as a multimer, the capture molecule and the carrier material affinity member can be made into a multimer through a combination of two substances having binding ability. Examples of the combination of the two substances having the binding ability include a combination such as biotin and avidin (streptavidin). When an antibody of unknown sequence is used as the capture molecule, biotin--is obtained by using a fusion protein in which biotin is labeled on the antibody and streptavidin is genetically linked to a cellulose binding domain used as a carrier material affinity member. An antibody of unknown sequence and a cellulose-binding domain can be configured as a multimeric capturing body member via streptavidin.

《Capturer fixed carrier》
The capturing body fixing carrier in the present invention comprises at least a capturing body member and a carrier. The capturing body member is fixed on the carrier. As described above, the capturing body member is a member including at least a part of both the capturing molecule and the carrier material affinity member, and the above-described capturing molecule and carrier material affinity member may be used. it can. As described below, a material having a structure capable of absorbing and diffusing the specimen liquid is used for the carrier of the capturing body fixing carrier. Since such a carrier functions as a stationary phase of the chromatography method, a capture body-immobilized carrier in which a capture body member capable of capturing a target substance is immobilized on this carrier material is suitable as a biosensor element used in immunochromatography. Can be used. The capturing body fixing carrier is provided in a state where the capturing body member is fixed to the carrier. However, the capturing body member and the carrier can be separately provided and provided as a test kit. The test kit is a test kit that includes at least a capturing body member and a carrier, and the capturing body member and the carrier are individually separated.

<Carrier>
In the present invention, the carrier is a solid phase that serves as a scaffold for immobilizing the capture molecule, and may have a structure capable of diffusing the sample liquid into the inside of the carrier. And the like having fine gaps. Examples of such a structure include a porous structure, a fibrous structure aggregate, and a fine powder aggregate. A wide range of materials can be applied to pore walls made of porous materials, and metal-oxides such as silica, titanium oxide, and alumina, and recently, organic-inorganic with organic groups introduced into the pore walls. Synthesis of hybrid porous materials has also been reported. Further, as the aggregate of fibrous structures, not only aggregates of inorganic materials such as glass fibers but also aggregates of organic materials such as cellulose fibers can be used. As the aggregate of organic fibers, typically, one having a woven fabric structure can be used. Further, it may be a piece of paper such as filter paper.

  In the present invention, the material constituting the porous structure, the fibrous structure aggregate, and the fine powder aggregate is not limited, but in combination with the capture body member used in the present invention, the capture body member A material that is recognized by the contained carrier affinity member and has the binding ability of the carrier affinity member is used.

  The carrier in the present invention has fine gaps on the surface and inside, and diffusion of the sample liquid proceeds on the carrier surface, so that it can be suitably used as a stationary phase for chromatography. Further, the shape of the carrier is preferably a flat plate shape such as a substrate or a film shape, but is not limited thereto.

<Immobilization of capture member on carrier>
As a capturing body immobilization carrier of the present invention, as a method for immobilizing a capturing body member on and inside the carrier, immobilization utilizing molecular recognition by a carrier affinity material can be used. When the solution containing the capturing body member is applied to the carrier by immobilizing the capturing body member to the carrier using the molecular recognition, the solute is dissolved when the solvent of the capturing body member solution diffuses inside the carrier. The capturing body member can be immobilized on the carrier material in the vicinity of the coated region rather than the range where the solvent diffuses, by the action of the carrier material-affinitive member and the carrier material of the capturing body member. As a result, in the area where the capturing body member-containing solution is applied to the carrier, bleeding due to solvent diffusion of the solution may occur, but the immobilization area of the capturing body member, which is a solute, is narrower than the solvent diffusion area. It can be expected to be tightly fixed in the region where the formation is desired (FIG. 2). By suppressing the diffusion of the capture molecules from the application region, it can be expected that the end of the capture member member immobilization region becomes clearer. Further, when a plurality of capture body members are immobilized on the same test piece in order to detect a plurality of target substances, contamination with adjacent capture body members may occur due to elution and diffusion of the capture body members. However, by using the capturing body member of the present invention, since the bleeding and elution of the capturing body member on the carrier can be suppressed, a plurality of capturing body members can be more closely fixed to the carrier (FIG. 3). Throughput can be expected.

  By drying the carrier to which the capturing body member is fixed as described above, it is possible to promote stronger adsorption of the capturing body member to the carrier material. Further, by applying a chemical cross-linking agent to the immobilization region of the capturing body member, the capturing body member, the carrier material, and the chemical cross-linking agent are allowed to react with each other in a covalent bond, and the capturing body member is eluted from the carrier material. It is also possible to suppress diffusion. Furthermore, the capturing body fixing carrier can be treated with a blocking agent. The blocking treatment can be expected to suppress the non-specific adsorption of impurities contained in the sample liquid in the detection region. Further, it can be expected that the target substance contained in the sample liquid is prevented from nonspecifically adsorbing to the capturing body-immobilized carrier outside the detection region by the blocking treatment.

<Combination of carrier and capture member>
≪Cellulose fibrous carrier and capture body member≫
When cellulose fibers are used as the carrier material used in the present invention, a cellulose-binding domain can be used as the carrier material affinity member contained in the capturing member. The cellulose binding domain is a substrate binding site possessed by the enzyme cellulase. The enzyme cellulase is isolated from various cells such as microorganisms, animals, and plants, but the origin of the enzyme cellulase is not particularly limited as the cellulose-binding domain used in the present invention. It is sufficient if it has affinity, and those having high affinity are preferable. The cellulose-binding domain can be a sequence isolated from natural cellulase, or a mutant cellulose-binding domain whose affinity for cellulose is improved by genetic recombination.

  The structure of the capturing body member having the cellulose binding domain is not particularly limited as long as it does not inhibit the functional expression of the capturing molecule and the cellulose binding domain. The capture molecule and the cellulose binding domain can be made into one capture member by fusing the amino terminus or / and carboxyl terminus of the cellulose binding domain directly to the carboxyl terminus or / and amino terminus of the capture molecule. . Although it is possible to fuse both directly as described above, the capture molecule and the cellulose-binding domain express their functions by forming independent tertiary structures, so that they function without interfering with each other. In order to facilitate this, both can be fused with a linker. The sequence of the linker is not particularly limited, and for example, a sequence such as GGGGS can be used. Further, in order to strengthen the fixation of the capturing body member to the carrier, the number of cellulose binding domains contained in the capturing body member may be plural. The increase in the number of cellulose binding domains increases the probability that the cellulose binding domain contacts the cellulose fibers when the capturing body member comes into contact with the cellulose fibers as the carrier material, and the immobilization rate to the cellulose carrier is increased. In addition, it can be expected to be more firmly fixed by improving the affinity. As a result, it is possible to suppress the capture body member from diffusing from the region to be fixed on the carrier material and bleeding of the fixed end.

≪Glass fiber carrier and capture body member≫
When glass fiber is used as the carrier material used in the present invention, a silicon dioxide-affinity peptide can be used as the carrier material affinity member contained in the capturing member. Material affinity peptides such as silicon dioxide affinity peptides can be obtained by methods known in the art such as the phage display.

  The structure of the capturing body member having the silicon dioxide affinity peptide is not particularly limited as long as it does not inhibit the function expression of the capturing molecule and the silicon dioxide affinity peptide. The capture molecule and the silicon dioxide affinity peptide are combined into a single capture member by fusing the amino terminus or / and carboxyl terminus of the cellulose binding domain directly to the carboxyl terminus or / and amino terminus of the capture molecule. Can do. Although it is possible to fuse both directly as described above, since the capture molecule expresses its function by forming a unique tertiary structure, the capture molecule and the silicon dioxide affinity peptide interfere with each other. In order to facilitate functional expression, both can be fused with a linker. The sequence of the linker is not particularly limited, and for example, a sequence such as GGGGS can be used. Further, in order to strengthen the fixation of the capturing body member to the carrier, the number of silicon dioxide affinity peptides contained in the capturing body member may be plural. By increasing the number of silicon dioxide-affinity peptides, the probability that the silicon dioxide-affinity peptide will contact the glass fibers increases when the capturing member contacts the glass fibers that are the carrier material. Since the immobilization speed is increased and the affinity is improved, it can be expected that the immobilization is more firmly fixed. Therefore, the capture body member diffuses from the region where the capture body member is immobilized on the carrier material and is fixed. It is possible to suppress bleeding of the chemical edge.

<Sample detection method>
The capture body fixing carrier used in the present invention suppresses the capture body member from diffusing inside the capture body fixing carrier when the capture body member is immobilized on the carrier. Even when the solution containing the solution is dripped (spotted) onto the carrier preferentially and the moisture in the sample liquid infiltrates and diffuses inside the carrier, the capturing body member is immobilized near the dripped region. The

《Target substance detection method》
As a method for detecting a target substance using the capturer-immobilized carrier in the present invention, for example, a sandwich type detection method using immunochromatography can be used. A labeled antibody is prepared by applying a detectable label to an antibody that recognizes a target substance in a sample liquid, and the labeled antibody is mixed in the sample liquid, whereby the target substance and the label in the sample liquid are mixed. To form a complex of conjugated antibodies. By bringing the complex into contact with the capture body-immobilized carrier in the present invention, the capture molecule having affinity for the target substance immobilized in advance on the capture body-immobilized carrier reacts with the complex via the target substance. To do. As a result, a detectable label is immobilized at the detection site on the capturing body immobilizing carrier of the present invention, and the amount of the target substance in the sample liquid can be indirectly measured by detecting this label. When immobilizing a capturing body member having a capturing molecule in a specific region (hereinafter referred to as capturing body fixing region) serving as a detection site on the carrier, the solution containing the complex is brought into contact with a region other than the capturing body fixing region. Then, the complex reaches the capturing body fixing region by infiltrating and diffusing inside the carrier. As the detectable label, colloidal gold, latex particles, fluorescent substances, luminescent substances, and the like can be used, and detection thereof can be performed by visual confirmation, concentration detection, light reflection or transmission.

  Hereinafter, the present invention will be described in more detail with reference to examples. However, the present invention is not limited to these examples, and biomaterials having similar functions and effects such as materials, composition conditions, reaction conditions, and the like. It can be changed freely as long as the sensor is available.

Example 1
The present example is an example of immobilizing a fusion polypeptide of a green fluorescent protein (Green Fluorescens Protein: GFP) and a cellulose affinity domain on a cellulose carrier.

Preparation of fusion polypeptide of GFP and cellulose affinity domain (1-1) Production process of fusion polypeptide GFP described in sequence 1 (gene sequence described in sequence 2) and cellulose affinity domain described in sequence 3 ( PGEX-6P is a gene expression vector for expressing a gene encoding a fusion polypeptide in which a gene sequence is described in sequence 4) via a linker (amino acid sequence and gene sequence are described in sequences 5 and 6, respectively). -1 (Amersham Biosciences) vector.

  The gene (sequence 8) encoding the polypeptide (sequence 7) is obtained by overlap PCR which is often performed when obtaining a gene fragment. BamHI is added to the 5 ′ end of the obtained amplified gene fragment, and an EcoRI restriction enzyme site is added to the 3 ′ end, and the amplified gene fragment is introduced so as to match the frame of the pGEX-6P-1 vector. Thereafter, the sequence is confirmed with a DNA sequencer.

E. coli Escherichia coli BL21 is transformed with the gene expression vector constructed as described above. After incubation for 16 hours, single colonies were picked from the culture plate and 3 ml of 2 × YT medium (tryptone; 16 wt%, yeast extract; 10 wt%, sodium chloride; 5 wt%) (final concentration of 100 μg / ml ampicillin). And incubate at 37 ° C. with shaking (pre-culture). After 12 hours, 3 ml of the culture is added to 250 ml of 2 × YT medium (containing ampicillin at a final concentration of 100 μg / ml) and cultured with shaking at 28 ° C. When the absorbance OD ₆₀₀ of the culture solution reaches 0.8, IPTG (isopropyl-β-D-galactopyranoside) is added to the culture solution to a final concentration of 1 mM to induce polypeptide production, Incubate for 12 hours.

(1-2) Fusion polypeptide purification step IPTG-derived E. coli was collected (8,000 × g, 2 minutes, 4 ° C.), and 1/10 amount of 4 ° C. phosphate buffered saline ((PBS)) NaCl; 8 g, Na ₂ HPO ₄ ; 1.44 g, KH ₂ PO ₄ ; 0.24 g, KCl; 0.2 g, purified water; 1000 ml). The cells are disrupted by freezing and thawing and sonication, and centrifuged (8,000 × g, 10 minutes, 4 ° C.) to remove solid impurities. The induced and expressed GST fusion polypeptide is purified by glutathione sepharose 4B (manufactured by Amersham Biosciences) by a method recommended by the manufacturer. The glutathione sepharose to be used is subjected to a treatment for suppressing nonspecific adsorption in advance. That is, glutathione-sepharose was washed three times with the same amount of PBS (8,000 × g, 1 minute, 4 ° C.), then added with the same amount of 4% bovine serum albumin-containing PBS, and treated at 4 ° C. for 1 hour. . After treatment, wash twice with the same volume of PBS and resuspend in 1/2 volume of PBS. 40 μl of pretreated glutathione sepharose is added to 1 ml of cell-free extract and gently stirred at 4 ° C. The GST fusion polypeptide is adsorbed to glutathione sepharose by the above procedure. After adsorption, the glutathione sepharose is collected by centrifugation (8,000 × g, 1 minute, 4 ° C.) and washed 3 times with 400 μl of PBS. Thereafter, 40 μl of 10 mM glutathione is added and stirred at 4 ° C. for 1 hour to elute the adsorbed fusion polypeptide. The supernatant is collected by centrifugation (8,000 × g, 2 minutes, 4 ° C.), then dialyzed against PBS to remove glutathione, and the GST fusion polypeptide is purified. The band of the GST fusion polypeptide is confirmed by SDS-PAGE.

  The GST fusion polypeptide was digested with PreScission protease (Amersham Pharmacia Biotech Co., Ltd., 5 U) according to the method recommended by the manufacturer, passed through glutathione sepharose to remove the protease and GST, and then SDS- The 32 kDa band is confirmed by PAGE.

(1-3) Immobilization of fusion polypeptide The polypeptide obtained as described above is spotted on a cellulose membrane, and the diffusion region of the solution is confirmed. By irradiating an excitation light of 460 nm to the cellulose membrane on which the polypeptide is immobilized, fluorescence of 508 nm is observed. It can be visually confirmed that the region where the fluorescence is observed is narrower than the diffusion diameter of the solution generated when the polypeptide is immobilized on the cellulose membrane.

(1-4) Preparation of GFP The same procedure is performed except that the fusion polypeptide produced and purified in (1-1) and (1-2) is changed to a single GFP (sequence 1) to obtain GFP. . The gene sequence of GFP is shown in Sequence 2.

(1-5) Control experiment: Immobilization of GFP Single GFP obtained as described above is spotted on a cellulose membrane, and a diffusion region of the solution is confirmed. By irradiating an excitation light of 460 nm to the cellulose membrane on which the polypeptide is immobilized, fluorescence of 508 nm is observed. It can be confirmed that the region where the fluorescence is observed is equivalent to the diffusion diameter of the solution generated when the polypeptide is immobilized on the cellulose membrane. From the results of (1-3) and (1-5), in the case of GFP alone, compared with the case where the fusion polypeptide with the cellulose-binding domain is spotted on the cellulose membrane, the region emitting green fluorescence is large and blurring. It can be confirmed that diffusion occurs.

  From the above results, by using a fusion polypeptide with a cellulose affinity domain, it is possible to provide a molecule that can suppress diffusion and bleeding of the polypeptide on the cellulose carrier and can limit the immobilization region.

(Example 2)
This example is an example of immobilizing a fusion polypeptide of green fluorescent protein (GFP) and a silicon dioxide affinity peptide on a glass filter.

Preparation of fusion polypeptide of GFP and silicon dioxide affinity peptide (2-1) Production process of fusion polypeptide GFP described in sequence 1 (gene sequence described in sequence 2) and affinity for silicon dioxide described in sequence 9 PGEX is a gene expression vector for expressing a gene encoding a fusion polypeptide in which a peptide (gene sequence is described in sequence 10) is fused via a linker (amino acid sequence and gene sequence are described in sequence 5 and sequence 6, respectively). -6P-1 (Amersham Biosciences) vector is used for construction.

  The gene (sequence 12) encoding the polypeptide (sequence 11) is obtained by overlap PCR which is often performed when obtaining a gene fragment. BamHI is added to the 5 ′ end of the obtained amplified gene fragment, and an EcoRI restriction enzyme site is added to the 3 ′ end, and the amplified gene fragment is introduced so as to match the frame of the pGEX-6P-1 vector. Thereafter, the sequence is confirmed with a DNA sequencer.

E. coli Escherichia coli BL21 is transformed with the gene expression vector constructed as described above. After incubation for 16 hours, single colonies were picked from the culture plate and 3 ml of 2 × YT medium (tryptone; 16 wt%, yeast extract; 10 wt%, sodium chloride; 5 wt%) (final concentration of 100 μg / ml ampicillin). And incubate at 37 ° C. with shaking (pre-culture). After 12 hours, 3 ml of the culture is added to 250 ml of 2 × YT medium (containing ampicillin at a final concentration of 100 μg / ml) and cultured with shaking at 28 ° C. When the absorbance OD ₆₀₀ of the culture solution reaches 0.8, IPTG (isopropyl-β-D-galactopyranoside) is added to the culture solution to a final concentration of 1 mM to induce polypeptide production, Incubate for 12 hours.

(2-2) Fusion polypeptide purification step IPTG-derived E. coli was collected (8,000 × g, 2 minutes, 4 ° C.), and 1/10 amount of 4 ° C. phosphate buffered saline ((PBS)) NaCl; 8 g, Na ₂ HPO ₄ ; 1.44 g, KH ₂ PO ₄ ; 0.24 g, KCl; 0.2 g, purified water; 1000 ml). The cells are disrupted by freezing and thawing and sonication, and centrifuged (8,000 × g, 10 minutes, 4 ° C.) to remove solid impurities. The induced and expressed GST fusion polypeptide is purified by glutathione sepharose 4B (manufactured by Amersham Biosciences) by a method recommended by the manufacturer. The glutathione sepharose to be used is subjected to a treatment for suppressing nonspecific adsorption in advance. That is, glutathione-sepharose was washed three times with the same amount of PBS (8,000 × g, 1 minute, 4 ° C.), then added with the same amount of 4% bovine serum albumin-containing PBS, and treated at 4 ° C. for 1 hour. . After treatment, wash twice with the same volume of PBS and resuspend in 1/2 volume of PBS. 40 μl of pretreated glutathione sepharose is added to 1 ml of cell-free extract and gently stirred at 4 ° C. The GST fusion polypeptide is adsorbed to glutathione sepharose by the above procedure. After adsorption, the glutathione sepharose is collected by centrifugation (8,000 × g, 1 minute, 4 ° C.) and washed 3 times with 400 μl of PBS. Thereafter, 40 μl of 10 mM glutathione is added and stirred at 4 ° C. for 1 hour to elute the adsorbed fusion polypeptide. The supernatant is collected by centrifugation (8,000 × g, 2 minutes, 4 ° C.), then dialyzed against PBS to remove glutathione, and the GST fusion polypeptide is purified. The band of the GST fusion polypeptide is confirmed by SDS-PAGE.

  The GST fusion polypeptide was digested with PreScission protease (Amersham Pharmacia Biotech Co., Ltd., 5 U) according to the method recommended by the manufacturer, passed through glutathione sepharose to remove the protease and GST, and then SDS- A 42 kDa band is confirmed by PAGE.

(2-3) Immobilization of fusion polypeptide The polypeptide obtained as described above is spotted on a cellulose membrane, and the diffusion region of the solution is confirmed. By irradiating the glass filter on which the polypeptide is immobilized with excitation light of 460 nm, fluorescence of 508 nm is observed. It can be visually confirmed that the region where the fluorescence is observed is narrower than the diffusion diameter of the solution generated when the polypeptide is immobilized on the glass filter.

(2-4) Preparation of GFP The same operation is performed except that the fusion polypeptide produced and purified in (2-1) and (2-2) is changed to a single GFP to obtain GFP.

(2-5) Control Experiment: Immobilization of GFP Single GFP obtained as described above is spotted on a glass filter, and the diffusion region of the solution is confirmed. By irradiating the glass filter on which the polypeptide is immobilized with excitation light of 460 nm, fluorescence of 508 nm is observed. It can be confirmed that the region where the fluorescence is observed is equivalent to the diffusion diameter of the solution generated when the polypeptide is immobilized on the glass filter. From the results of (2-3) and (2-5), in the case of GFP alone, a region emitting green fluorescence is larger than when the fusion polypeptide with the silicon dioxide affinity peptide is spotted on a glass filter. It can be confirmed that bleeding and diffusion occur.

  From the above results, by using a silicon dioxide affinity peptide and a fusion polypeptide, it is possible to provide a molecule that can suppress diffusion and bleeding of the polypeptide on the glass filter carrier and can limit the immobilization region.

Example 3
Detection of HEL (Hen Egg Lysozyme) by immunochromatography using a carrier on which a capture molecule of the present invention is immobilized (3-1) Anti-HEL antibody fragment (HyHEL10) (single chain Fv: scFv) and cellulose affinity domain Example 1 (1), (1) except that the GFP of Example 1 was changed to the anti-HEL antibody fragment HyHEL10 (amino acid sequence and gene sequence are shown in Sequence 13 and Sequence 14, respectively). The same operation as 2) is performed to obtain a fusion polypeptide. The amino acid sequence of the fusion polypeptide is shown in Sequence 15, and the gene sequence of the fusion polypeptide is shown in Sequence 16.

(3-2) Preparation of fusion polypeptide of streptavidin and cellulose affinity domain Example except that GFP of Example 1 is changed to streptavidin (amino acid sequence and gene sequence are shown in sequence 25 and sequence 26, respectively) The same operations as in (1-1) and (1-2) are performed to obtain a fusion polypeptide. The amino acid sequence of the fusion polypeptide is shown in Sequence 27, and the gene sequence of the fusion polypeptide is shown in Sequence 28.

(3-3) Preparation of test piece The fusion polypeptide obtained in (3-1) is prepared to a concentration of 1 mg / ml. The fusion polypeptide solution is line-coated on Cellulose Fiber Sample Pad (CFSP: manufactured by Millipore) using BioDot XYZ3050 and BioJetQianti 3000 dispenser manufactured by Nipple Technocluster. A biotinylated anti-IgG (Fc) antibody (manufactured by RCK, Rabbit, Goat-Poly) is mixed with the streptavidin-cellulose affinity domain fusion polypeptide solution prepared in (3-2), and biotin-streptavidin binding To form a multimeric capture member comprising an anti-IgG (Fc) antibody and a cellulose affinity domain. This captured body member is used as a member for a control line. The control line capturing body member is applied to the rear of the test line in the development direction so as to be a control line. After drying at room temperature for 12 hours, one end of the membrane is immersed in blocking buffer (0.5% casein, 50 mM boric acid (pH 8.5)), and after the blocking buffer is sucked up to the upper end, the entire membrane is immersed in the blocking buffer. Let stand for 30 minutes. The membrane is immersed in a washing / stabilizing buffer (0.5% sucrose, 0.05% sodium cholate, 50 mM Tris-HCl (pH 7.5)), and gently shaken for 30 minutes. The membrane is placed on a paper towel and dried overnight at room temperature to prepare a sample development / detection pad.

  An anti-chicken egg white lysozyme antibody (manufactured by RCK, Rabbit-Poly, HRP labeled) used as a labeled antibody is prepared to a concentration of 1 mg / ml, and 1 ml is collected. Add 2 ml of coating buffer (20 mM Tris-HCl (pH 8.2), 0.05% PEG 20000, 5% sucrose) to this antibody solution, and gently stir. The above-mentioned mixed solution is evenly applied to Glass Fiber Conjugate Pad (GFCP: manufactured by Millipore). The conjugate pad is prepared by drying under reduced pressure all day and night in a desiccator.

  The test line prepared above and the sample development / detection pad applied with the control line are attached to a backing sheet (manufactured by Adhesives Research) with the control line side facing up. Adhere the water absorption pad (Millipore) to the upper edge of the backing sheet. At this time, the attached sample development / detection pad and the water absorption pad are placed so as to overlap each other by about 5 mm. The conjugate pad to which the HRP-labeled anti-chicken egg white lysozyme antibody has been applied is placed on the bottom side of the backing sheet so as to overlap the sample development / detection pad by about 2 mm. A sample pad (GFCP: manufactured by Millipore) is attached to the bottom of the backing sheet so as to overlap with the conjugate pad by about 4 mm. The sheet prepared as described above is cut to a width of 5 mm with a cutting module CM4000 (manufactured by Nipple Techno Cluster) and set in a housing case.

(3-4) Detection of chicken egg white lysozyme Dissolve chicken egg white lysozyme (HEL) in a sample buffer (20 mM Tris-HCl (pH 8.0)) to a concentration of 10 μg / ml. Apply to the test piece prepared in 3-3) and develop the sample solution for 15 minutes.HRP label captured on the test line via HEL on the test piece using ECL Western Blotting Detection Reagent (manufactured by GE Healthcare) In the biosensor element of this embodiment, since the bleeding and diffusion of the capturing body member are suppressed, the end of the test line becomes clear, and the target substance can be detected with high sensitivity. By setting the concentration to 1/10 and 1/100, the emission intensity is also reduced to the HEL concentration. It can be confirmed that there is a correlation.

Example 4
A test sample having the same configuration as in Example 3 except that the cellulose-binding domain of Example 3 is changed to a peptide having affinity for silicon dioxide and the sample development / detection pad is changed to Glass Fiber Conjugate Pad. Develop and evaluate as a liquid. The amino acid sequence and gene sequence of the obtained fusion polypeptide are shown in sequences 17 and 18, respectively. In the biosensor element of the present embodiment, the capturing member is prevented from bleeding and diffusing, so that the end of the test line becomes clear and the target substance can be detected with high sensitivity. By setting the concentration of hen egg white lysozyme to 1/10 or 1/100, it can be confirmed that the emission intensity also correlates with the HEL concentration.

(Example 5) Detection of a plurality of antigens Fusion polypeptide 1 (amino acid sequence and gene sequence are shown in sequences 15 and 16 respectively) fused with an anti-HEL antibody fragment (HyHEL10 scFv) and a cellulose-binding domain, Bovine serum albumin (BSA) antibody scFv (amino acid sequence, gene sequence is shown in sequences 19 and 20, respectively) and a cellulose-binding domain fused polypeptide 2 (amino acid sequence and gene sequence are shown in sequences 21 and 22, respectively) ) And prepare. Fusion polypeptide 2 is the same as (1-1) and (1-2) of Example 1 except that (GFP) of (1-1) and (1-2) of Example 1 is changed to anti-BSA antibody scFv. Create. The fusion polypeptide 1 and fusion polypeptide 2 prepared as described above are immobilized in different regions on the same pad of the sample development / detection pad in the same manner as in (3-2) of Example 3. . First, the fusion polypeptide 1 is immobilized, and the fusion polypeptide 2 is immobilized behind the development direction. Further, an anti-IgG (Fc) antibody (manufactured by RCK) Rabbit, Goat-Poly) serving as a control line is applied on the rear side in the development direction, and fixed by physical adsorption. After drying at room temperature for 12 hours, one end of the membrane is immersed in blocking buffer (0.5% casein, 50 mM boric acid (pH 8.5)), and after the blocking buffer is sucked up to the upper end, the entire membrane is immersed in the blocking buffer. Let stand for 30 minutes. The membrane is immersed in a washing / stabilizing buffer (0.5% sucrose, 0.05% sodium cholate, 50 mM Tris-HCl (pH 7.5)), and gently shaken for 30 minutes. The membrane is placed on a paper towel and dried overnight at room temperature to prepare a sample development / detection pad.

  Anti-chicken egg white lysozyme antibody (RCK, Rabbit-Poly, HRP label) and anti-bovine serum albumin antibody (RCK, Rabbit-Poly, FITC label) used as labeling antibodies are prepared at a concentration of 1 mg / ml and 1 ml. Sort out. Add 2 ml of coating buffer (20 mM Tris-HCl (pH 8.2), 0.05% PEG 20000, 5% sucrose) to this antibody solution, and gently stir. The above-mentioned mixed solution is evenly applied to Glass Fiber Conjugate Pad (GFCP: manufactured by Millipore). The conjugate pad is prepared by drying under reduced pressure all day and night in a desiccator.

  The test line prepared above and the sample development / detection pad applied with the control line are attached to a backing sheet (manufactured by Adhesives Research) with the control line side facing up. Adhere the water absorption pad (Millipore) to the upper edge of the backing sheet. At this time, the attached sample development / detection pad and the water absorption pad are placed so as to overlap each other by about 5 mm. The conjugate pad coated with the above HRP-labeled anti-chicken egg white lysozyme antibody is placed on the bottom side of the backing sheet so as to overlap with the sample pad by about 2 mm. A sample pad (GFCP: manufactured by Millipore) is attached to the bottom of the backing sheet so as to overlap with the conjugate pad by about 4 mm. The sheet prepared as described above is cut to a width of 5 mm with a cutting module CM4000 (manufactured by Nipple Techno Cluster) and set in a housing case.

  Development is performed in the same manner as in (3-4) of Example 3 except that a mixed solution of HEL and BSA is used instead of the sample solution used in (3-4) of Example 3. The detection of HEL is performed in the same manner as (3-4) of Example 3, and the detection of BSA detects the fluorescence of FITC. By setting the HEL and BSA concentrations to 1/10 and 1/100, it can be confirmed that the emission intensity and the fluorescence intensity are also correlated with the HEL and BSA concentrations. In the biosensor element of the present embodiment, the capturing body member can be prevented from bleeding and diffusing, so that the end of the test line becomes clear and contamination with the adjacent capturing body member can be suppressed. Since it becomes possible to immobilize the capturing body members for a plurality of target substances close to each other, a plurality of target substances can be detected in the same test time.

(Example 6)
The fusion polypeptide 1 used in Example 5 and the cellulose-binding domain of fusion polypeptide 2 were changed to silicon dioxide affinity peptides, and fusion polypeptide 3 (amino acid sequence and gene sequence are shown in sequence 17 and sequence 18, respectively) And fusion polypeptide 4 (amino acid sequence and gene sequence are shown in sequence 21 and sequence 22, respectively). Fusion polypeptide 4 is prepared in the same manner as (1-1) and (1-2) in Example 1. The same sample development / detection pad as in Example 4 except that the fusion polypeptide 3 and fusion polypeptide 4 prepared as described above are changed to the Glass Fiber Conjugate Pad for the sample development / detection pad. The test pieces are prepared by immobilizing in different regions above and evaluated using the same method as the method evaluated in Example 5. In the biosensor element of the present embodiment, the capturing member is prevented from spreading and spreading, so that the end of the test line becomes clear, and it becomes possible to immobilize the capturing member for a plurality of target substances close to each other, Multiple target substances can be detected in the same test time.

It is a schematic diagram which shows an example (lateral flow type) of an immunochromatography. It is a schematic diagram which shows the bleed prevention mechanism using the capturing body member and sensor element in this invention. It is a schematic diagram which shows the example of fixation of several capture body members.

Explanation of symbols

1 Capture antibody 2 Control antibody (secondary antibody, etc.)
3 Test line 4 Control line 5 Labeled antibody
6 Labeling agent
7 Membrane (development part and detection part)
8 Sample pad (sample inhalation part)
9 Absorption pad (absorption part)
10 Target substance
11 Sample liquid
12 Schematic diagram immediately after applying capture molecules
13 Schematic diagram of diffusion of capture molecule solution
14 Schematic after solvent drying
15 Solvent
16 Capturing member
17 Carrier
18 Carrier material compatible parts
19 Capture molecule
20 Example of fixing multiple capture members (lateral flow type)
21 Capturing member
22 Example of immobilization of multiple capture members (array type)
23 Capturing member

Claims (3)

A capturing body immobilizing carrier for detecting a target substance in a sample liquid,
A capturing body member for capturing the target substance;
A carrier on which the capturing body member is fixed,
The capture member is at least
A capture molecule having affinity for the target substance;
A carrier material affinity member having an affinity for the material constituting the carrier,
The carrier-fixed carrier, wherein the carrier has a structure in which the sample liquid can diffuse into the carrier.   The carrier-fixing carrier according to claim 1, wherein the carrier material affinity member is a cellulose-binding domain.   The carrier-fixing carrier according to claim 1, wherein the carrier material affinity member is a silicon dioxide affinity peptide.