JP2013007759A - Method for immobilizing protein to carrier, carrier with immobilized protein, and measurement reagent of test substance using carrier with immobilized protein - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for expressing protein having a signal sequence in a base sequence of a gene at a state of having the signal sequence and immobilizing the protein having the signal sequence to a carrier, the carrier to which the protein in which secretory protein having the signal sequence in the base sequence of the gene is expressed at the state of having the signal sequence is immobilized by the method, and a measurement reagent of a test substance using the carrier.SOLUTION: There is provided a method for immobilizing protein for expressing the protein having a signal sequence in a base sequence of a gene at a state of having the signal sequence, and immobilizing the protein having the signal sequence to the carrier, the carrier to which the protein in which the protein having the signal sequence in the base sequence of the gene is expressed at the state of having the signal sequence is immobilized, and a measurement reagent of a test substance containing the carrier to which the protein in which the protein having the signal sequence in the base sequence of the gene is expressed at the state of having the signal sequence is immobilized.

Description

The present invention relates to a method for expressing a protein having a signal sequence in the base sequence of a gene in a state having the signal sequence and immobilizing the protein on a carrier, a protein having a signal sequence in the base sequence of the gene as a signal sequence A test substance using a carrier in which a protein expressed in a state having a signal is immobilized, and a carrier in which a protein having a signal sequence in a base sequence of a gene is expressed in a state having a signal sequence is immobilized It relates to the measurement reagent.
The present invention is useful in the field of life science such as clinical examination, immunology, and medicine, the field of chemistry such as analytical chemistry, the field of food hygiene, and the field of environmental health.

Known protein immobilization methods include chemical bonding methods such as covalent bonding methods and crosslinking methods, and physical adsorption methods.
Among these immobilization methods, the covalent bond method has the advantage that protein detachment from the carrier is extremely small. However, the amount of protein immobilized on the carrier is relatively small, and the protein is immobilized by a chemical reaction during immobilization. It tends to denature or decrease the physiological activity of the protein. In addition, the cross-linking method requires a large amount of protein and is not efficient, and the physiological activity of the protein is likely to be lowered during immobilization.
On the other hand, the physical adsorption method is easy to operate and can immobilize the protein on the carrier in a short time, can evenly immobilize the protein on the carrier, and has a relatively large amount of protein immobilized on the carrier. In general, it is widely used.

On the other hand, a measuring method or a measuring reagent for measuring a test substance in a sample using a carrier on which a protein that is a specific binding substance for the test substance is immobilized is frequently used. For example, a small amount of test substance contained in a sample using a reaction between substances having specific affinity such as antigen and antibody, sugar and lectin, nucleotide chain and complementary nucleotide chain, and ligand and receptor. Various measuring methods and measuring reagents are known.
This is a measurement of the presence or absence of a test substance contained in a sample by measuring the presence or absence of binding between the test substance contained in the sample and a specific binding substance for this test substance [ Qualitative measurement] or measurement (quantitative measurement) of its content (concentration).
For example, in an immunological measurement method (immunological measurement reagent) using an antigen-antibody reaction (immune reaction, immunological reaction) between an antigen and an antibody, a latex turbidimetric measurement method using latex particles as a carrier (Measurement reagent): Indirect agglutination reaction measurement method using latex particles, organic polymer particles, inorganic particles, metal particles, or red blood cells as a carrier (measurement reagent); microtiter plate, beads, particles, test tube, or container Labeled immunoassay (measuring reagent) such as enzyme immunoassay and luminescence immunoassay using as a carrier; or immunochromatography (measuring reagent) using cellulose or non-woven fabric as a carrier is frequently used. ing.
In these measurement methods or reagents, in many cases, a carrier on which a protein that is a specific binding substance for a test substance is immobilized is used. However, when a physical adsorption method is used to immobilize a protein that is a specific binding substance to a test substance on a carrier, the immobilization to the carrier may not be possible depending on the type of protein that is a specific binding substance to the test substance. Some were difficult. Moreover, the protein once immobilized may be detached.
If the protein, which is a specific binding substance for the test substance, is not sufficiently immobilized on the carrier, the test substance in the sample is measured using this immobilized carrier. There was a risk that the sensitivity of the measurement would be reduced due to a decrease in the amount of signal generated during the reaction. In this case, there is a possibility that an erroneous measurement result (false negative) that the test substance is not contained may be obtained even in the measurement of the sample containing the test substance. The risk of misdiagnosis was included. (There are cases where it is difficult to perform accurate measurement because a certain measurement sensitivity cannot be obtained.)

As described above, among the protein immobilization methods, the covalent bond method has a relatively small amount of protein immobilized on a carrier, and the protein is denatured by the chemical reaction during immobilization, or the physiological activity of the protein. There was a problem that the reduction was likely to occur. In addition, the chemical bonding method using a crosslinking reagent or the like has a problem that a large amount of protein is required and the efficiency is low, and the physiological activity of the protein is likely to be lowered when the protein is immobilized.
Furthermore, the physical adsorption method also has a problem that it is difficult to immobilize it on a carrier depending on the type of protein. In addition, there is a problem that once immobilized proteins are detached.
Therefore, an object of the present invention is to easily fix a protein on a carrier in a method for immobilizing a protein on a carrier, a carrier on which a protein is immobilized, and a measurement reagent for a test substance using the carrier on which a protein is immobilized. It is to provide means for immobilization and immobilization for a long time without reducing or denaturing the physiological activity of the protein.

  As a result of diligent research, the present inventors have expressed a protein having a signal sequence in the base sequence of a gene in a state having the signal sequence and immobilized it on a carrier. It was found that the immobilization can be kept stable for a long period of time, and the present invention has been completed.

  That is, the present invention is a protein immobilization method characterized by expressing a protein having a signal sequence in the base sequence of a gene in a state having the signal sequence, and immobilizing the protein having the signal sequence on a carrier. is there.

  Further, the present invention is a carrier on which a protein is immobilized, wherein the protein is a protein in which a protein having a signal sequence in a base sequence of a gene is expressed in a state having a signal sequence, A carrier on which a protein is immobilized.

  Furthermore, the present invention provides a measurement reagent for a test substance containing a carrier on which a protein is immobilized, wherein the protein has a protein having a signal sequence in the base sequence of a gene expressed in a state having the signal sequence. This is a reagent for measuring a test substance.

  In the present invention, the protein is preferably a membrane protein or a secreted protein.

  In the present invention, it is preferable that the protein is a specific binding substance for a test substance.

  Furthermore, in the present invention, it is preferable that the test substance is an antibody against Treponema paridum and the specific binding substance to the test substance is an antigen of Treponema paridum.

In the method for immobilizing a protein on a carrier, the carrier on which the protein is immobilized, and the test reagent for the test substance using the carrier on which the protein is immobilized, a protein having a signal sequence is used as the protein. Therefore, the protein can be easily immobilized on a carrier in a large amount.
In addition, during the storage of the carrier on which the protein of the present invention is immobilized and the measurement reagent, the protein is prevented from being detached from the carrier on which the protein is immobilized, and the protein is stably immobilized on the carrier for a long period of time. It can be left. Accordingly, the measurement reagent can be prevented from being lowered in sensitivity and stabilized, and the measurement result of the test substance can be obtained for a long period of time.
Furthermore, in the present invention, the protein is immobilized on a carrier using a protein expressed in a state having a signal sequence that the protein originally has. For this reason, there is little possibility that the structure or function of the protein will be impaired due to steric hindrance or the like that is considered in the case of a protein that is not associated with the protein, and the protein originally has Enzyme reactions or antigen-antibody reactions can be sufficiently exhibited even after immobilization.
In the present invention, since the protein can be easily immobilized on the carrier, the amount of the protein immobilized on the carrier can be reduced.

It is the figure which measured the antibody with respect to Treponema pallidum in a sample using the measuring reagent of this invention and a comparative example, and showed the result. It is the figure which measured the antibody with respect to Treponema paridam in a sample using this invention, the comparative example, and the measurement reagent of a control | contrast, and showed the result. It is the figure which showed the result of the storage stability of the measuring reagent of this invention.

[1] Basic Requirements of the Invention In the “method for immobilizing a protein on a carrier”, “a carrier on which a protein is immobilized”, and “a reagent for measuring a test substance containing a carrier on which a protein is immobilized”, The protein immobilized on the carrier is a protein having a signal sequence in the base sequence of the gene, and the protein expressed in a state having this signal sequence is immobilized on the carrier.

[2] Protein having a signal sequence in the base sequence of the gene In the present invention, the protein immobilized on the carrier is a protein having a signal sequence in the base sequence of the gene.
As a protein having a signal sequence in the base sequence of this gene, any protein may be used as long as it has a signal sequence in the base sequence of the gene, that is, a signal sequence on the N-terminal side of the mature protein. Various types of proteins can also be used.
The signal sequence is about 15 to 30 amino acid residues necessary for the protein to pass through the lipid bilayer of the cytoplasmic membrane on the N-terminal side. There is a hydrophobic region in the center of this signal sequence, where hydrophobic amino acid residues are continuous, a basic amino acid residue is present on the N-terminal side, and a hydrophilic side chain is compared on the C-terminal side. Small amino acid residues are present. The portion of the sequence consisting of this amino acid residue is called a signal peptide.
This signal sequence is thought to play a leading role when the protein passes through the lipid bilayer of the cytoplasmic membrane, and is usually cleaved and removed by the signal peptidase after the protein passes through the cytoplasmic membrane.
Therefore, the mature protein produced in vivo and attached to the surface of the cytoplasmic membrane or buried inside, or secreted outside the cytoplasm does not have a signal sequence.

In the present invention, the protein is preferably a membrane protein or a secreted protein.
Here, the membrane protein refers to a protein constituting a biological membrane. A protein attached to the surface of the biological membrane is a membrane surface protein, and a protein embedded inside is a membrane integral protein. Call.
Examples of the membrane protein include various enzymes, receptors, transporters, ion channels, and antigen proteins.
A secreted protein is a protein that is secreted outside the cytoplasmic membrane and is synthesized on a ribosome in the cytoplasm and then secreted outside the cytoplasm to become a mature protein.
Examples of the secreted protein include various digestive enzymes, peptide hormones, antibody protein, milk protein, egg white protein and the like.

In the present invention, the protein is preferably an antigenic protein of Treponema Pallidum, which is known as a causative bacterium for syphilis. Known antigenic proteins of Treponema pallidum include 15K Dalton antigen protein, 17K Dalton antigen protein, or 47K Dalton antigen protein, which are surface antigenic proteins of Treponema pallidum. These surface antigen proteins are membrane surface proteins.
In addition, the amino acid sequences and base sequences of these surface antigen proteins have already been reported. (15K Dalton antigen protein: Molecular Microbiology 4, 1371-1379, 1990, 17K Dalton antigen protein: Infection and Immunity 61, 1202-1210, 1993, 47K Dalton antigen protein: Infection and Immunity 60, 1568 To 1576, 1992)

  Among proteins localized in the endoplasmic reticulum, Golgi apparatus, and lysosome, those having a signal sequence in the base sequence of the gene can also be referred to as proteins in the present invention.

[3] Carrier In the present invention, it is preferable that at least the surface of the carrier where the protein is immobilized is hydrophobic.
In the carrier, the surface being hydrophobic means that the surface is made of a hydrophobic material, or a hydrophobic substance or functional group is bonded to or coated on the surface to impart a hydrophobic property. Can be mentioned.
Examples of such carriers include carriers usually used in immunological measurement methods, immunological measurement reagents and the like as described below.

Examples of the carrier in the present invention include latex particles used in latex turbidimetry or particles that can be used in latex turbidimetry.
Examples of such latex particles include polystyrene / latex particles, styrene / styrene sulfonate copolymer / latex particles, acrylonitrile / butadiene / styrene copolymer / latex particles, vinyl chloride / acrylate copolymer / Latex particles, vinyl acetate-acrylic acid copolymer / latex particles, polyacrolein / latex particles, styrene-methacrylic acid copolymer / latex particles, styrene-glycidyl (meth) acrylic acid copolymer / latex particles, heavy methacrylate Examples thereof include latex particles in which synthetic polymer particles such as coalescence / latex particles or acrylic acid polymer / latex particles are uniformly suspended.
There is no restriction | limiting in particular about the particle size of this latex particle.
When measuring a test substance using the latex turbidimetric method as a measurement principle, the degree to which latex particles are bonded through the test substance to form an aggregate, the ease of measurement of the generated aggregate, etc. For this reason, the average particle size of the latex particles is preferably 0.04 to 1 μm.
In addition, the concentration of the test substance measurement reagent includes “latex particles with a specific binding substance immobilized on the test substance”. This is the concentration of the test substance, the concentration in the sample, the test substance Since the optimum concentration differs depending on various conditions such as the kind of specific binding substance to the substance and the distribution density on the surface of the latex particles, the particle size of the latex particles, the mixing ratio of the sample and the measuring reagent, it cannot be generally stated.
However, normally, the sample and the measurement reagent for the test substance are mixed and the “specific binding substance for the test substance” immobilized on the latex particles is specific to the “test substance” contained in the sample. In the measurement reaction in which a typical binding reaction is performed, the concentration of “latex particles with a specific binding substance immobilized on the test substance” is 0.005 to 1% (w / v) in the reaction mixture. In this case, the measurement of the test substance is carried out with “latex particles having a specific binding substance immobilized on the test substance” having such a concentration in the reaction mixture. Include in reagent.

In addition, as the carrier in the present invention, for example, particles used in indirect agglutination reaction measurement methods such as particle agglutination reaction measurement method using latex particles or latex agglutination reaction measurement method, or used in this indirect agglutination reaction measurement method And particles that can be used.
Examples of such particles include particles made of an organic polymer material such as polystyrene, liposome, latex, gelatin, polyacrylamide, microcapsule or emulsion, and particles made of an inorganic polymer material such as glass, silica gel, carbon or bentonite. Or other artificial carriers can be mentioned.
Further, as the particles, particles colored by coating a dye or by dispersing or encapsulating the dye in the particles may be used.
There are no particular restrictions on the particle size of these particles.
However, the average particle size of these particles is preferably in the range of 0.01 to 100 μm, and more preferably in the range of 0.5 to 10 μm.
Further, the specific gravity of these particles is preferably in the range of 1 to 10, more preferably in the range of 1 to 2.

Examples of the carrier in the present invention include a container used for the indirect agglutination reaction measurement method, a container that can be used for the indirect agglutination reaction measurement method, and the like.
Examples of such containers include test tubes, microplates (microtiter plates) or trays made of glass, polystyrene, polyvinyl chloride, polymethacrylate, or the like.
In addition, it is preferable that the bottom surface of the solution storage portion (such as a well of a microplate) of the container has an inclined shape from the center to the periphery of the bottom surface such as a U shape, a V shape, or a UV shape.

The carrier in the present invention is used for immunoassay using a labeling substance such as enzyme immunoassay, fluorescent immunoassay, radioimmunoassay, or luminescent immunoassay, that is, labeled immunoassay. Or a carrier that can be used in this labeled immunoassay.
Examples of such a carrier include materials such as polystyrene, polycarbonate, polyvinyl toluene, polypropylene, polyethylene, polyvinyl chloride, nylon, polymethacrylate, polyacrylamide, latex, liposome, gelatin, agarose, cellulose, sepharose, and glass. There may be mentioned particles, microcapsules, beads, microplates (microtiter plates), test tubes, sticks or test pieces.

In addition, as the carrier in the present invention, for example, a specific binding substance for a test substance (test substance) described in JP-A-9-229936 and JP-A-10-132919 is immobilized and coated. A carrier having a flat surface and a carrier used in a measurement method using particles in which a specific binding substance to a test substance (test substance) is immobilized, or a carrier that can be used in this measurement method, etc. be able to.
Examples of such a carrier include non-water-absorbing materials such as polystyrene, glass, polyvinyl chloride, polyacrylate, polypropylene, nylon, polyethylene, polycarbonate, and polymethacrylate.

Also, a magnetic carrier prepared by coating the above-described carrier with a ferromagnetic material or containing a ferromagnetic material at the time of carrier molding can be used.
In the present invention, the carrier is preferably polystyrene.

[4] Method for expressing a protein having a signal sequence in the base sequence of a gene in a state having a signal sequence In the present invention, a protein having a signal sequence in a base sequence of a gene is expressed in a state having a signal sequence For this purpose, for example, a normal method such as a method known as genetic engineering technology may be used.
For example, a protein gene (having a signal sequence on the N-terminal side of a mature protein gene) is first cloned, and the obtained gene is incorporated into an expression vector such as a plasmid. By introducing this expression vector into a host cell such as Escherichia coli and culturing the resulting transformant, a protein having a signal sequence in the base sequence of the gene can be expressed in a state having the signal sequence.
Examples of the method for cloning a gene base sequence include a PCR method, a recombinant PCR method, a ligation method, and a linker ligation method. For example, when the PCR method is used, the base sequence of a gene can be obtained with a signal sequence by amplifying the base sequence of the gene of a natural protein using a primer.
For example, a method for expressing a Treponema paridum surface antigen protein having a signal sequence in the base sequence of a gene in a state having a signal sequence will be described using a 17 K Dalton antigen protein as an example. By amplifying only the “base sequence of the 17K dalton antigen protein gene having a signal sequence” in the base sequence of the natural 17K dalton antigen protein gene using a primer, the “17K dalton antigen protein having a signal sequence” The base sequence of the gene ”is obtained. The thus obtained “base sequence of a 17K Dalton antigen protein gene having a signal sequence” is incorporated into an expression vector by a ligation method or the like, this expression vector is introduced into Escherichia coli, and the resulting transformant is cultured. Thus, a 17 K Dalton antigen protein having a signal sequence in the base sequence of the gene can be expressed in a state having the signal sequence.

[5] Method of immobilizing a protein having a signal sequence on a carrier In the present invention, a protein having a signal sequence can be immobilized on a carrier by bringing the protein having a signal sequence into contact with the carrier. .

  For example, when the carrier is a container, a protein having a signal sequence dissolved in a buffer solution or the like is added to the solution storage part of the container and brought into contact with the inner wall surface, or when the carrier is a particle, After the protein having the sequence and the particles are mixed and brought into contact in a solution such as a buffer solution, this is performed at about 2 ° C. to about 40 ° C. for about 10 minutes to about 1 day, thereby immobilizing the carrier. be able to.

Furthermore, if it is necessary to carry out treatment to suppress non-specific reaction or spontaneous aggregation of the carrier, proteins such as bovine serum albumin (BSA), casein, gelatin, ovalbumin or salts thereof, surfactants or You may perform the blocking process (masking process) of the support | carrier which fix | immobilized the protein which has a signal sequence by well-known methods, such as making non-fat dry milk etc. contact and coat | cover.
In the present invention, since the protein to be immobilized on the carrier has a signal sequence, the hydrophobicity of the signal sequence portion strongly and stably binds and adsorbs on the surface of the carrier due to the hydrophobic interaction. It is estimated that it is difficult to cause separation.

[6] Reagent for measuring a test substance The test reagent for a test substance of the present invention is a test reagent for a test substance containing a carrier on which a protein is immobilized, and the protein contains a signal sequence in the base sequence of the gene. It is characterized in that it is a protein in which a protein having a signal sequence is expressed, and as “a carrier on which a specific binding substance for a test substance is immobilized”, “a protein having a signal sequence is immobilized. It is a measurement reagent for measuring a test substance in a sample using a “supported carrier”.

[7] Test substance Test substance In the reagent for measuring a test substance of the present invention, the test substance is a substance whose presence or content (concentration) is to be measured in a sample.

  Any test substance can be used as long as it can be measured using a test substance measurement reagent containing a carrier on which a protein having a signal sequence as a specific binding substance for the test substance is immobilized. It may be a thing.

  Examples of this test substance include organic substances such as proteins, carbohydrates, lipids, or nucleic acids.

  The test substance is preferably a virus-related antigen or antibody, or a bacteria-related antigen or antibody, and particularly preferably an antibody against Treponema paridum (anti-treponema paridum antibody).

2. Sample In the present invention, a sample refers to a substance in which the above-mentioned test substance may exist and the presence / absence of the test substance or the content (concentration) is to be measured.

  For example, human or animal blood, serum, plasma, urine, semen, spinal fluid, saliva, sweat, tears, ascites, amniotic fluid, or other body fluids; human or animal brain or other organs, hair, skin, nails, muscles, or Examples include extracts of nerve tissues and the like; extracts or suspensions of human or animal stool; extracts of cells or cells.

[8] Specific binding substance for the test substance In the measurement reagent for the test substance of the present invention, the specific binding substance for the test substance has a specific affinity and binds to the test substance. It is a substance that can be used.
In the reagent for measuring a test substance of the present invention, it is indispensable to use a carrier on which a “protein having a signal sequence” is immobilized as a “specific binding substance to the test substance”. Different “specific binding substances for the test substance” may be used in combination.

  The specific binding substance for this test substance is, for example, an antibody against this antigen when the test substance is an antigen, and an antigen against this antibody or an antibody against this antibody when the test substance is an antibody. , A ligand when the test substance is a protein or the like, a lectin that binds to this sugar when the test substance is a sugar, or a substance for this receptor when the test substance is a receptor Etc.

  That is, any substance can be used in the present invention as a specific binding substance for a test substance without particular limitation as long as the substance has a specific affinity for and binds to the test substance.

  In the present invention, it is preferable that the test substance is an antigen and the specific binding substance for the test substance is an antibody against the antigen.

Here, the antibody may be either a polyclonal antibody or a monoclonal antibody, and may be a fragment thereof (such as F (ab) ′ ₂ or Fab ′).

  In the present invention, it is preferable that the test substance is an antibody and the specific binding substance for the test substance is an antigen for the antibody or an antibody for the antibody.

  When the test substance is an antibody and the specific binding substance to the test substance is an antigen against the antibody, the “antigen against the antibody” may be artificially prepared by a genetic recombination method or the like. Good.

  The “antigen against the antibody” artificially prepared by this genetic recombination method may be fused with another protein. Examples of the other protein to be fused include glutathione-S. -Transferase (GST), maltose binding protein (MBP), thioredoxin, β-galactosidase, lactase, biotinylated protein, protein A, or gene 10 can be mentioned.

  More specifically, for example, when the test substance is an antibody against Treponema paridum, as a specific binding substance for the test substance, a 17K dalton antigen of Treponema paridum having a signal sequence is combined with maltose binding protein ( MBP) is fused, or Glutathione-S-transferase (GST) is fused to the 47K Dalton antigen of Treponema pallidum having a signal sequence.

  In the present invention, it is preferable that the test substance is an antibody against Treponema paridum and the specific binding substance to the test substance is an antigen of Treponema paridum.

  Examples of the antigen of Treponema pallidum include 15K dalton antigen, 17K dalton antigen, or 47K dalton antigen.

  These antigens of Treponema pallidum may be artificially prepared by a genetic recombination method or the like.

  The antigens of Treponema paridum artificially prepared by this genetic recombination method include, for example, glutathione-S-transferase (GST), maltose binding protein (MBP), thioredoxin, β-galactosidase, lactase, biotinylated protein, It may be fused with other protein such as protein A or gene 10.

  In the present invention, it is necessary that at least one of the antigens of Treponema pallidum used as a specific binding substance for the above test substance to be immobilized on a carrier has a signal sequence.

[9] Measuring reagent Measuring principle of measuring reagent (measuring method)
The carrier on which a protein is immobilized in the present invention can be used as a measurement reagent for measuring a test substance in a sample.
For example, a specific binding reaction between a “protein having a signal sequence” as a “specific binding substance to a test substance” immobilized on a carrier and a “test substance” contained in a sample is used. Thus, it can be used as a measurement reagent whose measurement principle is a method of measuring a test substance in a sample.

  The specific binding reaction between the “specific binding substance to the test substance” and the “test substance” includes, for example, an antigen-antibody reaction (immune reaction) between an antigen and an antibody, Examples include a reaction with a ligand, a reaction between a sugar and a lectin, or a reaction between a receptor and a substance corresponding thereto.

  And as an example of the method of measuring the test substance in a sample using such a binding reaction, an immunological measuring method etc. can be mentioned, for example.

  In addition, as this immunological measurement method, for example, latex turbidimetry; indirect aggregation reaction measurement method such as latex agglutination measurement method; enzyme immunoassay, fluorescence immunoassay, radioimmunoassay, or luminescence immunoassay Immunoassay using a labeling substance such as the labeling method, that is, labeled immunoassay; Western blotting; ELSA method (enzyme-linked ligand sorbent assay) shown by Dahlbeack et al. [Thromb. Haemost. 79, 767-772, 1998, and WO 98/23963]; or JP-A-9-229936, JP-A-10-132919, and the like. Using a carrier having an immobilized coated surface and a particle on which a specific binding substance to the test substance is immobilized, the particle is fixed on the surface coated with the specific binding substance to the test substance on the carrier. A method of performing measurement depending on whether or not they gather can be mentioned.

  The labeled immunoassay is the above-described method, and the present invention can be applied to any method such as the sandwich method, the competitive method, or the homogeneous method (homogeneous method).

2. Measurement Reagent A test reagent for a test substance in the present invention can be based on the measurement method described above, and a protein having a signal sequence in the base sequence of a gene in a state having a signal sequence. A reagent for measuring a test substance comprising a carrier on which a protein having an expressed signal sequence is immobilized.

Note that the reagent for measuring a test substance in the present invention can be sold alone or used for measurement of a test substance in a sample.
In addition, the measurement reagent for a test substance in the present invention can be sold in combination with other reagents or used for measurement of a test substance in a sample.

  Examples of the other reagent include a buffer solution, a sample diluent, a reagent diluent, a reagent containing a labeling substance, a reagent containing a substance that generates a signal such as color development, or calibration (calibration). The reagent of the substance containing these substances can be mentioned.

  The other reagent is the first reagent and the test substance measurement reagent in the present invention is the second reagent, or the test substance measurement reagent in the present invention is the first reagent, Can be sold and used in various combinations as appropriate.

  In addition, various aqueous solvents can be used as the solvent of the measurement reagent for the test substance in the present invention.

  Examples of the aqueous solvent include purified water, physiological saline, or various buffer solutions such as tris (hydroxymethyl) aminomethane buffer, phosphate buffer, or phosphate buffered saline. .

  About the pH of this buffer solution, an appropriate pH may be appropriately selected and used. Although there is no particular limitation, it is general to select and use a pH within the range of pH 3-12.

  In addition, it is preferable to use the pH in the alkaline region as the pH of the measurement reagent for the test substance in the present invention because the effect of stabilizing the measurement reagent can be enhanced. For example, the pH is more preferably 7.3 or higher.

  In addition, in the reagent for measuring a test substance in the present invention, bovine serum albumin (BSA), human serum albumin (HSA), in addition to a carrier on which a protein that is a specific binding substance for the test substance is immobilized, Proteins such as casein or salts thereof; various salts other than the above cations and anions; various sugars; skim milk powder; various animal sera such as normal rabbit serum; various preservatives such as sodium azide or antibiotics; One or more of a reaction promoting substance, a sensitivity increasing substance such as polyethylene glycol, and a nonspecific reaction inhibiting substance may be appropriately contained.

  And the density | concentration at the time of making these contain in the measuring reagent of a test substance is although it does not specifically limit, 0.001-10% (w / v) is preferable, Especially 0.01-5% (w / v) v) is preferred.

[10] Measurement of a test substance in a sample In order to measure a test substance in a sample using the test substance measurement reagent in the present invention, measurement is based on the measurement principle of the measurement reagent of the test substance. What is necessary is just to perform measurement operation according to the operation method of a method.

  This measurement may be performed by a method or using an apparatus such as an analyzer.

  In addition, this measurement may be performed by a one-step method (one-reagent method) or by a method performed by a plurality of operation steps such as a two-step method (two-reagent method).

The present invention will be further described below with reference to examples.
In addition, this invention is not limited by these.

[Example 1]
(Preparation of a carrier on which 17K Dalton antigen protein of Treponema pallidum having a signal sequence is immobilized, and preparation of a reagent for measuring antibody against Treponema pallidum containing this carrier)

1. Preparation of 17K Dalton Antigen Protein Immobilized Plate with Signal Sequence

(1) Preparation of 17K Dalton antigen protein having a signal sequence in the base sequence of the gene Treponema paridum cells were extracted from the crushed rabbit testicles subcultured with Treponema paridum, and the base sequence of the gene was extracted. . The base sequence of 17K Dalton antigen protein is already known as described above, and the entire base sequence and amino acid sequence thereof are as shown in SEQ ID NO: 1 and SEQ ID NO: 2 in the Sequence Listing. The 22nd sequence (tgt / Cys) in SEQ ID NO: 1 is the N-terminal of the mature 17K Dalton antigen protein, and the 1st to 21st sequences (atg / Met to ttg / Leu) are signal sequences.
Oligonucleotides of about 20 bases at both ends of the base sequence of the 17 K Dalton antigen protein having the signal sequence shown in SEQ ID NO: 1 were synthesized as primers. The base sequence having the signal sequence extracted here was used as a template, and the base sequence of the 17 K Dalton antigen protein having the signal sequence was amplified by PCR using the above primers. Next, the base sequence obtained above was inserted into a vector (Pharmacia; pGEX4T3) containing the base sequence of glutathione-S-transferase (GST). This vector was introduced into Escherichia coli, and the cells were cultured. Thereafter, Escherichia coli in which the 17K dalton antigen protein having the signal sequence was sufficiently expressed was disrupted by sonication, centrifuged, and the supernatant was applied to a glutathione sepharose 4B column. After washing, a 17K dalton antigen protein having a signal sequence fused with GST was eluted by flowing a glutathione solution. This was dialyzed to remove glutathione, and then thrombin was added to cleave the 17 K dalton antigen protein having GST and a signal sequence. It was again applied to a glutathione sepharose 4B column, and the flow-through fraction was collected to obtain a 17 K dalton antigen protein having a signal sequence.

(2) Preparation of washing solution Phosphate buffered saline containing 0.02% (w / v) Tween 20 was prepared and used as a washing solution.

(3) Preparation of blocking solution Phosphate buffered saline containing 0.5% (w / v) sodium caseinate was prepared and used as a blocking solution.

(4) Preparation of 17K Dalton Antigen Protein-Immobilized Plate Having Signal Sequence 17K Dalton antigen having the signal sequence of (1) above was mixed with 0 μg / mL, 0.03125 μg / mL, 0.0625 μg / mL, 0.125 μg / It added to the phosphate buffered saline so that it might become mL, 0.25 microgram / mL, 0.5 microgram / mL, 1 microgram / mL, and 2 microgram / mL. This was dispensed into each well of ELISA plate S (manufactured by Sumitomo Bakelite Co., Ltd.) in an amount of 50 μL, and then allowed to stand overnight at 4 ° C. to immobilize the 17 K dalton antigen protein having a signal sequence on the inner wall of each well. .

Next, the liquid in each well of the ELISA plate S was removed, and the plate was washed twice with the washing liquid (2).
Thereafter, 200 μL of the blocking solution (3) was dispensed into each well, and each well was blocked.
Then, the liquid in each well of the plate was removed, and the plate was washed twice with the washing liquid.
What was prepared by the above operation was used as the 17K dalton antigen protein fixed plate which has a signal sequence.

2. Preparation of POD-labeled antibody (1) POD-labeled antibody diluent Phosphate buffered saline containing 0.5% (w / v) sodium caseinate was prepared and used as a POD-labeled antibody diluent.

(2) Preparation of POD-labeled antibody A POD-labeled rabbit anti-human IgG antibody solution (manufactured by Dako; P0406) was prepared by diluting 1000 times with the POD-labeled antibody diluent prepared in (1) above.

[Example 2]
(Measurement of antibody against Treponema pallidum by ELISA method)
Using the test substance measurement reagent of the present invention, an antibody against Treponema paridum, which is the test substance, was measured.

1. Measurement: Fixation of 17 K Dalton antigen protein having the signal sequence prepared in 1 of Example 1 with 50 μL of TP antibody-positive serum diluted 2000-fold with phosphate buffered saline containing 0.5% (w / v) sodium caseinate Was added to each well of the reaction plate and allowed to react at 37 ° C. for 4 hours.
Thereafter, the serum solution in the wells of the plate was removed and the plate was washed with the washing solution.
Further, 50 μL of the POD-labeled rabbit anti-human IgG antibody solution prepared in 2 of Example 1 was added to each well and reacted at room temperature for 4 hours.
Next, each well was washed with the washing solution.

Thereafter, 100 μL of a coloring solution (33 μL of 0.3% hydrogen peroxide added immediately before use) to 1 mL of 103 mM disodium hydrogen phosphate-49 mM citrate buffer containing 4 mM ο-phenylenediamine substrate solution was added. After adding to each well and reacting for 20 minutes at room temperature, 50 μL of 4N sulfuric acid aqueous solution was added to each well to stop the reaction.
Thereafter, the absorbance at 490 nm of each well was measured using a microplate reader (Biorad, Model 3550).

2. Measurement Results Table 1 and FIG. 1 show the absorbance obtained by measuring the test substance (antibody against Treponema pallidum) in the sample.

  In this figure, the horizontal axis represents the concentration of 17 K Dalton antigen protein when immobilized on the well of the plate, and the vertical axis represents the measured value of absorbance at 490 nm.

[Comparative Example]
A measurement reagent was prepared in the same manner as in Example 1 except that the base sequence of the 17 K Dalton antigen protein having the signal sequence in Example 1 was used instead of the base sequence of the 17 K Dalton antigen protein having the signal sequence. Then, an antibody against Treponema paridum, which is a test substance, was measured according to the description in Example 2 using a measuring reagent including a plate on which the 17K Dalton antigen protein of Treponema paridum without this signal sequence was immobilized. . The measurement results are shown in Table 1 and FIG.

◎ Consideration of measurement results in Example 2 and Comparative Example From Table 1 and FIG. 1, in the measurement reagent including the 17K Dalton antigen protein-immobilized plate which does not have the signal sequence of Comparative Example, Even when the concentration of 17K Dalton antigen protein contacted with the well is increased, no increase in absorbance is observed. That is, it is difficult to immobilize 17K Dalton antigen protein that does not have a signal sequence for the plate, and since it is not sufficiently performed, the amount of signal (absorbance) generated during the measurement reaction is small, and the measurement It turns out that the sensitivity has fallen.

  In contrast, in the measurement reagent including the 17K dalton antigen protein-immobilized plate having the signal sequence of the present invention, the absorbance obtained according to the concentration of 17K dalton antigen protein brought into contact with the well of the plate for immobilization. It can be seen that 17K Dalton antigen protein having a signal sequence on the plate is easily and abundantly immobilized.

Therefore, it was confirmed that by using 17K Dalton antigen protein having a signal sequence for immobilization on the plate, the antigen protein can be sufficiently immobilized on the plate, and the test substance in the sample can be measured with high sensitivity. It was.
Then, it was confirmed that the antibody against Treponema pallidum in the sample can be accurately measured by the measuring reagent of the present invention.

Example 3
(Preparation of 17K Dalton antigen protein-immobilized particles of Treponema pallidum having a signal sequence, and preparation of an antibody measurement reagent for Treponema pallidum containing the particles)

1. Preparation of 17K Dalton Antigen Protein Immobilized Particles with Signal Sequence

(1) Preparation of cleaning solution The cleaning solution was prepared in the same manner as in Example 1.

(2) Preparation of blocking solution Prepared in the same manner as in Example 1.

(3) Preparation of 17K Dalton Antigen Protein-Immobilized Particles Having Signal Sequence 17K Dalton Antigen Having Signal Sequence Obtained in Example 1 and Magnetic Particle [Dynal; Dynabeads M-450 uncoated, Particle Size: 4. 5 μm, C.D. V. 5% or less, specific gravity 1.5, concentration 6% (w / v)] were mixed so that the antigen protein concentration was 200 μg / mL and the particle concentration was 6%, and reacted at room temperature for 1 hour. Thereafter, the supernatant was removed, and the blocking solution (2) was added in an amount of about 20 times and left standing at 37 ° C. overnight for blocking.
Thereafter, the obtained particles were washed with the washing liquid (1). The particles were dispersed in 50 mM glycine buffer (pH 9.5) so that the particle concentration was 0.2% (w / v) to prepare 17 K Dalton antigen protein-immobilized particles having a signal sequence.

2. Preparation of POD-labeled antibody

(1) POD-labeled antibody diluent Prepared in the same manner as in Example 1.

(2) Preparation of POD-labeled antibody A POD-labeled rabbit anti-human IgG antibody solution (manufactured by Dako; P0406) was prepared by diluting 500 times with the POD-labeled antibody diluent prepared in (1) above.

Example 4
(Measurement of antibody against Treponema pallidum by ELISA method)
Using the test substance measurement reagent of the present invention, an antibody against Treponema paridum, which is the test substance, was measured.

1. Measurement: 500 μL of TP antibody-positive serum diluted 2000-fold with phosphate buffered saline containing 0.5% (w / v) sodium caseinate and 17K Dalton antigen protein immobilization having the signal sequence prepared in Example 1 The particles were mixed with 150 μL and reacted at room temperature for 1 hour. Thereafter, only the particles were taken out by centrifugation, and the particles were washed with the washing solution.
Furthermore, 500 μL of the POD-labeled rabbit anti-human IgG antibody solution prepared in 2 of Example 3 was added and reacted at room temperature for 1 hour.
Next, only the particles were removed by centrifugation, and the particles were washed with a washing solution.

Thereafter, 100 μL of a coloring solution (33 μL of 0.3% hydrogen peroxide added immediately before use) to 1 mL of 103 mM disodium hydrogen phosphate-49 mM citrate buffer containing 4 mM ο-phenylenediamine substrate solution was added. After addition and reaction at 37 ° C. for 20 minutes, 50 μL of 4N sulfuric acid aqueous solution was added to stop the reaction.
Thereafter, the absorbance at 490 nm of the supernatant was measured using a microplate reader (Biorad; Model 3550).

2. Measurement Results Table 1 and FIG. 2 show the absorbance obtained by measuring the test substance (antibody against Treponema pallidum) in the sample.

  In this figure, the vertical axis represents the measured value of absorbance at 490 nm.

[Comparative Example]
A measurement reagent was prepared in the same manner as in Example 3 except that the base sequence of the 17 K Dalton antigen protein having the signal sequence in Example 1 was used instead of the base sequence of the 17 K Dalton antigen protein having the signal sequence removed. Then, an antibody against Treponema pallidum, which is a test substance, was measured according to the description of Example 4 using a measuring reagent containing particles to which the 17K Dalton antigen protein of Treponema pallidum that does not have this signal sequence was immobilized. . The measurement results are shown in Table 2 and FIG.

[Control]
Further, as a control, following the procedure of Example 3, magnetic particles not immobilized with 17 kDalton antigen protein and measurement reagents such as POD-labeled antibodies were prepared, and this measurement reagent was used as described in Example 4. Then, an antibody against Treponema paridum, which is a test substance, was measured. The measurement results are shown in Table 2 and FIG.

◎ Consideration of Measurement Results in Example 4 and Comparative Example From Table 2 and FIG. 2, the absorbance obtained using the measurement reagent of the comparative example is 37 of the absorbance obtained using the measurement reagent of the present invention. It remains at%. That is, the 17K Dalton antigen protein that does not have a signal sequence for the particles is difficult to immobilize and is not sufficient, so that only a low signal (absorbance) is generated in the measurement reaction, resulting in a decrease in measurement sensitivity. I understand that

  On the other hand, in the measurement reagent containing the 17K dalton antigen protein-immobilized particles having the signal sequence of the present invention, the obtained absorbance is high, and the 17K dalton antigen protein having the signal sequence in the particles can be easily obtained. It can be seen that a large amount is immobilized.

Therefore, it is confirmed that the antigen protein can be sufficiently immobilized on the particle by using the 17 K Dalton antigen protein having a signal sequence for immobilization on the particle, and the test substance in the sample can be measured with high sensitivity. It was.
Then, it was confirmed that the antibody against Treponema pallidum in the sample can be accurately measured by the measuring reagent of the present invention.

[Example 5] (Confirmation of storage stability of reagent for measuring antibody against Treponema pallidum)

  A measurement reagent containing 17K Dalton antigen protein-immobilized particles having a signal sequence prepared in the same manner as in Example 3 was stored at 5 ° C. for a long period of time, and stability during storage was confirmed.

1. Storage of measurement reagent The measurement reagents prepared in 1 and 2 of Example 3 were stored at 5 ° C for 1 year.

2. Confirmation of the stability of the measurement reagent Measure the test substance (antibodies against Treponema pallidum) in the sample using each of the measurement reagents immediately after preparation and after storage, and confirm the stability of the measurement reagent when stored Went.

3. Reagents The following measurement reagents were used.
(1) The measurement reagent after 1 year of storage at 5 ° C. in 1 above.
(2) Immediately after the measurement reagent is prepared, that is, before the start of storage

4). Sample Human serum containing an antibody against Treponema pallidum was used as a sample.

5. Measurement of test substance (antibodies against Treponema pallidum) in the sample Using the measurement reagents (1) and (2) above, antibodies against Treponema pallidum, which is the test substance, were measured according to the description in Example 4. .

6). Measurement Results Table 5 and FIG. 3 show the absorbance obtained by measuring the test substance (antibody against Treponema pallidum) in the sample.

7). Summary From Table 3 and FIG. 3, in the measurement reagent in which the 17K Dalton antigen protein having the signal sequence was immobilized on the particles, the absorbance value obtained after 1 year of storage at 5 ° C. was immediately after preparation (before the start of storage). It is 100.6% of the absorbance value, and it can be seen that the measurement reagent is stable even after long-term storage and an accurate measurement value can be obtained.

  Therefore, by using a 17 K Dalton antigen protein having a signal sequence, not only can it be easily and in large quantities immobilized on the particle (carrier), but the protein can be stably immobilized on the particle (carrier) for a long period of time. I was able to confirm that

SEQ ID NO: 1: Base sequence of 17K Dalton antigen protein of Treponema pallidum SEQ ID NO: 2: Amino acid sequence of 17K Dalton antigen protein of Treponema pallidum

Claims (12)

A method for immobilizing a protein, comprising expressing a protein having a signal sequence in the base sequence of a gene in a state having the signal sequence, and immobilizing the protein having the signal sequence on a carrier. The protein immobilization method according to claim 1, wherein the protein is a membrane protein or a secreted protein. The protein immobilization method according to claim 1 or 2, wherein the protein is a specific binding substance for a test substance. The method for immobilizing a protein according to claim 3, wherein the test substance is an antibody against Treponema pallidum, and the specific binding substance to the test substance is an antigen of Treponema pallidum. A carrier on which a protein is immobilized, wherein the protein is a protein in which a protein having a signal sequence in a base sequence of a gene is expressed in a state having a signal sequence. . The protein-immobilized carrier according to claim 5, wherein the protein is a membrane protein or a secreted protein. The protein-immobilized carrier according to claim 5 or 6, wherein the protein is a specific binding substance for a test substance. The carrier on which the protein according to claim 7 is immobilized, wherein the test substance is an antibody against Treponema paridum, and the specific binding substance to the test substance is an antigen of Treponema paridum. A reagent for measuring a test substance containing a carrier on which a protein is immobilized, wherein the protein is a protein in which a protein having a signal sequence in a base sequence of a gene is expressed in a state having the signal sequence. A reagent for measuring a test substance. The reagent for measuring a test substance according to claim 9, wherein the protein is a membrane protein or a secreted protein. The reagent for measuring a test substance according to claim 9 or 10, wherein the protein is a specific binding substance for the test substance. The test substance for a test substance according to claim 11, wherein the test substance is an antibody against Treponema pallidum, and the specific binding substance for the test substance is an antigen of Treponema pallidum.

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