JP2005281316A - Immunologic measurement method, reagent for immunologic measurement and method for producing the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for efficiently immobilizing an antigen or antibody on the surface of a carrier, in a method for mixing a specimen with the carrier having the antigen or antibody corresponding to a target antibody or antigen in the specimen, and then measuring an immunologic coagulation reaction-developing state in the mixing process. <P>SOLUTION: An antigen or antibody can efficiently be immobilized on the surface of a pigment by screening an amino acid sequence having a binding ability to a carrier, such as a pigment, from a peptide library, and then fusing a peptide having the amino acid sequence with an antigen or antibody by a genetic engineering method. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to an object to be measured in a sample by detecting or determining an aggregation state after mixing an antibody or antigen corresponding to the object to be measured with an antigen or an antibody and causing an immunological aggregation reaction. This invention relates to a measurement method for the above, a measurement reagent used therefor, and a method for producing the same.

  In the field of clinical tests, various diseases are diagnosed by measuring various components contained in biological samples such as blood and urine. As a method for measuring these, various measuring methods have been developed and used. For example, an enzymatic measuring method using an enzyme reaction, an immunological measuring method using an antigen-antibody reaction, etc. are widely used. . Particularly in recent years, immunoassay methods using highly specific antigen-antibody reactions have been actively used due to their high measurement accuracy and the like.

  On the other hand, in the fields of environmental analysis and food analysis, conventionally, a method of extracting and concentrating a substance to be measured from a sample and then measuring using an analytical instrument such as high performance liquid chromatography or gas chromatography has been generally used. However, in recent years, as seen in problems such as dioxins and endocrine disruptors (so-called environmental hormones), there is a need for measurement of extremely low concentrations of measurement objects, measurement of a wide variety of measurement objects, and rapid measurement of multiple samples. Increasingly, it is becoming difficult to cope with the conventional method. Thus, in recent years, immunoassays using antigen-antibody reactions have begun to be used in the fields of environmental analysis and food analysis.

  Examples of immunoassay methods include immunoagglutination methods (immunoturbidimetry, latex agglutination methods), enzyme immunoassays, radioimmunoassays, and the like, which are properly used according to the purpose. In particular, an immunoagglutination method that detects or determines the degree of aggregation after immobilizing an antigen or antibody on a water-insoluble carrier and mixing it with a specimen to cause an immunological agglutination reaction is not necessarily a large and expensive measuring device. This is widely used as an on-site simple measurement method and the like because it can be easily determined visually. However, in the immunoaggregation method, polystyrene latex or gelatin particles are generally used as the insoluble carrier fine particles (Patent Document 1, Patent Document 2), and the color tone is white or transparent, so let's make a visual judgment. In such cases, there were issues such as requiring specialized skills and training.

As a method for solving the above-described problems, attempts have been made to use a water-insoluble carrier having a color tone that is easy to visually check, such as a pigment or a dye. In JP-A-4-274762, a phthalocyanine dye is used as an insoluble carrier, and an immobilized antibody in which an antibody is immobilized on the fine particles by a physical adsorption method is used, whereby the aggregate after the immunological agglutination reaction is blue. An immunoagglutination measurement method that exhibits color tone and is easy to visually determine is disclosed (Patent Document 3).
JP-A-11-295313 JP 2000-338108 A JP-A-4-274762

  However, when an antigen or antibody is immobilized on an insoluble carrier such as a pigment by physical adsorption, sufficient adsorption power may not be obtained, and there are problems in the production efficiency and storage stability of the reagent for measurement. there were. In general, when an antigen or antibody is to be immobilized by physical adsorption, the hydrophobicity of the antigen or antibody becomes a problem, and it is often difficult to always fix a certain amount. This is a major factor that affects the performance of the measurement system, and also causes a production lot difference.

  For this reason, a method in which the antibody is partially denatured to increase the hydrophobicity and then adsorbed to an insoluble carrier has been attempted (Biochemical Experimental Method 11 Enzyme Immunoassay, Tokyo Chemical Dojin, p270, 1989). However, this method has a problem that the antibody may lose its activity due to denaturation and cannot be applied to all kinds of antibodies.

  An object of the present invention is to perform immunoassay, that is, an antibody or a carrier having an antigen corresponding to an antigen to be measured, and a carrier having the antibody, and an immunological aggregation reaction. An object of the present invention is to provide a measuring method of a measuring object in a specimen, a measuring reagent used therefor, and a method for producing the same, which measure the presence or amount of an antibody or antigen that is a measuring object depending on the state.

  The present inventors screened an amino acid sequence capable of binding to a carrier such as a pigment from a peptide library, and fused the peptide of this amino acid sequence to an antigen or antibody using a genetic engineering technique. As a result of the presentation, it was found that the antigen or the antibody can be efficiently immobilized on the pigment surface, and the present invention has been completed.

  That is, the present invention is a method for measuring the presence or amount of an antibody or antigen of interest in a specimen, comprising preparing a carrier on which an antibody or antigen corresponding to the antigen or antigen in the specimen is immobilized; A step of mixing a carrier and the specimen, and a step of measuring an occurrence state of an immunological agglutination reaction by the mixing step, wherein the antigen or antibody immobilized on the carrier binds to the carrier It is related with the measuring method couple | bonded with the said support | carrier via the amino acid sequence which has an ability.

  The present invention also relates to a measurement reagent for measuring the presence or amount of a target antibody or antigen in a sample, wherein the antigen or antibody corresponding to the antibody or antigen is immobilized on a carrier, and the carrier The antigen or antibody immobilized on the carrier is bound to the carrier via an amino acid sequence capable of binding to the carrier.

  The present invention is also a method for producing a measurement reagent for measuring the presence or amount of a target antibody or antigen in a sample, wherein the antigen or antibody corresponding to the antibody or antigen is immobilized on a carrier. The antigen or antibody having a step and immobilized on the carrier is bound to the carrier via an amino acid sequence having binding ability to the carrier. The present invention relates to a method for producing a measurement reagent.

  Here, the measurement of the presence or amount of the antibody or antigen in the present invention is a measurement of the aggregation state, and includes both the case where the presence or absence of aggregation is measured and the case where the degree of aggregation is measured.

  According to the present invention, since the antigen or antibody can be efficiently immobilized on the surface of the carrier, the production process is extremely efficient.

  As the carrier in the present invention, any conventionally known carrier for antigen or antibody used in antigen-antibody reaction can be used without any particular limitation. For example, organic polymer substances such as organic polymer latex obtained by emulsion polymerization of polystyrene, styrene-butadiene copolymer, styrene-methacrylic acid copolymer, polyglycidyl methacrylate, acrolein-ethylene glycol dimethacrylate copolymer, etc. And inorganic oxides such as silica, silica-alumina, and alumina. Furthermore, pigments can be used as carriers. The pigment is not particularly limited. For example, black pigments such as carbon black, copper oxide, manganese dioxide, aniline black, activated carbon, non-magnetic ferrite, magnetite, yellow lead, zinc yellow, yellow iron oxide, cadmium yellow, mineral fast yellow , Nickel Titanium Yellow, Navels Yellow, Naphthol Yellow S, Hanser Yellow G, Hanser Yellow 10G, Benzidine Yellow G, Benzidine Yellow GR, Quinoline Yellow Lake, Permanent Yellow NCG, Tartrazine Lake, Red Yellow Lead , Molybdenum Orange, Permanent Orange GTR, Pyrazolone Orange, Vulcan Orange, Benzidine Orange G, Indanthrene Brilliant Orange RK, Indanthrene Brilliant Orange GK, etc. Orange Pigment, Ben La, cadmium red lead, mercury sulfide, cadmium, permanent red 4R, risor red, pyrazolone red, watching red, calcium salt, lake red C, lake red D, brilliant carmine 6B, brilliant carmine 3B, oxine lake, rhodamine lake B, red pigments such as alizarin lake, bitumen, cobalt blue, alkali blue lake, Victoria blue lake, phthalocyanine blue, metal-free phthalocyanine blue, phthalocyanine blue partially chlorinated compounds, first sky blue, indanthrene blue BC and other blue pigments, manganese Purple pigments such as purple, fast violet B, methyl violet lake, chromium oxide, chrome green, pigment green B, malachite green lake, phi Use green pigments such as le yellow green G, white pigments such as zinc white, titanium oxide, antimony white, zinc sulfide, and extender pigments such as barite powder, barium carbonate, clay, silica, white carbon, talc, and alumina white. However, it is not limited to these.

  Further, as the carrier, the above organic polymer substance or the like can be used by coloring with a pigment or dye. However, it is desirable to enclose the dye in the polymer so that the dye component does not elute from the carrier when colored with the dye.

  The shape of the carrier can be appropriately selected according to the application. For example, when the measurement target is an antigen and the antibody corresponding to the carrier surface is immobilized, the particle size within the range of 1 nm to 10 μm is used. It is preferable to use particles having a particle diameter in the range of 50 nm to 1 μm.

  In the present invention, the antigen or antibody to the measurement target is not particularly limited, and is a protein or peptide derived from an organism that causes an antigen-antibody reaction with the measurement target, or a protein or peptide partially modified from this. Artificially designed proteins or peptides, or fragments thereof can be used. For example, when the measurement target is an antigen and an antibody corresponding to the pigment surface is immobilized, both the monoclonal antibody and the polyclonal antibody can be used as the antibody. When the measurement target is an antibody and an antigen corresponding to the pigment surface is immobilized, the antigen may be either a protein or a peptide.

  For example, for the purpose of diagnosing hepatitis C virus infection, when using a hepatitis C virus antibody in blood as a measurement target, a known hepatitis C virus antigen that causes an antigen-antibody reaction with the hepatitis C virus antibody Proteins, for example, the group consisting of structural proteins such as hepatitis C virus core protein and envelope protein, NS1 protein, NS2 protein, NS3 protein, NS4 protein, NS5 protein and non-structural proteins and fragments thereof (Proc. Natl Acad.Sci.USA, 89, 10011-10015, 1992) can be used as an antigen or an antibody against the measurement object. Further, even if a part of the amino acid sequence of the antigen protein is modified by addition, deletion, substitution, deletion, insertion, or the like of the amino acid, the antigenicity is provided to a degree sufficient for carrying out the method of the present invention. Can be used.

  In order to obtain an amino acid sequence capable of binding to the carrier of the present invention, for example, the phage display peptide library method described below can be used. As a method for constructing a phage random peptide library, for example, a random synthetic gene may be linked to the N-terminal gene of a surface protein (eg, gene III protein) of an M13 phage. As the method, Scott JK. and Smith GP. , Science, 249, 386, 1990 and Cwirla SE et al. , Proc. Natl. Acad. Sci. USA, 87, 6378, 1990 etc. are reported. The size of the gene to be inserted is not particularly limited as long as the peptide can be stably expressed. However, in order for the prepared library to cover all random sequences and to have binding ability, the length corresponding to 6 to 40 amino acids. (Corresponding to a molecular weight of about 600 to 4,000) is suitable, with 7 to 18 amino acids being preferred.

  In order to select phages that bind to the target carrier, the carrier is immobilized on a column or plate, the library is brought into contact with the carrier, and unbound phages are washed away, leaving bound phages. The phage remaining after washing is eluted with acid or the like, neutralized with a buffer solution, and then infected with E. coli to amplify the phage. When this selection is repeated a plurality of times, a plurality of clones capable of binding to the target carrier are concentrated. Here, in order to obtain a single clone, colonies are made on a medium plate in a state of being again infected with E. coli. After culturing each single colony in a liquid medium, the phage present in the medium supernatant is precipitated and purified with polyethylene glycol or the like, and the base sequence is analyzed to determine the peptide structure. As a method for preparing a peptide library having a random amino acid sequence, a chemically synthesized peptide can be used in addition to the method using a phage as described above. As the method, for example, a method using beads (Lam KS et al., Nature, 354, 82, 1991), a liquid phase focusing method (Houghton RA et al., Nature, 354, 84, 1991), a microplate method ( Fodor SPA et al., Science, 251, 767, 1991) and the like have been reported and any of them can be used in the present invention.

  When two or more types of amino acid sequences capable of binding to the carrier are obtained by screening the phage display peptide library, a sequence in which all or part of these amino acid sequences are connected in series in an appropriate combination is used. Alternatively, it may be used as an amino acid sequence having binding ability to the carrier. In this case, it is desirable to provide an appropriate spacer sequence between the two types of amino acid sequences. The spacer sequence is preferably 3 amino acids to about 400 amino acids, and the spacer sequence may contain any amino acid. Most preferably, the spacer sequence does not inhibit the action between the antigen or antibody and the analyte, and does not prevent the antigen or antibody from binding to the carrier.

  The amino acid sequence capable of binding to the carrier of the present invention can be an amino acid sequence rationally designed according to the chemical nature of the carrier, in addition to the amino acid sequence determined by screening a random peptide library.

  The amino acid sequence having the binding ability to the carrier obtained by the above method is used by fusing to an antigen or antibody for the measurement object using a normal genetic engineering technique. An amino acid sequence capable of binding to a carrier can be expressed by linking to the N-terminus or C-terminus of the protein or peptide that is the antigen or antibody described above. It can also be expressed by inserting an appropriate spacer sequence.

  The spacer sequence is preferably a sequence consisting of 3 amino acids to about 400 amino acids, and the spacer sequence may contain any amino acid. Most preferably, the spacer sequence does not interfere with the action between the antigen or antibody and the analyte, nor does it prevent the antigen or antibody from binding to the carrier.

  As described above, any method for separating and purifying an antigen or antibody containing an amino acid sequence capable of binding to a carrier can be used as long as it retains the activity of the antigen or antibody. it can.

  The step of immobilizing the antigen or antibody for the measurement object on a carrier is achieved by bringing the antigen or antibody containing an amino acid sequence having an ability to bind to the carrier into contact with the carrier in an aqueous medium.

  The composition of the aqueous medium in this step is not particularly limited, but various buffer solutions can be used, for example. Examples of the buffer include general buffers used for biochemical reactions, such as acetate buffer, phosphate buffer, potassium phosphate buffer, 3- (N-morpholino) propanesulfonic acid (MOPS) buffer, N-Tris. (Hydroxymethyl) methyl-3-aminopropane sulfonic acid (TAPS) buffer, Tris hydrochloric acid buffer, glycine buffer, 2- (cyclohexylamino) ethane sulfonic acid (CHES) buffer, etc. are preferably used. The buffer solution can be used at a common concentration, that is, in the range of 5 mM to 1.0 M, and preferably 10 to 200 mM. Further, the pH is adjusted to 5.5 to 9.0, preferably 7.0 to 8.5, but depending on the conditions used, it is not excluded to set conditions other than the above range.

  In addition, in order to maintain the dispersion state of the carrier in the aqueous medium, an appropriate surfactant may be added as long as the type and concentration do not interfere with the subsequent steps. Examples of such surfactants include anionic interfaces such as sodium oleate, sodium dodecyl sulfonate, sodium dodecyl sulfate, sodium dodecyl-N-sarcosine, sodium cholate, sodium deoxycholate, sodium taurodeoxycholate, etc. Activating agent, cationic surfactant such as cetyltrimethylammonium bromide, dodecylpyridinium chloride, 3-[(cholamidopropyl) dimethylammonio] -1-propanesulfonic acid (CHAPS), 3-[(3-cholamidopropyl) ) Dimethylammonio] -2-hydroxy-1-propanesulfonic acid (CHAPSO), palmitoyl lysolecithin, dodecyl-β-alanine and other zwitterionic surfactants, octyl glucoside, octyl thioglucoside, f Tylthioglucoside, decanoyl-N-methylglucamide (MEGA-10), polyoxyethylene dodecyl ether (Brij, Lubrol), polyoxyethylene-i-octylphenyl ether (Triton X), polyoxyethylene nonylphenyl ether (Nonidet) Nonionic surfactants such as P-40, Triton N), polyoxyethylene fatty acid ester (Span), and polyoxyethylene soribitol ester (Tween) can be mentioned.

  In addition, in order to maintain the dispersion state of the powdery carrier in the aqueous medium, an appropriate auxiliary solvent may be added as long as the type and concentration do not interfere with the subsequent steps. As the auxiliary solvent, for example, one kind selected from linear aliphatic hydrocarbons such as hexane, monohydric alcohols such as methanol and ethanol, polyhydric alcohols such as glycerol, fatty acid ethers, and derivatives such as carboxylic acid esters Alternatively, two or more types can be selected and used.

  Immobilization of the antigen or antibody on a carrier is achieved by mixing the carrier and the antigen or antibody in a predetermined aqueous medium so as to have a predetermined concentration. At this time, it is desirable that the reaction vessel is shaken or stirred with an appropriate strength so that the antigen or antibody is evenly adsorbed on the surface of the carrier.

  The amount of immobilization is measured by, for example, adding an antigen or antibody solution of known concentration to the carrier, performing immobilization treatment, measuring the protein concentration in the solution, and determining the amount of immobilization by a subtraction method, etc. May be used.

  The immobilized antigen or the immobilized antibody produced by the above method can be used as it is, but can also be used after lyophilization or the like. The time for immobilizing the antigen or antibody is desirably 1 minute to 24 hours, more desirably 10 minutes to 1 hour. Excessive standing or standing is not preferable because it causes a decrease in the activity of the antigen or antibody.

  In the present invention, an additive such as an organic solvent, a pigment, a dye, an indicator, a surfactant, a preservative, a protective agent, an inorganic salt, or the like may be used together with the above-described constituent elements.

  According to the present invention, since the antigen or antibody can be efficiently immobilized on the surface of the carrier, the production process is extremely efficient.

  In addition, when a coloring material such as a pigment is used as a carrier, it can be a measurement reagent with high quantitativeness and easy visual judgment and high reliability when used for clinical examination, environmental analysis, food analysis, etc. .

  Hereinafter, the present invention will be described more specifically with reference to examples. However, the examples described below are examples of the embodiments of the present invention, and the technical scope of the present invention is not limited to these examples.

  In the following examples, a pigment is used as a carrier. However, the pigment is not limited to a pigment, and any conventional antigen or antibody carrier used in antigen-antibody reactions can be used without any particular limitation. can do.

(Reference Example 1)
Acquisition of antigen protein against hepatitis C virus antibody Guanidium thiocyanate solution (4M guanidinium thiocyanate, 10 mM EDTA, 0.1M 2-mercaptomethanol) in 0.5 ml of human serum that is negative for hepatitis B virus and has a GPT value of 100 units or more 2.5 ml of 2% sarkosyl, 50 mM Tris-HCl pH 7.6) was added, phenol / chloroform extraction was performed, and total RNA in serum was purified by ethanol precipitation using glycogen as a carrier. Next, according to the method of Okamoto et al. (Japan J. Exp. Med., 60 (3), 167-177, 1990), the cDNA of RNA obtained using a random hexamer as a primer was converted into a cDNA synthesis system (Boehringer Mannheim). Manufactured). Using this cDNA as a template, PCR was performed to amplify the target DNA fragment. The 5 ′ end of the amplified DNA fragment was phosphorylated with T4 polynucleotide kinase and then cloned by ligation with pUC18 plasmid (Amersham Pharmacia Biotech) digested with restriction enzyme SmaI. A nucleotide sequence containing a gene encoding the target antigen protein NS4 from the nucleotide sequence determined by the sequence sequence kit (manufactured by United States Biochemical), the nucleotide sequence of the hepatitis C virus cDNA cloned in the plasmid Was inserted into the SmaI cleavage site of plasmid pUC18 according to a conventional method to construct plasmid pUC18-NS.

(Example 1)
Acquisition of amino acid sequence having binding ability to copper phthalocyanine (1) Copper phthalocyanine (α type, manufactured by Tokyo Chemical Industry Co., Ltd.) TBS buffer containing 0.1% Tween-20 (50 mM Tris-HCl pH 7.5, 150 mM) (NaCl) to a concentration of 5 mg / ml. 10 μl of this was added to an Eppendorf tube, and diluted by adding 990 μl TBST buffer (TBS buffer + 0.1% Tween-20).
(2) Ph. D. A 4 × 10 10 pfu equivalent of a −12 phage display peptide library (New England BioLabs) was added to the tube and allowed to stand at 25 ° C. for 10 minutes.
(3) After centrifuging the tube (20,630 × g, 5 minutes), the supernatant was discarded and the pigment was recovered as a precipitate. The collected pigment was suspended again in TBST buffer and centrifuged repeatedly to wash the pigment 10 times with TBST buffer.
(4) Add 100 μl of elution buffer (0.2 M Glycine-HCl pH 2.2, 1 mg / ml BSA) and let stand for 1 minute, then centrifuge (20,630 × g, 5 minutes) and remove supernatant. It was transferred to another Eppendorf tube and neutralized by adding 15 μl of 1M Tris-HCl (pH 9.1) to obtain eluted phages.
(5) The eluted phages were infected with E. coli ER2537 (manufactured by New England BioLabs) in the early logarithmic growth and amplified. The culture was performed at 37 ° C. for 4.5 hours. The phage was then separated from the cells by centrifugation and purified by polyethylene glycol precipitation. The purified and amplified phages were suspended in TBS buffer, and titers were measured by infecting E. coli with an appropriate dilution series.
(6) The above (1) to (5) were repeated three more times using the amplified phage. However, the washing conditions were tightened by increasing the concentration of Tween-20 in the TBST buffer used to 0.5%.

  From the second time, the same operation was performed on the Eppendorf tube as a control. The titers of phage eluted in each cycle are shown in Table 1.

  The final eluted phage was cloned by infecting a large excess of E. coli. After each clone is infected with Escherichia coli and amplified, ssDNA is prepared, the nucleotide sequence of the random region is decoded, and the amino acid sequence of the displayed peptide is determined, so that the amino acid sequence has binding ability to copper phthalocyanine Acquired. The resulting amino acid sequence and frequency are shown in Table 2.

(Example 2)
Acquisition of amino acid sequence having binding ability to carbon black (1) 5 mg of carbon black (manufactured by Sigma Aldrich Japan) in TBS buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl) containing 0.1% Tween-20 Suspended to a concentration of / ml. 10 μl of this was added to an Eppendorf tube, and diluted by adding 990 μl TBST buffer (TBS buffer + 0.1% Tween-20).
(2) Ph. D. A 4 × 10 10 pfu equivalent of a −12 phage display peptide library (New England BioLabs) was added to the tube and allowed to stand at 25 ° C. for 10 minutes.
(3) After centrifuging the tube (20,630 × g, 5 minutes), the supernatant was discarded and the pigment was recovered as a precipitate. The collected pigment was suspended again in TBST buffer and centrifuged repeatedly to wash the pigment 10 times with TBST buffer.
(4) Add 100 μl of elution buffer (0.2 M Glycine-HCl pH 2.2, 1 mg / ml BSA) and let stand for 1 minute, then centrifuge (20,630 × g, 5 minutes) and remove supernatant. It was transferred to another Eppendorf tube and neutralized by adding 15 μl of 1M Tris-HCl (pH 9.1) to obtain eluted phages.
(5) The eluted phages were infected with E. coli ER2537 (manufactured by New England BioLabs) in the early logarithmic growth and amplified. The culture was performed at 37 ° C. for 4.5 hours. The phage was then separated from the cells by centrifugation and purified by polyethylene glycol precipitation. The purified and amplified phages were suspended in TBS buffer, and titers were measured by infecting E. coli with an appropriate dilution series.
(6) The above (1) to (5) were repeated three more times using the amplified phage. However, the washing conditions were tightened by increasing the concentration of Tween-20 in the TBST buffer used to 0.5%.

  From the second time, the same operation was performed on the Eppendorf tube as a control. The titers of phage eluted in each cycle are shown in Table 3.

  The final eluted phage was cloned by infecting a large excess of E. coli. After each clone was infected with E. coli and amplified, ssDNA was prepared, and the amino acid sequence capable of binding to carbon black was obtained by decoding the base sequence of the random region. The resulting amino acid sequence and frequency are shown in Table 4.

(Example 3)
Production of antigenic protein having binding ability to copper phthalocyanine With respect to the base sequence encoding antigenic protein NS4 in pUC18-NS of plasmid reference example 1, an upstream primer (5′-TTCACAGATCCCACTGAGCTCGATGCCCCAC-3 ′) and a downstream primer ( 5'-GATCTGGGCTCGAGCCGACTAGTAGGTCGCT-3 '), PCR was performed using plasmid pUC18-NS as a template, and a DNA fragment containing the antigen protein NS4 gene having BamHI and SacI restriction sites upstream and SpeI and XhoI restriction sites downstream. Obtained.

  Each purified PCR amplification product was digested with BamHI and XhoI, and inserted into the corresponding site of plasmid pGEX-6P-1 (Amersham Pharmacia Biotech) to obtain vector pGEX-NS.

  On the other hand, for each amino acid sequence of Example 1 (SEQ ID NO: 1 to SEQ ID NO: 15), an Escherichia coli expression vector expressed by fusing to the N-terminus of the antigen protein NS4 via the spacer sequence GS is as follows. And built. In order to prepare DNAs encoding these amino acid sequences as double-stranded synthetic DNAs, a set of synthetic oligonucleotides listed in Table 5 below was prepared.

  Two synthetic DNAs listed in Table 5 for each amino acid sequence were phosphorylated using T4 polynucleotide kinase (Gibco) according to the manufacturer's instructions. Subsequently, equimolar amounts of the two synthetic DNAs were mixed, heated at 80 ° C. for 5 minutes, and then slowly cooled to room temperature to form double-stranded DNA fragments. The formed double-stranded DNA fragment was directly used for subsequent cloning.

  Plasmid pGEX-NS was digested with BamHI and SacI, and any one of the double-stranded DNA fragments was inserted. Using this vector, E. coli (JM109), which is a host microorganism, was transformed, and a strain for expression was obtained as a transformant. The strain was confirmed by sequencing using a base sequence of pGEX 5 ′ Sequencing Primer (manufactured by Amersham Pharmacia Biotech) using a plasmid DNA prepared using Miniprep (Wizard Minipreps DNA Purification Systems, manufactured by PROMEGA). By making a decision. The obtained strain was precultured overnight with 10 ml of LB-Amp medium, and 0.1 ml thereof was added to 10 ml of LB-Amp medium and cultured with shaking at 37 ° C. and 170 rpm for 3 hours. Thereafter, IPTG was added (final concentration 1 mM), and the culture was continued at 37 ° C. for 4 to 12 hours.

  E. coli induced by IPTG was collected (8,000 × g, 2 minutes, 4 ° C.) and resuspended in 1/10 amount of PBS at 4 ° C. The cells were disrupted by freeze-thawing and sonication, and centrifuged (8,000 × g, 10 minutes, 4 ° C.) to remove solid impurities. After confirming that the target expressed protein was present in the supernatant by SDS-PAGE, the induced and expressed GST fusion protein was purified by glutathione Sepharose 4B (Glutathion Sepharose 4B beads: manufactured by Amersham Pharmacia Biotech).

  The glutathione sepharose used was previously treated to suppress nonspecific adsorption. That is, glutathione sepharose was washed three times with the same amount of PBS (8,000 × g, 1 minute, 4 ° C.), and then the same amount of 4% BSA-containing PBS was added and treated at 4 ° C. for 1 hour. After the treatment, it was washed twice with the same amount of PBS and resuspended in 1/2 amount of PBS. 40 μl of pretreated glutathione sepharose was added to 1 ml of cell-free extract and gently stirred at 4 ° C. Thereby, GST fusion protein was made to adsorb | suck to glutathione sepharose.

  After adsorption, the mixture was centrifuged (8,000 × g, 1 minute, 4 ° C.) to collect glutathione sepharose and washed 3 times with 400 μl of PBS. Thereafter, 40 μl of 10 mM glutathione was added and stirred at 4 ° C. for 1 hour to elute the adsorbed GST fusion protein. The supernatant was collected by centrifugation (8,000 × g, 2 minutes, 4 ° C.) and then dialyzed against PBS to purify the GST fusion protein. It was confirmed by SDS-PAGE that a single band was exhibited.

  Each GST fusion protein (500 μg) was digested with PreScission protease (manufactured by Amersham Pharmacia Biotech, 5 U), and then passed through glutathione sepharose to remove PreScission protease and GST. The flow-through fraction was further applied to a Sephadex G200 column equilibrated with PBS to obtain a final purified product of the expressed protein. It was confirmed by SDS-PAGE that a single band was exhibited.

Example 4
Production of antigen protein having binding ability to carbon black With respect to the base sequence encoding antigen protein NS4 in plasmid pUC18-NS, an upstream primer (5'-TTCCAGGATCCACTGAGCTCGATGCCCCAC-3 ') and a downstream primer (5'-GATCTGGGCTCGAGCCGACTAGTAGTAGTCCT -3 '), PCR was performed using the plasmid pUC18-NS as a template to obtain a DNA fragment containing the antigen protein NS4 gene having BamHI and SacI restriction sites upstream and SpeI and XhoI restriction sites downstream.

  Each purified PCR amplification product was digested with BamHI and XhoI, and inserted into the corresponding site of plasmid pGEX-6P-1 (Amersham Pharmacia Biotech) to obtain vector pGEX-NS.

  On the other hand, for each amino acid sequence of Example 2 (SEQ ID NO: 16 to SEQ ID NO: 40), an Escherichia coli expression vector expressed by fusing to the N-terminus of the antigen protein NS4 via the spacer sequence GS is as follows. And built. In order to prepare DNAs encoding these amino acid sequences as double-stranded synthetic DNAs, a set of synthetic oligonucleotides listed in Table 6 below was prepared.

  Two synthetic DNAs listed in Table 6 for each amino acid sequence were phosphorylated using T4 polynucleotide kinase (Gibco) according to the manufacturer's instructions. Subsequently, equimolar amounts of the two synthetic DNAs were mixed, heated at 80 ° C. for 5 minutes, and then slowly cooled to room temperature to form double-stranded DNA fragments. The formed double-stranded DNA fragment was directly used for subsequent cloning.

  Plasmid pGEX-NS was digested with BamHI and SacI, and the double-stranded DNA fragment was inserted. Escherichia coli (JM109) was transformed with this vector to obtain an expression strain. The strain was confirmed by sequencing using a base sequence of pGEX 5 ′ Sequencing Primer (manufactured by Amersham Pharmacia Biotech) using a plasmid DNA prepared using Miniprep (Wizard Minipreps DNA Purification Systems, manufactured by PROMEGA). By making a decision. The obtained strain was precultured overnight with 10 ml of LB-Amp medium, and 0.1 ml thereof was added to 10 ml of LB-Amp medium and cultured with shaking at 37 ° C. and 170 rpm for 3 hours. Thereafter, IPTG was added (final concentration 1 mM), and the culture was continued at 37 ° C. for 4 to 12 hours.

  E. coli induced by IPTG was collected (8,000 × g, 2 minutes, 4 degrees) and resuspended in 1/10 volume of 4 ° C. PBS. The cells were disrupted by freeze-thawing and sonication, and centrifuged (8,000 × g, 10 minutes, 4 ° C.) to remove solid impurities. After confirming that the target expressed protein was present in the supernatant by SDS-PAGE, the induced and expressed GST fusion protein was purified by glutathione Sepharose 4B (Glutathion Sepharose 4B beads: manufactured by Amersham Pharmacia Biotech).

  The glutathione sepharose used was previously treated to suppress nonspecific adsorption. That is, glutathione sepharose was washed three times with the same amount of PBS (8,000 × g, 1 minute, 4 ° C.), and then the same amount of 4% BSA-containing PBS was added and treated at 4 ° C. for 1 hour. After the treatment, it was washed twice with the same amount of PBS and resuspended in 1/2 amount of PBS. 40 μl of pretreated glutathione sepharose was added to 1 ml of cell-free extract and gently stirred at 4 ° C. Thereby, GST fusion protein was made to adsorb | suck to glutathione sepharose.

  After adsorption, the mixture was centrifuged (8,000 × g, 1 minute, 4 ° C.) to collect glutathione sepharose and washed 3 times with 400 μl of PBS. Thereafter, 40 μl of 10 mM glutathione was added and stirred at 4 ° C. for 1 hour to elute the adsorbed GST fusion protein. The supernatant was collected by centrifugation (8,000 × g, 2 minutes, 4 degrees), and then dialyzed against PBS to purify the GST fusion protein. It was confirmed by SDS-PAGE that a single band was exhibited.

  Each GST fusion protein (500 μg) was digested with PreScission protease (manufactured by Amersham Pharmacia Biotech, 5 U), and then passed through glutathione sepharose to remove PreScission protease and GST. The flow-through fraction was further applied to a Sephadex G200 column equilibrated with PBS to obtain a final purified product of the expressed protein. It was confirmed by SDS-PAGE that a single band was exhibited.

(Example 5)
Production of antigenic protein having binding ability to copper phthalocyanine Two amino acid sequences having binding ability to copper phthalocyanine, Lys-Tyr-Asp-Ser-Arg-His-Leu-His-Thr-His-Ser-His (SEQ ID NO: : 1) and Pro-Asn-Arg-Leu-Gly-Arg-Arg-Pro-Val-Arg-Trp-Glu (SEQ ID NO: 2) all together with the spacer sequence (Gly-Gly-Gly-Ser-Gly- Gly-Gly-Ser), a sequence connected in series in this order, Lys-Tyr-Asp-Ser-Arg-His-Leu-His-Thr-His-Ser-His-Gly-Gly-Gly-Ser- Gly-Gly-Gly-Ser-Pro-Asn-Arg-Leu-Gly -An Escherichia coli expression vector that is expressed by fusing Arg-Arg-Pro-Val-Arg-Trp-Glu (SEQ ID NO: 41) to the N-terminus of the antigen protein NS4 via the spacer sequence GS as follows. And built. DNA encoding this amino acid sequence, two types of synthetic oligonucleotides was phosphorylated using 5'-GATCCAAATATGATAGCCGTCATCTGCATACCCATAGCCATGGCGGCGGCAGCGGCGGCGGCAGCCCGAACCGTCTGGGCCGTCGTCCGGTGCGTTGGGAAGAGCT-3 'and 5'-CTTCCCAACGCACCGGACGACGGCCCAGACGGTTCGGGCTGCCGCCGCCGCTGCCGCCGCCATGGCTATGGGTATGCAGATGACGGCTATCATATTTG-3', respectively T4 polynucleotide kinase (manufactured by Gibco), etc. Double-stranded D by mixing in moles and heating at 80 ° C. for 5 minutes, followed by slow cooling to room temperature It was formed as an NA fragment. The formed double-stranded DNA fragment was inserted into the BamHI / SacI site of plasmid pGEX-NS in the same manner as in Example 3, and Escherichia coli (JM109) was transformed with this vector to obtain a strain for expression. . In the same manner as in Example 3, the expressed protein in which the amino acid sequence of SEQ ID NO: 41 was fused to the N-terminus was purified.

(Example 6)
Production of antigenic protein having binding ability to carbon black Two types of amino acid sequences having binding ability to carbon black, Trp-Pro-His-Ala-Trp-Lys-Val-Trp-Trp-Pro-Ala-Ser (SEQ ID NO: : 16) and Asn-Trp-Trp-Trp-Pro-Pro-Tyr-Ile-Arg-His-Gln-Pro (SEQ ID NO: 17) all of the spacer sequence (Gly-Gly-Gly-Ser-Gly- Gly-Gly-Ser) in this order in series, Trp-Pro-His-Ala-Trp-Lys-Val-Trp-Trp-Pro-Ala-Ser-Gly-Gly-Gly-Ser- Gly-Gly-Gly-Ser-Asn-Trp-Trp-Trp-P An E. coli expression vector that expresses ro-Pro-Tyr-Ile-Arg-His-Gln-Pro (SEQ ID NO: 42) via the spacer sequence GS and fused to the N-terminus of antigen protein NS4 is as follows: And built. DNA encoding this amino acid sequence, two types of synthetic oligonucleotides was phosphorylated using 5'-GATCCTGGCCGCATGCGTGGAAAGTGTGGTGGCCGGCGAGCGGCGGCGGCAGCGGCGGCGGCAGCAACTGGTGGTGGCCGCCGTATATTCGTCATCAGCCGGAGCT-3 'and 5'-CCGGCTGATGACGAATATACGGCGGCCACCACCAGTTGCTGCCGCCGCCGCTGCCGCCGCCGCTCGCCGGCCACCACACTTTCCACGCATGCGGCCAG-3', respectively T4 polynucleotide kinase (manufactured by Gibco), etc. Double-stranded D by mixing in moles and heating at 80 ° C. for 5 minutes, followed by slow cooling to room temperature It was formed as an NA fragment. The formed double-stranded DNA fragment was inserted into the BamHI / SacI site of the plasmid pGEX-NS in the same manner as in Example 4, and Escherichia coli (JM109) was transformed with this vector to obtain a strain for expression. . In the same manner as in Example 4, the expressed protein in which the amino acid sequence of SEQ ID NO: 42 was fused to the N-terminus was purified.

(Comparative Example 1)
Production of antigen protein Using the upstream primer (5'-TTCACAGGATCCACTGAGCTCGATGCCCCAC-3 ') and the downstream primer (5'-GATCTGGGCTCGAGCCGACTAGTAGTCTCCT-3') with respect to the base sequence encoding the antigen protein NS4 in the plasmid pUC18-NS Then, PCR was performed using plasmid pUC18-NS as a template to obtain a DNA fragment containing the antigen protein NS4 gene having BamHI and SacI restriction sites upstream and SpeI and XhoI restriction sites downstream.

  Each purified PCR amplification product was digested with BamHI and XhoI and inserted into the corresponding site of plasmid pGEX-6P-1 (Amersham Pharmacia Biotech). Escherichia coli (JM109) was transformed with this vector pGEX-NS to obtain an expression strain. The strain was confirmed by DNA fragments obtained by treating plasmid DNA prepared in large quantities using Miniprep (Wizard Minipreps DNA Purification Systems, manufactured by PROMEGA) with BamHI and XhoI.

  The obtained strain was precultured overnight with 10 ml of LB-Amp medium, and 0.1 ml thereof was added to 10 ml of LB-Amp medium and cultured with shaking at 37 ° C. and 170 rpm for 3 hours. Thereafter, IPTG was added (final concentration 1 mM), and the culture was continued at 37 ° C. for 4 to 12 hours.

  E. coli induced by IPTG was collected (8,000 × g, 2 minutes, 4 degrees) and resuspended in 1/10 volume of 4 ° C. PBS. The cells were disrupted by freeze-thawing and sonication, and centrifuged (8,000 × g, 10 minutes, 4 ° C.) to remove solid impurities. After confirming that the target expressed protein was present in the supernatant by SDS-PAGE, the induced and expressed GST fusion protein was purified by glutathione Sepharose 4B (Glutathion Sepharose 4B beads: manufactured by Amersham Pharmacia Biotech).

  The glutathione sepharose used was previously treated to suppress nonspecific adsorption. That is, glutathione sepharose was washed three times with the same amount of PBS (8,000 × g, 1 minute, 4 ° C.), and then the same amount of 4% BSA-containing PBS was added and treated at 4 ° C. for 1 hour. After the treatment, it was washed twice with the same amount of PBS and resuspended in 1/2 amount of PBS. 40 μl of pretreated glutathione sepharose was added to 1 ml of cell-free extract and gently stirred at 4 ° C. Thereby, GST fusion protein was made to adsorb | suck to glutathione sepharose.

  After adsorption, the mixture was centrifuged (8,000 × g, 1 minute, 4 ° C.) to collect glutathione sepharose and washed 3 times with 400 μl of PBS. Thereafter, 40 μl of 10 mM glutathione was added and stirred at 4 ° C. for 1 hour to elute the adsorbed GST fusion protein. The supernatant was collected by centrifugation (8,000 × g, 2 minutes, 4 degrees), and then dialyzed against PBS to purify the GST fusion protein. It was confirmed by SDS-PAGE that a single band was exhibited.

  Each GST fusion protein (500 μg) was digested with PreScission protease (manufactured by Amersham Pharmacia Biotech, 5 U), and then passed through glutathione sepharose to remove PreScission protease and GST. The flow-through fraction was further applied to a Sephadex G200 column equilibrated with PBS to obtain a final purified product of the expressed protein. It was confirmed by SDS-PAGE that a single band was exhibited.

(Example 7)
Immobilization of antigen protein on copper phthalocyanine particles Copper phthalocyanine particles were suspended in a TBS buffer containing 0.1% Tween-20 so as to be 0.5% (w / v). 10 ml of this was placed in a centrifuge tube made of Teflon (registered trademark), and 50 μg of the fusion protein prepared in Example 3 or the protein prepared in Comparative Example 1 was added thereto, followed by shaking at room temperature for 30 minutes. Copper phthalocyanine particles were recovered as a precipitate by centrifugation (10,000 × g, 4 ° C., 10 minutes), and separated from the supernatant containing the enzyme that did not bind to the copper phthalocyanine particles. The copper phthalocyanine particles were suspended again in a TBS buffer containing 0.1% Tween-20, and the copper phthalocyanine particles were washed by repeating the centrifugal operation. The protein concentration of the washed copper phthalocyanine particle suspension was measured with a micro BCA protein quantitative reagent kit (Pierce Chemical Co., Ltd.). Table 7 shows the measurement results.

  Compared with the case of using the protein of Comparative Example 1, the case of using the fusion protein of Example 3 and Example 5 in which the copper phthalocyanine binding sequence is fused has a higher protein concentration, and the antigenic protein is more effectively applied to the pigment surface. It was confirmed that it can be fixed to.

(Example 8)
Immobilization of antigen protein on carbon black particles Carbon black particles were suspended in a TBS buffer containing 0.1% Tween-20 so as to be 0.5% (w / v). 10 ml of this was placed in a centrifuge tube made of Teflon (registered trademark), and 50 μg of the fusion protein prepared in Example 6 or the protein prepared in Comparative Example 1 was added thereto, followed by shaking at room temperature for 30 minutes. The carbon black particles were collected as a precipitate by centrifugation (10,000 × g, 4 ° C., 10 minutes), and separated from the supernatant containing the enzyme that did not bind to the carbon black particles. The carbon black particles were washed again by suspending them in a TBS buffer containing 0.1% Tween-20 and repeating the centrifugal operation. The protein concentration of the washed carbon black particle suspension was measured with a micro BCA protein quantitative reagent kit (Pierce Chemical Co., Ltd.). Table 8 shows the measurement results.

  Compared with the case of using the protein of Comparative Example 1, the case of using the fusion protein of Example 4 and Example 6 in which the carbon black binding sequence is fused has a higher protein concentration, and the antigenic protein is effectively applied to the pigment surface. It was confirmed that it can be fixed to.

Example 9
Measurement of hepatitis C virus antibody by immunoagglutination method 0.1 M potassium phosphate buffer (pH 6.5), 1.0% bovine serum albumin (manufactured by Sigma-Aldrich Japan), 0.05% NaN3 blocking buffer The procedure of suspending the pigment particles immobilized with the fusion protein produced in Example 8 to Example 11 in 10 ml and collecting the pigment particles by centrifugation (10,000 × g, 4 ° C., 10 minutes) is 3 This was washed repeatedly and suspended in 4 ml of blocking buffer.

  Comparison between the measurement reagent prepared as described above and a diagnostic reagent using a commercially available immunoaggregation method (Ortho HCV Ab PAII, manufactured by Ortho Diagnostics Systems, hereinafter referred to as “commercial product”) The test was performed using serum from a patient positive for hepatitis B virus antibody. The test method followed the manual for the commercial product. The results are shown in Table 9 and Table 10.

  From the results of Table 9 and Table 10, it was found that the measurement reagent of the present invention has a sensitivity equal to or higher than that of a commercially available product. Furthermore, the measuring reagent of the present invention has a color tone because the insoluble carrier is a pigment, and visual determination is very easy.

In the above embodiment, the measurement is performed visually, but the aggregation state may be measured using visual image recognition means such as a CCD. In this case, the aggregation state can be measured without using a coloring material for the carrier.
[Sequence Listing]
SEQ ID NO: 1
Sequence length: 12
Sequence Type: Amino Acid Topology: Linear Sequence Type: Peptide Sequence Lys Tyr Asp Ser Arg His Leu His
1 5
Thr His Ser His
10
SEQ ID NO: 2
Sequence length: 12
Sequence type: Amino acid Topology: Linear sequence type: Peptide sequence Pro Asn Arg Leu Gly Arg Arg Pro
1 5
Val Arg Trp Glu
10
SEQ ID NO: 3
Sequence length: 12
Sequence Type: Amino Acid Topology: Linear Sequence Type: Peptide Sequence Lys Cys Cys Tyr Tyr Asp His Ser
1 5
His Ala Leu Ser
10
SEQ ID NO: 4
Sequence length: 12
Sequence type: Amino acid Topology: Linear sequence type: Peptide sequence Glu Tyr Leu Ser Ala Ile Val Ala
1 5
Gly Pro Trp Pro
10
SEQ ID NO: 5
Sequence length: 12
Sequence Type: Amino Acid Topology: Linear Sequence Type: Peptide Sequence Lys Leu Trp Ile Leu Glu Pro Thr
1 5
Val Thr Pro Thr
10
SEQ ID NO: 6
Sequence length: 12
Sequence type: Amino acid Topology: Linear sequence type: Peptide sequence Gln Ser Asn Leu Lys Val Ile Pro
1 5
Ser Trp Trp Phe
10
SEQ ID NO: 7
Sequence length: 12
Sequence type: Amino acid Topology: Linear sequence type: Peptide sequence Trp Ile Pro Pro Gln Trp Ser Arg
1 5
Leu Ile Glu Pro
10
SEQ ID NO: 8
Sequence length: 12
Sequence Type: Amino Acid Topology: Linear Sequence Type: Peptide Sequence Asp His Pro Gln Ala Lys Pro Asn
1 5
Trp Tyr Gly Val
10
SEQ ID NO: 9
Sequence length: 12
Sequence type: Amino acid Topology: Linear sequence type: Peptide sequence Gly Leu Pro Pro Tyr Ser Pro His
1 5
Arg Leu Ala Gln
10
SEQ ID NO: 10
Sequence length: 12
Sequence Type: Amino Acid Topology: Linear Sequence Type: Peptide Sequence Lys Leu Thr Thr Gln Tyr Met Ala
1 5
Arg Ser Ser Ser
10
SEQ ID NO: 11
Sequence length: 12
Sequence Type: Amino Acid Topology: Linear Sequence Type: Peptide Sequence Lys Val Trp Met Leu Pro Pro Leu
1 5
Pro Gln Ala Thr
10
SEQ ID NO: 12
Sequence length: 12
Sequence type: Amino acid Topology: Linear sequence type: Peptide sequence Asn Val Thr Ser Thr Ala Phe Ile
1 5
Asp Thr Pro Trp
10
SEQ ID NO: 13
Sequence length: 12
Sequence type: Amino acid Topology: Linear sequence type: Peptide sequence Arg Leu Asn Leu Asp Ile Ile Ala
1 5
Val Thr Ser Val
10
SEQ ID NO: 14
Sequence length: 12
Sequence type: Amino acid Topology: Linear sequence type: Peptide sequence Thr Leu Pro Ser Pro Leu Ala Leu
1 5
Leu Thr Val His
10
SEQ ID NO: 15
Sequence length: 12
Sequence Type: Amino Acid Topology: Linear Sequence Type: Peptide Sequence Thr Asn Arg His Asn Pro His His
1 5
Leu His His Val
10
SEQ ID NO: 16
Sequence length: 12
Sequence type: Amino acid Topology: Linear sequence type: Peptide sequence Trp Pro His Ala Trp Lys Val Trp
1 5
Trp Pro Ala Ser
10
SEQ ID NO: 17
Sequence length: 12
Sequence type: Amino acid Topology: Linear sequence type: Peptide sequence Asn Trp Trp Trp Pro Pro Tyr Ile
1 5
Arg His Gln Pro
10
SEQ ID NO: 18
Sequence length: 12
Sequence type: Amino acid Topology: Linear sequence type: Peptide sequence Trp His Trp Ser Trp Thr Pro Trp
1 5
Pro Ser His His
10
SEQ ID NO: 19
Sequence length: 12
Sequence type: Amino acid Topology: Linear sequence type: Peptide sequence Trp Pro Trp Ala Trp His Pro Ser
1 5
Arg Asp Val Tyr
10
SEQ ID NO: 20
Sequence length: 12
Sequence Type: Amino Acid Topology: Linear Sequence Type: Peptide Sequence Trp His Gly Tyr Trp Tyr Ser Asn
1 5
Leu Asn Thr Thr
10
SEQ ID NO: 21
Sequence length: 12
Sequence Type: Amino Acid Topology: Linear Sequence Type: Peptide Sequence Trp Trp Thr Pro Trp Met Ser His
1 5
Ala Tyr Pro Val
10
SEQ ID NO: 22
Sequence length: 12
Sequence type: Amino acid Topology: Linear sequence type: Peptide sequence Trp Pro Asn Pro Tyr Trp Gly Trp
1 5
Phe Ala Ala Val
10
SEQ ID NO: 23
Sequence length: 12
Sequence type: Amino acid Topology: Linear sequence type: Peptide sequence Thr Ser Trp His Thr Trp Trp Trp
1 5
Arg Gln Pro Pro
10
SEQ ID NO: 24
Sequence length: 12
Sequence Type: Amino Acid Topology: Linear Sequence Type: Peptide Sequence Asn Ala Trp His Lys Tyr Trp Trp
1 5
Pro Ile Thr Lys
10
SEQ ID NO: 25
Sequence length: 12
Sequence Type: Amino Acid Topology: Linear Sequence Type: Peptide Sequence His Pro Asn Asn Asp Trp Ser Lys
1 5
Ala Pro Gln Phe
10
SEQ ID NO: 26
Sequence length: 12
Sequence type: Amino acid Topology: Linear sequence type: Peptide sequence Trp Trp Thr Pro Gln Pro Trp Trp
1 5
Ser Phe Pro Ile
10
SEQ ID NO: 27
Sequence length: 12
Sequence type: Amino acid Topology: Linear sequence type: Peptide sequence Trp Pro His Thr Ser Trp Trp Gln
1 5
Thr Pro Leu Thr
10
SEQ ID NO: 28
Sequence length: 12
Sequence type: Amino acid Topology: Linear sequence type: Peptide sequence Trp His Val Asn Trp Asp Pro Met
1 5
Ala Trp Tyr Arg
10
SEQ ID NO: 29
Sequence length: 12
Sequence Type: Amino Acid Topology: Linear Sequence Type: Peptide Sequence Ser Trp Pro Trp Trp Thr Ala Tyr
1 5
Arg Val His Ser
10
SEQ ID NO: 30
Sequence length: 12
Sequence Type: Amino Acid Topology: Linear Sequence Type: Peptide Sequence Trp His Ser Asn Trp Tyr Gln Ser Ile 1 5
Pro Gln Val
10
SEQ ID NO: 31
Sequence length: 12
Sequence type: Amino acid Topology: Linear sequence type: Peptide sequence Gly Tyr Trp Pro Trp Lys Phe Glu
1 5
His Ala Thr Val
10
SEQ ID NO: 32
Sequence length: 12
Sequence type: Amino acid Topology: Linear sequence type: Peptide sequence Ala Trp Trp Pro Thr Thr Phe Pro
1 5
Pro Tyr Tyr Tyr
10
SEQ ID NO: 33
Sequence length: 12
Sequence Type: Amino Acid Topology: Linear Sequence Type: Peptide Sequence Asn Pro Trp Trp Ser His Tyr Tyr Pro
1 5
Arg Ser Val
10
SEQ ID NO: 34
Sequence length: 12
Sequence type: Amino acid Topology: Linear sequence type: Peptide sequence Trp Pro His Asn Tyr Pro Leu Asn
1 5
His Ser Asn Pro
10
SEQ ID NO: 35
Sequence length: 12
Sequence type: Amino acid Topology: Linear sequence type: Peptide sequence Thr Trp Ala His Pro Leu Glu Ser
1 5
Asp Tyr Leu Arg
10
SEQ ID NO: 36
Sequence length: 12
Sequence Type: Amino Acid Topology: Linear Sequence Type: Peptide Sequence His Thr Tyr Tyr His Asp Gly Trp
1 5
Arg Leu Ala Pro
10
SEQ ID NO: 37
Sequence length: 12
Sequence type: Amino acid Topology: Linear sequence type: Peptide sequence Thr Phe Val Gln Thr Pro Leu Ser
1 5
His Leu Ile Ala
10
SEQ ID NO: 38
Sequence length: 12
Sequence Type: Amino Acid Topology: Linear Sequence Type: Peptide Sequence Arg Val Pro Pro Ser Lys Leu Thr
1 5
Arg Pro Pro Phe
10
SEQ ID NO: 39
Sequence length: 12
Sequence Type: Amino Acid Topology: Linear Sequence Type: Peptide Sequence His Ser Ile Tyr Ser Val Thr Pro
1 5
Ser Thr Ala Ser
10
SEQ ID NO: 40
Sequence length: 12
Sequence Type: Amino Acid Topology: Linear Sequence Type: Peptide Sequence Leu Asn Thr Gln Asn His His Ala Pro
1 5
Leu Pro Ser Ile
10
SEQ ID NO: 41
Sequence length: 32
Sequence Type: Amino Acid Topology: Linear Sequence Type: Peptide Sequence Lys Tyr Asp Ser Arg His Leu His
1 5
Thr His Ser His Gly Gly Gly Ser
10 15
Gly Gly Gly Ser Pro Asn Arg Leu
20
Gly Arg Arg Pro Val Arg Trp Glu
25 30
SEQ ID NO: 42
Sequence length: 32
Sequence type: Amino acid Topology: Linear sequence type: Peptide sequence Trp Pro His Ala Trp Lys Val Trp
1 5
Trp Pro Ala Ser Gly Gly Gly Ser
10 15
Gly Gly Gly Ser Asn Trp Trp Trp
20
Pro Pro Tyr Ile Arg His Gln Pro
25 30

Claims (2)

The amino acid sequence is
Lys-Tyr-Asp-Ser-Arg-His-Leu-His-Thr-His-Ser-His (SEQ ID NO: 1)
Pro-Asn-Arg-Leu-Gly-Arg-Arg-Pro-Val-Arg-Trp-Glu (SEQ ID NO: 2)
Lys-Cys-Cys-Tyr-Tyr-Asp-His-Ser-His-Ala-Leu-Ser (SEQ ID NO: 3)
Glu-Tyr-Leu-Ser-Ala-Ile-Val-Ala-Gly-Pro-Trp-Pro (SEQ ID NO: 4)
Lys-Leu-Trp-Ile-Leu-Glu-Pro-Thr-Val-Thr-Pro-Thr (SEQ ID NO: 5)
Gln-Ser-Asn-Leu-Lys-Val-Ile-Pro-Ser-Trp-Trp-Phe (SEQ ID NO: 6)
Trp-Ile-Pro-Pro-Gln-Trp-Ser-Arg-Leu-Ile-Glu-Pro (SEQ ID NO: 7)
Asp-His-Pro-Gln-Ala-Lys-Pro-Asn-Trp-Tyr-Gly-Val (SEQ ID NO: 8)
Gly-Leu-Pro-Pro-Tyr-Ser-Pro-His-Arg-Leu-Ala-Gln (SEQ ID NO: 9)
Lys-Leu-Thr-Thr-Gln-Tyr-Met-Ala-Arg-Ser-Ser-Ser (SEQ ID NO: 10)
Lys-Val-Trp-Met-Leu-Pro-Pro-Leu-Pro-Gln-Ala-Thr (SEQ ID NO: 11)
Asn-Val-Thr-Ser-Thr-Ala-Phe-Ile-Asp-Thr-Pro-Trp (SEQ ID NO: 12)
Arg-Leu-Asn-Leu-Asp-Ile-Ile-Ala-Val-Thr-Ser-Val (SEQ ID NO: 13)
Thr-Leu-Pro-Ser-Pro-Leu-Ala-Leu-Leu-Thr-Val-His (SEQ ID NO: 14)
Thr-Asn-Arg-His-Asn-Pro-His-His-Leu-His-His-Val (SEQ ID NO: 15)
Trp-Pro-His-Ala-Trp-Lys-Val-Trp-Trp-Pro-Ala-Ser (SEQ ID NO: 16)
Asn-Trp-Trp-Trp-Pro-Pro-Tyr-Ile-Arg-His-Gln-Pro (SEQ ID NO: 17)
Trp-His-Trp-Ser-Trp-Thr-Pro-Trp-Pro-Ser-His-His (SEQ ID NO: 18)
Trp-Pro-Trp-Ala-Trp-His-Pro-Ser-Arg-Asp-Val-Tyr (SEQ ID NO: 19)
Trp-His-Gly-Tyr-Trp-Tyr-Ser-Asn-Leu-Asn-Thr-Thr (SEQ ID NO: 20)
Trp-Trp-Thr-Pro-Trp-Met-Ser-His-Ala-Tyr-Pro-Val (SEQ ID NO: 21)
Trp-Pro-Asn-Pro-Tyr-Trp-Gly-Trp-Phe-Ala-Ala-Val (SEQ ID NO: 22)
Thr-Ser-Trp-His-Thr-Trp-Trp-Trp-Arg-Gln-Pro-Pro (SEQ ID NO: 23)
Asn-Ala-Trp-His-Lys-Tyr-Trp-Trp-Pro-Ile-Thr-Lys (SEQ ID NO: 24)
His-Pro-Asn-Asn-Asp-Trp-Ser-Lys-Ala-Pro-Gln-Phe (SEQ ID NO: 25)
Trp-Trp-Thr-Pro-Gln-Pro-Trp-Trp-Ser-Phe-Pro-Ile (SEQ ID NO: 26)
Trp-Pro-His-Thr-Ser-Trp-Trp-Gln-Thr-Pro-Leu-Thr (SEQ ID NO: 27)
Trp-His-Val-Asn-Trp-Asp-Pro-Met-Ala-Trp-Tyr-Arg (SEQ ID NO: 28)
Ser-Trp-Pro-Trp-Trp-Thr-Ala-Tyr-Arg-Val-His-Ser (SEQ ID NO: 29)
Trp-His-Ser-Asn-Trp-Tyr-Gln-Ser-Ile-Pro-Gln-Val (SEQ ID NO: 30)
Gly-Tyr-Trp-Pro-Trp-Lys-Phe-Glu-His-Ala-Thr-Val (SEQ ID NO: 31)
Ala-Trp-Trp-Pro-Thr-Thr-Phe-Pro-Pro-Tyr-Tyr-Tyr (SEQ ID NO: 32)
Asn-Pro-Trp-Trp-Ser-His-Tyr-Tyr-Pro-Arg-Ser-Val (SEQ ID NO: 33)
Trp-Pro-His-Asn-Tyr-Pro-Leu-Asn-His-Ser-Asn-Pro (SEQ ID NO: 34)
Thr-Trp-Ala-His-Pro-Leu-Glu-Ser-Asp-Tyr-Leu-Arg (SEQ ID NO: 35)
His-Thr-Tyr-Tyr-His-Asp-Gly-Trp-Arg-Leu-Ala-Pro (SEQ ID NO: 36)
Thr-Phe-Val-Gln-Thr-Pro-Leu-Ser-His-Leu-Ile-Ala (SEQ ID NO: 37)
Arg-Val-Pro-Pro-Ser-Lys-Leu-Thr-Arg-Pro-Pro-Phe (SEQ ID NO: 38)
His-Ser-Ile-Tyr-Ser-Val-Thr-Pro-Ser-Thr-Ala-Ser (SEQ ID NO: 39)
Leu-Asn-Thr-Gln-Asn-His-Ala-Pro-Leu-Pro-Ser-Ile (SEQ ID NO: 40)
A peptide comprising at least one sequence selected from the group consisting of: An antigen protein or antibody protein comprising at least one of the amino acid sequences according to claim 1.