JP2011184372A - ANTIBODY AGAINST CANINE TSH alpha-CHAIN PEPTIDE - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an antibody which specifically recognizes a cannine TSH antibody without procuring a cannine TSH antigen in a large quantity. <P>SOLUTION: There are provided the antibody generated by administering polyhapten including polypeptide including an amino-acid sequence represented by Phe-Pro-Asp-Gly-Glu-Phe at an N-terminal side, as an antigen to an immune animal, and a fragment of the antibody. <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

  The present invention relates to an antibody against canine TSHα chain peptide, a hybridoma producing the antibody, a reagent using the antibody, and a measuring method.

  Hormone is a substance that exists in a small amount in blood in the living body and controls various physiological functions. Thyroid-stimulating hormone (TSH), a type of thyroid hormone, is often measured in clinical settings to determine and understand thyroid function. Under normal conditions, the thyroid is stimulated by thyrotropin (TSH) and thyroxine (T4) is secreted. However, when thyroid function such as Hashimoto's disease is reduced, a situation occurs in which T4 is not secreted even though TSH is secreted. Therefore, measuring the blood concentration of TSH is necessary when thyroid function abnormality is suspected.

  However, since hormones such as TSH are present only in trace amounts in the blood, a reagent and a device capable of quantitative determination with high sensitivity are required. In immunodiagnostic reagents using antigen-antibody reactions, a method that does not use a labeling reaction, that is, as an unlabeled method, an immunodiffusion method using a precipitation reaction, an immunoturbidimetric method, an immunonephelometric method (nepherometry), an aggregation reaction There are hemagglutination method and latex method using the method, and as a method using a labeling reaction, an enzyme immunoassay method (EIA method) or a radioimmunoassay method (RIA method) depending on the kind and property of the labeled substance In addition, fluorescence immunoassay (FIA method), chemiluminescence immunoassay (CLIA method), bioluminescence immunoassay (BLIA method) and the like are currently used. As a method for measuring TSH, for example, Patent Document 1 includes using a commercially available antibody against human TSH, and measuring aggregation produced by contacting TSH with a carrier to which the anti-TSH antibody is bound. In the TSH measurement method, multiple types of anti-TSH antibodies that recognize different epitopes of TSH are independently carried on individual single bodies, and the carrier carrying each TSH antibody is brought into contact with a specimen containing TSH with a time difference. A method of measuring TSH, which is characterized in that it is performed, is described.

  In the immunodiagnosis of TSH as described above, a TSH antibody is necessary. However, since the canine TSH antigen is present in a very small amount in the living body, it is difficult to ensure a sufficient amount as an immunogen for producing a canine TSH antibody. Currently, dog cTSH antibodies are not commercially available. Furthermore, since it is difficult to construct a recombinant canine TSH protein expression system, it is difficult to obtain canine TSH protein in large quantities. As described above, since a large amount of canine TSH protein cannot be obtained, normal immunization cannot be performed, and it was difficult to produce an anti-dog TSH antibody.

JP 2009-85703 A

  The problem to be solved by the present invention is to provide an antibody that specifically recognizes canine TSH antibody without obtaining a large amount of canine TSH antigen. The present invention further provides a hybridoma that produces the antibody, an immunoassay reagent using the antibody, and a method for measuring dog TSH using the antibody.

  As a result of intensive studies to solve the above problems, the present inventors have used a peptide consisting of 11 amino acid residues at the N-terminal α chain, which is a part of the canine TSH antigen, as an immunogen. The present inventors have succeeded in producing an anti-dog TSH antibody and have completed the present invention.

That is, according to the present invention, an antibody produced by administering a polyhapten containing a polypeptide containing the amino acid sequence represented by Phe-Pro-Asp-Gly-Glu-Phe on the N-terminal side to an immunized animal as an antigen or That fragment is provided.
Preferably, the polypeptide comprising the amino acid sequence represented by Phe-Pro-Asp-Gly-Glu-Phe on the N-terminal side is a polypeptide comprising any of the following amino acid sequences represented on the N-terminal side.
Phe-Pro-Asp-Gly-Glu-Phe-Thr-Met-Gln-Gly- (Xaa) n
Phe-Pro-Asp-Gly-Glu-Phe-Thr-Met-Gln- (Xaa) n
Phe-Pro-Asp-Gly-Glu-Phe-Thr-Met- (Xaa) n
Phe-Pro-Asp-Gly-Glu-Phe-Thr- (Xaa) n
Phe-Pro-Asp-Gly-Glu-Phe- (Xaa) n
(In the formula, Phe represents the N-terminal amino acid of the peptide chain, Xaa represents an amino acid residue or linker, n represents an integer of 0 to 3, and 2 or 3 when n is 2 or 3. And the groups represented by Xaa may independently be the same or different.)

Preferably, the amino acid residue represented by Xaa is Cys.
Preferably, the linker represented by Xaa is a linker containing an ethylene glycol unit.
Preferably, in the antibody of the present invention or a fragment thereof, a polypeptide containing the amino acid sequence represented by Phe-Pro-Asp-Gly-Glu-Phe on the N-terminal side is added to hemocyanin protein (KLH) or bovine serum albumin (BSA). Produced by administering the conjugated polyhapten as an antigen to an immunized animal.

Preferably, the antibody or fragment thereof of the invention binds to canine TSH.
Preferably, the antibody of the present invention or a fragment thereof is selected by screening using dog TSH.
Preferably, the antibody or fragment thereof of the present invention is a monoclonal antibody.

The present invention further provides a hybridoma that produces the above-described antibody of the present invention.
The present invention further provides an immunoassay reagent comprising the above-described antibody of the present invention or a fragment thereof.
The present invention further provides an immunoassay reagent containing the above-described antibody of the present invention or a fragment thereof labeled with latex particles containing a dye or a fluorescent dye.
The present invention further provides an immunoassay reagent comprising the above-described antibody of the present invention or a fragment thereof labeled with an enzyme.
The immunoassay reagent of the present invention is preferably for dog TSH measurement.
The present invention further provides a method for measuring dog TSH in a sample, which comprises bringing the sample into contact with the above-described immunoassay reagent for measuring dog TSH of the present invention.

  The antibody of the present invention can specifically recognize canine TSH. Since the antibody of the present invention is produced using a peptide consisting of 11 amino acid residues at the N-terminal α chain, which is a part of the dog TSH antigen, as an immunogen, the antibody can be produced without obtaining a large amount of the dog TSH antigen. be able to.

FIG. 1 shows the results of primary screening of anti-dog TSH monoclonal antibody. FIG. 2 shows the results of primary screening of anti-dog TSH monoclonal antibody. FIG. 3 shows the results of primary screening of anti-dog TSH monoclonal antibody. FIG. 4 shows the results of primary screening of anti-dog TSH monoclonal antibody. FIG. 5 shows the results of primary screening of anti-dog TSH monoclonal antibody. FIG. 6 shows the results of primary screening of anti-dog TSH monoclonal antibody. FIG. 7 shows the results of primary screening of anti-dog TSH monoclonal antibody. FIG. 8 shows the results of primary screening of anti-dog TSH monoclonal antibody. FIG. 9 shows the results of secondary screening after limiting dilution of anti-dog TSH monoclonal antibody. FIG. 10 shows the result of secondary screening after limiting dilution of anti-dog TSH monoclonal antibody.

Hereinafter, the present invention will be described more specifically.
The antibody of the present invention is an antibody or fragment thereof produced by administering a polyhapten containing a polypeptide containing the amino acid sequence represented by Phe-Pro-Asp-Gly-Glu-Phe to the N-terminal side as an antigen to an immunized animal It is. Preferably, a polyhapten in which a polypeptide containing an amino acid sequence represented by Phe-Pro-Asp-Gly-Glu-Phe on the N-terminal side is bound to hemocyanin protein (KLH) or bovine serum albumin (BSA) can be used. . The antibody of the present invention may be a polyclonal antibody or a monoclonal antibody, but is preferably a monoclonal antibody. The antibody referred to in the present invention means not only the whole antibody molecule but also a fragment thereof (for example, Fab, F (ab ′) ₂ or Fab ′ fragment).

  The amino acid sequence of the polypeptide constituting the polyhapten used in the present invention only needs to contain the amino acid sequence represented by Phe-Pro-Asp-Gly-Glu-Phe on the N-terminal side, and is derived from the canine TSHα chain peptide. As long as the amino acid sequence is included, the length (number of amino acids) of the polypeptide is not particularly limited. The length of the polypeptide constituting the polyhapten used in the present invention is preferably 6 amino acids to 20 amino acids, and more preferably 6 amino acids to 15 amino acids.

Preferable examples of the polypeptide comprising the amino acid sequence represented by Phe-Pro-Asp-Gly-Glu-Phe on the N-terminal side include a polypeptide comprising any of the following amino acid sequences represented on the N-terminal side: Can
Phe-Pro-Asp-Gly-Glu-Phe-Thr-Met-Gln-Gly- (Xaa) n
Phe-Pro-Asp-Gly-Glu-Phe-Thr-Met-Gln- (Xaa) n
Phe-Pro-Asp-Gly-Glu-Phe-Thr-Met- (Xaa) n
Phe-Pro-Asp-Gly-Glu-Phe-Thr- (Xaa) n
Phe-Pro-Asp-Gly-Glu-Phe- (Xaa) n
In the above, Phe represents the N-terminal amino acid of the peptide chain, Xaa represents an amino acid residue (preferably Cys) or a linker (preferably a linker containing an ethylene glycol unit), and n represents an integer of 0 to 3. In the case where n is 2 or 3, the groups represented by 2 or 3 Xaas may be the same or different from each other.

  In the above, the linker is a group introduced between the carrier protein and the peptide chain for the purpose of providing a space from the carrier protein (for example, KLH, BSA). It is preferred that the linker comprises an ethylene glycol unit. Also preferred as the linker is a group having an amino group and a carboxyl group, or a dehydration condensate thereof. More preferably, it contains 2 or more carbon atoms between the amino group and the carboxyl group, and most preferably contains an ethylene glycol unit between the amino group and the carboxyl group.

  In order to obtain a polyclonal antibody, the hapten described above is used as an antigen and administered to a mammal such as a rat, mouse, rabbit or the like. The dose of the antigen per animal is 0.1 to 100 mg when no adjuvant is used, and 1 to 100 μg when an adjuvant is used. Examples of adjuvants include Freund's complete adjuvant (FCA), Freund's incomplete adjuvant (FIA), and aluminum hydroxide adjuvant. Immunization is performed mainly by injecting intravenously, subcutaneously, intraperitoneally, or the like. The immunization interval is not particularly limited, and immunization is performed 1 to 10 times, preferably 2 to 5 times at intervals of several days to several weeks, preferably 2 to 5 weeks. And 6 to 60 days after the last immunization, the antibody titer is measured by enzyme immunoassay (ELISA (enzume-linked immunosorbent assy) or EIA (enzyme immunoassay)), radioimmunoassay (RIA), etc. On the day when the maximum antibody titer is shown, blood can be collected to obtain antiserum. When purification of antibody from antiserum is required, it should be purified by selecting a known method such as ammonium sulfate salting-out method, ion exchange chromatography, gel filtration, affinity chromatography, or a combination thereof. Can do. The polyclonal antibody of the present invention can be obtained by the above method.

  Monoclonal antibodies can be obtained by the following method. That is, the antigen is injected into the abdominal cavity several times with adjuvant, and the spleen cells are taken out and fused with mouse myeloma cells using polyethylene glycol or the like. Then, antibody-producing cells are cloned from the fused cells and propagated as monoclonal cells. Ascites fluid and serum containing monoclonal antibodies can be obtained by further intraperitoneal injection of proliferating cells into mice. More specifically, it can be performed as follows.

  First, the antigen peptide described above is administered as an antigen to a mammal such as a rat, mouse, rabbit or the like. The dose of the antigen per animal is 0.1 to 100 mg when no adjuvant is used, and 1 to 2000 μg when an adjuvant is used. Examples of adjuvants include Freund's complete adjuvant (FCA), Freund's incomplete adjuvant (FIA), and aluminum hydroxide adjuvant. Immunization is performed mainly by injecting intravenously, subcutaneously or intraperitoneally. The immunization interval is not particularly limited, and immunization is performed 1 to 10 times, preferably 2 to 5 times at intervals of several days to several weeks, preferably 2 to 5 weeks. Then, antibody-producing cells are collected 1 to 60 days after the last immunization day, preferably 1 to 14 days later. Examples of antibody-producing cells include spleen cells, lymph node cells, peripheral blood cells, etc., but spleen cells or local lymph node cells are preferred.

  In order to obtain a cell fusion hybridoma, cell fusion between antibody-producing cells and myeloma cells is performed. As myeloma cells to be fused with antibody-producing cells, generally available cell lines of animals such as mice can be used. The cell line to be used has drug selectivity and cannot survive in a HAT selection medium (including hypoxanthine, aminopterin, and thymidine) in an unfused state, but can survive only in a state fused with antibody-producing cells. Those having the following are preferred. Examples of myeloma cells include mouse myeloma cell lines such as P3X63-Ag.8.U1 (P3U1) and NS-I.

Next, the myeloma cell and the antibody-producing cell are fused. Cell fusion is performed in animal cell culture media such as serum-free DMEM and RPMI-1640 media, and 1 × 10 ⁶ to 1 × 10 ⁷ antibody-producing cells and 2 × 10 ⁵ to 2 × 10 ^6. Per ml of myeloma cells (cell ratio of antibody-producing cells to myeloma cells is preferably 5: 1), and a fusion reaction is performed in the presence of a cell fusion promoter. As the cell fusion promoter, polyethylene glycol having an average molecular weight of 1000 to 6000 daltons can be used. Alternatively, antibody-producing cells and myeloma cells can be fused using a commercially available cell fusion device utilizing electrical stimulation (for example, electroporation).

The target hybridoma is selected from the cells after cell fusion treatment. As a method, the cell suspension is appropriately diluted with, for example, fetal bovine serum-containing RPMI-1640 medium, then plated on a microtiter plate at about 3 × 10 ⁵ cells / well, a selective medium is added to each well, and thereafter appropriate. The culture is carried out with the selective medium changed. As a result, cells that grow from about 14 days after the start of culture in the selective medium can be obtained as hybridomas.

  Next, it is screened whether the target antibody exists in the culture supernatant of the hybridoma which has proliferated. Hybridoma screening is not particularly limited, and may be performed according to ordinary methods. For example, a part of the culture supernatant contained in a well grown as a hybridoma can be collected and screened by enzyme immunoassay (such as ELISA) or radioimmunoassay. In the present invention, the antibody can be preferably selected by screening using canine TSH as an antigen. Preferably, the antibody of the present invention is characterized by binding to canine TSH.

  Cloning of the fused cells is performed by limiting dilution or the like, and finally, a hybridoma that is a monoclonal antibody-producing cell can be established.

As a method for collecting the monoclonal antibody from the established hybridoma, a normal cell culture method or ascites formation method can be employed. In the cell culture method, the hybridoma is cultured in an animal cell culture medium such as RPMI-1640 medium containing 10% fetal bovine serum, MEM medium, or serum-free medium under normal culture conditions (for example, 37 ° C., 5% CO ₂ concentration). Cultivate for 7 to 14 days and obtain antibody from the culture supernatant.

In the case of the ascites formation method, about 1 × 10 ⁷ hybridomas are administered into the abdominal cavity of a mammal derived from a myeloma cell and the same strain, and the hybridomas are proliferated in large quantities. And ascitic fluid is collected after 1-2 weeks. When antibody purification is required in the antibody collection method, a known method such as ammonium sulfate salting-out, ion exchange chromatography, gel filtration, affinity chromatography (protein A-agarose, etc.) is appropriately selected. Or by combining them.

Examples of the antibody subclass include IgG1, IgG2a, IgG2b, and the like. In the present invention, IgG1 has good antibody fragmentation efficiency when preparing an enzyme-antibody complex. The obtained IgG1 can be further reduced to F′ab ′) ₂ from which the Fc site has been removed with a protease such as activated papain or pepsin to be induced into a Fab ′ fragment.

In a preferred embodiment, Fab ′ fragments are used as antibodies. An intact antibody (IgG) has Fab (antigen binding site) and Fc (complement binding site). When using an enzyme-labeled antibody by binding an intact antibody to an enzyme, if the sample is a blood sample, the complement component in the blood binds to the Fc part, causing steric hindrance and inhibiting the enzyme activity. Become. Even when the sample is not a blood sample, the Fc portion adsorbs nonspecifically to the pores of the porous member constituting the vessel wall of the reaction vessel or the immune reaction layer, the surface of the internal voids, etc. The activity is apparently reduced, causing noise during measurement. In order to remove these noises, it is desirable to use Fab ′, F (ab ′) ₂ or Fab fragments that do not contain Fc sites as antibodies. Of these, the Fab ′ fragment having a free SH group is most preferably used as an antibody for the convenience of binding to an enzyme.

  The above-described antibody of the present invention, the latex reagent labeled with the antibody or a fragment thereof, and the antibody or fragment thereof labeled with an enzyme (for example, Bacillus subtilis α-amylase) are used as an immunoassay reagent (preferably an immunity for dog TSH measurement). Analysis reagent).

  The enzyme used as the label can be selected in consideration of the combination with the enzyme substrate used in the subsequent enzyme reaction. In the present invention, the reactivity to an enzyme that reacts with an enzyme substrate is suppressed by steric hindrance due to the formation of a matrix-like structure of the enzyme, antibody, and antigen. Therefore, the combination of the enzyme and the substrate is influenced by such steric hindrance. It is preferable to select a system that is easy to detect. That is, a relatively high molecular weight enzyme substrate is preferable in terms of sensitivity. For example, a substrate having a molecular weight of about 20,000 or more, preferably a molecular weight of about 100,000 or more is used. Examples of such a substrate include starch as a substrate for enzyme amylase; substrate cellulose for enzyme cellulase; protein such as gelatin and hemocyanin for protease; and various fats and oils for lipase. Reports on the selection of the above enzyme and substrate are disclosed in detail in JP-A-60-108756, 60-171461 and 60-171460. In this, the amylase which uses starch as a substrate is preferable. Further, these substrates are water-insoluble substrates, and steric hindrance due to the matrix-like structure of the enzyme / antibody / antigen appears remarkably, and it is particularly preferable to use these substrates.

  Examples of amylase include α-amylase, β-amylase, glucoamylase and the like, and those that are not substantially contained in the specimen are preferable from the viewpoint of noise prevention. The origin of amylase covers a wide range of animals (saliva, pancreatic juice, etc.), plants and microorganisms. Therefore, when analyzing body fluids such as humans and animals, blood, etc., it is preferable not to use these higher animal-derived amylases.

  Examples of amylases derived from microorganisms and plants include glucoamylase derived from the genus Aspergillus, Rhizopus, Saccharomyces yeast, etc .; β-amylase derived from barley malt, wheat, soybean, etc .; Bacillus Subtilis, Examples include α-amylase derived from Streptomyces griseus, Pseudomonas stutzeri, Thermoactiomyces vulgaris and the like. Of these, α-amylase derived from Bacillus Subtilis, which has a strong liquefaction power and excellent thermal stability, is most preferred.

  These enzymes are preferably unaffected by interfering factors present in any sample, and it is preferable that there is no competing enzyme of the same type in the sample. However, when an enzyme of the same type as the labeling enzyme is contained in the sample, this enzyme inhibitor may be used. The enzyme inhibitor only needs to have a greater degree of inhibiting the enzyme in the sample than inhibiting the activity of the labeling enzyme. Most preferably, the enzyme inhibitor completely inactivates the enzyme in the specimen, but does not inhibit the labeled enzyme at all. However, in practice, it is not necessary to simply increase the blank value at the time of measurement, and the enzyme activity in the specimen may be recovered after the measurement by deactivating the enzyme inhibitor. The enzyme inhibitor is not particularly limited as long as it does not inhibit the enzyme of the enzyme-labeled antibody, and the enzyme in the free state may be inhibited. As this enzyme inhibitor, a known enzyme inhibitor having the above-described specificity may be selected and used. Or you may make the antibody with respect to the enzyme which becomes a problem in a test substance, and may use this as an enzyme inhibitor.

  When α-amylase is used as oxygen, carboxymethylated starch, starch, amylose, amylopectin and the like can be used as a substrate. In particular, if water-insoluble starch or the like is used, the enzyme reaction becomes a reaction at the surface of the substrate particle, that is, at the solid-liquid interface. This is preferable. Moreover, you may make it detect the die | dye (pigment) attached to soluble amylose which is an enzyme degradation product using a water-insoluble die starch. As such a water-insoluble blue starch polymer, a commercially available product such as Neoamylase (Daiichi Chemical) can be used.

  The method of binding an enzyme and an antibody can be performed using functional groups (amino group, carboxyl group, thiol group, etc.) of two substances. Typical binding methods include the glutaraldehyde method, the periodic acid method, the pyridyl disulfide method, the maleimide-succinimide method, and the like. The binding method is not limited to these examples. For example, “Method in Immunology and Immunochemistry” Vol. 1, (CAWilliams, MWChase, Academic Press, 1967) or Ishikawa, Kawai, edited by Miyai, “Enzyme immunization” It can be used by appropriately selecting from methods described in a book such as “Measurement Method” (Medical School, issued in 1978). Among these binding methods, the maleimide-succinimide method, in which the thiol group of the antibody hinge part and the amino group of the enzyme are cross-linked, is excellent in that the reaction efficiency is good and the antibody activity can be retained.

  In the maleimide-succinimide method, for example, an enzyme and Fab ′ are bound as follows. First, the amino group of the enzyme is maleimidated with a maleimide-succinimide reagent. This is purified by gel filtration and then subjected to conjugation with an antibody having a thiol group (Fab ′). At this time, two or more kinds of antibodies (Fab ') having different epitopes may be used in combination, and in that case, these antibody fragments are subjected to a binding reaction together. This complexing reaction is preferably carried out in the range of 3 to 7 mol of antibody per 1 mol of enzyme. When Fab ′ (molecular weight of about 50,000) is used as an antibody and α-amylase (molecular weight of about 50,000) is used as an enzyme, the weight of α-amylase is 1/3 to 1/7 of the total Fab ′ amount. It is preferable to perform the binding reaction in a range. This binding reaction usually proceeds at 4 ° C. to room temperature.

  The produced enzyme-antibody complex (enzyme-labeled antibody) is purified by gel filtration and, if necessary, dried by freeze-drying or the like. The binding ratio between the enzyme and each antibody is not limited to 1: 1 and can be any ratio depending on the purpose. Since ordinary enzymes have a large number of amino groups, a plurality of maleimide groups are introduced, and a plurality of antibody molecules are introduced into one enzyme molecule. Since at least one antibody must be bound to one enzyme molecule, the molar ratio of the antibody to the enzyme in the complex must be 1 or more. The ratio is preferably in the range of 2-5. When Fab ′ (molecular weight of about 50,000) is used as an antibody and α-amylase (molecular weight of about 50,000) is used as an enzyme, the complex has a molecular weight of 150,000 daltons or more, preferably 250,000 to 300,000 daltons Is preferable in terms of high detection sensitivity.

  Next, a dog TSH measurement method (wet method) will be described. First, a conjugate of an antigen and an enzyme-antibody complex contained in a specimen is brought into contact in a solution. At that time, the temperature of the solution is about 20 to 45 ° C., and the pH is usually within the range of about 4.0 to about 8.5. If necessary, a buffer solution such as a phosphate buffer or an acetate buffer may be used to keep the pH constant. The contact time between the antigen and the enzyme-antibody complex may be such that it can sufficiently react. For example, when the temperature of the solution is 37 ° C., 20 to 30 minutes is appropriate. Thereafter, an enzyme substrate is added, and the enzyme activity of the enzyme-antibody complex is measured. If the test antigen is present in the sample, it can be detected as inhibition of enzyme activity. If a calibration curve is drawn in advance with a solution containing a known amount of the test antigen, the amount of the test antigen in the sample can be quantified.

  Note that only the reaction between the antigen and the enzyme-antibody complex may be performed in a solution system, and the reaction solution after the reaction may be subjected to dry analysis. That is, a dry analytical element having a substrate layer containing an enzyme substrate for a labeled enzyme may be prepared, and the enzyme activity may be measured by spotting a reaction solution after an immune reaction.

  The antibody of the present invention can also be used in a dry analytical element. As a specific example of the configuration of the dry analytical element, the same configuration as that shown in FIGS. 1 and 2 of Japanese Patent No. 3115080 can be exemplified. That is, as an example, a detection layer (or reagent layer) and an immune reaction layer are laminated on a light transmissive support. The immune reaction layer is composed of a water-permeable layer, and contains the enzyme-labeled antibody of the present invention and a non-diffusible substrate that is a substrate of the labeled enzyme. The reagent layer is composed of a water-permeable layer and contains a reagent composition for detecting an enzyme reaction product (diffusible substance) that has diffused and migrated from the immune reaction layer. When the enzyme reaction product is a substance that can be directly detected, such as a colored substance, the detection layer (or reagent layer) does not need to contain a detection reagent composition, and in this case, the detection layer (or The reagent layer) functions as a detection layer.

  The specimen (antigen) in the liquid sample supplied by spotting the elements reacts with the enzyme-labeled antibody and the antigen-antibody in the immune reaction layer to form a matrix structure. For this reason, the enzyme activity with respect to the substrate contained in the same reaction immune layer is suppressed. As a result, the amount of antigen in the sample can be known from the amount of the enzyme reaction product detected in the reagent layer (or detection layer).

  In another example, the enzyme-labeled antibody and the enzyme substrate may be contained in separate layers. In this case, a water-permeable substrate layer containing an enzyme substrate is disposed on the reagent layer (or detection layer), and an immune reaction layer containing an enzyme-labeled antibody is further disposed thereon. In this case, the specimen (antigen) in the liquid sample spotted and supplied to the element makes an antigen-antibody binding reaction with the enzyme-labeled antibody in the immune reaction layer to form a matrix structure and becomes substantially immobile. The enzyme-labeled antibody that does not bind to the antigen (or that has a matrix structure that is small enough not to be trapped by the layer structure) moves to the next substrate layer.

  In any of the above embodiments, the enzyme immunoreaction can proceed within the element simply by spotting a liquid sample on the element.

  The dog TSH measurement method in the present invention is not particularly limited. For example, a device capable of detecting fluorescence intensity, specifically, a microplate reader, or a bio for performing fluorescence detection (SPF) by surface plasmon excitation. The fluorescence intensity can be detected using a sensor or the like. The detection of the fluorescence intensity is usually completed after a certain time after the antigen-antibody reaction, for example, several minutes to several hours. By detecting the degree of formation of the immune complex as fluorescence intensity, the concentration of the substance to be detected can be quantified from the relationship between the fluorescence intensity and the concentration of the substance to be detected. The form of fluorescence measurement may be plate reader measurement or flow measurement. The fluorescence detection method (SPF method) by surface plasmon excitation can be measured with higher sensitivity than the fluorescence detection method by epi-illumination excitation.

  Examples of the surface plasmon fluorescence (SPF) biosensor include an optical waveguide formed of a material that transmits excitation light having a predetermined wavelength, as described in Japanese Patent Application Laid-Open No. 2008-249361, and the optical waveguide. A metal film formed on one surface of the substrate, a light source that generates a light beam, and the light beam that passes through the optical waveguide and is incident on the interface between the optical waveguide and the metal film at an incident angle that generates surface plasmon. A sensor including an optical system and fluorescence detection means for detecting fluorescence generated by being excited by an evanescent wave enhanced by the surface plasmon can be used.

  The present invention further relates to a method for measuring canine TSH in a sample, which comprises bringing the sample into contact with the above-described immunoassay reagent for measuring dog TSH of the present invention. The type of sample is not particularly limited, and examples thereof include blood (whole blood, plasma, serum), lymph, urine, etc., preferably blood (whole blood, plasma, serum), more preferably serum, plasma. Particularly preferred is serum.

  The present invention will be specifically described by the following examples, but the present invention is not limited to the examples.

Example 1: Immunization (1) Preparation of immunogen
The following sequences were selected as peptide antigens as sequences having low homology between species among the sequences presented on the surface of TSH antigen.
Canine TSHα chain peptide origin: Phe-Pro-Asp-Gly-Glu-Phe-Thr-Met-Gln-Gly-Cys (FPDGEFTMQGC) (N-terminal: 11 residues) (SEQ ID NO: 1)
Since the above sequence is shorter than the sequence used for normal peptide immunization, a sequence (FPDGEFTMQGXXC) via a PEG spacer (two introduced) having the following structure was designed.
_{X = H 2 N- (CH 2} ) 2 O- (CH 2) 2 O-CH 2 -COOH
The sequence represented by FPDGEFTMQGXXC was synthesized by Fmoc solid phase synthesis and purified by HPLC.

Next, a peptide KLH conjugate was made as follows.
20 mg KLH was dissolved in 2 mL milliQ water. 2.0 mg of maleimidobenzoyloxysuccinimide (MBS: crosslinker) was dissolved in 500 μl of DMF. While stirring the KLH solution, 400 μl of MBS solution was added dropwise. Then, it stirred slowly for 60 minutes at room temperature. The solution was applied to a gel filtration column (Sephadex G-25) equilibrated with 50 mM phosphate buffer (pH 7.4), and the absorbance was measured at 280 nm to recover protein components. The concentration of the recovered KLH-MBS was adjusted to 5 mg / mL, and an appropriate amount was used for the reaction with the peptide. The peptide was dissolved in milli-Q water or 1 × PBS so as to be 10 mg / mL. Equal amounts of the prepared KLH-MBS solution and peptide solution were mixed and reacted at room temperature for 2 hours while stirring with a stirrer. Thereafter, phosphate buffer (1 × PBS) was added to adjust the concentration to 1 mg / mL.

(2) Immunization of mouse 10 mg of sensitizing antigen (peptide-KLH) was used.
Sensitized antigen was immunized subcutaneously on the back of mice. The first time, an emulsion mixed with complete adjuvant (FCA) was administered. The 2nd to 4th time, an emulsion mixed with incomplete adjuvant (FIA) was administered. Immunization was performed 5 times at 2-week intervals, and blood was collected and the antibody titer was measured in the week following the third and fourth immunizations. A part of the collected blood serum (100 to 200 μL) was used for ELISA measurement. If an increase in antibody titer is observed, the fifth immunization is the final immunization. For final immunization, antigen A was diluted with PBS (−) and administered intraperitoneally. The spleen was removed 3 days after the final immunization.
(1) Animals: BALB / c mice, rabbits, 7-9 weeks old (when in stock)
(2) Number of animals: 3
(3) Immunity: 100-200 μg / animal / time
(4) Immune site: Dorsal subcutaneous
(5) Antibody titer measurement: ELISA
As a result of the antibody titer measurement, the spleen was removed from the selected mice (1 to 3 mice), and cell fusion was performed.

Example 2: Preparation of hybridoma Spleen cells extracted from immunized mice were mixed with myeloma to effect cell fusion.
Fusion method: Cells using PEG method: Spleen cells myeloma 3 days after final immunization: P3-X63-Ag8-U1
Cell ratio, spleen cells: myeloma = 5-10: 1
Cell seeding: 0.5-1.0 x 10 ⁵ cells / well as spleen cells seeded in a 96-well plate Medium used: RPMI-1640 + 10% FBS + HAT (additional HCF or IL-6 in some cases)

Example 3: Primary screening of monoclonal antibody (screening after cell fusion)
The antigen for measurement was diluted with PBS (−) to 1 μg / mL and immobilized, and the culture supernatant of the hybridoma grown in selective culture in HAT medium was screened by ELISA. An anti-mouse IgG HRP-labeled antibody was used as a secondary antibody. The screening positive hybridoma was cultured in a 24-well plate, and the culture supernatant was used for each 1 mL evaluation. Cloning wells were selected based on the results of SPR selection tests.

  Results of primary screening using canine TSH (cTSH; Whole) antigen are shown in FIGS.

  The response (ΔRU value) with cTSH-immobilized Fc is as follows.

(Condition details)
BiacoreT100
Chip: CM5
cTSH (Scrips) immobilized Fc: RU2000 (Evaluation of reactivity with cTSH)
captureAb immobilized Fc: RU2000 (quantitative determination of antibody concentration in supernatant)

Example 4: Secondary screening of monoclonal antibodies (screening after limiting dilution)
The secondary screening of monoclonal antibodies (screening after limiting dilution) was performed in the same manner as the screening after cell fusion in Example 3.

The results of the secondary screening of monoclonal antibodies (screening after limiting dilution) are shown in FIGS.
A high signal was observed except for 26G8B6. SPR evaluation alone does not give superiority or inferiority among each parental clone. Therefore, the final clone was selected after confirming the growth status and subclass of the cells. The subclass was determined using an Isotyping kit (Roche). The results are shown in the table below.

  Among the parent clones, strains with high SPR signal and good cell growth status were selected from the top two (18 clones in total of 10 clones). IgM was 1 strain, IgG2b was 2 strains, and all others were IgG1.

Claims (14)

An antibody or fragment thereof produced by administering to an immunized animal a polyhapten containing a polypeptide containing an amino acid sequence represented by Phe-Pro-Asp-Gly-Glu-Phe on the N-terminal side as an antigen. The polypeptide comprising an amino acid sequence represented by Phe-Pro-Asp-Gly-Glu-Phe on the N-terminal side is a polypeptide comprising an amino acid sequence represented by any of the following on the N-terminal side: The antibody or fragment thereof described.
Phe-Pro-Asp-Gly-Glu-Phe-Thr-Met-Gln-Gly- (Xaa) n
Phe-Pro-Asp-Gly-Glu-Phe-Thr-Met-Gln- (Xaa) n
Phe-Pro-Asp-Gly-Glu-Phe-Thr-Met- (Xaa) n
Phe-Pro-Asp-Gly-Glu-Phe-Thr- (Xaa) n
Phe-Pro-Asp-Gly-Glu-Phe- (Xaa) n
(In the formula, Phe represents the N-terminal amino acid of the peptide chain, Xaa represents an amino acid residue or linker, n represents an integer of 0 to 3, and 2 or 3 when n is 2 or 3. And the groups represented by Xaa may independently be the same or different.) The antibody or fragment thereof according to claim 1 or 2, wherein the amino acid residue represented by Xaa is Cys. The antibody or fragment thereof according to any one of claims 1 to 3, wherein the linker represented by Xaa is a linker containing an ethylene glycol unit. A polyhapten obtained by binding a polypeptide containing the amino acid sequence represented by Phe-Pro-Asp-Gly-Glu-Phe on the N-terminal side to hemocyanin protein (KLH) or bovine serum albumin (BSA) is administered to an immunized animal as an antigen. The antibody or fragment thereof according to any one of claims 1 to 4, produced by The antibody or fragment thereof according to any one of claims 1 to 5, which binds to canine TSH. The antibody or fragment thereof according to any one of claims 1 to 6, which has been selected by screening using dog TSH. The antibody or fragment thereof according to any one of claims 1 to 7, which is a monoclonal antibody. A hybridoma that produces the antibody according to any one of claims 1 to 8. An immunoassay reagent comprising the antibody or fragment thereof according to any one of claims 1 to 8. An immunoassay reagent comprising the antibody or fragment thereof according to any one of claims 1 to 8, which is labeled with latex particles containing a dye or a fluorescent dye. An immunoassay reagent comprising the antibody or fragment thereof according to any one of claims 1 to 8, which is labeled with an enzyme. The immunoassay reagent according to any one of claims 10 to 12, wherein the reagent is for dog TSH measurement. A method for measuring dog TSH in a sample, comprising bringing the sample into contact with the immunoassay reagent for measuring dog TSH according to claim 13.

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