JP2012068151A - Dry analytical element for measurement of canine crp - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a dry analytical element for measurement of canine CRP capable of expanding a measurement region; to provide an immunoanalytical reagent or kit for the measurement of the canine CRP; and to provide a method for measuring the canine CRP.SOLUTION: This dry analytical element for the measurement of the canine CRP includes, on a light-transmissive support, an immune reaction layer including: a monoclonal antibody fragment prepared by labeling with an enzyme a first monoclonal antibody fragment that satisfies a prescribed property and recognizes the canine CRP; a second monoclonal antibody that recognizes the canine CRP; and a monoclonal antibody fragment prepared by labeling with an enzyme a third monoclonal antibody fragment that recognizes the canine CRP.

Description

  The present invention relates to a dry analytical element for measuring canine CRP using a monoclonal antibody that recognizes canine CRP, a reagent or kit for measuring canine CRP, and a method for measuring canine CRP.

  C-reactive protein (CRP) is a protein whose production amount is known to correlate with the strength of inflammatory reaction. Therefore, by measuring the amount of CRP in serum, it can be used as an index of inflammatory reaction. That is, if the serum CRP level is high, it indicates that inflammation is strong.

  Patent Document 1 describes that an anti-canine CRP monoclonal antibody is prepared by a conventionally known monoclonal antibody production technique, and an antigen (canine CRP) is purified by a known affinity chromatography technique using the obtained antibody. Yes. The characteristic of the anti-canine CRP monoclonal antibody described in Patent Document 1 is that it captures canine CRP, and does not describe the quantification of canine CRP.

  Non-Patent Document 1 describes that an antibody used in an immunoturbidimetric test kit for human CRP measurement has reactivity with canine CRP. The antibody described in Non-Patent Document 1 is not intentionally prepared in consideration of reactivity with canine CRP, and there is no description of a monoclonal antibody. Further, Non-Patent Document 1 does not describe a method for stably producing this antibody.

  Patent Document 2 describes a dry immunoassay element for measuring human CRP, but does not describe a reaction with canine CRP. Even if canine serum is measured using the dry immunoassay element for human CRP measurement described in Patent Document 2, a result showing quantitativeness cannot be obtained, and it cannot be used for canine CRP quantification. This is because the specificity of the antibody used in Patent Document 2 is limited to human CRP and does not substantially recognize canine CRP.

  Patent Document 3 describes a simple detection reagent and a simple detection method for canine CRP in which an anti-canine CRP antibody is sensitized to latex particles and the antibody-sensitized particles are present in a buffer solution. The method described in Patent Document 3 has high sensitivity and is a latex agglutination method that can be used as a specimen sample without any treatment of collected blood, but has a drawback of poor quantitativeness.

  Patent Document 4 describes a canine CRP and an immunoassay reagent for human CRP measurement or a dry analysis element containing a monoclonal antibody or a fragment thereof that recognizes canine CRP and human CRP. Can be measured simultaneously and is highly sensitive to canine CRP and human CRP, but there is a problem that the measurement range is narrow.

JP-A-62-210984 Japanese Patent No. 3115080 JP 62-155299 A JP 2010-006774 A

Vet.clin.Pathol.2003,32: 81-87

  The present invention has been made to solve the above-described problems of the prior art. That is, an object of the present invention is to provide a dry analytical element for canine CRP measurement, an immunoanalysis reagent or kit for canine CRP measurement, and a canine CRP measurement method capable of expanding the measurement area.

  The present inventors conducted intensive research to solve the above-mentioned problems. First, immunization is performed on animals such as mice and rats, and after confirming an increase in antibody titer against the serum immunogen, lymphocytes and spleen cells are collected. A hybridoma was prepared by fusing with myeloma cells. When culturing the fused cells in a selective medium and screening using the hybridoma culture supernatant, screening is performed using a method that sensitively reflects antibody binding constants in addition to the conventional qualitative screening. By doing so, it was possible to reliably select hybridomas producing useful antibodies. The canine CRP antibody-producing hybridoma-derived antibody thus obtained had a high affinity for the antigen.

Furthermore, the inventors of the canine CRP antibody obtained as described above,
(1) the binding constant of the first monoclonal antibody, the second monoclonal antibody, and the third monoclonal antibody to canine CRP is 10 ⁷ M ⁻¹ to 10 ⁹ M ⁻¹ ;
(2) The subclass of the first monoclonal antibody, the second monoclonal antibody and the third monoclonal antibody is IgG1,
(3) The molar ratio of the second monoclonal antibody to the total of the first monoclonal antibody and the third monoclonal antibody is 0.1 to 30,
(4) The third monoclonal antibody fragment in the total weight of the monoclonal antibody fragment obtained by labeling the first monoclonal antibody fragment with an enzyme and the monoclonal antibody fragment obtained by labeling the third monoclonal antibody fragment with an enzyme. The weight of the monoclonal antibody fragment labeled with the enzyme is 50% by weight or less,
(5) The binding constant of the third monoclonal antibody to canine CRP is 0.01 to 0.8 times the binding constant of the first monoclonal antibody to canine CRP.
Using a combination of three types of monoclonal antibodies that satisfy all of the above characteristics, a dry analysis element can be created according to the description of Japanese Patent No. 3151080, thereby expanding the measurement area and at the same time allowing a practically acceptable signal / It was found that a dry analytical element for canine CRP measurement capable of accurately quantifying canine CRP by noise ratio (S / N ratio) can be provided. The present invention has been completed based on these findings.

That is, according to the present invention, a monoclonal antibody fragment obtained by labeling a fragment of a first monoclonal antibody recognizing canine CRP with an enzyme, a second monoclonal antibody recognizing canine CRP, and a first antibody recognizing canine CRP. A dry analytical element for canine CRP measurement comprising an immune reaction layer comprising a monoclonal antibody fragment obtained by labeling three fragments of a monoclonal antibody with an enzyme on a light-transmitting support,
(1) the binding constant of the first monoclonal antibody, the second monoclonal antibody, and the third monoclonal antibody to canine CRP is 10 ⁷ M ⁻¹ to 10 ⁹ M ⁻¹ ;
(2) The subclass of the first monoclonal antibody, the second monoclonal antibody and the third monoclonal antibody is IgG1,
(3) The molar ratio of the second monoclonal antibody to the total of the first monoclonal antibody and the third monoclonal antibody is 0.1 to 30,
(4) The third monoclonal antibody fragment in the total weight of the monoclonal antibody fragment obtained by labeling the first monoclonal antibody fragment with an enzyme and the monoclonal antibody fragment obtained by labeling the third monoclonal antibody fragment with an enzyme. The weight of the monoclonal antibody fragment labeled with the enzyme is 50% by weight or less,
(5) The binding constant of the third monoclonal antibody to canine CRP is 0.01 to 0.8 times the binding constant of the first monoclonal antibody to canine CRP.
A dry analytical element for canine CRP measurement is provided.

Preferably, the enzyme is Bacillus subtilis α-amylase.
Preferably, the dry analytical element for measuring canine CRP of the present invention further comprises an amylase inhibitor that inhibits amylase derived from dogs.

Preferably, the dry analytical element for canine CRP measurement of the present invention is provided on a light-transmitting support.
(A) a detection layer for detecting an enzyme reaction product diffused / transferred from the immune reaction layer;
(B) a substrate layer comprising a substrate of the enzyme described in (c) below; and (c) a monoclonal antibody fragment obtained by labeling the first monoclonal antibody fragment recognizing canine CRP with an enzyme, and canine CRP. An immune reaction layer comprising a second monoclonal antibody and a monoclonal antibody fragment obtained by labeling a fragment of the third monoclonal antibody recognizing canine CRP with an enzyme;
Are included in this order from the light-transmitting support side.
More preferably, the detection layer is a reagent layer containing a reagent composition for detecting an enzyme reaction product (diffusible substance) diffused / transferred from the immune reaction layer.

According to the present invention, further, a monoclonal antibody fragment obtained by labeling a fragment of a first monoclonal antibody that recognizes canine CRP with an enzyme, a second monoclonal antibody that recognizes canine CRP, and a third antibody that further recognizes canine CRP. An immunoanalytical reagent or kit for canine CRP measurement, comprising a monoclonal antibody fragment obtained by labeling a fragment of the monoclonal antibody with an enzyme,
(1) the binding constant of the first monoclonal antibody, the second monoclonal antibody, and the third monoclonal antibody to canine CRP is 10 ⁷ M ⁻¹ to 10 ⁹ M ⁻¹ ;
(2) The subclass of the first monoclonal antibody, the second monoclonal antibody and the third monoclonal antibody is IgG1,
(3) The molar ratio of the second monoclonal antibody to the total of the first monoclonal antibody and the third monoclonal antibody is 0.1 to 30,
(4) The third monoclonal antibody fragment in the total weight of the monoclonal antibody fragment obtained by labeling the first monoclonal antibody fragment with an enzyme and the monoclonal antibody fragment obtained by labeling the third monoclonal antibody fragment with an enzyme. The weight of the monoclonal antibody fragment labeled with the enzyme is 50% by weight or less,
(5) The binding constant of the third monoclonal antibody to canine CRP is 0.01 to 0.8 times the binding constant of the first monoclonal antibody to canine CRP.
An immunoassay reagent or kit for canine CRP measurement is provided.

Preferably, the enzyme is Bacillus subtilis α-amylase.
Preferably, the immunoassay reagent or kit for canine CRP measurement of the present invention further comprises an amylase inhibitor that inhibits canine-derived amylase.

  The present invention further provides a method for measuring canine CRP using the above-described dry analytical element for canine CRP measurement or immunoassay reagent or kit for canine CRP measurement of the present invention.

  According to the dry analytical element for canine CRP measurement, the immunoanalysis reagent or kit for canine CRP measurement, and the canine CRP measurement method according to the present invention, the measurement region can be expanded and the CRP concentration in the specimen is 0.05 to 20 mg / dL. A good signal / noise ratio (S / N ratio) that is practically acceptable can be achieved over the entire range. That is, according to the present invention, canine CRP can be accurately quantified up to a high concentration region.

FIG. 1 shows the results of measuring CRP in canine serum using the dry analytical elements of the present invention and comparative examples.

Hereinafter, the present invention will be described more specifically.
In the present invention, a first monoclonal antibody fragment that recognizes canine CRP is labeled with an enzyme, a second monoclonal antibody that recognizes canine CRP, and a third monoclonal antibody that recognizes canine CRP. A monoclonal antibody fragment obtained by labeling an antibody fragment with an enzyme is used. The three types of antibodies used in the present invention are:
(1) the binding constant of the first monoclonal antibody, the second monoclonal antibody, and the third monoclonal antibody to canine CRP is 10 ⁷ M ⁻¹ to 10 ⁹ M ⁻¹ ;
(2) The subclass of the first monoclonal antibody, the second monoclonal antibody and the third monoclonal antibody is IgG1,
(3) The molar ratio of the second monoclonal antibody to the total of the first monoclonal antibody and the third monoclonal antibody is 0.1 to 30,
(4) The third monoclonal antibody fragment in the total weight of the monoclonal antibody fragment obtained by labeling the first monoclonal antibody fragment with an enzyme and the monoclonal antibody fragment obtained by labeling the third monoclonal antibody fragment with an enzyme. The weight of the monoclonal antibody fragment labeled with the enzyme is 50% by weight or less,
(5) The binding constant of the third monoclonal antibody to canine CRP is 0.01 to 0.8 times the binding constant of the first monoclonal antibody to canine CRP.
This condition is satisfied. 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).

  According to one aspect of the present invention, a monoclonal antibody fragment obtained by labeling a fragment of a first monoclonal antibody that recognizes canine CRP with an enzyme, a second monoclonal antibody that recognizes canine CRP, and a first antibody that recognizes canine CRP. A monoclonal antibody fragment obtained by labeling three fragments of a monoclonal antibody with an enzyme can be used in a dry analytical element for canine CRP measurement. In this case, the dry analytical element for canine CRP measurement has a configuration including an immune reaction layer, and the antibody is included in the immune reaction layer.

  According to another aspect of the present invention, a monoclonal antibody fragment obtained by labeling a fragment of a first monoclonal antibody that recognizes canine CRP with an enzyme, a second monoclonal antibody that recognizes canine CRP, and canine CRP are recognized. By combining a fragment of the third monoclonal antibody with a monoclonal antibody fragment labeled with an enzyme, an immunoassay reagent or kit for canine CRP measurement can be constructed. That is, a first monoclonal antibody fragment that recognizes canine CRP is labeled with an enzyme, a second monoclonal antibody that recognizes canine CRP, and a third monoclonal antibody fragment that recognizes canine CRP. The antibody-labeled monoclonal antibody fragment may be mixed together to serve as an immunoassay reagent for canine CRP measurement, or the first monoclonal antibody fragment that recognizes canine CRP may be mixed with an enzyme-labeled monoclonal antibody fragment, A second monoclonal antibody that recognizes CRP and a third antibody that recognizes canine CRP are separately prepared with an enzyme-labeled monoclonal antibody fragment and combined to prepare an immunoassay kit for canine CRP measurement. It is good.

  The monoclonal antibody used in the present invention can be obtained by a usual 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, CRP (such as canine CRP) is administered as an antigen to mammals such as rats, mice, rabbits and 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. Here, in the present invention, a hybridoma that produces a useful antibody can be reliably selected by screening using a method that sensitively reflects the binding constant of the antibody. The measurement method reflecting the antibody binding constant is a concentration of antibody and antigen at the time of reaction that is the same as the reciprocal of the target binding constant or about 1 to 2 orders of magnitude, for example, the target binding constant is 10 ⁹ M ⁻¹ , each antigen is brought into contact with each antigen protein or antigen derivative of about 10 ⁻¹⁰ M to 10 ⁻⁶ M, more preferably about 10 ⁻⁹ M to 10 ⁻⁷ M, and It can be performed by combining measurement methods having sufficient sensitivity.

  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 monoclonal 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 monoclonal antibody of the present invention or the above-mentioned monoclonal antibody fragment labeled with an enzyme (for example, Bacillus subtilis α-amylase) can be used as an immunoassay reagent or kit for canine CRP measurement.

  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, JP-A-60-171461, and JP-A-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 amylase is used as the labeling enzyme, α-amylase exists as a competing similar enzyme in the sample. Therefore, it is preferable to inhibit amylase in the specimen using an enzyme inhibitor. The inhibitor that inhibits amylase in the sample may be any one that inhibits the enzyme in the sample to a greater extent than inhibits the activity of the labeled enzyme.
In particular, in dogs, since there are not a few specimens having an α-amylase activity value of 2500 U / L or more, an inhibitor that effectively inhibits a small amount is preferable.

  Amylase inhibitors are said to have different specificities depending on their origins, and examples include kidney beans, wheat, and fungi. Among these, those derived from bacteria are preferable. For example, an amylase inhibitor derived from Streptomyces nitrosporeus specifically inhibits α-amylase derived mainly from higher animals.

  Examples of amylase inhibitors include α-Amylase Inhibitor (CALZYME Laboratories, Inc.), α-Amylase Inhibitor from Triticum aestivum (wheat seed) (Sigma), α-Amylase Inhibitor (Streptomyces nitrosporeus) (Wako Pure Chemicals) Company), but is not limited to these.

  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 different antibody must be bound to one enzyme molecule, the molar ratio of the antibody to the enzyme in the complex needs to be 1 or more. In order to reliably increase detection sensitivity, The molar ratio is preferably in the range of 2 to 5, and most preferably in the molar ratio of 2 to 3. 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 molecular weight of the complex is 100,000 daltons or more, preferably 200 to 500,000 daltons. Most preferably, a substance of 200 to 300,000 daltons is preferable in terms of high detection sensitivity.

In the analysis element for canine CRP measurement of the present invention, the above-described monoclonal antibody fragment obtained by labeling the fragment of the first monoclonal antibody that recognizes canine CRP with an enzyme, the second monoclonal antibody that recognizes canine CRP, and Then, a third monoclonal antibody fragment that recognizes canine CRP is used with a monoclonal antibody fragment obtained by labeling with an enzyme. The monoclonal antibody that recognizes the second canine CRP has a binding constant for canine CRP of 10 ⁷ M ⁻¹ to 10 ⁹ M ⁻¹ , has a subclass of IgG1, and may be the same as or different from the first monoclonal antibody. Good, but not a fragment or labeled antibody.

  The labeled first monoclonal antibody, second monoclonal antibody, and third monoclonal antibody used in the analytical element for canine CRP measurement of the present invention can be used by appropriately changing the amount used. However, the molar ratio of the second monoclonal antibody to the sum of the first monoclonal antibody and the third monoclonal antibody is 0.1 to 30, preferably 1 to 20, and most preferably 5 to 15. If the amount of the second monoclonal antibody is too much relative to the total amount of the labeled first monoclonal antibody and the third monoclonal antibody, the second monoclonal antibody binds to most of the canine CRP and is labeled. The first monoclonal antibody and the labeled third monoclonal antibody do not work effectively, and detection sensitivity cannot be obtained. On the other hand, if the amount of the second monoclonal antibody is too small relative to the total amount of the labeled first monoclonal antibody and the third monoclonal antibody, the labeled first monoclonal antibody The labeled third monoclonal antibody reacts with canine CRP to some extent, but the dynamic range cannot be obtained.

  In the present invention, in addition to a monoclonal antibody fragment obtained by labeling a fragment of a first monoclonal antibody that recognizes canine CRP with an enzyme and a second monoclonal antibody that recognizes canine CRP, a third antibody that further recognizes canine CRP is used. A monoclonal antibody fragment obtained by labeling a fragment of this monoclonal antibody with an enzyme is used.

  In the present invention, the measurement region can be expanded by using a monoclonal antibody fragment obtained by labeling the third monoclonal antibody fragment with an enzyme.

  The amount of the monoclonal antibody fragment obtained by labeling the third monoclonal antibody fragment with an enzyme can be appropriately set. The monoclonal antibody fragment obtained by labeling the first monoclonal antibody fragment with an enzyme and the third monoclonal antibody fragment can be appropriately set. The weight of the monoclonal antibody fragment obtained by labeling the third monoclonal antibody fragment with the enzyme in the total weight of the antibody fragment labeled with the enzyme-labeled monoclonal antibody fragment is 50% by weight or less, preferably 1% by weight. It is more than 50 weight%, More preferably, it is 10 weight% or more and 50 weight% or less, More preferably, it is 25 weight% or more and 50 weight% or less.

  The monoclonal antibody fragment obtained by labeling the third monoclonal antibody fragment with an enzyme preferably has a smaller binding constant to canine CRP than the first monoclonal antibody. That is, the binding constant of the third monoclonal antibody to canine CRP is 0.01 to 0.8 times, preferably 0.05 to 0.8 times, more preferably 0.1 to 0.6 times the binding constant of the first monoclonal antibody to canine CRP. It is. Moreover, the condition that the subclass of the third monoclonal antibody is IgG1 is satisfied.

  The dry analytical element for canine CRP measurement of the present invention will be described. 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 has a matrix structure that is small enough not to be captured 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 dry analytical element of the present invention includes an immune reaction layer (or an immune reaction layer and a substrate layer), a reagent layer (or a detection layer), a support, a development layer, a detection layer, a light shielding layer, an adhesive layer, a water absorption layer. A multilayer including a layer, an undercoat layer and other layers may be used. Examples of such analytical elements include JP-A-49-53888 (corresponding US Pat. No. 3,992,158), JP-A-51-40191 (corresponding US Pat. No. 4,042,335), and JP-A-55-164356 (corresponding US Pat. No. 4,292,272). JP-A 61-4959 (corresponding EPC published patent 0166365A).

In the case of using a light-permeable water-impermeable support, the dry immunoassay element of the present invention can practically have the following configuration. However, the content of the present invention is not limited to this.
(1) One having a reagent layer on a support and an immune reaction layer thereon.
(2) One having a reagent layer, a substrate layer, and an immune reaction layer in this order on a support.
(3) One having a reagent layer, an adhesive layer, and an immune reaction layer in this order on a support.
(4) One having a reagent layer, an adhesive layer, a substrate layer, and an immune reaction layer in this order on a support.
(5) One having a detection layer, a reagent layer, and an immune reaction layer in this order on a support.
(6) One having a detection layer, a reagent layer, a substrate layer, and an immune reaction layer in this order on a support.
(7) One having a reagent layer, a light reflection layer, and an immune reaction layer in this order on a support.
(8) One having a reagent layer, a light reflecting layer, a substrate layer, and an immune reaction layer in this order on a support.
(9) One having a detection layer, a reagent layer, a light reflection layer, and an immune reaction layer in this order on a support.
(10) A substrate having a detection layer, a reagent layer, a light reflection layer, a substrate layer, and an immune reaction layer in this order on a support.
(11) One having a detection layer, a light reflection layer, a reagent layer, and an immune reaction layer in this order on a support.
(12) A substrate having a detection layer, a light reflection layer, a reagent layer, a substrate layer, and an immune reaction layer in this order on a support.
(13) One having a second reagent layer, a light reflecting layer, a first reagent layer, and an immune reaction layer in this order on a support.
(14) One having a second reagent layer, a light reflection layer, a first reagent layer, a substrate layer, and an immune reaction layer in this order on a support.
(15) A substrate having a detection layer, a second reagent layer, a light reflection layer, a first reagent layer, and an immune reaction layer in this order on a support.
(16) One having a detection layer, a second reagent layer, a light reflection layer, a first reagent layer, a substrate layer, and an immune reaction layer in this order on a support.

  In the above (1) to (12), the reagent layer may be composed of a plurality of different layers. A water absorption layer may be provided between the support and the reagent layer or the detection layer. Moreover, you may provide a filtration layer between each layer. Further, a developing layer may be provided on the substrate layer, or the substrate layer may have a developing action and function as a developing layer. In addition, when there are solid components such as blood cells in the specimen, an appropriate filtration layer may be provided in the uppermost layer of the analysis element.

  The immune reaction layer and the substrate layer are composed of a water-permeable layer. In order to ensure the water permeability of these layers, it is preferable to use a porous layer made of a porous medium or a layer made of a hydrophilic polymer binder.

  The porous layer may be fibrous or non-fibrous. Examples of the fibrous material include filter paper, non-woven fabric, woven fabric (for example, plain woven fabric), knitted fabric (for example, tricot knitted fabric), glass fiber filter paper, and the like. Examples of non-fibrous materials include membrane filters made of cellulose acetate described in JP-A-49-53888, JP-A-49-53888, JP-A-55-90859 (corresponding US Pat. No. 4,258,001), JP-A-58- 70163 (corresponding US Pat. No. 4,486,537) and the like may be any of the continuous void-containing granular structure layer composed of inorganic or organic fine particles. JP 61-4959 (corresponding European published EP 0166365A), JP 62-116258, JP 62-138756 (corresponding European published EP 0226465A), JP 62-138757 (corresponding European published EP 0226465A), special A laminate of a plurality of partially bonded porous layers described in, for example, Kokai 62-138758 (corresponding European publication EP 0226465A) is also suitable.

  The porous layer may be a spreading layer having a so-called metering action that spreads the liquid in an area approximately proportional to the amount of liquid supplied. Of these, a woven fabric, a knitted fabric and the like are preferable as the spreading layer. A woven fabric or the like may be subjected to glow discharge treatment as described in JP-A-57-66359. In the development layer, in order to adjust the development area, the development speed, etc., JP-A-60-222770 (corresponding to: EP 0162301A), JP-A-63-219397 (corresponding to West German Patent Publication DE 37 17 913A), JP-A-63. -112999 (corresponding: DE 37 17 913A) and JP-A 62-182652 (corresponding: DE 37 17 913A) may contain a hydrophilic polymer or a surfactant.

  As a useful method of incorporating the substrate into the porous layer, for example, paper, cloth, a porous membrane made of a polymer is impregnated or coated with the substrate in advance, and then another water-permeable layer provided on the support, for example, It is also a useful method to adhere on the reagent layer by a method as disclosed in JP-A-55-164356. As another method, the porous layer may be adhered to another water-permeable layer (for example, a reagent layer) by the method described above, and then the composition containing the substrate may be applied to the porous layer. A known method can be used for impregnating or coating the porous layer. For coating, for example, dip coating, doctor coating, hopper coating, curtain coating, etc. are appropriately selected and used. The thickness of the substrate layer thus produced is not particularly limited, but when it is provided as a coating layer, a range of about 1 μm to 50 μm, preferably 2 μm to 30 μm is appropriate. In the case of a method other than coating, such as lamination by lamination, the thickness can vary greatly in the range of several tens to several hundreds of μm.

  In the case where the immune reaction layer and the substrate layer are constituted by a water-permeable layer made of a hydrophilic polymer binder, examples of the hydrophilic polymer that can be used include the following. Gelatin and derivatives thereof (for example, phthalated gelatin), cellulose derivatives (for example, hydroxyethylcellulose), agarose, sodium alginate, acrylamide copolymer, methacrylamide copolymer, and copolymers of acrylamide or methacrylamide with various vinyl monomers Polyhydroxyethyl methacrylate, polyvinyl alcohol, polyvinylpyrrolidone, sodium polyacrylate, copolymers of acrylic acid and various vinyl monomers, and the like.

The substrate layer composed of a hydrophilic polymer binder is disclosed in JP-B-53-21677 (corresponding US Pat. No. 3,992,158), JP-A 55-164356 (corresponding US Pat. No. 4,292,272), JP-A 54-101398 (corresponding US patent) 4,132,528), an aqueous solution or aqueous dispersion containing a substrate or other reagent composition and a hydrophilic polymer according to the method described in the specification of JP-A-61-292063 (Chemical Abstracts, 106 , 210567y), etc. It can be provided by coating on another layer such as a layer and drying. The substrate layer with a hydrophilic polymer as a binder has a dry thickness of about 2 μm to about 50 μm, preferably about 4 μm to about 30 μm, and a coverage of about 2 g / m ² to about 50 g / m ² , preferably Is in the range of about 4 g / m ² to about 30 g / m ² .

In addition to the non-diffusible substrate, the substrate layer has an enzyme activator, a coenzyme, a surfactant, for the purpose of improving various properties such as coating characteristics, diffusibility of the diffusible compound, reactivity, and storage stability. Various additives consisting of organic substances or inorganic substances can be added, such as pH buffer compositions, fine powders, antioxidants, and the like. Examples of buffering agents that can be incorporated into the substrate layer include the Chemical Society of Japan "Basic Manual" (Tokyo, Maruzen Co., Ltd., 1966), pages 1312-1320, RMCDawson et al., "Data for Biochemical Research 2nd edition (Oxford at the Clarendon Press, 1969) 476-508, “Biochemistry” 5 , 467-477 (1966), “Analytical Biochemistry” 104 , 300-310 (1980) There is a pH buffer system described in. Buffers containing tris (hydroxymethyl) aminomethane (Tris) as specific examples of pH buffers; buffers containing phosphate; buffers containing borate; buffers containing citric acid or citrate; containing glycine Buffers: Buffers containing bicine (Bicine); Buffers containing HEPES; Good buffers such as buffers containing MES.

  An immune reaction layer can also be comprised similarly to the said substrate layer. In order to contain the substrate and the enzyme-labeled antibody in one layer or two adjacent layers in a substantially dry state or substantially in the absence of water, the enzyme-labeled antibody is added to alcohol (eg, ethanol) or the like. The water-permeable layer may be impregnated by dissolving or dispersing in a non-aqueous solvent.

  The reagent layer contains a reagent composition for detecting a diffusible substance that has diffused and migrated from the immune reaction layer (or substrate layer). If necessary, the reagent composition contains a low molecular enzyme, and a detection reagent composition for detecting a low molecular product produced by reducing the molecular weight of the diffusible substance. The reagent layer is composed of a water-permeable layer, and is preferably a continuous layer made of a hydrophilic polymer binder among the water-permeable layers described in the description of the substrate layer. The hydrophilic polymer binder to be used is determined in consideration of the diffusible product produced in the substrate layer, the coloring reagent contained in the reagent layer, and the like.

  If the diffusible substance that has diffused or migrated from the immune reaction layer (or substrate layer) is directly detectable, the reagent layer does not need to contain a detection reagent composition, and the reagent layer functions as a detection layer. To do. Also when it is set as a detection layer, it is preferable to make it the continuous layer which consists of a hydrophilic polymer binder among the water-permeable layers described by the description of the said substrate layer.

  As the support, any of light-opaque (opaque), light-semi-transmissive (semi-transparent), and light-transmissive (transparent) can be used, but generally light-transmissive and water-impermeable. These supports are preferred. Preferable materials for the light transmissive water-impermeable support are polyethylene terephthalate and polystyrene. In order to firmly adhere the hydrophilic layer, an undercoat layer is usually provided or a hydrophilic treatment is performed.

  The dry immunoassay element of the present invention comprises a diffusible substance decomposed by a labeling enzyme from a non-diffusible substrate, as in JP-A-3-295466 (corresponding EP 0451848A), 4-128655, and 4-2765551. Increasing the sensitivity can be achieved by including in the reagent layer a low-molecular-weight enzyme to be converted. In these combinations, the enzyme acts on a non-diffusible substrate to generate a diffusible substance, and this diffusible product can be easily detected by generating a low molecular weight product by a low molecular weight enzyme described later. You can choose from such combinations.

  That is, an antibody labeling enzyme is selected that decomposes a non-diffusible substrate made of a polymer and generates a diffusible product that causes a low-molecular-weight product to further generate a low-molecular product. As the non-diffusible substrate, a substrate that is non-diffusible (insoluble) with respect to the aqueous specimen solution and does not diffuse or migrate from the immune reaction layer (or substrate layer) to the reagent layer itself is selected. Further, the low molecular-weight enzyme is selected from those that make the diffusible product produced from the non-diffusible substrate by the antibody labeling enzyme into a further detectable low-molecular product. These examples will be specifically described below.

  Such an enzyme is preferably a degrading enzyme that generates a diffusible oligomer from a non-diffusible substrate made of a polymer. Among the above-mentioned labeling enzymes, for example, a carbohydrate hydrolase is preferable. Examples of such saccharide hydrolase include α-amylase, β-amylase, glucoamylase, lysozyme and the like.

  Examples of substrates for the aforementioned α-amylase, β-amylase, and glucoamylase include carboxymethylated starch and starch. When carboxymethylated starch or starch is used as a non-diffusible substrate, a combination using α-amylase as a labeling enzyme and glucoamylase or α-glucosidase described later as a depolymerizing enzyme is possible.

  This low molecular weight enzyme may be the same type of enzyme as the labeling enzyme. In this case, the labeling enzyme is an endo-active enzyme that cleaves from the inside of the molecule to produce an oligomer, and the depolymerizing enzyme acts from the end of the molecule to produce a monomer. It is preferable to have. For example, when the non-diffusible substrate is a polymer (eg, starch), a substance capable of decomposing a diffusible oligomer (eg, maltose) produced by a labeling enzyme into a monomer (eg, glucose) is used. Examples of such a low molecular weight enzyme include sugar hydrolase, and more specifically, α-amylase, β-amylase, glucoamylase, α-glucosidase and the like. When carboxylmethylcellulose and cellulase are used as the non-diffusible substrate and the labeling enzyme, C1 enzyme can be used as the depolymerizing enzyme.

  Combinations of these labeling enzymes, non-diffusible substrates, and low molecular weight enzymes are known in the literature (eg, “Enzyme Handbook” (supervised by Bunji Maruo, Nobuo Tamiya, Asakura Shoten, 1982), “Biochemical Handbook” (Nobumasa Imura). , Other editions, published in Maruzen 1984).

The low molecular product produced by the depolymerizing enzyme in the reagent layer can be optically detected by a known detection system reagent. As a method for detecting glucose finally produced by the low molecular weight enzyme, for example, a method for detecting hydrogen peroxide produced by oxidizing glucose in the presence of glucose oxidase (for example, Ann. Clin. Biochem., 6 , 24 (1964), J. Clin. Pathol., 22 , 246 (1969), Trinder reagent, JP 49-50991 (corresponding U.S. Patent 3,886,045), U.S. Patent 3,992,158, JP 55-164356 (Compatible US Patent 4,292,272) etc.Trinder reagent, JP-A-53-26188 (Corresponding US Patent 4,089,747), JP-A-58-45557 etc. No. 59-193352 (corresponding European Patent Publication EP 0122641A), JP-A-60-224677 (corresponding US Pat. No. 4,665,023) and the like, a method using a reagent containing a diaryl-monoaralkyl-substituted imidazole leuco dye, Generate in the presence of NAD Known methods such as a method for detecting NADH and a method for detecting glucose-6-phosphate produced in the presence of hexokinase can be used. Among these detection methods, a method of detecting hydrogen peroxide generated by oxidizing glucose in the presence of glucose oxidase using a peroxidase and a leuco dye is most desirable in terms of sensitivity.

  These detection reagents may be contained in the reagent layer of the analysis element together with the depolymerizing enzyme, but may be contained in another layer (for example, the second reagent layer or the detection layer) provided in the lower layer of the reagent layer. You may make it detect in this layer. In addition, when using a leuco dye, it is preferable on the stability of the produced | generated pigment | dye to make it the dispersion in the hydrophilic binder of the solution of a water immiscible solvent.

  The dry immunoassay element of the present invention can be prepared by known methods described in the aforementioned patent specifications. The analysis element of the present invention is cut into small pieces such as a square having a side of about 15 mm to about 30 mm or a circle of the same size, and the like, and Japanese Patent Publication No. 57-28331 (corresponding US Pat. No. 4,169,751), Japanese Utility Model Publication No. 56-142454 (corresponding US Pat. ), Japanese Unexamined Patent Publication No. 57-63452, Japanese Utility Model Publication No. 58-32350, Special Table No. 58-501144 (corresponding international publication: WO 83/00391), etc. From the viewpoint of packaging, transportation, storage, measurement operation and the like. Depending on the purpose of use, it can be used in the form of a long tape stored in a cassette or magazine, or a small piece can be attached to or stored in a card with an opening.

  The dry analysis element of the present invention can quantitatively analyze a macromolecular antigen that is a test substance in a liquid sample by the same operations as those described in the aforementioned patent specifications. For example, an aqueous liquid sample solution such as plasma, serum, urine or the like in the range of about 5 μL to about 30 μL, preferably 8 to 15 μL is spotted on the substrate layer. The spotted analytical element is incubated at a constant temperature in the range of about 20 ° C. to about 45 ° C., preferably at a constant temperature in the range of about 30 ° C. to about 40 ° C. for 1 to 10 minutes. The color development or discoloration in the element is reflected and photometrically measured from the side of the light-transmitting support, and the amount of macromolecular antigen in the sample can be determined based on the principle of the colorimetric measurement method using a calibration curve prepared in advance. Quantitative analysis can be performed with high accuracy by keeping the amount of liquid sample to be spotted, incubation time and temperature constant. The measurement operation can be carried out with high accuracy and quantitative analysis by an extremely easy operation using a chemical analyzer described in JP-A-60-125543, 60-220862, 61-294367, etc. Depending on the purpose and required accuracy, semi-quantitative measurement may be performed by visually judging the degree of color development. If no enzyme-labeled antibody is contained in the analysis element, mix the aqueous sample solution with the solution containing the enzyme-labeled antibody before spotting the element, and allow the binding reaction to take place. Just wear it.

  The present invention further relates to a method for measuring CRP in a sample, which comprises bringing the sample into contact with the above-described immunoassay reagent for canine CRP and human CRP measurement of the present invention or the dry analytical element for canine CRP and human CRP measurement. . 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: Preparation of monoclonal antibody-producing hybridoma whose antibody subclass is IgG1 and has reactivity with canine CRP (1) Preparation of antibody-producing fusion cell (hybridoma):
After immunizing mice (BALBc) with canine CRP isolated and purified from fresh beagle serum using an affinity column as an antigen and confirming an increase in antibody titer against canine CRP, splenocytes were collected and myeloma (P3U1 ) And the PEG method. A number of hybridomas were obtained by selective culture using HAT medium.

(2) Hybridoma selection (1): Confirmation of antigen specificity and confirmation of antibody type and subclass In the hybridoma culture supernatant obtained in (1) above, the protein antigen of canine CRP should be 1 μg / mL. Dilute with PBS (-), perform solid phase, and use anti-mouse IgG HRP-labeled goat antibody (No. AP308P Funakoshi Co., Ltd.) as the secondary antibody. The reactivity with the antigen was confirmed by ELISA using a microplate, and several hybridomas producing antibodies that react with the antigen of canine CRP could be selected. Moreover, about these hybridomas, the isotype and subclass of the antibody in the culture supernatant were identified using a commercially available identification kit reagent.

(3) Selection of hybridoma (2): Confirmation of binding constant to antigen For culture supernatant confirmed to be reactive with canine CRP, the antibody produced is IgG using Biacore equipment (Biacore3000). The binding constant was measured. Rabbit IgG of anti-mouse IgG is immobilized on the sensor chip, and this is brought into contact with the culture supernatant under certain conditions to immobilize mouse IgG in the culture supernatant. Next, 4 μg / mL (3.5 × 10 ⁻⁸ M) of each antigen protein is brought into contact with the target binding constant of 10 ⁹ M ⁻¹ . Based on the increase in signal when the culture supernatant is brought into contact (antibody signal: equivalent to the amount of IgG in the culture supernatant), the increase in signal when the antigen protein is brought into contact (antigen signal: the antigen capture capacity of the antibody) The ratio of antibody binding constants in the culture supernatant can be estimated. The measurement results of the culture supernatant are shown in Table 1. The signal ratio under the same conditions with the antibody prepared by the mouse ascites method using the three types of hybridomas 3A6, 11C6, and 16H4 and purified with the protein A column is in good agreement with the culture supernatant. These results show that they are effective for antibody selection. By this method, hybridoma 16H4 could be selected as an antibody-producing hybridoma having a sufficient binding constant of 10 ⁷ M ⁻¹ to 10 ⁹ M ⁻¹ for canine CRP and having a subclass of IgG1. Furthermore, 3A6 and 11C6 were obtained as hybridomas.

  Hybridoma 16H4 (indication for identification given by the depositor: MM44097-16H4F5) is independent as of April 18, 2008 as the accession number FERM P-21571 (reception number FERM AP-21571) It is deposited with the Patent Organism Depositary Center (National Institute of Advanced Industrial Science and Technology (1st, 1st, Higashi 1-chome, Tsukuba, Ibaraki, Japan)). The deposit with deposit number FERM P-21571 was transferred to an international deposit on May 27, 2009 and deposited under the provisions of the Budapest Treaty as deposit number FERM BP-11132.

*1：クローニング時に脱落。
*2：固定時の抗原濃度１μg/mL
*3：抗原濃度４μg/mL(約3.5×10^-8M)

* 1: Dropped during cloning.
* 2: Antigen concentration at the time of fixation 1 μg / mL
* 3: Antigen concentration 4 μg / mL (approximately 3.5 × 10 ⁻⁸ M)

Example 2: Preparation of monoclonal antibodies:
Hybridoma 16H4 is thawed and cultured in RPMI 1640 + 10% FBS, and 0.5 mL (5 × 10 ^ 6 cells / mouse) of the proliferated cells is transplanted into 20 mice. After a certain period of time, ascites is collected from the peritoneal cavity, centrifuged, and the supernatant is purified by the protein A-affinity chromatography method shown below. Ascites supernatant diluted with the same buffer is run on a protein A column equilibrated with 1.5 M glycine-NaOH (pH 8.9) -3 M NaCl to adsorb IgG. Elute with elution buffer (100 mM citrate buffer, pH 3.0). Immediately add neutralization solution (2 M Tris-HCl, pH 9.0) to adjust to pH 7.0-7.5, then dialyzed against PBS for purified antibody Got.

Example 3: Preparation of analytical element for canine CRP measurement Table 2 shows binding constants of canine CRP antibodies to canine CRP. As a comparative example, binding constants with IgG2b antibody are shown. According to the same method as that described in Japanese Patent No. 3151080, the labeled antibody was prepared as an anti-canine CRP • IgGFab ′ labeled with an enzyme using Bacillus subtilis α-amylase and the canine CRP antibody described in Table 1. As a comparative example, anti-canine CRP • IgGFab ′ labeled with an enzyme using an IgG2b antibody was also prepared. Using the materials shown in Table 2, an analytical element for canine CRP measurement was prepared. The canine CRP antibody shown in Table 2 was used as the second antibody.

Creating a dry analytical element for CRP measurement:
An aqueous solution having the following composition was coated on a 180 μm polyethylene terephthalate colorless and transparent smooth film coated with gelatin so that the thickness after drying was 14 μm and dried.
Gelatin 14.1g / m ²
Peroxidase 12.0KU / m ²
Glucose oxidase 6.0KU / m ²
Glucoamylase 5.0KU / m ²
Leuco dye 0.5g / m ²
Surfactant 1.0g / m ²

  Here, the surfactant is polyoxy (2-hydroxy) propylene nonylphenyl ether (Surfactant 10G, manufactured by Aurin), and the leuco dye is 2- (3,5-dimethoxy-4-hydroxyphenyl) -4- ( 4-Dimethylaminophenyl) -5-phenethylimidazole acetate was used.

Next, an aqueous solution having the following composition was applied onto the film so that the thickness after drying was 10 μm and dried.
Gelatin 10.2g / m ²
Surfactant 0.5g / m ²

Next, an aqueous solution adjusted to pH = 6.4 having the following composition was applied onto the film so that the thickness after drying was 8 μm and dried.
Hydroxypropyl cellulose 4.7 g / m ²
Carboxymethyl starch 3.5 g / m ²
PIPES 0.9 g / m ²
Mannitol 2.3 g / m ²
Surfactant 1.2 g / m ²
Amylase inhibitor 1020KU / m ²

Next, water is supplied over the film at a supply rate of about 60 g / m ² and moistened, and then a tricot knitted fabric knitted with 36 gauge polyethylene terephthalate spun yarn equivalent to 50 denier is laminated with light pressure. And dried.
On the above fabric, ethanol was applied to 200 g / m ² (= OC1 coating), dried, and then an ethanol solution having the following composition was added so that each component had the following amount, and the thickness after drying was It was applied so as to have a thickness of 5 μm (= OC2 application) and dried to produce an integrated multilayer analytical element.

Amylase-labeled anti-canine CRP antibody LA 19.0 KU / m ² (1.2 mg / m ² , molecular weight 30 KDa)
Anti-canine CRP antibody A 5.4 mg / m ² (molecular weight 15 KDa)
Polyvinylpyrrolidone 5.6 g / m ²
Surfactant 0.2 g / m ²
Amylase inhibitor 2080 KU / m ²

(Calculation of antibody molar ratio)
Antibody LA 1.2 mg / m ² ÷ Molecular weight 30 KDa = 0.04 x 10 ^-3 mmol / m ²
Antibody A 5.4 mg / m ² ÷ Molecular weight 15 KDa = 0.36 x 10 ^-3 mmol / m ²
Molar ratio = 0.36 ÷ 0.04 = 9

  The above integrated multilayer analysis element was cut into 12 mm x 13 mm square chips and placed in a slide frame (described in JP-A-57-63452) to produce a dry slide for CRP analysis (dry analysis element No 101). .

  Further, in the above, the dry analytical element shown in Table 3 was prepared in the same manner as described above except that a part of the amylase-labeled anti-canine CRP antibody LA was changed as shown in Table 3. Here, anti-canine CRP antibody A was used as the second antibody, and the molar ratio (sum of the second antibody / labeled antibody) was 9. The molecular weight of the amylase-labeled anti-canine CRP antibody LC is 30 KDa.

Example 4: Sensitivity performance evaluation test A diluted solution of the following composition (*) and dog serum with CRP concentrations of 0, 1.1, 3.0, 7.1, 14, and 20 mg / dL tested by immunoturbidimetric method were used in a diluted solution. The diluted solution was spotted at 10 μL on the dry slide for CRP analysis prepared in the above example.

  Then, while incubating at 37 ° C. for 5 minutes, the reflection density at 650 nm was measured with Fuji Dry Chem 7000 (manufactured by Fuji Photo Film Co., Ltd.) approximately every 10 seconds. FIG. 1 shows the result of obtaining the amount of increase in reflection density per minute (ΔODr) from the reflection density for 3 minutes to 5 minutes at that time. Table 4 shows values obtained by multiplying the decrease amount of ΔODr per CRP unit concentration (1 mg / dL) obtained from FIG. Since ΔODr with respect to the same CRP concentration varies to some extent, the value in Table 4 needs to be 4 or more in order to obtain a significant measurement value of the CRP concentration.

* Diluent composition
MES (* 1) 5mg
Casein aqueous solution (* 2) 100mg
Sodium azide 0.2mg
Purified water 1.0ml
* 1 MES: 2- (N-morpholino) ethanesulfonic acid monohydrate
* 2 For example, product name: Block Ace

  From the results shown in Table 4 and FIG. 1, a part of the monoclonal antibody fragment obtained by labeling the first monoclonal antibody fragment of the dry analytical element of the present invention with an enzyme and a part of the third monoclonal antibody fragment labeled with an enzyme are used. With the dry analytical elements 102 and 103 substituted with the antibody fragment, a sufficient amount of change in ΔODr was obtained corresponding to the change in concentration in canine serum up to canine CRP = 20 mg / dL. On the other hand, in the dry analysis element 101, the necessary ΔODr change amount corresponding to the change in concentration cannot be obtained in the canine CRP = 7.1 to 20.0 mg / dL. In the dry analysis element 104, in the canine CRP = 0 to 7.1 mg / dL The required amount of change in ΔODr corresponding to the concentration change cannot be obtained.

Claims (7)

Enzyme-labeled first monoclonal antibody fragment that recognizes canine CRP with enzyme, second monoclonal antibody that recognizes canine CRP, and third monoclonal antibody fragment that recognizes canine CRP A dry analytical element for canine CRP measurement comprising an immunoreactive layer comprising a monoclonal antibody fragment labeled with a light transmissive support,
(1) the binding constant of the first monoclonal antibody, the second monoclonal antibody, and the third monoclonal antibody to canine CRP is 10 ⁷ M ⁻¹ to 10 ⁹ M ⁻¹ ;
(2) The subclass of the first monoclonal antibody, the second monoclonal antibody and the third monoclonal antibody is IgG1,
(3) The molar ratio of the second monoclonal antibody to the total of the first monoclonal antibody and the third monoclonal antibody is 0.1 to 30,
(4) The third monoclonal antibody fragment in the total weight of the monoclonal antibody fragment obtained by labeling the first monoclonal antibody fragment with an enzyme and the monoclonal antibody fragment obtained by labeling the third monoclonal antibody fragment with an enzyme. The weight of the monoclonal antibody fragment labeled with the enzyme is 50% by weight or less,
(5) The binding constant of the third monoclonal antibody to canine CRP is 0.01 to 0.8 times the binding constant of the first monoclonal antibody to canine CRP.
Dry analytical element for canine CRP measurement. The dry analytical element for canine CRP measurement according to claim 1, wherein the enzyme is Bacillus subtilis α-amylase. The dry analytical element for canine CRP measurement according to claim 1 or 2, further comprising an amylase inhibitor that inhibits amylase derived from dogs. Enzyme-labeled first monoclonal antibody fragment that recognizes canine CRP with enzyme, second monoclonal antibody that recognizes canine CRP, and third monoclonal antibody fragment that recognizes canine CRP An immunoassay reagent or kit for canine CRP measurement, comprising a monoclonal antibody fragment labeled with
(1) the binding constant of the first monoclonal antibody, the second monoclonal antibody, and the third monoclonal antibody to canine CRP is 10 ⁷ M ⁻¹ to 10 ⁹ M ⁻¹ ;
(2) The subclass of the first monoclonal antibody, the second monoclonal antibody and the third monoclonal antibody is IgG1,
(3) The molar ratio of the second monoclonal antibody to the total of the first monoclonal antibody and the third monoclonal antibody is 0.1 to 30,
(4) The third monoclonal antibody fragment in the total weight of the monoclonal antibody fragment obtained by labeling the first monoclonal antibody fragment with an enzyme and the monoclonal antibody fragment obtained by labeling the third monoclonal antibody fragment with an enzyme. The weight of the monoclonal antibody fragment labeled with the enzyme is 50% by weight or less,
(5) The binding constant of the third monoclonal antibody to canine CRP is 0.01 to 0.8 times the binding constant of the first monoclonal antibody to canine CRP.
An immunoassay reagent or kit for canine CRP measurement. The immunoassay reagent or kit for canine CRP measurement according to claim 4, wherein the enzyme is Bacillus subtilis α-amylase. The immunoassay reagent or kit for canine CRP measurement according to claim 4 or 5, further comprising an amylase inhibitor that inhibits amylase derived from dogs. A method for measuring canine CRP using the dry analytical element for measuring canine CRP according to any one of claims 1 to 3, or the immunoassay reagent or kit for measuring canine CRP according to any one of claims 4 to 6.

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