JP2004208693A - Anti-hyaluronic acid antibody, reagent kit containing the sane, method for producing the anti-hyaluronic acid antibody and method for measuring hyaluronic acid - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an anti-hyaluronic acid antibody applicable to accurately and simply measuring hyaluronic acids having any molecular weight, to provide a reagent kit containing the antibody, to provide a method for producing the anti-hyaluronic acid antibody and to provide a method for measuring the hyaluronic acids. <P>SOLUTION: The anti-hyaluronic acid antibody is produced by immunizing an animal with an antibody, wherein the antibody has 20,000-4,000,000 viscosity-average molecular weight and is a solid unmodified hyaluronic acid (MH). The anti-hyaluronic acid antibody is preferably a monoclonal antibody. The method for producing the anti-hyaluronic acid antibody comprises an immunizing process immunizing the animal with the solid unmodified hyaluronic acid (MH) having 20,000-4,000,000 viscosity-average molecular weight. <P>COPYRIGHT: (C)2004,JPO&NCIPI

Description

  The present invention relates to an anti-hyaluronic acid antibody, a reagent kit containing the same, a method for producing an anti-hyaluronic acid antibody, and a method for measuring hyaluronic acid. More specifically, the present invention relates to an anti-hyaluronic acid antibody optimal for clinical examination, a reagent kit containing the same, a method for producing an anti-hyaluronic acid antibody, and a method for measuring hyaluronic acid.

  An anti-hyaluronic acid monoclonal antibody for immunoassay is obtained by immunizing an animal with a covalent conjugate formed by introducing hemocyanin, phosphatidylethanolamine or phosphatidylserine into low-molecular-weight sodium hyaluronate. Monoclonal antibodies are known (Patent Documents 1 and 2). In addition, as proteins for immunoassay, hyaluronic acid-binding proteins (proteoglycan, link protein and hyaluronectin) and CD44 (cell membrane penetrating protein) are known (Patent Documents 3 and 4).

JP-A-11-155566 JP-A-9-12600 Japanese Patent Publication No. 6-41952 JP-A-9-229930

  However, the monoclonal antibody against low molecular weight hyaluronic acid has a problem in that it cannot measure high molecular weight (particularly, viscosity average molecular weight of 180,000 to 4,000,000) hyaluronic acid existing in the living body. In addition, since hyaluronic acid-binding protein, CD44, and the like are present as trace components in the living body, there is a problem that they cannot be measured without extracting and purifying them from a sample collected from the living body. That is, an object of the present invention is to provide an anti-hyaluronic acid antibody capable of simply and accurately measuring hyaluronic acid of all molecular weights, a reagent kit containing this antibody, a method for producing this anti-hyaluronic acid antibody, and a method for measuring hyaluronic acid. That is.

  The present inventors have conducted intensive studies to solve the above problems, and as a result, have found that an anti-hyaluronic acid antibody that can be produced by a specific immunization method can achieve the above object, and have reached the present invention. That is, the present invention relates to an anti-hyaluronic acid antibody that can be produced by immunizing an animal with an antigen, wherein the antigen has a viscosity average molecular weight of 20,000 to 4,000,000 and is solid unmodified hyaluronic acid (MH). An anti-hyaluronic acid antibody, a reagent kit containing the antibody, and a method for quantifying hyaluronic acid using the kit. The present invention also provides a method for producing an anti-hyaluronic acid antibody, comprising an immunization step of immunizing an animal with a solid unmodified hyaluronic acid (MH) having a viscosity average molecular weight of 20,000 to 4,000,000.

  The anti-hyaluronic acid antibody of the present invention can easily and accurately measure hyaluronic acid of all molecular weights. The anti-hyaluronic acid antibody of the present invention has enabled not only a competitive immunoassay but also a sandwich immunoassay which could not be realized conventionally. Furthermore, this hyaluronic acid antibody is most suitable for a reagent kit for measuring hyaluronic acid, and among them, a reagent kit containing the same anti-hyaluronic acid monoclonal antibody (for sandwich measurement method) can be quantified with extremely high sensitivity. Therefore, when measurement is performed using a reagent kit containing the anti-hyaluronic acid antibody of the present invention, hyaluronic acid can be quantified extremely simply and with high accuracy. The method for producing an anti-hyaluronic acid antibody of the present invention can easily prepare an anti-hyaluronic acid antibody that enables the above-described simple and accurate measurement by including an immunization step by a specific immunization method.

  In the present invention, hyaluronic acid means hyaluronic acid and a hyaluronic acid salt unless otherwise specified. Examples of the hyaluronate include alkali metal (such as lithium, sodium and potassium) salts, alkaline earth metal (such as magnesium and calcium) salts, and ammonium salts. Of these, alkali metal salts are preferred, and sodium salts are particularly preferred. It is salt.

The viscosity average molecular weight of the antigen used for immunization is preferably 20,000 or more, more preferably 40,000 or more, particularly preferably 100,000 or more, most preferably 180,000 or more, and preferably 4,000,000 or less, more preferably 300,000 or less. It is at most 10,000, particularly preferably at most 2.5 million, most preferably at most 2 million. Within this range, an antibody that can react with hyaluronic acid of all molecular weights is easily obtained. The viscosity average molecular weight is measured according to the method described in Biopolymer 9,799 (1970). That is, the intrinsic viscosity is determined, and the value is expressed by the formula [h] = 2.28 × 10 ⁻⁴ × M ^0.816 where [h] is the intrinsic viscosity (dl / g) and M is the viscosity average molecular weight. . Substitute into｝ to determine the viscosity average molecular weight (the same applies hereinafter).

  The form of the antigen used for immunization may be liquid (including a molten state and a dissolved state) or solid (including a dispersed state), but is preferably a solid. When it is in a solid state, an antibody that can react with hyaluronic acid of all molecular weights is easily obtained. Here, the form means the state of the substance at 10 to 40 ° C., and is at least the state of the substance itself at the start of immunization. The shape and size of the solid can be freely set within a range that does not hinder the immunization operation. Examples of the shape include a powder, a sand (granular), a sheet, a fiber, and the like, and preferably a sand. Since the immunization operation usually uses an injection needle having an inner diameter of about 0.4 to 1.2 mm, the size is preferably a size that passes through the injection needle, for example, injection having an inner diameter of about 0.4 to 1.2 mm. When immunization is performed using a needle, the volume average particle diameter is preferably 1/10 to 1/2 of the inner diameter of the injection needle to be used, and more preferably 1/5 to 1/3 of the inner diameter of the injection needle. It is an average particle size. The volume average particle size is measured by a laser diffraction type particle size distribution analyzer (eg, SALD-1100 manufactured by Shimadzu Corporation) having a measurement principle {light scattering method (25 ° C.)} described in JIS Z8825-1: 2001. Desired.

  The antigen used for immunization can be used without limitation as long as it has the above characteristics. As the commercially available hyaluronic acid (trade name), sodium hyaluronate (derived from pig skin) ｛Seikagaku Corporation Manufactured by Seikagaku Corporation, viscosity average molecular weight: 800,000-1.2 million, hyaluronic acid Kyowa-HP (derived from microorganisms) Kyowa Hakko Kogyo Co., Ltd., viscosity average molecular weight 1.5 million to 2 million yen, sodium biohyaluronate (microorganism origin) {Shiseido Co., Ltd., viscosity average molecular weight 600,000 to 1.5 million yen, denka hyaluronic acid (microorganism origin) {Viscosity average molecular weight 600,000 to 1,500,000 produced by Denki Kagaku Kogyo Co., Ltd., and hyaluronic acid HA-Q (derived from chicken crown)} Ten thousand}, and the like.

  In addition to this, using the umbilical cord as a raw material, after crushing finely, most of the protein is removed by decomposition with pepsin or trypsin, and the remainder is shaken with chloroform-amyl alcohol to remove as a gel, and then with ammonium sulfate in the presence of pyridine. Separate precipitation is performed to remove other polysaccharides (other methods include extraction with sodium acetate, phenol, and trichloroacetic acid, and precipitation with alcohol). And it can also be obtained by fractionating and collecting into a preferable viscosity average molecular weight range by affinity chromatography, gel permeation chromatography or the like. The antigen used for immunization is preferably unmodified hyaluronic acid that has not been modified with hemocyanin, phosphatidylethanolamine, phosphatidylserine, or the like. When unmodified hyaluronic acid is used, an antibody capable of reacting with hyaluronic acid of all molecular weights is easily obtained.

  The animal used for immunization is not particularly limited, and can be appropriately selected from those conventionally used for producing antibodies, and examples include mouse, rat, rabbit, sheep, goat, and cow. Of these, mice and rats are preferable, and mouse is more preferable, from the viewpoint of easy preparation of monoclonal antibodies.

  Usually, when immunizing an animal with an antigen, it is dissolved in an appropriate solvent (such as physiological saline), then emulsified with an immunopotentiator (such as Complete Freund's adjuvant), and administered to the animal subcutaneously or intraperitoneally. General. However, in the case of unmodified hyaluronic acid, this method has a problem that a large amount of antigen cannot be immunized due to the high viscosity of the hyaluronic acid solution, and a small amount of antigen makes it difficult to obtain a sufficient antibody titer. In order to immunize unmodified hyaluronic acid and efficiently obtain anti-hyaluronic acid antibodies, a water-in-oil emulsion prepared by mixing and emulsifying an immunopotentiator (complete Freund's adjuvant, etc.) and physiological saline in a conventional manner. And solid unmodified hyaluronic acid (MH) are mixed and dispersed, and immediately administered (preferably within about 30 minutes, more preferably within 15 minutes, particularly preferably within 5 minutes) to an animal. . By immunizing an animal with the solid unmodified hyaluronic acid (MH) mixed with the water-in-oil emulsion, a sufficient amount of antigen can be administered and a sufficient antibody titer can be obtained. Cheap.

  The weight ratio of the immunity enhancer and physiological saline (immune enhancer: physiological saline) may be the same as in the case of normal immunization, and is preferably 1: 0.6 to 1, more preferably 1: 0. 8 to 1, particularly preferably 1: 1. The content (% by weight) of the solid unmodified hyaluronic acid (MH) is preferably 0.01 or more, more preferably 0.05 or more, based on the total weight of the immunopotentiator and physiological saline. It is particularly preferably at least 0.1, more preferably at most 30, still more preferably at most 20, particularly preferably at most 10.

  The amount of unmodified hyaluronic acid (MH) to be administered per dose can be appropriately set depending on the type and size of the animal, but is preferably 20 μg or more, more preferably 40 μg or more, and particularly preferably 100 μg per 1 g of animal body weight. It is preferably at least 5 mg, more preferably at most 2 mg, particularly preferably at most 1 mg. Within this range, a sufficient antibody titer can be easily obtained. That is, as an example of a preferred immunization method, when immunizing a mouse weighing 60 g, 0.2 g of a water-in-oil emulsion and 1.2 to 300 mg of solid unmodified hyaluronic acid (MH) are mixed and administered intraperitoneally. And the like. The number of immunizations can be appropriately set, but it is preferable to immunize at least three times at intervals of two weeks to one month. The second and subsequent immunization doses may usually be the same as the first immunization, but can be increased or decreased appropriately within the above range.

  When obtaining a polyclonal antibody, after immunization, blood is collected according to a conventional method to obtain a polyclonal antibody. For example, when the immunized animal is a rabbit, the lateral ear vein is incised and blood is collected. If the immunized animal is a mouse, the tail vein is incised and blood is collected. In each case, a polyclonal antibody is obtained by separating serum from the obtained blood and collecting an antibody fraction (usually an IgG fraction) using ammonium sulfate precipitation, an ion exchange column or the like. The reactivity of the obtained polyclonal antibody can be confirmed, for example, by an enzyme-linked immunosorbent assay (ELISA). That is, after reacting the obtained polyclonal antibody with an insoluble carrier to which hyaluronic acid is bound, a conjugate of the obtained antibody recognizing the polyclonal antibody and a label [for example, the obtained anti-hyaluronic acid polyclonal antibody is rabbit In the case of an antibody, a peroxidase-conjugated anti-rabbit antibody (porcine) or the like] is reacted, and the reactivity between the obtained polyclonal antibody and hyaluronic acid can be confirmed by detecting the label. At this time, the reactivity to hyaluronic acid having a different molecular weight or the like can be confirmed by changing the molecular weight or the like of hyaluronic acid bound to the insoluble carrier.

  On the other hand, when obtaining a monoclonal antibody, the spleen of the immunized animal is collected, and cell fusion is performed according to a conventional method using spleen cells prepared from the spleen to prepare a fused cell. Then, cells that produce an antibody that reacts with hyaluronic acid are selected from these by the above-mentioned enzyme-linked immunosorbent assay, and the selected fused cells are cloned by a limiting dilution method or the like. The cloned fused cells are grown in the mouse intraperitoneal cavity or in a serum-free medium, and purified from the obtained ascites or culture solution with protein A or the like to obtain a monoclonal antibody. The reactivity of the monoclonal antibody can also be confirmed by the enzyme immunoassay.

  The anti-hyaluronic acid antibody of the present invention can be produced as described above. However, the present invention is not limited to this method, and can be obtained by a method obtained by arranging the above methods or a completely different method. For example, a method of contacting spleen cells with hyaluronic acid, sensitizing the cells, and then performing cell fusion to prepare a monoclonal antibody is included. The anti-hyaluronic acid antibody of the present invention includes a polyclonal antibody and a monoclonal antibody. When used for measuring hyaluronic acid, monoclonal is preferred from the viewpoint of measurement accuracy.

  The anti-hyaluronic acid antibody of the present invention is most suitable as a reagent for a reagent kit for measuring hyaluronic acid. That is, when a reagent kit containing the anti-hyaluronic acid antibody of the present invention is used, hyaluronic acid of all molecular weights can be measured simply and accurately. The hyaluronic acid to be measured can be any hyaluronic acid contained in a living body, and includes hyaluronic acid contained in connective tissues such as skin and joints, organs such as liver, and body fluids such as blood. Among these, it is suitable for hyaluronic acid contained in body fluids such as blood, and is most suitable for hyaluronic acid in serum. It is known that hyaluronic acid in a living body exists in various molecular weights, and the reagent kit of the present invention can be applied to measurement of hyaluronic acid of all molecular weights. Among these molecular weights, it is suitable for a viscosity average molecular weight of 20,000 or more, further suitable for 40,000 or more, particularly 100,000 or more, most preferably 180,000 or more, and for a viscosity average molecular weight of 4,000,000 or less. It is suitable for 3 million or less, especially 2.5 million or less, most preferably 2 million or less. Within this range, a plurality of anti-hyaluronic antibodies can bind to these hyaluronic acids, so that measurement sensitivity and measurement accuracy are easily improved.

  The reagent kit of the present invention is not limited in the form of the reagent and the configuration of the reagent kit, as long as the above-mentioned anti-hyaluronic acid antibody is included. For example, the anti-hyaluronic acid antibody binds to an insoluble carrier and / or Thus, it may be contained in the reagent kit, or may be contained alone as the anti-hyaluronic acid antibody. That is, the reagent kit of the present invention can be applied to a homogeneous immunoassay such as a nephelometric method, a latex agglutination method, and a turbidimetric method, as well as to a heterogeneous immunoassay using an insoluble carrier. Among them, a reagent kit for heterogeneous immunoassay is preferable in terms of measurement sensitivity.

  The reagent kit can include a reaction buffer, a B / F separation buffer, a standard hyaluronic acid buffer, and the like. As the reaction buffer and the B / F separation buffer, buffers and the like conventionally used for immunoassays can be used, and phosphate buffers and Goods containing proteins, salts and / or surfactants and the like can be used. Can be used. Proteins include albumin (bovine serum albumin, rabbit serum albumin, mouse serum albumin, ovalbumin, conalbumin, lactalbumin, etc.), antibodies (antibodies that do not bind to hyaluronic acid such as normal rabbit IgG and normal mouse IgG) and gelatin And the like. Examples of the salt include sodium chloride, potassium chloride and lithium bromide. Examples of the surfactant include nonionic surfactants such as sorbitan lauric acid monoester ethylene oxide adduct (trade name: Tween 20, Tween 40, ICI America). The standard hyaluronic acid buffer is used to create a calibration curve for calculating the concentration. Then, a known amount of hyaluronic acid is dissolved in the buffer used for the reaction buffer and the B / F separation buffer.

  The reagent kit of the present invention preferably contains a ligand together with the anti-hyaluronic acid antibody. As the ligand, a protein ligand capable of binding to hyaluronic acid and hyaluronic acid itself can be used. Examples of the protein ligand capable of binding to hyaluronic acid include the anti-hyaluronic acid antibody of the present invention, a hyaluronic acid-binding protein, CD44, and the like. Examples of the hyaluronic acid binding protein include proteoglycans, link protein, hyaluronectin, and the like described in Analytical Biochemistry 109, 386-394, (1980) and the like. Examples of CD44 include standard CD44 (CD44H), CD44 mutant molecule (CD44V), CD44E, and soluble CD44 (sCD44) described in Patent Document 4 and the like. Among these ligands, a protein ligand capable of binding to hyaluronic acid (corresponding to a reagent kit for sandwich assay) is preferable, more preferably the anti-hyaluronic acid antibody of the present invention, and particularly preferably the anti-hyaluronic acid of the present invention. Acid antibody (monoclonal antibody).

  When the ligand is a protein ligand capable of binding to hyaluronic acid, a sandwich-like complex of [anti-hyaluronic acid antibody-hyaluronic acid-ligand] is formed in the measurement of hyaluronic acid, and this complex is formed. Hyaluronic acid can be quantified by measuring (reagent kit for sandwich measurement method). In this case, it is suitable for measuring hyaluronic acid having a high molecular weight (viscosity average molecular weight of 20,000 to 4,000,000), and is most suitable for measuring hyaluronic acid having a viscosity average molecular weight of 100,000 to 2,500,000, particularly 180,000 to 2,000,000. It is.

  On the other hand, when the ligand is hyaluronic acid itself, the anti-hyaluronic acid antibody of the present invention, the hyaluronic acid reacts competitively with the measured hyaluronic acid, the ligand of the ligand reacted with the anti-hyaluronic acid antibody By measuring the amount, hyaluronic acid can be quantified (competitive assay reagent kit). In this case, it is suitable for measuring hyaluronic acid having a low molecular weight (viscosity average molecular weight of 2,000 to 20,000).

In the reagent kit of the present invention, it is preferable that at least one of the anti-hyaluronic acid antibody and the ligand is labeled with a labeling compound. Conventionally known compounds can be used as the labeling compound, and radioisotopes, fluorescent substances, luminescent substances, enzymes and the like are used. The isotopes, ¹²⁵ I and the like, and the fluorescent substance include europium complex, etc., as the light-emitting material include N- methyl acridinium ester, etc., as the enzyme horseradish peroxidase, alkaline phosphatase and β- galactosidase And the like. Of these labeled compounds, enzymes are preferred, more preferably horseradish peroxidase, alkaline phosphatase and β-galactosidase, particularly preferably horseradish peroxidase and alkaline phosphatase.

As a method of labeling a labeled compound with an anti-hyaluronic acid antibody or a ligand, a conventionally known method or the like can be applied, and “Seizoku Chemistry Experimental Course 5 Immunobiochemical Experimental Method” (edited by The Japanese Biochemical Society, Tokyo Chemical Dojin, 1986) For example, the methods described on page 102-112 are used. For example, the following methods (1)-(4) can be applied.
(1) A method in which, when the labeled compound is an isotope, radioactive iodine is introduced into a tyrosine residue of an anti-hyaluronic acid antibody or a ligand using chloramine T as an oxidizing agent.
(2) When the labeling compound is a fluorescent substance, a method of reacting fluorescein isothiocyanate with an anti-hyaluronic acid antibody or a ligand in a buffer to bind to the lysine residue of the anti-hyaluronic acid antibody or the ligand.
(3) When the labeling substance is a luminescent substance, a trade name “Acridinium Derivative-I” (manufactured by Dojindo Laboratories) is reacted with an anti-hyaluronic acid antibody or a ligand in a buffer, and the anti-hyaluronic acid antibody or ligand is reacted. A method of binding to an amino group of a ligand.
(4) When the labeling substance is an enzyme, a method in which an amino group of the enzyme and a thiol group of an anti-hyaluronic acid antibody or a ligand are bound with a di-crosslinking reagent such as N-succinimidyl-6-maleide hexanoate. .

  In the reagent kit of the present invention, one of the anti-hyaluronic acid antibody and the ligand is preferably bound to a water-insoluble carrier, and the other is preferably labeled with a labeling compound. That is, [water-insoluble carrier-anti-hyaluronic acid antibody-hyaluronic acid-ligand-labeled compound] or [water-insoluble carrier-ligand-hyaluronic acid-anti-hyaluronic acid antibody-labeled compound] (sandwich measurement method) or [ (A water-insoluble carrier-anti-hyaluronic acid antibody-hyaluronic acid-labeled compound) (competitive assay).

  As the water-insoluble carrier, carriers described in JP-A-2-205774 can be used, and inorganic substances such as glass and organic substances such as polystyrene can be used. The shape of the water-insoluble carrier can be freely determined according to the purpose of use, and examples include a spherical or disk-shaped bead, a plate- or stick-shaped stick, a test tube, and a nonwoven fabric or a filter strip. Of these, beads are preferred, and more preferably spherical beads.

The size of the water-insoluble carrier can be freely determined according to the purpose of use, and is a size that can be put into a reaction vessel having a diameter of about 10 mm (excluding the test tube). In the case of beads, the diameter (mm) is preferably 1 to 10, more preferably 2 to 8, and particularly preferably 3 to 7. In the case of a stick, the length (mm) is preferably 2 to 10, more preferably 3 to 8, and particularly preferably 4 to 7. The cross-sectional area (mm ² ) of the stick is preferably 1 to 25, more preferably 2 to 16, and particularly preferably 3 to 9. The cross-sectional area means a cross-sectional area of a cut portion when cut perpendicular to the long axis direction. In the case of a test tube, the length (mm) is preferably 5 to 100, more preferably 8 to 80, and particularly preferably 10 to 20. Further, the inner diameter (mm) of the test tube is preferably 5 to 20, more preferably 6 to 16, and particularly preferably 8 to 12. In the case of a strip, the length (mm) is preferably 5 to 100, more preferably 10 to 80, and particularly preferably 10 to 50. Further, the width (mm) of the strip is preferably 1 to 20, more preferably 2 to 16, and particularly preferably 3 to 10.

  As a method of binding the anti-hyaluronic acid antibody or ligand to the water-insoluble carrier, a conventionally known method such as a method of chemically binding or a method of physical adsorption can be used. As a method of chemically bonding, a functional group such as an amino group and a sulfhydryl group introduced on the surface of a water-insoluble carrier and a functional group such as an amino group and a sulfhydryl group of an anti-hyaluronic acid antibody or a ligand are used as a binder. (Glutaraldehyde, succinaldehyde, m-maleimidobenzoyl-N-hydrosuccinimide ester, o-phenylenebismaleimide, etc.) (US Pat. Nos. 4,280,992 and 365,2761) and the like. Can be As a method by physical adsorption, when the water-insoluble carrier is polystyrene, the water-insoluble carrier is added to a carbonate buffer (pH 9.0) containing 0.001 to 0.04% (W / V) of an anti-hyaluronic acid antibody or a ligand. (Biosim Biofiz Acta, Vol. 251, 427, 1971). This method can be applied to the case where the carrier is a substance other than polystyrene, such as polypropylene, silicon, glass, and cellulose.

  The reagent kit of the present invention is suitable for a method for quantifying hyaluronic acid by a competitive measurement method and a sandwich measurement method, and is particularly suitable for a method for quantifying hyaluronic acid by a sandwich measurement method. Hyaluronic acid, which is the analyte, can be quantified by measuring the amount of the labeled compound. The measurement of the labeled compound can be performed by a conventionally known method or the like depending on the type of the labeled compound. When the labeling compound is a fluorescent substance, for example, the amount of fluorescence generated by irradiation with excitation light having an appropriate wavelength is quantified using a photomultiplier. When the labeling compound is a chemiluminescent substance, for example, in the case of acridinium ester, the amount of luminescence generated by adding an alkaline solution is quantified by a photomultiplier.

  When the labeled compound is an enzyme, the enzyme activity can be measured as an absorbance (absorbance measurement method), a fluorescence amount (fluorescence measurement method) or a luminescence amount (chemiluminescence measurement method) by reacting an appropriate substrate. For example, when the enzyme is peroxidase, the substrate is 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium (ABTS) (absorbance measurement method) and luminol / peroxide (chemiluminescence). Quantity measurement method) can be selected. When the enzyme is alkaline phosphatase, p-nitrophenyl phosphate (absorbance measurement method), 4-methylumbelliferyl phosphate (4-MUP) (fluorescence measurement method) and 3- (2′-spiroadamantane) are used as substrates. ) -4-methoxy-4- (3 ″ phosphoryloxy) phenyl-1,2-dioxetane disodium (AMPPD) (chemiluminescence assay) and the like. The absorbance is determined by a spectrophotometer, and the amount of fluorescence and the amount of chemiluminescence are determined by a photomultiplier. Among these, a chemiluminescence measurement method is preferable (that is, a method for quantifying hyaluronic acid using the reagent kit of the present invention is preferably a chemiluminescence enzyme immunoassay method), and more preferably peroxidase and luminol / peroxidase. This is a method for measuring the amount of chemiluminescence based on a combination of a substance and a combination of alkaline phosphatase and AMPPD.

  Luminol includes luminol, isoluminol, N-aminohexyl-N-ethylisoluminol (AHEI), N-aminobutyl-N-ethylisoluminol (ABEI), and metal salts thereof. As these metal salts, alkali metal (such as sodium and potassium) salts and alkaline earth metal (such as calcium and magnesium) salts can be used. Of these, luminol and a metal salt of luminol are preferred, more preferably a metal salt of luminol, and particularly preferably a sodium salt of luminol. As the peroxide, either an inorganic peroxide or an organic peroxide can be used. Inorganic peroxides include hydrogen peroxide, sodium perborate, potassium perborate, peroxide, carbon dioxide, dicarbonate, hypochlorous acid, potassium hypochlorite, chlorite, chlorine Acids, sodium chlorate, perchloric acid, perbronic acid, peroxosulfuric acid, peroxophosphoric acid, and the like. Examples of the organic peroxide include peracetic acid, perpropionic acid, dimethyl sulfoxide (DMSO), triethylamine oxide, methyldiethylamine oxide, and phthaloyl peroxide. Among these, from the viewpoint of storage stability and the like, inorganic peroxides are preferable, and hydrogen peroxide is more preferable.

Specific examples (steps 1 to 6) of the method for determining hyaluronic acid (sandwich measurement method) using the reagent kit of the present invention are shown below.
Step 1. The sample is reacted with the water-insoluble carrier to which the anti-hyaluronic acid antibody is bound to obtain a reaction mixture (including the complex 1).
Step 2. The unreacted material is removed from the reaction mixture of Step 1 (B / F separation) to obtain a complex 1.
Step 3. The complex 1 obtained in the step 2 is reacted with the ligand (B) labeled with the labeling compound to obtain a reaction mixture (including the complex 2).
Step 4. Step 3 Unreacted substances are removed from the reaction mixture (B / F separation) to obtain a complex 2.
Step 5. The amount of the labeled compound of the complex 2 is measured.
Step 6. Using the amount of the labeled compound, the amount of hyaluronic acid is calculated from a separately prepared calibration curve.
Similarly, when the ligand (B) is hyaluronic acid itself, it is a specific example of a method for quantifying hyaluronic acid using the reagent kit of the present invention (competitive measurement method).

  Next, an embodiment applied to the homogeneous measurement of the present invention will be exemplified. Make a buffer to which a certain amount of anti-hyaluronic acid antibody is added. The amount of the anti-hyaluronic acid antibody to be added can take various values depending on the measurement conditions and the concentration range of the hyaluronic acid to be measured, but is usually 0.1 to 100 mg / L based on the volume of the buffer solution. A certain amount of the buffer solution containing the anti-hyaluronic acid antibody and a certain amount of the sample are mixed and reacted, and quantification is performed by turbidimetry or nephelometry. In the case of turbidimetry, the change in turbidity due to the reaction is measured by a spectrophotometer or a photoelectric colorimeter. In the case of the nephelometry, the change in the scattered light caused by the reaction is measured with a nephelometer. Both the nephelometry and the nephelometry have narrow measurable concentration ranges, and are usually applied to specimens containing a high concentration (0.2 mg / L or more) of hyaluronic acid.

  The anti-hyaluronic acid antibody used in the turbidimetric method can be used in a form bound to fine particles such as latex. The binding between the latex particles and the anti-hyaluronic acid antibody can be performed by the above-mentioned physical adsorption method. The anti-hyaluronic acid antibody bound to the latex is allowed to react with the sample in a buffer solution, and the change in turbidity is measured with a spectrophotometer or a photoelectric colorimeter using near infrared rays (normally 940 nm). This method has a wider measurable concentration range than the turbidimetric method using no latex, and can be applied to measurement in a concentration range of 0.1 mg / L or more.

  The hyaluronic acid measurement kit of the present invention can easily and accurately measure hyaluronic acid of all molecular weights, and is therefore most suitable for diagnosing a disease and determining a therapeutic effect. Hyaluronic acid is widely distributed in the body such as the vitreous body, synovial fluid, and skin. In diseases such as liver disease, cancer and rheumatoid arthritis, the high molecular weight (viscosity average molecular weight of 20,000 to 2,000,000) hyaluronic acid present in the lesion exudes or decomposes, and the amount of hyaluronic acid in body fluids such as blood and urine is reduced. It is known to increase [eg, Hepatology, 5, 638-642, 1985, liver, 32 (8), 767-774, 1991, clinicopathology, 36 (5), 536-540, 1991. etc]. In particular, it is known that the amount of high-molecular-weight hyaluronic acid in blood correlates well with pathological conditions such as rheumatoid arthritis, cancer and liver disease. For example, in the case of rheumatoid arthritis, the serum concentration is 50 ng / mL or less in healthy individuals, 50 to 200 ng / mL in inactive chronic rheumatic patients, and 200 ng / mL or more in active chronic rheumatic patients. . It is almost the same for liver disease, and the value is 50 ng / mL or less for healthy subjects, 50 to 200 ng / mL for chronic hepatitis patients, and 200 ng / mL or more for liver cirrhosis patients. As described above, by measuring high-molecular-weight hyaluronic acid in blood, it can be applied to diagnosis of these diseases or judgment of therapeutic effects.

  Conventionally, in clinical measurement of high molecular weight hyaluronic acid, ligand measurement using hyaluronic acid binding protein has been almost exclusively performed. In this method, it is necessary to purify the hyaluronic acid-binding protein from the cartilage or the like of a living body when producing the reagent, and it is necessary to devise a method for stabilizing the quality of the hyaluronic acid-binding protein and the quality of the reagent. By using the anti-hyaluronic acid antibody of the present invention, it has become possible to provide a new method for measuring hyaluronic acid and a reagent kit of stable quality.

Hereinafter, the present invention will be further described with reference to Examples, but the present invention is not limited thereto.
<Example 1> Preparation of anti-hyaluronic acid polyclonal antibody 1 Preparation of antigen for immunization 1 part by weight of Complete Freund's adjuvant (manufactured by Difco) and 1 part by weight of a 0.85% by weight aqueous sodium chloride solution were mixed and emulsified by a homogenizer. To 1 part by weight of this emulsion, 4/1000 parts by weight of hyaluronic acid sodium salt (derived from human umbilical cord, viscosity average molecular weight 1,000,000, sand-like particles) [manufactured by Seikagaku Corporation] was mixed with a stirring rod, and the antigen emulsion for immunization was mixed. It was set to 1. The antigen emulsion for immunization 1 was allowed to stand at 37 ° C. for 1 hour, 8 hours, or 24 hours, and then 10 mL of acetone was added to 1 g of the emulsion 1, and the form of the precipitated hyaluronic acid was visually observed. did. When an aqueous solution of sodium hyaluronate (the above-mentioned sodium hyaluronate was added and dissolved in an amount of 0.1 mg per 1 mL of deionized water) was similarly precipitated, a flocculent precipitate was deposited. No sandy particles were observed. That is, it was confirmed that hyaluronic acid sodium salt did not dissolve in the emulsion but existed in a solid state. However, after standing for 24 hours, a flocculent precipitate was slightly observed on the surface of the sand-like particles, and it was confirmed that the particle surfaces were gradually dissolved.

2. Immunization of Mice Immediately after obtaining the immunizing antigen emulsion 1, 0.2 g of the immunizing antigen emulsion 1 (sodium hyaluronate: 0.8 mg) was intraperitoneally administered to each of three mice (Balb / c). They were similarly administered three times at one-month intervals (a total of four times). In addition, the immunization antigen emulsion 1 was adjusted immediately before immunization, respectively, without storing. Five months after the first immunization, 0.1 mL of blood was collected per mouse from the tail vein of the mouse. After mixing (0.3 mL) the blood of the three animals, they were centrifuged to obtain 0.15 mL of antiserum 1.

3. Purification of anti-hyaluronic acid antibody 0.15 mL of antiserum 1 was passed through a DEAE-cellulose column (2 mL in volume) equilibrated with phosphate buffer (pH 8.0, 0.0125 M). It was fractionated as a fraction containing. This solution was diluted with a phosphate buffer (pH 7.0, 0.0125 M) to a protein concentration of 10 μg / mL to obtain a buffer containing anti-hyaluronic acid polyclonal antibody 1.

<Example 2> Preparation of anti-hyaluronic acid polyclonal antibody 2 Instead of antigen emulsion for immunization 1, hyaluronic acid sodium salt (derived from human umbilical cord) was added to 1 part by weight of the emulsion [Seikagaku Corporation] 6/1000 parts by weight A buffer containing anti-hyaluronic acid polyclonal antibody 2 was obtained in the same manner as in Example 1 except that the antigen emulsion 2 for immunization was used. When the shape of hyaluronic acid in the antigen emulsion for immunization 2 was confirmed in the same manner as in Example 1, the results were the same as those in Example 1.

<Example 3> Preparation of anti-hyaluronic acid polyclonal antibody 3 Instead of antigen emulsion 1 for immunization, hyaluronic acid sodium salt (derived from human umbilical cord) [manufactured by Seikagaku Corporation] 10/1000 parts by weight was added to 1 part by weight of the emulsion. Was prepared in the same manner as in Example 1 except that the antigen emulsion 3 for immunization was used to obtain a buffer solution containing the anti-hyaluronic acid polyclonal antibody 3. When the shape of hyaluronic acid in the antigen emulsion for immunization 3 was confirmed in the same manner as in Example 1, the results were the same as those in Example 1.

<Example 4> Preparation of anti-hyaluronic acid polyclonal antibody 4 In place of antigen emulsion 1 for immunization, sodium salt of hyaluronate (derived from human umbilical cord) was added to 1 part by weight of the emulsion [manufactured by Seikagaku Corporation] 20/1000 parts by weight A buffer containing anti-hyaluronic acid polyclonal antibody 4 was obtained in the same manner as in Example 1, except that the antigen emulsion for immunization 4 was used. When the shape of hyaluronic acid in the immunizing antigen emulsion 4 was confirmed in the same manner as in Example 1, the results were the same as those in Example 1.

<Comparative Example 1> Preparation of anti-hyaluronic acid polyclonal antibody 5 Instead of antigen emulsion 1 for immunization, 1 / 10,000 parts by weight of hyaluronic acid sodium salt (derived from human umbilical cord) [manufactured by Seikagaku Corporation] was 0.85 parts by weight. The same procedure as in Example 1 was carried out except that 1 part by weight of a 1% by weight aqueous sodium chloride solution was used, and 1 part by weight of the solution dissolved by stirring and 1 part by weight of Complete Freund's adjuvant were mixed and emulsified to prepare an antigen antigen emulsion 5 for immunization. Thus, a buffer solution containing anti-hyaluronic acid polyclonal antibody 5 was obtained. When the shape of hyaluronic acid in emulsion 5 was confirmed in the same manner as in Example 1, only flocculent precipitation was observed, confirming that sodium hyaluronate was dissolved in the emulsion.

<Comparative Example 2> Preparation of anti-hyaluronic acid polyclonal antibody 6 In place of antigen emulsion 1 for immunization, 2 / 10,000 parts by weight of hyaluronic acid sodium salt (derived from human umbilical cord) [manufactured by Seikagaku Corporation] was 0.85 parts by weight. The same procedure as in Example 1 was carried out except that 1 part by weight of a 1% by weight aqueous solution of sodium chloride, 1 part by weight of a solution dissolved by stirring and 1 part by weight of Complete Freund's adjuvant were mixed and emulsified to prepare an antigen emulsion 6 for immunization. Thus, a buffer containing anti-hyaluronic acid polyclonal antibody 6 was obtained. When the shape of hyaluronic acid in the emulsion 6 was confirmed in the same manner as in Example 1, only flocculent precipitates were observed, confirming that sodium hyaluronate was dissolved in the emulsion.

<Comparative Example 3> Preparation of anti-hyaluronic acid polyclonal antibody 7 A low-molecular-weight sodium hyaluronate was prepared in the same manner as in the example described in Patent Document 1. That is, 0.1 g of hyaluronic acid sodium salt (derived from human umbilical cord, viscosity average molecular weight of 1,000,000) used in Example 1 was dissolved in 10 ml of sodium acetate buffer (pH 5.0). Subsequently, bovine testis-derived hyaluronidase (Sigma) was added to a final concentration of 60 U / ml, followed by digestion at 37 ° C. overnight. After heating at 100 ° C. for 10 minutes to inactivate the enzyme, the precipitate was removed by centrifugation. Next, sodium acetate (crystal) was added to a concentration of 1% (w / v) to obtain a sodium hyaluronate solution, and then low molecular weight sodium hyaluronate was separated and precipitated by acetone precipitation. That is, acetone was gradually added dropwise to the sodium hyaluronate solution with stirring, and when precipitate 1 was formed, precipitate 1 was removed by centrifugation. Acetone was gradually added dropwise to the supernatant from which the precipitate 1 had been removed to form a precipitate 2. After acetone was added dropwise until no increase in precipitate 2 was observed, precipitate 2 was centrifuged. The precipitate 2 was washed with acetone and dried under vacuum to obtain a low molecular weight sodium hyaluronate as a white powder. The viscosity average molecular weight of this hyaluronic acid sodium salt was 5,500.

  Instead of the antigen emulsion for immunization 1, 1 part by weight of a solution prepared by dissolving 4/1000 parts by weight of low molecular weight sodium hyaluronate in 1 part by weight of an aqueous solution of 0.85% by weight of sodium chloride and 1 part by weight of Complete Freund's adjuvant are mixed. A buffer solution containing an anti-hyaluronic acid polyclonal antibody 7 was obtained in the same manner as in Example 1, except that the immunizing antigen emulsion 7 prepared by emulsification was used. When the shape of hyaluronic acid in the emulsion 7 was confirmed in the same manner as in Example 1, only flocculent precipitation was observed, confirming that sodium hyaluronate was dissolved in the emulsion.

Comparative Example 4 Preparation of Antihyaluronic Acid Polyclonal Antibody 8 A conjugate of low molecular weight hyaluronic acid and hemocyanin was prepared in the same manner as in the example described in Patent Document 1. That is, after dissolving 15 mg of the low molecular weight hyaluronic acid / sodium salt obtained in Comparative Example 3 in 0.5 ml of 50 mM sodium borate buffer (pH 8.3), 1.0 mg of sodium borohydride was added, and room temperature ( (25-30 ° C.) for 6 hours to convert the reducing terminal N-acetyl-D-glucosamine structure (GlcNAc) into a sugar alcohol. The excess sodium borohydride was then decomposed at room temperature (15-25 ° C.) by adding 50 ml of acetone, and at the same time the hyaluronic acid sugar alcohol was precipitated. This hyaluronic acid sugar alcohol was separated and precipitated by the acetone precipitation method in the same manner as in Comparative Example 3 to obtain white powdered hyaluronic acid sugar alcohol.

  After dissolving 10 mg of the obtained hyaluronic acid sugar alcohol in 0.5 ml of imidazole buffer (pH 6.5), 3.0 mg of sodium periodate is added, and the mixture is allowed to react at 0 ° C. for 1 hour. The aldehyde was obtained. Hyaluronic aldehyde was separated and precipitated by an ethanol precipitation method. That is, ethanol was gradually dropped into the solution containing hyaluronic acid aldehyde while stirring, and when precipitate 1 was formed, precipitate 1 was removed by centrifugation. Ethanol was gradually added dropwise to the supernatant from which the precipitate 1 had been removed to form a precipitate 2. After ethanol was added dropwise until no increase in precipitate 2 was observed, precipitate 2 was centrifuged. The precipitate 2 was washed with ethanol and dried under vacuum to obtain hyaluronic aldehyde as a white powder.

  1.0 mg of hyaluronic acid aldehyde and 10 mg of hemocyanin (from Keyhole Limpets, manufactured by Sigma) obtained by the above method are dissolved in 0.4 ml of distilled water, and stirred at room temperature (25 to 30 ° C.) for 30 minutes. A base was formed. Subsequently, after adding 20 mg of sodium cyanoborohydride and stirring at room temperature (25 to 30 ° C.) for 7 hours, adding 5.0 mg of sodium borohydride and stirring overnight at a temperature of 2 to 10 ° C. Reduced the Schiff base. Acetone was gradually added dropwise to the reaction solution with stirring, and when precipitate 1 was formed, precipitate 1 was removed by centrifugation. Acetone was gradually added dropwise to the supernatant from which the precipitate was removed to form precipitate 2. After acetone was added dropwise until the increase of the precipitate 2 was not observed, the precipitate 2 was centrifuged to obtain a hemocyanin-bound hyaluronic acid sodium salt in which low-molecular-weight hyaluronic acid and hemocyanin were chemically bonded (light red powder).

  Instead of the immunizing antigen emulsion 1, 4/1000 parts by weight of hemocyanin-bound sodium hyaluronate was added to 1 part by weight of a 0.85% by weight aqueous sodium chloride solution, and the mixture was stirred and dissolved, and 1 part by weight of the solution and 1 part by weight of Complete Freund's adjuvant were used. A buffer solution containing an anti-hyaluronic acid polyclonal antibody 8 was obtained in the same manner as in Example 1, except that the immunizing antigen emulsion 8 prepared by mixing and emulsifying the parts was used. When the shape of hyaluronic acid in the emulsion 8 was confirmed in the same manner as in Example 1, only flocculent precipitates were observed, confirming that sodium hyaluronate was dissolved in the emulsion.

<Evaluation of antibody titer>
1) Preparation of reagent for measurement Sodium hyaluronate (viscosity average molecular weight 1,000,000, derived from human umbilical cord, manufactured by Seikagaku Corporation) was dissolved in a 0.1 M carbonate buffer (pH 9.0) to a concentration of 20 µg / mL. Then, 500 beads of 6.35 mm diameter polystyrene beads (trade name: Immunobeads, manufactured by Immunochemical Co., Ltd.) were immersed in 500 mL of this solution, and allowed to stand at 2 to 10 ° C. for 10 hours. Thereafter, the solution was removed by suction with an aspirator, and the polystyrene beads were immersed in a phosphate buffer solution (pH 7.0) containing 0.1% by weight of bovine serum albumin to obtain sodium hyaluronate-bonded beads (A). The prepared hyaluronic acid sodium salt-bound beads (A) were stored at 4 to 10 ° C. until use.

2) Preparation of a labeled anti-mouse IgG antibody solution A peroxidase-labeled anti-mouse IgG rabbit antibody (manufactured by Dako, product code P160) was diluted 2000-fold with a phosphate buffer solution (pH 6.0) containing 0.1% by weight of bovine serum albumin. This was used as a labeled antibody solution. It was stored frozen at -20 ° C until use.

3) Preparation of Coloring Solution To 100 mL of 0.1 M citrate buffer (pH 4.8), 300 mg of o-phenylenediamine and 70 μL of a 30% by weight hydrogen peroxide solution were added and dissolved to prepare a coloring solution. Stored refrigerated from light until use (can be used only on the day of creation).

4) Measurement procedure One test tube (A) containing sodium hyaluronate (A), 300 μL of a 1% by weight bovine serum albumin-containing phosphate buffer (pH 7.0) and a sample for measurement were placed in a test tube (inner diameter 1.2 cm, length 10 cm). (A buffer solution containing an anti-hyaluronic acid antibody) 50 μL was added, and the mixture was allowed to react at 37 ° C. for 4 hours. The reaction solution was removed by suction using an aspirator, and after adding 1 mL of a 0.85% by weight aqueous sodium chloride solution, the beads were washed by removing the aqueous sodium chloride solution using an aspirator. After performing this washing operation two more times, 300 μL of the labeled antibody solution was added, and the mixture was allowed to react at 37 ° C. for 1 hour. After the beads were washed three times in the same manner as described above, 500 μL of the coloring solution was added, and the mixture was allowed to react at 37 ° C. for 30 minutes. The reaction was stopped by adding 1 mL of 1.5 N sulfuric acid, and the absorbance (optical path length: 10 mm) was measured at a measurement wavelength of 492 nm.

  The antibody titers were evaluated using the buffers containing the anti-hyaluronic acid polyclonal antibodies 1 to 8 obtained in Examples 1 to 4 and Comparative Examples 1 to 4 as measurement samples, and the results are shown in Table 1. The measurement blank used was an anti-hyaluronic acid polyclonal antibody similarly obtained from a non-immunized mouse (normal mouse) diluted to 10 μg / mL.

  From Table 1, it can be seen that the anti-hyaluronic acid polyclonal antibodies of Examples 1 to 4 have clearly higher antibody titers (activity) than the anti-hyaluronic acid polyclonal antibodies of Comparative Examples 1 to 4.

<Example 5> Preparation of anti-hyaluronic acid monoclonal antibody Preparation of Hybridoma The spleen was excised from three mice immunized in Example 4, and an anti-hyaluronic acid monoclonal antibody was prepared according to a conventional method as follows. The following operation was performed for each animal. After spleen cells were collected from the spleen and washed with RPMI1640 medium, the total amount of spleen cells was mixed with 2 × 10 ⁷ myeloma cells (P3-NS1 / 1-Ag4.1), and 42.5% by weight (37 ° C.) 1 in 1 mL of RPMI 1640 medium containing polyethylene glycol 4000 (concentration based on the weight of the medium) of polyethylene glycol 4000 (number average molecular weight 4000, obtained from Nacalai Tesque, Inc.) and 7.5% by weight (concentration based on the weight of the medium) of dimethyl sulfoxide. Fused for minutes. One minute later, the cell suspension was gradually diluted with 5 mL of RPMI1640 medium. After the cells are centrifuged and washed, HAT medium (RPMI1640 medium containing hypoxanthine, aminopterin, and 10% by weight of fetal bovine serum) is added to a volume of 20 mL, and 0.2 mL is added to a 96-well microplate. After incubating for 2 weeks, the antibody titer of the culture supernatant in the well in which the cells proliferated was measured. The measurement of the antibody titer was carried out according to the evaluation of the anti-hyaluronic acid polyclonal antibody. The culture supernatant was directly used as a measurement sample.

  Next, a well showing an absorbance of 5 times or more the absorbance of the blank was defined as a well having an antibody titer, and cells in the well were cloned by a limiting dilution method. Of the three mice, two hybridomas were obtained from each of the two mice, that is, two hybridomas producing an anti-hyaluronic acid monoclonal antibody. No hybridoma producing an anti-hyaluronic acid monoclonal antibody was obtained from the remaining one animal.

2. Purification of anti-hyaluronic acid monoclonal antibody One of the two hybridomas was cultured for 1 week in 1 L of a serum-free medium (ASF medium 104, obtained from Ajinomoto Co., Ltd.), and the culture supernatant was subjected to an ultrafiltration membrane (Ultrafilter UK- 50, manufactured by Toyo Filter Paper Co., Ltd.). These concentrates were applied to a gel filtration column (Superdex 200 Prep column, manufactured by Pharmacia) using a 0.02 M phosphate buffer (pH 7.0) containing 0.85% by weight of sodium chloride as a mobile phase, and fractionated into each fraction. The antibody titer of each fraction was measured, and the fractions showing an absorbance at least three times the absorbance of the blank were pooled. The measurement of the antibody titer was carried out in accordance with the evaluation of the anti-hyaluronic acid polyclonal antibody, and each fraction was directly used for the measurement. The pooled fractions were concentrated with an ultrafiltration membrane (Ultrafilter UK-50, manufactured by Toyo Roshi Kaisha) to a protein concentration of 1 mg / mL to obtain a buffer solution of the anti-hyaluronic acid monoclonal antibody HAS1. In addition, the other one of the two hybridomas was purified in the same manner to obtain a buffer solution of the anti-hyaluronic acid monoclonal antibody HAS2.

3. Confirmation of reactivity of anti-hyaluronic acid monoclonal antibodies HAS1 and HAS2 An anti-hyaluronic acid monoclonal antibody was evaluated in the same manner as in the evaluation of anti-hyaluronic acid polyclonal, except that hyaluronic acid-bound beads prepared using hyaluronic acid described in Table 2 were used. Was evaluated for antibody titer. Table 2 shows the results. However, for the measurement, an anti-hyaluronic acid monoclonal antibody diluted to 10 μg / mL with a 1% by weight bovine serum albumin-containing phosphate buffer (pH 7.0) was used. As a control, an anti-hyaluronic acid polyclonal antibody purified from the serum of a non-immunized mouse was similarly tested. The sodium salt of hyaluronic acid having a viscosity average molecular weight of 180,000 in Table 2 was obtained by using a 1 mg / mL aqueous solution of sodium hyaluronate (viscosity average molecular weight of 1,000,000, derived from human umbilical cord, manufactured by Seikagaku Corporation) using an ultrasonic dispersing machine. (Teraoka Co., Ltd., model number UH-50), and prepared by sonication until the molecular weight reached 180,000.

  Both the anti-hyaluronic acid monoclonal antibodies HA1 and HA2 were found to bind to high molecular weight hyaluronic acid. It was also found that the binding to low molecular weight hyaluronic acid was relatively weaker than that of high molecular weight hyaluronic acid, but was clearly bound.

<Example 6> Reagent kit 1
Anti-hyaluronic acid monoclonal antibody-conjugated beads (HASB1), peroxidase-labeled anti-hyaluronic acid monoclonal antibody (PHAS1), buffer solution (first solution), oxidizing agent solution (second solution) and standard sodium hyaluronate as follows: Reagent kit 1 consisting of solutions 0, 10, 100 and 1000 was prepared.

1) Preparation of anti-hyaluronic acid monoclonal antibody-conjugated beads (HASB1) The buffer solution of anti-hyaluronic acid monoclonal antibody HAS1 obtained in Example 5 was added to a 0.1 M carbonate buffer (pH 9) so that the concentration of HAS1 was 20 μg / mL. Added. To 50 mL of this solution, 2,000 polystyrene beads having a diameter of 3.2 mm (manufactured by Immunochemical Co., Ltd.) were added and allowed to stand for 48 hours to physically adsorb the anti-hyaluronic acid monoclonal antibody HAS1 to the polystyrene beads. Thereafter, the buffer solution was removed by suction with an aspirator, and the beads were washed twice with 50 mL of a phosphate buffer containing 0.1% by weight of bovine serum albumin to prepare anti-hyaluronic acid monoclonal antibody-conjugated beads (HASB1). The anti-hyaluronic acid monoclonal antibody-conjugated beads (HASB1) were immersed again in 50 mL of a phosphate buffer containing 0.1% by weight of bovine serum albumin, and refrigerated (2 to 10 ° C.) in the immersion state.

2) Preparation of Peroxidase-Labeled Substance N-succinimidyl-3- ( 2-pyridyldithio) propionate {N-Succinimidyl3- (2-pyridyldithio) propionate} (trade name: SPDP, manufactured by Dojin Chemical Co., Ltd.) was reacted to introduce a mercapto group. A mercapto group-introduced peroxidase and an anti-hyaluronic acid monoclonal antibody (HAS1) were prepared by the method described in Journal of Biochemistry (J. Biochem), 92, 1413 (1982), using a double-crosslinking reagent N- (4-maleimidobutyryloxy). Succiniimide {N- (4-Maleimidobutyryloxy) succinimide} [GMBS, manufactured by Dojindo] was combined to prepare a peroxidase-labeled anti-hyaluronic acid monoclonal antibody (PHAS1), which was stored frozen (-30 ° C).

3) Preparation of buffer solution (first solution) 0.18 g of commercially available luminol sodium (manufactured by Wako Pure Chemical Industries, Ltd.) and 0.1 g of 4- (cyanomethylthio) phenol were mixed with 0.1 M-N, N- It was dissolved in 1 L of bis (2-hydroxyethyl) glycine / sodium hydroxide buffer (pH 8.5) to prepare a buffer solution (first solution), which was stored refrigerated (2 to 10 ° C.) until used for measurement.

4) Preparation of oxidizing agent solution (second liquid) 200 μl of 35% by weight hydrogen peroxide solution was dissolved in 1 liter of deionized water to prepare an oxidizing agent solution (second liquid) and refrigerated until used for measurement ( 2-10 ° C).

5) Preparation of Standard Solution of Hyaluronic Acid Sodium Salt Hyaluronic acid / sodium salt (viscosity average molecular weight 1,000,000, derived from human umbilical cord, manufactured by Seikagaku Corporation) was added to 0.1% by weight bovine serum albumin-containing phosphate buffer solution. The respective solutions were dissolved at a concentration of 10, 100, and 1000 ng / mL to prepare hyaluronic acid sodium salt standard solutions 0, 10, 100, and 1000, respectively.

<Example 7> Reagent kit 2
A peroxidase-labeled anti-hyaluronic acid monoclonal antibody (PHAS2) was prepared in the same manner as in Example 6 except that an anti-hyaluronic acid monoclonal antibody (HAS2) was used instead of the anti-hyaluronic acid monoclonal antibody (HAS1). Then, a reagent kit 2 was prepared in the same manner as in Example 6, except that the peroxidase-labeled anti-hyaluronic acid monoclonal antibody (PHAS1) was replaced with this peroxidase-labeled anti-hyaluronic acid monoclonal antibody (PHAS2).

<Example 8> Reagent kit 3
In the same manner as in Example 6 except that a hyaluronic acid binding protein (HABP; manufactured by Seikagaku Corporation, including proteoglycan and link protein) was used instead of the anti-hyaluronic acid monoclonal antibody (HAS1). A peroxidase-labeled hyaluronic acid binding protein (PHABP) was prepared. Then, a reagent kit 3 was prepared in the same manner as in Example 6, except that the peroxidase-labeled anti-hyaluronic acid monoclonal antibody (PHAS1) was replaced with peroxidase-labeled hyaluronic acid-binding protein (PHABP).

<Example 9> Reagent kit 4
Anti-hyaluronic acid monoclonal antibody-bound beads (HASB2) were prepared in the same manner as in Example 6, except that an anti-hyaluronic acid monoclonal antibody (HAS2) was used instead of the anti-hyaluronic acid monoclonal antibody (HAS1). Then, a reagent kit 4 was prepared in the same manner as in Example 6, except that the anti-hyaluronic acid monoclonal antibody-bound beads (HASB1) were replaced with anti-hyaluronic acid monoclonal antibody-bound beads (HASB2).

<Example 10> Reagent kit 5
Anti-hyaluronic acid monoclonal antibody-conjugated beads (HASB1) were obtained in the same manner as in Example 9, and peroxidase-labeled anti-hyaluronic acid monoclonal antibody (PHAS1) was obtained in the same manner as in Example 7 on anti-hyaluronic acid monoclonal antibody-conjugated beads (HASB2). Reagent kit 5 was prepared in the same manner as in Example 6, except that the obtained peroxidase-labeled anti-hyaluronic acid monoclonal antibody (PHAS2) was used.

<Example 11> Reagent kit 6
Anti-hyaluronic acid monoclonal antibody-conjugated beads (HASB1) were obtained in the same manner as in Example 9, and peroxidase-labeled anti-hyaluronic acid monoclonal antibody (PHAS1) was obtained in the same manner as in Example 8 on anti-hyaluronic acid monoclonal antibody-conjugated beads (HASB2). Reagent kit 6 was prepared in the same manner as in Example 6, except that the obtained peroxidase-labeled hyaluronic acid protein (PHABP) was used.

<Example 12> Reagent kit 7
Hyaluronic acid-binding protein-conjugated beads (HABPB) were used in the same manner as in Example 6 except that hyaluronic acid-binding protein (HABP; manufactured by Seikagaku Corporation) was used instead of the anti-hyaluronic acid monoclonal antibody (HAS1). ) Was adjusted. Reagent kit 7 was prepared in the same manner as in Example 6, except that the anti-hyaluronic acid monoclonal antibody-bound beads (HASB1) were replaced with hyaluronic acid-binding protein-bound beads (HABPB).

<Example 13> Reagent kit 8
Anti-hyaluronic acid monoclonal antibody-conjugated beads (HASB1) were obtained in the same manner as in Example 12, and peroxidase-labeled anti-hyaluronic acid monoclonal antibody (PHAS1) was obtained in the same manner as in Example 7 on hyaluronic acid-binding protein-conjugated beads (HABPB). Reagent kit 8 was prepared in the same manner as in Example 6, except that the obtained peroxidase-labeled anti-hyaluronic acid monoclonal antibody (PHAS2) was used.

<Comparative Example 5> Reagent kit 9
Anti-hyaluronic acid monoclonal antibody-conjugated beads (HASB1) were obtained in the same manner as in Example 12, and peroxidase-labeled anti-hyaluronic acid monoclonal antibody (PHAS1) was obtained in the same manner as in Example 8 on hyaluronic acid-binding protein-conjugated beads (HABPB). A comparative reagent kit 9 was prepared in the same manner as in Example 6, except that the obtained peroxidase-labeled hyaluronic acid binding protein (PHABP) was used instead.

<Quantification of hyaluronic acid by kit>
In a test tube (1.0 cm inside diameter, 8.5 cm length), 50 μL of hyaluronic acid sodium salt standard solution 0, 300 μL of 1% by weight bovine serum albumin-containing phosphate buffer, and one bead in the reagent kit At 37 ° C. for 1 hour. After removing the reaction solution with an aspirator, physiological saline was added to wash the beads, and the washing solution was removed with an aspirator. Next, the peroxidase-labeled anti-hyaluronic acid antibody (or peroxidase-labeled hyaluronic acid-binding protein) in the reagent kit was diluted with a 1% by weight bovine serum albumin-containing phosphate buffer to a protein concentration of 1 μg / mL, and A diluent was prepared. 300 μL of this labeled body dilution was added to the washed beads, and allowed to react at 37 ° C. for 1 hour. The reaction solution was removed with an aspirator, physiological saline was added to wash the beads, and the washing solution was removed with an aspirator. The enzyme activity of the washed beads was measured as follows.

A test tube containing the washed beads is set in a sample holder of a luminescence reader BLR-201 manufactured by Aloka, and 200 μL of a buffer solution (first solution) and 200 μL of an oxidizing agent solution (second solution) in a reagent kit are added. The chemiluminescent reaction was started.
From 40 seconds after the start of the chemiluminescence reaction, the amount of luminescence for 10 seconds (integrated value for 10 seconds, enzyme activity) was measured. The luminescence was measured in the same manner for hyaluronic acid sodium salt standard solutions 10, 100 and 1000. Table 3 shows the results of measuring the luminescence amounts of the reagent kits 1 to 9 of Examples 6 to 13 and Comparative Example 5.

The values in parentheses in Table 3 are S / N ratios (values obtained by dividing each light emission amount by a light emission amount of 0 ng / mL).

  It has been found that the reagent kits 1 to 8 of the present invention have a higher S / N ratio than the comparative reagent kit 9 and can perform highly sensitive measurement. Particularly, the reagent kit 5 for sandwich measurement using the same antibody was able to perform measurement with extremely high sensitivity. Also, from the results in Table 3, it can be seen that when HABP was used for the beads or the label, the luminescence amount of 0 ng / mL was high. This means that the non-specific reaction is large. Therefore, for example, when HASB2 and HABPB are applied to the competitive method, nonspecific adsorption of labeled hyaluronic acid (eg, a product obtained by reacting and binding peroxidase with hyaluronic acid aldehyde prepared in Comparative Example 4) is small in HASB2, It is thought that there are many. Reagent kits with many nonspecific adsorptions are considered to have low measurement sensitivity even in the competition method, and reagent kits with low nonspecific adsorption are considered to have high measurement sensitivity in the competition method, so the reagent kit of the present invention has high sensitivity even in the competition method. Measurement can be expected.

<Example 14> Preparation of anti-hyaluronic acid polyclonal antibody 9 Hyaluronic acid sodium salt (derived from human umbilical cord, viscosity average molecular weight 1,000,000, sandy particles) [Seikagaku Corporation] was converted to hyaluronic acid sodium salt (microorganism derived, viscosity averaged) A buffer solution containing anti-hyaluronic acid polyclonal antibody 9 was obtained in the same manner as in Example 1, except that the molecular weight was 2,000,000 and sand-like particles were used (manufactured by Kyowa Hakko Co., Ltd.). In the same manner as in Example 1, hyaluronic acid was precipitated from the antigen emulsion for immunization, and the shape was confirmed. The results were the same as those in Example 1.

<Example 15> Preparation of anti-hyaluronic acid polyclonal antibody 10 Hyaluronic acid sodium salt (derived from human umbilical cord, viscosity average molecular weight 1,000,000, sandy particles) [Seikagaku Corporation] 4/1000 parts by weight of hyaluronic acid sodium salt ( Microbial origin, viscosity average molecular weight 2,000,000, sand-like particles) [manufactured by Kyowa Hakko Co., Ltd.] A buffer solution containing anti-hyaluronic acid polyclonal antibody 10 was obtained in the same manner as in Example 1 except that the weight was changed to 6/1000 parts by weight. . In the same manner as in Example 1, hyaluronic acid was precipitated from the antigen emulsion for immunization, and the shape was confirmed. The results were the same as those in Example 1.

<Example 16> Preparation of anti-hyaluronic acid polyclonal antibody 11 Hyaluronic acid sodium salt (derived from human umbilical cord, viscosity average molecular weight 1,000,000, sandy particles) [Seikagaku Corporation] 4/1000 parts by weight of hyaluronic acid sodium salt ( Microbial origin, viscosity average molecular weight 2,000,000, sand-like particles) [manufactured by Kyowa Hakko Co., Ltd.] A buffer solution containing anti-hyaluronic acid polyclonal antibody 11 was obtained in the same manner as in Example 1 except that the amount was changed to 10/1000 parts by weight. . In the same manner as in Example 1, hyaluronic acid was precipitated from the antigen emulsion for immunization, and the shape was confirmed. The results were the same as those in Example 1.

<Example 17> Preparation of anti-hyaluronic acid polyclonal antibody 12 Hyaluronic acid sodium salt (derived from human umbilical cord, viscosity average molecular weight 1,000,000, sand-like particles) [Seikagaku Corporation] 4/1000 parts by weight of hyaluronic acid sodium salt ( Microbial origin, viscosity average molecular weight 2,000,000, sand-like particles) [manufactured by Kyowa Hakko Co., Ltd.] A buffer solution containing anti-hyaluronic acid polyclonal antibody 12 was obtained in the same manner as in Example 1 except that the weight was changed to 20/1000 parts by weight. . In the same manner as in Example 1, hyaluronic acid was precipitated from the antigen emulsion for immunization, and the shape was confirmed. The results were the same as those in Example 1.

<Comparative Example 6> Preparation of anti-hyaluronic acid polyclonal antibody 13 Instead of antigen emulsion 1 for immunization, 1 / of hyaluronic acid sodium salt (microorganism-derived, viscosity average molecular weight 2,000,000, sand-like particles) [manufactured by Kyowa Hakko Co., Ltd.] An antigen emulsion for immunization 13 prepared by adding 10000 parts by weight to 1 part by weight of a 0.85% by weight aqueous sodium chloride solution, mixing and emulsifying 1 part by weight of a solution dissolved and stirred and 1 part by weight of Complete Freund's adjuvant was used. A buffer solution containing anti-hyaluronic acid polyclonal antibody 13 was obtained in the same manner as in Example 1 except for the above. In addition, when the shape of the hyaluronic acid in the emulsion 13 was confirmed in the same manner as in Example 1, only a flocculent precipitate was observed, confirming that sodium hyaluronate was dissolved in the emulsion. .

<Comparative Example 7> Preparation of anti-hyaluronic acid polyclonal antibody 14 Instead of antigen emulsion 1 for immunization, 2 / of hyaluronic acid sodium salt (microorganism-derived, viscosity average molecular weight 2,000,000, sandy particles) [manufactured by Kyowa Hakko Co., Ltd.] An antigen emulsion 14 for immunization prepared by adding 10,000 parts by weight to 1 part by weight of a 0.85% by weight aqueous sodium chloride solution, mixing and emulsifying 1 part by weight of a solution dissolved by stirring and 1 part by weight of Complete Freund's adjuvant was used. A buffer containing anti-hyaluronic acid polyclonal antibody 14 was obtained in the same manner as in Example 1 except for the above. In addition, when the shape of the hyaluronic acid in the emulsion 14 was confirmed in the same manner as in Example 1, only a flocculent precipitate was found, confirming that sodium hyaluronate was dissolved in the emulsion. .

<Evaluation of antibody titer>
1) Preparation of reagent for measurement Hyaluronic acid sodium salt (derived from microorganisms, viscosity average molecular weight 2,000,000, sand-like particles, manufactured by Kyowa Hakko Co., Ltd.) was added to a concentration of 20 μg / mL in 0.1 M carbonate buffer (pH 9.0). After dissolving, 500 beads of 6.35 mm diameter polystyrene beads (trade name: Immunobeads, manufactured by Immunochemical Co., Ltd.) were immersed in 500 mL of this solution, and left at 2 to 10 ° C. for 10 hours. Thereafter, the solution was removed by suction with an aspirator, and the polystyrene beads were immersed in a phosphate buffer solution (pH 7.0) containing 0.1% by weight of bovine serum albumin to obtain sodium hyaluronate salt-bound beads (B). The prepared hyaluronic acid sodium salt-bound beads (B) were stored at 4 to 10 ° C until use.

2) Measuring operation One hyaluronic acid sodium salt-bound bead (B) was placed in a test tube (inner diameter 1.2 cm, length 10 cm), 300 μL of 1% by weight bovine serum albumin-containing phosphate buffer (pH 7.0) and a sample for measurement (A buffer solution containing an anti-hyaluronic acid antibody) 50 μL was added, and the mixture was allowed to react at 37 ° C. for 4 hours. The reaction solution was removed by suction using an aspirator, and after adding 1 mL of a 0.85% by weight aqueous sodium chloride solution, the beads were washed by removing the aqueous sodium chloride solution using an aspirator. After performing this washing operation two more times, 300 μL of the labeled antibody solution was added, and the mixture was allowed to react at 37 ° C. for 1 hour. After the beads were washed three times in the same manner as described above, 500 μL of the coloring solution was added, and the mixture was allowed to react at 37 ° C. for 30 minutes. The reaction was stopped by adding 1 mL of 1.5 N sulfuric acid, and the absorbance (optical path length: 10 mm) was measured at a measurement wavelength of 492 nm. Note that the same standard antibody solution and color developing solution as described above were used.

  Antibody titers were evaluated using the buffers containing the anti-hyaluronic acid polyclonal antibodies 9 to 14 obtained in Examples 14 to 17 and Comparative Examples 6 to 7 as measurement samples, and the results are shown in Table 4. The measurement blank used was an anti-hyaluronic acid polyclonal antibody similarly obtained from a non-immunized mouse (normal mouse) diluted to 10 μg / mL.

  From Table 4, it can be seen that the anti-hyaluronic acid polyclonal antibodies of Examples 14 to 17 have clearly higher antibody titers (activity) than the anti-hyaluronic acid polyclonal antibodies of Comparative Examples 6 and 7.

<Preparation of sodium salt of hyaluronic acid having viscosity average molecular weight of 180,000>
A 1 mg / mL aqueous solution of hyaluronic acid sodium salt (viscosity average molecular weight of 1,000,000, derived from human umbilical cord, manufactured by Seikagaku Corporation) was treated with an ultrasonic disperser (Teraoka Co., Ltd., model number UH-50) to have a viscosity average molecular weight of 180,000. It was prepared by sonication until Next, an aqueous solution of sodium hyaluronate having a viscosity average molecular weight of 180,000 was dried with a freeze dryer to prepare solid sodium hyaluronate (sand-like particles).

<Example 18> Preparation of anti-hyaluronic acid polyclonal antibody 15 Hyaluronic acid sodium salt (viscosity of human umbilical cord, viscosity average molecular weight 1,000,000, sandy particles) [Seikagaku Corporation] A buffer solution containing anti-hyaluronic acid polyclonal antibody 15 was obtained in the same manner as in Example 1 except that the average molecular weight was changed to 180,000 and sand-like particles. In the same manner as in Example 1, hyaluronic acid was precipitated from the antigen emulsion for immunization, and the shape was confirmed. The results were the same as those in Example 1.

<Example 19> Preparation of anti-hyaluronic acid polyclonal antibody 16 Hyaluronic acid sodium salt (derived from human umbilical cord, viscosity average molecular weight 1,000,000, sand-like particles) [Seikagaku Corporation] 4/1000 parts by weight of ultrasonically treated hyaluronic acid A buffer solution containing anti-hyaluronic acid polyclonal antibody 16 was obtained in the same manner as in Example 1, except that the acid sodium salt (viscosity average molecular weight: 180,000) was changed to 6/1000 parts by weight. When hyaluronic acid was precipitated from the antigen emulsion for immunization and the shape was confirmed in the same manner as in Example 1, the results were the same as those in Example 1.

<Example 20> Preparation of anti-hyaluronic acid polyclonal antibody 17 Hyaluronic acid sodium salt (derived from human umbilical cord, viscosity average molecular weight 1,000,000, sand-like particles) [Seikagaku Corporation] 4/1000 parts by weight of ultrasonically treated hyaluronic acid A buffer solution containing anti-hyaluronic acid polyclonal antibody 17 was obtained in the same manner as in Example 1, except that the acid sodium salt was changed to 10/1000 parts by weight (viscosity average molecular weight: 180,000). When hyaluronic acid was precipitated from the antigen emulsion for immunization and the shape was confirmed in the same manner as in Example 1, the results were the same as those in Example 1.

<Example 21> Preparation of anti-hyaluronic acid polyclonal antibody 18 Hyaluronic acid sodium salt (derived from human umbilical cord, viscosity average molecular weight 1,000,000, sand-like particles) [Seikagaku Corporation] 4/1000 parts by weight of ultrasonically treated hyaluronic acid A buffer containing anti-hyaluronic acid polyclonal antibody 18 was obtained in the same manner as in Example 1, except that the acid sodium salt (viscosity average molecular weight: 180,000) was changed to 20/1000 parts by weight. When hyaluronic acid was precipitated from the antigen emulsion for immunization and the shape was confirmed in the same manner as in Example 1, the results were the same as those in Example 1.

<Comparative Example 8> Preparation of anti-hyaluronic acid polyclonal antibody 19 Instead of antigen emulsion 1 for immunization, 1 / 10,000 parts by weight of ultrasonically treated sodium salt of hyaluronic acid (viscosity average molecular weight: 180,000) was 0.85% by weight chloride. The same procedure as in Example 1 was repeated except that 1 part by weight of a sodium aqueous solution was used, and 1 part by weight of a solution dissolved by stirring and 1 part by weight of Complete Freund's adjuvant were mixed and emulsified to prepare an antigen emulsion 19 for immunization. A buffer solution containing hyaluronic acid polyclonal antibody 19 was obtained. When the shape of hyaluronic acid in the emulsion 19 was confirmed in the same manner as in Example 1, only flocculent precipitation was recognized, confirming that sodium hyaluronate was dissolved in the emulsion.

<Comparative Example 9> Preparation of anti-hyaluronic acid polyclonal antibody 20 Instead of antigen emulsion 1 for immunization, 2 / 10,000 parts by weight of ultrasonically treated sodium salt of hyaluronic acid (viscosity average molecular weight 180,000) was 0.85% by weight chloride. The same procedure as in Example 1 was repeated except that 1 part by weight of a sodium aqueous solution was used, and 1 part by weight of a solution dissolved by stirring and 1 part by weight of Complete Freund's adjuvant were mixed and emulsified to prepare an antigen antigen for immunization 20. A buffer solution containing hyaluronic acid polyclonal antibody 6 was obtained. In addition, when the shape of the hyaluronic acid in the emulsion 20 was confirmed in the same manner as in Example 1, only a flocculent precipitate was observed, confirming that the sodium salt of hyaluronic acid was dissolved in the emulsion.

<Evaluation of antibody titer>
1) Preparation of Reagent for Measurement Ultrasonic sodium salt of hyaluronic acid (viscosity average molecular weight: 180,000) was dissolved in 0.1 M carbonate buffer (pH 9.0) to a concentration of 20 μg / mL. 500 pieces of 35 mm polystyrene beads (trade name: Immunobeads, manufactured by Immunochemical Co., Ltd.) were immersed and left at 2 to 10 ° C for 10 hours. Thereafter, the solution was removed by suction using an aspirator, and the polystyrene beads were immersed in a phosphate buffer solution (pH 7.0) containing 0.1% by weight of bovine serum albumin to obtain hyaluronic acid sodium salt-bound beads (C). The prepared hyaluronic acid sodium salt-bound beads (C) were stored at 4 to 10 ° C until use.

2) Measurement operation One test tube (inner diameter 1.2 cm, length 10 cm), hyaluronic acid sodium salt-conjugated beads (C), 300 μL of 1% by weight bovine serum albumin-containing phosphate buffer (pH 7.0) and a sample for measurement (A buffer solution containing an anti-hyaluronic acid antibody) 50 μL was added, and the mixture was allowed to react at 37 ° C. for 4 hours. The reaction solution was removed by suction using an aspirator, and after adding 1 mL of a 0.85% by weight aqueous sodium chloride solution, the beads were washed by removing the aqueous sodium chloride solution using an aspirator. After performing this washing operation two more times, 300 μL of the labeled antibody solution was added, and the mixture was allowed to react at 37 ° C. for 1 hour. After the beads were washed three times in the same manner as described above, 500 μL of the coloring solution was added, and the mixture was allowed to react at 37 ° C. for 30 minutes. The reaction was stopped by adding 1 mL of 1.5 N sulfuric acid, and the absorbance (optical path length: 10 mm) was measured at a measurement wavelength of 492 nm.

  The antibody titers were evaluated using the buffers containing the anti-hyaluronic acid polyclonal antibodies 15 to 20 obtained in Examples 18 to 21 and Comparative Examples 8 to 9 as measurement samples, and the results are shown in Table 5. The measurement blank used was an anti-hyaluronic acid polyclonal antibody similarly obtained from a non-immunized mouse (normal mouse) diluted to 10 μg / mL.

  From Table 5, it can be seen that the anti-hyaluronic acid polyclonal antibodies of Examples 18 to 21 have clearly higher antibody titers (activity) than the anti-hyaluronic acid polyclonal antibodies of Comparative Examples 8 to 9.

<Example 22> Preparation of anti-hyaluronic acid monoclonal antibody Preparation of Hybridoma Spleens were respectively excised from one mouse immunized in Example 17 and one mouse immunized in Example 21, and an anti-hyaluronic acid monoclonal antibody was prepared according to a conventional method as follows. The following operation was performed for each animal. After spleen cells were collected from the spleen and washed with RPMI1640 medium, the total amount of spleen cells was mixed with 2 × 10 ⁷ myeloma cells (P3-NS1 / 1-Ag4.1), and 42.5% by weight (37 ° C.) 1 in 1 mL of RPMI 1640 medium containing polyethylene glycol 4000 (concentration based on the weight of the medium) of polyethylene glycol 4000 (number average molecular weight 4000, obtained from Nacalai Tesque, Inc.) and 7.5% by weight (concentration based on the weight of the medium) of dimethyl sulfoxide. Fused for minutes. One minute later, the cell suspension was gradually diluted with 5 mL of RPMI1640 medium. After the cells are centrifuged and washed, HAT medium (RPMI1640 medium containing hypoxanthine, aminopterin, and 10% by weight of fetal bovine serum) is added to a volume of 20 mL, and 0.2 mL is added to a 96-well microplate. After incubating for 2 weeks, the antibody titer of the culture supernatant in the well in which the cells proliferated was measured. The measurement of the antibody titer was carried out according to the evaluation of the anti-hyaluronic acid polyclonal antibody. The culture supernatant was directly used as a measurement sample.

  Next, a well showing an absorbance of 5 times or more the absorbance of the blank was defined as a well having an antibody titer, and cells in the well were cloned by a limiting dilution method. From each mouse immunized in Example 17 and Example 21, one hybridoma (17 and 21) was obtained, that is, two hybridomas (17 and 21) producing an anti-hyaluronic acid monoclonal antibody were obtained.

2. Purification of anti-hyaluronic acid monoclonal antibody Hybridoma (17) obtained by immunization in Example 17 was cultured in 1 L of serum-free medium ASF medium 104, obtained from Ajinomoto Co. for 1 week, and the culture supernatant was subjected to ultrafiltration membrane. (Ultra Filter UK-50, manufactured by Toyo Roshi Kaisha, Ltd.), and concentrated to 1/100 volume. These concentrates were applied to a gel filtration column (Superdex 200 Prep column, manufactured by Pharmacia) using a 0.02 M phosphate buffer (pH 7.0) containing 0.85% by weight of sodium chloride as a mobile phase, and fractionated into each fraction. The antibody titer of each fraction was measured, and the fractions showing an absorbance at least three times the absorbance of the blank were pooled. The measurement of the antibody titer was carried out in accordance with the evaluation of the anti-hyaluronic acid polyclonal antibody, and each fraction was directly used for the measurement. The pooled fractions were concentrated with an ultrafiltration membrane (Ultrafilter UK-50, manufactured by Toyo Roshi Kaisha) to a protein concentration of 1 mg / mL to obtain a buffer solution of the anti-hyaluronic acid monoclonal antibody HAS3.
Hybridoma (21) obtained by immunization in Example 21 was purified in the same manner to obtain a buffer solution of anti-hyaluronic acid monoclonal antibody HAS4.

3. Confirmation of reactivity of anti-hyaluronic acid monoclonal antibodies HAS3 and HAS4 An anti-hyaluronic acid monoclonal antibody was evaluated in the same manner as in the evaluation of anti-hyaluronic acid polyclonal except that hyaluronic acid-bound beads prepared using hyaluronic acid shown in Table 5 were used. Was evaluated for antibody titer. Table 6 shows the results. However, for the measurement, an anti-hyaluronic acid monoclonal antibody diluted to 10 μg / mL with a 1% by weight bovine serum albumin-containing phosphate buffer (pH 7.0) was used. As a control, an anti-hyaluronic acid polyclonal antibody purified from the serum of a non-immunized mouse was similarly tested.

  Both anti-hyaluronic acid monoclonal antibodies HAS3 and HAS4 were found to bind to high molecular weight hyaluronic acid. It was also found that the binding to low molecular weight hyaluronic acid was relatively weaker than that of high molecular weight hyaluronic acid, but was clearly bound.

<Example 23> Reagent kit 10
Anti-hyaluronic acid monoclonal antibody-bound beads (HASB3), peroxidase-labeled anti-hyaluronic acid monoclonal antibody (PHAS3), buffer solution (first solution), oxidizing agent solution (second solution) and standard sodium hyaluronate as follows Reagent kits consisting of solutions 0, 10, 100 and 1000 were prepared.
That is, a peroxidase-labeled anti-hyaluronic acid monoclonal antibody (PHAS3) was prepared in the same manner as in Example 6 except that an anti-hyaluronic acid monoclonal antibody (HAS3) was used instead of the anti-hyaluronic acid monoclonal antibody (HAS1). Then, a reagent kit 10 was prepared in the same manner as in Example 6, except that the peroxidase-labeled anti-hyaluronic acid monoclonal antibody (PHAS1) was replaced with this peroxidase-labeled anti-hyaluronic acid monoclonal antibody (PHAS3).

<Example 24> Reagent kit 11
A peroxidase-labeled anti-hyaluronic acid monoclonal antibody (PHAS4) was prepared in the same manner as in Example 6 except that an anti-hyaluronic acid monoclonal antibody (HAS4) was used instead of the anti-hyaluronic acid monoclonal antibody (HAS1). Then, a reagent kit 11 was prepared in the same manner as in Example 6, except that the peroxidase-labeled anti-hyaluronic acid monoclonal antibody (PHAS1) was replaced with this peroxidase-labeled anti-hyaluronic acid monoclonal antibody (PHAS4).

<Quantification of hyaluronic acid by kit>
The luminescence was measured for the reagent kits 10 and 11 of Examples 23 and 24 in the same manner as the measurement of the luminescence for the reagent kits 1 to 9 of Examples 6 to 13 and Comparative Example 5, and the results are shown in Table 7. Show.

The values in parentheses in Table 7 are S / N ratios (values obtained by dividing each light emission amount by a light emission amount of 0 ng / mL).
It has been found that the reagent kits 10 and 11 of the present invention have a higher S / N ratio than the comparative reagent kit 9 and can perform highly sensitive measurement.

<Example 25> Preparation of anti-hyaluronic acid polyclonal antibody 21 Hyaluronic acid sodium salt (derived from human umbilical cord, viscosity average molecular weight 1,000,000, sand-like particles) [Seikagaku Corporation] 4/1000 parts by weight 2/1000 parts by weight A buffer solution containing the anti-hyaluronic acid polyclonal antibody 21 was obtained in the same manner as in Example 1 except for changing the parts. When hyaluronic acid was precipitated from the antigen emulsion for immunization and the shape was confirmed in the same manner as in Example 1, the results were the same as those in Example 1.

<Example 26> Preparation of anti-hyaluronic acid polyclonal antibody 22 Hyaluronic acid sodium salt (derived from human umbilical cord, viscosity average molecular weight 1,000,000) [manufactured by Seikagaku Corporation] 4/100 parts by weight was changed to 100/1000 parts by weight. A buffer containing an anti-hyaluronic acid polyclonal antibody 22 was obtained in the same manner as in Example 1 except for the above. When hyaluronic acid was precipitated from the antigen emulsion for immunization and the shape was confirmed in the same manner as in Example 1, the results were the same as those in Example 1.

<Evaluation of antibody titer>
Anti-hyaluronic acid polyclonal obtained in Example 25 or 26 in the same manner as in the evaluation of the antibody titer of the buffer containing anti-hyaluronic acid polyclonal antibodies 1 to 8 obtained in Examples 1 to 4 and Comparative Examples 1 to 4 The antibody titer was evaluated using a buffer containing each of the antibodies 21 and 22 as a measurement sample, and the results are shown in Table 8. The measurement blank used was an anti-hyaluronic acid polyclonal antibody similarly obtained from a non-immunized mouse (normal mouse) diluted to 10 μg / mL.

  From Table 8, it can be seen that the anti-hyaluronic acid polyclonal antibodies 21 and 22 of Examples 25 and 26 have clearly higher antibody titers (activity) than the anti-hyaluronic acid polyclonal antibodies 5 and 6 of Comparative Examples 1 and 2.

<Example 27> Preparation of anti-hyaluronic acid monoclonal antibody Preparation of Hybridoma Spleens were respectively excised from one mouse immunized in Example 25 and one mouse immunized in Example 26, and an anti-hyaluronic acid monoclonal antibody was prepared according to a conventional method as follows. The following operations were performed for each mouse. After spleen cells were collected from the spleen and washed with RPMI1640 medium, the total amount of spleen cells was mixed with 2 × 10 ⁷ myeloma cells (P3-NS1 / 1-Ag4.1), and 42.5% by weight (37 ° C.) 1 in 1 mL of RPMI 1640 medium containing polyethylene glycol 4000 (concentration based on the weight of the medium) of polyethylene glycol 4000 (number average molecular weight 4000, obtained from Nacalai Tesque, Inc.) and 7.5% by weight (concentration based on the weight of the medium) of dimethyl sulfoxide. Fused for minutes. One minute later, the cell suspension was gradually diluted with 5 mL of RPMI1640 medium. After the cells are centrifuged and washed, HAT medium (RPMI1640 medium containing hypoxanthine, aminopterin, and 10% by weight of fetal bovine serum) is added to a volume of 20 mL, and 0.2 mL is added to a 96-well microplate. After incubating for 2 weeks, the antibody titer of the culture supernatant in the well in which the cells proliferated was measured. The measurement of the antibody titer was carried out according to the evaluation of the anti-hyaluronic acid polyclonal antibody. The culture supernatant was directly used as a measurement sample.

  Next, a well showing an absorbance of 5 times or more the absorbance of the blank was defined as a well having an antibody titer, and cells in the well were cloned by a limiting dilution method. From each mouse immunized in Example 25 and Example 26, one hybridoma (25 and 26), that is, two hybridomas (25 and 26) producing an anti-hyaluronic acid monoclonal antibody were obtained.

2. Purification of anti-hyaluronic acid monoclonal antibody The hybridoma (25) obtained by immunization in Example 25 was cultured in 1 L of serum-free medium ASF medium 104, obtained from Ajinomoto Co. for 1 week, and the culture supernatant was subjected to ultrafiltration membrane. (Ultra Filter UK-50, manufactured by Toyo Roshi Kaisha, Ltd.), and concentrated to 1/100 volume. These concentrates were applied to a gel filtration column (Superdex 200 Prep column, manufactured by Pharmacia) using a 0.02 M phosphate buffer (pH 7.0) containing 0.85% by weight of sodium chloride as a mobile phase, and fractionated into each fraction. The antibody titer of each fraction was measured, and the fractions showing an absorbance at least three times the absorbance of the blank were pooled. The measurement of the antibody titer was carried out in accordance with the evaluation of the anti-hyaluronic acid polyclonal antibody, and each fraction was directly used for the measurement. The pooled fraction was concentrated with an ultrafiltration membrane (Ultrafilter UK-50, manufactured by Toyo Roshi Kaisha) to a protein concentration of 1 mg / mL to obtain a buffer solution of the anti-hyaluronic acid monoclonal antibody HAS5.
Hybridoma (26) obtained by immunization in Example 26 was purified in the same manner to obtain a buffer solution of anti-hyaluronic acid monoclonal antibody HAS6.

3. Confirmation of reactivity of anti-hyaluronic acid monoclonal antibodies HAS5 and HAS6 An anti-hyaluronic acid monoclonal antibody was evaluated in the same manner as in the evaluation of anti-hyaluronic acid polyclonal except that hyaluronic acid-bound beads prepared using hyaluronic acid described in Table 5 were used. Was evaluated for antibody titer. Table 9 shows the results. However, for the measurement, an anti-hyaluronic acid monoclonal antibody diluted to 10 μg / mL with a 1% by weight bovine serum albumin-containing phosphate buffer (pH 7.0) was used. As a control, an anti-hyaluronic acid polyclonal antibody purified from the serum of a non-immunized mouse was similarly tested.

  Both anti-hyaluronic acid monoclonal antibodies HAS5 and HAS6 were found to bind to high molecular weight hyaluronic acid. It was also found that the binding to low molecular weight hyaluronic acid was relatively weaker than that of high molecular weight hyaluronic acid, but was clearly bound.

<Example 29> Reagent kit 12
A peroxidase-labeled anti-hyaluronic acid monoclonal antibody (PHAS5) was prepared in the same manner as in Example 6 except that an anti-hyaluronic acid monoclonal antibody (HAS5) was used instead of the anti-hyaluronic acid monoclonal antibody (HAS1). Then, a reagent kit 12 was prepared in the same manner as in Example 6, except that the peroxidase-labeled anti-hyaluronic acid monoclonal antibody (PHAS1) was replaced with this peroxidase-labeled anti-hyaluronic acid monoclonal antibody (PHAS5).

<Example 30> Reagent kit 13
A peroxidase-labeled anti-hyaluronic acid monoclonal antibody (PHAS6) was prepared in the same manner as in Example 6, except that the anti-hyaluronic acid monoclonal antibody (HAS1) was used instead of the anti-hyaluronic acid monoclonal antibody (HAS1). Then, a reagent kit 13 was prepared in the same manner as in Example 6, except that the peroxidase-labeled anti-hyaluronic acid monoclonal antibody (PHAS1) was replaced with this peroxidase-labeled anti-hyaluronic acid monoclonal antibody (PHAS6).

<Quantification of hyaluronic acid by kit>
The luminescence was measured for the reagent kits 12 and 13 of Examples 29 and 30 in the same manner as the measurement of the luminescence for the reagent kits 1 to 9 of Examples 6 to 13 and Comparative Example 5, and the results are shown in Table 10. Show.

The values in parentheses in Table 10 are S / N ratios (values obtained by dividing each light emission amount by a light emission amount of 0 ng / mL).
It was found that the reagent kits 12 and 13 of the present invention had a higher S / N ratio than the comparative reagent kit 9 and were capable of high-sensitivity measurement.

Claims (12)

An anti-hyaluronic acid antibody which can be produced by immunizing an animal with an antigen, wherein the antigen has a viscosity average molecular weight of 20,000 to 4,000,000 and is solid unmodified hyaluronic acid (MH). Acid antibody. The anti-hyaluronic acid antibody according to claim 1, wherein the viscosity average molecular weight of the antigen is 100,000 to 2,000,000. 3. The anti-hyaluronic acid antibody according to claim 1, which is a monoclonal antibody. A reagent kit for measuring hyaluronic acid, comprising the anti-hyaluronic acid antibody according to claim 1. The reagent kit according to claim 4, further comprising a ligand. The reagent kit according to claim 5, wherein the anti-hyaluronic acid antibody and / or the ligand are labeled with a labeling compound. The reagent kit according to claim 5, wherein one of the anti-hyaluronic acid antibody and the ligand is constituted by being bound or adsorbed to a water-insoluble carrier, and the other is labeled with a labeling compound. 8. The reagent kit according to claim 6, wherein the labeling compound is an enzyme. A method for producing an anti-hyaluronic acid antibody, comprising an immunization step of immunizing an animal with a solid unmodified hyaluronic acid (MH) having a viscosity average molecular weight of 20,000 to 4,000,000. The production method according to claim 9, further comprising an immunization step of immunizing an animal in a state where solid unmodified hyaluronic acid (MH) is mixed with a water-in-oil emulsion. A method for quantifying hyaluronic acid by a sandwich method using the reagent kit for measuring hyaluronic acid according to any one of claims 4 to 8. A method for quantifying hyaluronic acid by a chemiluminescent enzyme immunoassay using the reagent kit according to claim 8.