JP2535426B2 - How to measure chondrocalcin - Google Patents

Description

TECHNICAL FIELD The present invention relates to an immunological assay method using an enzyme-labeled mammalian chondrocalcin or anti-chondrocalcin antibody, which uses a mammalian body fluid as a sample and is in solution. And a method for measuring chondrocalcin in the mammal, which comprises measuring the amount of chondrocalcin in the mammal.

Further, the present invention relates to a measurement reagent and a kit used in the above measurement method.

Chondrocalcin is a protein that was isolated and purified in 1983 from Etch Choi et al., Starting from fetal bovine epiphyseal cartilage, and is a molecule with a molecular weight of 69,000 consisting of two subunits with a molecular weight of 35,000. Known (HU Choi, et al., J. Biol. Chem., 258 , 655-661, (19
See 83)). Subsequent partial amino acid sequence studies reported that chondrocalcin is identical to the C-propeptide of type 2 procollagen (MVDRes
t, et al., Biochem. J., 237 , 923-925, (1986)).
Regarding the biodistribution of this protein, a histochemical study mainly on cattle has been reported by AR Poole et al. (ARPoole et al., J. Cell Biol., 98 , 54).
−65 (9184); Clinical Orthopaedics and Related Res
earch, 208 , 114-118 (1986)). According to it, it is said that the most abundant is cartilage where calcification is progressing, especially in the deep cartilage region, and is not present in mature bones, teeth and other tissues. It is presumed that its physiological action is deeply involved in calcification in cartilage due to the characteristics of such biodistribution and the binding of this protein with calcium ions and hydroxyapatite. As an experimental means in these histochemical research processes, immunological techniques such as tissue staining are used, in which polyclonal antibodies obtained by immunizing rabbits with bovine chondrocalcin were used to The organization is being studied.

On the other hand, there are few reports of pathological studies on chondrocalcin, and future development is expected. AR Poole et al. State that they are focusing on chondrocalcin in relation to pathology in diseases related to development, endochondral ossification, and abnormal calcification (ARPoole et al., Clinical O
rthopaedics and Related Research 208 , 114−118 (198
6)). Therefore, the measurement of chondrocalcin plays a very important role in the study of basic medicine and clinical medicine of chondrocalcin.

In 1987, A. Heinek et al. Measured chondrocalcin in cartilage extract and chondrocyte culture supernatant by radio immunoassay (A. Hinek et al., J. Cell Biol. 104) .
1435-1441 (1987)). However, the lower limit of measurement described here was 100 ng per 1 ml of the sample, and this sensitivity was insufficient for measuring chondrocalcin in body fluids (blood, joint fluid, etc.). In addition, the samples used for the measurement were a liquid in which cartilage was extracted with a 4M guanidine hydrochloride aqueous solution (pH 5.8) and a culture supernatant liquid in which chondrocytes were cultured in DME medium, which interfered with the specific reaction or was nonspecific. It does not describe the measurement of body fluids containing factors that cause a reaction. Therefore, development of a highly sensitive measurement method, measurement reagent, and kit that enables the measurement of chondrocalcin in body fluids is extremely important in clinical examinations in terms of diagnosis of diseases and monitoring of treatment.

The immunological measurement method utilizing the antigen-antibody reaction utilizes the fact that the antigen and the antibody specifically react in the body fluid containing many components. However, in practice, when a body fluid is measured as a sample, a non-specific reaction other than the intended antigen-antibody reaction or an interfering reaction occurs, so if a known amount of the antigen to be measured is added to the sample However, there is a problem that the added amount cannot be measured correctly, or the measurement sensitivity necessary for measuring the antigen cannot be obtained sufficiently. The cause of reactions other than such specific reactions is not always fully understood, but it is considered as follows. That is, it occurs due to the presence of a substance similar in antigenicity to the target substance to be measured in the body fluid, or due to the presence of a rheumatoid factor that reacts nonspecifically with the Fc portion of IgG, which is an antibody. Such is the case. Therefore, in clinical tests, it is always a very important task to correctly measure a target substance in body fluid.

The present inventors have solved the above problems and completed the present invention capable of measuring chondrocalcin in body fluids.

DISCLOSURE OF THE INVENTION That is, the present invention relates to an immunological assay method using an enzyme-labeled mammalian chondrocalcin or anti-chondrocalcin antibody, which uses a mammalian body fluid as a sample and utilizes an immune reaction in the solution. Then, the amount of chondrocalcin in the mammal is measured to measure chondrocalcin.

Further, the present invention comprises an anti-chondrocalcin antibody reagent immobilized on an insoluble carrier and an anti-chondrocalcin antibody reagent labeled with an enzyme, and determines the amount of chondrocalcin present in the body fluid of a mammal as a sample. Reagent for measuring chondrocalcin for immunological measurement, anti-chondrocalcin antibody immobilized on an insoluble carrier, labeled anti-chondrocalcin antibody, lysing agent, detergent, substrate for measuring enzyme activity A kit for immunologically measuring the amount of chondrocalcin present in the body fluid of a mammal as a specimen, which comprises a reaction-terminating agent.

Furthermore, the present invention includes a monoclonal antibody characterized by recognizing mammalian chondrocalcin.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a calibration curve of bovine chondrocalcin by a measurement reagent using the monoclonal antibody “Chon-5G” and the HRP-labeled monoclonal antibody “Chon-8G”.

Fig. 2 shows the results of measurement using bovine thyroid gland using the monoclonal antibody "Chon-3F" and HRP-labeled polyclonal antibody.
It is a calibration curve of chondrocalcin.

FIG. 3 is a calibration curve of bovine chondrocalcin by a measuring reagent using a polyclonal antibody and an HRP-labeled monoclonal antibody “Chon-4H”.

FIG. 4 is a calibration curve for measuring human chondrocalcin in a sample.

FIG. 5 is a bovine chondrocalcin calibration curve using the measurement reagent of the present invention using the anti-chondrocalcin polyclonal antibody and the HRP-labeled polyclonal antibody.

FIG. 6 shows the effect of adding skim milk in the chondrocalcin assay system. In Fig. 6-●-,
− ○ − is the final bovine chondrocalcin concentration of 5n
Indicates g / ml and Ong / ml.

FIG. 7 is a calibration curve of bovine chondrocalcin with a measurement reagent using anti-bovine chondrocalcin polyclonal antibody and peroxidase-labeled anti-bovine chondrocalcin polyclonal antibody Fab ′.

Fig. 8 shows the calibration of bovine chondrocalcin with the measurement reagent using anti-bovine chondrocalcin polyclonal antibody and peroxidase-labeled anti-bovine chondrocalcin monoclonal antibody "Chon-4H" Fab '. It is a line.

FIG. 9 shows the effect of skim milk addition in the chondrocalcin assay system using Fab ′. In the figure,-●-and-○-indicate the final bovine chondrocalcin concentrations of 1 ng / ml and Ong / ml, respectively.

FIG. 10 shows a measurement reagent using anti-bovine chondrocalcin polyclonal antibody and peroxidase-labeled anti-bovine chondrocalcin polyclonal antibody Fab ′. It is a calibration curve for the measurement of human chondrocalcin in a sample.

FIG. 11 shows a measurement reagent using anti-bovine chondrocalcin polyclonal antibody immobilized on mirror-polished polystyrene beads and anti-bovine chondrocalcin F (ab ′) ₂ multilabeled with peroxidase. FIG. 3 is a calibration curve for measuring human chondrocalcin in liquid.

BEST MODE FOR CARRYING OUT THE INVENTION First, the reagent for measuring chondrocalcin of the present invention will be described. The assay reagent of the present invention comprises an anti-chondrocalcin antibody reagent immobilized on an insoluble carrier and an anti-chondrocalcin antibody reagent labeled with an enzyme. , Anti-chondrocalcin monoclonal antibody.

As a principle, chondrocalcin as an antigen for obtaining the polyclonal antibody used in the present invention is a naturally-occurring chondrocalcin extracted from a natural material. Those having properties may be obtained by protein engineering or genetic engineering techniques. For example, it may be a hapten, that is, a peptide containing the antigenic determinant site of natural chondrocalcin.

Examples of the material for obtaining natural chondrocalcin include, for example, mammalian fetal cartilage and developing mammalian cartilage, but fetal bovine cartilage is preferred because it is relatively easy to obtain. Separation / purification can be carried out by a combination of commonly used protein separation techniques, such as salting out, extraction, centrifugation, ultrafiltration and various chromatographies. The above method is one example.

Monoclonal antibodies were obtained using the cell fusion method of Koehler and Milstein (G. Khler and Milstein, Nature (Lon
don). 256 , 495-497 (1975)), which was prepared by culturing the hybridoma, secreting it, and separating it from the culture solution. That is, after immunizing a mouse with mammalian-derived chondrocalcin, lymphocytes of this mouse were fused with mouse myeloma cells to prepare a hybridoma. The hybridoma thus obtained produces various monoclonal antibodies depending on each of various fused lymphocytes, and thus the hybridoma producing the desired monoclonal antibody is isolated as a cloned hybridoma by cloning. did. This cloned hybridoma was cultured in vitro to secrete the monoclonal antibody. The anti-chondrocalcin monoclonal antibody was separated from this culture supernatant.

In addition, the polyclonal antibody is a commonly used method,
For example, "The Biochemical Society of Japan, Second Biochemistry Laboratory, Vol. 5, 1-
P. 10, Tokyo Kagaku Dojin, 1986 ”.

The immunized animal is not particularly limited as long as it is different from the mammal used as the material of chondrocalcin as an antigen. For example, when bovine chondrocalcin is used as an antigen, goat, rabbit, guinea pig, rat Or a mouse etc. are mentioned preferably.

The histological staining of human fetal articular cartilage and normal human articular cartilage was performed by a conventional method using the anti-bovine chondrocalcin polyclonal antibody and the monoclonal antibody used in the assay reagent of the present invention, and the former gave a stained image. However, the latter was not obtained. From these results, it can be seen that the antibody of the present invention recognizes human chondrocalcin.

In the present invention, the above polyclonal antibody, the monoclonal antibody is used as at least a labeled anti-chondrocalcin antibody, but when using an anti-chondrocalcin antibody further immobilized on an insoluble carrier or immobilized on an insoluble carrier, The combination may be a polyclonal antibody, a monoclonal antibody, or one may be a polyclonal antibody and the other a monoclonal antibody.

In particular, Fab ', F (ab') ₂ or Facb fragment may be preferable as the labeled antibody for the following reasons. That is, firstly, in the body fluid to be measured,
Often, there is a component that binds to the Fc portion of an antibody, such as a rheumatoid factor, and such a component causes a reaction to a nonspecific antibody other than the desired antigen-antibody reaction. As a result, it may be difficult to correctly measure chondrocalcin in body fluid, but when the fragment is labeled and used, the reaction does not occur and an accurate value can be obtained. Secondly, in order to obtain the measurement sensitivity necessary for measuring chondrocalcin using a body fluid as a sample, it is required to reduce the nonspecific reaction to the insoluble carrier as much as possible. When using
The reaction is suppressed and preferable.

In the case of F (ab ′) ₂ or Fab ′, such a fragment is obtained by digesting the polyclonal antibody or monoclonal antibody obtained as described above by a known method, for example, by cleaving the antibody with pepsin to obtain F (ab ′) 2. ₂ fragment, or by further reducing the F (ab ') ₂ fragment to give a Fab' fragment (A. Nisonoff et al., Arch. Biochem. Biophys., 89 , 230).
(1960): P. Parham., J. Immunol., 131 , 2895 (1983), etc.).

As a labeling substance, radioisotopes are well known, but considering industrial production, there is a possibility that it may lead to pollution, there is a deadline for use, and special training of users is required, Enzymes that do not have these drawbacks and have a broadening effect are suitable.

Examples of the enzyme bound to the anti-chondrocalcin antibody include lysozyme, malate dehydrogenase, glucose-6-phosphate dehydrogenase, peroxidase, glucose oxidase,
Examples thereof include alkaline phosphatase, luciferase, β-galactosidase, alcohol dehydrogenase, invertase and the like. The binding method of the binding antibody between these enzymes and the anti-chondrocalcin antibody is
Conventional methods such as the glutaraldehyde method, the periodate method, and the maleimide method can be followed. For example, it can be carried out by reacting the maleimidated antibody or antibody fragment with the SH-enzyme in a solution. Maleimidation of an antibody or antibody fragment is performed by, for example, succinimidyl 4- (N-maleimidomethyl) cyclohexane carbonate (SMCC), sulfosuccinimidyl 4-.
It can be maleimidized with (N-maleimidomethyl) cyclohexane carbonate (sulfo SMCC), succinimidyl-metamaleimidobenzoate (MBS), succinimidyl 6-maleimidohexanoate (EMCS) and the like. The SH group can be introduced into the enzyme according to a known method (Ishikawa, "Enzyme-linked immunosorbent assay", Medical Institute).
For example, enzyme and S-acetylmercaptosuccinic anhydride (AM
SA) or N-succinimidyl-3- (2-pyridylthio) propionate (SPDP) and the like.

The number of labels of the enzyme in the binding antibody of the enzyme thus obtained and the anti-chondrocalcin antibody, that is, the enzyme-labeled antibody can be determined by measuring the molecular weight, the absorbance, the enzyme activity, or the like. For example, when the enzyme is peroxidase, it is derived from antibody and peroxidase.
The number of labels can be determined by measuring the absorbance at 280 nm and the absorbance at 403 nm derived from peroxidase (E. Ishikawa et al., J. Immunoassay., 4 , 209-327,
(1983), p243).

That is, at 403 nm, there is no absorbance of the antibody, and since the absorbance is derived from only peroxidase, the concentration of peroxidase is calculated from the absorbance. Since the absorbance at 280 nm is derived from both the antibody and peroxidase, the concentration of the antibody is calculated from the measured absorbance at 280 nm, the concentration of peroxidase calculated by the absorbance at 403 nm, and the molecular extinction coefficient of peroxidase at 280 nm. The value obtained by subtracting the contribution of the absorbance at 280 nm from
Calculated from the molecular extinction coefficient of the antibody at nm. The number of peroxidase labeled on the antibody can be determined from the concentration of peroxidase and antibody thus obtained.

Thus, the labeled number of the labeled anti-chondrocalcin antibody of the present invention was determined, and the correlation with the measurement sensitivity was investigated. As a result, the Fab ', F (ab') ₂ or Facb fragment was found to have an enzyme at an average of 1.0 molecule per antibody molecule. Although those having the above-mentioned binding are generally used, it has been clarified that the binding of 1.5 molecules or more (multi-labeled antibody) produces more preferable results. In order to obtain sufficient measurement sensitivity for measuring chondrocalcin in body fluids, the multi-labeled antibody should have an enzyme Fab ', F (a
An average of 1.5 or more molecules is preferably bound to one molecule of b ′) ₂ or Facb fragment, more preferably 2 or more molecules. However, this is not limited to the fragment.

Such a multi-labeled antibody is produced, for example, by reacting the maleimidated antibody or antibody fragment with an SH-enzyme. The reaction conditions include, for example, a molar ratio of the antibody fragment to the enzyme of 1: 1 or more,
Preferably 1: 3 or more, reaction temperature 4 to 40 ° C, preferably 4
The conditions can be as follows: -30 ° C, reaction pH 5.5-7.5, reaction time 5-48 hours, but not limited to the above reaction conditions as long as the multilabeled antibody of the present invention can be obtained. The multi-labeled antibody thus obtained can be separated and purified from the reaction solution by using, for example, gel chromatography.

The thus-obtained enzyme-labeled antibody of the present invention suppresses non-specific reactions or interfering reactions other than the intended antigen-antibody reaction that occurs when a body fluid is used as a sample, and is useful for measuring chondrocalcin in the body fluid. It can be measured correctly with the required measurement sensitivity.

On the other hand, the anti-chondrocalcin antibody immobilized on the insoluble carrier can be obtained as follows.

Examples of the insoluble carrier include polymers obtained from nature and derivatives thereof, and synthetic polymers and derivatives thereof. The former includes polysaccharides and their derivatives such as cellulose, sephadex, sepharose, carboxymethyl cellulose, nitrocellulose, cellulose acetate, dextran, and inorganic polymers such as glass and silk gel. The latter includes vinyl polymers such as polystyrene, polyethylene, polypropylene, ABS,
Polyvinyl fluoride, polyamine-methyl vinyl ether-maleic acid copolymer, ethylene-maleic acid copolymer, etc .; condensation type polymer, for example, polyamide such as 6-nylon, 6,6-nylon, polyester such as polyethylene terephthalate, There are amino acid polymers and the like. Also,
The shape is not particularly limited, such as a test tube, microtiter plate, beads or membrane. As the antibody immobilized on the insoluble carrier which becomes fragile, the antibody molecule of the anti-chondrocalcin antibody as described above, a fragment thereof which does not lose the antigen binding ability, for example, F (ab ') ₂ , Fab', Fab, Facb etc .; or It is a derivative of an antibody molecule or a fragment thereof that does not lose its antigen-binding ability. A method for immobilizing these antibodies on an insoluble carrier is a physical adsorption method, for example, a method in which a polystyrene carrier is immersed in a solution of an anti-chondrocalcin antibody; an ionic bond method, for example, an ion exchange resin or an amino group or a carboxylic acid group. , A method of using a carrier having an ionizable functional group such as a sulfonic acid group or a phosphoric acid group;
Alternatively, a covalent bond method by a chemical reaction, for example, a carboxy chloride method, a carbodiimide method, a maleic anhydride derivative method, an isocyanate derivative method, a cyanogen bromide activated polysaccharide method, a diazo method, an active ester method, a carrier bonding method by a crosslinking reagent (crosslinking As reagents, glutaraldehyde, hexamethylene isocyanate, succinimide, maleimide compounds, etc.); Furthermore, it has no binding ability for chondrocalcin but can bind to anti-chondrocalcin antibody through biological reaction. A method of binding via a substance, for example, a method of using a protein A binding carrier and the like.

As the insoluble carrier, it is preferable to use an insoluble carrier having a material whose surface is mirror-finished, because non-specific adsorption is low and the measurement sensitivity is improved. Such a mirror-finished insoluble carrier has a center line average roughness (Ra) of 1.5 on its surface.
Examples include those having a size of μm or less. The center line average roughness can be measured by a surface roughness profile measuring device, for example, Surfcom570A (manufactured by Tokyo Seimitsu Co., Ltd.).

The mirror-finished insoluble carrier is not particularly limited as long as it is the above-mentioned various polymers or derivatives thereof and has Ra of 1.5 μm or less, but polystyrene or the like is preferably used.

When the thus obtained anti-chondrocalcin antibody immobilized on the insoluble carrier of the present invention is used, a minute amount of chondrocalcin can be accurately measured using a body fluid as a sample.

Next, a book characterized by using a body fluid of a mammal as a sample using the measurement reagent thus obtained, and measuring the amount of chondrocalcin of the mammal by utilizing an immune reaction in the solution The method for measuring chondrocalcin of the invention will be described.

Examples of the immunological measurement method of the present invention include so-called competition method and sandwich method.

The sandwich method is roughly classified into a one-step sandwich method and a two-step sandwich method. The one-step sandwich method is a sample containing an antigen to be measured,
The solid-phase antibody immobilized on the insoluble carrier and the labeled antibody labeled with the enzyme are allowed to perform antigen / antibody in the same reaction system.
This is a method of forming an antigen-labeled antibody complex, washing the mixture, and then measuring the amount of the labeling substance. In this case, the sample, the solid phase antibody and the labeled antibody may be allowed to react at the same time, or the sample and the solid phase antibody may be reacted first and then the labeled antibody may be added and reacted. Alternatively, the solid phase antibody may be added after reacting the sample with the labeled antibody. In any case, it is only necessary to form the solid phase antibody / antigen / labeled antibody complex before the washing operation. On the other hand, in the two-step sandwich method, a sample and a solid phase antibody are first reacted to form a solid phase antibody / antigen complex, and then the sample is removed and washed, and then a labeling substance is added to the solid phase antibody / antigen complex. Allows formation of the antigen-labeled antibody complex. Then, after the washing operation, the amount of the labeling substance is measured.

In the present invention, the two-step sandwich method can be used when the body fluid contains an interfering substance that causes an erroneous measurement or a substance that causes a nonspecific reaction. In this method, once the antigen in the sample is reacted with the solid phase antibody, the sample is removed and washed, and then the labeled antibody is reacted so that the reaction of the labeled antibody is not affected by the above substance, It is excellent in terms of accuracy and sensitivity, but on the other hand, compared to the one-step sandwich method, the number of times of washing increases, so that the operation is complicated and the operation time becomes long. On the other hand, the one-step sandwich method can also be used. This method is a preferred embodiment because it is easy to operate and the reaction time is easily shortened, but it is generally susceptible to body fluids. That is, the nonspecific reaction or immune reaction is suppressed, so that the measurement sensitivity is lowered and becomes insufficient.

In the present invention, for example, a mirror-finished insoluble carrier as the insoluble carrier, especially the center line average roughness of the surface is 1.5
When beads with a size of μm or less are used and a multi-labeled F (ab ') ₂ antibody is used as a labeled antibody, non-specific adsorption to the beads is hindered and the sensitivity is increased by the multi-labeled F (ab') ₂ In particular, a one-step sandwich method is possible.

In the measuring method of the present invention, the molecular weight of the immune reaction solution is
It is non-specific to adjust the final concentration of these immunoreaction solutions to be 0.02 to 0.9% by weight by allowing the presence of a protein having 16,000 to 50,000 and an isoelectric point of 1.0 to 5.0 or a mixture containing it. Adsorption is suppressed, so that the background is remarkably lowered and high sensitivity is easily obtained, which is preferable. Such a protein or a mixture containing the protein may be contained in the immunoassay reagent of the present invention in the immunoreaction solution so that the predetermined amount is obtained. Such proteins include
Examples include casein, pepsin, ovoglycoprotein, orosomucoid and the like. Examples of such a mixture include 10 to 60% by weight, preferably 20 to 50% by weight, of the above-mentioned protein as a main component, and 30 to 80% sugar (eg, lactose).
% By weight, preferably 40-60% by weight, other fats (eg
0.5 to 2% by weight), ash (eg 5 to 12% by weight), water (eg 2 to 8% by weight) and the like can be contained. A typical example of such a mixture is skim milk.
Skim milk contains casein as a protein, but skim milk has better dispersibility in an immune reaction solution, and NBS (Non-specific binding) per unit weight of protein, as compared with the case of using casein alone.
It is characterized by high effect and good storage stability at a temperature of 4 ° C (precipitation does not easily occur). The skim milk used in the present invention may be milk of any origin as long as it is defatted milk. One of the most typical is the commercially available skim milk from Difco.

In the present invention, the "molecular weight" of a protein means a molecular weight measured by an osmotic pressure method. Specifically, a polymer solution, a pure solvent and a solvent molecule are freely permeable, but an eluting polymer is not permeable. The osmotic pressure difference between both solutions when contacting with a membrane as a boundary is used to measure the molecular weight of a protein by utilizing the fact that it serves as a parameter of the molecular weight of a polymer.
It is a value measured at 4 ° C. using a urea solution. The "isoelectric point" refers to the value measured by the chromatofocusing method that separates proteins according to their isoelectric point. Specifically, a column (0.5 cmφ x 45 cm) packed with PBE94 (Pharmacia) gel is used as the eluent. The value measured with 0.025M imidazole hydrochloric acid (pH 7.4).

The solvent used in the above-mentioned immune reaction may be any of various ordinary solvents that do not adversely affect the reaction. For example, it is preferable to use a phosphate buffer solution, a Tris / hydrochloric acid buffer solution, an acetate buffer solution or the like having a pH of about 6.0 to 8.0.

There are no particular restrictions on the temperature of the immune reaction during the measurement as long as it does not denature the properties of the protein as a constituent and does not significantly suppress the immune reaction, but it is generally 50 ° C or lower, preferably about 4 ° C to 45 ° C. The reaction may be performed under the temperature conditions of about 5 minutes to 20 hours.

Further, as the body fluid of the mammal in the measuring method of the present invention,
A living liquid component of a living body, that is, a body fluid of a mammal, such as a human or a horse, which contains a factor that interferes with a specific reaction or causes a non-specific reaction, and is a normal clinical sample such as blood in a serum or plasma form. , Joint fluid, lymph,
It may be any body fluid such as thymus fluid, ascites fluid, amniotic fluid, cell tissue fluid, bone marrow fluid, and urine.

Further, in the present invention, the amount of chondrocalcin is measured by combining the anti-chondrocalcin antibody immobilized on an insoluble carrier and the labeled anti-chondrocalcin antibody with other substances necessary for immunological measurement. A kit for can be configured. As such substances, there are known ones such as a lysing agent, a detergent, a substrate for measuring an enzyme activity and a reaction terminator thereof.

Thus, according to the immunoassay method or the like of the present invention, a sample containing a minute amount of mammalian chondrocalcin such as a clinical sample is used as a specimen, and chondrocalcin in the specimen is highly sensitive, highly accurate, and simple. It can be quantified by operation.

INDUSTRIAL APPLICABILITY The measurement value of chondrocalcin in body fluids by the immunological measurement method and reagent of the present invention can be used to measure cartilage diseases in mammals such as horse joint disease and human chondrocyte-related diseases. It is useful for diagnosis and treatment monitoring. Particularly, it is useful for diagnosis and treatment monitoring of human osteoarthritis, and for differential diagnosis of osteoarthritis and rheumatoid arthritis.

 The present invention will be described in detail below with reference to examples and reference examples.

 In the examples, "%" indicates "% by weight".

Example 1 (1) Preparation of bovine chondrocalcin According to the method of Etch You Choi et al., Bovine chondrocalcin was extracted from cartilage of 20 long bones of fetal limbs of fetal bovines.
I got 10.0 mg. As a result of examination by SDS-PAGE electrophoresis, a single band was shown at a position of a molecular weight of 35,000.

(2) Preparation of antigen-stimulated lymphocytes Male Balb / c mice were intraperitoneally injected with bovine chondrocalcin.
An emulsion of 90 μg and Freund's complete adjuvant was administered. Thereafter, an emulsion of 40 to 50 μg of bovine chondrocalcin and incomplete adjuvant was intraperitoneally administered 5 times at 3 to 4 week intervals. Ten days after the fifth administration, 60 μg of chondrocalcin was dissolved in 1.0 ml of physiological saline and administered intravenously. Four days later, the spleen was aseptically removed from the mouse and passed through a stainless mesh to obtain L.
-Glutamine 0.39 g / l, kanamycin sulfate 0.2 g / l and NaHC
A splenocyte suspension suspended in RPMI-1640 medium (GIBCO) supplemented with O ₃ 2.0 g / l was prepared. The suspension cells were washed three times with the medium described above, and then a splenocyte suspension was prepared.

(3) Cell fusion Mouse myeloma cells P3U1 were cultured in a GIT synthetic medium (manufactured by Daigo Nutrition Chemical Co., Ltd.) supplemented with kanamycin sulfate 0.2 g / l. Myeloma cells were in the exponential phase of cell division at the time of cell fusion. The spleen cells and the myeloma cells have a cell number of 3: 1
Suspended in serum-free RPMI-1640 medium for 5 minutes at 1300
Centrifuge at rpm. After removing the medium, 1 ml of a 50% polyethylene glycol solution (pH 7.2) having an average molecular weight of 1,500 was gently added to the precipitated cells to form a suspension. Then 9 ml of serum free RPMI-1640 medium was added and the cells were shaken carefully. After that, the mixture was centrifuged at 1,000 rpm for 5 minutes to remove the supernatant medium, and the cells were collected as a precipitate and suspended in 40 ml of HAT medium.

(4) Acquisition of cloned hybridoma The fused cell solution was distributed on a 96-microplate (200 μl per well). The plate was incubated at 37 ° C. in a 5% CO ₂ atmosphere. After 1 day and 2 days, half of the medium was replaced with a new HAT medium and cultured at intervals of 2 days thereafter. After 11 days, screening was performed for the antibody against bovine chondrocalcin in the culture supernatant of the hybridoma by the enzyme immunoassay. The antigen used for the screening was bovine chondrocalcin, and the second antibody was goat anti-mouse IgG antibody labeled with alkaline phosphatase.

In 102 wells out of 192 wells into which the hybridomas were distributed, colonies of fused cells were observed, and 7 wells thereof were antibody-positive.

The hybridoma thus obtained in each antibody-positive well was diluted to 0.9 cells per well of 96 microplate, and thymocytes of Balb / c mice were added as feeder cells and distributed to the plate and kanamycin sulfate 0.2 g / l
The cells were cultured in GIT medium supplemented with. Observation under a microscope confirmed that it was definitely a single colony. Screening was carried out by an enzyme immunoassay for antibodies to bovine chondrocalcin in the culture supernatant of hybridomas. Each well was antibody-positive and produced anti-bovine chondrocalcin monoclonal antibody.

(5) these cloned hybridoma culture of hybridomas, two weeks ago pristane (Wako Pure Chemical) 0.1 ml in Balb / c mice were intraperitoneally administered was administered cell count 10 ⁶ to 10 ⁷ cells / mouse intraperitoneally . Thereafter, on the 7th to 10th day, 2-3 ml / animal ascites fluid was collected.

(6) Purification of monoclonal antibody The collected ascites fluid was collected by the method of Ey et al. (PLEy et al., Immuno
chemistry, 15 , 429-436 (1978)). That is, Protein A-Sepharose column (gel volume 5 ml) equilibrated with 0.1 M phosphate buffer (pH 8.0)
2 to 3 ml of ascites fluid, and then a buffer solution of 0.1 M sodium citrate having a pH of 6.0, 5.0, 4.0 and 3.0 was sequentially passed to elute the moroclonal antibody from the column, and purified monoclonal antibody, namely Chon-3F , Chon-5G, Chon-8G and Chon-4H were obtained.

(7) Class of Purified Monoclonal Antibody A specific class of purified monoclonal antibody was determined in an Octa Loney gel diffusion test using a class-specific anti-mouse antiserum. The results are shown in Table 1 below.

(8) Search for difference in recognition site of monoclonal antibody 2 μg / ml bovine chondrocalcin PBS solution was distributed to 96 microplate at 150 μl per well, left overnight at 4 ° C., washed with PBS, and added with 0.5% BSA-PBS. Further, the plate was left overnight to prepare an antigen-immobilized plate. 5μ on this plate
150 μl of g / ml monoclonal antibody PBS solution (first antibody) was added to each well, and the mixture was allowed to stand at 37 ° C. for 75 minutes. After washing the wells, 0.5% BSA-PB of horseradish peroxidase (HRP) -labeled monoclonal antibody (second antibody)
150 μl of S solution was added to each well and reacted at 37 ° C. for 60 minutes. After washing with saline, HRP substrate solution (0.1M phosphoric acid /
2,2'-azinodi [3-] in citrate buffer (pH 4.5)
Ethylbenzthiazoline sulfonate (6)] diammonium salt (50 mg / dl and 2 MH ₂ O ₂ 50 μl / dl) (150 μl) was added and color was developed at 25 ° C. for 6 minutes. 50μ of 0.1M oxalic acid solution
Then, the reaction was stopped by adding 1 of each of them, and measured at 415 nm with a plate reader.

From the results shown in Table 2, the obtained four monoclonal antibodies are three types of antibodies having different epitopes, and Chon-5G and Chon-4H have the same epitope or close epitopes at the same time. It is clearly an antibody that cannot bind.

Example 2 An emulsion of 1 mg of bovine chondrocalcin purified in Example 1 and Freund's complete adjuvant was subcutaneously administered to a rabbit. 19 days later bovine chondrocalcin 450
μg was subcutaneously administered in the same manner. 16 days later, an emulsion of 450 μg of bovine chondrocalcin and Freund's complete adjuvant was subcutaneously administered, and blood was collected on the 10th day to make serum. Then, an enzyme immunoassay method was conducted, that is, bovine chondrocalcin was used as an antigen. 2 antibody with goat anti-rabbit IgG antibody labeled with horseradish peroxidase.
The antibody titer against chondrocalcin was measured and confirmed to be 400,000 times. After that, whole blood was collected and used as serum, which was precipitated with 40% saturated ammonium sulfate, protein A-Sepharose-4B.
Purification by column was performed to obtain a polyclonal antibody against bovine chondrocalcin.

Example 3 The monoclonal antibody "Chon-5G" described in Example 1 was
0.1M phosphate / citrate buffer (pH 4.0) with a concentration of 20 μg / ml
As a solution of 100 μl, 100 μl was distributed per well in a microplate, and left at 4 ° C. for one day to be immobilized. PB this
After washing with S, add 100 μl of 0.5% BSA-PBS to each well and leave it at 4 ° C overnight for after-coating.
The plate was washed with PBS to prepare a "Chon-5G" fixed plate.

Next, 15,10,5,1.5, On of purified bovine chondrocalcin
A g / ml dilution series was prepared in PBS, 100 μl per well was added to the “Chon-5G” fixed plate, and the mixture was reacted at 37 ° C. for 1 hour. After washing with PBS, 0.5% BSA-P of horseradish peroxidase (HRP) -labeled monoclonal antibody "Chon-8G" (described in Example 1)
100 μl of the BS solution was distributed per well and reacted at 37 ° C. for 1 hour. After washing with PBS, HRP substrate solution (TMB) 100μ
Add 1 liter and develop color at 37 ° C for 30 minutes, 100 μl of 1N sulfuric acid solution
Was added to stop the reaction, and the absorbance was measured with a plate reader at 450 nm.

 The calibration curve obtained as a result is shown in FIG.

Example 4 The monoclonal antibody "Chon-3F" described in Example 1 was prepared with 0.1 M phosphoric acid / citrate buffer so that the protein concentration was 20 μg / ml, and polystyrene balls were added thereto and immersed for 3 days and nights. Fixed. After washing with PBS, it was immersed in 0.5% PSA-PBS overnight for after-coating, and further washed with PBS to obtain "Chon-3F" fixed balls.

Next, purified bovine chondrocalcin 15,10,5,1.67, O
A ng / ml dilution series was prepared in PBS and reacted with "Chon-3F" fixed balls at 37 ° C for 1 hour. After washing with PBS, 400 μl of 0.5% BSA-PBS solution of horseradish peroxidase (HRP) -labeled polyclonal antibody (described in Example 2) was added and reacted at 37 ° C. for 1 hour. After washing with PBS, substrate solution for HRP (0.1M phosphate / citrate buffer (pH 4.
(5) containing 0.125% 3,3 ', 5,5'-tetramethylbenzidine and 0.03% hydrogen peroxide) (400 μl) was added and color was developed at 37 ° C for 30 minutes. The termination reaction was carried out with 1.0 ml of 1N-sulfuric acid, and the absorbance of the color-developing solution at 450 nm was measured with a spectrophotometer.

 The calibration curve obtained as a result is shown in FIG.

Example 5 A ball to which a polyclonal antibody against bovine chondrocalcin (described in Example 2) was immobilized was prepared by the method described in Example 4.

Next, purified bovine chondrocalcin 15,10,5,1.67, O
A dilution series of ng / ml was prepared in PBS, added to each 400 μl glass tube, and reacted with the fixed balls at 37 ° C. for 1 hour. PB
After washing with S, horseradish peroxidase (HR
P) labeled monoclonal antibody "Chon-4H" (Example 1
Described in 1.), 400 μl of 0.5% BSA-PBS solution was added, and the mixture was reacted at 37 ° C. for 1 hour. Hereinafter, the calibration curve of FIG. 3 was obtained in the same manner as in Example 4.

Example 6 Purified bovine chondrocalcin 10.0,5.0,1.0,0.6,0.
Sepharose-4B in which a dilution series of 4,0.2,0.1,0.0 ng / ml was prepared in PBS and a polyclonal antibody against bovine chondrocalcin (described in Example 2) was immobilized on each of them.
5.0, 2.5, 0.5, 0.3, 0.2, 0.1, 0.05, 0.0ng / m in human plasma diluted twice by adding an equal volume of normal human plasma that has passed through the column
A dilution series containing l of bovine chondrocalcin was prepared.

This diluted series of bovine chondrocalcin solution was measured using "Chon-4H" (described in Example 1) fixed plate prepared in the same manner as in Example 3 and HRP-labeled polyclonal antibody (described in Example 2). Then, the calibration curve of FIG. 4 was created.

Next, an equal volume of PBS was added to the sample of serum or synovial fluid, and the absorbance was measured in the same manner as in Example 3 as a sample solution of 2-fold dilution. From the calibration curve of FIG. 4, bovine chondrocalcin-converted human Chondrocalcin concentration was measured. The results are summarized in Table 3.

Example 7 The anti-bovine chondrocalcin polyclonal antibody described in Example 2 was dispensed as a solution of 0.1 M phosphate / citrate buffer (pH 4.0) at a concentration of 20 μg / ml into a microplate at 100 μl per well. It was left to stand overnight at 4 ° C. for immobilization. After washing this with PBS, add 0.5% BSA-PBS to each well for 10
After adding 0 μl of each, the mixture was left to stand at 4 ° C. overnight for after-coating, and then washed with PBS to prepare an anti-bovine chondrocalcin polyclonal antibody-immobilized plate.

Next, 15,10,5,1.5, On of purified bovine chondrocalcin
A dilution series of g / ml was prepared with 1% BSA-final concentration 0.065% skim milk-PBS, and 100 per well was added to the antibody-immobilized plate.
Each μl was added and reacted at 37 ° C. for 1 hour. After washing with PBS, a 0.5% BSA-0.1% skimmed milk-PBS solution of HRP-labeled anti-bovine chondrocalcin polyclonal antibody was distributed in 100 µl per well and reacted at 37 ° C for 1 hour. After washing with PBS, HRP substrate solution (2.5 mM H ₂ O ₂ , 0.0225
% 3,3'.5,5'-Tetrabenzidine) 100 μl was added at 37 ° C.
Color was developed for 30 minutes, 100 μl of 1N-sulfuric acid solution was added to stop the reaction, and the absorbance was measured with a plate reader at 450 nm.

 The calibration curve obtained as a result is shown in FIG.

The lower limit of measurement is 1 ng / ml, and the lower limit of measurement by the radioimmunoassay of A. Heinek et al.
The sensitivity was remarkably higher than that of al, J. Cell Biol., 104 , 1435-1441 (1987).

Example 8 Buffer for immunoassay (0.01M phosphoric acid containing 1% BSA, 0.85%
Nacl buffer solution pH = 7.2 and later abbreviated as 1% BSA-PBS),
Bovine chondrocalcin was diluted to make a 0,10 ng / ml solution, and 50 μl of it was added to the same buffer solution to give a final concentration of 0,0.00625,0.01.
50 μl of skim milk-containing solution adjusted to 25, 0.025, 0.05, 0.1, 0.2, 0.4 and 0.8% was added and mixed well, and the mixture was added to the anti-bovine chondrocalcin polyclonal antibody-immobilized microplate described in Example 2. 100 μl of the same solution was added and reacted at 37 ° C. for 2 hours (primary reaction). Then
After washing with PBS-0.05% Tween-20, HRP-labeled anti-bovine chondrocalcin antibody obtained by adjusting in the same manner as in Example 3 was immunoassayed with skim milk to give the same final concentration as the primary reaction. Dilute with buffer solution for 100 μl to each well
The mixture was allowed to react at 37 ° C. for 1 hour (secondary reaction). PBS-0.05
After washing with 10% Tween-20, 10% of the HRP substrate solution described in Example 3 was used.
Add 0 μl, develop color at 37 ℃ for 0.5 hours, and add 1N-sulfuric acid solution 25μ
The reaction was terminated by adding 1 and the absorbance was measured with a plate reader at 450 nm. The results are shown in FIG. As can be seen from this figure, skim milk concentration 0.0062
Non-specific adsorption can be prevented with a skim milk concentration of 5% or more.

Example 9 The Fab ′ fragment of the anti-bovine chondrocalcin polyclonal antibody (anti-chondrocalcin PCA) described in Example 2 was bound to peroxidase by binding to peroxidase by a conventional method according to the method of Nisonov. Obtained Fab '(edited by The Biochemical Society of Japan, sequel to biochemistry experimental course, Volume 5, 109-11)
2 pages, Tokyo Kagaku Dojin, 1986). That is, 1250 μg of anti-chondrocalcin PCA was added to 0.2 M sodium acetate buffer (pH
4.5) Dissolved in 1.25 ml and dialyzed against the same buffer. twenty five
μg of pepsin was added and digested and decomposed at 37 ° C. for 24 hours. 1N-NaOH was added dropwise to the reaction solution to adjust the pH to 8 to stop the reaction.
TSKgel3000SW column-HPLC eluate 5 mM EDTA-0.1M
A sodium phosphate buffer (pH 6.0) was used to separate an F (ab ') ₂ fragment having a molecular weight of 92,000 from this reaction solution. This was concentrated by an ultrafilter to obtain 2.2 ml of a liquid having a F (ab ') ₂ concentration of 122.3 µg / ml. 0.1M2 to this
-245 μl of mercaptoethylamine was added and reduction treatment was carried out at 37 ° C. for an hour. The reaction solution was concentrated by an ultrafilter and then subjected to HPLC in the same manner as F (ab ') ₂ to give a molecular weight of 46,00.
The Fab 'fragment of 0 was isolated. 83.7 μg / ml Fab ′
2.0 ml of the liquid of the fragment was obtained.

On the other hand, 4.0 mg of horseradish peroxidase (manufactured by Toyobo Co., Ltd. (hereinafter, HRP)) was dissolved in 0.6 ml of 0.1 M sodium phosphate buffer (pH 7.0), and N-succinimidyl-3-
40 parts of maleimidobenzoate in dimethylformamide
μl (31.4 mg / ml concentration) was added dropwise, and the mixture was reacted at 30 ° C. for 1 hour. Add this reaction solution to a Sephacryl S-200 column,
Maleimide-HRP was separated by flushing with 0.1 M sodium phosphate buffer (pH 6.0).

3.10 mg / ml maleimide-HRP 470μ thus obtained
1 was added dropwise to the above Fab ′ fragment solution and reacted at 4 ° C. for 20 hours. After concentrating the reaction solution with an ultrafilter, TSK gel
PBS (pH 7.2) was separated as an eluent by 3000 SW column-HPLC to obtain HRP-labeled Fab 'having a molecular weight of 86,000. This labeled Fab 'was used for preparing the following calibration curve.

The anti-chondrocalcin PCA described in Example 2 was added at a concentration of 20 μg.
Dispense 100 μl per well into a microplate as a solution of 0.1 M phosphate / citrate buffer (pH 3.5) / ml,
It was left to stand overnight at 4 ° C. for immobilization. After washing this with PBS
After adding 100 μl of 1.0% BSA-PBS per well and leaving it at 4 ° C. for one day to perform after-coating, the plate was washed with PBS to prepare an anti-chondrocalcin PCA-fixed plate.

Next, 0,10,50,100,50 of purified bovine chondrocalcin
Dilute 0,1000,5000,10000 pg / ml with 1% BSA-final concentration
Prepare with 0.1% skimmed milk-PBS (pH7.2), add 100 μl per well to the antibody-immobilized plate, and add 5 μl at 37 ° C.
The reaction was allowed to proceed for 4 hours and then overnight at 4 ° C. PBS-0.05% Tween
After washing with −20, Fa of HRP-labeled anti-chondrocalcin PCA
Adjust the concentration of Fab ′ component of b ′ to 60ng / ml with 1.0% BSA.
-Final concentration of 0.1% skim milk-diluted with PBS (pH7.2), the prepared Fab 'solution was distributed in 100 μl per well
The reaction was carried out at 20 ° C for 4 hours. After washing with PBS-0.05Tween-20, HRP substrate for liquid _{(2.5mM H 2 O 2, 0.0225} % 3,3 ', 5,5'-
Tetramethylbenzidine) (100 μl) was added and color was developed for 2 hours at 20 ° C, and 25 μl of 1N sulfuric acid solution was added to stop the reaction.
Absorbance was measured with a plate reader at 0 nm.

 The calibration curve obtained as a result is shown in FIG.

As it is clear from FIG. 7, the measurement lower limit is a 10 pg / ml, measured by er-Haineku et radio Iminoassei lower 100ng / ml (A.Hinek et al. , J.Cell.Biol. 104, 1
430-1441 (1987)) and was significantly more sensitive.

Example 10 The anti-bovine chondrocalcin MCA "Cho described in Example 1
The Fab 'fragment of n-4H "was prepared according to the method of Parham (edited by the Biochemical Society of Japan, ed.
Page, Tokyo Kagaku Dojin, 1986), and HRP in the same manner as in Example 8.
Labeled Fab 'was obtained. That is, 480 μg of "Chon-4H"
2.0 ml dissolved in 0.1M sodium citrate buffer (pH 4.1)
As a result, it was dialyzed against the same buffer solution. 56.25 μg of pepsin was added and digested and decomposed at 37 ° C. for 90 minutes. 1N-Na in the reaction solution
OH was added dropwise to adjust the pH to 8 to stop the reaction. TSK gel 3000
Using SW column-HPLC, 5mM EDTA-0.1M sodium phosphate buffer (pH 6.0) was used as the eluent, and the molecular weight was 9
2,000 F (ab ') ₂ fragments were isolated. This was concentrated to obtain 7.5 ml of a liquid having an F (ab ') ₂ concentration of 19.6 μg / ml. To this, 0.1M 2-mercaptoethanolamine 835μ
1 was added and reduction treatment was carried out at 37 ° C. for 2 hours. From this reaction solution, 5.5 ml of a 9.5 μg / ml Fab 'fragment solution was obtained in the same manner as in Example 9.

To the Fab ′ fragment solution, 146 μl of 3.10 mg / ml maleimide-HRP obtained in Example 9 was added dropwise and reacted at 4 ° C. for 20 hours. From this reaction solution, HRP-labeled Fab 'was obtained in the same manner as in Example 8. This labeled Fab 'was used for preparing the following calibration curve.

 The fixing plate used in Example 9 was used.

Next, 0,10,50,100,50 of purified bovine chondrocalcin
Make a dilution series of 0,1000,5000,10000 pg / ml with 1% BSA 0.1% skim milk-PBS (pH7.2), add 100 µl per well to the antibody-immobilized plate, and allow to react at 37 ° C for 2 hours. It was After washing with PBS-0.05% Tween20, "Chon-"
The concentration of Fab 'component of HRP-labeled Fab' obtained from 4H "is 300ng / ml
1% BSA-final concentration 0.1% skim milk-PBS as
The labeled Fab 'solution diluted with (pH7.2) was prepared per well
The solution was distributed in 100 μl portions and reacted at 37 ° C. for 1 hour. PBS-0.
After washing with 05% Tween 20, substrate solution for HRP 2.5 mM H ₂ O ₂ , 0.022
5%) 3,3 ', 5,5'-Tetramethylbenzidine) 100 μl was added and color was developed for 30 minutes at 20 ° C, 20 μl of 1N-sulfuric acid solution was added to stop reaction, and the absorbance was measured with a plate reader at 450 nm did.

The calibration curve obtained as a result is shown in FIG. Lower limit of measurement is 50
pg / ml, 100 ng / m by the method of A Heinek et al.
The sensitivity was significantly higher than the lower limit of measurement of l.

Example 11 Immunoassay buffer (0.01% phosphoric acid containing 1% BSA, 0.85%)
NaCl buffer pH = 7.2 and thereafter abbreviated as 1% BSA-PBS),
Bovine chondrocalcin was diluted to make a 0.2 ng / ml solution, and 50 ml of it was added to the same buffer solution to give a final concentration of 0,0.00625,0.0125
50 μg of skim milk-containing solution prepared to be 0.025, 0.05, 0.1, 0.2, 0.4 and 0.8% was added and mixed well, and 100 ml of the same solution was placed in the anti-chondrocalcin PCA fixed plate prepared in the same manner as in Example 8, The reaction was carried out at 37 ° C for 2 hours (primary reaction). Then, after washing with PBS-0.05% Tween-20, HRP-labeled Fab 'prepared in the same manner as in Example 8 was diluted with an immunoassay buffer containing skim milk to give a final concentration of the same concentration as in the primary reaction, and each well was diluted. Then, 100 ml of the solution was added and reacted at 37 ° C. for 1 hour (secondary reaction). After washing with PBS-0.05% Tween-20, 100 µl of the substrate solution for HRP described in Example 8 was added, and the mixture was incubated at 37 ° C.
Color was developed for 0.5 hour, 25 μl of 1N-sulfuric acid solution was added to stop the reaction, and the absorbance was measured with a plate reader at 450 nm. The results are shown in FIG. As is clear from this figure, non-specific adsorption can be prevented at a skim milk concentration of 0.00625% or more.

Example 12 Purified bovine chondrocalcin 0,7.5,37.5,75,375,75
0,3750,7500 pg / ml dilution series with 1% BSA-final concentration 0.00625
Created with skim milk-PBS (pH7.2),
A dilution series of bovine chondrocalcin containing human serum diluted 5-fold by adding 1/4 volume of normal human plasma passed through a Sepharose-4B column immobilized with anti-chondrocalcin PCA described in Example 2 was used. Prepared. This dilution series contained 0,30,150,300,150 bovine chondrocalcin per ml in human serum.
It will contain 0,3000,15000,30000 pg / ml.

This diluted series of bovine chondrocalcin solution was used as a polyclonal antibody-immobilized plate prepared in the same manner as in Example 9.
The calibration curve shown in FIG. 10 was prepared by measurement using HRP-labeled Fab ′.

Next, in serum or joint fluid samples, 1% BSA-final concentration 0.00625
% Skimmed milk-PBS (pH 7.2) was added in a 4-fold volume, and the absorbance was measured as a 5-fold diluted sample solution in the same manner as in Example 9. From the calibration curve in FIG. 10, bovine chondrocalcin-converted human Chondrocalcin concentration was measured. Table 4 summarizes the results.

As is clear from the calibration curve in FIG. 10, the lower limit of measurement according to this example was 30 pg / ml, which was remarkably higher than the lower limit of measurement of 100 ng / ml of the radioimino assay of A. Heinek et al.

Example 13 Peroxidase-labeled anti-chondrocalcin F
Preparation of (ab ′) ₂ labeled antibody 1. Anti-chondrocalcin rabbit polyclonal antibody F
Preparation of (ab ′) ₂ fragment 0.1 mg of pepsin was added to 2.5 ml of an acetate buffer (pH 4.5) of the anti-chondrocalcin rabbit polyclonal antibody (6.0 mg) described in Example 2 and the mixture was incubated at 37 ° C. overnight, followed by 1N NaOH. PH to 6.0 with HPLC (column: TSK Gel G-
With 3000 SW, eluent PBS pH 7.2), 3.6 mg of F (ab ') ₂ fragment was obtained.

2.F (ab ') ₂ fragments of maleimidated F (ab') succinimidyl in PBS for ₂ fragment (3.6 mg) - meta - maleimide benzoate (MBS)
0.09 ml of DMF solution (10 mg / ml) was added dropwise at 25 ° C. After stirring for 30 minutes, the column of Sephadex G-25 (1 cm x 45 c
Using 0.1 M phosphate buffer as the eluent in m), 4.0 mg of gel-passed maleimidated F (ab ') ₂ fragment was obtained.

3. Introduction of thiol group of peroxidase To 5.4 ml of 0.1M phosphate buffer (pH6.5) solution of peroxidase (Toyobo: 54 mg), 0.2 ml of DMF solution of S-acetylmercaptosuccinic anhydride (6.0 mg / ml) (25 mg). With stirring,
The mixture was gradually added dropwise and the reaction was continued for another 1.5 hours. In this solution
2.1 ml of 0.1 M Tris-HCl buffer (pH 7.0) and 0.1 M EDTA solution
Add 0.4 ml and 4.3 ml of 1M hydroxylamine solution and add 5 at 30 ℃.
Stir for minutes. The solution was placed in a dialysis tube and dialyzed at 0.1C phosphate buffer pH 6.0-5 mM EDTA as an external solution at 4 ° C for two nights to obtain a SH group-introduced peroxidase solution (2.54 mg / ml).

4. maleimidated F (ab ') coupling the maleimide of introduced peroxidase ₂ fragments and thiol groups were F (ab') ₂ introduced peroxidase solution fragments 4.0mg and thiol group (2.54mg / ml) 2.8ml and Were mixed and left overnight at 25 ° C. The reaction mixture was analyzed by HPLC (column TSK Gel G-3000SW, eluent PB
Separation with S pH 7.2) and peroxidase-bound F (ab ')
Fractions of ₂ fragments were collected to give 3.1 mg as F (ab ') ₂ fragment.

The molar ratio of F (ab ') ₂ fragment to peroxidase in the obtained fraction was 1: 2.7.

Example 14 The anti-chondrocalcin polyclonal antibody described in Example 2 was added to 0.01 M phosphate buffered saline (hereinafter abbreviated as PBS).
Adjusted to a concentration of 20 μg / ml and mirror-polished polystyrene beads (Immunochemical: Immunobeads, Surfcom 570A
Centerline average roughness of surface (R manufactured by Tokyo Seimitsu Co., Ltd.)
a): 1.3 μm) was soaked and left at 4 ° C. overnight. Bees in PBS
After washing 3 times with, the mixture was allowed to stand at room temperature for 2 hours with 1% BSA-PBS (pH 7.2). It was washed 3 times with PBS again and stored in PBS until use at 4 ° C.

A 0.75% BSA-0.01% skim milk-PBS (pH7.2) solution of chondrocalcin 0, 0.025, 0.3125, 0.625, 1.25, 5 ng was prepared, and 0.2 ml of this solution was placed in a polypropylene plastic test tube. Next, the peroxidase-labeled anti-chondrocalcin F (ab ') ₂ fragment prepared in Example 13 was added to 0.5% BSA-0.2% skim milk-PBS (pH 7.2) to give an F (ab') ₂ fragment concentration of 3 µg / ml. ,
After adding 0.2 ml each to each tube, immediately add one anti-chondrocalcin-immobilized bead to each tube at 37 ℃.
And incubated for 90 minutes.

After washing 3 times with a solution containing 0.05% Tween 20 in PBS (abbreviated as PBS-T), 2.5 mM H ₂ O ₂ -0.025% was used as a color former.
Add 0.4 ml of 3,3 ', 5,5' tetramethylbenzidine solution and incubate at 37 ℃ for 30 minutes.
The color reaction was stopped by adding 1 ml, and the absorbance at 450 nm was measured.

The calibration curve obtained as a result is shown in FIG. The lower limit of measurement is 25
pg / ml, lower limit of 100 ng / ml (A.Hinek et al., J. Cel.
l.Biol. 104 , 1435-1441 (1987)).

Next, 0.75% BSA-0.01% skimmed milk-10 mM PBS (pH7.2) was added to the serum or joint fluid sample in a 3-fold amount to prepare a 4-fold diluted sample solution, and the absorbance was measured in the same manner as in the preparation of the calibration curve. The concentration of bovine chondrocalcin-converted human chondrocalcin was measured from the calibration curve in FIG.

 The results are shown in Table 5.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Kenji Hosoda, 3-3-12 Asahi-cho, Kawagoe-shi, Saitama Prefecture (72) Masayoshi Shinmei 4--4-4, Nakaarai, Tokorozawa-shi, Saitama Prefecture (72) Inventor Shigeo Matsuyama 1-17502 Hirosawa, Wako-shi, Saitama Prefecture (56) Reference JP 62-116262 (JP, A) JP 62-24149 (JP, A) JP 60-130825 (JP) , A) The Journal of Cell Biology, 98 (1), P. 54-65, (1984) The Journal of Cell Biology, 104 (5), P. 1435-1441, (1987).

Claims (16)

(57) [Claims] 1. An immunological assay method using an enzyme-labeled mammalian chondrocalcin or anti-chondrocalcin antibody, which uses a mammalian body fluid as a sample and utilizes an immune reaction in the solution. In the method for measuring chondrocalcin, which comprises measuring the amount of chondrocalcin in the mammal, the immune reaction solution has a molecular weight of 16,000 to
A chondrolide characterized by allowing a protein having a 50,000 and an isoelectric point of 1.0 to 5.0 or a mixture containing the protein to be present and adjusting the final concentration of the protein or the mixture in the immune reaction solution to be 0.02 to 0.9% by weight. How to measure calcin. 2. A sandwich method using an anti-chondrocalcin antibody immobilized on an insoluble carrier and a labeled anti-chondrocalcin antibody, wherein a body fluid of a mammal is used as a sample. The method for measuring chondrocalcin as described. 3. The immune reaction is carried out by allowing an anti-chondrocalcin antibody immobilized on an insoluble carrier, a labeled anti-chondrocalcin antibody and a body fluid of a mammal as a specimen to exist in the same reaction system and reacting. The method for measuring chondrocalcin according to claim 2. 4. At least one of the anti-chondrocalcin antibody immobilized on an insoluble carrier and the labeled anti-chondrocalcin antibody recognizes human chondrocalcin obtained by using mammalian chondrocalcin as an immunogen. The method for measuring chondrocalcin according to claim 2, which is a monoclonal antibody. 5. At least one of the anti-chondrocalcin antibody immobilized on an insoluble carrier and the labeled anti-chondrocalcin antibody is obtained by immunizing a mammal different from the mammal with chondrocalcin. The method for measuring chondrocalcin according to claim 2, which is a polyclonal antibody that recognizes human chondrocalcin. 6. The method for measuring chondrocalcin according to claim 2, wherein the insoluble carrier is a mirror-finished bead. 7. The labeled anti-chondrocalcin antibody is a Fab ′ fragment or F (ab ′) _{2 of the} anti-chondrocalcin antibody.
The method for measuring chondrocalcin according to claim 1, which is a fragment. 8. The method for measuring chondrocalcin according to claim 1, characterized in that the labeled anti-chondrocalcin antibody is labeled with an average of 1.5 molecules or more of enzyme per molecule of antibody. . 9. The method for measuring chondrocalcin according to any one of claims 1 to 8, wherein the mixture containing the protein is skim milk. 10. An anti-chondrocalcin antibody reagent immobilized on an insoluble carrier, an anti-chondrocalcin antibody reagent labeled with an enzyme, and its final concentration in an immunoreaction solution of 0.02 to 0.
Molecular weight of 16,000 to 50,000 and isoelectric point of 9% by weight
A reagent for measuring chondrocalcin for immunologically measuring the amount of chondrocalcin present in a body fluid of a mammal as a sample, which comprises a protein of 1.0 to 5.0 or a mixture containing the protein. 11. At least one of the anti-chondrocalcin antibody immobilized on an insoluble carrier and the labeled anti-chondrocalcin antibody recognizes human chondrocalcin obtained by using mammalian chondrocalcin as an immunogen. 11. The chondrocalcin assay reagent according to claim 10, which is a monoclonal antibody. 12. At least one of an anti-chondrocalcin antibody immobilized on an insoluble carrier and an anti-chondrocalcin antibody labeled with the antibody is obtained by immunizing a mammal different from the mammal with chondrocalcin of a mammal. 11. The assay reagent for chondrocalcin according to claim 10, which is a polyclonal antibody that recognizes the obtained human chondrocalcin. 13. The chondrocalcin assay reagent according to claim 10, wherein the insoluble carrier is a bead having a mirror surface. 14. The labeled anti-chondrocalcin antibody is a Fab ′ fragment or F (a of the anti-chondrocalcin antibody.
11. The chondrocalcin assay reagent according to claim 10, which is b ′) ₂ fragment. 15. The measuring reagent according to claim 10, wherein the labeled anti-chondrocalcin antibody is labeled with an average of 1.5 molecules or more of enzyme per molecule of antibody. 16. The chondrocalcin assay reagent according to claim 10, wherein the mixture containing the protein is skim milk.