Detection method for collagen

Abstract

To provide a new detection method for collagen contained in a composition by an immunoassay using a collagen antibody.SOLUTION: A detection method for collagen contained in a composition by an immunoassay using a collagen antibody, comprises a stepwise solidification process using a basic solution. The stepwise solidification process includes a step of solidifying collagen contained in collagen solution as an antigen on a plate a plurality of times and diluting the collagen solution using the basic solution. The stepwise solidification process includes a first-stage solidification step, and second and subsequent-stage solidification steps. The first-stage solidification step is performed using a collagen solution with a concentration higher than that of a collagen solution used in the second and subsequent-stage solidification steps, and the second and subsequent-stage solidification steps are performed using the collagen solution with the concentration lower than those of the first-stage solidification steps.SELECTED DRAWING: Figure 3

Description

[Technical field]

[0001] The present invention relates to, for example, a method for detecting collagen contained in a composition by an immunological assay using an antibody against collagen. [Background technology]

[0002] Collagen is known to exist everywhere in animal tissues, such as skin, blood vessels, internal organs, and bones. For example, 70% of the protein in the dermis and 90% of the protein in bones are collagen, and the fascia that encases each muscle is also made of collagen, which is said to account for one-third of the protein that makes up humans. Collagen in the body is distributed in large amounts in connective tissues, cartilage tissues, and skin tissues, combining with acidic mucopolysaccharides such as hyaluronic acid and chondroitin sulfate, and is useful for maintaining the function and morphology of cells. It is one of the important and basic components that surrounds the cell body and serves as a scaffolding to support the cells. It is said that collagen takes longer to turn over than normal proteins, and that the cycle slows down with age (aging). When the cycle slows down, the degeneration (aging) of collagen itself also progresses, leading to a decrease in the flexibility and elasticity of the skin. It is thought that when collagen ages, its function as a structural support is reduced, which inhibits the proliferation, differentiation, and migration of fibroblasts present in the skin base, further slowing down the skin turnover cycle, resulting in a vicious cycle. (Patent Document 1).

[0003] [Prior art documents] [Patent documents]

[0004] [Patent Document 1] Patent Publication No. 2012-153614 Summary of the Invention [Problem to be solved by the invention]

[0005] The problem to be solved by the present invention relates to providing a new method for detecting collagen contained in a composition by an immunological assay using an antibody against collagen. [Means for solving the problem]

[0006] Therefore, the inventors of the present invention have conducted extensive research and have completed the present invention by gradually changing the concentration of the basic solution used. Specifically, the present invention includes the following items. [Item 1] A method for detecting collagen contained in a composition by an immunoassay method using an antibody against collagen, comprising: The method includes a stepwise solidification step using a basic solution, The stepwise solidification step includes a step of solidifying collagen contained in a collagen solution as an antigen on a plate multiple times, diluting the collagen solution with the basic solution; The stepwise solidification process includes a first solidification process and a second or subsequent solidification process, The first solidification step is carried out using a collagen solution having a higher concentration than the collagen solution used in the second or subsequent solidification steps, The method, wherein the second and subsequent solidification steps are carried out using a collagen solution having a lower concentration than that in the first solidification step. [Item 2] The method described in [Item 1], wherein the basic solution has a pH of greater than 8. Effect of the Invention

[0007] The present invention can provide a new method for detecting collagen contained in a composition by an immunological assay using an antibody against collagen. [Brief description of the drawings]

[0008] [Figure 1] FIG. 1 is a diagram showing the steps for producing the composition of the embodiment. [Diagram 2]FIG. 2 shows the method of immobilizing antigens shown in Test 1 and Test 2. [Diagram 3] FIG. 3 is a graph showing the values ​​shown in Table 2, with the horizontal axis representing the sample concentration (ng / mL) and the vertical axis representing absorbance. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0009] Hereinafter, an embodiment of the present invention will be described.

[0010] (collagen) Examples of collagen include fibrous collagen and non-fibrous collagen. Fibrous collagen refers to collagen that is the main component of collagen fibers, and specifically includes type I collagen, type II collagen, type III collagen, etc. Examples of non-fibrous collagen include type IV collagen.

[0011] (Anti-collagen antibody) The anti-collagen antibody used in the present invention may be a monoclonal antibody or a polyclonal antibody, and these antibodies can be obtained by a conventional antibody production method. In addition, not only IgG but also antibody fragments (e.g., F(ab')2 fragment, Fab' fragment, etc.) may be used as the antibody.

[0012] For example, polyclonal anti-collagen antibodies can be obtained by immunizing a mammal with collagen as an antigen, collecting blood from the mammal, and isolating and purifying the antibody from the collected blood. For example, mammals such as mice, hamsters, guinea pigs, chickens, rats, rabbits, dogs, goats, sheep, and cows can be immunized. Methods of administering antigens are known to those skilled in the art, and the administration route is not particularly limited, and subcutaneous administration, intradermal administration, intraperitoneal administration, intravenous administration, intramuscular administration, and the like can be appropriately selected. In addition, adjuvants can be used as necessary. After raising the immunized mammal for an appropriate period of time, a small amount of serum from the mammal is sampled from an ear vein or the like, and the antibody titer is measured. When the antibody titer increases, an antigen may be administered as a booster depending on the situation. One to two months after the final administration, blood is collected from the immunized animal by a conventional method, and the blood is separated and purified by a conventional method, for example, centrifugation, precipitation using ammonium sulfate or polyethylene glycol, or chromatography such as gel filtration chromatography, ion exchange chromatography, or affinity chromatography, to obtain a polyclonal anti-collagen antibody as a polyclonal antiserum.

[0013] Monoclonal antibodies can be obtained, for example, by preparing hybridomas by cell fusion between antibody-producing cells and myeloma cell lines. As antibody-producing cells, spleen cells, lymph node cells, B lymphocytes, etc. from immunized animals can be used. For example, spleen cells are obtained as antibody-producing cells from immunized animals, and then fused with myeloma cells by a known method (G. Kohler et al., Nature, 256 495 (1975)) to prepare hybridomas. Examples of myeloma cell lines used for cell fusion include P3X63Ag8, P3U1, and Sp2 / 0 strains in mice. When performing cell fusion, a fusion promoter such as polyethylene glycol or Sendai virus is used, and hypoxanthine-aminopterin-thymidine (HAT) medium can be used in the usual manner to select hybridomas after cell fusion. Hybridomas obtained by cell fusion are cloned by limiting dilution or the like. Furthermore, by screening using an enzyme immunoassay method using collagen, a cell line producing a monoclonal antibody that specifically recognizes a desired collagen can be obtained. To produce a desired monoclonal antibody from a hybridoma, the hybridoma is cultured by a conventional cell culture method or ascites formation method, and the monoclonal antibody is purified from the culture supernatant or ascites. The monoclonal antibody can be purified from the culture supernatant or ascites by a conventional method. For example, ammonium sulfate fractionation, gel filtration, ion exchange chromatography, affinity chromatography, etc. can be used in appropriate combination.

[0014] The anti-collagen antibody used as the labeled antibody is labeled with a labeling substance, preferably a non-radioactive labeling substance. As the non-radioactive label, an enzyme label, a fluorescent label, a luminescent label, and the like can be used. As the enzyme label, for example, alkaline phosphatase (ALP), β-D-galactosidase, horseradish peroxidase (HRP), and the like can be used as the labeling enzyme. For the detection of these enzymes, the respective enzyme substrates, 4-methylumbelliferyl phosphate (4-MUP), a combination of hydrogen peroxide and 3,3',5,5'-tetramethylbenzidine, 2-nitrophenyl-β-D-galactoside, and the like, can be used. These enzyme substrates are changed (e.g., cleaved) by the action of the enzyme, and thus develop color, which can be detected.

[0015] (Solid phase process) The solid-phase step is a step of solidifying collagen contained in a collagen solution as an antigen onto a plate. The stepwise solid-phase step is a step including a step of solidifying collagen contained in a collagen solution as an antigen onto a plate multiple times.

[0016] In the present invention, the solidification step is carried out multiple times (2 or more times, preferably 3 or more times, more preferably 4 or more times, more preferably 5 or more times, and even more preferably 6 or more times). In the present invention, the solidification step is carried out in a step carried out in a stepwise manner, including a first solidification step and a second or subsequent solidification steps, the first solidification step is carried out using a collagen solution having a higher concentration than the collagen solution used in the solidification in the second or subsequent solidification steps, and the second or subsequent solidification steps are carried out using a collagen solution having a lower concentration than the first step.

[0017] (About immunoassays (including explanation of solid phase)) The immunoassay used in the present invention may be any method well known to those skilled in the art, such as a competitive method, an agglutination method, a Western blot method, an immunostaining method, or a sandwich method. For example, an ELISA method (Enzyme-Linked Immunosorbent Assay) is one of the immunoassay methods using antibodies. The ELISA method is a method in which a target antigen or antibody contained in a sample solution is captured by a specific antibody or antigen, and is detected and quantified using an enzyme reaction.

[0018] (solid phase) Solid-phase immobilization refers to immobilizing a target protein or a target antibody on a plate (such as a microplate) used in an immunoassay such as ELISA (URL: https: / / www.jstage.jst.go.jp / article / kagakutoseibutsu1962 / 23 / 7 / 23_7_462 / _pdf / -char / ja). Immunoassays (antigen-antibody reactions) using solid-phase plates include, for example, the direct method, the indirect method, the sandwich method, and the competitive method (URL: https: / / ruo.mbl.co.jp / bio / support / method / elisa.html).

[0019] The direct method is carried out in the following procedure. The target protein (or target antibody) is immobilized on a plate, and an antibody (or an antigen to which the target antibody specifically binds) for the enzyme-labeled target protein is reacted. After this reaction, the plate is washed as required, and the enzyme activity remaining on the plate is detected. The indirect method is carried out in the following procedure. The target protein is immobilized on a plate, and then an antibody against the target protein is reacted with the antibody, followed by an enzyme-labeled secondary antibody against that antibody. After this reaction, the plate is washed as required, and the enzyme activity remaining on the plate is detected. The sandwich method is carried out in the following procedure. ·An antibody against the target protein is immobilized on the plate, and the target protein is reacted. Following this reaction, another antibody against the target protein (the antibody labeled with an enzyme) is reacted. After this reaction, the plate is washed as required, and the enzyme activity remaining on the plate is detected. The competitive method is carried out according to the following procedure. The antibody against the target protein is immobilized on a plate, and the target protein and enzyme-labeled antigen are reacted simultaneously on the same plate. After this reaction, the enzyme activity remaining on the plate is detected.

[0020] The plate used in the present invention may be subjected to a predetermined processing as necessary for reaction purposes (binding purposes), for example, to make it hydrophobic, to make it hydrophilic, or to make it covalently bond with amino groups, carboxyl groups, etc.

[0021] In the present invention, the time and temperature of immobilization are preferably set under conditions that do not denature the target protein. The time of immobilization is, for example, 5 hours or more and 72 hours or less, more preferably 10 hours or more and 36 hours or less, and even more preferably 15 hours or more and 24 hours or less. The temperature of immobilization is, for example, 4°C or more and 25°C or less, more preferably 4°C or more and 8°C or less.

[0022] In the following examples of the present invention, the collagen contained in the composition was immobilized using the above-mentioned indirect method, and the collagen contained in the composition was detected. In the present invention, when a primary antibody and a secondary antibody are used, the dilution ratios of the primary antibody and the secondary antibody are preferably as follows: Dilution rate of primary antibody: 500 to 2000 times dilution, more preferably 1000 to 2000 times dilution, and even more preferably 1000 times dilution. Dilution rate of secondary antibody: 5,000 to 20,000 times dilution, more preferably 10,000 to 15,000 times dilution, and even more preferably 10,000 times dilution.

[0023] (basic solution) The basic solution used in the present invention can be obtained, for example, by mixing water and a basic substance. The basic solution used in the present invention may be a commercially available basic solution such as ammonia water. Examples of the basic substance include substances that donate ammonium ions. In the basic solution used in the present invention, examples of the substances that donate ammonium ions include ammonia, ammonium carbonate, and ammonium bicarbonate. In the basic solution used in the present invention, the basic substance in the free reagent may be one type or two or more types. The basic solution used in the present invention preferably has a pH of more than 8.0 and 11.0 or less.

[0024] (composition) The composition used in the present invention (oral composition, skin topical composition, etc.) may be in any form, for example, liquid, solid, powder, paste, jelly, gel, tablet, for example, solid form, liquid form. The solid form includes, for example, powder, tablet, pill, capsule, cachet, lozenge, suppository and dispersible granule. For example, in powder, the carrier is generally a finely divided solid that is a mixture with finely divided active ingredient. For example, in tablet, the active ingredient is generally mixed with a carrier having the necessary binding capacity in an appropriate ratio and molded into the desired shape and size. Suitable carriers may include, but are not limited to, for example, magnesium carbonate, magnesium stearate, talc, sugar, lactose, pectin, dextrin, starch, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose, low melting wax, cocoa butter, etc.

[0025] The present invention will now be described in more detail with reference to the following examples, but the present invention is not limited to these examples. In the following examples, the unit % of the numerical values ​​showing the amount of each component added means % by mass. EXAMPLES

[0026] Examples of the present invention will be described below.

[0027] [Example 1: Production of a powder mixture of proteoglycan and collagen (cartilage component mixture, collagen-containing composition)] 400 g of nasal cartilage extracted from the head of a chum salmon stored frozen at -30°C to -20°C was prepared as the starting material. 2000 mL of acetic acid aqueous solutions of various concentrations according to Comparative Example 1 and Example 1 below were added to this, and extraction was performed for 48 to 72 hours at an extraction temperature of 30°C to 40°C. The various concentrations (%), extraction temperatures (°C), and extraction times (hr, hours) according to Comparative Example 1 and Example 1 are as shown in Table 1.

[0028] The extract was filtered through a No. 26 filter paper (110 mm) to remove insoluble matter. Next, 2% powdered cellulose (manufactured by Nippon Paper Industries Co., Ltd., product name "KC Flock W-400G") was added to the liquid volume, and the mixture was stirred for 30 minutes and then filtered. The filtrate was concentrated using a hollow fiber membrane with a molecular weight cutoff of 50,000 until the liquid volume was reduced to 1 / 10. Concentration and purification were repeated while diluting with water, and finally 500 to 800 g of a concentrated liquid (pH 6 to 7) was obtained. The obtained concentrated liquid was then freeze-dried to obtain 10 to 20 g of a mixture of proteoglycan and collagen (lyophilized product).

[0029] The following items were measured for the obtained freeze-dried product, and the results are shown in Table 1. PG yield (%): Value (%) calculated by ((proteoglycan content in the obtained lyophilized product) / (weight of the nasal cartilage 400 (g))) x 100. ·PG molecular weight (×10 4 ): Proteoglycan molecular weight. For example, the entry "56" in Example 1 of Table 1 indicates a molecular weight of 560,000 (560,000). PG content ratio: the proteoglycan content in the lyophilized product of Comparative Example 1 is taken as 100, and the proteoglycan content in the lyophilized product of each Example is a relative value (relative value shown in comparison with Comparative Example 1). Collagen content (%): Collagen content in the lyophilized material. - "-": Indicates that no measurement has been performed.

[0030] (Method for quantifying proteoglycan (PG)) About 1 g of the dried product obtained by the manufacturing method according to Comparative Example 1 and Example 1 was precisely weighed, and phosphate buffer (pH 6.8) was added to make exactly 10 mL to prepare a sample solution. Each sample solution was passed through a 0.45 μm membrane filter, and then HPLC was performed under the following operating conditions to determine the amount of proteoglycan from the calibration curve of the standard product. The calibration curve was prepared by precisely weighing a proteoglycan standard product (derived from salmon nasal cartilage, Fujifilm Wako Pure Chemical Industries, 162-22131) that had been dried in a reduced pressure desiccator (silica gel) at room temperature for 3 hours and then dissolving it in the same phosphate buffer as the sample to prepare a standard solution for preparing the calibration curve. In addition, the molecular weight of the peak top was determined from the calibration curve prepared using Shodex STANDARD P-82 (manufactured by Showa Denko KK) as a molecular weight marker.

[0031] Operating conditions Analyzer: HPLC analyzer Detector: Differential refractive index detector (RID-10A, Shimadzu Corporation) Column: Gel filtration column (TSKgel G5000PWXL manufactured by Tosoh Corporation) Column temperature: 40℃ Sample injection volume: 50 μL Mobile phase: Phosphate buffer (pH 6.8) Flow rate: 0.5mL / min

[0032] (Method of quantifying collagen) Collagen is characterized by containing hydroxyproline and hydroxylysine, which are not contained in general proteins. Hydroxyproline is said to account for about 10% of all amino acids in collagen, and it is possible to estimate the amount of collagen by quantifying this hydroxyproline (Hikagagaku, vol. 56, No. 2, p71-79, 2010, "Functionality of Natural Collagen"). The collagen content of dried product samples obtained by the manufacturing methods of Comparative Example 1 and Example 1 was calculated by measuring the amount of this hydroxyproline.

[0033] [Table 1]

[0034] As shown in Table 1, it was confirmed that, compared with Comparative Example 1, Example 1 had improved proteoglycan yield and the like.

[0035] [Test 1 and Test 2: Detection of type II collagen] Type II collagen was detected using a liquid containing type II collagen (K41 type II collagen liquid derived from bovine articular cartilage, Collagen Technology Workshop, collagen concentration: 0.3%). The reagents used in Test 1 and Test 2 are as follows. 96-well plate for ELISA: F96 CERT. Maxisorp NUNC-Immunoplate (Thermo Scientific, 439454) Collagen dilution solvent (pH 9.5): Solvent containing Na2CO3 (final concentration 29 mM) and NaHCO3 (final concentration 71 mM) ·Primary antibody: Anti-Collagen II (ab34712) Primary antibody dilution solution: Can Get Signal Solution 1 (Toyobo) ·Secondary antibody: Goat Anti-Rabbit IgG H&L HRP (ab205718) Secondary antibody diluent: Can Get Signal Solution 2 (Toyobo) Blocking buffer: PVDF Blocking Reagent for Can Get Signal (Toyobo) ·Washing solution: PBS containing 0.05% Tween-20 Coloring solution: TMB solution (Fujifilm Wako Pure Chemical Industries, 208-17371) Reaction stop solution: Sulfuric acid (98%, Kishida Chemical) diluted to 0.5M (0.5mol / L) ·PBS: Phosphate Buffered Saline Powder (0.01 mol / l, pH7.2~7.4) TWEEN20: SIGMA, P9416-100ML

[0036] 1. Immobilization of antigen The method of solidification is shown in Figure 2. A solution containing type II collagen was diluted to a predetermined concentration (0.3 mg / ml dilution 1 in Test Example 1, and 0.3 mg / ml dilution 1 in Test Example 2). As shown in Figure 2, each prepared solution was dispensed into a 96-well plate for ELISA at 100 µL / well (Solidification 1, Solidification 2, Solidification 3, Solidification 4, Solidification 5, Solidification 6, Solidification 7, and Solidification 8 in both Test Example 1 and Test Example 2). After dispensing, the plate was covered with a plate seal. After the lid was placed, the plate was left to stand at 4°C for 16 hours to perform solidification.

[0037] 2. Washing After the immobilization, the solution was removed from the plate, and the plate was washed four times with a washing solution.

[0038] 3. Blocking After washing, 300 µL of blocking buffer was dispensed into each well. After dispensing, the plate was shielded from light with aluminum foil. After shielding from light, the plate was shaken at 25°C for 1 hour using a microplate shaker.

[0039] 4. Primary antibody reaction After shaking for 1 hour, the blocking buffer was removed. After removal, the primary antibody was diluted 1000-fold with primary antibody dilution solution. 100 µL of the primary antibody solution was dispensed per well. After dispensing, the plate was shielded from light. After shielding from light, the plate was left to stand at 25°C for 1 hour.

[0040] 5. Washing After the plate was left to stand, the solution was removed, and the plate was washed four times with a washing solution.

[0041] 6. Secondary antibody reaction After washing, the secondary antibody was diluted 10,000 times with secondary antibody dilution solution. 100 µL of the secondary antibody solution was dispensed into each well. After dispensing, the plate was shielded from light. After shielding from light, the plate was left to stand at 25°C for 1 hour.

[0042] 7. Washing After the immobilization, the solution was removed from the plate, and the plate was washed four times with a washing solution.

[0043] 8. Measurement After the washing, the TMB solution was dispensed into the plate at 100µL / well. After the dispensing, the plate was left to stand for 20 minutes in the dark (color reaction). After the reaction, the stop solution was dispensed into the plate at 100µL / well. After the dispensing, the absorbance of each well was measured using a microplate reader. The wavelengths of the absorbance were 450nm for measurement and 620nm for correction. The measurement results are shown in Table 2 and Figure 3. Figure 3 is a graph showing the values ​​shown in Table 2, with the horizontal axis representing the sample concentration (ng / mL) and the vertical axis representing the absorbance. As shown in Table 2 and Figure 3, compared to Test Example 1, Test Example 2, which had a more stepped solidification, showed a greater increase in absorbance, and it was confirmed that the detection accuracy of collagen was improved.

[0044] [Table 2]

[0045] [Test 3: Detection of type I collagen] Detection of type I collagen was carried out for the composition of Example 1. The reagents used in Test 1 are as follows. 96-well plate for ELISA: F96 CERT. Maxisorp NUNC-Immunoplate (Thermo Scientific, 439454) Collagen dilution solvent (pH 9.5): Solvent containing Na2CO3 (final concentration 29 mM) and NaHCO3 (final concentration 71 mM) Primary antibody: Purified Rabbit Anti-Salmon Collagen Type I (CEDARLANE, CL50171AP-S) Primary antibody diluent: Can Get Signal Solution 1 (Toyobo) ·Secondary antibody: Goat Anti-Rabbit IgG H&L HRP (ab205718) Secondary antibody diluent: Can Get Signal Solution 2 (Toyobo) Blocking buffer: PVDF Blocking Reagent for Can Get Signal (Toyobo) ·Washing solution: PBS containing 0.05% Tween-20 Coloring solution: TMB solution (Fujifilm Wako Pure Chemical Industries, 208-17371) Reaction stop solution: Sulfuric acid (98%, Kishida Chemical) diluted to 0.5M Collagen extraction solution: Pepsin 1:10000, derived from porcine gastric mucosa (Fujifilm Wako Pure Chemical Industries, 161-24482, working concentration: 1mg / ml) + 0.5M acetic acid solution ·PBS: Phosphate Buffered Saline Powder (0.01 mol / l, pH7.2~7.4) TWEEN20: SIGMA, P9416-100ML

[0046] 1. Immobilization of antigen A collagen extraction solution was used to prepare a collagen-extracted solution from the composition of Example 1 described above. The solution was centrifuged (13000 rpm, 4°C, 5 min). The supernatant after the centrifugation was collected. The supernatant was dispensed using each collagen dilution solvent, with the dilution ratio set (specimen concentration set) as in the above-mentioned Test Example 2 (Test Example 2 in Figure 2). Each prepared solution was dispensed into a 96-well plate for ELISA at 100 µL / well. After dispensing, the plate was covered with a plate seal. After the cover was placed, the plate was left to stand at 4°C for 16 hours to perform solid-phase formation.

[0047] 2. Washing After the immobilization, the solution was removed from the plate, and the plate was washed four times with a washing solution.

[0048] 3. Blocking After washing, 300 µL of blocking buffer was dispensed into each well. After dispensing, the plate was shielded from light with aluminum foil. After shielding from light, the plate was shaken at 25°C for 1 hour using a microplate shaker.

[0049] 4. Primary antibody reaction After shaking for 1 hour, the blocking buffer was removed. After removal, the primary antibody was diluted 2000-fold with the primary antibody dilution solution. 100 µL of the primary antibody solution was dispensed per well. After dispensing, the plate was shielded from light. After shielding from light, the plate was left to stand at 25°C for 1 hour.

[0050] 5. Washing After the plate was left to stand, the solution was removed, and the plate was washed four times with a washing solution.

[0051] 6. Secondary antibody reaction After washing, the secondary antibody was diluted 10,000 times with secondary antibody dilution solution. 100 µL of the secondary antibody solution was dispensed into each well. After dispensing, the plate was shielded from light. After shielding from light, the plate was left to stand at 25°C for 1 hour.

[0052] 7. Washing The solution was removed from the plate, after which the plate was washed four times with washing solution.

[0053] 8. Measurement After the washing, the TMB solution was dispensed to the plate at 100 μL / well. After the dispensing, the plate was left to stand for 20 minutes in the dark (color reaction). After the reaction, a stop solution was dispensed to the plate at 100 μL / well. After the dispensing, the absorbance of each well was measured using a microplate reader. The wavelengths of the absorbance were set to a measurement wavelength of 450 nm and a correction wavelength of 620 nm. The absorbance of the blank was 0.166, and the absorbance of the sample using the composition of Example 1 was 0.572. The absorbance of this sample could be measured, which indicated that collagen (type I collagen) of the sample could be detected.

[0054] [Test 4: Detection of type II collagen] Detection of type II collagen was carried out for the composition of Example 1. The reagents used in this test 4 are as follows. 96-well plate for ELISA: F96 CERT. Maxisorp NUNC-Immunoplate (Thermo Scientific, 439454) Collagen dilution solvent (pH 9.5): Solvent containing Na2CO3 (final concentration 29 mM) and NaHCO3 (final concentration 71 mM) ·Primary antibody: Anti-Collagen II (ab34712) Primary antibody diluent: Can Get Signal Solution 1 (Toyobo) ·Secondary antibody: Goat Anti-Rabbit IgG H&L HRP (ab205718) Secondary antibody diluent: Can Get Signal Solution 2 (Toyobo) Blocking buffer: PVDF Blocking Reagent for Can Get Signal (Toyobo) ·Washing solution: PBS containing 0.05% Tween-20 Coloring solution: TMB solution (Fujifilm Wako Pure Chemical Industries, 208-17371) Reaction stop solution: Sulfuric acid (98%, Kishida Chemical) diluted to 0.5M Collagen extraction solution: Pepsin 1:10000, derived from porcine gastric mucosa (Fujifilm Wako Pure Chemical Industries, 161-24482, working concentration: 1 mg / ml) + 0.5M acetic acid solution ·PBS: Phosphate Buffered Saline Powder (0.01 mol / l, pH7.2~7.4) TWEEN20: SIGMA, P9416-100ML

[0055] 1. Immobilization of antigen A collagen extraction solution was used to prepare a collagen-extracted solution from the composition of Example 1 described above. The solution was centrifuged (13000 rpm, 4°C, 5 min). The supernatant after the centrifugation was collected. The supernatant was dispensed using each collagen dilution solvent, with the dilution ratio set (sample concentration set) as in the above-mentioned Test Example 2 (Test Example 2 in Figure 2). Each prepared solution was dispensed into a 96-well plate for ELISA at 100 µL / well. After dispensing, the plate was covered with a plate seal. After the cover was placed, the plate was left to stand at 4°C for 16 hours to perform solid-phase formation.

[0056] 2. Washing After the immobilization, the solution was removed from the plate, and the plate was washed four times with a washing solution.

[0057] 3. Blocking After washing, blocking buffer was dispensed into each well at 300 µL / well. After dispensing, the plate was shielded from light with aluminum foil. After shielding from light, the plate was left to stand at 25°C for 1 hour.

[0058] 4. Primary antibody reaction After leaving it for 1 hour, the blocking buffer was removed. After removal, the primary antibody was diluted 1000-fold with the primary antibody dilution solution. 100µL of the primary antibody solution was dispensed per well. After dispensing, the plate was shielded from light. After shielding from light, the plate was allowed to stand at 25℃ for 1 hour.

[0059] 5. Washing After the plate was left to stand, the solution was removed, and the plate was washed four times with a washing solution.

[0060] 6. Secondary antibody reaction After washing, the secondary antibody was diluted 10,000 times with secondary antibody dilution solution. 100 µL of the secondary antibody solution was dispensed into each well. After dispensing, the plate was shielded from light. After shielding from light, the plate was left to stand at 25°C for 1 hour.

[0061] 7. Washing The solution was removed from the plate, after which the plate was washed four times with washing solution.

[0062] 8. Measurement After the washing, the TMB solution was dispensed into the plate at 100 μL / well. After the dispensing, the plate was left to stand for 20 minutes in the dark (color reaction). After the reaction, a stop solution was dispensed into the plate at 100 μL / well. After the dispensing, the absorbance of each well was measured using a microplate reader. The wavelengths of the absorbance were set to a measurement wavelength of 450 nm and a correction wavelength of 620 nm. The absorbance of the blank was 0, and the absorbance of the sample using the composition of Example 1 was 0.735 (sample concentration was 38.6 μg / ml). The absorbance of this sample could be measured, which showed that collagen type II collagen could be detected in the sample.

[0063] The above describes the embodiment of the present invention (including examples) with reference to the drawings. However, the specific configuration of the present invention is not limited to this, and even if there are design changes, etc., within the scope that does not deviate from the gist of the present invention, they are included in the present invention. [Industrial Applicability]

[0064] It is also possible to provide a new method for detecting collagen contained in a composition by an immunological assay method using an antibody against collagen.

Claims

1. A method for detecting collagen contained in a composition by an immunoassay method using an antibody against collagen, comprising the steps of: The method includes a stepwise solidification step using a basic solution, The stepwise solidification step includes a step of solidifying collagen contained in a collagen solution as an antigen on a plate multiple times, diluting the collagen solution with the basic solution; The stepwise solidification process includes a first solidification process and a second or subsequent solidification process, The first solidification step is carried out using a collagen solution having a higher concentration than the collagen solution used in the second or subsequent solidification steps, The method, wherein the second and subsequent solidification steps are carried out using a collagen solution having a lower concentration than that in the first solidification step.

2. The method of claim 1 , wherein the basic solution has a pH greater than 8.

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