A kit for detecting collagen integrin functional domain based on receptors and its preparation and application

Abstract

The present invention belongs to the field of biotechnology detection, and specifically relates to a reagent kit for detecting collagen integrin functional domains based on receptors, and its preparation and application. The present invention detects whether collagen contains integrin binding domains by a competitive method and thereby assists in determining whether collagen has corresponding efficacy. Compared with the sandwich method, the false negative rate of the detection method of the present invention is significantly reduced; it fills the gap in the field of biological function detection of collagen products; the kit of the present invention is low-cost and the results can be obtained in as fast as 15 minutes; the kit of the present invention is of great significance for the research and development, production, quality control, and detection and evaluation of collagen products.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology detection, and specifically relates to a reagent kit for detecting collagen integrin functional domains based on receptors, and the preparation and application thereof. Background Art

[0002] In recent years, with the continuous deepening of research on the structure and function of collagen, the demand for collagen products has grown rapidly worldwide. Collagen has been widely used in food, health food and cosmetics. The application of collagen in the medical field, such as dressings, implant materials and tissue engineering medical products, is also growing. Collagen is the main protein component of connective tissue and is a functional protein closely related to the functions of cells, tissues and organs of the body. It has good biocompatibility and biodegradability, and also has properties such as high tensile strength, low immunity, hemostasis and promotion of cell growth.

[0003] Recombinant collagen refers to a recombinant protein prepared by DNA recombination technology, containing the full-length or partial gene sequence of a specific type of human collagen gene, or a human collagen analog prepared by gene editing, combination, assembly, editing, etc. containing a partial sequence of a specific or different type of human collagen gene, or a collagen analog prepared by gene editing, combination, assembly, etc. containing a partial gene code of a specific or different type of human collagen and an artificially designed gene code, and having the physical and chemical properties and biological functions of collagen or similar to collagen. The purity and integrity of collagen molecules (including functional regions) are key determinants of their polymerization ability and the microstructure, physical and biological properties of self-assembled products.

[0004] With the rapid development of the collagen market, the quality of products varies greatly, and the demand for its identification and detection is also growing rapidly. At present, the main methods for detecting collagen products are: SDS-PAGE gel electrophoresis, ultraviolet spectroscopy, etc. can identify the type and purity of collagen, high performance liquid chromatography-mass spectrometry, hydroxyproline colorimetry, immunological detection, etc. can quantitatively detect collagen, circular dichroism, Fourier transform infrared spectroscopy, Raman spectroscopy, etc. can detect the structure of collagen. The Chinese invention patent with publication number CN115586340A discloses a natural active collagen rapid identification kit, which can quickly and qualitatively detect active collagen with a complete triple helix structure. Many collagen products on the market claim that they have biological activities such as moisturizing, whitening, and anti-aging, but there is no detection method in the prior art that can quickly detect or assist in detecting the biological functions of collagen products. Summary of the invention

[0005] To solve the above problems, the present invention provides a kit for detecting collagen integrin functional domain based on receptors and a preparation method thereof, which assists in determining whether collagen has relevant efficacy by quickly detecting whether the integrin functional domain is contained.

[0006] The first object of the present invention is to provide a collagen functional domain detection kit, wherein the kit comprises a test strip, and the test strip comprises a labeled integrin and an integrin antibody.

[0007] Preferably, the labeled integrin is gold-labeled integrin.

[0008] Preferably, the integrin antibody is an integrin β1 polyclonal antibody.

[0009] Preferably, the labeled integrin is a gold-labeled integrin and the integrin antibody is an integrin β1 polyclonal antibody.

[0010] Preferably, the test strip comprises a PVC bottom plate, on which a sample pad, a gold pad, a nitrocellulose membrane (NC membrane) and a blotting paper are overlapped from left to right. The gold pad is fixed with a gold-labeled integrin, and the NC membrane includes a detection line (T line) and a quality control line (C line) in the chromatography direction, the T line is coated with a recombinant collagen containing an integrin binding domain, and the C line is coated with an integrin β1 polyclonal antibody.

[0011] In a preferred embodiment, the labeled integrin is a gold-labeled integrin, and the preparation steps of the kit are as follows:

[0012] S1. Preparation of colloidal gold:

[0013] Deionized water and chloroauric acid solution, stir and boil, then quickly add the sodium citrate solution into the flask, continue stirring until the solution returns to room temperature, then dilute the colloidal gold solution with deionized water and set aside.

[0014] S2. Preparation of gold-labeled integrin

[0015] Add the above colloidal gold solution and stir, transfer K2CO3 solution dropwise and add it into the colloidal gold solution and stir, then add integrin to the solution, add BSA solution and stir to seal, centrifuge, then remove the supernatant, and use a re-solution (Tris-HCl buffer containing sucrose, BSA, Tween-20, and preservative) to re-dissolve the precipitate.

[0016] S3. Preparation of gold pad:

[0017] A Tris-HCl buffer solution containing BSA, Tween-20, preservatives, and sucrose was coated on the glass fiber and dried, and then the gold-labeled integrin was sprayed on the treated glass fiber and baked.

[0018] S4. Preparation of nitrocellulose membrane:

[0019] PBS buffer containing BSA was used as coating buffer, recombinant collagen containing integrin binding domain was coated on NC membrane as T line, integrin β1 polyclonal antibody was diluted with coating buffer and coated on NC membrane as C line, and then dried.

[0020] S5. Preparation of sample pad:

[0021] The sample pad is obtained by coating the glass fiber with Tris-HCl buffer containing PVP, Tween-20, preservatives and sucrose and drying the mixture.

[0022] S6. Preparation of test strips:

[0023] Lay the sample pad in step S5, the gold pad in step S3, the nitrocellulose membrane and the absorbent paper in step S4 on the PVC bottom plate from left to right, and cut them into the required width to obtain the test strip;

[0024] S7. Preparation of the test kit: Place the test strip into the card slot in an environment with humidity <30% RH, and provide a bottled sample diluent and instructions.

[0025] Preferably, the colloidal gold in step S1 is 4 / 10,000 colloidal gold with a particle size of 40 nm, the mass percentage of the chloroauric acid solution is 10%, and the mass percentage of the sodium citrate solution is 10%, and the chloroauric acid solution and sodium citrate solution used to prepare 100 mL of the colloidal gold are 1.2 mL and 3.84 mL respectively.

[0026] Preferably, in step S2, the concentration of the K2CO3 solution is 0.2M, the amount of integrin added is 4-10 μg / mL colloidal gold, the final concentration of BSA after the addition of the BSA solution is 0.5%-2%, the reconstituted solution contains 1%-5% (w / v) sucrose, 0.5%-2% (w / v) BSA, 0.05%-2% (v / v) Tween-20, 0.04%-1% (v / v) preservative, the preservative is Proclin 300, the concentration of the Tris-HCl buffer is 0.02-0.1M, and the pH is 7.0-9.0.

[0027] Preferably, the gold pad in step S3 is pre-treated with a Tris-HCl buffer solution containing 1% to 5% (w / v) sucrose, 0.05% to 0.2% (v / v) Tween-20, 0.05% to 0.2% (w / v) BSA, and 0.04% to 0.1% (v / v) preservative at a concentration of 0.02 to 0.1 M and a pH of 7.0 to 9.0, and then dried. The labeled integrin is then sprayed on the dried glass fiber at a spray rate of 1.0 to 5.0 μL / cm and baked. The preservative is Proclin 300.

[0028] Preferably, the spraying process in step S3 is: sucking the composite into a gold spray film scratching instrument, setting the spraying volume of the gold spray film scratching instrument to 3.0 μL / cm, the spraying speed to 100 mm / s, and drying at 40°C and <30% RH for 12 to 24 hours after spraying to obtain the gold pad.

[0029] Preferably, the coating buffer in step S4 is a PBS buffer containing 0.05% to 2% (w / v) BSA, a concentration of 0.01 to 0.1 M, and a pH of 7.0 to 9.0, the recombinant collagen is diluted with the coating buffer to 0.1 to 2 mg / mL as a T-line coating solution, and the integrin β1 polyclonal antibody is diluted with the coating buffer to 0.1 to 2 mg / mL as a C-line coating solution, and the coating amount of the T-line coating solution and the C-line coating solution is 0.8 to 2 μL / cm.

[0030] Preferably, the sample pad in step S5 is prepared by treating glass fiber with a Tris-HCl buffer solution containing 1% to 5% (w / v) sucrose, 0.05% to 0.2% (v / v) Tween-20, 0.05% to 2% (w / v) PVP, and 0.04% to 0.1% (v / v) preservative at a concentration of 0.02 to 0.1 M and a pH of 7.0 to 9.0, and then drying, wherein the preservative is Proclin 300.

[0031] In step S7, the sample diluent is preferably a Tris-HCl buffer containing Tween-20 and Proclin 300; the sample diluent is more preferably a Tris-HCl buffer containing 0.05% to 0.2% (v / v) Tween-20, 0.04% to 0.2% (v / v) Proclin300, a concentration of 0.02 to 0.2M, and a pH of 7.0 to 9.0; the sample diluent is most preferably a Tris-HCl buffer containing 0.1% (v / v) Tween-20, 0.04% (v / v) Proclin 300, a concentration of 0.05M, and a pH of 8.0.

[0032] The integrins of the present invention include but are not limited to integrin α1β1, α2β1, α10β1 and α11β1.

[0033] The second object of the present invention is to provide the application of the above-mentioned kit, wherein the kit is used in detecting collagen functional domains to assist in detecting the biological function of collagen.

[0034] More preferably, the collagen is recombinant collagen.

[0035] The present invention has the following advantages: it fills the blank in the field of biological function detection of collagen products; the preparation cost of the kit of the present invention is low and the results can be obtained in as fast as 5 minutes; the kit of the present invention is of great significance for the research and development, production, quality control, detection and evaluation of collagen products.

[0036] In order to achieve the purpose of the present invention, the inventors have simultaneously developed reagents for the sandwich method and the competitive method. The sandwich method uses collagen antibodies for T-line coating, and the competitive method uses collagen containing integrin functional domains for T-line coating. Considering that most of the recombinant collagen in the industry is human collagen fragments and different manufacturers use different fragment positions and sizes to achieve different promotional functions, but collagen antibodies can only bind to specific immune sites and cannot cope with all types of recombinant collagen, so it is easy to produce false negatives. The competitive method only requires collagen to contain integrin functional domains to effectively avoid this design defect. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 Schematic diagram of the structure and detection principle of the kit of the present invention.

[0038] Figure 2 It is a schematic diagram of determining the results of the test kit of the present invention for samples.

[0039] Figure 3 This is a diagram of the actual measurement results of the test kit of the present invention detecting samples.

[0040] Figure 4 The results are as follows: The test results of the kit of the present invention on cosmetics containing collagen.

[0041] Figure 5 This is a graph showing the actual test results of samples detected by the comparative test kit (sandwich method). DETAILED DESCRIPTION

[0042] Example 1 Preparation of colloidal gold kit T1 of the present invention

[0043] The test strip in the kit of the present invention comprises a PVC base plate, on which a sample pad, a gold pad, a nitrocellulose membrane (NC membrane) and a water-absorbing paper are overlapped in sequence from left to right. Figure 1As shown. The gold pad is fixed with gold-labeled integrin, and the NC membrane includes a detection line (T line) and a quality control line (C line) in the chromatography direction. The T line is coated with recombinant collagen containing integrin binding domain, and the C line is coated with integrin β1 polyclonal antibody. The specific preparation method and steps are as follows:

[0044] S1. Preparation of colloidal gold:

[0045] Take a clean 1000mL round-bottom flask, put in a suitable stirrer, and then add 300mL of deionized water and 1.2mL of 10% (w / v) chloroauric acid (G4022, Sigma-Aldrich LLC.) solution in turn, turn on the stirring function of an intelligent magnetic stirrer (ZNCL-TS-1L, Shanghai Yiming Industrial Co., Ltd.) to boil and maintain boiling for 1min, then use a pipette to transfer 3.84mL of 10% (w / v) sodium citrate (S189183, Shanghai Aladdin Biochemical Technology Co., Ltd.) solution at one time and quickly add it to the flask, continue heating for 5min (the solution first turns black and then red during this process), then turn off the heating function of the intelligent magnetic stirrer and continue stirring until the solution returns to room temperature, then dilute the colloidal gold solution to 300mL with deionized water for use.

[0046] S2. Preparation of gold-labeled integrin

[0047] A clean 200 mL beaker was placed on a magnetic stirrer (MS-PB, Beijing Dalong Xingchuang Experimental Instrument Co., Ltd.), and then 100 mL of the above colloidal gold solution was added and the stirring function was continuously turned on. 800 μL of 0.2M K2CO3 (P111556, Shanghai Aladdin Biochemical Technology Co., Ltd.) solution was pipetted and added dropwise to the colloidal gold solution. After stirring for 30 min, 600 μg of integrin α2β1 (IT1-H52W6, Beijing Biopsies Biotechnology Co., Ltd.) was added to the solution. After stirring for 1 h, 10 mL of 10% (w / v) BSA (GHB034, Hangzhou Tianlai Biotechnology Co., Ltd.) solution was added and stirred for 1 h. The mixture was then divided into centrifuge tubes and centrifuged at 10,000 rpm for 30 min at 4°C using a centrifuge (2-16R, Hunan Hengnuo Instrument Equipment Co., Ltd.). The supernatant was then removed and the solution was resuspended with 10 mL of a reconstitution solution (containing 5% (w / v) sucrose, 1% (w / v) BSA, 0.1% (v / v) Tween-20, 0.1% (v / v) Proclin 300 in 50 mM The precipitate was redissolved in Tris buffer (pH 8.0).

[0048] S3. Preparation of gold pad:

[0049] A Tris-HCl buffer solution containing 1% (w / v) BSA, 0.1% (v / v) Tween-20, 0.05% (v / v) Proclin 300, 2% (w / v) sucrose, and pH 8.0 was coated on the glass fiber and dried, and then the gold-labeled integrin was sprayed on the treated glass fiber and baked. The spraying process was as follows: the complex was sucked into a gold spray film scratcher (HM3030, Shanghai Jinbiao Biotechnology Co., Ltd.), and the gold spray film scratcher was set to a spraying amount of 3.0 μL / cm and a spraying speed of 100 mm / s. After spraying, the gold pad was dried at 40°C and <30% RH for 12 to 24 hours to obtain the gold pad.

[0050] S4. Preparation of nitrocellulose membrane:

[0051] A 10 mM PBS buffer containing 1% (w / v) BSA and pH 7.4 was used as the coating buffer. Recombinant collagen containing the integrin binding domain (VN72, produced by Shenzhen Liying Biotechnology Co., Ltd.) was coated on the NC membrane as the T line. Integrin β1 polyclonal antibody (A2217, Wuhan Abotek Biotechnology Co., Ltd.) was diluted 5 times with the coating buffer and then coated on the NC membrane as the C line. The membrane was dried at 40°C and <30% RH for 12 to 24 hours.

[0052] S5. Preparation of sample pad:

[0053] The glass fiber was coated with a Tris-HCl buffer solution containing 1% PVP, 0.05% Tween-20, 0.05% preservative, 2% sucrose, and pH 8.0, and then dried at 40° C. and <30% RH for 12 to 24 hours to obtain the sample pad.

[0054] S6. Preparation of test strips:

[0055] The sample pad in step S5, the gold pad in step S3, the nitrocellulose membrane and the absorbent paper in step S4 are overlapped in sequence from left to right on the PVC bottom plate as shown in the figure, and the test strip is obtained by cutting into the required width.

[0056] S7. Preparation of the kit

[0057] In an environment with humidity less than 30% RH, the test strip is placed in a plastic card inner slot that matches its width and fastened, and placed together with a desiccant in a suitable aluminum foil bag, and heat-sealed with a fully automatic film sealer to complete the inner packaging. The inner-packaged test strip and the matching bottled sample diluent together constitute the kit of the present invention. The sample diluent is a Tris-HCl buffer solution containing 0.1% (v / v) Tween-20, 0.04% (v / v) Proclin 300, a concentration of 0.05M, and a pH of 8.0.

[0058] Example 2 Preparation of the Colored Microsphere Kit T2 of the Present Invention

[0059] This embodiment provides another preparation method and steps of the kit of the present invention, which are as follows:

[0060] S1. Preparation of labeled integrins

[0061] 25 μL of colored microspheres (DR01, Changsha Meiniu Biotechnology Co., Ltd.) were added to 1 mL of deionized water, and 5 μL of EDC solution (10 mg / mL, E106172, Shanghai Aladdin Biochemical Technology Co., Ltd.) and 5 μL of NHS solution (10 mg / mL, H109330, Shanghai Aladdin Biochemical Technology Co., Ltd.) were added for activation for 30 minutes, and then 25 μg of integrin α2β1 was added for labeling for 2 hours, followed by addition of 100 μL of 10% (w / v) BSA solution for blocking for 1 hour, followed by centrifugation at 12000 rpm for 10 minutes at 4°C, the supernatant was removed, and 250 μL of reconstitution solution (50 mM Tris buffer containing 5% (w / v) sucrose, 1% (w / v) BSA, 0.1% (v / v) Tween-20, 0.1% (v / v) Proclin 300, pH 8.0) was added to reconstitute the precipitate.

[0062] S2. Preparation of gold pad:

[0063] A Tris-HCl buffer solution containing 1% (w / v) BSA, 0.1% (v / v) Tween-20, 0.05% (v / v) Proclin 300, 2% (w / v) sucrose, and pH 8.0 was coated on the glass fiber and dried, and then the gold-labeled integrin was sprayed on the treated glass fiber and baked. The spraying process was as follows: the complex was sucked into a gold spray film scratcher (HM3030, Shanghai Jinbiao Biotechnology Co., Ltd.), and the gold spray film scratcher was set to a spraying amount of 2.1 μL / cm and a spraying speed of 100 mm / s. After spraying, the gold pad was dried at 40°C and <30% RH for 12 to 24 hours to obtain the gold pad.

[0064] S3. Preparation of nitrocellulose membrane:

[0065] A 10 mM PBS buffer containing 1% (w / v) BSA and pH 7.4 was used as the coating buffer. Recombinant collagen containing the integrin binding domain (VN72, produced by Shenzhen Liying Biotechnology Co., Ltd.) was coated on the NC membrane as the T line. Integrin β1 polyclonal antibody (A2217, Wuhan Abotek Biotechnology Co., Ltd.) was diluted 5 times with the coating buffer and then coated on the NC membrane as the C line. The membrane was dried at 40°C and <30% RH for 12 to 24 hours.

[0066] S4. Preparation of sample pad:

[0067] The glass fiber was coated with a Tris-HCl buffer solution containing 1% PVP, 0.05% Tween-20, 0.05% preservative, 2% sucrose, and pH 8.0, and then dried at 40° C. and <30% RH for 12 to 24 hours to obtain the sample pad.

[0068] S5. Preparation of test strips:

[0069] The sample pad in step S5, the gold pad in step S3, the nitrocellulose membrane and the absorbent paper in step S4 are overlapped in sequence from left to right on the PVC bottom plate as shown in the figure, and the test strip is obtained by cutting into the required width.

[0070] S6. Preparation of the kit

[0071] In an environment with humidity less than 30% RH, the test strip is placed in a plastic card inner slot that matches its width and fastened, and placed together with a desiccant in a suitable aluminum foil bag, and heat-sealed with a fully automatic film sealer to complete the inner packaging. The inner-packaged test strip and the matching bottled sample diluent together constitute the kit of the present invention. The sample diluent is a Tris-HCl buffer solution containing 0.1% (v / v) Tween-20, 0.04% (v / v) Proclin 300, a concentration of 0.05M, and a pH of 8.0.

[0072] Example 3 Detection method of the kit of the present invention

[0073] You can perform the corresponding operations according to the instructions, including the following steps:

[0074] 1. Equilibrate the test card, sample diluent and sample to be tested to room temperature (15-30°C);

[0075] 2. Open the aluminum foil bag of the test card and place the test card on a flat table;

[0076] 3. Take 50 μL of the sample to be tested and the sample diluent, mix them evenly, and pipette 50 μL (2 to 3 drops) of the mixed solution into the sample well (S) of the test card;

[0077] 4. Leave at room temperature for 5 to 10 minutes;

[0078] 5. Observe and interpret the results. Interpretation is based on Figure 2 shown.

[0079] Collagen with integrin binding domain was prepared into samples (1 μg / mL, 10 μg / mL, 100 μg / mL), and blank control was set up. The colloidal gold kit described in Example 1 was used for detection. Figure 3It can be seen that the minimum detection limit of the kit of the present invention is <10 μg / mL.

[0080] Example 4 Detection results of the kit of the present invention on market products

[0081] Collect collagen-related products on the market for testing, the results are as follows Figure 4 As shown, the inventors have conducted functional verification and found that the results are consistent with those of the detection kit, indicating that the kit can accurately detect the functional domains of collagen products, thereby assisting in determining whether the collagen has the corresponding efficacy.

[0082] Comparative Example 1 Preparation and Detection of Sandwich Kit

[0083] The test strip in the sandwich test kit includes a PVC base plate, on which a sample pad, a gold pad, a nitrocellulose membrane (NC membrane) and a water-absorbing paper are overlapped in sequence from left to right. Figure 1 As shown. The gold pad is fixed with gold-labeled integrin, and the NC membrane includes a detection line (T line) and a quality control line (C line) in the chromatography direction. The T line is coated with recombinant collagen containing an integrin binding domain, and the C line is coated with an integrin β1 polyclonal antibody. The specific preparation method refers to the preparation method and steps of the kit in Example 1, the difference being:

[0084] S4. Preparation of nitrocellulose membrane:

[0085] A 10 mM PBS buffer containing 1% (w / v) BSA and pH 7.4 was used as the coating buffer. Type III collagen rabbit monoclonal antibody (A0817, Wuhan Abotek Biotechnology Co., Ltd.) was diluted with the coating buffer and coated on the NC membrane as the T line. Integrin β1 polyclonal antibody (A2217, Wuhan Abotek Biotechnology Co., Ltd.) was diluted 5 times with the coating buffer and coated on the NC membrane as the C line. The membrane was dried at 40°C and <30% RH for 12 to 24 hours.

[0086] The kit was used to test samples containing recombinant collagen, and the results are shown in the figure below. Figure 5 As shown, the sandwich test kit showed false negative results for recombinant collagen, which shows that this method is prone to missed detection.

Claims

1. A collagen functional domain detection kit, characterized in that: The kit comprises a test strip, wherein the test strip comprises a labeled integrin and an integrin antibody; The integrin antibody is an integrin β1 polyclonal antibody; The test strip comprises a bottom plate, on which a sample pad, a gold pad, an NC membrane and absorbent paper are overlapped in sequence from left to right, the gold pad is fixed with a labeled integrin, the NC membrane comprises a T line and a C line in sequence according to the chromatography direction, the T line is coated with collagen containing an integrin binding domain, and the C line is coated with an integrin β1 polyclonal antibody; The labeled integrin is a gold-labeled integrin, and the preparation of the kit comprises the following steps: S1. Preparation of colloidal gold: deionized water and chloroauric acid solution are mixed, stirred and boiled, then sodium citrate solution is quickly added to a flask, and stirring is continued until the solution returns to room temperature, and then the colloidal gold solution is fixed to volume with deionized water for later use; S2. Preparation of gold-labeled integrin: add the colloidal gold solution and stir, pipette K2CO3 solution and add dropwise to the colloidal gold solution and stir, then add integrin to the solution, add BSA solution and stir to seal, centrifuge, then remove the supernatant, and redissolve the precipitate with a re-solution, wherein the re-solution contains sucrose, BSA, Tween-20, and a preservative Tris-HCl buffer; S3, preparation of gold pad: coating the glass fiber with Tris-HCl buffer containing BSA, Tween-20, preservatives, and sucrose and drying it, then spraying the labeled integrin on the treated glass fiber and baking it; S4. Preparation of NC membrane: Use PBS buffer containing BSA as coating buffer, coat the recombinant collagen containing integrin binding domain on the NC membrane as T line, dilute integrin β1 polyclonal antibody with coating buffer and coat it on the NC membrane as C line, and dry it; S5. Preparation of sample pad: coating glass fiber with Tris-HCl buffer containing PVP, Tween-20, preservative, and sucrose, and drying to obtain the sample pad; S6. Preparation of test strips: overlapping the sample pad in step S5, the gold pad in step S3, the NC film in step S4 and the absorbent paper from left to right on the PVC bottom plate, and cutting into the required width to obtain the test strips; S7. Preparation of the test kit: Place the test strip into the card slot in an environment with humidity <30%RH, and provide a bottle of sample diluent and instructions.

2. The kit according to claim 1, characterized in that The colloidal gold in step S1 is 4 / 10,000 colloidal gold with a particle size of 40 nm, the mass percentage of the chloroauric acid solution is 10%, and the mass percentage of the sodium citrate solution is 10%. The chloroauric acid solution and sodium citrate solution used to prepare 100 mL of the colloidal gold are 1.2 mL and 3.84 mL respectively.

3. The kit according to claim 1, characterized in that In step S2, the concentration of the K2CO3 solution is 0.2 M, the amount of integrin added is 4-10 μg / mL colloidal gold, the final concentration of BSA after the addition of the BSA solution is 0.5%-2%, the reconstituted solution contains 1%-5% w / v sucrose, 0.5%-2% w / v BSA, 0.05%-2% v / v Tween-20, 0.04%-1% v / v preservative, the preservative is Proclin 300, the concentration of the Tris-HCl buffer is 0.02-0.1 M, and the pH is 7.0-9.

0.

4. The kit according to claim 1, characterized in that The gold pad in step S3 is pre-treated with a Tris-HCl buffer containing 1% to 5% w / v sucrose, 0.05% to 0.2% v / v Tween-20, 0.05% to 0.2% w / v BSA, and 0.04% to 0.1% v / v preservative at a concentration of 0.02 to 0.1 M and a pH of 7.0 to 9.0, and then the labeled integrin is sprayed on the dried glass fiber at a spray rate of 1.0 to 5.0 μL / cm and baked; the sample pad in step S5 is pre-treated with a Tris-HCl buffer containing 1% to 5% w / v sucrose, 0.05% to 0.2% v / v Tween-20, 0.05% to 2% w / v PVP, and 0.04% to 0.1% v / v preservative at a concentration of 0.02 to 0.1 The glass fiber is treated with Tris-HCl buffer with a pH of 7.0 to 9.0 and then dried, and the preservative is Proclin 300.

5. The kit according to claim 1, characterized in that The coating buffer in step S4 is a PBS buffer containing 0.05%~2% w / v BSA, a concentration of 0.01~0.1 M, and a pH of 7.0~9.

0. The recombinant collagen is diluted to 0.1~2 mg / mL with the coating buffer as a T-line coating solution, and the integrin β1 polyclonal antibody is diluted to 0.1~2 mg / mL with the coating buffer as a C-line coating solution. The coating volume of the T-line coating solution and the C-line coating solution is 0.8~2 μL / cm.

6. Use of the kit according to any one of claims 1 to 5 in detecting collagen functional domains for non-disease diagnosis purposes, thereby assisting in detecting the biological functions of collagen.

7. The use according to claim 6, characterized in that: The collagen is recombinant collagen.

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