Cleaning solution, cleaning method of container bottle, squamous cell carcinoma antigen detection kit and preparation method of squamous cell carcinoma antigen detection kit

Abstract

The invention discloses a cleaning solution, a cleaning method of a container bottle, a squamous cell carcinoma antigen detection kit and a preparation method of the squamous cell carcinoma antigen detection kit, and relates to the technical field of immunoassay. The container bottle cleaning solution comprises the following components in percentage by mass: 0.3%-3% of sodium hypochlorite, 0.1%-2% of sodium alkyl benzene sulfonate, 0.1%-2% of sodium tripolyphosphate, 0.01%-0.05% of a chelating agent, 0.01%-0.05% of a complexing agent, 0.1%-0.5% of an enzyme inhibitor and the balance of water. The container bottle cleaning solution provided by the invention can effectively clean container bottles for squamous cell carcinoma antigen detection kits, effectively reduces pollution caused in the production process of the kits, well controls the stability of the kits in batches, and improves the detection accuracy of the kits.

Description

Technical Field

[0001] The invention relates to the technical field of immunoassay analysis, and in particular to a cleaning liquid, a cleaning method for a container bottle, and a squamous cell carcinoma antigen detection kit and a preparation method thereof. Background Art

[0002] Squamous cell carcinoma antigen (SCC) was first isolated from the squamous epithelium of the cervix. It is a glycoprotein with a relative molecular mass of 48kd and belongs to the family of serine protease inhibitors. Under physiological conditions, SCC antigen is expressed in small amounts in normal squamous epithelium and pseudostratified columnar epithelium, but is overexpressed in many malignant tumor tissues derived from stratified squamous epithelial cells. The serum SCC antigen content is related to tumor tissue grade, lymph node infiltration and treatment response, and is negatively correlated with patient prognosis. Therefore, SCC antigen is an important tumor marker that reflects the biological characteristics of squamous cell carcinoma.

[0003] Squamous cells are the main component of the epithelium and are also found in the digestive tract, lungs, and other parts of the body. Since squamous cell carcinoma antigens are present in sweat, saliva, hair, and dandruff, they can easily be distributed in aerosols and dust through behaviors such as sneezing, contaminating containers, bottles, disposable utensils, or instruments, which can lead to false increases in squamous cell carcinoma antigen test values. Therefore, the current detection method has a high risk of contamination, which can cause squamous cell carcinoma antigen test values ​​to be falsely increased. Therefore, this poses a great challenge to the production process control of the squamous cell carcinoma antigen test kit and the subsequent inspection operations.

[0004] Therefore, the prior art still needs to be improved and developed. Summary of the invention

[0005] Based on the above-mentioned deficiencies of the prior art, the purpose of the present invention is to provide a cleaning liquid, a cleaning method for a container bottle, a squamous cell carcinoma antigen detection kit and a preparation method thereof, aiming to provide a cleaning liquid for a container bottle of a squamous cell carcinoma antigen detection kit, reduce the contamination of the container bottle, and improve the detection accuracy of the kit.

[0006] The technical solution of the present invention is as follows:

[0007] In a first aspect of the present invention, a container bottle cleaning liquid is provided, wherein the container bottle cleaning liquid comprises the following components in percentage by weight:

[0008] Sodium hypochlorite 0.3%-3%, sodium alkylbenzene sulfonate 0.1%-2%, sodium tripolyphosphate 0.1%-2%, chelating agent 0.01%-0.05%, complexing agent 0.01%-0.05%, enzyme inhibitor 0.1%-0.5% and the balance to 100% water.

[0009] Optionally, the chelating agent includes at least one of disodium ethylenediaminetetraacetate, dimercaprol and mercaptoethylamine.

[0010] Optionally, the complexing agent includes at least one of tartaric acid, citric acid and oxalic acid;

[0011] And / or, the enzyme inhibitor comprises proteinase K.

[0012] The second aspect of the present invention provides a method for cleaning a container bottle for a squamous cell carcinoma antigen detection kit, which comprises the following steps:

[0013] Placing a container bottle for a squamous cell carcinoma antigen detection kit in a container bottle cleaning solution, and filling the container bottle with the container bottle cleaning solution, and performing ultrasound for a preset time;

[0014] Next, replace the container bottle with a new cleaning solution, and fill the container bottle of the squamous cell carcinoma antigen detection kit with the new cleaning solution, soak for 15±2h, rinse with tap water for several times, rinse with running purified water for several times, and then dry at a temperature of 50±5°C, thus completing the cleaning of the container bottle of the squamous cell carcinoma antigen detection kit;

[0015] The container bottle cleaning liquid is the container bottle cleaning liquid or sodium hypochlorite aqueous solution as described above in the present invention, and the mass content of sodium hypochlorite in the sodium hypochlorite aqueous solution is 0.3% to 3%.

[0016] The third aspect of the present invention provides a method for preparing a squamous cell carcinoma antigen detection kit, which comprises the following steps:

[0017] Providing a container bottle, and using the cleaning method of the present invention as described above to clean the container bottle to obtain a cleaned container bottle;

[0018] Filling the squamous cell carcinoma antigen detection reagents into different cleaned container bottles to obtain the squamous cell carcinoma antigen detection kit;

[0019] Wherein, the squamous cell carcinoma antigen detection reagent includes reagent R1 and reagent R2;

[0020] Reagent R1 comprises a complex, wherein the complex comprises streptavidin magnetic particles and a biotin-labeled squamous cell carcinoma antigen-specific mouse monoclonal antibody linked to the streptavidin magnetic particles;

[0021] Reagent R2 comprises an acridinium ester-labeled squamous cell carcinoma antigen-specific mouse monoclonal antibody.

[0022] Optionally, the reagent R1 is prepared by mixing a solid phase premix reagent and a biotin premix reagent, wherein the solid phase premix reagent comprises the following components:

[0023] 10-30 mmol / L of a second buffer, 100-300 mmol / L of salts, and streptavidin magnetic particles with a mass content of 0.01% to 0.08%;

[0024] The biotin premix reagent includes the following components:

[0025] 100-400mmol / L of a second buffer, 100-300mmol / L of salts, 0.1%-2% by weight of a protective agent, 0.1%-2% by weight of a preservative, 0.1%-2% by weight of a surfactant, water, and a biotin-labeled squamous cell carcinoma antigen-specific mouse monoclonal antibody; in the biotin-labeled squamous cell carcinoma antigen-specific mouse monoclonal antibody, the molar ratio of the squamous cell carcinoma antigen-specific mouse monoclonal antibody to biotin is 1:5-1:20, and the concentration of the squamous cell carcinoma antigen-specific mouse monoclonal antibody is 0.5-2.0μg / mL;

[0026] The reagent R2 includes the following components:

[0027] 100-400mmol / L of a third buffer, 100-300mmol / L of salts, 0.1%-2% by weight of a protective agent, 0.1%-2% by weight of a preservative, 0.1%-2% by weight of a surfactant and water, and acridinium ester-labeled squamous cell carcinoma antigen-specific mouse monoclonal antibody; in the acridinium ester-labeled squamous cell carcinoma antigen-specific mouse monoclonal antibody, the molar ratio of the squamous cell carcinoma antigen-specific mouse monoclonal antibody to the acridinium ester is 1:1-1:10, and the concentration of the squamous cell carcinoma antigen-specific mouse monoclonal antibody is 0.1-0.8μg / mL.

[0028] Optionally, the method for preparing a biotin-labeled squamous cell carcinoma antigen-specific mouse monoclonal antibody comprises the following steps:

[0029] Put squamous cell carcinoma antigen-specific mouse monoclonal antibody into a centrifuge tube, add carbonate buffer, mix well, add biotin, centrifuge, shake and mix well in the dark, add blocking solution, shake and mix well, and collect the blocked biotin-labeled squamous cell carcinoma antigen-specific mouse monoclonal antibody after purification;

[0030] The method for preparing acridinium ester-labeled squamous cell carcinoma antigen-specific mouse monoclonal antibody comprises the following steps:

[0031] The squamous cell carcinoma antigen-specific mouse monoclonal antibody is placed in a centrifuge tube, carbonate buffer is added, acridinium ester is added after mixing, centrifugation is performed, oscillation is performed in the dark, blocking solution is added, oscillation is performed, and the blocked acridinium ester-labeled squamous cell carcinoma antigen-specific mouse monoclonal antibody is purified and collected.

[0032] Optionally, the squamous cell carcinoma antigen detection reagent also includes a squamous cell carcinoma antigen calibrator and a squamous cell carcinoma antigen quality control product; the squamous cell carcinoma antigen calibrator includes squamous cell carcinoma antigen solutions with concentrations of 0.00 ng / mL and 25 ng / mL, respectively; the squamous cell carcinoma antigen quality control product includes squamous cell carcinoma antigen solutions with concentrations of 5 ng / mL and 25 ng / mL, respectively.

[0033] Optionally, the squamous cell carcinoma antigen detection reagent further comprises a chemiluminescent excitation liquid, and the chemiluminescent excitation liquid comprises an acidic excitation liquid and an alkaline excitation liquid.

[0034] A fourth aspect of the present invention provides a squamous cell carcinoma antigen detection kit, wherein the kit is prepared by the preparation method described in any one of the above items of the present invention.

[0035] Beneficial effects: The container bottle cleaning liquid provided by the present invention can effectively clean the container bottle for the squamous cell carcinoma antigen detection kit (the container bottle is used to hold various reagents in the kit), effectively reducing the contamination caused by the production process of the kit, so that the stability of the kit batches is well controlled, and the detection accuracy of the kit is improved. DETAILED DESCRIPTION

[0036] The present invention provides a cleaning solution, a cleaning method for a container bottle, and a squamous cell carcinoma antigen detection kit and a preparation method thereof. To make the purpose, technical scheme and effect of the present invention clearer and more specific, the present invention is further described in detail below. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0037] Unless otherwise defined, all technical terms and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art of the present invention. The terms used in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0038] If there are descriptions involving "first", "second", etc. in the embodiments of the present invention, the descriptions of "first", "second", etc. are only used for descriptive purposes and cannot be understood as indicating or implying their relative importance or implicitly indicating the number of the indicated technical features.

[0039] Since squamous cell carcinoma antigen (SCC, hereinafter referred to as SCC) exists in sweat, saliva, hair and dandruff, it is very easy to be distributed in aerosol and dust through sneezing and other behaviors, contaminating container bottles (such as reagent bottles, calibration bottles and quality control bottles, etc.), disposable utensils or instruments, thereby causing a false increase in SCC detection values. Therefore, the control of the production process is extremely important. It is necessary not only to ensure the protection of personnel during the preparation process to avoid contamination by sweat, dandruff and saliva, but also to thoroughly clean the container bottles. Based on this, an embodiment of the present invention provides a container bottle cleaning solution, wherein the container bottle cleaning solution includes the following components in percentage by mass:

[0040] Sodium hypochlorite 0.3%-3%, sodium alkylbenzene sulfonate 0.1%-2%, sodium tripolyphosphate 0.1%-2%, chelating agent 0.01%-0.05%, complexing agent 0.01%-0.05%, enzyme inhibitor 0.1%-0.5% and the balance to 100% water.

[0041] The container bottle cleaning liquid provided by the present invention can effectively clean the container bottle for the SCC detection kit (the container bottle is used to contain various reagents in the kit), effectively reducing the pollution caused in the production process of the kit, so that the stability between batches of the kit is well controlled, and at the same time improving the detection accuracy of the kit.

[0042] In the present invention, sodium alkylbenzene sulfonate and sodium tripolyphosphate play a role in improving the cleaning effect in the container bottle cleaning liquid, the chelating agent and the complexing agent play a role in binding metal ions, and the enzyme inhibitor plays a role in enzymatically hydrolyzing proteins. Sodium hypochlorite, as a main cleaning component, synergistically acts with sodium alkylbenzene sulfonate (as an anionic surfactant, sodium tripolyphosphate (as a builder), the chelating agent, the complexing agent and the enzyme inhibitor, and can better remove metal ions, impurities and protein contaminants on the container bottle, so that the container bottle cleaning liquid has a good cleaning effect on the container bottle used to hold the SCC detection reagent, and reduces the contamination of the SCC detection reagent.

[0043] In some embodiments, the chelating agent includes at least one of disodium ethylenediaminetetraacetate (EDTA-2Na), dimercaptopropanol, and mercaptoethylamine, but is not limited thereto.

[0044] In some embodiments, the complexing agent includes at least one of tartaric acid, citric acid, and oxalic acid, but is not limited thereto.

[0045] In some embodiments, the enzyme inhibitor includes proteinase K, but is not limited thereto.

[0046] The embodiment of the present invention also provides a method for cleaning a container bottle for an SCC detection kit, which comprises the following steps:

[0047] S11, placing the container bottle for the SCC detection kit in the container bottle cleaning solution, and making the interior of the container bottle for the SCC detection kit be filled with the container bottle cleaning solution, and performing ultrasound for a preset time (e.g., 30 minutes);

[0048] S12. Next, replace the new container bottle cleaning liquid, and fill the inside of the container bottle for the SCC detection kit with the new container bottle cleaning liquid, soak for 15±2h (e.g., 13h, 14h, 15h, 16h or 17h, etc.), rinse with tap water for several times (e.g., 10 times), and then rinse with running purified water for several times (e.g., 10 times), and then dry at a temperature of 50±5°C (e.g., 45°C, 46°C, 47°C, 48°C, 49°C, 50°C, 51°C, 52°C, 53°C, 54°C or 55°C, etc.), and the cleaning of the container bottle for the SCC detection kit is completed;

[0049] Wherein, the container bottle cleaning liquid is the container bottle cleaning liquid or sodium hypochlorite aqueous solution as described above in the present invention, and the mass content of sodium hypochlorite in the sodium hypochlorite aqueous solution is 0.3% to 3% (for example, the mass content of sodium hypochlorite in the sodium hypochlorite aqueous solution can be 0.3%, 0.5%, 1%, 1.5%, 2%, 2.5% or 3%, etc.).

[0050] The present invention achieves a better cleaning effect by combining a specific container bottle cleaning liquid with ultrasonic technology, effectively reduces the contamination caused in the production process of the SCC detection kit, makes the stability between batches of the SCC detection kit well controlled, and improves the detection accuracy of the SCC detection kit.

[0051] The present invention also provides a method for preparing a SCC detection kit, which comprises the following steps:

[0052] S21, providing a container bottle, and cleaning the container bottle using the cleaning method of the present invention to obtain a cleaned container bottle;

[0053] S22, filling the SCC detection reagents into different cleaned container bottles respectively to obtain the SCC detection kit;

[0054] Wherein, the SCC detection reagents include reagent R1 and reagent R2;

[0055] Reagent R1 comprises a complex, wherein the complex comprises streptavidin magnetic particles and a biotin-labeled SCC-specific mouse monoclonal antibody linked to the streptavidin magnetic particles;

[0056] Reagent R2 comprises an acridinium ester-labeled SCC-specific mouse monoclonal antibody.

[0057] Reagent R1 and reagent R2 can be filled into different containers or the same container. Other test reagents such as quality control products or calibration products need to be filled into containers different from the containers for filling reagents R1 and R2.

[0058] In the present invention, the SCC detection kit uses a sandwich method based on chemiluminescent immunoassay technology to detect SCC. Specifically, acridinium ester-labeled SCC-specific mouse monoclonal antibody and biotin-labeled SCC-specific mouse monoclonal antibody react with SCC in the sample to form an antibody-antigen-antibody complex, which is bound to the magnetic particles through biotin and streptavidin. The SCC content in the sample is proportional to the relative light unit (RLU) detected by the system.

[0059] The SCC detection kit prepared by the preparation method provided by the present invention has the advantages of high sensitivity, good specificity, wide linear range, good long-term stability and thermal stability, simple operation, and small batch-to-batch variability. In the present invention, the SCC detection kit is based on chemiluminescence immunoassay and can complete the detection of SCC using a fully automatic chemiluminescence immunoassay instrument as a detection tool. Therefore, the kit is matched with the instrument, shortening the time required for clinical detection and having high detection accuracy.

[0060] In addition, in the present invention, the complex including streptavidin magnetic particles and biotin-labeled SCC-specific mouse monoclonal antibodies connected to the streptavidin magnetic particles is used as the main component of the reagent R1, which can reduce the interference of biotin contained in the sample on the detection.

[0061] In some embodiments, the reagent R1 is prepared by mixing a solid phase premix reagent and a biotin premix reagent, wherein the solid phase premix reagent includes the following components:

[0062] 10-30 mmol / L of a first buffer (the final concentration of the first buffer in the solid phase premix reagent is 10-30 mmol / L), 100-300 mmol / L of salts, and streptavidin magnetic particles with a mass content of 0.01% to 0.08%;

[0063] The biotin premix reagent includes the following components:

[0064] 100-400 mmol / L of a second buffer (the final concentration of the second buffer in the biotin premix reagent is 100-400 mmol / L), 100-300 mmol / L of salts, 0.1%-2% by weight of a protective agent, 0.1%-2% by weight of a preservative, 0.1%-2% by weight of a surfactant, water, and a biotin-labeled SCC-specific mouse monoclonal antibody; in the biotin-labeled SCC-specific mouse monoclonal antibody, the molar ratio of the SCC-specific mouse monoclonal antibody to biotin is 1:5-1:20, and the concentration of the SCC-specific mouse monoclonal antibody is 0.5-2.0 μg / mL.

[0065] In some embodiments, the step of mixing the solid phase premix reagent and the biotin premix reagent to obtain reagent R1 specifically includes:

[0066] The solid phase premix reagent and the biotin premix reagent are measured in a volume ratio of 1:1, and the solid phase premix reagent is magnetically separated on a magnetic separator; after the supernatant is visually clear and transparent, the supernatant is aspirated and discarded to obtain separated magnetic beads. The biotin premix reagent is added to the above separated magnetic beads at one time, inverted and mixed, and then placed in a mixer (360-degree flip mixer) or a blood mixer for premixing to obtain reagent R1.

[0067] Such premixing can reduce the interference of biotin contained in the sample on the detection.

[0068] As an example, in the solid phase premix reagent, the concentration of the first buffer is 10mmol / L, 15mmol / L, 20mmol / L, 25mmol / L or 30mmol / L, etc.; the concentration of the salt is 100mmol / L, 150mmol / L, 200mmol / L, 250mmol / L or 300mmol / L, etc.; the mass content of the streptavidin magnetic particles is 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07% or 0.08%, etc.

[0069] In some embodiments, the first buffer includes but is not limited to at least one of HEPES buffer (wherein HEPES is 4-hydroxyethylpiperazineethanesulfonic acid), PIPES buffer (wherein PIPES is piperazine-1,4-diethanesulfonic acid), MOPS buffer (wherein MOPS is 3-(N-morpholinyl)propanesulfonic acid sodium salt), disodium hydrogen phosphate-citrate buffer, phosphate buffer, BISTRIS buffer (wherein BISTRIS is bis(2-hydroxyethyl)amino(trihydroxymethyl)methane), TRIS buffer (wherein TRIS stands for trishydroxymethylaminomethane), and sodium barbital-hydrochloric acid buffer.

[0070] In some embodiments, the salt in the solid phase premix reagent includes but is not limited to at least one of NaCl, KCl and MgCl2.

[0071] In some embodiments, in the streptavidin magnetic particles, the particle size of the magnetic particles is 1 to 3 μm, for example, 1 μm, 2 μm or 3 μm.

[0072] In some embodiments, the method for preparing the streptavidin magnetic particles comprises the following steps:

[0073] Take 0.5-1 mL of 50-100 mg / mL streptavidin magnetic particle solution, add 5-10 mL of TBST solution and mix thoroughly for 10-15 min, place on a magnetic separator until the supernatant is clear, discard the supernatant, and keep the magnetic particles. After repeated washing 3 times, use a buffer containing 50 mM MES (i.e., 2-morpholineethanesulfonic acid), 0.05% (mass content) Tween-20, 0.05% (mass content) Proclin300, pH 6.5 to prepare a reagent with a magnetic bead mass concentration (i.e., mass content) of 0.01% to 1% (the reagent is used to prepare a solid phase premix reagent), and store at 2°C to 8°C.

[0074] For example, in the biotin premix reagent, the concentration of the second buffer is 100mmol / L, 150mmol / L, 200mmol / L, 250mmol / L, 300mmol / L, 350mmol / L or 400mmol / L, etc.; the concentration of the salt is 100mmol / L, 150mmol / L, 200mmol / L, 250mmol / L or 300mmol / L, etc.; the mass content of the protective agent is 0.1%, 0.2%, 0.3%, 0.4%, etc. %, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9% or 2%, etc.; the mass content of the preservative is 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9% or 2%, etc.; the mass content of the preservative is 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9% or 2%. 2%, etc.; the mass content of the surfactant is 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9% or 2%, etc.; the molar ratio of SCC-specific mouse monoclonal antibody to biotin is 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, the concentration of SCC-specific mouse monoclonal antibody is 0.5μg / mL, 0.6μg / mL, 0.7μg / mL, 0.8μg / mL, 0.9μg / mL, 1.0μg / mL, 1.1μg / mL, 1.2μg / mL, 1.3μg / mL, 1.4μg / mL, 1.5μg / mL, 1.6μg / mL, 1.7μg / mL, 1.8μg / mL, 1.9μg / mL or 2.0μg / mL, etc.

[0075] In some embodiments, the second buffer includes but is not limited to at least one of HEPES buffer, PIPES buffer, MOPS buffer, disodium hydrogen phosphate-citrate buffer, phosphate buffer, BISTRIS buffer, TRIS buffer and sodium barbital-hydrochloric acid buffer.

[0076] In some embodiments, the salts in the biotin premix reagent include but are not limited to at least one of NaCl, KCl and MgCl 2. These salts can balance the osmotic pressure of the reagent R1.

[0077] In some embodiments, the protective agent in the biotin premix reagent includes, but is not limited to, at least one of casein, bovine serum albumin, and human serum albumin. These protective agents can maintain the activity of the SCC-specific mouse monoclonal antibody and maintain the stability of the spatial structure of the SCC-specific mouse monoclonal antibody.

[0078] In some embodiments, the preservatives in the biotin premix reagent include but are not limited to at least one of ProClin300 (i.e., 3% 5-chloro-2-methyl-4-isothiazoline-3-one and 2-methyl-4-isothiazoline-3-one solution, purchased from MERCK), pc950 (the main active ingredient is 2-methyl-4-isothiazoline-3-one, purchased from Shaanxi Proanti Biotechnology Development Co., Ltd.), GML-2 (bis(hydroxymethyl)imidazolidinyl urea) and gentamicin.

[0079] In some embodiments, the surfactant in the biotin premix reagent includes but is not limited to at least one of polyoxyethylene lauryl ether, Triton X-100 and Tween (eg, Tween-20, Tween-80, etc.).

[0080] In some embodiments, the method for preparing the biotin-labeled SCC-specific mouse monoclonal antibody comprises the following steps:

[0081] The SCC-specific mouse monoclonal antibody is placed in a centrifuge tube, carbonate buffer is added, biotin is added after mixing, centrifuged, and oscillated in the dark (for example, placed in an air bath constant temperature oscillator for mixing). After adding blocking solution, oscillated (for example, placed in an air bath constant temperature oscillator for mixing), the blocked biotin-labeled SCC-specific mouse monoclonal antibody is collected after purification.

[0082] In some embodiments, the method for preparing a biotin-labeled SCC-specific mouse monoclonal antibody comprises the following steps:

[0083] (1) Replacement: The SCC-specific mouse monoclonal antibody is taken out and equilibrated to room temperature, mixed thoroughly, added to an ultrafiltration centrifuge tube, centrifuged at 1500 rpm for 5 to 10 min (for example, 5 min, 6 min, 7 min, 8 min, 9 min or 10 min, etc.), and then the liquid in the lower tube is discarded. A replacement buffer is added to the upper tube of the ultrafiltration centrifuge tube to dilute the SCC-specific mouse monoclonal antibody in the upper tube to 2 mg / mL, centrifuged at 1500 rpm for 5 to 10 min, and the liquid in the lower tube is discarded after centrifugation; the replacement buffer is added to the upper tube again to dilute the SCC-specific mouse monoclonal antibody in the upper tube to 2 mg / mL, centrifuged at 1500 rpm for 5 to 10 min (for example, 5 min, 6 min, 7 min, 8 min, 9 min or 10 min, etc.), and the liquid in the lower tube is discarded after centrifugation; the replacement buffer is added to the upper tube again to dilute the SCC-specific mouse monoclonal antibody in the upper tube to 2 mg / mL, centrifuged at 1500 rpm for 5 to 10 min, and the liquid in the lower tube is discarded after centrifugation (i.e., the steps of adding replacement buffer for dilution, centrifugation, and discarding the liquid in the lower tube are repeated three times), and the SCC-specific mouse monoclonal antibody in the upper tube is collected.

[0084] (2) Labeling: Place SCC-specific mouse monoclonal antibody (250-1000 μg) in a centrifuge tube, add carbonate buffer solution, mix thoroughly, add 0.75 μL-5 μL (for example, 0.75 μL, 1 μL, 2 μL, 3 μL, 4 μL or 5 μL, etc.) of 2 mg / mL biotin in N,N-dimethylformamide (DMF) solution, and centrifuge for 0.5-1 min at room temperature. Seal the centrifuge tube with sealing film and place it in a light-proof dark box, then place the dark box in an air bath constant temperature oscillator (23°C-25°C) and mix for 3-4 hours. Add 0.5 mL-1 mL of 20%-30% lysine blocking solution, place in an air bath constant temperature oscillator (23°C-25°C), mix at medium speed, and the blocking time is 1-2 hours.

[0085] (3) Purification: The blocked SCC-specific mouse monoclonal antibody was purified on a superdex200 column, eluted with phosphate buffer (PB), and collected in steps. The collected SCC-specific mouse monoclonal antibody concentrated solution was stored at 2°C to 8°C.

[0086] When used (such as when preparing a biotin premix reagent), the purified SCC-specific mouse monoclonal antibody concentrated solution is diluted to a final concentration of 0.1 μg / mL with a buffer solution containing 50 mM sodium methyl ester sulfonate (MES), 0.05% Tween (mass content), 0.05% (mass content) Proclin300, pH 6.5, and stored at 2°C to 8°C.

[0087] In some embodiments, the reagent R2 includes the following components:

[0088] 100-400 mmol / L of a third buffer, 100-300 mmol / L of salts, 0.1%-2% by weight of a protective agent, 0.1%-2% by weight of a preservative, 0.1%-2% by weight of a surfactant and water (e.g., 1 L), and an acridinium ester-labeled SCC-specific mouse monoclonal antibody; in the acridinium ester-labeled SCC-specific mouse monoclonal antibody, the molar ratio of the SCC-specific mouse monoclonal antibody to the acridinium ester is 1:1-1:10, and the concentration of the SCC antibody is 0.1-0.8 μg / mL.

[0089] For example, in reagent R2, the concentration of the third buffer is 100mmol / L, 150mmol / L, 200mmol / L, 250mmol / L, 300mmol / L, 350mmol / L or 400mmol / L, etc.; the concentration of the salt is 100mmol / L, 150mmol / L, 200mmol / L, 250mmol / L or 300mmol / L, etc.; the mass content of the protective agent is 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9% or 2%, etc.; the mass content of the preservative is 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 1.1% , 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9% or 2%, etc.; the mass content of the surfactant is 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9% or 2%, etc.; the mass content of the acridinium ester label is 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9% or 2%, etc. In the SCC-specific mouse monoclonal antibody, the molar ratio of the SCC-specific mouse monoclonal antibody to the acridinium ester can be 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9 or 1:10, etc., and the concentration of the SCC antibody can be 0.1 μg / mL, 0.2 μg / mL, 0.3 μg / mL, 0.4 μg / mL, 0.5 μg / mL, 0.6 μg / mL, 0.7 μg / mL or 0.8 μg / mL, etc.

[0090] In some embodiments, the third buffer includes but is not limited to at least one of HEPES buffer, PIPES buffer, MOPS buffer, disodium hydrogen phosphate-citrate buffer, phosphate buffer, BISTRIS buffer, TRIS buffer, and sodium barbital-hydrochloric acid buffer.

[0091] In some embodiments, the salt in the reagent R2 includes but is not limited to at least one of NaCl, KCl and MgCl2.

[0092] In some embodiments, the protective agent in the reagent R2 includes but is not limited to at least one of casein, bovine serum albumin, and human serum albumin. In some embodiments, the selection of the preservative in the reagent R2 refers to the preservative in the biotin premix reagent.

[0093] In some embodiments, the surfactant in the reagent R2 includes but is not limited to at least one of polyoxyethylene lauryl ether, Triton X-100 and Tween (eg, Tween-20, Tween-80, etc.).

[0094] In the reagent R2, in some embodiments, the method for preparing the acridinium ester-labeled SCC-specific mouse monoclonal antibody comprises the following steps:

[0095] The SCC-specific mouse monoclonal antibody is placed in a centrifuge tube, carbonate buffer is added, acridinium ester is added after mixing, centrifuged, and oscillated in the dark (for example, placed in an air bath constant temperature oscillator for mixing). After adding blocking solution, oscillated (for example, placed in an air bath constant temperature oscillator for mixing), the blocked acridinium ester-labeled SCC-specific mouse monoclonal antibody is purified, collected, and then diluted in a buffer.

[0096] In some specific embodiments, the method for preparing the acridinium ester-labeled SCC-specific mouse monoclonal antibody comprises the following steps:

[0097] (1) Replacement: For specific steps, refer to the replacement step in the method for preparing biotin-labeled SCC-specific mouse monoclonal antibody above.

[0098] (2) Labeling: Place SCC-specific mouse monoclonal antibody (250-1000 μg) in a centrifuge tube, add carbonate buffer solution, mix thoroughly, add 0.75 μL-5 μL (for example, 0.75 μL, 1 μL, 2 μL, 3 μL, 4 μL or 5 μL, etc.) of 2 mg / mL acridinium ester DMF solution, and centrifuge for 0.5-1 min at room temperature. Seal the centrifuge tube with sealing film and place it in a light-proof dark box, then place the dark box in an air bath constant temperature oscillator (23°C-25°C) and mix for 3h-4h. Add 0.5-1 mL of 20%-30% lysine blocking solution, place in an air bath constant temperature oscillator (23°C-25°C), mix at medium speed, and the blocking time is 1-2h.

[0099] (3) Purification: The blocked acridinium ester-labeled SCC-specific mouse monoclonal antibody was purified on a superdex200 column, eluted with PB buffer, and collected in steps. The collected acridinium ester-labeled SCC-specific mouse monoclonal antibody concentrated solution was stored at 2°C to 8°C.

[0100] When used (for example, when preparing reagent R2), the purified acridinium ester-labeled SCC-specific mouse monoclonal antibody concentrated solution is diluted to a final concentration of 0.1 μg / mL with 50 mM sodium methyl ester sulfonate (MES), 0.05% (mass content) Tween, 0.05% (mass content) Proclin 300, pH 6.5 buffer and stored at 2°C to 8°C.

[0101] In some embodiments, the SCC detection reagent further includes at least one of a SCC calibrator and a SCC antigen quality control product.

[0102] In some embodiments, the SCC antigen calibrator comprises SCC antigen solutions having concentrations of 0.00 ng / mL and 25 ng / mL, respectively; and the SCC antigen quality control comprises SCC solutions having concentrations of 5 ng / mL and 25 ng / mL, respectively.

[0103] In some embodiments, the SCC detection reagent further comprises a chemiluminescent excitation liquid, and the chemiluminescent excitation liquid comprises an acidic excitation liquid and an alkaline excitation liquid. In some specific embodiments, the acidic excitation liquid is a mixed solution of nitric acid and hydrogen peroxide (wherein the concentration of HNO3 is 0.02-0.04 mol / L, and the mass content of H2O2 is 1%-2%), and the alkaline excitation liquid is a sodium hydroxide solution (wherein the concentration of sodium hydroxide is 0.25-0.45 mol / L).

[0104] The present invention also provides an SCC detection kit, which is prepared by the preparation method described in the present invention. The SCC detection kit includes a plurality of container bottles (specifically, the bottle mouth of the container bottle is a cross design, so that the reagents therein are not easy to spill out, foreign objects are not easy to enter, and the risk of contamination caused by human operation during the test is reduced) and SCC detection reagents located in the container bottles, different reagents are placed in different container bottles, and the SCC detection reagents include reagent R1 and reagent R2 (the specific selection of reagent R1 and reagent R2 is described above).

[0105] In some specific embodiments, the SCC detection reagent further includes at least one of a SCC calibrator and a SCC quality control product (for specific selection of the calibrator and the quality control product, see the above description).

[0106] In some specific embodiments, the SCC detection reagent further comprises a chemiluminescent excitation solution (the specific selection of the chemiluminescent excitation solution is described above).

[0107] The present invention will be further described below by means of specific examples.

[0108] Unless otherwise specified, the raw materials, reagents, etc. used in the following examples are all commercially available products.

[0109] Example 1

[0110] The reagents in the SCC detection kit include reagent R1 and reagent R2, SCC calibrators, SCC quality controls, and chemiluminescence excitation solution;

[0111] Wherein, the preparation method of reagent R1 is as follows:

[0112] The solid phase premix reagent and the biotin premix reagent were measured in a volume ratio of 1:1, and the solid phase premix reagent was magnetically separated on a magnetic separator; after the supernatant was visually clear and transparent, the supernatant was aspirated and discarded to obtain separated magnetic beads. The biotin premix reagent was added to the above separated magnetic beads at one time, inverted and mixed, and then placed in a 360-degree flip mixer for premixing to obtain reagent R1.

[0113] Among them, the solid phase premix reagent includes:

[0114] TRIS buffer 25mmol / L;

[0115] NaCl 150mmol / L;

[0116] Streptavidin magnetic particles 0.072% (mass content).

[0117] The particle size of the magnetic particles is 3 μm.

[0118] Biotin premix includes:

[0119]

[0120] Biotin-labeled SCC-specific mouse monoclonal antibody, the molar ratio of SCC-specific mouse monoclonal antibody to biotin was 1:10, and the concentration of SCC-specific mouse monoclonal antibody was 0.7 μg / mL.

[0121] Reagent R2 includes:

[0122]

[0123] The SCC-specific mouse monoclonal antibody was labeled with acridinium ester, the molar ratio of the SCC-specific mouse monoclonal antibody to acridinium ester was 1:5, and the concentration of the SCC-specific mouse monoclonal antibody was 0.2 μg / mL.

[0124] The SCC calibrators include SCC solutions with concentrations of 0 ng / mL and 25 ng / mL;

[0125] SCC quality control products include SCC solutions with concentrations of 5 ng / mL and 25 ng / mL;

[0126] The chemiluminescent excitation liquid includes an acidic excitation liquid and an alkaline excitation liquid. The acidic excitation liquid is a nitric acid and sodium hydroxide solution (wherein the concentration of HNO3 is 0.03 mol / L and the mass content of H2O2 is 1.5%), and the alkaline excitation liquid is a sodium hydroxide solution (wherein the concentration of sodium hydroxide is 0.35 mol / L).

[0127] Magnetic particle preparation:

[0128] Take 0.5 mL of 100 mg / mL streptavidin magnetic particle solution (available for purchase), add 10 mL of TBST solution and mix thoroughly (mixing time is 15 min), place on a magnetic separator until the supernatant is clear, discard the supernatant, and keep the magnetic particles. Repeat the washing 3 times, and use a buffer containing 50 mM MES, 0.05% Tween-20, 0.05% Proclin300 and pH = 6.5 to prepare a reagent with a magnetic bead mass content of 0.072%, and store at 2°C to 8°C.

[0129] Preparation of biotinylated SCC-specific mouse monoclonal antibody:

[0130] (1) Replacement: The SCC-specific mouse monoclonal antibody was balanced to room temperature and mixed thoroughly. The mixed antibody was added to an ultrafiltration centrifuge tube, centrifuged at 1500 rpm for 8 min, and the liquid in the lower tube was discarded. The replacement buffer was added to the upper tube of the ultrafiltration centrifuge tube, the antibody in the upper tube was diluted to 2 mg / mL, and centrifuged at 1500 rpm for 8 min. After centrifugation, the liquid in the lower tube was discarded, and the replacement buffer was added to the upper tube again. The antibody in the upper tube was diluted to 2 mg / mL, and centrifuged at 1500 rpm for 8 min. Repeat three times and collect the antibody in the upper tube.

[0131] (2) Labeling: Place SCC-specific mouse monoclonal antibody (250 μg) in a centrifuge tube, then add carbonate buffer solution and mix thoroughly. After mixing, add 2.37 μL of 2 mg / mL biotin DMF solution and centrifuge at room temperature for 1 min. Seal the centrifuge tube with sealing film and place it in a light-proof dark box. Then place the dark box in an air bath constant temperature oscillator (23°C to 25°C) and mix for 4 hours. Add 1 mL of 25% lysine blocking solution and place it in an air bath constant temperature oscillator (23°C to 25°C). Mix at medium speed and the blocking time is 2 hours.

[0132] (3) Purification: The blocked antibody was purified on a superdex200 column, eluted with PB buffer, and collected in steps. The collected SCC-specific mouse monoclonal antibody concentrated solution was stored at 2°C to 8°C. When used, the purified SCC-specific mouse monoclonal antibody concentrated solution was diluted with a buffer containing 50mM MES, 0.05% Tween, 0.05% Proclin300 and pH = 6.5 to a final concentration of 0.1μg / mL and stored at 2°C to 8°C.

[0133] Preparation of SCC-specific mouse monoclonal antibody labeled with acridinium ester:

[0134] (1) Replacement: The same replacement step as in the preparation of the biotin-labeled SCC-specific mouse monoclonal antibody in Example 1.

[0135] (2) Labeling: Place SCC-specific mouse monoclonal antibody (250 μg) in a centrifuge tube, add carbonate buffer solution, mix thoroughly, add 1.7 μL of 2 mg / mL acridinium ester DMF solution after mixing, and centrifuge for 1 min at room temperature. Seal the centrifuge tube with sealing film and place it in a light-proof dark box, then place the dark box in an air bath constant temperature oscillator (23°C to 25°C) and mix for 4 h. Add 1 mL of 25% lysine blocking solution, place in an air bath constant temperature oscillator (23°C to 25°C), mix at medium speed, and the blocking time is 2 h.

[0136] (3) Purification: The blocked acridinium ester-labeled SCC-specific mouse monoclonal antibody was purified on a superdex200 column, eluted with PB buffer, and collected in steps. The collected acridinium ester-labeled SCC-specific mouse monoclonal antibody concentrated solution was stored at 2°C to 8°C. When used, the purified SCC-specific mouse monoclonal antibody concentrated solution was diluted with 50mM MES, 0.05% Tween, 0.05% Proclin300, pH 6.5 buffer to a final concentration of 0.1μg / mL.

[0137] Example 2

[0138] This embodiment provides a container bottle cleaning liquid A, comprising the following components:

[0139] Sodium hypochlorite 0.3%, sodium alkylbenzene sulfonate 0.1%, sodium tripolyphosphate 2%, EDTA-2Na 0.01%, citric acid 0.05%, proteinase K 0.2% and the balance to 100% is water.

[0140] Example 3

[0141] This embodiment provides a container bottle cleaning liquid B, including the following components:

[0142] Sodium hypochlorite 3%, sodium alkylbenzene sulfonate 2%, sodium tripolyphosphate 0.1%, EDTA-2Na 0.03%, citric acid 0.03%, proteinase K 0.1% and the balance to 100% is water.

[0143] Example 4

[0144] This embodiment also provides a container bottle cleaning liquid C, including the following components:

[0145] Sodium hypochlorite 1.5%, sodium alkylbenzene sulfonate 1%, sodium tripolyphosphate 0.8%, EDTA-2Na 0.05%, citric acid 0.01%, proteinase K 0.5% and the balance to 100% is water.

[0146] Example 5

[0147] This embodiment provides a method for cleaning a container bottle, comprising the following steps:

[0148] Prepare a cleaning solution using the container bottle cleaning solution a in Example 2;

[0149] Prepare water containing sweat and dandruff (the source is the collected sweat and hand washing water diluted), i.e. contaminated water;

[0150] Soak the container bottle in the contaminated water for 2 hours, take it out and put it in an oven to dry;

[0151] Place the dried container bottle into an ultrasonic cleaning machine, pour in the container cleaning solution, ensure that the inside of the container bottle is filled with the container cleaning solution, and ultrasonicate for 30 minutes.

[0152] After the ultrasonic treatment, replace the new container cleaning solution to ensure that the container bottle is filled with the new container cleaning solution, soak for 15 hours, rinse the soaked container bottle with tap water 10 times, and then rinse with running purified water 10 times, drain the water in the container bottle, and there should be no dust or obvious stains. Place the cleaned container bottle in a 50℃ dryer for 5 hours, and after drying, pack the container bottle in a ziplock bag and use it within 3 days to avoid contamination caused by long-term storage.

[0153] Since SCC testing is easily contaminated by saliva, sweat, and dander, you should wear clean work clothes and a work hat before operation, and your hair, mouth, nose, and personal clothing should not be exposed. You should also clean your hands. At the same time, the preparation of container cleaning fluid and the cleaning of container bottles should be carried out in a fixed cleaning room to avoid contact with acidic solutions to produce toxic gases.

[0154] The above steps were repeated, but the difference was that different cleaning solutions were used for cleaning, namely, the container bottle cleaning solution b in Example 3, the container bottle cleaning solution c in Example 4, anhydrous ethanol, 0.3% sodium hypochlorite aqueous solution, 3% sodium hypochlorite aqueous solution, and cleaning solution (special for the instrument, purchased from Dirui Medical Technology Co., Ltd.) were used for cleaning.

[0155] Take the reagents in the SCC detection kit in Example 1 (including reagent R1, reagent R2, SCC calibrator, and SCC quality control), use some container bottles as reagent bottles for filling reagent R1 and reagent R2 (reagent R1 and reagent R2 are placed in the same reagent bottle), use some container bottles as calibration quality control bottles (i.e., calibration bottles and quality control bottles, respectively), for filling SCC calibrator and SCC quality control, and fill the above reagents with reagent bottles and calibration quality control bottles, respectively, to obtain an SCC detection kit. And use container bottles that are not cleaned to fill the reagents in the SCC detection kit as a control group.

[0156] test:

[0157] (1) Reagent performance evaluation:

[0158] The SCC test kits cleaned with different cleaning solutions were used to test the primary calibrator, Bio-Rad quality control, and clinical samples. The results are shown in Tables 1 and 2. The 0-value light intensity of the container bottle measured after being treated with 1% hydrochloric acid, anhydrous ethanol, and cleaning solution was high, resulting in inaccurate low-value measurements. The 0-value of the container bottle test results after being treated with 0.3% sodium hypochlorite and 3% sodium hypochlorite, as well as container bottle cleaning solution a, container bottle cleaning solution b, and container bottle cleaning solution c was low, and the low-value samples were less different from the competing products.

[0159] Table 1. Results of reagent performance evaluation (Part I)

[0160]

[0161] Table 2. Reagent evaluation results (Part II)

[0162]

[0163] (2) Bottle-to-bottle performance evaluation

[0164] Test the inter-bottle performance of the reagent bottles and calibration quality control bottles separately, specifically:

[0165] Reagent bottle room: select 10 bottles of each of the reagent bottles treated with container bottle cleaning solution a, container bottle cleaning solution b, container bottle cleaning solution c, 1% hydrochloric acid, anhydrous ethanol, 0.3% sodium hypochlorite aqueous solution, 3% sodium hypochlorite solution, and cleaning solution (special for the instrument), and test a bottle of calibration quality control product at the same time. The results are shown in Table 3. There is no obvious difference in the test H (calibrator high value), C1 (quality control level 1) and C2 (quality control level 2) points of several reagent bottles. The zero value of the test L (calibrator low value) point is 1% hydrochloric acid, The reagents treated with anhydrous ethanol There are obvious differences in the measured zero values ​​of the bottles, and the CV is relatively large; the light intensity at point L of the reagent bottles treated with container bottle cleaning liquid a, container bottle cleaning liquid b, container bottle cleaning liquid c, 0.3% sodium hypochlorite, and 3% sodium hypochlorite is relatively stable, but when testing the zero value of point L, the CV of the reagent bottles treated with 0.3% sodium hypochlorite and 3% sodium hypochlorite is larger than that of the reagent bottles treated with container bottle cleaning liquid a, container bottle cleaning liquid b, and container bottle cleaning liquid c, indicating that the container bottle cleaning liquid provided by the present invention has a better cleaning effect than using sodium hypochlorite alone.

[0166] Table 3. Performance results of reagent bottles

[0167]

[0168]

[0169] Calibration bottle and quality control bottle room: select 10 calibration bottles and quality control bottles treated with container bottle cleaning solution a, container bottle cleaning solution b, container bottle cleaning solution c, 1% hydrochloric acid, anhydrous ethanol, 0.3% sodium hypochlorite, 3% sodium hypochlorite, and cleaning solution (special for instrument), and use a bottle of qualified reagent for testing. The results are shown in Table 4.

[0170] There was no significant difference in the H, C1, and C2 points of the test of several calibration bottles and quality control bottles. The zero value of the calibration bottles treated with 1% hydrochloric acid, anhydrous ethanol, and cleaning solution at the L point was significantly different; the light intensity at the L point of the calibration bottles treated with container bottle cleaning solution a, container bottle cleaning solution b, container bottle cleaning solution c, 0.3% sodium hypochlorite, and 3% sodium hypochlorite was relatively stable. Although it was found that the use of sodium hypochlorite alone can also improve the cleanliness of the reagent bottle to a certain extent, the blank light intensity of individual calibration bottles was close to 1000. The container bottle cleaning solution containing surfactants, builders, chelating agents, complexing agents, and protein inhibitors has a better cleaning effect. Specifically, as shown in Table 4, the L point zero value CV of the calibrant tested after cleaning with 3% sodium hypochlorite alone can reach 23.83%, and the blank light quantity of some calibrant bottles is close to 1000; while the blank light quantities of the calibrant bottles tested after cleaning with container bottle cleaning solution a, container bottle cleaning solution b and container bottle cleaning solution c are all below 500, and the CV is significantly improved, indicating that container cleaning solutions a, b and c can effectively enhance the container cleaning effect.

[0171] Table 4. Evaluation results of calibration quality control bottles

[0172]

[0173]

[0174] Example 5

[0175] The container bottles were cleaned with the container bottle cleaning solution c in Example 4 (the method is shown in Example 2), and the reagents in Example 1 were respectively filled into different cleaned container bottles to obtain an SCC detection kit.

[0176] Evaluation of the performance indicators of the SCC detection kit in Example 5:

[0177] Linear evaluation: The high-value samples close to the upper limit of the linear range were diluted to at least 5 concentrations according to a certain ratio, and the low-value concentration samples must be close to the lower limit of the linear range. The SCC test kit was used to test each concentration of the sample three times, and the average value was calculated. The average value of the measured concentration was fitted with the theoretical concentration or dilution ratio using the least squares method, and the linear correlation coefficient r was calculated. The results are shown in Table 5, and the linear correlation coefficient r is 0.9993.

[0178] Table 5. Linear evaluation results

[0179]

[0180] Blank limit evaluation: The number of photons was measured 20 times for the zero-value calibrator, and the average value minus twice the standard deviation was substituted into the standard curve to obtain the blank limit. The results are shown in Table 6. The blank limit of the SCC detection kit is 0.01 ng / mL.

[0181] Table 6. Evaluation results of blank limit

[0182]

[0183]

[0184] Repeatability evaluation: The same batch of SCC test kits were used to test two samples with low repeatability (5 ng / mL±2 ng / mL) and high repeatability (50 ng / mL±10 ng / mL) 10 times each, and the mean (M) and standard deviation (SD) of the 10 measured concentration results were calculated. The results are shown in Table 7. The CV (coefficient of variation, i.e., the ratio of the standard deviation to the mean) of the 10 tests of the low concentration sample was 1.65%, and the CV of the 10 tests of the high concentration sample was 1.56%.

[0185] Table 7. Repeatability evaluation results

[0186]

[0187]

[0188] Evaluation of biotin interference: The test kit in Example 3 of Chinese invention patent CN109142753A (referred to as the control kit) and the SCC detection kit of the present invention were used to simultaneously test samples with different concentrations of biotin interference, with biotin concentrations of 70 ng / mL, 100 ng / mL, 1000 ng / mL and 3500 ng / mL, respectively. The results are shown in Table 8. The control kit verifies that the interference rate of the 70 ng / mL biotin sample is less than 5%, but as the biotin concentration in the biotin interference sample increases, the interference rate gradually increases, affecting the measured value. The SCC detection kit in the embodiment of the present invention shows a good anti-interference effect, and the biotin interference of each concentration has no obvious interference on the SCC low-value and high-value samples.

[0189] Table 8. Biotin interference results

[0190]

[0191]

[0192] Long-term stability evaluation: The SCC detection kit was stored at 2°C-8°C, and the appearance, blank limit, linearity and repeatability were evaluated at 3 months, 6 months, 9 months, 12 months and 15 months. The results are shown in Table 9. The blank limit, linearity and repeatability of reagents with different validity periods all met the detection requirements, indicating that the reagents in the SCC detection kit have good shelf life stability and are convenient for long-term storage.

[0193] Table 9. Long-term stability evaluation

[0194]

[0195] Thermal stability evaluation: The SCC detection kit within the validity period was placed at 37°C for 3 days, 7 days and 10 days, and then placed in a CM-320 fully automatic chemiluminescence immunoassay analyzer to test the appearance blank limit, linearity and repeatability. The results are shown in Table 10. After the SCC detection kit was placed at high temperature, the blank limit, linearity and repeatability of the test met the detection requirements, indicating that the reagents in the SCC detection kit have good thermal stability and are convenient for transportation.

[0196] Table 10. Thermal stability evaluation

[0197]

[0198]

[0199] It should be understood that the application of the present invention is not limited to the above examples. For ordinary technicians in this field, improvements or changes can be made based on the above description. All these improvements and changes should fall within the scope of protection of the claims attached to the present invention.

Claims

1. A container bottle cleaning liquid, characterized in that: The container bottle cleaning liquid comprises the following components in percentage by weight: Sodium hypochlorite 0.3%-3%, sodium alkylbenzene sulfonate 0.1%-2%, sodium tripolyphosphate 0.1%-2%, chelating agent 0.01%-0.05%, complexing agent 0.01%-0.05%, enzyme inhibitor 0.1%-0.5% and the balance to 100% water.

2. The container bottle cleaning liquid according to claim 1, characterized in that: The chelating agent includes at least one of disodium ethylenediaminetetraacetate, dimercaptopropanol and mercaptoethylamine.

3. The container bottle cleaning liquid according to claim 1 or 2, characterized in that: The complexing agent includes at least one of tartaric acid, citric acid and oxalic acid; And / or, the enzyme inhibitor comprises proteinase K.

4. A method for cleaning a container bottle for a squamous cell carcinoma antigen detection kit, characterized in that: The steps include: Placing a container bottle for a squamous cell carcinoma antigen detection kit in a container bottle cleaning solution, and filling the container bottle with the container bottle cleaning solution, and performing ultrasound for a preset time; Next, replace the container bottle with a new cleaning solution, and fill the container bottle of the squamous cell carcinoma antigen detection kit with the new cleaning solution, soak for 15±2h, rinse with tap water for several times, rinse with running purified water for several times, and then dry at a temperature of 50±5°C, thus completing the cleaning of the container bottle of the squamous cell carcinoma antigen detection kit; Wherein, the container bottle cleaning liquid is the container bottle cleaning liquid or the sodium hypochlorite aqueous solution according to any one of claims 1 to 3, and the mass content of sodium hypochlorite in the sodium hypochlorite aqueous solution is 0.3% to 3%.

5. A method for preparing a squamous cell carcinoma antigen detection kit, characterized in that: The steps include: Providing a container bottle, and cleaning the container bottle using the cleaning method of claim 4 to obtain a cleaned container bottle; Filling the squamous cell carcinoma antigen detection reagents into different cleaned container bottles to obtain the squamous cell carcinoma antigen detection kit; Wherein, the squamous cell carcinoma antigen detection reagent includes reagent R1 and reagent R2; Reagent R1 comprises a complex, wherein the complex comprises streptavidin magnetic particles and a biotin-labeled squamous cell carcinoma antigen-specific mouse monoclonal antibody linked to the streptavidin magnetic particles; Reagent R2 comprises an acridinium ester-labeled squamous cell carcinoma antigen-specific mouse monoclonal antibody.

6. The preparation method according to claim 5, characterized in that: The reagent R1 is prepared by mixing a solid phase premix reagent and a biotin premix reagent, wherein the solid phase premix reagent includes the following components: 10-30 mmol / L of a first buffer, 100-300 mmol / L of salts, and streptavidin magnetic particles with a mass content of 0.01% to 0.08%; The biotin premix reagent includes the following components: 100-400mmol / L of a second buffer, 100-300mmol / L of salts, 0.1%-2% by weight of a protective agent, 0.1%-2% by weight of a preservative, 0.1%-2% by weight of a surfactant, water, and a biotin-labeled squamous cell carcinoma antigen-specific mouse monoclonal antibody; in the biotin-labeled squamous cell carcinoma antigen-specific mouse monoclonal antibody, the molar ratio of the squamous cell carcinoma antigen-specific mouse monoclonal antibody to biotin is 1:5-1:20, and the concentration of the squamous cell carcinoma antigen-specific mouse monoclonal antibody is 0.5-2.0μg / mL; The reagent R2 includes the following components: 100-400mmol / L of a third buffer, 100-300mmol / L of salts, 0.1%-2% by weight of a protective agent, 0.1%-2% by weight of a preservative, 0.1%-2% by weight of a surfactant and water, and acridinium ester-labeled squamous cell carcinoma antigen-specific mouse monoclonal antibody; in the acridinium ester-labeled squamous cell carcinoma antigen-specific mouse monoclonal antibody, the molar ratio of the squamous cell carcinoma antigen-specific mouse monoclonal antibody to the acridinium ester is 1:1-1:10, and the concentration of the squamous cell carcinoma antigen-specific mouse monoclonal antibody is 0.1-0.8μg / mL.

7. The preparation method according to claim 6, characterized in that: The method for preparing a biotin-labeled squamous cell carcinoma antigen-specific mouse monoclonal antibody comprises the following steps: Put squamous cell carcinoma antigen-specific mouse monoclonal antibody into a centrifuge tube, add carbonate buffer, mix well, add biotin, centrifuge, shake and mix well in the dark, add blocking solution, shake and mix well, and collect the blocked biotin-labeled squamous cell carcinoma antigen-specific mouse monoclonal antibody after purification; The method for preparing acridinium ester-labeled squamous cell carcinoma antigen-specific mouse monoclonal antibody comprises the following steps: The squamous cell carcinoma antigen-specific mouse monoclonal antibody is placed in a centrifuge tube, carbonate buffer is added, acridinium ester is added after mixing, centrifugation is performed, oscillation is performed in the dark, blocking solution is added, oscillation is performed, and the blocked acridinium ester-labeled squamous cell carcinoma antigen-specific mouse monoclonal antibody is purified and collected.

8. The preparation method according to claim 6, characterized in that: The squamous cell carcinoma antigen detection reagent also includes a squamous cell carcinoma antigen calibrator and a squamous cell carcinoma antigen quality control product; the squamous cell carcinoma antigen calibrator includes squamous cell carcinoma antigen solutions with concentrations of 0.00 ng / mL and 25 ng / mL, respectively; the squamous cell carcinoma antigen quality control product includes squamous cell carcinoma antigen solutions with concentrations of 5 ng / mL and 25 ng / mL, respectively.

9. The preparation method according to claim 8, characterized in that: The squamous cell carcinoma antigen detection reagent also includes a chemiluminescent excitation liquid, and the chemiluminescent excitation liquid includes an acidic excitation liquid and an alkaline excitation liquid.

10. A squamous cell carcinoma antigen detection kit, characterized in that: The preparation method is described in any one of claims 5 to 9.

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