A squamous cell carcinoma antigen assay kit and use thereof

Abstract

The application belongs to the technical field of biomedicine, and specifically discloses a squamous cell carcinoma antigen determination kit and application, which comprises anti-reagent A, anti-reagent B and magnetic micro-particle reagent, wherein the anti-reagent A is a biotin-labeled squamous cell carcinoma antigen antibody; the anti-reagent B is an alkaline phosphatase-labeled squamous cell carcinoma antigen antibody; the squamous cell carcinoma antigen determination kit can effectively detect the concentration of squamous cell carcinoma antigen (SCC) in serum and plasma samples, and is mainly used for the treatment monitoring of cervical cancer, non-small cell carcinoma and the like, and has a wide application prospect.

Description

A squamous cell carcinoma antigen assay kit and its application Technical Field

[0001] This invention relates to the field of medical testing technology, specifically to a squamous cell carcinoma antigen assay kit and its application. Background Technology

[0002] Squamous cell carcinoma antigen (SCC) is a tumor-associated protein first isolated from squamous cell carcinoma tissue of the cervix in 1977. Its serum levels are widely used for the diagnosis and management of squamous cell carcinoma in various tissues and organs. To date, two genes (SCC Ag-1 and SCC Ag-2) have been identified.

[0003] Clinical studies have found that serum SCC levels and positivity rates in cervical cancer patients were higher before radiotherapy than after radiotherapy. One-way ANOVA showed that elevated serum SCC levels before treatment were correlated with tumor differentiation, lymph node metastasis, and tumor diameter. During carcinogenesis, squamous cell carcinoma tissue grows rapidly and secretes large amounts of SCC, which then spreads into the bloodstream. This is the main reason for the high serum SCC levels and positivity rates in cervical cancer patients before treatment. Conversely, if serum levels remain high after treatment or decrease and then rapidly increase again, it indicates ineffective treatment and may suggest well-differentiated tumors and lymph node metastasis. In non-small cell lung cancer, the median expression levels of squamous cell carcinoma antigen (SCC), carcinoembryonic antigen (CEA), cancer antigen 125 (SCC), and carbohydrate antigen 19-9 (CA19-9) were significantly higher than in the benign control group. Furthermore, SCC levels were associated with chemotherapy efficacy, disease progression, recurrence, and metastasis. Commonly used immunoassay methods in clinical practice include enzyme-linked immunosorbent assay (ELISA) and chemiluminescent immunoassay (CLIA). Summary of the Invention

[0004] This invention provides a squamous cell carcinoma antigen assay kit and its application. The squamous cell carcinoma antigen assay kit of this invention can effectively detect the concentration of squamous cell carcinoma antigen (SCC) in serum and plasma samples. Clinically, it is mainly used for the treatment monitoring of cervical cancer, non-small cell carcinoma, etc., and has broad application prospects.

[0005] The present invention solves its technical problem by adopting the following technical solution:

[0006] A squamous cell carcinoma antigen assay kit includes anti-reagent A, anti-reagent B, and magnetic microparticle reagent;

[0007] The anti-reagent A is a biotin-labeled squamous cell carcinoma antigen antibody;

[0008] The anti-reagent B is an alkaline phosphatase-labeled squamous cell carcinoma antigen antibody.

[0009] As a preferred embodiment of the present invention, the preparation method of the biotin-labeled squamous cell carcinoma antigen antibody is as follows: 100 μg of squamous cell carcinoma antigen antibody, 10-50 μg of biotin, and 200-400 μg of phosphate buffer with a concentration of 0.02-0.1 mol / L are mixed evenly, and then 10-20 μg of K2Cr2O7, 5-20 μg of N-methylpyrrolidone, and 5-20 μg of sodium borohydride are added, stirred evenly, and diluted 100 times with a stabilizing diluent to obtain the biotin-labeled squamous cell carcinoma antigen antibody.

[0010] As a preferred embodiment of the present invention, the method for preparing the alkaline phosphatase-labeled squamous cell carcinoma antigen antibody is as follows: 100 μg of squamous cell carcinoma antigen antibody, 20-40 μg of alkaline phosphatase, and 200-400 μg of phosphate buffer with a concentration of 0.02-0.1 mol / L are mixed evenly, and then 10-20 μg of K2Cr2O7, 5-20 μg of N-methylpyrrolidone, and 5-20 μg of sodium borohydride are added, stirred evenly, and diluted 100 times with a stabilizing diluent to obtain the alkaline phosphatase-labeled squamous cell carcinoma antigen antibody.

[0011] As a preferred embodiment of the present invention, it further includes a magnetic microparticle reagent, wherein the magnetic microparticle reagent is magnetic microparticles coupled with streptavidin. The preparation method of the magnetic microparticles coupled with streptavidin is as follows: 10 mg of zinc oxide iron magnetic microparticles are resuspended in MES buffer, and then 100-200 μL of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide solution with a concentration of 5-15 mg / mL is added, stirred evenly, 0.5-2 mg of streptavidin is added, and the mixture is reacted at room temperature for 1-3 h. After magnetic separation of the supernatant, the mixture is washed, and then a blocking agent is added for blocking. Finally, the volume is adjusted to 500 mL with a stabilizing diluent to obtain magnetic microparticles coupled with streptavidin.

[0012] As a preferred embodiment of the present invention, the stabilizing diluent comprises the following components in parts by weight: 0.5-2% dimethyl silicone oil, 0.1-0.4% fish gelatin, 0.5-1.8% polyvinyl alcohol, 0.2-1% polyvinylpyrrolidone, 0.5-1.5% hydroxyethyl starch, 0.2-1.2% L-cysteine, 0.2-0.8% calcium oxide, 1-4% bovine serum albumin, 0.1-0.5% reduced glutathione, and the balance being phosphate buffer.

[0013] As a preferred embodiment of the present invention, it further includes a first quality control sample and a second quality control sample;

[0014] The first quality control sample is prepared by using an antigen diluent to prepare a squamous cell carcinoma antigen with a concentration of 2-6 ng / mL.

[0015] The second quality control sample is prepared by using an antigen diluent to prepare a squamous cell carcinoma antigen with a concentration of 20-60 ng / mL.

[0016] As a preferred embodiment of the present invention, it further includes a first calibrator, a second calibrator, and a third calibrator;

[0017] The first calibrator is prepared by using an antigen diluent to prepare a squamous cell carcinoma antigen with a concentration of 0 ng / mL.

[0018] The second calibrator is prepared by using an antigen diluent to prepare a solution with a concentration of 0.5-2 ng / mL for squamous cell carcinoma antigen.

[0019] The preparation method of the third calibrator is as follows: squamous cell carcinoma antigen is prepared with antigen dilution solution to a concentration of 8~15ng / mL to obtain the third calibrator.

[0020] As a preferred embodiment of the present invention, the invention further includes a substrate solution, which is an enzyme-catalyzed luminescent substrate. The luminescent substrate is 3-(2-spirodalane)-4-methoxy-4-(3-phosphoryl)-phenyl-1,2-dioxane disodium salt.

[0021] As a preferred embodiment of the present invention, a cleaning solution is also included, wherein the cleaning solution is a Tris buffer solution.

[0022] The present invention also provides the application of a squamous cell carcinoma antigen assay kit in detecting the concentration of squamous cell carcinoma antigen in human serum and / or plasma.

[0023] The beneficial effects of the present invention are as follows: (1) The squamous cell carcinoma antigen assay kit of the present invention can effectively detect the concentration of squamous cell carcinoma antigen (SCC) in serum and plasma samples. It is mainly used in clinical practice for the treatment monitoring of cervical cancer, non-small cell carcinoma, etc., and has a wide range of application prospects; (2) The squamous cell carcinoma antigen assay kit of the present invention has a low detection limit, not higher than 0.10 ng / mL; it has good linearity, and the method is stable, highly precise, reproducible, and homogeneous. Detailed Implementation

[0024] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0025] In this invention, the technical features described in an open-ended manner include both closed-ended technical solutions composed of the listed features and open-ended technical solutions that include the listed features.

[0026] In this invention, numerical ranges are involved. Unless otherwise specified, the numerical ranges are considered continuous and include the minimum and maximum values ​​of the range, as well as every value between the minimum and maximum values. Furthermore, when the range refers to integers, it includes every integer between the minimum and maximum values ​​of the range. Additionally, when multiple ranges are provided to describe features or characteristics, the ranges may be merged. In other words, unless otherwise specified, all ranges disclosed herein should be understood to include any and all subranges to which they are included.

[0027] In this invention, there are no particular limitations on the specific dispersion and stirring methods.

[0028] Unless otherwise specified, all reagents or instruments used in this invention are commercially available conventional products. Unless otherwise specified, the raw materials used in each comparative example and the parallel experiments of each embodiment are the same commercially available products.

[0029] The squamous cell carcinoma antigen assay kit of the present invention uses the sandwich method principle for detection:

[0030] (1) The sample, biotin-labeled SCC monoclonal antibody and alkaline phosphatase-labeled SCC monoclonal antibody are reacted under incubation conditions to form an antibody-antigen-antibody complex and bind to magnetic microparticles through the reaction of biotin and streptavidin.

[0031] (2) Detection and reading: After incubation, a magnetic field is applied to precipitate the material, the supernatant is removed, the precipitate complex is washed with a cleaning solution, and the waste liquid is dried to remove any substances not bound to the magnetic particles. The reaction vessel is then sent into the measurement chamber. The instrument automatically pumps in the chemiluminescent substrate 3-(2-spiroadamantane)-4-methoxy-4-(3-phosphoyl)-phenyl-1,2-dioxane disodium salt (AMPPD). The luminescent substrate generates a chemiluminescent signal under the catalysis of alkaline phosphatase, and the luminescence intensity is measured by an optical detection system.

[0032] (3) The instrument automatically calculates the test results through the working curve.

[0033] The testing instruments can be the POClia chemiluminescence analyzer manufactured by Taizhou Zecheng Biotechnology Co., Ltd., models: POClia 8, POClia minus, POClia plus, POClia auto, or the CIA 600, CIA 1200, CIA 1200M, CIA 1800, CIA 2800 manufactured by Taizhou Zecheng Biotechnology Co., Ltd., or the Shine i1900 and Shine i2000 manufactured by Shenzhen Yingkai Biotechnology Co., Ltd.

[0034] The samples were human serum and plasma collected according to standard operating procedures (heparin sodium anticoagulation, heparin lithium anticoagulation, EDTA anticoagulation).

[0035] Sample collection and processing: (1) Sample collection: Blood samples were collected in accordance with standard operating procedures using collection tubes without anticoagulants or collection tubes containing anticoagulants (sodium heparin anticoagulant, lithium heparin anticoagulant, EDTA anticoagulant).

[0036] (2) Sample processing

[0037] The collected blood samples were left to stand at room temperature, and then the serum or plasma fraction was separated by centrifugation (3000 rpm for 5 minutes).

[0038] If the serum or plasma sample is turbid or contains visible flocculent fibrin, the sample should be centrifuged at 3000 rpm for 15 minutes, and the supernatant should be used.

[0039] Sample Transportation and Preservation: Samples should be centrifuged immediately after blood collection and tested on the same day. If immediate testing is not possible, samples should be stored in cryovials without rubber stoppers. They can be stored for 7 days at 2℃~8℃ or for 6 months at -20±5℃. Repeated freeze-thaw cycles should be avoided. Samples should be transported at -20±5℃ for no more than 5 days. Samples must be equilibrated to room temperature before testing.

[0040] Example 1

[0041] A method for preparing a squamous cell carcinoma antigen assay kit includes the following steps:

[0042] (1) Preparation of anti-reagent A: Mix 100 μg squamous cell carcinoma antigen antibody, 40 μg biotin, and 300 μg phosphate buffer with a concentration of 0.05 mol / L evenly, then add 15 μg K2Cr2O7, 10 μg N-methylpyrrolidone and 12 μg sodium borohydride, stir evenly, and dilute 100 times with a stabilizing diluent to obtain biotin-labeled squamous cell carcinoma antigen antibody.

[0043] (2) Preparation of anti-reagent B: The alkaline phosphatase-labeled squamous cell carcinoma antigen antibody is prepared by mixing 100 μg of squamous cell carcinoma antigen antibody, 30 μg of alkaline phosphatase, and 300 μg of 0.05 mol / L phosphate buffer. Then, add 15 μg of K2Cr2O7, 10 μg of N-methylpyrrolidone, and 12 μg of sodium borohydride, stir evenly, and dilute 100 times with a stabilizing diluent to obtain alkaline phosphatase-labeled squamous cell carcinoma antigen antibody.

[0044] (3) Preparation of magnetic microparticle reagent: 10 mg of zinc oxide iron magnetic microparticles were resuspended in MES buffer (pH 5), and then 150 μL of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide solution with a concentration of 10 mg / mL was added. The mixture was stirred evenly, and 1 mg of streptavidin was added. The mixture was reacted at room temperature for 2 h. After magnetic separation of the supernatant, the supernatant was washed, and then a blocking agent was added to block it. Finally, the volume was adjusted to 500 mL with a stabilizing diluent to obtain magnetic microparticles coupled with streptavidin.

[0045] The blocking agent is a phosphate buffer solution with a pH of 7.5 containing 5% BSA and 0.1% ProClin300.

[0046] (4) The high-value quality control product is prepared by diluting squamous cell carcinoma antigen with antigen diluent to a concentration of 40 ng / mL.

[0047] The low-value quality control material is prepared by diluting squamous cell carcinoma antigen with antigen diluent to a concentration of 5 ng / mL.

[0048] (5) The calibrator H is prepared by diluting squamous cell carcinoma antigen with antigen diluent to a concentration of 0 ng / mL.

[0049] The calibrator M is prepared by diluting squamous cell carcinoma antigen with antigen diluent to a concentration of 1 ng / mL.

[0050] The calibrator L is prepared by diluting squamous cell carcinoma antigen with antigen diluent to a concentration of 10 ng / mL.

[0051] The antigen diluent is a phosphate buffer containing 0.5% BSA and 0.05% Prolin 300.

[0052] (6) The substrate solution is an enzyme-catalyzed luminescent substrate. The luminescent substrate is the disodium salt of 3-(2-spirodalane)-4-methoxy-4-(3-phosphoryl)-phenyl-1,2-dioxane.

[0053] (7) Cleaning solution, wherein the cleaning solution is Tris buffer.

[0054] The stabilizing diluent described in this embodiment comprises the following components in parts by weight: 1% dimethyl silicone oil, 0.2% fish gelatin, 1% polyvinyl alcohol, 0.8% polyvinylpyrrolidone, 1% hydroxyethyl starch, 0.6% L-cysteine, 0.5% calcium oxide, 3% bovine serum albumin, 0.4% reduced glutathione, and the balance being phosphate buffer.

[0055] The concentration of the phosphate buffer mentioned in this invention is 50 mM.

[0056] Each reagent can be prepared according to the specifications in Table 1.

[0057] Table 1

[0058]

[0059] Example 2

[0060] The stabilizing diluent in this embodiment is different from that in Example 1, but everything else is the same.

[0061] The stabilizing diluent comprises the following components in parts by weight: 0.5% dimethyl silicone oil, 0.4% fish gelatin, 0.5% polyvinyl alcohol, 1% polyvinylpyrrolidone, 0.5% hydroxyethyl starch, 1.2% L-cysteine, 0.2% calcium oxide, 1% bovine serum albumin, 0.5% reduced glutathione, and the balance being phosphate buffer.

[0062] Example 3

[0063] The stabilizing diluent in this embodiment is different from that in Example 1, but everything else is the same.

[0064] The stabilizing diluent comprises the following components in parts by weight: 2% dimethyl silicone oil, 0.1% fish gelatin, 1.8% polyvinyl alcohol, 0.2% polyvinylpyrrolidone, 1.5% hydroxyethyl starch, 0.2% L-cysteine, 0.8% calcium oxide, 4% bovine serum albumin, 0.1% reduced glutathione, and the balance being phosphate buffer.

[0065] Comparative Example 1

[0066] Comparative Example 1 uses an equal amount of pure water to replace the stabilizing diluent, while everything else remains the same.

[0067] Comparative Example 2

[0068] The difference between Comparative Example 2 and Example 1 is that the stabilizing diluent is different, but everything else is the same.

[0069] The stabilizing diluent described in this comparative example comprises the following components in parts by weight: 1% polyvinyl alcohol, 0.8% polyvinylpyrrolidone, 1% hydroxyethyl starch, 0.6% L-cysteine, 0.5% calcium oxide, 3% bovine serum albumin, 0.4% reduced glutathione, and the balance being phosphate buffer.

[0070] Comparative Example 3

[0071] The difference between Comparative Example 3 and Example 1 is that the stabilizing diluent is different, but everything else is the same.

[0072] The stabilizing diluent described in this comparative example comprises the following components in parts by weight: 1% dimethyl silicone oil, 0.2% fish gelatin, 1% polyvinyl alcohol, 0.8% polyvinylpyrrolidone, 1% hydroxyethyl starch, 3% bovine serum albumin, 0.4% reduced glutathione, and the balance being phosphate buffer.

[0073] Comparative Example 4

[0074] The difference between Comparative Example 4 and Example 1 is that the stabilizing diluent is different, but everything else is the same.

[0075] The stabilizing diluent described in this comparative example comprises the following components in parts by weight: 0.2% dimethyl silicone oil, 1% fish gelatin, 0.2% polyvinyl alcohol, 0.1% polyvinylpyrrolidone, 2% hydroxyethyl starch, 0.1% L-cysteine, 1% calcium oxide, 5% bovine serum albumin, 0.05% reduced glutathione, and the balance being phosphate buffer.

[0076] Comparative Example 5

[0077] The difference between Comparative Example 5 and Example 1 is that the stabilizing diluent is different, but everything else is the same.

[0078] The stabilizing diluent described in this comparative example comprises the following components in parts by weight: 4% dimethyl silicone oil, 0.1% fish gelatin, 2% polyvinyl alcohol, 2% polyvinylpyrrolidone, 0.2% hydroxyethyl starch, 1.5% L-cysteine, 0.1% calcium oxide, 0.5% bovine serum albumin, 0.8% reduced glutathione, and the balance being phosphate buffer.

[0079] Test case

[0080] 1. Appearance: All components are complete, both inner and outer packaging are intact, labels are clear, and liquid reagents are leak-free; after shaking, the magnetic particle reagent is a uniform suspension with no obvious agglomeration; other solution components are clear and free of foreign matter, precipitates, and flocculent matter.

[0081] 2. Limit of Detection (LOD) Experiment: Using a stable diluent as the sample, perform the test 20 times, obtaining the RLU values ​​(relative luminescence values) of the 20 measurements. Calculate the mean (M) and standard deviation (SD) of these values ​​to obtain M+2SD. Based on the calibration curve equation of the calibrators used in the kit, or based on the concentration-RLU value results between zero concentration and adjacent calibrators, perform a two-point regression fitting to obtain a linear equation. Substitute the RLU value corresponding to M+2SD into the above equation to calculate the corresponding concentration, which is the LOD. The LOD should be ≤0.10 ng / mL.

[0082] The detection limit of Example 1 was 0.01 ng / mL, the detection limit of Example 2 was 0.02 ng / mL, and the detection limit of Example 3 was 0.02 ng / mL.

[0083] The limits of detection (LODs) for Comparative Examples 1–5 were higher than 0.10 ng / mL. Specifically, the LOD for Comparative Example 1 was 0.45 ng / mL, the LOD for Comparative Example 2 was 0.26 ng / mL, the LOD for Comparative Example 3 was 0.25 ng / mL, the LOD for Comparative Example 4 was 0.19 ng / mL, and the LOD for Comparative Example 5 was 0.15 ng / mL.

[0084] 3. Linearity Experiment: Dilute the high-value linear reference material (close to the upper limit of the linear range) at a certain ratio to prepare samples of 5 concentrations, where the low-concentration samples must be close to the lower limit of the linear interval. Follow the kit instructions and repeat the test 3 times for each concentration. Calculate the average value, and fit the average value to the theoretical concentration using the least squares method to obtain a linear relationship. Calculate the linear correlation coefficient r.

[0085] ;

[0086] The results showed that, within the range of 0.15 ng / mL to 70.00 ng / mL, the correlation coefficients (r) of Examples 1 to 3 were not less than 0.9900, which met the requirements.

[0087] The correlation coefficients of comparative examples 1 to 5 are below 0.9900, which does not meet the requirements.

[0088] 4. Accuracy Experiment: Accuracy reference standards with concentrations of (2.00±0.20) ng / mL and (20.00±2.00) ng / mL were selected from the manufacturer for testing. The tests were repeated three times, and the relative deviation was calculated. The calculation formula is:

[0089] ;

[0090] In the formula:

[0091] --Relative deviation;

[0092] --Measurement results;

[0093] --Calibrated concentration.

[0094] The results showed that the relative accuracy deviations of Examples 1-3 and Comparative Examples 2-5 were within ±10.0%.

[0095] The accuracy of Comparative Example 1 does not meet the requirements, being higher than 10%. The accuracy of Comparative Example 1 is 12.5%.

[0096] 5. Repeatability test: Samples at two concentration levels of (2.00±0.20) ng / mL and (20.00±2.00) ng / mL were tested 10 times each, and the average value of the 10 measurements was calculated. and standard deviation Calculate the coefficient of variation using the following formula. .

[0097] ;

[0098] In the formula:

[0099] --Coefficient of variation;

[0100] --Standard deviation of 10 measurements;

[0101] --The average of 10 measurements.

[0102] The results showed that the coefficient of variation (CV) of Examples 1-3 and Comparative Examples 2-5 was no greater than 10.0%.

[0103] The coefficient of variation of Comparative Example 1 does not meet the requirements, as it is higher than 10%. The coefficient of variation of Comparative Example 1 is 11.9%.

[0104] 6. Inter-batch variation test: Samples with concentrations in the range of (2.00±0.20) ng / mL and (20.00±2.00) ng / mL were tested using kits from three different batches, with each test repeated 10 times. The average value of the 30 measurements was calculated. and standard deviation The coefficient of variation is obtained according to the following formula. .

[0105] ;

[0106] In the formula:

[0107] --Coefficient of variation;

[0108] --Standard deviation of 30 measurements;

[0109] --The average of 30 measurements.

[0110] The results showed that the coefficient of variation (CV) of Examples 1-3 and Comparative Examples 1-5 was no greater than 15%.

[0111] 7. Accuracy test: Test the working calibrator separately using reagents from the same batch, repeating 3 times for each bottle.

[0112] ;

[0113] In the formula:

[0114] --Relative deviation;

[0115] --Measure the concentration value;

[0116] -- Target value of the calibrator.

[0117] The results showed that the relative deviations of Examples 1-3 should be within ±10.0%.

[0118] The relative deviations of comparative examples 1 to 5 were higher than 10%, with the relative deviations of comparative example 1 being 14.9%, comparative example 2 being 12.5%, comparative example 3 being 12.2%, comparative example 4 being 11.5%, and comparative example 5 being 10.8%.

[0119] 8. Homogeneity Test: Randomly select 10 or 15 bottles of calibrators from the same batch (excluding zero-concentration calibrators), and measure each bottle once. Calculate the average of the 10 or 15 measurement results using the following formula ( ) and standard deviation ( ); In addition, use one bottle of the above calibrators to perform 10 or 15 consecutive measurements, and calculate the average value of the results ( ) and standard deviation ( ); Calculate the coefficient of variation (CV) for inter-bottle repeatability using the following formulas.

[0120] The calculation formula is:

[0121] ;

[0122] ;

[0123] ;

[0124] ;

[0125] when season

[0126] In the formula

[0127] --Measure the mean;

[0128] S -- Standard deviation;

[0129] n -- Number of measurements;

[0130] --Specify the i-th measurement value of the parameter.

[0131] Note: The sampling rule is that when the total number of samples is ≤500 bottles, the sampling quantity is 10 bottles; when the total number of samples is >500 bottles, the sampling quantity is 15 bottles.

[0132] The results showed that the coefficient of variation (CV) was no greater than 10.0%.

[0133] The results showed that the relative deviations of Examples 1-3 should be within ±10.0%.

[0134] The relative deviations of comparative examples 1 to 5 were higher than 10%, with the relative deviations of comparative example 1 being 15.7%, comparative example 2 being 12.9%, comparative example 3 being 12.5%, comparative example 4 being 11.8%, and comparative example 5 being 11.2%.

[0135] 9. Uniformity Test: Take 10 or 15 bottles each of Level 1 and Level 2 quality control samples from the same batch (randomly numbered 1-10 or 1-15). Test each bottle 3 times on the compatible testing system, using a different order for each measurement. For example, the measurement order could be: 1, 3, 5, 7, 9, 2, 4, 6, 8, 10, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, 2, 4, 6, 8, 10, 1, 3, 5, 7, 9; or 1, 3, 5, 7, 9, 11, 13, 15, 2, 4, 6, 8, 10, 12, 14, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, 2, 4, 6, 8, 10, 12, 14, 1, 3, 5, 7, 9, 11, 13, 15.

[0136] Record the measurement results and calculate F using the following formulas (10)-(20). , and .

[0137] …………(10);

[0138] …………(11);

[0139] …………(12);

[0140] …………(13);

[0141] …………(14);

[0142] …………(15);

[0143] …………(16);

[0144] …………(17);

[0145] …………(18);

[0146] …………(19);

[0147] …………(20);

[0148] In the formula:

[0149] SS -- Variance;

[0150] --Specify parameter number The measurement value or calculation result;

[0151] --Overall average;

[0152] --sample The One result;

[0153] --Degrees of freedom;

[0154] --Degrees of freedom between bottles;

[0155] --Degrees of freedom within the bottle;

[0156] --mean square;

[0157] -- Test value;

[0158] --Total number of tests;

[0159] --No. The number of times the bottle was measured repeatedly;

[0160] --Number of valid measurements;

[0161] --Number of samples drawn;

[0162] --Standard deviation between bottles;

[0163] --Intra-bottle standard deviation (repeatability standard deviation).

[0164] when At that time, with replace calculate The results should meet the requirements of 2.3.4;

[0165] when At that time, the test results showed no significant difference in uniformity between the bottles, and the calculation... The results should meet the requirements of 2.3.4;

[0166] when , When the value is ≤0.3δ, the uniformity between bottles is considered good. Calculation The results should meet the requirements of 2.3.4;

[0167] when , When the value is greater than 0.3δ, the uniformity between bottles is considered poor and does not meet the requirements.

[0168] Note 1: δ is the target standard deviation.

[0169] Note 2: The sampling rule is that when the total number of samples is ≤500 bottles, the sampling quantity is 10 bottles; when the total number of samples is >500 bottles, the sampling quantity is 15 bottles.

[0170] The results showed that the relative deviations of Examples 1-3 should be within ±10.0%.

[0171] The relative deviations of comparative examples 1 to 5 were higher than 10%, with the relative deviations of comparative example 1 being 15.6%, comparative example 2 being 13.1%, comparative example 3 being 12.8%, comparative example 4 being 11.9%, and comparative example 5 being 11.5%.

[0172] Finally, it should be noted that the above embodiments are used to illustrate the technical solutions of the present invention and not to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. A squamous cell carcinoma antigen assay kit, characterized in that, The reagent includes anti-reagent A, anti-reagent B, and magnetic microparticle reagent; anti-reagent A is a biotin-labeled squamous cell carcinoma antigen-antibody; anti-reagent B is an alkaline phosphatase-labeled squamous cell carcinoma antigen-antibody; the preparation method of the biotin-labeled squamous cell carcinoma antigen-antibody is as follows: 100 μg of squamous cell carcinoma antigen-antibody, 10-50 μg of biotin, and 200-400 μg of phosphate buffer with a concentration of 0.02-0.1 mol / L are mixed evenly, and then 10-20 μg of K2Cr2O7 and 5-20 μg of... N-methylpyrrolidone and 5-20 μg sodium borohydride were stirred evenly and diluted 100-fold with a stabilizing diluent to obtain biotin-labeled squamous cell carcinoma antigen antibody; the preparation method of the alkaline phosphatase-labeled squamous cell carcinoma antigen antibody was as follows: 100 μg squamous cell carcinoma antigen antibody, 20-40 μg alkaline phosphatase, and 200-400 μg phosphate buffer with a concentration of 0.02-0.1 mol / L were mixed evenly, and then 10-20 μg K2Cr2O7 and 5-20 μg sodium borohydride were added. N-methylpyrrolidone and 5-20 μg sodium borohydride were stirred until homogeneous and diluted 100-fold with a stabilizing diluent to obtain alkaline phosphatase-labeled squamous cell carcinoma antigen-antibody. The mixture also included magnetic microparticle reagents, which were streptavidin-coupled magnetic microparticles. The preparation method of the streptavidin-coupled magnetic microparticles was as follows: 10 mg of zinc oxide iron magnetic microparticles were resuspended in MES buffer, and then 100-200 μL of a 5-15 mg / mL 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide solution was added and stirred until homogeneous. Add 0.5-2 mg of streptavidin and react at room temperature for 1-3 h. After magnetic separation of the supernatant, wash and then add a blocking agent to block. Finally, adjust the volume to 500 mL with a stabilizing diluent to obtain magnetic microparticles coupled with streptavidin. The stabilizing diluent comprises the following components in parts by weight: 0.5-2% dimethyl silicone oil, 0.1-0.4% fish gelatin, 0.5-1.8% polyvinyl alcohol, 0.2-1% polyvinylpyrrolidone, 0.5-1.5% hydroxyethyl starch, 0.2-1.2% L-cysteine, 0.2-0.8% calcium oxide, 1-4% bovine serum albumin, 0.1-0.5% reduced glutathione, and the balance being phosphate buffer.

2. The squamous cell carcinoma antigen assay kit according to claim 1, characterized in that, It also includes a first quality control and a second quality control; the first quality control is prepared by using an antigen diluent to prepare a squamous cell carcinoma antigen with a concentration of 2-6 ng / mL to obtain the first quality control; the second quality control is prepared by using an antigen diluent to prepare a squamous cell carcinoma antigen with a concentration of 20-60 ng / mL to obtain the second quality control.

3. The squamous cell carcinoma antigen assay kit according to claim 1, characterized in that, It also includes a first calibrator, a second calibrator, and a third calibrator; the first calibrator is prepared by dissolving squamous cell carcinoma antigen in an antigen diluent to a concentration of 0 ng / mL; the second calibrator is prepared by dissolving squamous cell carcinoma antigen in an antigen diluent to a concentration of 0.5~2 ng / mL; the third calibrator is prepared by dissolving squamous cell carcinoma antigen in an antigen diluent to a concentration of 8~15 ng / mL.

4. The squamous cell carcinoma antigen assay kit according to claim 1, characterized in that, It also includes a substrate solution, which is an enzyme-catalyzed luminescent substrate.

5. The squamous cell carcinoma antigen assay kit according to claim 1, characterized in that, It also includes a cleaning solution, which is a Tris buffer solution.

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