Kit for determining squamous cell carcinoma antigen and application thereof

Abstract

The invention belongs to the technical field of biomedicine, and particularly discloses a squamous epithelial cell carcinoma antigen determination kit and application, the squamous epithelial cell carcinoma antigen determination kit comprises an anti-reagent A, an anti-reagent B and a magnetic particle reagent, the anti-reagent A is a biotin-labeled squamous epithelial 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, is mainly used for treatment monitoring of cervical cancer, non-small cell carcinoma and the like clinically, and has a wide application prospect.

Description

Technical Field

[0001] The present invention relates to the technical field of medical detection, and in particular to a kit for determining squamous cell carcinoma antigens and its application. Background Art

[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 in 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 positive rates are higher in cervical cancer patients before radiotherapy than after treatment. One-way analysis of variance revealed that elevated pre-treatment serum SCC levels are correlated with tumor differentiation, lymph node metastasis, and tumor diameter. Rapid growth of squamous cell carcinoma tissue during carcinogenesis secretes large amounts of SCC, which spread into the bloodstream. This is the primary reason for the high pre-treatment serum SCC and positive rates in cervical cancer patients. Conversely, if serum levels remain elevated after treatment or decrease and then rapidly rise again, it indicates treatment ineffectiveness and is likely associated with high tumor differentiation 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 (CCC), and carbohydrate antigen 19-9 (CA19-9) are significantly higher than those in benign controls. SCC levels are also associated with chemotherapy efficacy, disease progression, recurrence, and metastasis. Commonly used immunoassays in clinical practice include enzyme-linked immunosorbent assay (ELISA) and chemiluminescent immunoassay (CLIA). Summary of the Invention

[0004] The present invention provides a squamous cell carcinoma antigen assay kit and its application. 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 clinically for treatment monitoring of cervical cancer, non-small cell carcinoma, etc., and has broad application prospects.

[0005] The present invention solves its technical problems by adopting the following technical solutions: A squamous cell carcinoma antigen detection kit, comprising anti-reagent A, anti-reagent B, and magnetic particle reagent; 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; 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 0.02-0.1 mol / L phosphate buffer are mixed uniformly, and then 10-20 μg of K2Cr2O7, 5-20 μg of N-methylpyrrolidone, and 5-20 μg of sodium borohydride are added, and the mixture is stirred uniformly. The mixture is diluted 100-fold with a stable diluent to obtain a biotin-labeled squamous cell carcinoma antigen antibody.

[0006] As a preferred embodiment of the present invention, the preparation method of 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 0.02-0.1 mol / L phosphate buffer are mixed uniformly, and then 10-20 μg of K2Cr2O7, 5-20 μg of N-methylpyrrolidone, and 5-20 μg of sodium borohydride are added, stirred uniformly, and diluted 100-fold with a stable diluent to obtain the alkaline phosphatase-labeled squamous cell carcinoma antigen antibody.

[0007] As a preferred embodiment of the present invention, it also includes a magnetic particle reagent, which is magnetic particles coupled with streptavidin. The preparation method of the magnetic particles coupled with streptavidin is as follows: 10 mg of zinc oxide ferromagnetic particles are resuspended with MES buffer, and then 100-200 μL of 5-15 mg / mL 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide solution is added and stirred evenly. 0.5-2 mg of streptavidin is added and reacted at room temperature for 1-3 hours. The supernatant is magnetically separated and 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 particles coupled with streptavidin.

[0008] As a preferred embodiment of the present invention, the stabilizing diluent comprises the following components in parts by mass: 0.5-2% dimethicone, 0.1-0.4% fish gelatin, 0.5-1.8% polyvinyl alcohol, 0.2-1% polyvinyl pyrrolidone, 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 phosphate buffer.

[0009] As a preferred embodiment of the present invention, it also includes a first quality control product and a second quality control product; The preparation method of the first quality control product is: squamous cell carcinoma antigen is prepared with antigen diluent to a concentration of 2-6 ng / mL to obtain the first quality control product.

[0010] The second quality control product is prepared by preparing the squamous cell carcinoma antigen with an antigen diluent to a concentration of 20-60 ng / mL to obtain the second quality control product.

[0011] As a preferred embodiment of the present invention, it further comprises a first calibrator, a second calibrator and a third calibrator; The first calibrator is prepared by: preparing a squamous cell carcinoma antigen with an antigen diluent to a concentration of 0 ng / mL to obtain a first calibrator; The second calibrator is prepared by: preparing a squamous cell carcinoma antigen with an antigen diluent to a concentration of 0.5-2 ng / mL to obtain a second calibrator; The preparation method of the third calibrator is as follows: squamous cell carcinoma antigen is prepared with antigen diluent to a concentration of 8-15 ng / mL to obtain the third calibrator.

[0012] As a preferred embodiment of the present invention, the method further comprises a substrate solution, wherein the substrate solution is an enzymatic luminescent substrate, wherein the luminescent substrate is 3-(2-spiroadamantane)-4-methoxy-4-(3-phosphoyl)-phenyl-1,2-dioxetane disodium salt.

[0013] As a preferred embodiment of the present invention, it further comprises a cleaning solution, which is a Tris buffer.

[0014] The present invention also provides a use of a squamous cell carcinoma antigen determination kit in detecting the concentration of squamous cell carcinoma antigen in human serum and / or plasma.

[0015] 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, and is mainly used clinically for treatment monitoring of cervical cancer, non-small cell carcinoma, etc., and has broad application prospects; (2) The squamous cell carcinoma antigen assay kit of the present invention has a low detection limit of no more than 0.10 ng / mL; it has good linearity, and the method is stable, with high precision, good reproducibility, and good uniformity. DETAILED DESCRIPTION

[0016] To make the objectives, technical solutions, and advantages of the embodiments of the present invention more clear, the technical solutions in the embodiments of the present invention are clearly and completely described below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. All other embodiments obtained by ordinary technicians in this field based on the embodiments of the present invention without making any creative efforts shall fall within the scope of protection of the present invention.

[0017] In the present invention, the technical features described in an open manner include closed technical solutions composed of the listed features, and also include open technical solutions containing the listed features.

[0018] In the present invention, when referring to numerical ranges, 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 such minimum and maximum values. Furthermore, when a range refers to an integer, every integer between the minimum and maximum values of the range is included. In addition, when multiple ranges are provided to describe a feature or characteristic, the ranges can be combined. In other words, unless otherwise specified, all ranges disclosed herein should be understood to include any and all subranges subsumed therein.

[0019] In the present invention, there is no particular limitation on the specific dispersion and stirring treatment methods.

[0020] The reagents or instruments used in the present invention without indicating the manufacturer are all conventional products that can be obtained commercially. The raw materials used in the comparative examples and the raw materials used in the parallel experiments of the examples are the same commercially available products unless otherwise specified.

[0021] The squamous cell carcinoma antigen detection kit of the present invention adopts the sandwich method principle for detection: (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, which is then bound to the magnetic particles through the reaction of biotin and streptavidin.

[0022] (2) Detection reading: After the incubation is completed, a magnetic field is applied to precipitate the solution, the supernatant is removed, the precipitated complex is washed with a cleaning solution, the waste liquid is aspirated to remove the substances not bound to the magnetic particles, and the reaction cup is then placed into the measurement chamber. The instrument automatically pumps in the chemiluminescent substrate 3-(2-spiroadamantane)-4-methoxy-4-(3-phosphoinoyl)-phenyl-1,2-dioxetane 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.

[0023] (3) The instrument automatically calculates the test results through the working curve.

[0024] The detection instrument can be the POClia chemiluminescence analyzer (models: POClia 8, POClia minus, POClia plus, POClia auto) produced by Taizhou Zecheng Biotechnology Co., Ltd., or the CIA 600, CIA 1200, CIA 1200M, CIA 1800, CIA 2800 produced by Taizhou Zecheng Biotechnology Co., Ltd., or the Shine i1900 and Shine i2000 produced by Shenzhen Yingkai Biotechnology Co., Ltd.

[0025] The samples are human serum and plasma (anticoagulant: sodium heparin, lithium heparin, or EDTA).

[0026] Sample collection and processing: (1) Sample collection: Use collection tubes without anticoagulants or collection tubes containing anticoagulants (sodium heparin anticoagulant, lithium heparin anticoagulant, EDTA anticoagulant) to collect blood samples according to standard procedures.

[0027] (2) Sample processing The collected blood samples were allowed to stand at room temperature and then centrifuged (3000 rpm for 5 minutes) to separate the serum or plasma fraction.

[0028] If the serum or plasma sample is turbid or contains visible flocculent fibrin, centrifuge the sample at 3000 rpm for 15 minutes and use the supernatant.

[0029] Sample transportation and storage: Samples should be centrifuged immediately after blood collection and tested the same day. If immediate testing is not possible, samples should be stored in cryotubes without rubber stoppers at 2°C to 8°C for 7 days and at -20±5°C for 6 months. Avoid repeated freezing and thawing. Samples should be transported at -20±5°C for no more than 5 days. Samples should be equilibrated to room temperature before testing.

[0030] Example 1 A method for preparing a kit for detecting squamous cell carcinoma antigens comprises the following steps: (1) Preparation of anti-reagent A: Mix 100 μg of squamous cell carcinoma antigen antibody, 40 μg of biotin, 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 stable diluent to obtain biotin-labeled squamous cell carcinoma antigen antibody.

[0031] (2) Preparation of anti-reagent B: The preparation method of the alkaline phosphatase-labeled squamous cell carcinoma antigen antibody is as follows: 100 μg of squamous cell carcinoma antigen antibody, 30 μg of alkaline phosphatase, and 300 μg of 0.05 mol / L phosphate buffer are mixed evenly, and then 15 μg of K2Cr2O7, 10 μg of N-methylpyrrolidone and 12 μg of sodium borohydride are added, stirred evenly, and diluted 100 times with a stable diluent to obtain the alkaline phosphatase-labeled squamous cell carcinoma antigen antibody.

[0032] (3) Preparation of magnetic particle reagent: Resuspend 10 mg of zinc oxide ferromagnetic particles with MES buffer (pH 5), then add 150 μL of 10 mg / mL 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide solution, stir evenly, add 1 mg of streptavidin and react at room temperature for 2 h, magnetically separate the supernatant and wash, then add blocking agent to block, and finally dilute to 500 mL with a stable diluent to obtain magnetic particles coupled with streptavidin.

[0033] The blocking agent is a phosphate buffer with a pH of 7.5 containing 5% BSA and 0.1% ProClin300.

[0034] (4) The high-value quality control product is: squamous cell carcinoma antigen is prepared with antigen diluent to a concentration of 40 ng / mL.

[0035] The low-value quality control product is: squamous cell carcinoma antigen is prepared with antigen diluent to a concentration of 5 ng / mL.

[0036] (5) The calibrator H is prepared by using an antigen diluent to prepare squamous cell carcinoma antigen to a concentration of 0 ng / mL.

[0037] The calibrator M is prepared by using squamous cell carcinoma antigen with antigen diluent to prepare a concentration of 1 ng / mL.

[0038] The calibrator L is prepared by using squamous cell carcinoma antigen with antigen diluent to prepare a concentration of 10 ng / mL.

[0039] The antigen diluent is a phosphate buffer solution containing 0.5% BSA and 0.05% prolin300.

[0040] (6) The substrate solution is an enzymatic luminescent substrate, wherein the luminescent substrate is 3-(2-spiroadamantane)-4-methoxy-4-(3-phosphoinoyl)-phenyl-1,2-dioxetane disodium salt.

[0041] (7) A cleaning solution, wherein the cleaning solution is a Tris buffer.

[0042] The stabilizing diluent described in this embodiment includes the following components in parts by weight: 1% dimethyl silicone oil, 0.2% fish gelatin, 1% polyvinyl alcohol, 0.8% polyvinyl pyrrolidone, 1% hydroxyethyl starch, 0.6% L-cysteine, 0.5% calcium oxide, 3% bovine serum albumin, 0.4% reduced glutathione, and the balance phosphate buffer.

[0043] Wherein, the concentration of the phosphate buffer mentioned in the present invention is 50mM.

[0044] Among them, each reagent can be loaded according to the specifications in Table 1.

[0045] Table 1

[0046] Example 2 The stabilizing diluent of this embodiment is different from that of embodiment 1, and the other aspects are the same.

[0047] 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% polyvinyl pyrrolidone, 0.5% hydroxyethyl starch, 1.2% L-cysteine, 0.2% calcium oxide, 1% bovine serum albumin, 0.5% reduced glutathione, and the balance phosphate buffer.

[0048] Example 3 The stabilizing diluent of this embodiment is different from that of embodiment 1, and the other aspects are the same.

[0049] 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% polyvinyl pyrrolidone, 1.5% hydroxyethyl starch, 0.2% L-cysteine, 0.8% calcium oxide, 4% bovine serum albumin, 0.1% reduced glutathione, and the balance phosphate buffer.

[0050] Comparative Example 1 In Comparative Example 1, an equal amount of pure water was used to replace the stabilizing diluent, and all other conditions were the same.

[0051] Comparative Example 2 The difference between Comparative Example 2 and Example 1 is that the stabilizing diluent is different, and all other aspects are the same.

[0052] The stabilizing diluent described in this comparative example includes the following components in parts by mass: 1% polyvinyl alcohol, 0.8% polyvinyl pyrrolidone, 1% hydroxyethyl starch, 0.6% L-cysteine, 0.5% calcium oxide, 3% bovine serum albumin, 0.4% reduced glutathione, and the balance phosphate buffer.

[0053] Comparative Example 3 The difference between Comparative Example 3 and Example 1 is that the stabilizing diluent is different, and all other aspects are the same.

[0054] The stabilizing diluent described in this comparative example includes the following components in parts by mass: 1% dimethyl silicone oil, 0.2% fish gelatin, 1% polyvinyl alcohol, 0.8% polyvinyl pyrrolidone, 1% hydroxyethyl starch, 3% bovine serum albumin, 0.4% reduced glutathione, and the balance phosphate buffer.

[0055] Comparative Example 4 The difference between Comparative Example 4 and Example 1 is that the stabilizing diluent is different, and all other aspects are the same.

[0056] The stabilizing diluent described in this comparative example includes the following components in parts by mass: 0.2% dimethyl silicone oil, 1% fish gelatin, 0.2% polyvinyl alcohol, 0.1% polyvinyl pyrrolidone, 2% hydroxyethyl starch, 0.1% L-cysteine, 1% calcium oxide, 5% bovine serum albumin, 0.05% reduced glutathione, and the balance phosphate buffer.

[0057] Comparative Example 5 The difference between Comparative Example 5 and Example 1 is that the stabilizing diluent is different, and all other aspects are the same.

[0058] The stabilizing diluent described in this comparative example includes the following components in parts by mass: 4% dimethyl silicone oil, 0.1% fish gelatin, 2% polyvinyl alcohol, 2% polyvinyl pyrrolidone, 0.2% hydroxyethyl starch, 1.5% L-cysteine, 0.1% calcium oxide, 0.5% bovine serum albumin, 0.8% reduced glutathione, and the balance phosphate buffer.

[0059] Test Case 1. Appearance: All components are complete, the inner and outer packaging are intact, the labels are clear, and there is no leakage of liquid reagents; after shaking, the magnetic particle reagent is a uniform suspension with no obvious agglomeration; other solution components are clear, without foreign matter, sediment, or flocculent matter.

[0060] 2. Minimum Detection Limit Experiment: Use a stable diluent as the sample for testing, repeat the measurement 20 times, and obtain the RLU values (relative luminescence values) of the 20 measurements. Calculate the mean (M) and standard deviation (SD) 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 the zero concentration and the adjacent calibrators, perform a two-point regression fit 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 minimum detection limit. The minimum detection limit is required to be ≤0.10 ng / mL.

[0061] Among them, the minimum detection limit of Example 1 is 0.01 ng / mL, the detection limit of Example 2 is 0.02 ng / mL, and the detection limit of Example 3 is 0.02 ng / mL.

[0062] The detection limits of comparative examples 1 to 5 were higher than 0.10 ng / mL, among which the detection limit of comparative example 1 was 0.45 ng / mL, the detection limit of comparative example 2 was 0.26 ng / mL, the detection limit of comparative example 3 was 0.25 ng / mL, the detection limit of comparative example 4 was 0.19 ng / mL, and the detection limit of comparative example 5 was 0.15 ng / mL.

[0063] 3. Linearity Test: Dilute a high-value reference sample near the upper limit of the linear range at a specific ratio to create five sample concentrations. The low-value sample concentration must be close to the lower limit of the linear range. Follow the kit instructions and repeat the test three times for each concentration. Calculate the average value. Use the least squares method to fit the average value to the theoretical concentration and calculate the linear correlation coefficient, r.

[0064] ; 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.

[0065] The correlation coefficients of Comparative Examples 1 to 5 were lower than 0.9900, which did not meet the requirements.

[0066] 4. Accuracy test: Select the enterprise accuracy reference materials with concentration values of (2.00±0.20) ng / mL and (20.00±2.00) ng / mL, perform the test, repeat the test 3 times, and calculate the relative deviation. The calculation formula is: ; Where: --relative deviation; --Measurement results; --Calibration concentration.

[0067] The results show that the relative deviations of the accuracy of Examples 1 to 3 and Comparative Examples 2 to 5 are within the range of ±10.0%.

[0068] The accuracy of Comparative Example 1 does not meet the requirement and is higher than 10%. The accuracy of Comparative Example 1 is 12.5%.

[0069] 5. Repeatability test: Use samples with concentrations of (2.00 ± 0.20) ng / mL and (20.00 ± 2.00) ng / mL, repeat the test 10 times, and calculate the average of the 10 measurement results. and standard deviation , calculate the coefficient of variation according to the following formula .

[0070] ;

[0071] Where: --coefficient of variation; --Standard deviation of 10 measurements; --The average value of 10 measurements.

[0072] The results showed that the coefficient of variation (CV) of Examples 1 to 3 and Comparative Examples 2 to 5 was no more than 10.0%.

[0073] The coefficient of variation of comparative example 1 does not meet the requirement and is higher than 10%. The coefficient of variation of comparative example 1 is 11.9%.

[0074] 6. Inter-batch difference experiment: Use three batches of kits to test samples with concentrations within the range of (2.00±0.20) ng / mL and (20.00±2.00) ng / mL, repeat the test 10 times, and calculate the average of the 30 measurement results. and standard deviation The coefficient of variation is obtained according to the following formula .

[0075] ; Where: --coefficient of variation; --Standard deviation of 30 measurements; --The average value of 30 measurements.

[0076] The results showed that the coefficient of variation (CV) of Examples 1 to 3 and Comparative Examples 1 to 5 was no more than 15%.

[0077] 7. Accuracy test: Test the working calibrator with the same batch of reagents separately, repeating each bottle three times.

[0078] ; Where: --relative deviation; --Measurement of concentration value; --Target value of the calibrator.

[0079] The results show that the relative deviations of Examples 1 to 3 should be within the range of ±10.0%.

[0080] The relative deviations of Comparative Examples 1 to 5 are higher than 10%, among which the relative deviation of Comparative Example 1 is 14.9%, the relative deviation of Comparative Example 2 is 12.5%, the relative deviation of Comparative Example 3 is 12.2%, the relative deviation of Comparative Example 4 is 11.5%, and the relative deviation of Comparative Example 5 is 10.8%.

[0081] 8. Homogeneity test: Randomly select 10 or 15 bottles of calibrators (excluding zero-concentration calibrators) of the same batch number, measure each bottle once, and calculate the average value of the 10 or 15 measurement results according to the following formula ( ) and standard deviation ( ); Use one bottle of the above calibration material to measure 10 or 15 times continuously, and calculate the average value of the measurement results ( ) and standard deviation ( ); Calculate the coefficient of variation (CV%) of repeatability between bottles according to the following formulas.

[0082] The calculation formula is: ; ; ; ; when season

[0083] In the formula --Determine the mean; S--standard deviation; n--number of measurements; --Specify the parameter's i-th measurement value.

[0084] Note: The sampling rule is that when the total sample volume is ≤500 bottles, the sampling volume is 10 bottles; when the total sample volume is >500 bottles, the sampling volume is 15 bottles.

[0085] The results showed that the coefficient of variation (CV) was no more than 10.0%.

[0086] The results show that the relative deviations of Examples 1 to 3 should be within the range of ±10.0%.

[0087] The relative deviations of Comparative Examples 1 to 5 are higher than 10%, among which the relative deviation of Comparative Example 1 is 15.7%, the relative deviation of Comparative Example 2 is 12.9%, the relative deviation of Comparative Example 3 is 12.5%, the relative deviation of Comparative Example 4 is 11.8%, and the relative deviation of Comparative Example 5 is 11.2%.

[0088] 9. Homogeneity test: Take 10 or 15 bottles of quality control product level 1 and level 2 from the same batch (randomly numbered 1-10 or 1-15) and test each bottle three times on the adapted detection system. The three measurements should be performed in different orders, for example, the measurement order is: 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.

[0089] Record the measurement results and calculate F, 、 and .

[0090] …………(10); …………(11); …………(12); …………(13); …………(14); …………(15); …………(16); …………(17); …………(18); …………(19); …………(20); Where: SS--variance; --Specify parameter Secondary measurement value or calculation result; --total average value; --sample No. results; -- degrees of freedom; --Freedom between bottles; --Degrees of freedom inside the bottle; -- mean square; -- Test value; --Total number of tests; --No. Number of replicate measurements of the bottle; --Effective measurement times; --Number of samples taken; --Standard deviation between bottles; --Intra-bottle standard deviation (repeatability standard deviation).

[0091] when When replace calculate , the results should meet the requirements of 2.3.4; when When the test results show that there is no significant difference in the uniformity between bottles, calculate , the results should meet the requirements of 2.3.4; when 、 When ≤0.3δ, the uniformity between bottles is considered good and the calculation , the results should meet the requirements of 2.3.4; when 、 When δ is greater than 0.3, it is considered that the uniformity between bottles is poor and does not meet the requirements.

[0092] Note 1: δ is the target standard deviation.

[0093] Note 2: The sampling rule is that when the total sample volume is ≤500 bottles, the sampling volume is 10 bottles; when the total sample volume is >500 bottles, the sampling volume is 15 bottles.

[0094] The results show that the relative deviations of Examples 1 to 3 should be within the range of ±10.0%.

[0095] The relative deviations of Comparative Examples 1 to 5 are higher than 10%, among which the relative deviation of Comparative Example 1 is 15.6%, the relative deviation of Comparative Example 2 is 13.1%, the relative deviation of Comparative Example 3 is 12.8%, the relative deviation of Comparative Example 4 is 11.9%, and the relative deviation of Comparative Example 5 is 11.5%.

[0096] Finally, it should be noted that the above embodiments are intended to illustrate the technical solutions of the present invention rather than 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 the technical solutions of the present invention may be modified or replaced by equivalents without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. A kit for determining squamous cell carcinoma antigen, characterized in that: Including anti-reagent A, anti-reagent B, magnetic particle reagent; 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.

2. The squamous cell carcinoma antigen assay kit according to claim 1, characterized in that The preparation method of the biotin-labeled squamous cell carcinoma antigen antibody comprises: uniformly mixing 100 μg of the squamous cell carcinoma antigen antibody, 10-50 μg of biotin, and 200-400 μg of 0.02-0.1 mol / L phosphate buffer, then adding 10-20 μg of K2Cr2O7, 5-20 μg of N-methylpyrrolidone, and 5-20 μg of sodium borohydride, stirring evenly, and diluting 100 times with a stable diluent to obtain the biotin-labeled squamous cell carcinoma antigen antibody.

3. The squamous cell carcinoma antigen assay kit according to claim 1, characterized in that The preparation method of the alkaline phosphatase-labeled squamous cell carcinoma antigen antibody comprises: uniformly mixing 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, then adding 10-20 μg of K2Cr2O7, 5-20 μg of N-methylpyrrolidone, and 5-20 μg of sodium borohydride, stirring evenly, and diluting 100 times with a stable diluent to obtain the alkaline phosphatase-labeled squamous cell carcinoma antigen antibody.

4. The squamous cell carcinoma antigen assay kit according to claim 1, characterized in that: It also includes a magnetic particle reagent, which is magnetic particles coupled with streptavidin. The preparation method of the magnetic particles coupled with streptavidin is as follows: 10 mg of zinc oxide ferromagnetic particles are resuspended with MES buffer, and then 100-200 μL of 5-15 mg / mL 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide solution is added and stirred evenly. 0.5-2 mg of streptavidin is added and reacted at room temperature for 1-3 hours. The supernatant is magnetically separated and 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 particles coupled with streptavidin.

5. The squamous cell carcinoma antigen assay kit according to claim 1, characterized in that: 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% polyvinyl pyrrolidone, 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 phosphate buffer.

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

7. The squamous cell carcinoma antigen assay kit according to claim 1, characterized in that: Also included are a first calibrator, a second calibrator, and a third calibrator; The first calibrator is prepared by: preparing a squamous cell carcinoma antigen with an antigen diluent to a concentration of 0 ng / mL to obtain a first calibrator; The second calibrator is prepared by: preparing a squamous cell carcinoma antigen with an antigen diluent to a concentration of 0.5-2 ng / mL to obtain a second calibrator; The preparation method of the third calibrator is as follows: squamous cell carcinoma antigen is prepared with antigen diluent to a concentration of 8-15 ng / mL to obtain the third calibrator.

8. The squamous cell carcinoma antigen assay kit according to claim 1, characterized in that The invention also comprises a substrate solution, which is an enzymatic luminescent substrate.

9. The squamous cell carcinoma antigen assay kit according to claim 1, characterized in that: The method further comprises a cleaning solution, which is a Tris buffer solution.

10. Use of the squamous cell carcinoma antigen assay kit according to any one of claims 1 to 9 for detecting the concentration of squamous cell carcinoma antigen in human serum and / or plasma.

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