A magnetic microparticle chemiluminescence-based carcinoembryonic antigen-related cell adhesion molecule 6 assay kit and its preparation method

Abstract

The present invention discloses a carcinoembryonic antigen-related cell adhesion molecule 6 assay kit based on magnetic microparticle chemiluminescence and a preparation method thereof. The assay kit comprises: R1 reagent: magnetic microparticles coated with streptavidin or CEACAM6 monoclonal antibody, R3 reagent: CEACAM6 monoclonal antibody labeled with acridinium ester, alkaline phosphatase or terpyridine ruthenium, and R4 reagent: CEACAM6 monoclonal antibody labeled with biotin or diluent, wherein the R3 reagent and / or R4 reagent are dissolved in the diluent, and the diluent is a citric acid buffer containing a biosurfactant. In the present invention, the R3 reagent and the R4 reagent contain a nonionic surfactant and / or a biosurfactant, which can enhance the stability of the reagent and further improve the dispersibility of the reagent, thereby improving the sensitivity of the reagent and shortening the reaction time.

Description

Technical Field

[0001] The present invention relates to the field of detection technology, and in particular to a magnetic particle chemiluminescence-based carcinoembryonic antigen-related cell adhesion molecule 6 assay kit and a preparation method thereof. Background Art

[0002] Carcinoembryonic Antigen-related Cell Adhesion Molecule 6 (CEACAM6) is a member of the carcinoembryonic antigen family of immunoglobulin cell adhesion molecules.

[0003] CEACAM6 is linked to the cell membrane through glycosylphosphatidylinositol (GPI), and the encoded protein contains 344 amino acid residues. CEACAM6 is involved in regulating cell processes such as cell adhesion, cell proliferation, angiogenesis, and tumorigenesis. CEACAM6 is highly expressed in tumors, and is involved in promoting tumor cell proliferation, migration, and invasion, and inhibiting their apoptosis. CEACAM6 is an important biological marker for many invasive tumors. CEACAM6 has potential application value in assisting the diagnosis of tumors such as bile duct cancer, pancreatic cancer, gastric cancer, colon cancer, and lung cancer, and in monitoring their development. The latest study found that the level of CEACAM6 in the cerebrospinal fluid of patients with LUAD-LM (lung cancer leptomeningeal metastasis) was significantly higher than that of the normal population. The level of CEACAM6 in serum can better distinguish LM from non-LM in LUAD patients compared with CEA.

[0004] At present, the main method for detecting CEACAM6 is enzyme-linked immunosorbent assay. Enzyme-linked immunosorbent assay has the disadvantages of low sensitivity, complex operation, long reaction time, and large amount of specimens. Therefore, it is very necessary to find a direct, rapid, highly sensitive method with low reagent price and simple operation steps. Summary of the invention

[0005] Purpose of the invention: In order to overcome the deficiencies in the prior art, the present invention provides a magnetic microparticle chemiluminescence-based carcinoembryonic antigen-related cell adhesion molecule 6 assay kit and a preparation method thereof, the kit can quantitatively measure the concentration of CEACAM6 in serum, plasma, whole blood and cerebrospinal fluid, with short detection time, wide linear range, good precision, high sensitivity, high accuracy and good specificity.

[0006] On the one hand, the present invention adopts magnetic particle chemiluminescence immunoassay, which can solve the shortcomings of enzyme-linked immunosorbent assay such as long detection time, narrow linear range, poor precision and low sensitivity; on the other hand, the diluent of R3 reagent and R4 reagent contains biological surfactant, which can enhance the stability of the reagent and further improve the dispersibility of the reagent, thereby improving the sensitivity of the reagent and shortening the reaction time.

[0007] Technical solution: To achieve the above purpose, the technical solution adopted by the present invention is:

[0008] In a first aspect, the present invention provides a magnetic microparticle chemiluminescence-based carcinoembryonic antigen-related cell adhesion molecule 6 assay kit, the assay kit comprising:

[0009] R1 reagent: magnetic particles coated with streptavidin or magnetic particles coated with CEACAM6 monoclonal antibody,

[0010] R3 reagent: CEACAM6 monoclonal antibody labeled with acridinium ester or alkaline phosphatase or terpyridine ruthenium, wherein the R3 reagent is dissolved in a diluent, and

[0011] R4 reagent: diluent. When the R1 reagent is a magnetic particle coated with streptavidin, the R4 reagent also includes a biotin-labeled CEACAM6 monoclonal antibody, wherein:

[0012] The R3 reagent is dissolved in a diluent, and the diluent is a citric acid buffer containing a biosurfactant.

[0013] The diluent used for the R3 reagent and the R4 reagent in the present invention adopts a biosurfactant or a combination thereof with a conventional surfactant because the biosurfactant has the functions of emulsification, solubilization and reducing surface tension, and can enhance the stability of the reagent, which is beneficial to further improve the sensitivity and shorten the reaction time.

[0014] Optionally, in one embodiment of the present invention, the diluent further comprises a citric acid buffer solution of a non-ionic surfactant.

[0015] Optionally, in one embodiment of the present invention, the nonionic surfactant includes one or a combination of two of Tween 20, Tween 80 or polyethylene glycol octylphenyl ether.

[0016] Optionally, in one embodiment of the present invention, the biosurfactant includes one or more of rhamnolipids, sophorolipids, mannosyl erythritol lipids or surfactin.

[0017] Optionally, in one embodiment of the present invention, the concentration of the biosurfactant is 0.05%-0.5% w / v.

[0018] Optionally, in one embodiment of the present invention, the concentration of the non-ionic surfactant is 0.1%-1% v / v.

[0019] Optionally, in one embodiment of the present invention, the concentration of the R3 reagent dissolved in the diluent is 0.05-2.0 μg / mL.

[0020] Optionally, in one embodiment of the present invention, the concentration of the biotin-labeled CEACAM6 monoclonal antibody dissolved in the diluent in the R4 reagent is 0.5-4.0 μg / mL.

[0021] Optionally, in one embodiment of the present invention, the concentration of the citric acid buffer is 50-100 mM, and the pH is 6.0-7.0.

[0022] Optionally, in one embodiment of the present invention, the citrate buffer contains 1%-2% w / v BSA and 50-250 μg / mL blocking agent.

[0023] Further optionally, in one embodiment of the present invention, the R3 reagent is dissolved in a diluent, wherein the citrate buffer contains 50 μg / mL of blocking agent.

[0024] Further optionally, in one embodiment of the present invention, in the diluent of the R4 reagent, the citrate buffer contains 250 μg / mL of blocking agent.

[0025] Optionally, in one embodiment of the present invention, the citrate buffer contains 0.01%-0.1% v / v ProClin 300 preservative.

[0026] Optionally, in one embodiment of the present invention, the particle size of the magnetic microparticles in the R1 reagent is 1.0-3.0 μm.

[0027] Optionally, in one embodiment of the present invention, the R1 reagent is dissolved in a buffer solution at a concentration of 0.5 mg / mL-1.5 mg / mL.

[0028] Optionally, in one embodiment of the present invention, the assay kit further comprises calibrators and quality control products.

[0029] In a second aspect, the present invention provides a method for preparing a magnetic microparticle chemiluminescence-based carcinoembryonic antigen-related cell adhesion molecule 6 assay kit, the method comprising the following steps:

[0030] Prepare R1 reagent,

[0031] Prepare R3 reagent,

[0032] Prepare R4 reagent, wherein

[0033] The R1 reagent is streptavidin-coated magnetic microparticles or CEACAM6 monoclonal antibody-coated magnetic microparticles;

[0034] The preparation of the R3 reagent comprises: coupling the CEACAM6 monoclonal antibody with an acridinium ester to obtain an acridinium ester-labeled CEACAM6 monoclonal antibody, and diluting the antibody with a diluent;

[0035] The preparation of the R4 reagent comprises: a diluent, or when the R1 reagent is a magnetic particle coated with streptavidin, coupling the CEACAM6 monoclonal antibody with biotin to obtain a biotin-labeled CEACAM6 monoclonal antibody, and diluting it with the diluent;

[0036] The diluent is a citric acid buffer containing a biosurfactant.

[0037] Optionally, in one embodiment of the present invention, the concentration of the biosurfactant is 0.05%-0.5% w / v.

[0038] Optionally, in one embodiment of the present invention, the biosurfactant includes one or more of rhamnolipids, sophorolipids, mannosyl erythritol lipids or surfactin.

[0039] Optionally, in one embodiment of the present invention, the diluent further comprises a citric acid buffer solution of a non-ionic surfactant.

[0040] Optionally, in one embodiment of the present invention, the concentration of the non-ionic surfactant is 0.1%-1% v / v.

[0041] Optionally, in one embodiment of the present invention, the nonionic surfactant includes one or a combination of two of Tween 20, Tween 80 or polyethylene glycol octylphenyl ether.

[0042] Optionally, in one embodiment of the present invention, the R3 reagent is diluted to 0.05-2.0 μg / mL with a diluent.

[0043] Optionally, in one embodiment of the present invention, the biotin-labeled CEACAM6 monoclonal antibody is diluted to 0.5-4.0 μg / mL with a diluent.

[0044] Optionally, in one embodiment of the present invention, the concentration of the citric acid buffer is 50-100 mM, and the pH is 6.0-7.0.

[0045] Optionally, in one embodiment of the present invention, the citrate buffer contains 1%-2% w / v BSA and 50-250 μg / mL blocking agent.

[0046] Further optionally, in one embodiment of the present invention, the R3 reagent is dissolved in a diluent, wherein the citrate buffer contains 50 μg / mL of blocking agent.

[0047] Further optionally, in one embodiment of the present invention, in the diluent of the R4 reagent, the citrate buffer contains 250 μg / mL of blocking agent.

[0048] Optionally, in one embodiment of the present invention, the citrate buffer contains 0.01%-0.1% v / v ProClin 300 preservative.

[0049] Optionally, in one embodiment of the present invention, the preparation of R1 reagent includes: adding 0.5-1.5 mg / mL of magnetic particles, 0.05-0.15 mg / mL of streptavidin or CEACAM6 monoclonal antibody, and 10 mg / mL 1-ethyl-(3-dimethylaminopropyl)carbodiimide to a 10-100 mM pH 5-6 2-morpholineethanesulfonic acid solution to prepare magnetic particles coated with streptavidin or CEACAM6 monoclonal antibody, and preparing the magnetic particles coated with streptavidin or CEACAM6 monoclonal antibody at a concentration of 0.5 mg / mL-1.5 mg / mL using 100-200 mM Tris-HCl buffer.

[0050] Optionally, in one embodiment of the present invention, the method further comprises: preparing a calibrator.

[0051] Further optionally, in one embodiment of the present invention, the step of preparing a calibrator comprises: diluting the CEACAM6 antigen into a calibrator using a 50-200 mM PBS buffer containing 1%-2% w / v BSA, 0.3%-0.7% v / v glycerol, 0.05-0.15% v / v Tween 20, and 0.1% v / v ProClin 300 preservative, at a pH of 7.0-7.5.

[0052] Further optionally, in one embodiment of the present invention, the calibrator includes calibrator 1 and calibrator 2, the calibrator 1 is 100.00±20.00 pg / mL, and the calibrator 2 is 5000±1000 pg / mL.

[0053] Optionally, in one embodiment of the present invention, the method further comprises: preparing a quality control product.

[0054] Further optionally, in one embodiment of the present invention, the step of preparing the quality control product includes: diluting the CEACAM6 antigen into a quality control product using 50-200mM PBS buffer containing 1%-2% w / v BSA, 10%-30% v / v newborn calf serum, 0.05-0.15% v / v Tween 20, and 0.1% v / v ProClin300 preservative, with a pH of 7.0-7.5.

[0055] Further optionally, in one embodiment of the present invention, the quality control product includes quality control product 1 and quality control product 2, the quality control product 1 is 100.00±20.00pg / mL, and the quality control product 2 is 5000±1000pg / mL.

[0056] In a third aspect, the present invention provides an assay kit for use in the assay of carcinoembryonic antigen-related cell adhesion molecule 6, wherein the assay kit is an assay kit as described in any one of the above, or an assay kit prepared by the method described in any one of the above.

[0057] In a fourth aspect, the present invention provides a method for detecting carcinoembryonic antigen-related cell adhesion molecule 6 based on magnetic microparticle chemiluminescence, which comprises: adding a sample to be tested to an assay kit for incubation and then testing, wherein the method is not directly intended for diagnosis or treatment of a disease, and the assay kit is an assay kit as described in any one of the above items, or an assay kit prepared by the method described in any one of the above items.

[0058] Optionally, in one embodiment of the present invention, the blank limit of the assay kit is lower than 5.0 pg / mL, and the detection limit is lower than 5.5 pg / mL.

[0059] Further optionally, in one embodiment of the present invention, the blank limit of the assay kit is 4.638 pg / mL and the detection limit is 5.019 pg / mL.

[0060] Optionally, in one embodiment of the present invention, the assay kit achieves an optimal signal-to-noise ratio within 10 minutes.

[0061] Further optionally, in one embodiment of the present invention, the assay kit reaches an optimal signal-to-noise ratio in 6 minutes.

[0062] Optionally, in one embodiment of the present invention, the total coefficient of variation CV of the assay kit is <2%.

[0063] Further optionally, in one embodiment of the present invention, the total coefficient of variation CV of the assay kit is <1.6%.

[0064] Optionally, in one embodiment of the present invention, the inter-day precision of the assay kit is an inter-day total coefficient of variation CV < 5%.

[0065] Further optionally, in one embodiment of the present invention, the inter-day precision of the assay kit is an inter-day total coefficient of variation CV < 3.5%.

[0066] Optionally, in one embodiment of the present invention, the detection method of the carcinoembryonic antigen-related cell adhesion molecule 6 determination kit of the present invention comprises the following steps:

[0067] 1. Aspirate the sample, add R1 reagent: magnetic microparticles coated with streptavidin or CEACAM6 monoclonal antibody, R4 reagent: biotin-labeled CEACAM6 monoclonal antibody or diluent, and R3 reagent: acridinium ester, alkaline phosphatase or terpyridine ruthenium-labeled CEACAM6 monoclonal antibody, incubate for 6 minutes, CEACAM6 in the sample forms an antigen-antibody sandwich complex with biotin-labeled antibody and acridinium ester labeled antibody and binds to the magnetic microparticles through the reaction between biotin and streptavidin / CEACAM6 monoclonal antibody.

[0068] 2. Detection reading: After the incubation, add magnetic field for precipitation, remove the supernatant, wash the precipitated complex with cleaning solution, absorb the waste liquid, remove the substances not bound to the magnetic particles, and then send the reaction cup into the measurement chamber. The instrument automatically pumps in the excitation solution to make the complex produce chemiluminescent signals, and the luminescence intensity is measured by the photomultiplier.

[0069] 3. The instrument automatically calculates the test results through the working curve.

[0070] Beneficial effects: Compared with the traditional enzyme-linked immunosorbent assay, the magnetic particle chemiluminescence immunoassay method used in the present invention has the characteristics of short detection time, wide linear range, good precision, small sample dosage, high sensitivity, high accuracy, good specificity, etc.; the diluent of R3 reagent and R4 reagent adopts a combination of biological surfactants and / or conventional surfactants to further improve the sensitivity and shorten the reaction time. DETAILED DESCRIPTION

[0071] The present invention will be further described below in conjunction with examples. According to the following examples, the present invention can be better understood. However, it is easy for those skilled in the art to understand that the specific material ratios, process conditions and results described in the examples are only used to illustrate the present invention, and should not and will not limit the present invention described in detail in the claims.

[0072] The preferred embodiments of the present invention are described below, using streptavidin-coated magnetic particles as R1 reagent, acridinium ester-labeled CEACAM6 monoclonal antibody as R3 reagent, and a diluent containing biotin-labeled CEACAM6 monoclonal antibody as R4 reagent for illustrative purposes. The preferred embodiments described herein are only used to illustrate and explain the present invention and are not intended to limit the present invention.

[0073] Preparation of a carcinoembryonic antigen-related cell adhesion molecule 6 assay kit for the control group

[0074] This control group is intended to provide a diluent without a biosurfactant as a control, and this diluent only contains a conventional non-ionic surfactant: Tween 20. Specifically, the R3 diluent used in this control group is a 50mM citric acid buffer with a pH of 6.5 containing 1% w / v BSA, 0.5% v / v Tween 20, 0.05% v / v ProClin 300 preservative and 50μg / mL blocking agent; the R4 diluent used in this control group is a 50mM citric acid buffer with a pH of 6.5 containing 1% w / v BSA, 0.5% v / v Tween 20, 0.05% v / v ProClin 300 preservative and 250μg / mL blocking agent.

[0075] (1) Preparation of Streptavidin-coated Magnetic Microparticles (R1 Reagent)

[0076] To 200 mL of 0.01 mol / L 2-morpholineethanesulfonic acid solution (MES solution), add 200 mg of carboxyl magnetic particles and 20 mg of streptavidin (SA) in sequence, react at 37 °C for 30 min, then add 2 mL of 10 mg / mL 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC), react for 2 h, adsorb using a magnetic stand, let stand for 5 min, remove the liquid, add 200 mL of 0.01 mol / L Tris buffer, repeat the above process, wash 3 times in total, and finally dilute to 320 mL with 0.01 mol / L Tris buffer and store at 2-8 °C.

[0077] (2) Preparation of acridinium ester-labeled CEACAM6 monoclonal antibody (R3 reagent)

[0078] The two were mixed at a molar ratio of acridinium ester:CEACAM6 antibody = 10:1, reacted at 37°C for 1 hour, and then dialyzed and purified with phosphate buffer to obtain acridinium ester-labeled CEACAM6 antibody. The antibody was diluted to 0.1 μg / mL with 50 mM citric acid buffer at pH 6.5 containing 1% w / v BSA, 0.5% v / v Tween 20, 0.05% v / v ProClin 300 preservative, and 50 μg / mL blocking agent.

[0079] (3) Preparation of biotin-labeled CEACAM6 monoclonal antibody (R4 reagent)

[0080] The biotin-CEACAM6 antibody was mixed at a molar ratio of 10:1, reacted at 37°C for 30 min, and then dialyzed with phosphate buffer to obtain biotin-labeled CEACAM6 antibody, which was diluted to 1.0 μg / mL with 50 mM citrate buffer at pH 6.5 containing 1% w / v BSA, 0.5% v / v Tween 20, 0.05% v / v ProClin 300 preservative, and 250 μg / mL blocking agent.

[0081] (4) Preparation of calibrators

[0082] CEACAM6 antigen was diluted into calibrator 1 (100.00±20.00 pg / mL) and calibrator 2 (5000±1000 pg / mL) using 100 mM PBS buffer, pH 7.4, containing 2% w / v BSA, 0.5% v / v glycerol, 0.05% v / v Tween 20, 0.1% v / v ProClin 300 preservative.

[0083] (5) Preparation of quality control products

[0084] CEACAM6 antigen was diluted into control 1 (100.00±20.00 pg / mL) and control 2 (5000±1000 pg / mL) using 100 mM PBS buffer, pH 7.4, containing 1% w / v BSA, 10% v / v newborn calf serum, 0.1% v / v Tween 20, and 0.1% v / v ProClin300 preservative.

[0085] Example 1 Preparation of a Carcinoembryonic Antigen-Related Cell Adhesion Molecule 6 Assay Kit

[0086] (1) Preparation of Streptavidin-coated Magnetic Microparticles (R1 Reagent)

[0087] To 200 mL of 0.01 mol / L 2-morpholineethanesulfonic acid solution (MES solution), add 200 mg of carboxyl magnetic particles and 20 mg of streptavidin (SA) in sequence, react at 37 °C for 30 min, then add 2 mL of 10 mg / mL 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC), react for 2 h, adsorb using a magnetic stand, let stand for 5 min, remove the liquid, add 200 mL of 0.01 mol / L Tris buffer, repeat the above process, wash 3 times in total, and finally dilute to 320 mL with 0.01 mol / L Tris buffer and store at 2-8 °C.

[0088] (2) Preparation of acridinium ester-labeled CEACAM6 monoclonal antibody (R3 reagent)

[0089] The two were mixed at a molar ratio of acridinium ester:CEACAM6 antibody = 10:1, reacted at 37°C for 1 hour, and then dialyzed and purified with phosphate buffer to obtain acridinium ester-labeled CEACAM6 antibody. The antibody was diluted to 0.1 μg / mL with 50 mM citrate buffer at pH 6.5 containing 1% w / v BSA, 0.5% v / v Tween 20, 0.05% v / v ProClin 300 preservative, 0.1% w / v rhamnolipid, 0.1% w / v surfactin, and 50 μg / mL blocking agent.

[0090] (3) Preparation of biotin-labeled CEACAM6 monoclonal antibody (R4 reagent)

[0091] The biotin-CEACAM6 antibody was mixed at a molar ratio of 10:1, reacted at 37°C for 30 min, and then dialyzed with phosphate buffer to obtain biotin-labeled CEACAM6 antibody. The antibody was diluted to 1.0 μg / mL in 50 mM citrate buffer at pH 6.5 containing 1% w / v BSA, 0.5% v / v Tween 20, 0.05% v / v ProClin 300 preservative, 0.1% w / v rhamnolipid, 0.1% w / v surfactin, and 250 μg / mL blocking agent.

[0092] (4) Preparation of calibrators

[0093] CEACAM6 antigen was diluted into calibrator 1 (100.00±20.00 pg / mL) and calibrator 2 (5000±1000 pg / mL) using 100 mM PBS buffer, pH 7.4, containing 2% w / v BSA, 0.5% v / v glycerol, 0.05% v / v Tween 20 and 0.1% v / v ProClin 300 preservative.

[0094] (5) Preparation of quality control products

[0095] CEACAM6 antigen was diluted into control 1 (100.00±20.00 pg / mL) and control 2 (5000±1000 pg / mL) using 100 mM PBS buffer, pH 7.4, containing 1% w / v BSA, 10% v / v newborn calf serum, 0.1% v / v Tween 20 and 0.1% v / v ProClin300 preservative.

[0096] (6) Composition of semi-finished products and finished products

[0097] The product obtained in the above steps is a semi-finished product, which is assembled into a carcinoembryonic antigen-related cell adhesion molecule 6 assay kit after master curve determination, calibration product value assignment and quality control product value determination.

[0098] Test example: determination of carcinoembryonic antigen-related cell adhesion molecule 6

[0099] The method for measuring CEACAM6 using the measuring kit prepared in Example 1 is as follows:

[0100] (1) 8 μL of serum, plasma, whole blood or cerebrospinal fluid sample was aspirated, and streptavidin-coated magnetic microparticles, biotin-labeled CEACAM6 monoclonal antibody and acridinium ester-labeled CEACAM6 monoclonal antibody were added and incubated for 6 min. The CEACAM6 protein in the sample formed an antigen-antibody sandwich complex with the biotin-labeled antibody and the acridinium ester-labeled antibody and then bound to the magnetic microparticles through the reaction between biotin and streptavidin.

[0101] (2) Detection reading: After the incubation, the magnetic field is applied to precipitate, the supernatant is removed, the precipitated complex is washed with a cleaning solution, and the waste liquid is aspirated to remove the substances not bound to the magnetic particles, and then the reaction cup is sent to the measurement chamber. The instrument automatically pumps in the excitation solution to make the complex produce a chemiluminescent signal, and the luminescence intensity is measured by a photomultiplier.

[0102] (3) The instrument automatically calculates the test results through the working curve.

[0103] According to the above-mentioned assay method, the following performance evaluation was performed on the assay kit.

[0104] (1) Response time

[0105] Take a batch of control group reagents and Example 1 reagents, set the incubation time to 6 min, 10 min and 15 min respectively, test the blank dilution and the sample, read the luminescence value, and calculate the signal-to-noise ratio.

[0106] The test data of the control group are shown in Table 1, and the test data of Example 1 are shown in Table 2. The results show that the reagent of Example 1 can reach the optimal signal-to-noise ratio in 6 minutes, while the reagent of the control group needs 15 minutes to reach the optimal signal-to-noise ratio.

[0107] Table 1 Test results of different reaction times of control group reagents

[0108]

[0109] Table 2 Test results of different reaction times of reagents in Example 1

[0110]

[0111] (2) Limit of Blank (LOB) and Limit of Detection (LOD) Analysis

[0112] LOB: Test 5 blank samples, repeat the test 4 times for each sample, and test for 3 consecutive days. Perform a normal distribution test on the test data, and use Medcale software to determine whether the data is normally distributed. If the test data is normally distributed, use the parametric analysis method to calculate the blank limit. If the test data is non-normally distributed, use the non-parametric analysis method to calculate the blank limit.

[0113] LOD: Test 5 samples at low concentration levels, repeat the test 4 times for each sample, and test for 3 consecutive days. Perform a normal distribution test on the test data (or its logarithm), use Medcalc software to determine whether the data is normally distributed, and use Medcalc software to perform a variance homogeneity test on the two batches of reagents.

[0114] If the test results of low-concentration samples are normally distributed and have equal variance, the detection limit is calculated using the parametric analysis method. If the data are non-normally distributed, the detection limit is calculated using the non-parametric analysis method.

[0115] Tables 3, 4, 5 and 6 are the test results of the control group reagent, and Tables 7, 8, 9 and 10 are the test results of the Example 1 reagent. The results show that the blank limit of the control group reagent is 14.57 pg / mL, and the detection limit is 15.43 pg / mL; the blank limit of the Example 1 reagent is 4.638 pg / mL, and the detection limit is 5.019 pg / mL. The Example 1 reagent has higher sensitivity.

[0116] Table 3 Test results of blank samples of control group reagents

[0117] Unit: pg / mL The data were in accordance with normal distribution and the blank limit was calculated by parametric method.

[0118] Table 4 Calculation results of blank limit data of control group reagent Unit: pg / mL

[0119] parameter average value 12.93 Standard Deviation 0.992 k 1.653 LoB 14.57

[0120] Table 5 Test results of samples with low concentration level of control group reagent

[0121] Unit: pg / mL

[0122]

[0123] Table 6 Calculation results of the test data of the control group reagent Unit: pg / mL

[0124] median 15.43 Proportion of results smaller than LOB 33.3％ LOD 15.43

[0125] Table 7 Test results of reagent blank sample of Example 1 Unit: pg / mL

[0126]

[0127] The data were in accordance with normal distribution and the blank limit was calculated by parametric method.

[0128] Table 8 Calculation results of reagent blank limit data of Example 1 Unit: pg / mL

[0129] parameter average value 3.812 Standard Deviation 0.5002 k 1.653 LoB 4.638

[0130] Table 9 Test results of low concentration level samples of reagents in Example 1 Unit: pg / mL

[0131]

[0132] Table 10 Calculation results of reagent test data of Example 1 Unit: pg / mL

[0133] median 5.019 Proportion of results smaller than LOB 21.7％ LOD 5.019

[0134] (3) Precision

[0135] (i) Intra-batch precision

[0136] Take a batch of control group reagents and Example 1 reagents, use low and high value quality control products and low and high value cerebrospinal fluid specimens, respectively, test 10 times each, and calculate the coefficient of variation of the measured concentration. The concentration coefficient of variation characterizes the dispersion of the reagent to a certain extent. In the results of Tables 11 and 12, the total coefficient of variation CV characterizing the precision of Example 1 is significantly better than that of the control group, regardless of testing quality control product 1, quality control product 2, cerebrospinal fluid 1 and cerebrospinal fluid 2. This result shows that the reagent in Example 1 has better dispersion and total coefficient of variation CV because of the additional addition of biosurfactants.

[0137] Table 11 Results of intra-batch precision test of control group

[0138] Serial number Quality control product 1 (pg / mL) Quality control product 2 (pg / mL) Cerebrospinal fluid 1 (pg / mL) Cerebrospinal fluid 2 (pg / mL) 1 84.38 14957 78.32 6936 2 88.64 15665 89.18 7039 3 89.50 15807 80.39 7654 4 90.73 16012 91.13 7631 5 83.55 14819 88.77 6821 6 74.53 13320 86.96 7368 7 84.33 14948 91.32 7466 8 85.84 15199 79.32 6812 9 91.64 16163 81.56 6731 10 79.17 14091 90.96 7165 AVG 85.23 15098 85.79 7162 SD 5.346 888.17 5.294 347.63 <![CDATA[CV 批内 ]]> 6.27％ 5.88％ 6.17％ 4.85％

[0139] Table 12 Intra-batch precision test results of Example 1

[0140]

[0141]

[0142] (ii) Inter-day precision

[0143] The control group and the reagent of Example 1 were used to test quality control product 1, quality control product 2, low-value samples and high-value samples, respectively. The test was performed once a day, and each sample was repeated 5 times each time. The test was performed for 5 days in total, and the total coefficient of variation CV was calculated.

[0144] The results in Tables 13 and 14 show that the inter-day precision of the reagent in Example 1 is significantly better than that of the reagent in the control group.

[0145] Table 13 Results of intra-day precision test of control group Unit: pg / mL

[0146]

[0147]

[0148] Table 14 Example 1 Daytime precision test results Unit: pg / mL

[0149]

[0150]

[0151] (4) Opening stability

[0152] After opening the bottles of the reagents of the control group and Example 1, the reagents were placed in a fully automatic chemiluminescence tester (2-8°C), and the corresponding quality control products 1 and 2 were tested respectively, and the tests were performed simultaneously on days 1, 3, 5, 7, 14, 21, 28, and 30 after opening the bottles. The test was repeated 3 times, the average value was taken, and the test results after opening the bottles were compared with the results on day 0 to calculate the relative deviation (%).

[0153] The results in Tables 15 and 16 show that the control group is stable within 14 days after opening the bottle (with a deviation of 5% as the judgment standard), and Example 1 is stable within 30 days after opening the bottle, and the stability of Example 1 is better, which indicates that the biosurfactant added in Example 1 enhances the stability of the reagent.

[0154] In summary, the sensitivity, precision and opening stability of the reagent in Example 1 are better, so the diluent formula of the reagent in Example 1 was selected to further test its performance. The following data are all obtained by testing the diluent formula of the reagent in Example 1 unless otherwise specified.

[0155] Table 15 Results of stability test of control group after opening vial Unit: pg / mL

[0156]

[0157]

[0158] Table 16 Opening stability test results of Example 1 Unit: pg / mL

[0159]

[0160] (5) Accuracy

[0161] A high-value CEACAM6 sample (A) was added to a low-value CEACAM6 sample (B), and the volume ratio between the added sample A and the added sample B was 1:9. The measurement was repeated three times and the results were calculated according to formula (1).

[0162]

[0163] Where:

[0164] R——recovery rate;

[0165] C——the detection concentration of sample A after adding liquid B;

[0166] V0——the volume of sample B;

[0167] V——volume of liquid A added;

[0168] C0——detection concentration of sample B;

[0169] Cs——concentration of liquid A.

[0170] The results in Table 17 show that the accuracy recovery rates of the reagents in Example 1 are all between 85% and 115%, and the accuracy is good.

[0171] Table 17 Accuracy test results Unit: pg / mL

[0172]

[0173] (6) Linear range

[0174] The high and low value samples close to the upper and lower limits of the linear range are mixed in a certain proportion to form at least 5 concentrations, and the average value of the concentration measured in Example 1 and the theoretical concentration are linearly fitted using the least squares method, and the linear correlation coefficient is calculated.

[0175] The results in Table 18 show that the linear range of the kit of Example 1 is between 5.00-25000 pg / mL, and the correlation coefficient is 0.998, which indicates that the linear correlation of the reagent of Example 1 is good within the linear range.

[0176] Table 18 Linear range test results Unit: pg / mL

[0177]

[0178] (7) Specificity

[0179] 1000ng / mL CEA, 5000U / mL CA125, 300U / mL CA15-3, 1000U / mL CA19-9, 300U / mL CA72-4 samples were measured. The above samples were prepared to the specified concentration using sample diluent and measured three times in parallel.

[0180] The results in Table 19 show that the reagent in Example 1 has no obvious cross-reaction with the above-mentioned interferents and has good specificity.

[0181] Table 19 Specificity experimental data

[0182]

[0183] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A magnetic microparticle chemiluminescence-based carcinoembryonic antigen-related cell adhesion molecule 6 assay kit, characterized in that: The assay kit includes: R1 reagent: streptavidin-coated magnetic particles, R3 reagent: acridinium ester-labeled CEACAM6 monoclonal antibody, prepared as follows: acridinium ester:CEACAM6 antibody=10:1 molar ratio was mixed, reacted at 37°C for 1 hour, dialyzed with phosphate buffer to obtain acridinium ester-labeled CEACAM6 antibody, and diluted to 0.1 μg / mL with 50 mM citric acid buffer at pH 6.5 containing 1% w / v BSA, 0.5% v / v Tween 20, 0.05% v / v ProClin 300 preservative, 0.1% w / v rhamnolipid, 0.1% w / v surfactin and 50 μg / mL blocking agent; R4 reagent: includes diluent and biotinylated CEACAM6 monoclonal antibody, The preparation method of the R4 reagent is as follows: biotin:CEACAM6 antibody is mixed at a molar ratio of 10:1, reacted at 37°C for 30 minutes, dialyzed and purified with phosphate buffer to obtain biotin-labeled CEACAM6 antibody, and diluted to 1.0 μg / mL with 50 mM citric acid buffer at pH 6.5 containing 1% w / v BSA, 0.5% v / v Tween 20, 0.05% v / v ProClin 300 preservative, 0.1% w / v rhamnolipid, 0.1% w / v surfactin and 250 μg / mL blocking agent.

2. The assay kit according to claim 1, characterized in that The particle size of the magnetic particles in the R1 reagent is 1.0-3.0 μm.

3. The assay kit according to claim 1, characterized in that The assay kit also includes calibrators and quality control products.

4. A method for preparing a magnetic microparticle chemiluminescence-based carcinoembryonic antigen-related cell adhesion molecule 6 assay kit according to any one of claims 1 to 3, characterized in that: The method comprises the following steps: Prepare R1 reagent, Prepare R3 reagent, Prepare R4 reagent, wherein The R1 reagent is a magnetic particle coated with streptavidin; The preparation of the R3 reagent comprises: coupling the CEACAM6 antibody with an acridinium ester to obtain an acridinium ester-labeled CEACAM6 monoclonal antibody, and diluting the antibody with a diluent; The preparation of the R4 reagent comprises: coupling the CEACAM6 antibody with biotin to obtain a biotin-labeled CEACAM6 monoclonal antibody, and diluting it with a diluent.

5. The method according to claim 4, characterized in that The preparation of the R1 reagent comprises: adding 0.5-1.5 mg / mL magnetic particles, 0.05-0.15 mg / mL streptavidin, and 10 mg / mL 1-ethyl-(3-dimethylaminopropyl)carbodiimide to a 10-100 mM 2-morpholineethanesulfonic acid solution with a pH of 5-6 to prepare magnetic particles coated with streptavidin, and using 100-200 mM Tris-HCl buffer to prepare the concentration of the magnetic particles coated with streptavidin to be 0.5 mg / mL-1.5 mg / mL.

6. The method according to claim 4, characterized in that The method further comprises: Preparation of calibrators, and / or Prepare quality control products.

7. The method according to claim 6, characterized in that The step of preparing the calibrator comprises: diluting the CEACAM6 antigen into the calibrator using a 50-200 mM PBS buffer solution containing 1%-2% w / v BSA, 0.3%-0.7% v / v glycerol, 0.05-0.15% v / v Tween 20 and 0.1% v / v ProClin 300 preservative, with a pH of 7.0-7.

5.

8. The method according to claim 6, characterized in that The step of preparing the quality control product comprises: diluting the CEACAM6 antigen into the quality control product using a 50-200 mM PBS buffer solution containing 1%-2% w / v BSA, 10%-30% v / v newborn calf serum, 0.05-0.15% v / v Tween 20 and 0.1% v / v ProClin 300 preservative, with a pH of 7.0-7.

5.

9. Use of an assay kit in the assay of carcinoembryonic antigen-related cell adhesion molecule 6, wherein the assay kit is the assay kit according to any one of claims 1 to 3, or the assay kit prepared by the method according to any one of claims 5 to 8, and the use is not for the purpose of treating a disease.

10. A method for detecting carcinoembryonic antigen-related cell adhesion molecule 6 based on magnetic microparticle chemiluminescence, characterized in that: It includes: The sample to be tested is added to the assay kit for incubation and then tested. The method is not directly intended for diagnosis or treatment of a disease. The assay kit is an assay kit as described in any one of claims 1 to 3, or an assay kit prepared by the method as described in any one of claims 5 to 8.

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