Kit, preparation method and application thereof, and method for detecting antigen in sample

Abstract

The invention relates to a kit, a preparation method and application thereof and a method for detecting an antigen in a sample. The kit comprises a kit based on electrochemical luminescence immunoassay, the kit comprises a reagent R1, and the reagent R1 comprises a biotinylated antigen and free biotin. The detection sensitivity and the anti-biotin capability can be improved.

Description

Technical Field

[0001] The present application relates to the field of biotechnology, and particularly relates to a kit, a preparation method thereof, an application, and a method for detecting an antigen in a test sample. Background Art

[0002] Streptavidin (SA) is a secreted protein derived from Streptomyces Avidinii. Its non-specific binding is much lower than that of avidin. Streptavidin exists in the form of a homotetramer, and each mole of the tetramer molecule can bind four moles of biotin molecules. This property combined with the biotin-conjugated antigen / antibody technology is widely used in signal amplification, and the streptavidin-biotin (SA-Biotin) system has an extremely high binding affinity (K = 10^(-15)). This rapid, tight and specific binding can withstand extreme pH values, temperatures, organic solvents and other conditions, making it widely used in the biological field. Using protein conjugation technology to covalently link SA to the surface of a solid support can efficiently bind ligand molecules such as biotinylated antibodies, nucleic acids, and proteins.

[0003] Electrochemiluminescence immunoassay (ECLIA) is a labeled immunoassay method that combines immunoassay technology and electrochemiluminescence. In this assay method, SA is covalently linked to magnetic microspheres, and biotin-labeled antigens, antibodies and other molecules and ruthenium-labeled antigens, antibodies and other molecules are used to detect specific analytes.

[0004] It is generally considered that SA-coated magnetic microspheres are relatively stable and there are no obvious changes affecting the test results during the detection process. However, it has been found in actual applications that about 6% of SA falls off during the storage of SA-coated magnetic microspheres and becomes free SA. For a detection reagent system with an excess of reagents, the change of SA-coated magnetic microspheres does not affect the test results. For a detection reagent system with a limited amount of reagents, a change of about 6% of SA will significantly affect the test results. Therefore, how to effectively reduce free SA and thus reduce the change of SA-coated magnetic microspheres has become a key link in the preparation of high-quality electrochemiluminescence immunoassay reagents.

[0005] Currently, new raw materials with higher sensitivity and stronger anti-interference ability are screened to reduce free SA, but the workload is large, the experimental process is complex, and the newly screened raw materials need to be evaluated for performance and meet relevant requirements; or the SA-coated magnetic microspheres are simply treated (such as washed), but there is still a situation of falling off after treatment, which affects the performance of the kit within the shelf life. Summary of the Invention

[0006] Based on this, an embodiment of the present application provides a kit, a preparation method thereof, an application, and a method for detecting an antigen in a test sample, which can improve the detection sensitivity and anti-biotin ability.

[0007] The technical solution includes:

[0008] A kit, the kit includes a kit based on electrochemiluminescence immunoassay, the kit includes reagent R1, and the reagent R1 includes biotinylated antigen and free biotin.

[0009] In one embodiment, the working concentration of the free biotin is 0.00001 ppm to 0.001 ppm.

[0010] In one embodiment, the working concentration of the free biotin is 0.00008 ppm to 0.00011 ppm.

[0011] In one embodiment, the kit further includes reagent RM and reagent R2, the reagent RM includes SA-coated magnetic microspheres, and the reagent R2 includes ruthenium (II) tris(bipyridine) labeled antibody.

[0012] In one embodiment, the kit meets one or more of the following conditions:

[0013] (1) The working concentration of the biotinylated antigen is 3 ng / mL to 10 ng / mL;

[0014] (2) The working concentration of the SA-coated magnetic microspheres is 0.3 mg / mL to 1.0 mg / mL;

[0015] (3) The working concentration of the ruthenium (II) tris(bipyridine) labeled antibody is 20 ng / mL to 500 ng / mL; and

[0016] (4) The ruthenium (II) tris(bipyridine) labeled antibody includes ruthenium (II) tris(bipyridine) labeled anti-triiodothyronine antibody, ruthenium (II) tris(bipyridine) labeled anti-thyroxine antibody or ruthenium (II) tris(bipyridine) labeled anti-thyroglobulin antibody, and the biotinylated antigen includes biotinylated triiodothyronine, biotinylated thyroxine or biotinylated thyroglobulin.

[0017] In one embodiment, the kit further includes a buffer, and the buffer includes 20 mmol / L to 100 mmol / L of a basic buffer.

[0018] In one embodiment, the basic buffer includes one or more of PBS buffer, Tris buffer, HEPES buffer and MOPS buffer.

[0019] In one embodiment, the buffer further includes one or more of inorganic salts, surfactants, protectants and preservatives;

[0020] In one embodiment, the inorganic salt includes one or more of sodium chloride, sodium sulfate, and potassium chloride.

[0021] In one embodiment, the surfactant includes one or more of Tween 20, PEG6000, and Triton X-100.

[0022] In one embodiment, the protective agent includes one or more of casein, horse serum, human serum albumin, and bovine serum albumin.

[0023] In one embodiment, the preservative includes one or more of proclin 300 and sodium azide.

[0024] In one embodiment, the buffer satisfies one or more of the following conditions:

[0025] a. The mass percentage content of the inorganic salt is 0.1% to 5%;

[0026] b. The mass percentage content of the surfactant is 0.02% to 1%; and,

[0027] c. The mass percentage content of the protective agent is 0.05% to 3%.

[0028] Use of free biotin in the preparation of a kit for electrochemiluminescence immunoassay.

[0029] The preparation method of the kit includes mixing the biotinylated antigen with the free biotin to prepare the reagent R1.

[0030] A method for detecting an antigen in a sample, the method includes using the kit to detect the antigen in the sample.

[0031] In one embodiment, the method includes the following steps:

[0032] Mix the sample to be detected with the reagent R2 and perform the first incubation;

[0033] Add the reagent RM and the reagent R1, mix well, perform the second incubation, measure the luminescence value, and determine the antigen content in the sample according to the luminescence value.

[0034] Compared with the traditional technology, the present application has the following beneficial effects:

[0035] In the process of developing a kit with limited reagents, by adding free biotin to the biotin-labeled antigen reagent of the kit for electrochemiluminescence immunoassay, the ability to resist biotin interference and the detection sensitivity can be simply and effectively improved, and this method is stable and reliable. Detailed implementation mode

[0036] To make the above objects, features, and advantages of the present application more apparent and understandable, the following provides a detailed description of the specific embodiments of the present application. Many specific details are set forth in the following description to facilitate a thorough understanding of the present application. However, the present application can be implemented in many other ways different from those described herein, and those skilled in the art can make similar improvements without departing from the connotation of the present application. Therefore, the present application is not limited by the specific embodiments disclosed below.

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

[0038] The term "and / or" as used herein includes any and all combinations of one or more of the related listed items.

[0039] The "limit of blank (now uniformly named as limit of blank)" herein refers to the highest measurement result that may be observed in a blank sample. This performance is the main performance of in vitro diagnostic kits.

[0040] During the development of reagent-limited kits, the inventors of the present application found that the SA-coated magnetic microspheres used had poor ability in terms of anti-biotin interference, and a small amount of free SA that fell off in the SA-coated magnetic microspheres significantly affected the detection results. The main reason is that during the process of coating SA on the SA magnetic beads, some SA is non-covalently adsorbed on the magnetic beads. As the storage time prolongs, this part of SA falls off to form free SA. In reagent-limited products, due to the binding of some biotinylated antigens to free SA, the amount of biotinylated antigens actually bound to the magnetic beads decreases, resulting in a decrease in sensitivity. Moreover, when there is a small amount of free biotin in the sample, the free biotin in the sample preferentially binds to free SA, making the amount of biotinylated antigens bound to the magnetic beads increase significantly, that is, showing poor anti-biotin interference ability.

[0041] Through research of the present application, it is found that by adding a certain amount of free biotin to the biotin-labeled antigen reaction solution, the ability of anti-biotin interference and sensitivity can be significantly improved.

[0042] Therefore, an embodiment of the present application provides a kit, including reagent R1, and reagent R1 includes biotinylated antigen and free biotin.

[0043] In a specific example, the working concentration of free biotin is 0.00001 ppm to 0.001 ppm. Optionally, the working concentration of free biotin is 0.00001 ppm, 0.00005 ppm, 0.00008 ppm, 0.0001 ppm, 0.00011 ppm, 0.0002 ppm, 0.0005 ppm, 0.0008 ppm, 0.001 ppm, or the range between any two of the above values. In a specific example, the working concentration of free biotin is 0.00008 ppm to 0.00011 ppm.

[0044] In this article, "ppm" refers to the mass percentage content, and 1 ppm = 0.0001%.

[0045] In a specific example, the kit further includes reagent RM and reagent R2. Reagent RM includes SA-coated magnetic microspheres, and reagent R2 includes ruthenium (II) tris(bipyridine) labeled antibody.

[0046] In a specific example, the SA-coated magnetic microspheres include SA magnetic microspheres coupled through carboxyl groups and SA magnetic microspheres coupled through Tosyl (tosyl) groups.

[0047] In a specific example, the working concentration of biotinylated antigen is 3 ng / mL to 10 ng / mL, and can be optionally 3 ng / mL, 4 ng / mL, 5 ng / mL, 6 ng / mL, 7 ng / mL, 8 ng / mL, 9 ng / mL or 10 ng / mL.

[0048] In a specific example, the working concentration of SA-coated magnetic microspheres is 0.3 mg / mL to 1.0 mg / mL, and can be optionally 0.3 mg / mL, 0.4 mg / mL, 0.5 mg / mL, 0.6 mg / mL, 0.7 mg / mL, 0.8 mg / mL, 0.9 mg / mL or 1.0 mg / mL.

[0049] In a specific example, the working concentration of ruthenium (II) tris(bipyridine) labeled antibody is 20 ng / mL to 500 ng / mL, and can be optionally 20 ng / mL, 50 ng / mL, 100 ng / mL, 150 ng / mL, 200 ng / mL, 300 ng / mL, 400 ng / mL or 500 ng / mL.

[0050] In a specific example, the ruthenium (II) tris(bipyridine) labeled antibody includes ruthenium (II) tris(bipyridine) labeled anti-triiodothyronine (T3) antibody, ruthenium (II) tris(bipyridine) labeled anti-thyroxine (T4) antibody or ruthenium (II) tris(bipyridine) labeled anti-thyroglobulin antibody.

[0051] In a specific example, the biotinylated antigen includes biotin-labeled triiodothyronine (T3), biotinylated thyroxine (T4), or biotinylated thyroglobulin.

[0052] In a specific example, the kit further includes a buffer, and the buffer includes a basic buffer.

[0053] In a specific example, the buffer includes 20 mmol / L to 100 mmol / L of the basic buffer. Optionally, the buffer includes 20 mmol / L, 30 mmol / L, 40 mmol / L, 50 mmol / L, 60 mmol / L, 70 mmol / L, 80 mmol / L, 90 mmol / L, or 100 mmol / L of the basic buffer.

[0054] In a specific example, the basic buffer includes one or more of PBS buffer, Tris buffer, HEPES buffer, and MOPS buffer.

[0055] In a specific example, the buffer further includes one or more of inorganic salts, surfactants, protectants, and preservatives.

[0056] In a specific example, the inorganic salts include one or two of sodium chloride, sodium sulfate, and potassium chloride. In a specific example, in the buffer, the mass percentage of the inorganic salts is 0.1% to 5%. Optionally, in the buffer, the mass percentage of the inorganic salts is 0.1%, 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, or the range between any two of the above values.

[0057] In a specific example, the surfactants include one or more of Tween 20, PEG6000, and Triton X-100. In a specific example, in the buffer, the mass percentage of the surfactants is 0.02% to 1%. Optionally, in the buffer, the mass percentage of the surfactants is 0.02%, 0.05%, 0.08%, 0.1%, 0.3%, 0.5%, 0.8%, 1%, or the range between any two of the above values.

[0058] In a specific example, the protectants include one or more of casein, horse serum, human serum albumin, and bovine serum albumin. In a specific example, in the buffer, the mass percentage of the protectants is 0.05% to 3%. Optionally, in the buffer, the mass percentage of the protectants is 0.05%, 0.1%, 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, or the range between any two of the above values.

[0059] In a specific example, the preservative includes at least one of proclin 300 and sodium azide. In a specific example, in the buffer, the mass percentage content of the preservative is 0.05% to 0.5%. Optionally, in the buffer, the mass percentage content of the preservative is 0.05%, 0.1%, 0.2%, 0.3%, 0.4% or 0.5%.

[0060] One embodiment of the present application also provides an application of free biotin in the preparation of a kit for electrochemiluminescence immunoassay. By adding a certain amount of free biotin to the biotin-labeled antigen reaction solution, the ability to resist biotin interference and the sensitivity can be significantly improved simply, at low cost and with high efficiency. Moreover, this method has good stability and little influence on the main performance.

[0061] One embodiment of the present application also provides a preparation method of the kit, including: mixing the biotinylated antigen and the free biotin to prepare reagent R1.

[0062] In a specific example, the biotinylated antigen, free biotin and buffer are mixed to prepare reagent R1. Optionally, after mixing, the concentrations of the biotinylated antigen and free biotin are the working concentrations.

[0063] In a specific example, it further includes the step of preparing reagent RM. In a specific example, the SA-coated magnetic microspheres and buffer are mixed to prepare reagent RM. Optionally, after mixing, the concentration of the SA-coated magnetic microspheres is the working concentration.

[0064] In a specific example, it further includes the step of preparing reagent R2. In a specific example, the ruthenium tris(bipyridine) labeled antibody and buffer are mixed to prepare reagent R2. In a specific example, after mixing, the concentration of the ruthenium tris(bipyridine) labeled antibody is the working concentration.

[0065] One embodiment of the present application also provides a method for detecting an antigen in a sample. The method includes detecting the antigen in the sample using the kit. Electrochemical detection using reagent R1 containing free biotin can reduce background interference, make the detection result more stable and reliable, improve the detection accuracy and sensitivity, and has strong anti-biotin interference ability.

[0066] In a specific example, the method includes the following steps: mixing the sample to be detected with reagent R2, and then performing the first incubation; after incubation, adding reagent RM and reagent R1 and mixing them evenly, then performing the second incubation, measuring the luminescence value, and determining the antigen content in the sample according to the luminescence value.

[0067] An embodiment of the present application also provides a method for improving the sensitivity or anti-biotin interference ability of an electrochemiluminescence kit, including the step of adding free biotin to the reagent containing biotinylated antigen in the electrochemiluminescence kit.

[0068] It should be noted that the electrochemiluminescence kit can be a commercially available product, including a reagent containing biotinylated antigen, SA-coated magnetic microspheres, and a ruthenium tris(bipyridine)-labeled antibody.

[0069] When in use, adding a corresponding concentration of free biotin to the reagent containing biotinylated antigen in the electrochemiluminescence kit (commercially available) can improve the sensitivity or anti-biotin interference ability of the electrochemiluminescence kit.

[0070] The present application ingeniously uses free biotin to reduce the detection background signal, improve the detection sensitivity, and eliminate the interference of free biotin in the sample.

[0071] The following will describe the implementation schemes of the present application in detail with reference to examples. It should be understood that these examples are only used to illustrate the present application and not to limit the scope of the present application. For the experimental methods without specific conditions noted in the following examples, the guidelines given in the present application are preferably referred to, and it is also possible to follow the experimental manuals or conventional conditions in the art, or the conditions recommended by the manufacturer, or refer to the experimental methods known in the art.

[0072] In the following specific examples, for the measurement parameters of raw material components, if not otherwise specified, there may be slight deviations within the weighing accuracy range. For temperature and time parameters, acceptable deviations caused by instrument test accuracy or operation accuracy are allowed.

[0073] Example 1

[0074] 1. Prepare the buffer: The buffer consists of potassium chloride, PEG6000, casein, proclin 300, sucrose, and phosphate (PBS) buffer, where: the mass percentage content of potassium chloride is 2%, the mass percentage content of PEG6000 is 0.1%, the mass percentage content of casein is 0.1%, the mass percentage content of proclin 300 is 0.1%, the mass percentage content of sucrose is 1%, and the concentration of the phosphate buffer is 0.1 mol / L.

[0075] 2. Dilute SA-coated magnetic particles, ruthenium tris(bipyridine)-labeled antibody, and biotinylated antigen to the working concentration (Table 1) with the above buffer, and add 0.00008 ppm of free biotin to the working solution of biotinylated antigen.

[0076] Table 1

[0077]

[0078] 3. Combine the above three working fluids to obtain the required kit product.

[0079] Example 2

[0080] 1. Prepare the buffer: The buffer is composed of sodium chloride, PEG6000, bovine serum albumin, proclin 300 and MOPS buffer. Among them: the mass percentage of sodium chloride is 0.5%, the mass percentage of PEG6000 is 0.08%, the mass percentage of bovine serum albumin is 1%, the mass percentage of proclin 300 is 0.2%, and the concentration of 3-(N-morpholino)propanesulfonic acid (MOPS) buffer is 50 mmol / L.

[0081] 2. Dilute SA-coated magnetic particles, ruthenium-labeled antibody and biotinylated antigen to the working concentration (Table 2) with the above buffer respectively, and add 0.00011 ppm of free biotin to the working fluid of biotinylated antigen. Combine the above three working fluids to obtain the required kit product.

[0082] Table 2

[0083]

[0084] It should be noted that the biotinylated antigen is biotinylated T3, T4 or other biotinylated antigens used in the competitive method; the ruthenium-labeled antibody is ruthenium-labeled anti-T3 antibody, anti-T4 antibody or other corresponding ruthenium-labeled antibodies used in the competitive method. The biotinylated antigen and ruthenium-labeled antibody can be obtained commercially.

[0085] Test

[0086] Use the SE1200 type automatic electrochemiluminescence immunoassay analyzer produced by Jiangsu Sanlian Biological Co., Ltd. to detect the samples. The working process of the SE1200 type automatic electrochemiluminescence immunoassay analyzer is as follows:

[0087] Based on the kit combined in Example 2, the instrument automatically aspirates the quality control sample and ruthenium-labeled antibody into the reaction cup and mixes them evenly. After incubating at 37°C for 9 minutes, the instrument automatically aspirates the SA magnetic particles and biotinylated antigen into the reaction cup and mixes them evenly (the group without adding free biotin is the control group, and the group adding free biotin is the experimental group). After incubating again for 9 minutes, the instrument washes and separates the magnetic particles after the reaction, and performs electrochemiluminescence in the measurement cell to give the analysis result.

[0088] a. Evaluate the minimum detection limit of two groups of total triiodothyronine (TT3) reagents, that is, test zero-concentration samples respectively, repeat the measurement 20 times, calculate the mean M and standard deviation SD of the 20 luminescence values (RLU, RLU is the relative luminescence unit), and evaluate the concentration value corresponding to the RLU value of M - 2SD, which is the minimum detection limit, as shown in Table 3 (the method is from the industry standard of the TT3 kit: "YY / T 1222-2014 Total Triiodothyronine Quantitative Labeled Immunoassay Kit").

[0089] Table 3

[0090]

[0091] b. Evaluate the ability of two groups of free triiodothyronine (FT3) reagents to resist biotin interference, that is, select two samples (concentrations are about 3.15 pmol / L and 35.2 pmol / L) and divide each into two parts, add 50 ng / mL of biotin as experimental samples, and add the corresponding amount of DMSO as control samples. The results are shown in Tables 4 - 5.

[0092] Table 4

[0093]

[0094] Table 5

[0095]

[0096] c. Evaluate the sensitivity (Table 6) and the situation of biotin interference resistance (Table 7) of two groups of reagents for cleaning magnetic beads and adding free biotin after 21 days of acceleration: Use the TT3 reagents before and after acceleration to test zero-concentration samples respectively, repeat the measurement 20 times, calculate the mean M and standard deviation SD of the 20 luminescence values (RLU, RLU is the relative luminescence unit), and evaluate the concentration value corresponding to the RLU value of M - 2SD, which is the minimum detection limit, and the results are shown in Table 4.

[0097] Table 6

[0098]

[0099] Use the FT3 reagents before and after acceleration to test the ability of the reagents to resist biotin interference, that is, select two samples (concentrations are about 3.15 pmol / L and 35.2 pmol / L) and divide each into two parts, add 50 ng / mL of biotin as experimental samples, and add the corresponding amount of DMSO as control samples, and the results are shown in Tables 7-1 to 7-8.

[0100] Tables 7-1 to 7-8 Biotin Interference Resistance Ability (unit: pmol / L)

[0101]

[0102] The situation of falling still exists after simple treatment (such as washing) of the magnetic beads, which may affect the detection performance within the validity period of the kit. However, the detection performance within the validity period of the kit of the present application (including the biotinylated antigen reagent added with free biotin) is not affected.

[0103] The technical features of the above-described embodiments can be combined arbitrarily. For the sake of concise description, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, it should be considered as the scope recorded in this specification.

[0104] The above-described embodiments only express several implementation manners of the present application. The description is relatively specific and detailed, but it cannot be understood as a limitation to the scope of the invention patent. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present application, several deformations and improvements can still be made, and these all belong to the protection scope of the present application. Therefore, the protection scope of the patent of the present application shall be subject to the appended claims, and the specification can be used to interpret the content of the claims.

Claims

1. Kit, characterized in that, The kit includes a kit based on electrochemiluminescence immunoassay, the kit includes reagent R1, and the reagent R1 includes biotinylated antigen and free biotin.

2. The kit according to claim 1, wherein The working concentration of the free biotin is 0.00001 ppm to 0.001 ppm; Optionally, the working concentration of the free biotin is 0.00008 ppm to 0.00011 ppm.

3. The kit according to claim 1 or 2, characterized in that, The kit further includes reagent RM and reagent R2, the reagent RM includes SA-coated magnetic microspheres, and the reagent R2 includes ruthenium (II) tris(bipyridyl) labeled antibody.

4. The kit according to any one of claims 3, characterized in that, The kit meets one or more of the following conditions: (1) The working concentration of the biotinylated antigen is 3 ng / mL to 10 ng / mL; (2) The working concentration of the SA-coated magnetic microspheres is 0.3 mg / mL to 1.0 mg / mL; (3) The working concentration of the ruthenium (II) tris(bipyridyl) labeled antibody is 20 ng / mL to 500 ng / mL; And (4) The ruthenium (II) tris(bipyridyl) labeled antibody includes ruthenium (II) tris(bipyridyl) labeled anti-triiodothyronine antibody, ruthenium (II) tris(bipyridyl) labeled anti-thyroxine antibody or ruthenium (II) tris(bipyridyl) labeled anti-thyroglobulin antibody, and the biotinylated antigen includes biotinylated triiodothyronine, biotinylated thyroxine or biotinylated thyroglobulin.

5. The kit according to any one of claims 1 or 2, characterized in that, The kit further includes a buffer, and the buffer includes 20 mmol / L to 100 mmol / L of a basic buffer; Optionally, the basic buffer includes one or more of PBS buffer, Tris buffer, HEPES buffer and MOPS buffer; Optionally, the buffer further includes one or more of inorganic salts, surfactants, protectants and preservatives; Optionally, the inorganic salts include one or more of sodium chloride, sodium sulfate and potassium chloride; Optionally, the surfactants include one or more of Tween 20, PEG6000 and Triton X-100; Optionally, the protectants include one or more of casein, horse serum, human serum albumin and bovine serum albumin; Optionally, the preservatives include one or more of proclin 300 and sodium azide.

6. The kit according to claim 5, wherein The buffer meets one or more of the following conditions: a. The mass percentage content of the inorganic salt is 0.1% to 5%; b. The mass percentage content of the surfactant is 0.02% to 1%; and, c. The mass percentage content of the protectant is 0.05% to 3%.

7. Application of free biotin in the preparation of a kit for electrochemiluminescence immunoassay.

8. A method for preparing the kit according to any one of claims 1 to 6, characterized in that, It includes mixing the biotinylated antigen with the free biotin to prepare the reagent R1.

9. A method for detecting an antigen in a test sample, characterized in that, The method includes detecting an antigen in a sample by using the kit according to any one of claims 1 to 6.

10. The method according to claim 9, wherein The method includes the following steps: Mixing the sample to be detected with the reagent R2 and performing the first incubation; Adding the reagent RM and the reagent R1, mixing them, performing the second incubation, measuring the luminescence value, and determining the antigen content in the sample according to the luminescence value.

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