Chemiluminescence reaction system of ceria-based alkaline phosphatase, chemiluminescence immunoassay kit and application of chemiluminescence reaction system and chemiluminescence immunoassay kit

Abstract

The invention relates to the technical field of immunofluorescence detection reagents, in particular to a chemiluminescence reaction system of cerium dioxide-based alkaline phosphatase, a chemiluminescence immunodetection kit and application of the chemiluminescence reaction system and the chemiluminescence immunodetection kit. The chemical reaction system comprises coated magnetic beads and a growth differentiation factor 15 antibody containing a ceria-based alkaline phosphatase label, and the surface of each coated magnetic bead is coated with an anti-growth differentiation factor 15 antibody and streptavidin; wherein the weight m1 of the coated magnetic beads and the weight m2 of the antibody marked with the ceria-based alkaline phosphatase and used for marking the growth differentiation factor 15 meet the relational expression that m1: m2 is smaller than or equal to 400: 1. According to the chemiluminescence reaction system, ceria-based alkaline phosphatase is used for replacing traditional alkaline phosphatase, and the stability and accuracy of chemiluminescence immunodetection of the growth differentiation factor 15 can be synchronously improved.

Description

Technical Field

[0001] The present application relates to the technical field of immuno-fluorescence detection reagents, and in particular to a chemiluminescence reaction system of cerium dioxide-based alkaline phosphatase-like, a chemiluminescence immunoassay kit and its application. Background Art

[0002] Growth differentiation factor 15 (GDF-15) was initially identified as macrophage inhibitory cytokine 1 or MIC-1. GDF-15 has a conserved interchain disulfide bond and is usually secreted in the form of a dimer protein, consisting of 224 amino acids with a molecular weight of approximately 25 kDa. In most cases, only the mature GDF-15 is secreted, but in some cases, especially in cancer, the full-length protein is not separated from its propeptide and is also secreted from cells. However, different from the mature protein, this unprocessed full-length protein does not circulate in the serum. Existing studies have shown that GDF-15 is an independent inflammatory biochemical marker that can reflect cardiovascular function and diseases, and is closely related to the occurrence and development of acute coronary syndrome (ACS). Currently, the detection techniques related to GDF-15 are mainly latex enhanced immunoturbidimetry and chemiluminescence immunoassay. However, the detection accuracy of the current latex enhanced immunoturbidimetry is poor. Therefore, chemiluminescence immunoassay is currently used to detect GDF-15. However, the enzymes used in traditional chemiluminescence immunoassay are generally natural enzymes, and natural enzymes are sensitive to temperature and pH and are prone to losing activity. In addition, the properties of natural enzymes are unstable and are easily degraded by proteases during the chemiluminescence process and the detection stage. Moreover, the preparation and purification processes of natural enzymes are relatively complex and difficult, which undoubtedly increases the use cost of chemiluminescence immunoassay.

[0003] In view of the above defects, at the present stage, nanozymes with unique physical and chemical properties can be used to make up for the defects of traditional immunoassays. Based on the advantages of easy preparation, low cost, biodegradability, good stability, etc. of nanozymes, using nanozymes instead of natural enzymes can improve the sensitivity and stability of traditional immunoassays. For example, existing studies have confirmed that noble metal nanozymes, metal oxide nanozymes, metal-organic framework nanozymes, and various metal mixed nanozymes can all exhibit alkaline phosphatase-like activity. In addition, nano-materials based on elements such as gold, silver, platinum, and palladium have also been reported to show peroxidase-like activity and can catalyze the colorless substrate of 3,3',5,5'-tetramethylbenzidine. Therefore, the application of nanozymes in chemiluminescence immunoassay can greatly improve the detection accuracy and stability, simplify the operation steps of chemiluminescence immunoassay, and shorten the detection time, thus promoting the development of immunoassay towards a more rapid, sensitive, and specific direction.

[0004] However, currently in the chemiluminescent immunoassay of growth differentiation factor 15, traditional mimic enzymes are generally used. Mimic enzymes are prone to inactivation during the chemiluminescent immunoassay stage, and their catalytic performance is limited, which affects the accuracy of the detection results of chemiluminescent immunoassay.

[0005] In the current detection techniques related to GDF-15, (1) latex enhanced immunoturbidimetry is a commonly used immunoassay method for detecting the concentration of specific antigens or antibodies in serum or body fluids. This technique combines immunoturbidimetry and latex enhancement technology, and generates turbidity changes through the binding reaction of latex particles with antigens or antibodies, thereby indirectly determining the concentration of the target substance. The detection principle of this technique is as follows: during the detection process, the surface of the latex particles will be covered with specific antigens or antibodies. If the target substance exists in the sample, the antigens and antibodies covering the surface of the latex particles will bind to these target substances to form immune complexes. As these immune complexes are generated, the latex particles will aggregate and form a turbid solution. During this process, by measuring the change in the optical density or turbidity of the solution, the concentration of the target substance can be indirectly reflected. Therefore, latex enhanced immunoturbidimetry is a simple, economical and efficient method. It does not require complex instrument equipment and is very convenient for the processing of large-scale samples. Multiple samples can be processed simultaneously, thereby improving the experimental efficiency. In addition, latex enhanced immunoturbidimetry also has high specificity and can be used to detect low concentrations of antigens or antibodies. (2) Chemiluminescent immunoassay detects and analyzes substances by generating visible light through chemical reactions. Existing research shows that the light intensity generated by chemiluminescent immunoassay is relatively high, so very low concentrations of target substances can be detected, improving the detection sensitivity. This makes chemiluminescent immunoassay have advantages in many applications, such as bioanalysis and drug screening. In addition, the high-sensitivity chemiluminescent immunoassay also means that the sample volume can be reduced, thereby saving costs and reducing experimental time. In addition, chemiluminescent immunoassay has a wide linear range, which refers to the concentration range of the target substance that the kit can reliably measure. Therefore, chemiluminescent immunoassay can accurately measure samples with low to high concentrations. This characteristic makes chemiluminescent immunoassay kits very flexible in many applications and can adapt to samples with different concentration ranges. In addition, the reactions involved in the detection process of chemiluminescent immunoassay are usually completed within a few seconds, and the results can be obtained quickly. Therefore, compared with spectroscopic analysis or chromatography, chemiluminescent immunoassay kits can save a large amount of experimental time, which is particularly important for laboratories or clinical laboratories that require high-throughput analysis. For example, the magnetic microsphere electrochemiluminescent immunoassay system currently used combines the high specificity of antibody-antigen reaction with the high sensitivity of ruthenium tris(bipyridine) luminescence. Its principle is to use the photons generated by ruthenium tris(bipyridine) under DBAE to detect the product concentration. Summary of the Invention

[0006] The present application provides a chemiluminescence reaction system based on cerium dioxide-based alkaline phosphatase-like, a chemiluminescence immunoassay kit and its application, so as to solve the following technical problems: how to improve the stability and accuracy of chemiluminescence immunoassay for growth factors synchronously.

[0007] In the first aspect, the present application provides a chemiluminescence reaction system based on cerium dioxide-based alkaline phosphatase-like, and the chemical reaction system includes: coated magnetic beads and growth differentiation factor 15 antibody labeled with cerium dioxide-based alkaline phosphatase-like, and the surface of the coated magnetic beads is coated with anti-growth differentiation factor 15 antibody and streptavidin; wherein, the weight m1 of the coated magnetic beads and the weight m2 of the labeled growth differentiation factor 15 antibody containing cerium dioxide-based alkaline phosphatase-like satisfy the relationship: m1:m2 ≤ 400:1.

[0008] Optionally, the weight m1 of the coated magnetic beads and the weight m2 of the labeled growth differentiation factor 15 antibody containing cerium dioxide-based alkaline phosphatase-like satisfy the relationship: m1:m2 = (400:1) to (200:1).

[0009] Optionally, the weight m3 of the anti-growth differentiation factor 15 antibody and the weight m4 of the streptavidin satisfy the relationship: m3:m4 = (1:1) to (1:5).

[0010] Optionally, the coated magnetic beads include a magnetic bead matrix, and the surface of the magnetic bead matrix is coated with the anti-growth differentiation factor 15 antibody and the streptavidin; the particle size of the magnetic bead matrix is 100 nm to 300 nm.

[0011] In the second aspect, the present application provides a method for preparing the chemiluminescence reaction system described in the first aspect, and the method includes:

[0012] Preparing a biotinylated anti-growth differentiation factor 15 antibody buffer solution using a labeling buffer;

[0013] Preparing a streptavidin magnetic bead mother liquor;

[0014] Mixing the biotinylated anti-growth differentiation factor 15 antibody buffer solution and the streptavidin, and then diluting the mixed solution to obtain coated magnetic beads;

[0015] Activating the cerium dioxide-based alkaline phosphatase-like to obtain activated alkaline phosphatase-like;

[0016] Coupling and blocking reactions are sequentially carried out on the growth differentiation factor 15 antibody and the activated alkaline phosphatase-like to obtain a growth differentiation factor 15 antibody labeled with cerium dioxide-based alkaline phosphatase-like;

[0017] Dispense the coated magnetic beads and the growth differentiation factor 15 antibody labeled with cerium dioxide-based alkaline phosphatase mimetics to obtain a chemiluminescence reaction system.

[0018] Optionally, the amount of substance n1 of the growth differentiation factor 15 antibody and the amount of substance n2 of the activated alkaline phosphatase mimetic satisfy the relational expression: n1:n2 = (1:3) - (1:8).

[0019] In a third aspect, the present application provides a chemiluminescence immunoassay kit, and the chemiluminescence immunoassay kit includes the chemiluminescence reaction system described in the first aspect.

[0020] Optionally, the chemiluminescence immunoassay kit further includes a 1,2-dioxetane derivative.

[0021] In a fourth aspect, the present application provides a chemiluminescence immunoassay method for non-disease diagnosis and treatment purposes, and the chemiluminescence immunoassay method is implemented based on the chemiluminescence immunoassay kit described in the third aspect. The chemiluminescence immunoassay method includes:

[0022] Specifically bind the sample to be tested with the coated magnetic beads to obtain an antibody complex;

[0023] Specifically bind the growth differentiation factor 15 antibody labeled with cerium dioxide-based alkaline phosphatase mimetics and the antibody complex to obtain a binding product;

[0024] Perform a decomposition reaction on the chemiluminescent substrate and the binding product, and detect the photoelectric signal intensity of the decomposition reaction product during the decomposition reaction to obtain the light signal intensity of the binding product;

[0025] Plot a standard curve of the light signal intensity versus the growth differentiation factor 15;

[0026] According to the standard curve and the light signal intensity of the binding product, obtain the mass concentration of the growth differentiation factor 15 in the sample to be tested.

[0027] Optionally, the temperature of the decomposition reaction is 25°C - 37°C, and the time of the decomposition reaction is 10 min - 20 min.

[0028] The above technical solutions provided by the embodiments of the present application have the following advantages compared with the prior art:

[0029] A chemiluminescence reaction system based on cerium dioxide-based alkaline phosphatase-like provided by an embodiment of the present application uses cerium dioxide-based alkaline phosphatase-like to replace traditional alkaline phosphatase in a labeled antibody. The cerium dioxide-based alkaline phosphatase-like has excellent alkaline phosphatase-like activity and will form a large number of ester bonds with the carboxyl group of the growth differentiation factor 15 antibody. These ester bonds will improve the binding stability between the growth differentiation factor 15 antibody and the cerium dioxide-based alkaline phosphatase-like, so that the growth differentiation factor 15 antibody stably exists on the surface of the cerium dioxide-based alkaline phosphatase-like to improve the catalytic activity of the cerium dioxide-based alkaline phosphatase-like. In addition, the weight m1 of the coated magnetic beads and the weight m2 of the labeled growth differentiation factor 15 antibody containing cerium dioxide-based alkaline phosphatase satisfy the relational expression: m1:m2 ≤ 400:1, which can promote the effectively catalytic binding between the cerium dioxide-based alkaline phosphatase-like with improved catalytic activity and the coated magnetic beads, and further improve the sensitivity and accuracy of the chemiluminescence reaction system detection. Therefore, using cerium dioxide-based alkaline phosphatase-like to replace traditional alkaline phosphatase can simultaneously improve the stability and accuracy of chemiluminescence immunoassay for growth factors. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] The accompanying drawings herein are incorporated into the specification and constitute a part of this specification, showing embodiments consistent with the present application and, together with the specification, are used to explain the principles of the present application.

[0031] To more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the following will briefly introduce the accompanying drawings required for use in the description of the embodiments or the prior art. Obviously, for those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.

[0032] Figure 1 It is a product schematic diagram of a chemiluminescence reaction system based on cerium dioxide-based alkaline phosphatase-like provided by an embodiment of the present application; wherein, 1 is the coated magnetic beads, and 2 is the growth differentiation factor 15 antibody labeled with cerium dioxide-based alkaline phosphatase-like.

[0033] Figure 2 It is a schematic flow chart of a method for preparing the chemiluminescence reaction system provided by an embodiment of the present application;

[0034] Figure 3 It is a schematic flow chart of a chemiluminescence immunoassay method for non-disease diagnosis and treatment purposes provided by an embodiment of the present application;

[0035] Figure 4 It is a sample correlation graph of a chemiluminescence immunoassay kit provided by an embodiment of the present application. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0036] To make the objectives, technical solutions, and advantages of the embodiments of this application clearer, the following will clearly and completely describe the technical solutions in the embodiments of this application with reference to the accompanying drawings in the embodiments of this application. Obviously, the described embodiments are some, but not all, of the embodiments of this application. All other embodiments obtained by those of ordinary skill in the art based on the embodiments in this application without creative efforts fall within the scope of protection of this application.

[0037] The various embodiments of this application may exist in the form of a range; it should be understood that the description in the form of a range is only for convenience and brevity and should not be construed as a rigid limitation on the scope of this application; therefore, it should be considered that the described range description has specifically disclosed all possible sub-ranges and individual values within that range; for example, it should be considered that the range description from 1 to 6 has specifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., and individual numbers within that range, such as 1, 2, 3, 4, 5, and 6, and this applies regardless of the range; in addition, whenever a numerical range is indicated herein, it means including any cited number (fraction or integer) within the indicated range.

[0038] In this document, terms including "comprising" etc. mean "including but not limited to". Unless otherwise specifically stated, various raw materials, reagents, instruments, and equipment used in this application can be obtained through market purchases or can be prepared by existing methods.

[0039] It should be noted that regarding the prior art (1) described in the background art, the inventor found that the latex enhanced immunoturbidimetry method also has some disadvantages: 1) The latex enhanced immunoturbidimetry method may have agglutination reactions, and the agglutination reactions may be interfered by other factors, such as the pH value, ionic strength, and temperature of the detection solution, and these factors may lead to inaccurate results of the agglutination reaction; 2) Due to the inconsistent size and shape of the latex particles in the latex enhanced immunoturbidimetry method, it will cause errors in the measurement results. At the same time, the stability of the latex particles is poor, and aggregation or precipitation occurs during long-term storage, affecting the reliability of the detection results; 3) The latex enhanced immunoturbidimetry method may be insensitive to certain specific types of antigens or antibodies, limiting its application in certain detection items.

[0040] Regarding the prior art (2) described in the background art, the inventors found that: Since the catalytic efficiency and activity of enzymes in the chemiluminescence system are extremely important for the sensitivity of immunoassays, however, when antibodies conjugated with alkaline phosphatase specifically bind to antigens, most natural alkaline phosphatases have some inherent deficiencies. For example, these enzymes are sensitive to temperature and pH, are prone to losing activity, and are unstable and easily degraded by proteases. In addition, the preparation and purification of natural alkaline phosphatase are relatively difficult, which undoubtedly increases the cost of chemiluminescent immunoassays.

[0041] Figure 1 Exemplarily shown is a schematic diagram of a product of a chemiluminescence reaction system of a cerium dioxide-based alkaline phosphatase-like provided by an embodiment of the present application;

[0042] As Figure 1 shown, an embodiment of the present application provides a chemiluminescence reaction system of a cerium dioxide-based alkaline phosphatase-like. The chemical reaction system includes: coated magnetic beads and a growth differentiation factor 15 antibody labeled with a cerium dioxide-based alkaline phosphatase-like, a calibrator, and a quality control product. The surface of the coated magnetic beads is coated with an anti-growth differentiation factor 15 antibody and streptavidin; wherein, the weight m1 of the coated magnetic beads and the weight m2 of the labeled growth differentiation factor 15 antibody containing the cerium dioxide-based alkaline phosphatase-like satisfy the relationship: m1:m2 ≤ 400:1.

[0043] It should be noted that the cerium dioxide-based alkaline phosphatase-like can be nanoscale cerium dioxide, and the particle size of the cerium dioxide is 3 nm to 5 nm.

[0044] In some alternative embodiments, the weight m1 of the coated magnetic beads and the weight m2 of the labeled growth differentiation factor 15 antibody containing the cerium dioxide-based alkaline phosphatase-like satisfy the relationship: m1:m2 = (400:1) to (200:1);

[0045] In these embodiments, the weight m1 of the coated magnetic beads and the weight m2 of the labeled growth differentiation factor 15 antibody containing the cerium dioxide-based alkaline phosphatase-like can satisfy the relationship: m1:m2 = (400:1) to (200:1), which can promote the effective catalysis of the binding between the cerium dioxide-based alkaline phosphatase-like and the growth differentiation factor 15 antibody by the coated magnetic beads, and thus can improve the sensitivity and accuracy of the detection of the chemiluminescence reaction system.

[0046] The weight m1 of the coated magnetic beads and the weight m2 of the labeled growth differentiation factor 15 antibody containing the cerium dioxide-based alkaline phosphatase-like can satisfy the relationship: m1:m2 = 400:1, 350:1, 300:1, 250:1, or 200:1.

[0047] In some alternative embodiments, the weight m3 of the anti-growth differentiation factor 15 antibody and the weight m4 of the streptavidin satisfy the relational expression: m3:m4 = (1:1) to (1:5);

[0048] In these embodiments, the weight m3 of the anti-growth differentiation factor 15 antibody and the weight m4 of the streptavidin may satisfy the relational expression: m3:m4 = (1:1) to (1:5), which can ensure that there is a sufficient amount of streptavidin and anti-growth differentiation factor 15 antibody in the coated magnetic beads. The sufficient amount of streptavidin and anti-growth differentiation factor 15 antibody can effectively bind to the growth differentiation factor 15 in the sample to be detected, so as to improve the accuracy of the chemiluminescence immunoassay method.

[0049] The weight m3 of the anti-growth differentiation factor 15 antibody and the weight m4 of the streptavidin may satisfy the relational expression: m3:m4 = 1:1, 1:2, 1:3, 1:4 or 1:5.

[0050] In some alternative embodiments, the coated magnetic beads include a magnetic bead matrix, and the surface of the magnetic bead matrix is coated with the anti-growth differentiation factor 15 antibody and the streptavidin; the particle size of the magnetic bead matrix is 100 nm to 300 nm;

[0051] In these embodiments, the coated magnetic beads may include a magnetic bead matrix, and the particle size of the magnetic bead matrix is 100 nm to 300 nm, which indicates that the magnetic bead matrix has a sufficient specific surface area. The magnetic bead matrix with a sufficiently large specific surface area can carry a sufficient amount of streptavidin and anti-growth differentiation factor 15 antibody. The sufficient amount of streptavidin and anti-growth differentiation factor 15 antibody can effectively bind to the growth differentiation factor 15 in the sample to be detected, so as to improve the accuracy of the chemiluminescence immunoassay method.

[0052] The particle size of the magnetic bead matrix may be 100 nm, 150 nm, 200 nm, 240 nm, 280 nm or 300 nm.

[0053] Figure 2 Exemplarily, a schematic flow chart of a method for preparing the chemiluminescence reaction system provided by the embodiments of the present application is shown;

[0054] Based on a general inventive concept, as Figure 2 shown, the embodiments of the present application provide a method for preparing the chemiluminescence reaction system, and the method includes:

[0055] S1. Prepare a biotinylated anti-growth differentiation factor 15 antibody buffer solution using a labeling buffer;

[0056] S2. Prepare a streptavidin magnetic bead mother liquor;

[0057] S3. Mix the biotinylated anti-growth differentiation factor 15 antibody buffer and the streptavidin, and then dilute the mixed solution to obtain coated magnetic beads.

[0058] S4. Activate the cerium dioxide-based alkaline phosphatase-like enzyme to obtain the activated alkaline phosphatase-like enzyme.

[0059] S5. Perform coupling and blocking reactions on the growth differentiation factor 15 antibody and the activated alkaline phosphatase-like enzyme in sequence to obtain the growth differentiation factor 15 antibody labeled with cerium dioxide-based alkaline phosphatase-like enzyme.

[0060] S6. Aliquot the coated magnetic beads and the growth differentiation factor 15 antibody labeled with cerium dioxide-based alkaline phosphatase-like enzyme to obtain a chemiluminescence reaction system.

[0061] This method is a preparation method for the above chemiluminescence reaction system. The specific composition of the chemiluminescence reaction system can refer to the above embodiments. Since this method adopts some or all of the technical solutions of the above embodiments, it has at least all the beneficial effects brought by the technical solutions of the above embodiments, which will not be elaborated here one by one.

[0062] It should be noted that the activation of the cerium dioxide-based alkaline phosphatase-like enzyme is different from that of ordinary alkaline phosphatase in that the activation of the cerium dioxide-based alkaline phosphatase-like enzyme is generally achieved through an inorganic catalytic mechanism, which is the reason why the cerium dioxide-based alkaline phosphatase-like enzyme has the characteristics of broad-spectrum pH tolerance, ultra-high stability and low cost. While ordinary alkaline phosphatase depends on the biological active center, although it is maturely applied in traditional detections, limited by the biological active center, there are certain differences in its stability, cost and operation complexity compared with the cerium dioxide-based alkaline phosphatase-like enzyme.

[0063] It should be noted that this activation can be carried out at room temperature.

[0064] In some optional embodiments, the amount of substance n1 of the growth differentiation factor 15 antibody and the amount of substance n2 of the activated alkaline phosphatase-like enzyme satisfy the relationship: n1:n2 = (1:3) - (1:8).

[0065] In these embodiments, the amount of substance n1 of the growth differentiation factor 15 antibody and the amount of substance n2 of the activated alkaline phosphatase-like enzyme can satisfy the relationship: n1:n2 = (1:3) - (1:8), which can promote the activated alkaline phosphatase-like enzyme to fully carry enough growth differentiation factor 15 antibodies, and sufficient activated alkaline phosphatase-like enzyme and growth differentiation factor 15 antibodies can form sufficient growth differentiation factor 15 antibodies labeled with cerium dioxide-based alkaline phosphatase-like enzyme, so as to effectively bind to the coated magnetic beads, thereby improving the sensitivity and accuracy of the chemiluminescence immunoassay method.

[0066] The amount of substance n1 of the growth differentiation factor 15 antibody and the amount of substance n2 of the activated alkaline phosphatase-like enzyme can satisfy the relationship: n1:n2 = 1:3, 1:4, 1:5, 1:6, 1:7 or 1:8.

[0067] Based on a general inventive concept, the embodiments of the present application provide a chemiluminescent immunoassay kit, and the chemiluminescent immunoassay kit includes the chemiluminescent reaction system.

[0068] The chemiluminescent immunoassay kit is implemented based on the above chemiluminescent reaction system. The specific composition of the chemiluminescent reaction system can refer to the above embodiments. Since this method adopts some or all of the technical solutions of the above embodiments, it at least has all the beneficial effects brought by the technical solutions of the above embodiments, which will not be elaborated here one by one.

[0069] In some alternative embodiments, the chemiluminescent immunoassay kit further includes a 1,2-dioxetane derivative;

[0070] In these embodiments, the chemiluminescent immunoassay kit may further include a 1,2-dioxetane derivative. By adding the 1,2-dioxetane derivative to the chemiluminescent immunoassay kit, a phosphate group of the 1,2-dioxetane derivative can be decomposed and removed by the cerium dioxide-based alkaline phosphatase-like enzyme, so that the 1,2-dioxetane derivative forms an unstable excited-state intermediate, and when the unstable excited-state intermediate returns to the ground state, a light signal is generated. Detecting the intensity of this light signal can directly determine the mass concentration of the growth differentiation factor 15 in the test sample.

[0071] Figure 3 Exemplarily shown is a schematic flow chart of a chemiluminescent immunoassay method provided by the embodiments of the present application for non-disease diagnosis and treatment purposes;

[0072] Based on a general inventive concept, as Figure 3 shown, the embodiments of the present application provide a chemiluminescent immunoassay method for non-disease diagnosis and treatment purposes. The chemiluminescent immunoassay method is implemented based on the chemiluminescent immunoassay kit, and the chemiluminescent immunoassay method includes:

[0073] S1. Specifically bind the test sample with the coated magnetic beads to obtain an antibody complex;

[0074] S2. Specifically bind the growth differentiation factor 15 antibody labeled with the cerium dioxide-based alkaline phosphatase-like enzyme and the antibody complex to obtain a binding product;

[0075] S3. Perform a decomposition reaction on the chemiluminescent substrate and the binding product, and detect the photoelectric signal intensity of the decomposition reaction product during the decomposition reaction to obtain the light signal intensity of the binding product;

[0076] S4. Plot a standard curve of the light signal intensity versus growth differentiation factor 15;

[0077] S5. Obtain the mass concentration of growth differentiation factor 15 in the sample to be tested based on the standard curve and the light signal intensity of the binding product.

[0078] This chemiluminescent immunoassay method is implemented based on the above-mentioned chemiluminescent immunoassay kit. The specific composition of the chemiluminescent immunoassay kit can refer to the above-mentioned embodiments. Since this chemiluminescent immunoassay method adopts some or all of the technical solutions of the above-mentioned embodiments, it at least has all the beneficial effects brought by the technical solutions of the above-mentioned embodiments, which will not be elaborated one by one here.

[0079] In some optional embodiments, the temperature of the decomposition reaction is 25°C to 37°C, and the time of the decomposition reaction is 10 min to 20 min;

[0080] In these embodiments, the temperature of the decomposition reaction can be 25°C to 37°C, and the time of the decomposition reaction is 10 min to 20 min, which promotes the full chemical reaction of the chemiluminescent substrate and the cerium dioxide-based alkaline phosphatase of the binding product to decompose and remove a phosphate group of the 1,2-dioxetane derivative, enabling the 1,2-dioxetane derivative to form an unstable excited-state intermediate. When the unstable excited-state intermediate returns to the ground state, a light signal is generated. Detecting the intensity of this light signal can directly determine the mass concentration of growth differentiation factor 15 in the sample to be tested.

[0081] The temperature of this decomposition reaction can be 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C or 37°C.

[0082] The time of this decomposition reaction is 10 min, 11 min, 12 min, 13 min, 14 min, 15 min, 16 min, 17 min, 18 min, 19 min or 20 min.

[0083] The following further elaborates the present application in combination with specific embodiments. The experimental methods without specific conditions noted in the following embodiments are generally determined according to industry standards; if there is no corresponding industry standard, they are carried out according to general international standards, conventional conditions or the conditions recommended by the manufacturer.

[0084] Example 1

[0085] 1. Experimental materials:

[0086] (1) Anti-growth differentiation factor 15 antibody: Anti-growth differentiation factor 15 monoclonal antibody a, anti-growth differentiation factor 15 monoclonal antibody b, with purity > 95%, purchased from Wuhan Huamei Biological Engineering Co., Ltd.

[0087] (2) Growth differentiation factor 15 recombinant protein: with purity > 95%, purchased from Wuhan Huamei Biological Engineering Co., Ltd.

[0088] (3) Streptavidin-coated magnetic beads, biotin, cerium dioxide-based alkaline phosphatase.

[0089] 2. Experimental methods and procedures:

[0090] (1) Preparation of growth differentiation factor 15 magnetic bead antibody working solution: Mix anti-growth differentiation factor 15 monoclonal antibody a and biotin at a molar ratio concentration of 1:4 and react at room temperature for 1 h to biotinylate the antibody. Couple the biotinylated antibody with streptavidin-coated magnetic beads at a mass ratio of 1.0:0.5 to obtain streptavidin magnetic beads conjugated with biotinylated anti-growth differentiation factor 15 antibody. Dilute the streptavidin magnetic beads conjugated with biotinylated anti-growth differentiation factor 15 antibody and magnetic bead diluent at a mass ratio of 1:9 to obtain the growth differentiation factor 15 magnetic bead antibody working solution.

[0091] (2) Preparation of growth differentiation factor 15 enzyme-labeled antibody working solution: Activate cerium dioxide-based alkaline phosphatase and anti-growth differentiation factor 15 monoclonal antibody b respectively, and then couple and react at a mass ratio of 1:1.2 at 30 °C for 1 h, and then store it after blocking to obtain anti-growth differentiation factor 15 antibody labeled with cerium dioxide-based alkaline phosphatase. Dilute the anti-growth differentiation factor 15 antibody labeled with cerium dioxide-based alkaline phosphatase and enzyme diluent at a mass ratio of 1:1000 to obtain the growth differentiation factor 15 enzyme-labeled antibody working solution.

[0092] (3) Preparation of calibrators and quality control products: Dissolve growth differentiation factor 15 recombinant protein in calibrator diluent and prepare calibrators with concentrations of 0.0 pg / mL, 1000.0 pg / mL, 2500.0 pg / mL, 5000.0 pg / mL, 10000.0 pg / mL, 20000.0 pg / mL, as well as quality control products with concentrations of 750.0 pg / mL and 7500.0 pg / mL by gradient dilution.

[0093] 3. Related experiments:

[0094] Using the SZY-CL2100 automatic chemiluminescence analyzer as the detection tool, detect the main performance indicators of Example 1 and Comparative Example 1 respectively. The specific detection methods are as follows:

[0095] (1) Minimum detection limit: Use the zero-concentration calibrator or sample diluent as the sample for detection, repeat the determination 20 times, obtain the RLU values (relative luminescence values) of the 20 determination results, calculate their average value (M) and standard deviation (SD), obtain M + 2SD, and perform two-point regression fitting on the calibration curve equation of the calibrator used in the kit or the concentration-RLU value results between the zero-concentration calibrator and the adjacent calibrator to obtain a linear equation. Substitute the RLU value of M + 2SD into the above equation to find the corresponding concentration value, which is the minimum detection limit.

[0096] (2) Repeatability: Use samples with concentrations of (750.0 ± 75.0) pg / mL and (7500.0 ± 750.0) pg / mL to repeat the detection 10 times respectively, and calculate their coefficient of variation (CV).

[0097] (3) Accuracy: Add the growth differentiation factor 15 sample with a concentration of approximately 20000.0 pg / mL (allowable deviation of ±20%) to sample B in serum or other corresponding matrices. The volume of A added should not exceed 10% of the total volume (A + B). Repeat the determination 3 times and calculate the recovery rate R.

[0098] (4) Linearity: Dilute the high-value sample close to the upper limit of the linear range to at least 5 concentrations in a certain proportion, and the low-value concentration sample must be close to the lower limit of the linear range. Repeat the detection 3 times for each concentration, calculate the average value, perform linear fitting on the result average value and the dilution ratio by the least squares method, and calculate the linear correlation coefficient R.

[0099] (5) Thermal stability: Place the kit at 4°C and 37°C for 7 days respectively, detect the concentrations of the quality control product and low, medium, and high-concentration clinical samples, and calculate the retention rate of the kit at 37°C compared to that at 4°C.

[0100] (6) Long-term stability: Place the kit at 4°C, and detect the concentrations of the quality control product and low, medium, and high-concentration clinical samples at 0d, 15d, 30d, 2 months, 3 months, 4.5 months, and 6 months respectively, and calculate the retention rate of the kit when stored at 4°C for a long time.

[0101] Comparative Example 1

[0102] On the basis of the content disclosed in Example 1, the following further modifications are made:

[0103] Use traditional alkaline phosphatase to replace cerium dioxide-based alkaline phosphatase.

[0104] 4. Related experimental data and results:

[0105] 1. The chemiluminescence detection kits obtained in Example 1 and Comparative Example 1 were actually tested, and the results are shown in Tables 1 and 2.

[0106] Table 1 Detection results of the minimum detection limit, repeatability, accuracy, linearity, and thermal stability of the kits in Example 1 and Comparative Example 1

[0107]

[0108]

[0109] Note: Some of the recovery rate data in Table 1 above 100% are determined by various factors; in the technical field of the kit, a recovery rate between 85% and 115% is acceptable.

[0110] Table 2 Detection results of the long-term stability of the kits in Example 1 and Comparative Example 1

[0111]

[0112] As can be seen from Tables 1 and 2, in the verification and comparison of the performances such as the minimum detection limit, repeatability, accuracy, linearity, thermal stability, and long-term stability between Example 1 and Comparative Example 1, the repeatability CVs of both Example 1 and Comparative Example 1 are < 5%, the recoveries of low, medium, and high clinical samples are between 85% and 115%, and the linear Rs are all > 0.9900; however, compared with Comparative Example 1, Example 1 has a relatively lower background and higher sensitivity; the thermal stability retention rate of Example 1 is > 95%, and the thermal stability retention rate of Comparative Example 1 is only about 87%; in addition, the retention rate of the 6-month long-term stability measured by the kit of Example 1 is > 90%, and the retention rate of the 6-month long-term stability measured by the kit of Comparative Example 1 is only about 80%.

[0113] 2. Sample correlation of the kit:

[0114] Clinical samples within the linear range of 400.0 pg / mL to

[0115] 20000.0 pg / mL were tested using Example 1 and the comparison kit REF: IM4406155 respectively to verify the sample correlation of the kit. According to the verification results, the sample correlation map of the kit of Example 1 and the comparison kit was drawn, and the results are as Figure 4 shown. The results show that the kit provided in the embodiment of the present application has good sample correlation.

[0116] In summary, a chemiluminescence reaction system based on cerium dioxide-based alkaline phosphatase provided in the embodiment of the present application uses cerium dioxide-based alkaline phosphatase to replace traditional alkaline phosphatase, and can simultaneously improve the stability and accuracy of chemiluminescence immunoassay for growth factors.

[0117] In addition, a chemiluminescence reaction system based on cerium dioxide-based alkaline phosphatase-like provided in an embodiment of the present application uses cerium dioxide-based alkaline phosphatase-like to replace traditional alkaline phosphatase, which can not only solve the problems of easy loss of activity, limited catalytic performance and high cost existing in the mimic enzyme in the traditional chemiluminescence immunoassay process of growth differentiation factor 15, but also can enhance the sensitivity of chemiluminescence immunoassay to a certain extent.

[0118] In addition, a chemiluminescence immunoassay method provided in an embodiment of the present application aims at non-disease diagnosis and treatment. The chemiluminescence immunoassay method is easy to operate, the materials used are easy to obtain, and the cost of chemiluminescence immunoassay is low.

[0119] The above are only specific embodiments of the present application, enabling those skilled in the art to understand or implement the present application. Various modifications to these embodiments will be obvious to those skilled in the art, and the general principles defined in the present application can be implemented in other embodiments without departing from the spirit or scope of the present application. Therefore, the present application will not be limited to these embodiments shown in the present application, but will conform to the widest scope consistent with the principles and novel features claimed in the present application.

Claims

1. A chemiluminescence reaction system based on cerium dioxide-based alkaline phosphatase-like, characterized in that, The chemical reaction system includes: coated magnetic beads and a growth differentiation factor 15 antibody labeled with cerium dioxide-based alkaline phosphatase-like. The surface of the coated magnetic beads is coated with an anti-growth differentiation factor 15 antibody and streptavidin; wherein, the weight m1 of the coated magnetic beads and the weight m2 of the labeled growth differentiation factor 15 antibody containing cerium dioxide-based alkaline phosphatase-like satisfy the relationship: m1:m2 ≤ 400:

1.

2. The chemiluminescence reaction system according to claim 1, wherein The weight m1 of the coated magnetic beads and the weight m2 of the labeled growth differentiation factor 15 antibody containing cerium dioxide-based alkaline phosphatase-like satisfy the relationship: m1:m2 = (400:1) - (200:1).

3. The chemiluminescence reaction system according to claim 1, wherein The weight m3 of the anti-growth differentiation factor 15 antibody and the weight m4 of the streptavidin satisfy the relationship: m3:m4 = (1:1) - (1:5).

4. The chemiluminescence reaction system according to claim 1, wherein The coated magnetic beads include a magnetic bead matrix, and the surface of the magnetic bead matrix is coated with the anti-growth differentiation factor 15 antibody and the streptavidin; the particle size of the magnetic bead matrix is 100 nm - 300 nm.

5. A method for preparing the chemiluminescence reaction system according to any one of claims 1 to 4, characterized in that, The method includes: Preparing a biotinylated anti-growth differentiation factor 15 antibody buffer solution using a labeling buffer. Preparing a streptavidin magnetic bead mother liquor. Mixing the biotinylated anti-growth differentiation factor 15 antibody buffer solution and the streptavidin, and then diluting the mixed solution to obtain coated magnetic beads. Activating cerium dioxide-based alkaline phosphatase-like to obtain activated alkaline phosphatase-like. Sequentially performing a coupling reaction and a blocking reaction on the growth differentiation factor 15 antibody and the activated alkaline phosphatase-like to obtain a growth differentiation factor 15 antibody labeled with cerium dioxide-based alkaline phosphatase-like. Subpackaging the coated magnetic beads and the growth differentiation factor 15 antibody labeled with cerium dioxide-based alkaline phosphatase-like to obtain a chemiluminescence reaction system.

6. The method according to claim 5, characterized in that, The amount of substance n1 of the growth differentiation factor 15 antibody and the amount of substance n2 of the activated alkaline phosphatase-like satisfy the relationship: n1:n2 = (1:3) - (1:8).

7. A chemiluminescence immunoassay kit, characterized in that, The chemiluminescence immunoassay kit includes the chemiluminescence reaction system according to any one of claims 1 - 4.

8. The chemiluminescent immunoassay kit according to claim 7, wherein The chemiluminescence immunoassay kit further includes a 1,2-dioxetane derivative.

9. A chemiluminescence immunoassay method for non-disease diagnosis and treatment purposes, characterized in that, The chemiluminescence immunoassay method is implemented based on the chemiluminescence immunoassay kit according to claim 7 or 8. The chemiluminescence immunoassay method includes: Specifically binding a test sample with the coated magnetic beads to obtain an antibody complex. Specifically binding the growth differentiation factor 15 antibody labeled with cerium dioxide-based alkaline phosphatase-like and the antibody complex to obtain a binding product. Performing a decomposition reaction on a chemiluminescent substrate and the binding product, and detecting the photoelectric signal intensity of the decomposition reaction product during the decomposition reaction to obtain the light signal intensity of the binding product. Plotting a standard curve of the light signal intensity versus the growth differentiation factor 15. Based on the standard curve and the light signal intensity of the binding product, obtaining the mass concentration of the growth differentiation factor 15 in the test sample.

10. The chemiluminescent immunoassay method according to claim 9, wherein The temperature of the decomposition reaction is 25°C to 37°C, and the time of the decomposition reaction is 10 min to 20 min.

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