GDF15 detection method and kit based on homogeneous light-activated chemiluminescence immunoassay technology

Abstract

The invention discloses a GDF15 detection method based on a homogeneous-phase light-excited chemiluminescence immunoassay technology and a kit. The detection method comprises the following steps: preparing donor microspheres coupled with a GDF15 capture antibody and receptor microspheres coupled with a GDF15 detection antibody, uniformly mixing and incubating a sample to be detected and the coupled donor microspheres and receptor microspheres, and performing fluorescence detection to obtain the GDF15 concentration of the sample to be detected. The kit comprises donor microspheres coupled with a GDF15 capture antibody, receptor microspheres coupled with a GDF15 detection antibody, a GDF15 standard substance solution and an analysis buffer solution. According to the invention, the carboxyl modified microspheres are used, so that the coupling efficiency with the antibody is higher; the GDF15 content in a to-be-detected sample can be rapidly detected by applying a double-antibody sandwich immunoassay and light-activated chemiluminescence characteristics, the detection range is wide, the sensitivity and the accuracy are high, the detection is rapid, simple and convenient, and the GDF15 detection kit can be clinically used for early auxiliary diagnosis of cardiovascular diseases such as HF and MI and has a good application prospect.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology detection, and relates to an immunoassay analysis technology, and specifically to a GDF15 detection method based on homogeneous photochemiluminescence immunoassay technology, which can detect the concentration of GDF15 in a sample to be tested, and a kit used for the detection method. Background Art

[0002] Amplified luminescent proximity homogeneous assay linked immunosorbent assay (AlphaLISA) is an immunological research technique based on the interaction between biological molecules, fluorescence resonance energy transfer luminescence, silica microspheres as carriers, and time-resolved fluorescence as the detection mode. The intermolecular interaction (antigen-antibody specific interaction) will bring the donor and acceptor microspheres closer to the singlet oxygen diffusion range, that is, within 200nm, thereby stimulating the cascade amplified chemiluminescence reaction. The luminescence principle is photoinduced chemiluminescence, the surface of the donor microsphere is coated with the photosensitizer phthalocyanine, and the surface of the acceptor microsphere is coated with the luminescent agent dimethylthiophene derivative and chelated with the rare earth atom europium. Therefore, when irradiated with a 680nm laser, the photosensitizer on the surface of the donor microspheres decomposes the oxygen in the environment to form monomeric oxygen molecules (oxygen free radicals), which diffuse to the acceptor microspheres and ultimately transfer the energy to the rare earth atom europium. The excitation light has an excitation wavelength of 615nm and a half-life of 0.3s, which can be detected by a fluorescence scanner.

[0003] Cardiovascular disease (CVD) is the result of endothelial cell dysfunction. Common cardiovascular diseases include heart failure (HF), myocardial infarction (MI), atrial fibrillation (AF), atherosclerosis, hypertension and stroke. Among them, HF (heart failure) is a common health problem. It is a syndrome characterized by the inability of the heart to pump sufficient blood and oxygen to support the metabolic needs of other organs. It is caused by complex structural and functional cardiac abnormalities, such as inflammation, cardiomyopathy, neurohormonal activation, myocyte damage and other complex interactions. These abnormalities can lead to reduced cardiac output or excessive intracardiac pressure. HF is a typical age-related disease. Due to the aging population and the increase in the burden of risk factors, the incidence and mortality of HF are high, and the lifetime risk of HF is also high, about 20%-45%. Over time, although the hospitalization rate for heart failure has dropped significantly, its mortality rate has not improved significantly. Therefore, the demand for early diagnosis of heart failure is increasing. Patients with heart failure often present with nonspecific signs and symptoms and have a broad differential diagnosis, but diagnosis based on clinical manifestations alone is challenging. Although noninvasive imaging studies such as echocardiography can provide clues to the underlying cause of heart failure, such testing is expensive and time-consuming and is not suitable for early diagnosis of HF.

[0004] MI (myocardial infarction) is a life-threatening cardiovascular disease characterized by sudden cardiac death. It is caused by atherosclerotic clots or arterial spasm that leads to the blockage of one or more coronary arteries of the myocardium, resulting in damage and death of cardiac tissue. It is a dynamic and progressive pathophysiological process under the combined action of endothelial dysfunction and inflammation. In the United States, more than half of cardiovascular deaths are caused by myocardial infarction, and MI (myocardial infarction) is also the main cause of HF (heart failure). In addition, MI is the most deadly disease with high mortality, suddenness and strong concealment. Although most MI patients are characterized by significant and often severe symptoms, some patients are accompanied by mild or no discomfort. Therefore, there are problems such as delayed diagnosis, misdiagnosis and missed diagnosis in clinical practice. At present, coronary angiography is the standard for the diagnosis of myocardial infarction, but as an invasive detection method, it limits its application in the diagnosis of MI. Traditional methods for the assessment and management of HF and MI are limited by subjective interpretation, time consumption, cost, or invasiveness. In contrast, biomarkers provide convenient, objective, safe, and biologically relevant insights. In the past 20 years, biomarkers have made great contributions to improving the diagnosis and prognosis of cardiovascular diseases and optimizing treatment. In particular, in HF and MI, biomarkers have been incorporated into guidelines.

[0005] In recent years, growth differentiation factor 15 (GDF15) has gained increasing attention as a valuable biomarker in the cardiovascular field. GDF15 is a distant relative of the bone morphogenetic protein (BMP) subfamily of the transforming growth factor-β (TGF-β) superfamily. It is secreted by cells exposed to a variety of stressors and has multiple nicknames, including macrophage inhibitory cytokine-1 (MIC-1), prostate-derived factor (PDF), placental TGF-b (PTGF-b), placental bone morphogenetic protein (PLAB), and nonsteroidal anti-inflammatory drug-activated gene-1 (NAG-1). GDF15 is a marker of cell damage and inflammation. Under physiological conditions, it is expressed in a variety of cells and tissues, including the placenta, kidney, pancreas, liver, lung, heart, and brain, with the highest expression in the placenta. In terms of the heart, GDF15 is usually not expressed in adult myocardium, but it is also significantly induced in myocardial injury or heart failure. Therefore, basic research also calls GDF15 a cardiac factor. It not only plays a physiological role in maintaining cardiac homeostasis and responding to myocardial injury, but also can be used as a biomarker to evaluate cardiac function, which is helpful for clinical diagnosis. Studies have shown that there is an association between GDF15 and cardiac fibrosis during HF and MI, and there is a correlation with the severity of fibrosis. Bouchot et al. analyzed GDF15 in HF serum samples and found that the serum GDF15 level of HF patients was 2 times higher than that of non-HF patients. In addition, the time factor plays a vital role for HF and MI patients, especially MI patients. Data show that for every hour of delay in MI (myocardial infarction) patients, the risk of mortality and heart failure increases by 10-18%. The existing GDF15 detection methods are mainly ELISA (enzyme-linked immunosorbent assay) and IFMA (immunofluorescence measurement method), and the detection process is complicated and time-consuming, which cannot meet the needs of rapid diagnosis. In contrast, AlphaLISA (homogeneous photoluminescence immunoassay) is a sensitive, rapid, accurate, uniform, non-radioimmunoassay method that does not require a large number of washing processes and has great application potential. In addition, compared with IFMA, the biggest advantage of AlphaLISA is that it eliminates the interference of the multi-step washing process in IFMA on the accuracy, avoids the influence of the external environment, improves the detection sensitivity and accuracy, and greatly shortens the reaction time. It only takes 15 minutes to obtain the GDF15 concentration value of the sample to be tested. In summary, GDF15 (growth differentiation factor 15) is a promising serum marker for identifying HF and MI. Therefore, it is of great significance to use AlphaLISA to accurately and quickly detect the concentration of GDF15 in human serum.

[0006] Homogeneous photoinduced chemiluminescence immunoassay (AlphaLISA) technology uses time resolution as the detection platform, has low background impact, can perform large-scale rapid detection of samples in a short time, has high detection sensitivity and strong stability, can realize the rapid assessment of cardiovascular diseases such as HF and MI, and has a relatively broad application space. Summary of the invention

[0007] The purpose of the present invention is to overcome the shortcomings of the prior art and provide a GDF15 detection method based on homogeneous photochemiluminescence immunoassay technology that can detect the GDF15 content in a sample to be tested, with high efficiency, high sensitivity, good stability and fast detection, as well as a kit used for the detection method.

[0008] The technical solution of the present invention is as follows:

[0009] The invention discloses a GDF15 detection method based on homogeneous photochemiluminescence immunoassay technology. The detection method comprises the following steps: selecting a pair of mouse anti-human GDF15 monoclonal antibodies as GDF15 capture antibody and GDF15 detection antibody respectively; selecting carboxyl-modified donor microspheres and acceptor microspheres, activating and cleaning the same; coupling the GDF15 capture antibody to the donor microspheres (the solid phase carrier is the donor microspheres coupled with the GDF15 capture antibody, and the GDF15 antigen is covalently coupled to the surface of the donor microspheres); and coupling the GDF15 detection antibody to the acceptor microspheres at the same time, and using a double antibody sandwich method to form a fluorescent immune complex (fluorescent immune complex) of the donor microspheres coupled with the GDF15 capture antibody-GDF15 antigen-acceptor microspheres coupled with the GDF15 detection antibody, irradiating the photosensitizer on the donor microspheres with excitation light to generate singlet oxygen, and the luminescent agent on the acceptor microspheres receives the energy transfer of the singlet oxygen to generate fluorescence, and finally performing detection and analysis according to the fluorescence intensity.

[0010] Furthermore, the GDF15 detection method comprises the following steps: covalently coupling the GDF15 capture antibody and the GDF15 detection antibody to the surface of the donor microsphere and the acceptor microsphere, respectively, in an MES labeling buffer environment to form a microsphere-antibody conjugate (microsphere-antibody complex): the GDF15 capture antibody is covalently coupled to the surface of the donor microsphere, and the GDF15 detection antibody is covalently coupled to the surface of the acceptor microsphere; after the microsphere-antibody conjugate is formed, washing and separation are performed using Tris-HCl washing solution to remove unbound antibodies; thereafter, the concentration and dosage of the microspheres coupled with the antibodies are optimized to determine the appropriate microsphere concentration and dosage (the appropriate concentration of the donor microspheres coupled with the GDF15 capture antibody is 70-80ug / ml, and the appropriate dosage is 20-30uL; the appropriate concentration of the acceptor microspheres coupled with the GDF15 detection antibody is 90-110ug / ml, and the appropriate dosage is 20-30uL), and using an analysis buffer (analysis buffer composition: 50mmol / L The microsphere-antibody conjugate was diluted with Tris-HCl, 0.2% BSA, 0.9% NaCl, 0.05% (v / v) Proclin300, pH 7.8) to achieve an appropriate microsphere concentration and avoid excessive detection microspheres; then, the GDF15 capture antibody coupled to the donor microspheres, the GDF15 antigen in the test sample, and the GDF15 detection antibody coupled to the acceptor microspheres were used for detection by a double antibody sandwich method: the donor microspheres coupled with the GDF15 capture antibody, the acceptor microspheres coupled with the GDF15 detection antibody and the test sample (serum) at an appropriate dose were added to a white, non-transparent well plate for mixing and incubation (the mixing and incubation conditions were 36.5°C-37.5°C, light-proof and oscillating incubation for 15min-25min), without washing, and finally the GDF15 capture antibody coupled to the donor microspheres was formed. A fluorescent immune complex of antibody-GDF15 antigen-GDF15 detection antibody coupled to acceptor microspheres is obtained. The fluorescent immune complex is irradiated with excitation light to generate singlet oxygen on the photosensitizer on the donor microspheres, and the luminescent agent on the acceptor microspheres receives the energy transfer of singlet oxygen to generate fluorescence. Finally, fluorescence detection and analysis are performed on a fluorescence scanning analyzer to obtain the fluorescence intensity of the sample to be tested, and then the content of the analyte in the sample to be tested is calculated according to the fluorescence intensity of the sample to be tested (a standard curve is drawn according to the concentration of the standard and its corresponding fluorescence intensity, and a standard curve equation is obtained according to the standard curve. Then, the fluorescence intensity of the sample to be tested is substituted into the corresponding standard curve equation to calculate the GDF15 content in the sample to be tested).

[0011] Furthermore, the GDF15 detection method is carried out according to the following steps: the donor microspheres and the acceptor microspheres are activated and washed respectively with the labeling buffer MES (4-morpholineethanesulfonic acid), NHS (N-hydroxysuccinimide) and EDC (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride), and then coated and incubated with the purified GDF15 capture antibody and GDF15 detection antibody to form a microsphere-antibody conjugate (microsphere-antibody complex): the GDF15 capture antibody is covalently coupled to the surface of the donor microsphere, and the GDF15 detection antibody is covalently coupled to the acceptor microsphere. The microsphere-antibody conjugate was diluted with an analytical buffer solution to make the concentration of the donor microspheres coupled with the GDF15 capture antibody 70-80ug / ml and the concentration of the acceptor microspheres coupled with the GDF15 detection antibody 90-110ug / ml. The GDF15 capture antibody coupled with the donor microspheres, the GDF15 antigen in the sample to be tested, and the GDF15 detection antibody coupled with the acceptor microspheres were used to perform the test by a double antibody sandwich method: the donor microspheres, the acceptor microspheres and the sample to be tested were mixed with each other. The appropriate dosage of the sample is 20-30uL. The appropriate dosage of donor microspheres coupled with GDF15 capture antibodies, acceptor microspheres coupled with GDF15 detection antibodies and the sample to be tested are added to a white, non-transparent well plate at a volume ratio of 1:1:1 and mixed and incubated (the mixing and incubation conditions are 36.5°C-37.5°C in the dark and incubated for 15min-25min). The GDF15 capture antibodies coupled to the donor microspheres, the GDF15 antigen in the sample to be tested, and the GDF15 detection antibodies coupled to the acceptor microspheres form fluorescent immune complexes, which are irradiated by the excitation light to the donor. The photosensitizer on the microspheres produces singlet oxygen, and the luminescent agent on the acceptor microspheres receives the energy transfer from the singlet oxygen to produce fluorescence. Finally, the fluorescence intensity of the sample to be tested is obtained by fluorescence detection and analysis using a fluorescence scanning analyzer. A standard curve is drawn based on the concentration of the standard and its corresponding fluorescence intensity, and then the content of the analyte in the sample to be tested is obtained based on the fluorescence intensity of the sample to be tested and the standard curve (a standard curve is drawn based on the concentration of the standard and its corresponding fluorescence intensity, and a standard curve equation is obtained based on the standard curve. Then, the fluorescence intensity of the sample to be tested is substituted into the corresponding standard curve equation to calculate the GDF15 content in the sample to be tested).

[0012] Preferably, the donor microspheres and the acceptor microspheres are activated and washed with labeling buffers MES, NHS and EDC, respectively, and then coated and incubated with purified GDF15 capture antibody and GDF15 detection antibody, respectively, to form microsphere-antibody conjugates, and then the donor microspheres coupled with GDF15 capture antibody and the acceptor microspheres coupled with GDF15 detection antibody are placed in BSA blocking solution and incubated in the dark for 0.5-2 hours (preferably 1 hour) at room temperature with shaking; then washed and separated with Tris-HCl washing solution to remove unbound antibodies.

[0013] Preferably, the coating ratios of the donor microspheres and the GDF15 capture antibody to form a conjugate, and the acceptor microspheres and the GDF15 detection antibody to form a conjugate are: 1 mg microspheres labeled (coated) 0.05 mg-0.2 mg antibody, and the coating incubation (coupling) conditions are: incubation at room temperature in the dark with shaking for 0.5-3 hours.

[0014] More preferably, the coating ratios in the process of forming a conjugate between the donor microspheres and the GDF15 capture antibody, and in the process of forming a conjugate between the acceptor microspheres and the GDF15 detection antibody are: 1 mg of microspheres labeled (coated) with 0.1 mg of antibody, and every 0.1 mg of antibody is coupled to 1 mg of microspheres; that is, the content of GDF15 capture antibody coupled to 1 mg of donor microspheres is 0.1 mg, and the content of GDF15 detection antibody coupled to 1 mg of acceptor microspheres is 0.1 mg.

[0015] More preferably, the coating and incubation (coupling) conditions for the process of forming a conjugate between the donor microspheres and the GDF15 capture antibody, and the process of forming a conjugate between the acceptor microspheres and the GDF15 detection antibody are: incubation at room temperature in the dark with shaking for 2 hours.

[0016] Preferably, during the formation of fluorescent immune complexes: the concentration of donor microspheres coupled to GDF15 capture antibodies is 75ug / ml, and the dosage is 25uL; the concentration of acceptor microspheres coupled to GDF15 detection antibodies is 100ug / ml, and the dosage is 25uL; the mixing and incubation conditions are incubation at 37°C in the dark with shaking for 15 minutes.

[0017] Preferably, the white, non-transparent well plate used in the sample preparation process can be a polypropylene well plate, an ELISA plate, or a 96-well plate.

[0018] Preferably, the analysis buffer comprises: 50 mmol / L Tris-HCl, 0.2% BSA, 0.9% NaCl, 0.05% (v / v) Proclin 300, pH 7.8.

[0019] Preferably, the concentration of the labeling buffer MES is 50 mmol / L, pH 6.0, the concentration of the NHS solution is 20 mg / mL, and the concentration of the EDC solution is 20 mg / mL.

[0020] The fluorescence detection analysis refers to the use of the long-life fluorescence characteristic of lanthanide rare earth chelates. After the sample is excited by pulsed light and before the fluorescence signal is collected, the fluorescence decay time of different fluorescent substances contained in the sample is different. After the short-life background fluorescence in the sample to be tested is completely quenched during this time period, the long-life rare earth chelate specific fluorescence signal is collected and detected. The time-resolved delay can effectively eliminate the interference from other non-specific fluorescence from samples, reagents, instruments, etc., thereby greatly improving the sensitivity of the detection.

[0021] The GDF15 detection method based on homogeneous photochemiluminescence immunoassay technology of the present invention covalently couples the capture antibody and detection antibody of GDF15 to the surface of the donor microsphere and the acceptor microsphere, respectively, and uses the GDF15 capture antibody coupled to the donor microsphere, the GDF15 antigen in the sample to be tested, and the GDF15 detection antibody coupled to the acceptor microsphere to perform detection by a double antibody sandwich method. The fluorescent immune complex is irradiated with excitation light to generate singlet oxygen on the photosensitizer on the donor microsphere, and the luminescent agent on the acceptor microsphere receives the energy transfer of singlet oxygen to generate fluorescence, and the content of the test object is calculated according to its fluorescence intensity. Detection and analysis are performed according to the fluorescence intensity, that is, the fluorescence intensity in the final product is determined by time-resolved fluorescence measurement, thereby effectively improving the sensitivity of detection.

[0022] The present invention discloses a GDF15 antigen detection kit used in a GDF15 detection method based on homogeneous photochemiluminescence immunoassay technology, comprising a kit body and a plurality of drug boxes placed in the kit body; wherein the plurality of drug boxes include: a drug box containing donor microspheres coupled to GDF15 capture antibodies, a drug box containing acceptor microspheres coupled to GDF15 detection antibodies, a drug box containing GDF15 standard solution and a drug box containing buffer solution; the kit body comprises a box body and a box cover connected thereto.

[0023] Preferably, the buffer is an assay buffer at pH 7.8.

[0024] More preferably, the analysis buffer comprises: 50 mmol / L Tris-HCl, 0.2% BSA, 0.9% NaCl, 0.05% (v / v) Proclin 300, pH 7.8.

[0025] Preferably, an expandable polystyrene foam layer is provided in the body of the test kit.

[0026] Preferably, the box body and the box cover are connected by a connecting shaft.

[0027] More preferably, the box body and the box cover are connected by a connecting shaft made of phenolic plastic.

[0028] More preferably, an ice trough for storing crushed ice is prefabricated at the bottom of the box body of the reagent kit.

[0029] Beneficial effects of the present invention:

[0030] The GDF15 detection method based on homogeneous photochemiluminescence immunoassay technology of the present invention can quickly detect the content of GDF15 in the sample to be tested, has the characteristics of strong specificity, high sensitivity, good stability, rapid detection, etc., and can be used for clinical auxiliary diagnosis of HF and MI, and has good application prospects. The GDF15 antigen detection kit of the present invention uses expandable polystyrene foam material, the box body and the box cover are connected by a connecting shaft of phenolic plastic material, and the bottom of the kit is prefabricated with an ice trough for storing crushed ice, which has a simple structure and strong practicality.

[0031] The GDF15 detection method based on homogeneous photochemiluminescence immunoassay technology of the present invention is based on the interaction between biological molecules and fluorescence resonance energy transfer luminescence, and uses silica microspheres as carriers. It eliminates nonspecific interference in the sample by identifying and utilizing time-resolved fluorescence measurement and spectral separation technology, and determines the fluorescence intensity in the final product, thereby effectively improving the detection sensitivity and detection efficiency.

[0032] Compared with the prior art, the GDF15 detection method based on homogeneous photochemiluminescence immunoassay technology of the present invention has the following advantages:

[0033] (1) The present invention uses carboxyl-modified donor microspheres and acceptor microspheres, which are activated and washed with labeling buffers MES, NHS and EDC respectively, and then coupled with capture antibodies and detection antibodies respectively. After the carboxyl group is activated, succinimidyl ester-modified microspheres are obtained. The succinimidyl ester group on the microsphere reacts with the amino group on the antibody to make the coupling efficiency higher; the activated carboxyl group is directly coupled to the microsphere, and the reaction step is only one step, which greatly shortens the reaction time and only takes 15 minutes. The previously developed AlphaLISA method often uses donor microspheres modified with streptavidin and introduces streptavidin and biotin signal amplification systems. Although it can provide specificity and sensitivity, it results in two reaction steps and the reaction time generally takes 30-105 minutes.

[0034] (2) The present invention activates and washes the donor microspheres and the acceptor microspheres with labeling buffers MES, NHS and EDC respectively, and then coats and incubates them with purified GDF15 capture antibody and GDF15 detection antibody respectively; after the microsphere-antibody complex is formed, it is washed and separated with Tris-HCl washing solution to remove unbound antibodies, and the operation is simple and fast.

[0035] (3) The present invention selects a pair of mouse anti-human GDF15 monoclonal antibodies as GDF15 capture antibody and GDF15 detection antibody, respectively, which can specifically bind to GDF15 in the sample to be tested without being interfered by other components.

[0036] (4) In terms of dosage, the serum dosage used in the present invention is 20-30uL, and the preferred serum dosage is 25uL; the preferred donor microsphere concentration is 75ug / ml, and the preferred dosage is 25uL; the preferred acceptor microsphere concentration is 100ug / ml, and the preferred dosage is 25uL; it can effectively reduce the amount of serum used and the cost. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 This is a standard curve diagram of GDF15 detection in Example 1 of the present invention.

[0038] Figure 2 It is the serum GDF15 concentration of the healthy control group, heart failure patients and myocardial infarction patients in Example 5 of the present invention. DETAILED DESCRIPTION

[0039] In order to make the objects, methods and advantages of the present invention more clear, the present invention is further described in detail below in conjunction with specific embodiments and drawings. The embodiments are only used to illustrate the present invention rather than to limit the present invention. Any modifications, equivalent substitutions, etc. made on the basis of the present invention are within the protection scope of the present invention.

[0040] The present invention discloses a GDF15 detection method based on homogeneous photochemiluminescence immunoassay technology as follows: a pair of mouse anti-human GDF15 monoclonal antibodies are selected as GDF15 capture antibody and GDF15 detection antibody respectively; carboxyl-modified donor microspheres and acceptor microspheres are selected, and activated and washed; the donor microspheres and acceptor microspheres are activated and washed respectively with labeling buffers MES, NHS and EDC, and then coated and incubated with purified GDF15 capture antibody and GDF15 detection antibody respectively, 1 mg of microspheres are coated with 0.05 mg-0.2 mg (preferably 0.1 mg) of antibody, and incubated at room temperature in the dark for 0.5-3 h (preferably 2 h), Forming microsphere-antibody conjugates: GDF15 capture antibody is covalently coupled to the surface of donor microspheres, and GDF15 detection antibody is covalently coupled to the surface of acceptor microspheres; then, the donor microspheres coupled with GDF15 capture antibody and the acceptor microspheres coupled with GDF15 detection antibody are placed in BSA blocking solution and incubated at room temperature in the dark for 0.5-2 hours (preferably 1 hour); then, they are washed and separated with Tris-HCl washing solution to remove unbound antibodies; then, the analysis buffer (composition of the analysis buffer: 50mmol / LTris-HCl, 0.2% BSA, 0.9% NaCl, 0.05% (v / v) Proclin300, pH 7.8) diluting the microsphere-antibody conjugate so that the concentration of the donor microspheres coupled with the GDF15 capture antibody is 70-80ug / ml (preferably 75ug / ml), and the concentration of the acceptor microspheres coupled with the GDF15 detection antibody is 90-110ug / ml (preferably 100ug / ml); then, using the GDF15 capture antibody coupled to the donor microspheres, the GDF15 antigen in the sample to be tested, and the GDF15 detection antibody coupled to the acceptor microspheres, Detection by double antibody sandwich method: The appropriate dosage of donor microspheres, acceptor microspheres and samples to be tested is 20-30uL (preferably 25uL), and the appropriate dosage of donor microspheres coupled with GDF15 capture antibody, acceptor microspheres coupled with GDF15 detection antibody and samples to be tested (serum) are added to a white, non-transparent well plate (which can be a polypropylene well plate, an ELISA plate or a 96-well plate) at a volume ratio of 1:1:1, mixed and incubated, and the mixing and incubation conditions are 36.5℃-37.Incubate at 5°C (preferably 37°C) in the dark for 15-25 minutes (preferably 20 minutes), and the GDF15 capture antibody coupled to the donor microspheres, the GDF15 antigen in the sample to be tested, and the GDF15 detection antibody coupled to the acceptor microspheres form a fluorescent immune complex. The fluorescent immune complex irradiates the photosensitizer on the donor microspheres with excitation light to produce singlet oxygen, and the luminescent agent on the acceptor microspheres receives the energy transfer of singlet oxygen to produce fluorescence. Finally, the fluorescence intensity of the sample to be tested is obtained by fluorescence detection and analysis using a fluorescence scanning analyzer (CL-96i reader produced by Zhejiang Baotai Intelligent Technology Co., Ltd., and other brands of fluorescence scanning analyzers can also be used). A standard curve is drawn according to the concentration of the standard and its corresponding fluorescence intensity, and then the content of the analyte in the sample to be tested is obtained according to the fluorescence intensity of the sample to be tested and the standard curve (a standard curve is drawn according to the concentration of the standard and its corresponding fluorescence intensity, and the standard curve equation is obtained according to the standard curve, and then the fluorescence intensity of the sample to be tested is substituted into the corresponding standard curve equation to calculate the GDF15 content in the sample to be tested). .

[0041] The following is combined with Figure 1 , Attachment Figure 2 The present application is further described in detail with Examples 1 to 6.

[0042] Example 1

[0043] This embodiment provides a GDF15 detection method based on homogeneous photochemiluminescence immunoassay technology, which is specifically carried out in the following steps:

[0044] Step 1: Microsphere activation and cleaning

[0045] The microsphere raw material to be coupled (the microsphere raw material is purchased, and the purchased microsphere raw material is a carboxyl-modified microsphere, which is then activated to become a succinimide ester-modified microsphere, thereby improving the coupling efficiency) donor microspheres and acceptor microspheres (microsphere addition amount is 100uL) are added to 2mL centrifuge tubes, 900μL labeling buffer (MES, concentration is 50mmol / L, pH 6.0) are added, centrifuged at 17000rpm for 20min, the supernatant is discarded, 1mL labeling buffer (MES) is added for ultrasonic 15min, and the mixture is shaken and mixed. Then, 10μL of 20mg / mL NHS solution and 5μL of 20mg / mL EDC solution are added, and the mixture is shaken at room temperature and away from light for 20min. Subsequently, the activated microspheres are centrifuged at 17000rpm for 20min, the supernatant is discarded, and 1000μL of labeling buffer (MES) is added for ultrasonic mixing, and repeated twice.

[0046] The present invention uses carboxyl-modified donor microspheres and acceptor microspheres as raw materials. The carboxyl-modified donor microspheres and acceptor microspheres are activated and washed with labeling buffers MES, NHS and EDC in sequence and then coupled with capture antibodies and detection antibodies respectively. After the carboxyl groups are activated, succinimidyl ester-modified microspheres are obtained. The succinimidyl ester groups on the microspheres react with the amino groups on the antibodies, so that the coupling efficiency is higher.

[0047] Step 2 Antibody purification

[0048] A pair of mouse anti-human GDF15 monoclonal antibodies were selected as GDF15 capture antibody and GDF15 detection antibody respectively; the antibody raw materials to be coupled, GDF15 capture antibody and GDF15 detection antibody (the amount of antibody added was 0.1 mg, and the GDF15 capture antibody added was 41 uL according to the antibody concentration, and the GDF15 detection antibody was 48 uL, wherein the concentration of GDF15 capture antibody was 2.45 mg / mL, and the concentration of GDF15 detection antibody was 2.08 mg / mL. The antibody raw materials were purchased from Wuhan Aokebot Biotechnology Co., Ltd.) were added to a 50KD centrifuge tube with a filter membrane for centrifugal concentration, and the antibody raw materials after centrifugation and concentration were added with 300 μL of labeling buffer (MES buffer), centrifuged at 10000 rpm for 5 minutes, the filtrate was discarded, and 300 μL of MES buffer was added, and the above centrifugation steps were repeated 8 times. The centrifuge tube after several centrifugations was taken out, the filtrate was discarded, the filter membrane of the centrifuge tube was reversed, centrifuged at 3000 rpm for 1 minute, and the filtrate was collected. Then, the filter membrane was turned over again, 50 μL of MES buffer was added, and the membrane was allowed to stand for a while to allow the membrane to absorb the labeled buffer and dissolve the residual antibody material on the membrane. The membrane was then turned over again and centrifuged at 3000 rpm for 1 min to collect the liquid on the membrane, so that as much antibody material as possible on the membrane was recovered, and the desired concentrated purified antibody material (including GDF15 capture antibody and GDF15 detection antibody) was obtained. The optimal volume of collected antibodies in the whole process is about 100 μL.

[0049] The above steps 1 and 2 can be performed simultaneously.

[0050] Step 3: Formation of microsphere-antibody conjugates (donor microspheres coupled to GDF15 capture antibodies, acceptor microspheres coupled to GDF15 detection antibodies)

[0051] 1 mg of donor microspheres activated and washed with labeling buffer (MES), NHS and EDC in step 1 were coated and incubated with 0.1 mg of capture antibody purified in step 2. 1 mg of donor microspheres were coated with 0.1 mg of capture antibody and incubated at room temperature in the dark for 2 hours to form a donor microsphere conjugate coupled to the capture antibody.

[0052] 1 mg of the receptor microspheres activated and washed with labeling buffer (MES), NHS and EDC in step 1 were coated and incubated with 0.1 mg of the detection antibody purified in step 2. 1 mg of the receptor microspheres were coated with 0.1 mg of the detection antibody and incubated at room temperature in the dark for 2 hours to form a receptor microsphere conjugate coupled to the detection antibody.

[0053] Subsequently, 100 μL of BSA blocking solution was added to the above-mentioned donor microsphere conjugate coupled with the capture antibody and the acceptor microsphere conjugate coupled with the detection antibody, and incubated at room temperature for 1 hour in the dark; finally, the microspheres were washed and separated with Tris-HCl solution to remove the unbound antibodies, and stored in a refrigerator at 4°C.

[0054] Step 4: Form a fluorescent immune complex (GDF15 capture antibody coupled to donor microspheres - GDF15 antigen - GDF15 detection antibody coupled to acceptor microspheres), and detect the fluorescence intensity on a microscope

[0055] First, the microsphere-antibody conjugate was diluted with assay buffer (assay buffer composition: 50mmol / L Tris-HCl, 0.2% BSA, 0.9% NaCl, 0.05% (v / v) Proclin300, pH 7.8) so that the concentration of the donor microspheres coupled to the GDF15 capture antibody was 75ug / ml, and the concentration of the acceptor microspheres coupled to the GDF15 detection antibody was 100ug / ml.

[0056] Then, 25 μL of donor microspheres coupled with GDF15 capture antibody, acceptor microspheres coupled with GDF15 detection antibody and the sample to be tested (serum) were added together (the addition ratio of the three was 1:1:1) into a polypropylene well plate, and the incubation conditions were 37°C with shaking in the dark for 15 minutes to obtain a fluorescent immune complex of GDF15 capture antibody coupled to donor microspheres-GDF15 antigen-GDF15 detection antibody coupled to acceptor microspheres. The fluorescent immune complex was irradiated with excitation light, and the photosensitizer on the donor microspheres produced singlet oxygen, and the luminescent agent on the acceptor microspheres accepted the energy transfer of singlet oxygen to produce fluorescence. Finally, the fluorescence intensity of the sample to be tested was detected on a fluorescence scanning analyzer CL-96ireader (produced by Zhejiang Baotai Intelligent Technology Co., Ltd.).

[0057] Step 5: Determine the content of the test index

[0058] A standard curve is drawn according to the concentration of the standard and its corresponding fluorescence intensity, and then the content of the analyte GDF15 in the sample to be tested is obtained according to the standard curve. The content of the analyte in the sample to be tested can be calculated according to the fluorescence intensity of the sample to be tested (the fluorescence intensity of the sample to be tested is substituted into the corresponding standard curve equation to calculate the content of the analyte in the sample to be tested).

[0059] Prepare multiple groups of GDF15 antigen standards of different concentrations, and use the GDF15-AlphaLISA detection kit of Example 4 of the present invention to detect them respectively, to obtain multiple groups of GDF15 concentration values ​​(standard concentrations) of different concentrations and corresponding fluorescence intensity values, and use the multiple groups of fluorescence intensity values ​​and GDF15 concentration values ​​to draw a standard curve to obtain a standard curve for GDF15 antigen detection, such as Figure 1 As shown, according to the standard curve, the linear equation (standard curve equation) for GDF15 antigen detection can be obtained as follows: Log 10 Y=0.8727*Log 10 X+3.900, R 2 =0.9923, linear range is 0.072ng / mL~200ng / mL.

[0060] Linear range, sensitivity, specificity, precision and recovery of GDF15-AlphaLISA detection.

[0061] The fluorescence intensity value of the 0 ng / mL reference standard was measured in parallel 20 times, and the mean (mean) and standard deviation (SD) were calculated. Mean + 1.645 * SD was substituted into the standard curve equation, and the blank limit (LOB) of this kit for GDF15 detection was calculated to be 0.003 ng / mL.

[0062] The fluorescence intensity value of the 0 ng / mL reference standard was measured in parallel 20 times, and the mean (mean) and standard deviation (SD) were calculated. Mean + 3.29 * SD was substituted into the standard curve equation, and the detection limit (LOD) of this kit for GDF15 detection was calculated to be 0.029 ng / mL.

[0063] Six different concentrations of GDF15 recombinant protein samples (0.03125 ng / mL, 0.0625 ng / mL, 0.125 ng / mL, 0.25 ng / mL, 0.5 ng / mL and 1 ng / mL) were tested in parallel 10 times, and the corresponding CV values ​​(standard deviation SD of fluorescence intensity values / mean of fluorescence intensity values) were calculated. A power function curve was prepared with GDF15 concentration as the horizontal axis and the corresponding CV value as the vertical axis. CV=20% was substituted into the power function curve, and the quantification limit (LOQ) of this kit was calculated to be 0.072 ng / mL.

[0064] The antigens of Galectin-3, ST2 and cTnI were measured three times, and their concentrations were calculated by the standard curve. Cross-reaction rate (%) = detection concentration / actual concentration. The results are shown in Table 1. It was calculated that the kit had almost no cross-reaction rate for Galectin-3, ST2 and cTnI, and had good specificity.

[0065] Table 1 GDF15 detection specificity

[0066]

[0067] The low-value samples, medium-value samples, and high-value samples of GDF15 were tested 10 times each. The results are shown in Table 2. The intra-batch coefficient of variation of GDF15 detection was 1.88%-4.40% (<10%), and the inter-batch coefficient of variation was 0.47%-3.26% (<15%).

[0068] Table 2 GDF15 analysis precision

[0069]

[0070] Three GDF15 antigen standards (10 ng / mL, 50 ng / mL, and 100 ng / mL) were mixed with a serum sample with a known concentration of 27.65 ng / mL in a ratio of 1:9. The AlphaLISA signal of the mixed solution was calculated using the standard curve to determine the actual concentration of the mixed solution. The serum and antigen standard solutions were mixed in proportion to prepare mixed solutions with theoretical concentrations of 25.89 ng / mL, 29.89 ng / mL, and 34.89 ng / mL. The recovery rate (%) was calculated as follows: Recovery rate (%) = actual concentration / theoretical concentration. The results are shown in Table 3, and the recovery rate was 96.06%-106.50%.

[0071] Table 3 GDF15 detection recovery

[0072]

[0073] In summary, the GDF15 detection method based on homogeneous photochemiluminescence immunoassay technology (GDF15-AlphaLISA detection method) of the present invention can detect the GDF15 antigen content in the sample to be tested, has high efficiency, high sensitivity, good stability, and rapid detection, can be used for HF and MI diagnosis and screening, and has good application prospects.

[0074] Example 2

[0075] This embodiment is a GDF15 detection method based on homogeneous photochemiluminescence immunoassay technology, which is basically the same as that of Example 1, except that:

[0076] In step 3:

[0077] 1 mg of donor microspheres were coated with 0.2 mg of capture antibody and incubated at room temperature in the dark for 0.5 h to form a donor microsphere conjugate coupled to capture antibody. 1 mg of acceptor microspheres were coated with 0.2 mg of detection antibody and incubated at room temperature in the dark for 0.5 h to form an acceptor microsphere conjugate coupled to detection antibody.

[0078] Subsequently, 100 μL of BSA blocking solution was added to the above-mentioned donor microsphere conjugate coupled with capture antibody and the acceptor microsphere conjugate coupled with detection antibody, respectively, and incubated at room temperature in the dark for 2 hours; finally, Tris-HCl washing solution was used for washing and separation to remove unbound antibodies.

[0079] In step 4:

[0080] First, the microsphere-antibody conjugate was diluted with assay buffer (assay buffer composition: 50mmol / L Tris-HCl, 0.2% BSA, 0.9% NaCl, 0.05% (v / v) Proclin300, pH 7.8) so that the concentration of the donor microspheres coupled to the GDF15 capture antibody was 70ug / ml, and the concentration of the acceptor microspheres coupled to the GDF15 detection antibody was 90ug / ml.

[0081] Then, 30 μL of donor microspheres coupled with GDF15 capture antibody, acceptor microspheres coupled with GDF15 detection antibody and the sample to be tested (serum) were added together (the addition ratio of the three was 1:1:1) to the ELISA plate, and the incubation conditions were 36.5°C with shaking in the dark for 25 minutes to obtain a fluorescent immune complex of GDF15 capture antibody coupled to donor microspheres-GDF15 antigen-GDF15 detection antibody coupled to acceptor microspheres.

[0082] Example 3

[0083] This embodiment is a GDF15 detection method based on homogeneous photochemiluminescence immunoassay technology, which is basically the same as that of Example 1, except that:

[0084] In step 3:

[0085] 1 mg of donor microspheres were coated with 0.05 mg of capture antibody and incubated at room temperature in the dark for 3 h to form a donor microsphere conjugate coupled to capture antibody. 1 mg of acceptor microspheres were coated with 0.05 mg of detection antibody and incubated at room temperature in the dark for 3 h to form an acceptor microsphere conjugate coupled to detection antibody.

[0086] Subsequently, 100 μL of BSA blocking solution was added to the above-mentioned donor microsphere conjugate coupled with capture antibody and the acceptor microsphere conjugate coupled with detection antibody, and incubated at room temperature in the dark for 0.5 hours; finally, Tris-HCl washing solution was used for washing and separation to remove unbound antibodies.

[0087] In step 4:

[0088] First, the microsphere-antibody conjugate was diluted with assay buffer (assay buffer composition: 50mmol / L Tris-HCl, 0.2% BSA, 0.9% NaCl, 0.05% (v / v) Proclin300, pH 7.8) so that the concentration of the donor microspheres coupled to the GDF15 capture antibody was 80ug / ml, and the concentration of the acceptor microspheres coupled to the GDF15 detection antibody was 110ug / ml.

[0089] Then, 20 μL of donor microspheres coupled with GDF15 capture antibody, acceptor microspheres coupled with GDF15 detection antibody and the sample to be tested (serum) were added together (the addition ratio of the three was 1:1:1) into a 96-well plate, and the incubation conditions were 37.5°C with shaking in the dark for 15 minutes to obtain a fluorescent immune complex of GDF15 capture antibody coupled to donor microspheres-GDF15 antigen-GDF15 detection antibody coupled to acceptor microspheres.

[0090] Example 4

[0091] The present invention discloses a GDF15 antigen detection kit based on homogeneous photochemiluminescence immunoassay technology (a GDF15-AlphaLISA detection kit), comprising a kit body and a plurality of medicine boxes placed in the kit body; wherein the plurality of medicine boxes include a medicine box containing donor microspheres coupled to GDF15 capture antibodies, a medicine box containing acceptor microspheres coupled to GDF15 detection antibodies, a medicine box containing GDF15 standard solution, and a medicine box containing buffer solution; an expandable polystyrene foam layer is provided in the kit body, the kit body comprises a box body and a box cover, the box body and the box cover are connected by a connecting shaft made of phenolic plastic material, and an ice trough for storing crushed ice is prefabricated at the bottom of the kit body.

[0092] The buffer in the kit is an analytical buffer with a pH of 7.8 (analytical buffer composition: 50mmol / LTris-HCl, 0.2% BSA, 0.9% NaCl, 0.05% (v / v) Proclin300, pH 7.8) (The buffer in the kit does not contain labeling buffer MES, NHS and EDC. Labeling buffer MES, NHS and EDC are buffers used to couple microspheres and antibodies. The kit provides a microsphere-antibody coupling complex, so labeling buffer MES, NHS and EDC are no longer provided.)

[0093] The GDF15-AlphaLISA detection kit of the present invention contains the following items:

[0094] Donor microspheres coupled to GDF15 capture antibody concentration: 1 mg / mL, dose: 1 mL

[0095] Concentration of receptor microspheres coupled to GDF15 detection antibody: 1 mg / mL, dosage: 1 mL

[0096] GDF15 standard solution The concentrations of the six GDF15 standard solutions are:

[0097] 1ng / mL, 5ng / mL, 10ng / mL, 50ng / mL, 100ng / mL, 20ng / mL,

[0098] The dosage of each concentration of GDF15 standard solution is 1mL

[0099] Buffer Analysis Buffer

[0100] Ingredients: 50mmol / L Tris-HCl, 0.2% BSA, 0.9% NaCl, 0.05% (v / v) Proclin300,

[0101] pH 7.8, dosage: 10mL

[0102] The method for performing GDF15-AlphaLISA antigen detection using the GDF15-AlphaLISA detection kit of the present invention is as follows:

[0103] First, the microsphere-antibody conjugate was diluted with an analytical buffer so that the concentration of the donor microspheres coupled to the GDF15 capture antibody was 75 ug / ml and the concentration of the acceptor microspheres coupled to the GDF15 detection antibody was 100 ug / ml.

[0104] Then, 25 μL of donor microspheres coupled with GDF15 capture antibody, acceptor microspheres coupled with GDF15 detection antibody and the sample to be tested (serum) were added together (the addition ratio of the three was 1:1:1) into a polypropylene well plate (which can be replaced by an ELISA plate and a 96-well plate, as long as it is a white, non-transparent well plate), and the incubation condition was 37°C with shaking in the dark for 15 minutes to obtain a fluorescent immune complex of GDF15 capture antibody coupled to donor microspheres-GDF15 antigen-GDF15 detection antibody coupled to acceptor microspheres. The fluorescent immune complex was irradiated with excitation light, and the photosensitizer on the donor microspheres produced singlet oxygen, and the luminescent agent on the acceptor microspheres received the energy transfer of singlet oxygen to produce fluorescence, and finally the fluorescence intensity of the sample to be tested was detected on the fluorescence scanning analyzer CL-96i reader.

[0105] Example 5 (Application Example)

[0106] This example is based on the GDF15-AlphaLISA detection method of Example 1 of the present invention and the GDF15-AlphaLISA detection kit of Example 4, and detects the serum GDF15 concentration of heart failure patients, myocardial infarction patients and healthy control patients, and is carried out according to the following steps:

[0107] Step 1: Collect serum samples

[0108] Blood samples were collected from 78 healthy controls, 57 heart failure patients, and 129 myocardial infarction patients in the hospital. Each blood sample was collected in a vacuum tube containing sodium citrate (sodium citrate has an anticoagulant effect and can prevent blood from clotting) and centrifuged at 1500rpm. After complete separation, the serum was carefully aspirated and stored at -20℃.

[0109] Step 2: Use the GDF15-AlphaLISA method to detect the fluorescence intensity of the GDF15 fluorescent immune complex in the serum sample

[0110] Use the GDF15-AlphaLISA detection kit of the present invention to perform GDF15-AlphaLISA detection:

[0111] First, the microsphere-antibody conjugate was diluted with an analytical buffer so that the concentration of the donor microspheres coupled to the GDF15 capture antibody was 75 ug / ml and the concentration of the acceptor microspheres coupled to the GDF15 detection antibody was 100 ug / ml.

[0112] Then, donor microspheres coupled with GDF15 capture antibodies, acceptor microspheres coupled with GDF15 detection antibodies, and standards or serum samples of different concentrations were added in a ratio of 1:1:1 (donor microsphere concentration 75 μg / mL, acceptor microsphere concentration 100 μg / mL, and the added volume was 25 μL) to a polypropylene well plate (which can be replaced by an ELISA plate and a 96-well plate, as long as it is a white, non-transparent well plate). The incubation condition is 37°C with shaking in the dark for 15 minutes to obtain a fluorescent immune complex of GDF15 capture antibody-GDF15 antigen coupled to donor microspheres-GDF15 detection antibody coupled to acceptor microspheres. The fluorescent immune complex is irradiated with excitation light, and the photosensitizer on the donor microspheres produces singlet oxygen, and the luminescent agent on the acceptor microspheres receives the energy transfer of singlet oxygen to produce fluorescence. Finally, the fluorescence intensity of the sample to be tested is detected on a fluorescence scanning analyzer CL-96i reader.

[0113] Step 3: Draw a standard curve according to the concentration of the standard and its corresponding fluorescence intensity, and then obtain the content of the GDF15 analyte in the serum sample according to the standard curve. The content of the analyte in the sample can be calculated according to the fluorescence intensity of the sample (substitute the fluorescence intensity of the sample into the corresponding standard curve equation to calculate the content of the analyte in the sample), that is, determine the content of the detection index.

[0114] The test results obtained are as follows Figure 2 As shown ( Figure 2 HC: healthy control group; HF: heart failure group; MI: myocardial infarction group). The test results showed that the concentration of GDF15 in patients with heart failure

[0115] [9.366 (1.983-27.68) ng / mL] and GDF15 concentration in patients with myocardial infarction

[0116] [11.29 (0.7565-30.92) ng / mL] was significantly higher than the GDF15 concentration in the healthy control group

[0117] [1.380(0.1307-7.962)ng / mL] (P<0.0001).

[0118] Example 6 (Comparative Example)

[0119] Comparison of GDF15-AlphaLISA and GDF15-TRFIA of the present invention

[0120] Using the same GDF15 antigen, GDF15 capture antibody and GDF15 detection antibody as in Example 1, GDF15 was detected using time-resolved fluorescence immunoassay (TRFIA), and the performance of the GDF15-TRFIA detection method was evaluated.

[0121] The GDF15-TRFIA detection method is carried out as follows:

[0122] Step 1 Preparation of solid phase GDF15 capture antibody

[0123] The GDF15 capture antibody was diluted with 100 mmol / L Na 2 CO 3 -NaHCO 3The buffer solution was diluted to 2.0 μg / mL of coating solution, and 100 μL of coating solution was added to each well of a 96-well plate. Incubate at 4°C overnight, discard the coating solution, wash the plate once, add 150 μL of blocking solution (50 mmol / L Tris-HCl, 0.9% NaCl, 0.05% (v / v) Proclin300 and 1% BSA, pH = 7.8) to each well, block at room temperature for 2 h, discard the blocking solution, dry by vacuum, seal the plate with strips, and store in a frozen state at -20°C.

[0124] Step 2Eu 3+ - Preparation of GDF15 antibody

[0125] Take 0.3mg GDF15 detection antibody and add it to Ultracel-50K ultrafiltration tube, centrifuge at 10000rpm for 5min at 4℃, discard the filtrate, add 300μL labeling buffer, centrifuge at 10000rpm for 5min, discard the filtrate, add 300μL elution buffer, and repeat the above centrifugation steps 8 times. Take out the centrifuge tube after several centrifugations, discard the filtrate, reverse the filter membrane of the centrifuge tube, centrifuge at 3000rpm for 1min, and collect the filtrate. Then reverse the filter membrane again, add 50μL labeling buffer, let it stand for 2min, let the filter membrane absorb the labeling buffer, dissolve the residual antibody raw material left on the filter membrane, and then reverse the filter membrane again and centrifuge at 3000rpm for 1min, collect the liquid on the filter membrane, and recover as much antibody raw material as possible on the filter membrane. The collected antibodies were mixed with 0.06 mg Eu3+DTTA chelate, and after shaking culture at 28°C overnight, the mixture was chromatographed on a Sephadex-G50 column equilibrated with an eluent containing 0.2% BSA. The effluent (2 mL / tube) was collected, and the fluorescence intensity value was detected tube by tube using an automatic time-resolved fluorescence immunoassay analyzer. The protein peak was collected and the purified Eu3+DTTA was purified. 3+ -GDF15 antibody was aliquoted and stored at -20°C.

[0126] Step 3: GDF15-TRFIA detection and performance evaluation

[0127] The GDF15-TRFIA detection is carried out as follows: 50 μL of standard or serum sample is added to each well of the GDF15 capture antibody solid phase coated plate obtained in step 1 above for reaction, and then 50 μL of Eu obtained in step 2 above is diluted with an assay buffer (the assay buffer here is different from the assay buffer in Examples 1-5, and the components of the assay buffer here are 50 mmol / L Tris-HCl, 0.9% NaCl, 0.2% BSA, 0.0008% DTPA, 0.0005% PHloxine B, 0.01% (v / v) Tween-20 and 0.05% (v / v) Proclin300, pH 7.8) is added to each well.3+ -GDF15 antibody (dilution ratio of 1:200), incubate with shaking at 37°C for 1 hour, wash the plate 6 times, and finally add 100 μL of enhancement solution to each well. After shaking for 3 minutes, the plate was tested by time-resolved immunofluorescence analyzer to obtain the fluorescence intensity in the standard and sample wells, thereby calculating the GDF15 content in the corresponding sample. The reaction time was 1 hour.

[0128] Performance evaluation of the GDF15-TRFIA assay:

[0129] According to the above-mentioned GDF15-TRFIA detection method, GDF15 low-value samples, median-value samples, and high-value samples were detected 10 times each. The results showed that the intra-batch precision of GDF15 detection was 3.27%-4.54% (<10%), and the inter-batch precision was 5.84%-10.41% (<15%).

[0130] Three GDF15 antigen standards (10 ng / mL, 50 ng / mL, and 100 ng / mL) were mixed with a serum sample with a known concentration of 53.07 ng / mL in a ratio of 1:9. The fluorescence intensity value of the mixed solution was calculated using the standard curve to determine the actual concentration of the mixed solution. The serum and antigen standard solutions were mixed in proportion to prepare mixed solutions with theoretical concentrations of 48.76 ng / mL, 52.76 ng / mL, and 57.76 ng / mL. The recovery rate (%) was calculated as follows: Recovery rate (%) = actual concentration / theoretical concentration. The results showed that the recovery rate was 88.15%-112.36%.

[0131] Step 4 Comparison of GDF15-TRFIA and GDF15-AlphaLISA

[0132] GDF15-TRFIA and GDF15-AlphaLISA were compared in terms of reaction time, recovery, inter-batch and intra-batch precision, and the results are shown in Table 4.

[0133] Table 4 Comparison of GDF15-TRFIA and GDF15-AlphaLISA

[0134]

[0135] Beneficial effects of the embodiments:

[0136] The present invention uses carboxyl-modified donor microspheres and acceptor microspheres, which are activated and cleaned with labeling buffers MES, NHS and EDC respectively, and then coupled with capture antibodies and detection antibodies respectively. After the carboxyl group is activated, succinimidyl ester-modified microspheres are obtained, and the succinimidyl ester group on the microsphere reacts with the amino group on the antibody to make the coupling efficiency higher; the antibody is directly coupled to the microsphere through the activated carboxyl group, and the reaction step is only one step, which greatly shortens the reaction time and only takes 15 minutes. The present invention selects a pair of mouse anti-human GDF15 monoclonal antibodies as GDF15 capture antibodies and GDF15 detection antibodies, respectively, which can specifically bind to GDF15 in the sample to be tested without interference from other components. The serum dosage used in the present invention is 20-30uL, which can effectively reduce the amount of serum and cost.

[0137] The present invention utilizes the characteristics of double antibody sandwich immunoassay and photoinduced chemiluminescence to rapidly detect the GDF15 content in the sample to be tested, has a wide detection range, high sensitivity, high accuracy, and is rapid and easy to detect. It can be used clinically for early auxiliary diagnosis of cardiovascular diseases such as heart failure and myocardial infarction, and has good application prospects.

[0138] Finally, it should be noted that the present invention is not limited to the above embodiments, and there are many variations. All variations that can be directly derived or associated with the content disclosed by ordinary technicians in this field should be considered as the protection scope of the present invention.

Claims

1. A GDF15 detection method based on homogeneous photochemiluminescence immunoassay technology, characterized in that: A pair of mouse anti-human GDF15 monoclonal antibodies are selected as GDF15 capture antibody and GDF15 detection antibody respectively; carboxyl-modified donor microspheres and acceptor microspheres are selected, activated and washed; the GDF15 capture antibody is coupled to the donor microspheres, and the GDF15 detection antibody is coupled to the acceptor microspheres at the same time, and a double antibody sandwich method is used to form a fluorescent immune complex of donor microspheres coupled with GDF15 capture antibody-GDF15 antigen-acceptor microspheres coupled with GDF15 detection antibody, and the photosensitizer on the donor microspheres is irradiated with excitation light to generate singlet oxygen, and the luminescent agent on the acceptor microspheres receives the singlet oxygen energy transfer to generate fluorescence, and finally detection and analysis are performed according to the fluorescence intensity.

2. The GDF15 detection method according to claim 1, characterized in that The GDF15 detection method comprises the following steps: covalently coupling a GDF15 capture antibody and a GDF15 detection antibody to the surface of a donor microsphere and an acceptor microsphere, respectively, in an MES labeling buffer environment to form a microsphere-antibody conjugate: the GDF15 capture antibody is covalently coupled to the surface of the donor microsphere, and the GDF15 detection antibody is covalently coupled to the surface of the acceptor microsphere; then, washing and separation are performed using a Tris-HCl washing solution to remove unbound antibodies; then, the concentration and dosage of the microspheres coupled with the antibodies are optimized to determine a suitable microsphere concentration and dosage, and the microsphere-antibody conjugate is diluted with an analysis buffer to achieve a suitable microsphere concentration; then, the GDF15 capture antibody coupled to the donor microsphere and the GDF15 antibody in the sample to be tested are used to detect the GDF15 in the sample; The GDF15 antigen-coupled GDF15 detection antibody is coupled to the acceptor microspheres and detected by the double antibody sandwich method: the donor microspheres coupled to the GDF15 capture antibody, the acceptor microspheres coupled to the GDF15 detection antibody and the sample to be tested are added to a white, non-transparent well plate with appropriate doses, mixed and incubated, and finally a fluorescent immune complex of the GDF15 capture antibody coupled to the donor microspheres-GDF15 antigen-GDF15 detection antibody coupled to the acceptor microspheres is formed. The fluorescent immune complex is irradiated with excitation light to generate singlet oxygen by the photosensitizer on the donor microspheres, and the luminescent agent on the acceptor microspheres receives the energy transfer of the singlet oxygen to generate fluorescence. Finally, the fluorescence intensity of the sample to be tested is obtained by fluorescence detection and analysis by a fluorescence scanning analyzer, and then the content of the analyte in the sample to be tested is obtained according to the fluorescence intensity.

3. The GDF15 detection method according to claim 2, characterized in that: The GDF15 detection method is carried out according to the following steps: the donor microspheres and the acceptor microspheres are activated and washed respectively with labeling buffers MES, NHS and EDC, and then coated and incubated with purified GDF15 capture antibody and GDF15 detection antibody respectively, 1 mg of microspheres are coated with 0.05 mg-0.2 mg of antibody, and incubated for 10 min-3 h at room temperature in the dark with shaking to form microsphere-antibody conjugates, and then washed and separated with Tris-HCl washing solution to remove unbound antibodies; then, the microsphere-antibody conjugates are diluted with analysis buffer to make the concentration of the donor microspheres coupled with the GDF15 capture antibody 70-80 ug / ml, and the concentration of the acceptor microspheres coupled with the GDF15 detection antibody 90-110 ug / ml; then, the GDF15 capture antibody coupled to the donor microspheres, the GDF15 antigen in the sample to be tested and the GDF15 detection antibody coupled to the acceptor microspheres are used to perform double antibody sandwich assay. The method is used for detection: 20-30uL of donor microspheres coupled with GDF15 capture antibodies, acceptor microspheres coupled with GDF15 detection antibodies and samples to be tested are added to a white, non-transparent well plate at a volume ratio of 1:1:1, mixed and incubated, and the mixing and incubation conditions are 36.5℃-37.5℃ in the dark and shaken for 15min-25min. The GDF15 capture antibodies coupled to the donor microspheres, the GDF15 antigen in the sample to be tested, and the GDF15 detection antibodies coupled to the acceptor microspheres form fluorescent immune complexes. The fluorescent immune complexes produce singlet oxygen by irradiating the photosensitizer on the donor microspheres with excitation light, and the luminescent agent on the acceptor microspheres receives the energy transfer of singlet oxygen to produce fluorescence. Finally, the fluorescence intensity of the sample to be tested is obtained by fluorescence detection and analysis using a fluorescence scanning analyzer. A standard curve is drawn according to the concentration of the standard and its corresponding fluorescence intensity. Then, the GDF15 content of the analyte in the sample to be tested is obtained according to the fluorescence intensity of the sample to be tested and the standard curve.

4. The GDF15 detection method according to claim 2 or 3, characterized in that: After the microsphere-antibody conjugate is formed, the donor microspheres coupled with the GDF15 capture antibody and the acceptor microspheres coupled with the GDF15 detection antibody are placed in BSA blocking solution and incubated at room temperature in the dark for 0.5-2 hours; then washed and separated with Tris-HCl washing solution to remove unbound antibodies.

5. The GDF15 detection method according to claim 4, characterized in that: The coating ratio and coating incubation conditions in the process of forming a conjugate between the donor microspheres and the GDF15 capture antibody, as well as the coating ratio and coating incubation conditions in the process of forming a conjugate between the acceptor microspheres and the GDF15 detection antibody, are all: 1 mg of microspheres are coated with 0.1 mg of antibody, and incubated at room temperature in the dark with shaking for 2 hours; after the microsphere-antibody conjugate is formed, the donor microspheres coupled with the GDF15 capture antibody and the acceptor microspheres coupled with the GDF15 detection antibody are placed in BSA blocking solution and incubated at room temperature in the dark with shaking for 1 hour; in the process of forming a fluorescent immune complex: the concentration of the donor microspheres coupled with the GDF15 capture antibody is 75ug / ml, and the dosage is 25uL; the concentration of the acceptor microspheres coupled with the GDF15 detection antibody is 100ug / ml, and the dosage is 25uL; the mixing and incubation conditions are incubated at 37°C in the dark with shaking for 15min.

6. The GDF15 detection method according to claim 2 or 3, characterized in that: The white, non-transparent well plate is a polypropylene well plate, or an ELISA plate, or a 96-well plate; the analysis buffer composition is: 50mmol / L Tris-HCl, 0.2% BSA, 0.9% NaCl, 0.05% (v / v) Proclin300, pH 7.

8.

7. A GDF15 detection kit based on the GDF15 detection method according to any one of claims 1 to 6, characterized in that: It includes a test kit body and multiple medicine boxes placed in the test kit body; wherein the multiple medicine boxes include: a medicine box containing donor microspheres coupled to GDF15 capture antibodies, a medicine box containing acceptor microspheres coupled to GDF15 detection antibodies, a medicine box containing GDF15 standard solution and a medicine box containing buffer solution; the test kit body includes a connected box body and a box cover.

8. The GDF15 detection kit according to claim 7, characterized in that The box body and the box cover are connected by a connecting shaft; the buffer solution is an analytical buffer solution with a pH of 7.

8.

9. The GDF15 detection kit according to claim 8, characterized in that The box body and the box cover are connected by a connecting shaft made of phenolic plastic material; the analysis buffer composition is: 50mmol / L Tris-HCl, 0.2% BSA, 0.9% NaCl, 0.05% (v / v) Proclin300, pH 7.

8.

10. The GDF15 detection kit according to claim 7, 8 or 9, characterized in that: An expandable polystyrene foam layer is arranged in the test kit body; an ice trough for storing crushed ice is prefabricated at the bottom of the box body of the test kit body.

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