A kit for combined detection of GDF-15, NSE, and CYFRA21-1 and its application in diagnosis of pulmonary nodules

Abstract

The invention provides a kit for the combined detection of GDF-15, NSE and CYFRA 21-1, comprising: an acridinium ester-labeled antibody working solution and a magnetic bead-coated antibody working solution; the acridinium ester-labeled antibody working solution contains acridinium ester-labeled GDF-15, NSE and CYFRA 21-1 antibodies, and the magnetic bead-coated antibody working solution contains magnetic bead-coated GDF-15, NSE and CYFRA 21-1 antibodies; by screening the components and the contents of the components of the acridinium ester-labeled working solution and the magnetic bead-coated working solution, a stable working environment can be provided for the acridinium ester-labeled GDF-15, NSE and CYFRA 21-1 antibodies and the magnetic bead-coated GDF-15, NSE and CYFRA 21-1 antibodies, and specific recognition and detection of GDF-15, NSE and CYFRA 21-1 are achieved, thereby achieving GDF-15, NSE and CYFRA 21-1 specific recognition and detection. The 21-1 triple test is used to determine whether lung nodules are benign or malignant and for the early diagnosis of non-small cell lung cancer.

Description

Technical Field

[0001] The present invention belongs to the field of medical technology, and specifically relates to a kit for the combined detection of GDF-15, NSE and CYFRA 21-1 and an application thereof in the detection of biomarkers of pulmonary nodules. Background Art

[0002] Pulmonary nodules mainly refer to single lesions on the lungs with clear boundaries and a diameter of no more than 3 cm, surrounded by air-filled lung tissue, and not accompanied by atelectasis or pleural effusion. Images show small focal, rounded, and increased density shadows. Most pulmonary nodules are benign and may be caused by infection, inflammation, or benign tumors. However, patients with pulmonary nodules are usually advised to follow up regularly to monitor changes in nodules, because a small number of pulmonary nodules may be malignant, especially in people with risk factors for lung cancer. Follow-up includes regular imaging examinations and sometimes tumor marker testing. The evaluation of the benign and malignant nature of pulmonary nodules mainly relies on the following methods: low-dose spiral computed tomography (LDCT) is the preferred method for discovering and evaluating pulmonary nodules. LDCT can observe the size, morphology, density, calcification, lobes and other characteristics of the nodules, providing important clues for the evaluation of benign and malignant nodules. Puncture biopsy is the gold standard for diagnosing the benign and malignant nature of pulmonary nodules. Tissue samples obtained by puncture and pathological examination can confirm the diagnosis. However, the imaging manifestations of many benign nodules and early lung cancer are highly similar, and it is difficult to make an accurate judgment based on imaging alone. An increase in tumor markers does not necessarily mean a tumor, and it may also appear in some benign diseases. A puncture biopsy has a certain risk of complications, such as pneumothorax and bleeding.

[0003] Cytokeratin fragment antigen 21-1 (CYFRA 21-1) is a non-organ-specific tumor marker mainly used for the diagnosis of lung cancer. It is a soluble fragment of cytokeratin-19 (CK19) and is widely used in epithelial cells. In the diagnosis of lung cancer, the increased concentration of CYFRA 21-1 is positively correlated with the clinical stage of the tumor, especially in squamous cell carcinoma of the lung. After treatment, the level of CYFRA 21-1 in patients with non-small cell lung cancer (NSCLC) decreased significantly, indicating that it can be used as an indicator of treatment effect.

[0004] Neuron-specific enolase (NSE) is an enolase found in neural and neuroendocrine tissues, which plays an important role in the glycolytic pathway. NSE is overexpressed in tumors related to neuroendocrine tissue origin, such as small cell lung cancer (SCLC), resulting in significantly elevated serum NSE levels. This enzyme has become a valuable biomarker due to its high expression in certain tumor types, especially in the diagnosis, monitoring, evaluation of treatment response, and prediction of recurrence of small cell lung cancer. Therefore, in clinical practice, the determination of NSE is of great significance for the diagnosis and treatment monitoring of nervous system diseases and certain specific types of tumors.

[0005] Growth Differentiation Factor 15 (GDF-15) is a biomarker associated with a variety of disease processes and has shown potential clinical value in the diagnosis and prognosis of NSCLC in recent years. Studies have shown that GDF-15 levels are often elevated in patients with non-small cell lung cancer, which may be related to tumor growth, metastasis, and the patient's overall inflammatory state. Therefore, GDF-15, as a blood biomarker, may be helpful in assisting the diagnosis of NSCLC, especially when traditional diagnostic methods are not sensitive or specific enough to determine whether lung nodules are benign or malignant.

[0006] Studies have shown that the levels of these markers in lung cancer patients are significantly higher than those in healthy people and patients with benign lung lesions, making them a powerful tool for distinguishing lung cancer from non-lung cancer lesions. Therefore, the combined detection of these biomarkers provides important information for the evaluation of benign and malignant lung nodules and the early diagnosis and treatment monitoring of non-small cell lung cancer, which helps to improve the prognosis of patients.

[0007] Chemiluminescence immunoassay (CLIA) is a highly sensitive and specific diagnostic technique used to detect and quantify disease biomarkers in biological samples. By combining the target antigen with a specific antibody, which then reacts with a chemiluminescent substrate to produce light, accurate diagnosis of the target disease can be achieved by measuring the luminescence intensity. However, when detecting biomarkers, the accuracy and stability of chemiluminescence may be affected by various factors. In addition, by optimizing antibody labeling and signal amplification strategies, the detection ability of low-abundance biomarkers can be effectively improved, which is of great significance for early disease diagnosis. Summary of the invention

[0008] The object of the present invention is to provide a kit for the combined detection of GDF-15, NSE and CYFRA 21-1, comprising: an acridinium ester labeled antibody working solution and a magnetic bead coated antibody working solution; the acridinium ester labeled antibody working solution contains acridinium ester labeled GDF-15, NSE and CYFRA 21-1 antibodies, and the magnetic bead coated antibody working solution contains magnetic bead coated GDF-15, NSE and CYFRA 21-1 antibodies;

[0009] The magnetic bead-coated antibody working solution is composed of: 0.5-1.0 mg / mL magnetic beads coated with GDF-15, NSE, CYFRA 21-1 antibodies, 20-50 mM Tris, 100-200 mM NaCl, 20-30 mM MES, 1-2% BSA, 1-2% sucrose, 2-5% glycerol, 0.5-1% bilirubin oxidase, 0.05-0.1% ascorbic acid oxidase and 0.005-0.01% Proclin300, and the pH of the working solution is 6.5-6.8;

[0010] The acridinium ester-labeled antibody working solution is composed of: 0.8-1.2 μg / mL acridinium ester-labeled GDF-15, NSE, CYFRA 21-1 antibodies, 20-50 mM Tris, 100-200 mM NaCl, 1-2% BSA, 1-2% sucrose, 2-5% glycerol, 0.5-1% bilirubin oxidase, 0.05-0.1% ascorbic acid oxidase, 0.1-0.5% glycine and 0.005-0.01% Proclin300, and the pH of the working solution is 6.5-6.8.

[0011] The present invention has conducted in-depth research on the working solution of magnetic bead-coated antibodies and the working solution of acridinium ester-labeled antibodies. The working solution includes Tris buffer, NaCl, BSA, sucrose, glycerol, bilirubin oxidase, ascorbic acid oxidase, Proclin300 and other ingredients, which provide a stable detection environment, can effectively avoid the mutual interference of GDF-15, NSE, and CYFRA 21-1 antigen detection, improve the sensitivity and accuracy of the three detections, and can be used for early screening of patients with pulmonary nodules. More importantly, the applicant has found that by adding bilirubin oxidase and ascorbic acid oxidase to the working solution, the interference of bilirubin and ascorbic acid is effectively avoided, the reaction system is prevented from being turbid, and the substrate stability and the anti-interference ability of the reagent are enhanced. In particular, the sensitivity of GDF-15 can be effectively improved. Preferably, the weight ratio of bilirubin oxidase and ascorbic acid oxidase is 5 to 10:1. Studies have found that too large or too small a ratio will affect the stability of the substrate. More preferably, the weight ratio of the two is 10:1. In addition, 20-50 mM MES is added to the magnetic bead-coated antibody working solution to maintain the stability of the magnetic bead-coated antibody, and 0.05-0.1% glycine is added to the acridinium ester-labeled antibody working solution to maintain the activity and stability of the acridinium ester-labeled antibody, and the detection kit has good long-term storage stability. More preferably, in the acridinium ester-labeled antibody working solution, the weight ratio of bilirubin oxidase, ascorbic acid oxidase and glycine is 5-10:1:2.5.

[0012] In addition, the applicant's research also found that the pH of the working solution will also affect antibody detection. The pH of the magnetic bead-coated antibody working solution is alkaline, which will affect the sensitivity, repeatability and recovery rate of antibody detection. Preferably, the pH of the magnetic bead-coated antibody working solution is 6.5-6.8, and the pH of the acridinium ester-labeled antibody working solution is 6.5-6.8.

[0013] In some embodiments, the magnetic bead-coated antibody working solution is composed of: 0.5-1.0 mg / mL magnetic beads coated with GDF-15, NSE, CYFRA 21-1 antibodies, 20-30 mM Tris, 100-150 mM NaCl, 25-30 mM MES, 1.5-2% BSA, 1.5-2% sucrose, 2-3% glycerol, 0.5-1% bilirubin oxidase, 0.05-0.1% ascorbic acid oxidase and 0.005-0.01% Proclin300.

[0014] In some embodiments, the acridinium ester-labeled antibody working solution is composed of: 0.8-1.2 μg / mL acridinium ester-labeled GDF-15, NSE, CYFRA 21-1 antibodies, 20-30 mM Tris, 100-150 mM NaCl, 1.5-2% BSA, 1.5-2% sucrose, 2-3% glycerol, 0.5-1% bilirubin oxidase, 0.05-0.1% ascorbic acid oxidase, 0.1-0.5% glycine and 0.005-0.01% Proclin300.

[0015] In some embodiments, the kit further includes a treatment solution, which is composed of: 20-30 mM Tris, 100-150 mM NaCl, 2-3% urea, 1.5-2% BSA, 1.5-2% sucrose, 2-3% glycerol and 0.005-0.01% Proclin300; the pH of the treatment solution is 6.5-7.0. Pretreatment of the sample can effectively eliminate blood cells in plasma, avoid the possibility of blood cells swallowing magnetic beads, and thus improve the accuracy of the test. In particular, the addition of 2-3% urea can quickly eliminate red blood cells in plasma.

[0016] In some embodiments, the volume ratio of the treatment liquid to the sample to be tested is ≤ 3, preferably 1: 1. Studies have found that if the volume ratio of the treatment liquid to the sample to be tested is too large, the sample concentration is low, which affects the detection sensitivity of the sample.

[0017] In some embodiments, the sample-containing processing solution, magnetic bead-coated GDF-15, NSE, CYFRA 21-1 antibody working solution, and acridinium ester-labeled GDF-15, NSE, CYFRA 21-1 antibody working solution are prepared in a volume ratio of 1:1:1.

[0018] In some embodiments, the combined detection kit further comprises a substrate solution, and the substrate solution is a chemiluminescent substrate solution.

[0019] The present invention also provides a preparation process of magnetic beads coated with GDF-15, NSE, and CYFRA 21-1 antibodies, comprising the following steps:

[0020] 1) Dilute GDF-15, NSE, and CYFRA 21-1 antibodies with dialysate, then dialyze at 2-8°C for 16-24 hours, and mix the three antibodies to obtain a mixed coating antibody solution;

[0021] 2) Rinse the magnetic beads with coating buffer, add NHS and EDC solution to the washed magnetic beads, mix well and incubate for 0.5-1 hour to wash the magnetic beads;

[0022] 3) Add the magnetic beads from step 2) to the mixed coated antibody solution from step 1), rotate and mix well at 25°C, incubate for 1-3 hours, and wash the magnetic beads with magnetic bead washing solution 1;

[0023] 4) Add magnetic bead blocking solution, rotate and mix at 25°C, incubate for 2-4 hours, and wash the magnetic beads with magnetic bead washing solution 2 to obtain magnetic beads coated with GDF-15, NSE, and CYFRA 21-1 antibodies.

[0024] In some embodiments, the dialysate in step 1) is 0.1 M PBS buffer with a pH of 7.8.

[0025] In some embodiments, the coating buffer in step 2) is 100 mM MES buffer at pH 5.0.

[0026] In some embodiments, the concentration of the NHS solution in step 2) is 0.02-0.1 mg / mL, and the concentration of the EDC solution is 0.02-0.1 mg / mL.

[0027] In some embodiments, in step 3), the magnetic beads: antibodies are mixed at a weight ratio of 40:1. The inventors of the present application have also tried different ratios during the experiment, among which a larger ratio resulted in poor antibody labeling effect, and a smaller ratio affected the substrate signal. In general, a weight ratio of 40:1 is preferred for good results.

[0028] In some embodiments, the magnetic bead washing solution 1 in step 3) consists of the following ingredients: 25 mM Tris, 150 mM NaCl, and 2% gelatin.

[0029] In some embodiments, the magnetic bead blocking solution in step 4) consists of the following components: 50 mM Tris, 1% BSA.

[0030] In some embodiments, the magnetic bead washing solution 2 described in step 4) consists of the following components: 25 mM Tris, 150 mM NaCl.

[0031] In some embodiments, the magnetic beads are magnetic microparticles comprising carboxyl functional groups.

[0032] The present invention also provides a preparation process of magnetic beads coated with GDF-15, NSE, and CYFRA 21-1 antibodies, comprising the following steps:

[0033] (1) Dilute GDF-15, NSE, and CYFRA 21-1 antibodies to 1 mg / mL with PBS buffer (0.1 M, pH 7.8), then dialyze with PBS buffer at 2-8°C for 16-24 hours, and mix GDF-15, NSE, and CYFRA 21-1 antibodies in a mass ratio of 2:1:1 to obtain a mixed coating antibody solution;

[0034] (2) Rinse the magnetic beads with pH 5.0 100 mM MES buffer, add NHS (concentration 0.1 mg / mL) and EDC (concentration 0.1 mg / mL) solutions to the washed magnetic beads, mix well, rotate and incubate at 25°C for 30 minutes, and wash the magnetic beads again;

[0035] (3) Add the mixed coating antibody solution at a ratio of 40:1 (mass ratio) and incubate at 25°C for 2 hours with rotation mixing. Wash the magnetic beads with a washing solution of 25 mM Tris, 150 mM NaCl, and 2% gelatin.

[0036] (4) Add magnetic bead blocking solution, rotate and mix well, and incubate at 25°C for 3 hours. The magnetic bead blocking solution is 50 mM Tris, 1% BSA, pH 8.0. After incubation, the magnetic beads are washed again with a magnetic bead washing solution of 25 mM Tris pH 7.8, 150 mM NaCl. The washed magnetic beads are resuspended to a concentration of 10 mg / mL and stored at 2-8°C for use. The magnetic beads are coated with GDF-15, NSE, and CYFRA21-1 antibodies.

[0037] The present invention also provides a process for preparing acridinium ester-labeled GDF-15, NSE, and CYFRA 21-1 antibodies, comprising the following steps:

[0038] A) diluting the GDF-15, NSE, and CYFRA 21-1 antibodies to be labeled with dialysate, and then dialyzing them at 2-8°C for 16-24 hours, and mixing the three antibodies to obtain a mixed labeled antibody solution;

[0039] B) diluting the acridinium ester solution with the labeling buffer, mixing the diluted acridinium ester solution with the mixed antibody solution to be labeled, and reacting at 25°C with shaking for 2-3 hours;

[0040] C) Add magnetic bead blocking solution and block the reaction at 25°C for 20-50 min;

[0041] D) Purification by chromatography column was performed to obtain acridinium ester labeled GDF-15, NSE, and CYFRA 21-1 antibodies.

[0042] In some embodiments, the dialysate in step A) is 0.1 M PBS buffer with a pH of 7.8.

[0043] In some embodiments, in step A), GDF-15, NSE, and CYFRA 21-1 antibodies are mixed at a mass ratio of 2:1:1.

[0044] In some embodiments, the labeling buffer in step B) is 0.1 M PBS buffer with a pH of 7.8.

[0045] In some embodiments, in step B), the weight ratio of antibody to be labeled: acridinium ester is 4:1. The inventors of the present application have also tried different ratios during the experiment. A larger weight ratio resulted in poor antibody labeling effect, while a smaller weight ratio affected the substrate signal. Generally speaking, a weight ratio of 4:1 is preferred for better results.

[0046] In some embodiments, the blocking solution in step C) is a PBS solution containing 1% glycine, with a pH of 7.8. The inventors of the present application have also tried PBS solutions containing different proportions of glycine in the blocking agent during the experiment, and the glycine content was too high, such as 2%, and the signal value was generally not as good as the effect of low concentration.

[0047] In some embodiments, the purification buffer in step D) is 0.1 M PBS buffer containing 0.25% glycine, with a pH of 7.8.

[0048] Furthermore, the present invention also provides a process for preparing acridinium ester-labeled GDF-15, NSE, and CYFRA 21-1 antibodies, comprising the following steps:

[0049] (1) Dilute the GDF-15, NSE, and CYFRA 21-1 antibodies to be labeled with PBS buffer (0.1 M, pH 7.8) to 0.5 mg / mL, then dialyze with PBS buffer (0.1 M, pH 7.8) at 2-8°C for 16-24 hours, and mix the GDF-15, NSE, and CYFRA 21-1 antibodies in a mass ratio of 2:1:1 to obtain a mixed antibody solution to be labeled;

[0050] (2) Dilute the acridinium ester DMSO solution with PBS buffer (0.1 M, pH 7.8) and use it within 5 minutes after preparation. Mix the acridinium ester solution with the mixed antibody solution at a ratio of 4:1 (mass ratio) and shake at 25°C for 120 minutes. Protect from light throughout the process.

[0051] (3) Add PBS solution (0.1 M, pH 7.8) containing 1% glycine to a final concentration of 0.25% and incubate the reaction at 25°C for 30 min.

[0052] (4) Purification was performed using a chromatography column, and the purification buffer was a PBS buffer (0.1 M, pH 7.8) containing 0.25% glycine. The purified acridinium ester-labeled antibody was resuspended to a concentration of 25 ug / mL and stored at 2-8°C until use. Thus, acridinium ester-labeled GDF-15, NSE, and CYFRA 21-1 antibodies were obtained.

[0053] Furthermore, the present invention also provides a method for using a combined detection kit for GDF-15, NSE, and CYFRA 21-1, comprising the following steps:

[0054] (1) Add the treatment solution to the sample to be tested and treat at 25°C for 5 min;

[0055] (2) mixing the treatment solution containing the sample to be tested, the working solution of GDF-15, NSE, and CYFRA 21-1 magnetic bead-coated antibody, and the working solution of GDF-15, NSE, and CYFRA 21-1 acridinium ester-labeled antibody at a volume ratio of 1:1:1, and reacting at 25°C for 5 min;

[0056] (3) subjecting the reaction solution to magnetic separation and collecting magnetic beads;

[0057] (4) Wash the magnetic beads, then add the chemiluminescent substrate solution, mix well, and detect the luminescence value.

[0058] In some embodiments, the treatment solution consists of: 20-30 mM Tris, 100-150 mM NaCl, 2-3% urea, 1.5-2% BSA, 1.5-2% sucrose, 2-3% glycerol and 0.005-0.01% Proclin300, and the pH of the treatment solution is 6.5-7.0.

[0059] In another aspect, the present invention provides a kit for the combined detection of lung nodule biomarkers GDF-15, NSE, and CYFRA 21-1, wherein the kit is as defined above.

[0060] On the other hand, the present invention provides the use of GDF-15, NSE, and CYFRA 21-1 as biomarkers in the preparation of a detection reagent or a kit for diagnosing benign or malignant pulmonary nodules.

[0061] Beneficial effects:

[0062] 1. The present invention provides a kit for the joint detection of GDF-15, NSE and CYFRA 21-1. The components and contents of the acridinium ester-labeled working solution and the magnetic bead-coated working solution are screened to provide a stable working environment for the acridinium ester-labeled GDF-15, NSE and CYFRA 21-1 antibodies and the magnetic bead-coated GDF-15, NSE and CYFRA 21-1 antibodies, thereby achieving specific recognition and detection of GDF-15, NSE and CYFRA 21-1, wherein the detection sensitivity is high, the detection range is wide, and the linear range is good; meanwhile, the repeatability and recovery rate of the detection results of the kit meet the requirements, thereby achieving the triple simultaneous detection of GDF-15, NSE and CYFRA21-1, and the accuracy and detection efficiency are greatly improved.

[0063] 2. The GDF-15, NSE and CYFRA 21-1 detection kits provided by the present invention preferably pre-treat the samples to quickly eliminate blood cells in the plasma and effectively avoid the possibility of blood cells swallowing magnetic beads.

[0064] 3. The present invention provides a method for preparing a working solution of magnetic beads coated with GDF-15, NSE, and CYFRA 21-1 antibodies. During the preparation process, NHS and EDC are first used to activate the magnetic beads, thereby improving the coupling efficiency of GDF-15, NSE, and CYFRA 21-1 antibodies with the magnetic beads; the magnetic beads are dissolved with MES buffer to ensure the dispersion stability of the magnetic beads, effectively preventing the detection signal from being affected by the coagulation of the magnetic beads, thereby ensuring the accuracy of the detection results; when the kit is used for plasma detection, the blood has complex components. The preparation method provided by the present invention adds a magnetic bead blocking solution after coupling the GDF-15, NSE, and CYFRA 21-1 antibodies with the magnetic beads, thereby avoiding non-specific binding that may occur when the samples are detected later, thereby further ensuring the accuracy of the detection results. The use of a magnetic bead blocking solution to simultaneously achieve effective blocking of the magnetic beads can ensure the activity of the GDF-15, NSE, and CYFRA 21-1 antibodies and improve the detection performance of the kit.

[0065] 4. The present invention also provides a method for preparing a working solution of acridinium ester-labeled antibodies of GDF-15, NSE, and CYFRA 21-1. During the preparation process, a PBS solution containing glycine is added to terminate the activation reaction, thereby effectively avoiding the occurrence of nonspecific reactions. At the same time, a PBS solution containing glycine is used for purification during the purification process, thereby ensuring the stability of the conjugate of the antibody and acridinium ester and the activity of the antibody. DETAILED DESCRIPTION

[0066] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention.

[0067] Unless otherwise specified, the reagents or consumables used in the present invention are commercially available.

[0068] Unless otherwise specified herein, % refers to mass percentage.

[0069] Example 1: Preparation of magnetic bead-coated antibody working solution and acridinium ester-labeled antibody working solution

[0070] (1) Preparation of magnetic bead-coated GDF-15, NSE, and CYFRA 21-1 antibody working solution:

[0071] The GDF-15, NSE, and CYFRA 21-1 coating antibodies were diluted to 1 mg / mL with PBS buffer (0.1 M, pH 7.8), and then dialyzed with PBS buffer (0.1 M, pH 7.8) at 2-8°C for 16-24 hours. The GDF-15, NSE, and CYFRA 21-1 antibodies were mixed at a mass ratio of 2:1:1 to obtain a mixed coating antibody solution.

[0072] The magnetic beads were rinsed three times with pH 5.0 100 mM MES buffer. NHS (N-hydroxysuccinimide, concentration 0.1 mg / mL) and EDC (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, concentration 0.1 mg / mL) solutions were added to the washed magnetic beads, mixed and incubated at 25°C for 30 minutes, and the magnetic beads were washed again. The mixed coating antibody solution was added at a ratio of magnetic beads: antibody = 40:1 (mass ratio), incubated at 25°C for 2 hours, and the magnetic beads were washed again with magnetic bead washing solution (25 mM Tris, 150 mM NaCl, 2% gelatin). Then, add magnetic bead blocking solution and incubate at 25°C for 2 hours with rotation mixing. The magnetic bead blocking solution is 50 mM Tris, 1% BSA, pH 8.0. After incubation, the magnetic beads are washed three times with magnetic bead washing solution (25mM Tris pH 7.8, 150 mM NaCl). Resuspend the washed magnetic beads to a concentration of 10 mg / mL and store at 2-8°C for use. The magnetic beads are coated with GDF-15, NSE, and CYFRA 21-1 antibodies.

[0073] The magnetic bead-coated antibody working solution is composed of: 0.5 mg / mL magnetic beads coated with GDF-15, NSE, CYFRA 21-1 antibodies, 20 mM Tris, 100 mM NaCl, 25 mM MES, 1.5% BSA, 1.5% sucrose, 2% glycerol, 0.5% bilirubin oxidase, 0.1% ascorbic acid oxidase and 0.005% Proclin300, and the pH of the magnetic bead-coated antibody working solution is 6.5-6.8.

[0074] (2) Preparation of acridinium ester labeled GDF-15, NSE, and CYFRA 21-1 antibodies:

[0075] The GDF-15, NSE, and CYFRA 21-1 antibodies to be labeled were diluted to 0.5 mg / mL with PBS buffer (0.1 M, pH 7.8), and then dialyzed at 2-8°C for 16-24 hours. The GDF-15, NSE, and CYFRA 21-1 antibodies were mixed in a mass ratio of 2:1:1 to obtain a mixed antibody solution to be labeled.

[0076] Take 2mg / mL acridinium ester DMSO solution, dilute the acridinium ester DMSO solution with PBS buffer (0.1M, pH 7.8) (use within 5 minutes after preparation, light will reduce the performance of acridinium ester), label the antibody at a mass ratio of 4:1, mix the diluted acridinium ester DMSO solution with the mixed antibody solution to be labeled, shake and react at 25℃ for 120 minutes, and protect from light throughout the process. Add PBS (0.1M, pH 7.8) containing 1% glycine to a final concentration of 0.25%, and block at room temperature (25℃) for 30 minutes. Finally, use a chromatography column filled with G-25 for purification, and the purification buffer is PBS buffer (0.1M, pH7.8) containing 0.25% glycine. The purified acridinium ester-labeled antibody was resuspended to a concentration of 25 μg / mL and stored at 2-8° C. for later use, thereby obtaining acridinium ester-labeled GDF-15, NSE, and CYFRA 21-1 antibodies.

[0077] The acridinium ester-labeled antibody working solution is composed of: 0.8 μg / mL acridinium ester-labeled GDF-15, NSE, CYFRA 21-1 antibodies, 20 mM Tris, 100 mM NaCl, 1.5% BSA, 1.5% sucrose, 2% glycerol, 0.5% bilirubin oxidase, 0.1% ascorbic acid oxidase, 0.25% glycine and 0.005% Proclin300, and the pH of the acridinium ester-labeled antibody working solution is 6.5-6.8.

[0078] Example 2

[0079] (1) Preparation of magnetic bead-coated GDF-15, NSE, and CYFRA 21-1 antibody working solution:

[0080] The GDF-15, NSE, and CYFRA 21-1 coating antibodies were diluted to 1 mg / mL with PBS buffer (0.1 M, pH 7.8), and then dialyzed with PBS buffer (0.1 M, pH 7.8) at 2-8°C for 16-24 hours. The GDF-15, NSE, and CYFRA 21-1 antibodies were mixed at a mass ratio of 2:1:1 to obtain a mixed coating antibody solution.

[0081] The magnetic beads were rinsed three times with pH 5.0 100 mM MES buffer. NHS (N-hydroxysuccinimide, concentration 0.1 mg / mL) and EDC (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, concentration 0.1 mg / mL) solutions were added to the washed magnetic beads, mixed and incubated at 25°C for 30 minutes, and the magnetic beads were washed again. The mixed coating antibody solution was added at a ratio of magnetic beads: antibody = 40:1 (mass ratio), incubated at 25°C for 2 hours, and the magnetic beads were washed again with magnetic bead washing solution (25 mM Tris, 150 mM NaCl, 2% gelatin). Then, add magnetic bead blocking solution and rotate and mix at 25℃ for 2 hours. The magnetic bead blocking solution is 50 mM Tris, 1% BSA, pH 8.0. After incubation, the magnetic beads are washed three times with magnetic bead washing solution (25mM Tris pH 7.8, 150 mM sodium chloride). Resuspend the washed magnetic beads to a concentration of 10 mg / mL and store at 2-8℃ for use. The magnetic beads are coated with GDF-15, NSE, and CYFRA 21-1 antibodies.

[0082] The magnetic bead-coated antibody working solution is composed of: 1.0 mg / mL magnetic beads coated with GDF-15, NSE, CYFRA 21-1 antibodies, 30 mM Tris, 150 mM NaCl, 30 mM MES, 2% BSA, 2% sucrose, 3% glycerol, 1% bilirubin oxidase, 0.1% ascorbic acid oxidase and 0.005% Proclin300, and the pH of the magnetic bead-coated antibody working solution is 6.5-6.8.

[0083] (2) Preparation of acridinium ester labeled GDF-15, NSE, and CYFRA 21-1 antibodies:

[0084] The GDF-15, NSE, and CYFRA 21-1 antibodies to be labeled were diluted to 0.5 mg / mL with PBS buffer (0.1 M, pH 7.8), and then dialyzed at 2-8°C for 16-24 hours. The GDF-15, NSE, and CYFRA 21-1 antibodies were mixed in a mass ratio of 2:1:1 to obtain a mixed antibody solution to be labeled.

[0085] Take 2mg / mL acridinium ester DMSO solution, dilute the acridinium ester DMSO solution with PBS buffer (0.1M, pH 7.8) (use within 5 minutes after preparation, light will reduce the performance of acridinium ester), label the antibody at a mass ratio of 4:1, mix the diluted acridinium ester DMSO solution with the mixed antibody solution to be labeled, shake and react at 25℃ for 120 minutes, and protect from light throughout the process. Add PBS (0.1M, pH 7.8) containing 1% glycine to a final concentration of 0.25%, and block at room temperature (25℃) for 30 minutes. Finally, use a chromatography column filled with G-25 for purification, and the purification buffer is PBS buffer (0.1M, pH7.8) containing 0.25% glycine. The purified acridinium ester-labeled antibody was resuspended to a concentration of 25 μg / mL and stored at 2-8° C. for later use, thereby obtaining acridinium ester-labeled GDF-15, NSE, and CYFRA 21-1 antibodies.

[0086] The acridinium ester labeling working solution is composed of: 1.2 μg / mL acridinium ester labeled GDF-15, NSE, CYFRA21-1 antibodies, 30 mM Tris, 150 mM NaCl, 2% BSA, 2% sucrose, 3% glycerol, 1% bilirubin oxidase, 0.1% ascorbic acid oxidase, 0.25% glycine and 0.01% Proclin300, and the pH of the acridinium ester labeling working solution is 6.5-6.8.

[0087] Comparative Example 1:

[0088] The only difference from Example 1 is that the pH of the working solution of the magnetic beads coated with GDF-15, NSE, and CYFRA 21-1 antibodies is 7.0-8.0.

[0089] The only difference from Example 1 is that the pH of the acridinium ester labeled GDF-15, NSE, CYFRA 21-1 antibody working solution is 7.0-8.0.

[0090] Comparative Example 2

[0091] The only difference from Example 1 is that the magnetic bead-coated antibody working solution is composed of: 0.5 mg / mL magnetic beads coated with GDF-15, NSE, CYFRA 21-1 antibodies, 20 mM Tris, 100 mM NaCl, 1.5% BSA, 1.5% sucrose, 2% glycerol and 0.005% Proclin300, and the pH of the magnetic bead-coated antibody working solution is 6.5-6.8.

[0092] The acridinium ester labeled antibody working solution is composed of: 0.8 μg / mL acridinium ester labeled GDF-15, NSE, CYFRA 21-1 antibodies, 20 mM Tris, 100 mM NaCl, 1.5% BSA, 1.5% sucrose, 2% glycerol and 0.005% Proclin300, and the pH of the acridinium ester labeled antibody working solution is 6.5-6.8.

[0093] Test Example 1: Determination of samples

[0094] The method of using the GDF-15, NSE, and CYFRA 21-1 detection kit includes the following steps:

[0095] (1) Add a treatment solution to the sample to be tested and treat it at a temperature of 25°C for 5 min; wherein the volume ratio of the treatment solution to the sample to be tested is ≤3, and preferably, the volume ratio of the treatment solution to the sample to be tested is 1:1. The treatment solution is composed of: 20 mM Tris, 150 mM NaCl, 3% urea, 2% BSA, 2% sucrose, 3% glycerol and 0.01% Proclin300, and the pH of the treatment solution is 6.5-7.0.

[0096] (2) mixing the treatment solution containing the sample to be tested, the working solution of GDF-15, NSE, and CYFRA 21-1 magnetic bead-coated antibody, and the working solution of GDF-15, NSE, and CYFRA 21-1 acridinium ester-labeled antibody at a volume ratio of 1:1:1, and reacting at a temperature of 25°C for 5 minutes;

[0097] (3) subjecting the reaction solution to magnetic separation and collecting magnetic beads;

[0098] (4) Wash the magnetic beads, then add the chemiluminescent substrate solution, mix well, and detect the luminescence value.

[0099] Test Example 2: Performance Evaluation

[0100] (1) Linear detection

[0101] GDF-15, NSE, and CYFRA 21-1 were diluted with standard diluent and prepared into calibration solutions of different concentrations. Then, the GDF-15, NSE, and CYFRA 21-1 calibration products were detected using the kit in the embodiment, and the luminescence intensity values ​​corresponding to each calibration product were read respectively. The standard curve was obtained by fitting with the concentration as the abscissa and the luminescence intensity as the ordinate. The results showed that the linear correlation coefficients of GDF-15, NSE, and CYFRA 21-1 were all >0.99.

[0102] (2) Minimum detection limit detection

[0103] The zero-concentration calibrator was used as a sample for detection, and the measurement was repeated 20 times to obtain the relative luminescence intensity (RLU) value of the 20 measurement results, and the mean value (M) and standard deviation (SD) were calculated to obtain M+2SD. A linear equation was obtained by performing a two-point regression fitting based on the concentration-RLU between the zero-concentration calibrator and the adjacent calibrator. The RLU value of M+2SD (positive reaction) or M-2SD (negative reaction) was substituted into the above equation to obtain the corresponding concentration value, which is the minimum detection limit. The test results are shown in Table 1.

[0104] Table 1 Test results

[0105] Minimum detection limit GDF-15(pg / mL) NSE (ng / mL) CYFRA21-1 (ng / mL) Example 1 321.135 1.563 0.489 Example 2 318.467 1.476 0.405 Comparative Example 1 430.368 1.753 0.693 Comparative Example 2 545.568 1.844 0.934

[0106] (3) Repeatability test

[0107] Use 2 samples with different concentration levels (high value and low value), and repeat the test 10 times for each concentration sample. Calculate the mean (M) and standard deviation (SD) of the 10 measurement results, and then calculate the coefficient of variation (CV) according to the formula CV = SD / M × 100%. Among them, CV is the coefficient of variation, SD is the standard deviation of the 10 measurement results, and M is the mean of the 10 measurement results. The results show that CV is within ≤8%.

[0108] Table 2 GDF-15 repeatability test results

[0109]

[0110] Table 3 NSE repeatability test results

[0111]

[0112] Table 4 CYFRA 21-1 repeatability test results

[0113]

[0114] (4) Accuracy test

[0115] Using samples of GDF-15, NSE, and CYFRA 21-1 standards at high and low concentration levels, the measurements were repeated three times, and the relative deviation between the average value (denoted as M) and the labeled value was calculated. The relative deviation B of the measured concentration was calculated according to the formula B = (MT) / T × 100%, where B is the relative deviation, M is the average value of the measured concentration, and T is the labeled value. The results showed that the relative deviation was ≤10%.

[0116] Table 5 GDF-15 accuracy test results

[0117]

[0118] Table 6 NSE accuracy test results

[0119]

[0120] Table 7 CYFRA 21-1 accuracy test results

[0121]

[0122] (5) Recovery rate test

[0123] High-concentration sample A was added to low-concentration sample B. (The volume ratio between sample A and sample B was 1:9, and the total concentration after addition was within the linear range of the kit), and the mixed sample A+B, sample A and sample B were measured respectively, and each was repeated 3 times, and the average value was calculated, and the recovery rate was calculated according to the formula.

[0124]

[0125] R is the recovery rate, V is the volume of sample A solution, V0 is the volume of sample B solution, C is the average detection concentration of sample B solution after adding A solution, C0 is the average detection concentration of sample B solution, C s is the average detection concentration of sample A. The results show that the recovery rate is in the range of 85% to 115%.

[0126] Table 8 GDF-15 recovery determination results

[0127]

[0128] Table 9 NSE recovery determination results

[0129]

[0130] Table 10 CYFRA 21-1 recovery determination results

[0131]

[0132] (6) Thermal acceleration stability test

[0133] The working solution in the kit was placed in a 37°C thermal acceleration test for 7 days, and the luminescence values ​​before and after the test were measured. The results showed that compared with the kit without the thermal acceleration test, the RLU measured by the kit of the embodiment of the present invention was only reduced by 5%, while the RLU in Comparative Example 2 was reduced by 12%. This shows that the kit of the present invention has good stability.

[0134] Test Case 3: Clinical Diagnosis of Non-Small Cell Lung Cancer in Patients with Pulmonary Nodules

[0135] The data of patients with lung nodules were analyzed using the method in Test Example 1, and the diagnostic performance of different indicators and combinations for non-small cell lung cancer in patients with lung nodules was analyzed by the receiver operating characteristic (ROC) curve. The samples were placed in a fully automatic chemiluminescence immunoassay analyzer, and the concentration of each biomarker was measured and recorded. At the same time, the samples were added to a single marker reagent component, and the concentration of each biomarker was measured and recorded. In patients with lung nodules, early diagnosis of benign and malignant diseases and timely treatment are crucial to improving prognosis. Through clinical research, the levels of GDF-15, NSE, and CYFRA 21-1 in blood samples of 75 patients with benign lung nodules found in the initial LDCT screening and pathologically confirmed, and 75 patients with lung nodules found in the initial LDCT screening and diagnosed with early non-small cell lung cancer were analyzed, and the imaging examination data of the patients were collected.

[0136] A logistic regression model was used to predict the occurrence of lung cancer. The model formula is as follows:

[0137]

[0138] Among them, p is the probability that the lung nodule is a malignant tumor, i.e., lung cancer. GDF, NSE, and CYFRA represent the concentration values ​​detected by the chemiluminescent immunoassay of GDF-15, NSE, and CYFRA 21-1, respectively. β0 is the natural constant obtained by regression. β1, β2, and β3 are the regression coefficients of GDF-15, NSE, and CYFRA 21-1, respectively. Β4, β5, β6, and β7 are the regression coefficients of Smoker, Location, Diameter, and Spiculation, respectively. If the subject is a former or current smoker (in 1997, WHO defined "a person who has smoked continuously or cumulatively for 6 months or more in his life" as a smoker), Smoker is 1 (otherwise 0), and the other three parameters are the results of the patient's imaging examination: Diameter is the size of the lung nodule (in millimeters). If the lung nodule is spiculated (otherwise 0), Spiculation is 1, and if the lung nodule is located in the upper lobe (otherwise zero), Location is 1.

[0139] β0, β1, β2 and β3 are -0.381, 0.442, 0.579 and 0.198 respectively.

[0140] β4, β5, β6 and β7 are 0.351, 0.637, 0.602 and 0.776 respectively.

[0141] The experimental results showed that the average GDF-15 of lung cancer patients was 1395.44 pg / mL, the average NSE was 18.32 ng / mL, and the average CYFRA 21-1 was 5.52 ng / mL. Relatively speaking, the levels of GDF-15, NSE, and CYFRA21-1 in patients with benign pulmonary nodules were significantly lower, at 495.44 pg / mL, 11.34 ng / mL, and 2.64 ng / mL, respectively. Through ROC curve analysis, the AUC of the combined diagnosis of the model established by the combined detection and imaging examination (LDCT (low-dose chest CT)) data reached 0.889, the specificity was 87.15%, and the sensitivity was 91.31%, as shown in Table 11. The results show that the GDF-15, NSE, and CYFRA 21-1 combined detection kit of the present invention has high accuracy in the judgment of benign and malignant lung nodules and the diagnosis of non-small cell lung cancer, and is better than the performance of single indicator detection.

[0142] Table 11 Early diagnosis results of lung cancer in patients with pulmonary nodules

[0143]

[0144] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and substitutions can be made without departing from the technical principles of the present invention. These improvements and substitutions should also be regarded as the scope of protection of the present invention.

Claims

1. A kit for the combined detection of GDF-15, NSE and CYFRA 21-1, characterized in that: The kit comprises: an acridinium ester labeled antibody working solution and a magnetic bead coated antibody working solution; the acridinium ester labeled antibody working solution contains acridinium ester labeled GDF-15, NSE, CYFRA 21-1 antibodies, and the magnetic bead coated antibody working solution contains magnetic bead coated GDF-15, NSE, CYFRA 21-1 antibodies; The magnetic bead-coated antibody working solution is composed of: 0.5-1.0 mg / mL magnetic beads coated with GDF-15, NSE, CYFRA21-1 antibodies, 20-50 mM Tris, 100-200 mM NaCl, 20-30 mM MES, 1-2% BSA, 1-2% sucrose, 2-5% glycerol, 0.5-1% bilirubin oxidase, 0.05-0.1% ascorbic acid oxidase and 0.005-0.01% Proclin300, and the pH of the working solution is 6.5-6.8; The acridinium ester-labeled antibody working solution is composed of: 0.8-1.2 μg / mL acridinium ester-labeled GDF-15, NSE, CYFRA 21-1 antibodies, 20-50 mM Tris, 100-200 mM NaCl, 1-2% BSA, 1-2% sucrose, 2-5% glycerol, 0.5-1% bilirubin oxidase, 0.05-0.1% ascorbic acid oxidase, 0.1-0.5% glycine and 0.005-0.01% Proclin300, and the pH of the working solution is 6.5-6.8; The preparation process of the magnetic beads coated with GDF-15, NSE, and CYFRA21-1 antibodies comprises the following steps: 1) Dilute GDF-15, NSE, and CYFRA 21-1 antibodies with dialysate, then dialyze at 2-8°C for 16-24 hours, and mix the three antibodies to obtain a mixed coating antibody solution; 2) Rinse the magnetic beads with coating buffer, add NHS and EDC solution to the washed magnetic beads, mix well and incubate for 0.5-1 hour to wash the magnetic beads; 3) Add the magnetic beads from step 2) to the mixed coated antibody solution from step 1), rotate and mix at 25°C for 1-3 hours, and wash the magnetic beads with magnetic bead washing solution 1, wherein the magnetic beads: antibody are mixed at a mass ratio of 40:1; 4) Add magnetic bead blocking solution, rotate and mix, incubate at 25°C for 2-4 hours, and wash the magnetic beads with magnetic bead washing solution 2 to obtain magnetic beads coated with GDF-15, NSE, and CYFRA 21-1 antibodies; The preparation process of the acridinium ester-labeled GDF-15, NSE, and CYFRA 21-1 antibodies comprises the following steps: A) diluting the GDF-15, NSE, and CYFRA 21-1 antibodies to be labeled with dialysate, and then dialyzing them at 2-8°C for 16-24 hours, and mixing the three antibodies to obtain a mixed labeled antibody solution; B) diluting the acridinium ester solution with a labeling buffer, mixing the diluted acridinium ester solution with the antibody solution to be labeled, and reacting with shaking at 25° C. for 2-3 hours, wherein the mass ratio of the antibody to be labeled to the acridinium ester is 4:1; C) adding magnetic bead blocking solution to block the reaction at 25°C for 20-50 minutes, wherein the blocking solution is a PBS solution containing 1% glycine, with a pH of 7.8; D) Purification by chromatography column was performed to obtain acridinium ester labeled GDF-15, NSE, and CYFRA 21-1 antibodies.

2. A kit for the combined detection of GDF-15, NSE and CYFRA 21-1 according to claim 1, characterized in that: The magnetic bead-coated antibody working solution is composed of: 0.5-1.0 mg / mL magnetic beads coated with GDF-15, NSE, CYFRA21-1 antibodies, 20-30 mM Tris, 100-150 mM NaCl, 25-30 mM MES, 1.5-2% BSA, 1.5-2% sucrose, 2-3% glycerol, 0.5-1% bilirubin oxidase, 0.05-0.1% ascorbic acid oxidase and 0.005-0.01% Proclin300.

3. A kit for the combined detection of GDF-15, NSE and CYFRA 21-1 according to claim 1, characterized in that: The acridinium ester-labeled antibody working solution is composed of: 0.8-1.2 μg / mL acridinium ester-labeled GDF-15, NSE, CYFRA 21-1 antibodies, 20-30 mM Tris, 100-150 mM NaCl, 1.5-2% BSA, 1.5-2% sucrose, 2-3% glycerol, 0.5-1% bilirubin oxidase, 0.05-0.1% ascorbic acid oxidase, 0.1-0.5% glycine and 0.005-0.01% Proclin300.

4. A kit for the combined detection of GDF-15, NSE and CYFRA 21-1 according to claim 1, characterized in that: The kit also includes a treatment solution, which is composed of: 20-30 mM Tris, 100-150 mM NaCl, 2-3% urea, 1.5-2% BSA, 1.5-2% sucrose, 2-3% glycerol and 0.005-0.01% Proclin300.

5. A kit for the combined detection of GDF-15, NSE and CYFRA 21-1 according to claim 4, characterized in that: The pH of the treatment liquid is 6.5-7.

0.

6. A kit for the combined detection of GDF-15, NSE and CYFRA 21-1 according to claim 1, characterized in that: The dialysate in step A) is 0.1 M PBS buffer with a pH of 7.

8.

7. A kit for the combined detection of GDF-15, NSE and CYFRA 21-1 according to claim 1, characterized in that: The labeling buffer in step B) is 0.1 M PBS buffer with a pH of 7.

8.

8. A kit for the combined detection of GDF-15, NSE and CYFRA 21-1 according to claim 1, characterized in that: The purification buffer in step D) is 0.1 M PBS buffer containing 0.25% glycine, pH 7.8.

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