A kit for detecting or aiding in the detection of a lung cancer marker and a system comprising the same

Abstract

The application discloses a kit for detecting or assisting in detecting a lung cancer index and a system comprising the same. The kit provided by the application comprises a kit for detecting lung cancer and a data processing device, the data processing device comprises a data acquisition module, a data comparison module and a result determination module, and the kit comprises a magnetic bead working solution, a metal ion binder suspension and a calibrant containing CEA, CYFRA211, NSE, SCC and ProGRP. The method of the application uses element mass spectrometry, greatly shortens the reaction time, and has the advantages of high accuracy, strong specificity, high sensitivity, wide linear range, stable detection and convenience. The kit can also realize large-throughput detection and meet the detection requirements of different city passenger flows.

Description

Technical Field

[0001] This invention relates to the field of in vitro diagnostic reagent technology, specifically to a reagent kit and system comprising lung cancer markers for detection or auxiliary detection. Background Technology

[0002] Statistics show that lung cancer is the leading cause of death among malignant tumors, with approximately 70,000 to 80,000 new cases each year. Early symptoms of lung cancer are not obvious, and patients usually seek medical attention only after symptoms such as hemoptysis appear, by which time it is usually already in an advanced stage. Furthermore, lung cancer tissue diagnosis is an invasive screening procedure, which is not well accepted by patients and is difficult to obtain.

[0003] With the development of molecular diagnostics, serological markers have provided valuable support for the classification and screening of lung cancer. NSE and ProGRP are ideal markers for the auxiliary diagnosis of SCLC, while elevated levels of CEA, SCC, and CYFRA21-1 in patient serum are helpful in the diagnosis of NSCLC. SCC and CYFRA21-1 are generally considered to have high specificity for squamous cell carcinoma of the lung. It is hypothesized that combined detection of markers such as NSE, CYFRA21-1, ProGRP, CEA, and SCC could improve the accuracy of differentiating between SCLC and NSCLC. Summary of the Invention

[0004] The present invention aims to provide a detection method that can reduce detection costs, shorten detection time, improve detection sensitivity and linear range, and simultaneously detect the content of CEA, CYFRA211, NSE, SCC and ProGRP.

[0005] This invention provides a system for detecting or assisting in the detection of lung cancer indicators, comprising a reagent kit for detecting lung cancer and a data processing device, wherein the data processing device includes:

[0006] Data acquisition module: The data acquisition module receives the concentration data of the sample to be tested, which is obtained from the reagent kit, and transmits the concentration data to the data comparison module.

[0007] Furthermore, the concentration data refers to the concentration values ​​of at least three of CEA, CYFRA211, NSE, SCC, and ProGRP detected by inductively coupled plasma mass spectrometry.

[0008] Data comparison module: The data comparison module receives the concentration value data transmitted by the data acquisition module, compares the concentration value data with the corresponding cutoff value, and transmits the comparison result to the result determination module;

[0009] Furthermore, the cutoff value for detecting CEA antigen was 4 ng / mL; the cutoff value for detecting CYFRA211 antigen was 2 ng / mL; the cutoff value for detecting NSE antigen was 11 ng / mL; the cutoff value for detecting SCC antigen was 1.5 ng / mL; and the cutoff value for detecting ProGRP antigen was 64 pg / mL.

[0010] Result determination module: The structure determination module receives the comparison result from the data comparison module and determines the result.

[0011] The kit includes:

[0012] (1) Magnetic bead working solution, wherein the magnetic bead working solution comprises magnetic beads connected with biotinylated CEA antibody, magnetic beads connected with biotinylated CYFRA211 antibody, magnetic beads connected with biotinylated NSE antibody, magnetic beads connected with biotinylated SCC antibody, magnetic beads connected with biotinylated Pro-GRP antibody and magnetic bead buffer.

[0013] (2) A metal ion conjugate suspension, wherein the metal ion conjugate suspension includes a CEA antibody labeled with lanthanide A1, a CYFRA211 antibody labeled with lanthanide A2, an NSE antibody labeled with lanthanide A3, an SCC antibody labeled with lanthanide A4, and a ProGRP antibody labeled with lanthanide A5.

[0014] (3) Calibrators containing CEA, CYFRA211, NSE, SCC, and ProGRP;

[0015] The lanthanide elements A1, A2, A3, A4, and A5 are different lanthanide elements;

[0016] Each liter of the magnetic bead buffer contains 40g of polyvinylpyrrolidone, 11.56g of MOPSO, 50g of sucrose, 10g of bovine serum albumin, 8g of sodium chloride, 750μL of Tween-20 liquid, 1000μL of Proclin-300, and 750μL of Triton X-100, with the pH adjusted to 6.5 using NaOH.

[0017] The CEA antibody marker labeled with lanthanide element A1 is obtained by reacting lanthanide element A1-chelating agent with CEA antibody.

[0018] The CYFRA211 antibody marker labeled with lanthanide element A2 is obtained by reacting lanthanide element A2-chelating agent with CYFRA211 antibody;

[0019] The NSE antibody marker labeled with lanthanide element A3 is obtained by reacting lanthanide element A3-chelating agent with NSE antibody;

[0020] The SCC antibody marker labeled with lanthanide element A4 is obtained by reacting lanthanide element A4-chelating agent with SCC antibody;

[0021] The ProGRP antibody marker labeled with lanthanide A5 is obtained by reacting a lanthanide A5 chelating agent with a ProGRP antibody.

[0022] The lanthanide elements A1, A2, A3, A4, and A5 are selected from lutetium, holmium, thulium, europium, and terbium.

[0023] The chelating agent is diethyltriaminepentaacetic acid.

[0024] The kit also includes at least one of the following: calibrator diluent, concentrated cleaning solution, and dissociation solution.

[0025] Wherein, the CEA antibody is a murine CEA antibody; the CYFRA211 antibody is a murine CYFRA211 antibody; the NSE antibody is a murine NSE antibody; the SCC antibody is a murine SCC antibody; and the ProGRP antibody is a murine ProGRP antibody.

[0026] The aforementioned kit for detecting lung cancer marker levels should also be within the scope of protection of this invention.

[0027] The application of the above-described system or reagent kit in any of the following situations shall be within the scope of protection of this invention:

[0028] P1. Detect the content of lung cancer markers in the sample to be tested;

[0029] P2. Prepare products for detecting lung cancer marker content in samples to be tested;

[0030] P3. Prepare products for diagnosing the content of lung cancer markers in test samples;

[0031] The above-mentioned lung cancer markers are at least three of the following: CEA, CYFRA211, NSE, SCC, and ProGRP.

[0032] Compared with existing technologies, the advantages of this invention are as follows: This method uses elemental mass spectrometry, which greatly shortens the reaction time, and also has the advantages of high accuracy, high specificity, high sensitivity, wide linear range, and stable and convenient detection. This kit can also achieve high-throughput detection, meeting the detection needs of population flow in different cities.

[0033] Compared with existing technologies, the elemental mass spectrometry detection kit for detecting CEA, CYFRA211, NSE, SCC, and ProGRP in the present invention can simultaneously detect the contents of CEA, CYFRA211, NSE, SCC, and ProGRP in the sample, especially the contents of CEA, CYFRA211, and NSE. It saves time while significantly improving sensitivity and detection range. Attached Figure Description

[0034] Figure 1 This is the ROC result for CEA.

[0035] Figure 2 The ROC results are for CYFRA211.

[0036] Figure 3 The ROC results are for NSE.

[0037] Figure 4 The ROC results are for ProGRP.

[0038] Figure 5 This is the ROC result for SCC. Detailed Implementation

[0039] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0040] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0041] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0042] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0043] In a specific embodiment of the present invention, the streptavidin magnetic beads are specifically from Suzhou Beaver Biomedical Engineering Co., Ltd., catalog number 22305-100; the activated biotin is specifically a product of SIGMA, catalog number 89689-52-1; the CEA-coated antibody is specifically a product of Guangdong Feipeng Biotechnology Co., Ltd., catalog number 3CEA-23; the CEA-labeled antibody is specifically a product of Guangdong Feipeng Biotechnology Co., Ltd., catalog number CEA-100; the CEA antigen is specifically a product of Guangdong Feipeng Biotechnology Co., Ltd., catalog number CEA-100; the CYFRA211-coated antibody is specifically a product of Beijing Keyue Zhongkai Biotechnology Co., Ltd., catalog number CY11N005; the CYFRA211-labeled antibody is specifically a product of Beijing Keyue Zhongkai Biotechnology Co., Ltd., catalog number CY11N007; the CYFRA211 antigen is specifically a product of Guangdong Feipeng Biotechnology Co., Ltd., catalog number 20190420-2; the... The NSE-coated antibody is a product of Nanjing Jingda Biotechnology Co., Ltd., catalog number M9158; the NSE-labeled antibody is a product of Nanjing Jingda Biotechnology Co., Ltd., catalog number M9159; the NSE antigen is a product of Medix, catalog number RDR-463; the SCC-coated antibody is a product of Hangzhou Huakui Jinpei Biotechnology Co., Ltd., catalog number 15B1; the SCC-labeled antibody is a product of Hangzhou Huakui Jinpei Biotechnology Co., Ltd., catalog number 3C9; the SCC antigen is a product of Abcam, catalog number GR23054-16; the ProGRP-coated antibody is a product of Guangdong Feipeng Biotechnology Co., Ltd., catalog number 20200528-2; the ProGRP-labeled antibody is a product of Guangdong Feipeng Biotechnology Co., Ltd., catalog number 20190429-2; and the ProGRP antigen is a product of Guangdong Feipeng Biotechnology Co., Ltd., catalog number 20200117-2.

[0044] In a first aspect, the present invention claims a combined elemental mass spectrometry detection kit for detecting the contents of CEA, CYFRA211, NSE, SCC, and ProGRP.

[0045] The elemental mass spectrometry combined detection kit for detecting the contents of CEA, CYFRA211, NSE, SCC, and ProGRP claimed in this invention contains:

[0046] (1) Magnetic bead working solutions labeled with biotinylated CEA-coated antibody, biotinylated CYFRA211-coated antibody, biotinylated NSE-coated antibody, biotinylated SCC-coated antibody, and biotinylated ProGRP-coated antibody, respectively.

[0047] (2) Metal ion conjugates of CEA antibody markers labeled with lanthanide A1, CYFRA211 antibody markers labeled with lanthanide A2, NSE antibody markers labeled with lanthanide A3, SCC antibody markers labeled with lanthanide A4, and ProGRP antibody markers labeled with lanthanide A5.

[0048] The lanthanide elements A1, A2, A3, A4, and A5 are different lanthanide elements.

[0049] The CEA antibody marker labeled with lanthanide element A1 is obtained by reacting a lanthanide element A1 chelating agent with an anti-human labeled CEA antibody (i.e., labeling the labeled CEA antibody with lanthanide element A1 using a chelating agent); the CYFRA211 antibody marker labeled with lanthanide element A2 is obtained by reacting a CYFRA211 labeled antibody with a lanthanide element A2 chelating agent (i.e., labeling the CYFRA211 labeled antibody with a chelating agent using a chelating agent); and the NSE antibody marker labeled with lanthanide element A3 is... The SCC antibody marker is obtained by reacting lanthanide A3 with an NSE-labeled antibody (i.e., by labeling the NSE-labeled antibody with the lanthanide A3 using a chelating agent); the SCC antibody marker labeled with lanthanide A4 is obtained by reacting lanthanide A4 with an SCC-labeled antibody (i.e., by labeling the SCC antibody with the lanthanide A4 using a chelating agent); the ProGRP antibody marker labeled with lanthanide A5 is obtained by reacting lanthanide A5 with a ProGRP-labeled antibody (i.e., by labeling the ProGRP antibody with the lanthanide A5 using a chelating agent).

[0050] The lanthanide elements include, but are not limited to, lutetium (Lu), holmium (Ho), thulium (Tm), europium (Eu), and terbium (Tb). The chelating agents include, but are not limited to, diethyltriaminepentaacetic acid (DTPA).

[0051] In a specific embodiment of the present invention, the lanthanide element A1 is lutetium (Lu); the lanthanide element A2 is holmium (Ho); the lanthanide element A3 is thulium (Tm); the lanthanide element A4 is europium (Eu); the lanthanide element A5 is terbium (Tb); and the chelating agent is diethyltriaminepentaacetic acid (DTPA).

[0052] In a specific embodiment of the present invention, the CEA antibody labeled with lanthanide element A1 is obtained by reacting the CEA antibody with DTPA-Lu. During the reaction, the ratio of CEA antibody to DTPA-Lu is 1:5 (molar ratio), the reaction temperature is 25°C, and the reaction time is 18 hours.

[0053] In a specific embodiment of the present invention, the CYFRA211 antibody marker labeled with lanthanide element A2 is obtained by reacting the CYFRA211 antibody with DTPA-Ho. During the reaction, the ratio of CYFRA211 antibody to DTPA-Ho is 1:5 (molar ratio), the reaction temperature is 25°C, and the reaction time is 18 hours.

[0054] In a specific embodiment of the present invention, the NSE antibody marker labeled with lanthanide element A3 is obtained by reacting the NSE antibody with DTPA-Tm. During the reaction, the amount of NSE antibody and DTPA-Tm added is...

[0055] The ratio was 1:20 (molar ratio), the reaction temperature was 25℃, and the reaction time was 18h.

[0056] In a specific embodiment of the present invention, the SCC antibody marker labeled with lanthanide element A4 is obtained by reacting the SCC antibody with DTPA-Eu. During the reaction, the ratio of SCC antibody to DTPA-Eu is 1:5 (molar ratio), the reaction temperature is 25°C, and the reaction time is 18 hours.

[0057] In a specific embodiment of the present invention, the ProGRP antibody marker labeled with lanthanide element A5 is obtained by reacting the ProGRP antibody with DTPA-Tb. During the reaction, the molar ratio of ProGRP antibody to DTPA-Tb is 1:10, the reaction temperature is 25°C, and the reaction time is 18 hours.

[0058] The DTPA-Lu, DTPA-Ho, DTPA-Tm, DTPA-Eu, and DTPA-Tb can be prepared by a method comprising the following steps: reacting a chelating agent (DTPA) with TmCl3, LuCl3, Ho(NO3)3, EuCl3, and TbCl3 in a molar ratio of 1:2 to obtain DTPA-Tm, DTPA-Lu, DTPA-Ho, DTPA-Eu, and DTPA-Tb. The reaction conditions can be: 25°C for 30 min.

[0059] Furthermore, the kit may also include all or part of the following:

[0060] (3) Calibration diluent;

[0061] The composition of the calibrator diluent is as follows: each liter of calibrator buffer contains 25g BSA, 50g sucrose, 2.5mL Proclin-300 liquid, and the remainder is a Tris-HCl solution with a pH of 7.8.

[0062] In a specific embodiment of the present invention, the Tris-HCl solution with a pH of 7.8 is prepared by a method comprising the following steps: 5-6g (e.g., 6.057g) of tris(hydroxymethyl)aminomethane (tris) and 8-10g (e.g., 9g) of sodium chloride are added to 800ml of deionized water and stirred thoroughly until completely dissolved. Hydrochloric acid solution is added to adjust the pH of the solution to 7.8, and deionized water is added to bring the volume to 1L to obtain the Tris-HCl solution.

[0063] (4) Concentrated cleaning solution (10x concentrated cleaning solution);

[0064] The solvent of the concentrated cleaning solution is water, and the solutes and their concentrations are: tris(hydroxymethyl)aminomethane (tris) 60.57 g / L, sodium chloride 90 g / L, Tween 20 500 μL / L, and Triton X-100 500 μL / L; the pH value is adjusted to 7.8 with hydrochloric acid solution.

[0065] (5) Dissociation solution;

[0066] The dissociation solution is a Re solution with a concentration of 1 ng / mL prepared using 1% (v / v) dilute nitric acid. The rhenium (Re) is an internal standard element for inductively coupled plasma mass spectrometry (ICP-MS).

[0067] In the kit, the magnetic beads are streptavidin magnetic beads; the biotin is activated biotin; the CEA-coated antibody can be a murine CEA antibody; the CEA-labeled antibody can be a murine CEA antibody; the CYFRA211-coated antibody can be a murine CYFRA211 antibody; the CYFRA211-labeled antibody can be a murine CYFRA211 antibody; the NSE-coated antibody can be a murine NSE antibody; the NSE-labeled antibody can be a murine NSE antibody; the SCC-coated antibody can be a murine SCC antibody; the SCC-labeled antibody can be a murine SCC antibody; the NSE-coated antibody can be a murine NSE antibody; the NSE-labeled antibody can be a murine NSE antibody; the ProGRP-coated antibody can be a murine ProGRP antibody; the ProGRP-labeled antibody can be a murine ProGRP antibody.

[0068] Secondly, the present invention claims a system for detecting whether a sample to be tested contains the contents of CEA, CYFRA211, NSE, SCC, and ProGRP.

[0069] The system claimed in this invention for detecting whether a sample contains CEA, CYFRA211, NSE, SCC, and ProGRP includes the reagent kit and data processing device described above.

[0070] The data processing device may include the following modules:

[0071] (a1) Data acquisition module; the data acquisition module is configured to receive the concentration values ​​of CEA, CYFRA211, NSE, SCC and ProGRP in the sample to be tested using the kit described above and inductively coupled plasma mass spectrometry; the cutoff value for CEA antigen is 4 ng / mL; the cutoff value for CYFRA211 antigen is 2 ng / mL; the cutoff value for NSE antigen is 11 ng / mL; the cutoff value for SCC antigen is 1.5 ng / mL; and the cutoff value for ProGRP antigen is 64 pg / mL.

[0072] (a2) Data comparison module; the data comparison module is configured to receive the concentration values ​​of CEA, CYFRA211, NSE, SCC and ProGRP of the subject from the data acquisition module and compare them with the corresponding cutoff values.

[0073] (a3) Result determination module; the structure determination module is configured to receive the comparison result from the data comparison module, and then determine the result according to predetermined conditions.

[0074] Further, the predetermined conditions are as follows: if the CEA antigen concentration of the test subject is ≥4 ng / mL, the CEA antigen of the test sample is determined to be positive; if the CYFRA211 antigen concentration of the test subject is ≥2 ng / mL, the CYFRA211 antigen of the test sample is determined to be positive; if the NSE antigen concentration of the test subject is ≥11 ng / mL, the NSE antigen of the test sample is determined to be positive; if the SCC antigen concentration of the test subject is ≥1.5 ng / mL, the SCC antigen of the test sample is determined to be positive; if the ProGRP antigen concentration of the test subject is ≥64 pg / mL, the ProGRP antigen of the test sample is determined to be positive.

[0075] Thirdly, the present invention claims protection for the use of the reagent kit or system described above in any of the following:

[0076] P1. Detect whether the sample contains CEA, CYFRA211, NSE, SCC, and ProGRP.

[0077] P2. Prepare products for detecting the content of CEA, CYFRA211, NSE, SCC, and ProGRP in the sample to be tested;

[0078] P3. Prepare products for diagnosing the levels of CEA, CYFRA211, NSE, SCC, and ProGRP in test samples.

[0079] Among them, the application described in P1 can be a non-disease diagnosis and treatment application, such as simply detecting the content of CEA, CYFRA211, NSE, SCC and ProGRP in blood products (such as serum).

[0080] In all of the above aspects, the sample to be tested may be an isolated human serum sample.

[0081] Luttium (Lu)-labeled CEA antibody, holmium (Ho)-labeled CYFRA211 antibody, thulium (Tm)-labeled NSE antibody, europium (Eu)-labeled SCC antibody, and terbium (Tb)-labeled ProGRP antibody must be clear and transparent in appearance, without precipitation or turbidity. Functionality: When paired with other qualified components, it must ensure compliance with product technical requirements. Store at 4°C.

[0082] Example 1: Preparation of an elemental mass spectrometry combined detection kit for CEA, CYFRA211, NSE, SCC, and ProGRP content.

[0083] The elemental mass spectrometry combined detection kit for CEA, CYFRA211, NSE, SCC, and ProGRP content provided by this invention includes: magnetic bead working solution labeled with biotinylated CEA-coated antibody, biotinylated CYFRA211-coated antibody, biotinylated NSE-coated antibody, biotinylated SCC-coated antibody, and biotinylated NSE-coated antibody; metal ion conjugate suspensions of lanthanide-labeled CEA antibody, lanthanide-holmium-labeled CYFRA211 antibody, lanthanide-thulium-labeled NSE antibody, lanthanide-europium-labeled SCC antibody, and lanthanide-terbium-labeled ProGRP antibody; concentrated washing solution; dissociation solution; magnetic bead buffer; and calibrator dilution solution.

[0084] I. Preparation of various solutions

[0085] 1. Calibration diluent

[0086] ① Weigh 6.057g of tris(hydroxymethyl)aminomethane (tris) and 9g of sodium chloride, add them to 800ml of deionized water and stir thoroughly until completely dissolved to obtain a solution;

[0087] ② Adjust the pH of the solution to 7.8 with hydrochloric acid solution, and add deionized water to make up to 1L to obtain Tris-HCl solution;

[0088] ③ Weigh out 25g of BSA and 50g of sucrose, add them to Tris-HCl and stir until fully dissolved;

[0089] ④ Add 2.5 mL of Proclin-300 liquid to the solution, stir well, and let it dissolve completely;

[0090] ⑤ Add Tris-HCl solution to bring the volume to 1L.

[0091] 2. Concentrated cleaning solution

[0092] ① Weigh 60.57g of tris(hydroxymethyl)aminomethane (tris) and 90g of sodium chloride, add them to 800ml of deionized water and stir thoroughly until completely dissolved to obtain a solution;

[0093] ② Adjust the pH of the solution to 7.8 by adding hydrochloric acid solution;

[0094] ③ Add 500 μL of Tween 20 liquid and 500 μL of Triton-100 to the solution in step ②, stir well and let it dissolve completely;

[0095] ④ Add deionized water to a final volume of 1L and store at room temperature.

[0096] The above preparation is a 10x concentrated cleaning solution. When using it, dilute the 10x concentrated cleaning solution with purified water at a ratio of 1:10 (100mL of concentrated cleaning solution to 900mL of purified water) to obtain a 1× cleaning solution. If crystals form in the cleaning solution, place it at room temperature or 37°C until the crystals dissolve before dilution.

[0097] 3. Dissociation solution

[0098] The dissociation buffer was a Re solution with a concentration of 1 ng / mL, prepared using 1% (v / v) dilute nitric acid. Rhenium (Re) was used as an internal standard element for inductively coupled plasma mass spectrometry (ICP-MS).

[0099] 4. Magnetic bead buffer solution

[0100] ① Weigh 11.56g of MOPSO, 50g of sucrose, 10g of bovine serum albumin, 8g of sodium chloride, and 40g of polyvinylpyrrolidone into 800mL of deionized water, stir thoroughly until completely dissolved to obtain a solution;

[0101] ② Add NaOH to adjust the pH of the buffer solution to 6.5;

[0102] ③ Add 750 μL of Tween-20 liquid, 1000 μL of Proclin-300, and 750 μL of Triton X-100 to the solution in step ②, and stir until fully dissolved.

[0103] ④ Make up to 1L with deionized water and store at 2-8℃.

[0104] 5. Probe preparation

[0105] The chelating agent (DTPA) was added to TmCl3, LuCl3, Ho(NO3)3, EuCl3 and TbCl3 in a molar ratio of 1:2, and reacted at 25℃ for 30 min to obtain DTPA-Tm, DTPA-Lu, DTPA-Ho, DTPA-Eu and DTPA-Tb.

[0106] II. Preparation of biotinylated CEA-coated antibody, biotinylated CYFRA211-coated antibody, biotinylated NSE-coated antibody, biotinylated SCC-coated antibody, and biotinylated ProGRP-coated antibody

[0107] Functionality: When paired with other qualified components, it ensures the precision, limit of detection, linearity, repeatability, and specificity of the lung cancer five-item kit. Store at 4°C.

[0108] (1) Preparation of biotinylated CEA-coated antibody

[0109] ① Add the CEA-coated antibody and activated biotin at a ratio of 1:2 (molar ratio) and mix well. Adjust the volume with 0.01M pH7.4 PBS buffer to make the antibody concentration 2mg / mL. Mix by rotating for 30 minutes at room temperature.

[0110] ② Dialyze the biotin-labeled coated antibody overnight with 0.01M pH7.4 PBS buffer. Change the buffer for the first time after 2 hours, and for the second time after 4 hours (each time, pour out 2 / 3 of the total volume and then add 0.01M PBS buffer to make up to 1L). Dialyze for 24 hours and take out the antibody. After dialysis, make up to 1ml with 0.01M PBS and store at 2-8℃ for later use.

[0111] (2) Preparation of biotinylated CYFRA211-coated antibody

[0112] ① Add the CYFRA211-coated antibody and activated biotin at a ratio of 1:1.5 (molar ratio) and mix well. Adjust the volume with 0.01M pH7.4 PBS buffer to make the antibody concentration 2mg / mL. Mix by rotating at room temperature for 30 minutes.

[0113] ② Dialyze the biotin-labeled coated antibody overnight with 0.01M pH7.4 PBS buffer. Change the buffer for the first time after 2 hours, and for the second time after 4 hours (each time, pour out 2 / 3 of the total volume and then add 0.01M PBS buffer to make up to 1L). Dialyze for 24 hours and take out the antibody. After dialysis, make up to 1ml with 0.01M PBS and store at 2-8℃ for later use.

[0114] (3) Preparation of biotinylated NSE-coated antibody

[0115] ① Add NSE-coated antibody and activated biotin at a ratio of 1:5 (molar ratio) and mix well. Adjust the volume with 0.01M pH7.4 PBS buffer to make the antibody concentration 2mg / mL. Mix by rotating for 30 minutes at room temperature.

[0116] ② Dialyze the biotin-labeled coated antibody overnight with 0.01M pH7.4 PBS buffer. Change the buffer for the first time after 2 hours, and for the second time after 4 hours (each time, pour out 2 / 3 of the total volume and then add 0.01M PBS buffer to make up to 1L). Dialyze for 24 hours and take out the antibody. After dialysis, make up to 1ml with 0.01M PBS and store at 2-8℃ for later use.

[0117] (4) Preparation of biotinylated SCC-coated antibody

[0118] ① Add the SCC-coated antibody and activated biotin at a ratio of 1:10 (molar ratio) and mix well. Adjust the volume with 0.01M pH7.4 PBS buffer to make the antibody concentration 2mg / mL. Mix by rotating for 30 minutes at room temperature.

[0119] ② Dialyze the biotin-labeled coated antibody overnight with 0.01M pH7.4 PBS buffer. Change the buffer for the first time after 2 hours, and for the second time after 4 hours (each time, pour out 2 / 3 of the total volume and then add 0.01M PBS buffer to make up to 1L). Dialyze for 24 hours and take out the antibody. After dialysis, make up to 1ml with 0.01M PBS and store at 2-8℃ for later use.

[0120] (5) Preparation of biotinylated ProGRP-coated antibodies

[0121] ① Add the ProGRP-coated antibody and activated biotin at a ratio of 1:5 (molar ratio) and mix well. Adjust the volume with 0.01M pH7.4 PBS buffer to make the antibody concentration 2mg / mL. Mix by rotating at room temperature for 30 minutes.

[0122] ② Dialyze the biotin-labeled coated antibody overnight with 0.01M pH7.4 PBS buffer. Change the buffer for the first time after 2 hours, and for the second time after 4 hours (each time, pour out 2 / 3 of the total volume and then add 0.01M PBS buffer to make up to 1L). Dialyze for 24 hours and take out the antibody. After dialysis, make up to 1ml with 0.01M PBS and store at 2-8℃ for later use.

[0123] III. Biotinylated CEA-coated antibody, biotinylated CYFRA211-coated antibody, biotinylated NSE-coated antibody, biotinylated SCC-coated antibody, and biotinylated ProGRP-coated antibody linked to magnetic beads.

[0124] ① After mixing the magnetic beads, conjugate them with biotinylated CEA antibody at volume ratios of 1:0.05, 1:0.25, 1:0.5, 1:0.75, 1:1.0, 1:1.25, and 1:1.5, respectively, and perform immunoassay to determine the optimal conjugation ratio of magnetic beads to biotinylated antibody. The determined optimal ratio is 1:0.25. Based on this ratio, determine the volume of magnetic beads to be used. After determining the volume, wash the magnetic beads three times with 10 times their volume of washing buffer (mix for 3 seconds after adding the washing buffer, let stand for 2 minutes, allow the magnetic beads to magnetically adsorb, and remove the washing buffer after it becomes clear and transparent). Add biotinylated CEA antibody according to the determined ratio, and use 0.01M... Adjust the volume of PBS to 2.5 times the volume of magnetic beads, and rotate to mix at 25°C for 1 hour. After the reaction is complete, wash the magnetic beads 3 times with 10 times the volume of washing buffer (mix for 3 seconds after adding the washing buffer, let stand for 2 minutes, and remove the washing buffer after the magnetic beads are magnetically adsorbed and the washing buffer is clear and transparent). Finally, add 2.5 times the volume of magnetic bead buffer to obtain 4 mg / mL of magnetic bead biotinylated antibody, and store at 2-8°C for later use.

[0125] ② After mixing the magnetic beads, conjugate them with biotinylated CYFRA211 antibody at volume ratios of 1:0.05, 1:0.25, 1:0.5, 1:0.75, 1:1.0, 1:1.25, and 1:1.5, respectively. Immunoassay was performed to determine the optimal conjugation ratio of magnetic beads to biotinylated antibody, finding it to be 1:0.25. Based on this ratio, the volume of magnetic beads to be used was determined. After determining the volume, the magnetic beads were washed three times with 10 times their volume of washing buffer (mix for 3 seconds after adding the washing buffer, then let stand for 2 minutes). After the magnetic beads were washed with a clear and transparent wash solution, the wash solution was removed. Biotinylated CYFRA211 antibody was added in a predetermined ratio, and the volume was adjusted to 2.5 times the volume of the magnetic beads with 0.01M PBS. The mixture was rotated and stirred at 25°C for 1 hour. After the reaction was completed, the magnetic beads were washed 3 times with 10 times the volume of the wash solution (mix for 3 seconds after adding the wash solution, let stand for 2 minutes, and remove the wash solution after the magnetic beads were washed with a clear and transparent wash solution). Finally, 2.5 times the volume of the magnetic bead buffer was added to obtain 4 mg / mL of biotinylated magnetic bead antibody, which was stored at 2-8°C for later use.

[0126] ③ After mixing the magnetic beads, conjugate them with biotinylated NSE antibody at volume ratios of 1:0.05, 1:0.25, 1:0.5, 1:0.75, 1:1.0, 1:1.25, and 1:1.5, respectively, and perform immunoassay to determine the optimal conjugation ratio of magnetic beads to biotinylated antibody. The determined conjugation ratio is 1:0.25. Based on this ratio, determine the volume of magnetic beads to be used. After determining the volume, wash the magnetic beads three times with 10 times their volume of washing buffer (mix for 3 seconds after adding the washing buffer, let stand for 2 minutes, allow the magnetic beads to magnetically adsorb, and remove the washing buffer after it becomes clear and transparent). Add biotinylated CEA antibody according to the determined ratio, and use 0.01M... Adjust the volume of PBS to 2.5 times the volume of magnetic beads, and rotate to mix at 25°C for 1 hour. After the reaction is complete, wash the magnetic beads 3 times with 10 times the volume of washing buffer (mix for 3 seconds after adding the washing buffer, let stand for 2 minutes, and remove the washing buffer after the magnetic beads are magnetically adsorbed and the washing buffer is clear and transparent). Finally, add 2.5 times the volume of magnetic bead buffer to obtain 4 mg / mL of magnetic bead biotinylated antibody, and store at 2-8°C for later use.

[0127] ④ After mixing the magnetic beads, conjugate them with biotinylated SCC antibody at volume ratios of 1:0.05, 1:0.25, 1:0.5, 1:0.75, 1:1.0, 1:1.25, and 1:1.5, respectively, and perform immunoassay to determine the optimal conjugation ratio of magnetic beads to biotinylated antibody. The determined conjugation ratio is 1:0.25. Based on this ratio, determine the volume of magnetic beads to be used. After determining the volume, wash the magnetic beads three times with 10 times their volume of washing buffer (mix for 3 seconds after adding the washing buffer, let stand for 2 minutes, allow the magnetic beads to magnetically adsorb, and remove the washing buffer after it becomes clear and transparent). Add the biotinylated SCC antibody according to the determined ratio, and use 0.01M... Adjust the volume of PBS to 2.5 times the volume of magnetic beads, and rotate to mix at 25°C for 1 hour. After the reaction is complete, wash the magnetic beads 3 times with 10 times the volume of washing buffer (mix for 3 seconds after adding the washing buffer, let stand for 2 minutes, and remove the washing buffer after the magnetic beads are magnetically adsorbed and the washing buffer is clear and transparent). Finally, add 2.5 times the volume of magnetic bead buffer to obtain 4 mg / mL of magnetic bead biotinylated antibody, and store at 2-8°C for later use.

[0128] ⑤ After mixing the magnetic beads, conjugate them with biotinylated ProGRP antibody at volume ratios of 1:0.05, 1:0.25, 1:0.5, 1:0.75, 1:1.0, 1:1.25, and 1:1.5, respectively, and perform immunoassay to determine the optimal conjugation ratio of magnetic beads to biotinylated antibody. The determined conjugation ratio is 1:0.25. Based on this ratio, determine the volume of magnetic beads to be used. After determining the volume, wash the magnetic beads three times with 10 times their volume of washing buffer (mix for 3 seconds after adding the washing buffer, let stand for 2 minutes, allow the magnetic beads to magnetically adsorb, and remove the washing buffer after it becomes clear and transparent). Add the biotinylated ProGRP antibody according to the determined ratio, and use 0.01M... Adjust the volume of PBS to 2.5 times the volume of magnetic beads, and rotate to mix at 25°C for 1 hour. After the reaction is complete, wash the magnetic beads 3 times with 10 times the volume of washing buffer (mix for 3 seconds after adding the washing buffer, let stand for 2 minutes, and remove the washing buffer after the magnetic beads are magnetically adsorbed and the washing buffer is clear and transparent). Finally, add 2.5 times the volume of magnetic bead buffer to obtain 4 mg / mL of magnetic bead biotinylated antibody, and store at 2-8°C for later use.

[0129] ⑥ Mix the five types of biotinylated antibodies for magnetic beads to prepare a working solution for magnetic beads, and store at 2-8℃ for later use.

[0130] IV. Preparation of lanthanide-labeled metal ion conjugates

[0131] Functionality: When paired with other qualified components, it ensures the precision, limit of detection, linearity, repeatability, and specificity of the lung cancer five-item kit. Store at 4°C.

[0132] (1) CEA antibody labeled with lutetium (Lu), a lanthanide element

[0133] ① Mix CEA antibody with 0.1M carbonate buffer and DTPA-Lu, with the ratio of CEA-labeled antibody to lutetium being 1:5 (molar ratio), and react at 25℃ for 18 hours.

[0134] ② After the labeled solution was left overnight, it was purified by gel column chromatography equilibrated with 0.01M pH7.4 PBS buffer. The first peak was collected by A280 detection and aliquoted.

[0135] (2) CYFRA211 antibody labeled with holmium (Ho), a lanthanide element

[0136] ① Mix CYFRA211 antibody with 0.1M carbonate buffer and DTPA-Ho, with the ratio of CYFRA211-labeled antibody to thulium being 1:5 (molar ratio), and react at 25℃ for 18 hours.

[0137] ② After the labeled solution was left overnight, it was purified by gel column chromatography equilibrated with 0.01M pH7.4 PBS buffer. The first peak was collected by A280 detection and aliquoted.

[0138] (3) NSE antibodies labeled with thulium (Tm), a lanthanide element

[0139] ① Mix NSE antibody with 0.1M carbonate buffer and DTPA-Ho, with the ratio of NSE-labeled antibody to thulium being 1:20 (molar ratio), and react at 25℃ for 18 hours.

[0140] ② After the labeled solution was left overnight, it was purified by gel column chromatography equilibrated with 0.01M pH7.4 PBS buffer. The first peak was collected by A280 detection and aliquoted.

[0141] (4) SCC antibody labeled with europium (Eu), a lanthanide element

[0142] ① Mix NSE antibody with 0.1M carbonate buffer and DTPA-Eu, and add SCC-labeled antibody to thulium in a ratio of 1:5 (molar ratio). React at 25℃ for 18 hours.

[0143] ② After the labeled solution was left overnight, it was purified by gel column chromatography equilibrated with 0.01M pH7.4 PBS buffer. The first peak was collected by A280 detection and aliquoted.

[0144] (5) ProGRP antibody labeled with terbium (Tb), a lanthanide element

[0145] ① Mix NSE antibody with 0.1M carbonate buffer and DTPA-Tb, and add ProGRP-labeled antibody to thulium in a ratio of 1:10 (molar ratio). React at 25℃ for 18 hours.

[0146] ② After the labeled solution was left overnight, it was purified by gel column chromatography equilibrated with 0.01M pH7.4 PBS buffer. The first peak was collected by A280 detection and aliquoted.

[0147] (6) Mix Lu-CEA, Ho-CYFRA211, Tm-NSE, Eu-SCC and Tb-ProGRP to prepare a metal ion conjugate and store it at 2-8℃.

[0148] V. Inkjet printing, labeling, and assembly into finished reagent kits.

[0149] Example 2: Optimization of magnetic bead buffer solution for an elemental mass spectrometry combined detection kit for CEA, CYFRA211, NSE, SCC, and ProGRP content.

[0150] I. Traditional buffer formulations are as follows:

[0151] The buffer solution is water, and each liter of conventional buffer contains 11.56g MOPSO, 50g sucrose, 10g bovine serum albumin, 8g sodium chloride, 750μL Tween-20 liquid, 1000μL Proclin-300, and 750μL Triton X-100. The pH is adjusted to 6.5 using NaOH.

[0152] Experiments revealed that the traditional formulation resulted in excessive variation in the zero-point signal value. Therefore, 40g of polyvinylpyrrolidone was added to each liter of the traditional buffer solution. That is, each liter of the magnetic bead buffer solution of this invention contains 40g of polyvinylpyrrolidone, 11.56g of MOPSO, 50g of sucrose, 10g of bovine serum albumin, 8g of sodium chloride, 750μL of Tween-20 liquid, 1000μL of Proclin-300, and 750μL of Triton X-100. The pH was adjusted to 6.5 using NaOH.

[0153] The magnetic bead buffer solution of the present invention was tested and compared with the conventional magnetic bead buffer solution. The results are shown in Tables 1-1 to 1-5. The results show that the magnetic bead buffer solution of the present invention can improve the stability and accuracy of the zero-point signal value.

[0154] Table 1-1 Comparison of CEA's Traditional Buffer and Self-Developed Buffer

[0155]

[0156] Table 1-2 Comparison of CYFRA211 conventional buffer and self-developed buffer.

[0157]

[0158] Table 1-3 Comparison of Traditional NSE Buffer and Self-Developed Buffer

[0159]

[0160] Table 1-4 Comparison of SCC conventional buffer and self-developed buffer

[0161]

[0162] Table 1-5 Comparison of ProGRP conventional buffer and self-developed buffer.

[0163]

[0164] As shown in Tables 1-1, 1-2, 1-3, 1-4, and 1-5, traditional buffer solutions cause significant skipping of the zero-point signal value, resulting in excessive variation. After adding polyvinylpyrrolidone, the variation is significantly reduced, and the signal value does not change significantly.

[0165] Example 3: Instructions for using the elemental mass spectrometry combined detection kit for CEA, CYFRA211, NSE, SCC, and ProGRP content.

[0166] 1. Equilibrate all components of the kit at room temperature (18-25℃).

[0167] 2. Solution preparation: Dilute the 10x concentrated washing solution with purified water at a ratio of 1:10 (100mL concentrated washing solution to 900mL purified water) to obtain a 1× washing solution. If crystals are present in the washing solution, place it at room temperature or 37℃ until the crystals dissolve before further dilution.

[0168] III. Sample Analysis Process

[0169] 1. Preparation of calibrator gradient points: Take 1 ml of calibrator from the kit. Specifically, the calibrator refers to a mixed solution prepared by reconstituted pure CEA, CYFRA211, NSE, SCC, and PROGRP antigens using calibrator buffer. The concentrations are: CEA 243 ng / mL, CYFRA211 162 ng / mL, NSE 405 ng / mL, SCC 121.5 ng / mL, and PROGRP 4860 pg / mL. The antigens themselves are commercially available. Label this mixed solution S5. Then, use calibrator diluent to dilute sequentially by 3 times to obtain calibrators S4, S3, S2, and S1. S0 is the calibrator diluent. The specific dilution process is as follows: 0.5 ml S5 + 1 ml diluent → S4, 0.5 ml S4 + 1 ml diluent → S3, 0.5 ml S3 + 1 ml diluent → S2, 0.5 ml S2 + 1 ml diluent → S1.

[0170] 2. Place the microplate on a horizontal table, arrange the six calibrators S0-S5 and the test sample in order, and use a pipette to add 50 μL to the corresponding microwell;

[0171] 3. Using a 20-200 μL pipette, add 50 μL of the pre-mixed metal ion conjugate to the microwells above;

[0172] 4. Using a 20-200μL pipette, add 50μL of the pre-mixed magnetic bead working solution to the microwells and gently vortex horizontally for 5-10 seconds.

[0173] 5. Seal the plate with sealing film to keep it horizontal and place it in a constant temperature incubator (37±0.5℃) for 60 minutes;

[0174] 6. Take out the microplate after the incubation is complete, remove the sealing film, put the microplate into the semi-automatic magnetic bead washing machine and fix it in place. Perform the washing operation according to the operating procedures of the semi-automatic magnetic bead washing machine.

[0175] 7. After cleaning, remove the microplate, add 120 μL of dissociation solution to each well, gently shake for 5 to 10 seconds, and let stand for 5 minutes.

[0176] 8. After dissociation, the sample is tested. That is, the sample processed in step 9 is introduced into inductively coupled plasma mass spectrometry (ICP-MS) for detection. The response values ​​of lanthanide elements Lu, Ho, Tm, Eu, Tb and internal standard element Re are collected—the corresponding ion counts per second (cps). Based on the calibrator response value and the sample response value, the contents of CEA, CYFRA211, NSE, SCC and ProGRP in the sample to be tested are obtained.

[0177] Example 4: Application of an elemental mass spectrometry combined detection kit for CEA, CYFRA211, NSE, SCC, and ProGRP content.

[0178] 1. Test samples: Calibrators S2 and S4 prepared in Example 3 were used as samples;

[0179] The instrument used for the following tests was an inductively coupled plasma mass spectrometer (ICP-MS). Model: 7850. Manufacturer: Agilent Technologies.

[0180] 2. Precision design requirements:

[0181] Precision S2: n = 20;

[0182] Precision S4: n = 20;

[0183] Precision reference CV value: The positive detection rate should be 100%, and the CV value should be ≤10% (n=20).

[0184] 3. Test methods

[0185] Intra-assay precision: Within the dose-response curve range of the kit, with two different concentrations of quality control samples, the coefficient of variation (CV) of the measured results should not exceed 10.0%.

[0186] Coefficient of variation: CV = SD / AV × 100%;

[0187] Where CV is the coefficient of variation, SD is the standard deviation of the test results, and AV is the average value of the test results.

[0188] 4. Results

[0189] The precision test data are shown in Table 2. The results of the test using the method of this invention are compared with those of the hospital's reagent test, and the results are shown in Table 3. Paired T-tests were performed using GraphPad, and the test results are as follows. The CEA paired T-test results are shown in Table 4-1, and the CEAROC results are as follows. Figure 1As shown in Table 4-2, the paired T-detection results of CYFRA211 are shown in Table 4-2, and the ROC results of CYFRA211 are shown in Table 4-2. Figure 2 As shown; the paired T-detection results of NSE are shown in Table 4-3, and the ROC results of NSE are shown in Table 4-3. Figure 3 As shown in Table 4-4, the paired T-detection results of ProGRP are shown in Table 4-4, and the ROC results of ProGRP are shown in Table 4-4. Figure 4 As shown; the paired T-detection results of SCC are shown in Tables 4-5, and the ROC results of SCC are shown in Tables 4-5. Figure 5 As shown in the figure; based on the above test results, P is greater than 0.05, and there is no significant difference between the hospital's measured values ​​and the actual measured values.

[0190] Table 2. Precision Test Data

[0191]

[0192]

[0193] Note: In the table, S2 and S4 indicate that the quality control samples in the kit were tested 20 times each.

[0194] Table 3. Compliance of Partial Clinical Samples

[0195]

[0196]

[0197] Table 4-1. Results of CEA-paired T test

[0198] Paired t test P value 0.2924 P value summary ns Significantlydifferent(P<0.05)? No One- or two-tailed P value? Two-tailed t, df t = 1.083, df = 19 Number of pairs 20 Correlation coefficient (r) 0.9985

[0199] Table 4-2, Results of CYFRA211 Paired T Test

[0200] Paired t test P value 0.3658 P value summary ns Significantlydifferent(P<0.05)? No One- or two-tailed P value? Two-tailed t, df t = 0.9265, df = 19 Number of pairs 20 Correlation coefficient (r) 0.9973

[0201] Table 4-3. NSE Paired-T Detection Results

[0202] Paired t test P value 0.5866 P value summary ns Significantlydifferent(P<0.05)? No One- or two-tailed P value? Two-tailed t, df t = 0.5532, df = 19 Number of pairs 20 Correlation coefficient (r) 0.9994

[0203] Table 4-4 ProGRP Paired-T Detection Results

[0204] Paired t test P value 0.2379 P value summary ns Significantlydifferent(P<0.05)? No One- or two-tailed P value? Two-tailed t, df t = 1.219, df = 19 Number of pairs 20 Correlation coefficient (r) 0.9875

[0205] Table 4-5. SCC Paired T Detection Results

[0206] Paired t test P value 0.1993 P value summary ns Significantlydifferent(P<0.05)? No One- or two-tailed P value? Two-tailed t, df t = 1.330, df = 19 Number of pairs 20 Correlation coefficient (r) 0.9999

[0207] The present invention has been described in detail above. For those skilled in the art, the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. Although specific embodiments have been given, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein. Some of the essential features can be applied within the scope of the following appended claims.

Claims

1. A system for detecting or assisting in the detection of lung cancer markers, characterized in that, The invention includes a kit for detecting lung cancer and a data processing device. The data processing device comprises: a data acquisition module, which receives concentration data of a sample to be tested obtained by the kit and transmits the concentration data to a data comparison module; a data comparison module, which receives the concentration data transmitted by the data acquisition module, compares the concentration data with the corresponding cutoff value, and transmits the comparison result to a result determination module; and a result determination module, which receives the comparison result from the data comparison module and determines the result. The kit includes magnetic bead working solution, metal ion conjugate suspension, and calibrators containing lung cancer markers. In the kit, the magnetic bead working solution includes magnetic beads linked to biotinylated CEA antibody, magnetic beads linked to biotinylated CYFRA211 antibody, magnetic beads linked to biotinylated NSE antibody, magnetic beads linked to biotinylated SCC antibody, magnetic beads linked to biotinylated Pro-GRP antibody, and magnetic bead buffer; the metal ion conjugate suspension includes CEA antibody labeled with lanthanide A1, CYFRA211 antibody labeled with lanthanide A2, NSE antibody labeled with lanthanide A3, SCC antibody labeled with lanthanide A4, and ProGRP antibody labeled with lanthanide A5. The lanthanide elements A1, A2, A3, A4, and A5 are different lanthanide elements; Each liter of the magnetic bead buffer solution contains 40g of polyvinylpyrrolidone, 11.56g of MOPSO, 50g of sucrose, 10g of bovine serum albumin, 8g of sodium chloride, 750μL of Tween-20 solution, 1000μL of Proclin-300, and 750μL of Triton X-100, with the pH adjusted to 6.5 using NaOH. The lung cancer markers are CEA, CYFRA211, NSE, SCC, and ProGRP. The concentration values ​​in the data acquisition module are the concentrations of CEA, CYFRA211, NSE, SCC, and ProGRP detected by inductively coupled plasma mass spectrometry. In the data comparison module, the cutoff value for CEA antigen is 4 ng / mL; the cutoff value for CYFRA211 antigen is 2 ng / mL; the cutoff value for NSE antigen is 11 ng / mL; the cutoff value for SCC antigen is 1.5 ng / mL; and the cutoff value for ProGRP antigen is 64 pg / mL.

2. The system according to claim 1, characterized in that: The CEA antibody marker labeled with lanthanide element A1 is obtained by reacting lanthanide element A1-chelating agent with CEA antibody; The CYFRA211 antibody marker labeled with lanthanide element A2 is obtained by reacting lanthanide element A2-chelating agent with CYFRA211 antibody; The NSE antibody marker labeled with lanthanide element A3 is obtained by reacting lanthanide element A3-chelating agent with NSE antibody; The SCC antibody marker labeled with lanthanide element A4 is obtained by reacting lanthanide element A4-chelating agent with SCC antibody; The ProGRP antibody marker labeled with lanthanide A5 is obtained by reacting a lanthanide A5 chelating agent with a ProGRP antibody.

3. The system according to claim 2, characterized in that: The lanthanide elements A1, A2, A3, A4, and A5 are selected from lutetium, holmium, thulium, europium, and terbium; the chelating agent is diethyltriaminepentaacetic acid.

4. The system according to any one of claims 1-3, characterized in that: The kit also includes at least one of the following: calibrator diluent, concentrated cleaning solution, and dissociation solution.

5. The system according to any one of claims 1-4, characterized in that: The CEA antibody is a murine CEA antibody; the CYFRA211 antibody is a murine CYFRA211 antibody; the NSE antibody is a murine NSE antibody; the SCC antibody is a murine SCC antibody; and the ProGRP antibody is a murine ProGRP antibody.

6. The kit for detecting lung cancer marker levels according to any one of claims 1-5.

7. The use of the system according to any one of claims 1-5 or the kit according to claim 6 in any of the following: P1. Prepare products for detecting lung cancer markers in samples to be tested; P2. Prepare products for diagnosing the levels of lung cancer markers in test samples.

8. The application according to claim 7, wherein the lung cancer markers are CEA, CYFRA211, NSE, SCC, and ProGRP.

Images