Method for quantitatively detecting serum sp70 of non-small cell lung cancer patient

Abstract

The application discloses a method for quantitatively detecting serum SP70 of non-small cell lung cancer patients, which comprises the following steps: preparing a working concentration washing solution, washing a plate, adding a monoclonal antibody, adding an enzyme-labeled secondary antibody, incubating, adding a chromogenic solution, adding a termination solution, and determining an OD value. Compared with the prior art, the method has the advantages that it can be used in early diagnosis of tumors, the tumor marker has high sensitivity and specificity, and the operation requirement is simple and convenient based on serum detection.

Description

Technical Field

[0001] This invention relates to serum detection methods, specifically a method for quantitatively detecting serum SP70 in patients with non-small cell lung cancer. Background Technology

[0002] SP70 refers to a cell line containing a monoclonal antibody against human non-small cell lung cancer. It is named SP70 because the relative molecular mass of the antigen it specifically binds to is approximately 70,000. SP70 is mainly found in the cell membrane and cytoplasm of cancer cells. Functional studies have revealed that SP70 has anti-apoptotic, proliferative, and metastatic effects, and it is considered a molecular target in the clinical diagnosis and detection of lung cancer, as well as a target for anti-cancer therapy. Current methods for detecting SP70 mainly rely on imaging, histology, and pathology. Imaging methods have a low diagnostic rate in early-stage lung cancer, while histology, although having a certain diagnostic accuracy, can still lead to misdiagnosis and missed diagnosis due to various reasons. Summary of the Invention

[0003] The technical problem to be solved by the present invention is the aforementioned technical problem, and to provide a method for quantitative detection of serum SP70 in patients with non-small cell lung cancer with a high detection level that can be used for early diagnosis.

[0004] To solve the above-mentioned technical problems, the technical solution provided by the present invention is: a method for quantitatively detecting serum SP70 in patients with non-small cell lung cancer, comprising the following steps:

[0005] Step 1: Remove the reagent kit from the refrigerator and allow it to equilibrate to room temperature for 30 minutes before conducting the experiment;

[0006] Step 2: Prepare working concentration washing buffer: Add 30mL of concentrated washing solution to a 1L reagent bottle containing 570mL of pure water at room temperature, mix thoroughly, and store at 2℃~8℃.

[0007] Step 3: Remove the anti-SP70 enzyme-labeled reaction plate, put the remaining microplate strips into a sealed bag, and store them in a refrigerator at 2℃~8℃.

[0008] Step 4: Dissolve the standard: Use a pipette to add 120 μL of pure water to the standard tube containing the lyophilized powder to dissolve the SP70 lyophilized standard and mix thoroughly to obtain the SP70 standard.

[0009] Step 5: Use a pipette to add 100 μL of SP70 standard and sample to the corresponding detection wells. Complete the sample addition process within 10 minutes. Place the reaction plate on the shelf of a 37°C water bath and incubate at 37°C for 60 minutes. Discard the incubation liquid.

[0010] Step 6: Washing the plate: Machine wash: Add 250 μL of working concentration washing buffer to each well and wash a total of 4 times; Manual wash: Discard the liquid in the well, add 250 μL of working concentration washing buffer to each well, discard the liquid, shake dry, pat dry on absorbent paper, and repeat 4 times.

[0011] Step 7: Add monoclonal antibody: Dilute the required monoclonal antibody 100 times with antibody diluent to prepare monoclonal antibody working solution. Add 100 μL of antibody working solution to each well and place the reaction plate on the shelf of a 37°C water bath. Incubate at 37°C for 30 minutes and discard the incubation liquid.

[0012] Step 8: Washing the plate: Machine wash: Add 250 μL of working concentration washing buffer to each well and wash a total of 4 times; Manual wash: Discard the liquid in the well, add 250 μL of working concentration washing buffer to each well, discard the liquid, shake dry, pat dry on absorbent paper, and repeat 4 times.

[0013] Step 9: Add enzyme-labeled secondary antibody: Dilute the required enzyme-labeled secondary antibody 100 times with antibody dilution buffer to obtain enzyme-labeled secondary antibody working solution, and add 100 μL of enzyme-labeled secondary antibody working solution to each well;

[0014] Step 10: Incubation: Place the reaction plate on the shelf of a 37°C water bath and incubate at 37°C for 30 minutes. Discard the incubation liquid.

[0015] Step 11: Wash the plate: Perform the same operation as before, wash 5 times;

[0016] Step 12: Add colorimetric solution: Add 100 μL of TMB to each well and place the reaction plate in a dark place at room temperature for 15 minutes.

[0017] Step 13: Add stop solution: Add 50 μL of stop solution to each well, gently shake the edge of the plate, mix for 30 seconds, check and remove air bubbles in each well.

[0018] Step 14: OD value determination: Measure the OD value of each well using an ELISA reader at a wavelength of 450nm. Complete the ELISA reader reading within 15 minutes after termination.

[0019] The advantages of this invention compared to existing technologies are: it can be used in the early diagnosis of tumors; the tumor markers have high sensitivity and specificity; it can be based on serum detection; and the operation requirements are simple. Attached Figure Description

[0020] Figure 1 This is the precision verification result of the method for quantitative detection of serum SP70 in patients with non-small cell lung cancer according to the present invention.

[0021] Figure 2 This is the verification result of the accuracy of the method for quantitative detection of serum SP70 in patients with non-small cell lung cancer according to the present invention.

[0022] Figure 3 This is the reference interval validation result of the method for quantitative detection of serum SP70 in patients with non-small cell lung cancer according to the present invention. Detailed Implementation

[0023] The present invention will now be described in further detail with reference to the accompanying drawings.

[0024] Combined with appendix Figure 1 Appendix Figure 2 and attached Figure 3 A method for quantitatively detecting serum SP70 in patients with non-small cell lung cancer, comprising the following steps:

[0025] Step 1: Remove the reagent kit from the refrigerator and allow it to equilibrate to room temperature for 30 minutes before conducting the experiment;

[0026] Step 2: Prepare working concentration washing buffer: Add 30mL of concentrated washing solution to a 1L reagent bottle containing 570mL of pure water at room temperature, mix thoroughly, and store at 2℃~8℃.

[0027] Step 3: Remove the anti-SP70 enzyme-labeled reaction plate, put the remaining microplate strips into a sealed bag, and store them in a refrigerator at 2℃~8℃.

[0028] Step 4: Dissolve the standard: Use a pipette to add 120 μL of pure water to the standard tube containing the lyophilized powder to dissolve the SP70 lyophilized standard and mix thoroughly to obtain the SP70 standard.

[0029] Step 5: Use a pipette to add 100 μL of SP70 standard and sample to the corresponding detection wells. Complete the sample addition process within 10 minutes. Place the reaction plate on the shelf of a 37°C water bath and incubate at 37°C for 60 minutes. Discard the incubation liquid.

[0030] Step 6: Washing the plate: Machine wash: Add 250 μL of working concentration washing buffer to each well and wash a total of 4 times; Manual wash: Discard the liquid in the well, add 250 μL of working concentration washing buffer to each well, discard the liquid, shake dry, pat dry on absorbent paper, and repeat 4 times.

[0031] Step 7: Add monoclonal antibody: Dilute the required monoclonal antibody 100 times with antibody diluent to prepare monoclonal antibody working solution. Add 100 μL of antibody working solution to each well and place the reaction plate on the shelf of a 37°C water bath. Incubate at 37°C for 30 minutes and discard the incubation liquid.

[0032] Step 8: Washing the plate: Machine wash: Add 250 μL of working concentration washing buffer to each well and wash a total of 4 times; Manual wash: Discard the liquid in the well, add 250 μL of working concentration washing buffer to each well, discard the liquid, shake dry, pat dry on absorbent paper, and repeat 4 times.

[0033] Step 9: Add enzyme-labeled secondary antibody: Dilute the required enzyme-labeled secondary antibody 100 times with antibody dilution buffer to obtain enzyme-labeled secondary antibody working solution, and add 100 μL of enzyme-labeled secondary antibody working solution to each well;

[0034] Step 10: Incubation: Place the reaction plate on the shelf of a 37°C water bath and incubate at 37°C for 30 minutes. Discard the incubation liquid.

[0035] Step 11: Wash the plate: Perform the same operation as before, wash 5 times;

[0036] Step 12: Add colorimetric solution: Add 100 μL of TMB to each well and place the reaction plate in a dark place at room temperature for 15 minutes.

[0037] Step 13: Add stop solution: Add 50 μL of stop solution to each well, gently shake the edge of the plate, mix for 30 seconds, check and remove air bubbles in each well.

[0038] Step 14: OD value determination: Measure the OD value of each well using an ELISA reader at a wavelength of 450nm. Complete the ELISA reader reading within 15 minutes after termination.

[0039] In the specific implementation of this invention, according to CLSI EP15-A, two concentration levels of SP70 serum samples, with concentrations of 29.63 mg / dL and 9.89 mg / dL respectively, were selected for precision verification. According to CLSI EP15-A2, two sets each of high and low concentration levels of standard products from the manufacturer were selected for accuracy verification. Twenty sets of 0 ng / mL standard products were selected for limit of detection verification. Serum samples from 20 healthy individuals were selected, including 10 males and 10 females, for reference interval verification. According to CLSI EP6-A, one low-value serum sample (L) and one high-value serum sample (H) were taken and diluted according to the ratios of 1L, 4L+1H, 3L+2H, 2L+3H, 1L+4H, and 1H to form a series of evaluation samples with different concentration levels in 6 tubes for linear range verification.

[0040] The working principle of this invention is as follows: The microwells of the reaction plate in the kit are pre-coated with anti-SP70 polyclonal antibody. After adding serum, the SP70 antigen in the serum binds to the antibody coated in the reaction wells to form an antigen-antibody complex. Then, mouse anti-human SP70 monoclonal antibody NJ001 is added and binds to the complex. By adding horseradish peroxidase (HRP)-labeled rabbit anti-mouse IgG antibody, the substrate tetramethylbenzidine (TMB) is catalyzed by HRP and turns blue. After terminating the reaction, the optical density (OD value) is measured by an enzyme-linked immunosorbent assay (ELISA) reader at a wavelength of 450 nm. Since the concentration level of SP70 in the serum sample is directly proportional to the OD value, the sample concentration can be obtained from the standard curve.

[0041] The present invention and its embodiments have been described above. This description is not restrictive, and the accompanying drawings are only one embodiment of the present invention; the actual structure is not limited thereto. In conclusion, if those skilled in the art are inspired by this description and design similar structures and embodiments without departing from the spirit of the invention, such designs should fall within the protection scope of the present invention.

Claims

1. A method for quantitatively detecting serum SP70 of non-small cell lung cancer patient, characterized in that The method comprises the following steps: Step 1: Take the kit out of the refrigerator and place it at room temperature for 30 minutes before performing the experiment; Step 2: Prepare the working concentration of the washing buffer: add 30 mL of concentrated washing solution to a 1 L reagent bottle containing 570 mL of pure water at room temperature, mix well, and store at 2-8°C; Step 3: Take out the anti-SP70 enzyme-labeled reaction plate, and place the remaining microplate strip in a sealed bag and store it in the refrigerator at 2-8°C; Step 4: Dissolve the standard: use a pipette to take 120 μL of pure water and add it to the standard tube containing the freeze-dried powder, dissolve the SP70 freeze-dried standard, and mix well to obtain the SP70 standard; Step 5: Use a pipette to take 100 μL of SP70 standard and sample and add them to the corresponding detection holes, complete the sample addition process within 10 minutes, place the reaction plate on the shelf of a 37°C water bath, incubate at 37°C for 60 minutes, and discard the incubation liquid; Step 6: Wash the plate: machine washing: add 250 μL of working concentration of washing buffer to each hole, wash a total of 4 times; manual washing: discard the liquid in the holes, add 250 μL of working concentration of washing buffer to each hole, discard the liquid, spin dry, and pat dry on a blotting paper, repeat 4 times; Step 7: Add monoclonal antibody: dilute the required monoclonal antibody 100 times with antibody diluent to obtain a monoclonal antibody working solution, add 100 μL of the antibody working solution to each hole, and place the reaction plate on the shelf of a 37°C water bath, incubate at 37°C for 30 minutes, and discard the incubation liquid; Step 8: Wash the plate: machine washing: add 250 μL of working concentration of washing buffer to each hole, wash a total of 4 times; manual washing: discard the liquid in the holes, add 250 μL of working concentration of washing buffer to each hole, discard the liquid, spin dry, and pat dry on a blotting paper, repeat 4 times; Step 9: Add enzyme-labeled secondary antibody: dilute the required enzyme-labeled secondary antibody 100 times with antibody diluent to obtain an enzyme-labeled secondary antibody working solution, add 100 μL of the enzyme-labeled secondary antibody working solution to each hole; Step 10: Incubate: place the reaction plate on the shelf of a 37°C water bath, incubate at 37°C for 30 minutes, and discard the incubation liquid; Step 11: Wash the plate: the operation is the same as before, wash 5 times; Step 12: Add color developing solution: add 100 μL of TMB to each hole, and place the reaction plate in a dark place at room temperature for 15 minutes; Step 13: Add stop solution: add 50 μL of stop solution to each hole, gently shake the edge of the plate, mix for 30 seconds, check and remove the bubbles in each hole; Step 14: Measure the OD value: use an enzyme-labeled instrument to measure the OD value of each hole at 450 nm wavelength, and complete the reading of the enzyme-labeled instrument within 15 minutes after termination.

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