Kit and detection method for evaluating immune prognosis of small cell lung cancer

Abstract

The invention discloses a kit for evaluating immunoprognosis of small cell lung cancer and a detection method, belongs to the field of molecular biology, relates to medical and biotechnology, and aims to solve the problems of long detection time and tedious detection steps of small cell lung cancer indexes in the prior art. The kit comprises a fluorescent microsphere and an antibody pair, the antibody pair comprises a capture antibody and a detection antibody, the capture antibody is fixed on the surface of the fluorescent microsphere, and the tail end of the detection antibody is connected with report fluorescence. Fluorescein is adopted to carry out fluorescence coding on microspheres with the particle size of 5.5 microns, and a capture antibody is connected to the fluorescent microspheres through chemical reaction; a detection target is captured through the fluorescent microspheres, and then a signal is displayed through the detection antibody, so that the detection of the prognosis index is completed. A single reaction system can detect various targets, and multiple repeated tests can be carried out, so that the detection flux is greatly improved, and the detection difficulty of a hospital end or an industrial end on inflammatory factors is reduced.

Description

Technical Field

[0001] The present invention belongs to the technical field of molecular biomedicine, and in particular relates to a kit and a detection method for evaluating the immune prognosis of small cell lung cancer. Background Art

[0002] Small cell lung cancer (SCLC) is a high-grade neuroendocrine cancer that primarily develops in current or former smokers and carries a very poor prognosis. SCLC accounts for approximately 15% of cancer cases. Patients with SCLC typically present with respiratory symptoms, including cough and dyspnea. Imaging studies reveal a central lung mass, and thoracic lymph node involvement is common. Two-thirds of patients have distant metastatic disease at the time of initial diagnosis. The most common sites of metastasis include the contralateral lung, brain, liver, adrenal gland, and bone. SCLC has a high propensity for metastasis, with the number of circulating tumor cells (CTCs) in SCLC being the highest of all solid tumors. Despite emerging insights into the biological subtypes of SCLC based on transcription factor expression profiles, the current clinical approach to treating SCLC is consistent regardless of subtype. For the rare patient with very early-stage disease at diagnosis, treatment may include surgery and adjuvant platinum-based chemotherapy. More commonly, patients with early-stage or locally advanced disease receive both radiation therapy and platinum-based chemotherapy. Patients with metastatic disease receive systemic chemotherapy with or without immunotherapy. Small cell lung cancer initially responds exceptionally well to cytotoxic therapy—up to 25% of patients with early-stage small cell lung cancer achieve long-term disease control with concurrent chemoradiotherapy (CRT), and response rates consistently exceed 60% even in patients with metastatic disease. However, in the vast majority of patients, these responses are short-lived, resulting in median survival of <2 years for patients with early-stage disease and approximately 1 year for patients with metastatic disease.

[0003] Methods for assessing the prognosis of small cell lung cancer primarily include computed tomography (CT) and magnetic resonance imaging (MRI) of the tumor site, and tumor marker measurement. However, because CT and MRI are imaging technologies, CT is limited by spatial resolution and cannot accurately diagnose small lesions. Furthermore, CT cannot directly reflect the metabolic and proliferation of tumor cells, functional characteristics that are crucial for prognostic assessment. This inability to provide this information makes it difficult to accurately predict a patient's prognosis based solely on CT scans. Furthermore, due to radiation dose limitations, frequent examinations are difficult; prolonged intervals can miss key milestones in tumor progression or remission. Furthermore, changes in tumor size on CT images can lag behind changes in the biological behavior of tumor cells. For example, tumor cells may have undergone metabolic changes after treatment, but the tumor size shown on CT scans has not changed significantly, which can affect the accurate assessment of prognosis.

[0004] Magnetic resonance imaging (MRI) is not as effective as CT in imaging the lung parenchyma. Because the lungs contain a large amount of gas and have a low hydrogen proton density, the spatial resolution and contrast of MRI images are inferior to those of CT in lung imaging. For small cell lung cancer, a disease that primarily occurs in the lungs, MRI is inadequate in displaying the details of the primary lung tumor (such as tumor margins, internal structures, etc.), which affects the accurate assessment of tumor characteristics and is therefore not conducive to prognosis. In addition, if a patient has metal implants (pacemakers, stents, etc.), MRI examinations will not be possible. For these patients, MRI cannot be used to assess the condition of small cell lung cancer, limiting its versatility as a prognostic evaluation method.

[0005] While tumor marker testing has some value in disease diagnosis and monitoring, its specificity is limited. Benign diseases (e.g., brain injury, which can lead to elevated NSE, and gastric inflammation, which can affect progastrin-releasing peptide (ProGRP) levels) can also cause these markers to rise, potentially leading to misjudgments of tumor recurrence or progression. Tumor markers are primarily tested in the blood, which only indirectly reflect tumor cell secretion and cannot provide information on the tumor's location, size, morphology, or relationship to surrounding tissues.

[0006] Cytokines are a class of small proteins with a wide range of biological activities that are expressed and secreted by immune cells and some non-immune cells upon stimulation. They typically regulate cell growth, differentiation, and effects by binding to corresponding receptors, ultimately modulating immune responses. Some cytokines play a crucial role in the diagnosis of certain diseases.

[0007] The CD70 protein is a type II transmembrane protein and a member of the tumor necrosis factor (TNF) superfamily. It is primarily expressed on activated T and B cells and interacts with the CD27 receptor, participating in the regulation of immune responses. By binding to its receptor, CD27, CD70 activates the NF-κB and c-Jun kinase pathways, thereby promoting the activation, proliferation, and differentiation of T and B cells. The CD70-CD27 signaling pathway plays a crucial role in normal physiological function. In T cell activation, upon antigen stimulation, T cells express CD70. Binding of CD70 to CD27 enhances T cell proliferation, survival, and effector function, promoting the production of cytokines (such as interferon-γ and interleukin-2), and contributing to the effective elimination of pathogens. CD70 expression levels have potential diagnostic value in certain tumors. For example, in renal cell carcinoma, detecting CD70 expression in tumor tissue can aid in diagnosis. High CD70 expression may indicate a higher degree of tumor malignancy. In terms of prognosis, for many tumors (such as non-Hodgkin's lymphoma), patients with positive expression of CD70 often have a poor prognosis and may have a shorter survival period. This is because high expression of CD70 may be related to the tumor's aggressiveness, metastatic ability, and resistance to immunotherapy. In hematological tumors, such as acute lymphoblastic leukemia, detecting CD70 expression in leukemia cells can assist in diagnosis, and the expression level of CD70 is associated with the risk of recurrence of the disease. Patients with high expression of CD70 are more likely to experience disease recurrence, which is an important indicator for judging prognosis.

[0008] CD83 is a type I transmembrane glycoprotein with a molecular weight of approximately 45 kDa and belongs to the immunoglobulin superfamily. It consists of an extracellular immunoglobulin-like domain, a transmembrane region, and a relatively short intracellular domain. This structural feature enables it to perform recognition and signaling functions on the cell surface. In the immune system, CD83 is primarily expressed on mature dendritic cells (DCs) and is a key marker of mature DCs. DCs are the most powerful antigen-presenting cells in the body and play a key role in the initiation and regulation of immune responses. After DCs take up antigens, they undergo a maturation process during which CD83 expression is upregulated. CD83 interacts with receptors on the surfaces of other immune cells, enhancing antigen presentation and promoting T cell activation and differentiation. For example, in T cell-dependent immune responses, mature CD83-positive DCs can effectively present antigen peptide-MHC complexes to T cells and provide co-stimulatory signals, thereby inducing T cell proliferation and functional differentiation, initiating specific immune responses. CD83 expression in tumor tissue can serve as a reference marker for tumor diagnosis and prognosis. In studies of some tumors (such as colon cancer and lung cancer), it was found that the number and distribution of CD83-positive cells in tumor tissue are related to factors such as the type and stage of the tumor. For example, in patients with colon cancer, if the number of CD83+DC cells around the tumor tissue is small, it may indicate that the tumor has a strong immunosuppressive microenvironment and a relatively poor prognosis. CD83 may also have a certain value in predicting the effect of immunotherapy. In patients receiving immunotherapy (such as immune checkpoint inhibitor therapy), the expression changes of CD83 in tumor tissue are detected before and after treatment. If CD83 expression increases after treatment and is accompanied by increased immune cell infiltration, it may indicate a better treatment effect and prognosis.

[0009] CCL3 belongs to the chemokine family (C-C motif chemokine ligand 3), also known as macrophage inflammatory protein-1α (MIP-1α). It is a small secreted protein with a molecular weight of approximately 8-10 kDa. Its molecular structure is primarily composed of multiple β-sheets, which form a specific spatial conformation through disulfide bonds. This structure is crucial for receptor binding and biological function. In the early stages of an inflammatory response, damaged tissues or activated immune cells (such as macrophages and monocytes) secrete CCL3. Under the influence of a concentration gradient, CCL3 can attract a variety of immune cells to the site of inflammation. For example, CCL3 can recruit monocytes, T lymphocytes, and natural killer (NK) cells. After migrating to the site of inflammation, monocytes can further differentiate into macrophages, participating in pathogen clearance and tissue repair; T lymphocytes and NK cells can exert cellular immunity to kill infected cells or pathogens. Clinically, CCL3 expression levels are correlated with tumor prognosis. For example, in various tumors, such as breast and lung cancer, patients with high CCL3 expression in their tumor tissues often have a poor prognosis and are more likely to experience tumor metastasis and recurrence. This is because CCL3 promotes tumor cell metastasis and the formation of the tumor microenvironment. Therefore, CCL3 has the potential to become a target for cancer treatment. By inhibiting CCL3 secretion or blocking its binding to receptors, it is possible to interfere with the metastatic process of tumor cells and the formation of the tumor microenvironment, thereby improving the prognosis of cancer patients.

[0010] CCL4 (also known as macrophage inflammatory protein-1β, MIP-1β) is also a member of the CC motif chemokine ligand family. Its molecular weight is similar to that of CCL3, approximately 8-10 kDa. Its molecular structure also has the typical β-pleated structure of chemokines, and there are conserved cysteine residues used to form disulfide bonds, thereby stabilizing its three-dimensional structure and ensuring its specificity and functional activity in binding to the receptor. In cancer patients, the expression levels of CCL3 and CCL4 are closely correlated with tumor prognosis. In various tumors (such as breast cancer, lung cancer, melanoma, etc.), patients with high expression of CCL3 and CCL4 in tumor tissue often have a poor prognosis and are more likely to experience tumor metastasis and recurrence. This is because they promote the metastasis of tumor cells and the formation of the tumor microenvironment, which plays a role in promoting tumor progression.

[0011] Cytometric BeadArray (CBA) liquid-based chip technology (hereinafter referred to as CBA) is a detection method based on the capture of labeled microspheres and the detection of targets in patient fluids through flow cytometry. It can simultaneously perform qualitative and quantitative detection of multiple indicators in a sample. Furthermore, it has the advantages of requiring a small sample volume, high sensitivity, excellent stability, a short detection process, simple operation, and a high detection rate. Currently, this detection method has been widely used in fields such as cell biology, immunology, and antibody drug screening, providing data support for life science research. Summary of the Invention

[0012] To enable rapid and convenient detection of prognostic indicators related to small cell lung cancer, the present invention provides a test kit for assessing the immune prognosis of small cell lung cancer based on liquid-based chip technology. Fluorescein is used to fluorescently encode 5.5 μm microspheres, and capture antibodies are attached to the fluorescent microspheres through a chemical reaction. The fluorescent microspheres capture the detection target, and the detection antibody displays the signal, thereby completing the detection of prognostic indicators. This overcomes the drawbacks of previous small cell lung cancer indicator detection, which has been long and complex.

[0013] In a first aspect, the present invention provides a kit for evaluating the immune prognosis of small cell lung cancer, the kit comprising fluorescent microspheres and an antibody pair, the antibody pair comprising a capture antibody and a detection antibody, the capture antibody being immobilized on the surface of the fluorescent microspheres, and the detection antibody being terminally connected to a reporter fluorescence.

[0014] In some preferred examples in this aspect, the number of fluorescent microspheres is equal to the logarithm of the antibody pairs, the capture antibodies representing different targets correspond one to one with the multiple fluorescent microspheres, and the multiple fluorescent microspheres are coded with different concentrations of fluorescein, and the fluorescein gradient is 1 mg / mL, 0.2 mg / mL, 0.04 mg / mL, and 0.008 mg / mL.

[0015] In some preferred examples of this aspect, the particle size of the plurality of fluorescent microspheres is 5.5 μm.

[0016] In some preferred examples of this aspect, the fluorescein is allophycocyanin dye.

[0017] In some preferred examples of this aspect, the capture antibodies included in the four antibody pairs are CD70 monoclonal antibody, CD83 monoclonal antibody, CCL3 monoclonal antibody and CCL4 monoclonal antibody respectively.

[0018] In some preferred examples of this aspect, the CD70 monoclonal antibody, CD83 monoclonal antibody, CCL3 monoclonal antibody and CCL4 monoclonal antibody are respectively coupled to the surface of their corresponding fluorescent microspheres through chemical reactions.

[0019] In some preferred examples of this aspect, the chemical reaction process for antibody coupling is as follows: This preferred example antibody labeling method uses the carbodiimide labeling (EDC) method. The antibody is first dissolved in a PBS solution, and the fluorescein powder is dissolved in a DMSO solution. An appropriate amount of EDC and N-hydroxysuccinimide (NHS) is added to the fluorescein solution to activate the carboxyl group of the fluorescein. The activated fluorescein solution is then added to the antibody solution and reacted overnight at 4°C and pH = 7.2. After the reaction is completed, the labeled antibody is purified by semipermeable membrane dialysis.

[0020] In some preferred examples of this aspect, the reporter fluorescence is phycoerythrin.

[0021] In some preferred examples of this aspect, the detection antibodies included in the four antibody pairs are CD70 monoclonal antibody, CD83 monoclonal antibody, CCL3 monoclonal antibody and CCL4 monoclonal antibody linked to phycoerythrin, respectively.

[0022] In some preferred examples of this aspect, the kit further comprises a freeze-dried detection target standard mixture, a sample diluent and a washing buffer.

[0023] In a second aspect, the present invention provides a detection method for a kit for evaluating the immune prognosis of small cell lung cancer, the detection method comprising:

[0024] Step 1: Prepare the sample. Serum or plasma samples do not need to be diluted. Cell culture medium and cell culture supernatant need to be diluted with sample diluent.

[0025] Step 2: Centrifuge the fluorescent microspheres coupled with the capture antibody in the dark.

[0026] Step 3: Prepare the standard. Centrifuge the lyophilized standard mixture and dissolve it in sample diluent, labeling it S1. Then, take seven EP tubes, label them S2-S8, and add 75 μL of sample diluent to each tube. Use a pipette to transfer 25 μL of S1 to S2 and shake evenly. Then, transfer 25 μL of S2 to S3 and shake evenly. Treat the remaining tubes S4-S7 as described above. Add sample diluent to S8 as a control.

[0027] Step 4: Add the fluorescent microsphere mixture coupled with CD70 monoclonal antibody, CD83 monoclonal antibody, CCL3 monoclonal antibody and CCL4 monoclonal antibody to the flow cytometer tube, add the diluted gradient standard and test sample in the EP tube to the flow cytometer tube, and then add the detection antibody in sequence. After incubation at room temperature, centrifuge to remove the supernatant, add wash buffer to wash the fluorescent microspheres twice, each time for 10 minutes. After washing, add wash buffer again and analyze on the flow cytometer;

[0028] Step 5: Data acquisition and analysis. Set up the allophycocyanin dye detection channel and the phycoerythrin detection channel in the flow cytometer, and set up the FSC graph, SSC graph, and cross-analysis graph of the allophycocyanin dye detection channel and the phycoerythrin detection channel in the flow cytometer software interface. Start by loading the S8 onto the machine, adjust the voltage in the FSC / SSC graph, circle the initial microsphere group and set the gate, then circle the corresponding targets and set the gate according to the microsphere fluorescence coding and the microsphere fluorescence intensity in the flow cytometer graph, adjust the PE channel voltage so that the MFI of the PE channel of all detection targets is near 0, then load all samples onto the machine for detection, and export all data for analysis on the corresponding software.

[0029] In some preferred examples in this aspect, the capture antibody in step 4 specifically binds to the target protein to be detected during the sample incubation stage, and the detection antibody is subsequently added to continue incubation. After the detection antibody specifically binds to the target protein, the target protein not bound to the microspheres is washed away with a washing buffer, and the amount of target protein captured on the fluorescent microspheres is reflected by the fluorescence level of the reporter fluorescence.

[0030] In some preferred examples of this aspect, the phycoerythrin signal generated by the standards S1-S7 in step 5 is compared with a standard curve generated by the signal generated by a purified protein standard of known concentration to determine the target protein content.

[0031] Compared with the prior art, the present invention has the following advantages:

[0032] 1. The kit of the present application is a detection reagent using the CBA method, which distinguishes different targets by the difference in APC fluorescence intensity; each group of fluorescent microspheres has a unique capture antibody label, so that during the detection process, it can quickly, accurately and specifically bind to specific inflammatory factor targets. A single reaction system can detect multiple targets and can perform repeated tests multiple times, greatly improving the detection throughput and reducing the difficulty of detecting inflammatory factors on the hospital or industrial side.

[0033] 2. The detection kit for evaluating the immune prognosis of small cell lung cancer based on liquid-based chip technology described in this application can be adapted to most 2-light 6-color flow cytometers on the market and has high versatility.

[0034] 3. Compared to other small cell lung cancer prognosis detection methods, the small cell lung cancer prognosis reagent described in this application requires only 25 μL of sample per test, while the sample volume for enzyme-linked immunosorbent assay and western blotting is 200 μL or even higher. This can significantly reduce the sample volume required for testing.

[0035] 4. The difference between wells detected by the kit is small and the CV value is small.

[0036] 5. The kit of the present application can avoid the fluorescence crosstalk caused by fluorescence overlap in current detection methods, greatly improving the sensitivity and accuracy of detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 This is a schematic diagram of the principle of the kit of the present invention for detecting samples. DETAILED DESCRIPTION

[0038] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the embodiments and accompanying drawings. It should be understood that the specific embodiments described are only for explaining the specific application of the present invention and are not intended to limit the scope of the present invention. Through these embodiments, it is intended to help understand the principles, operating methods and advantages of the present invention, but it is not excluded that appropriate adjustments and modifications can be made during the implementation of the present invention.

[0039] Example 1

[0040] The detection kit for evaluating the immune prognosis of small cell lung cancer based on liquid-based chip technology provided in this embodiment includes fluorescent microspheres with different fluorescence intensities and four pairs of antibodies. Among them, the fluorescent microspheres with different fluorescence (APC) intensities are fluorescent microspheres with a particle size of 5.5μm, and the four pairs of antibodies include capture antibodies and detection antibodies, wherein the capture antibodies are fixed to the surface of the fluorescent microspheres through a chemical reaction, and the capture antibodies representing different targets correspond one-to-one to the fluorescent microspheres with different fluorescence codes. The ends of the detection antibodies are chemically linked to the reporter fluorescent phycoerythrin (PE).

[0041] The fluorescent microspheres described in this embodiment are microspheres that have been coded with different concentrations of fluorescein, which is allophycocyanin dye (APC).

[0042] The capture antibodies for the four antibody pairs are monoclonal antibodies against CD70, CD83, CCL3, and CCL4, respectively. The detection antibodies are monoclonal antibodies against CD70, CD83, CCL3, and CCL4 conjugated with PE (phycoerythrin) dye. See Table 1 below for details.

[0043] Table 1 Sources of capture antibodies

[0044] name source CD70 Biolegend CD83 Biolegend CCL3 Biolegend CCL4 Biolegend

[0045] The kit also includes a reaction system, which includes a freeze-dried detection target standard mixture, a sample diluent, and a washing buffer solution.

[0046] Example 2

[0047] Each fluorescent microsphere set with the same concentration of fluorescent labeling has only one capture antibody on its surface. The capture antibody specifically binds to the target protein being detected during the sample incubation phase. A detection antibody labeled with PE dye is then added and incubation continues. After the detection antibody specifically binds to the target protein, any target protein not bound to the microspheres is washed away with a wash buffer. Individual proteins are identified using a specific microsphere-fluorescence combination, and the PE fluorescence level reflects the amount of protein captured on the microspheres. The PE signal generated by the standard is compared with a standard curve generated by signals generated by purified protein standards of known concentrations to determine the target protein content.

[0048] The liquid-based chip technology detection kit for evaluating the immune prognosis of small cell lung cancer described in this application can be combined with a flow cytometer with 488nm and 638nm lasers and 6 detection channels for detection. The flow cytometer can perform high-speed quantitative measurement of multiple fluorescent microspheres at the same time.

[0049] The working principle of the detection using the kit in Example 1 is as follows:

[0050] 1. Sample preparation: Serum or plasma samples do not require dilution. Cell culture medium and cell culture supernatant need to be diluted with sample diluent. The recommended dilution factor is 2-4 times.

[0051] 2. Preparation before the experiment: Centrifuge the fluorescent microspheres immobilized with the capture antibody in a dark environment for 1 min.

[0052] 3. Prepare standards: Centrifuge the lyophilized standard mixture at 12,000 rpm for 1 minute, then dissolve it in sample diluent (labeled S1). Then, pipette 25 μL of S1 into seven 1.5 mL EP tubes labeled S2-S8 and vortex thoroughly. Then, pipette 25 μL of S2 into S3 and vortex thoroughly. Repeat the above procedure for tubes S4-S7. Add sample diluent to tube S8 as a control.

[0053] 4. Experimental Procedure: Add the microsphere mixture linked to monoclonal antibodies against CD70, CD83, CCL3, and CCL4 to the flow cytometer according to experimental requirements. Subsequently, add the diluted gradient standard and test sample in the EP tube to the flow cytometer, followed by the detection antibody. Incubate at room temperature for 2 hours. After centrifugation to remove the supernatant, add wash buffer to wash the fluorescent microspheres. After washing, add 200 μL of wash buffer, and then analyze on a flow cytometer.

[0054] 5. Data acquisition and analysis: Data were collected by flow cytometer and analyzed by analysis software (Flowjo, FCAP).

[0055] The following are specific examples of this application. The reagents used in the following examples are described as follows: 5.5 μm microspheres were produced by NanoMicro, and the capture antibodies were derived from the antibodies listed in Table 1 above. The lyophilized protein standards were obtained from BioLegend. The detection antibodies were all PE-dyed antibodies to the target. Details are shown in the table below:

[0056] Table 2 Sources of detection antibodies

[0057] name source CD70-PE Biolegend CD83-PE Biolegend CCL3-PE Biolegend CCL4-PE Biolegend

[0058] Table 3 Sources of protein standards

[0059] name source CD70 Biolegend CD83 Biolegend CCL3 Biolegend CCL4 Biolegend

[0060] The liquid-based chip technology detection kit described in this example for evaluating the immune prognosis of small cell lung cancer includes a single particle size (5.5 μm), a mixture of fluorescent microspheres coupled with capture antibodies (CD70, CD83, CCL3, CCL4) and fluorescein-encoded, a sample diluent, a wash buffer, a detection antibody, and a lyophilized protein standard.

[0061] Example 3

[0062] Performance evaluation: The inflammatory factor detection kit of the above embodiment was used to evaluate its performance including sensitivity, standard sample recovery rate, and intra-batch and inter-batch precision.

[0063] The experimental steps are as follows:

[0064] 1. Sample preparation: Serum or plasma samples do not require dilution. Cell culture medium and cell culture supernatant need to be diluted with sample diluent. The recommended dilution factor is 2x.

[0065] 2. Preparation before the experiment: Centrifuge the fluorescent microspheres immobilized with the capture antibody in a dark environment for 1 min.

[0066] 3. Prepare standards: Centrifuge the lyophilized standard mixture at 12,000 rpm for 1 minute, then dissolve it in sample diluent (labeled S1). Then, pipette 25 μL of S1 into seven 1.5 mL EP tubes labeled S2-S8 and vortex thoroughly. Then, pipette 25 μL of S2 into S3 and vortex thoroughly. Repeat the above procedure for tubes S4-S7. Add sample diluent to tube S8 as a control.

[0067] 4. Experimental procedure: Add the microspheres linked to the capture antibody to the flow cytometer according to the experimental requirements. Then, add the standard dilution gradient and the test sample to the flow cytometer, and then add the detection antibody in sequence. Incubate at room temperature for 2 hours. After centrifugation to remove the supernatant, add wash buffer to wash the fluorescent microspheres. After washing, add 200 μL of wash buffer, and then analyze on a flow cytometer.

[0068] 5. Data acquisition and analysis: Set up APC and PE detection channels in the flow cytometer, and set up FSC (forward scatter), SSC (side scatter) graphs, and APC channel and PE channel cross-analysis graphs in the flow cytometer software interface. First, load the S8 sample onto the machine, adjust the voltage in the FSC / SSC graph, circle the initial microsphere group and set the gate, then circle the corresponding targets and set the gate according to the microsphere fluorescence coding and the microsphere fluorescence intensity in the flow cytometer graph, and adjust the PE channel voltage so that the MFI (mean fluorescence intensity) of the PE channel of all detection targets is around 0. All samples were then tested on the machine, and the total number of microspheres read was 1800. All data were exported as FCS files and analyzed in the corresponding software.

[0069] In the above test, the corresponding relationship between the gate and the corresponding target protein is as follows:

[0070] Table 4. Correspondence between gates and target proteins

[0071] Gating Target protein P1 CD70 P2 CD83 P3 CCL3 P4 CCL4

[0072] 1. Sensitivity test

[0073] According to the above experimental process, each target protein standard was diluted with sample diluent, and the sensitivity test (detection limit) of the inflammatory factor detection kit was performed. The detection limit test results are shown in the following table:

[0074] Table 5 Detection limit test results

[0075] Target protein Detection limit concentration (pg / ml) CD70 3.55 CD83 4.35 CCL3 6.45 CCL4 2.37

[0076] 2. Cross-reactivity

[0077] No detectable or significant cross-reactivity was detected between targets.

[0078] 3. Calibration recovery rate

[0079] The target protein was diluted to a fixed concentration, and the standard diluted to a fixed concentration was tested according to the above experimental steps. The test concentration was compared with the theoretical concentration to calculate the recovery rate. The analysis results are shown in Table 6 below.

[0080] Table 6 Recovery test results

[0081] Target protein Recovery rate (%) CD70 98.3 CD83 99.7 CCL3 107.5 CCL4 110.8

[0082] 4. Intra-batch precision

[0083] The target protein was diluted to medium and lower concentrations using sample diluent, and the test was performed according to the above experimental method. A single concentration was repeated 10 times to obtain the intra-assay precision or coefficient of variation (CV) of the sample. The data results are shown in Table 7

[0084] Table 7 Coefficient of variation of precision within sample batches

[0085]

[0086] 5.Batch-to-batch precision

[0087] The target protein was diluted to medium and lower concentrations using sample diluent, and the test was performed according to the above experimental method. A single concentration was repeated 10 times to obtain the intra-assay precision or coefficient of variation (CV) of the sample. The data results are shown in Table 8

[0088] Table 8 Coefficient of variation of precision between sample batches

[0089]

[0090] The above test results show that the kit provided in this application has a low detection limit concentration, among which the target protein concentration with the highest detection limit is only 2.37 pg / mL (CCL4), with high detection sensitivity and no cross-reactivity between targets.

[0091] Through the calculation and analysis of intra-batch precision and inter-batch precision, the detection performance of the kit provided in this application is stable, and it is a stable and reliable detection method.

[0092] Compared with existing detection methods, the present invention has high detection sensitivity, short detection time and detection steps, and is more conducive to practical application.

[0093] The above descriptions are merely a few embodiments of the present application and do not constitute any form of limitation to the present application. Although the present application discloses the preferred embodiments as above, they are not intended to limit the present application. Any technical personnel familiar with the present profession, without departing from the scope of the technical solution of the present application, using the technical content disclosed above to make slight changes or modifications are equivalent to equivalent implementation cases and fall within the scope of the technical solution.

Claims

1. A kit for evaluating the immune prognosis of small cell lung cancer, characterized in that: The kit includes fluorescent microspheres and an antibody pair. The antibody pair includes a capture antibody and a detection antibody. The capture antibody is fixed on the surface of the fluorescent microspheres, and the detection antibody terminal is connected to a reporter fluorescence.

2. A kit for evaluating the immune prognosis of small cell lung cancer according to claim 1, characterized in that: The number of fluorescent microspheres is equal to the number of antibody pairs. The capture antibodies representing different targets correspond one to one with multiple fluorescent microspheres. The multiple fluorescent microspheres are coded with different concentrations of fluorescein, and the fluorescein gradient is 1 mg / mL, 0.2 mg / mL, 0.04 mg / mL, and 0.008 mg / mL.

3. A kit for evaluating the immune prognosis of small cell lung cancer according to claim 2, characterized in that: The particle size of the multiple fluorescent microspheres is 5.5 μm.

4. A kit for evaluating the immune prognosis of small cell lung cancer according to claim 2, characterized in that: The capture antibodies included in the four antibody pairs are CD70 monoclonal antibody, CD83 monoclonal antibody, CCL3 monoclonal antibody and CCL4 monoclonal antibody respectively.

5. The kit for evaluating the immune prognosis of small cell lung cancer according to claim 4, characterized in that: The CD70 monoclonal antibody, CD83 monoclonal antibody, CCL3 monoclonal antibody and CCL4 monoclonal antibody are respectively coupled to the surface of their corresponding fluorescent microspheres through chemical reactions.

6. The kit for evaluating the immune prognosis of small cell lung cancer according to claim 4, characterized in that: The detection antibodies included in the four pairs of antibodies are CD70 monoclonal antibody, CD83 monoclonal antibody, CCL3 monoclonal antibody and CCL4 monoclonal antibody connected to phycoerythrin.

7. The kit for evaluating the immune prognosis of small cell lung cancer according to claim 1, characterized in that: The kit also includes a lyophilized detection target standard mixture, a sample diluent, and a wash buffer.

8. A detection method for a kit for evaluating the immune prognosis of small cell lung cancer, characterized in that: The method is achieved by using the kit according to any one of claims 1 to 9, wherein the detection method comprises: Step 1: Prepare the sample. Serum or plasma samples do not need to be diluted. Cell culture medium and cell culture supernatant need to be diluted with sample diluent. Step 2: Centrifuge the fluorescent microspheres coupled with the capture antibody in the dark. Step 3: Prepare the standard. Centrifuge the lyophilized standard mixture and dissolve it in sample diluent, labeling it S1. Then, take seven EP tubes, label them S2-S8, and add 75 μL of sample diluent to each tube. Use a pipette to transfer 25 μL of S1 to S2 and shake evenly. Then, transfer 25 μL of S2 to S3 and shake evenly. Treat the remaining tubes S4-S7 as described above. Add sample diluent to S8 as a control. Step 4: Add the fluorescent microsphere mixture coupled with CD70 monoclonal antibody, CD83 monoclonal antibody, CCL3 monoclonal antibody and CCL4 monoclonal antibody to the flow cytometer tube, add the dilution gradient standard and test sample in the EP tube to the flow cytometer tube, and add the detection antibody in sequence. After incubation at room temperature, centrifuge to remove the supernatant, add wash buffer to wash the fluorescent microspheres twice, each time for 10 minutes. After washing, add wash buffer again and analyze on the flow cytometer; Step 5: Data acquisition and analysis. Set up the allophycocyanin dye detection channel and the phycoerythrin detection channel in the flow cytometer, and set up the FSC graph, SSC graph, and cross-analysis graph of the allophycocyanin dye detection channel and the phycoerythrin detection channel in the flow cytometer software interface. Start by loading the S8 onto the machine, adjust the voltage in the FSC / SSC graph, circle the initial microsphere group and set the gate, then circle the corresponding targets and set the gate according to the microsphere fluorescence coding and the microsphere fluorescence intensity in the flow cytometer graph, adjust the PE channel voltage so that the MFI of the PE channel of all detection targets is near 0, then load all samples onto the machine for detection, and export all data for analysis on the corresponding software.

9. The detection method according to claim 8, characterized in that In step 4, the capture antibody specifically binds to the target protein to be detected during the sample incubation stage, and the detection antibody is subsequently added to continue incubation. After the detection antibody specifically binds to the target protein, the target protein that is not bound to the microspheres is washed away with the washing buffer, and the amount of target protein captured on the fluorescent microspheres is reflected by the fluorescence level of the reporter fluorescence.

10. The detection method according to claim 9, characterized in that: The phycoerythrin signal generated by standards S1-S7 in step 5 is compared with the standard curve generated by the signal generated by the purified protein standards of known concentration to determine the target protein content.

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