A kit and method for detecting T lymphocyte subsets in lung cancer

Abstract

The present invention belongs to the field of medical detection technology, and discloses a kit for detecting lung cancer T lymphocyte subpopulations, which includes: a vertical flow immunochromatography box and three signal probes; the vertical flow immunochromatography box includes a box body, and a water-absorbing pad, a filter membrane and a detection cover stacked on the box body from bottom to top, and a sample addition hole is provided on the detection cover; the three signal probes include three red latex microspheres coupled with three antibodies respectively, wherein the three antibodies are CD3 antibody, CD4 antibody and CD8 antibody, and the labeled amount of the antibody in the red latex microspheres is 11μg / 25μL to 14μg / 25μL. The kit for detecting lung cancer T lymphocyte subpopulations can detect the number of lung cancer T lymphocyte subpopulations and evaluate the health status of lung cancer patients.

Description

Technical Field

[0001] The invention belongs to the technical field of medical detection, and in particular relates to a kit and a method for detecting T lymphocyte subsets of lung cancer. Background Art

[0002] The immune system is composed of immune organs, immune cells and immune active substances, and has the functions of immune defense, immune surveillance and immune stability. Among them, cellular immunity plays an important role in the body's anti-tumor activities, and lymphocytes are an important part of cellular immunity. Lymphocyte subsets can be divided into: T lymphocytes, B lymphocytes and natural killer cells (NK cells) according to their biological functions and cell surface antigens. Among them, T lymphocytes are the most important form of immune expression and play an important role in immune surveillance. T lymphocytes can be divided into helper T cells (Helper T cells, Th), cytotoxic T cells (Cytotoxic T cells, Tc), regulatory T cells (Regulatory cells, Treg), and effector T cells (Effector T cells, Te) according to their functions. Helper T cells express CD4 molecules on their surface, also known as CD4 + T cells secrete cytokines to positively regulate anti-tumor effects and can also regulate B cells to secrete antibodies to assist in cellular and humoral immunity. The surface of cytotoxic T cells express CD8 molecules, also known as CD8 + T cells kill tumor cells by secreting cytotoxic granules. + T lymphocytes, CD4 + T lymphocytes, CD8 + Maintaining a dynamic balance between the number of T lymphocytes and their ratio is an important condition for ensuring the normal cellular immune function of the body. If there is a significant change in their number or ratio, it indicates an imbalance in the body's immune function and indicates that the body is in a diseased state.

[0003] Lung cancer is the most common primary lung tumor. Over the past half century, the incidence and mortality of lung cancer have risen rapidly, making it one of the tumors with the highest incidence in the world. Lung cancer has an insidious onset and lacks typical clinical manifestations in the early stages of the disease. It generally presents common respiratory disease symptoms such as cough, sputum, and fever, which also makes early detection difficult, resulting in missed opportunities for surgical cure. Currently, the main treatments for lung cancer include: surgical resection, radiotherapy, drug therapy (including chemotherapy, targeted therapy, and immunotherapy), etc. For patients with advanced lung cancer, comprehensive treatment or systemic treatment is often used to achieve cure or a relatively long symptom-free survival period, among which chemotherapy is a very important treatment method. Early and clear diagnosis or prediction of the prognosis of advanced lung cancer can help improve prognosis and help clinicians adjust medication regimens in real time to maximize patient benefits.

[0004] However, while chemotherapy drugs kill tumor cells, they also have a certain impact on the human immune system. If the immune system is unbalanced and immune surveillance is low, the tumor invasiveness may increase. The lymphocytes and NK cells in the peripheral blood reflect the body's immune function. The number and ratio of these cells reflect the body's immune status. According to studies, while chemotherapy drugs kill tumor cells, they have a certain degree of cytotoxicity, which may cause adverse reactions such as bone marrow suppression, infection, anemia, etc., and the number of cells will change to a certain extent.

[0005] In 2021, Hou et al. studied the role of T lymphocytes in blood tumors (diffuse large B-cell lymphoma). The study showed that the cell counts of T lymphocyte subsets in patients with diffuse large B-cell lymphoma were lower than those in the healthy control group before treatment. Due to the toxic effects of chemotherapy drugs (cyclophosphamide, etc.), the CD3, CD4, and CD8 cell counts gradually decreased. The number of T cells recovered after 6 cycles of treatment, and the function of T cells recovered after 2 cycles of chemotherapy. The results of the study reflect that the recovery of the body's immune function is related to reasonable treatment. The goal of immunotherapy is to restore CD4 + and CD8 + T cell effector cells and memory cells respond to specific cancer antigens to trigger anti-tumor immune responses. Monitoring changes in the number and ratio of immune cells helps monitor and regulate anti-tumor responses. Studies have shown that the prognosis of malignant tumors is closely related to the composition and function of the immune system. This study suggests that peripheral CD4 + The number of T lymphocytes is associated with overall survival of lung cancer. + T lymphocytes were not related to overall survival. Patients with high levels of CD3 cell counts had longer overall survival (P < 0.05), patients with high levels of CD4 cell counts and CD4 / CD8 ratios had longer survival, and patients with high levels of CD8 cell counts had shorter disease-free survival. Chemotherapy is an important treatment for cancer tumors. However, while chemotherapy drugs kill tumor cells, they also affect the body's immune status, especially T cell-mediated cellular immunity, which plays a key role in the body's anti-tumor immune response mechanism. Therefore, regular testing of the number of T lymphocyte subsets during lung cancer chemotherapy is of great value in evaluating tumor efficacy and adjusting medication.

[0006] At present, flow cytometry is the "gold standard" for clinical T lymphocyte counting, but there are still some problems in its clinical application, such as reliance on large and expensive instruments and equipment, the need for professional technicians, and high cost of detection reagents, which is not conducive to the implementation of T lymphocyte counting projects in some areas with limited medical resources. Microscope counting is cost-effective and the equipment is simple, but the operation is complicated and the accuracy is not high. The enzyme-linked immunosorbent assay requires repeated washing and is time-consuming, which is not convenient to operate. Therefore, it is necessary to develop a simple, economical, and widely applicable method for detecting the number of T lymphocytes in some areas with relatively scarce medical resources or some special clinical application scenarios. Summary of the invention

[0007] The purpose of the present invention is to provide a kit capable of instantly and rapidly quantitatively detecting changes in the number of CD3, CD4 and CD8 cells in T lymphocyte subsets of lung cancer.

[0008] The above-mentioned purpose is achieved by the following technical solutions.

[0009] The first aspect of the present invention provides a kit for detecting T lymphocyte subsets in lung cancer, the kit comprising: a vertical flow immunochromatography box and three signal probes;

[0010] The vertical flow immunochromatography box comprises a box body, and a water-absorbing pad, a filter membrane and a detection cover which are sequentially stacked on the box body from bottom to top, and the detection cover is provided with a sample addition hole;

[0011] The three signal probes include three red latex microspheres coupled with three antibodies respectively, wherein the three antibodies are CD3 antibody, CD4 antibody and CD8 antibody respectively, and the labeled amount of the antibodies in the red latex microspheres is 11μg / 25μL to 14μg / 25μL.

[0012] In some embodiments, the monoclonal numbers of the CD3 antibody, the CD4 antibody and the CD8 antibody are SK7, SK3 and HIT8a, respectively.

[0013] In some embodiments, when the signal probe is prepared, the labeled amount of the antibody in the red latex microsphere is 12μg~13μg / 25μL; preferably, when the signal probe is prepared, the labeled amount of the antibody in the red latex microsphere is 12.4μg~12.6μg / 25μL.

[0014] In some embodiments, when the signal probe is prepared, the coupling reaction between the antibody and the red latex microspheres is carried out in a PBS labeling buffer, wherein the pH value of the PBS labeling buffer corresponding to the CD3 antibody is 7.3-7.7, the pH value of the PBS labeling buffer corresponding to the CD4 antibody is 7-7.5, and the pH value of the PBS labeling buffer corresponding to the CD8 antibody is 6.3-6.7.

[0015] In some embodiments, the method for preparing the signal probe comprises the following steps:

[0016] Adding 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide to the red latex microspheres for activation reaction to obtain activated red latex microspheres, and adding PBS labeling buffer to the activated red latex microspheres to obtain an activated microsphere suspension;

[0017] adding the antibody to the obtained activated microsphere suspension for incubation;

[0018] Adding blocking solution to the incubated solution to perform blocking reaction to obtain precipitation;

[0019] The obtained precipitate is added into the labeling solution and mixed evenly to obtain the signal probe.

[0020] In some embodiments, the labeling reconstitution solution contains 7-9% sucrose, 1-2% trehalose, 1-2% caseinate sodium, 0.3-0.7% BSA, 0.1M-0.2M pH8.5 Gly-NaOH, 0.3-0.7% ProClin300 and 3.8%-4.2% w / v PVP-K30.

[0021] In some embodiments, the filter membrane is a nitrocellulose membrane, and the pore size of the filter membrane is 4 μm to 6 μm; and / or,

[0022] The red latex microspheres are polystyrene red microspheres; and / or,

[0023] The absorbent pad is provided with two layers.

[0024] The second aspect of the present invention provides a reagent for detecting the number of T lymphocyte subsets in a sample; preferably, the use of the kit for detecting T lymphocyte subsets as described above in the preparation of a product for detecting lung cancer. The product may be a reagent, a kit, etc.

[0025] The third aspect of the present invention provides a method for detecting T lymphocyte subpopulations of lung cancer, using the above-mentioned kit for detecting T lymphocyte subpopulations, the method comprising the following steps:

[0026] The collected peripheral blood sample is processed by density gradient centrifugation to obtain a mononuclear cell precipitate, and a buffer is added to the mononuclear cell precipitate to obtain a mononuclear cell fluid;

[0027] The three signal probes are added to the mononuclear cell fluid respectively for incubation, and after incubation, they are added to the sample addition holes of the vertical flow immunochromatography box respectively for chromatography treatment, and colorimetric analysis is performed after the treatment is completed.

[0028] In some embodiments, the density gradient centrifugation method comprises the following steps:

[0029] The collected peripheral blood sample is added to the lymphocyte separation solution and centrifuged at a centrifugal force of 1200×g to 1300×g for 20min to 25min to obtain a layered solution;

[0030] The buffy coat layer in the layered solution is aspirated and centrifuged for a second time to obtain a precipitate, the centrifugal force is 500×g to 600×g, and the centrifugation is performed for 8min to 12min. The obtained precipitate is the mononuclear cell.

[0031] In some embodiments, the usage ratio of the signal probe containing the CD3 antibody to the mononuclear cell fluid is 2.3 μL to 2.7 μL: 50 μL, and the CD3 cell concentration in the mononuclear cell fluid is 1000 to 1500 cells / μL; the usage ratio of the signal probe containing the CD4 antibody to the mononuclear cell fluid is 2.3 μL to 2.7 μL: 50 μL, and the CD4 cell concentration in the mononuclear cell fluid is 1000 to 1500 cells / μL; the usage ratio of the signal probe containing the CD8 antibody to the mononuclear cell fluid is 2.6 μL to 2.8 μL: 50 μL, and the CD8 cell concentration in the mononuclear cell fluid is 500 to 800 cells / μL; and / or,

[0032] The incubation time is 9 min to 11 min.

[0033] In the present invention, the inventors found that the changes in the number of CD3, CD4 and CD8 cells during the treatment of lung cancer are valuable in the prognosis assessment of lung cancer, and specifically labeled the mononuclear cells separated by gradient density of peripheral blood using red latex microspheres modified with CD3 antibodies, CD4 antibodies and CD8 antibodies, respectively, optimized the appropriate antibodies and antibody labeling amounts, and achieved the separation and detection of specific CD3 cells, CD4 cells and CD8 cells through the designed vertical flow immunochromatography box, thereby constructing a detection method for the number of specific T lymphocyte subsets, which can achieve quantitative detection of CD3 cells, CD4 cells and CD8 cells, and can obtain accurate T lymphocyte subset count analysis, making T lymphocyte subset number monitoring low-cost and universal, and achieving the evaluation of the health status of lung cancer patients. In the process of lung cancer treatment, especially chemotherapy, the patient's own condition and the changes in the detected T lymphocyte subset cell numbers can be combined to predict the patient's efficacy, and the treatment plan can be adjusted in a timely and effective manner. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 This is a scatter plot analysis of T lymphocyte subsets in tumor patients before and after chemotherapy. (A) Comparative analysis of CD3 cell numbers; (B) Comparative analysis of CD4 cell numbers; (C) Comparative analysis of CD8 cell numbers; D. CD4 / CD8 ratio analysis.

[0035] Figure 2 This is a schematic diagram of the 3D printing synthesis device; A. Detection cover; B. Box body; C. Assembled vertical flow immunochromatography box (green is the absorbent pad, white is the filter membrane); D. Mobile phone holder black box.

[0036] Figure 3 This is a scanning electron microscope of NC membrane detection cells; where: (A) nitrocellulose membrane; (B) CD3 cells; (C) PS-CD3Ab signal probe; (D) cells combined with PS-CD3Ab signal probe.

[0037] Figure 4 This is a graph showing the potential analysis results of the microspheres before and after antibody labeling.

[0038] Figure 5 It is a bar graph and a line graph of the screening marker antibody; among them, (A) CD3 antibody capture efficiency; (B) CD4 antibody capture efficiency; (C) CD8 antibody capture efficiency. Note: SNR signal-to-noise ratio, which is positive value / negative value.

[0039] Figure 6 It is a flow cytometric analysis of T lymphocytes in PBMC; among them, (A) flow cytometric analysis of lymphocyte ratio; (B) flow cytometric analysis of CD3 cell ratio; (C) flow cytometric analysis of CD4 cell ratio; (D) flow cytometric analysis of CD8 cell ratio.

[0040] Figure 7 It is to optimize the amount of labeled antibodies; among them, (A) optimizes the amount of CD3 labeled antibodies; (B) optimizes the amount of CD4 labeled antibodies; (C) optimizes the amount of CD8 labeled antibodies.

[0041] Figure 8 It is to optimize the pH of the labeling buffer of the coupled antibody; wherein, (A) pH of the CD3 antibody labeling buffer; (B) pH of the CD4 antibody labeling buffer; (C) pH of the CD8 antibody labeling buffer.

[0042] Fig. 9 The method is to optimize the amount of signal probe added for each test; (A) optimize the amount of CD3 cell signal probe added; (B) optimize the amount of CD4 cell signal probe added; (C) optimize the amount of CD8 cell signal probe added.

[0043] Fig.10 is the incubation time for the PS-CDxAb signal probe to bind to the cells.

[0044] Fig.11 The PVP ratio of the labeled complex solution is optimized.

[0045] Fig.12 The standard curve and physical picture of PS-VFA detection of T lymphocyte subsets; (A) linear range and physical picture of CD3 cells; (B) linear range and physical picture of CD4 cells; (C) linear range and physical picture of CD8 cells.

[0046] Fig.13 This is the precision analysis of PS-VFA detection of lymphocyte counts; among them, (A) precision detection of CD3 cells; (B) precision detection of CD4 cells; (C) precision detection of CD8 cells.

[0047] Fig.14 This is a comparison between PS-VFA and flow cytometry for detecting cell numbers; (A) comparison of methods for detecting CD3 cell numbers; (B) comparison of methods for detecting CD4 cell numbers; (C) comparison of methods for detecting CD8 cell numbers.

[0048] Fig.15 It is the consistency and correlation analysis between PS-VFA and flow cytometry cell numbers; among them, (AB) Bland-Altman analysis and correlation analysis of CD3 cells; (CD) Bland-Altman and correlation analysis of CD4 cells; (EF) Bland-Altman and correlation analysis of CD8 cells. DETAILED DESCRIPTION

[0049] In order to facilitate the understanding of the present invention, the present invention will be described more fully below. The present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present invention more thorough and comprehensive.

[0050] The experimental methods in the following examples without specifying specific conditions are usually carried out under conventional conditions or conditions recommended by the manufacturers. The various commonly used chemical reagents used in the examples are all commercially available products.

[0051] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those commonly understood by those skilled in the art to which the present invention belongs. The terms used in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention. The term "and / or" used in the present invention includes any and all combinations of one or more of the related listed items.

[0052] Example 1 Correlation analysis of peripheral blood T lymphocyte count and therapeutic efficacy in lung cancer

[0053] 1.1 Research subjects and materials

[0054] 1.1.1 Research subjects

[0055] A retrospective analysis of the number of peripheral blood lymphocyte subsets before treatment and after four cycles of chemotherapy was performed in 28 patients with lung cancer who were treated for the first time in our hospital from 2019 to 2022. Inclusion criteria: ① pathological diagnosis of lung cancer; ② first treatment in our hospital, chemotherapy lasting at least four courses; ③ age > 18 years old. Exclusion criteria: ① history of other malignant tumors or serious underlying diseases; ② history of long-term use of hormone drugs; ③ history of radiotherapy during the analysis course; ④ related autoimmune diseases.

[0056] 1.1.2 Clinical data

[0057] ①Baseline data: gender, age. ②Disease and treatment: pathological type, presence or absence of metastasis, treatment method. ③Laboratory indicators: T lymphocyte subset values ​​(CD3 cell count, CD4 cell count, CD8 cell count, CD4 / CD8 ratio).

[0058] 1.1.3 Peripheral blood T lymphocyte count

[0059] Mindray Bricyte E6 flow cytometer was used to analyze the cell number of T lymphocyte subsets.

[0060] 1.1.4 Statistical analysis

[0061] SPSS26 statistical software was used for analysis, and the data were consistent with the normal distribution. The inter-group analysis was performed with two independent sample t-tests; the intra-group analysis was performed with paired t-tests; the count data were expressed as case numbers and rates, and the general data of patients were expressed with chi-square tests; P < 0.05 was considered statistically significant.

[0062] 1.2 Research Results

[0063] 1.2.1 General information of the research subjects

[0064] A total of 28 lung cancer patients were included in this study, as shown in Table 1-1, including 25 males and 3 females, with an average age of 60.11 years. A total of 28 healthy subjects were included, including 23 males and 5 females, with an average age of 57.29 years. There was no statistically significant difference in age and gender between the two groups of patients (P>0.05).

[0065] Table 1-1 Comparison of general conditions between the control group and the observation group

[0066] Control group Observation Group P-value age 57.29(51.25,60.00) 60.11(54.25,69.50) 0.115 Gender (n, %) / / 0.445 male 23(82.1％) 25(89.3％) / female 5(17.9％) 3(10.7％) /

[0067] 1.2.2 Comparison of T lymphocyte counts between patients and healthy controls before treatment

[0068] The number of T lymphocyte subsets was analyzed in the observation group before chemotherapy and in the control group after physical examination. The results are shown in Table 1-2. The number of CD3 cells, CD4 cells, and CD8 cells in the cancer patients in the observation group was lower than that in the healthy control group, and the difference was statistically significant (P < 0.05). There was no statistically significant difference in the CD4 / CD8 ratio between the two groups.

[0069] Table 1-2 Comparison of T lymphocyte subsets in the observation group before treatment and those in the healthy control group

[0070]

[0071] 1.2.3 Comparison of T lymphocyte counts before and after treatment

[0072] The results of T lymphocyte subset cell number analysis before and after chemotherapy in the observation group are shown in Table 1-3. The CD3 cell number in the observation group was 1180.29 cells / μL before chemotherapy and 1370.79 cells / μL after chemotherapy; the CD4 cell number in the observation group was 749.61 cells / μL before chemotherapy and 868.00 cells / μL after chemotherapy; the CD8 cell number in the observation group was 377.89 cells / μL before chemotherapy and 445.75 cells / μL after chemotherapy. There were statistically significant differences in the CD3, CD4, and CD8 cell numbers before and after chemotherapy. There was no statistically significant difference in the CD4 / CD8 ratio analysis before and after chemotherapy.

[0073] Table 1-3 Comparison of T lymphocyte subsets in the observation group before and after treatment

[0074]

[0075]

[0076] The scatter plot analysis of the number of T lymphocyte subsets in the observation group before and after chemotherapy showed that Figure 1 As shown in the data, the number of CD3 cells, CD4 cells, and CD8 cells in the observation group increased after 4 courses of chemotherapy compared with before chemotherapy (P<0.05), and there was no significant difference in CD4 / CD8 levels (P>0.05). This suggests that chemotherapy can increase the number of immune cells and restore immune function to a certain extent. Monitoring the number of T lymphocyte subsets is helpful for evaluating the body's immune function and the prognosis of chemotherapy.

[0077] Example 2 Construction of T lymphocyte subset detection method

[0078] 2.1 Experimental reagents are as follows Table 2.1

[0079] 2.1 Experimental Reagents

[0080]

[0081]

[0082] 2.2 Preparation of main solution

[0083] (1) According to conventional methods, phosphate buffer (PBS, 1×) 0.015 mol / L pH 7.4, 1 mol / L NaOH solution, 0.1 mol / L, pH = 6.0 MES buffer, 10 mg / mL EDC solution (prepared and used immediately), 50 mg / mL NHS solution (prepared and used immediately), 10% ProClin TM 300 preservatives, 10% (w / v) BSA solution, 0.1 mol / L glycine-NaOH buffer at pH=8.5.

[0084] (2) The preparation of the labeled solution is shown in Table 2.2

[0085] 2.2 Labeling solution components

[0086] Reagent name concentration quantity unit sucrose 8％ 2 g Trehalose 2％ 0.5 g BSA 0.5％ 0.125 g Casein Sodium Salt 1％ 0.25 g <![CDATA[10%ProClin TM 300]]> 0.5％ 0.125 mL 0.1M pH8.5 Gly-NaOH - 25 mL PVP-K30 4%(W / V) 1.00 g

[0087] The above prepared solutions, the group activator EDC and NHS solution must be used immediately and cannot be stored for a long time; except for 1X PBS, the rest should be stored in a 4℃ refrigerator.

[0088] 2.3 Experimental process

[0089] 2.3.1 Electron microscopic analysis of filter membrane

[0090] Nitrocellulose membrane was selected as the filter membrane in this study, PBS was used as the negative control and cell dilution solution, and CD3 cells with a concentration of 2000 cells / μL were selected as positive samples. A total of 4 groups of experiments were set up, namely ①PBS, ②PBS+PSCD3Ab signal probe, ③CD3 cells, and ④CD3 cells+PSCD3Ab signal probe. After incubation, 100μL of sample was added to the test box and washed with 100μL of 1X PBS. Dry at room temperature for 15min, add 1 drop of scanning electron microscope fixative, and place it in a 4℃ refrigerator for 2.5h. Take out the excess fixative and absorb it with absorbent paper, and wash it 3 times with 1X PBS buffer. Prepare different concentrations of alcohol with pure water and anhydrous alcohol, and dehydrate the cells from low to high concentrations: 30%, 50%, 70%, 80%, 90%, 95%, and 100%. After dehydration, the sample was dried and vacuum-dry. Finally, the sample was sprayed with gold and then examined by microscope.

[0091] 2.3.2 Potential analysis of labeled antibodies

[0092] Biolegend's CD3 antibody and Color Rich red latex microspheres (polystyrene red microspheres, PS microspheres) were selected for potential analysis before and after labeling and grouped into CD3 antibody, non-activated PS microspheres, activated PS microspheres, and antibody-labeled PS microspheres.

[0093] 2.3.3 Screening of labeled antibodies

[0094] Three CD3 antibodies from Biolegend were selected, with clone numbers SK7, OKT3, and UCTH1; three CD4 antibodies from Biolegend were selected, with clone numbers RPAT4, SK3, and OKT4; and three CD8 antibodies from Biolegend were selected, with clone numbers SK1, RPAT8, and HIT8a. The above 9 antibodies were selected to couple with Color Rich red latex microspheres (0.41 μm) to prepare labeled probes, and the cell concentration was measured after labeling negative samples and positive samples, and the most suitable labeled antibody was selected according to the signal-to-noise ratio of the positive / negative signal.

[0095] 2.3.4 Preparation of signal probe

[0096] The most suitable CD3 antibody, CD4 antibody, and CD8 antibody screened out above were respectively used with red latex microspheres modified with carboxyl groups to prepare labeled probes through microsphere immunolabeling antibody technology. The carboxyl groups on the surface of the red latex microspheres reacted with the amino groups on the antibodies under the activation of EDC / NHS to form a stable amide bond. The specific steps are as follows:

[0097] (1) Cleaning microspheres:

[0098] Add 50 μL (4% solid) red latex microspheres to 950 μL PBS (1X pH 7.4), vortex and mix well, place in a handheld centrifuge and shake gently for 2-5 seconds to allow the solution on the centrifuge tube cover to flow to the bottom; take out and place in a low-temperature high-speed centrifuge for centrifugation for 30 minutes, 4°C, 18880×g, and discard the supernatant after centrifugation. Add 1mL 0.1mol / L MES (pH 6.0) solution to the above precipitate, resuspend and mix by ultrasound (power 5%, start 2s, stop 3s), repeat 10 times until the precipitate is evenly dispersed into a solution.

[0099] (2) Activated microspheres:

[0100] Add 10 μL of EDC activation solution (concentration of 10 mg / mL) and 10 μL of NHS solution (concentration of 50 mg / mL) to the above solution, vortex mix, and rotate and activate on a disc rotating mixer at room temperature for 30 minutes. Then place in a low-temperature high-speed centrifuge, and the centrifugation conditions are the same as step (1). Discard the supernatant, add 1 mL of PBS buffer to the precipitate, and resuspend and mix by ultrasound. The ultrasound mixing is the same as step (1).

[0101] (3) Microsphere-coupled antibodies:

[0102] Add 50 μL (0.5 mg / mL) of SK7 antibody, SK3 antibody or HIT8a antibody to the above suspension, wrap with tin foil to avoid light and incubate with rotation for 3 hours at room temperature.

[0103] (4) Closure:

[0104] Add 10 μL of 10% BSA blocking solution to the above solution, rotate at room temperature in the dark for 30 min, and centrifuge after the blocking reaction is complete. The centrifugation conditions are the same as those in step (1).

[0105] (5) Redissolution:

[0106] The supernatant was discarded, and 250 μL of labeled complex solution (8% sucrose-2% trehalose-1% sodium caseinate-0.5% BSA-0.1 mol / L Gly-NaOH pH 8.5-0.5% ProClin300) was added to the precipitate, and the precipitate was thoroughly mixed by ultrasound to successfully prepare the signal probes of T lymphocyte subsets labeled with red microspheres (PS-CD3Ab, PS-CD4Ab, PS-CD8Ab), which were stored in a 4°C refrigerator away from light for future use.

[0107] 2.3.5 Preparation of vertical flow immunochromatography cassette

[0108] like Figure 2 As shown in the figure, the design of the vertical flow immunochromatography box and the black box of the mobile phone holder is modeled by SOLIDWORKS, and the model is printed by a 3D printer before use. The box body is a plastic model printed by a 3D thermal printer, with specifications of 44.2mm in length, 29.2mm in width, 5mm in height, and 4mm in thickness. The detection cover is a resin model printed by a nova photosensitive printer, with specifications of 45.5mm in length, 25.2mm in width, 4mm in height, and 3mm in thickness. There is a sample addition hole in the center of the detection cover, which gradually decreases from top to bottom. The diameter of the color development area is 2mm, and the height of the protrusion to the inside is 1.6mm. Several small triangles are designed on the edge to fix the filter membrane and the absorbent pad. The absorbent pad is a Shanghai Jie Ning H2 strong absorbent pad, and the specifications used in the experiment are 25-28mm in length, 24mm in width, and 1.8mm in thickness. The filter membrane is whatman's nitrocellulose membrane AE98 hydrophilic filter paper with an average pore size of 5μm. The specifications used in this study are 22mm in length and 12.5mm in width.

[0109] Assembly of vertical flow immunochromatography box: Take 2 layers of H2 absorbent pads and place them at the bottom layer inside the box body. The double-layer absorbent pads strengthen the capillary attraction; the filter membrane is placed directly above the center of the absorbent pad; the detection cover covers the top of the filter membrane, and the middle of the sample injection hole is aligned with the center of the filter membrane. Pressurize the detection cover to fit closely with the box body. The raised height inside the detection cover fixes the filter membrane and the absorbent pad to prevent the sample to be tested from overflowing and prevent the waste liquid from flowing back.

[0110] 2.3.6 Preparation of standard lymphocyte fluid

[0111] Material preparation: Samples include fresh EDTA anticoagulated blood from healthy subjects, TBD lymphocyte separation fluid from Tianjin Haoyang, and 0.9% sterile saline. The specific steps are as follows:

[0112] (1) Take 2 mL of EDTA-anticoagulated whole blood (try to use fresh blood that is 2-6 hours old; blood that has been stored for a long time is prone to hemolysis), add 2 mL of normal saline, and use a rubber-tipped pipette to gently pipette and mix to make a whole blood diluent (the ratio of whole blood to normal saline is 1:1).

[0113] (2) Take 5 mL of lymphocyte separation solution and add it to the bottom of a 15 mL centrifuge tube. Tilt the centrifuge tube and use a rubber-tipped pipette to draw up the whole blood diluent and slowly add it to the upper layer of the lymphocyte separation solution. Make sure the layers are clearly separated. Be careful to tilt the tube to avoid mixing.

[0114] (3) Centrifugation: room temperature, 1250×g for 22 min.

[0115] (4) After centrifugation, you can see that there are four distinct layers in the centrifuge tube, from top to bottom: plasma and saline mixed layer, mononuclear cell buffy coat layer, TBD lymphocyte separation fluid layer, granulocyte and red blood cell layer. Carefully aspirate the buffy coat layer with a pipette and place it in a new 15 mL centrifuge tube. Be careful not to blow down the granulocyte / red blood cell layer at the bottom during the aspiration process. Add 1X PBS buffer to the new centrifuge tube to make up to 10 mL and gently pipette to mix.

[0116] (5) Second centrifugation: room temperature, 560×g for 10 min.

[0117] (6) After centrifugation, carefully remove the centrifuge tube, open the lid and tilt it 90° to pour out the supernatant, retaining the cell pellet. If there is still liquid, be careful not to pour it out again; add 6 mL of 1X PBS buffer to the pellet and gently pipette to mix. Repeat step (5) and centrifuge again.

[0118] (7) After centrifugation, tilt the tube 90° to discard the supernatant, add an appropriate amount of PBS buffer to the precipitate, gently pipette to mix and resuspend the cells to complete the preparation of mononuclear cell solution. Store in a 4°C refrigerator away from light and use within 1 week.

[0119] 2.3.7 Flow cytometry to detect T lymphocyte count

[0120] Take out the mononuclear cell solution prepared above from the 4°C refrigerator; BD Trucount absolute cell counting tube (number of microbeads: 47600); 4 kinds of flow cytometry fluorescent antibodies, namely APC-anti-human CD 45 Antibody, FITC-anti-human CD3 antibody, Pacific Blue-anti-human CD4 antibody, PerCP-anti-human CD8 antibody, take out for about 15 minutes to balance and restore to room temperature for use. Take 50μL of cell fluid into an absolute counting tube, add 5μL APC-anti-CD 45 Antibodies: 5 μL FITC-anti-CD3 antibody, 5 μL PacificBlue-anti-CD4 antibody, 5 μL PerCP-anti-CD8 antibody, mix well and incubate at room temperature in the dark for 15 min. After incubation, add 150 μL 1X PBS buffer to the absolute counting tube, mix well and test on the machine. For flow cytometry analysis, select the APC channel to filter out some mixed cells, and set the gate based on white blood cells (CD 45-APC fluorescence), select the lymphocyte population and the known microbead group (Beads) in the upper right corner respectively; set up the CD3 cell gate in the lymphocyte population, respectively CD 45 + CD3 - , CD 45 + CD3 + There are two cell groups in total. Circle the latter cell group. Continue to use the CD3 cell group to set up the CD4 cell group, which are CD 45 + CD3 - , CD 45 + CD3 + CD4 - , CD 45 + CD3 + CD4 + Three cell populations, select CD 45 + CD3 + CD4 + The cell population is the proportion of CD4 cells; CD3 cell population is used to establish CD8 cell population, which are CD 45 + CD3 - , CD 45 + CD3 + CD8 - , CD 45 + CD3 + CD8 + Three cell populations, select CD 45 + CD3 + CD8 + The cell population is the proportion of CD8 cells. After the gate is closed, the specific number of cells in the T lymphocyte subsets is calculated by combining the Beads population in the white blood cell gate and the known number of Beads. The conversion formula is as follows:

[0121]

[0122] Through the above steps, the cell concentrations (cells / μL) of CD3 cells, CD4 cells, and CD8 cells can be obtained.

[0123] 2.3.8 Optimization of experimental conditions for labeling antibodies

[0124] 2.3.8.1 Optimization of labeling antibody quantity

[0125] Different masses of CD3 or CD4 or CD8 antibodies (all at a concentration of 0.5 mg / mL) were added to a certain volume (475 μL) of activated red latex microsphere suspension, and the remaining steps were the same as the preparation of the aforementioned signal probes. The PS-CD3Ab signal probe, PS-CD4Ab signal probe, and PS-CD8Ab signal probe were labeled and stored in a 4°C refrigerator for later use. The prepared lymphocyte fluid was diluted with 1X PBS buffer to prepare negative and positive group samples, and the diluted cell fluids with CD3 cell concentrations of 0 and 1300 cells / μL; the diluted cell fluids with CD4 cell concentrations of 0 and 1200 cells / μL; and the diluted cell fluids with CD8 cell concentrations of 0 and 800 cells / μL were prepared respectively. CD3 cells were divided into 10 groups according to the amount of CD3 labeled antibody, 5 negative control groups and 5 positive samples. 300 μL of cell fluid sample was taken from each group, 15 μL of PS-CD3Ab labeled antibody was added and mixed, and then incubated at room temperature for 15 min. AE98 filter membrane and H2 absorbent pad were used to prepare the test kit. After incubation, 25 μL × 2 samples were added for each test, and the liquid was allowed to flow through completely for about 1-5 min. 1X PBS 100 μL was added for each test to wash and remove the excess unbound background interference color. According to the RGB analysis of the smartphone Color Picker application, each test has different R values, G values, and B values. The colorimetric analysis of this study is red, and the G value changes significantly, so the R / G value is selected as the analysis value. The R / G values ​​of the five groups of data with different amounts of CD3 labeled antibodies were analyzed. The signal value of the negative group is the background interference signal, and the lower the better. Therefore, the highest signal-to-noise ratio of the positive value / negative value is the optimal ratio of colored microspheres to labeled antibodies. The experimental process of CD4 and CD8 labeled antibodies is the same as that of CD3 antibody labeling.

[0126] 2.3.8.2 Optimization of pH conditions of labeling buffer

[0127] Prepare 5 different pH 1X PBS labeling buffers in advance, namely 6.0, 6.5, 7.0, 7.5, and 8.0. Label PS-CD3Ab or PS-CD4Ab or PS-CD8Ab, first wash the microspheres: add 25μL red latex microspheres to 475μL 1X PBS (pH 7.4), vortex to mix, place in a handheld centrifuge and shake gently for 2-5s, take out and place in a low-temperature high-speed centrifuge for centrifugation for 30min, 4℃, 18880×g, and discard the supernatant after centrifugation.

[0128] 500 μL of 1X PBS labeling buffer with 5 different pH values ​​was added to the precipitate, and ultrasound (5% power, 2s on, 3s off) was repeated 10 times until the precipitate was evenly dispersed into an activated red latex microsphere solution. 12.5 μg of CD3 or CD4 or CD8 antibody was added to the above activated microsphere suspension, mixed and incubated in dark for 3 hours; the subsequent labeling steps were the same as the preparation of the above signal probes; the labeling of PS-CD3Ab signal probe, PS-CD4Ab signal probe, and PS-CD8Ab signal probe was completed, and stored in a 4°C refrigerator for standby use. The prepared lymphocyte fluid was diluted with 1X PBS buffer to prepare negative group (cell concentration was 0 cells / μL) and positive group samples, and the CD3 cell concentration was 1300 cells / μL, the CD4 cell concentration was 1200 cells / μL, and the CD8 cell concentration was 800 cells / μL, respectively.

[0129] According to the pH of the labeling buffer, negative group, and positive group, the samples were divided into 5 groups of negative controls and 5 groups of positive samples. Each group took 300 μL of sample, added 15 μL of PS-CDxAb signal probe, mixed and incubated at room temperature for 15 min. Prepare the test kit with AE98 filter membrane and H2 absorbent pad. Add 25 μL × 2 samples for each test, and wash with 1X PBS 100 μL to remove excess unbound background interference colors. RGB analysis was performed according to the smartphone Color Picker application, and the R / G value was selected as the analysis value. The highest signal-to-noise ratio of the positive value / negative value was taken as the optimal antibody labeling buffer pH. According to the experimental results, the most suitable PS-CD3Ab signal probe optimal pH, PS-CD4Ab signal probe optimal pH, and PS-CD8Ab signal probe optimal pH were selected.

[0130] 2.3.8.3 Optimization of signal probe addition conditions

[0131] According to the above experimental conditions, the labeled antibody amount of CD3, CD4, and CD8 was 12.5 μg / 25 μL microspheres, the optimal labeling buffer pH was 7.5, and the antibody was re-labeled. Lymphocyte dilution was prepared, and the CD3 cell concentration of the negative group (cell concentration was 0 cells / μL) and the CD4 cell concentration of the positive group samples was 1300 cells / μL, 1200 cells / μL, and 600 cells / μL.

[0132] Take 10 1.5mL EP tubes and number them 1-10. Add 300μL of PBS to tubes 1-5 and 300μL of sample to tubes 6-10. Add 2μL / test of PS-CD3Ab signal probe to EP tubes 1 and 6, 2.25μL / test of PS-CD3Ab signal probe to EP tubes 2 and 7, 2.5μL / test of PS-CD3Ab signal probe to EP tubes 3 and 8, 2.75μL / test of PS-CD3Ab signal probe to EP tubes 4 and 9, and 3μL / test of PS-CD3Ab signal probe to EP tubes 5 and 10. Mix well and incubate at room temperature for 15min. Prepare the test kit by taking AE98 filter membrane and H2 absorbent pad. Add 25μL×2 samples for each test and wash with 100μL of 1X PBS. Perform RGB analysis according to the smartphone ColorPicker application and select the R / G value as the analysis value. The highest signal-to-noise ratio of positive value / negative value is taken as the optimal signal probe addition amount for CD3 cells. The experimental operation steps are the same, and the optimal signal probe addition amount for CD4 and CD8 cells is selected according to the experimental results.

[0133] 2.3.8.4 Optimization of reaction time conditions

[0134] Prepare lymphocyte dilution solution, negative group (cell concentration is 0 cells / μL), positive group samples CD4 cell concentration is 800 cells / μL. Take 4 negative groups and 4 positive groups, each with 300 μL sample volume, add 15 μL PS-CD4Ab signal probe to each group, and incubate for 5 min, 10 min, 15 min, 20 min respectively. Prepare the detection box with AE98 filter membrane and H2 absorbent pad. After incubation, add 25 μL×2 and wash with 1X PBS100 μL. Perform RGB analysis according to the smartphone Color Picker application, and select R / G value as the analysis value. The highest signal-to-noise ratio of positive value / negative value is the optimal incubation time for CD4 cells.

[0135] 2.3.8.5 Optimization of the PVP ratio in the labeled reconstituted solution

[0136] Prepare the basic labeling solution, add different mass / volume ratios of PVP-K30 powder, the ratios are 0%, 0.5%, 1%, 2%, and 4%. After preparing the signal probe, resuspend it with the labeling solution with different PVP ratios by ultrasonication for subsequent negative control and positive sample detection.

[0137] 2.1.5 Analytical performance of PS-VFA (a method for detecting the number of T lymphocyte subsets)

[0138] 2.1.5.1 Preparation of signal probe after optimization conditions

[0139] According to the above optimized experimental conditions, the amount of labeled antibody is 12.5μg / 25μL microspheres, the pH of the labeling buffer is 7.5, and the antibody is re-labeled as follows:

[0140] 1) Wash the microspheres: Take three 1.5 mL centrifuge tubes and number them ①②③ in sequence. Add 475 μL PBS (1X pH 7.4) and 25 μL red polystyrene microspheres to the 1.5 mL centrifuge tubes, vortex and mix well, then centrifuge in a low-temperature high-speed centrifuge for 30 min at 4°C and 18880×g. Discard the supernatant after centrifugation. Add 0.5 mL 0.1 mol / L MES (pH 6.0) solution to the above precipitate, mix thoroughly with ultrasound, and evenly disperse the precipitate into an activated microsphere solution.

[0141] 2) Activation of microspheres: Add 5 μL EDC (concentration of 10 mg / mL) and 5 μL NHS (concentration of 50 mg / mL) to each tube, vortex to mix, and activate by rotation at room temperature for 30 minutes. Then place in a low-temperature high-speed centrifuge and centrifuge for 30 minutes at 4°C and 18880×g. Discard the supernatant, add 1 mL 1X PBS (pH 7.5) buffer to the precipitate and mix thoroughly by ultrasound.

[0142] 3) Microsphere-coupled antibody: add 12.5 μg CD3-SK7 antibody to ①, add 12.5 μg CD4-SK3 antibody to ②, add 12.5 μg CD8-HIT8a antibody to ③, and incubate with rotation at room temperature in the dark for 3 hours.

[0143] 4) Blocking: Add 5 μL of 10% BSA blocking solution to each tube, rotate at room temperature in the dark for 30 minutes, and centrifuge after blocking.

[0144] 5) Redissolution: discard the supernatant, add 250 μL of labeling solution (8% sucrose-2% trehalose-1% sodium caseinate-0.5% BSA-0.1 mol / L Gly-NaOH pH 8.5-0.5% ProClin300) to each tube of precipitate, and thoroughly mix the precipitate by ultrasonication to successfully prepare the optimized red microsphere signal probe (PS-CD3Ab, PS-CD4Ab, PS-CD8Ab), and store in a refrigerator at 4°C away from light.

[0145] 2.1.5.2 Establishment of the linear range of PS-VFA detection of CD3 lymphocyte count

[0146] Prepare lymphocyte dilution solution. The CD3 cell concentration of the cell stock solution was 2767 cells / μL by flow cytometry. Dilute with 1X PBS to CD3 cell concentrations of 0, 50, 100, 200, 400, 600, 800, 1000, and 1400 cells / μL. Take 9 clean 1.5mL EP tubes numbered 0-8, add 300μL of samples with different CD3 cell concentrations (from low to high), add 15μL PS-CD3Ab signal probe to each group, mix gently, and incubate at room temperature in the dark for 10 minutes. Prepare the detection box with AE98 filter membrane and H2 absorbent pad. After incubation, add 50μL of sample and wash with 1X PBS100μL. Within 10 minutes after the reaction, under the illumination of the fixed light source in the black box of the mobile phone holder, use the smartphone vivo X90 to take photos and record the experimental results at the conditions of sensitivity ISO 200, shutter speed 1 / 60s, and shooting distance 10cm. According to the smartphone Color Picker application for RGB analysis, the Δ(G+B) value is selected as the analysis value, that is, the difference between (G+B) and (G0+B0) for each test, which can accurately reflect the signal value added by the experiment.

[0147] 2.1.5.3 Establishment of the linear range of PS-VFA detection of CD4 lymphocyte count

[0148] Prepare lymphocyte dilution solution. The CD4 cell concentration of the cell stock solution was 1520 cells / μL by flow cytometry. Use 1X PBS to dilute the CD4 cell concentration to 0, 50, 100, 200, 400, 600, 800, 1000, 1200, and 1400 cells / μL. Take 10 clean 1.5mL EP tubes numbered 0-9, add 300μL of samples with different CD4 cell concentrations (from low to high), add 15μL PS-CD4Ab signal probe to each group, mix gently, and incubate at room temperature in the dark for 10 minutes. Prepare the detection box with AE98 filter membrane and H2 absorbent pad. After incubation, add 50μL of sample and wash with 1X PBS100μL.

[0149] 2.1.5.4 Establishment of the linear range of PS-VFA detection of CD8 lymphocyte count

[0150] Prepare lymphocyte dilution solution, and determine the CD8 cell concentration of the cell stock solution by flow cytometry to be 1021 cells / μL. Use 1X PBS to dilute the CD8 cell concentration to 0, 25, 50, 100, 200, 400, 600, and 800 cells / μL. Take 8 clean 1.5mL EP tubes numbered 0-7, add 300μL of samples with different CD8 cell concentrations (from low to high), add 15μL PS-CD8Ab signal probe to each group, mix gently, and incubate at room temperature in the dark for 10 minutes. Prepare the detection box with AE98 filter membrane and H2 absorbent pad. After incubation, add 50μL of sample and wash with 1X PBS100μL.

[0151] 2.1.5.5 Precision evaluation

[0152] Prepare low, medium and high concentrations of lymphocyte fluid, and determine the concentration by flow cytometry. Take 4 1.5mL EP tubes numbered 0-3, add 600μL of negative, low, medium and high concentration cell fluid samples respectively, add 30μL PS-CD3Ab signal probe to each tube, incubate at room temperature in the dark for 10 minutes, add 50μL, and wash with 100μL PBS. Repeat the test 10 times for each concentration. Take a photo with a mobile phone to record the results and read the RGB analysis results through Color Picker, using the Δ(G+B) value as the analysis value.

[0153] 2.1.5.6 Methodological comparison, consistency and correlation analysis

[0154] A total of 28 healthy people and patients with advanced lung cancer in our hospital in December 2023 were selected, and the remaining blood samples after the patients completed the physical examination or disease test were used for methodological comparative analysis. Take 1mL of EDTA anticoagulated blood to extract mononuclear cell fluid according to the above steps, store it at 4℃ after extraction, and use it as soon as possible; take 50μL of cell fluid and perform flow cytometry after fluorescent antibody labeling to determine the concentration of cells to be tested. Each sample was labeled with a signal probe, incubated, added, and the results were collected. The PS-CD3Ab signal probe was 2.5μL / test, the PS-CD4Ab signal probe was 2.5μL / test, and the PS-CD8Ab signal probe was 2.75μL / test. The results were recorded by mobile phone photography and the RGB analysis results were read by Color Picker. The Δ(G+B) value was used as the analysis value, and a bar graph, Bland-Altman consistency analysis, and correlation analysis were performed.

[0155] 2.2 Experimental Results

[0156] 2.2.1 Electron microscopic analysis of filter membrane

[0157] Figure 3This is a scanning electron microscope characterization of the negative control and positive samples added to the nitrocellulose membrane AE98. Figure 3 A is the PBS negative control, the nitrocellulose membrane has a smooth surface and a relatively uniform pore size, and no cells or microspheres are observed; Figure 3 B is the addition of unlabeled CD3 cell solution, and it can be seen that the pores are evenly filled, which is obviously caused by the cells being blocked; Figure 3 C only has the PSCD3Ab signal probe, and a small number of microspheres with small diameters, uniformity, and regular round shapes can be seen; Figure 3 D is the complex of CD3 cell fluid and PSCD3Ab signal probe labeling. It can be seen that the pores are obviously covered with round or quasi-round cells, and some cells are connected to one or several microspheres, indicating that the microspheres are successfully combined with the cells and are intercepted on the filter membrane surface.

[0158] 2.2.2 Potential analysis of labeled antibodies

[0159] Figure 4 This is the potential analysis before and after CD3 antibody was coupled to PS microspheres. It can be seen that the average potential of the antibody is -11.4mV, the average potential of the unactivated PS microspheres is -67.6mV, the average potential of the activated PS microspheres is -70.7mV, and the average potential of the PS microspheres after antibody labeling is -41.3mV, indicating that CD3 antibody was successfully coupled to PS microspheres.

[0160] 2.2.3 Screening of labeled antibodies

[0161] Figure 5 To screen the results of antibodies with different clone numbers, the binding efficiency of antibodies to cells was determined based on the capture efficiency. Figure 5 A is the capture efficiency of three CD3 antibodies, whose clone numbers are SK7, OKT3, and UCTH1. The results show that the CD3 antibody with clone number SK7 has the highest capture efficiency, and the signal-to-noise ratio of the positive / negative value is 2.1, so SK7 is selected as the labeled antibody for the subsequent signal probe. Figure 5 B is the capture efficiency of three CD4 antibodies, namely RPAT4, SK3, and OKT4. The results show that the CD4 antibody with clone number SK3 has the highest capture efficiency, and the signal-to-noise ratio of the positive / negative value is 3.7, so SK3 is selected as the labeled antibody for the subsequent signal probe. Figure 5 C is the capture efficiency of three CD8 antibodies, namely SK1, RPAT8, and HIT8a. The results show that the CD8 antibody with clone number HIT8a has the highest capture efficiency, and the signal-to-noise ratio of the positive / negative value is 4.9, so HIT8a is selected as the labeling antibody for the subsequent signal probe.

[0162] 2.2.4 Detection of T lymphocyte count in PBMC by flow cytometry

[0163] Flow cytometry analysis of T lymphocyte counts, such as Figure 6 As shown in the figure, the threshold value is used to select the APC channel to filter out some mixed cells, and the gate is set as white blood cells (CD 45 -APC fluorescence), select the lymphocyte population and the known microbead population (Beads) in the upper right corner respectively; set up a CD3 cell gate in the lymphocyte population and circle the CD 45 + CD3 + Cell group; continue to set up the CD4 cell group with the CD3 cell group, circle the CD 45 + CD3 + CD4 + Cell group; Use the CD3 cell group to set up the CD8 cell group, circle the CD in the upper right corner 45 + CD3 + CD8 + After the gating is completed, the specific number of cells in the T lymphocyte subsets is calculated by combining the beads group in the leukocyte gate and the known number of beads.

[0164] 2.2.5 Optimization of experimental conditions for labeling antibodies

[0165] 2.2.5.1 Optimization of the amount of labeled antibody

[0166] Figure 7 To optimize the results of the labeled antibody amount, the coupling efficiency of the antibody and the microspheres was determined by the signal-to-noise ratio. Different amounts of CDx antibodies were added to 25 μL red latex microspheres, namely 5 μg, 7.5 μg, 10 μg, 12.5 μg, and 15 μg. The analysis results showed that the coupling efficiency of 12.5 μg of CD3, CD4, and CD8 antibodies added to 25 μL red polystyrene microspheres was the highest.

[0167] 2.2.5.2 Optimization of pH conditions of labeling buffer

[0168] Figure 8 To optimize the pH value of the labeling buffer, the optimal pH for antibody and microsphere labeling was determined by the signal-to-noise ratio. Different antibodies have the most suitable coupling pH, so this study selected five common pH PBS buffers, namely 6.0, 6.5, 7.0, 7.5, and 8.0. The analysis results showed that the optimal labeling buffer pH for CD3 and CD4 antibodies was 7.5, which was close to the pH value of the buffer recommended by the manufacturer; the optimal labeling buffer pH for CD8 antibodies was 6.5.

[0169] 2.2.5.3 Optimization of signal probe addition conditions

[0170] As the amount of signal probe added increases, the background interference signal of the negative group increases accordingly, so it is necessary to select the optimal amount of signal probe added for each test. Fig. 9 In order to optimize the result of adding the signal probe for each test, the greater the signal-to-noise ratio, the greater the signal difference between the positive sample and the negative group. In this study, the addition amount of each signal probe was selected as 2.0, 2.25, 2.5, 2.75, and 3.0 μL (12.5 μg CDx antibody / 25 μL polystyrene microspheres), where the sample volume for each test was 50 μL, the cell concentration of the negative group was 0 cells / μL, and the CD3 cell concentration of the positive group sample was 1300 cells / μL, the CD4 cell concentration was 1200 cells / μL, and the CD8 cell concentration was 600 cells / μL.

[0171] Analysis results: The best signal probe addition amount for CD3 cells is 2.5 μL / test, the best signal probe addition amount for CD4 cells is 2.5 μL / test, and the best signal probe addition amount for CD8 cells is 2.75 μL / test.

[0172] 2.2.5.4 Optimization of reaction time conditions

[0173] It takes a certain amount of time for antigens and antibodies to bind, but the longer the binding time is not necessarily better. Fig.10 To optimize the incubation time of PS-CDxAb signal probe and cells, this study selected incubation time of 5, 10, 15, and 20 min, and determined the optimal incubation time based on the signal-to-noise ratio. Fig.10 As shown, the analysis results show that the signal-to-noise ratio is the highest when the signal probe is incubated with cells for 10 minutes, which is the optimal incubation time. The subsequent incubation time for labeling target cells is 10 minutes.

[0174] 2.2.5.5 Optimization of the PVP ratio in the labeled reconstituted solution

[0175] The labeled signal probe was resuspended and stored with different ratios (w / v) of PVP-K30 labeled solution. Fig.11 As shown in the figure, the intra-group error of the test was the smallest when the PVP ratio was 0% and 4%, indicating that the stability of this labeled antibody was the best; the negative control signal values ​​of the two were almost the same, and the signal value of the positive sample increased significantly when the PVP ratio was 4%, which could clearly distinguish the number of cells. Therefore, the labeling solution formula with a PVP ratio of 4% (w / v) was selected to prepare and store the signal probe.

[0176] 2.2.6 Analytical performance of PS-VFA

[0177] 2.2.6.1 Establishment of standard curve for PS-VFA detection of T lymphocyte subsets

[0178] Mononuclear cell fluid was prepared and diluted with PBS to form a series of cell fluids with a concentration gradient. The absolute number of CD3 cells, CD4 cells, and CD8 cells was detected according to the above flow cytometry steps. The results were read and analyzed using a homemade detection kit and a smartphone. The cell dilution concentration was used as the horizontal axis and Δ(G+B)valve was used as the vertical axis. Linear fitting was performed and graphed using GraphPad Prism software. The results are shown in the figure. Fig.12 As shown: Fig.12 A is the standard curve of CD3 cell detection by PS-VFA, the linear range is 50~1400 cells / μL, the lowest detection concentration is 50 cells / μL, the correlation R 2 =0.9685; Fig.12 B is the standard curve of CD4 cell detection of PS-VFA, the linear range is 100~1400 cells / μL, the lowest detection concentration is 100 cells / μL, the correlation R 2 =0.9754; Fig.12 C is the standard curve of CD8 cell detection by PS-VFA, the linear range is 25~800 cells / μL, the lowest detection concentration is 25 cells / μL, and the correlation R 2 =0.9660.

[0179] 2.2.6.2 Precision evaluation

[0180] Mononuclear cell fluid was prepared, and flow cytometry was used to determine the low, medium, and high concentrations of cells. In order to test the precision of the methodology established in this study, each concentration was tested 10 times. GraphPad Prism software was used for graphical analysis, with the cell concentration level as the horizontal axis and Δ(G+B)valve as the vertical axis to make a bar graph; SPSS26 software was used for normality test and intra-group coefficient of variation (CV) analysis. The results are shown in the figure. Fig.13 As shown, Fig.13 A is the precision analysis of CD3 cells, CV < 4.5%; Fig.13 B is the precision analysis of CD4 cells, CV < 5%; Fig.13 C is the precision analysis of CD8 cells, CV < 6%, and CV values ​​are all < 15%, which can meet the detection needs of the industry.

[0181] 2.2.6.3 Methodological comparison

[0182] Eleven patients with advanced lung cancer (No. 1-11) and 17 healthy subjects (No. 12-28) from Guangdong Second People's Hospital were selected for PS-VFA analysis and flow cytometry analysis of 28 EDTA-anticoagulated whole blood samples to compare the difference in cell number detected by the two methods. The bar graph was made with the sample number as the horizontal axis and the cell concentration as the vertical axis, and each sample was repeated 3 times. The results are shown in the figure. Fig.14 The results of T lymphocyte subset counting by flow cytometry were basically consistent with those by PS-VFA. Fig.14 Comparison of T lymphocyte counts in lung cancer patients 1-11 Fig.14 The levels of No. 12-28 in healthy people are reduced, and the immune function of lung cancer patients suffers a certain degree of damage.

[0183] 2.2.6.4 Consistency and correlation analysis of PS-VFA

[0184] EDTA-anticoagulated whole blood from 11 patients with advanced lung cancer and 17 healthy subjects at Guangdong Second People's Hospital was analyzed by PS-VFA and flow cytometry, and the consistency of the two methods was compared by Bland-Altman. Flow cytometry was used as the gold standard as the horizontal axis of the correlation analysis, and the number of cells detected by PS-VFA was used as the vertical axis. Fig.15 As shown:

[0185] Fig.15 A shows that the average deviation of the two detection methods of CD3 cells is -52.07 cells / μL, and the 95% confidence interval is -253.3 to 149.1 cells / μL. Almost all sample analyses are within the 95% confidence interval; Fig.15 B shows that the two methods have a good correlation (R2=0.9021), indicating that the PS-VFA method of this study can be used for the analysis of CD3 cell counts in clinical samples.

[0186] Fig.15 C shows that the average deviation of the two detection methods of CD4 cells is 91.36 cells / μL, and the 95% confidence interval is -77.53 to 260.3 cells / μL. The sample analysis is basically within the 95% confidence interval; Fig.15 D shows that the two methods have a good correlation (R2=0.8852), indicating that the PS-VFA method of this study can be used to analyze the number of CD4 cells in clinical samples.

[0187] Fig.15 E shows that the average deviation of the two detection methods of CD8 cells is -9.32 cells / μL, and the 95% confidence interval is -75.92 to 57.29 cells / μL. Almost all sample analyses are within the 95% confidence interval; Fig.15F shows that the two methods have a good correlation (R2=0.9179), indicating that the PS-VFA method of this study can be used to analyze the number of CD8 cells in clinical samples.

[0188] In summary, this embodiment successfully constructs a PS-VFA method for detecting the number of T lymphocyte subsets, wherein the experimental results show that the linear range of CD3 cell detection is 50-1400 cells / μL, and the minimum detection concentration is 50 cells / μL; the linear range of CD4 cell detection is 100-1400 cells / μL, and the minimum detection concentration is 100 cells / μL; the linear range of CD8 cell detection is 25-800 cells / μL, and the minimum detection concentration is 25 cells / μL. The detection limit is based on the "blocking competition" principle. The detection concentration of the lateral immunochromatography fluorescent test strip (137 cells / L) is lower. 28 real sample tests were analyzed, and the test results were basically consistent with the flow cytometry analysis results; the performance verification of the precision test, the CV of the repeatability test of low, medium and high concentrations is less than 6%, which meets the industry requirements (CV <15%); the performance of Bland-Altman consistency analysis and correlation analysis is good.

[0189] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0190] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A kit for detecting T lymphocyte subsets in lung cancer, characterized in that: The kit comprises: a vertical flow immunochromatography box and three signal probes; The vertical flow immunochromatography box comprises a box body, and a water-absorbing pad, a filter membrane and a detection cover which are stacked on the box body from bottom to top, and the detection cover is provided with a sample addition hole; the filter membrane is a nitrocellulose membrane, and the pore size of the filter membrane is 4mm~6mm; The three signal probes include three red latex microspheres coupled with three antibodies respectively, wherein the three antibodies are CD3 antibody, CD4 antibody and CD8 antibody, and the labeled amount of the antibodies in the red latex microspheres is 12 mg / 25 mL to 13 mg / 25 mL; the red latex microspheres are carboxyl-modified polystyrene red microspheres; The CD3 antibody, CD4 antibody and CD8 antibody are from Biolegend, and their clone numbers are SK7, SK3 and HIT8a respectively; The method for preparing the signal probe comprises the following steps: Adding 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide to the red latex microspheres for activation reaction to obtain activated red latex microspheres, and adding PBS labeling buffer to the activated red latex microspheres to obtain an activated microsphere suspension; adding the antibody to the obtained activated microsphere suspension for incubation; Adding blocking solution to the incubated solution to perform blocking reaction to obtain precipitation; The obtained precipitate is added into the labeling complex solution and mixed evenly to obtain the signal probe; The labeling reconstitution solution contains 8% w / v sucrose, 2% w / v trehalose, 1% w / v sodium caseinate, 0.5% w / v BSA, 0.1M pH 8.5 Gly-NaOH, 0.5% v / v ProClin 300 and 4% w / v PVP-K30; When the signal probe is prepared, the coupling reaction between the antibody and the red latex microspheres is carried out in a PBS labeling buffer, wherein the pH value of the PBS labeling buffer corresponding to the CD3 antibody is 7.3-7.7, the pH value of the PBS labeling buffer corresponding to the CD4 antibody is 7-7.5, and the pH value of the PBS labeling buffer corresponding to the CD8 antibody is 6.3-6.

7.

2. The kit according to claim 1, characterized in that When the signal probe is prepared, the labeled amount of the antibody in the red latex microspheres is 12.5 mg / 25 mL.

3. The kit according to claim 1, characterized in that The pH value of the PBS labeling buffer corresponding to the CD3 antibody is 7.5, the pH value of the PBS labeling buffer corresponding to the CD4 antibody is 7.5, and the pH value of the PBS labeling buffer corresponding to the CD8 antibody is 6.

5.

4. The kit according to any one of claims 1 to 3, characterized in that The absorbent pad is provided with two layers.

5. Use of the kit for detecting T lymphocyte subsets according to any one of claims 1 to 4 in the preparation of a product for detecting lung cancer.

6. A method for detecting lung cancer T lymphocyte subsets for non-disease diagnosis purposes, characterized in that: The kit for detecting T lymphocyte subsets according to any one of claims 1 to 4 is used, and the method comprises the following steps: The collected peripheral blood sample is processed by density gradient centrifugation to obtain a mononuclear cell precipitate, and a buffer is added to the mononuclear cell precipitate to obtain a mononuclear cell fluid; The three signal probes are added to the mononuclear cell fluid respectively for incubation, and after incubation, they are added to the sample addition holes of the vertical flow immunochromatography box respectively for chromatography treatment, and colorimetric analysis is performed after the treatment is completed.

7. The method according to claim 6, characterized in that The density gradient centrifugation method comprises the following steps: The collected peripheral blood samples were added to lymphocyte separation solution and centrifuged at a centrifugal force of 1200×g~1300×g for 20min~25min to obtain a layered solution; The buffy coat layer in the layered solution is aspirated and centrifuged a second time to obtain a precipitate, the centrifugal force is 500×g~600×g, and the centrifugation is performed for 8 min~12 min. The obtained precipitate is the mononuclear cell.

8. The method according to claim 6, characterized in that The incubation time is 9 min to 11 min.

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