Preparation method of programmed death receptor 1 (PD-1) antibody magnetic beads

Abstract

The invention belongs to the technical field of biological medicine, and provides a preparation method of programmed death receptor 1 (PD-1) antibody magnetic beads, which comprises the following steps: respectively coupling 14 anti-human PD-1 monoclonal antibodies with carboxyl magnetic beads to prepare PD-1 antibody magnetic beads; the cloning numbers of the 14 anti-human PD-1 monoclonal antibodies are respectively 7E5, 1H8, 1C4, 9A5, 9B4, 7C9, 3E5, 7G12, 9E11, 5B2, 5H7, 2H7, BIC4 and 2C4; by optimizing the coupling scheme of the antibody and the magnetic bead, the coupling amount of the antibody on the surface of the magnetic bead reaches that 1mL of the magnetic bead is combined with 1mg of the antibody, the fluorescence signal on the surface of the magnetic bead is enhanced after the antibody is coupled, and the prepared antibody magnetic bead can capture PD-1 positive cells.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to a method for preparing programmed death receptor 1 (PD-1) antibody magnetic beads. Background Art

[0002] The immune system can recognize and eliminate foreign components that express new antigens to maintain homeostasis of the internal environment. Abnormal cells that have become cancerous are also targets of elimination. T cell-mediated cellular immune responses play an important regulatory role, forming an anti-tumor immune cycle consisting of a multi-step cascade reaction. There are multiple factors in the anti-tumor immune cycle that can enhance or inhibit immune clearance of tumor cells, including programmed death factor 1 (PD-1) and its ligand, which form an important immunosuppressive checkpoint, namely the PD-1 / PD-L1 immune checkpoint.

[0003] Programmed death factor PD-1 belongs to the CD28 family. PD-1 is an induced protein, that is, T cells have almost no expression of PD-1 when they are not activated. Only after T cells are activated, PD-1 will be induced to express. In addition to being expressed on activated mature T cells, PD-1 is also lowly expressed on double negative (CD4-CD87) T cells in the thymus, activated NK cells, monocytes and immature Langerhans cells. PD-1 has 20% homology with cytotoxic T lymphocyte-associated antigen 4 (CTLA-4). PD-1 is a type I transmembrane glycoprotein composed of 268 amino acids. Its structure mainly includes an extracellular immunoglobulin variable region (IgV)-like structure, a hydrophobic transmembrane region and an intracellular region. There are two independent tyrosine residues at the tail of the intracellular region. The tyrosine residue at the nitrogen end participates in the formation of an immunoreceptor tyrosine inhibition motif, and the tyrosine residue at the carbon end participates in the formation of an immunoreceptor tyrosine conversion motif. After PD-1 and PD-L1 bind to activated T cells, they promote the phosphorylation of tyrosine in the ITSM domain of PD-1, which in turn causes the dephosphorylation of downstream protein kinases such as PI3K, inhibits the activation of downstream pathways such as AKT and ERK, and ultimately inhibits the transcription and translation of genes and cytokines required for T cell activation, thereby playing a role in negatively regulating T cell activity.

[0004] PD-1 and its ligand PD-L1 are a pair of immune co-stimulatory factors. In recent years, the PD-1 / PD-L1 immune checkpoint has attracted much attention due to its involvement in the tumor immune escape mechanism. PD-1 expressed by tumor-infiltrating T lymphocytes interacts with PD-L1 expressed by tumor cells to activate the PD-1 / PD-L1 signaling pathway, which can inhibit the anti-tumor activity of effector T cells, leading to the formation of an immunosuppressive tumor microenvironment, and ultimately allowing tumor cells to escape immune clearance. Almost all types of human tumors express PD-L1, such as melanoma, renal cell carcinoma, lung cancer, head and neck cancer, gastrointestinal cancer, bladder cancer, ovarian cancer, and hematological malignancies. Blocking the PD-1 / PD-L1 immune checkpoint can effectively improve the tumor immune microenvironment and enhance the immune surveillance and clearance of tumor cells by effector T cells. Immunotherapy based on antibody drugs that block this immune checkpoint has become a new strategy for tumor treatment. Summary of the invention

[0005] The purpose of the present invention is to provide a method for preparing programmed death receptor 1 (PD-1) antibody magnetic beads. The present invention optimizes the coupling scheme of antibodies and magnetic beads so that the coupling amount of antibodies on the surface of magnetic beads reaches 1 mL of magnetic beads combined with 1 mg of antibodies, and the fluorescence signal on the surface of magnetic beads is enhanced after antibody coupling.

[0006] A method for preparing programmed death receptor 1 (PD-1) antibody magnetic beads, comprising coupling 14 anti-human PD-1 monoclonal antibodies to carboxyl magnetic beads respectively to prepare PD-1 antibody magnetic beads;

[0007] The covalent coupling of the carboxyl magnetic beads and the anti-human PD-1 monoclonal antibody specifically comprises the following steps:

[0008] S1. Remove the supernatant from the magnetically activated carboxyl magnetic beads, add 50 g to 200 g of biological ligand and mix gently; the biological ligand is one of the 14 anti-human PD-1 monoclonal antibodies;

[0009] S2, coupling at 25°C for 2 hours, or coupling at 25°C for 1 hour and then standing at 4°C overnight, keeping the magnetic beads suspended during the coupling period;

[0010] S3, remove the supernatant, add 400 μL PBST solution to resuspend the magnetic beads, react at 25° C. for 1 h to block the unreacted activated carboxyl groups on the surface of the magnetic beads, and keep the magnetic beads suspended during this period; the pH of the PBST solution is 7.2, and the PBST solution contains 1% (w / v) BSA;

[0011] S4. Remove the supernatant, wash three times with 400 μL PBS solution or PBS storage solution supplemented with 1% (w / v) BSA each time, resuspend in PBS storage solution supplemented with 1% (w / v) BSA, and store at 4°C to obtain PD-1 antibody magnetic beads; the pH of the PBS solution is 7.2.

[0012] Preferably, the clone numbers of the 14 anti-human PD-1 monoclonal antibodies are 7E5, 1H8, 1C4, 9A5, 9B4, 7C9, 3E5, 7G12, 9E11, 5B2, 5H7, 2H7, BIC4, and 2C4, respectively.

[0013] Preferably, the amount of the biological ligand in S1 is 400 μg-600 μg, the concentration of the biological ligand is 5-10 mg / mL, the pH of the solution is maintained at ≈ 8.0, and 0.05% volume fraction of Tween 20 is also added to S1.

[0014] Preferably, the supernatant is removed in S3 and S4 by placing a centrifuge tube on a magnetic separation rack for magnetic separation.

[0015] Preferably, the amount of the PBS storage solution containing 1% (w / v) BSA added in S4 is 100-400 μL.

[0016] Preferably, the coupling amount of the antibody on the surface of the magnetic beads is: 1 mL of magnetic beads is combined with 1 mg of antibody, and the diameter of the carboxyl magnetic beads is 2 μm.

[0017] Preferably, the method for preparing the carboxyl magnetic beads comprises the following steps:

[0018] a. After mixing the Mag beads, take 100 μL of Mag beads into a 1 mL centrifuge tube, remove the supernatant by magnetic separation, wash twice with 200 μL of MEST solution, and then remove the supernatant;

[0019] b. Quickly add 100 μL of the prepared 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride solution and 100 μL of N-hydroxysuccinimide solution to the centrifuge tube containing the magnetic beads, vortex mix to fully suspend the magnetic beads, and activate at 25°C for 30 minutes. Keep the magnetic beads suspended during the activation period.

[0020] Preferably, the MEST solution contains a concentration of 100 m M 2-(N-morpholine)ethanesulfonic acid, pH of MEST solution 5.0, MEST solution contains 0.05% Tween 20 by volume;

[0021] The concentration of the 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride solution is 10-50 mg / mL, and the 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride solution uses MEST / ethanol / ultrapure water as a dispersant; the concentration of the N-hydroxysuccinimide solution is 10-50 mg / mL, and the N-hydroxysuccinimide solution uses MEST / ethanol / ultrapure water solution as a dispersant.

[0022] Preferably, the magnetic beads are kept in suspension during the activation and coupling periods by inverting and mixing using a vertical mixer.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] 1. The preparation of the immunomagnetic beads of the present invention does not mean that the more antibodies coupled to the magnetic beads, the better. While ensuring the antibody structure, the appropriate antibody ratio is the key to the preparation of functional magnetic beads. The coupling scheme of antibodies and magnetic beads is optimized so that the coupling amount of antibodies on the surface of magnetic beads is: 1 mL of magnetic beads is combined with 1 mg of antibodies.

[0025] 2. The present invention can detect the efficiency of antibody coupling to magnetic beads through fluorescent labeling technology. After the antibody is bound to the magnetic beads, the secondary antibody labeled with fluorescein can specifically bind to the antibody, so that the antibody magnetic beads are labeled with fluorescence. Compared with the control group, the fluorescence signal on the surface of the magnetic beads is enhanced after coupling with the antibody. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 The standard curve diagram of the Bradford assay antibody concentration of the present invention;

[0027] Figure 2 171B is a bright field control image of the antibody magnetic beads, and 171F is a fluorescence image of the antibody magnetic beads;

[0028] Figure 3 172B is a bright field control image of the antibody magnetic beads, and 172F is a fluorescence image of the antibody magnetic beads;

[0029] Figure 4 173B is a bright field control image of the antibody magnetic beads, and 173F is a fluorescence image of the antibody magnetic beads;

[0030] Figure 5 174B is a bright field control image of the antibody magnetic beads; 174F is a fluorescence image of the antibody magnetic beads;

[0031] Figure 6175B is a bright field control image of the antibody magnetic beads, and 175F is a fluorescence image of the antibody magnetic beads;

[0032] Figure 7 190B is a bright field control image of blank magnetic beads, and 190F is a fluorescence image of blank magnetic beads;

[0033] Figure 8 191B is a bright field control image of the antibody magnetic beads, and 191F is a fluorescence image of the antibody magnetic beads;

[0034] Fig. 9 192B is a bright field control image of the antibody magnetic beads, and 192F is a fluorescence image of the antibody magnetic beads;

[0035] Fig.10 193B is a bright field control image of the antibody magnetic beads, and 193F is a fluorescence image of the antibody magnetic beads;

[0036] Fig.11 194B is a bright field control image of the antibody magnetic beads, and 194F is a fluorescence image of the antibody magnetic beads;

[0037] Fig.12 195B is the immunofluorescence image of the PD-1 antibody 5B2 magnetic beads of the present invention; 195F is the antibody magnetic bead bright field control image, and 195F is the antibody magnetic bead fluorescence image;

[0038] Fig.13 196B is the immunofluorescence image of the PD-1 antibody 5H7 magnetic beads of the present invention; 196F is the antibody magnetic bead bright field control image and 196F is the antibody magnetic bead fluorescence image. DETAILED DESCRIPTION

[0039] The following embodiments of the present invention are described in further detail in conjunction with the accompanying drawings and examples. The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention.

[0040] As attached Figure 1 To Attachment Fig.13 As shown:

[0041] Embodiment: The present invention provides a method for preparing programmed death receptor 1 (PD-1) antibody magnetic beads, comprising coupling 14 anti-human PD-1 monoclonal antibodies to carboxyl magnetic beads, respectively, to prepare PD-1 antibody magnetic beads; the clone numbers of the 14 anti-human PD-1 monoclonal antibodies are 7E5, 1H8, 1C4, 9A5, 9B4, 7C9, 3E5, 7G12, 9E11, 5B2, 5H7, 2H7, BIC4, and 2C4, respectively.

[0042] The covalent coupling of carboxyl magnetic beads and anti-human PD-1 monoclonal antibodies specifically includes the following steps:

[0043] S1. Remove the supernatant from the magnetically activated carboxyl magnetic beads, add 50 g to 200 g of biological ligand and mix gently; the biological ligand is one of 14 anti-human PD-1 monoclonal antibodies; the amount of biological ligand in S1 is 400 μg-600 μg, the concentration of biological ligand is 5-10 mg / mL, the solution pH is maintained at ≈ 8.0, and 0.05% volume fraction of Tween 20 is also added to S1.

[0044] S2, coupling at 25°C for 2 hours, or coupling at 25°C for 1 hour and then standing at 4°C overnight, keeping the magnetic beads suspended during the coupling period;

[0045] S3. Remove the supernatant, add 400 μL PBST solution to resuspend the magnetic beads, and react at 25°C for 1 h to block the unreacted activated carboxyl groups on the surface of the magnetic beads, while keeping the magnetic beads suspended; the pH of the PBST solution is 7.2, and the PBST solution contains 1% (w / v) BSA; the amount of the PBS storage solution with 1% (w / v) BSA added is 100-400 μL.

[0046] S4. Remove the supernatant, wash three times with 400 μL PBS solution or PBS storage solution supplemented with 1% (w / v) BSA each time, resuspend in PBS storage solution supplemented with 1% (w / v) BSA, and store at 4°C to obtain PD-1 antibody magnetic beads; the pH of the PBS solution is 7.2.

[0047] The supernatant from S3 and S4 was removed and placed in a centrifuge tube on a magnetic separation rack for magnetic separation.

[0048] The coupling amount of the antibody on the surface of the magnetic beads is: 1mL of magnetic beads is combined with 1㎎ of antibody, and the diameter of the carboxyl magnetic beads is 2μm.

[0049] The preparation method of carboxyl magnetic beads comprises the following steps:

[0050] a. After mixing the Mag beads, take 100 μL of Mag beads into a 1 mL centrifuge tube, remove the supernatant by magnetic separation, wash twice with 200 μL of MEST solution, and then remove the supernatant;

[0051] b. Quickly add 100 μL of the prepared 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride solution and 100 μL of N-hydroxysuccinimide solution to the centrifuge tube containing the magnetic beads, vortex mix to fully suspend the magnetic beads, and activate at 25°C for 30 minutes. Keep the magnetic beads suspended during the activation period.

[0052] The MEST solution contains 100 mM 2-(N-morpholine)ethanesulfonic acid, the pH of the MEST solution is 5.0, and the volume fraction of the MEST solution is 0.05% Tween 20;

[0053] The concentration of the 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride solution is 10-50 mg / mL, and the 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride solution uses MEST / ethanol / ultrapure water as a dispersant; the concentration of the N-hydroxysuccinimide solution is 10-50 mg / mL, and the N-hydroxysuccinimide solution uses MEST / ethanol / ultrapure water solution as a dispersant.

[0054] During activation and coupling, the magnetic beads were kept suspended by inverting and mixing using a vertical mixer.

[0055] Experimental Example 1: Preparation of PD-1 Antibody Magnetic Beads

[0056] 1. EDC / NHS activated carboxyl magnetic beads coupled to mouse anti-human PD-1 monoclonal antibody

[0057] 14 anti-human PD-1 monoclonal antibody strains successfully developed by the anogen-yes team: 7E5, 1H8, 1C4, 9A5, 9B4, 7C9, 3E5, 7G12, 9E11, 5B2, 5H7, 2H7, BIC4, 2C4. They were coupled with 2μm diameter carboxyl magnetic beads developed by Beaver biomedical to prepare PD-1 antibody magnetic beads.

[0058] A. Activation of carboxyl groups on the surface of magnetic beads

[0059] 1. After mixing the magnetic beads, take 100 μL Mag beads into a 1 mL centrifuge tube, remove the supernatant by magnetic separation, and wash twice with 200 μL MEST solution (100 mM MES, pH 5.0, 0.05% Tween 20), and then remove the supernatant;

[0060] 2. Quickly add freshly prepared 100mL EDC solution (10-50mg / mL, with MEST / ethanol / ultrapure water as dispersant) and 100HL NHS (10-50mg / mL, with MEST / ethanol / ultrapure water solution as dispersant) solution to the centrifuge tube containing magnetic beads, vortex mix to fully suspend the magnetic beads, and activate at 25℃ for 30min, during which the magnetic beads are kept suspended (vertical mixer can be used for inversion mixing); after the above steps, the carboxyl groups on the surface of the magnetic beads have been activated and can be covalently coupled with antibodies with primary amino groups.

[0061] B. Covalent coupling of magnetic beads and antibodies

[0062] 1. Remove the supernatant by magnetic separation, add 50μg~200μg of biological ligand (the appropriate amount and concentration need to be optimized according to the specific experiment, keep the solution pH ≈ 8.0, add 0.05% Tween 20 to improve the dispersion of magnetic beads, and avoid the presence of reagents containing primary amino groups other than biological ligands in the buffer system), and mix gently;

[0063] 2. Coupling at 25℃ for 2h, or coupling at 25℃ for 1h and then standing at 4℃ overnight. Keep the magnetic beads suspended during coupling and use a vertical mixer to mix by inversion;

[0064] 3. Place the centrifuge tube on a magnetic separation rack to remove the supernatant by magnetic separation, add 400L PBST solution (pH 7.2, containing 1% BSA) to resuspend the magnetic beads (ultrasound can be performed as needed), react at 25°C for 1h to block the unreacted activated carboxyl groups on the surface of the magnetic beads, and keep the magnetic beads suspended during this period;

[0065] 4. Place the centrifuge tube on a magnetic separator to remove the supernatant by magnetic separation, wash three times with 400 μL PBS solution (pH 7.2) or preservation solution each time, resuspend in preservation solution (the amount of preservation solution added can be determined as needed to adjust the concentration of coupled ligand magnetic beads), and store at 4°C.

[0066] The preparation of immunomagnetic beads does not mean that the more antibodies coupled to the beads, the better. While ensuring the antibody structure, the appropriate antibody ratio is the key to the preparation of functional magnetic beads. Optimize the coupling scheme of antibodies and magnetic beads so that the coupling amount of antibodies on the surface of magnetic beads is: 1mL magnetic beads are combined with 1mg of antibodies.

[0067] Table 1 Antibody coupling effects of different antibody concentrations and coupling systems

[0068]

[0069] 2. Identification of the number of antibodies coupled to carboxyl magnetic beads

[0070] The antibody lyophilized powder was dissolved in double distilled water, and samples were collected before and after coupling with magnetic beads. Due to the special nature of EDC / NHS activated magnetic beads and coupling methods, the Bradford method, which is more accurate for protein quantification, was used to determine the sample antibody content and calculate the coupling efficiency: M 偶联抗体量 / 100μMegbead=C 偶联前抗体浓度 V 加入体积 -C 偶联后悬液抗体浓度 V 偶联后上层液体积 .

[0071] 2.1 Bradford protein quantification

[0072] The Bradford method is based on the binding of Coomassie Brilliant Blue G-250 dye to protein in an acidic solution, which changes the position of the dye's maximum absorption peak from 465nm to 595nm, and the color of the solution changes from brown-black to blue. Studies have shown that the dye mainly binds to basic amino acids (especially arginine) and aromatic amino acid residues in proteins. The absorbance value A595 measured at 595nm is proportional to the protein concentration.

[0073] Standard Methods

[0074] (1) Take 16 test tubes, 1 as a blank, 3 as unknown samples, and divide the remaining test tubes into two groups. Add samples, water and reagents in the order in the table. Add 0, 0.02, 0.04, 0.06, 0.08, 0.1 mL of 1.0 mg / mL standard antibody solution to each test tube, and then add deionized water to 0.1 mL. At the same time, take 100 μL of unknown sample with a suitable dilution ratio into the test tube. Finally, add 5.0 mL of staining reagent (100 mg Coomassie Brilliant Blue G-250, dissolved in 50 mL 95% ethanol, then add 100 mL 85% phosphoric acid, and dilute with water to 1 liter) to each test tube. After adding each tube, mix it immediately on a vortex mixer (be careful not to mix too vigorously to avoid generating a large number of bubbles that are difficult to eliminate).

[0075] (2) 2 to 5 minutes after adding the reagent, you can start using a cuvette to measure the light absorption value A595 of each sample at 595nm on a spectrophotometer. The blank control is 0.1mL H 2 Add 5.0mL of staining reagent to the sample. Note: Do not use quartz cuvettes (because the stain is not easy to wash off). Plastic or glass cuvettes can be used. Immediately rinse with a small amount of 95% ethanol after use to wash off the stain. Plastic cuvettes should not be soaked in ethanol or acetone for a long time.

[0076] (3) Use the standard antibody concentration (mg / mL) as the horizontal axis and the absorbance value A595 as the vertical axis to draw a graph to obtain a standard curve. Based on this standard curve, the protein content of the unknown sample can be found according to the measured A595 value of the unknown sample.

[0077] 2.2 Immunofluorescence

[0078] Take 10 μL of 1% BSA-blocked PD-1 antibody magnetic beads in an eppendorf tube, add 200 μL of 1:200 diluted FITC-labeled goat anti-mouse secondary antibody, and incubate at room temperature for two hours in the dark. Separate the magnetic beads by magnetic force and wash them with PBST five times. Resuspend the magnetic beads with PBS, and select blank carboxyl magnetic beads as a control during the experiment. Observe the surface fluorescence labeling of PD-1 antibody magnetic beads coupled under different conditions under a fluorescence microscope.

[0079] This fluorescent labeling can indicate that after the antibody is bound to the magnetic beads, the secondary antibody labeled with fluorescein can specifically bind to the antibody, so that the antibody magnetic beads are labeled with fluorescence. Compared with the control group, the fluorescence signal on the surface of the magnetic beads is enhanced after the antibody is coupled.

[0080] Experimental Example 2: Using immunomagnetic beads to separate and eliminate PD-1 positive T cells

[0081] Isolation of peripheral blood mononuclear cells (PBMCs)

[0082] Dilute 10 mL of anticoagulated whole blood to 21 mL with 1X PBS + 3% FCS. Add 6 mL of Ficoll to three 15 mL conical vials. Add 7 mL of diluted blood to the Ficoll at a very steep angle using a 10 mL pipette. Then slowly add the diluted blood to cover the Ficoll. Centrifuge the cells at 1500 RPM for 30 minutes. Aspirate the supernatant, combine the samples, and wash with PBS + 5% FCS. Resuspend the cell pellet, count using a hemacytometer, and then dilute to a final concentration of 1x10 in each tube. 6 cells / 100 μL for subsequent experiments.

[0083] Antibody magnetic beads capture and isolate PD-1 positive cells

[0084] Pipette 200 μL of PD-1 antibody magnetic bead suspension and transfer it to a centrifuge tube. Separate the magnetic beads with a magnetic stand and remove the upper layer of liquid. Add an equal amount of the above-separated PBMC to the centrifuge tube containing the magnetic beads and pipette gently and repeatedly. Place the centrifuge tube on a vertical mixer and mix at room temperature for 2 hours. Then separate the magnetic beads with a magnetic stand and take the upper layer of liquid to analyze the proportion of PD-1+ cells using a flow cytometer.

[0085] Flow cytometry analysis of the proportion of PD-1+ cells in the suspension after immunomagnetic bead capture

[0086] After capture by immunomagnetic beads, different fluorescently labeled PD-1 / CD3 / CD4 / CD8 antibodies (CD3-FITC, CD4-PE, CD8-APC, PD-1-PE / Cy7 commercial antibodies) were added to the suspension, incubated at room temperature for 1 hour, and the expression of cell surface markers PD-1 / CD3 / CD4 / CD8 was analyzed by flow cytometry.

[0087] Case 1 was from a lung cancer patient at Shanghai Ruijin Hospital. Peripheral blood mononuclear cells were isolated to evaluate the ability of PD-1 antibody magnetic beads to capture PD-1 positive cells.

[0088] Table 2 Ability of PD-1 antibody magnetic beads to capture PD-1 positive cells in patient 1

[0089]

[0090] As shown in the table, the proportion of PD-1 positive cells in the sample itself (the sample in the control group that was not treated with magnetic beads) was only 3.49%, and the proportion of PD-1+ positive cells was relatively low. Antibody magnetic beads can capture PD-1 positive cells to varying degrees, among which 7G12 and 9E11 were more effective, reducing the proportion of positive cells by 80.27% and 78.26%, respectively. Due to limited patient samples, in order to screen a variety of magnetic beads, the working concentration of the magnetic beads was temporarily determined based on experience and literature, and may not be optimal. This experiment shows that some antibody magnetic beads are still relatively excellent, and more patient samples are needed for verification and further optimization of the working concentration.

[0091] Case 2 was from a lung cancer patient at Shanghai Ruijin Hospital. Peripheral blood mononuclear cells were isolated to evaluate the ability of PD-1 antibody magnetic beads to capture PD-1 positive cells.

[0092] Table 3 Capacity of PD-1 antibody magnetic beads to capture PD-1 positive cells in patient 2

[0093]

[0094] The samples were from lung cancer patients after chemotherapy. The proportion of PD-1 positive cells in peripheral blood mononuclear cells of patient 2 was 12.40%. From the data in the table, it can be seen that 7G12 and 9A5 have better ability to capture PD-1 positive cells. Compared with the magnetic bead verification data of patient 1, this test showed that 9E11's ability to capture PD-1 was average, while 9A5 performed well. However, the two patient samples confirmed that 7G12 was the best.

[0095] Importantly, it should be noted that the construction and arrangement of the present application shown in a plurality of different exemplary embodiments are only exemplary. Although only a few embodiments are described in detail in this disclosure, it should be readily understood by those who refer to this disclosure that many modifications are possible without substantially departing from the novel teachings and advantages of the subject matter described in the application. Without departing from the scope of the present invention, other replacements, modifications, changes and omissions may be made in the design, operating conditions and arrangement of the exemplary embodiments. Therefore, the present invention is not limited to specific embodiments, but extends to a variety of modifications still falling within the scope of the appended claims.

[0096] Additionally, in order to provide a concise description of exemplary embodiments, all features of an actual embodiment (ie, those features that are not relevant to the best mode presently contemplated for carrying out the invention or those that are not relevant to implementing the invention) may not be described.

[0097] It will be appreciated that in the development of any actual implementation, as in any engineering or design project, numerous implementation-specific decisions may be made. Such a development effort may be complex and time-consuming, but will be a routine task of design, fabrication, and production for those of ordinary skill having the benefit of this disclosure without undue experimentation.

[0098] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention may be modified or replaced by equivalents without departing from the spirit and scope of the technical solutions of the present invention, which should all be included in the scope of the claims of the present invention.

Claims

1. A method for preparing programmed death receptor 1 (PD-1) antibody magnetic beads, characterized in that: The method comprises coupling 14 anti-human PD-1 monoclonal antibodies to carboxyl magnetic beads respectively to prepare PD-1 antibody magnetic beads; The covalent coupling of the carboxyl magnetic beads and the anti-human PD-1 monoclonal antibody specifically comprises the following steps: S1. Remove the supernatant from the magnetically activated carboxyl magnetic beads, add 50 g to 200 g of biological ligand and mix gently; the biological ligand is one of the 14 anti-human PD-1 monoclonal antibodies; S2, coupling at 25°C for 2 hours, or coupling at 25°C for 1 hour and then standing at 4°C overnight, keeping the magnetic beads suspended during the coupling period; S3, remove the supernatant, add 400 μL PBST solution to resuspend the magnetic beads, react at 25° C. for 1 h to block the unreacted activated carboxyl groups on the surface of the magnetic beads, and keep the magnetic beads suspended during this period; the pH of the PBST solution is 7.2, and the PBST solution contains 1% (w / v) BSA; S4. Remove the supernatant, wash three times with 400 μL PBS solution or PBS storage solution supplemented with 1% (w / v) BSA each time, resuspend in PBS storage solution supplemented with 1% (w / v) BSA, and store at 4°C to obtain PD-1 antibody magnetic beads; the pH of the PBS solution is 7.

2.

2. The method for preparing programmed death receptor 1 (PD-1) antibody magnetic beads according to claim 1, characterized in that: The clone numbers of the 14 anti-human PD-1 monoclonal antibodies are 7E5, 1H8, 1C4, 9A5, 9B4, 7C9, 3E5, 7G12, 9E11, 5B2, 5H7, 2H7, BIC4, and 2C4, respectively.

3. The method for preparing programmed death receptor 1 (PD-1) antibody magnetic beads according to claim 1, characterized in that: The amount of biological ligand in the S1 is 400 μg-600 μg, the concentration of biological ligand is 5-10 mg / mL, and the solution pH is maintained at ≈ 8.

0. 0.05% volume fraction of Tween 20 is also added to the S1.

4. The method for preparing programmed death receptor 1 (PD-1) antibody magnetic beads according to claim 1, characterized in that: The supernatant removed from S3 and S4 is placed in a centrifuge tube on a magnetic separation rack for magnetic separation.

5. The method for preparing programmed death receptor 1 (PD-1) antibody magnetic beads according to claim 1, characterized in that: The amount of the PBS storage solution containing 1% (w / v) BSA added in S4 is 100-400 μL.

6. The method for preparing programmed death receptor 1 (PD-1) antibody magnetic beads according to claim 1, characterized in that: The coupling amount of the antibody on the surface of the magnetic beads is: 1 mL of magnetic beads is combined with 1 mg of antibody, and the diameter of the carboxyl magnetic beads is 2 μm.

7. The method for preparing programmed death receptor 1 (PD-1) antibody magnetic beads according to claim 1, characterized in that: The preparation method of the carboxyl magnetic beads comprises the following steps: a. After mixing the Mag beads, take 100 μL of Mag beads into a 1 mL centrifuge tube, remove the supernatant by magnetic separation, wash twice with 200 μL MEST solution, and then remove the supernatant; b. Quickly add 100 μL of the prepared 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride solution and 100 μL of N-hydroxysuccinimide solution to the centrifuge tube containing the magnetic beads, vortex mix to fully suspend the magnetic beads, and activate at 25°C for 30 minutes, keeping the magnetic beads suspended during the activation period.

8. The method for preparing programmed death receptor 1 (PD-1) antibody magnetic beads according to claim 7, characterized in that: The MEST solution contains 100 mM 2-(N-morpholine)ethanesulfonic acid, the pH of the MEST solution is 5.0, and the volume fraction of the MEST solution is 0.05% Tween 20; The concentration of the 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride solution is 10-50 mg / mL, and the 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride solution uses MEST / ethanol / ultrapure water as a dispersant; the concentration of the N-hydroxysuccinimide solution is 10-50 mg / mL, and the N-hydroxysuccinimide solution uses MEST / ethanol / ultrapure water solution as a dispersant.

9. The method for preparing programmed death receptor 1 (PD-1) antibody magnetic beads according to claim 1 or 7, characterized in that: During the activation and coupling periods, the magnetic beads were kept in suspension by inverting and mixing using a vertical mixer.

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