Anti-human CD14 antibody and use thereof

Abstract

Provided is an anti-human CD14 antibody, comprising a heavy chain and a light chain. An amino acid sequence of a heavy chain variable region comprises a sequence as shown in SEQ ID NO: 2, and an amino acid sequence of a light chain variable region comprises a sequence as shown in SEQ ID NO: 4. The coupling of the anti-human CD14 antibody and magnetic beads enables the simple and rapid sorting of high-purity CD14+ monocytes from fresh or cryopreserved peripheral blood mononuclear cells, with good specificity.

Description

Anti-human CD14 antibodies and uses thereof TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological medicine, and particularly relates to an anti-human CD14 antibody and application thereof in sorting CD14 monocytes. BACKGROUND

[0002] CD14 (cluster of differentiation 14), that is, an LPS receptor, is a surface antigen and belongs to one of the members of the cell surface glycoprotein family. CD14 exists in two forms, one is cell membrane CD14 (mCD14) which is fixed on the membrane through a glycosylphosphatidylinositol tail, and the other is a soluble form (sCD14). The expression of CD14 in the human body has tissue specificity and cell specificity. mCD14 is mainly distributed on the cell surface of monocytes, macrophages and dendritic cells, in addition, mCD14 also exists in neutrophils. sCD14 exists in the plasma of normal humans and animals, and sCD14 also exists in urine in trace amounts.

[0003] In vitro culture of monocytes is not only crucial for immunology-related research, but also has important uses in cell and gene therapy. In a study by Julia K Bialek-Waldmann, genetically modified patient-derived monocytes were used as a strategy for treating relapsed acute myeloid leukemia (AML) (Cancer Immunology, Immunotherapy, Volume 68, pages 1891-1899, 2019). Therefore, it is of great significance to sort out monocytes with high purity.

[0004] The sorting methods of monocytes mainly include the following:

[0005] (1) Flow cytometry sorting technology: monocytes and other white blood cell components are distinguished according to cell size and intracellular particle size properties, and monocytes are sorted out using anti-CD14 antibodies.

[0006] (2) Density gradient centrifugation method: cells are separated according to the density difference between different cells by density gradient centrifugation.

[0007] (3) Magnetic bead sorting method: CD14+ sorting magnetic beads are used to sort monocytes. Compared with other methods, CD14+ sorting magnetic beads can quickly obtain a larger number of monocytes, and is one of the important methods.

[0008] The characteristics of the above three methods are as follows:

[0009] (1) Flow cytometry sorting technology: the flow cytometer is high in price and high in operation and maintenance cost, in addition, professional operators are needed; and due to the need for pressure in the cell sorting process, the cell activity is greatly affected, which is not conducive to subsequent experiments;

[0010] (2) Density gradient centrifugation method: this method is low in cost and simple in operation, and does not need special equipment, but due to its principle, it cannot accurately separate multiple cells with similar densities, and the purity of the separated CD14+ monocytes is not high, which is not conducive to subsequent experiments;

[0011] (3) Magnetic bead sorting method: the cell stimulation is small, the cell activity rate is high, and the method has the characteristics of high sensitivity, high purity and easy operation, and has become the preferred method for cell sorting, and has very strong potential application prospect. However, as shown in FIG. 8, using the existing commercially available product to sort CD14+ monocytes, monocytes only account for 76.61% of the total cells sorted, and other types of cells account for 16.39% of the proportion, and the sorting specificity is poor.

[0012] The disclosure of the above background art content is only used to assist in understanding the inventive concept and technical solutions of the present application, and it does not necessarily belong to the prior art of the present patent application. In the absence of explicit evidence that the above content has been disclosed before the filing date of the present patent application, the above background art should not be used to evaluate the novelty and inventiveness of the present application. SUMMARY

[0013] Therefore, the purpose of the present application is to provide a sequence of an anti-human CD14 antibody, an anti-human CD14 antibody, and an antibody-conjugated magnetic bead and application, to realize efficient sorting of CD14+ monocytes.

[0014] In order to achieve the above purpose, the technical scheme adopted by the present application is as follows:

[0015] The present application provides an anti-human CD14 antibody, which comprises a heavy chain and a light chain, the amino acid sequence of the variable region of the heavy chain is the sequence shown in SEQ ID NO. 2, and the amino acid sequence of the variable region of the light chain is the sequence shown in SEQ ID NO. 4.

[0016] The present application provides a nucleic acid encoding the anti-human CD14 antibody as described above.

[0017] According to some preferred embodiments of the present application, the nucleotide sequence encoding the variable region of the antibody heavy chain is the sequence shown in SEQ ID NO. 1, and the nucleotide sequence encoding the variable region of the antibody light chain is the sequence shown in SEQ ID NO. 3.

[0018] The present application provides an anti-human CD14 antibody, comprising a heavy chain and a light chain, wherein the nucleotide sequence of the heavy chain variable region is the sequence shown in SEQ ID NO. 1, and the nucleotide sequence of the light chain variable region is the sequence shown in SEQ ID NO. 3.

[0019] The present application also provides a nucleic acid encoding the anti-human CD14 antibody as described above.

[0020] The present application also provides a recombinant expression vector comprising the nucleic acid as described above.

[0021] According to some preferred embodiments of the present application, the expression vector comprising the heavy chain antibody has a nucleotide sequence corresponding to an amino acid sequence shown in SEQ ID NO. 7.

[0022] Preferably, the nucleotide sequence of the expression vector of the heavy chain antibody has an amino acid sequence obtained by constructing the anti-human CD14 antibody heavy chain variable region amino acid sequence shown in SEQ ID NO. 2 and the human IgG1-LALA amino acid sequence shown in SEQ ID NO. 5 into a pcDNA3.4 vector.

[0023] According to some preferred embodiments of the present application, the expression vector comprising the light chain antibody has a nucleotide sequence corresponding to an amino acid sequence shown in SEQ ID NO. 8.

[0024] Preferably, the nucleotide sequence of the expression vector has an amino acid sequence obtained by constructing the anti-human CD14 antibody light chain variable region amino acid sequence shown in SEQ ID NO. 4 and the human Kappa amino acid sequence shown in SEQ ID NO. 6 into a pcDNA3.4 vector.

[0025] The present application also provides a cell transformed with the recombinant expression vector as described above.

[0026] The present application also provides the use of the antibody as described above, the nucleic acid as described above, the recombinant expression vector as described above, and the cell as described above in the manufacture of a medicament or a related kit for treating diseases related to endotoxemia, endotoxin shock, autoimmune diseases, atherosclerosis, sepsis, etc.

[0027] The present application also provides a pharmaceutical composition comprising the antibody as described above, and / or the nucleic acid as described above, and / or the recombinant expression vector as described above, in combination with one or more pharmaceutically acceptable excipients.

[0028] The application further provides a magnetic bead coupled with the anti-human CD14 antibody, and application of the coupled magnetic bead in sorting CD14+ monocytes, and the magnetic bead is coupled to coat the CD14 antibody on the magnetic microparticle to form an immunomagnetic composite microparticle, so that CD14+ monocytes with high purity are sorted from fresh or frozen peripheral blood mononuclear cells simply and quickly, and the specificity is better than that of the existing products on the market.

[0029] The application further provides a preparation method of the magnetic bead, comprising the following steps:

[0030] FeCl3·6H2O and FeCl2·4H2O are dissolved in deionized water, NH4OH is added, and the magnetic separation is washed with deionized water and dried to obtain the nano magnetic bead;

[0031] The carboxyl dextran aqueous solution and the nano magnetic bead are mixed and heated to react, the magnetic bead is screened by magnetic separation, and the carboxyl dextran coated magnetic bead is obtained;

[0032] The buffer solution is used as a cleaning solution, the carboxyl dextran coated magnetic bead is washed by magnetic separation, EDC and NHS are added to activate the carboxyl group, the supernatant is removed by magnetic separation, the buffer solution is used for resuspension again, the anti-human CD14 antibody is added for incubation, magnetic separation is used, the blocking solution is added, the storage solution is used for washing and storage in the storage solution.

[0033] The application further provides a sorting method of CD14 monocytes, comprising the following steps:

[0034] Peripheral blood mononuclear cells are extracted, resuspended, the magnetic bead is added, mixed, incubated in the dark, the buffer solution is added to clean the cells, the supernatant is discarded after centrifugation, the buffer solution is used for resuspension to obtain a cell suspension;

[0035] The sorting column is placed on a magnetic stand, and the sorting column is washed; the cell suspension is added to the sorting column in the magnetic field, at this time, the cells flowing out of the sorting column are non-target cells; the residual cells are washed with PBS, when the liquid drops at the lower end of the sorting column no longer drop, the washing liquid is added to the sorting column, and the non-target cells flowing out are collected; when the liquid is completely drained, the sorting column is taken off from the magnetic stand and placed in a new collection tube, PBS is added to the sorting column, the piston is inserted into the sorting column body and pushed to the bottom of the sorting column, at this time, the collected liquid is the target cells;

[0036] The collected target cells and non-target cells are detected by flow cytometry to determine the proportion of CD14 monocytes.

[0037] The application provides an amino acid sequence of an anti-human CD14 antibody which is specifically combined with human CD14, comprising an amino acid sequence of a variable region of a heavy chain and an amino acid sequence of a variable region of a light chain; the amino acid sequence of the variable region of the heavy chain comprises a sequence shown in SEQ ID NO. 2; and the amino acid sequence of the variable region of the light chain comprises a sequence shown in SEQ ID NO. 4.

[0038] The application provides a nucleotide sequence of an anti-human CD14 antibody which is specifically combined with human CD14, comprising a nucleotide sequence for coding a variable region of a heavy chain and a nucleotide sequence for coding a variable region of a light chain; the nucleotide sequence for coding the variable region of the heavy chain comprises a sequence shown in SEQ ID NO. 1; and the nucleotide sequence for coding the variable region of the light chain comprises a sequence shown in SEQ ID NO. 3.

[0039] Compared with the prior art, the anti-human CD14 antibody of the application is coupled with magnetic beads, and can be used to simply and quickly sort CD14+ mononuclear cells with higher purity from fresh or frozen peripheral blood mononuclear cells, and has better specificity. BRIEF DESCRIPTION OF DRAWINGS

[0040] In order to more clearly illustrate the technical solutions in the embodiments of the application, the drawings needed in the embodiment description will be briefly introduced. Obviously, the drawings in the following description are only some embodiments of the application, and other drawings can be obtained by those skilled in the art without creative effort.

[0041] Fig. 1 is a schematic diagram of a control result of flow identification of CD14 monoclonal antibodies in the embodiment 1 of the application;

[0042] Fig. 2 is a non-reducing gel electrophoresis diagram of SDS-PAGE of the anti-human CD14 antibody in the embodiment 2 of the application;

[0043] Fig. 3 is an affinity curve of the anti-human CD14 antibody and human CD14 binding in the embodiment 2 of the application;

[0044] Fig. 4 is a FACS identification result of the anti-human CD14 antibody binding analysis in the embodiment 2 of the application: negative control;

[0045] Fig. 5 is a FACS identification result of the anti-human CD14 antibody binding analysis in the embodiment 2 of the application: CD14 antibody (to-be-tested sample);

[0046] Fig. 6 is a FACS identification result of the anti-human CD14 antibody after coupling with magnetic beads in the embodiment 3 of the application: before sorting;

[0047] Figure 7 is the FACS identification result of the anti-human CD14 antibody coupled with magnetic beads in the embodiment 3 of the present application: after sorting.

[0048] Figure 8 is the FACS identification result of the existing product on the market: after sorting. DETAILED DESCRIPTION

[0049] In order to make the person skilled in the art better understand the technical solutions of the present application, the technical solutions in the embodiments of the present application will be described clearly and completely below in combination with the drawings in the embodiments of the present application. Obviously, the described embodiments are only a part of the embodiments of the present application, not all. Based on the embodiments in the present application, all other embodiments obtained by the person skilled in the art without creative labor should belong to the protection scope of the present application.

[0050] The current magnetic bead separation product separates mononuclear cells with a large proportion of other types of cells being sorted out. The present application obtains an antibody coupled magnetic bead capable of sorting CD14+ mononuclear cells with high purity by screening suitable antibodies and coupling with nano magnetic beads. Specifically, the present application obtains an anti-human CD14 antibody by screening a yeast library, and couples with nano magnetic beads, so as to form a magnetic bead coating material capable of simply and quickly sorting CD14+ mononuclear cells with high purity from peripheral blood mononuclear cells. The mononuclear cells account for 98.55% of the total cells, which shows a better effect of separating mononuclear cells, and a better specificity than other antibody sequences and existing products on the market.

[0051] Example 1 Screening of Anti-human CD14 Antibody

[0052] 1.1 Library Magnetic Bead Enrichment

[0053] (1) Take 100 OD of yeast library strains (human antibody scfv yeast display library) from a-80℃ refrigerator, add to 100 mL of SD-Trp deficient medium, and culture at 30℃, 220 rpm for 3-4 h.

[0054] (2) Measure the absorbance of OD 600, centrifuge to remove the deficient medium, collect the bacteria, and add the bacteria to SG-CAA medium. The bacteria concentration is adjusted to 0.5 OD / mL, and the bacteria are cultured at 20℃, 220 rpm for 48 h. Centrifuge to collect 500 OD of yeast cells.

[0055] (3) Resuspend the yeast cells with 40 mL of PBSA (PBS+1% BSA).

[0056] (4) Centrifuge at 3000 rpm for 3 min, discard the supernatant.

[0057] (5) Centrifuge at 3000 rpm for 3 min, discard the supernatant.

[0058] (6) Add 40 mL PBSA, centrifuge at 3000 rpm for 3 min.

[0059] (7) Repeat step (6) twice.

[0060] (8) Resuspend the cells with 5 mL PBSA, then add 40 μL SA-beads, mix well.

[0061] (9) Incubate for 20 min.

[0062] (10) Place the magnetic column on the magnetic stand, and pass the incubated cells through the column, then wash the column with 5 mL PBSA for 3 times.

[0063] (11) Take the magnetic column out of the magnetic stand, and elute the cells with 5 mL SD-Trp-deficient medium.

[0064] (12) Incubate the eluted cells at 30°C, 220 rpm overnight.

[0065] (13) After overnight incubation, replace the medium with SG-CAA medium, and incubate at 20°C, 220 rpm for 48 h before FACS enrichment.

[0066] 1.2 Library FACS enrichment

[0067] (1) Take 10 OD of bacterial solution in a 50 mL centrifuge tube, centrifuge and discard the supernatant.

[0068] (2) Add 5 mL PBSA, centrifuge at 3000 rpm for 3 min, and discard the supernatant.

[0069] (3) Repeat step (2) twice.

[0070] (4) Add 1 mL of 1:1000 diluted anti-FLAG antibody solution and human CD14-Biotin, with a concentration of 50 nM, and incubate at room temperature for 1 h.

[0071] (5) After incubation, centrifuge at 3000 rpm for 3 min, and discard the supernatant.

[0072] (6) Add 40 mL PBSA to the bacterial cells, centrifuge at 3000 rpm for 3 min, and discard the supernatant.

[0073] (7) Repeat step (6) twice.

[0074] (8) Prepare a mixture of PE-labeled streptavidin (SA-PE) and Alexa Fluor 647-labeled goat anti-mouse antibody (goat anti mouse-647) at a ratio of 1:1000 with PBSA, and add 1 mL to the cells treated in step (7), and incubate at room temperature for 1 h.

[0075] (9) After incubation, centrifuge at 3000 rpm for 3 min and discard the supernatant.

[0076] (10) Repeat step (6) twice.

[0077] (11) Resuspend the bacterial cells with 1 mL PBSA, and flow sort the cell population with PE and 647 double positive cells.

[0078] 1.3 FACS identification of clones

[0079] (1) Spread the enriched yeast cells on an SD-Trp plate, and culture in a 30 °C incubator until single colonies grow.

[0080] (2) Pick the colonies and inoculate in an SD-Trp liquid medium, and culture at 30 °C overnight with shaking.

[0081] (3) Transfer the yeast cells cultured overnight to an SG-CAA medium at a concentration of 0.1 OD / mL, and culture at 20 °C for 48 h.

[0082] (4) Perform FACS detection according to step 1.2, wherein the concentration of human CD14-biotin in the experimental group is 50 nM, and the concentration of human CD14-biotin in the control group is 0 nM.

[0083] (5) The results of flow identification of single colonies are shown in FIG. 1, wherein FIG. 1A is the experimental group, and the average fluorescence intensity (MFI) of PE-labeled streptavidin (SA-PE) and 647 double positive cells is 16802; and FIG. 1B is the control group, and the average fluorescence intensity (MFI) of PE-labeled streptavidin (SA-PE) and 647 double positive cells is 783. The results show that the antibody can bind to human CD14.

[0084] 1.4 Sequencing of yeast clones

[0085] (1) Yeast clone plasmid extraction according to the yeast plasmid extraction kit.

[0086] (2) Transform the plasmid into Top10 E. coli competent cells.

[0087] (3) Pick the clones for sequencing to obtain the anti-human CD14 antibody sequence. The obtained anti-human CD14 antibody heavy chain variable region nucleotide sequence is shown as SEQ ID NO. 1; the anti-human CD14 antibody heavy chain variable region amino acid sequence is shown as SEQ ID NO. 2; the anti-human CD14 antibody light chain variable region nucleotide sequence is shown as SEQ ID NO. 3; and the anti-human CD14 antibody light chain variable region amino acid sequence is shown as SEQ ID NO. 4.

[0088] Example 2 Expression and verification of the anti-human CD14 antibody

[0089] 2.1 Expression and purification of the anti-human CD14 antibody

[0090] (1) The anti-human CD14 antibody heavy chain variable region amino acid sequence shown as SEQ ID NO. 2 and the human IgG1-LALA amino acid sequence shown as SEQ ID NO. 5 are constructed into a pcDNA3.4 vector to obtain an amino acid sequence shown as SEQ ID NO. 7. The amino acid sequence is transformed to obtain the nucleotide sequence of the expression frame corresponding to the expression vector of the heavy chain antibody.

[0091] (2) The anti-human CD14 antibody light chain variable region amino acid sequence shown as SEQ ID NO. 4 and the human Kappa sequence amino acid sequence shown as SEQ ID NO. 6 are constructed into a pcDNA3.4 vector to obtain an amino acid sequence shown as SEQ ID NO. 8. The amino acid sequence is transformed to obtain the nucleotide sequence of the expression frame corresponding to the expression vector of the light chain antibody.

[0092] (3) The light chain plasmid and the heavy chain plasmid are transformed into 293 cells at a ratio of 1:1, and then the antibody is expressed and purified.

[0093] 2.2 SDS-PAGE of the anti-human CD14 antibody

[0094] (1) Take 4 μL of loading buffer and add it to 16 μL of sample, denature at 100 °C for 5-10 min.

[0095] (2) Take 10 μL of sample and load it, 150 V, 45 min, run SDS-PAGE electrophoresis.

[0096] (3) After the end of electrophoresis, turn off the power, take out the glue, put it into a large culture dish, stain, use fast staining solution, room temperature, 10 min, discard the staining solution, decolorize with distilled water overnight.

[0097] (4) Obtain purified CD14 antibody, the results are shown in Figure 2, indicating the size of the anti-human CD14 antibody molecular weight. Band M is Marker; Band 1 is purified anti-human CD14 antibody. In Figure 2, Marker from top to bottom are: 270 kDa, 175 kDa, 130 kDa, 95 kDa, 65 kDa, 50 kDa, 35 kDa, 30 kDa, 15 kDa, 5 kDa.

[0098] 2.3 Anti-human CD14 antibody and human CD14 binding analysis

[0099] (1) Coating: dilute human CD14 antigen protein with coating solution to 1 μg / mL, 100 μL / well, 37 ℃ incubate for 1 h.

[0100] (2) Washing: wash three times with 300 μL / well, 0.05% Tween 20 containing PBS washing solution, shake off the liquid in the plate.

[0101] (3) Blocking: block with 300 μL / well, 2.5% BSA blocking solution, 37 ℃ for 1 h.

[0102] (4) Washing: wash three times with 300 μL / well, 0.05% Tween 20 containing PBS washing solution, shake off the liquid in the plate.

[0103] (5) Dilute the antibody gradient, and sequentially take 100 μL to add to the above antigen-coated wells, use irrelevant antibodies as negative controls, 37 ℃ incubate for 1 h.

[0104] (6) Washing: wash three times with 200 μL / well, 0.05% Tween 20 containing PBS washing solution, shake off the liquid in the plate.

[0105] (7) Add HRP labeled anti-human IgG Fc (anti-Human-Fc-HRP) to the wells with previously incubated samples according to 100 μL / well, 37 ℃ incubate for 1 h.

[0106] (8) Washing: wash three times with 200 μL / well, 0.05% Tween 20 containing PBS washing solution, shake off the liquid in the plate.

[0107] (9) Color development: add 100 μL TMB to each well, and incubate at 37°C for 10 min in the dark.

[0108] (10) Reading: add 50 μL stop solution to each well to stop the color development reaction, and read the OD 450 nm value.

[0109] (11) Data analysis As shown in FIG. 3, the anti-human CD14 antibody and human CD14 antigen have strong binding.

[0110] 2.4 Anti-human CD14 antibody and human CD14-expressing cell binding analysis

[0111] (1) Take 2.00E+06 human CD14 cells (plasmid-transfected 293 cells expressing human CD14 sequences) in a centrifuge tube, centrifuge at 1000 g for 5 min, remove the supernatant, and wash with 1x PBSA three times.

[0112] (2) Add 50 nM of the antibody to the experimental group, and do not add the antibody to the control group, and incubate at 400 rpm for 1 h at room temperature.

[0113] (3) After incubation, centrifuge at 1000 g for 3 min to discard the supernatant, and wash with PBSA three times.

[0114] (5) Add 500 μL of PE-labeled goat anti-human IgG Fc (diluted 1:1000), and incubate at 400 rpm for 1 h at room temperature.

[0115] (6) After incubation, centrifuge at 1000 g for 3 min to discard the supernatant, and wash with PBSA three times.

[0116] (7) Finally, resuspend with 300 μL of PBSA, and perform flow detection.

[0117] (8) Data analysis As shown in FIG. 4: negative control, and FIG. 5: sample to be tested. The results show that the anti-human CD14 antibody has binding ability with human CD14-transfected cells.

[0118] Example 3 Anti-human CD14 antibody and magnetic bead coupling and sorting effect analysis

[0119] 3.1 Magnetic bead preparation

[0120] (1) 2.2 g FeCl3·6H2O and 0.8 g FeCl2·4H2O were dissolved in 40 mL deionized water in a 2:1 ratio, respectively, and sealed in a three-necked flask under the condition of vigorous stirring and inert nitrogen environment. 5 mL of NH4OH was added dropwise to the reaction container, and the reaction was carried out at 85°C for 1 hour. After magnetic separation, the magnetic beads were washed with deionized water three times and dried to obtain the magnetic beads.

[0121] (2) Carboxymethyl dextran coating on the surface of magnetic beads: according to the mass ratio of carboxymethyl dextran to nanometer magnetic beads of 1:5 or 1:10, carboxymethyl dextran aqueous solution and nanometer magnetic beads were added to the flask, stirred and heated at 50°C for 4h, and the magnetic beads were screened by magnetic separation to obtain carboxymethyl dextran coated magnetic beads.

[0122] (3) Coupling of anti-human CD14 antibody: the carboxymethyl dextran coated magnetic beads were washed by magnetic separation using 25 mM pH 6.0 MES buffer as washing solution, 10 μL of EDC (10 mg / mL) and 20 μL of NHS (10 mg / mL) were added, and the carboxyl group was activated at 37°C for 0.5 h. The supernatant was removed by magnetic separation, and the magnetic beads were resuspended to 1 mL using 25 mM pH 7.4 MES buffer. 60 μg of anti-human CD14 antibody was added, and the reaction was carried out at 37°C for 6 h in a rotary incubator. The magnetic beads were washed by magnetic separation, and the blocking solution (PBS solution containing 2% BSA) was added. The magnetic beads were washed with the storage solution (PBS solution containing 1% Tween 20) and stored in the storage solution.

[0123] 3.2 Cell sorting and effect verification

[0124] (1) 30 mL of peripheral blood was taken, and PBMC was extracted by density gradient centrifugation, and the cells were counted. 1.00E+07 PBMC cells were taken in 3 tubes, and the cells were resuspended in 90 μL / tube of PBS, and 2 tubes were added with the magnetic beads of the above step or the existing commercially available magnetic beads, respectively, and the third tube was used as a blank control. Incubate at 4°C for 20 min in the dark. Wash the cells with 1 mL of buffer, centrifuge at 300 x g for 5 min, and resuspend with 500 μL of buffer after discarding the supernatant.

[0125] (2) Place the MS sorting column on the magnetic stand and add 1 mL of PBS to rinse the sorting column. Add the cell suspension to the sorting column in the magnetic field, at this time the cells flowing out of the sorting column are non-target cells. Add 750 μL of PBS to wash the residual cells, and when the liquid drops at the lower end of the sorting column stop, add the washing liquid to the sorting column, repeat twice, and collect the non-target cells flowing out at the same time. When the liquid is completely drained, remove the sorting column from the magnetic stand and place it in a new 1.5 mL centrifuge tube. Add 0.5 mL of PBS to the sorting column, quickly insert the piston into the column body, and push it to the bottom of the sorting column. At this time, the collected liquid is the target cells.

[0126] (3) The collected target cells and non-target cells are detected by flow to obtain the proportion of CD14+ monocytes, and the sorting effect is shown in Figure 6: before sorting, Figure 7: after sorting (the embodiment), and Figure 8: after sorting (commercial product).

[0127] The results show that, based on the same magnetic beads and sorting method, different antibodies, and using the existing commercial product to sort CD14+ monocytes, the monocytes only account for 76.61% of the total sorted cells, and other types of cells account for 16.39% of the total cells. In the embodiment, the corresponding monocytes account for 98.55% of the total cells, indicating that the CD14+ magnetic beads of the embodiment sort out CD14+ monocytes with higher purity, and show better separation effect for monocyte sorting, and better specificity than the existing commercial product.

[0128] In order to obtain monocytes with higher purity, the application screens a yeast library to obtain an anti-human CD14 antibody with a specific sequence, and couples the antibody with the above-mentioned nano-magnetic beads based on the coupling process, so as to form a magnetic bead coating, which can simply and quickly sort out CD14+ monocytes with higher purity from peripheral blood mononuclear cells, and the monocytes account for 98.55% of the total cells, showing excellent effect for separating monocytes, and can be used for basic and clinical research.

[0129] The above embodiments are only for illustrating the technical concept and characteristics of the application, and the purpose is to enable those skilled in the art to understand the content of the application and implement it, and cannot limit the protection scope of the application. Any equivalent changes or modifications made according to the spirit and essence of the application should be covered within the protection scope of the application.

[0130] The corresponding table of the sequence number and specific sequence of the application is as follows:

[0131]

Claims

1. An anti-human CD 14 antibody, characterized in that, comprises a heavy chain and a light chain, an amino acid sequence of the heavy chain variable region comprises a sequence shown in SEQ ID NO. 2, and an amino acid sequence of the light chain variable region comprises a sequence shown in SEQ ID NO.

4.

2. A nucleic acid encoding the anti-human CD14 antibody according to claim 1.

3. The nucleic acid of claim 2, wherein An amino acid sequence of the heavy chain variable region of the antibody comprises a sequence shown in SEQ ID NO. 2, and an amino acid sequence of the light chain variable region of the antibody comprises a sequence shown in SEQ ID NO.

4.

4. An anti-human CD 14 antibody, characterized in that, An amino acid sequence of the heavy chain variable region of the antibody comprises a sequence shown in SEQ ID NO. 2, and an amino acid sequence of the light chain variable region of the antibody comprises a sequence shown in SEQ ID NO.

4.

5. A nucleic acid encoding the anti-human CD14 antibody according to claim 4.

6. A recombinant expression vector comprising the nucleic acid according to claim 2, claim 3 or claim 5.

7. The recombinant expression vector according to claim 6, characterized in that, An amino acid sequence of the heavy chain variable region of the antibody comprises a sequence shown in SEQ ID NO. 2, and an amino acid sequence of the light chain variable region of the antibody comprises a sequence shown in SEQ ID NO.

4.

8. The recombinant expression vector according to claim 7, characterized in that, An amino acid sequence of the heavy chain variable region of the antibody comprises a sequence shown in SEQ ID NO. 2, and an amino acid sequence of the light chain variable region of the antibody comprises a sequence shown in SEQ ID NO.

4.

9. The recombinant expression vector according to claim 8, characterized in that, An amino acid sequence of the heavy chain variable region of the antibody comprises a sequence shown in SEQ ID NO. 2, and an amino acid sequence of the light chain variable region of the antibody comprises a sequence shown in SEQ ID NO.

4.

10. The recombinant expression vector according to claim 9, characterized in that, An amino acid sequence of the heavy chain variable region of the antibody comprises a sequence shown in SEQ ID NO. 2, and an amino acid sequence of the light chain variable region of the antibody comprises a sequence shown in SEQ ID NO.

4.

11. A cell transformed with the recombinant expression vector according to any one of claims 6-10.

12. Use of the antibody according to claim 1 or 4, the nucleic acid according to claim 2, 3 or 5, the recombinant expression vector according to any one of claims 6-10, or the cell according to claim 11 in the preparation of a medicament or a kit for treating a related disease.

13. A pharmaceutical composition comprising the antibody according to claim 1 or 4, and / or the nucleic acid according to claim 2, 3 or 5, and / or the recombinant expression vector according to any one of claims 6-10, in combination with one or more pharmaceutically acceptable excipients.

14. A magnetic bead, characterized by The magnetic beads are coupled with the anti-human CD14 antibody according to claim 1 or 4.

15. A method of producing magnetic beads as claimed in claim 14, characterized in that The method comprises the following steps: FeCl3·6H2O and FeCl2·4H2O are dissolved in deionized water, NH4OH is added, and after magnetic separation, the magnetic beads are washed with deionized water and dried to obtain the nano magnetic beads; The carboxyl dextran aqueous solution and the nano magnetic beads are mixed and heated to react, and the magnetic beads are screened by magnetic separation to obtain the carboxyl dextran-coated magnetic beads; Using buffer as washing liquid, using magnetic separation to wash carboxyl dextran coated magnetic beads, adding EDC and NHS to activate carboxyl; removing supernatant by magnetic separation, resuspending with buffer again; Adding anti-human CD14 antibody as claimed in claim 1 or 4, incubating the reaction; using magnetic separation, adding blocking liquid, washing with storage liquid and storing in storage liquid.

16. Use of the magnetic beads as claimed in claim 14 in sorting CD14 monocytes.

17. A method of sorting CD14 monocytes, characterized by, The method comprises the following steps: Extracting peripheral blood mononuclear cells, resuspending, adding magnetic beads as claimed in claim 14, mixing, incubating in dark, adding buffer to wash cells, centrifuging to remove supernatant, resuspending with buffer to obtain cell suspension; Placing the sorting column on a magnetic stand, wetting the sorting column; adding cell suspension to the sorting column in magnetic field, at this time the cells flowing out of the sorting column are non-target cells; adding PBS to wash residual cells, when the liquid drops at the lower end of the sorting column stop dropping, adding washing liquid to the sorting column, repeating, while collecting the non-target cells flowing out; when the liquid is completely drained, taking the sorting column off the magnetic stand, placing it in a new collection tube, adding PBS to the sorting column, inserting the piston into the sorting column body and pushing it to the bottom of the sorting column, at this time the collected liquid is target cells; Collecting the target cells and non-target cells and detecting the proportion of CD14 monocytes by flow cytometry.

18. An amino acid sequence of an anti-human CD14 antibody that specifically binds to human CD14, characterized in that, The amino acid sequence of the variable region of the heavy chain and the amino acid sequence of the variable region of the light chain; the amino acid sequence of the variable region of the heavy chain comprises the sequence shown in SEQ ID NO. 2; the amino acid sequence of the variable region of the light chain comprises the sequence shown in SEQ ID NO.

4.

19. A nucleotide sequence of an anti-human CD14 antibody which specifically binds to human CD14, characterized in that, The nucleotide sequence encoding the variable region of the heavy chain and the nucleotide sequence encoding the variable region of the light chain; the nucleotide sequence encoding the variable region of the heavy chain comprises the sequence shown in SEQ ID NO. 1, and the nucleotide sequence encoding the variable region of the light chain comprises the sequence shown in SEQ ID NO. 3.

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