CN116444673A - Humanized membrane CD14 monoclonal antibody and preparation method and application thereof - Google Patents
Humanized membrane CD14 monoclonal antibody and preparation method and application thereof Download PDFInfo
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- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
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Abstract
The invention relates to the technical field of antibodies, in particular to a humanized membrane CD14 monoclonal antibody, a preparation method and application thereof. Antibodies provided by the inventionThe amino acid sequences of the heavy chain complementarity determining regions CDR1, CDR2 and CDR3 of the antigen binding fragment are shown as SEQ ID NO.1-3 respectively, and the amino acid sequences of the light chain complementarity determining regions CDR1, CDR2 and CDR3 are shown as SEQ ID NO.4-6 respectively. The human membrane CD14 antibody can specifically recognize and bind human membrane CD14 protein and CD14 + Cells with high affinity for CD14 + Flow cytometry detection of cells on human membrane CD14 protein and CD14 + Has wide application prospect in cell detection.
Description
Technical Field
The invention relates to the technical field of antibodies, in particular to a humanized membrane CD14 monoclonal antibody, a preparation method and application thereof.
Background
CD14, LPS (Lipopolysaccharide) acceptor, is one kind of glycoprotein with biological function of mainly recognizing, combining LPS or LPS/LBP complex, mediating LPS induced cell reaction and important function in LPS inflammatory reaction, endotoxin shock, etc. CD14 includes both membrane CD14 (mCD 14) and soluble CD14 (sCD 14) types. Wherein mCD14 is mainly distributed on the surfaces of monocytes, macrophages, neutrophils and the like, can be combined with LPS/LBP complex, and mediates the stimulation of LPS on cells.
Monoclonal antibody technology (monoclonal antibody technique) was invented in 1975 by the uk scientist Milstein and Kohler on the principle that: b lymphocytes are capable of producing antibodies, but are unable to divide indefinitely in vitro; while tumor cells can be passaged indefinitely in vitro, they are unable to produce antibodies. The hybridoma obtained by fusing the two cells has the characteristics of the two parent cells. The monoclonal antibody obtaining process comprises animal immunization, cell fusion, cell screening, cloning, characteristic identification and the like, can recognize specific single antigen epitope and has high specificity.
For human membrane CD14 (hmCD 14), there is still a lack of human membrane CD14 antibodies that can bind to it with high efficiency and are suitable for detection by flow cytometry and the like.
Disclosure of Invention
The invention provides a humanized membrane CD14 antibody, a preparation method and application thereof.
The invention provides an antibody or an antigen binding fragment thereof, wherein the amino acid sequences of a heavy chain complementarity determining region CDR1, a CDR2 and a CDR3 of the antibody or the antigen binding fragment thereof are respectively shown in SEQ ID NO.1-3, and the amino acid sequences of a light chain complementarity determining region CDR1, a CDR2 and a CDR3 are respectively shown in SEQ ID NO. 4-6.
The above-mentioned antibody or antigen-binding fragment thereof is capable of specifically recognizing and binding to human membrane CD14 protein and cells expressing human membrane CD14 protein (CD 14) + Cells) with a higher affinity.
Preferably, the amino acid sequence of the heavy chain variable region of the antibody or antigen binding fragment thereof is shown as SEQ ID NO.7 or has at least 80% similarity to the amino acid sequence shown as SEQ ID NO.7, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO.8 or has at least 80% similarity to the amino acid sequence shown as SEQ ID NO. 8.
In some embodiments of the invention, the amino acid sequence of the heavy chain variable region of the antibody or antigen binding fragment thereof is shown in SEQ ID NO.7 and the amino acid sequence of the light chain variable region is shown in SEQ ID NO. 8.
In the case of the heavy chain complementarity determining regions CDR1, CDR2, CDR3 and light chain complementarity determining regions CDR1, CDR2, CDR3 described above, the amino acid sequence of the heavy chain variable region is an amino acid sequence corresponding to an amino acid sequence having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5% sequence similarity to the amino acid sequence shown as SEQ ID No.7, and the amino acid sequence of the light chain variable region is an antibody or antigen binding fragment thereof corresponding to an amino acid sequence having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 99.5% sequence similarity to the amino acid sequence shown as SEQ ID No. 8.
The antibody or antigen-binding fragment thereof described above is any one selected from the group consisting of monoclonal antibodies, fab ', F (ab') 2, fd, fv, dAb, complementarity determining region fragments, and single chain antibodies.
Among them, monoclonal antibodies include animal-derived antibodies (e.g., murine antibodies), chimeric antibodies, humanized antibodies, and the like.
The present invention provides bispecific or multispecific antibodies comprising an antibody or antigen-binding fragment thereof described above.
The present invention provides a nucleic acid molecule encoding an antibody or antigen binding fragment thereof as described above.
Based on the amino acid sequence and codon regularity of the above-described antibodies or antigen-binding fragments thereof, one skilled in the art can obtain the nucleotide sequence of a nucleic acid molecule encoding the above-described antibodies or antigen-binding fragments thereof. Because of the degeneracy of the codons, the nucleotide sequences of the nucleic acid molecules encoding the antibodies or antigen binding fragments thereof are not unique, and all nucleic acid molecules capable of encoding the antibodies or antigen binding fragments thereof are within the scope of the invention.
In some embodiments of the invention, the nucleotide sequence of the nucleic acid molecule encoding the heavy chain variable region is set forth in SEQ ID NO. 9; the nucleotide sequence of the nucleic acid molecule encoding the light chain variable region is shown in SEQ ID NO. 10.
The present invention provides a biological material comprising the nucleic acid molecule described above, which biological material is an expression cassette, a vector or a host cell.
The expression cassette can be obtained by operably linking the nucleic acid molecules described above with regulatory elements such as promoters, terminators, and the like.
The vectors described above include, but are not limited to, plasmid vectors, viral vectors, and the like.
The host cells described above include microbial cells or animal cells. Wherein the microbial cells include, but are not limited to, E.coli, yeast, etc., and the animal cells include, but are not limited to, CHO cells, 293T cells, etc.
The invention provides an antibody conjugate, which is obtained by coupling the antibody or antigen binding fragment thereof or the bispecific antibody or multispecific antibody with a label or protein; the marker is one or more selected from chemiluminescent dye markers, enzyme markers, biotin markers, fluorescent dye markers, colloidal gold markers and radioactive markers.
The antibodies or antigen-binding fragments thereof provided herein can be prepared by methods conventional in the art, including: chemical synthesis, host expression, and the like.
The present invention provides a method of preparing an antibody or antigen-binding fragment thereof as described above, the method comprising: culturing a host cell capable of expressing the antibody or antigen-binding fragment thereof, and isolating the antibody or antigen-binding fragment thereof.
The present invention provides the use of any of the antibodies or antigen binding fragments thereof or the bispecific or multispecific antibodies or the nucleic acid molecules or the biological material or the antibody conjugates described above:
(1) Use in the preparation of a product for detecting the presence or level of human membrane CD14 protein or cells expressing human membrane CD14 protein in a sample;
(2) Use in detecting the presence or level of human membrane CD14 protein in a sample;
(3) Use in detecting the presence or level of cells expressing human membrane CD14 protein in a sample;
(4) Use in the manufacture of a medicament for alleviating an inflammatory response mediated by LPS and/or endotoxin shock.
In the above (1), the product may be a detection reagent or a kit.
In the above (1), (2) and (3), the sample may be a sample derived from a living human or animal (including blood or the like), or may be a sample derived from a non-living human or animal such as cells or cell culture medium cultured in vitro.
For the above (2) and (3), detection for the purpose of diagnosis and treatment of non-disease is preferable.
In the applications described in (1), (2) and (3), the method for detecting an antibody or an antigen-binding fragment thereof provided by the present invention is preferably flow cytometry.
The present invention provides a product comprising an antibody or antigen binding fragment thereof as described above, or comprising said bispecific or multispecific antibody, or comprising said antibody conjugate; the product is a detection reagent or a pharmaceutical composition.
The invention has the beneficial effects that: the human-derived membrane CD14 antibody provided by the invention can specifically recognize and bind human-derived membrane CD14 protein and CD14 + Cells with high affinity for CD14 + Cell flow cytometry and other detection has high sensitivity and specificity, and can be used for detecting human membrane CD14 protein and CD14 + Has wide application prospect in cell detection.
Drawings
In order to more clearly illustrate the invention or the technical solutions of the prior art, the following description will briefly explain the drawings used in the embodiments or the description of the prior art, and it is obvious that the drawings in the following description are some embodiments of the invention, and other drawings can be obtained according to the drawings without inventive effort for a person skilled in the art.
FIG. 1 shows the results of the use of hmCD14 monoclonal antibodies in flow cytometry in example 2 of the present invention, wherein A, B, C, D is the result of the A, B, C, D four-tube assay, respectively.
Detailed Description
For the purpose of making the objects, technical solutions and advantages of the present invention more apparent, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings, and it is apparent that the described embodiments are some embodiments of the present invention, not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Specifically, the invention provides a humanized membrane CD14 (hmCD 14) monoclonal antibody, and the antibody is obtained by animal immunization (taking recombinant hmCD14 protein as an antigen), cell fusion, screening and cloning of positive hybridoma cells, ascites purification and flow cytometry verification. The humanized membrane CD14 monoclonal antibody provided by the invention can effectively bind to hmCD14 and CD14 + Cells, with stable performance, can be used for detecting CD14 by flow cytometry + And (3) cells.
In the invention, the screening of positive hybridoma cells is carried out through double screening of recombinant hmCD14 protein and human microglial cells (HMC 3 cells), and finally, the flow cytometry is adopted to verify, so that the monoclonal antibody can effectively identify natural hmCD14 protein, the problem of later large-scale screening is avoided, the research and development period of the monoclonal antibody is shortened, and the operation is simple.
The reagents and formulations used in the following examples are shown in table 1.
TABLE 1
EXAMPLE 1 acquisition of humanized Membrane CD14 monoclonal antibodies
The invention provides an hmCD14 monoclonal antibody which is obtained by the following steps:
1. immunization of animals
The purified recombinant hmCD14 was diluted to 0.4mg/mL and mixed with water-soluble fast adjuvant (Boolon Quick antibody mouse w) in equal volume, and 6 female Balb/c mice 6 weeks old were inoculated in multiple points on leg muscle at an antigen dose of 20 μg/mouse. After 21d, the injection was again performed, and the amount of immunity was the same as that of the first time. 3d before cell fusion, the recombinant hmCD14 without adjuvant was intraperitoneally injected for immune impact, followed by cell fusion.
2. Establishment of recombinant hmCD14 monoclonal antibody screening method
1. Establishment of recombinant hmCD14 indirect ELISA method
(1) Recombinant hmCD14 was diluted to 1, 5, 10, 15, 20 μg/mL,100 μl/well with carbonate buffer (pH 9.6), respectively, and incubated at 37 ℃ for 1h at 4 ℃ overnight.
(2) Spin-drying the coating liquid and washing the PBST for 3 times.
(3) 5% skim milk blocking solution, 250. Mu.L/well, was added and incubated at 37℃for 1h.
(4) The same PBST washing 3 times.
(5) Negative and positive serum were diluted 1:1000, 1:2000, 1:4000, 1:8000, 1:16000, 1:32000, 1:64000 and 1:128000 with 5% skim milk, 100 μl/well, respectively, and incubated at 37 ℃ for 1h.
(6) The same PBST was washed 3 times.
(7) Goat anti-mouse IgG-HRP was diluted 1:10000 times with skim milk, 100. Mu.L/well, and incubated at 37℃for 0.5h.
(8) PBST was washed 4 times.
(9) Each well was added with a freshly prepared TMB developing solution at 100. Mu.L/well and developed for 10min at room temperature in the dark.
(10) Add 1M H 2 SO 4 The reaction was stopped at 50. Mu.L/well.
(11) Measuring OD of each well by enzyme labeling instrument 450 。
The optimal coating concentration of recombinant hmCD14 protein, as well as the optimal dilutions of negative and positive serum, were determined by the above experiments.
2. Establishment of HMC3 cell indirect ELISA method
(1) HMC3 cells were diluted to 4X 10 with DMEM medium containing 10% FBS, respectively 6 3X 10 per mL 6 Per mL, 2X 10 6 Individual/mL and 1X 10 6 Each 100. Mu.L/well of 96-well culture plate was added thereto, and the mixture was placed at 37℃and 5% CO 2 Overnight in a saturated humidity incubator.
(2) Spin-drying the culture solution, adding cell fixing solution, 100 μl/well, and standing at room temperature for 15min.
(3) Spin-drying 4% formaldehyde solution, and washing with PBST for 3 times.
(4) 5% skim milk blocking solution, 250. Mu.L/well, was added and incubated at 37℃for 1h.
(5) Negative and positive serum were diluted 1:1000, 1:2000, 1:4000, 1:8000, 1:16000, 1:32000, 1:64000 and 1:128000 with 5% skim milk, 100 μl/well, respectively, and incubated at 37 ℃ for 1h, as per PBST wash 3 times above.
(6) The same PBST was washed 3 times.
(7) Goat anti-mouse IgG-HRP was diluted 1:10000 with skim milk, 100. Mu.L/well, and incubated at 37℃for 0.5h.
(8) PBST was washed 5 times.
(9) Each well was added with a freshly prepared TMB developing solution at 100. Mu.L/well and developed at 37℃for 30min in the dark.
(10) Add 1M H 2 SO 4 The reaction was stopped at 50. Mu.L/well.
(11) Automatic enzyme-labeled instrument for measuring OD of each hole 450 。
The optimal coating concentration of HMC3 cells, as well as the optimal dilutions of negative and positive serum, were determined by the above experiments.
3. Preparation of sp2/0 myeloma cells
Taking out sp2/0 cells in liquid nitrogen tank, thawing in 37deg.C water, centrifuging at 1000rpm for 5min, discarding supernatant, re-suspending with DMEM complete culture solution, transferring into cell bottle, thawing at 37deg.C, and concentrating with 5% CO 2 Culturing in a saturated humidity incubator. The sp2/0 cells with uniform cell morphology, clear boundary and good growth state are taken in the day of fusion, the culture solution is discarded, the culture solution is washed for 2 times, then 20mL of the DMEM culture solution is used for resuspension of the cells, and then the cells are counted and placed at 4 ℃ for standby.
4. Preparation of feeder cells
Before fusion, selecting 2 non-immunized 10-week-old Kunming female mice, killing cervical dislocation, soaking in 75% alcohol for 5min, sterilizing, cutting off abdominal skin under aseptic condition, and fully exposing abdomen; lifting the peritoneum with forceps, injecting 8mL of HAT culture solution into the abdominal cavity with a syringe, gently pressing the abdominal cavity with an alcohol cotton ball, finally sucking the culture solution in the abdominal cavity with the syringe, counting cells, and re-suspending with HAT culture solution to regulate cell number to 2×10 5 Each 100. Mu.L/well of 96-well plates were incubated at 37℃in 5% CO 2 The mixture was placed in a saturated humidity incubator overnight, and whether contamination was observed.
5. Preparation of spleen cells
The titers of the serum of immunized mice were determined by indirect ELISA, and when the hmCD14 antibody titers in the serum reached 16000, the recombinant hmCD14 was boosted once. After 3d, the mice were collected from their eyeballs, and the blood was centrifuged at 37℃for 0.5h, 4℃for 1h,4000rpm for 5min, and the separated serum was used as positive serum. Mice were sacrificed, and after 5min of sterilization by 75% alcohol, spleens were removed under aseptic conditions and ground on a 70 μm cell sieve. The obtained spleen cells were washed 2 times with DMEM medium (1000 rpm, centrifugation for 8 min), and then the cells were suspended in 20mL of DMEM medium and counted.
6. Cell fusion
1. The prepared sp2/0 myeloma cells and spleen cells are mixed in a ratio of 1:5-1:10, placed in a 50mL centrifuge tube, washed 3 times with DMEM culture solution, centrifuged at 1000rpm for 8min, and the supernatant is sucked dry after the last centrifugation, and the bottom of the centrifuge tube is tapped with fingers to loosen the cells.
2. The centrifuge tube was placed in a 37℃water bath, 1mL of 37℃preheated PEG1450 (Sigma) was pipetted into the centrifuge tube at constant speed, gently stirred while adding, completed within 1min, and allowed to stand for 1min.
3. 30mL of DMEM medium is slowly added, 1mL is added, 2 mL is added, 3min is added, 5mL is added, 4min is added, 7mL is added, and all 5min is added.
4. Centrifuge at 1000rpm for 8min and discard supernatant.
5. The HAT culture solution is used for suspending the fused cells, the action is gentle, the cells which are just fused are prevented from being blown away, 60-100mL of HAT culture solution is added, 96-well cell culture plates are paved, and 6-10 cells can be paved.
6. The cell culture plate was placed at 37℃in 5% CO 2 Culturing in saturated humidity incubator, mixing for 3d, and changing with HAT culture solution for half amount, and changing into HT culture solution for 10 d.
7. Establishment of hmCD14 hybridoma cell line
1. Screening of positive hybridoma cells
And 10d, HT culture solution is used instead, and detection is carried out when the hybridoma cells grow to 1/4 of the bottom of the hole. Coating the recombinant hmCD14 and HMC3 cells on an ELISA plate and a 96-well culture plate according to the optimal coating concentration respectively, taking the culture supernatant of the hybridoma cells in the same well after 2d of liquid exchange, respectively reacting with the recombinant hmCD14 and HMC3 cells, taking sp2/0 cell culture supernatant as a negative control, taking positive mouse serum as a positive control, and selecting the hybridoma cell well reacted with both the recombinant hmCD14 and HMC3 cells as a positive cell strain for next subcloning.
2. Subcloning cells
Preparing feeder cells according to the fourth step, and plating 96-well plates with 100 μl/well of feeder cells obtained by washing abdominal cavity of mice with HT nutrient solution, placing 96-well plates at 37deg.C and 5% CO 2 Culturing overnight in a saturated humidity incubator. The cells in the hybridoma cell wells detected as positive were blown up and mixed uniformly, 10. Mu.L of cells were taken, the cells were counted after 10-fold dilution, 100 cells were calculated from the corresponding wells and added to 10mL of HT medium, and the cells were inoculated into 96-well cell culture plates plated with feeder cells at 100. Mu.L/well, i.e., about 1 cell per well. The cell plates were exposed to 5% CO at 37 ℃ 2 Culturing in a saturated humidity incubator. After 4d, observing the clone number of each hole, when 7-10d cells grow to 1/4 hole bottom, performing ELISA detection, and taking 2 times of monoclonal holes positive in detection for subcloning. After 3 subcloning until the detection positive rate of all cloned cell holes is 100%, the hybridoma cell strain capable of stably secreting monoclonal antibodies can be determined.
3. Cryopreservation and resuscitation of hmCD14 hybridoma cells
And (3) performing amplification culture on the final clone which is positive and is observed to be a single clone hole under a microscope, blowing off cells in the hole into a 6-hole plate, and transferring the cells into a 50mL cell bottle for culture after the cells in the 6-hole plate are full. After the cells grow to 80% -90%, subculturing, and simultaneously freezing hybridoma cell strains, wherein each strain is frozen for 3 tubes. Taking cells in good state in growth log phase, blowing off, mixing, centrifuging at 1000rpm for 8min, discarding supernatant, and re-suspending cells with frozen stock solution to reach cell number of 1×10 6 Transferring the mixture into a freezing tube, placing 1 mL/tube into a program cooling box at-70 ℃ overnight, and transferring the mixture into liquid nitrogen for preservation the next day.
4. Ascites preparation
Taking 10-week-old Balb/c female mice, and injecting into abdominal cavity0.5mL of liquid paraffin was introduced. After 7d, the abdominal cavity was inoculated with 1X 10 6 Individual/hybridoma cells. Before inoculation, hybridoma cells were washed twice with DMEM medium at a concentration of 1X 10 7 Each mL was taken as 0.1mL of the cell suspension, and injected into the abdominal cavity of the mouse. After 7d, ascites can be generated, and the ascites can be collected by suction of a syringe. The collected ascites was centrifuged at 3000rpm for 5min, and the supernatant was collected.
8. Purification of hmCD14 monoclonal antibody
1. The ascites is centrifuged at 12000rpm for 5min, the supernatant is collected and the precipitate is discarded.
2. The centrifuged ascites was filtered with a 0.22 μm filter.
3. The Protein A column was rinsed with 10 volumes of PBS of 1mL/min flow rate.
4. The ascites after filtration is added into a chromatographic column with the flow rate of 1mL/min.
5. The Protein A column was rinsed with 10 volumes of PBS of 1mL/min flow rate.
6. Eluting with glycine-hydrochloric acid (pH 3.0, 0.1M), collecting eluate with 1.5mL centrifuge tube at a flow rate of 1mL/min, collecting 0.5mL each tube, continuously collecting 12 tubes, adding 1M Tris-HCl (pH 8.8) 20 μl each tube after collection, and neutralizing, and performing PAGE-SDS electrophoresis.
7. The purified monoclonal antibody was dialyzed against PBS and dialyzed overnight at 4℃and quantified using the BCA quantification kit, and the concentration was adjusted to 1mg/mL.
Sequencing the hmCD14 monoclonal antibody, and sequencing the results show that the amino acid sequences of the heavy chain complementarity determining regions CDR1, CDR2 and CDR3 of the hmCD14 monoclonal antibody are shown as SEQ ID NO.1-3, the amino acid sequences of the light chain complementarity determining regions CDR1, CDR2 and CDR3 are shown as SEQ ID NO.4-6, the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO.7, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 8. The nucleotide sequence of the nucleic acid molecule encoding the heavy chain variable region is shown in SEQ ID NO. 9; the nucleotide sequence of the nucleic acid molecule encoding the light chain variable region is shown in SEQ ID NO. 10.
8. hmCD14 monoclonal antibody titer detection
(1) Recombinant hmCD14 was diluted to 1 μg/mL with carbonate buffer (pH 9.6), 100 μl/well, incubated at 37 ℃ for 1h, overnight at 4 ℃.
(2) Spin-drying the coating liquid and washing the PBST for 3 times.
(3) 5% skim milk blocking solution, 250. Mu.L/well, was added and incubated at 37℃for 1h.
(4) The same PBST washing 3 times.
(5) 1mg/mL of hmCD14 monoclonal antibody and 1mg/mL of human CD4 polyclonal antibody (negative control) were diluted with 5% skim milk, 100. Mu.L/well, respectively, and incubated at 37℃for 1h at 1:1000, 1:2000, 1:4000, 1:8000, 1:16000, 1:32000, 1:64000 and 1:128000.
(6) The same PBST was washed 3 times.
(7) Sheep anti-mouse IgG-HRP was diluted 1:10000 times with skim milk, 100. Mu.L/well and incubated at 37℃for 0.5h.
(8) PBST was washed 4 times.
(9) Each well was added with a freshly prepared TMB developing solution at 100. Mu.L/well and developed for 10min at room temperature in the dark.
(10) Add 1M H 2 SO 4 The reaction was stopped at 50. Mu.L/well.
(11) Measuring OD of each well by enzyme labeling instrument 450 。
The results are shown in Table 2, with hmCD14 monoclonal antibody titers of 1:128000.
TABLE 2
EXAMPLE 2 humanized Membrane CD14 monoclonal antibodies for flow cytometry detection
The humanized membrane CD14 monoclonal antibody prepared in example 1 is used for flow cytometry detection, and the specific method is as follows:
1. 4 centrifuge tubes (1.5 mL) were used, A, B, C, D labeled, and the PBMC concentration was adjusted to 1X 10 6 Each tube was filled with 0.5mL of PBS (1 mL), centrifuged at 350 and g for 5min, and the supernatant was removed.
2. The cells were resuspended in 200. Mu.L PBS by addition to the A-tube.
3. The B tube was resuspended in 100. Mu.L PBS, then 2. Mu.L anti-mouse IgG-PE flow antibody (BioLegend), thoroughly mixed, incubated in the dark for 15min, 1mL PBS stopped, centrifuged for 5min at 350g, the supernatant removed, and 200. Mu.L PBS was added to resuspend the cells.
4. 100. Mu.L of PBS was added to the C tube to resuspend the cells, then 2. Mu.L of anti-hCD14-PE flow antibody (BioLegend) was added, mixed well, incubated for 15min in the dark, 1mL of PBS stopped, centrifuged for 5min at 350g, the supernatant removed, and 200. Mu.L of PBS was added to resuspend the cells.
5. The D tube was resuspended in 100. Mu.L PBS, 1. Mu.L hmCD14 monoclonal antibody was added, mixed well and incubated for 30min at room temperature. Adding 1mLPBS, mixing, centrifuging at 350g for 5min, removing supernatant, and washing for 2 times. 100 u L PBS heavy suspension cells, adding 2 u L anti-mouse IgG-PE flow antibody, fully mixing, light shielding incubation for 15min, 1mL PBS termination reaction, 350g centrifugal 5min, supernatant, adding 200 u L PBS heavy suspension cells.
6. 4-tube cells were mechanically examined.
The detection results are shown in fig. 1, and the results show that: the ratio of the positive cells of the B tube is 0.61%, the ratio of the positive cells of the D tube is 1.73%, and the calculated true ratio of the positive cells of the D tube is 1.12%, which is approximately equal to the result of the C tube (1.20%), thus indicating that the monoclonal antibody of the hmCD14 can effectively recognize the CD14 + Cells, which can be used for flow cytometry detection.
Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present invention, and are not limiting; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit and scope of the technical solutions of the embodiments of the present invention.
Claims (10)
1. The antibody or antigen binding fragment thereof is characterized in that the amino acid sequences of the heavy chain complementarity determining regions CDR1, CDR2 and CDR3 of the antibody or antigen binding fragment thereof are respectively shown in SEQ ID NO.1-3, and the amino acid sequences of the light chain complementarity determining regions CDR1, CDR2 and CDR3 are respectively shown in SEQ ID NO. 4-6.
2. The antibody or antigen-binding fragment thereof of claim 1, wherein the heavy chain variable region of the antibody or antigen-binding fragment thereof has an amino acid sequence as shown in SEQ ID No.7 or at least 80% similarity to the amino acid sequence as shown in SEQ ID No.7, and the light chain variable region has an amino acid sequence as shown in SEQ ID No.8 or at least 80% similarity to the amino acid sequence as shown in SEQ ID No. 8.
3. The antibody or antigen-binding fragment thereof according to claim 1 or 2, wherein the antibody or antigen-binding fragment thereof is selected from the group consisting of monoclonal antibodies, fab ', F (ab') 2 Fd, fv, dAb, a complementarity determining region fragment, a single chain antibody.
4. A bispecific or multispecific antibody comprising an antibody or antigen-binding fragment thereof according to any one of claims 1 to 3.
5. A nucleic acid molecule encoding the antibody or antigen-binding fragment thereof of any one of claims 1-3.
6. A biological material comprising the nucleic acid molecule of claim 5, wherein the biological material is an expression cassette, a vector or a host cell.
7. An antibody conjugate, which is characterized in that the antibody or the antigen binding fragment thereof according to any one of claims 1 to 3 or the bispecific antibody or the multispecific antibody according to claim 4 is conjugated with a label or a protein;
the marker is one or more selected from chemiluminescent dye markers, enzyme markers, biotin markers, fluorescent dye markers, colloidal gold markers and radioactive markers.
8. A method of producing an antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, comprising: culturing a host cell capable of expressing the antibody or antigen-binding fragment thereof, and isolating the antibody or antigen-binding fragment thereof.
9. Use of the antibody or antigen binding fragment thereof of any one of claims 1-3 or the bispecific or multispecific antibody of claim 4 or the nucleic acid molecule of claim 5 or the biological material of claim 6 or the antibody conjugate of claim 7 for any one of the following:
(1) Use in the preparation of a product for detecting the presence or level of human membrane CD14 protein or cells expressing human membrane CD14 protein in a sample;
(2) Use in detecting the presence or level of human membrane CD14 protein in a sample;
(3) Use in detecting the presence or level of cells expressing human membrane CD14 protein in a sample;
(4) Use in the manufacture of a medicament for alleviating an inflammatory response mediated by LPS and/or endotoxin shock.
10. A product comprising the antibody or antigen-binding fragment thereof of any one of claims 1-3, or comprising the bispecific or multispecific antibody of claim 4, or comprising the antibody conjugate of claim 7;
the product is a detection reagent or a pharmaceutical composition.
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