CN116970082B - Humanized CD7 monoclonal antibody and preparation method and application thereof - Google Patents
Humanized CD7 monoclonal antibody and preparation method and application thereof Download PDFInfo
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- C—CHEMISTRY; METALLURGY
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- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- 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
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
- C07K2317/565—Complementarity determining region [CDR]
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Abstract
The invention relates to the technical field of antibodies, in particular to a humanized CD7 monoclonal antibody, a preparation method and application thereof. The antibody or antigen binding fragment thereof provided by the invention specifically defines the amino acid sequences of heavy chain complementarity determining regions CDR-H1, CDR-H2, CDR-H3 and light chain complementarity determining regions CDR-L1, CDR-L2, CDR-L3. The human CD7 antibody can specifically recognize and bind human CD7 protein and CD7 + Cells with higher affinity for CD7 + Flow cytometry detection of cells, in human CD7 proteins and CD7 + Has wide application prospect in cell detection.
Description
Technical Field
The invention relates to the technical field of antibodies, in particular to a humanized CD7 monoclonal antibody, a preparation method and application thereof.
Background
CD7 (T cell antigen 7, also known as GP40, TP41, LEU-9) is a single-chain transmembrane glycoprotein, 40kDa, comprising 240 amino acid residues, belonging to the immunoglobulin superfamily members. CD7 is typically expressed in 85% of peripheral blood T cells and NK cells and their precursors, a costimulatory receptor protein that aids T cell activation and interactions with other immune subpopulation 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 CD7 (hCD 7), there is still a lack of human CD7 antibodies capable of binding thereto with high efficiency and suitable for detection by flow cytometry and the like.
Disclosure of Invention
In view of the problems in the background art, the invention provides a humanized CD7 antibody, and a preparation method and application thereof.
Specifically, the technical scheme of the invention is as follows:
in a first aspect, the present invention provides an antibody or antigen binding fragment thereof having the amino acid sequence of heavy chain complementarity determining region CDR-H1 shown in SEQ ID NO.1, the amino acid sequence of heavy chain complementarity determining region CDR-H2 shown in SEQ ID NO.2, and the amino acid sequence of heavy chain complementarity determining region CDR-H3 shown in SEQ ID NO. 3; the amino acid sequence of the light chain complementarity determining region CDR-L1 is shown as SEQ ID NO.4, the amino acid sequence of the light chain complementarity determining region CDR-L2 is shown as SEQ ID NO.5, and the amino acid sequence of the light chain complementarity determining region CDR-L3 is shown as SEQ ID NO. 6.
The above-mentioned antibody or antigen-binding fragment thereof is capable of specifically recognizing and binding to human CD7 protein and cells expressing human CD7 protein (CD 7) + Cells), has a high 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 CDR-H1, CDR-H2, CDR-H3 and light chain complementarity determining regions CDR-L1, CDR-L2, CDR-L3 described above, the amino acid sequence of the heavy 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 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 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 99.5% sequence similarity to the amino acid sequence shown as shown in 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.
In a second aspect, the invention provides a bispecific or multispecific antibody comprising an antibody or antigen-binding fragment thereof as described above.
In a third aspect, the 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 a fourth aspect, the invention provides a biological material comprising a nucleic acid molecule as described above, said biological material being 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.
In a fifth aspect, the present invention provides an antibody conjugate obtained by coupling an antibody or antigen-binding fragment thereof as described above or the bispecific or multispecific antibody with a label or protein.
Preferably, the label is selected from one or more of chemiluminescent dye label, enzyme label, biotin label, fluorescent dye label, colloidal gold label, and radioactive label.
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.
In a sixth aspect, 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.
In a seventh aspect, the 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 a human CD7 protein or a cell expressing a human CD7 protein in a sample;
(2) Use in detecting the presence or level of a CD7 protein of human origin in a sample;
(3) Use in detecting the presence or level of cells expressing a human CD7 protein in a sample.
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.
In an eighth aspect, the invention provides a product comprising an antibody or antigen binding fragment thereof as described above, or comprising the bispecific or multispecific antibody, or comprising the antibody conjugate; the product is a detection reagent or a pharmaceutical composition.
The invention has the beneficial effects that: the human CD7 antibody provided by the invention can specifically recognize and bind human CD7 protein and CD7 + Cells with higher affinity for CD7 + Cell flow cytometry and other detection has high sensitivity and specificity, and can be used for detecting human CD7 protein and CD7 + 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 hCD7 monoclonal antibodies in flow cytometry in example 2 of the present invention, wherein A, B, C, D is the result of the detection in A, B, C, D four tubes, 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 human CD7 (hCD 7) monoclonal antibody, and the antibody is obtained by animal immunization (taking recombinant hCD7 protein as an antigen), cell fusion, screening and cloning of positive hybridoma cells, and ascites purification and flow cytometry verification. The humanized CD7 monoclonal antibody provided by the invention can effectively bind hCD7 and CD7 + The cells have stable performance and can be used for detecting CD7 by flow cytometry + And (3) cells.
In the invention, positive hybridoma cells are screened by recombinant hCD7 protein and then verified by flow cytometry, so that the monoclonal antibody can effectively identify natural hCD7 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 human CD7 monoclonal antibodies
The hCD7 monoclonal antibody provided by the invention is obtained by the following steps:
recombinant hCD7 expression purification
(1) hCD7 recombinant plasmid was transfected into HEK293 cells at 100ml with 5% CO 2 Constant temperature shaking table, 37 ℃, 120rpm constant temperature shaking culture 6dThe culture was stopped.
(2) The cell fluid was placed at 1000rpm, centrifuged for 5min, the supernatant was aspirated, and filtered through a 0.45um filter.
(3) The supernatant pH was adjusted to 7.4 using 1M Tris-HCl (pH 8.8).
(4) The pure instrument was turned on and the column was rinsed with 10mL of 1 XPBS.
(5) The sample was started at a loading flow rate of 1mL/min.
(6) The column was washed with 10mL of 1 XPBS.
(7) hCD7 was eluted and collected, the concentration was measured by a protein quantification apparatus, and hCD7 solutions of high concentration were combined.
(8) The purified hCD7 solution was placed in 1 XPBS and dialyzed overnight at 4 ℃.
(9) hCD7 solution was collected and quantified using BCA protein quantification kit.
2. Immunization of animals
Purified recombinant hCD7 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 spots 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 hCD7 without adjuvant is injected into the abdominal cavity for immune impact, and then cell fusion is carried out.
3. Establishment of recombinant hCD7 indirect ELISA method
(1) Recombinant hCD7 was diluted to 1, 5, 10, 15, 20. Mu.g/mL, 100. Mu.L/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 hCD7 protein, as well as the optimal dilutions of negative and positive serum, were determined by the above experiments.
4. 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.
5. 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.
6. Preparation of spleen cells
The titers of the serum of immunized mice were determined by indirect ELISA, and when the hCD7 antibody titers in the serum reached 16000, recombinant hCD7 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.
7. 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 c water bath, 1ml of 37 c pre-heated PEG1450 (Sigma ) was added at a constant speed to the centrifuge tube, gently stirred while being added, and allowed to stand for 1min after the addition was completed within 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.
8. Establishment of hCD7 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 an ELISA plate with recombinant hCD7 according to the optimal coating concentration, taking hybridoma cell culture supernatant after 2d of liquid exchange, reacting with the recombinant hCD7, taking sp2/0 cell culture supernatant as negative control, taking positive mouse serum as positive control, and selecting hybridoma cell holes reacted with the recombinant hCD7 as positive cell strains 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 hCD7 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
10-week-old Balb/c female mice were intraperitoneally injected with 0.5mL of liquid paraffin. 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 0.1mL of cells were taken per mLThe suspension was injected into the abdominal cavity of the mice. 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.
9. hCD7 monoclonal antibody purification
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 hCD7 monoclonal antibody, wherein the sequencing result shows that the amino acid sequence of a heavy chain complementarity determining region CDR-H1 of the hCD7 monoclonal antibody is shown as SEQ ID NO.1, the amino acid sequence of a heavy chain complementarity determining region CDR-H2 is shown as SEQ ID NO.2, and the amino acid sequence of a heavy chain complementarity determining region CDR-H3 is shown as SEQ ID NO. 3; the amino acid sequence of the light chain complementarity determining region CDR-L1 is shown as SEQ ID NO.4, the amino acid sequence of the light chain complementarity determining region CDR-L2 is shown as SEQ ID NO.5, the amino acid sequence of the light chain complementarity determining region CDR-L3 is shown as SEQ ID NO.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.
8. hCD7 monoclonal antibody titer detection
(1) Recombinant hCD7 was diluted to 1. Mu.g/mL with carbonate buffer (pH 9.6), 100. Mu.L/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 hCD7 monoclonal antibody and 1mg/mL of hCD14 monoclonal 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 hCD7 monoclonal antibody titers of 1:128000.
TABLE 2
EXAMPLE 2 use of humanized CD7 monoclonal antibodies for flow cytometry detection
The human CD7 monoclonal antibody prepared in the 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 0.5mL of PBS was added to each tube at a concentration of 0.5 mL/mL, and the mixture was centrifuged at 350 and g for 5min to remove the supernatant.
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, bioBioLegend, bioBioBioBioBioSec), thoroughly mixed, incubated in the dark for 15 min,1mL PBS stopped, centrifuged for 5min at 350g, the supernatant removed, 200. Mu.L PBS resuspended in cells, 2. Mu.L 7AAD staining solution (BioLegend, bioBioBioSec), thoroughly mixed, incubated in the dark for 15 min.
4. 100. Mu.L PBS was added to the C tube to resuspend the cells, then 2. Mu.L anti-hCD7-PE flow antibody (BioLegend, bioBeech Co., ltd.) was added, mixed well, incubated in the dark for 15 min,1mL PBS stopped the reaction, 350g centrifuged for 5min, the supernatant removed, 200. Mu.L PBS was added to resuspend the cells, then 2. Mu.L 7AAD staining solution (BioLegend, bioBeech Co., ltd.) was added, mixed well, incubated in the dark for 15 min.
5. The D-tube was resuspended in 100. Mu.L PBS, 1. Mu.L of hCD7 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. Mu.L PBS was used to resuspend cells, 2. Mu.L anti-mouse IgG-PE flow antibody was added, mixed well, incubated in the dark for 15 min,1mL PBS stopped, 350g centrifuged for 5min, the supernatant removed, 200. Mu.L PBS was added to resuspend cells, 2. Mu.L 7AAD staining solution (BioLegend, hundred Biotechnology Co., USA) was added, mixed well, and incubated in the dark for 15 min.
6. 4-tube cells were mechanically examined.
The detection results are shown in fig. 1, and the results show that: the positive cell ratio of the D tube is 33.3 percent, which is approximately equal to the result of the C tube (33 percent), which shows that the hCD7 monoclonal antibody can effectively recognize CD7 + 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. An antibody or antigen binding fragment thereof targeting CD7, wherein the amino acid sequence of a heavy chain complementarity determining region CDR-H1 of the antibody or antigen binding fragment thereof is shown as SEQ ID NO.1, the amino acid sequence of a heavy chain complementarity determining region CDR-H2 is shown as SEQ ID NO.2, and the amino acid sequence of a heavy chain complementarity determining region CDR-H3 is shown as SEQ ID NO. 3; the amino acid sequence of the light chain complementarity determining region CDR-L1 is shown as SEQ ID NO.4, the amino acid sequence of the light chain complementarity determining region CDR-L2 is shown as SEQ ID NO.5, and the amino acid sequence of the light chain complementarity determining region CDR-L3 is shown as SEQ ID NO. 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, 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 an antibody or antigen binding fragment thereof according to any one of claims 1-3 or a bispecific or multispecific antibody according to claim 4 or a nucleic acid molecule according to claim 5 or a biological material according to claim 6 or an antibody conjugate according to claim 7 for the preparation of a product for detecting the presence or level of a human CD7 protein or a cell expressing a human CD7 protein in a sample.
10. A reagent or pharmaceutical composition for detecting a human CD7 protein, wherein the reagent or pharmaceutical composition for detecting a human CD7 protein comprises the antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, or comprises the bispecific or multispecific antibody according to claim 4, or comprises the antibody conjugate according to claim 7.
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