WO2019129178A1 - Composition containing anti-cd45 monoclonal antibody, and method for using same - Google Patents

Composition containing anti-cd45 monoclonal antibody, and method for using same Download PDF

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WO2019129178A1
WO2019129178A1 PCT/CN2018/124695 CN2018124695W WO2019129178A1 WO 2019129178 A1 WO2019129178 A1 WO 2019129178A1 CN 2018124695 W CN2018124695 W CN 2018124695W WO 2019129178 A1 WO2019129178 A1 WO 2019129178A1
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mab
antibody
cells
blood
mabs
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PCT/CN2018/124695
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French (fr)
Chinese (zh)
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钱其军
叶真龙
马硕
王欣玥
张晓霞
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上海白泽医学检验所有限公司
上海细胞治疗研究院
上海细胞治疗集团有限公司
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Publication of WO2019129178A1 publication Critical patent/WO2019129178A1/en

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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
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/536—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase
    • 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
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • 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
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
    • G01N33/54326—Magnetic particles
    • 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
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens

Definitions

  • the method of directly capturing by using CTCs surface markers is also called positive phase enrichment. This method is currently the most widely used positive phase enrichment method. The most typical representative is the Cellsearch system. Because CTCs belong to non-blood-derived epithelial cells, some CTCs express epithelial cell-specific molecules, Epithelial cell adhesion molecule (EpCAM), so this method mainly uses epithelial cell surface marker EpCAM antibody and even Linked to a carrier, such as a magnetic bead, to capture EpCAM-positive CTCs directly from the blood sample. However, the data suggest that not all solid tumors have EpCAM-positive expression, such as EpCAM, which is either low or negative on bladder cancer and malignant melanoma cells.
  • EpCAM Epithelial cell adhesion molecule
  • the tumor cells in the primary tumor tissue lose their polar epithelial cell characteristics (adhesive, flaky structure) and transform into interstitial cell characteristics with migration and invasion ability. (No cell polarity, loss of tight junctions between cells and cells).
  • these tumor cells undergo obvious cytoskeletal remodeling, which changes the expression of various EMT-related transcription factors and cell surface receptors. Therefore, the direct capture method using EpCAM is not high in specificity, and can not effectively capture CTCs of various tumors, and the capture efficiency and recovery rate are low.
  • cell surface EpCAM protein antigens are extremely active signaling-inducing factors, so the use of EpCAM antibody-coupled magnetic beads to capture CTCs is likely to trigger and activate a series of intracellular signaling pathways. Therefore, most of the CTCs captured by this method may not be CTCs in the natural state, and the captured CTCs have low cell activity, unable to carry out cell culture in the later stage, and are not conducive to cell separation, and are used in downstream technology. Research.
  • Density gradient centrifugation is used instead of red blood cell lysate to eliminate red blood cells, which greatly reduces the damage to CTCs, and retains the characteristics and characteristics of CTCs. Although this method has high sensitivity, it can effectively separate CTCs in the natural state, but the residual white blood cells are excessive.
  • SE-iFISH is a method in which red blood cells and plasma are removed by density gradient centrifugation, and immunomagnetic beads coupled with various leukocyte subclass antibodies are added to incubate to bind white blood cells, and magnetic force is used to remove white blood cells. effect. However, the final leukocyte remaining amount used to remove leukocytes is usually as high as 5x10 3 -1.5x10 4 /mL.
  • one of the main criteria for identifying CTCs is chromosome 8 polyploid, while macrophages in leukocytes are also polyploids of chromosome 8, and polyploid macrophages are likely to be present in such high leukocyte residuals.
  • the presence of this it will easily lead to the occurrence of false positive CTCs; and excessive white blood cell residue, when the microscope automatically scans and filters the white blood cells with CD45 positive expression, if there is CTC in the white blood cell agglomeration, it is easy to be automatically determined by the scanning system. A false negative thus filters out the cells.
  • the excessive number of residual white blood cells will bring greater difficulties and background effects to downstream CTCs cell sequencing and CTCs culture.
  • the MINDEC method is another negative enrichment method. Compared with the SE-iFISH method, the number of white blood cells remaining in the process using MINDEC is significantly less.
  • This method first uses density gradient centrifugation to remove red blood cells and plasma, and then uses a combination of antibodies against different leukocyte subclasses to capture white blood cells to enrich CTCs. In contrast to the SE-iFISH method, this method is first incubated with a biotin-labeled antibody in a mononuclear cell suspension after removal of red blood cells and plasma, followed by streptavidin. The labeled magnetic beads allow it to bind to the antibody.
  • the MINDEC method has more magnetic bead removal times than SE-iFISH, which has a better effect of removing magnetic beads and white blood cells, and at the same time avoids the risk of CTCs being removed by magnetic beads. Recovery rate of CTCs.
  • the application of MINDEC method has higher leukocyte removal efficiency, and the final number of remaining leukocyte cells is 437 ⁇ 350/mL, which effectively reduces the possibility of false positive and false negative detection. However, because of the different combinations of antibodies against the leukocyte subclass, the amount of antibody required is higher and the cost is higher.
  • the antibody used by MINDEC is a murine monoclonal antibody, and the antibody extracted from the mouse has low affinity for human leukocytes, so the demand for the mouse monoclonal antibody is large in the MINDEC method.
  • a first aspect of the invention provides a composition comprising an antibody comprising two or more anti-CD45 rabbit monoclonal antibodies.
  • the antigenic determinants on the CD45 antigen molecule recognized by the two or more anti-CD45 mAbs are each different.
  • the anti-CD45 mAb is a rabbit anti-human monoclonal antibody.
  • the anti-CD45 mAb has an affinity coefficient ⁇ 1.0 x 10 -10 M.
  • the affinity of at least one of the anti-CD45 mAbs is ⁇ 4.0 x 10 -11 M.
  • the antibody binds to an affinity tag; the affinity tag is preferably biotin.
  • the mass ratio of the highest affinity anti-CD45 rabbit monoclonal antibody to the total content of all other anti-CD45 rabbit monoclonal antibodies in the anti-CD45 mAb of the composition is 1-10: 1; preferably 5:1.
  • the antibody-containing composition is used with anti-CD16 mAb, anti-CD19 mAb, and anti-CD235a mAb for enrichment of circulating tumor cells.
  • the antibody-containing composition comprises an anti-CD16 mAb, an anti-CD19 mAb, an anti-CD235a mAb, and two or more anti-CD45 mAbs, wherein the two or The antigenic determinants on the CD45 antigen molecule recognized by the two or more anti-CD45 mAbs are different; preferably, the mAbs are all coupled with an affinity tag; preferably, the affinity tag is a biotin molecule.
  • the two or more anti-CD45 mAbs have an affinity coefficient of less than 1.0 x 10 -10 M; preferably, the two or more anti-CD45 mAbs At least one of the affinity coefficients is less than 4.0 x 10 -11 M.
  • the species source of the monoclonal antibodies in the composition is the same or a different species source; preferably, the species source of one of the two or more anti-CD45 mAbs is The source of the rabbit, other anti-CD45 mAb species is a murine source; more preferably, the species source of the two or more anti-CD45 mAbs is a rabbit source.
  • the anti-CD16 mAb, anti-CD19 mAb, and anti-CD235a mAb are each independently a rabbit anti-human monoclonal antibody or a murine anti-human monoclonal antibody.
  • the two or more anti-CD45 mAbs are rabbit anti-human CD45 monoclonal antibodies
  • the anti-CD16 mAb is a murine anti-human CD16 monoclonal antibody
  • the anti-CD19 The monoclonal antibody is a murine anti-human CD19 monoclonal antibody
  • the anti-CD235a monoclonal antibody is a murine anti-human CD235a monoclonal antibody.
  • the invention provides a kit comprising the antibody-containing composition described herein.
  • the kit further comprises magnetic beads coupled to a ligand capable of binding to the affinity tag of the preceding embodiments; preferably, the ligand is Streptavidin.
  • the antibody-containing composition and magnetic beads are each placed in separate containers.
  • the invention provides a combination of two or more CD45 monoclonal antibodies in combination with anti-CD16 mAb, anti-CD19 mAb and anti-CD235a mAb in leukocytes isolated from blood or in enriched peripheral blood circulating tumor cells Applications, or in the preparation of reagents or kits for isolating leukocytes in blood or for enriching peripheral blood circulating tumor cells.
  • the anti-CD45 mAb composition comprises two or more anti-CD45 mAbs that target different antigenic determinants of the CD45 molecule, one of which is a rabbit source, the other one For the mouse source.
  • the ratio of the highest affinity anti-CD45 rabbit monoclonal antibody in the anti-CD45 mAb composition to the sum of the masses of the other anti-CD45 rabbit monoclonal antibodies is 1-10:1; preferably 5 :1.
  • the present invention also provides a method of separating leukocytes in blood, the method comprising the step of contacting blood with a composition described herein.
  • the blood is blood from which red blood cells and plasma are removed.
  • the present invention provides a method of enriching circulating tumor cells, the method comprising:
  • the step (4) comprises mixing the liquid obtained in the step (3) with the antibody-containing composition, incubating for 10 to 30 minutes, and then centrifuging.
  • the step (5) comprises adding streptavidin-coupled magnetic beads for 10 to 20 minutes.
  • the step (6) comprises, after the end of the incubation, placing the container containing the liquid and the magnetic beads on a magnetic stand, allowing to stand, so that the magnetic beads are adsorbed on the magnet, and the suction is not performed.
  • a liquid containing a magnetic bead portion is not performed.
  • Figure 1 Total number of residual white blood cells after treatment with high affinity and low affinity CD45 antibody at a mass ratio of 1:1, 5:1, 10:1.
  • Figure 2 Total number of CTCs detected after treatment with high affinity and low affinity CD45 antibody at a mass ratio of 1:1, 5:1, 10:1.
  • Figure 3 Detection rate of CTCs by the method of the invention and the control method.
  • Figure 5 Total number of suspected cells during the identification of CTCs by the method of the invention and the control method.
  • PT.1 (Patient 1) in Figure 1-5 represents Patient 1, and so on.
  • the present invention aims to solve the problem that the circulating tumor cell recovery rate is not high, the white blood cell identification is incomplete, and the active circulating tumor cells cannot be accurately obtained due to excessive residual leukocyte cells in the existing circulating tumor cell isolation method.
  • an antibody-containing composition also referred to as "antibody composition”
  • the antibody comprising the antibody comprising two or more anti-CD45
  • Monoclonal antibodies in combination with anti-CD16 mAb, anti-CD19 mAb and anti-CD235a mAb, are used for enrichment and identification of CTCs.
  • Anti-CD45 mAb, anti-CD16 mAb, anti-CD19 mAb, and anti-CD235a mAb suitable for use herein can be a variety of anti-CD45 mAb, anti-CD16 mAb, anti-CD19 mAb, and anti-CD235a mAb, which are well known in the art.
  • the present invention can be practiced using commercially available anti-CD45 mAb, anti-CD16 mAb, anti-CD19 mAb, and anti-CD235a mAb, as can be obtained from Abcam, Thermo Fisher.
  • these monoclonal antibodies can be prepared by themselves using techniques well known in the art, such as hybridoma technology.
  • two or more anti-CD45 mAbs are rabbit anti-human CD45 mAb.
  • the affinity coefficient of the two or more anti-CD45 mAbs is ⁇ 1.0 x 10 -10 M. More preferably, the affinity coefficient of at least one of the two or more anti-CD45 mAbs is ⁇ 4.0 ⁇ 10 -11 M, preferably ⁇ 2.0 ⁇ 10 -11 M, more preferably ⁇ 1.0 ⁇ 10 -11 M.
  • the affinity coefficients of the two or more anti-CD45 mAbs are ⁇ 3.0 x 10 -11 M.
  • the antigenic determinants on the CD45 antigen molecule recognized by the two or more anti-CD45 mAbs are different.
  • the two or more anti-CD45 mAbs are rabbit anti-human CD45 mAbs with different affinities, preferably having an affinity coefficient of ⁇ 4.0 x 10 -11 M.
  • the ratio of the mass of the most affinity-resistant anti-CD45 mAb to the sum of the masses of the other anti-CD45 mAbs in the composition may range from 1 to 10:1, preferably from 3 to 7:1. In certain embodiments, the ratio is 5:1.
  • the amount of the antibody can be measured in micrograms.
  • the anti-CD16 mAb, anti-CD19 mAb, and anti-CD235a mAb may be rabbit anti-human mAb or murine anti-human mAb.
  • the affinity coefficient of rabbit anti-human monoclonal antibody suitable for use herein is ⁇ 4.0 ⁇ 10 -11 M, preferably ⁇ 2.0 ⁇ 10 -11 M, more preferably ⁇ 1.0 ⁇ 10 -11 M.
  • the antibody in the antibody-containing compositions described herein, may be two or more rabbit anti-human CD45 mAb, murine anti-human CD16 mAb, murine anti-human CD19 mAb, and mouse anti- Human CD235a monoclonal antibody.
  • the mass ratio of two or more anti-CD45 mAb, anti-CD16 mAb, anti-CD19 mAb and anti-CD235a mAb in the composition may range from 1:1 to 5:1 to 5:1 to 5, It is preferably in the range of 1:2 to 4:2 to 4:2 to 4.
  • the mass ratio of two or more anti-CD45 mAb, anti-CD16 mAb, anti-CD19 mAb, and anti-CD235a mAb is 1:3:3:3.
  • the antibody in the antibody-containing compositions described herein, is two or more rabbit anti-human CD45 mAb, murine anti-human CD16 mAb, murine anti-human CD19 mAb, and murine anti-human
  • the CD235a monoclonal antibody has a mass ratio in the range of 1:1 to 5:1 to 5:1 to 5, preferably in the range of 1:2 to 4:2 to 4:2 to 4, more preferably 1:3. :3:3.
  • Suitable solvents may be included in the compositions including, but not limited to, PBS, EDTA, and BSA.
  • kits comprising a composition described herein.
  • the kit may also contain magnetic beads.
  • the composition and magnetic beads are dispensed in separate containers.
  • the magnetic beads can be various magnetic beads known in the art for cell separation, including magnetic beads of any suitable size and material.
  • streptavidin or other suitable molecule is coupled to the magnetic beads to bind to an antibody that is coupled to biotin or a corresponding molecule to separate leukocytes captured by the antibody molecules.
  • the antibody compositions herein can be used to separate leukocytes in blood or enrich for circulating tumor cells.
  • a method of isolating leukocytes in blood comprising the step of contacting an antibody composition described herein with the blood.
  • the contacting can be carried out according to the technical means conventional in the art, and then the magnetic beads coupled with the corresponding binding molecules are added to adsorb the leukocytes captured by the antibody molecules onto the magnetic beads, and the blood beads are separated from the blood by separating them.
  • White blood cells are isolated.
  • blood is usually referred to as human peripheral blood unless otherwise stated.
  • plasma and red blood cells in the blood can be removed using conventional techniques in the art. For example, plasma can be removed by conventional centrifugation and red blood cells can be removed by density gradient centrifugation.
  • a method of enriching circulating tumor cells comprising:
  • the magnetic beads are removed to enrich the circulating tumor cells.
  • the blood When serum is removed, the blood can be mixed and centrifuged, and the supernatant is washed away.
  • the precipitate obtained after plasma removal can be mixed with a washing buffer and a density gradient separation solution for density gradient centrifugation.
  • the mixture After centrifugation, the mixture is usually layered in three layers, the red precipitated layer being red blood cells and the middle being a white film layer. Usually, the middle white film layer is removed first, and then the remaining liquid is taken up. The liquid is washed several times and centrifuged to remove impurities such as proteins and platelets in the solution. These steps can be accomplished using techniques conventional in the art. For example, the time, the rotation speed, and the like of the centrifugation can be carried out under normal conditions.
  • the centrifugation conditions for removing plasma may be 150 to 300 g for 10 to 20 minutes; the centrifugation conditions for removing red blood cells may be 300 to 500 g for 20 to 30 minutes; and the centrifugation conditions for removing impurities such as proteins and platelets may be 500 to 700 g, 3 ⁇ 8 minutes.
  • the antibody composition of the present invention is added, and after incubation for a period of time, the magnetic beads coupled with the corresponding molecules are added, and then incubated for a period of time, and the magnetic beads bound to the white blood cells are separated by magnetic force, thereby enriching the circulating tumor cells.
  • the amount of the antibody composition to be added and the amount of the magnetic beads to be added can be determined according to actual conditions.
  • the incubation time after mixing with the antibody composition is usually in the range of 10 to 30 minutes, and the incubation temperature may be in the range of 2 to 8 °C.
  • the separation washing solution may be added, mixed, and centrifuged to remove the antibody that does not bind the blood cells. It can be centrifuged for 5 to 15 minutes under conditions of 200 to 400 g.
  • the incubation time with the magnetic beads is usually in the range of 10 to 20 minutes, and the incubation temperature is usually 2 to 8 °C.
  • the cells can be resuspended by separating the cleaning solution, and gently mixed with a gun head, then placed on a magnetic stand for magnetic separation, and the supernatant is aspirated.
  • the supernatant can be centrifuged at 2 to 8 ° C, and the centrifugation conditions can be 200 to 400 g for 5 to 15 minutes. It should be understood that the entire process of isolating white blood cells is usually carried out at a temperature of 2 to 8 °C.
  • the present invention is based on two or more high affinity anti-CD45 mAbs (the affinity for human leukocytes is much higher than the commonly used murine monoclonal antibody, up to 3.6 ⁇ 10 -11 M), when When the single cell suspension is incubated, it can bind to leukocytes more efficiently, so the anti-CD45 monoclonal antibody required by the method of the present invention can be reduced by at least half compared to the amount of murine monoclonal antibody of MINDEC. After that, the white blood cells in the suspension can be removed more effectively by combining the magnetic beads. Under the same antibody dosage, the method of the invention can control the remaining white blood cell residual amount to 200 ⁇ 50/6.0mL, further increasing the recovery rate of CTCs. Reduce the appearance of suspected cells and reduce the difficulty of identifying CTCs.
  • the method of the invention can better label various leukocyte subclasses due to the high affinity of the antibody combination, thereby better removing white blood cells and enriching CTCs more efficiently. It does not damage CTC cells, so that CTC cells maintain a good natural state and cell morphology, and can perform genomic, transcriptome and proteomic analysis on subsequent single (multiple) circulating tumor cells or culture of circulating tumor cells after enrichment. .
  • the recovery rate of the circulating tumor cells of the present invention is as high as 95% or more; the present invention also has the advantage of high sensitivity, and can stably detect circulating tumor cells in samples with extremely small blood volume.
  • combinations of two or more of the high affinity anti-CD45 mAbs described herein are also provided herein for isolating leukocytes in the blood together with anti-CD16 mAb, anti-CD19 mAb, and anti-CD235a mAb or enrichment Use in peripheral blood circulating tumor cells, or in the preparation of reagents or kits for isolating leukocytes in blood or for enriching peripheral blood circulating tumor cells.
  • CTCs circulating tumor cells
  • CD45 common antigen on the surface of leukocytes
  • CD16 NK cells and neutrophil antigens
  • CD19 B cell antigen
  • CD235a blood glycoprotein a.
  • Magnetic beads Dynabeads MyOne 1 ⁇ m, streptavidin coated, Thermo Fisher.
  • DAPI 4',6-diamidino-2-phenylindole
  • Step 1 Aspirate the corresponding amount of the magnetic bead mixture from the magnetic bead mixture labeled with streptavidin into a 2 mL EP tube, and place the EP tube on the magnetic stand for 3 minutes, the configuration of the magnetic beads.
  • the amount is 100 ⁇ L per 1 ⁇ 10 7 cells;
  • Step 2 After the magnetic beads are adsorbed on the magnetic frame, use a vacuum pump to aspirate the magnetic bead preservation solution, add at least 1 mL of the separated cleaning solution, mix and mix slowly, place on a magnetic stand for 3 minutes, and use a vacuum pump to aspirate the cleaning solution. ;
  • Step 3 Add an equal volume of separation cleaning solution to the magnetic beads mixed with the first extraction, mix slowly, and let stand at room temperature for subsequent operations.
  • Step 1 Collect 6.0 mL of peripheral blood, mix the gently-tailed tail of the blood collection tube and centrifuge, and centrifuge for 200 g for 15 minutes; the supernatant (plasma) after centrifugation is vacuumed with a vacuum pump;
  • Step 2 Add an equal volume of cell cleaning solution to the blood cells precipitated in the blood collection tube, and gently mix the head and tail;
  • Step 3 Add 3 mL of density gradient separation solution Ficoll-Pague (1.086 g/mL) to the labeled 15 mL centrifuge tube A;
  • Step 4 slowly add the above blood cell mixture to the upper part of the separation liquid in the centrifuge tube A, and centrifuge in a centrifuge, and centrifuge at 400 g for 20-30 minutes;
  • Step 6 Add 5 times the cell volume of the above liquid volume to the centrifuge tube B, centrifuge the cells for washing, centrifuge at 600 g for 5 minutes, discard the supernatant to 100 ⁇ L, and mix and resuspend the cells by pipetting.
  • Step 1 100 ⁇ L of the cell suspension obtained in the above procedure was transferred to a new 15 mL centrifuge tube C, and a biotin-labeled high-affinity antibody mixture (rabbit anti-human CD45 mAb, mouse anti-human CD16 mAb, mouse anti-mouse) was added.
  • Step 2 Mix gently and mix, incubate at 4 ° C for 20 minutes, mix once every 10 minutes;
  • Step 3 After the end of the incubation, the separation washing solution is added to remove the unbound blood cells, the head and tail are mixed upside down several times, and the supernatant is centrifuged to 100 ⁇ L, and the centrifugation condition is 300 g for 10 minutes (2 ° C - 8 ° C);
  • Step 4 Add 3 mL of separation washing solution and mix and resuspend the cells;
  • Step 6 After the end of the incubation, add 3 mL of the separated washing solution to the centrifuge tube C; resuspend the magnetic beads-bound cells, mix gently with a pipette tip, and remember not to generate air bubbles; then place on a magnetic stand for 3 minutes;
  • Step 7 Carefully avoid the magnetic beads transfer supernatant to a new 15 mL centrifuge tube D;
  • Step 9 Carefully avoid the magnetic beads transfer supernatant to the 15mL centrifuge tube D;
  • Step 11 Transfer all the liquid from the centrifuge tube D to the labeled 15 mL centrifuge tube E; discard the supernatant to 50 ⁇ L and centrifuge at 300 g for 10 minutes (4 ° C).
  • Step 2 Add 1 ⁇ L of each fluorescent antibody (CD45-AF594, tumor-labeled fluorescent antibody), incubate at room temperature for 20 minutes in the dark, and gently mix once every 10 minutes using an oscillating mixer;
  • each fluorescent antibody CD45-AF594, tumor-labeled fluorescent antibody
  • Step 3 Add the cell washing solution to 14 mL, trim, mix by inversion, centrifuge at room temperature, centrifuge at 950 g for 4 minutes, discard the supernatant to 100 ⁇ L;
  • Step 4 Add 100 ⁇ L of 4% paraformaldehyde to the above 100 ⁇ L of cell suspension, and gently mix and mix using a pipette tip;
  • Step 8 gently add 200 ⁇ L of 1 ⁇ PBS along the inner corner of the specimen frame, and immediately discard it for 2 times;
  • Step 9 gently add 200 ⁇ L of 1 ⁇ PBS along the inner corner of the specimen frame, let stand for 2 minutes, and discard it for a total of two times;
  • Step 10 gently add 200 ⁇ L of absolute ethanol along the inner corner of the specimen frame, and immediately discard it, repeating 2 times;
  • Step 11 Insert the slide into the dyeing tank containing absolute ethanol for 2 minutes, remove the slide, erect on the filter paper, completely absorb the residual liquid from the slide, and gently slide the slide to the surface completely using a micro-hair dryer. Rear;
  • Step 12 The following operations should be protected from light. Immediately after drying the slide, 10 ⁇ L of the chromosome 18 FISH probe was added to the center of the specimen frame, and then the cover slip was placed on the probe solution using forceps to make the liquid. Spread to the entire specimen frame; if necessary, for example, there are air bubbles in the specimen frame, use a forceps to gently press the cover slip so that the probe fluid covers the entire specimen frame and discharge the air bubbles;
  • Step 13 Mounting: Cut off the tip of the 1 mL sampler, and seal each side of the coverslip with 250 ⁇ L of sealant after each slide, and put it directly into the hybridization instrument;
  • Step 14 Hybridization: denaturation at 76 ° C for 10 minutes; hybridization at 37 ° C for 4 hours;
  • Step 15 After the hybridization is completed, remove the slide; gently press the corner of the cover glass by hand, use a pair of tweezers to remove the sealant, place the slide on the dyeing tank preheated to room temperature, and let stand for 1 minute, then gently shake the dyeing tank. Until the coverslip falls off. After the coverslip is detached, the slide is continuously left in the cylinder for 1 minute at room temperature, and then the residual liquid flowing down the slide is dried by using a filter paper, and the liquid outside the specimen frame is wiped off;
  • Step 16 After gently dropping 200 ⁇ L of 2x SSC/0.1% NP-40 along the inner corner of the specimen frame, immediately aspirate the solution and repeat 2 times;
  • Step 18 Take 10 ⁇ L of fluorescent preservation droplets and add them to the center of the specimen frame. After placing the coverslips, fix the coverslips in one hand to prevent them from sliding. The other hand gently squeezes and absorbs the excess liquid overflowing with vacuum pump tips or filter paper. Sealed specimens;
  • the blood of the three patients was divided into 3 tubes, 2 ml each, and the total number of residual white blood cells and CTC detected by the high-affinity and low-affinity CD45 antibody mass ratios of 1:1, 5:1, and 10:1, respectively. Compare.
  • the total number of CTCs detected for each sample was counted and the results are shown in Fig. 2.
  • the results show that the mass ratio of 5:1 is better than 1:1 and 10:1.
  • the possible reason is that the amount of high-affinity antibodies in the 1:1 dosage is too low, and the white blood cells remain too much, causing the CTC cells to be missed; and in the ratio of 10:1, the amount of high-affinity antibodies is too high, so that the CTC cells It is removed by leukocyte agglomeration, resulting in missed detection.
  • the total number of cells per sample was scanned and the results are shown in FIG.
  • the results show that the total amount of residual blood-derived white blood cells of the present invention is lower than that of the control, indicating that the present invention has the ability to efficiently enrich CTCs.

Abstract

The present invention provides a method for enriching and identifying circulating tumor cells (CTCs) on the basis of two or more high affinity anti-CD45 rabbit monoclonal antibodies, and application thereof. Specifically, the present invention provides a composition containing two or more CD45 rabbit monoclonal antibodies, wherein the antibodies can recognize different antigenic determinants on CD45 antigen molecules. The present invention also comprises a method for separating leukocytes and enriching and identifying CTCs using said composition together with anti-CD16 monoclonal antibody, anti-CD19 monoclonal antibody, and anti-CD235a monoclonal antibody. The antibody combination in the present invention involves rabbit monoclonal antibodies with high affinity and specificity, can better label various leukocyte subclasses, thereby better removing leukocytes and more efficiently enriching and identifying CTCs, and will not damage CTC cells, so that the CTC cells can maintain a good natural state and cell form.

Description

一种含抗CD45单抗的组合物及其使用方法Composition containing anti-CD45 monoclonal antibody and using method thereof 技术领域Technical field
本发明属分子生物学领域，涉及基于两种及两种以上高亲和力抗CD45兔单抗用于富集和鉴定循环肿瘤细胞的方法及其应用。The present invention belongs to the field of molecular biology and relates to a method for enriching and identifying circulating tumor cells based on two or more high affinity anti-CD45 rabbit monoclonal antibodies and uses thereof.
背景技术Background technique
循环肿瘤细胞(Circulating Tumor Cells,CTCs)是指在肿瘤生长过程中从肿瘤原发灶脱落并进入外周血循环***的实体瘤上皮细胞。CTCs作为原发灶和转移灶的链接会通过血液循环***依附到远端组织上生长形成新的肿瘤组织，即转移灶。大量临床数据表明肿瘤转移是直接导致肿瘤患者死亡的直接原因，因而CTCs数目检测能实现对肿瘤发展的实时监测，对CTCs数目的实时监测不仅能判断机体免疫力的状态还对辅助诊断疾病、预后、指导临床治疗、治疗效果评估、耐药性和转移复发等有着很重要的临床研究价值。现有数据表明，CTCs早在肿瘤初期就可以在外周血中被检测到，而研究发现在肿瘤早期就被发现的癌症患者治愈率可高达90％，越晚期的肿瘤患者的生存率则越低。另外世界卫生组织也指出，癌症的预防治疗应秉持早发现、早诊断、早治疗的原则，因此CTCs数目检测对肿瘤的早筛也具有很重要的临床价值意义。Circulating Tumor Cells (CTCs) refer to solid tumor epithelial cells that shed from the primary tumor of the tumor and enter the peripheral blood circulatory system during tumor growth. The links of CTCs as primary tumors and metastases are attached to the distal tissues through the blood circulation system to form new tumor tissues, ie, metastases. A large number of clinical data indicate that tumor metastasis is the direct cause of death in tumor patients. Therefore, the detection of CTCs can realize real-time monitoring of tumor development. The real-time monitoring of the number of CTCs can not only judge the state of immunity but also assist in the diagnosis of diseases and prognosis. It has important clinical research value to guide clinical treatment, evaluation of treatment effect, drug resistance and metastasis and recurrence. Available data indicate that CTCs can be detected in peripheral blood as early as the early stage of the tumor, and studies have found that cancer patients found early in the tumor can cure up to 90%, and the survival rate of patients with advanced cancer is lower. . In addition, the World Health Organization also pointed out that the prevention and treatment of cancer should adhere to the principles of early detection, early diagnosis and early treatment. Therefore, the detection of the number of CTCs has important clinical value for early screening of tumors.
但如何从外周血中有效、准确的分离出CTCs是非常关键的，同时也是非常复杂的，因为血液中存在着大量的血液细胞，其中红细胞含量约为5x10 12个/L、白细胞含量约为9x10 9个/L、血小板含量约为3x10 11个/L。在如此多的血液细胞中，CTCs的含量及其稀少，肿瘤患者血液中每10 6～10 7(一百万～一千万)个白细胞中才会发现一个CTC。 However, how to effectively and accurately separate CTCs from peripheral blood is very important and complicated. Because there are a lot of blood cells in the blood, the red blood cell content is about 5x10 12 / L, and the white blood cell content is about 9x10. 9 / L, platelet content of about 3 x 10 11 / L. In so many blood cells, the content of CTCs is rare, and a CTC is found in every 16 to 107 (one million to ten million) white blood cells in the blood of tumor patients.
随着检测技术的不断进步，捕获CTCs的方法有很多，主要分为以下三大类：利用CTCs与血液细胞的体积大小差异进行过滤捕获，利用CTCs表面标记物直接进行捕获，和利用针对血液中的各种细胞抗体磁珠的组合进行捕获。With the continuous improvement of detection technology, there are many methods for capturing CTCs, which are mainly divided into the following three categories: using CTCs and blood cell volume difference for filtering and capturing, using CTCs surface markers for direct capture, and utilizing for blood. A combination of various cellular antibody magnetic beads is captured.
利用CTCs与血液细胞体积大小差异进行过滤捕获的方法的基本原理来自于CTCs与肿瘤细胞的大小相似这一假设。此方法利用细胞筛过滤掉体积较小的白细胞从而留下体积较大的CTCs从而达到捕获CTCs的目的。此方法优点在于可以方便并迅速地将CTCs与血细胞分离开，但是越来越多的研究发现，有部分CTCs与白细胞大小类似，甚至小于白细胞，这是因为肿瘤细胞在上皮细胞间质转型(epithelial-mesenchymal transition，EMT)转化过程中由于细胞骨架的重建从而导致了CTCs的体积变小。小细胞CTCs在临床上的意义也得到了越来越多的关注，因此过滤捕获的方法会导致大量的小细胞CTCs丢失。另外，因为捕获到的CTCs是在滤膜上面，对于后期的CTCs培养和下游的技术研究也会造成很大的困难。The rationale for the method of filtering capture using CTCs and blood cell volume differences comes from the assumption that CTCs are similar in size to tumor cells. This method uses a cell sieve to filter out smaller white blood cells to leave larger CTCs for the purpose of capturing CTCs. The advantage of this method is that CTCs can be separated from blood cells conveniently and quickly, but more and more studies have found that some CTCs are similar in size to white blood cells, even smaller than white blood cells, because tumor cells are in epithelial cell interstitial transition (epithelial). -mesenchymal transition, EMT) The volume of CTCs is reduced due to the reconstruction of the cytoskeleton during the transformation. The clinical significance of small cell CTCs has also received more and more attention, so the method of filtration and capture will lead to the loss of a large number of small cell CTCs. In addition, because the captured CTCs are above the filter, it can be very difficult for later CTCs culture and downstream technical research.
利用CTCs表面标记物直接进行捕获的方法又称正相富集，此方法是目前应用最为广泛的正相富集方法，最典型的代表就是Cellsearch***。因CTCs属于非血源性上皮细胞，部分CTCs会表达上皮细胞的特有蛋白——上皮细胞粘附分子(Epithelial cell adhesion molecule，EpCAM)，所以此方法主要利用上皮细胞的表面标记物EpCAM抗体并偶联在载体上，如磁珠，从而直接从血液样本中捕获EpCAM阳性的CTCs。然而研究数据表明，不是所有实体肿瘤都会有EpCAM阳性表达，如EpCAM在膀胱癌和恶行黑色素肿瘤细胞上就表现为低表达或阴性。而且通常认为，肿瘤在前转移阶段，原发肿瘤组织中的肿瘤细胞失去其本具有的极性上皮细胞特性(粘附性强、片状结构)，转化为具有迁移侵袭能力的***特性(无细胞极性、失去细胞与细胞之间的紧密连接)。这些肿瘤细胞在EMT转化过程中，发生了明显的细胞骨架重建，改变多种与EMT相关的转录因子、细胞表面受体的表达。因而使用EpCAM的直接捕获方法特异性不高，不能有效捕获多种肿瘤的CTCs，捕获效率和回收率较低。另外，细胞表面的EpCAM蛋白抗原是极为活跃的信号传导诱发因子，所以使用EpCAM抗体偶联磁珠来捕获CTCs很有可能会触发和激活一系列的细胞内信号传导通路的发生。因此，使用此方法捕获到的CTCs很大一部分程度上可能已经不是自然状态下的CTCs并且捕获的CTCs细胞活性较低，无法进行后期的细胞培养，同时也不利于进行细胞分离，用于下游技术的研究。The method of directly capturing by using CTCs surface markers is also called positive phase enrichment. This method is currently the most widely used positive phase enrichment method. The most typical representative is the Cellsearch system. Because CTCs belong to non-blood-derived epithelial cells, some CTCs express epithelial cell-specific molecules, Epithelial cell adhesion molecule (EpCAM), so this method mainly uses epithelial cell surface marker EpCAM antibody and even Linked to a carrier, such as a magnetic bead, to capture EpCAM-positive CTCs directly from the blood sample. However, the data suggest that not all solid tumors have EpCAM-positive expression, such as EpCAM, which is either low or negative on bladder cancer and malignant melanoma cells. Moreover, it is generally believed that in the pre-metastasis stage of the tumor, the tumor cells in the primary tumor tissue lose their polar epithelial cell characteristics (adhesive, flaky structure) and transform into interstitial cell characteristics with migration and invasion ability. (No cell polarity, loss of tight junctions between cells and cells). During the transformation of EMT, these tumor cells undergo obvious cytoskeletal remodeling, which changes the expression of various EMT-related transcription factors and cell surface receptors. Therefore, the direct capture method using EpCAM is not high in specificity, and can not effectively capture CTCs of various tumors, and the capture efficiency and recovery rate are low. In addition, cell surface EpCAM protein antigens are extremely active signaling-inducing factors, so the use of EpCAM antibody-coupled magnetic beads to capture CTCs is likely to trigger and activate a series of intracellular signaling pathways. Therefore, most of the CTCs captured by this method may not be CTCs in the natural state, and the captured CTCs have low cell activity, unable to carry out cell culture in the later stage, and are not conducive to cell separation, and are used in downstream technology. Research.
利用针对血液中各种细胞抗体磁珠组合进行负向富集的方法先根据细胞的不同密度，利用密度梯度离心来分选多种细胞从而有效地分离去除红细胞和血浆，再利用多种针对白细胞的抗体组合偶联磁珠抓取白细胞从而富集出CTCs。SE-iFISH(赛特生物；美国专利号US8,969,021)所使用subtraction enrichment就是这样一种方法。这种从血液中一个个剔除血源性细胞从而留下CTCs的方法被称为负向富集方法。此方法对CTCs的细胞损伤极小，所用的抗体为针对血源性细胞，因此不会触发细胞内的信号通路机制。使用密度梯度离心替代红细胞裂解液来剔除红细胞，极大的减少了对CTCs的损伤，很好的保留了CTCs细胞的特性和特征。虽然此方法具有很高的灵敏度，能有效的分离出自然状态下的CTCs，但是残余的白细胞数量过多。SE-iFISH(赛特生物)的方法是在用密度梯度离心去除红细胞和血浆后，加入偶联了各种白细胞亚类抗体的免疫磁珠孵育来结合白细胞，并通过磁力来起到去除白细胞的效果。但使用这种方法来去除白细胞最后的白细胞剩余量通常仍高达5x10 3-1.5x10 4个/mL。另外，鉴定CTCs的一个主要标准为八号染色体多倍体，而白细胞中的巨噬细胞也为八号染色体多倍体，在如此高的白细胞残余量中很可能会有多倍体巨噬细胞的存在，这样就会容易导致假阳性的CTCs出现；而过多的白细胞残余，在显微镜自动化扫描过滤有CD45阳性表达的白细胞的时候，如果在白细胞团聚处有CTC，容易被扫描***自动判定成假阴性从而过滤掉此细胞。另外，残余的白细胞数量过高会给下游的CTCs细胞测序和CTCs培养等操作带来较大的困难和背景过高的影响。 Using a method for negative enrichment of various cellular antibody magnetic beads in blood, firstly sorting various cells by density gradient centrifugation according to different densities of cells, thereby effectively separating and removing red blood cells and plasma, and then using various kinds of white blood cells The antibody combination is coupled to the magnetic beads to capture white blood cells to enrich CTCs. Subtraction enrichment used by SE-iFISH (US Patent No. US 8,969,021) is one such method. This method of removing blood-derived cells from the blood to leave CTCs is called a negative enrichment method. This method has minimal cell damage to CTCs, and the antibodies used are directed against blood-derived cells and therefore do not trigger signaling mechanisms within the cell. Density gradient centrifugation is used instead of red blood cell lysate to eliminate red blood cells, which greatly reduces the damage to CTCs, and retains the characteristics and characteristics of CTCs. Although this method has high sensitivity, it can effectively separate CTCs in the natural state, but the residual white blood cells are excessive. SE-iFISH is a method in which red blood cells and plasma are removed by density gradient centrifugation, and immunomagnetic beads coupled with various leukocyte subclass antibodies are added to incubate to bind white blood cells, and magnetic force is used to remove white blood cells. effect. However, the final leukocyte remaining amount used to remove leukocytes is usually as high as 5x10 3 -1.5x10 4 /mL. In addition, one of the main criteria for identifying CTCs is chromosome 8 polyploid, while macrophages in leukocytes are also polyploids of chromosome 8, and polyploid macrophages are likely to be present in such high leukocyte residuals. The presence of this, it will easily lead to the occurrence of false positive CTCs; and excessive white blood cell residue, when the microscope automatically scans and filters the white blood cells with CD45 positive expression, if there is CTC in the white blood cell agglomeration, it is easy to be automatically determined by the scanning system. A false negative thus filters out the cells. In addition, the excessive number of residual white blood cells will bring greater difficulties and background effects to downstream CTCs cell sequencing and CTCs culture.
MINDEC方法是另一种负向富集方法。与SE-iFISH(赛特生物)的方法相比，采用MINDEC的方法最终所残余的白细胞数量明显更少。此方法首先利用密度梯度离心来剔除红细胞和血浆，之后也是利用针对不同白细胞亚类的抗体组合来抓取白细胞从而富集CTCs。与SE-iFISH(赛特生物)方法不同的是，此方法先用生物素标记过的抗体加入去除掉红细胞和血浆之后的单核细胞悬液中进行孵育，之后再加入用链霉亲和素标记的磁珠让其与抗体结合。MINDEC方法的磁珠去除次数相比SE-iFISH(赛特生物)的方法要多，起到了更好的去除磁珠和白细胞的效果，并且同时避免了CTCs被磁珠包裹去除的风险，增加了 CTCs的回收率。应用MINDEC方法白细胞去除效率更高，最后的白细胞残余细胞数量在437±350个/mL，有效的降低了检测结果的假阳性和假阴性出现的可能性。然而因为使用不同针对白细胞亚类的抗体组合，所需要用到的抗体量较多，成本也会较高。MINDEC所使用的抗体为鼠单克隆抗体，从鼠类身上提取的抗体对人类的白细胞亲和力不高，因此在MINDEC方法中鼠单抗的需求量很大。The MINDEC method is another negative enrichment method. Compared with the SE-iFISH method, the number of white blood cells remaining in the process using MINDEC is significantly less. This method first uses density gradient centrifugation to remove red blood cells and plasma, and then uses a combination of antibodies against different leukocyte subclasses to capture white blood cells to enrich CTCs. In contrast to the SE-iFISH method, this method is first incubated with a biotin-labeled antibody in a mononuclear cell suspension after removal of red blood cells and plasma, followed by streptavidin. The labeled magnetic beads allow it to bind to the antibody. The MINDEC method has more magnetic bead removal times than SE-iFISH, which has a better effect of removing magnetic beads and white blood cells, and at the same time avoids the risk of CTCs being removed by magnetic beads. Recovery rate of CTCs. The application of MINDEC method has higher leukocyte removal efficiency, and the final number of remaining leukocyte cells is 437±350/mL, which effectively reduces the possibility of false positive and false negative detection. However, because of the different combinations of antibodies against the leukocyte subclass, the amount of antibody required is higher and the cost is higher. The antibody used by MINDEC is a murine monoclonal antibody, and the antibody extracted from the mouse has low affinity for human leukocytes, so the demand for the mouse monoclonal antibody is large in the MINDEC method.
此外，在CTC鉴定过程中，我们发现有部分染色体正常，但不表达CD45的疑似细胞存在，可能的原因是这类白细胞表面的CD45分子没有与使用的CD45抗体结合。因此需要更好的CTC鉴定抗体组合及其方法。In addition, during the CTC identification process, we found that some cells with normal chromosomes but no CD45 were present, possibly because the CD45 molecules on the surface of such leukocytes did not bind to the CD45 antibody used. There is therefore a need for better CTC identification antibody combinations and methods therefor.
发明内容Summary of the invention
本发明第一方面提供一种含有抗体的组合物，所述抗体包括两种或两种以上抗CD45兔单抗。A first aspect of the invention provides a composition comprising an antibody comprising two or more anti-CD45 rabbit monoclonal antibodies.
在一个或多个实施方案中，所述两种或两种以上抗CD45单抗所识别CD45抗原分子上的抗原决定簇各不相同。In one or more embodiments, the antigenic determinants on the CD45 antigen molecule recognized by the two or more anti-CD45 mAbs are each different.
在一个或多个实施方案中，所述抗CD45单抗为兔抗人单克隆抗体。In one or more embodiments, the anti-CD45 mAb is a rabbit anti-human monoclonal antibody.
在一个或多个实施方案中，所述抗CD45单抗的亲和力系数≤1.0×10 -10M。 In one or more embodiments, the anti-CD45 mAb has an affinity coefficient < 1.0 x 10 -10 M.
在一个或多个实施方案中，所述抗CD45单抗中至少一个单抗的亲和力≤4.0×10 -11M。 In one or more embodiments, the affinity of at least one of the anti-CD45 mAbs is < 4.0 x 10 -11 M.
在一个或多个实施方案中，所述抗体结合了亲和标记；所述亲和标记优选为生物素。In one or more embodiments, the antibody binds to an affinity tag; the affinity tag is preferably biotin.
在一个或多个实施方案中，所述组合物的抗CD45单抗中，最高亲和力抗CD45兔单抗的含量与所有其它抗CD45兔单抗的总含量之间的质量比为1-10:1；优选为5:1。In one or more embodiments, the mass ratio of the highest affinity anti-CD45 rabbit monoclonal antibody to the total content of all other anti-CD45 rabbit monoclonal antibodies in the anti-CD45 mAb of the composition is 1-10: 1; preferably 5:1.
在一个或多个实施方案中，所述含有抗体的组合物与抗CD16单抗、抗CD19单抗和抗CD235a单抗一起使用，用于循环肿瘤细胞的富集。In one or more embodiments, the antibody-containing composition is used with anti-CD16 mAb, anti-CD19 mAb, and anti-CD235a mAb for enrichment of circulating tumor cells.
在一个或多个实施方案中，所述含有抗体的组合物含有抗CD16单抗、抗CD19单抗、抗CD235a单抗和两种或两种以上抗CD45单抗，其中，所述两 种或两种以上抗CD45单抗所识别CD45抗原分子上的抗原决定簇各不相同；优选地，所述单抗均偶联有亲和标记；优选地，所述亲和标记为生物素分子。In one or more embodiments, the antibody-containing composition comprises an anti-CD16 mAb, an anti-CD19 mAb, an anti-CD235a mAb, and two or more anti-CD45 mAbs, wherein the two or The antigenic determinants on the CD45 antigen molecule recognized by the two or more anti-CD45 mAbs are different; preferably, the mAbs are all coupled with an affinity tag; preferably, the affinity tag is a biotin molecule.
在一个或多个实施方案中，所述两种或两种以上抗CD45单抗的亲和力系数均低于1.0×10 -10M；优选地，所述两种或两种以上抗CD45单抗中的至少一种的亲和力系数低于4.0×10 -11M。 In one or more embodiments, the two or more anti-CD45 mAbs have an affinity coefficient of less than 1.0 x 10 -10 M; preferably, the two or more anti-CD45 mAbs At least one of the affinity coefficients is less than 4.0 x 10 -11 M.
在一个或多个实施方案中，组合物中的单抗的物种来源为相同的或不同的物种来源；优选地，所述两种或两种以上抗CD45单抗中的一种的物种来源为兔源，其它抗CD45单抗的物种来源为鼠源；更优选地，所述两种或两种以上抗CD45单抗的物种来源均为兔源。In one or more embodiments, the species source of the monoclonal antibodies in the composition is the same or a different species source; preferably, the species source of one of the two or more anti-CD45 mAbs is The source of the rabbit, other anti-CD45 mAb species is a murine source; more preferably, the species source of the two or more anti-CD45 mAbs is a rabbit source.
在一个或多个实施方案中，所述抗CD16单抗、抗CD19单抗和抗CD235a单抗各自独立为兔抗人单克隆抗体或鼠抗人单克隆抗体。In one or more embodiments, the anti-CD16 mAb, anti-CD19 mAb, and anti-CD235a mAb are each independently a rabbit anti-human monoclonal antibody or a murine anti-human monoclonal antibody.
在一个或多个实施方案中，所述两种或两种以上抗CD45单抗均为兔抗人CD45单克隆抗体，所述抗CD16单抗为鼠抗人CD16单克隆抗体、所述抗CD19单抗为鼠抗人CD19单克隆抗体和所述抗CD235a单抗为鼠抗人CD235a单克隆抗体。In one or more embodiments, the two or more anti-CD45 mAbs are rabbit anti-human CD45 monoclonal antibodies, the anti-CD16 mAb is a murine anti-human CD16 monoclonal antibody, the anti-CD19 The monoclonal antibody is a murine anti-human CD19 monoclonal antibody and the anti-CD235a monoclonal antibody is a murine anti-human CD235a monoclonal antibody.
在一个或多个实施方案中，所述组合物中所述两种或两种以上抗CD45单抗、抗CD16单抗、抗CD19单抗和抗CD235a单抗之间的质量比为1：1～5：1～5：1～5；优选为1：2～4：2～4：2～4；更优选为1：3：3：3。In one or more embodiments, the mass ratio between the two or more anti-CD45 mAbs, anti-CD16 mAb, anti-CD19 mAb, and anti-CD235a mAb in the composition is 1:1. ～5:1 to 5:1 to 5; preferably 1:2 to 4:2 to 4:2 to 4; more preferably 1:3:3:3.
另一方面，本发明提供一种试剂盒，所述试剂盒含有本文所述的含抗体的组合物。In another aspect, the invention provides a kit comprising the antibody-containing composition described herein.
在一个或多个实施方案中，所述试剂盒还含有磁珠，所述磁珠偶联了能够与前述实施方案中所述亲和标记相结合的配体；优选地，所述配体为链霉亲和素。In one or more embodiments, the kit further comprises magnetic beads coupled to a ligand capable of binding to the affinity tag of the preceding embodiments; preferably, the ligand is Streptavidin.
在一个或多个实施方案中，试剂盒中，所述含抗体的组合物和磁珠分别置于不同的容器中。In one or more embodiments, in the kit, the antibody-containing composition and magnetic beads are each placed in separate containers.
再一方面，本发明提供抗两种或两种以上的CD45单抗组合与抗CD16单抗、抗CD19单抗和抗CD235a单抗一起在分离血液中的白细胞或在富集外周血循环肿瘤细胞中的应用，或在制备用于分离血液中的白细胞或用于富集外周 血循环肿瘤细胞的试剂或试剂盒中的应用。In a further aspect, the invention provides a combination of two or more CD45 monoclonal antibodies in combination with anti-CD16 mAb, anti-CD19 mAb and anti-CD235a mAb in leukocytes isolated from blood or in enriched peripheral blood circulating tumor cells Applications, or in the preparation of reagents or kits for isolating leukocytes in blood or for enriching peripheral blood circulating tumor cells.
在一个或多个实施方案中，所述两种或两种以上的抗CD45单抗组合包括靶向CD45分子不同的抗原决定簇的两种以上抗CD45单抗，其中所有的抗CD45单抗均为兔源。In one or more embodiments, the two or more anti-CD45 mAb combinations comprise two or more anti-CD45 mAbs that target different antigenic determinants of the CD45 molecule, wherein all anti-CD45 mAbs are For the rabbit source.
在一个或多个实施方案中，所述抗CD45单抗组合物包括靶向CD45分子不同的抗原决定簇的两种以上抗CD45单抗，其中的一种抗CD45单抗为兔源，其他的为鼠源。In one or more embodiments, the anti-CD45 mAb composition comprises two or more anti-CD45 mAbs that target different antigenic determinants of the CD45 molecule, one of which is a rabbit source, the other one For the mouse source.
在一个或多个实施方案中，所述抗CD45单抗组合物中最高亲和力的抗CD45兔单抗的质量与其他抗CD45兔单抗质量之和的比例为1-10:1；优选为5:1。In one or more embodiments, the ratio of the highest affinity anti-CD45 rabbit monoclonal antibody in the anti-CD45 mAb composition to the sum of the masses of the other anti-CD45 rabbit monoclonal antibodies is 1-10:1; preferably 5 :1.
再一方面，本发明还提供一种分离血液中白细胞的方法，所述方法包括使血液与本文所述的组合物接触的步骤。In still another aspect, the present invention also provides a method of separating leukocytes in blood, the method comprising the step of contacting blood with a composition described herein.
在一个或多个实施方案中，所述血液是除去了红细胞和血浆的血液。In one or more embodiments, the blood is blood from which red blood cells and plasma are removed.
再一方面，本发明提供一种富集循环肿瘤细胞的方法，所述方法包括：In still another aspect, the present invention provides a method of enriching circulating tumor cells, the method comprising:
(1)离心，除去血浆；(1) centrifugation to remove plasma;
(2)离心，除去红细胞；(2) centrifugation to remove red blood cells;
(3)洗涤步骤(2)获得的液体；(3) the liquid obtained in the washing step (2);
(4)在步骤(3)获得的液体中加入本文所述的含抗体的组合物，孵育，离心清洗；(4) adding the antibody-containing composition described herein to the liquid obtained in the step (3), incubating, and centrifuging;
(5)在步骤(4)获得的细胞中加入偶联了链霉亲和素的磁珠，孵育；和(6)孵育结束后，除去磁珠，从而富集循环肿瘤细胞。(5) adding magnetic beads to which streptavidin is conjugated to the cells obtained in the step (4), and incubated; and (6) after the end of the incubation, the magnetic beads are removed to enrich the circulating tumor cells.
在一个或多个实施方案中，所述步骤(4)包括，将步骤(3)获得的液体与所述含抗体的组合物混合，孵育10～30分钟后，离心清洗。In one or more embodiments, the step (4) comprises mixing the liquid obtained in the step (3) with the antibody-containing composition, incubating for 10 to 30 minutes, and then centrifuging.
在一个或多个实施方案中，所述步骤(5)包括，加入链霉亲和素偶联的磁珠，孵育10～20分钟。In one or more embodiments, the step (5) comprises adding streptavidin-coupled magnetic beads for 10 to 20 minutes.
在一个或多个实施方案中，所述步骤(6)包括，孵育结束后，将含所述液体和磁珠的容器置于磁力架上，静置，使磁珠吸附在磁铁上，吸取未含有磁珠部分的液体。In one or more embodiments, the step (6) comprises, after the end of the incubation, placing the container containing the liquid and the magnetic beads on a magnetic stand, allowing to stand, so that the magnetic beads are adsorbed on the magnet, and the suction is not performed. A liquid containing a magnetic bead portion.
附图说明DRAWINGS
图1：使用质量比为1:1、5:1、10:1的高亲和力和低亲和力CD45抗体处理后的残余白细胞总数。Figure 1: Total number of residual white blood cells after treatment with high affinity and low affinity CD45 antibody at a mass ratio of 1:1, 5:1, 10:1.
图2：使用质量比为1:1、5:1、10:1的高亲和力和低亲和力CD45抗体处理后的CTCs检出总数。Figure 2: Total number of CTCs detected after treatment with high affinity and low affinity CD45 antibody at a mass ratio of 1:1, 5:1, 10:1.
图3：本发明方法与对照方法的CTCs检出率。Figure 3: Detection rate of CTCs by the method of the invention and the control method.
图4：本发明方法与对照方法的残余白细胞总数。Figure 4: Total number of residual white blood cells in the method of the invention and the control method.
图5：本发明方法与对照方法的CTCs鉴定过程中疑似细胞总数。Figure 5: Total number of suspected cells during the identification of CTCs by the method of the invention and the control method.
图1-5中的PT.1(Patient 1)表示患者1，依次类推。PT.1 (Patient 1) in Figure 1-5 represents Patient 1, and so on.
具体实施方式Detailed ways
应理解，在本发明范围中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成优选的技术方案。It is to be understood that within the scope of the present invention, the various technical features of the present invention and the technical features specifically described in the following (as in the embodiments) may be combined with each other to constitute a preferred technical solution.
本发明旨在解决现有循环肿瘤细胞分离方法存在的因白细胞残余量过多而导致的循环肿瘤细胞回收率不高、白细胞鉴定不完全以及不能准确获得有活性的循环肿瘤细胞的问题。The present invention aims to solve the problem that the circulating tumor cell recovery rate is not high, the white blood cell identification is incomplete, and the active circulating tumor cells cannot be accurately obtained due to excessive residual leukocyte cells in the existing circulating tumor cell isolation method.
为了高效的去除各种不同类型的白细胞，本发明提供了一种含抗体的组合物(也称为“抗体组合物”)，该含抗体组合物中的抗体包括两种及两种以上抗CD45单抗，与抗CD16单抗、抗CD19单抗和抗CD235a单抗共同用于CTCs的富集和鉴定。In order to efficiently remove various types of leukocytes, the present invention provides an antibody-containing composition (also referred to as "antibody composition"), the antibody comprising the antibody comprising two or more anti-CD45 Monoclonal antibodies, in combination with anti-CD16 mAb, anti-CD19 mAb and anti-CD235a mAb, are used for enrichment and identification of CTCs.
适用于本文的抗CD45单抗、抗CD16单抗、抗CD19单抗和抗CD235a单抗可以是本领域周知的各种抗CD45单抗、抗CD16单抗、抗CD19单抗和抗CD235a单抗。例如，可使用市售的抗CD45单抗、抗CD16单抗、抗CD19单抗和抗CD235a单抗来实施本发明，如可从Abcam、赛默飞世尔购得这类单抗。或者，也可采用本领域公知技术，如杂交瘤技术，自行制备这些单抗。Anti-CD45 mAb, anti-CD16 mAb, anti-CD19 mAb, and anti-CD235a mAb suitable for use herein can be a variety of anti-CD45 mAb, anti-CD16 mAb, anti-CD19 mAb, and anti-CD235a mAb, which are well known in the art. . For example, the present invention can be practiced using commercially available anti-CD45 mAb, anti-CD16 mAb, anti-CD19 mAb, and anti-CD235a mAb, as can be obtained from Abcam, Thermo Fisher. Alternatively, these monoclonal antibodies can be prepared by themselves using techniques well known in the art, such as hybridoma technology.
在优选的实施方案中，该含抗体的组合物中，两种或两种以上抗CD45单抗是兔抗人CD45单抗。优选的是，所述两种或两种以上抗CD45单抗的亲和 力系数≤1.0×10 -10M。更优选地，所述两种或两种以上抗CD45单抗中至少有一种的亲和力系数≤4.0×10 -11M，优选≤2.0×10 -11M，更优选≤1.0×10 -11M。优选地，所述两种或两种以上抗CD45单抗的亲和力系数都≤3.0×10 -11M。 In a preferred embodiment, in the antibody-containing composition, two or more anti-CD45 mAbs are rabbit anti-human CD45 mAb. Preferably, the affinity coefficient of the two or more anti-CD45 mAbs is ≤ 1.0 x 10 -10 M. More preferably, the affinity coefficient of at least one of the two or more anti-CD45 mAbs is ≤ 4.0 × 10 -11 M, preferably ≤ 2.0 × 10 -11 M, more preferably ≤ 1.0 × 10 -11 M. Preferably, the affinity coefficients of the two or more anti-CD45 mAbs are < 3.0 x 10 -11 M.
在某些实施方案中，所述两种或两种以上抗CD45单抗所识别的CD45抗原分子上的抗原决定簇各不相同。优选地，所述两种或两种以上抗CD45单抗是亲和力不同的兔抗人CD45单抗，优选它们的亲和力系数都≤4.0×10 -11M。 In certain embodiments, the antigenic determinants on the CD45 antigen molecule recognized by the two or more anti-CD45 mAbs are different. Preferably, the two or more anti-CD45 mAbs are rabbit anti-human CD45 mAbs with different affinities, preferably having an affinity coefficient of < 4.0 x 10 -11 M.
组合物中，亲和力最高的抗CD45单抗的质量与其它抗CD45单抗的质量之和之比可在1～10：1的范围内，优选在3～7：1的范围内。在某些实施方案中，该比例为5：1。本文中，抗体的用量可以微克计。The ratio of the mass of the most affinity-resistant anti-CD45 mAb to the sum of the masses of the other anti-CD45 mAbs in the composition may range from 1 to 10:1, preferably from 3 to 7:1. In certain embodiments, the ratio is 5:1. Herein, the amount of the antibody can be measured in micrograms.
本文中，抗CD16单抗、抗CD19单抗和抗CD235a单抗可以是兔抗人单抗，也可以是鼠抗人单抗。优选的是，适用于本文的兔抗人单抗的亲和力系数≤4.0×10 -11M，优选≤2.0×10 -11M，更优选≤1.0×10 -11M。 Herein, the anti-CD16 mAb, anti-CD19 mAb, and anti-CD235a mAb may be rabbit anti-human mAb or murine anti-human mAb. Preferably, the affinity coefficient of rabbit anti-human monoclonal antibody suitable for use herein is ≤ 4.0 × 10 -11 M, preferably ≤ 2.0 × 10 -11 M, more preferably ≤ 1.0 × 10 -11 M.
在某些实施方案中，本文所述的含抗体的组合物中，抗体可以是两种或两种以上兔抗人CD45单抗，鼠抗人CD16单抗，鼠抗人CD19单抗和鼠抗人CD235a单抗。In certain embodiments, in the antibody-containing compositions described herein, the antibody may be two or more rabbit anti-human CD45 mAb, murine anti-human CD16 mAb, murine anti-human CD19 mAb, and mouse anti- Human CD235a monoclonal antibody.
组合物中两种或两种以上抗CD45单抗、抗CD16单抗、抗CD19单抗和抗CD235a单抗的质量比可以在1：1～5：1～5：1～5的范围内，优选在1：2～4：2～4：2～4的范围内。在某些实施方案中，两种或两种以上抗CD45单抗、抗CD16单抗、抗CD19单抗和抗CD235a单抗的质量比为1：3：3：3。The mass ratio of two or more anti-CD45 mAb, anti-CD16 mAb, anti-CD19 mAb and anti-CD235a mAb in the composition may range from 1:1 to 5:1 to 5:1 to 5, It is preferably in the range of 1:2 to 4:2 to 4:2 to 4. In certain embodiments, the mass ratio of two or more anti-CD45 mAb, anti-CD16 mAb, anti-CD19 mAb, and anti-CD235a mAb is 1:3:3:3.
在某些实施方案中，本文所述的含抗体的组合物中，抗体是两种或两种以上兔抗人CD45单抗，鼠抗人CD16单抗、鼠抗人CD19单抗和鼠抗人CD235a单抗，其质量比在1：1～5：1～5：1～5的范围内，优选在1：2～4：2～4：2～4的范围内，更优选为1：3：3：3。In certain embodiments, in the antibody-containing compositions described herein, the antibody is two or more rabbit anti-human CD45 mAb, murine anti-human CD16 mAb, murine anti-human CD19 mAb, and murine anti-human The CD235a monoclonal antibody has a mass ratio in the range of 1:1 to 5:1 to 5:1 to 5, preferably in the range of 1:2 to 4:2 to 4:2 to 4, more preferably 1:3. :3:3.
通常，组合物中抗体偶联有生物素，以使其可与磁珠上偶联的链霉亲和素结合。Typically, the antibody is conjugated to biotin in the composition such that it binds to streptavidin coupled to the magnetic beads.
组合物中可含有合适的溶剂，包括但不限于PBS、EDTA和BSA。Suitable solvents may be included in the compositions including, but not limited to, PBS, EDTA, and BSA.
本文还提供一种试剂盒，该试剂盒含有本文所述的组合物。在某些实施方案中，试剂盒中还可含有磁珠。通常，组合物和磁珠分装在不同的容器中。磁 珠可以是本领域已知的各种用于细胞分离的磁珠，包括具有任何合适大小和材质的磁珠。通常，磁珠上偶联有链霉亲和素或其它合适的分子，以与偶联了生物素或相应分子的抗体结合，从而将被抗体分子俘获的白细胞分离出来。Also provided herein is a kit comprising a composition described herein. In certain embodiments, the kit may also contain magnetic beads. Typically, the composition and magnetic beads are dispensed in separate containers. The magnetic beads can be various magnetic beads known in the art for cell separation, including magnetic beads of any suitable size and material. Typically, streptavidin or other suitable molecule is coupled to the magnetic beads to bind to an antibody that is coupled to biotin or a corresponding molecule to separate leukocytes captured by the antibody molecules.
如前文所述，本文的抗体组合物可用于分离血液中的白细胞或富集循环肿瘤细胞。因此，本文提供一种分离血液中的白细胞的方法，包括使本文所述的抗体组合物与该血液接触的步骤。可按本领域常规的技术手段进行所述接触，然后加入偶联了相应结合分子的磁珠，将被抗体分子俘获的白细胞吸附到磁珠上，并通过分离出这类磁珠而从血液中分离出白细胞。本文中，除非另有说明，否则血液通常指人的外周血。在分离血液中的白细胞之前，可先采用本领域常规的技术手段除去血液中的血浆和红细胞。例如，可通过常规的离心而除去血浆，可采用密度梯度离心除去红细胞。As described above, the antibody compositions herein can be used to separate leukocytes in blood or enrich for circulating tumor cells. Accordingly, provided herein is a method of isolating leukocytes in blood comprising the step of contacting an antibody composition described herein with the blood. The contacting can be carried out according to the technical means conventional in the art, and then the magnetic beads coupled with the corresponding binding molecules are added to adsorb the leukocytes captured by the antibody molecules onto the magnetic beads, and the blood beads are separated from the blood by separating them. White blood cells are isolated. Herein, blood is usually referred to as human peripheral blood unless otherwise stated. Prior to the separation of leukocytes in the blood, plasma and red blood cells in the blood can be removed using conventional techniques in the art. For example, plasma can be removed by conventional centrifugation and red blood cells can be removed by density gradient centrifugation.
在某些方面，本文提供一种富集循环肿瘤细胞的方法，所述方法包括：In certain aspects, provided herein is a method of enriching circulating tumor cells, the method comprising:
(1)离心，除去血浆；(1) centrifugation to remove plasma;
(2)采用密度梯度离心除去红细胞；(2) removing red blood cells by density gradient centrifugation;
(3)洗涤步骤(2)获得的液体；(3) the liquid obtained in the washing step (2);
(4)在步骤(3)获得的液体中加入本文所述的含抗体的组合物，孵育，离心清洗；(4) adding the antibody-containing composition described herein to the liquid obtained in the step (3), incubating, and centrifuging;
(5)在步骤(4)获得的细胞沉淀物中加入偶联了链霉亲和素的磁珠，孵育；和(5) adding a magnetic bead coupled with streptavidin to the cell pellet obtained in the step (4), and incubating;
(6)孵育结束后，除去磁珠，从而富集循环肿瘤细胞。(6) After the end of the incubation, the magnetic beads are removed to enrich the circulating tumor cells.
除去血清时，可将血液混匀后离心，洗去上清即血浆。可将除去血浆后获得的沉淀与清洗缓冲液和密度梯度分离液混合，进行密度梯度离心。离心后混合物通常分层三层，红色沉淀层为红细胞，中间为白膜层。通常，先去除中间的白膜层，然后吸取余下的液体。对该液体进行洗涤数次，离心以除去溶液中的蛋白和血小板等杂质。这些步骤可采用本领域常规的技术完成。例如，对于离心的时间、转速等，均可采取常规的条件实施。例如，除去血浆的离心条件可以是150～300g，10～20分钟；除去红细胞的离心条件可以是300～500g，20～30分钟；除去蛋白和血小板等杂质的离心条件可以是500～700g，3～8 分钟。When serum is removed, the blood can be mixed and centrifuged, and the supernatant is washed away. The precipitate obtained after plasma removal can be mixed with a washing buffer and a density gradient separation solution for density gradient centrifugation. After centrifugation, the mixture is usually layered in three layers, the red precipitated layer being red blood cells and the middle being a white film layer. Usually, the middle white film layer is removed first, and then the remaining liquid is taken up. The liquid is washed several times and centrifuged to remove impurities such as proteins and platelets in the solution. These steps can be accomplished using techniques conventional in the art. For example, the time, the rotation speed, and the like of the centrifugation can be carried out under normal conditions. For example, the centrifugation conditions for removing plasma may be 150 to 300 g for 10 to 20 minutes; the centrifugation conditions for removing red blood cells may be 300 to 500 g for 20 to 30 minutes; and the centrifugation conditions for removing impurities such as proteins and platelets may be 500 to 700 g, 3 ~8 minutes.
之后加入本文抗体组合物，孵育一段时间后再加入偶联了相应分子的磁珠，再孵育一段时间，利用磁力分离出结合了白细胞的磁珠，从而实现循环肿瘤细胞的富集。抗体组合物的加入量以及磁珠的加入量可根据实际情况加以确定。Thereafter, the antibody composition of the present invention is added, and after incubation for a period of time, the magnetic beads coupled with the corresponding molecules are added, and then incubated for a period of time, and the magnetic beads bound to the white blood cells are separated by magnetic force, thereby enriching the circulating tumor cells. The amount of the antibody composition to be added and the amount of the magnetic beads to be added can be determined according to actual conditions.
本文中，与抗体组合物混合后的孵育时间通常在10～30分钟的范围内，孵育温度可在2～8℃的范围内。孵育结束后，可加入分离清洗液，混匀后离心，以除去未结合血细胞的抗体。可在200～400g的条件下离心5～15分钟。与磁珠的孵育时间通常在10～20分钟的范围内，孵育温度通常在2～8℃。孵育结束后，可加入分离清洗液重悬磁珠结合的细胞，用枪头轻轻吹打混匀，然后放置在磁力架上进行磁力分离，吸取上清。可在2～8℃对上清进行离心处理，离心条件可为200～400g，5～15分钟。应理解的是，在分离白细胞的整个过程中，通常在2～8℃的温度条件下进行。Herein, the incubation time after mixing with the antibody composition is usually in the range of 10 to 30 minutes, and the incubation temperature may be in the range of 2 to 8 °C. After the incubation is completed, the separation washing solution may be added, mixed, and centrifuged to remove the antibody that does not bind the blood cells. It can be centrifuged for 5 to 15 minutes under conditions of 200 to 400 g. The incubation time with the magnetic beads is usually in the range of 10 to 20 minutes, and the incubation temperature is usually 2 to 8 °C. After the incubation, the cells can be resuspended by separating the cleaning solution, and gently mixed with a gun head, then placed on a magnetic stand for magnetic separation, and the supernatant is aspirated. The supernatant can be centrifuged at 2 to 8 ° C, and the centrifugation conditions can be 200 to 400 g for 5 to 15 minutes. It should be understood that the entire process of isolating white blood cells is usually carried out at a temperature of 2 to 8 °C.
本发明的有益效果在于：本发明基于两种及两种以上高亲和力抗CD45单抗(对人类白细胞的亲和力比常用的鼠单抗要高很多，最高达到3.6×10 -11M)，当与单细胞悬液进行孵育时，能够更加有效地结合在白细胞上，因此本发明的方法所需的抗CD45单抗相比MINDEC的鼠单抗用量至少能减少一半。此后再结合磁珠能够更加有效地去除掉悬液中的白细胞，同等抗体用量下，本发明方法能把最后的白细胞残余数量控制在200±50个/6.0mL，进一步增加了CTCs的回收率，降低疑似细胞的出现，降低CTCs鉴定的难度。 The beneficial effects of the present invention are: the present invention is based on two or more high affinity anti-CD45 mAbs (the affinity for human leukocytes is much higher than the commonly used murine monoclonal antibody, up to 3.6×10 -11 M), when When the single cell suspension is incubated, it can bind to leukocytes more efficiently, so the anti-CD45 monoclonal antibody required by the method of the present invention can be reduced by at least half compared to the amount of murine monoclonal antibody of MINDEC. After that, the white blood cells in the suspension can be removed more effectively by combining the magnetic beads. Under the same antibody dosage, the method of the invention can control the remaining white blood cell residual amount to 200±50/6.0mL, further increasing the recovery rate of CTCs. Reduce the appearance of suspected cells and reduce the difficulty of identifying CTCs.
本发明的方法与传统循环肿瘤细胞阴性富集方法相比较，因抗体组合的高亲和力，能更好的标记各种白细胞亚类，从而能够更好的去除白细胞，更高效地富集CTCs，同时不会损伤CTC细胞，使得CTC细胞维持良好的自然状态和细胞形态，可对后续的单(多)个循环肿瘤细胞进行基因组、转录组和蛋白组分析或对富集后的循环肿瘤细胞进行培养。本发明的循环肿瘤细胞的回收率高达95％以上；本发明还具有高灵敏度的优点，可以在血量极少的样品中稳定检出循环肿瘤细胞。Compared with the traditional circulating tumor cell negative enrichment method, the method of the invention can better label various leukocyte subclasses due to the high affinity of the antibody combination, thereby better removing white blood cells and enriching CTCs more efficiently. It does not damage CTC cells, so that CTC cells maintain a good natural state and cell morphology, and can perform genomic, transcriptome and proteomic analysis on subsequent single (multiple) circulating tumor cells or culture of circulating tumor cells after enrichment. . The recovery rate of the circulating tumor cells of the present invention is as high as 95% or more; the present invention also has the advantage of high sensitivity, and can stably detect circulating tumor cells in samples with extremely small blood volume.
因此，本文也提供本文所述的两种或两种以上的高亲和力抗CD45单抗的组合在与抗CD16单抗、抗CD19单抗和抗CD235a单抗一同分离血液中的白 细胞或在富集外周血循环肿瘤细胞中的应用，或在制备用于分离血液中的白细胞或用于富集外周血循环肿瘤细胞的试剂或试剂盒中的应用。Accordingly, combinations of two or more of the high affinity anti-CD45 mAbs described herein are also provided herein for isolating leukocytes in the blood together with anti-CD16 mAb, anti-CD19 mAb, and anti-CD235a mAb or enrichment Use in peripheral blood circulating tumor cells, or in the preparation of reagents or kits for isolating leukocytes in blood or for enriching peripheral blood circulating tumor cells.
下文将以具体实施例的方式阐述本发明。应理解，这些实施例仅仅是阐述性的，并非限制本发明的范围。实施例中所用到的方法和材料，除非另有说明，否则为本领域常规的方法和材料。The invention will be elucidated below in the context of specific embodiments. It is to be understood that the examples are merely illustrative and not limiting of the scope of the invention. The methods and materials used in the examples are routine methods and materials in the art unless otherwise indicated.
本发明涉及的缩略语和关键术语定义：The abbreviations and key terms defined in the present invention:
CTCs：循环肿瘤细胞；CTCs: circulating tumor cells;
CD45：白细胞表面共同抗原；CD45: common antigen on the surface of leukocytes;
DAPI：4',6-二脒基-2-苯基吲哚；DAPI: 4',6-diamidino-2-phenylindole;
CD16：NK细胞和中性粒细胞抗原；CD16: NK cells and neutrophil antigens;
CD19：B细胞抗原；CD19: B cell antigen;
CD235a：血型糖蛋白a。CD235a: blood glycoprotein a.
实施例Example
材料与方法Materials and Methods
兔抗人CD45单抗：Abcam(高、低亲和力：3.6x 10 -11M、1.0x 10 -10M)； Rabbit anti-human CD45 mAb: Abcam (high and low affinity: 3.6 x 10 -11 M, 1.0 x 10 -10 M);
鼠抗人CD45单抗：赛默飞世尔；Mouse anti-human CD45 mAb: Thermo Fisher;
鼠抗人CD16单抗：赛默飞世尔；Mouse anti-human CD16 mAb: Thermo Fisher;
鼠抗人CD19单抗：赛默飞世尔；Mouse anti-human CD19 mAb: Thermo Fisher;
鼠抗人CD235a单抗：赛默飞世尔；Mouse anti-human CD235a mAb: Thermo Fisher;
磁珠：Dynabeads MyOne 1μm，链霉亲和素包被，赛默飞世尔。Magnetic beads: Dynabeads MyOne 1μm, streptavidin coated, Thermo Fisher.
实施例中涉及到的试剂为：The reagents involved in the examples are:
DAPI：4',6-二脒基-2-苯基吲哚；DAPI: 4',6-diamidino-2-phenylindole;
细胞清洗液:PBS+2mM EDTA+0.5％BSA；Cell washing solution: PBS + 2 mM EDTA + 0.5% BSA;
分离清洗液:PBS+2mM EDTA+0.1％BSA；Separate the cleaning solution: PBS + 2 mM EDTA + 0.1% BSA;
FCR阻断液：Militenyi Biotec公司；FCR blocking solution: Milientyi Biotec;
2x SSC/0.1％NP-40：Abbott公司。2x SSC/0.1% NP-40: Abbott.
按以下步骤分离CTCs并鉴定CTCs。Follow the steps below to isolate CTCs and identify CTCs.
1、准备磁珠1, prepare the magnetic beads
步骤1：从标记好链霉亲和素的磁珠混合液中吸取相应分量的磁珠混合液到2mL EP管中，并将该EP管放置在磁力架上静置3分钟，磁珠的配置量为100μL每1x10 7个细胞； Step 1: Aspirate the corresponding amount of the magnetic bead mixture from the magnetic bead mixture labeled with streptavidin into a 2 mL EP tube, and place the EP tube on the magnetic stand for 3 minutes, the configuration of the magnetic beads. The amount is 100 μL per 1×10 7 cells;
步骤2：待磁珠都吸附在磁力架后，使用真空泵吸弃磁珠保存液，加入至少1mL的分离清洗液缓慢吹打混匀，放置在磁力架上静置3分钟，使用真空泵吸弃清洗液；Step 2: After the magnetic beads are adsorbed on the magnetic frame, use a vacuum pump to aspirate the magnetic bead preservation solution, add at least 1 mL of the separated cleaning solution, mix and mix slowly, place on a magnetic stand for 3 minutes, and use a vacuum pump to aspirate the cleaning solution. ;
步骤3：加入与最开始吸取的磁珠混合液等体积的分离清洗液，缓慢吹打混匀，静置在室温等待后续操作。Step 3: Add an equal volume of separation cleaning solution to the magnetic beads mixed with the first extraction, mix slowly, and let stand at room temperature for subsequent operations.
2、红细胞，血浆分离2, red blood cells, plasma separation
步骤1：采集6.0mL外周血，将采血管轻柔头尾颠倒混匀并离心，离心条件为200g，15分钟；将离心结束后的上清(血浆)使用真空泵吸弃；Step 1: Collect 6.0 mL of peripheral blood, mix the gently-tailed tail of the blood collection tube and centrifuge, and centrifuge for 200 g for 15 minutes; the supernatant (plasma) after centrifugation is vacuumed with a vacuum pump;
步骤2：加入与采血管中沉淀的血细胞等体积的细胞清洗液，轻柔头尾颠倒混匀；Step 2: Add an equal volume of cell cleaning solution to the blood cells precipitated in the blood collection tube, and gently mix the head and tail;
步骤3：在标记好的15mL离心管A中加入3mL密度梯度分离液Ficoll-Pague(1.086g/mL)；Step 3: Add 3 mL of density gradient separation solution Ficoll-Pague (1.086 g/mL) to the labeled 15 mL centrifuge tube A;
步骤4：缓慢将上述血细胞混合液加叠至离心管A中分离液的上方，放入离心机内离心，离心条件为室温400g,20-30分钟；Step 4: slowly add the above blood cell mixture to the upper part of the separation liquid in the centrifuge tube A, and centrifuge in a centrifuge, and centrifuge at 400 g for 20-30 minutes;
步骤5：离心结束后，离心管中的液体分成三层，先吸取中间的白膜层后，再吸取红细胞沉淀上方的所有液体，并一同加入到50mL离心管B中；Step 5: After the end of centrifugation, the liquid in the centrifuge tube is divided into three layers. After sucking the middle white membrane layer, all the liquid above the red blood cell pellet is sucked and added to the 50 mL centrifuge tube B;
步骤6：加入5倍于上述液体体积的细胞清洗液至离心管B中，离心清洗细胞，离心条件为室温600g，5分钟，弃上清至100μL，吹打混匀重悬细胞。Step 6: Add 5 times the cell volume of the above liquid volume to the centrifuge tube B, centrifuge the cells for washing, centrifuge at 600 g for 5 minutes, discard the supernatant to 100 μL, and mix and resuspend the cells by pipetting.
3、白细胞去除3, white blood cell removal
步骤1：在上述步骤得到的100μL细胞悬液转移至新的15mL离心管C 中，并加入生物素标记的高亲和力抗体混合液(兔抗人CD45单抗、鼠抗人CD16单抗、鼠抗人CD19单抗、鼠抗人CD235a单抗，抗体用量比例为1:3:3:3；添加量分别为1μL、3μL、3μL、3μL)至离心管C中；Step 1: 100 μL of the cell suspension obtained in the above procedure was transferred to a new 15 mL centrifuge tube C, and a biotin-labeled high-affinity antibody mixture (rabbit anti-human CD45 mAb, mouse anti-human CD16 mAb, mouse anti-mouse) was added. Human CD19 monoclonal antibody, mouse anti-human CD235a monoclonal antibody, the ratio of antibody dosage is 1:3:3:3; the addition amount is 1 μL, 3 μL, 3 μL, 3 μL) to the centrifuge tube C;
步骤2：轻柔吹打混匀，4℃孵育20分钟，每10分钟混匀一次；Step 2: Mix gently and mix, incubate at 4 ° C for 20 minutes, mix once every 10 minutes;
步骤3：孵育结束后，加入分离清洗液来去除掉未结合血细胞的抗体，头尾颠倒混匀数次，离心弃上清至100μL，离心条件为300g,10分钟(2℃-8℃)；Step 3: After the end of the incubation, the separation washing solution is added to remove the unbound blood cells, the head and tail are mixed upside down several times, and the supernatant is centrifuged to 100 μL, and the centrifugation condition is 300 g for 10 minutes (2 ° C - 8 ° C);
步骤4：加入3mL分离清洗液吹打混匀重悬细胞；Step 4: Add 3 mL of separation washing solution and mix and resuspend the cells;
步骤5：加入上述步骤配置好的磁珠悬液，持续一边摇晃离心管C一边滴加磁珠悬液，混匀并放置在混匀器上孵育15分钟(2℃-8℃)；Step 5: Add the magnetic bead suspension configured in the above steps, continue to shake the centrifuge tube C while dropping the magnetic bead suspension, mix and place on the mixer for 15 minutes (2 ° C - 8 ° C);
步骤6：孵育结束后加入3mL分离清洗液到离心管C中；重悬磁珠结合的细胞，用枪头轻轻吹打混匀，切记勿产生气泡；然后放置在磁力架上静置3分钟；Step 6: After the end of the incubation, add 3 mL of the separated washing solution to the centrifuge tube C; resuspend the magnetic beads-bound cells, mix gently with a pipette tip, and remember not to generate air bubbles; then place on a magnetic stand for 3 minutes;
步骤7：小心避开磁珠转移上清至新的15mL离心管D中；Step 7: Carefully avoid the magnetic beads transfer supernatant to a new 15 mL centrifuge tube D;
步骤8：加入4mL分离清洗液至装有磁珠的离心管C中；轻柔吹打混匀，切记勿产生气泡，并放置在磁力架上静置3分钟；Step 8: Add 4 mL of separation cleaning solution to the centrifuge tube C containing the magnetic beads; gently mix and mix, remember not to generate air bubbles, and place on a magnetic stand for 3 minutes;
步骤9：小心避开磁珠转移上清至15mL离心管D中；Step 9: Carefully avoid the magnetic beads transfer supernatant to the 15mL centrifuge tube D;
步骤10：吹打混匀离心管D中的液体并放置离心管D在磁力架上静置3分钟；Step 10: Blow the liquid in the mixing tube D and place the centrifuge tube D on the magnetic stand for 3 minutes;
步骤11：转移离心管D中的所有液体到标记好的15mL离心管E中；离心弃上清至50μL，离心条件为300g，10分钟(4℃)。Step 11: Transfer all the liquid from the centrifuge tube D to the labeled 15 mL centrifuge tube E; discard the supernatant to 50 μL and centrifuge at 300 g for 10 minutes (4 ° C).
4、CTCs鉴定4. Identification of CTCs
步骤1：加入25μL FCR阻断液到实施例3步骤得到的50μL细胞悬液中，使用振荡混合仪轻柔振荡混匀沉淀细胞，室温静置10分钟；Step 1: Add 25 μL of FCR blocking solution to 50 μL of the cell suspension obtained in the step of Example 3, gently mix the precipitated cells with a shaking mixture, and let stand for 10 minutes at room temperature;
步骤2：各加入1μL荧光抗体(CD45-AF594，瘤标荧光抗体)，室温避光孵育20分钟，每10分钟使用震荡混合仪轻柔混匀一次；Step 2: Add 1 μL of each fluorescent antibody (CD45-AF594, tumor-labeled fluorescent antibody), incubate at room temperature for 20 minutes in the dark, and gently mix once every 10 minutes using an oscillating mixer;
步骤3：补加细胞清洗液至14mL，配平，颠倒混匀，室温离心，离心条件为950g，4分钟，弃上清至100μL；Step 3: Add the cell washing solution to 14 mL, trim, mix by inversion, centrifuge at room temperature, centrifuge at 950 g for 4 minutes, discard the supernatant to 100 μL;
步骤4：加入100μL 4％多聚甲醛于以上100μL细胞悬液中，并使用加样器枪头轻柔吹打混匀；Step 4: Add 100 μL of 4% paraformaldehyde to the above 100 μL of cell suspension, and gently mix and mix using a pipette tip;
步骤5：将离心管E中的所有标本液体滴加涂于载玻片标本框内；Step 5: Apply all the sample liquid in the centrifuge tube E to the slide specimen box;
步骤6：将标本放置30-32℃干燥箱过夜，第二天关闭干燥箱温度后将标本继续静置于干燥箱中30分钟凉至室温，立即进行后续检测；Step 6: Place the specimen in a drying oven at 30-32 ° C overnight, and then turn off the temperature of the drying oven on the next day, and then continue to place the specimen in a dry box for 30 minutes to cool to room temperature, and immediately perform subsequent testing;
步骤7：加入200μL新鲜配置的固定液(甲醇：冰乙酸＝3：1)于玻片上避免气泡,室温静置10分钟；Step 7: Add 200 μL of freshly prepared fixative solution (methanol: glacial acetic acid = 3:1) to the slide to avoid air bubbles, and let stand at room temperature for 10 minutes;
步骤8：沿标本框内侧边角轻柔滴加200μL 1xPBS后，立即吸弃，共重复2次；Step 8: gently add 200 μL of 1×PBS along the inner corner of the specimen frame, and immediately discard it for 2 times;
步骤9：沿标本框内侧边角轻柔滴加200μL 1xPBS，静置2分钟，吸弃，共重复两次；Step 9: gently add 200 μL of 1×PBS along the inner corner of the specimen frame, let stand for 2 minutes, and discard it for a total of two times;
步骤10：沿标本框内侧边角轻柔滴加200μL无水乙醇后，立即吸弃，共重复2次；Step 10: gently add 200 μL of absolute ethanol along the inner corner of the specimen frame, and immediately discard it, repeating 2 times;
步骤11：将玻片***装有无水乙醇的染色缸中静置2分钟，取出玻片，竖立在滤纸上完全吸干玻片流下残留液，并使用微型吹风机将玻片轻柔吹至完全干燥后；Step 11: Insert the slide into the dyeing tank containing absolute ethanol for 2 minutes, remove the slide, erect on the filter paper, completely absorb the residual liquid from the slide, and gently slide the slide to the surface completely using a micro-hair dryer. Rear;
步骤12：以下操作均须避光，将载玻片吹干后立即滴加10μL八号染色体FISH探针于标本框中央，后立即使用镊子将盖玻片盖放在探针液上，使液体向四周蔓延至整个标本框；如果需要，例如标本框内有气泡，需使用镊子轻压盖玻片以使探针液覆盖整个标本框并把气泡排出；Step 12: The following operations should be protected from light. Immediately after drying the slide, 10 μL of the chromosome 18 FISH probe was added to the center of the specimen frame, and then the cover slip was placed on the probe solution using forceps to make the liquid. Spread to the entire specimen frame; if necessary, for example, there are air bubbles in the specimen frame, use a forceps to gently press the cover slip so that the probe fluid covers the entire specimen frame and discharge the air bubbles;
步骤13：封片：剪去1mL加样器尖头，每张玻片使用250μL封片胶封住盖玻片四边边缘后，直接放入杂交仪；Step 13: Mounting: Cut off the tip of the 1 mL sampler, and seal each side of the coverslip with 250 μL of sealant after each slide, and put it directly into the hybridization instrument;
步骤14：杂交：变性76℃，10分钟；杂交37℃，4小时；Step 14: Hybridization: denaturation at 76 ° C for 10 minutes; hybridization at 37 ° C for 4 hours;
步骤15：杂交完毕，取出玻片；用手轻按盖玻片一角，使用镊子撕去封片胶，将玻片置于预热至室温的染色缸中，静置1分钟后轻柔晃动染色缸，直至盖玻片脱落。盖玻片脱落后，将玻片继续在缸中室温静置1分钟后取出，使用滤纸吸干玻片流下的残留液，并擦干标本框***液体；Step 15: After the hybridization is completed, remove the slide; gently press the corner of the cover glass by hand, use a pair of tweezers to remove the sealant, place the slide on the dyeing tank preheated to room temperature, and let stand for 1 minute, then gently shake the dyeing tank. Until the coverslip falls off. After the coverslip is detached, the slide is continuously left in the cylinder for 1 minute at room temperature, and then the residual liquid flowing down the slide is dried by using a filter paper, and the liquid outside the specimen frame is wiped off;
步骤16：沿标本框内侧边角轻柔滴加200μL 2x SSC/0.1％NP-40后，立 即吸弃溶液，共重复2次；Step 16: After gently dropping 200 μL of 2x SSC/0.1% NP-40 along the inner corner of the specimen frame, immediately aspirate the solution and repeat 2 times;
步骤17：沿标本框内侧边角轻柔滴加200μL 2x SSC/0.1％NP-40，室温静置2分钟，吸弃溶液；Step 17: gently add 200 μL of 2x SSC/0.1% NP-40 along the inner corner of the specimen frame, let stand for 2 minutes at room temperature, and discard the solution;
步骤18：取10μL荧光保存液滴加于标本框中央，置放盖玻片后，一手固定盖玻片防止其滑动，另一手用真空泵吸头或滤纸轻柔挤压并吸干溢出的多余液体，封干标本；Step 18: Take 10 μL of fluorescent preservation droplets and add them to the center of the specimen frame. After placing the coverslips, fix the coverslips in one hand to prevent them from sliding. The other hand gently squeezes and absorbs the excess liquid overflowing with vacuum pump tips or filter paper. Sealed specimens;
步骤19：立即在荧光扫描显微镜下观察或于4℃避光保存。为避免FISH信号及抗体染色荧光减弱，标本应在一周内完成检测。在荧光显微镜上反复观察标本会使荧光淬灭。Step 19: Immediately observe under a fluorescent scanning microscope or store at 4 ° C in the dark. In order to avoid the attenuation of FISH signal and antibody staining, the specimen should be completed within one week. Repeated observation of the specimen on a fluorescence microscope will quench the fluorescence.
实施例1Example 1
对3位患者血液分别分成3管，每管2ml，分别采用高亲和力、低亲和力两种CD45抗体质量比为1:1、5:1、10:1的量进行残余白细胞总数、CTC检出数目进行比较。The blood of the three patients was divided into 3 tubes, 2 ml each, and the total number of residual white blood cells and CTC detected by the high-affinity and low-affinity CD45 antibody mass ratios of 1:1, 5:1, and 10:1, respectively. Compare.
结果如图1所示。结果显示，高亲和力、低亲和力两种CD45抗体质量量比为1:1、5:1、10:1时，残余白细胞总数依次减少，说明去除白细胞的能力逐渐递增。The result is shown in Figure 1. The results showed that the high-affinity, low-affinity CD45 antibody mass ratio was 1:1, 5:1, 10:1, the total number of residual white blood cells decreased, indicating that the ability to remove white blood cells gradually increased.
对每个样品的CTC的检出总数进行统计，结果如图2所示。结果显示质量比为5:1的效果优于1:1和10:1。可能的原因是，1:1的用量中高亲和力抗体的量太低，白细胞剩余太多，导致CTC细胞被漏检；而10:1的用量比例中，高亲和力抗体的量太高，使CTC细胞被白细胞团聚去除掉，导致漏检。The total number of CTCs detected for each sample was counted and the results are shown in Fig. 2. The results show that the mass ratio of 5:1 is better than 1:1 and 10:1. The possible reason is that the amount of high-affinity antibodies in the 1:1 dosage is too low, and the white blood cells remain too much, causing the CTC cells to be missed; and in the ratio of 10:1, the amount of high-affinity antibodies is too high, so that the CTC cells It is removed by leukocyte agglomeration, resulting in missed detection.
实施例2Example 2
对同一肿瘤患者采取两管等量外周血，重复三次实验操作，即共采取15位不同肿瘤患者的各两管外周血。分别按照对照(赛特生物；美国专利号：US8,969,021)所述的方法和本文“材料与方法”部分所述的方法进行CTC检测，对最终检出率进行评价。两种CD45抗体质量比为5:1。两种检测方法对CTC检出数量如图3所示。Two equal volumes of peripheral blood were taken from the same tumor patient, and three experimental operations were repeated, that is, a total of 15 peripheral blood samples of 15 different tumor patients were taken. The CTC test was carried out according to the method described in the control (Scitech Bio; US Pat. No. 8,969,021) and the method described in the "Materials and Methods" section, respectively, and the final detection rate was evaluated. The ratio of the two CD45 antibodies was 5:1. The number of detection methods for the two CTCs is shown in Figure 3.
对每个样本的总细胞数进行扫描计数，结果如图4所示。结果显示，本发明残留的血源性白细胞总数低于对照，表明本发明具有高效富集CTCs的能力。The total number of cells per sample was scanned and the results are shown in FIG. The results show that the total amount of residual blood-derived white blood cells of the present invention is lower than that of the control, indicating that the present invention has the ability to efficiently enrich CTCs.
对每个样品的疑似细胞总数进行统计和比较，结果如下图5所示。结果显示，本发明疑似细胞总数低于对照，表明本发明具有更高效鉴定CTCs的能力。Statistics and comparisons were made on the total number of suspected cells for each sample. The results are shown in Figure 5 below. The results show that the total number of suspected cells of the present invention is lower than the control, indicating that the present invention has the ability to more efficiently identify CTCs.

Claims (11)

  1. 一种含有抗体的组合物，其特征在于，其包括抗CD16单抗、抗CD19单抗、抗CD235a单抗和两种或两种以上抗CD45单抗，其中，所述两种或两种以上抗CD45单抗所识别CD45抗原分子上的抗原决定簇各不相同；优选地，所述单抗均偶联有亲和标记；优选地，所述亲和标记为生物素分子。An antibody-containing composition comprising an anti-CD16 mAb, an anti-CD19 mAb, an anti-CD235a mAb, and two or more anti-CD45 mAbs, wherein the two or more The antigenic determinants on the CD45 antigen molecule recognized by the anti-CD45 mAb are different; preferably, the mAb is coupled with an affinity tag; preferably, the affinity tag is a biotin molecule.
  2. 如权利要求1所述的含有抗体的组合物，其特征在于，所述两种或两种以上抗CD45单抗的亲和力系数均低于1.0×10 -10M；优选地，所述两种或两种以上抗CD45单抗中的至少一种的亲和力系数低于4.0×10 -11M。 The antibody-containing composition according to claim 1, wherein the two or more anti-CD45 mAbs have an affinity coefficient of less than 1.0 × 10 -10 M; preferably, the two or The affinity coefficient of at least one of the two or more anti-CD45 mAbs is less than 4.0 x 10 -11 M.
  3. 如权利要求2所述的含有抗体的组合物，其特征在于，以质量计，所述两种或两种以上抗CD45单抗中亲和力最高的抗CD45单抗的含量与所有其它抗CD45单抗的含量之和之比在1～10：1的范围内，优选在3～7：1的范围内，更优选为5：1。The antibody-containing composition according to claim 2, wherein the content of the anti-CD45 mAb having the highest affinity among the two or more anti-CD45 mAbs and all other anti-CD45 mAbs by mass The ratio of the sum of the contents is in the range of 1 to 10:1, preferably in the range of 3 to 7:1, and more preferably 5:1.
  4. 如权利要求1-3中任一项所述的含有抗体的组合物，其特征在于，组合物中的单抗的物种来源为相同的或不同的物种来源；优选地，所述两种或两种以上抗CD45单抗中的一种的物种来源为兔源，其它抗CD45单抗的物种来源为鼠源；更优选地，所述两种或两种以上抗CD45单抗的物种来源均为兔源。The antibody-containing composition according to any one of claims 1 to 3, wherein the species source of the monoclonal antibodies in the composition is the same or different species sources; preferably, the two or two The species source of one of the above anti-CD45 mAbs is a rabbit source, and the source of other anti-CD45 mAbs is a mouse source; more preferably, the species sources of the two or more anti-CD45 mAbs are Rabbit source.
  5. 如权利要求1－4中任一项所述的含有抗体的组合物，其特征在于，所述抗CD16单抗、抗CD19单抗和抗CD235a单抗各自独立为兔抗人单克隆抗体或鼠抗人单克隆抗体；The antibody-containing composition according to any one of claims 1 to 4, wherein the anti-CD16 monoclonal antibody, the anti-CD19 monoclonal antibody and the anti-CD235a monoclonal antibody are each independently a rabbit anti-human monoclonal antibody or a mouse. Anti-human monoclonal antibody;
    优选地，所述两种或两种以上抗CD45单抗均为兔抗人CD45单克隆抗体，所述抗CD16单抗为鼠抗人CD16单克隆抗体、所述抗CD19单抗为鼠抗人CD19单克隆抗体和所述抗CD235a单抗为鼠抗人CD235a单克隆抗体。Preferably, the two or more anti-CD45 mAbs are rabbit anti-human CD45 monoclonal antibodies, the anti-CD16 mAb is a murine anti-human CD16 monoclonal antibody, and the anti-CD19 mAb is a mouse anti-human The CD19 monoclonal antibody and the anti-CD235a monoclonal antibody are murine anti-human CD235a monoclonal antibodies.
  6. 如权利要求1－5中任一项所述的组合物，其特征在于，以质量计，所述组合物中所述两种或两种以上抗CD45单抗、抗CD16单抗、抗CD19单抗和抗CD235a单抗之间的含量比例为1：1～5：1～5：1～5；优选为1：2～4：2～4：2～4；更优选为1：3：3：3。The composition according to any one of claims 1 to 5, wherein the two or more anti-CD45 mAb, anti-CD16 mAb, anti-CD19 single in the composition are by mass. The content ratio between the anti-CD235a monoclonal antibody is 1:1 to 5:1 to 5:1 to 5; preferably 1:2 to 4:2 to 4:2 to 4; more preferably 1:3:3 :3.
  7. 一种试剂盒，其特征在于，所述试剂盒含有权利要求1－6中任一项所 述的含抗体的组合物，和任选的磁珠；A kit comprising the antibody-containing composition of any one of claims 1 to 6, and optionally a magnetic bead;
    优选地，试剂盒中，所述含抗体的组合物和磁珠分别置于不同的容器中；Preferably, in the kit, the antibody-containing composition and the magnetic beads are respectively placed in different containers;
    优选地，所述磁珠偶联了能与抗体上所偶联的亲和标记相结合的配体。Preferably, the magnetic beads are coupled to a ligand that binds to an affinity tag coupled to the antibody.
  8. 两种或两种以上抗CD45单抗、抗CD16单抗、抗CD19单抗和抗CD235a单抗的组合在分离血液中的白细胞或在富集外周血循环肿瘤细胞中的应用，或在制备用于分离血液中的白细胞或用于富集外周血循环肿瘤细胞的试剂或试剂盒中的应用；Combination of two or more anti-CD45 mAb, anti-CD16 mAb, anti-CD19 mAb and anti-CD235a mAb in the separation of leukocytes in blood or in enriched peripheral blood circulating tumor cells, or in preparation for Separating white blood cells in blood or applications in reagents or kits for enriching circulating blood cells of peripheral blood;
    优选地，所述两种或两种以上抗CD45单抗、抗CD16单抗、抗CD19单抗和抗CD235a单抗如权利要求2－5中任一项所述；Preferably, the two or more anti-CD45 mAb, anti-CD16 mAb, anti-CD19 mAb and anti-CD235a mAb are as claimed in any one of claims 2-5;
    优选地，所述应用中，以质量计，两种或两种以上抗CD45单抗、抗CD16单抗、抗CD19单抗和抗CD235a单抗之间的浓度比例为1：1～5：1～5：1～5，优选为1：2～4：2～4：2～4，更优选为1：3：3：3。Preferably, in the application, the concentration ratio between two or more anti-CD45 mAb, anti-CD16 mAb, anti-CD19 mAb and anti-CD235a mAb is 1:1 to 5:1 by mass. ～5:1 to 5, preferably 1:2 to 4:2 to 4:2 to 4, more preferably 1:3:3:3.
  9. 一种分离血液中白细胞的方法，其特征在于，所述方法包括：A method of separating white blood cells in blood, characterized in that the method comprises:
    (1)孵育血液与权利要求1－6中任一项所述的组合物的混合物，其中所述组合物中的抗体均偶联有亲和标记；和(1) A mixture of blood and a composition of any one of claims 1 to 6, wherein the antibody in the composition is coupled with an affinity tag;
    (2)孵育结束后，加入磁珠，孵育并分离出结合了白细胞的磁珠，其中，所述磁珠偶联了能与抗体上所偶联的亲和标记相结合的配体；(2) after the end of the incubation, adding magnetic beads, incubating and separating the magnetic beads bound to the white blood cells, wherein the magnetic beads are coupled with a ligand capable of binding to the affinity label coupled to the antibody;
    从而分离出血液中的白细胞；Thereby separating white blood cells in the blood;
    优选地，所述血液是除去了红细胞和血浆的血液。Preferably, the blood is blood from which red blood cells and plasma are removed.
  10. 一种富集循环肿瘤细胞的方法，其特征在于，所述方法包括：A method for enriching circulating tumor cells, the method comprising:
    (1)离心，除去血浆；(1) centrifugation to remove plasma;
    (2)离心，除去红细胞；(2) centrifugation to remove red blood cells;
    (3)洗涤步骤(2)获得的液体；(3) the liquid obtained in the washing step (2);
    (4)在步骤(3)获得的液体中加入权利要求1－6中任一项所述的含抗体的组合物，孵育，离心清洗，其中所述组合物中的抗体均偶联有亲和标记；(4) The antibody-containing composition according to any one of claims 1 to 6 is added to the liquid obtained in the step (3), incubated, and centrifuged, wherein the antibodies in the composition are coupled with an affinity. mark;
    (5)在步骤(4)获得的细胞中加入偶联了能与抗体上所偶联的亲和标记相结合的配体的磁珠，孵育；和(5) adding, to the cells obtained in the step (4), a magnetic bead coupled with a ligand capable of binding to the affinity tag coupled to the antibody, and incubating;
    (6)孵育结束后，除去磁珠，从而富集循环肿瘤细胞。(6) After the end of the incubation, the magnetic beads are removed to enrich the circulating tumor cells.
  11. 如权利要求10所述的方法，其特征在于，The method of claim 10 wherein:
    所述步骤(4)包括，将步骤(3)获得的液体与所述含抗体的组合物混合，孵育10～30分钟后，离心、清洗；The step (4) comprises: mixing the liquid obtained in the step (3) with the antibody-containing composition, incubating for 10 to 30 minutes, and then centrifuging and washing;
    所述步骤(5)包括，加入偶联了能与抗体上所偶联的亲和标记相结合的配体的磁珠，孵育10～20分钟；The step (5) comprises: adding a magnetic bead coupled with a ligand capable of binding to the affinity tag coupled to the antibody, and incubating for 10 to 20 minutes;
    所述步骤(6)包括，孵育结束后，将含所述液体和磁珠的容器置于磁力架上，静置，使磁珠吸附在磁铁上，吸取未含有磁珠部分的液体；和The step (6) includes, after the end of the incubation, placing the container containing the liquid and the magnetic beads on a magnetic stand, allowing to stand, so that the magnetic beads are adsorbed on the magnet, and the liquid not containing the magnetic bead portion is sucked;
    所述步骤(4)到步骤(6)在2～8℃的温度下进行。The steps (4) to (6) are carried out at a temperature of 2 to 8 °C.
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