CN116731962A - Kit and method for separating red blood cells from whole blood - Google Patents
Kit and method for separating red blood cells from whole blood Download PDFInfo
- Publication number
- CN116731962A CN116731962A CN202311013185.5A CN202311013185A CN116731962A CN 116731962 A CN116731962 A CN 116731962A CN 202311013185 A CN202311013185 A CN 202311013185A CN 116731962 A CN116731962 A CN 116731962A
- Authority
- CN
- China
- Prior art keywords
- red blood
- antibody
- blood cells
- coupled
- magnetic
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 210000003743 erythrocyte Anatomy 0.000 title claims abstract description 116
- 239000008280 blood Substances 0.000 title claims abstract description 58
- 210000004369 blood Anatomy 0.000 title claims abstract description 57
- 238000000034 method Methods 0.000 title claims description 14
- 102000006834 complement receptors Human genes 0.000 claims abstract description 45
- 108010047295 complement receptors Proteins 0.000 claims abstract description 45
- 239000002122 magnetic nanoparticle Substances 0.000 claims abstract description 43
- 239000002245 particle Substances 0.000 claims abstract description 41
- 239000000427 antigen Substances 0.000 claims abstract description 6
- 102000036639 antigens Human genes 0.000 claims abstract description 6
- 108091007433 antigens Proteins 0.000 claims abstract description 6
- 239000004005 microsphere Substances 0.000 claims description 29
- 239000004816 latex Substances 0.000 claims description 27
- 229920000126 latex Polymers 0.000 claims description 27
- 210000002381 plasma Anatomy 0.000 claims description 19
- 150000001875 compounds Chemical class 0.000 claims description 17
- 102100030886 Complement receptor type 1 Human genes 0.000 claims description 14
- 101000727061 Homo sapiens Complement receptor type 1 Proteins 0.000 claims description 14
- 229920000642 polymer Polymers 0.000 claims description 12
- 238000002156 mixing Methods 0.000 claims description 11
- 238000007664 blowing Methods 0.000 claims description 7
- 239000011325 microbead Substances 0.000 claims description 7
- 102100022002 CD59 glycoprotein Human genes 0.000 claims description 4
- 102100025680 Complement decay-accelerating factor Human genes 0.000 claims description 4
- -1 DAF Proteins 0.000 claims description 4
- 102000004190 Enzymes Human genes 0.000 claims description 4
- 108090000790 Enzymes Proteins 0.000 claims description 4
- 101000897400 Homo sapiens CD59 glycoprotein Proteins 0.000 claims description 4
- 101000856022 Homo sapiens Complement decay-accelerating factor Proteins 0.000 claims description 4
- 101001063392 Homo sapiens Lymphocyte function-associated antigen 3 Proteins 0.000 claims description 4
- 102100030984 Lymphocyte function-associated antigen 3 Human genes 0.000 claims description 4
- 102000010681 interleukin-8 receptors Human genes 0.000 claims description 4
- 108010038415 interleukin-8 receptors Proteins 0.000 claims description 4
- 230000005389 magnetism Effects 0.000 claims description 4
- 210000002966 serum Anatomy 0.000 claims description 4
- 206010057249 Phagocytosis Diseases 0.000 claims description 3
- GNBHRKFJIUUOQI-UHFFFAOYSA-N fluorescein Chemical compound O1C(=O)C2=CC=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 GNBHRKFJIUUOQI-UHFFFAOYSA-N 0.000 claims description 3
- 239000003112 inhibitor Substances 0.000 claims description 3
- 210000002540 macrophage Anatomy 0.000 claims description 3
- 230000008782 phagocytosis Effects 0.000 claims description 3
- 239000002096 quantum dot Substances 0.000 claims description 3
- 102000009410 Chemokine receptor Human genes 0.000 claims 1
- 108050000299 Chemokine receptor Proteins 0.000 claims 1
- 239000007850 fluorescent dye Substances 0.000 claims 1
- 238000000926 separation method Methods 0.000 abstract description 12
- 210000004027 cell Anatomy 0.000 abstract description 6
- 239000002105 nanoparticle Substances 0.000 abstract description 4
- 230000000694 effects Effects 0.000 abstract description 3
- 239000011324 bead Substances 0.000 description 19
- 238000001514 detection method Methods 0.000 description 8
- 239000006228 supernatant Substances 0.000 description 8
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 5
- 230000008878 coupling Effects 0.000 description 4
- 238000010168 coupling process Methods 0.000 description 4
- 238000005859 coupling reaction Methods 0.000 description 4
- 230000003213 activating effect Effects 0.000 description 3
- 210000000601 blood cell Anatomy 0.000 description 3
- 238000006243 chemical reaction Methods 0.000 description 3
- 230000004913 activation Effects 0.000 description 2
- 238000005119 centrifugation Methods 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 239000005482 chemotactic factor Substances 0.000 description 2
- 230000007547 defect Effects 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 102000005962 receptors Human genes 0.000 description 2
- 108020003175 receptors Proteins 0.000 description 2
- 230000035945 sensitivity Effects 0.000 description 2
- 239000000725 suspension Substances 0.000 description 2
- 108010047620 Phytohemagglutinins Proteins 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical group [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000002131 composite material Substances 0.000 description 1
- 239000011246 composite particle Substances 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- 239000002270 dispersing agent Substances 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 210000003617 erythrocyte membrane Anatomy 0.000 description 1
- 230000002349 favourable effect Effects 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 238000001917 fluorescence detection Methods 0.000 description 1
- 238000004108 freeze drying Methods 0.000 description 1
- 230000036737 immune function Effects 0.000 description 1
- 238000007885 magnetic separation Methods 0.000 description 1
- 230000014759 maintenance of location Effects 0.000 description 1
- 238000005457 optimization Methods 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 230000001885 phytohemagglutinin Effects 0.000 description 1
- 238000005070 sampling Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 239000012780 transparent material Substances 0.000 description 1
- 238000009777 vacuum freeze-drying Methods 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0634—Cells from the blood or the immune system
- C12N5/0641—Erythrocytes
-
- 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
- G01N33/543—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
- G01N33/54346—Nanoparticles
-
- 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
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56966—Animal cells
-
- 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
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
- G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label
-
- 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
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
- G01N33/585—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with a particulate label, e.g. coloured latex
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2509/00—Methods for the dissociation of cells, e.g. specific use of enzymes
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2469/00—Immunoassays for the detection of microorganisms
- G01N2469/10—Detection of antigens from microorganism in sample from host
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Immunology (AREA)
- Chemical & Material Sciences (AREA)
- Biomedical Technology (AREA)
- Hematology (AREA)
- Urology & Nephrology (AREA)
- Molecular Biology (AREA)
- Cell Biology (AREA)
- Biotechnology (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Microbiology (AREA)
- Pathology (AREA)
- General Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Physics & Mathematics (AREA)
- Medicinal Chemistry (AREA)
- Food Science & Technology (AREA)
- Zoology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Organic Chemistry (AREA)
- Genetics & Genomics (AREA)
- Wood Science & Technology (AREA)
- Virology (AREA)
- Tropical Medicine & Parasitology (AREA)
- General Engineering & Computer Science (AREA)
- Nanotechnology (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
The invention belongs to the technical field of cell separation, and provides a kit for separating red blood cells in whole blood, which comprises biological particles coupled with a red blood cell complement receptor antibody and magnetic nano particles coupled with an anti-RBC antibody. The red blood cells are provided with complement receptors, the biological particles coupled with the complement receptor antibodies of the red blood cells are specifically combined with the complement receptors of the red blood cells, so that the red blood cells do not occupy RBC antigen sites on the surfaces of the red blood cells, the red blood cells and the immunomagnetic nanoparticles are more convenient to be combined rapidly and effectively, the immunomagnetic nanoparticles are enriched in advance to form cell clusters, and then are combined with the immunomagnetic nanoparticles, the effect of amplifying and combining the red blood cells is achieved, the use amount of the magnetic nanoparticles is saved, the cost of the biological particles is lower than that of the magnetic nanoparticles, and the cost of a kit is saved.
Description
Technical Field
The invention belongs to the technical field of cell separation, and particularly relates to a kit and a method for separating red blood cells from whole blood.
Background
The traditional method for separating red blood cells from blood samples to obtain serum or plasma is centrifugal separation (more than 5 minutes), special centrifugal equipment is needed, the blood cells are possibly broken when the centrifugal operation is improper, the rapid detection is not easy to realize, the requirement of clinical immunodetection on rapid detection is difficult to meet, and the automation of red blood cell separation is difficult to realize by the centrifugal method, so that the miniaturization of the detection equipment is not easy to realize. The existence of red blood cells brings great interference to the detection of the existing blood markers, so that the blood sample is rapidly and accurately processed and detected, and the treatment time window is favorable for patients.
For a long time, it has been considered that erythrocytes have a simple structure "only carry oxygen function" but since the concept of "erythrocyte immune function" has been proposed by american geneticists, new understanding of erythrocytes has been achieved, and a number of experiments have been carried out to prove that erythrocyte membrane surfaces have many immune-related substances, such as: complement receptor 1 (CD 35), complement receptor 3, CD55, CD58, CD59, IL-8 receptor, DAF, SOD enzyme, and the like.
Chinese patent publication No. CN111308070a discloses a kit for separating red blood cells from whole blood and a method for separating red blood cells from whole blood using chemically coupled anti-RBC antibody magnetic beads, phytohemagglutinin and/or anti-RBC antibody for separation, and then separating red blood cells by magnetic attraction, but the method requires using the same volume of carboxyl magnetic beads as whole blood, equivalent to diluting plasma sample at the same time, thus necessarily affecting the sensitivity of sample detection; the cost of the carboxyl magnetic beads is relatively high, and the use of a large number of carboxyl magnetic beads causes the increase of the separation cost.
Disclosure of Invention
Aiming at the defects of the existing method for separating red blood cells in whole blood, the invention provides a kit and a method for separating red blood cells in whole blood, and the use amount of a magnetic nanoparticle solution can be effectively reduced by using the kit to separate the red blood cells from the whole blood, so that the cost of the kit is further reduced.
In order to achieve the above purpose, the technical scheme of the invention is realized in such a way that the kit for separating red blood cells in whole blood comprises biological particles coupled with complement receptor antibodies of the red blood cells and magnetic nano particles coupled with anti-RBC antibodies.
Further, the complement receptor in the biological particle coupled with the erythrocyte complement receptor antibody is one or more of CD35, complement receptor 3, CD55, CD58, CD59, IL-8 receptor, DAF, SOD enzyme, erythrocyte chemotactic factor receptor and macrophage phagocytosis inhibitor.
Further, the biological particles in the biological particles coupled with the erythrocyte complement receptor antibody are at least one of latex microspheres, fluorescein filled microspheres, time-resolved fluorescent microspheres and quantum dot fluorescent microspheres.
Further, the magnetic nanoparticles in the magnetic nanoparticles coupled with the anti-RBC antibody are magnetic polymer microbeads.
Further, the biological particles coupled with the erythrocyte complement receptor antibody have fluorescent markers.
In another aspect, the present invention also provides a method for separating red blood cells from whole blood using the kit according to any one of the above aspects, comprising at least the steps of:
1) Adding biological particles coupled with the erythrocyte complement receptor antibody into a test tube, then adding whole blood, gently blowing and uniformly mixing to ensure that the biological particles coupled with the erythrocyte complement receptor antibody are specifically combined with complement receptors on erythrocytes at first, and the biological particles rapidly enrich erythrocytes to form cell clusters;
2) Continuously adding magnetic nano particles coupled with the anti-RBC antibody into a test tube, so that the magnetic nano particles coupled with the anti-RBC antibody are specifically combined with RBC antigens on the surface of red blood cells, the magnetic nano particles coupled with the antibody are combined with red blood cells combined with a complement receptor antibody of the red blood cells to form a red blood cell double-antibody sandwich compound, and the magnetic nano particles coupled with the antibody can be combined with a small amount of free red blood cells to form a red blood cell-RBC antibody compound;
3) The annular electromagnetic sleeve is sleeved outside the test tube, the magnetic pole and magnetism of the electromagnetic sleeve are controlled by changing the direction and intensity of current, and the magnetic force is uniformly mixed and absorbed for 1min, so that the red blood cell double antibody sandwich compound and the red blood cell-RBC antibody compound combined with the magnetic nano particles are gathered towards the direction of the magnet.
In another aspect, the present invention also provides a method for separating red blood cells from whole blood using the kit according to the above technical solution, including at least the following steps:
1) Adding fluorescent-labeled biological particles of the coupled erythrocyte complement receptor antibody into a test tube, then adding whole blood, and gently blowing and uniformly mixing to ensure that the biological particles of the coupled erythrocyte complement receptor antibody are specifically combined with complement receptors on erythrocytes;
2) Continuously adding magnetic nano particles coupled with the anti-RBC antibody into a test tube, so that the magnetic nano particles coupled with the anti-RBC antibody are specifically combined with RBC antigens on the surface of red blood cells, the magnetic nano particles coupled with the antibody are combined with red blood cells combined with a complement receptor antibody of the red blood cells to form a red blood cell double-antibody sandwich compound, and the magnetic nano particles coupled with the antibody can be combined with a small amount of free red blood cells to form a red blood cell-RBC antibody compound;
3) The annular electromagnetic sleeve is sleeved outside the test tube, the magnetic pole and magnetism of the electromagnetic sleeve are controlled by changing the direction and intensity of current, and the magnetic force is uniformly mixed and absorbed for 1min, so that the red blood cell double antibody sandwich compound and the red blood cell-RBC antibody compound combined with the magnetic nano particles are gathered towards the direction of the magnet.
4) By detecting whether the excessive fluorescent biological particles are remained in the plasma/serum separated in the middle of the test tube by using the fluorescence detector through the transparent area of the test tube bottom, if the fluorescence value in the middle of the test tube is not over the threshold value, the subsequent blood sample can be sampled, and if the fluorescence value in the middle of the test tube is over the threshold value, the excessive fluorescent biological particles are remained, and the blood sample cannot be sampled.
The kit and the method for separating the red blood cells from the whole blood, which are obtained through the technical scheme, have the beneficial effects that:
1. because the red blood cells are provided with complement receptors, the biological particles coupled with the complement receptor antibodies of the red blood cells are specifically combined with the complement receptors of the red blood cells, do not occupy RBC antigen sites on the surfaces of the red blood cells, and are more convenient for the rapid and effective combination of the red blood cells and the immunomagnetic nano particles.
2. The biological particles are specifically combined with the red blood cells and mutually aggregated, so that the effect of enriching the red blood cells is achieved, the composite particles become large after the enrichment of the red blood cells, cell clusters are formed, the aggregated red blood cell composite is combined with the magnetic nano particles, and the combination effect of the magnetic nano particles is amplified.
3. The immune biological particles and the immune magnetic nano particles are combined with red blood cells at the same time to form a double-antibody sandwich structure, so that the sensitivity is higher, the addition amount of a magnetic nano particle solution can be remarkably saved, the defect that a sample is diluted is avoided, the cost of the biological particles is lower than that of carboxyl magnetic nano particles, and meanwhile, the use cost of the kit can be reduced.
4. The annular electromagnetic sleeve can uniformly mix magnetic nano particles with whole blood by controlling magnetic poles and attraction through current, and the phenomenon of red blood cell rupture is avoided without uniformly mixing by means of tools and external force.
5. The annular electromagnetic sleeve increases the magnetic attraction area, improves the efficiency of absorbing the magnetic compound, and red blood cells are adsorbed on the pipe wall, and separated serum/plasma is in the middle of the pipe, so that the subsequent sampling gun can be directly inserted into the test tube to absorb the plasma sample conveniently, and the problem of erroneously absorbing the bottom red blood cell sample is not required to be considered.
6. The separation of blood plasma and blood cells is carried out without centrifugation and filtration, so that the use of a blood plasma separation device is simplified, and compared with the traditional centrifugation method for separating the whole blood sample, the separation efficiency is improved, the separation time is shortened, and the whole blood can be efficiently separated within 1-2 min.
7. The immune magnetic ball combined with the red blood cells is adsorbed on the wall of the magnet sleeve, the bottom of the test tube is made of high transparent material, fluorescent labels are adopted to couple biological particles of the red blood cell complement receptor antibody, a fluorescent detector is used for detecting whether the separated blood plasma is detained by surplus fluorescent biological particles through irradiation of a transparent area of the bottom of the test tube, if the blood plasma is detained by the fluorescent biological particles, the instrument does not sample blood, and the interference of further detection of the blood sample can be reduced.
Drawings
FIG. 1 is a schematic representation of the present invention when whole blood is added to a tube containing a erythrocyte complement receptor coupled biological particle;
FIG. 2 is a schematic illustration of the present invention when magnetic nanoparticles conjugated with anti-RBC antibodies are added to the tube of FIG. 1;
FIG. 3 is a schematic view of the present invention when an annular electromagnetic sleeve is sleeved outside the test tube of FIG. 2.
Detailed Description
It should be noted that, without conflict, the embodiments of the present invention and features of the embodiments may be combined with each other.
Unless defined otherwise, technical terms used in the following examples have the same meaning as commonly understood by one of ordinary skill in the art to which the present invention pertains. The test reagents used in the following examples, unless otherwise specified, are all conventional biochemical reagents; the experimental methods are conventional methods unless otherwise specified.
The invention relates to the technical field of cell separation in general, and uses immune biological particles and immune magnetic nanoparticles to combine with erythrocytes simultaneously, so as to play a role in amplifying and combining erythrocytes and save the use amount of the magnetic nanoparticles.
The invention is further illustrated below with reference to examples and figures, it being understood that the invention is not limited to the specific embodiments described.
As shown in the figure, a kit for separating red blood cells in whole blood comprises biological particles coupled with a red blood cell complement receptor antibody and magnetic nanoparticles coupled with an anti-RBC antibody.
The complement receptor in the biological particles coupled with the erythrocyte complement receptor antibody is one or more of CD35, complement receptor 3, CD55, CD58, CD59, IL-8 receptor, DAF, SOD enzyme, erythrocyte chemotactic factor receptor and macrophage phagocytosis inhibitor.
The biological particles in the biological particles coupled with the erythrocyte complement receptor antibody are at least one of latex microspheres, fluorescein filled microspheres, time-resolved fluorescent microspheres and quantum dot fluorescent microspheres.
The magnetic nanoparticles in the magnetic nanoparticles coupled with the anti-RBC antibody are magnetic polymer microbeads.
The biological particles coupled with the erythrocyte complement receptor antibody are provided with fluorescent labels.
The following is a preparation method of the latex microsphere coupled with CD35 antibody/fluorescent latex microsphere coupled with CD35 antibody in one technical scheme of the invention:
1) Taking 100ul of latex microspheres/fluorescent latex microspheres (purchased from Suzhou Nami technologies Co., ltd., hereinafter referred to as latex spheres) into a 1.5mL centrifuge tube, centrifuging to remove the supernatant, and washing with MES solution for 2 times;
2) Activating: dispersing the washed latex balls in 80 mu LMES solution, rapidly adding newly prepared 10 mu LNHS and 10 mu LEDC solution (50 mg/mL, using the MES as a dispersing agent), blowing and mixing uniformly, placing on a rotator for activating for 5min at normal temperature, centrifuging, removing supernatant, and carrying out covalent coupling with an anti-CD 35 antibody;
3) Coupling antibody: adding CD35 antibody into the activated latex ball, gently mixing, keeping the antibody in PBS solution with pH of 7.0, and centrifuging to remove supernatant after the reaction;
4) Closing: adding a PBS solution containing 5% BSA to resuspend the reacted latex beads, reacting for 1h at room temperature, centrifuging to remove supernatant, and re-suspending in the PBS solution with pH of 7.0 to obtain a latex microsphere solution coupled with the CD35 antibody/a fluorescent latex microsphere solution coupled with the CD35 antibody.
5) And (3) freeze-drying: and (3) performing vacuum freeze-drying treatment on the latex microsphere solution of the coupled CD35 antibody at the pressure of 20Pa and the temperature of less than-30 ℃ to obtain freeze-dried latex microsphere of the coupled CD35 antibody/freeze-dried fluorescent latex microsphere of the coupled CD35 antibody.
The following is a preparation method of magnetic polymer microbeads coupled with anti-RBC antibodies in a technical scheme of the invention:
1) Taking 100ul of carboxyl magnetic polymer microbeads (purchased from Wuhan New longitudinal biotechnology Co., ltd., hereinafter referred to as magnetic beads) into a 1.5mL centrifuge tube, wherein the particle size of the magnetic beads is 10 μm, and removing the supernatant by magnetic separation;
2) Activating: dispersing the washed magnetic nano particles in MES solution, rapidly adding newly prepared NHS and EDC solution, blowing and mixing uniformly, placing on a rotary instrument for activation for 5min, keeping the magnetic beads in a suspension state during activation, and magnetically separating to remove supernatant;
3) Coupling antibody: adding an anti-RBC antibody into the activated magnetic beads, gently mixing, reacting for 1h at room temperature with a PBS solution of which the antibody retention solution is pH7.0, keeping the suspension state of the magnetic beads during coupling, and magnetically sucking the supernatant after the reaction is finished;
4) Closing: adding PBS solution with pH7.0 containing 5% BSA to re-suspend the reacted magnetic beads, and reacting for 1h at room temperature while keeping the suspended state of the magnetic beads; magnetically sucking the supernatant, re-suspending in PBS solution of pH7.0, and placing in a refrigerator at 4 ℃ to obtain the magnetic polymer microbeads coupled with the anti-RBC antibody.
Example 1:
whole blood 200ul was added to each of the 4 tubes, and magnetic polymer microbeads 20ul, 40ul, 80ul, 160ul, to which anti-RBC antibodies were conjugated, were added, the tubes were placed in an annular electromagnetic sleeve for magnetic attraction for 1min, and intermediate plasma was aspirated, and the amount of plasma in the tubes was shown in Table 1.
TABLE 1 amount of magnetic beads isolated from whole blood
As can be seen from Table 1, if only the magnetic polymer beads of the anti-RBC antibody were added, 80ul of the magnetic polymer beads of the anti-RBC antibody were required to be added to 200ul of whole blood, and red blood cells in the whole blood could be separated.
Example 2:
adding 200ul of whole blood and 10ug of freeze-dried latex microspheres coupled with CD35 antibody into 4 test tubes, gently blowing and uniformly mixing for 0.5-1min, respectively adding 10ul, 20ul, 40ul and 80ul of latex microspheres coupled with anti-RBC antibody, putting the test tubes into a ring-shaped electromagnetic sleeve for magnetic attraction for 1min, absorbing middle plasma, and obtaining the amount of plasma in the test tubes as shown in Table 2.
TABLE 2 amount of latex microspheres for Whole blood separation with latex spheres added
As can be seen from Table 2, the addition of the CD35 antibody-conjugated latex beads effectively reduced the amount of the anti-RBC antibody-conjugated magnetic polymer beads, and 20ul of the anti-RBC antibody-conjugated magnetic polymer beads were used to separate out the red blood cells from the whole blood after 10ug immune latex beads were added to 200ul of the whole blood.
Example 3:
the 4 test tubes are put into the freeze-dried fluorescent latex microspheres of which the amounts are 2.5ug, 5ug, 10ug and 20ug respectively, 200ul of whole blood is added, the mixture is gently blown and evenly mixed for reaction for 0.5 to 1min, then 20ul of magnetic polymer microspheres of the conjugated anti-RBC antibody is added, the test tubes are put into an annular electromagnetic sleeve for magnetic attraction for 1min, middle plasma is sucked, and the plasma is subjected to fluorescent detection by a fluorescent detector, wherein the plasma amount, the red blood cell free condition and the fluorescent condition in the plasma are shown in Table 3. (the fluorescence detector is conventional technology and is consistent with the signal carried by the fluorescent latex microsphere)
TABLE 3 optimization of the amount of whole blood latex spheres isolated
As can be seen from Table 3, it was found by fluorescence detection that adding 5ug of the lyophilized fluorescent latex microspheres coupled with CD35 antibody to 200ul of whole blood can satisfy the requirement of red blood cell binding without leaving excessive fluorescent latex microspheres in plasma, and can be used for subsequent detection.
The above technical solution only represents the preferred technical solution of the present invention, and some changes that may be made by those skilled in the art to some parts of the technical solution represent the principles of the present invention, and the technical solution falls within the scope of the present invention.
Claims (7)
1. A kit for separating red blood cells from whole blood, which is characterized by comprising biological particles coupled with red blood cell complement receptor antibodies and magnetic nanoparticles coupled with anti-RBC antibodies.
2. The kit for separating red blood cells from whole blood according to claim 1, wherein the complement receptor in the biological particles coupled with the antibody of the complement receptor of the red blood cells is one or more of CD35, complement receptor 3, CD55, CD58, CD59, IL-8 receptor, DAF, SOD enzyme, erythrocyte chemokine receptor, macrophage phagocytosis inhibitor.
3. The kit for separating red blood cells from whole blood according to claim 1, wherein the biological particles in the biological particles coupled with the antibody of the red blood cell complement receptor are at least one of latex microspheres, fluorescein-filled microspheres, time-resolved fluorescent microspheres and quantum dot fluorescent microspheres.
4. The kit for separating red blood cells from whole blood according to claim 1, wherein the magnetic nanoparticles in the magnetic nanoparticles coupled with anti-RBC antibodies are magnetic polymer microbeads.
5. The kit for separating red blood cells from whole blood according to any one of claims 1 to 4, wherein the biological particles coupled to the antibody to the complement receptor of red blood cells have a fluorescent label.
6. A method for separating erythrocytes in whole blood, characterized in that a kit according to any one of claims 1 to 4 is used, comprising at least the following steps:
1) Adding biological particles coupled with the erythrocyte complement receptor antibody into a test tube, then adding whole blood, and gently blowing and uniformly mixing to ensure that the biological particles coupled with the erythrocyte complement receptor antibody are specifically combined with complement receptors on erythrocytes;
2) Continuously adding magnetic nano particles coupled with the anti-RBC antibody into a test tube, so that the magnetic nano particles coupled with the anti-RBC antibody are specifically combined with RBC antigens on the surface of red blood cells, the magnetic nano particles coupled with the antibody are combined with red blood cells combined with a complement receptor antibody of the red blood cells to form a red blood cell double-antibody sandwich compound, and the magnetic nano particles coupled with the antibody can be combined with a small amount of free red blood cells to form a red blood cell-RBC antibody compound;
3) The annular electromagnetic sleeve is sleeved outside the test tube, the magnetic pole and magnetism of the electromagnetic sleeve are controlled by changing the direction and intensity of current, and the magnetic force is uniformly mixed and absorbed for 1min, so that the red blood cell double antibody sandwich compound and the red blood cell-RBC antibody compound combined with the magnetic nano particles are gathered towards the direction of the magnet.
7. A method for separating red blood cells from whole blood, comprising the steps of:
1) Adding fluorescent-labeled biological particles of the coupled erythrocyte complement receptor antibody into a test tube, then adding whole blood, and gently blowing and uniformly mixing to ensure that the biological particles of the coupled erythrocyte complement receptor antibody are specifically combined with complement receptors on erythrocytes;
2) Continuously adding magnetic nano particles coupled with the anti-RBC antibody into a test tube, so that the magnetic nano particles coupled with the anti-RBC antibody are specifically combined with RBC antigens on the surface of red blood cells, the magnetic nano particles coupled with the antibody are combined with red blood cells combined with a complement receptor antibody of the red blood cells to form a red blood cell double-antibody sandwich compound, and the magnetic nano particles coupled with the antibody can be combined with a small amount of free red blood cells to form a red blood cell-RBC antibody compound;
3) The annular electromagnetic sleeve is sleeved outside the test tube, the magnetic poles and magnetism of the electromagnetic sleeve are controlled by changing the direction and intensity of current, and the magnetic attraction is carried out for 1min after uniform mixing, so that the red blood cell double antibody sandwich compound and the red blood cell-RBC antibody compound combined with the magnetic nano particles are gathered towards the direction of the magnet;
4) By detecting whether the excessive fluorescent biological particles are remained in the plasma/serum separated in the middle of the test tube by using the fluorescence detector through the transparent area of the test tube bottom, if the fluorescence value in the middle of the test tube is not over the threshold value, the subsequent blood sample can be sampled, and if the fluorescence value in the middle of the test tube is over the threshold value, the excessive fluorescent biological particles are remained, and the blood sample cannot be sampled.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202311013185.5A CN116731962B (en) | 2023-08-14 | 2023-08-14 | Kit and method for separating red blood cells from whole blood |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202311013185.5A CN116731962B (en) | 2023-08-14 | 2023-08-14 | Kit and method for separating red blood cells from whole blood |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN116731962A true CN116731962A (en) | 2023-09-12 |
| CN116731962B CN116731962B (en) | 2023-11-03 |
Family
ID=87909971
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202311013185.5A Active CN116731962B (en) | 2023-08-14 | 2023-08-14 | Kit and method for separating red blood cells from whole blood |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN116731962B (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN118275671A (en) * | 2024-06-04 | 2024-07-02 | 天津中新科炬生物制药股份有限公司 | Multiple competition detection method and kit |
Citations (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101939649A (en) * | 2008-01-07 | 2011-01-05 | 卢米耐克斯公司 | Isolation and enumeration of cells from a complex sample matrix |
| CN103946373A (en) * | 2011-11-25 | 2014-07-23 | 美天旎生物技术有限公司 | Cell separation method |
| CN104271148A (en) * | 2012-02-15 | 2015-01-07 | 洛桑聚合联合学院 | Erythrocyte-binding therapeutics |
| CN104977284A (en) * | 2015-07-03 | 2015-10-14 | 石莹 | Capture and identification method for fetal nucleated red blood cells |
| CN106840828A (en) * | 2017-03-29 | 2017-06-13 | 天津中新科炬生物制药股份有限公司 | The method and separator of quick separating blood plasma in a kind of micro whole blood |
| CN109415697A (en) * | 2016-04-07 | 2019-03-01 | 梅索泰克斯公司 | Separate the method and its application of karyocyte and karyocyte group |
| WO2019084531A1 (en) * | 2017-10-27 | 2019-05-02 | The Children's Hospital Of Philadelphia | Engineered red blood cells having rare antigen phenotypes |
| CN111308070A (en) * | 2020-02-28 | 2020-06-19 | 厦门宝太生物科技有限公司 | Kit for separating red blood cells from whole blood and method for separating red blood cells from whole blood |
| TW202204608A (en) * | 2020-03-27 | 2022-02-01 | 美商血小板生源股份有限公司 | Novel anucleated cells and uses thereof |
-
2023
- 2023-08-14 CN CN202311013185.5A patent/CN116731962B/en active Active
Patent Citations (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101939649A (en) * | 2008-01-07 | 2011-01-05 | 卢米耐克斯公司 | Isolation and enumeration of cells from a complex sample matrix |
| CN103946373A (en) * | 2011-11-25 | 2014-07-23 | 美天旎生物技术有限公司 | Cell separation method |
| CN104271148A (en) * | 2012-02-15 | 2015-01-07 | 洛桑聚合联合学院 | Erythrocyte-binding therapeutics |
| CN104977284A (en) * | 2015-07-03 | 2015-10-14 | 石莹 | Capture and identification method for fetal nucleated red blood cells |
| CN109415697A (en) * | 2016-04-07 | 2019-03-01 | 梅索泰克斯公司 | Separate the method and its application of karyocyte and karyocyte group |
| CN106840828A (en) * | 2017-03-29 | 2017-06-13 | 天津中新科炬生物制药股份有限公司 | The method and separator of quick separating blood plasma in a kind of micro whole blood |
| WO2019084531A1 (en) * | 2017-10-27 | 2019-05-02 | The Children's Hospital Of Philadelphia | Engineered red blood cells having rare antigen phenotypes |
| CN111308070A (en) * | 2020-02-28 | 2020-06-19 | 厦门宝太生物科技有限公司 | Kit for separating red blood cells from whole blood and method for separating red blood cells from whole blood |
| TW202204608A (en) * | 2020-03-27 | 2022-02-01 | 美商血小板生源股份有限公司 | Novel anucleated cells and uses thereof |
Non-Patent Citations (1)
| Title |
|---|
| PRADEEP K DAGUR 等: "Collection, Storage, and Preparation of Human Blood Cells", CURR PROTOC CYTOM ., vol. 73, pages 1 - 5 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN118275671A (en) * | 2024-06-04 | 2024-07-02 | 天津中新科炬生物制药股份有限公司 | Multiple competition detection method and kit |
| CN118275671B (en) * | 2024-06-04 | 2024-09-03 | 天津中新科炬生物制药股份有限公司 | Multiple competition detection method and kit |
Also Published As
| Publication number | Publication date |
|---|---|
| CN116731962B (en) | 2023-11-03 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN116731962B (en) | Kit and method for separating red blood cells from whole blood | |
| CA1304006C (en) | Particle separation method | |
| US9885032B2 (en) | Multisort cell separation method | |
| US9435799B2 (en) | Methods and reagents for improved selection of biological materials | |
| EP1151297B1 (en) | Methods for enhancing binding interactions between members of specific binding pairs | |
| JP2762259B2 (en) | How to use magnetically responsive fluorescent polymer | |
| CN111826351B (en) | Magnetic red blood cell cluster for enriching circulating tumor cells based on magnetic separation method | |
| CN102527353A (en) | Preparation for nanometer magnetic immunomicrosphere for enriching circulating cancer cells | |
| WO2011144012A1 (en) | Magnetic kit for rapid detection of microorganisms and its preparation and usage | |
| CN103305464B (en) | Method for directly separating CD<4+> and CD<8+> lymphocytes | |
| CN103952397A (en) | Method for separating free nucleic acid from blood serum or blood plasma sample by using magnetic bead | |
| CN114231523A (en) | Exosome separation method based on CD63 antibody coupling magnetic beads | |
| CN111826349B (en) | Erythrocyte cluster based on size filtration method for enriching circulating tumor cells | |
| CN107655879B (en) | For detecting the micro-fluidic chemiluminescence detection system of the magnetic particle of sexual gland series | |
| CN111474356A (en) | Double-immunomagnetic-bead sorting reagent, preparation method thereof and application thereof in enrichment of humoral exosomes | |
| CN114958743A (en) | Separation detection method and application of neutrophil exosome | |
| CN115558629A (en) | Exosome extraction affinity magnetic bead and extraction kit thereof | |
| CN111308070A (en) | Kit for separating red blood cells from whole blood and method for separating red blood cells from whole blood | |
| CN215856071U (en) | Centrifugal tube for separating extracellular vesicles and kit for extracting extracellular vesicles | |
| CN117330481B (en) | Flow detection method for exosomes and application thereof | |
| CN111724954B (en) | Graphene oxide magnetic bead, antibody-coupled graphene oxide magnetic bead and application of graphene oxide magnetic bead in cell sorting | |
| JP2008089415A (en) | Examination reagent and examination method | |
| CN115825436B (en) | Brucella antibody gel detection reagent and Brucella antibody gel detection method | |
| CN115356477A (en) | Streptavidin magnetic bead and preparation method and application thereof | |
| CN115561147A (en) | Exosome protein detection method |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |