CN116731962B - 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 PDF

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CN116731962B
CN116731962B CN202311013185.5A CN202311013185A CN116731962B CN 116731962 B CN116731962 B CN 116731962B CN 202311013185 A CN202311013185 A CN 202311013185A CN 116731962 B CN116731962 B CN 116731962B
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red blood
antibody
blood cells
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magnetic
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CN116731962A (en
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范永刚
魏华英
李慧
王俊水
李轩
周洪锐
李昀地
赵娜
肖福磊
秦月
钱龙
孟佳
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Newscen Coast Bio Pharmaceutical Co ltd
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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

Kit and method for separating red blood cells from whole blood
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 (6)

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, wherein the complement receptor in the biological particles coupled with the red blood cell complement receptor antibodies is CD35.
2. 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.
3. 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.
4. A kit for separating erythrocytes in whole blood according to any one of claims 1 to 3, characterized in that the biological particles coupled to the erythrocyte complement receptor antibody have a fluorescent label.
5. A method for separating erythrocytes in whole blood, characterized in that a kit according to any one of claims 1 to 3 is used, comprising at least the following steps:
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.
6. 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.
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