CN111308070A - Kit for separating red blood cells from whole blood and method for separating red blood cells from whole blood - Google Patents

Abstract

The invention discloses a kit for separating red blood cells from whole blood and a method for separating the red blood cells from the whole blood. Magnetic beads comprising chemically coupled anti-RBC antibodies, lectins, and/or anti-RBC antibodies. And then separating red blood cells from whole blood by matching the obtained magnetic beads of the chemically coupled anti-RBC antibodies in the kit with phytohemagglutinin and/or free anti-RBC antibodies. The separation method is rapid and has good separation effect.

Description

Kit for separating red blood cells from whole blood and method for separating red blood cells from whole blood

Technical Field

The invention relates to the field of cell separation, in particular to a kit for separating red blood cells from whole blood and a method for separating red blood cells from whole blood.

Background

At present, the mode for separating the red blood cells is mainly a centrifugal method, the centrifugal method is difficult to realize the automation of the red blood cell separation and is not beneficial to the miniaturization of detection equipment, and the existence of the red blood cells brings great interference to the detection of the existing blood marker. The method of separating red blood cells from whole blood disclosed in chinese patent application No. 200710305363.6, which uses the addition of labeled phytohemagglutinin or anti-erythrocyte antibody microspheres to achieve the purpose of separating red blood cells. However, the separation takes 10min or more, and the time is long. Patent application No. 200810244503.8 discloses a method for rapidly separating supernatant of human whole blood or suspended red blood cells by using lyophilized preparation, which comprises coating microspheres with phytohemagglutinin or anti-red blood cell antibody, preparing lyophilized preparation, and mixing with whole blood to separate red blood cells. However, the separation of the technical scheme needs 2-3min, and the reagent is lyophilized. In both of the two technical schemes, magnetic beads are used for red blood cell separation, but phytohemagglutinin or anti-RBC antibodies are coupled with the magnetic beads through physical adsorption, so that the requirement on whole blood sample separation is high, for example, in chinese patent application 200710305363.6, no lectin or other reagents are added to the sample, the requirement on the sample detection time is provided, a part of patient samples are generally collected clinically, on-machine detection is performed at the same time, and the whole blood sample is limited in the detection time; the separation time of the Chinese patent application 200810244503.8 is relatively long, and needs 2-3 min.

Disclosure of Invention

The invention aims to provide a kit for separating red blood cells from whole blood, which is used for separating red blood cells from whole blood, and has the advantages of high speed and good separation effect.

To achieve the above object, the present invention provides a kit for separating red blood cells from whole blood, comprising magnetic beads of chemically coupled anti-RBC antibodies, lectin and/or anti-RBC antibodies.

Furthermore, the preparation method of the magnetic bead of the chemically coupled anti-RBC antibody comprises the following steps,

magnetic bead activation: adding a newly prepared EDC solution into carboxyl magnetic beads washed by the MEST solution, uniformly mixing to enable the magnetic beads to be fully suspended and then activated, and enabling the magnetic beads to be kept in a suspended state in the activation process;

adding an antibody for coupling: magnetically separating the activated magnetic beads, removing supernatant to obtain magnetic bead suspension, adding anti-RBC antibody, and gently mixing, wherein the anti-RBC antibody exists in PBS (pH8.0) containing 0.05% Tween 20; coupling is carried out, and the suspension state of the magnetic beads is kept during coupling;

washing: magnetically separating to remove supernatant, adding PBST solution to resuspend the magnetic beads, reacting, sealing unreacted activated carboxyl groups on the surfaces of the magnetic beads, and keeping the suspension state of the magnetic beads; and (4) magnetically separating to remove supernatant, washing the supernatant for 2 to 4 times by using a PBS (phosphate buffer solution) with the pH value of 7.2, and then resuspending the supernatant in a preservation solution to obtain the magnetic beads of the chemically coupled anti-RBC antibody.

Further, in the magnetic bead activation step, the carboxyl magnetic beads washed by the MEST solution are loaded into magnetic beads; preferably, the magnetic beads have a particle size of 1 to 4 μm; after addition of the MEST solution, washing by magnetic separation 1-3 times, preferably 2 times, and then removing the supernatant; for washing, a MEST solution 2 times the volume of the magnetic beads was used.

Optionally, the activation is at 20-30 ℃ for 20-40 min; preferably, the activation is 30min at 25 ℃;

further, in the step of adding the antibody for coupling, the adding amount of the antibody is that 50-200 μ g of anti-RBC antibody is added into 100 μ L of 10mg/mL magnetic bead suspension; preferably, 200 μ g of anti-RBC antibody is added.

Optionally, the coupling is at 23-27 ℃ for 1.5-2.5 h; preferably, it is 25 ℃ for 2 h.

Further, in the washing step, the reaction is carried out at 23-27 ℃ for 0.5-1.5 h; preferably, the reaction is carried out at 25 ℃ for 1 h.

Optionally, the MEST solution is 100mM MES, pH5.0, 0.05% Tween 20; the EDC solution is obtained by dispersing 10mg/mL EDC in the MEST; the PBST solution is PBS solution with pH7.2, 0.05% Tween20 and 1% BSA;

the preservation solution is PBS or MEST or HEPES or Tris-HCl or CBS buffer solution with the pH value of 30-50mM and the pH value of 5.0-8.5.

Furthermore, the preparation method of the magnetic bead of the chemically coupled anti-RBC antibody comprises the following steps,

magnetic bead activation: putting 100 mu L of 10mg/mL carboxyl magnetic beads into a 1mL centrifuge tube, carrying out magnetic separation to remove supernatant, carrying out magnetic separation and washing for 2 times by using 200 mu L MEST solution, and then removing the supernatant; adding 200 μ L EDC solution, mixing to make the magnetic beads fully suspended, activating at 25 deg.C for 30min, shaking by shaking table to make the magnetic beads maintain suspension state; through the steps, the carboxyl on the surface of the magnetic bead is activated and can be covalently coupled with the anti-RBC antibody;

adding an antibody for coupling: magnetically separating the activated magnetic beads, removing supernatant, adding 200 μ g of anti-RBC antibody, and mixing, wherein the antibody exists in PBS (pH8.0) containing 0.05% Tween 20; coupling for 2h at 25 ℃, and keeping the suspension state of the magnetic beads during coupling;

washing: removing supernatant by magnetic separation, adding 200 mu L of PBST solution to resuspend the magnetic beads, reacting for 1h at 25 ℃, sealing unreacted activated carboxyl groups on the surfaces of the magnetic beads, and keeping the suspension state of the magnetic beads in the process; and (3) magnetically separating to remove supernatant, washing the supernatant for 2 to 4 times by using 200 mu L PBS (phosphate buffer solution) with pH7.2, and then resuspending the supernatant in a preservation solution to obtain the magnetic beads of the chemically coupled anti-RBC antibody.

The present invention also provides a method for separating red blood cells from whole blood, which is characterized by using the kit for separating red blood cells from whole blood.

Further, the method comprises the following steps of,

taking the magnetic beads of the chemically coupled anti-RBC antibody in the kit for separating the red blood cells from the whole blood, magnetically separating the supernatant, adding the phytohemagglutinin and/or the anti-RBC antibody, gently and uniformly blowing, adding the whole blood, gently and uniformly blowing, performing magnetic adsorption, and sucking the supernatant;

the whole blood is EDTA-2Na or sodium citrate or heparin sodium anticoagulated whole blood or whole blood without anticoagulant.

Further, the dosage of the phytohemagglutinin is 50-500 mu g/mL of final concentration;

optionally, the anti-RBC antibody is administered at a final concentration of 0.5-5 μ g/mL.

The invention discloses a method for rapidly separating red blood cells in conventional whole blood, which is simple, easy to realize automation and beneficial to development of rapid detection equipment and reagents.

The invention uses a chemical coupling mode to directly or indirectly connect the anti-RBC to the magnetic bead, and the separation system containing free anti-RBC and/or phytohemagglutinin is matched, so that the red blood cells can be rapidly separated in a liquid state (within 2min, the optimal separation time can be controlled within 10-20 seconds), and no special requirement is required for the whole blood sample, and the whole blood sample containing common anticoagulation whole blood samples such as EDTA, sodium citrate, heparin sodium and the like and the whole blood sample without anticoagulant can be used.

The anti-erythrocyte antibody (anti-RBC antibody) is coupled with the magnetic particles for marking, the marked magnetic particles can be specifically combined on the surface of the erythrocyte through the erythrocyte antibody, and then the erythrocyte combined with the magnetic particles is quickly enriched towards the direction of the magnet through magnetic adsorption, thereby finally achieving the purpose of separation. In addition, free partial anti-RBC, phytohemagglutinin and the like are added into the red blood cells, which is beneficial to the enrichment of the red blood cells and achieves the aim of quickly separating blood plasma/serum. In the whole reaction process of the method, the main key components are magnetic beads which are directly or indirectly chemically coupled; there is also a need for free phytohemagglutinin and/or anti-RBC.

The principle of the method for separating the red blood cells from the whole blood is that magnetic beads and anti-RBC antibodies are chemically coupled, the coupled magnetic beads are specifically combined with antigens on the surface of the red blood cells through the anti-RBC antibodies and are mutually aggregated, meanwhile, free anti-RBC and phytohemagglutinin can also be specifically or non-specifically combined with the red blood cells and are mutually aggregated, and finally, the red blood cells combined with the magnetic beads are quickly aggregated towards the direction of a magnet through magnet adsorption to achieve the purpose of separation, which is shown in figure 1 and figure 2. FIG. 1 is a schematic diagram of erythrocyte aggregation, which includes a magnetic bead coupled with an anti-RBC antibody and the principle of erythrocyte aggregation, a free lectin and the principle of erythrocyte aggregation, and a principle of continuous mutual aggregation of the above three aggregates through an exposed epitope. FIG. 2 is a schematic diagram of red blood cell separation from whole blood, wherein three aggregates including magnetic beads, anti-RBC antibody, red blood cell, and phytohemagglutinin, or their combination are adsorbed by a magnet to complete red blood cell separation. Wherein, the first diagram of fig. 1 shows that the magnetic beads are chemically coupled with the anti-RBC antibody, the coupled magnetic beads are specifically combined with the surface antigen of the red blood cells through the anti-RBC antibody and are mutually aggregated, the second diagram shows that the free anti-RBC antibody is specifically combined with the red blood cells and are mutually aggregated, the third diagram shows that the free phytohemagglutinin is nonspecifically combined with the red blood cells and are mutually aggregated, and the fourth diagram shows that the magnetic beads, the anti-RBC antibody, the free anti-RBC antibody and the free phytohemagglutinin exist together, the magnetic beads, the anti-RBC antibody, the free anti-RBC antibody and the free phytohemagglutinin are combined. FIG. 2 shows that red blood cells directly or indirectly bound with magnetic beads are rapidly aggregated toward a magnet by magnetic adsorption to achieve the separation.

Drawings

FIG. 1 is a schematic diagram of red blood cell aggregation.

FIG. 2 is a schematic diagram of red blood cell separation from whole blood.

Detailed Description

Reference will now be made in detail to embodiments of the present invention, examples of which are illustrated in the accompanying drawings, wherein like or similar reference numerals refer to the same or similar elements or elements having the same or similar function throughout. The embodiments described below with reference to the drawings are illustrative and intended to be illustrative of the invention and are not to be construed as limiting the invention. The examples do not specify particular techniques or conditions, and are performed according to the techniques or conditions described in the literature in the art or according to the product specifications. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.

Example 1: anti-RBC coupled to carboxyl magnetic beads (1)

1. Taking 1mg of carboxyl magnetic beads (such as purchased from Suzhou Kong Co.) into a 1mL centrifuge tube, wherein the particle size of the magnetic beads is 1 mu m, washing the magnetic beads with 200 mu LMEST solution (100mMMES, pH5.0, 0.05% Tween20) for 2 times, and removing supernatant through magnetic separation;

2. quickly adding a newly prepared 200 mu LEDC solution (10mg/mL, using the MEST as a dispersing agent) into a centrifuge tube filled with magnetic beads, uniformly mixing by vortex to fully suspend the magnetic beads, activating at 25 ℃ for 30min, and shaking and uniformly mixing by using a shaking table during the period to keep the magnetic beads in a suspended state; through the steps, the carboxyl on the surface of the magnetic bead is activated and can be covalently coupled with the anti-RBC antibody.

3. Magnetically separating to remove supernatant, adding 200 μ g of anti-RBC antibody, wherein the antibody retaining solution is PBS (pH8.0), and contains 0.05% Tween20, and avoiding the existence of reagent containing primary amino group except the anti-RBC antibody in the buffer system, and gently mixing;

4. coupling for 2h at 25 ℃, and keeping the suspension state of the magnetic beads during coupling;

5. removing supernatant by magnetic separation, adding 200 mu of LPBST solution (pH7.2, 0.05% Tween20, 1% BSA) for resuspending the magnetic beads, reacting for 1h at 25 ℃, and blocking unreacted activated carboxyl groups on the surfaces of the magnetic beads, wherein the suspension state of the magnetic beads is kept;

6. the supernatant was removed by magnetic separation in a centrifuge tube, washed 3 times with 200. mu.L of PBS (pH7.2), resuspended in 10mM PBS pH7.2, and stored at 4 ℃. The preservative and 0.2% BSA were added for long-term storage, and the mixture was placed in a refrigerator at 4 ℃ and labeled as (1) magnetic beads.

Example 2: anti-RBC-streptavidin magnetic bead coupling (2)

1. Taking 1mg of streptavidin magnetic beads (purchased from Suzhou Kong Co., Ltd.) into a 1mL centrifuge tube, wherein the particle size of the magnetic beads is 1 mu m;

2. adding 1mL Buffer (40mM PBS, pH7.4, containing 0.05% Tween20, 0.01% -0.1% BSA) into the centrifuge tube, fully shaking the resuspended beads, and magnetically separating out the supernatant;

3. repeating the step 2 twice;

4. adding 1mL of biotinylated anti-RBC diluted by the Buffer, wherein the concentration of the magnetic beads is 1mg/mL, fully and uniformly mixing, and then rotating and mixing on a shaking table at room temperature for 60 min;

5. magnetically separating out the supernatant, and transferring the supernatant into a new centrifuge tube;

6. the beads were washed 5 times as in step 2 and 200ul buffer was added, at which point the binding of biotinylated antibody was complete and recorded as (2) beads.

Example 3: anti-RBC-dextran-carboxyl magnetic bead coupling (3)

1. Taking 1mg of carboxyl magnetic beads (such as purchased from Suzhou Kong Co.) into a 1mL centrifuge tube, wherein the particle size of the magnetic beads is 1 mu m, washing the magnetic beads with 200 mu LMEST solution (100mMMES, pH5.0, 0.05% Tween20) for 2 times, and removing supernatant through magnetic separation;

2. quickly adding a newly prepared 200 mu LEDC solution (10mg/mL, using the MEST as a dispersing agent) into a centrifuge tube filled with magnetic beads, uniformly mixing by vortex to fully suspend the magnetic beads, activating at 25 ℃ for 30min, and shaking and uniformly mixing by using a shaking table during the period to keep the magnetic beads in a suspended state;

3. adding 2.8 μ g of aminodextran, mixing gently, and coupling on a shaking table overnight;

4. magnetically separating supernatant, adding 200 μ L of blocking solution (pH7.2PBST, 0.05% Tween20, 1% BSA) for resuspending magnetic beads, performing blocking reaction on a shaking table for 1h, magnetically separating supernatant, adding 112 μ L of newly prepared 1mg/mL NaIO₄Mixing the solution in suspension for 30 minutes;

5. the supernatant was magnetically separated, 224. mu.g of anti-RBC antibody was added, 500. mu.L of CB (0.2mol/L carbonate buffer, pH9.5, anhydrous sodium carbonate 0.32g + sodium bicarbonate 0.586g +50mL of distilled or deionized water) was supplemented with PH9.50.02M, and the mixture was gently mixed for 2 hours at room temperature;

6. adding 500 μ L ethanolamine solution (1%, diluted with water), mixing, suspending at room temperature for 2 hr;

7. the supernatant was magnetically separated, washed 3 times with PBS, and then replaced with 200. mu.L of a preservation solution (10 mM pH7.2Tris, 0.5% Casein-Na, 0.2% Tween20, 0.8% BSA, 1% PEG20000, 1 ‰ proclin300), and the magnetic bead was designated (3).

Example 4:

respectively taking 100 mu L of the uniformly oscillated (1), (2) and (3) magnetic beads, respectively adding 100 mu L of EDTA-2Na, sodium citrate and heparin sodium anticoagulated whole blood and whole blood without the anticoagulant, lightly blowing, uniformly mixing, carrying out magnetic adsorption for 1min, and absorbing upper plasma/serum, wherein the result is shown in Table 1.

TABLE 1 results of experiments for separating whole blood by direct adsorption of magnetic beads

Example 5:

and respectively taking 100 mu L of coupling magnetic beads (1), (2) and (3) which are uniformly oscillated, adding 1 mu L of 10mg/mL phytohemagglutinin, slightly blowing, beating and uniformly mixing, respectively adding 100 mu L of EDTA-2Na, sodium citrate and heparin sodium anticoagulated whole blood and whole blood without the anticoagulation agent, slightly blowing, beating and uniformly mixing, and performing magnetic adsorption. Adsorbing for 1min, and sucking upper layer plasma/serum. The results are shown in Table 2.

TABLE 2 results of experiments on whole blood separation with addition of free phytohemagglutinin

Example 6:

and (3) respectively taking 100 mu L of the uniformly vibrated (1), (2) and (3) magnetic beads, adding 5 mu L of 0.1mg/mL anti-RBC antibody, slightly blowing, uniformly mixing, respectively adding 100 mu L of anticoagulated whole blood of DTA-2Na, sodium citrate and heparin sodium and whole blood without the anticoagulated whole blood, slightly blowing, uniformly mixing, and performing magnetic adsorption. Adsorbing for 1min, and sucking upper layer plasma/serum. The results are shown in Table 3.

Table 3 results of whole blood separation experiment with addition of free anti-RBC antibody

Example 7

And respectively taking 100 mu L of uniformly oscillated (1), (2) and (3) magnetic beads, adding 1 mu L of 10mg/mL phytohemagglutinin, adding 5 mu L of 0.1mg/mL anti-RBC antibody, lightly blowing, uniformly mixing, adding 100 mu L of EDTA-2Na, sodium citrate and heparin sodium anticoagulated whole blood and whole blood without the anticoagulant, lightly blowing, uniformly mixing, and carrying out magnetic adsorption. Adsorbing for 1min, and sucking upper layer plasma/serum. The results are shown in Table 4.

TABLE 4 results of whole blood separation experiment with addition of free phytohemagglutinin and anti-RBC antibody

As can be seen from the results of tables 1 to 4,

after the treatment of the phytohemagglutinin and the adsorption of the magnetic beads, the volume of the separated blood plasma/serum has an increase of 9.2 mu L on average compared with that of the blood plasma/serum without any substance, and the erythrocyte aggregate particles are enlarged through the agglutination of the phytohemagglutinin, and the number of the free erythrocytes in the separated blood plasma/serum is obviously reduced compared with that of the blood plasma/serum without any substance;

after the treatment of the anti-RBC antibody and the adsorption of the magnetic beads, the volume of the separated plasma/serum has an increase of 62 mu L on average compared with that of the plasma/serum without any substance, and the agglutination of the anti-RBC antibody causes the red blood cells to be more quickly aggregated and the particles to be more dense, so that the number of the free red blood cells in the separated plasma/serum is obviously reduced compared with that of the plasma/serum without any substance;

after the double treatment of the phytohemagglutinin and the anti-RBC antibody and the magnetic bead adsorption, the volume of the separated plasma/serum has an increase of 90 microliter compared with that without any substance, and the aggregation particles of the red blood cells are enlarged and faster due to the agglutination of the phytohemagglutinin and the anti-RBC antibody, and the number of the free red blood cells in the separated plasma/serum is further reduced compared with the case of adding the lectin or the anti-RBC antibody.

Example 8:

carboxylic magnetic beads (purchased from Suzhou Koch Co., Ltd.) having particle diameters of 350nm, 1 μm, 3 μm and 5 μm in an amount of 1mg each were labeled as follows, and the labeled magnetic beads were designated as (4) magnetic beads, (5) magnetic beads, (6) magnetic beads and (7) magnetic beads.

1. After mixing the magnetic beads, 1mg of carboxyl magnetic beads (in a 1mL centrifuge tube, removing the supernatant by magnetic separation, performing magnetic separation and washing 2 times with 200. mu. LMEST solution (100mM MES, pH5.0, 0.05% Tween20), and then removing the supernatant;

2. quickly adding newly prepared 200 μ L EDC solution (10mg/mL, using the MEST as dispersant) into a centrifuge tube filled with magnetic beads, mixing by vortex to fully suspend the magnetic beads, activating at 25 deg.C for 30min, and shaking by a shaking table to uniformly suspend the magnetic beads during the period; through the steps, the carboxyl on the surface of the magnetic bead is activated and can be covalently coupled with the anti-RBC.

3. Magnetically separating to remove supernatant, adding 200 μ g of anti-RBC antibody, adding 0.05% Tween20 in PBS (pH8.0), preventing reagent containing primary amino group except anti-RBC from existing in buffer system, and mixing;

4. coupling for 2h at 25 ℃, and keeping the suspension state of the magnetic beads during coupling;

5. removing supernatant by magnetic separation of a centrifuge tube, adding 200 mu L of PBST solution (pH7.2, 0.05% Tween20, 1% BSA) for resuspending magnetic beads, reacting for 1h at 25 ℃, and blocking unreacted activated carboxyl groups on the surfaces of the magnetic beads, wherein the suspension state of the magnetic beads is kept;

6. the tube was magnetically separated to remove the supernatant, washed 3 times with 200. mu.L PBS (pH7.2), resuspended in 10mM PBS (pH7.2), stored at 4 ℃ and preserved for a long period of time with preservative and 0.2% BSA, and placed in a refrigerator at 4 ℃.

Example 9:

the whole blood was separated using (4), (5), (6) and (7) magnetic beads, and 100. mu.L of each magnetic bead was added with 1. mu.L of 10mg/mL phytohemagglutinin, 5. mu.L of 0.1mg/mL anti-RBC antibody, and 100. mu.L of each of EDTA-2Na, sodium citrate, heparin sodium, and whole blood without anticoagulant, and the whole blood was gently pipetted and mixed to conduct magnetic adsorption. Adsorbing for 1min, and sucking upper layer plasma/serum.

TABLE 5 results of comparison experiments on the effect of separating whole blood with different particle size of carboxyl magnetic beads (containing phytohemagglutinin and free anti-RBC antibody)

As can be seen from the results in Table 5, the mean plasma/serum volumes obtained by adsorbing whole blood in (4) (5) (6) (7) were 44. mu.L, 119. mu.L, 95. mu.L and 94. mu.L, respectively, after the double treatment with phytohemagglutinin and anti-RBC antibody, there was no significant difference in the number of free erythrocytes in the separated plasma/serum. It can be seen that the magnetic beads with the particle size of 1-5 μm, especially 1 μm, have good effect of adsorbing whole blood under the same experimental conditions.

Example 10:

the amount of lectin used was confirmed by using (1) 100. mu.L of magnetic beads, and 0.1. mu.L, 0.5. mu.L, 1. mu.L, 5. mu.L, 10. mu.L, and 20. mu.L of 10mg/mL lectin was added thereto, the mixture was gently pipetted and mixed, 100. mu.L of anticoagulated whole blood with sodium citrate and heparin sodium and whole blood without anticoagulant were added thereto, the mixture was gently pipetted and mixed, magnetic adsorption was performed for 1min, and upper plasma/serum was aspirated, and the results are shown in Table 6.

Table 6 table of results of experiments for determining the amount of free phytohemagglutinin added

From table 6 it can be derived: in 200 mul reaction system, the addition of 10mg/mL phytohemagglutinin is 1 mul-10 mul, namely the final concentration of the system is 50-500 mug/mL phytohemagglutinin with the best dosage, the volume of the separated blood plasma/blood serum is the most, and the red blood cell separation is the most thorough.

Example 11:

the amount of free anti-RBC was confirmed by using (1) 100. mu.L of magnetic beads, and 0.1. mu.L, 0.5. mu.L, 1. mu.L, 5. mu.L, 10. mu.L, and 20. mu.L of 0.1mg/mL anti-RBC antibody was added thereto, gently pipetted and mixed, then 100. mu.L of anticoagulated whole blood with sodium citrate and heparin sodium and whole blood without anticoagulant were added thereto, gently pipetted and mixed, magnetic adsorption was performed for 1min, and the supernatant plasma/serum was aspirated, and the results are shown in Table 7.

Table 7 table of results of experiment for determining addition amount of free anti-RBC antibody

From table 7 it can be derived: in a 200 mu L reaction system, the adding amount of 0.1mg/mL anti-RBC is 1 mu L-10 mu L best, namely the final concentration of the system is 0.5-5 mu g/mL anti-RBC antibody, the volume of the separated blood plasma/blood serum is the most, and the separation is the most thorough.

Example 12:

the magnetic bead preservation solution confirmation was carried out using (1) magnetic beads, the magnetic bead protection solution was replaced with 50mM pH6.0 MEST buffer, 40mM pH7.0HEPES buffer, 30mM pH7.2 Tris-HCl buffer, and 50mM pH7.2 CBS buffer, 100. mu.L of each of the above preservation solution and 40mM pH7.2PBS preservation solution magnetic beads were taken, 1. mu.L of 10mg/mL phytohemagglutinin and 5. mu.L of 0.1mg/mL anti-RBC antibody were added, 100. mu.L of anticoagulated whole blood with sodium citrate and heparin and whole blood without anticoagulated were added, gently mixed by pipetting, magnetic adsorption was carried out for 0.5 to 1min, and upper plasma/serum was aspirated. The results are shown in Table 8.

Table 8 table of results of magnetic bead preservation solution confirmation experiment

Anticoagulant agent

Separation ratio

PBS

MEST

HEPES

Tris

CBS

EDTA-2Na anticoagulated whole blood

Blood plasma

125μL

121μL

123μL

122μL

121μL

Anticoagulated whole blood of sodium citrate

Blood plasma

126μL

123μL

121μL

124μL

126μL

Anticoagulant whole blood of heparin sodium

Blood plasma

123μL

122μL

120μL

123μL

123μL

Whole blood without anticoagulant

Serum

100μL

97μL

104μL

101μL

96μL

Note: PBS: namely 40mM PBS with pH7.2; MEST: namely 50mM pH6.0 MEST; HEPES (high efficiency particulate air): namely 40mM HEPES with pH 7.0; tris (Tris): namely 30mM Tris-HCl with pH7.2; and (3) CBS: this was 50mM pH7.2 CBS.

As can be seen from the results in Table 8, several preservative solutions used in this experiment all had good separation effect.

Example 13:

the magnetic bead preservation solution was confirmed using (1) magnetic beads by replacing the magnetic bead protective solution with 40mM PBS buffer (pH5.0), 40mM PBS buffer (pH7.0), and 40mM PBS buffer (pH8.5), and taking 100. mu.L of each of the magnetic beads in the preservation solution, adding 1. mu.L of 10mg/mL phytohemagglutinin and 5. mu.L of 0.1mg/mL anti-RBC antibody, adding 100. mu.L of anticoagulated whole blood containing DTA-2Na, sodium citrate, and heparin sodium and whole blood without anticoagulation, gently pipetting and mixing, performing magnetic adsorption for 0.5-1min, and aspirating the supernatant plasma/serum. The results are shown in Table 9.

TABLE 9 results of the effect of different pH values of the preservation solution of magnetic beads on the separation of whole red blood cells

Anticoagulant agent	Separation ratio	Ph＝5.0	Ph＝7.0	Ph＝8.5
					EDTA-2Na anticoagulated whole blood	Blood plasma	125μL	121μL	123μL
Anticoagulated whole blood of sodium citrate	Blood plasma	126μL	123μL	121μL
					Anticoagulant whole blood of heparin sodium	Blood plasma	123μL	122μL	120μL
Whole blood without anticoagulant	Serum	100μL	97μL	104μL

As can be seen from the results in Table 9, the pH of the preservation solution was between 5.0 and 8.5, and the separation effect was not clearly distinguished.

Example 14:

the magnetic bead preservation solution was confirmed using (1) magnetic beads by replacing the magnetic bead protective solution with 10mM pH7.0PBS buffer, 20mM pH7.0PBS buffer, 30mM pH7.0PBS buffer, 40mM pH7.0PBS buffer, 50mM pH7.0PBS buffer, and 60mM pH7.0PBS buffer, respectively, taking 100. mu.L of each magnetic bead of the preservation solution, adding 1. mu.L of 10mg/mL phytohemagglutinin and 5. mu.L of 0.1mg/mL anti-RBC antibody, adding 100. mu.L of whole anticoagulated blood of DTA-2Na, sodium citrate, and heparin sodium and whole blood without anticoagulation, gently pipetting and mixing, and performing magnetic adsorption for 0.5-1min, and aspirating the upper plasma/serum. The results are shown in Table 10.

TABLE 10 results of the effect of different salt ion concentration preserving solutions on the separation of whole red blood cells

Anticoagulant agent

Separation ratio

10mM

20mM

30mM

40mM

50mM

60mM

EDTA-2Na anticoagulated whole blood

Blood plasma (Shangqing)

65μL

72μL

122μL

124μL

124μL

71μL

Anticoagulated whole blood of sodium citrate

Blood plasma (Shangqing)

62μL

75μL

123μL

122μL

124μL

59μL

Anticoagulant whole blood of heparin sodium

Blood plasma (Shangqing)

63μL

77μL

121μL

123μL

123μL

63μL

Whole blood without anticoagulant

Serum (Shangqing)

60μL

67μL

114μL

111μL

109μL

57μL

As can be seen from the results in table 10, the PBS concentrations that gave the best separation were: 30-50 mM.

Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention, and that variations, modifications, substitutions and alterations can be made in the above embodiments by those of ordinary skill in the art without departing from the principle and spirit of the present invention.

Claims (9)

1. A kit for separating red blood cells from whole blood, comprising magnetic beads of chemically coupled anti-RBC antibodies, phytohemagglutinin and/or anti-RBC antibodies.

2. The kit according to claim 1, wherein the magnetic beads of chemically coupled anti-RBC antibodies are prepared by,

magnetic bead activation: adding a newly prepared EDC solution into carboxyl magnetic beads washed by the MEST solution, uniformly mixing to enable the magnetic beads to be fully suspended and then activated, and enabling the magnetic beads to be kept in a suspended state in the activation process;

adding an antibody for coupling: magnetically separating the activated magnetic beads, removing supernatant to obtain magnetic bead suspension, adding anti-RBC antibody, and gently mixing, wherein the anti-RBC antibody exists in PBS (pH8.0) containing 0.05% Tween 20; coupling is carried out, and the suspension state of the magnetic beads is kept during coupling;

washing: magnetically separating to remove supernatant, adding PBST solution to resuspend the magnetic beads, reacting, sealing unreacted activated carboxyl groups on the surfaces of the magnetic beads, and keeping the suspension state of the magnetic beads; and (4) magnetically separating to remove supernatant, washing the supernatant for 2 to 4 times by using a PBS (phosphate buffer solution) with the pH value of 7.2, and then resuspending the supernatant in a preservation solution to obtain the magnetic beads of the chemically coupled anti-RBC antibody.

3. The kit for separating red blood cells from whole blood according to claim 2, wherein in the magnetic bead activation step, the carboxyl magnetic beads washed with the MEST solution are bound to the magnetic beads; preferably, the magnetic beads have a particle size of 1 to 4 μm; after addition of the MEST solution, washing by magnetic separation 1-3 times, preferably 2 times, and then removing the supernatant; during washing, MEST solution with the volume 2 times that of the magnetic beads is used for washing;

optionally, the activation is at 20-30 ℃ for 20-40 min; preferably, the activation is at 25 ℃ for 30 min.

4. The kit for separating red blood cells from whole blood according to claim 2, wherein the antibody is added in the coupling step in an amount of 50-200 μ g of the anti-RBC antibody to 100 μ L of a suspension of 10mg/mL magnetic beads; preferably, 200 μ g of anti-RBC antibody is added;

optionally, the coupling is at 23-27 ℃ for 1.5-2.5 h; preferably, it is 25 ℃ for 2 h.

5. The kit for separating red blood cells from whole blood according to claim 2, wherein in the washing step, the reaction is carried out at 23-27 ℃ for 0.5-1.5 hours; preferably, the reaction is carried out for 1h at 25 ℃;

optionally, the MEST solution is 100mM MES, pH5.0, 0.05% Tween 20; the EDC solution is obtained by dispersing 10mg/mL EDC in the MEST; the PBST solution is PBS solution with pH7.2, 0.05% Tween20 and 1% BSA;

the preservation solution is PBS or MEST or HEPES or Tris-HCl or CBS buffer solution with the pH value of 30-50mM and the pH value of 5.0-8.5.

6. The kit for separating red blood cells from whole blood according to claim 1 or 2, wherein the magnetic beads of chemically coupled anti-RBC antibodies are prepared by,

magnetic bead activation: putting 100 mu L of 10mg/mL carboxyl magnetic beads into a 1mL centrifuge tube, carrying out magnetic separation to remove supernatant, carrying out magnetic separation and washing for 2 times by using 200 mu LMEST solution, and then removing the supernatant; adding 200 μ L EDC solution, mixing to make the magnetic beads fully suspended, activating at 25 deg.C for 30min, shaking by shaking table to make the magnetic beads maintain suspension state; through the steps, the carboxyl on the surface of the magnetic bead is activated and can be covalently coupled with the anti-RBC antibody;

adding an antibody for coupling: magnetically separating the activated magnetic beads, removing supernatant, adding 200 μ g of anti-RBC antibody, and mixing, wherein the antibody exists in PBS (pH8.0) containing 0.05% Tween 20; coupling for 2h at 25 ℃, and keeping the suspension state of the magnetic beads during coupling;

washing: removing supernatant by magnetic separation, adding 200 mu L of PBST solution to resuspend the magnetic beads, reacting for 1h at 25 ℃, sealing unreacted activated carboxyl groups on the surfaces of the magnetic beads, and keeping the suspension state of the magnetic beads in the process; and (3) magnetically separating to remove supernatant, washing the supernatant for 2 to 4 times by using 200 mu L PBS (phosphate buffer solution) with pH7.2, and then resuspending the supernatant in a preservation solution to obtain the magnetic beads of the chemically coupled anti-RBC antibody.

7. A method for separating red blood cells from whole blood, which comprises using the kit for separating red blood cells from whole blood according to any one of claims 1 to 6.

8. The method of claim 7 for separating red blood cells from whole blood, comprising the steps of,

taking the magnetic beads of the chemically coupled anti-RBC antibody in the kit for separating red blood cells from whole blood according to any one of claims 1 to 6, magnetically separating the supernatant, adding phytohemagglutinin and/or anti-RBC antibody, gently blowing and uniformly mixing, adding whole blood, gently blowing and uniformly mixing, performing magnetic adsorption, and sucking the supernatant;

the whole blood is EDTA-2Na or sodium citrate or heparin sodium anticoagulated whole blood or whole blood without anticoagulant.

9. The method of separating red blood cells from whole blood according to claim 8, wherein the lectin is used in an amount to give a final concentration of 50-500 μ g/mL;

optionally, the anti-RBC antibody is administered at a final concentration of 0.5-5 μ g/mL.

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