Method for preparing platelet-rich plasma through magnetic bead enrichment based on platelet antibody

Abstract

The invention relates to a method for preparing platelet-rich plasma based on magnetic bead enrichment of platelet antibodies, which comprises the following steps of: after carrying out primary centrifugation on a blood sample, sucking upper-layer plasma; then uniformly mixing magnetic beads coupled with monoclonal antibodies of anti-CD42b membrane protein consisting of a heavy chain variable region as shown in SEQ ID NO: 3 and a light chain variable region as shown in SEQ ID NO: 7 with upper-layer plasma sucked after primary centrifugation, continuing secondary centrifugation, and due to attachment of the magnetic beads, the effective mass of blood platelets is increased, so that the sedimentation coefficient of the blood platelets is increased; during the second centrifugation, more platelets are concentrated and precipitated to the bottom of the test tube, and white blood cells are positioned in a layer between the upper-layer plasma and platelets, so that three fourths of the upper-layer platelet-poor plasma is removed; and finally obtaining the high-quality platelet-rich plasma which is low in platelet loss degree, high in enrichment degree and capable of keeping platelet biological activity, wherein the lower layer of the platelet-rich plasma is one quartered.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine and blood product preparation, and specifically relates to a method for preparing platelet-rich plasma based on magnetic bead enrichment of platelet antibodies. The method can be used for the preparation of high-quality platelet-rich plasma (PRP) in clinical treatment, regenerative medicine and experimental research. Background Art

[0002] Platelet-Rich Plasma (PRP) is a type of plasma separated from whole blood, which is rich in platelets and is usually used in medical treatments such as accelerating tissue repair, reducing inflammatory responses, and promoting wound healing. The preparation method of PRP usually includes two centrifugations, in which the first centrifugation is used to separate platelets and red blood cells, and the second centrifugation is used to further remove white blood cells from the plasma, and finally obtain a platelet-rich plasma sample. In this process, the activity and concentration of platelets are the key factors in preparation, and different centrifugation speeds and times have an important influence on the recovery rate and function of platelets. In recent years, with the increase in clinical demand, the application of PRP in plastic surgery, dermatology, dentistry and other fields has gradually expanded, especially in the treatment of soft tissue injuries, joint diseases and facial beauty, which has shown significant therapeutic effects.

[0003] CD42b is part of the platelet surface and constitutes the GPIb-IX-V complex, which includes GPIbα, GPIbβ, GPIX and GPV. This complex plays an important role in the adhesion process of platelets, especially binding with von Willebrand factor (vWF) to help platelets adhere to sites of vascular damage; CD42b is not directly involved in the activation process of platelets. It mainly binds to vWF when platelets are not activated to help platelets adhere to damaged blood vessel walls. It maintains stable expression during platelet activation. CD42b is a very specific platelet marker, often used for platelet identification and separation, and is not affected by the activation process.

[0004] Platelet-rich plasma is widely used in the fields of tissue repair, trauma treatment and anti-aging because it contains rich platelets and multiple growth factors. However, the existing PRP preparation method usually separates platelets through centrifugation technology, which has the problems of complex operation, long time, platelet activity loss, low purity and low activity. Therefore, there is an urgent need for an efficient, fast and accurate preparation method to obtain high-quality platelet-rich plasma. Summary of the invention

[0005] In order to solve the problems of reduced platelet activity, low platelet recovery rate and low enrichment efficiency in the preparation process of platelet-rich plasma (PRP) in the prior art, the present invention provides a method for preparing platelet-rich plasma by magnetic bead enrichment based on platelet antibodies. The method efficiently separates and enriches platelets through the binding action of specific antibodies coupled to magnetic beads, while maintaining the activity of platelets, thereby obtaining high-quality platelet-rich plasma.

[0006] In certain embodiments, the method comprises the following steps: first, the blood sample is centrifuged for the first time to remove red blood cells and most white blood cells, and the upper plasma is drawn; then, the magnetic beads coupled with the monoclonal antibody against CD42b membrane protein are mixed with the upper plasma drawn after the first centrifugation, and the second centrifugation is continued. Through the second centrifugation, three quarters of the platelet-poor plasma in the upper layer are removed, and finally a quarter of the high-quality platelet-rich plasma in the lower layer is obtained. This method can effectively improve the enrichment of platelets, reduce the loss of platelets, and ensure the activity of platelets during the separation process.

[0007] In certain embodiments, the monoclonal antibody against CD42b membrane protein is composed of heavy chain CDR1-3 shown in SEQ ID NO: 4-5 and light chain CDR1-3 shown in SEQ ID NO: 8-10, and has high specificity and affinity, and is used to effectively recognize and bind to CD42b molecules on the surface of platelets. The specific binding of the antibody helps to minimize the contamination of non-platelet cells during the preparation of platelet-rich plasma, thereby improving the purity and activity of platelets.

[0008] The present invention also provides the monoclonal antibody against CD42b membrane protein, the heavy chain variable region and the light chain variable region of which are respectively composed of the sequences shown in SEQ ID NO: 3 and SEQ ID NO: 7, and have excellent specificity and efficient platelet enrichment effect.

[0009] In addition, the present invention also provides a platelet-rich plasma prepared based on the above method. The platelet-rich plasma has the characteristics of low platelet loss and high enrichment, and is suitable for a variety of clinical treatments, such as promoting tissue repair, reducing inflammatory reactions, etc.

[0010] In certain embodiments, the platelet-rich plasma has a low degree of platelet apoptosis and is not significantly activated, thereby ensuring the functional integrity of platelets during the treatment process.

[0011] The implementation method of the present invention can effectively improve the recovery rate and enrichment efficiency of platelets by combining immunomagnetic bead separation technology and centrifugation technology, providing a more accurate and efficient platelet-rich plasma preparation solution.

[0012] Compared with the prior art, the present invention has at least the following beneficial effects:

[0013] Platelet-rich plasma (PRP) is obtained by two centrifugations of platelets adsorbed with immunomagnetic beads, which involves the physical separation of platelets, specific binding, and maintenance of platelet activity, mainly in the following aspects;

[0014] On the one hand, the platelets adsorbed by the immunomagnetic beads have increased their effective mass and surface area due to the attachment of magnetic beads, and their sedimentation coefficient is usually larger than that of other leukocyte impurities, so they settle faster during centrifugation and are located at a lower level. This change makes the platelets adsorbed by the immunomagnetic beads easier to separate and collect during centrifugation;

[0015] On the other hand, the present invention selects CD42b membrane protein which is not directly involved in platelet activation as a specific target, so that the platelets in the prepared platelet-rich plasma still have strong biological activity after activation. At the same time, the specific anti-CD42b monoclonal antibody ensures that the magnetic beads can be tightly adsorbed on the surface of platelets, thereby changing their sedimentation coefficient, which is beneficial to subsequent separation.

[0016] In summary, the present invention utilizes the specific binding of immunomagnetic beads coupled with specific anti-CD42b monoclonal antibodies to platelets and the difference in sedimentation coefficients of different components during the centrifugation process to efficiently prepare high-quality PRP with low platelet loss, high enrichment, and maintained platelet biological activity. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 ABCpred was used to predict B cell epitopes that mimic CD42b.

[0018] Figure 2 SDS-PAGE detection of CD42b-S recombinant protein.

[0019] Figure 3 Titer and subtype detection of antibody 3H1.

[0020] Figure 4 The half effective concentration (EC50) of monoclonal antibody 3H1 was tested.

[0021] Figure 5 Analysis of the specific binding ability of antibody 3H1-coupled magnetic beads to platelets.

[0022] Figure 6 Detection of platelet recovery rate and enrichment factor of platelet-rich plasma prepared by different methods.

[0023] Figure 7 Detection of platelet apoptosis and activation in platelet-rich plasma prepared by different methods.

[0024] Figure 8Analysis of PDGF and VEGF activities in platelet-rich plasma prepared by different methods. DETAILED DESCRIPTION

[0025] In order to make the technical problems, technical solutions and advantages to be solved by the present invention more clear, a detailed description will be given below with reference to the accompanying drawings and specific embodiments.

[0026] Example 1 Preparation of monoclonal antibody against human platelet membrane protein CD42b

[0027] Preparation of recombinant CD42b antigen: The sequence information of human platelet membrane protein CD42b was retrieved using the NCBI database, and the protein sequence was selected as follows: Chain B, Platelet glycoprotein Ib alpha chain (Glycoprotein Ibalpha) (GP-Ib alpha) (GPIbA) (GPIb-alpha) (CD42B-alpha) (CD42B) [Homo sapiens], PDB: 1SQ0_B:

[0028] HPICEVSKVASHLEVNCDKRDLTALPPDLPKDTTILHLSENLLYTFSLATLMPYTRLTQLNLDRCELTKLQVDGTLPVLGTLDLSHNQLQSLPLLGQTLPALTVLDVSFNRLTSLPLGALRGLGELQELYLKGNELKTLPPGLLTP TPKLEKLSLANNDLTELPAGLLNGLEENLDTLLLQENSLYTIPKGFFGSHLLPFAFLHGNPWLCNCEILYFRRWLQDNAENVYVWKQGVDVKAMTSNVASVQCDNSDKFPVYKYPGKGCPTLGDEGDTDLYDYYPEEDTEGDK(SEQ IDNO:1).

[0029] ABCpred was used to predict the B cell epitopes of CD42b, with a threshold of 0.51. The differences between the CD42b sequence and the predicted B cell epitopes were compared, and the highly conserved epitope sequences located on the surface of the protein structure were predicted. Figure 1 .

[0030] Figure 1 The results showed the multi-epitope antigen sequence of CD42b. The antigen epitope peptide sequences with score values ​​greater than 0.80 were connected in sequence through the flexible linker "SGGGGS" as a linker to construct the "platelet membrane protein CD42b combined antigen", referred to as CD42b-S, whose amino acid sequence is as follows:

[0031] TDLYDYYPEEDTEGDKSGGGGSYPGKGCPTLGDEGDTDSGGGGSPTL

[0032] GDEGDTDLYDYYPSGGGGSSVQCDNSDKFPVYKYPSGGGGSPICEVSKVAS

[0033] HLEVNCSGGGGSSLYTIPKGFFGSHLLPSGGGGSGELQELYLKGNELKTLSG

[0034] GGGSGVDVKAMTSNVASVQCSGGGGSHLLPFAFLHGNPWLCNSGGGGSS

[0035] HLEVNCDKRDLTALP (SEQ ID NO:2).

[0036] According to the amino acid sequence of SEQ ID NO:2, reverse translation was performed into a nucleic acid sequence, and the codons were optimized in combination with the target host Escherichia coli. The pET-28a-CD42b-S recombinant expression plasmid with a His tag was provided by Beijing Saibaisheng Gene Technology Co., Ltd. The constructed expression plasmid was transformed into the expression host bacteria Escherichia coli BL21 (DE3); the positive clones containing the resistance marker kanamycin were screened by plating. The monoclonal colony was inoculated into LB medium containing antibiotics, and large-scale protein expression was performed in 500mL to 2L LB medium. The cells were cultured at 37°C with shaking until OD600 = 0.6-0.8, and the inducer IPTG 0.5 mM was added. The expression was induced at 16°C for 12 hours, and the cells were collected after induction (4°C, 5000×g, 10 minutes); the cells were resuspended in lysis buffer (50 mM Tris-HCl, 300 mM NaCl, 10 mM Imidazole, pH 8.0); the protease inhibitor PMSF was added, and the cells were lysed by an ultrasonic disruptor (under ice bath, ultrasonication for 3 seconds, interval of 6 seconds, cycle for 20-30 minutes), and the lysate was centrifuged at 4°C, 12000×g for 30 minutes, and the supernatant was collected. Prepare Ni-NTA resin, balance with equilibration buffer (50mM Tris-HCl, 300mMNaCl, 10mM Imidazole, pH 8.0), load the lysate, incubate at 4℃ for 1 hour to allow His-tagged protein to bind, wash to remove non-specific binding protein (buffer contains 20-40mM Imidazole), elute the target protein with elution buffer (buffer contains 250-300mM Imidazole), and further purify with Superdex 200Increase 10, and then detect the molecular weight by SDS-PAGE electrophoresis, see Figure 2 .

[0037] Figure 2 The results showed that the specific target band of CD42b-S recombinant protein had a high separation purity and the molecular weight was consistent with the expected 21.42kDa.

[0038] Preparation of monoclonal antibody against CD42b-S recombinant protein: 5 female 4-6 week old specific pathogen free (SPF) Balb / c mice were taken, CD42b-S recombinant protein immunogen was mixed with equal volumes of Freund's complete adjuvant, and the mice were injected subcutaneously at multiple points, with 0.30 mL injected per mouse. The mice were boosted 4 times with the same dose every 14 days, and the adjuvant was Freund's incomplete adjuvant. 3 days before fusion, a final immunization was performed with a higher dose, 0.35 mL per mouse, and no adjuvant was used for this immunization. 3 days later, cell fusion was performed, SP2 / 0 cells were mixed with spleen cells isolated from immunized mice in a 5:1 ratio in a 50 mL centrifuge tube, and fused under the action of PEG1500. The positive hybridoma cells were subcloned multiple times using the limiting dilution method, and cell lines with strong positivity and high inhibition rate were selected for further cloning. The cloning operation was repeated multiple times until two consecutive full positivity results were obtained with ideal sensitivity, thus obtaining the monoclonal cell line 3H1 that can stably secrete antibodies and has high specificity and is cryopreserved.

[0039] Ten 8- to 10-week-old female Balb / c mice were selected and each mouse was intraperitoneally injected with 0.5 mL of pristane. One week later, 1×10 6 ~2×10 6 After the abdominal cavity of the mouse showed obvious bulge (usually 5-7 days), the ascites was collected and purified by caprylic acid-ammonium sulfate precipitation method; the titer of the purified antibody 3H1 was determined by ELISA method, and the subtype of the monoclonal antibody was tested using an antibody subtype detection kit. Figure 3 .

[0040] Figure 3 The results showed that the titer of antibody 3H1 was 1:102400, and the subtype of antibody 3H1 was IgG3.

[0041] Total RNA of 3H1 cells was extracted using Trizol reagent. High-quality total RNA was extracted by processing according to the instructions for use of Trizol. RNA was reverse transcribed into cDNA using a reverse transcription kit; reaction system: total RNA (1 μg), primers, reverse transcription buffer, reverse transcriptase and RNase inhibitor; incubated at 42°C for 1 hour, then inactivated at 85°C for 5 minutes, primers were designed based on the conserved region of mouse IgG antibody, PCR amplified fragments were used, and the amplified products were inserted into the TA cloning vector. Competent Escherichia coli were transformed and positive clones were picked. Sanger sequencing of positive clones was performed using universal sequencing primers. VH and VL sequences were compared using the IgBlast database to determine the variable region sequence. CDR region and framework region sequences were analyzed according to the Kabat definition, see Table 1.

[0042] Table 13 Identification of variable region sequences of H1 monoclonal antibody

[0043]

[0044] Human CD42b was diluted into PBS at a concentration of 0.5 μg·mL-1, and 100 μL per well was coated on the microplate at 4°C overnight. The coating solution was discarded, and the plate was washed with PBST, and then blocked with 5% BSA-PBST blocking solution at 37°C for 1 hour; after washing, different concentration gradients of 3H1 monoclonal antibody were added to each well and incubated at 37°C for 1 hour. After the reaction was completed, the plate was washed, and 100 μL of HRP-labeled goat anti-human IgG diluted 1:10000 was added to each well, and reacted at 37°C for 1 hour. After washing the plate, TMB solution was added, and after reacting at room temperature in the dark for 10 minutes, 1 mol·L-1 H2SO4 was added to terminate the reaction. The absorbance was detected at a wavelength of 450 nm on an ELISA reader, and four-parameter curve fitting was performed using GraphPad Prism7 software to calculate the half effective concentration (EC50) value.

[0045] Figure 4 The results showed that the half effective concentration (EC50) value of the 3H1 monoclonal antibody was 0.93 ng / mL.

[0046] Example 2 Construction of 3H1 antibody-coupled magnetic beads and its specific detection

[0047] Construction of antibody-coupled immunomagnetic beads:

[0048] Take 10 mg of carboxyl magnetic beads and place them in a centrifuge tube; add 1 mL of MES buffer and mix well. Use a magnetic separation device to separate the magnetic beads, discard the supernatant, and repeat washing twice. Add EDC and NHS to the magnetic bead suspension to a final concentration of 2 mg / mL and 5 mg / mL, respectively, mix gently and react for 1 hour. After activation, separate the magnetic beads using a magnetic separation device and discard the supernatant. Wash the magnetic beads twice with MES buffer to remove unreacted EDC / NHS. Dilute 3H1 antibody to PBS buffer at a concentration of 2 mg / mL; add 3H1 antibody to the activated magnetic bead suspension with a volume ratio of 1:10 for antibody to magnetic beads. After mixing, react at room temperature for 2 hours, add a blocking agent (1% BSA solution) to block unbound active sites, and react at room temperature for 30 minutes. Wash the magnetic beads with PBS buffer and remove the supernatant using a magnetic separation device; repeat washing 3 times, suspend the coupled magnetic beads in an appropriate amount of PBS buffer (containing 0.1% NaN3 preservative) and store at 4°C.

[0049] Specificity detection of immunomagnetic beads:

[0050] Mix 3H1 antibody-coupled magnetic beads with platelet samples, using about 1-2 μg of anti-CD42b magnetic beads and 10 6 -10 7 Incubate platelets with 100 μg of magnetic beads; incubate at room temperature for 30 minutes, shaking the mixture to ensure adequate contact; use a magnetic rack to separate magnetic beads; separate platelets from unbound magnetic beads and retain platelets bound to magnetic beads; wash the platelets on the magnetic beads three times with PBS buffer to remove non-specifically bound impurities; after each wash, separate the magnetic beads using a magnetic rack and remove the washing solution; then resuspend the immunomagnetic beads with PBS as the experimental group for subsequent flow cytometry detection.

[0051] Mix the non-antibody-coupled magnetic beads with the platelet sample, using approximately 1-2 μg of magnetic beads and 10 6 -10 7 Incubate platelets with 100 μg / mL of magnetic beads; incubate at room temperature for 30 minutes, shaking the mixture to ensure adequate contact; use a magnetic rack to separate magnetic beads; separate platelets from unbound magnetic beads and retain platelets bound to magnetic beads; wash platelets on magnetic beads three times with PBS buffer to remove non-specifically bound impurities; after each wash, separate magnetic beads using a magnetic rack and remove the washing solution; then resuspend the immunomagnetic beads with PBS as a control group for subsequent flow cytometry detection.

[0052] Fluorescently labeled anti-platelet antibodies, namely FITC-anti-CD41 and PE-anti-CD61, were added to the experimental group and the control group respectively to identify the expression of platelets. Appropriate lasers and filters were set to collect fluorescent signals for flow cytometry detection and statistical experimental data. The data were quantitatively analyzed and the efficiency of magnetic beads binding to platelets was calculated. Figure 5 .

[0053] Figure 5 The results showed that the platelets bound to the immunomagnetic beads accounted for 96.45% of the total platelets, indicating that the magnetic beads coupled to the 3H1 antibody have good specificity and binding ability.

[0054] Example 3 Comparative Analysis of Different Platelet-Rich Plasma Separation Methods

[0055] Scheme A (single centrifugation): collect 50 mL of human peripheral blood sample and centrifuge it at 450×g RCF for 10 min; then use a 10 mL syringe to absorb the components above the buffy coat layer to obtain platelet-rich plasma A.

[0056] Scheme B (centrifugal separation + centrifugal concentration): 50 mL of human peripheral blood sample was collected and centrifuged at 650×gRCF for 15 min. Red blood cells, due to their highest density, were deposited at the bottom of the centrifuge tube, while plasma and buffy coat (containing platelets) remained in the upper layer. After the upper plasma (including the buffy coat) was removed, high-speed centrifugation was performed again, at 1500×g RCF for 10 min. Platelets were deposited at the bottom of the centrifuge tube due to their high density, while the lighter plasma floated on top, and finally two layers were obtained. The upper two-thirds was platelet-poor plasma with most of the platelets removed, and the lower one-third was concentrated platelets. By removing the platelet-poor plasma, platelet-rich plasma B was finally obtained.

[0057] Scheme C (centrifugation + magnetic bead enrichment):

[0058] Step 1: Collect 50 mL of human peripheral blood sample and centrifuge at 650×g RCF for 10 min; carefully aspirate the upper plasma, avoiding the white blood cell layer, and transfer the collected plasma to a new centrifuge tube for the next step of platelet enrichment;

[0059] Step 2: Take 5 mL of the 3H1 antibody-coupled magnetic beads prepared in Example 2, add 10 mL of PBS buffer, and repeat the washing process 2-3 times to ensure that impurities are removed. Resuspend the washed magnetic beads in PBS buffer. When resuspending, gently shake or rotate repeatedly to ensure that the magnetic beads are evenly distributed and adjust the magnetic bead concentration to 5×10 8 Magnetic beads / mL;

[0060] Step 3: Mix the plasma collected in step 1 and the 3H1 antibody-coupled magnetic beads after washing in step 2 in the same centrifuge tube, mix at a ratio of 1:10, and shake gently; incubate at low temperature for 20-30 minutes, and gently shake the mixture during this period to ensure that the platelets are fully bound to the 3H1 antibody-coupled magnetic beads; then place the mixture in a 50mL centrifuge tube and centrifuge at 2000×g RCF for 15 minutes; since the platelets adsorbed by the immunomagnetic beads have a higher density and are more easily deposited at the bottom of the centrifuge tube, the sedimentation coefficient is higher, and the lighter plasma floats on top, and finally two layers are obtained, the upper three-quarters is platelet-poor plasma with most platelets removed, and the lower one-quarter is concentrated platelets, and the platelet-rich plasma C is finally obtained by removing the platelet-poor plasma.

[0061] Platelet-rich plasma parameter detection: Use a blood cell counter to determine the number of platelets in human peripheral blood whole blood samples and platelet-rich plasma AC. After mixing them, take 50ul and use a blood analyzer to count the cells and analyze the platelet count. Use the following formula to calculate the platelet recovery rate and enrichment factor of different separation methods, see Figure 6 .

[0062] Platelet recovery rate = (platelet number in PRP) / (platelet count in blood sample) × 100%;

[0063] Enrichment factor = platelet concentration in PRP / platelet concentration in blood sample.

[0064] Figure 6 The results showed that compared with platelet-rich plasmas A and B, platelet-rich plasma C had significantly improved platelet recovery rate and enrichment coefficient index. That is, the extraction method based on 3H1 antibody-coupled immunomagnetic beads in scheme C had a significant effect on the platelet recovery rate and enrichment coefficient of platelet-rich plasma. The degree of platelet loss in platelet-rich plasma C was low, and the platelet concentration was high enough to meet the standards of high-quality PRP.

[0065] Analysis of platelet apoptosis and activation in platelet-rich plasma:

[0066] In order to detect the apoptosis of the extracted PRP, this study selected FITC-labeled Annexin V antibody and used flow cytometry to detect the phosphatidylserine (PS) externalization rate of PRP to evaluate the apoptosis of cells in PRP; at the same time, the expression of CD62p on the platelet surface was detected by flow cytometry to evaluate the activation degree of platelets before and after PRP preparation. Figure 7 .

[0067] Figure 7The results showed that there was no significant difference in the platelet PS eversion rate and CD62p expression in platelet-rich plasma C compared with human peripheral blood samples (P>0.05); this indicates that the PRP extraction method of scheme C did not cause an increase in platelet apoptosis rate and platelet activation.

[0068] Growth factor analysis of platelet-rich plasma:

[0069] ELISA kits were used to detect the concentrations of PDGF and VEGF in the growth factors in platelet-rich plasma A, B, C and blood sample supernatants, as well as the concentrations of growth factors after PRP activation. Figure 8 .

[0070] Figure 8 The results showed that the PDGF and VEGF in platelet-rich plasma C were significantly higher than those in human peripheral whole blood samples, and the PRP after activation with an activator increased by 2 times compared to before activation, indicating that the PRP extracted by scheme C can effectively release growth factors.

[0071] The above is a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for preparing platelet-rich plasma based on magnetic bead enrichment of platelet antibodies, characterized in that: The method comprises the steps of: performing a first centrifugation on a blood sample, aspirating the upper plasma, and then mixing the magnetic beads coupled with the monoclonal antibody against CD42b membrane protein with the upper plasma aspirated after the first centrifugation, and then performing a second centrifugation to remove three quarters of the platelet-poor plasma in the upper layer, and finally obtaining the lower quarter of high-quality platelet-rich plasma.

2. The method according to claim 1, characterized in that: The monoclonal antibody against CD42b membrane protein consists of heavy chain CDR1-3 shown in SEQ ID NO: 4-5 and light chain CDR1-3 shown in SEQ ID NO: 8-10.

3. The method according to claim 1 or 2, characterized in that: The monoclonal antibody against CD42b membrane protein consists of a heavy chain variable region shown in SEQ ID NO:3 and a light chain variable region shown in SEQ ID NO:

7.

4. A monoclonal antibody against CD42b membrane protein, characterized in that: The monoclonal antibody consists of heavy chain CDR1-3 shown in SEQ ID NOs: 4-5 and light chain CDR1-3 shown in SEQ ID NOs: 8-10.

5. The monoclonal antibody according to claim 4, characterized in that: The monoclonal antibody consists of a heavy chain variable region shown in SEQ ID NO:3 and a light chain variable region shown in SEQ ID NO:

7.

6. An immunomagnetic bead based on platelet antibodies, characterized in that: The monoclonal antibody consists of heavy chain CDR1-3 shown in SEQ ID NOs: 4-5 and light chain CDR1-3 shown in SEQ ID NOs: 8-10.

7. Platelet-rich plasma prepared by the method according to any one of claims 1 to 3, characterized in that: The platelet-rich plasma has the characteristics of low platelet loss and high platelet enrichment.

8. The platelet-rich plasma according to claim 7, characterized in that: The platelet apoptosis in the platelet-rich plasma was low and not significantly activated.

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