Anti-platelet antibody detection kit based on magnetic particle chemiluminescence and application

Abstract

The invention discloses an anti-platelet antibody detection kit based on magnetic particle chemiluminescence and application, and relates to the technical field of blood detection. Comprising a reagent M, which comprises magnetic particles coated with recombinant antigen proteins GPIIb / IIIa, GPIa / IIa, GPIb / IX and HLA antigen at the same time, and a magnetic particle protection solution containing Ca < 2 + >; the R1 reagent comprises a buffer solution; the R2 reagent comprises an anti-human IgG antibody marked by an enzyme or a luminescence agent; wherein the magnetic particles are treated by the following steps: adding EDC, NHS and NH2-PEG6000-COOH into the magnetic particles, and carrying out oscillatory reaction for more than 4 hours; according to the kit, magnetic particle immobilization coating is combined with a chemiluminescence immunoassay signal amplification system for detection, multiple synchronous or independent detection of multiple antigens is achieved, the detection range is wide, and the kit has good sensitivity, specificity and stability and good detection repeatability.

Description

Technical Field

[0001] The present invention relates to the technical field of blood detection, and particularly relates to a kit for detecting anti-platelet antibodies based on magnetic particle chemiluminescence and an application thereof. Background Art

[0002] Platelets have a complex antigen system, including platelet-specific antigens (human platelet antigen, HPA); human leukocyte antigens (human leukocyte antigen, HLA) shared with other tissues or cells; CD36 antigens shared with monocytes / macrophages and nucleated red blood cells, etc. Antibodies against the platelet antigen system can cause immune platelet transfusion refractoriness (PTR), fetal and neonatal alloimmune thrombocytopenia (FNAIT), febrile non-haemolytic transfusion reaction (FNHTR), post-transfusion purpura (PTP), idiopathic thrombocytopenic purpura (ITP), etc.

[0003] Platelet-specific membrane glycoproteins are the main targets of anti-platelet antibodies, including GPIIb, GPIIIa, GPIbα, GPIbβ, GPIa, and CD109. Among them: GPIIb / IIIa (CD41 / CD61) is the most abundant glycoprotein on the platelet surface, mediates fibrinogen binding during platelet activation, promotes platelet aggregation, and is the most common target antigen in ITP patients (accounting for about 40%); the GPIb / IX / V complex (CD42b / c / d) is involved in platelet adhesion and is related to chronic ITP and NAIT; GPIa / IIa (CD49b / CD29) is a collagen receptor and is related to some refractory ITP. Polymorphisms in the genes encoding platelet membrane glycoprotein antigens lead to the production of platelet alloantigens. So far, 33 HPAs expressed on different platelet glycoproteins have been described; for example, the HPA-1a / 1b and HPA-3a / 3b allele polymorphisms in humans are closely related to the onset of NAIT. In addition, platelet-related antigens, such as the presence of HLA class I molecules on the cell surface, FNHTR is mainly caused by HLA antibodies. FNHTR caused by HLA antibodies accounts for about 14.6% of the total transfusion reactions and is the main cause of platelet cross-matching incompatibility and platelet transfusion refractoriness (PTR).

[0004] Existing detection techniques for anti-platelet antibodies mainly include monoclonal antibody-specific solid-phase detection method (MAIPA), chemiluminescence method, flow cytometry (FCM), and enzyme-linked immunosorbent assay (ELISA). However, these methods have significant limitations: The MAIPA method relies on donor platelets as the antigen source, and the competitive effect between monoclonal antibodies and the antibodies to be detected results in low detection sensitivity of this method. Moreover, multiple washing and centrifugation steps are required, and there are problems such as difficult preservation of donor platelets and large batch-to-batch differences; The chemiluminescence method is a rapid screening method, suitable for preliminary detection, with relatively low cost, but it cannot accurately distinguish antibodies against specific glycoproteins; FCM has high sensitivity, but requires fresh platelet samples, expensive equipment, and is difficult to popularize; Some ELISA kits use recombinant antigens, but have many steps, long time consumption, low throughput, and manual operation is prone to introduce errors. In addition, conventional detection techniques generally require a high sample volume (≥5 mL of whole blood), long detection time (≥4 hours, at least 2 hours), and high experimental operation requirements, severely restricting clinical promotion.

[0005] Based on the chemiluminescence detection technology has the characteristics of automation and high throughput, can significantly reduce the detection time, and is of great significance for disease diagnosis, typing, treatment plan selection, and prognosis evaluation. However, existing anti-platelet antibody detection kits based on chemiluminescence detection usually use single antigens, with a narrow coverage of anti-platelet antibody targets, and the sensitivity, specificity, and stability of the kits still need to be improved. Summary of the Invention

[0006] The present invention is to solve the technical problems that existing anti-platelet antibody detection kits based on chemiluminescence detection usually use single antigens, with a narrow coverage of anti-platelet antibody targets, and the sensitivity, specificity, and stability of the kits still need to be improved. The purpose is to provide an anti-platelet antibody detection kit and application based on magnetic particle chemiluminescence. Detection is carried out by using magnetic particle solid-phase coating combined with a chemiluminescence immunoassay signal amplification system, realizing the multiplex synchronous or independent detection of recombinant antigens GPIIb / IIIa, GPIa / IIa, GPIb / IX complex, and HLA antibody. The detection range is wide, the kit has good sensitivity, specificity, and stability, and the detection repeatability is good.

[0007] The present invention is realized through the following technical solutions.

[0008] The first object of the present invention is to provide an anti-platelet antibody detection kit based on magnetic particle chemiluminescence, including: Reagent M, including magnetic particles simultaneously coated with recombinant antigen proteins GPIIb / IIIa complex, GPIa / IIa complex, GPIb / IX complex, and HLA antigen, and a magnetic particle protective liquid containing Ca 2+ ; Reagent R1, including a buffer solution; Reagent R2, including anti-human IgG antibody labeled with an enzyme or a luminescent agent; Among them, the magnetic particles are treated as follows: EDC, NHS, and NH2-PEG6000-COOH are added to the magnetic particles and oscillated for reaction for more than 4 h.

[0009] The amino acid sequences of the recombinant antigens involved in the present invention are as follows: the amino acid sequence of GPIIb is as shown in SEQ ID NO.1, the amino acid sequence of GPIIIa is as shown in SEQ ID NO.2, the amino acid sequence of GPIa is as shown in SEQ ID NO.3, the amino acid sequence of GPIIa is as shown in SEQ ID NO.4, the amino acid sequence of GPIb is as shown in SEQ ID NO.5, the amino acid sequence of GPⅨ is as shown in SEQ IDNO.6, and the amino acid sequence of HLA-A is as shown in SEQ ID NO.7.

[0010] The present invention uses recombinantly expressed human platelet-specific glycoprotein complexes as standardized antigens to eliminate inter-batch variations. At the same time, GPIIb / IIIa, GPIa / IIa, GPIb / IX complex, and HLA antigen are coated on the surface of magnetic bead carriers, independent of fresh donor platelet cells, and can cover more than 90% of anti-platelet antibody targets, with a wider detection range.

[0011] Meanwhile, in the present invention, the magnetic particles are treated with EDC, NHS, and NH2-PEG6000-COOH. After NH2-PEG6000-COOH binds to the magnetic particles, PEG6000 and carboxyl groups are introduced. Among them, PEG6000 serves as a spacer arm, which can reduce steric hindrance, fully expose the antigen epitopes, so as to improve the sensitivity, repeatability, and stability of detection. The introduced carboxyl groups increase the active sites of the magnetic particles. After activation with EDC and NHS, the reaction activity of the magnetic particles is improved, effectively enhancing the efficiency and stability of the covalent coupling between the magnetic particles and the recombinant antigen.

[0012] In addition, the present invention also adopts a magnetic particle protective solution containing Ca 2+ to maintain the GP conformation through a Ca 2+ buffer solution, solve the problem that the recombinant antigen is easily inactivated, and effectively improve the stability of the recombinant antigen.

[0013] Furthermore, the magnetic particles are specifically treated as follows: Add EDC and NHS to the magnetic particles and oscillate for reaction for 30 - 60 min, then add NH2-PEG6000-COOH and oscillate for reaction for 4 - 5 h, and finally add EDC and NHS again and oscillate for reaction for 30 - 60 min.

[0014] In the present invention, the magnetic particles are first chemically activated with EDC and NHS to carboxyl groups to achieve covalent coupling with the recombinant antigen, and then NH2-PEG6000-COOH is added to further modify the magnetic particles, introducing PEG6000 and carboxyl groups. PEG6000 serves as a spacer arm, which can reduce steric hindrance, fully expose the antigen epitopes, so as to improve the sensitivity, repeatability, and stability of detection. At the same time, carboxyl groups are further introduced into NH2-PEG6000-COOH and then activated with EDC and NHS, effectively increasing the active sites of the covalent coupling between the magnetic particles and the recombinant antigen. The magnetic particles modified by the method of the present invention can improve the coupling efficiency and stability between the magnetic particles and the recombinant antigen.

[0015] Furthermore, the magnetic particles specifically include the following steps: Suspend the magnetic particles in MES buffer solution, add 50 mM EDC and 25 mM NHS and oscillate for reaction for 30 - 60 min. After magnetic separation, wash with MES buffer solution multiple times to remove unreacted EDC and NHS; Then add NH2-PEG6000-COOH and oscillate for reaction for 4 - 5 h. After magnetic separation, wash with Tris buffer solution multiple times to remove unbound NH2-PEG6000-COOH; Finally, add 50 mM EDC and 25 mM NHS and react with shaking for 30 - 60 min. After magnetic separation, wash with MES buffer multiple times to remove unreacted EDC and NHS.

[0016] Furthermore, the mass ratio of NH2-PEG6000-COOH to magnetic particles is (4 - 6):1. For example, mass ratios of 4:1, 5:1, and 6:1 can be adopted.

[0017] Furthermore, the magnetic particle protection solution containing Ca 2+ is a Tris buffer solution containing Ca 2+

[0018] Furthermore, the magnetic particles are selected from any one or more of streptavidin magnetic beads, carboxyl magnetic beads, and tosyl magnetic beads.

[0019] Furthermore, the R1 reagent is a Tris buffer solution added with a stabilizer. Among them, the stabilizer can be selected from any one or more of glycine, bovine serum albumin, casein, trehalose, mannitol, Tween 20, Triton X100, and PEG.

[0020] Furthermore, the enzyme for labeling is selected from alkaline phosphatase or horseradish peroxidase, and the corresponding chemiluminescent substrates can be selected from 3-(2-spiroadamantane)-4-methoxy-4-(3-phosphoryl)-phenyl-1,2-dioxetane disodium salt (AMPPD) and luminol; the luminescent agent is selected from any one of acridinium ester, isoluminol, and tris(bipyridine)ruthenium, and the corresponding initiators can be selected from NaOH / H2O2 solution and tripropylamine.

[0021] Furthermore, the recombinant antigen proteins GPIIb / IIIa complex, GPIa / IIa complex, and GPIb / IX complex are all prepared by the following method: PCR amplify the target gene, recover the PCR product, ligate the PCR product with a dicistronic vector, then amplify through Escherichia coli, extract the plasmid, transfect it into HEK293t cells for expression, and finally extract and purify. Among them, the GPIIb / IIIa, GPIa / IIa, and GPIb / IX complexes are co-transfected by the dicistronic vector, which can effectively ensure correct folding and disulfide bond formation.

[0022] The second object of the present invention is to provide an application of an anti-platelet antibody detection kit based on magnetic particle chemiluminescence, and the kit is detected using an automatic chemiluminescence detection system; Before detection, incubate and react the M reagent, R1 reagent, and the serum sample to be detected for 8 - 15 min, then add the R2 reagent and incubate and react for 8 - 15 min. After washing, add the chemiluminescent substrate or initiator.​

[0023] In a specific embodiment, the operation steps are as follows: First, add M reagent, R1 reagent, and the serum sample to be tested into the reaction cup and incubate for 10 min. The autoantibodies in the sample to be tested specifically bind to the antigen in the M reagent to form a solid-phase antigen-antibody complex, and the sample impurities are removed by washing. Then, add R2 reagent to the reaction cup and incubate for 10 min. At this time, there is a solid-phase antigen-antibody-enzyme-labeled secondary antibody complex (or antigen-antibody-luminescent agent-labeled secondary antibody complex) in the reaction cup. After washing again, add a luminescent substrate (or activator), and a chemiluminescence reaction occurs to release photons. The concentration of the antibody in the sample to be tested is proportional to the number of photons generated.

[0024] The kit of the present invention is equipped with a fully automatic chemiluminescence detection system (Sichuan Xiguang Biotechnology Co., Ltd., Sharay8000): integrating all steps of sample loading, incubation, magnetic separation, washing, and detection. The single-sample detection time < 35 min. Compared with the prior art, which requires at least 2 hours at the shortest, the detection time is greatly shortened, making it suitable for clinical rapid diagnosis and large-scale population screening of HPA / HLA antibodies.

[0025] It should be understood that the kit of the present invention is also equipped with calibrators and quality control products. The preparation method is as follows: The raw materials are prepared by screening high-affinity IgG monoclonal antibodies using the recombinant protein of the target antigen of APA as the immunogen, and performing humanization modification to make it simulate the binding of APA antibodies in patient serum to the target antigen in the kit. The obtained APA human IgG monoclonal antibody is diluted with Tris buffer at a certain ratio to prepare calibrators and quality control products.

[0026] Compared with the prior art, the present invention has the following advantages and beneficial effects: 1. The present invention uses a recombinant human platelet-specific glycoprotein complex as a standardized antigen to eliminate inter-batch variation. At the same time, GPIIb / IIIa, GPIa / IIa, GPIb / IX complexes, and HLA antigens are coated on the surface of magnetic beads. It does not rely on fresh donor platelets and can cover more than 90% of anti-platelet antibody targets, with a wider detection range. Combining chemiluminescence technology, the detection time is shortened from at least 2 hours by the traditional method to within 35 minutes, and at the same time, the sample requirement is reduced from 5 mL to 0.02 mL, especially meeting the clinical needs of pediatric and critically ill patients; 2. The present invention treats magnetic particles with EDC, NHS, and NH2-PEG6000-COOH. After NH2-PEG6000-COOH binds to the magnetic particles, PEG6000 and carboxyl groups are introduced; among them, PEG6000 acts as a spacer arm, which can reduce steric hindrance and fully expose antigenic epitopes to improve the sensitivity, repeatability, and stability of detection; the introduced carboxyl groups increase the active sites of the magnetic particles. After activation with EDC and NHS, the reaction activity of the magnetic particles is improved, effectively enhancing the efficiency and stability of the covalent coupling of the magnetic particles with the recombinant antigen. 3. A magnetic particle protective solution containing Ca 2+ is used. The conformation of GP is maintained by the Ca 2+ buffer solution, solving the problem that the recombinant antigen is easily inactivated and effectively improving the stability of the recombinant antigen. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] In order to more clearly illustrate the technical solutions of the exemplary embodiments of the present invention, the drawings required for use in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as limiting the scope. For those of ordinary skill in the art, other related drawings can be obtained based on these drawings without creative efforts. In the drawings: Figure 1 It is a thermal stability curve graph of the M reagent coated with the recombinant antigen GPIIb / IIIa complex in Example 2; Figure 2 It is a thermal stability curve graph of the M reagent coated with the HLA antigen in Example 3; Figure 3 It is the result graph of the negative coincidence rate of the kit of the present invention in Example 4. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0028] To make the objectives, technical solutions, and advantages of the present invention clearer and more understandable, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the illustrative embodiments and their descriptions of the present invention are only used to explain the present invention and do not serve as a limitation to the present invention.

[0029] The following details the implementation manners of a kit for detecting anti-platelet antibodies based on magnetic particle chemiluminescence and its application according to the present invention. However, there will be cases where unnecessary detailed descriptions are omitted. For example, there are cases where the detailed descriptions of well-known matters and repeated descriptions are omitted. This is to avoid the following descriptions from becoming unnecessarily lengthy and to facilitate the understanding of those skilled in the art.

[0030] The "scope" disclosed by the present invention is defined in the form of a lower limit and an upper limit. A given scope is defined by selecting a lower limit and an upper limit, and the selected lower limit and upper limit define the boundary of a particular scope. The scope defined in this way can include or exclude the end values, and can be combined arbitrarily, that is, any lower limit can be combined with any upper limit to form a scope.

[0031] If there is no special instruction, all embodiments and optional embodiments of the present invention can be combined with each other to form a new technical solution.

[0032] If there is no special instruction, all technical features and optional technical features of the present invention can be combined with each other to form a new technical solution.

[0033] If there is no special instruction, the "including" and "comprising" mentioned in the present invention mean open-ended, and can also be closed-ended. For example, the "including" and "comprising" can mean that other substances not listed can also be included or comprised, or only the substances listed can be included or comprised.

[0034] If there is no special instruction, all steps of the present invention can be carried out in sequence or randomly, preferably in sequence. For example, the method includes steps (a) and (b), which means that the method can include steps (a) and (b) carried out in sequence, or can also include steps (b) and (a) carried out in sequence. For example, it is mentioned that the method may further include step (c), which means that step (c) can be added to the method in any order. For example, the method can include steps (a), (b) and (c), or can also include steps (a), (c) and (b), or can also include steps (c), (a) and (b), etc.

[0035] The technical solution of the present invention will be further described in detail below in conjunction with embodiments.

[0036] It should be noted that the experimental methods used in the embodiments are all conventional methods unless otherwise specified. The materials, reagents, methods and instruments used are all conventional materials, reagents, methods and instruments in this field, and those skilled in the art can obtain them through commercial channels without special instructions.

[0037] Example 1.

[0038] Preparation of the recombinant antigen complex protein (taking the GPIIb / IIIa antigen as an example, and the preparation methods of the other recombinant antigen complex proteins are the same), including the following steps.

[0039] 1. Construction of the bicistronic vector PCR amplified the target genes GPⅡb (ITGA2B) cDNA (NCBI accession number: NM_000419.5) and GPⅢa(ITGB3) cDNA (NCBI accession number: NM_000212.3). The target genes were commercially synthesized. The primers used for PCR amplification were: Forward primer for GPⅡb: 5'-GGA ATT C ATG GGG CTC CTG CTG-3' (EcoRI site underlined); Reverse primer for GPⅡb: 5'-CCG CTC GAG TCA GGC AGG TCA GGT-3' (XhoI site underlined); Forward primer for GPⅢa: 5'-CGC GGA TCC ATG GCT CCC GAG GAG-3' (BamHI site underlined); Reverse primer for GPⅢa: 5'-ATA GCG GCC GC TCA GGC TGG GAC TTC-3' (NotI site underlined); The PCR products were recovered using a DNA rapid purification and recovery kit. The PCR products and the pIRES2 vector (containing the CMV promoter, IRES sequence, Flag tag, and resistance gene) were digested with EcoRI, XhoI, BamHI, and NotI respectively and then ligated.

[0040] 2. Cloning of the bicistronic vector Pick a DH5α monoclonal from a fresh plate and culture it with shaking at 37°C overnight; add the overnight culture to fresh LB medium at a ratio of 1:100 and continue to culture at 37°C until the OD260 reaches 0.3 - 0.4; transfer the bacterial solution to a pre-cooled 50 mL centrifuge tube and centrifuge at 4°C at 1000g for 15 min to recover the bacteria; resuspend the pellet with ice-precooled 0.1M CaCl2 and centrifuge to collect the bacteria; resuspend with ice-precooled CaCl2 to obtain competent bacteria; take 200 μL of competent bacteria and 10 μL of DNA and add them to a cold EP tube, gently rotate and mix well, and place on ice for 30 min; then place the tube in a 42°C water bath and let it stand for 90 s. After rapid cooling, add 800 μL of LB medium to each tube and culture with gentle shaking at 37°C for 1 h to recover the bacteria; take 200 μL of the bacterial solution and spread it on an Amp plate containing X-gal and IPTG, place it at room temperature until the liquid is absorbed, and then incubate it inverted at 37°C overnight. Pick a white clone from the overnight culture plate and culture it with shaking at 37°C in LB medium containing 100 μL / ml Amp; take 1 μL of the culture as a template for PCR to identify whether there is an inserted fragment, inoculate the positively identified strain into 5 ml of LB medium containing ampicillin, shake vigorously at 37°C overnight, the next day, centrifuge to remove the supernatant, add 250 μL of cell resuspension to resuspend the bacteria, add 250 μL of cell lysate and 10 μL of alkaline protease solution, mix well, add 350 μL of neutralizing solution, and white flocculent precipitate can be seen, centrifuge at 12000×g for 10 min; insert the adsorption column into the collection tube, carefully aspirate the supernatant of the bacterial lysate into the adsorption column; centrifuge and wash three times at room temperature, transfer the adsorption column to a new EP tube, add 50 μL of sterile water preheated to 50°C to the adsorption column, and centrifuge at 12000×g for 1 min to collect the eluate, which is the plasmid.

[0041] 3. Transfection of the dicistronic vector into HEK293t cells Digest the cells with trypsin and count them. Seed the cells into 100-mm culture dishes to achieve a density of 60%-70% on the transfection day. For a cell culture dish with a bottom area of 100 mm, add a maximum volume of 24 μg of plasmid DNA to each dish. Dilute 80 μl of LIPOFECTAMINE 2000 reagent with 1.5 ml of serum-free medium. Mix it with the DNA within 5 minutes. Let the mixed plasmid DNA and LIPOFECTAMINE 2000 stand at room temperature for 20 minutes. Then add the above mixture evenly to the cells. Incubate at 37 °C, 5% CO2, and 100% saturated humidity for 6 hours. Add 12 ml of fresh DMEM medium containing 10% FBS to each culture dish and continue culturing. After 24 hours, replace the original old medium with fresh DMEM medium containing 10% FBS and continue culturing. Proteins and RNA can be extracted 48 hours after transfection. Collect the cells into a centrifuge tube and rinse the cells with PBS wash solution to remove the residual medium and cell debris in the culture dish; Add RIPA lysis buffer, shake for 15 s, then place on ice for 30 min. Centrifuge at 12,000×g for 30 min, collect the supernatant, and then purify it using a Flag purification kit to obtain the recombinant antigen GPIIb / IIIa complex.

[0042] Example 2.

[0043] Couple the recombinant antigen to magnetic beads. Taking the recombinant antigen GPIIb / IIIa complex as an example, coat the GPIIb / IIIa complex on the surface of carboxyl magnetic beads, including the following steps.

[0044] First, suspend the carboxyl magnetic beads in MES buffer at pH 5.0, add EDC and NHS, and react with shaking at room temperature for 30 minutes to activate the carboxyl groups to generate NHS esters; after magnetic separation, wash 3 times with cold MES buffer to remove the unreacted EDC / NHS; Dissolve NH2-PEG6000-COOH and mix it with the activated magnetic beads at a mass ratio of 5:1, and react with shaking at room temperature for 4 hours; after magnetic separation, wash 3 times with Tris buffer to remove the unbound PEG; Resuspend the PEG-modified magnetic beads in MES buffer with EDC and NHS to activate the -COOH groups at the end of PEG. After washing 3 times, replace it with Tris buffer containing Ca 2+ and add 0.5 mg / mL of the recombinant antigen GPIIb / IIIa complex and react for 2 hours.

[0045] After the reaction, block the unreacted active sites with 1% BSA, and finally wash with Tris buffer containing Ca 2+ and dilute it into a working solution of 0.2 mg / mL as the M reagent; Label alkaline phosphatase (AP) on the surface of anti-human antibody IgG and dilute it with MES buffer as the R2 reagent; Add 1% BSA as a stabilizer to Tris buffer as the R1 reagent; Calibrator & Quality Control: Use high-value human anti-GPIIb / IIIa antibody as the raw material for the calibrator; dilute the raw materials of the calibration quality control with Tris buffer at a certain ratio to 0, 5, 20, 50, 200, 400 RU / mL as the calibrator, which is used to calibrate the kit and calculate the concentration value of the sample to be tested.

[0046] Clinical sample verification: Use the whole set of kits to detect the samples with positive anti-platelet antibodies by solid-phase agglutination method. The detection results are shown in Table 1. Among 30 samples, 16 samples can be detected as positive.

[0047] Table 1. Detection results of GPIIb / IIIa antibody

[0048] Stability verification: Place the M reagent coated with recombinant antigen GPIIb / IIIa complex at 37 °C for heat destruction treatment and then detect 2 positive samples to investigate the accelerated stability of the M coupling process. Heat destruction at 37 °C for 1 day, 4 days, and 7 days can respectively represent the expiration stability at 4 °C for 1 month, 6 months, and 12 months. The results are shown in Figure 1 , after heat destruction at 37 °C for 1 day, 4 days, and 7 days, the signal retention rates of the M reagent are 99%, 95%, and 92% respectively. It can be seen that the M reagent prepared by the method of Example 2 for coating recombinant antigen GPIIb / IIIa complex has high stability.

[0049] Inter-batch coefficient of variation (CV) verification: Take 2 positive samples and detect them once in the morning and afternoon every day, with 4 replicates each time, for a total of 5 days, with 40 test numbers at each point. The results are shown in Table 2. Calculate that the CV is <5%, indicating good detection repeatability and stability.

[0050] Table 2. Verification results of inter-batch coefficient of variation (CV) of GPIIb / IIIa

[0051] Comparative Example 1.

[0052] The difference between this comparative example and Example 2 is that the Tris buffer containing Ca 2+ is replaced with pure Tris buffer, and the remaining steps remain unchanged.

[0053] Comparative Example 2

[0054] The difference between this comparative example and Example 2 is that the step of treatment with NH2-PEG6000-COOH is removed, and the remaining steps remain unchanged.

[0055] The detection rates and stability data of the M reagent of Comparative Examples 1-2 and Example 2 are compared as shown in Tables 3 and 4.

[0056] Table 3. Comparison of detection rates and titer data of the M reagent of Comparative Examples 1-2 and Example 2

[0057] Table 4. Comparison of stability data of the M reagent of Comparative Examples 1-2 and Example 2

[0058] It can be seen from the data in Tables 3 and 4 that when the M reagent is prepared by coating the recombinant antigen on the surface of the magnetic beads using the method of the present invention, the detection rate and the reagent stability are higher than those of the comparative examples. When the magnetic beads are not treated with NH2-PEG6000-COOH or without using the buffer solution containing Ca 2+ , the detection rate and stability decrease significantly.

[0059] Example 3

[0060] Coat HLA on the surface of tosyl (Tosyl) magnetic beads, and the steps are as follows: First, pretreat the magnetic beads with PBS buffer solution at pH 9.5. React 1 mg / mL of HLA antigen with Tosyl magnetic beads in an alkaline buffer solution at room temperature with shaking for 6 hours to form a thioether bond. Subsequently, block the residual active groups with 10 mM cysteine, wash with PBS buffer solution, and dilute to a working solution of 0.2 mg / mL as the M reagent; Label alkaline phosphatase (AP) on the surface of anti-human antibody IgG, and dilute with MES buffer solution to use as the R2 reagent; Add 1% BSA as a stabilizer in the PBS buffer solution to use as the R1 reagent; Calibrator & quality control product: Use high-value human anti-HLA antibody as the calibrator raw material; Dilute the raw materials of the calibration quality control product with Tris buffer solution at a certain ratio to 0, 5, 20, 50, 200, 400 RU / mL as the calibrator to calibrate the kit and calculate the concentration value of the test sample.

[0061] Use the whole set of kits to detect clinical samples that are positive for anti-platelet antibody detection by the solid-phase agglutination method. The detection results are shown in Table 5. Among 30 samples, 7 samples can be detected as positive.

[0062] Table 5. HLA antibody detection results

[0063] Stability verification: The M reagent coated with HLA antigen was placed at 37°C for heat destruction treatment, and then positive samples and quality control products were detected to investigate the accelerated stability of the M coupling process. Heat destruction at 37°C for 1 day, 4 days, and 7 days can respectively characterize the expiration stability at 4°C for 1 month, 6 months, and 12 months. The results are shown in Figure 2 , after heat destruction at 37°C for 1 day, 4 days, and 7 days, the signal retention rates of the M reagent were 101%, 98%, and 96% respectively.

[0064] Example 4 Magnetic beads coated with GPIa / IIa complex and GPIb / IX complex were obtained according to the method of Example 2 above.

[0065] This example provides a magnetic particle chemiluminescence-based anti-platelet antibody detection kit, including: M reagent: Coated with GPIIb / IIIa, GPIa / IIa, GPIb / IX, and HLA magnetic beads simultaneously and mixed according to the working solution concentration (0.2 mg / mL * 4); R2 reagent: Alkaline phosphatase (AP) was labeled on the surface of anti-human antibody IgG and diluted with MES buffer; R1 reagent: 1% BSA was added as a stabilizer in Tris buffer; Calibrator & quality control product: High-value human-derived anti-GPIIb / IIIa, GPIa / IIa, GPIb / IX, and HLA antibodies were used as the raw materials for the calibrator; the raw materials of the calibration quality control product were diluted with Tris buffer at a certain ratio to 0, 5, 20, 50, 200, 400 RU / mL as the calibrator, which was used to calibrate the kit and calculate the concentration value of the sample to be tested.

[0066] The whole set of the kit was used to detect clinical samples that were positive for anti-platelet antibody detection by the solid-phase agglutination method. The detection results are shown in Table 6, and all 30 samples could be detected.

[0067] Table 6. Detection results of the kit of the present invention

[0068] Negative coincidence rate: The kit of the present invention was used to measure 200 random healthy human physical examination samples. The results are as Figure 3 shown. The results indicate that the negative coincidence rate of the kit of the present invention is 99% (198 / 200), indicating that the kit of the present invention has high clinical specificity.

[0069] The amino acid sequences involved in the present invention are as follows.

[0070] SEQ ID NO.1: Amino acid sequence of GPIIb SEQ ID NO.2: Amino acid sequence of GPIIIa MRARPRPRPLWATVLALGALAGVGVGGPNICTTRGVSSCQQCLAVSPMCAWCSDEALPLGSPRCDLKENLLKDNCAPESIEFPVSEARVLEDRPLSDKGSGDSSQVTQVSPQRIALRLRPDDSKNFSIQVRQVEDYPVDIYYLMDLSYSMKDDLWSIQNLGTKLATQMRKLTSNLRIGFGAFVDKPVSPYMYISPPEALENPCYDMKTTCLPMFGYKHVLTLTDQVTRFNEEVKKQSVSRNRDAPEGGFDAIMQATVCDEKIGWRNDASHLLVFTTDAKTHIALDGRLAGIVQPNDGQCHVGSDNHYSASTTMDYPSLGLMTEKLSQKNINLIFAVTENVVNLYQNYSELIPGTTVGVLSMDSSNVLQLIVDAYGKIRSKVELEVRDLPEELSLSFNATCLNNEVIPGLKSCMGLKIGDTVSFSIEAKVRGCPQEKEKSFTIKPVGFKDSLIVQVTFDCDCACQAQAEPNSHRCNNGNGTFECGVCRCGPGWLGSQCECSEEDYRPSQQDECSPREGQPVCSQRGECLCGQCVCHSSDFGKITGKYCECDDFSCVRYKGEMCSGHGQCSCGDCLCDSDWTGYYCNCTTRTDTCMSSNGLLCSGRGKCECGSCVCIQPGSYGDTCEKCPTCPDACTFKKECVECKKFDRGALHDENTCNRYCRDEIESVKELKDTGKDAVNCTYKNEDDCVVRFQYYEDSSGKSILYVVEEPECPKGPDILVVLLSVMGAILLIGLAALLIWKLLITIHDRKEFAKFEEERARAKWDTANNPLYKEATSTFTNITYRGT SEQ ID NO.3: Amino acid sequence of GPIa SEQ ID NO.4: Amino acid sequence of GPIIa MNLQPIFWIGLISSVCCVFAQTDENRCLKANAKSCGECIQAGPNCGWCTNSTFLQEGMPTSARCDDLEALKKKGCPPDDIENPRGSKDIKKNKNVTNRSKGTAEKLKPEDITQIQPQQLVLRLRSGEPQTFTLKFKRAEDYPIDLYYLMDLSYSMKDDLENVKSLGTDLMNEMRRITSDFRIGFGSFVEKTVMPYISTTPAKLRNPCTSEQNCTSPFSYKNVLSLTNKGEVFNELVGKQRISGNLDSPEGGFDAIMQVAVCGSLIGWRNVTRLLVFSTDAGFHFAGDGKLGGIVLPNDGQCHLENNMYTMSHYYDYPSIAHLVQKLSENNIQTIFAVTEEFQPVYKELKNLIPKSAVGTLSANSSNVIQLIIDAYNSLSSEVILENGKLSEGVTISYKSYCKNGVNGTGENGRKCSNISIGDEVQFEISITSNKCPKKDSDSFKIRPLGFTEEVEVILQYICECECQSEGIPESPKCHEGNGTFECGACRCNEGRVGRHCECSTDEVNSEDMDAYCRKENSSEICSNNGECVCGQCVCRKRDNTNEIYSGKFCECDNFNCDRSNGLICGGNGVCKCRVCECNPNYTGSACDCSLDTSTCEASNGQICNGRGICECGVCKCTDPKFQGQTCEMCQTCLGVCAEHKECVQCRAFNKGEKKDTCTQECSYFNITKVESRDKLPQPVQPDPVSHCKEKDVDDCWFYFTYSVNGNNEVMVHVVENPECPTGPDIIPIVAGVVAGIVLIGLALLLIWKLLMIIHDRREFAKFEKEKMNAKWDTGENPIYKSAVTTVVNPKYEGK SEQ ID NO.5: Amino acid sequence of GPIb MPLLLLLLLLPSPLHPHPICEVSKVASHLEVNCDKRNLTALPPDLPKDTTILHLSENLLYTFSLATLMPYTRLTQLNLDRCELTKLQVDGTLPVLGTLDLSHNQLQSLPLLGQTLPALTVLDVSFNRLTSLPLGALRGLGELQELYLKGNELKTLPPGLLTPTPKLEKLSLANNNLTELPAGLLNGLENLDTLLLQENSLYTIPKGFFGSHLLPFAFLHGNPWLCNCEILYFRRWLQDNAENVYVWKQGVDVKAMTSNVASVQCDNSDKFPVYKYPGKGCPTLGDEGDTDLYDYYPEEDTEGDKVRATRTVVKFPTKAHTTPWGLFYSWSTASLDSQMPSSLHPTQESTKEQTTFPPRWTPNFTLHMESITFSKTPKSTTEPTPSPTTSEPVPEPAPNMTTLEPTPSPTTPEPTSEPAPSPTTPEPTSEPAPSPTTPEPTSEPAPSPTTPEPTPIPTIATSPTILVSATSLITPKSTFLTTTKPVSLLESTKKTIPELDQPPKLRGVLQGHLESSRNDPFLHPDFCCLLPLGFYVLGLFWLLFASVVLILLLSWVGHVKPQALDSGQGAALTTATQTTHLELQRGRQVTVPRAWLLFLRGSLPTFRSSLFLWVRPNGRVGPLVAGRRPSALSQGRGQDLLSTVSIRYSGHSL SEQ ID NO.6: Amino acid sequence of GPⅨ MPAWGALFLLWATAEATKDCPSPCTCRALETMGLWVDCRGHGLTALPALPARTRHLLLANNSLQSVPPGAFDHLPQLQTLDVTQNPWHCDCSLTYLRLWLEDRTPEALLQVRCASPSLAAHGPLGRLTGYQLGSCGWQLQASWVRPGVLWDVALVAVAALGLALLAGLLCATTEALD SEQ ID NO.7: Amino acid sequence of HLA-A MAVMAPRTLLLLLSGALALTQTWAGSHSMRYFFTSVSRPGRGEPRFIAVGYVDDTQFVRFDSDAASQRMEPRAPWIEQEGPEYWDQETRNVKAQSQTDRVDLGTLRGYYNQSEAGSHTIQIMYGCDVGSDGRFLRGYRQDAYDGKDYIALNEDLRSWTAADMAAQITKRKWEAAHEAEQLRAYLDGTCVEWLRRYLENGKETLQRTDPPKTHMTHHPISDHEATLRCWALGFYPAEITLTWQRDGEDQTQDTELVETRPAGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPKPLTLRWELSSQPTIPIVGIIAGLVLLGAVITGAVVAAVMWRRKSSDRKGGSYTQAASSDSAQGSDVSLTACKV。

[0071] Finally, it should be noted that the above specific embodiments are only used to elaborate in detail the purpose, technical solutions and beneficial effects of the present invention. It should be understood that the above description is only the specific implementation manners of the present invention and is not used to limit the protection scope of the present invention; although the present invention has been described in detail with reference to the foregoing specific embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions recorded in the foregoing embodiments, or perform equivalent replacements or improvements on some or all of the technical features; and these modifications, equivalent replacements and improvements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present invention, and they should all be covered by the scope of the claims and the description of the present invention.

Claims

1. A kit for detecting anti - platelet antibodies based on magnetic particle chemiluminescence, characterized in that, Comprising: Reagent M, comprising magnetic microparticles co-coated with recombinant antigen proteins GPIIb / IIIa complex, GPIa / IIa complex, GPIb / IX complex, HLA antigen, and a magnetic microparticle protective solution containing Ca 2+ ; R1 reagent, including a buffer solution; R2 reagent, including an anti-human IgG antibody labeled with an enzyme or a luminescent agent; Wherein, the magnetic particles are processed as follows: Add EDC, NHS and NH2-PEG6000-COOH to the magnetic particles and oscillate and react for more than 4 h.

2. The anti - platelet antibody detection kit based on magnetic particle chemiluminescence according to claim 1, wherein, The magnetic particles are specifically processed as follows: Add EDC and NHS to the magnetic particles and oscillate and react for 30-60 min, then add NH2-PEG6000-COOH and oscillate and react for 4-5 h, and finally add EDC and NHS again and oscillate and react for 30-60 min.

3. The anti - platelet antibody detection kit based on magnetic particle chemiluminescence according to claim 2, wherein, The magnetic particles specifically include the following steps: Suspend the magnetic particles in MES buffer solution, add EDC and NHS and oscillate and react for 30-60 min. After magnetic separation, wash with MES buffer solution multiple times to remove unreacted EDC and NHS; Then add NH2-PEG6000-COOH and oscillate and react for 4-5 h. After magnetic separation, wash with Tris buffer solution multiple times to remove unbound NH2-PEG6000-COOH; Finally, add EDC and NHS again and oscillate and react for 30-60 min. After magnetic separation, wash with MES buffer solution multiple times to remove unreacted EDC and NHS.

4. The anti - platelet antibody detection kit based on magnetic particle chemiluminescence according to claim 1, characterized in that, The mass ratio of NH2-PEG6000-COOH to the magnetic particles is (4-6):

1.

5. The anti - platelet antibody detection kit based on magnetic particle chemiluminescence according to claim 1, wherein, The Ca-containing 2+ magnetic particle protective solution is a Tris buffer solution containing Ca 2+ .

6. A kit for detecting anti - platelet antibodies based on magnetic particle chemiluminescence according to any one of claims 1 - 5, characterized in that, The magnetic particles are selected from any one or more of streptavidin magnetic beads, carboxyl magnetic beads, and tosyl magnetic beads.

7. A kit for detecting anti - platelet antibodies based on magnetic particle chemiluminescence according to any one of claims 1 - 5, characterized in that, The R1 reagent is a Tris buffer solution added with a stabilizer.

8. A kit for detecting anti - platelet antibodies based on magnetic particle chemiluminescence according to any one of claims 1 - 5, characterized in that, The enzyme for labeling is selected from alkaline phosphatase or horseradish peroxidase; the luminescent agent is selected from any one of acridinium ester, isoluminol, and ruthenium tris(bipyridyl).

9. The anti - platelet antibody detection kit based on magnetic particle chemiluminescence according to claim 1, wherein, The recombinant antigen proteins GPIIb / IIIa complex, GPIa / IIa complex, and GPIb / IX complex are all prepared by the following method: PCR amplify the target gene, recover the PCR product, ligate the PCR product with a dicistronic vector, then amplify through Escherichia coli, extract the plasmid, transfect it into HEK293t cells for expression, and finally extract and purify.

10. Use of the anti - platelet antibody detection kit based on magnetic particle chemiluminescence according to any one of claims 1 - 9, characterized in that, The kit is detected by an automatic chemiluminescence detection system; Before detection, incubate and react the M reagent, R1 reagent and the serum sample to be detected for 8-15 min, then add the R2 reagent and incubate and react for 8-15 min. After washing, add a luminescent substrate or an activator.

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