Method and kit for determining platelet antibody and cross matching type by chemiluminescence method

Abstract

The invention relates to the field of biological detection, and particularly discloses a method and a kit for determining platelet antibodies and cross-matching types by a chemiluminescence method. The detection method comprises the following steps: firstly, adding tool platelets / donor platelets into a reaction hole, adding a sample to be detected and magnetic bead particles coated with platelet antibodies into the reaction hole, and incubating to obtain an immune complex; mixing the immune complex with an acridinium ester labeled anti-human IgG antibody, and incubating to obtain a reaction complex; and mixing the reaction compound with a pre-excitation liquid and an excitation liquid of acridinium ester in sequence, carrying out a chemiluminescence reaction, and calculating the concentration of the platelet antibody in the sample to be detected. The kit not only can be used for effectively screening platelet antibodies in a to-be-detected plasma sample, but also can be used for carrying out cross-matching detection before platelet infusion, so that a more accurate basis is provided for evaluating the platelet infusion effect and formulating a treatment scheme.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biological detection, more specifically, it relates to a method and kit for determining platelet antibodies and cross matching by chemiluminescence. BACKGROUND

[0002] Currently, platelet transfusion refractoriness (PTR), autoimmune thrombocytopenia, and neonatal alloimmune thrombocytopenia (NAIT) are not uncommon in clinical practice. Both immune and non-immune factors can induce PTR. With the widespread use of leukocyte-reduced blood products, the incidence of immune PTR is usually less than 10%. Among patients receiving platelet transfusion, the incidence of immune PTR is about 4% to 8%, which hinders effective platelet transfusion in clinical practice. The mortality rate of neonatal alloimmune thrombocytopenia is about 10%, and about 25% or more of children have neurological complications.

[0003] The detection of platelet antibodies is of great significance in clinical medicine, especially in the prevention and management of platelet transfusion refractoriness (PTR), autoimmune thrombocytopenia, and neonatal alloimmune thrombocytopenia (NAIT). Effective diagnosis and treatment of these conditions rely on high-precision platelet antibody screening and matching detection. In recent years, with the advancement of biological detection technology, various enzyme-linked immunosorbent assays (ELISA), flow cytometry, and other immunological methods have gradually become popular, driving the development of precision medicine for platelet-related diseases. However, there are still many challenges in actual operation, and how to improve the detection efficiency and accuracy has become a core problem that needs to be solved.

[0004] The main problem existing in the prior art is that the traditional detection method cannot simultaneously meet the requirements of high efficiency, simplicity, and high sensitivity, especially when a quick result is needed. At the same time, in the process of screening platelet antibodies, the existing technology cannot perform cross matching detection before platelet transfusion, making it difficult to effectively prevent the occurrence of platelet transfusion refractoriness. SUMMARY

[0005] To solve the above problems, the present application provides a method and kit for determining platelet antibodies and cross matching by chemiluminescence. The kit can be used with a fully automatic chemiluminescence immunoassay analyzer, which not only can effectively screen platelet antibodies in the sample to be tested, but also can perform cross matching detection before platelet transfusion, thereby assisting in the diagnosis of autoimmune thrombocytopenia, neonatal alloimmune thrombocytopenia (NAIT), and platelet transfusion refractoriness, and providing more accurate basis for the evaluation of platelet transfusion effect and the formulation of treatment plan.

[0006] The present application adopts the following technical solutions: In a first aspect, the present application provides a method for determining platelet antibody and cross matching by chemiluminescence, comprising: (1) adding tool platelet / donor platelet to a reaction well, adding a sample to be tested and magnetic bead particles coated with platelet antibody in the reaction well, incubating at 35-39℃ for 22-26 min to obtain an immune complex with the structure of magnetic bead particles-platelet antibody-tool platelet / donor platelet-receptor platelet antibody; (2) mixing the washed immune complex with acridinium ester-labeled anti-human IgG antibody, incubating at 35-39℃ for 10-14 min to obtain a reaction complex with the structure of magnetic bead particles-platelet antibody-tool platelet / donor platelet-receptor platelet antibody-anti-human IgG antibody-acridinium ester; (3) mixing the washed reaction complex with pre-priming solution and priming solution of acridinium ester in sequence to perform chemiluminescence reaction, and calculating the concentration of platelet antibody in the sample to be tested by measuring the number of photons generated in the reaction.

[0007] By adopting the above technical solution, the platelet antibody in the sample to be tested can be effectively screened, and cross matching detection can be performed before platelet transfusion. Specifically, when the platelet antibody is effectively screened, the tool platelet is selected as the "connecting bridge", and the immune complex is formed by the magnetic bead particles coated with platelet antibody and the tool platelet and the receptor platelet antibody in the sample to be tested, so that the target antibody can be specifically captured. When the cross matching detection is performed before the platelet transfusion, multiple donor platelets can be selected for adaptation. The reaction complex formed after the further combination of the anti-human IgG antibody labeled with acridinium ester releases quantifiable light signals in the chemiluminescence reaction process, and the highly sensitive detection of the platelet antibody concentration is realized. If the chemiluminescence signal is weak, it indicates that there is no significant immune reaction between the donor platelet and the receptor platelet, and the matching is successful. Otherwise, it indicates that there is a greater risk of ineffective transfusion. This design not only can accurately capture low-concentration platelet antibodies, but also has high specificity, avoids non-specific interference, and thus ensures the reliability of the detection results. In addition, the whole process is simple and time-saving, and is suitable for rapid screening of platelet antibodies and related matching conditions in a clinical environment, and provides strong support for preventing ineffective platelet transfusion.

[0008] Further, the detection limit of the platelet antibody in the sample to be tested by the above method is 1 Ru / mL.

[0009] By adopting the above technical solution, the detection limit is 1 Ru / mL, which can significantly improve the sensitivity and accuracy of detection. This limit enables the method to effectively identify the presence of platelet antibodies in a lower concentration range, thereby better meeting the clinical demand for high-precision detection. In particular, when determining platelet transfusion compatibility, a clear detection limit helps to reduce the probability of false negative results and improves the success rate of cross matching.

[0010] Further, when the method is used to determine the platelet antibody cross matching between the blood donor and the blood recipient, the determination criteria are: If the concentration value of platelets in the sample to be tested is < 1 Ru / mL, it is determined to be negative, and the matching is successful. If the concentration value of platelets in the sample to be tested is ≥ 1 Ru / mL, it is determined to be positive, and the matching fails.

[0011] By adopting the above technical solution, accurate determination of platelet antibody cross matching between blood donors and recipients can be achieved. Specifically, when the concentration value of platelets in the sample to be tested is less than 1 Ru / mL, it indicates that there is no significant immune response, so it is determined to be negative and the matching is successful. Conversely, if the concentration value of platelets in the sample to be tested is greater than or equal to 1 Ru / mL, it indicates that there is a potential risk of immune rejection, so it is determined to be positive and the matching fails. This method not only improves the accuracy of the detection results, but also simplifies the operation process, which helps to quickly screen suitable platelet donors in the clinic and reduce the risk of ineffective platelet transfusion.

[0012] Further, the preparation method of the magnetic bead microparticles coated with platelet antibodies comprises: Take the magnetic beads, vortex to separate and remove the supernatant, and then wash. Add platelet antibodies and disperse in coating buffer, mix well and incubate at 35-40°C for 12-36h. After incubation, remove the supernatant, wash the magnetic beads, and re-disperse in storage solution I. After blocking incubation, it is obtained.

[0013] By adopting the above technical solution, the preparation method of the magnetic bead microparticles coated with platelet antibodies enables the magnetic bead surface to uniformly and stably bind platelet antibodies. This method controls the incubation temperature and time to ensure sufficient reaction between platelet antibodies and magnetic beads, thereby improving the efficiency and stability of immune complex formation. In addition, the magnetic bead microparticles after blocking treatment reduce the possibility of non-specific binding, improving the signal-to-noise ratio and specificity of the detection system. Ultimately, this method lays the foundation for subsequent efficient platelet antibody capture and detection, significantly enhancing the sensitivity and reliability of the entire determination system.

[0014] Further, the mass ratio of platelet antibodies to magnetic beads is (15-25) μg: 1 mg.

[0015] By adopting the technical scheme, the mass ratio of the platelet antibody and the magnetic beads is optimized to (15-25) μg: 1 mg, so that the magnetic beads surface is fully covered by the platelet antibody while avoiding the steric hindrance effect caused by excessive antibodies, thereby improving the efficiency and stability of the immune complex formation. The optimization significantly improves the capture capacity of the detection system for the target platelet antibody, enhances the sensitivity and specificity of the detection, and ensures the reliability of the detection result.

[0016] Further, the preparation method of the acridinium ester-labeled anti-human IgG antibody is as follows: The anti-human IgG antibody to be labeled and acridinium ester are mixed and dispersed in a labeling buffer according to a mass ratio of 1 mg:(25-35) μl, and then the mixture is placed in a constant-temperature and constant-humidity box at 35-37°C under light-proof conditions for 2-2.5 h of reaction, thereby obtaining an acridinium ester-labeled anti-human IgG antibody mixture; Subsequently, the obtained acridinium ester-labeled anti-human IgG antibody mixture is placed in an ultrafiltration tube for ultrafiltration to remove unreacted acridinium ester, thereby obtaining the acridinium ester-labeled anti-human IgG antibody.

[0017] By adopting the technical scheme, the preparation process of the acridinium ester-labeled anti-human IgG antibody is optimized, so that the labeling reaction is fully carried out under light-proof and suitable temperature conditions, and the labeling efficiency and uniformity are effectively improved. The acridinium ester-labeled anti-human IgG antibody prepared by the method has higher purity, and non-specific impurities are effectively removed, thereby significantly improving the sensitivity and specificity of the subsequent chemiluminescence detection. Specifically, the scheme enables the detection system to more accurately identify low-concentration platelet antibodies and reduce the false positive rate, thereby providing reliable protection for the safety and effectiveness of clinical platelet transfusion.

[0018] In a second aspect, the application provides a kit for determining platelet antibodies and cross matching by a chemiluminescence method, and the determination method adopts the kit, which comprises reagent R1, reagent R2, reagent R3 and reagent R5. The reagent R1 is magnetic bead particles coated with platelet antibodies, the magnetic bead particles are dispersed in a preservative solution I containing BSA and a preservative, and the concentration of the magnetic bead particles is 1-2 mg / mL. The reagent R2 is acridinium ester-labeled anti-human IgG antibody, the anti-human IgG antibody is dispersed in a preservative solution II containing sodium chloride and potassium chloride, and the concentration of the anti-human IgG antibody is 100-120 ng / mL. The reagent R3 is a buffer solution containing BSA and sodium chloride. The reagent R5 is a sample diluent.

[0019] The preparation of reagent R3: Tris, sodium chloride, BSA are weighed and put into a beaker, and then dissolved in ultrapure water, and then transferred into a centrifuge tube to obtain the reagent R3. The preparation of reagent R5: disodium hydrogen phosphate, potassium dihydrogen phosphate, sodium chloride are weighed and put into a beaker, and then dissolved in ultrapure water, and then transferred into a centrifuge tube to obtain the reagent R5.

[0020] By adopting the above technical solutions, the components in the kit cooperate with each other to realize efficient and accurate detection of platelet antibodies and cross matching. Specifically, the magnetic bead particles coated with platelet antibodies (reagent R1) are used as a solid carrier, and a specific antibody for platelets is coated on the magnetic bead particles. This highly specific binding can accurately capture the target platelet antibodies in the sample and reduce the background noise signal caused by non-specific binding. The aridin ester is used as a luminescent label, which has high sensitivity, good stability, and strong luminescent intensity, and can produce strong chemiluminescence signal under excitation conditions, thereby improving the sensitivity of detection. The buffer solution (reagent R3) provides suitable environmental conditions to ensure the stability and repeatability of the reaction system. The sample diluent (reagent R5) is used to process the sample to be tested, which optimizes the sample concentration range and ensures the accuracy of the detection results.

[0021] The optimized concentration of magnetic bead particles (1-2 mg / ml) ensures sufficient capture area and stability, neither too dense to cause a decrease in capture efficiency, nor too sparse to affect detection sensitivity. The concentration of aridin ester labeled anti-human IgG antibody (100-120 ng / ml) is also carefully optimized to achieve a balance between optimal signal strength and detection sensitivity.

[0022] The kit is used with a full-automatic chemiluminescence immunoassay analyzer to realize a full-automatic process from sample processing to result output. Not only can it detect platelet antibodies, but also can complete the cross matching detection of platelets on the instrument. The on-machine detection improves the detection efficiency and reduces the errors caused by human operation, thereby ensuring the accuracy and repeatability of the detection results. This comprehensive detection capability provides more information for the clinic, which helps doctors more accurately judge the types of diseases such as thrombocytopenia and platelet transfusion ineffective, the severity of the disease, and the overall coverage of the prognosis.

[0023] Further, the above-mentioned storage solution I is a phosphate buffer solution with a pH of 7.5-8.5, wherein the concentration of BSA is 8-12 mg / mL, and the concentration of preservative is 0.02-0.1 wt%.

[0024] By adopting the technical scheme, the composition and parameter range of the preservation solution I are optimized, so that the magnetic bead particles coated with platelet antibodies can be stably stored in a suitable environment, and the active time is effectively prolonged. The BSA concentration is limited to 8-12 mg / mL, which can play a good protein protection role and reduce the risk of inactivation of antibody molecules due to aggregation or denaturation; the preservative concentration is controlled in 0.02-0.1 wt%, which helps to inhibit the growth and reproduction of microorganisms and ensure the safety and effectiveness of long-term use.

[0025] Preferably, the preservation solution II is a phosphate buffer solution with a pH of 7.8-8.2, wherein the concentration of sodium chloride contained is 14-18 mg / mL; the concentration of potassium chloride contained is 0.2-0.6 mg / mL.

[0026] By adopting the technical scheme, the composition and parameter range of the preservation solution I are optimized, so that the magnetic bead particles coated with platelet antibodies can be stably stored in a suitable environment, and the active time is effectively prolonged. The BSA concentration is limited to 8-12 mg / mL, which can play a good protein protection role and reduce the risk of inactivation of antibody molecules due to aggregation or denaturation; the preservative concentration is controlled in 0.02-0.1 wt%, which helps to inhibit the growth and reproduction of microorganisms and ensure the safety and effectiveness of long-term use.

[0027] In a third aspect, the application provides a use of the above-mentioned kit in platelet antibody and cross matching detection, which comprises: (1) taking a sample to be tested, diluting it with reagent R5, and then adding it to a reaction well containing tool platelets / donor platelets together with reagent R1, incubating at 35-39°C for 22-26 min to obtain an immune complex; (2) mixing the washed immune complex with reagent R2 and reagent R3, incubating at 35-39°C for 10-14 min to obtain a reaction complex; (3) mixing the washed reaction complex with the pre-priming solution and priming solution of acridinium ester, performing a chemiluminescence reaction, measuring the number of photons generated in the reaction, and calculating the concentration of platelet antibodies in the sample to be tested.

[0028] Further, the volume ratio of the above-mentioned reagent R1, reagent R2, reagent R3 and reagent R5 is 1:(2-4):(2-4):(11-12).

[0029] By adopting the technical scheme, the reagent proportion is optimized to achieve the best synergistic effect among the components. Specifically, adjusting the volume ratio of the reagent R1, the reagent R2, the reagent R3 and the reagent R5 to 1:(2-4):(2-4):(11-12) can significantly improve the stability and sensitivity of the detection system. This ratio setting ensures that the magnetic bead particles coated with platelet antibodies can fully bind to the recipient platelet antibodies in the sample to be tested, and at the same time, the acridinium ester-labeled anti-human IgG antibody can also participate in the subsequent reaction efficiently, thereby maximizing the amplification of the chemiluminescence signal. Ultimately, more accurate quantitative analysis of the platelet antibody concentration is achieved, and the accuracy of the cross matching judgment is improved.

[0030] In summary, the present application has the following beneficial effects: 1. The present application uses magnetic bead particles coated with platelet antibodies to form an immune complex with tool platelets / donor platelets and recipient platelet antibodies in the sample to be tested, significantly improving the specificity and sensitivity of the detection, and accurately capturing low-concentration platelet antibodies; the introduction of acridinium ester-labeled anti-human IgG antibody into the reaction generates quantifiable light signals, making the detection process more stable and reliable, and achieving highly precise measurement of the platelet antibody concentration. Using the detection method and kit provided by the present application, not only can platelet antibodies be detected, but also cross matching detection before platelet transfusion can be performed, which provides a more comprehensive and accurate basis for the diagnosis of autoimmune thrombocytopenia and ineffective platelet transfusion, the evaluation of platelet transfusion effect and the development of treatment plan, and helps doctors to more accurately judge the disease and develop personalized treatment plans.

[0031] 2. In the prior art, the detection method in which platelet antigens are coated on magnetic beads mainly detects platelet antibodies according to the direct formation of a complex between platelet antigens and platelet antibodies in the sample to be tested. This method has the following disadvantages: first, it cannot be used for detecting and matching donor platelets and recipient platelets; second, the sensitivity is low, and it is difficult to meet the needs of precise detection of low-concentration platelet antibodies.

[0032] 3. The kit product of the present application is designed as independent packaging, single detection, and machine detection, which is simple and convenient to operate, can meet the instant detection needs of small amounts of scattered samples in clinical practice, improves the detection efficiency, and can complete the determination and cross matching analysis of platelet antibodies in a short time, greatly improving the clinical detection efficiency and providing a strong guarantee for preventing ineffective platelet transfusion. DETAILED DESCRIPTION

[0033] The embodiments of the present invention will be described in detail below with reference to the examples. However, those skilled in the art will understand that the following examples are only used to illustrate the present invention and should not be construed as limiting the scope of the present invention. Specific conditions not specified in the examples are carried out according to conventional conditions or conditions recommended by the manufacturer. Reagents or instruments used without indicating the manufacturer are all conventional products that can be purchased commercially.

[0034] The raw materials used in this embodiment are all commercially available materials unless otherwise specified in Table 1.

[0035] Table 1. Raw material Brand and manufacturer Purpose of use Specification Platelet antibody BIOVU Coagulant 5 mg / mL Magnetic bead Bio-Rad Indicator 2.5％ Acridine ester MEKATE Indicator 2％ Pre-priming solution Makoto Indicator 15 mL / bottle Priming solution Makoto Indicator 15 mL / bottle The following is a detailed description of the specific embodiments of the present invention. It should be understood that the specific embodiments described herein are only used to illustrate and explain the present invention and are not intended to limit the present invention.

[0036] First Preparation Example Preparation Example 1 This preparation example provides a platelet antibody-coated magnetic bead microparticle (reagent R1), the preparation method of which includes: (1) Prepare Preservation Solution I: Weigh 7.4 g Na2HPO4, 1 g NaH2PO4, and 8 g NaCl into a beaker, add 800 ml ultrapure water, and stir. Adjust the pH of the solution in the beaker to 8.0 with 1 M HCl solution. Weigh 10 g BSA and add 1 mL Proclin-300 to the solution. Mix thoroughly and dilute to 1000 ml to obtain Preservation Solution I. (2) When coating platelet antibodies on magnetic beads, the following steps are taken: 1. Magnetic bead washing: (1) Mix the magnetic beads until they are not sticky to the wall. Take a 2 mL centrifuge tube and pipette 30 mg of magnetic beads into each centrifuge tube. Place the rest back in the 4 °C refrigerator. (2) Vortex the magnetic beads for 30 seconds, place the centrifuge tube on a magnetic stand to separate the magnetic beads, and remove the supernatant; (3) Add 1 ml of coating buffer, vortex for 30 seconds, and clean the beads on an ultrasonic cleaner at 7% ultrasonic power for 1 minute. Place the beads on a magnetic stand and wait for 1 minute before removing the supernatant. (4) Vortex and wash the beads twice with the coating buffer prepared above, and remove the supernatant.

[0037] 2. Magnetic bead coating (1) Add the coating buffer to the above centrifuge tube, add 0.6 mg of platelet antibody (the concentration of platelet antibody is 20 μg / mg magnetic beads), vortex mix, and then add the catalyst and vortex mix evenly; The preparation method of the catalyst is as follows: 120 mL of ultrapure water is measured with a 500 mL graduated cylinder, poured into a 500 mL beaker, and stirred on a magnetic stirrer; 60 g of ammonium sulfate is weighed and completely dissolved in the beaker; the pH meter is calibrated, and the solution in the beaker is adjusted to pH 9.5 with 1M NaOH solution; the solution in the beaker is poured into a 500 mL graduated cylinder, diluted to 200 mL, poured into a bottle, and stored in a 2-8°C refrigerator; The preparation method of the coating buffer is as follows: 400 mL of ultrapure water is measured with a 500 mL graduated cylinder, poured into a 500 mL beaker, and stirred on a magnetic stirrer; 1 g of boric acid is weighed and completely dissolved in the beaker; the pH meter is calibrated, and the solution in the beaker is adjusted to pH 8.0 with 1M NaOH solution; the solution in the beaker is poured into a 500 mL graduated cylinder, diluted to 500 mL; poured into a bottle, and stored in a 2-8°C refrigerator.

[0038] (2) Incubate at 37°C for 24 hours in a rotary mixer at 40 rpm.

[0039] 3. Magnetic bead sealing (1) Take out the centrifugal tube, adsorb the magnetic beads, and remove the supernatant; (2) Wash the magnetic beads three times with 1 mL of storage solution I each time; (3) Add 1 mL of storage solution I to the centrifugal tube and incubate at 37°C with rotation (40 rpm).

[0040] 4. Magnetic bead washing and storage (1) Take out the magnetic beads, adsorb the magnetic beads, and remove the supernatant; (2) Wash three times with storage solution I and remove the supernatant.

[0041] (3) Dilute with storage solution I in the test tube, seal and store, and the concentration of the coated platelet antibody magnetic bead particles in storage solution I is 1 mg / ml, which is reagent R1.

[0042] Preparation Example 2 The difference between this preparation example and Preparation Example 1 is that the concentration of platelet antibody is 15 μg / mg of magnetic beads.

[0043] Preparation Example 3 The difference between this preparation example and Preparation Example 1 is that the concentration of platelet antibody is 25 μg / mg of magnetic beads.

[0044] Second Preparation Example Preparation Example 4 This preparation example provides an acridinium ester-labeled anti-human IgG antibody (reagent R2), and the preparation method comprises: 1. Prepare storage solution II: accurately weigh 0.024 g of potassium dihydrogen phosphate, 0.014 g of sodium phosphate dibasic, 0.8 g of sodium chloride, and 0.8 g of potassium chloride 0.02 g is put into a 100 ml beaker, 50 mL of ultrapure water is measured with a 100 ml measuring cylinder and added to the beaker to dissolve the solid, the solution is transferred to a 50 ml centrifuge tube, and stored in a 2-8 °C refrigerator to obtain storage solution II.

[0045] 2. The acridinium ester labeled anti-human IgG antibody is prepared using the following steps: 1. Take the materials: Take 80 ul of storage solution II, 2 ul of acridinium ester, and 0.1 mg of anti-human IgG antibody and add them to a centrifuge tube, wrap it with tin foil, and place it in a 37 °C constant temperature and humidity box for 2 hours of reaction.

[0046] 2. Ultrafiltration to remove free acridinium ester: Use a 50KD ultrafiltration tube to remove unreacted acridinium ester by ultrafiltration, add 0.2 mL of storage solution II, centrifuge at 8000 rpm for 5 min at 4 °C, and remove the ultrafiltration solution; repeat the above steps twice, and transfer the labeled antibody to a new centrifuge tube.

[0047] Dilute the labeled antibody with storage solution II, and the concentration of acridinium ester labeled anti-human IgG antibody in storage solution II is 100 ng / mL, and R2 is obtained.

[0048] Preparation Example 5 The difference between this preparation example and Preparation Example 4 is that the acridinium ester is 2.5 ul and the anti-human IgG antibody is 0.1 mg.

[0049] Preparation Example 6 The difference between this preparation example and Preparation Example 4 is that the acridinium ester is 1.5 ul and the anti-human IgG antibody is 0.1 mg. Example

[0050] Example 1 This example provides a kit for chemiluminescence determination of platelet antibodies and cross matching and its application, which is composed of reagent R1, reagent R2, reagent R3, and reagent R5, wherein reagent R1 is provided by Preparation Example 1, reagent R2 is provided by Preparation Example 4; The preparation method of reagent R3 is as follows: accurately weigh 3 g of Tris, 0.8 g of sodium chloride, and 1 g of BSA into a 100 ml beaker, add 50 mL of ultrapure water measured with a 100 ml measuring cylinder to the beaker to dissolve the solids, transfer the solution to a 50 ml centrifuge tube, and store it in a 2-8 °C refrigerator; The reagent R5 is prepared as follows: 800 mL of ultrapure water is measured with a 1000 mL measuring cylinder, poured into a 1000 mL beaker, and stirred on a magnetic stirrer; 2.5 g of Na2HPO4, 12.3 g of NaH2PO4, and 20 g of NaCl are weighed and completely dissolved in the beaker; the pH meter is calibrated, and the pH of the solution in the beaker is adjusted to 6.5 with a 1M HCl solution; 1 mL of Proclin-300 is measured with a pipette and added to the solution and mixed evenly; the solution in the beaker is poured into a 1000 mL measuring cylinder, and the volume is made up to 1000 mL, poured into a bottle, and stored in a 2-8℃ refrigerator.

[0051] The application method of the kit in determining platelet antibodies is as follows (cooperating with a full-automatic chemiluminescence immunoassay analyzer to perform sample testing): (1) The reagent card in the kit is taken out, tool platelets are added to the reaction wells of the reagent card, the reagent card is placed in the reagent card drawer, and the drawer is closed; (2) The sample to be tested is opened and placed on the sample holder, and the sample holder is placed in the machine; The tool platelets are three-person proportionally mixed O-type platelet suspensions, which are diluted to a concentration of 100 x 10 9 / L by a diluent, stored in an ep tube, and stored at 20-25℃ and detected within 8 hours.

[0052] (3) The test type is selected as “sample” on the test application interface, sample information is input, a test item is selected to apply for testing; (4) Click to start, and start testing; the reagent consumption required for each test is R1 50 μL, R2 100 μL, R3 100 μL, and R5 590 μL; the instrument automatically completes the sampling and mixing of the sample and the reagent R5, and then the diluted sample is taken to the R1 well to sample the magnetic beads, placed in the reaction well, mixed with the tool platelets in the reaction well, washed and mixed, and incubated at 37℃ for 24 min; after the incubation is completed, the immune complex is washed; (5) After washing, R2 and R3 are added, and incubated at 37℃ for 12 min; after the incubation is completed, the immune complex is washed; after washing, the pre-priming solution is added to the immune complex and incubated for 1 min; about 5 s after the priming solution is added, the luminescence signal value is detected; the time from sampling to the result is about 50 min.

[0053] Example 2 The present embodiment provides a kit for chemiluminescence determination of platelet antibodies and cross matching, and application thereof The composition of the kit is consistent with that of Example 1; The application method of the kit in determining platelet antibody cross matching is as follows: (1) Take out the reagent card in the kit, add donor platelets into the reaction hole of the reagent card, and then put the reagent card into the reagent card drawer and close the drawer; (2) After opening the cap of the sample to be matched, place it on the sample rack, and put the sample rack into the machine; (3) Select the test type as "sample" in the test application interface, input the sample information, select the test item to apply for the test; (4) Click start to begin the test, and the reagent consumption required for each test is R1 50 μL, R2 100 μL, R3 100 μL, and R5 590 μL; the instrument automatically completes the sample and reagent R5 suction and mixing, and then takes the diluted sample to the R1 hole to suck the magnetic beads, and places them into the reaction hole to mix with the donor platelets in the reaction hole, washes and mixes, and incubates at 37°C for 24 min; after the incubation is completed, the immune complex is washed; (5) After washing, add R2 and R3, and incubate at 37°C for 12 min; after the incubation is completed, the immune complex is washed, and after washing, the pre-priming solution is added to incubate with the immune complex for 1 min, and then the priming solution is added about 5 s later to detect the luminescence signal value; the time from sampling to result is about 50 min.

[0054] Example 3 The present embodiment provides a kit for determining platelet antibodies and cross matching by chemiluminescence method, which is different from example 1 in that: the reagent R1 is provided by preparation example 2, and the reagent R2 is provided by preparation example 5.

[0055] Example 4 The present embodiment provides a kit for determining platelet antibodies and cross matching by chemiluminescence method, which is different from example 1 in that: the reagent R1 is provided by preparation example 3, and the reagent R2 is provided by preparation example 4.

[0056] Example 5 The present embodiment provides an application of a kit for determining platelet antibodies and cross matching by chemiluminescence method, which is different from example 2 in that: in step (4), the diluted sample to be tested and the reagent R1 are added to the reaction hole containing donor platelets, and incubated at 39°C for 22 min to obtain the immune complex.

[0057] Example 6 The present embodiment provides an application of a kit for determining platelet antibodies and cross matching by chemiluminescence method, which is different from example 2 in that: in step (4), the diluted sample to be tested and the reagent R1 are added to the reaction hole containing donor platelets, and incubated at 35°C for 26 min to obtain the immune complex.

[0058] Example 7 The present example provides a kit for determining platelet antibodies and cross matching by chemiluminescence method, which is different from example 2 in that in step (5), the incubation condition after adding R2 and R3 is incubation at 39℃ for 10 min.

[0059] Example 8 The present example provides a kit for determining platelet antibodies and cross matching by chemiluminescence method, which is different from example 2 in that in step (5), the incubation condition after adding R2 and R3 is incubation at 35℃ for 14 min.

[0060] Example 9 The present example provides a kit for determining platelet antibodies and cross matching by chemiluminescence method, which is different from example 2 in that the addition amount of reagent R1, reagent R2, reagent R3 and reagent R5 is 50 μl, 150 μl, 150 μl and 570 μl.

[0061] Example 10 The present example provides a kit for determining platelet antibodies and cross matching by chemiluminescence method, which is different from example 2 in that the addition amount of reagent R1, reagent R2, reagent R3 and reagent R5 is 50 μl, 200 μl, 200 μl and 600 μl.

[0062] I. Methodological validation test: The kit and method provided in example 1 are used to perform methodological validation: The following reference samples are prepared: Reference sample M0: blank sample, i.e. phosphate solution without platelet antibodies.

[0063] Reference sample M1: phosphate solution of platelet antibodies with a concentration of 20% lower than the critical value (0.8 Ru / mL).

[0064] Reference sample M2: phosphate solution of platelet antibodies with a concentration of 20% higher than the critical value (1.2 Ru / mL).

[0065] Reference sample M3: phosphate solution of platelet antibodies with a concentration of 5 times the critical value (5 Ru / mL).

[0066] Reference sample M4: phosphate solution containing anti-A (IgM) monoclonal antibody (concentration of 5 Ru / mL).

[0067] Reference sample M5: phosphate solution containing anti-B (IgM) monoclonal antibody (concentration of 5 Ru / mL).

[0068] Reference sample M6: phosphate solution containing anti-D (IgM) monoclonal antibody (concentration of 5 Ru / mL).

[0069] Reference M7: phosphate solution containing anti-D (IgG) monoclonal antibody (concentration of 5 Ru / mL).

[0070] 1. Determination of blank limit: Reference M0 was used as the sample to be tested, and the detection was repeated 10 times.

[0071] The detection results are as follows: Table 2. As can be seen from the above Table 2, the reference M0 was repeatedly detected 10 times, and the results were all negative (<1 Ru / mL).

[0072] 2. Cut-off value (1) The anti-platelet antibody content of 80 clinical positive samples and 80 clinical negative samples was measured by the detection method of the present application (Example 1), and the minimum detection limit was determined as the cut-off value according to the results of the test samples, i.e. the best positive judgment value was 1 Ru / mL.

[0073] (2) 10 clinical positive samples and 10 clinical negative samples were taken and detected simultaneously with the platelet antibody detection kit (solid phase agglutination method) of Changchun Bode, and the results are as follows: Table 3. Note: "Negative 1" is the first clinical negative sample, "Positive 1" is the first clinical positive sample, and so on. As can be seen from the above Table 3, the results of the present method in detecting immune-mediated thrombocytopenia patient samples and normal human samples are more consistent with the actual situation. When there is immune-mediated thrombocytopenia, the content of anti-platelet antibody is high; the platelet function of normal people is normal, and the content of anti-platelet antibody in the sample is low.

[0074] (3) Reference M1, M2 and M3 were used as samples to be tested, and each was detected 10 times.

[0075] Result determination: The detection limit of the reagent is 1 Ru / mL, if the sample concentration value is <1 Ru / mL, it is judged as negative; if the sample concentration value is ≥1 Ru / mL, it is judged as positive.

[0076] The detection results are as follows: Table 4. From the above Table 4, it can be seen that the results of 10 repeated detections of Ml were all negative (<1 RU / mL); the results of 10 repeated detections of M2 and M3 were all positive (>1 RU / mL).

[0077] 3. Specificity Reference products M4, M5, M6 and M7 were used as the samples to be detected, and each was detected 3 times.

[0078] The detection results were as follows: M4, M5, M6 and M7 were each detected 3 times, and the results were all negative (<1 RU / mL), as shown in Table 5: Table 5. 4. Reproducibility Reference product M3 was used as the sample to be detected, and was detected 20 times.

[0079] The detection results were as follows: M3 was detected 20 times, and the results were all positive (>1 RU / mL), and the coefficient of variation CV was ≤15.0%.

[0080] 5. Batch reproducibility Three batches of reagent cards, each with 10 portions, were used, and reference product M3 was used as the sample to be detected, and was detected 20 times for each batch, and the results of the three batches of reagent cards were compared.

[0081] The detection results were as follows: the results of 20 detections of each of the three batches of reagent cards were all positive (>1 RU / mL), and the coefficient of variation was ≤15.0%.

[0082] 6. Stability The product was incubated at 37±1°C for 7 days, and then stability tests were performed on its physical properties, blank limit, critical value, specificity, reproducibility and batch reproducibility. The test results were consistent with the foregoing results.

[0083] 1. Detection of platelet antibody cross matching of clinical samples: The kit provided in Example 2 was used to detect the cross matching of 20 donor platelet samples with the platelets of a recipient, and the cross matching was also detected by the solid-phase agglutination method of the prior art.

[0084] The detection steps of the magnetic microparticle chemiluminescence method were as follows: (1) 20 donor platelet samples were placed in the reaction wells (the donor platelet samples were diluted with platelet diluent to a concentration of 100 x 10 9The magnetic bead particles coated with platelet antibody are added into the reaction well, mixed and incubated (37℃, 24 min) with the donor platelets in the reaction well, the platelets without binding platelet antibody are washed away, and then the anti-human IgG antibody labeled with acridinium ester is added, mixed and incubated (37℃, 12 min), the anti-human IgG antibody labeled with acridinium ester binds with IgG antibody to form a complex of magnetic bead-platelet antibody-donor platelet-receptor platelet antibody-anti-human IgG-acridinium ester; (2) After the reaction is completed, the magnetic field attracts the magnetic beads, the unbound substances are washed away by the washing solution, and then the pre-activation solution and the activation solution are added into the reaction mixture to generate a chemiluminescence reaction; (3) The number of photons generated in the reaction is measured by a photomultiplier tube, and the number of photons generated is proportional to the platelet antibody concentration in the plasma sample.

[0085] Result determination: the detection limit of the reagent is 1 Ru / mL, if the sample concentration value < 1 Ru / mL, it is judged as negative; if the sample concentration value ≥ 1 Ru / mL, it is judged as positive.

[0086] The determination results are shown in Table 6: Table 6. Note: "sample 1" represents the first example of donor platelets, "sample 2" represents the second example of donor platelets, and so on. As shown in Table 6, the results of the platelet cross matching test of the method for clinical samples are consistent with the results of the solid phase agglutination method, and the coincidence rate reaches 100%.

[0087] The specific embodiments are only an explanation of the present application, and are not a limitation of the present application, and those skilled in the art can make modifications to the embodiments without creative contribution after reading the present specification, but as long as the modifications are within the scope of the claims of the present application, they are protected by the patent law.

Claims

1. A method for determining platelet antibodies and cross-matching by chemiluminescence, characterized in that: It includes: First, add the tool platelets / donor platelets to the reaction wells, then add the test sample and magnetic beads coated with platelet antibodies to the reaction wells, and incubate at 35-39°C for 22-26 minutes to obtain an immune complex with the structure of magnetic beads-platelet antibodies-tool platelets / donor platelets-acceptor platelet antibodies; The washed immune complex is mixed with an acridinium ester-labeled anti-human IgG antibody, and incubated at 35-39° C. for 10-14 minutes to obtain a reaction complex having a structure of magnetic beads-platelet antibody-tool platelet / donor platelet-recipient platelet antibody-anti-human IgG antibody-acridinium ester; The washed reaction complex is sequentially mixed with acridinium ester pre-excitation solution and excitation solution to carry out chemiluminescence reaction. The concentration of platelet antibodies in the sample to be tested is calculated by measuring the number of photons generated in the reaction.

2. The method for determining platelet antibodies and cross-matching by chemiluminescence according to claim 1, characterized in that: The detection limit of the method for platelet antibodies in the sample to be tested is 1 Ru / mL.

3. The method for determining platelet antibodies and cross-matching by chemiluminescence according to claim 1, characterized in that: When the method is used to determine the platelet antibody cross-match between the blood donor and the blood recipient, the judgment criteria are: If the platelet concentration in the sample to be tested is less than 1 Ru / mL, it is judged as negative and the matching is successful; If the platelet concentration in the sample to be tested is ≥1 Ru / mL, it is judged to be positive and the matching fails.

4. The method for determining platelet antibodies and cross-matching by chemiluminescence according to any one of claims 1 to 3, characterized in that: The method for preparing the platelet antibody-coated magnetic bead microparticles comprises: Take the magnetic beads, vortex and separate, remove the supernatant, wash, add platelet antibody and disperse in coating buffer, mix well and incubate at 35-40℃ for 12-36h; After the incubation is completed, the supernatant is removed, the magnetic beads are washed, and redispersed in preservation solution I. After blocking and incubation, the result is obtained.

5. The method for determining platelet antibodies and cross-matching by chemiluminescence according to claim 4, characterized in that: The mass ratio of the platelet antibody to the magnetic beads is (15-25) μg:1 mg.

6. The method for determining platelet antibodies and cross-matching by chemiluminescence according to any one of claims 1 to 3, characterized in that: The method for preparing the acridinium ester-labeled anti-human IgG antibody is as follows: The anti-human IgG antibody to be labeled is mixed with acridinium ester at a mass ratio of 1 mg: (25-35) μl and dispersed in labeling buffer. The mixture is allowed to react in a constant temperature and humidity chamber at 35-37°C under light-proof conditions for 2-2.5 hours to obtain an anti-human IgG antibody mixture labeled with acridinium ester. The obtained acridinium ester-labeled anti-human IgG antibody mixture is then placed in an ultrafiltration tube for ultrafiltration to remove unreacted acridinium ester, thereby obtaining the acridinium ester-labeled anti-human IgG antibody.

7. A kit for determining platelet antibodies and cross-matching by chemiluminescence, wherein the method according to any one of claims 1 to 6 is carried out using the kit, characterized in that: It includes reagent R1, reagent R2, reagent R3 and reagent R5; The reagent R1 is magnetic beads coated with platelet antibodies, which are dispersed in a preservation solution I containing BSA and a preservative, and the concentration of the magnetic beads is 1-2 mg / mL; The reagent R2 is an acridinium ester-labeled anti-human IgG antibody, which is dispersed in a preservation solution II containing sodium chloride and potassium chloride. The concentration of the anti-human IgG antibody is 100-120 ng / mL; The reagent R3 is a buffer solution containing BSA and sodium chloride; The reagent R5 is a sample diluent.

8. The kit for determining platelet antibodies and cross-matching by chemiluminescence according to claim 7, characterized in that: The preservation solution I is a phosphate buffer solution with a pH of 7.5-8.5, wherein the concentration of BSA is 8-12 mg / mL and the concentration of the preservative is 0.02-0.1 wt %; The preservation solution II is a phosphate buffer solution with a pH of 7.8-8.2, wherein the concentration of sodium chloride contained therein is 14-18 mg / mL; the concentration of potassium chloride contained therein is 0.2-0.6 mg / mL.

9. Use of the kit according to any one of claims 7 or 8 in platelet antibody and cross-matching detection, characterized in that: It includes: Dilute the sample to be tested with reagent R5 and add it together with reagent R1 to the reaction well containing the tool platelets / donor platelets, and incubate at 35-39°C for 22-26 minutes to obtain an immune complex; Mixing the washed immune complex with the reagent R2 and the reagent R3, and incubating at 35-39° C. for 10-14 minutes to obtain a reaction complex; The washed reaction complex is mixed with acridinium ester pre-excitation solution and excitation solution to carry out chemiluminescence reaction. The concentration of platelet antibodies in the sample to be tested is calculated by measuring the number of photons generated in the reaction.

10. Use of the kit according to claim 9 in platelet antibody and cross-matching detection, characterized in that: The volume ratio of the reagent R1, reagent R2, reagent R3 and reagent R5 is 1:(2-4):(2-4):(11-12).

Images