Platelet antibody detection kit and application thereof

Abstract

The application discloses a platelet antibody detection kit and application thereof, and belongs to the technical field of detection kits.The kit comprises a micro-column gel card, an enzyme-labeled secondary antibody, an enzyme-labeled secondary antibody diluent, 20x washing solution, color developing solution, fresh platelet processing solution, platelet buffer solution, platelet preparation solution, freeze-dried platelet reconstitution solution, freeze-dried platelets and a negative control.The platelet antibody detection kit makes platelet cross matching possible;the micro-column gel immunization technology is combined with the enzyme immunization labeling technology, so that the limitation that the micro-column gel method can only carry out agglutination reaction is solved;the reaction card washing equipment realizes semi-automatic operation of the micro-column gel method detection;triglyceride in plasma is less than or equal to 5mg / mL, bilirubin is less than or equal to 20mg / dL, and hemoglobin is less than or equal to 5mg / mL, which will not affect the result;the kit has good stability, long validity period, high sensitivity, strong specificity and simple operation.

Description

Technical Field

[0001] This invention relates to the field of detection kit technology, and more specifically to a platelet antibody detection kit and its application. Background Technology

[0002] Platelet transfusion can be used to prevent and treat bleeding symptoms in patients with low platelet counts or impaired platelet function, restoring and maintaining normal hemostasis and coagulation functions. However, long-term platelet transfusion may lead to serious consequences such as platelet transfusion ineffectiveness. Detecting the presence of antiplatelet antibodies in patients can assist clinicians in taking appropriate preventive and therapeutic measures. In particular, it can facilitate cross-matching for patients with positive antiplatelet antibodies to screen for platelet-compatible donors, significantly reducing the occurrence of platelet transfusion ineffectiveness.

[0003] Current methods for platelet detection mainly rely on the following: indirect immunofluorescence, enzyme-linked immunosorbent assay (ELISA), hydrogel assay, flow cytometry, gene detection, microcolumn gel immunoassay, and solid-phase agglutination. However, each of these methods has its own limitations. ① Indirect immunofluorescence and ELISA can only detect anti-platelet antibodies in serum and cannot perform cross-matching. Furthermore, these methods are relatively complex to operate, requiring skilled technicians with specialized training; the reaction times for these kits are long, and their manufacturing costs are high. ② Hydrogel assay can only detect anti-platelet antibodies and cannot perform cross-matching. Current kits also have low stability and reproducibility. Their colorimetric principle relies on indicator red blood cells, resulting in high kit manufacturing costs. ③ Flow cytometry also cannot perform cross-matching and relies on expensive flow cytometers, requiring relatively expensive consumables, which hinders its large-scale application. ④ Genetic testing can determine the antigen type of platelets using PCR or sequencing methods, but it cannot detect platelet antibodies in serum, nor can it perform cross-matching. ⑤ Microcolumn gel immunoassay and solid-phase agglutination methods have been applied to platelet antibody detection, but they cannot perform platelet cross-matching.

[0004] In summary, existing platelet detection methods all have inherent problems in their underlying principles, limiting their large-scale clinical application. Current research on platelet detection methods mainly focuses on improvements to the aforementioned methods. For example, for antigen-antibody detection, immunomagnetic bead liquid chromatography chips have emerged; gene sequencing methods have matured and their prices are gradually decreasing; and the advent of dedicated flow cytometry has also reduced related costs. However, these improvements cannot resolve the inherent defects in their underlying principles, and none of these methods can achieve cross-matching.

[0005] In summary, how to provide a new platelet antibody detection kit that can achieve cross-matching is a problem that urgently needs to be solved by those skilled in the art. Summary of the Invention

[0006] In view of this, the present invention provides a platelet antibody detection kit and its application.

[0007] To achieve the above objectives, the present invention adopts the following technical solution:

[0008] A platelet antibody detection kit includes the following components: microcolumn gel card, enzyme-labeled secondary antibody, enzyme-labeled secondary antibody diluent, 20x washing buffer, chromogenic solution, fresh platelet processing solution, platelet buffer, platelet preparation solution, lyophilized platelet reconstitution solution, lyophilized platelets, and negative control.

[0009] Furthermore, the micropillar gel card includes dextran gel particles and polypropylene cards.

[0010] Furthermore, the enzyme-labeled secondary antibody is alkaline phosphatase-goat anti-human IgG, with an IgG content ≥1 mg / mL;

[0011] The colorimetric solution is an alkaline phosphatase substrate;

[0012] The lyophilized platelets were human type O platelets, with a concentration of 100 ± 10 × 10⁻⁶ before lyophilization. 9 / L.

[0013] Furthermore, the enzyme-labeled secondary antibody diluent comprises the following components at the following mass concentrations: NaCl 0.819%, KCl 0.02%, Na2HPO4·12H2O 0.358%, KH2PO4 0.024%, Tween-20 0.05%, and EDTA-Na2·2H2O 0.05%.

[0014] The 20x washing solution comprises the following components in mass concentrations: NaCl 16.38%, KCl 0.402%, Na2HPO4·12H2O 7.16%, KH2PO4 0.48%, Tween-20 1%, and EDTA-Na2·2H2O 1%.

[0015] The fresh platelet processing solution comprises the following components at the following mass concentrations: sheep blood albumin 30%, NaCl 0.629%, PC-300 0.1%, and glutaraldehyde 0.2%.

[0016] The platelet buffer comprises the following components in the indicated mass concentrations: Na₂HPO₄·12H₂O 0.358%, KCl 0.02%, NaCl 0.819%, KH₂PO₄ 0.024%, BSA 0.2145%, and EDTA-Na₂·2H₂O 0.0715%.

[0017] The platelet preparation solution comprises the following components at the following mass concentrations: sheep blood albumin 30%, NaCl 0.629%, and PC-300 0.1%;

[0018] The lyophilized platelet reconstituted solution contains 0.9% NaCl by mass.

[0019] The above-mentioned kit is used in the detection of platelet antibodies.

[0020] The above-mentioned kit is used in cross-matching detection.

[0021] The above-mentioned kit is used in the preparation of reagents for detecting platelet antibodies.

[0022] The above-mentioned kit is used in the preparation of cross-matching detection reagents.

[0023] Detection principle:

[0024] Platelet antigen-antibody reactions were detected using the principle of size exclusion chromatography and the principle of color development of enzyme-labeled secondary antibodies and enzyme reaction substrates.

[0025] The kit's microcolumn gel cards contain dextran gel particles. Platelet cells are incubated with the test plasma or serum in the reaction chamber. In a positive reaction, anti-platelet antibodies in the plasma or serum bind to platelet antigens, forming immune complexes. In a negative reaction, where anti-platelet antibodies are absent, no immune complexes are formed. Under a certain suction force, the platelet immune complexes formed in the positive reaction and the platelets in the negative reaction cannot pass through the dextran gel particles, while non-cellular components such as the test plasma or serum can pass through the gel particles. These are removed by adding washing buffer, thus separating the plasma or serum from the platelets in the reaction system, and washing both the platelet complexes and the test platelets. When alkaline phosphatase-labeled secondary antibody (sheep-anti-human immunoglobulin, S-AHG) is added, in a positive reaction, the alkaline phosphatase-labeled secondary antibody (Fab fragment) binds to the antibody (Fc fragment) in the immune complex and reacts with the chromogenic solution, showing a color change; in a negative reaction, there is no immune complex, and the alkaline phosphatase-labeled secondary antibody does not have a corresponding antibody that reacts with the chromogenic solution, resulting in no color change.

[0026] As can be seen from the above technical solutions, compared with the prior art, the beneficial effects achieved by the present invention are as follows: ① The platelet-related detection reagents used in clinical applications in the prior art cannot perform platelet cross-matching. The platelet antibody detection kit of the present invention makes platelet cross-matching possible for the first time; ② Products of microcolumn gel immunoassay technology can only be used for red blood cell agglutination tests, including blood typing and blood typing, etc. The platelet antibody detection kit of the present invention breaks through the limitations of this technology by combining microcolumn gel immunoassay technology with enzyme immunolabeling technology, solving the limitation that the microcolumn gel method can only perform agglutination reactions, and enabling this technology to be used for a variety of immunolabeling methods; ③ The reaction card washing device of the present invention innovatively realizes the semi-automatic operation of microcolumn gel method detection, providing support for the establishment of a fully automated detection system in the future; ④ Plasma triglycerides ≤5mg / mL, bilirubin ≤20mg / dL, and hemoglobin ≤5mg / mL will not affect the results; ⑤ Good stability, long shelf life, high sensitivity, strong specificity, and simple operation. Attached Figure Description

[0027] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0028] Figure 1 Image of the micropillar gel card in Embodiment 1 of the present invention;

[0029] Figure 2 Images showing the separation of the microcolumn gel card in Embodiment 1 of this invention;

[0030] Figure 3 This is a schematic diagram of the detection steps and principle in Embodiment 2 of the present invention;

[0031] Figure 4 This is an example of result determination in Embodiment 2 of the present invention;

[0032] Figure 5 This refers to the test results of the negative reference material from the enterprise in Experiment 1 of this invention;

[0033] Figure 6 This refers to the test results of the enterprise's positive reference material in Experiment 1 of this invention;

[0034] Figure 7 This is the detection limit result in Experiment 1 of the present invention. Detailed Implementation

[0035] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0036] The reagents required for this invention are conventional experimental reagents, purchased from commercially available channels; the experimental methods not mentioned are conventional experimental methods, and will not be described in detail here.

[0037] The instruments used in the following embodiments are as follows:

[0038] FYQ type reagent card incubator manufactured by Changchun Boyan Scientific Instruments Co., Ltd.

[0039] The Hw-L1 type liquid suction machine (plate washer) is manufactured by Changchun Houwei Scientific Instruments Co., Ltd.

[0040] Example 1

[0041] A platelet antibody detection kit includes the following components:

[0042] Microcolumn gel card, enzyme-labeled secondary antibody, enzyme-labeled secondary antibody dilution buffer, 20x washing buffer, chromogenic solution, fresh platelet processing solution, platelet buffer, platelet preparation solution, lyophilized platelet reconstitution solution, lyophilized platelets and negative control.

[0043] The micropillar gel card includes dextran gel particles and polypropylene cards, such as... Figure 1 As shown, the micropillar gel card adopts a design that allows for separation of the upper and lower parts, such as... Figure 2 As shown;

[0044] The enzyme-labeled secondary antibody is alkaline phosphatase-goat anti-human IgG, with an IgG content ≥1 mg / mL;

[0045] The enzyme-labeled secondary antibody dilution solution includes 327.6g NaCl, 8.04g KCl, 143.29g Na2HPO4·12H2O, 9.6g KH2PO4, 20mL Tween-20, 20g EDTA-Na2·2H2O, and 40L purified water;

[0046] The 20x washing solution comprises 327.6g NaCl, 8.04g KCl, 143.29g Na2HPO4·12H2O, 9.6g KH2PO4, 20mL Tween-20, 20g EDTA-Na2·2H2O, and 2L purified water;

[0047] The colorimetric solution is an alkaline phosphatase substrate;

[0048] The fresh platelet processing solution includes 300 mL of sheep blood albumin, 6.291 g of NaCl, 1 mL of PC-300, 2 mL of glutaraldehyde, and 699 mL of purified water.

[0049] The platelet buffer solution comprises 7.16g Na2HPO4·12H2O, 0.40g KCl, 16.38g NaCl, 0.48g KH2PO4, 4.29g BSA (bovine serum albumin), 1.43g EDTA-Na2·2H2O, and 2L purified water.

[0050] The platelet preparation solution includes 300 mL of sheep blood albumin, 6.291 g of NaCl, 1 mL of PC-300, and 699 mL of purified water;

[0051] The lyophilized platelet reconstituted solution comprises 9g NaCl and 1L purified water;

[0052] The lyophilized platelets were human type O platelets, with a concentration of 100 × 10⁻⁶ before lyophilization. 9 / L;

[0053] The negative control is human platelet antibody-negative plasma or serum with a PC-300 concentration of 1 / 1000.

[0054] Example 2

[0055] Antibody testing

[0056] 1. Sample Requirements

[0057] Whole blood samples collected using EDTA anticoagulant or separating gel. If the sample cannot be tested within a short period, it can be stored at 2–8°C for 7 days. The sample can be stored long-term at -20±5°C, and can be repeatedly frozen and thawed twice without affecting the test results.

[0058] 2. Reagent preparation

[0059] (1) 1x washing solution: 1 mL of 20x washing solution was added to 19 mL of purified water and mixed to obtain 20 mL of 1x washing solution.

[0060] (2) The ratio of enzyme-labeled secondary antibody preparation is 1:1000, that is, 1 μL of enzyme-labeled secondary antibody is added to 1 mL of enzyme-labeled secondary antibody dilution solution, mixed well and then used.

[0061] 3. Testing Steps

[0062] The detection steps and principles are as follows: Figure 3 As shown, the details are as follows:

[0063] (1) Sample processing: Whole blood samples collected with EDTA anticoagulant or separating gel are centrifuged at 1500g for 5 minutes to obtain plasma or serum samples for testing.

[0064] (2) Take lyophilized platelets (400 μL before lyophilization) and add lyophilized platelet reconstitution solution (400 μL) to reconstitute. Centrifuge at 950g for 5 minutes, discard the supernatant, add 400 μL of platelet preparation solution and mix thoroughly. Incubate at 37°C for 20 minutes.

[0065] (3) Mark the microcolumn gel card, separate the upper and lower parts, place the lower part on the card holder, place the upper part on the suction machine card slot, tear the film, aspirate for 0.3 seconds to remove the liquid (platelet preparation solution) on the gel, and then tightly splice the upper and lower parts and place them on the card holder.

[0066] (4) Add the reconstituted platelets to the reaction chamber of the microcolumn gel card, 50 μL / well. Add the test sample and negative control, 20 μL / well, to the corresponding reaction chamber of the microcolumn gel card and incubate at 37°C for 15 minutes.

[0067] (5) Separate the upper and lower parts of the microcolumn gel card, place the lower part on the card holder, and place the upper part on the suction machine card slot. Aspirate for 0.3 seconds, add 200μL of 1x washing solution to each well each time, aspirate for 0.6 seconds each time, repeat the washing 3 times, and then put the upper and lower parts tightly together on the card holder.

[0068] (6) Add 50 μL of enzyme-labeled secondary antibody per well, aspirate for 0.2 seconds to allow the enzyme-labeled secondary antibody to be gradually and evenly distributed in the gel, and place the upper and lower parts tightly together on the card tray. Incubate at 37°C for 10 minutes.

[0069] (7) Separate the upper and lower parts of the microcolumn gel card, place the lower part on the card holder, and place the upper part on the suction machine card slot. Aspirate for 0.1 seconds, add 200 μL of 1x washing solution to each well each time, aspirate for 0.6 seconds each time, repeat the washing 5 times, and then put the upper and lower parts tightly together on the card holder.

[0070] (8) Add 100 μL of color developer per well, aspirate for 0.3 seconds to gradually and evenly distribute the color developer in the gel, then place the upper and lower parts tightly together on the card holder and develop the color at room temperature in the dark for 10 to 12 minutes.

[0071] (9) Observe the experimental results with the naked eye.

[0072] Positive results: Strong positive results are blue-purple; weak positive results are light purple.

[0073] Negative result: A negative result is nearly white.

[0074] like Figure 4 As shown.

[0075] A positive result indicates that the sample contains platelet antibodies; a negative result indicates that the sample does not contain platelet antibodies.

[0076] If the negative control result is nearly white, the result is valid and can be interpreted. If the negative control result is invalid, the test must be repeated.

[0077] Example 3

[0078] Cross-matching

[0079] 1. Sample Requirements

[0080] Whole blood samples collected via apheresis platelets or EDTA anticoagulant and separating gel.

[0081] It must be used within 2 days of collection, otherwise it will affect the test results.

[0082] 2. Testing Steps

[0083] (1) Sample processing

[0084] ① Donor Sample: Apheresis platelets are used as donor platelets. Centrifuge the donor platelets (in EP tubes) at 950g for 5 minutes, remove the supernatant, add an equal volume of platelet buffer to the donor platelets, mix thoroughly, centrifuge at 950g for 5 minutes, remove the supernatant, and collect the precipitate, which is the fresh donor platelet to be tested.

[0085] ② Recipient Samples: Whole blood samples collected with EDTA anticoagulant or separating gel are centrifuged at 1500g for 5 minutes to obtain plasma or serum samples for testing.

[0086] (2) Add an equal volume of fresh platelet processing solution to the fresh platelets from the donor to be tested, mix thoroughly, incubate at 37°C for 20 minutes, centrifuge at 950g for 5 minutes, remove the supernatant, add an equal volume of platelet preparation solution to the fresh platelets from the donor, mix thoroughly, and then test.

[0087] (3) Take the microcolumn gel card and mark it. Separate the upper and lower parts. Place the lower part on the card holder and the upper part on the suction machine slot. Tear off the film and suction for 0.3 seconds to remove the liquid on the gel. Then, tightly splice the upper and lower parts together and place them on the card holder.

[0088] (4) Add 50 μL of donor platelets to be tested into the reaction chamber of the microcolumn gel card, and add 20 μL of recipient plasma or serum and negative control into the reaction chamber of the corresponding microcolumn gel card. Incubate at 37°C for 15 minutes.

[0089] (5)~(9) are the same as in Example 2.

[0090] A positive result indicates that the recipient and donor are not a crossmatch, while a negative result indicates that the recipient and donor are a crossmatch.

[0091] If the negative control result is nearly white, the result is valid and can be interpreted. If the negative control result is invalid, the test must be repeated.

[0092] Experiment 1

[0093] Product performance testing

[0094] 1. Negative reference sample compliance rate

[0095] Plasma or serum samples that tested negative for platelet antibodies using a commercially available platelet antibody test kit were used as raw materials for the negative reference sample. The reference sample raw material was centrifuged at 3000 rpm for 10 minutes, and the supernatant was separated. PC-300 was added at 0.1%, and the sample was filtered for sterilization. After passing the test, the sample was aliquoted into reagent bottles. The aliquot volume was 0.03 mL / vial, thus obtaining the company's negative reference sample.

[0096] Three negative reference samples N1 to N3 from the enterprise were used for testing using the kit of this invention. The testing method was the same as in Example 2.

[0097] The results are as follows Figure 5 As shown, the negative compliance rate is 100%.

[0098] 2. Conformity rate of positive reference materials

[0099] Plasma or serum samples that tested positive for platelet antibodies using a commercially available platelet antibody test kit were used as raw materials for the positive reference standard. The reference standard raw material was centrifuged at 3000 rpm for 10 minutes, and the supernatant was separated. The raw material was then diluted using the commercially available platelet antibody test kit, with the positive reference standard diluted to the point where the last two or three weakly positive dilutions appeared. This was used as a weakly positive reference standard. The dilutions of the strong and weakly positive reference standards were confirmed and diluted accordingly. 0.1% PC-300 was added, and the sample was filtered for sterilization. After passing the test, the sample was aliquoted into reagent bottles. The aliquot volume was 0.03 mL / vial, thus obtaining the company's positive reference standard.

[0100] Six positive reference samples P1 to P6 from the enterprise were used for testing using the kit of this invention. The testing method was the same as in Example 2.

[0101] The results are as follows Figure 6 As shown, the positive concordance rate is 100%. Among them, P1 to P3 should be strongly positive, and P4 to P6 should be weakly positive.

[0102] 3. Detection limit

[0103] Plasma or serum samples that tested positive for platelet antibodies using a commercially available platelet antibody detection kit were used as the raw material for the positive reference standard. The reference standard raw material was centrifuged at 3000 rpm for 10 minutes, and the supernatant was separated. The raw material was then diluted using the commercially available platelet antibody detection kit, with the positive reference standard diluted to the last weakly positive dilution factor, which was used as the limit of detection reference standard. 0.1% PC-300 was added, and the sample was filtered for sterilization. After passing the test, the sample was aliquoted into reagent bottles. The aliquot volume was 0.03 mL / vial.

[0104] The detection limit reference was used, and the kit of the present invention was used for detection. The detection method was the same as in Example 2.

[0105] The results are as follows Figure 7 As shown, the detection limit reference L1 showed a weak positive result.

[0106] 4. Validity period

[0107] Microcolumn gel cards should be stored at 2–25°C for one year. Other components, except for enzyme-labeled secondary antibodies, should be stored at 2–8°C for one year, and enzyme-labeled secondary antibodies should be stored at -20±5°C for one year.

[0108] The various embodiments in this specification are described in a progressive manner, with each embodiment focusing on the differences from other embodiments. The same or similar parts between the various embodiments can be referred to each other.

[0109] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A platelet antibody detection kit, characterized in that, The product includes the following components: microcolumn gel card, enzyme-labeled secondary antibody, enzyme-labeled secondary antibody dilution buffer, 20x washing buffer, chromogenic solution, fresh platelet processing solution, platelet buffer, platelet preparation solution, lyophilized platelet reconstitution solution, lyophilized platelets, and negative control. The micropillar gel card includes dextran gel particles and a polypropylene card; The enzyme-labeled secondary antibody is alkaline phosphatase-goat anti-human IgG, with an IgG content ≥1 mg / mL; The colorimetric solution is an alkaline phosphatase substrate; The lyophilized platelets were human type O platelets, with a concentration of 100 ± 10 × 10⁻⁶ before lyophilization. 9 / L; The enzyme-labeled secondary antibody dilution solution comprises the following components at the following mass concentrations: NaCl 0.819%, KCl 0.02%, Na₂HPO₄·12H₂O 0.358%, KH₂PO₄ 0.024%, Tween-20 0.05%, and EDTA-Na₂·2H₂O 0.05%. The 20x washing solution comprises the following components by mass concentration: NaCl 16.38%, KCl 0.402%, Na2HPO4·12H2O 7.16%, KH2PO4 0.48%, Tween-20 1%, and EDTA-Na2·2H2O 1%. The fresh platelet processing solution comprises the following components at the following mass concentrations: sheep blood albumin 30%, NaCl 0.629%, PC-300 0.1%, and glutaraldehyde 0.2%; The platelet buffer comprises the following components at the following mass concentrations: Na₂HPO₄·12H₂O 0.358%, KCl 0.02%, NaCl 0.819%, KH₂PO₄ 0.024%, BSA 0.2145%, and EDTA-Na₂·2H₂O 0.0715%. The platelet preparation solution comprises the following components at the following mass concentrations: sheep blood albumin 30%, NaCl 0.629%, and PC-300 0.1%; The lyophilized platelet reconstituted solution contains 0.9% NaCl by mass.

2. The use of the kit according to claim 1 in the detection of platelet antibodies.

3. The application of the kit described in claim 1 in cross-matching detection.

4. The use of the kit according to claim 1 in the preparation of reagents for detecting platelet antibodies.

5. The application of the kit according to claim 1 in the preparation of cross-matching detection reagents.

Images