Detection reagent and detection method for platelet HPA homogeneous antibody

Abstract

The invention provides a detection reagent and a detection method of a platelet HPA homogeneous antibody, and belongs to the technical field of antibody detection. According to the method for preparing the platelet suspension by eluting the platelet HLA antigen, the HLA antigen on the platelet is eluted through acid, and the HPA antigen is reserved. A microwell plate coated with the platelet suspension retaining the HPA antigen is further prepared, platelets are adsorbed by means of the non-specific adsorption capacity of phytolectin, the adsorbed platelets are directly freeze-dried to the bottom of the microwell plate, only detected serum / plasma needs to be added for incubation in clinical application, and the platelet suspension retaining the HPA antigen and the platelet suspension retaining the HPA antigen can be obtained. A detection result can be obtained by using an anti-human globulin reagent and indicating red blood cells, so that intermediate links of detection are reduced, and standardization and automation are facilitated.

Description

Technical Field

[0001] The present invention belongs to the technical field of antibody detection, and in particular relates to a detection reagent and a detection method for platelet HPA alloantibodies. Background Art

[0002] There are complex antigens on the surface of platelets, which are mainly divided into two categories: one is histocompatibility antigens, such as human leukocyte antigen (HLA); the other is human platelet antigens (HPA).

[0003] Fetal / neonatal alloimmune thrombocytopenia (FNAIT) is caused by a platelet incompatibility between the fetus and the mother. Immunized mothers produce alloimmune IgG antibodies that cross the placenta and attack the incompatible platelets in the fetus, leading to thrombocytopenia. Clinical manifestations include bleeding and petechiae, and in severe cases, intracranial hemorrhage or even death.

[0004] Platelet HPA alloantibodies are the primary pathological factor in FNAIT. Therefore, the detection of platelet HPA alloantibodies is an important indicator for the diagnosis and prevention of fetal / neonatal alloimmune thrombocytopenia (FNAIT).

[0005] Currently, there are two main methods for detecting platelet HPA alloantibodies: the monoclonal antibody specific immobilization of platelet antigen (MAIPA) method and the Luminex immunomagnetic bead method. MAIPA is the gold standard for detecting platelet antibodies, but the experiment is cumbersome and time-consuming, and each target is tested separately, requiring a large amount of plasma or serum. Therefore, MAIPA is only used in professional reference laboratories and is rarely used in clinical practice. The Luminex immunomagnetic bead method couples purified GP or platelet antigens to fluorescent microspheres to detect platelet HPA alloantibodies. Currently, the Luminex immunomagnetic bead method is rarely used in clinical practice in China because the kits on the market are all foreign products. Not only are they expensive, but the HPA gene frequencies in Asian and Caucasian populations are different, so there is a risk of missed detection. Summary of the Invention

[0006] The present invention provides a detection reagent and method for platelet HPA alloantibodies, which reduces the intermediate links of detection, has a simple detection method, strong specificity, high sensitivity, and is conducive to standardization and automation of detection.

[0007] The present invention provides a method for preparing a platelet suspension by eluting platelet HLA antigens, comprising the following steps: washing three O-type mixed platelets and adjusting the concentration to (10-500)×10 9 / L, after centrifugation, the collected cells are mixed with a chloroquine phosphate eluent of pH 2.0 to pH 5.0, react for a period of time, and then washed again. After centrifugation, the cells are resuspended in physiological saline or phosphate buffer to obtain a platelet suspension.

[0008] In a preferred embodiment of the present invention, the washing comprises washing with physiological saline or a phosphate buffer solution at pH 6.8 to pH 7.8.

[0009] In a preferred embodiment of the present invention, the reaction temperature is 24-37° C. and the reaction time is 1-120 min.

[0010] The present invention also provides a platelet suspension containing HPA antigen prepared by the above method.

[0011] The present invention also provides a microplate coated with HPA antigen, comprising a phytohemagglutinin coating solution and the platelet suspension.

[0012] The present invention also provides a method for preparing the above-mentioned microplate, comprising the following steps: adding a plant lectin coating solution to each well of the microplate and performing a first incubation; adding the above-mentioned platelet suspension after a first wash and performing a second incubation; adding a blocking solution after a second wash and performing blocking; then adding a freeze-drying protection solution and performing a third incubation and then freeze-drying to obtain the microplate.

[0013] In a preferred embodiment of the present invention, the lectin coating solution is a carbonate buffer solution containing lectin;

[0014] The washing solutions used in the first washing and the second washing are both phosphate buffer containing Tween-20;

[0015] The blocking solution comprises the following components: 1-5% (m / v) gelatin, 0.1-2% (m / v) BSA, 0.005-0.01 mol / L disodium hydrogen phosphate, 0.001-0.005 mol / L potassium dihydrogen phosphate and 0.1-0.5 mol / L sodium chloride.

[0016] In a preferred embodiment of the present invention, the freeze-drying protective solution comprises the following components: 0.01-0.1 mol / L of sodium chloride, 0.001-0.005 mol / L of potassium chloride, 0.01-0.05 mol / L of sodium bicarbonate, 0.001-0.005 mol / L of citric acid, 0.1-1.0 mmol / L of calcium chloride, 0.01-0.05 mol / L of sodium acetate, 0.01-0.05 mol / L of glucose, 0.001-0.05 mol / L of magnesium sulfate, 0.001-0.005 mol / L of trisodium citrate, 0.001-0.015 mol / L of trehalose and 1-5% (m / v) BSA.

[0017] The present invention also provides a kit for detecting platelet HPA alloantibodies, comprising the above-mentioned microplate, indicator red blood cells and anti-human globulin reagent, blocking solution, washing solution, negative quality control and positive quality control.

[0018] The present invention also provides a method for detecting platelet HPA alloantibodies for non-diagnostic purposes, comprising the following steps: adding the test sample to the above-mentioned microplate, incubating for a period of time and then washing; adding blocking solution to the washed microplate for secondary blocking, after removing the blocking solution, adding anti-human globulin reagent and indicator red blood cells, and observing the results after centrifugation.

[0019] Beneficial Effects: The present invention provides a method for eluting platelet HLA antigens to prepare a platelet suspension, utilizing acid elution to remove HLA antigens from platelets while retaining HPA antigens. The present invention also prepares a microplate coated with the platelet suspension retaining HPA antigens, utilizing the nonspecific adsorption capacity of plant lectins to adsorb platelets. The adsorbed platelets are then freeze-dried directly onto the bottom of the microplate. In clinical applications, test results can be obtained by simply adding the test serum for incubation, along with an anti-human globulin reagent and indicator red blood cells, reducing the number of intermediate steps in the test and facilitating standardization and automation. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 A complete flowchart for platelet HPA alloantibody testing;

[0021] Figure 2 The results of HLA antigen elution detected by flow cytometry are shown in Figure A, which shows the fluorescence intensity of HLA antigen on platelets before elution; and Figure B shows the fluorescence intensity of HLA antigen on platelets after elution.

[0022] Figure 3 This is a graph showing the optimization results of red blood cell concentration and anti-human globulin titer in Example 1;

[0023] Figure 4Graphs showing the specificity and sensitivity test results in Example 2, where A shows the results of detecting HPA antibodies and B shows the results of detecting HLA antibodies;

[0024] Figure 5 This is a diagram of the stability test results in Example 3. DETAILED DESCRIPTION

[0025] The present invention provides a method for preparing a platelet suspension by eluting platelet HLA antigens, comprising the following steps: washing three O-type mixed platelets and adjusting the concentration to (10-500)×10 9 / L, after centrifugation, the collected cells are mixed with a chloroquine phosphate eluent of pH 2.0 to pH 5.0, react for a period of time, and then washed again. After centrifugation, the cells are resuspended in physiological saline or phosphate buffer to obtain a platelet suspension.

[0026] The present invention selects three-person O-type mixed platelets based on the distribution frequency of platelet-specific antigens. The three-person mixed platelets can cover more HPA antigens, avoiding the high-frequency antigen dilution problem caused by mixing more people, thereby ensuring the capture ability and detection sensitivity of high-frequency platelet HPA alloantibodies. The platelet count in the three-person O-type mixed platelets of the present invention can reach (2.5-10)×10 11 .

[0027] The present invention washes the three-person O-type mixed platelets, and the washing solution used for the washing includes physiological saline or pH 6.8 to pH 7.8 phosphate buffer, and the three-person O-type mixed platelets are washed 1 to 5 times with physiological saline or pH 6.8 to pH 7.8 phosphate buffer, and the centrifugal speed during each washing is required to be 100 to 10000 g, such as 100 g, 200 g, 500 g, 800 g, 1000 g, 2000 g, 3000 g, 4000 g, 5000 g, 6000 g, 7000 g, 8000 g, 9 ... the centrifugation time is 1 to 30 min, such as 1 min, 2 min, 3 min, 4 min, 5 min, 6 min, 7 min, 8 min, 9 min, 10 min, 11 min, 12 min, 13 min, 14 min, 15 min, 16 min, 17 min, 18 min, 19 min, 20 min, 21 min, 22 min, 23 min, 24 min, 25 min, 26 min, 27 min, 28 min, 29 min or 30 min.

[0028] The present invention adjusts the concentration of washed platelets to (10-500)×10 9 / L, the adjustment can be performed using physiological saline or pH 6.8 to pH 7.8 phosphate buffer, and then centrifuged to discard the supernatant. The centrifugal speed is 100g to 10000g, and the centrifugal time is 1 to 30min. Since the exemplary data of the centrifugal speed and centrifugal time are the same as above, they are not repeated here.

[0029] After discarding the supernatant, the present invention adds a pH 2.0 to pH 5.0 chloroquine phosphate eluent of equal volume to the discarded supernatant and mixes well to elute the HLA antigen. The components of the chloroquine phosphate eluent include: 0.1 to 0.5 mol / L chloroquine phosphate, 0.005 to 0.010 mol / L EDTA-2Na, 0.01 to 0.05 mol / L Na2HPO4.2H2O and 0.1 to 0.5 mol / L NaCl, and the pH is adjusted to 2.0 to 5.0. In one embodiment, the components of the chloroquine phosphate eluent are specifically: 0.2 mol / L chloroquine phosphate, 0.005 mol / L EDTA-2Na, 0.01 mol / L Na2HPO4.2H2O and 0.4 mol / L NaCl. The present invention is accompanied by oscillation during the elution process. The elution temperature is 24 to 37° C., the elution time is 1 to 120 min, and the oscillation frequency is 1 to 300 rpm. After the elution, another wash is performed. The wash parameters are the same as those for the above wash and are not described in detail here. After the wash, an equal volume of physiological saline or pH 6.8 to pH 7.8 phosphate buffer is added, mixed, and set aside. This is the platelet suspension of the present invention in which the HLA antigens are eluted and the HPA antigens are retained.

[0030] The present invention removes HLA antigens adsorbed on the platelet surface by acid elution of platelets. Only platelet-specific antigens (HPA) exist on the eluted platelets, thereby distinguishing platelet HPA alloantibodies.

[0031] The present invention also provides a platelet suspension containing HPA antigen prepared by the above method.

[0032] The platelet suspension of the present invention contains almost no HLA antigen. The elution of HLA antigen was detected by flow cytometry, and the result showed that the elution rate of HLA antigen reached 99.21%.

[0033] The present invention also provides a microplate coated with HPA antigen, comprising a phytohemagglutinin coating solution and the platelet suspension.

[0034] The lectin coating solution of the present invention is a carbonate buffer solution (pH 8.0 to pH 10.0) containing lectin, and the concentration of the lectin in the lectin coating solution is 1 to 100 μg / mL, such as 1 μg / mL, 2 μg / mL, 5 μg / mL, 10 μg / mL, 20 μg / mL, 30 μg / mL, 40 μg / mL, 50 μg / mL, 60 μg / mL, 70 μg / mL, 80 μg / mL, 90 μg / mL or 100 μg / mL.

[0035] The present invention also provides a method for preparing the above-mentioned microplate, comprising the following steps: adding a plant lectin coating solution to each well of the microplate and performing a first incubation; adding the above-mentioned platelet suspension after a first wash and performing a second incubation; adding a blocking solution after a second wash and performing blocking; then adding a freeze-drying protection solution and performing a third incubation and then freeze-drying to obtain the microplate.

[0036] The present invention adds a lectin coating solution to each well of a microplate and then performs a first incubation. The amount of the lectin coating solution added is 10 to 200 μL / well, such as 10 μL / well, 20 μL / well, 30 μL / well, 40 μL / well, 50 μL / well, 60 μL / well, 70 μL / well, 80 μL / well, 90 μL / well or 100 μL / well. The temperature of the first incubation is 24 to 56° C., such as 24° C., 25° C., 28°C, 30°C, 32°C, 35°C, 36°C, 37°C, 38°C, 40°C, 42°C, 45°C, 46°C, 48°C, 50°C, 55°C or 56°C; the time is 1 to 120 min, such as 1 min, 5 min, 10 min, 20 min, 30 min, 40 min, 50 min, 60 min, 70 min, 80 min, 90 min, 100 min, 110 min or 120 min.

[0037] After the first incubation, a washing solution is added to each well for a first wash. The washing solution is a phosphate buffer solution at pH 6.8 to pH 7.8 containing Tween-20, wherein the concentration of Tween-20 in the washing solution is 0.01 to 0.05% (v / v). In one embodiment, 10 to 300 μL of the washing solution is added to each well, such as 10 μL / well, 20 μL / well, 50 μL / well, 100 μL / well, 150 μL / well, 200 μL / well, 2500 μL / well, or 300 μL / well. The washing solution is then removed and the remaining liquid is drained.

[0038] After the first wash, the platelet suspension is added to the microplate for a second incubation. The amount of the platelet suspension added can be 10 to 200 μL / well, such as 10 μL / well, 20 μL / well, 50 μL / well, 60 μL / well, 70 μL / well, 80 μL / well, 100 μL / well, 120 μL / well, 150 μL / well or 200 μL / well; the temperature of the second incubation is 24 to 56°C, and the time is 1 to 120 minutes.

[0039] After the second incubation, a second wash is performed by adding a wash solution to each well. The wash solution is a phosphate buffer solution at pH 6.8 to pH 7.8 containing Tween-20, wherein the concentration of Tween-20 in the wash solution is 0.01 to 0.05% (v / v). In one embodiment, 10 to 300 μL of the wash solution is added to each well, and then the wash solution is removed and the residual liquid is drained.

[0040] The present invention performs a blocking step after the second washing, and the blocking step comprises adding a blocking solution to each well. The blocking solution comprises the following components: 1-5% (m / v) gelatin, 0.1-2% (m / v) BSA, 0.005-0.01 mol / L of disodium hydrogen phosphate, 0.001-0.005 mol / L of potassium dihydrogen phosphate, and 0.1-0.5 mol / L of sodium chloride. In one embodiment, the specific composition of the blocking solution used is 2% (m / v) gelatin, 1% (m / v) BSA, 0.008 mol / L of disodium hydrogen phosphate, 0.001 mol / L of potassium dihydrogen phosphate, and 0.4 mol / L of sodium chloride. In the present invention, 10 to 300 μL of the blocking solution is added to each well for blocking, such as 10 μL / well, 20 μL / well, 50 μL / well, 100 μL / well, 150 μL / well, 200 μL / well, 2500 μL / well or 300 μL / well; the blocking temperature is 24 to 56°C, such as 24°C, 25°C, 26°C, 30°C, 35°C, 40°C, 42°C, 45°C, 48°C, 50°C, 51°C, 52°C, 53°C, 54°C, 55°C or 56°C; and the blocking time is 1 to 120 min, such as 1 min, 5 min, 10 min, 20 min, 30 min, 40 min, 50 min, 60 min, 70 min, 80 min, 90 min, 100 min, 110 min or 120 min.

[0041] After the first closure, the present invention can be used as a microplate for detecting platelet HPA alloantibodies. After obtaining the microplate, the present invention further includes adding a freeze-drying protective solution for a third incubation and then freeze-drying; the freeze-drying protective solution includes the following components: sodium chloride 0.01-0.1mol / L, potassium chloride 0.001-0.005mol / L, sodium bicarbonate 0.01-0.05mol / L, citric acid 0.001-0.005mol / L, calcium chloride 0.1-1.0mmol / L, sodium acetate 0.01-0.05mol / L, glucose 0.01-0.05mol / L, magnesium sulfate 0.001-0.005mol / L, trisodium citrate 0.001-0.0 15mol / L, trehalose 0.01-0.05mol / L and 1-5% (m / v) BSA. In one embodiment, the lyophilization protection solution used includes the following components: sodium chloride 0.05mol / L, potassium chloride 0.002mol / L, sodium bicarbonate 0.01mol / L, citric acid 0.004mol / L, calcium chloride 1.0mmol / L, sodium acetate 0.05mol / L, glucose 0.01mol / L, magnesium sulfate 0.002mol / L, trisodium citrate 0.001mol / L, trehalose 0.05mol / L and 1% (m / v) BSA.

[0042] According to the present invention, after removing the blocking solution, 10 to 200 μL of freeze-drying protection solution is added, such as 10 μL, 20 μL, 50 μL, 60 μL, 80 μL, 100 μL or 200 μL; and incubated at 24 to 56° C., such as 24° C., 25° C., 26° C., 30° C., 35° C., 40° C., 42° C., 45° C., 48° C., 50° C., 51° C., 52° C., 53° C., 54° C., 55° C. or 56° C.; the incubation time is 10 to 200 min, such as 10 min, 20 min, 50 min, 60 min, 80 min, 90 min, 100 min, 120 min, 150 min, 160 min, 180 min, 190 min or 200 min, and freeze-drying is performed after the incubation.

[0043] The present invention does not particularly limit the freeze-drying method. For example, freeze-drying can be performed by referring to the method shown in Table 1.

[0044] Table 1 Freeze-drying parameters

[0045]

[0046] The present invention increases the adsorption capacity for three mixed platelets through the nonspecific adsorption effect of plant lectins, thereby reducing the missed detection rate and improving the sensitivity of the product; through the blocking effect of the blocking liquid, other nonspecific adsorption sites for platelet antibodies on the solid phase carrier are blocked, and only the specific adsorption sites of platelet-specific antigens (HPA) for platelet HPA alloantibodies are retained, thereby improving the specificity of the product; and through freeze-drying technology, the platelet antigen structure is stabilized, thereby extending the product detection cycle.

[0047] The present invention also provides a kit for detecting platelet HPA alloantibodies, comprising the above-mentioned microplate, indicator red blood cells and anti-human globulin reagent, blocking solution, washing solution, negative quality control and positive quality control.

[0048] The indicator red blood cells of the present invention are a 0.1-2% (V / V) red blood cell suspension. In the specific preparation, three O-type RhD-positive red blood cells are used to prepare a 0.1%-10% (V / V) red blood cell suspension, and the anti-D IgG is diluted 16-4096 times. The red blood cell suspension and the diluted anti-D IgG are mixed in a ratio of 1:1-1:5, and then incubated at 24-56°C. The incubation temperature can be 24°C, 25°C, 26°C, 30°C, 35°C, 40°C, 42°C, 45°C, 48°C, 50°C, 51°C, 52°C, 53°C, 54°C, 55°C or 56°C; the incubation time can be 10-60 min, such as 10 min, 20 min, 30 min, 40 min, 50 min or 60 min. In one embodiment, the incubation used herein is water bath incubation, followed by washing with physiological saline 1 to 5 times, and after washing, a 0.1 to 2% red blood cell suspension is prepared, wherein the centrifugal speed of washing is 100 to 10000 rpm, such as 100 rpm, 200 rpm, 500 rpm, 1000 rpm, 2000 rpm, 3000 rpm, 4000 rpm, 5000 rpm, 6000 rpm, 7000 rpm, 8000 rpm, 9000 rpm or 10000 rpm; the centrifugation time is 1 to 10 min, such as 1 min, 2 min, 3 min, 4 min, 5 min, 6 min, 7 min, 8 min, 9 min or 10 min.

[0049] The blocking solution of the present invention comprises the following components: 1-5% (m / v) gelatin, 0.1-2% (m / v) BSA, 0.005-0.01 mol / L of disodium hydrogen phosphate, 0.001-0.005 mol / L of potassium dihydrogen phosphate, and 0.1-0.5 mol / L of sodium chloride. In one embodiment, the blocking solution used is specifically composed of 2% (m / v) gelatin, 1% (m / v) BSA, 0.008 mol / L of disodium hydrogen phosphate, 0.001 mol / L of potassium dihydrogen phosphate, and 0.4 mol / L of sodium chloride.

[0050] The washing solution of the present invention is a phosphate buffer solution with a pH value of 6.8 to 7.8 and containing Tween-20, wherein the concentration of Tween-20 in the washing solution is 0.01 to 0.05% (v / v).

[0051] The negative quality control described in the present invention is serum or plasma that does not contain platelet HPA alloantibodies.

[0052] The positive quality control of the present invention is serum or plasma containing platelet HPA alloantibodies.

[0053] The present invention also provides a method for detecting platelet HPA alloantibodies for non-diagnostic purposes, comprising the following steps: adding the test sample to the above-mentioned microplate, incubating for a period of time and then washing; adding blocking solution to the washed microplate for secondary blocking, after removing the blocking solution, adding anti-human globulin reagent and indicator red blood cells, and observing the results after centrifugation.

[0054] The complete process of detecting platelet HPA alloantibodies of the present invention is as follows: Figure 1 As shown, the test sample is added to the microplate or the freeze-dried microplate, the amount of the test sample added is 1 to 100 μL / well, and the sample is incubated. The amount of the test sample added can be 1 μL / well, 2 μL / well, 5 μL / well, 10 μL / well, 20 μL / well, 30 μL / well, 40 μL / well, 50 μL / well, 60 μL / well, 70 μL / well, 80 μL / well, 90 μL / well or 100 μL / well; the sample is incubated at a temperature of 24 to 56°C and for a time of 1 to 120 minutes. The exemplary figures for the incubation temperature and time are the same as above and are not repeated here. The test sample of the present invention can be serum or plasma anticoagulated with EDTA or sodium citrate, but samples that are hemolyzed, icteric or high in lipids cannot be used for detection.

[0055] The present invention performs a washing process after the sample is incubated. The washing process includes adding 10 to 300 μL of washing solution to each well, then removing the washing solution, and controlling the residual liquid.

[0056] The present invention performs a secondary blocking after the washing, comprising adding a blocking solution to each well, wherein the amount of the blocking solution added is 10 to 300 μL / well, the temperature of the secondary blocking is 24 to 56°C, and the time is 1 to 120 minutes. The exemplary figures for the amount of blocking solution added, the temperature, and the time in the blocking process are the same as above and will not be repeated here.

[0057] In the present invention, after removing the blocking solution, 10 to 200 μL of anti-human globulin reagent and 10 to 200 μL of 0.1 to 2% indicator red blood cells are added to each well, wherein the amount of the anti-human globulin reagent added can be 10 μL, 20 μL, 50 μL, 60 μL, 80 μL, 100 μL or 200 μL, and the amount of the indicator red blood cells added can be 10 μL, 20 μL, 50 μL, 60 μL, 80 μL, 100 μL or 200 μL; after adding the anti-human globulin reagent and indicator red blood cells, centrifugation is performed, and the centrifugal speed is 100 to 10000 g. The specific exemplary numbers of the centrifugation are the same as above and are not repeated here; the centrifugation time can be 1 to 30 min, such as 1 min, 2 min, 3 min, 4 min, 5 min, 6 min, 7 min, 8 min, 9 min, 10 min, 20 min or 30 min. The results of the present invention can be observed after the centrifugation. If platelet HPA alloantibodies are present, a uniform red bottom phenomenon of red blood cells adhering to the bottom of the well is formed, indicating a positive result; if platelet HPA alloantibodies are absent, red blood cells settle to form a small dot in the center, indicating a negative result.

[0058] It is worth noting that the numbers listed in the specification of the present invention are all exemplary numbers, and of course also include integer numbers not listed between any two given numbers, as well as other non-integer numbers in fractional or decimal form.

[0059] To further illustrate the present invention, a platelet HPA alloantibody detection reagent and detection method provided by the present invention are described in detail below with reference to the examples, but they should not be construed as limiting the scope of protection of the present invention.

[0060] Example 1—Optimization of indicator red blood cell concentration and anti-human globulin titer for detection

[0061] (1) Preparation of platelet suspension: Wash with pH 7.2 phosphate buffer (2.5-10) × 10 11 Three O-type platelets were pooled five times (centrifuged at 2000 g for 10 min) and the platelet concentration was adjusted to 500 × 10 9 / L, centrifuge, discard the supernatant, add an equal volume of pH 5.0 chloroquine phosphate eluent and mix well, react at 24°C for 60 min, shake continuously at 200 rpm during the reaction. After the reaction is completed, wash twice with pH 7.2 phosphate buffer (centrifugation speed is 2000g for 10 min), add an equal volume of pH 7.2 phosphate buffer, mix well and set aside.

[0062] (2) Preparation of indicator red blood cells: Prepare a 10% red blood cell suspension using three O-type RhD-positive red blood cells. Dilute anti-D IgG 4096-fold. Mix the red blood cell suspension and diluted anti-D IgG in a 1:2 ratio and incubate in a 37°C water bath for 10 minutes. Wash three times with physiological saline (centrifugation speed at 3000 rpm for 3 minutes). After washing, prepare red blood cell suspensions of 0.1%, 0.3%, 0.5%, 0.7%, 0.9%, 1.1%, 1.3%, and 1.5%.

[0063] (3) Coating: Add 50 μL of 5 μg / mL lectin coating solution (prepared in carbonate buffer) to each well and coat at 24°C for 120 min.

[0064] (4) Washing: Add 200 μL of pH 7.2 phosphate buffer containing 0.03% (v / v) Tween-20 to each well, then remove the washing solution and drain the remaining liquid.

[0065] (5) Platelet incubation: Add 50 μL of platelet suspension to each well and incubate at 37°C for 30 min.

[0066] (6) Washing: Add 200 μL of pH 7.2 phosphate buffer containing 0.03% (v / v) Tween-20 to each well, then remove the washing solution and drain the remaining liquid.

[0067] (7) Primary blocking: Add 100 μL of blocking solution to each well and block at 24°C for 30 min.

[0068] (8) Freeze-drying: Remove the blocking solution, add 200 μL of freeze-drying protection solution, incubate at 37°C for 30 min, and then freeze-dry.

[0069] (9) Sample incubation: Add 50 μL of the test sample to each well of the freeze-dried microplate and incubate at 37°C for 10 min.

[0070] (10) Washing: Add 200 μL of pH 7.2 phosphate buffer containing 0.03% (v / v) Tween-20 to each well, then remove the washing solution and drain the remaining liquid.

[0071] (11) Secondary blocking: Add 100 μL of blocking solution to each well and block at 24°C for 30 min.

[0072] (12) Detection: Remove the blocking solution, add 100 μL of anti-human globulin reagent (the dilution multiple of the anti-human globulin reagent ranges from 2 to 4096, and is a doubly diluted product) and 100 μL of indicator red blood cells (the concentration of indicator red blood cells is 0.1% to 1.5%) to each well, and centrifuge at 450 g for 5 minutes.

[0073] The results are as follows Figure 4 As shown, by using different concentrations of indicator red blood cells and different dilution multiples of anti-human globulin reagents to conduct orthogonal experiments and testing positive controls and negative controls, the optimal concentration of indicator red blood cells, 0.1%, and the optimal dilution multiple of anti-human globulin reagent, 64 times, were optimized.

[0074] Example 2—Specificity and Sensitivity

[0075] (1) Preparation of platelet suspension: Wash with pH 7.2 phosphate buffer (2.5-10) × 10 11 Three O-type platelets were pooled five times (centrifuged at 2000 g for 10 min) and the platelet concentration was adjusted to 500 × 10 9 / L, centrifuge, discard the supernatant, add an equal volume of pH 5.0 chloroquine phosphate eluent and mix well, react at 24°C for 60 min, shake continuously at 200 rpm during the reaction. After the reaction is completed, wash twice with pH 7.2 phosphate buffer (centrifugation speed is 2000g for 10 min), add an equal volume of pH 7.2 phosphate buffer, mix well and set aside.

[0076] HLA antigen elution was detected by flow cytometry. Figure 2 As shown in Table 2, according to the statistical data of the mean fluorescence intensity in Table 2, the elution rate of HLA antigen is 99.21%.

[0077] Table 2 Flow cytometry statistics (HLA antigens)

[0078] Test tube name P3% parent group P2GeoMeanPE-A Before elution 96.33％ 2635.5 After elution 1.39％ 20.8

[0079] (2) Preparation of indicator red blood cells: Prepare a 10% red blood cell suspension using three O-type RhD-positive red blood cells. Dilute anti-D IgG 4096-fold. Mix the red blood cell suspension and diluted anti-D IgG in a 1:2 ratio and incubate in a 37°C water bath for 10 min. Wash three times with physiological saline (centrifugation speed at 3000 rpm for 3 min). After washing, prepare a 0.1% red blood cell suspension.

[0080] (3) Coating: Add 50 μL of 5 μg / mL lectin coating solution (prepared in carbonate buffer) to each well and coat at 24°C for 120 min.

[0081] (4) Washing: Add 200 μL of pH 7.2 phosphate buffer containing 0.03% (v / v) Tween-20 to each well, then remove the washing solution and drain the remaining liquid.

[0082] (5) Platelet incubation: Add 50 μL of platelet suspension to each well and incubate at 37°C for 30 min.

[0083] (6) Washing: Add 200 μL of pH 7.2 phosphate buffer containing 0.03% (v / v) Tween-20 to each well, then remove the washing solution and drain the remaining liquid.

[0084] (7) Primary blocking: Add 100 μL of blocking solution to each well and block at 24°C for 30 min.

[0085] (8) Freeze-drying: Remove the blocking solution, add 200 μL of freeze-drying protection solution, incubate at 37°C for 30 min, and then freeze-dry.

[0086] (9) Sample incubation: Add 50 μl of the test sample (anticoagulant plasma or serum) to each well of the freeze-dried microplate and incubate at 37°C for 10 min.

[0087] (10) Washing: Add 200 μL of pH 7.2 phosphate buffer containing 0.03% (v / v) Tween-20 to each well, then remove the washing solution and drain the remaining liquid.

[0088] (11) Secondary blocking: Add 100 μL of blocking solution to each well and block at 24°C for 30 min.

[0089] (12) Detection: Remove the blocking solution, add 100 μL of anti-human globulin reagent and 100 μL of 0.1% indicator red blood cells to each well, and centrifuge at 450 g for 5 min.

[0090] The results are as follows Figure 3 As shown, when detecting HPA antibodies, the detection sensitivity before and after elution was consistent, and a 4+ result was still obtained after an 8-fold dilution of the HPA antibodies. When detecting HLA antibodies, a 4+ result was still obtained after a 128-fold dilution of the HLA antibodies before elution, but the test results after elution were all negative. This shows that the method of the present invention can specifically detect platelet HPA alloantibodies. At the same time, the chloroquine phosphate eluent does not affect the HPA antigens on the platelets and does not affect the sensitivity of detecting platelet HPA alloantibodies.

[0091] Example 3 - Testing stability

[0092] The specificity and sensitivity of the reaction plates for HPA antibody detection were compared among the unfreeze-dried reaction plates, the freeze-dried reaction plates, and the freeze-dried reaction plates stored at 2-8°C for 2 months, 4 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, and 12 months, respectively.

[0093] The test steps for the reaction plate that has not been lyophilized are as follows (1) to (7) and (9) to (12);

[0094] The reaction plate detection steps after freeze-drying are as follows (1) to (12):

[0095] (1) Preparation of platelet suspension: Wash with pH 7.2 phosphate buffer (2.5-10) × 10 11 Three O-type platelets were pooled five times (centrifuged at 2000 g for 10 min) and the platelet concentration was adjusted to 500 × 10 9 / L, centrifuge, discard the supernatant, add an equal volume of pH 5.0 chloroquine phosphate eluent and mix well, react at 24°C for 60 min, shake continuously at 200 rpm during the reaction. After the reaction is completed, wash twice with pH 7.2 phosphate buffer (centrifugation speed is 2000g for 10 min), add an equal volume of pH 7.2 phosphate buffer, mix well and set aside.

[0096] (2) Preparation of indicator red blood cells: Prepare a 10% red blood cell suspension using three O-type RhD-positive red blood cells. Dilute anti-D IgG 4096-fold. Mix the red blood cell suspension and diluted anti-D IgG in a 1:2 ratio and incubate in a 37°C water bath for 10 min. Wash three times with physiological saline (centrifugation speed at 3000 rpm for 3 min). After washing, prepare a 0.1% red blood cell suspension.

[0097] (3) Coating: Add 50 μL of 5 μg / mL lectin coating solution (prepared in carbonate buffer) to each well and coat at 24°C for 120 min.

[0098] (4) Washing: Add 200 μL of pH 7.2 phosphate buffer containing 0.03% (v / v) Tween-20 to each well, then remove the washing solution and drain the remaining liquid.

[0099] (5) Platelet incubation: Add 50 μL of platelet suspension to each well and incubate at 37°C for 30 min.

[0100] (6) Washing: Add 200 μL of pH 7.2 phosphate buffer containing 0.03% (v / v) Tween-20 to each well, then remove the washing solution and drain the remaining liquid.

[0101] (7) Primary blocking: Add 100 μL of blocking solution to each well and block at 24°C for 30 min.

[0102] (8) Freeze-drying: Remove the blocking solution, add 200 μL of freeze-drying protection solution, incubate at 37°C for 30 min, and then freeze-dry.

[0103] (9) Sample incubation: Add 50 μL of the test sample to each well of the freeze-dried microplate and incubate at 37°C for 10 min.

[0104] (10) Washing: Add 200 μL of pH 7.2 phosphate buffer containing 0.03% (v / v) Tween-20 to each well, then remove the washing solution and drain the remaining liquid.

[0105] (11) Secondary blocking: Add 100 μL of blocking solution to each well and block at 24°C for 30 min.

[0106] (12) Detection: Remove the blocking solution, add 100 μL of anti-human globulin reagent and 100 μL of 0.1% indicator red blood cells to each well, and centrifuge at 450 g for 5 min.

[0107] The results are as follows Figure 5 As shown, the freeze-dried reaction plates were tested according to the designed stability testing cycle. The specificity and sensitivity of HPA antibody detection in the freeze-dried plates were not reduced compared to the unfreeze-dried plates. The specificity and sensitivity of the freeze-dried plates for HPA antibody detection also remained unchanged after storage for 12 months. This demonstrates that freeze-drying stabilizes the platelet antigen structure, maintaining the sensitivity and specificity of the freeze-dried plates. Furthermore, the freeze-dried plates can be stored at 2-8°C for 12 months.

[0108] Although the above embodiment provides a detailed description of the present invention, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the scope of protection of the present invention.

Claims

1. A method for eluting platelet HLA antigens to prepare a platelet suspension, characterized in that: The following steps are involved: Wash three O-type mixed platelets and adjust the concentration to (10-500)×10 9 / L, after centrifugation, the collected cells are mixed with a chloroquine phosphate eluent of pH 2.0 to pH 5.0, react for a period of time, and then washed again. After centrifugation, the cells are resuspended in physiological saline or phosphate buffer to obtain a platelet suspension.

2. The method according to claim 1, characterized in that The washing includes using physiological saline or pH 6.8 to pH 7.8 phosphate buffer to perform the washing.

3. The method according to claim 1, characterized in that The reaction temperature is 24-37° C., and the reaction time is 1-120 min.

4. A platelet suspension containing HPA antigen prepared by the method according to any one of claims 1 to 3.

5. A microplate coated with HPA antigen, characterized in that: The invention comprises a phytohemagglutinin coating solution and the platelet suspension according to claim 4.

6. The method for preparing the microplate according to claim 5, characterized in that: The following steps are involved: After adding the lectin coating solution to each well of the microplate, a first incubation is performed. After the first wash, the platelet suspension according to claim 4 is added for a second incubation. After the second wash, a blocking solution is added for blocking. Then, a freeze-drying protection solution is added for a third incubation and then freeze-dried to obtain the microplate.

7. The preparation method according to claim 6, characterized in that: The lectin coating solution is a carbonate buffer solution containing lectin; The washing solutions used in the first washing and the second washing are both phosphate buffer containing Tween-20; The blocking solution comprises the following components: 1-5% (m / v) gelatin, 0.1-2% (m / v) BSA, 0.005-0.01 mol / L disodium hydrogen phosphate, 0.001-0.005 mol / L potassium dihydrogen phosphate and 0.1-0.5 mol / L sodium chloride.

8. The preparation method according to claim 6, characterized in that: The freeze-drying protective solution comprises the following components: 0.01-0.1 mol / L of sodium chloride, 0.001-0.005 mol / L of potassium chloride, 0.01-0.05 mol / L of sodium bicarbonate, 0.001-0.005 mol / L of citric acid, 0.1-1.0 mmol / L of calcium chloride, 0.01-0.05 mol / L of sodium acetate, 0.01-0.05 mol / L of glucose, 0.001-0.05 mol / L of magnesium sulfate, 0.001-0.005 mol / L of trisodium citrate, 0.001-0.015 mol / L of trehalose and 1-5% (m / v) of BSA.

9. A kit for detecting platelet HPA alloantibodies, characterized in that: The method comprises the microplate according to claim 5, indicator red blood cells and anti-human globulin reagent, blocking solution, washing solution, negative quality control and positive quality control.

10. A method for detecting platelet HPA alloantibodies for non-diagnostic purposes, characterized in that: The following steps are involved: Adding the sample to be tested to the microplate of claim 5, incubating for a period of time and then washing; After washing, blocking solution was added to the microplate for secondary blocking. After removing the blocking solution, anti-human globulin reagent and indicator red blood cells were added, and the results were observed after centrifugation.

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