An immunochromatographic detection kit for platelet P2Y12 receptor inhibition rate, a preparation method and a detection method

Abstract

The present invention relates to the field of immunochromatographic detection, and specifically to an immunochromatographic detection kit for platelet P2Y12 receptor inhibition rate, a preparation method, and a detection method. The kit comprises: a test strip A and a sample diluent, or in test strip A: the sample diluent, the sample pad, and / or the conjugate pad contain fluorescently labeled platelet surface-specific receptor antibodies, anti-erythrocyte antibodies, ADP, and prostaglandin E1; and a test strip B and a sample diluent, or in test strip B: the sample diluent and / or the sample-conjugate pad contain fluorescently labeled platelet surface-specific receptor antibodies, anti-erythrocyte antibodies, ADP, and prostaglandin E1. The kit has high detection accuracy and specificity, and a comparative test with the "gold standard" turbidimetric method (LTA method) showed that the detection results of the present invention were well consistent with those of the "gold standard."

Description

Technical Field

[0001] The present invention relates to the field of immunochromatographic detection, and in particular to an immunochromatographic detection kit for platelet P2Y12 receptor inhibition rate, a preparation method and a detection method. Background Art

[0002] Platelets play a key role in maintaining normal hemostasis. When exposed to damaged blood vessels, platelets adhere to the exposed subendothelial matrix. Subsequently, various factors (such as thrombin, adenosine diphosphate (ADP), or collagen) released or produced at the site of injury activate platelets. ADP, as an activator, binds to one of the platelet receptors, the P2Y12 receptor, thereby activating platelet activity through the P2Y12 receptor pathway. Following platelet activation, the glycoprotein GPIIb / IIIa receptor on the platelet undergoes a conformational change, allowing it to bind to adhesion proteins such as fibrinogen, leading to the recruitment of neighboring platelets to the site of injury and aggregation to form a hemostatic plug or thrombus.

[0003] Currently, the commonly used antiplatelet drugs on the market are mainly cyclooxygenase-1 inhibitors (mainly represented by aspirin) and P2Y12 receptor inhibitors (represented by clopidogrel, pralidoxime, and ticagrelor). P2Y12 receptor inhibitors block the P2Y12 receptor in a reversible or irreversible manner, preventing platelets from being activated through the P2Y12 receptor pathway. However, due to individual differences in platelet function and drug efficacy, patients taking P2Y12 receptor inhibitors may experience varying degrees of thrombosis, bleeding, and other risks. Therefore, clinical testing of patients' platelet aggregation function related to the P2Y12 receptor pathway or P2Y12 receptor inhibition rate is very necessary to guide their P2Y12 inhibitor treatment.

[0004] At present, the main clinical methods for detecting the degree of platelet P2Y12 receptor inhibition are: transmission turbidimetry (LTA method), electrical impedance method, and thromboelastography method. Among them, the LTA method is used as the "gold standard", but there are no reports on immunochromatographic detection products for the relevant platelet P2Y12 receptor inhibition rate. Summary of the Invention

[0005] Therefore, the technical problem to be solved by the present invention is to provide an immunochromatographic detection kit for platelet P2Y12 receptor inhibition rate, a preparation method and a detection method.

[0006] To this end, the present invention provides the following technical solutions:

[0007] An immunochromatographic detection kit for platelet P2Y12 receptor inhibition rate, comprising: a test strip and a sample diluent; or a test strip;

[0008] The test strip comprises a bottom plate and a sample pad, a conjugate pad, a chromatography membrane and a water-absorbing pad overlapped in sequence on the bottom plate, and the test strip is denoted as test strip A; or

[0009] The test strip comprises a bottom plate and a sample-binding integrated pad, a chromatography membrane and a water-absorbing pad overlapped in sequence on the bottom plate. The test strip is denoted as test strip B.

[0010] The test strip A and the sample diluent or the test strip A:

[0011] The sample diluent, sample pad and / or conjugate pad contain fluorescently labeled platelet surface specific receptor antibodies, anti-erythrocyte antibodies, ADP and prostaglandin E1;

[0012] The chromatographic membrane is provided with a detection line and a quality control line spaced apart from each other, the detection line is coated with fibrinogen, and the quality control line is coated with polyclonal antibodies;

[0013] The test strip B and the sample diluent or the test strip B:

[0014] The sample diluent and / or sample-binding integrated pad contains fluorescently labeled platelet surface specific receptor antibodies, anti-erythrocyte antibodies, ADP and prostaglandin E1;

[0015] The chromatographic membrane is provided with a detection line and a quality control line spaced apart from each other. The detection line is coated with fibrinogen, and the quality control line is coated with polyclonal antibodies.

[0016] Optionally, the platelet surface specific receptor antibody is a P2Y12 receptor antibody, a P-selectin receptor antibody, a prostaglandin receptor antibody, a platelet chemokine receptor antibody and / or a 5-hydroxytryptamine receptor antibody;

[0017] Optionally, the platelet surface specific receptor antibody is a P2Y12 receptor antibody, a P-selectin receptor antibody and / or a 5-hydroxytryptamine receptor antibody;

[0018] Optionally, the platelet surface specific receptor antibody is a P2Y12 receptor antibody and / or a P-selectin receptor antibody.

[0019] Optionally, in the test strip A and the sample diluent, the sample diluent or the sample pad contains fluorescently labeled platelet surface-specific receptor antibodies;

[0020] Optionally, in the test strip A and the sample diluent, the sample diluent contains fluorescently labeled platelet surface-specific receptor antibodies;

[0021] Optionally, in the test strip B and the sample diluent, the sample diluent contains fluorescently labeled platelet surface-specific receptor antibodies;

[0022] Optionally, in the test strip A and the sample diluent, the sample pad and / or the conjugate pad contain anti-erythrocyte antibodies;

[0023] Optionally, in the test strip B and the sample diluent, the sample-binding integrated pad contains anti-erythrocyte antibodies.

[0024] Optionally, the sample diluent further contains 0.01M-0.2M buffer salt, pH 7.4-7.5; 0.2wt%-5wt% protein protectant; 3.2-3.8wt% anticoagulant; 0.2wt%-3wt% thickener; 0.01wt%-0.1wt% preservative and / or 0.01wt%-0.5wt% surfactant;

[0025] Optionally, the buffer salt in the sample diluent is at least one of PBS, TRIS, HEPES, and boric acid;

[0026] Optionally, the protein protective agent is at least one of BSA, casein, gelatin and trehalose;

[0027] Optionally, in the sample diluent, the anticoagulant is sodium citrate;

[0028] Optionally, the thickener is at least one of PVP40, PEG20000 and PVA;

[0029] Optionally, the preservative is at least one of sodium azide, P300, and gentamicin;

[0030] Optionally, the surfactant is at least one of Tween 20, Triton 100, Tween 80, surfactant S9, and surfactant S7;

[0031] Optionally, when the sample diluent contains fluorescently labeled platelet surface specific receptor antibodies, the concentration of the fluorescently labeled platelet surface specific receptor antibodies is 0.01 wt % to 2 wt %.

[0032] A method for preparing the immunochromatographic detection kit for platelet P2Y12 receptor inhibition rate, comprising the preparation of the test strip and the preparation of the sample diluent or the preparation of the test strip; the test strip comprises test strip A or test strip B;

[0033] The preparation of the test strip A comprises:

[0034] soaking the sample pad with a sample pad treatment solution and drying;

[0035] soaking the conjugate pad in a conjugate pad treatment solution and drying the conjugate pad;

[0036] streaking the chromatographic membrane with a streaking solution containing fibrinogen to form a detection line, streaking the chromatographic membrane with a streaking solution containing polyclonal antibodies to form a quality control line, and drying;

[0037] The sample diluent, sample pad treatment solution and / or conjugate pad treatment solution contain fluorescently labeled platelet surface specific receptor antibodies, anti-erythrocyte antibodies, ADP and prostaglandin E1;

[0038] The sample pad, conjugate pad, chromatography membrane and absorbent pad are overlapped in sequence on the bottom plate; or

[0039] The preparation of the test strip B comprises:

[0040] soaking the sample-binding integrated pad with a sample-binding integrated pad treatment solution and drying;

[0041] streaking the chromatographic membrane with a streaking solution containing fibrinogen to form a detection line, streaking the chromatographic membrane with a streaking solution containing polyclonal antibodies to form a quality control line, and drying;

[0042] The sample diluent and / or sample-binding integrated pad treatment solution contains fluorescently labeled platelet surface specific receptor antibodies, anti-erythrocyte antibodies, ADP and prostaglandin E1;

[0043] The sample-binding integrated pad, the chromatography membrane and the water-absorbing pad are overlapped in sequence on the bottom plate.

[0044] Optionally, when the sample diluent contains fluorescently labeled platelet surface specific receptor antibodies, the concentration of the fluorescently labeled platelet surface specific receptor antibodies is 0.01 wt% to 2 wt%; and / or

[0045] When the sample pad treatment solution contains fluorescently labeled platelet surface specific receptor antibodies, the concentration of the fluorescently labeled platelet surface specific receptor antibodies is 0.01 wt% to 2 wt%; and / or

[0046] When the conjugate pad treatment solution contains fluorescently labeled platelet surface specific receptor antibodies, the concentration of the fluorescently labeled platelet surface specific receptor antibodies is 0.01 wt % to 2 wt %; and / or

[0047] When the sample-binding integrated pad treatment solution contains fluorescently labeled platelet surface specific receptor antibodies, the concentration of the fluorescently labeled platelet surface specific receptor antibodies is 0.01 wt % to 2 wt %; and / or

[0048] In the test strip A and the sample diluent, the sample pad and / or the conjugate pad contain anti-erythrocyte antibodies; and / or

[0049] In the test strip B and the sample diluent, the sample-binding integrated pad contains anti-erythrocyte antibodies.

[0050] Optionally, when the sample pad treatment fluid contains fluorescently labeled platelet surface-specific receptor antibodies, the sample pad treatment fluid contains an ADP concentration of 1-20 mM, a prostaglandin E1 concentration of 0.001 to 1 μM, and an anti-erythrocyte antibody concentration of 0.01 to 0.25 mg / mL;

[0051] Optionally, when the sample pad treatment fluid contains fluorescently labeled platelet surface-specific receptor antibodies, the sample pad treatment fluid further contains 0.01 to 0.5 M buffer salt, pH 7.4 to 7.5; 0.2 wt% to 0.5 wt% protein protectant; 0.01 wt% to 0.05 wt% preservative; 0.01 wt% to 0.5 wt% thickener; and / or 0.01 wt% to 0.2 wt% surfactant;

[0052] Optionally, when the sample pad treatment solution does not contain fluorescently labeled platelet surface-specific receptor antibodies, the sample pad treatment solution contains an ADP concentration of 1-20 mM, a prostaglandin E1 concentration of 0.001 to 1 μM, and an anti-erythrocyte antibody concentration of 0.01 to 0.5 mg / mL;

[0053] Optionally, when the sample pad treatment solution does not contain fluorescently labeled platelet surface-specific receptor antibodies, the sample pad treatment solution further contains 0.01 to 0.2 M buffer salt, pH 7.4 to 7.5; 0.2 wt% to 2 wt% protein protectant; 0.01 wt% to 0.05 wt% preservative; 0.01 wt% to 0.5 wt% thickener; and / or 0.01 wt% to 0.5 wt% surfactant;

[0054] Optionally, the buffer salt in the sample pad treatment solution is at least one of PBS, Tris, and HEPES;

[0055] Optionally, the protein protective agent in the sample pad treatment solution is at least one of BSA, casein and gelatin;

[0056] Optionally, the preservative in the sample pad treatment solution is at least one of sodium azide, P300, and gentamicin;

[0057] Optionally, the thickener in the sample pad treatment solution is at least one of PVP-40, NMP, PEG40000, PVA, and polylysine;

[0058] The surfactant in the sample pad treatment solution is at least one of S7, S9, Tween 20, Tween 80, and Triton;

[0059] Optionally, the concentration of the anti-erythrocyte antibody contained in the conjugate pad treatment solution is 0.01 to 0.5 mg / mL;

[0060] Optionally, the conjugate pad treatment solution further contains 0.01-0.2M buffer salt, pH 7.4-7.5; 1wt%-20wt% of carbohydrate; 0.01wt%-5wt% of thickener; and / or 0.01wt%-0.1wt% of surfactant;

[0061] Optionally, the buffer salt in the conjugate pad treatment solution is at least one of PBS, Tris, and HEPES;

[0062] Optionally, the sugar substance in the conjugate pad treatment solution is at least one of trehalose, sucrose, lactose, maltose, and glucose;

[0063] Optionally, the thickener in the conjugate pad treatment solution is at least one of PVP-40, NMP, PEG40000, PVA, and polylysine;

[0064] Optionally, the surfactant in the conjugate pad treatment solution is at least one of S7, S9, Tween 20, Tween 80, and Triton;

[0065] Optionally, in the fibrinogen-containing streaking solution, the concentration of fibrinogen is 0.5 to 2.0 mg / mL, and the rate value is 0.5 to 0.8 μL / mm;

[0066] Optionally, in the streaking solution containing polyclonal antibodies, the concentration of the polyclonal antibodies is 0.5 to 2.0 mg / mL, and the rate value is 0.5 to 1.0 μL / mm;

[0067] Optionally, the stripping solution further contains 0.01-0.1M buffer salt, pH 7.4-7.5; 1v / v%-5v / v% alcohol; 0.01wt%-0.05wt% carbohydrate; 0.001wt%-0.05wt% protein protective agent; and / or 0.001wt%-0.05wt% surfactant;

[0068] Optionally, the buffer salt in the streaking solution is at least one of PBS, HEPES, TRIS, and boric acid;

[0069] Optionally, the alcohol substance in the stripping solution is at least one of methanol, ethanol, and propylene glycol;

[0070] Optionally, the sugar substance in the streaking solution is at least one of trehalose, sucrose, lactose, maltose, and glucose;

[0071] Optionally, the protein protective agent in the streaking solution is at least one of BSA, casein, and gelatin;

[0072] Optionally, the surfactant in the stripping solution is at least one of S7, S9, Tween 20, Tween 80, and Triton.

[0073] Optionally, the sample-binding integrated pad treatment solution contains an ADP concentration of 1-20 mM, a prostaglandin E1 concentration of 0.001 to 1 μM, and an anti-erythrocyte antibody concentration of 0.01 to 0.5 mg / mL;

[0074] Optionally, the sample-binding integrated pad treatment solution further contains 0.01-0.2M buffer salt, pH 7.4-7.5; 1wt%-15wt% carbohydrate; 0.01wt%-0.05wt% preservative; 0.001wt%-0.05wt% protein protectant, 0.01wt%-5wt% thickener; and / or 0.01wt%-0.1wt% surfactant;

[0075] Optionally, the buffer salt in the sample-binding integrated pad treatment solution is at least one of PBS, HEPES, TRIS, and boric acid;

[0076] Optionally, the sugar substance in the sample-binding integrated pad treatment solution is at least one of trehalose, sucrose, lactose, maltose, and glucose;

[0077] Optionally, the preservative in the sample-binding integrated pad treatment solution is at least one of sodium azide, P300, and gentamicin;

[0078] Optionally, the protein protective agent in the sample-binding integrated pad treatment solution is at least one of BSA, casein, and gelatin;

[0079] Optionally, the thickener in the sample-binding integrated pad treatment solution is at least one of PVP-40, NMP, PEG40000, PVA, and polylysine;

[0080] Optionally, the surfactant in the sample-binding integrated pad treatment solution is at least one of S7, S9, Tween 20, Tween 80, and Triton;

[0081] Optionally, in the fibrinogen-containing streaking solution, the concentration of fibrinogen is 0.5 to 2.0 mg / mL, and the rate (spray volume) value is 0.5 to 0.8 μL / mm;

[0082] Optionally, in the streaking solution containing polyclonal antibodies, the concentration of the polyclonal antibodies is 0.5 to 2.0 mg / mL, and the rate value is 0.5 to 1.0 μL / mm;

[0083] Optionally, the stripping solution further contains 0.01-0.1M buffer salt, pH 7.5; 1v / v%-5v / v% alcohol; 0.01wt%-0.05wt% carbohydrate; 0.001wt%-0.05wt% protein protective agent and / or 0.001wt%-0.05wt% surfactant;

[0084] Optionally, the buffer salt in the streaking solution is at least one of PBS, HEPES, TRIS, and boric acid;

[0085] Optionally, the alcohol substance in the stripping solution is at least one of methanol, ethanol, and propylene glycol;

[0086] Optionally, the sugar substance in the streaking solution is at least one of trehalose, sucrose, lactose, maltose, and glucose;

[0087] Optionally, the protein protective agent in the streaking solution is at least one of BSA, casein, and gelatin;

[0088] Optionally, the surfactant in the stripping solution is at least one of S7, S9, Tween 20, Tween 80, and Triton.

[0089] A method for immunochromatographic detection of platelet P2Y12 receptor inhibition rate comprises the immunochromatographic detection kit for platelet P2Y12 receptor inhibition rate or the immunochromatographic detection kit for platelet P2Y12 receptor inhibition rate prepared by the preparation method.

[0090] Optional, including:

[0091] Quantitative detection method:

[0092] Take the sample to be tested and add it to the sample diluent or not, then add it dropwise to the sample pad of test strip A or the sample-binding integrated pad of test strip B. After 3-10 minutes, detect the fluorescence signals of the test line and the quality control line respectively, and calculate the platelet P2Y12 receptor inhibition rate using the following formula:

[0093] R = [1-T / (T+C)] × 100%;

[0094] In the formula, R represents the platelet P2Y12 receptor inhibition rate;

[0095] T represents the fluorescence signal of the detection line;

[0096] C represents the fluorescence signal of the quality control line;

[0097] Qualitative detection method:

[0098] Take the sample to be tested and add it to the sample diluent or not, then add it dropwise to the sample pad of test strip A or the sample-binding integrated pad of test strip B. After 3-10 minutes, compare the fluorescence signals of the test line and the quality control line, and interpret them according to the reference color card;

[0099] Optionally, the sample to be tested includes a whole blood sample, concentrated platelets, platelet-containing plasma, a diluted whole blood sample, a diluted concentrated platelet, or a diluted platelet-containing plasma;

[0100] Optionally, the sample to be tested contains platelet-containing plasma or diluted platelet-containing plasma.

[0101] The technical solution of the present invention has the following advantages:

[0102] 1. The present invention provides an immunochromatographic detection kit for platelet P2Y12 receptor inhibition rate, comprising: a test strip and a sample diluent; or a test strip; the test strip comprises a bottom plate and a sample pad, a conjugation pad, a chromatographic membrane and a water-absorbing pad overlapped in sequence on the bottom plate, and the test strip is referred to as test strip A; or the test strip comprises a bottom plate and a sample-binding integrated pad, a chromatographic membrane and a water-absorbing pad overlapped in sequence on the bottom plate, and the test strip is referred to as test strip B; the test strip A and the sample diluent or the test strip A: the sample diluent, the sample pad and / or the conjugation pad contain fluorescently labeled platelet surface specific receptor antibodies, anti-erythrocyte Antibody, ADP and prostaglandin E1; the chromatographic membrane is provided with a detection line and a quality control line spaced apart, the detection line is coated with fibrinogen, and the quality control line is coated with polyclonal antibodies; in the test strip B and the sample diluent: the sample diluent and / or the sample-binding integrated pad contains fluorescently labeled platelet surface specific receptor antibodies, anti-erythrocyte antibodies, ADP and prostaglandin E1; the chromatographic membrane is provided with a detection line and a quality control line spaced apart, the detection line is coated with fibrinogen, and the quality control line is coated with polyclonal antibodies; in the above-mentioned kit, the present invention has found through research that: about 3% to 5% of patients have platelet surface specific receptor antibodies due to platelet surface specific receptor antibodies. The platelet P2Y1 receptor is non-specifically activated by ADP, resulting in insufficient platelet inhibition in platelet function tests. After the addition of prostaglandin E1 (PGE1), the platelet aggregation rate is significantly reduced. Therefore, in the kit of the present invention, in the test strip A, the sample diluent, the sample pad and / or the conjugation pad contain the platelet P2Y12 receptor activation pathway activator ADP, and at the same time contain prostaglandin E1 to shield the activation of the platelet P2Y1 receptor by ADP, thereby ensuring the specificity of ADP activating the P2Y12 receptor. When the test is to be tested, after the sample is added, on the sample pad and / or the conjugation pad, ADP and prostaglandin E1 diffuse with the sample, and the platelet P2Y12 receptor is activated. The platelets in the sample are specifically activated by ADP through the P2Y12 receptor pathway. The activated platelets are captured by the coated fibrinogen on the test line, and the unactivated platelets are captured by the coated polyclonal antibodies on the quality control line. Since the sample diluent or sample pad or conjugate pad contains fluorescently labeled platelet surface-specific receptor antibodies, the fluorescently labeled platelet surface-specific receptor antibodies can fluorescently label the activated and unactivated platelets in the sample to be tested. Therefore, the test results can be signaled by collecting the fluorescent signals on the test line and the quality control line, and qualitative or quantitative detection can be performed based on the fluorescent signals on the test line and the quality control line. The same is true for test strip B.

[0103] In summary, the kit of the present invention has high detection accuracy and specificity. A comparative test was conducted with the "gold standard" turbidimetry (LTA method). The comparative results showed that the detection results of the present invention were well consistent with those of the "gold standard".

[0104] 2. The present invention provides an immunochromatographic detection kit for platelet P2Y12 receptor inhibition rate, wherein the platelet surface-specific receptor antibody is a P2Y12 receptor antibody, a P-selectin receptor antibody, a prostaglandin receptor antibody, a platelet chemokine receptor antibody, and / or a 5-hydroxytryptamine receptor antibody. After long-term research, the present invention specifically selects the above-mentioned platelet surface-specific receptor antibodies, and uses platelet surface-specific receptor antibodies labeled with fluorescent substances to achieve fluorescent labeling of platelets, thereby ensuring that both activated and unactivated platelets can be fluorescently labeled, thereby signaling the test results, and further enabling qualitative and quantitative analysis of the test results.

[0105] Furthermore, anti-erythrocyte antibodies are coated on the sample pad and / or the binding pad. The anti-erythrocyte antibodies can bind to the red blood cells in the whole blood sample to be tested, intercept the red blood cells, and prevent the red blood cells from being chromatographed onto the chromatographic membrane to generate background signals, affecting the detection of the test line and quality control line signals, thereby further improving the accuracy of the test. The same is true for test strip B. BRIEF DESCRIPTION OF THE DRAWINGS

[0106] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the specific embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0107] Figure 1 Schematic diagram of the structure of the test strip in Example 1 of the present invention;

[0108] Figure 2 Qualitative testing color chart;

[0109] Figure 3 is a graph showing the relationship between the aggregation rate by the LTA method in Experimental Example 1 of the present invention and the inhibition rate detected in Example 3;

[0110] Figure 4 is a graph showing the relationship between the aggregation rate by the LTA method in Experimental Example 1 of the present invention and the inhibition rate detected in Example 6;

[0111] Figure 5 is a graph showing the relationship between the aggregation rate by the LTA method in Experimental Example 1 of the present invention and the inhibition rate detected in Example 9;

[0112] Figure 6 is a graph showing the relationship between the aggregation rate by the LTA method in Experimental Example 1 of the present invention and the inhibition rate detected in Example 12;

[0113] Figure 7This is a graph showing the relationship between the aggregation rate using the LTA method in Experimental Example 1 of the present invention and the inhibition rate detected in Example 15.

[0114] Reference numerals:

[0115] 1-sample pad, 2-binding pad, 3-detection line, 4-quality control line, 5-chromatographic membrane, 6-absorbent pad, 7-substrate. DETAILED DESCRIPTION

[0116] The following examples are provided for a better understanding of the present invention and are not intended to limit the best mode of implementation. They do not limit the content and scope of protection of the present invention. Any product identical or similar to the present invention obtained by anyone under the guidance of the present invention or by combining the features of the present invention with other prior arts shall fall within the scope of protection of the present invention.

[0117] If no specific experimental steps or conditions are specified in the examples, the conventional experimental steps or conditions described in the literature in this field can be used. If the manufacturer of the reagents or instruments is not specified, they are all commercially available conventional reagents.

[0118] In the present invention, PVP-40 is poly (N-vinyl butyrolactam) in Chinese; BSA is bovine serum albumin in Chinese; surfactant S9 is Tetronic 1307; PBS is phosphate buffered saline; TRIS is tris (hydroxymethyl)aminomethane buffer; HEPES is 4-hydroxyethylpiperazineethanesulfonic acid; PEG20000 is polyethylene glycol 20000; PVA is polyvinyl alcohol; and NMP is N-methylpyrrolidone.

[0119] The anti-erythrocyte antibodies in the following examples were purchased from Feipeng Biotechnology Co., Ltd.;

[0120] Fluorescently labeled P2Y12 receptor antibody, fluorescently labeled P-selectin receptor antibody, fluorescently labeled prostaglandin receptor antibody, fluorescently labeled platelet chemokine receptor antibody, fluorescently labeled 5-hydroxytryptamine receptor antibody, and polyclonal antibody (goat anti-mouse antibody) used for quality control line were purchased from Feipeng Biotechnology Co., Ltd.

[0121] Fibrinogen was purchased from Sigma.

[0122] Examples 1-9

[0123] The sample dilution formulas for Examples 1-9 are shown in the table below.

[0124] Table 1. Concentration of each component in the sample diluent formula

[0125]

[0126]

[0127] Table 1. Specific composition of each component in the sample diluent formula

[0128]

[0129] Note: P1 represents PEG20000, P2 represents PVP-40, and P3 represents PVA; P1P2 represents PEG20000 and PVP-40 in a mass ratio of 4:1; P1P2P3 represents PEG20000, PVP-40 and PVA in a mass ratio of 1:1:1; P2P3 represents PVP-40 and PVA in a mass ratio of 1:1.

[0130] The formulas of the sample pad treatment solutions in Examples 1 to 9 are shown in the table below.

[0131] Table 2 Concentration of each component in the sample pad treatment solution formula

[0132]

[0133]

[0134] Table 2. Specific composition of each component in the sample pad treatment solution formula

[0135]

[0136]

[0137] The formulations of the conjugate pad treatment solutions of Examples 1 to 9 are shown in the table below.

[0138] Table 3 Concentration of each component in the conjugate pad treatment solution formulation

[0139]

[0140]

[0141] Table 3. Specific ingredients of each component in the combination pad treatment solution formula

[0142]

[0143]

[0144] The formulations of the stripping solutions used for the test lines of Examples 1 to 9 are shown in the table below.

[0145] Table 4. Concentration of each component in the formulation of the test line membrane solution

[0146]

[0147] Table 4. Specific ingredients of each component in the formulation of the test line stripping solution

[0148]

[0149]

[0150] The formulas of the streaking solutions for the quality control lines of Examples 1 to 9 are shown in the table below.

[0151] Table 5. Concentration of each component in the formula of the quality control line stripping solution

[0152]

[0153] Table 5. Specific ingredients of each component in the formula of the quality control line membrane solution

[0154]

[0155]

[0156] Example 1 A method for preparing an immunochromatographic detection kit for platelet P2Y12 receptor inhibition rate comprises the following steps:

[0157] The sample diluent was prepared according to the sample diluent formula in the above table, and the solvent was sterile purified water.

[0158] A method for preparing a test strip, comprising:

[0159] Sample pad preparation: Prepare the sample pad treatment solution according to the formula in the table above, using sterile purified water as the solvent. Then, immerse the entire sample pad (made of glass fiber) in the prepared sample pad treatment solution for 5 minutes. Then, carefully remove it and place it on a flat carrier. Transfer it to an electric blast drying oven at 45°C for 72 hours.

[0160] Preparation of the conjugate pad: Prepare the conjugate pad treatment solution according to the formula in the table above, using sterile purified water as the solvent. Then, immerse the entire conjugate pad (made of glass fiber) in the prepared conjugate pad treatment solution for 5 minutes. Then, carefully remove it and place it on a flat carrier. Transfer it to an electric blast drying oven at 45°C for 72 hours.

[0161] Preparation of nitrocellulose membrane (NC membrane): Prepare the stripping solution for the test line according to the table above, using sterile water as the solvent; prepare the stripping solution for the quality control line according to the table above, using sterile water as the solvent; select the coated surface of the nitrocellulose membrane and mark it. Apply the stripping solution for the test line and quality control line in parallel and evenly on the membrane, with a spacing of 4-6mm between the test line and the quality control line. The NC membrane stripping speed is 70s / 4cm. After stripping, carefully remove the membrane and place it on a flat carrier plate. Transfer it to an electric forced air drying oven at 45°C for 72h.

[0162] Assembly, strip cutting and assembly: The sample pad (15 mm long), conjugation pad (10 mm long), NC membrane (25 mm), and absorbent pad (30 mm) are overlapped in sequence (the overlap width of the sample pad and conjugation pad, and the overlap width of the conjugation pad and NC membrane is 2 mm; the overlap width of the NC membrane and the absorbent pad is 4 mm) and pasted on the substrate to assemble the large plate. The detection line is about 10 mm away from the edge of the conjugation pad on the side close to the detection line, and the quality control line is about 10 mm away from the edge of the absorbent pad on the side close to the quality control line. The finished large plate is cut into narrow strips with a width of 4.0 mm. The schematic diagram of the prepared test strip structure is shown in the figure. Figure 1 As shown, it can then be assembled on the card box or card sleeve of the test strip.

[0163] An immunochromatographic detection method for platelet P2Y12 receptor inhibition rate is performed using the above-mentioned kit. 50 μL of the sample to be tested is added to the sample diluent, mixed for 1 minute, and then dripped onto the sample pad. The time is set for 5 minutes, and the reading and result are reported by the supporting equipment (fluorescence immunoassay analyzer LEPU Quant-Fluo800).

[0164] The fluorescence signals of the test line and the quality control line were recorded respectively, and the platelet P2Y12 receptor inhibition rate was calculated by the following formula:

[0165] R = [1-T / (T+C)] × 100%;

[0166] In the formula, R represents the platelet P2Y12 receptor inhibition rate;

[0167] T represents the fluorescence signal of the detection line;

[0168] C represents the fluorescence signal of the quality control line.

[0169] Qualitative detection, compare the fluorescence signals of the test line and the quality control line, according to Figure 2 Refer to the reference color card shown. Figure 2 The percentages in the table represent the platelet P2Y12 receptor inhibition rate.

[0170] Example 2

[0171] The difference between this embodiment and embodiment 1 is that in the preparation of the sample pad: the sample pad is polyester, the soaking time is 10 minutes, the drying temperature is 65° C., and the drying time is 12 hours.

[0172] In the preparation of the conjugate pad: the conjugate pad is polyester, the soaking time is 10 minutes, the drying temperature is 65°C, and the drying time is 12 hours.

[0173] During the preparation of the NC membrane, the NC membrane climbing speed was 140 s / 4 cm, the drying temperature was 65°C, and the drying time was 12 h.

[0174] Panel assembly, strip cutting and assembly: Cut the finished large panels into 3.5mm narrow strips.

[0175] In the detection method: take 30μL of the sample to be tested and add it to the sample diluent, mix it for 5 minutes, and then add it dropwise to the sample pad. The time is 3 minutes, and the supporting equipment (fluorescence immunoassay analyzer LEPU Quant-Fluo 800) reads and reports the results.

[0176] Example 3

[0177] The difference between this embodiment and embodiment 1 is that in the preparation of the sample pad: the sample pad is polyester, the soaking time is 8 minutes, the drying temperature is 55° C., and the drying time is 42 hours.

[0178] In the preparation of the conjugate pad: the conjugate pad is polyester, the soaking time is 8 minutes, the drying temperature is 55°C, and the drying time is 42 hours.

[0179] During the preparation of the NC membrane, the NC membrane climbing speed was 105s / 4cm, the drying temperature was 55°C, and the drying time was 42h.

[0180] Panel assembly, strip cutting and assembly: Cut the finished large panels into 3.8mm narrow strips.

[0181] In the detection method: take 40μL of the sample to be tested and add it to the sample diluent, mix it for 3 minutes, and then add it dropwise to the sample pad. The time is 4 minutes, and the supporting equipment (fluorescence immunoassay analyzer LEPU Quant-Fluo800) reads and reports the results.

[0182] The difference between Example 4 and Example 7 and Example 1 is that in the detection method: 100-140 μL of the sample to be tested is added to the sample diluent or directly dripped into the sample addition well (in this embodiment, it is selected to be added to the sample diluent first), dripped into the sample addition well, and timed for 3-10 minutes. The supporting equipment reads the data and reports the results.

[0183] The difference between Example 5 and Example 8 and Example 2 is that in the detection method: 100-140 μL of the sample to be tested is added to the sample diluent or directly dripped into the sample addition well (in this embodiment, it is selected to be added to the sample diluent first), dripped into the sample addition well, and timed for 3-10 minutes. The supporting equipment reads the data and reports the results.

[0184] The difference between Example 6 and Example 9 and Example 3 is that in the detection method: 100-140 μL of the sample to be tested is added to the sample diluent or directly dripped into the sample addition well (in this embodiment, it is selected to be added to the sample diluent first), dripped into the sample addition well, and timed for 3-10 minutes. The supporting equipment reads the data and reports the results.

[0185] The sample dilution formulas for Examples 10-15 are shown in the table below.

[0186] Table 6. Concentration of each component in the sample diluent formula

[0187]

[0188] Table 6, continued, specific composition of each component in the sample diluent formula

[0189]

[0190]

[0191] Note: P1 represents PEG20000, P2 represents PVP-40, and P3 represents PVA; P1P2 represents PEG20000 and PVP-40 in a mass ratio of 4:1; P1P2P3 represents PEG20000, PVP-40 and PVA in a mass ratio of 1:1:1.

[0192] The formula of the sample-combined integrated pad treatment solution in Examples 10-15 is shown in the table below.

[0193] Table 7 Concentration of each component in the sample-combined integrated pad treatment solution formula

[0194]

[0195]

[0196] Table 7, continued. Specific ingredients of each component in the sample-combined integrated pad treatment solution formula

[0197]

[0198]

[0199] The formulations of the stripping solutions for the test lines of Examples 10 to 15 are shown in the table below.

[0200] Table 8. Concentration of each component in the formulation of the test line membrane solution

[0201]

[0202] Table 8 Continued. Specific ingredients of each component in the formulation of the test line stripping solution

[0203]

[0204]

[0205] The formulas of the streaking solutions for the quality control lines of Examples 10 to 15 are shown in the table below.

[0206] Table 9. Concentration of each component in the formula of the quality control line stripping solution

[0207]

[0208] Table 9. Specific composition of each component in the formula of the quality control line membrane solution

[0209]

[0210]

[0211] The difference between Example 10 and Example 1 is that in the sample pad preparation: the sample pad and the conjugate pad are combined into one pad, called a sample-conjugate integrated pad, and treated with a sample-conjugate integrated pad treatment solution;

[0212] The conjugate pad preparation step is omitted;

[0213] Panel assembly, strip cutting, and assembly: Assemble a large panel by overlapping the sample-combination integrated pad (25 mm long), NC membrane (25 mm), and absorbent pad (30 mm) in this order (the overlap width of the sample pad-combination integrated pad and the NC membrane is 3 mm; the overlap width of the NC membrane and the absorbent pad is 4 mm). The test line is about 10 mm from the edge of the sample-combination integrated pad on the side closest to the test line, and the quality control line is about 10 mm from the edge of the absorbent pad on the side closest to the quality control line. Cut the finished large panel into narrow strips with a width of 4.0 mm.

[0214] The difference between Example 11 and Example 2 is that in the sample pad preparation: the sample pad and the conjugate pad are combined into one pad, called a sample-conjugate integrated pad, and treated with a sample-conjugate integrated pad treatment solution;

[0215] The conjugate pad preparation step is omitted;

[0216] Panel assembly, strip cutting and assembly: same as Example 10.

[0217] The difference between Example 12 and Example 3 is that in the sample pad preparation: the sample pad and the conjugate pad are combined into one pad, called a sample-conjugate integrated pad, and treated with a sample-conjugate integrated pad treatment solution;

[0218] The conjugate pad preparation step is omitted;

[0219] Panel assembly, strip cutting and assembly: same as Example 10.

[0220] The difference between Example 13 and Example 4 is that in the sample pad preparation: the sample pad and the conjugate pad are combined into one pad, called a sample-conjugate integrated pad, and treated with a sample-conjugate integrated pad treatment solution;

[0221] The conjugate pad preparation step is omitted;

[0222] Panel assembly, strip cutting and assembly: same as Example 10.

[0223] The difference between Example 14 and Example 5 is that in the preparation of the sample pad: the sample pad and the conjugate pad are combined into one pad, called a sample-conjugate integrated pad, and treated with a sample-conjugate integrated pad treatment solution;

[0224] The conjugate pad preparation step is omitted;

[0225] Panel assembly, strip cutting and assembly: same as Example 10.

[0226] The difference between Example 15 and Example 6 is that in the preparation of the sample pad: the sample pad and the conjugate pad are combined into one pad, called a sample-conjugate integrated pad, and treated with a sample-conjugate integrated pad treatment solution;

[0227] The conjugate pad preparation step is omitted;

[0228] Panel assembly, strip cutting and assembly: same as Example 10.

[0229] Experimental Example 1

[0230] The kits and detection methods in Examples 3, 6, 9, 12, and 15 of the present invention were used for comparative testing with the "gold standard" turbidimetry (LTA method). The Helena AggRAM platelet aggregation analysis system (four-channel) was selected for the LTA method. The samples to be tested were 40 samples (sodium citrate anticoagulated venous whole blood samples). The inhibition rate obtained by the present invention was used as the horizontal axis, and the inhibition rate obtained by the LTA method was used as the vertical axis. The fitting curves were as follows: Figure 3-Figure 7 As shown in the figure, we can see that R 2 =0.974, R 2 =0.9626, R 2 =0.9303, R 2 =0.9337, R2 =0.9159, indicating that the present invention has good consistency with the "gold standard".

[0231] Experimental Example 2 Specificity

[0232] The kits and detection methods in Examples 3, 6, 9, 12, and 15 of the present invention were used to perform a specificity comparison test with the "gold standard" turbidimetry (LTA method). The Helena AggRAM platelet aggregation analysis system (four channels) was selected for the LTA method. The samples to be tested were 5 samples (sodium citrate anticoagulated venous whole blood samples). The test results are shown in the following table. By comparing whether PGE1 was added or not, it was judged that when PGE1 was added, the activator ADP activated the platelets through the P2Y1 pathway and was shielded, so that the platelets would not be pseudo-activated. The higher the aggregation rate, the better the platelet activity of the P2Y12 receptor pathway. The higher the inhibition rate of the present invention, the worse the platelet activity of the P2Y12 receptor pathway. The two are inversely proportional. As can be seen from the following table, the ADP+PGE1 aggregation rate of the LTA method and the inhibition rate of the present invention are close to each other in inverse proportion, indicating that the present invention has good specificity compared with the "gold standard".

[0233] Table 10 Specificity experiment of Example 3

[0234]

[0235] Table 11 Example 6 Specificity Experiment

[0236]

[0237] Table 12 Example 9 specificity experiment

[0238]

[0239] Table 13 Example 12 Specificity Experiment

[0240]

[0241] Table 14 Example 15 Specificity Experiment

[0242]

[0243] Obviously, the above embodiments are merely examples for clarity of explanation and are not intended to limit the implementation methods. Those skilled in the art will readily appreciate that other variations or modifications based on the above descriptions are possible. It is not necessary and impossible to enumerate all implementation methods here. Obvious variations or modifications arising therefrom remain within the scope of the present invention.

Claims

1. A method for preparing an immunochromatographic detection kit for platelet P2Y12 receptor inhibition rate, characterized in that: include: Methods for preparing test strips and sample diluents; The test strip comprises a bottom plate and a sample pad, a conjugate pad, a chromatography membrane and a water-absorbing pad overlapped in sequence on the bottom plate, and the test strip is denoted as test strip A; or The test strip comprises a bottom plate and a sample-binding integrated pad, a chromatography membrane and a water-absorbing pad overlapped in sequence on the bottom plate. The test strip is denoted as test strip B. In the test strip A and sample diluent: The sample diluent comprises: PBS, 0.1 mol / L, pH 7.4; BSA, 2 wt%; sodium citrate, 3.5 wt%; PEG2000, 1.2 wt%; PVP-40, 0.3 wt%; sodium azide, 0.05 wt%; Tween 20, 0.25 wt%; fluorescently labeled platelet surface specific receptor antibody P2Y12, 1 wt%; The sample pad was soaked in a sample pad treatment solution and dried; the sample pad treatment solution contained: PBS, 0.1 mol / L, pH 7.5; BSA, 1 wt%; sodium azide, 0.03 wt%; PVP-40, 0.25 wt%; S9, 0.25 wt%; ADP, 10 mM; prostaglandin E1, 0.5 μM; anti-erythrocyte antibody, 0.25 mg / mL; soaking the conjugate pad in a conjugate pad treatment solution and drying the conjugate pad; streaking the chromatographic membrane with a streaking solution containing fibrinogen to form a detection line, streaking the chromatographic membrane with a streaking solution containing polyclonal antibodies to form a quality control line, and drying; The sample pad, conjugate pad, chromatography membrane and absorbent pad are overlapped in sequence on the bottom plate; In the test strip B and sample diluent: The sample diluent comprises: PBS, 0.1 mol / L, pH 7.4; BSA, 2 wt%; sodium citrate, 3.5 wt%; PEG2000, 1.2 wt%; PVP-40, 0.3 wt%; sodium azide, 0.05 wt%; Tween 20, 0.25 wt%; fluorescently labeled platelet surface specific receptor antibody P2Y12, 1 wt%; The sample-binding integrated pad was soaked in a sample-binding integrated pad treatment solution and dried; the sample-binding integrated pad treatment solution contained: PBS, 0.1 mol / L, pH 7.5; BSA, 1 wt%; sodium azide, 0.03 wt%; PVP-40, 0.25 wt%; S9, 0.25 wt%; ADP, 10 mM; prostaglandin E1, 0.5 μM; and anti-erythrocyte antibody, 0.25 mg / mL; streaking the chromatographic membrane with a streaking solution containing fibrinogen to form a detection line, streaking the chromatographic membrane with a streaking solution containing polyclonal antibodies to form a quality control line, and drying; The sample-binding integrated pad, the chromatography membrane and the water-absorbing pad are overlapped in sequence on the bottom plate.

2. The method for preparing the immunochromatographic detection kit for platelet P2Y12 receptor inhibition rate according to claim 1, characterized in that: The concentration of the anti-erythrocyte antibody in the conjugate pad treatment solution is 0.01-0.5 mg / mL.

3. The method for preparing the immunochromatographic detection kit for platelet P2Y12 receptor inhibition rate according to claim 2, The conjugate pad treatment solution further contains 0.01-0.2 M buffer salt, pH 7.4-7.5; 1 wt%-20 wt% of saccharide; 0.01 wt%-5 wt% of thickener; and / or 0.01 wt%-0.1 wt% of surfactant.

4. The method for preparing the immunochromatographic detection kit for platelet P2Y12 receptor inhibition rate according to claim 3, The buffer salt in the conjugate pad treatment solution is at least one of PBS, Tris, and HEPES; and / or, the sugar substance in the conjugate pad treatment solution is at least one of trehalose, sucrose, lactose, maltose, and glucose; and / or, the thickener in the conjugate pad treatment solution is at least one of PVP-40, NMP, PEG40000, PVA, and polylysine; And / or, the surfactant in the conjugate pad treatment solution is at least one of S7, S9, Tween 20, Tween 80, and Triton.

5. A method for preparing the immunochromatographic detection kit for platelet P2Y12 receptor inhibition rate according to any one of claims 1 to 4, In the fibrinogen-containing streaking solution, the concentration of fibrinogen is 0.5-2.0 mg / mL, and the rate value is 0.5-0.8 μL / mm; And / or, in the streaking solution containing polyclonal antibodies, the concentration of the polyclonal antibodies is 0.5-2.0 mg / mL, and the rate value is 0.5-1.0 μL / mm.

6. A method for preparing the immunochromatographic detection kit for platelet P2Y12 receptor inhibition rate according to any one of claims 1 to 4, The stripping solution further contains 0.01-0.1M buffer salt, pH 7.4-7.5; 1v / v%-5v / v% alcohol; 0.01wt%-0.05wt% sugar; 0.001wt%-0.05wt% protein protective agent; and / or 0.001wt%-0.05wt% surfactant.

7. The method for preparing the immunochromatographic detection kit for platelet P2Y12 receptor inhibition rate according to claim 6, The buffer salt in the stripping solution is at least one of PBS, HEPES, TRIS, and boric acid; And / or, the alcohol substance in the stripping solution is at least one of methanol, ethanol, and propylene glycol; and / or, the sugar substance in the streaking solution is at least one of trehalose, sucrose, lactose, maltose, and glucose; And / or, the protein protective agent in the streaking solution is at least one of BSA, casein, and gelatin; And / or, the surfactant in the stripping solution is at least one of S7, S9, Tween 20, Tween 80, and Triton.

8. An immunochromatographic detection method for platelet P2Y12 receptor inhibition rate for non-disease diagnosis and treatment methods, characterized in that: The invention relates to an immunochromatographic detection kit for platelet P2Y12 receptor inhibition rate prepared by the method for preparing the immunochromatographic detection kit for platelet P2Y12 receptor inhibition rate according to any one of claims 1 to 7.

9. The immunochromatographic detection method for platelet P2Y12 receptor inhibition rate according to claim 8, wherein: include: Quantitative detection method: Take the sample to be tested and add it to the sample diluent or not, then add it dropwise to the sample pad of test strip A or the sample-binding integrated pad of test strip B. After 3-10 minutes, detect the fluorescence signals of the test line and the quality control line respectively, and calculate the platelet P2Y12 receptor inhibition rate using the following formula: R = [1-T / (T+C)] × 100%; In the formula, R represents the platelet P2Y12 receptor inhibition rate; T represents the fluorescence signal of the detection line; C represents the fluorescence signal of the quality control line; Qualitative detection method: Take the sample to be tested and add it to the sample diluent or not, then add it dropwise to the sample pad of test strip A or the sample-binding integrated pad of test strip B. After 3-10 minutes, compare the fluorescence signals of the test line and the quality control line, and make an interpretation based on the reference color card.

10. The immunochromatographic detection method for platelet P2Y12 receptor inhibition rate according to claim 9, wherein: The samples to be tested include whole blood samples, concentrated platelets, and platelet-containing plasma.

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