A kit for detecting platelet aggregation function based on CRET technology and a detection method thereof

Abstract

The application belongs to the technical field of biological detection, and particularly relates to a kit for detecting platelet aggregation function based on CRET technology and a detection method thereof. The kit comprises an anticoagulant, a platelet aggregation inducer, a donor probe, an acceptor probe, a chemiluminescent substrate, a positive control, a negative control and a buffer. The donor probe is an HRP-labeled CD61 antibody, and the acceptor probe is an amino-modified CdTe quantum dot-labeled PAC-1 antibody or CD62P antibody. The kit based on the CRET technology can realize rapid and accurate detection of platelet aggregation, is particularly suitable for low platelet samples, and is low in cost and anti-interference.

Description

Technical Field

[0001] This invention belongs to the field of biological detection technology, specifically relating to a reagent kit and detection method for detecting platelet aggregation function based on CRET technology. Background Technology

[0002] Platelet aggregation function is a core indicator for assessing the functional status of platelets during hemostasis and thrombosis. Its test results have important clinical guiding significance for the diagnosis of hemorrhagic diseases, monitoring the efficacy of antiplatelet drugs (such as aspirin and clopidogrel), preoperative risk assessment, and research on thrombotic diseases.

[0003] Currently, commonly used clinical methods for detecting platelet aggregation function mainly include flow cytometry, platelet aggregation test (PAT, or LTA), whole blood platelet aggregation test, platelet function analyzer test, point-of-care rapid test method, thromboelastography, etc. However, these methods all have obvious defects and are difficult to meet the clinical needs for accurate, rapid, convenient and adaptable testing in multiple scenarios.

[0004] Specifically, the shortcomings of existing technologies are mainly reflected in the following aspects: 1) Although flow cytometry, as a commonly used method for detecting platelet activation status, has high sensitivity and can perform multi-parameter analysis, it requires a cell count of at least 10⁻⁶ cells per cell. 5 The above points highlight several issues: 1) High reagent prices and numerous consumables; complex sample pretreatment procedures (including washing and centrifugation); lengthy incubation time; expensive and complex equipment, making routine implementation difficult in general hospitals; and the risk of artificial platelet activation during in vitro manipulation, affecting the accuracy of the test. 2) The optical transilluminance method is complex, requires fresh samples, demands high laboratory technical skills, and is easily affected by platelet concentration, making it unsuitable for testing patients with low platelet counts. 3) While platelet function analyzers are easy to operate and closely approximate the physiological environment, they cannot distinguish specific defective pathways, have limited sensitivity to certain diseases, and are costly. 4) Point-of-care rapid testing methods (such as VerifyNow) are expensive, have limited applicability, and insufficient accuracy. 5) Most existing testing methods can only achieve qualitative / quantitative detection of platelet activation markers, failing to directly reflect platelet aggregation function. They require indirect assessment using other methods, resulting in long testing chains and large errors. Furthermore, the core issues of inaccurate detection of low platelet concentration samples and interference with platelet activity during in vitro manipulation remain unresolved.

[0005] Ortho-touch chemiluminescence resonance energy transfer (CRET) is a novel detection technology that integrates immunology and molecular techniques. It eliminates the magnetic beads required in traditional chemiluminescence detection systems and successfully removes the magnetic separation and cleaning steps. This not only greatly simplifies the instrument structure and detection process, but also enables high-precision detection of the content of relevant markers in cells on a chemiluminescence technology platform, and the detection results are not affected by platelet concentration.

[0006] Currently, CRET technology has demonstrated significant advantages in the detection of platelet membrane molecules and activation function. However, there are no relevant technical solutions for its application in the specific detection of platelet aggregation function. Existing CRET-related technologies are limited to the detection of single biomarkers, failing to achieve direct and quantitative assessment of platelet aggregation function. Furthermore, they suffer from problems such as complex reagent preparation, high detection costs, and limited applicability, failing to meet the urgent clinical need for accurate, rapid, and convenient detection of platelet aggregation function. Therefore, developing a method and reagent kit based on CRET technology that overcomes the limitations of existing technologies, enables direct and accurate detection of platelet aggregation function, is suitable for patients with low platelet counts, is easy to operate, and has controllable costs, thus addressing many drawbacks of existing technologies. This has significant clinical value and application prospects, and forms the basis for the technical improvements of this invention. Summary of the Invention

[0007] Technical issues This invention aims to provide a dedicated reagent kit and detection method for detecting platelet aggregation function based on adjacent contact chemiluminescence resonance energy transfer (CRET) technology. This overcomes the limitations of existing platelet aggregation function detection methods, such as complex operation, long processing time, high cost, insufficient accuracy, and susceptibility to platelet concentration. Furthermore, existing CRET technologies can only detect a single activation marker and cannot directly reflect platelet aggregation function. This invention achieves rapid, accurate, and convenient detection of platelet aggregation function, particularly suitable for accurate assessment of platelet quality in patients with low platelet counts. It also simplifies reagent preparation and reduces detection costs. The invention is applicable to clinical scenarios such as the diagnosis of platelet-related hemorrhagic diseases and thrombotic diseases, monitoring the efficacy of antiplatelet therapy, and preoperative platelet function assessment, providing a reliable basis for clinical diagnosis and treatment, and filling the gap in the direct detection of platelet aggregation function using existing CRET technology.

[0008] Technical solution The first aspect of this invention provides a kit for detecting platelet aggregation function based on CRET technology, comprising an anticoagulant, a platelet aggregation inducer, a donor probe, a receptor probe, a chemiluminescent substrate, a positive control, a negative control, and a buffer solution; wherein the anticoagulant is a sodium citrate solution or a heparin solution containing vitamin C; the platelet aggregation inducer is selected from one or more of ADP, collagen, adrenaline, and arachidonic acid; the donor probe is an HRP-labeled CD61 antibody, and the receptor probe is an amino-modified CdTe quantum dot-labeled PAC-1 antibody or a CD62P antibody, preferably a PAC-1 antibody; the chemiluminescent substrate is a luminol-hydrogen peroxide mixture containing p-hydroxyphenylpropionic acid; the buffer solution is PBS at pH 7.4; the positive control is platelet-rich plasma with normal aggregation function, and the negative control is platelet-free plasma.

[0009] The anticoagulant in the kit contains vitamin C to inhibit artificial platelet activation in vitro. The donor and recipient probes in the kit specifically bind to platelet surface markers CD61 and PAC-1 or CD62P, respectively. These three markers are all specific markers that are essential during platelet aggregation. CD61 and PAC-1 are preferred because CD61 (GPIIIa) is the most abundant integrin on the platelet surface, with approximately 50,000-80,000 copies expressed per platelet, evenly distributed across the entire platelet surface. Its high expression level ensures sufficient probe binding. Meanwhile, PAC-1 is a specific antibody against the conformationally altered GPIIb / IIIa complex after platelet activation, exposing its binding site only after platelet activation and the conformational change of GPIIb / IIIa. The combination of these two markers allows for specific detection of aggregation function at the individual platelet level, unlike single activation marker detection methods. More specifically, when the donor probe and the receptor probe bind to CD61 and PAC-1 on the surfaces of different platelets in a non-aggregated state, respectively, the spatial distance between the two probes is much greater than the effective energy transfer distance of CRET (1nm-10nm), thus failing to trigger a CRET signal. Similarly, when the donor and receptor probes bind to a single activated platelet, the spatial distance between CD61 and PAC-1 on a single activated platelet is much greater than the effective CRET distance (>50nm), also failing to trigger a signal. Only when platelets aggregate, the CD61 and PAC-1 molecules belonging to different platelets approach each other due to close contact between platelets (aggregation time interval <5nm), at which point the donor and receptor probes enter the effective CRET distance, triggering a chemiluminescent signal. The quantum dot surface is modified with amino groups to avoid non-specific adsorption and extend the luminescence stability of the quantum dots, unlike existing quantum dot probes that are easily quenched and lack specificity. The chemiluminescent substrate contains p-hydroxyphenylpropionic acid, which can significantly amplify the luminescence signal of luminol, lower the detection limit, and is suitable for detecting low-concentration platelet aggregation signals. The buffer solution is used to dilute the sample and probe, maintaining the stability of the detection system. The negative control (platelet-free plasma) is used to assess the background signal level of the detection system, verify whether the chemiluminescent substrate and probe produce non-specific signals under platelet-free conditions, and is used for quality control; if the negative control signal is abnormally elevated, it indicates that there is non-specific interference or contamination in the system, and the detection result is invalid.

[0010] In some embodiments, the platelet aggregation inducer is ADP; the ADP reagent contains 0.005% w / v-0.1% w / v chitosan and 0.2% w / v-2.0% w / v trehalose. Chitosan and trehalose are used to improve the activity and stability of ADP and reduce nonspecific interference.

[0011] In some embodiments, the clone number of the CD61 antibody is 10E5; the clone number of the PAC-1 antibody is 4O21; and the emission wavelength of the CdTe quantum dots is 580nm-650nm. The screened antibodies further enhance probe binding specificity and avoid cross-reactivity.

[0012] In some embodiments, the storage conditions for each component of the kit are as follows: HRP-labeled CD61 antibody, amino-CdTe quantum dot-labeled PAC-1 or CD62P antibody are stored at -20°C protected from light; ADP inducer is stored at 2°C-8°C; luminol-hydrogen peroxide chemiluminescent substrate is stored at 2°C-8°C protected from light; positive control (platelet-rich plasma with normal aggregation function) and negative control (platelet-free plasma) are stored at -20°C; calibration reagent (gradient concentration platelet standards) are stored at -20°C; PBS buffer can be stored at room temperature.

[0013] In some embodiments, the corrective reagent contains 20 × 10 9 / L-200×10 9 / L gradient concentration platelet standard. The calibration reagent contains 20×10 9 / L, 50×10 9 / L, 100×10 9 / L, 200×10 9 A plasma standard containing / L platelets. The calibration reagent is used to construct a calibration curve to correct for fluctuations in platelet concentration and ensure the accuracy of testing samples with low platelet counts.

[0014] In some embodiments, the concentration of platelets in the sample detected by the kit is 20 × 10⁻⁶. 9 / L-300×10 9 / L.

[0015] In some embodiments, the method for preparing the donor probe includes: 1) diluting CD61 antibody with 0.008 mol / L to 0.012 mol / L PBS at pH 7.4 to 0.8 mg / mL to 1.2 mg / mL; 2) activating HRP with 0.08 mol / L to 0.12 mol / L sodium periodate at 2℃ to 6℃ in the dark for 20 min to 40 min, and terminating the reaction with ethylene glycol; 3) mixing activated HRP and CD61 antibody at a molar ratio of 4:1 to 6:1, adding sodium borohydride, and stirring at 2℃ to 6℃ in the dark for 1.5 h to 2.5 h; 4) purifying using an 8 kDa to 12 kDa ultrafiltration tube to obtain the donor probe.

[0016] In some embodiments, the preparation method of the receptor probe includes: 1) amylating CdTe quantum dots with APTES and dialyzing with 0.008 mol / L to 0.012 mol / L PBS at pH 7.4 for 12 h to 36 h; 2) crosslinking and activating the amylated quantum dots with 0.15% to 0.25% glutaraldehyde at 18 °C to 28 °C for 30 min to 1.5 h; 3) adding PAC-1 antibody at a quantum dot to antibody molar ratio of 8:1 to 12:1 and reacting at 2 °C to 6 °C in the dark for 8 h to 16 h; 4) filtration through a 0.20 μm to 0.25 μm filter and purification by gel chromatography to obtain the receptor probe.

[0017] The second aspect of this invention provides a detection method for a reagent kit based on CRET technology for detecting platelet aggregation function as described in any of the above claims, comprising the following steps: S1. Take a whole blood sample, add an anticoagulant containing vitamin C, mix well, and obtain a pretreated whole blood sample; or take a whole blood sample, add an anticoagulant containing vitamin C, mix well, centrifuge, and obtain a pretreated plasma sample; S2. Mix the pretreated whole blood or plasma sample with a platelet aggregation inducer, a donor probe, and a receptor probe to obtain a test solution, and then incubate at 35℃-38℃ for 1min-5min; S3. Add a chemiluminescent substrate to the test solution and detect the chemiluminescence intensity using a chemiluminescence detector; S4. Calculate the platelet aggregation rate based on the luminescence intensity and obtain the corrected aggregation rate using a concentration correction formula.

[0018] In some embodiments, the volume ratio of whole blood sample to anticoagulant in step S1 is 9:1; the anticoagulant contains 0.001% w / v-0.005% w / v vitamin C; when the anticoagulant is sodium citrate solution, the concentration of sodium citrate in the pretreated whole blood / plasma sample is 8 mmol / L-15 mmol / L; when the anticoagulant is heparin solution, the concentration of heparin in the pretreated whole blood / plasma sample is ≥1.45 IU / mL.

[0019] In some embodiments, the platelet-rich plasma is prepared by low-speed centrifugation at 800 r / min-1200 r / min for 4 min-6 min, which avoids damage to platelets caused by high-speed centrifugation.

[0020] In some embodiments, the platelet-free plasma is prepared as follows: platelet-rich plasma is centrifuged at 2800 r / min-3200 r / min for 13 min-17 min, the supernatant is collected, and then centrifuged again at 2800 r / min-3200 r / min for 8 min-12 min, and the supernatant is collected to obtain platelet-free plasma with a platelet count <5 × 10⁻⁶. 9 / L.

[0021] In some embodiments, when the platelet aggregation inducer in step S2 is selected from ADP, collagen, adrenaline, or arachidonic acid, the working concentrations are respectively: ADP 2μmol / L-50μmol / L, collagen 5μg / mL-50μg / mL, adrenaline 1μmol / L-10μmol / L, and arachidonic acid 50μmol / L-200μmol / L.

[0022] In some embodiments, the concentration ratio of the donor probe to the acceptor probe in step S2 is 1:1 to 1:3, and the working concentrations are 0.05 μg / mL to 0.5 μg / mL and 0.1 μg / mL to 1 μg / mL, respectively.

[0023] In some embodiments, the coupling rate between the CD61 antibody and HRP is ≥92%, and in the receptor probe, the coupling rate between the PAC-1 antibody and CdTe quantum dots is ≥92%.

[0024] In some embodiments, the antibody is purified to a purity of ≥95%.

[0025] In some embodiments, the working concentration of p-hydroxyphenylpropionic acid in the chemiluminescent substrate in step S3 is 0.002%-0.01%, the working concentration of luminol is 0.1mmol / L-1mmol / L, and the working concentration of hydrogen peroxide is 0.01mmol / L-0.1mmol / L; the detection wavelength of the chemiluminescence detector is 580nm-650nm, and the detection time is 10s-30s.

[0026] In some embodiments, the platelet aggregation rate in step S4 is calculated using the following formula: Platelet aggregation rate = (Sample chemiluminescence intensity - Blank control chemiluminescence intensity) / (Positive control chemiluminescence intensity - Blank control chemiluminescence intensity) × 100%, and the correction formula is: Corrected aggregation rate = Platelet aggregation rate × (1 + 0.001 × Platelet concentration); wherein, the blank control chemiluminescence intensity is the chemiluminescence intensity measured in the test solution prepared from platelet-free plasma or PBS instead of platelet samples and inducers, and the positive control chemiluminescence intensity is the chemiluminescence intensity measured in the test solution prepared from platelet-rich plasma with normal aggregation function. The introduction of the correction formula further eliminates the influence of platelet concentration fluctuations on the test results, ensuring the accuracy of the test results for samples with low platelet counts. Unless otherwise specified, "platelet concentration" in this invention refers to a concentration calculated using a 10⁻¹² m / s²· ... 9 / L is a numerical value. The negative control does not appear directly in the calculation formula. Its main functions are: 1) System background signal assessment: The negative control (platelet-free plasma) is used to assess the background signal level of the detection system; 2) Quality control: The signal of the negative control should be close to that of the blank control. If it is abnormally high, it indicates that there is non-specific interference in the system; 3) Result interpretation assistance: The signal of the negative control should be significantly lower than that of the positive control and the sample group, which is used to help determine whether the detection system is operating normally.

[0027] In some embodiments, a corrected aggregation rate ≥60% indicates normal aggregation function, 30%-60% indicates weakened aggregation function, and <30% indicates severely weakened aggregation function.

[0028] Technical effect 1. Direct, accurate, and quantitative detection of platelet aggregation function: This invention is the first to use CRET technology for the direct quantitative detection of platelet aggregation function, overcoming the limitation of existing CRET technologies that can only detect single platelet activation markers and cannot correlate with the overall platelet aggregation function. This invention uses a dual antibody pairing of CD61 and PAC-1 / CD62P, generating a CRET signal only when platelets aggregate, thus accurately reflecting platelet aggregation behavior, unaffected by simple activation states, and eliminating the need for other indirect methods. The consistency with the clinical gold standard (turbidimetric assay) is over 99%, with a detection error ≤2.5%; while existing flow cytometry has a consistency of 92.5% with the clinical gold standard and a detection error ≤5%.

[0029] 2. Inhibiting artificial activation in vitro from the source, the test results are closer to the true state in vivo: By adding vitamin C to the anticoagulant, artificial activation of platelets in vitro is inhibited during the sample pretreatment stage, avoiding false activation interference caused by the operation process.

[0030] 3. Applicable to the detection of samples with low platelet counts: This invention introduces a platelet concentration correction mechanism and formula, which can eliminate the influence of platelet concentration fluctuations on the results, and is suitable for platelet counts <50×10⁻⁶. 9 The accuracy rate of detecting low platelet count samples ( / L) is over 92%, which is much higher than that of flow cytometry (70%) and light transmittance turbidimetry (65%), filling the clinical gap in functional assessment of patients with low platelet count.

[0031] 4. Significantly optimized probes and systems result in higher sensitivity, lower non-specific interference, and improved detection accuracy and stability: The nucleic acid probe pairing method is abandoned, and direct coupling between antibodies and HRP / quantum dots is adopted, simplifying probe structure and preparation; combined with CD61 (10E5) and PAC-1 (4O21) clonal antibodies and aminated quantum dots, the specificity is enhanced, significantly reducing non-specific adsorption and background interference; p-hydroxyphenylpropionic acid (HPPA) is added to the chemiluminescent substrate, where HPPA generates a fluorescent product under HRP catalysis, synergistically enhancing the total signal with luminol chemiluminescence; chitosan and trehalose stabilizers are added to the ADP inducer to address the problem of unstable inducer activity and easy non-specific platelet activation.

[0032] 5. Extremely simple operation and extremely fast speed to meet the needs of rapid detection: This invention adopts the "one-step sample addition and rapid incubation" mode, which does not require complex pretreatment such as washing, centrifugation, magnetic separation or preparation of nucleic acid probes. The entire detection process takes ≤10 minutes, and the results are available in as little as 5 minutes, which is much faster than flow cytometry (60 minutes) and light transmission turbidimetry (30 minutes). The operation is simple and does not require professional personnel, so it can be widely used in scenarios with high time requirements.

[0033] 6. Significantly reduced costs and stable and easy-to-use reagents: The probe preparation process is simplified, there are no nucleic acid probes, and single-use packaging avoids reagent waste. The cost of single-sample testing is only 1 / 5 of that of flow cytometry and 1 / 3 of that of existing CRET protocols. The reagents are highly stable, can be stored and transported at room temperature, and the supporting equipment is lightweight and maintenance-free, greatly reducing the threshold for clinical application.

[0034] 7. Wider applicability and stronger clinical adaptability: Whole blood or platelet-rich plasma can be used as test samples, which is suitable for platelet aggregation function testing in humans and mammals. It is especially suitable for platelet quality assessment in patients with low platelet counts and is applicable to various bleeding / thrombotic disease diagnosis, antiplatelet drug efficacy monitoring, preoperative functional assessment and other scenarios. Attached Figure Description

[0035] Figure 1 This is a schematic diagram illustrating the principle of platelet aggregation detection based on CRET technology. Figure 2 This is a schematic diagram illustrating the correction effect of the correction formula of this invention; the horizontal axis represents platelet concentration (×10). 9 / L), with the vertical axis representing the detection error (%). Figure 3 This is a comparison chart of the detection time of the method of the present invention with that of flow cytometry and LTA (turbidimetric assay). Figure 4 This is a comparison of the detection accuracy of the method of the present invention with flow cytometry and LTA (light transmittance turbidimetry) for samples with different platelet concentrations. Detailed Implementation

[0036] To facilitate the implementation of the technical solutions applied for, the terms and expressions involved in this invention will first be explained and defined in general terms and expressions below.

[0037] The terms “comprising,” “including,” or any other variations thereof are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Without further limitation, an element defined by the phrase “comprising one…” does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes said element.

[0038] For experiments not specifically described in the examples, the procedures or conditions should be followed according to the conventional experimental procedures described in the literature in this field. Reagents or instruments whose manufacturers are not specified are all commercially available conventional reagent products.

[0039] The following provides a further description of a reagent kit and detection method for detecting platelet aggregation function based on CRET technology provided by the present invention.

[0040] Example 1: Reagent kit for detecting platelet aggregation function based on CRET technology A schematic diagram illustrating the principle of platelet aggregation function detection based on CRET technology is shown below. Figure 1 As shown. The kit includes the following reagents: 0.109 mol / L sodium citrate anticoagulant (containing 0.003% w / v vitamin C), ADP inducer (containing 150 μmol / L ADP, 0.02% w / v chitosan, and 1.0% w / v trehalose), donor probe (HRP-labeled CD61 antibody, concentration 1 μg / mL, conjugation rate 95%), receptor probe (amino-CdTe quantum dot-labeled PAC-1 antibody, concentration 2 μg / mL, conjugation rate 93%), luminol-hydrogen peroxide substrate containing 0.0375% w / v p-hydroxyphenylpropionic acid (luminol 7.5 mmol / L, hydrogen peroxide 0.75 mmol / L), positive control (platelet-rich plasma with normal aggregation function, platelet concentration 200 × 10⁻⁶). 9 / L-300×10 9 / L), negative control (platelet-free plasma, platelet concentration <5×10⁻⁶ / ...). 9 / L), PBS buffer (pH 7.4), calibration reagent (containing 20×10 9 / L, 50×10 9 / L, 100×10 9 / L, 200×10 9 / L platelet standard).

[0041] The preparation method of the donor probe is as follows: (1) CD61 antibody pretreatment: CD61 antibody was diluted to 1 mg / mL with 0.01 mol / L PBS (pH 7.4); (2) HRP activation: Horseradish peroxidase (HRP, Sigma, catalog number P8375) was activated with 0.1 mol / L sodium periodate (NaIO4) at 4°C in the dark for 30 min. The reaction was terminated with ethylene glycol after activation. (3) Antibody-HRP conjugation: Activated HRP and CD61 antibody were mixed at a molar ratio of 5:1, and 1 mg / mL sodium borohydride (NaBH4) reducing agent was added. The mixture was stirred at 4°C in the dark for 2 h. (4) Purification: Transfer the coupling reaction solution to a 10kDa ultrafiltration centrifuge tube, centrifuge at 5000g for 15min, wash with PBS 3 times to remove uncoupled HRP; (5) Coupling rate detection: A was determined by spectrophotometry. 403 (HRP) and A 280 (Antibody) absorbance, calculate the conjugation rate = (A) 403 (×dilution factor) / (HRP molar extinction coefficient×HRP concentration)×100%, coupling rate ≥92%; (6) Aliquoting and storage: Aliquot the purified HRP-CD61 antibody donor probe, adjust the concentration to 1 μg / mL, and store at -20℃ in the dark.

[0042] The preparation method of the receptor probe is as follows: (1) Aminoation modification of CdTe quantum dots: CdTe quantum dots with an emission wavelength of 620 nm (purchased from Nanjing Xianfeng Nanomaterials Technology Co., Ltd.) were reacted with 3-aminopropyltriethoxysilane (APTES) in an ethanol / water (1:1, v / v) mixed solution at 60 °C for 4 h to aminate the surface of the quantum dots; excess APTES was removed by dialyzing with 0.01 mol / L PBS (pH 7.4) for 24 h to obtain aminated CdTe quantum dots; (2) PAC-1 antibody pretreatment: PAC-1 antibody was diluted to 1 mg / mL with 0.01 mol / L PBS (pH 7.4); (3) Quantum dot-antibody conjugation: Aminated CdTe quantum dots were crosslinked with glutaraldehyde (final concentration 0.2%) at room temperature for 1 h, and then PAC-1 antibody was added. The molar ratio of quantum dots to antibody was 10:1. The reaction was carried out overnight at 4°C in the dark with stirring. (4) Purification: The coupling reaction solution was filtered through a 0.22 μm filter membrane to remove aggregates, and then purified by Sephadex G-200 gel chromatography column, and the first elution peak was collected; (5) Coupling rate detection: The fluorescence intensity of quantum dots (λex=480nm, λem=620nm) and A were determined by fluorescence spectroscopy. 280 (Antibody) absorbance, calculate the conjugation rate = (antibody binding amount / total antibody input) × 100%, conjugation rate ≥ 92%; (6) Aliquoting and storage: The purified amino-modified CdTe quantum dot-PAC-1 antibody receptor probe is aliquoted and the concentration is adjusted to 2 μg / mL. It is stored at -20℃ in the dark.

[0043] CD61 antibody and PAC-1 antibody were screened through the following process: 1) Screening of CD61 antibody clones Three CD61 antibody clones (10E5, VI-PL2, and Y2 / 51) were selected for screening, and the evaluation indicators included binding affinity, specificity, and conjugation efficiency.

[0044] Results: Clone 10E5 had a conjugation rate of 95% and a cross-reactivity rate of <2%, achieving the highest overall score; VI-PL2 had a conjugation rate of 88% and a cross-reactivity rate of 5%; Y2 / 51 had a conjugation rate of 82% and a cross-reactivity rate of 8%. Therefore, the CD61 antibody from clone 10E5 was selected for subsequent examples.

[0045] 2) Screening of PAC-1 antibody clones Three PAC-1 antibody clones (4O21, BL-H7, and PAC-1.1) were selected for screening.

[0046] Results: Clone 4O21 had a conjugation rate of 93%, an activated platelet recognition rate of 99.2%, and the highest overall score; BL-H7 had a conjugation rate of 87% and a recognition rate of 95.5%; PAC-1.1 had a conjugation rate of 85% and a recognition rate of 93.8%. Therefore, PAC-1 antibody from clone 4O21 was selected for subsequent examples.

[0047] Example 2: How to use the kit Instruments: Chemiluminescence detector (detection wavelength 580-650nm), constant temperature incubator (37℃), low speed centrifuge, pipette.

[0048] Testing steps: 1) Sample pretreatment: Collect 5 mL of whole blood from healthy volunteers, add 0.109 mol / L sodium citrate anticoagulant (blood to anticoagulant volume ratio 9:1), mix gently, take 2 mL of whole blood, centrifuge at 1000 r / min for 5 min to prepare platelet-rich plasma with normal aggregation function, and set aside; centrifuge the platelet-rich plasma at 3000 r / min for 15 min, take the supernatant, centrifuge again at 3000 r / min for 10 min, and take the supernatant to obtain platelet-free plasma (platelet count <5×10). 9 / L), for later use; at the same time, whole blood was collected from patients suspected of having platelet aggregation dysfunction (including 10 cases of low platelet count, with a platelet count of 20×10). 9 / L-50×10 9 Platelet plasma ( / L) was obtained by processing it in the same way and set aside for later use; 2) Construction of CRET detection system: In a 96-well plate, add 100 μL of PBS buffer and 10 μL of PBS (as a substitute for the inducing agent) to each well in the blank control group; add 100 μL of platelet-rich plasma with normal aggregation function (platelet concentration 200 × 10⁻⁶) to each well in the positive control group. 9 / L-300×10 9 10 μL of platelet-free plasma and 10 μL of ADP inducer were added to each well of the negative control group; 100 μL of test sample plasma and 10 μL of ADP inducer were added to each well of the sample group; then 10 μL of donor probe and 10 μL of recipient probe were added to each well of each group, gently mixed, and incubated at 37°C for 3 min.

[0049] 3) Chemiluminescence signal detection: Add 20 μL of diluted chemiluminescence substrate (luminol working concentration 0.5 mmol / L, hydrogen peroxide working concentration 0.05 mmol / L, p-hydroxyphenylpropionic acid working concentration 0.005% w / v) to each well, mix quickly, and use a chemiluminescence detector to detect the chemiluminescence intensity of each well (detection time 20 s). 4) Results Analysis: The platelet aggregation rate of each experimental group was calculated. The formula is: Platelet aggregation rate = (Sample chemiluminescence intensity - Blank control chemiluminescence intensity) / (Positive control chemiluminescence intensity - Blank control chemiluminescence intensity) × 100%; Then, using the calibration curve constructed with calibration reagents, the calibration formula was substituted: Corrected aggregation rate = Platelet aggregation rate × (1 + 0.001 × Platelet concentration), where 0.001 is the calibration coefficient, yielding the final test results: Healthy volunteer samples (50-300 × 10⁻⁶) 9 The corrected aggregation rate of platelet count ( / L) was 65%-75%, which is considered normal aggregation function; in samples from suspected abnormal patients, the normal platelet concentration (>50×10⁻⁶) was... 9The corrected aggregation rate for platelet counts ( / L) is 25%-35%, and for low platelet concentration samples (20-50×10⁻⁶ / L) it is 25%-35%. 9 The corrected aggregation rates ( / L) were 28%-36%, all indicating severely weakened aggregation function, suggesting a possible bleeding disorder or antiplatelet drug overdose in the patient. A diagram illustrating the correction effect is shown below. Figure 2 As shown, the detection error is significantly reduced after adding the correction formula, which is especially suitable for samples with low platelet concentration.

[0050] Example 3: Methodological Validation 1) Feasibility verification The kit of this invention was used to detect aggregated platelets and activated but non-aggregated platelets. The results showed that the CRET signal (RLU) of aggregated platelets was significantly higher than that of activated but non-aggregated platelets (P<0.001), confirming that the dual antibody pairing can specifically detect aggregation function rather than simply the activation state.

[0051] 2) Selective Validation Adding white blood cells or red blood cells (10 times the number of platelets, respectively) to the detection system showed that the CRET signal of non-target cells was not significantly different from that of the blank control (P>0.05), confirming that this kit has no cross-reactivity with non-platelet cells.

[0052] 3) Reproducibility verification For the same low platelet count sample (35×10), 9 The platelet count was repeated 10 times ( / L), with a CV of 3.8%; for normal platelet samples (150×10⁶ / L), the CV was 3.8%. 9 The test was repeated 10 times ( / L), and the CV was 2.5%, which met the requirement of CV < 5%.

[0053] 4) Stability verification The donor and recipient probes were stored at 4°C and detected every 24 hours for 7 consecutive days. The signal attenuation rates were 4.2% and 3.8%, respectively, which met the requirement that the signal attenuation was <5% after 7 days of storage at 4°C.

[0054] 5) Anti-interference capability verification Hemolysis (5g / LHb), lipemia (10mmol / LTG), and bilirubin (200μmol / L) were added to the samples, respectively. The results showed that the influence of each interfering substance on the aggregation rate detection results was <5%, which met the anti-interference requirements.

[0055] Example 4: Correction Coefficient Fitting Experiment To determine the source of the coefficient 0.001 in the correction formula, the following fitting experiment was conducted: (1) Sample preparation: Whole blood was collected from healthy volunteers, platelet-rich plasma was separated, and samples with different platelet concentrations (20×10⁻⁶) were prepared by diluting with platelet-free plasma. 9 / L, 30×10 9 / L, 40×10 9 / L, 50×10 9 / L, 100×10 9 / L, 150×10 9 / L, 200×10 9 / L, 250×10 9 / L, 300×10 9 / L, with 3 replicates for each concentration).

[0056] (2) Detection method: Platelet aggregation rate of each concentration sample was simultaneously detected by the CRET method and LTA (light transmission turbidimetric method) of the present invention, and the detection results of the two methods were recorded.

[0057] (3) Data processing: Calculate the deviation between the method of the present invention and the LTA detection results at each concentration. Deviation (%) = [(Result of the present invention - LTA result) / LTA result] × 100%.

[0058] (4) Fit analysis: using platelet concentration (×10) 9 Using platelet concentration ( / L) as the x-axis and deviation (%) as the y-axis, a linear regression analysis was performed, yielding the following fitted equation: Deviation (%) = 0.001 × platelet concentration (×10) 9 / L), correlation coefficient r 2 =0.985, P<0.001.

[0059] (5) Validation: Substituting the fitting coefficient of 0.001 into the correction formula, another batch of 60 samples (including 20 low platelet samples and 40 platelet-rich samples with normal aggregation function) were validated. After correction, the detection error decreased from an average of 5.2% to 1.8%. Low platelet samples (20-50×10 9 / L) detection accuracy improved from 82% to 92%. The fitting results are as follows: Figure 2 As shown, the correction formula can effectively eliminate the influence of platelet concentration on the test results.

[0060] Example 5: Comparative Experiment Verification Sixty clinical samples were selected (including 20 cases of low platelet count, with platelet counts ranging from 20 to 50 × 10⁻⁶). 9 / L; 40 normal platelet concentration samples were randomly divided into 3 groups of 20 cases each. Platelet aggregation function was detected by the CRET technology platform of this invention, the existing flow cytometry platform, and the optical transilluminance method, respectively. The detection results of the three methods were compared.

[0061] 1) Flow cytometry method: The activity percentage of PAC-1 and CD62P is detected by flow cytometer to observe platelet aggregation function at the molecular level.

[0062] 2) Light Transmittance Turbidimetric Assay (LTA): LTA is the internationally recognized gold standard for platelet aggregation detection. A Chrono-Log Model 700 LTA instrument and a platelet detection kit (Helena) were used, with ADP 10 μmol / L as the inducer, to detect platelet aggregation rate at 37°C. The detection time was set to 10 min, and the maximum aggregation rate (MAX%) and aggregation slope were recorded.

[0063] This invention strictly follows the formula: Platelet aggregation rate = (Sample chemiluminescence intensity - Blank control chemiluminescence intensity) / (Positive control chemiluminescence intensity - Blank control chemiluminescence intensity) × 100%. The results of the comparison of normal platelet aggregation rate detection data (unit: %) by different methods are shown in Tables 1-3.

[0064] Table 1: Comparison of Aggregation Rate Detection Data for Normal Platelet Concentration Samples

[0065] Note: Normal platelet concentration sample (platelet count 50-300 × 10⁻⁶) 9 / L, a total of 40 cases).

[0066] As shown in Table 1, there was no statistically significant difference in the average aggregation rate among the three methods (P>0.05), but the aggregation rate of this invention exhibited the smallest fluctuation range, demonstrating superior detection stability. Meanwhile, Figure 3 The graph shows a comparison of the detection time of the method of the present invention with that of flow cytometry and optical transilluminance turbidimetry, clearly demonstrating that the detection time of the method of the present invention is significantly shortened.

[0067] Table 2: Comparison of Aggregation Rate Detection Data for Samples with Low Platelet Concentration

[0068] Note: Samples with low platelet count (platelet count 20-50 × 10⁻⁶) 9 / L, a total of 20 cases).

[0069] As shown in Table 2, in the detection of samples with low platelet concentration, the aggregation rate fluctuation range of this invention was significantly smaller than that of the latter two methods (P<0.05), and it showed the highest consistency with the average aggregation rate (32.1%±3.2%) of the clinical gold standard (LTA), with an error of only 0.3%, far superior to the errors of flow cytometry (error 2.4%) and the clinical gold standard (LTA). Meanwhile, as... Figure 4As shown, the detection accuracy of the method of the present invention is over 92%, indicating that the method of the present invention still has a high detection accuracy in samples with low platelet concentration, which is significantly better than the flow cytometry method.

[0070] Table 3: Detection data of blank control group and positive control group

[0071] As shown in Table 3, the signal difference between the positive control and the blank control of this invention is the largest, which is 1440±74 RLU. This results in the highest signal recognition and effectively reduces the error in the aggregation rate calculation.

[0072] Experimental results show that the CRET technology platform of this invention has more stable detection results in platelet aggregation rate detection, whether for samples with normal or low platelet concentrations. The aggregation rate calculation strictly follows the given formula and has the highest consistency with the clinical gold standard. At the same time, it is significantly superior to existing technologies in terms of detection accuracy, detection speed, ease of operation, detection cost, and suitability for low platelet samples.

[0073] The above specific embodiments further illustrate the purpose, technical solution and beneficial effects of this application. It should be understood that the above are only specific embodiments of this application and are not intended to limit the scope of protection of this application. Any modifications, equivalent substitutions, improvements, etc., made on the basis of the technical solution of this application should be included within the scope of protection of this application.

Claims

1. A reagent kit for detecting platelet aggregation function based on CRET technology, characterized in that, Includes anticoagulants, platelet aggregation inducers, donor probes, recipient probes, chemiluminescent substrates, positive controls, negative controls, and buffer solutions; The anticoagulant is a sodium citrate solution or a heparin solution containing vitamin C. The platelet aggregation inducer is selected from one or more of ADP, collagen, adrenaline, and arachidonic acid; The donor probe is an HRP-labeled CD61 antibody, and the receptor probe is an amino-modified CdTe quantum dot-labeled PAC-1 antibody or a CD62P antibody. The chemiluminescent substrate is a mixture of luminol and hydrogen peroxide containing p-hydroxyphenylpropionic acid; The buffer solution is PBS with a pH of 7.4; The positive control is platelet-rich plasma with normal aggregation function, and the negative control is platelet-free plasma.

2. The reagent kit for detecting platelet aggregation function based on CRET technology according to claim 1, characterized in that, The platelet aggregation inducer is ADP; the ADP reagent contains 0.005% w / v-0.1% w / v chitosan and 0.2% w / v-2.0% w / v trehalose.

3. The reagent kit for detecting platelet aggregation function based on CRET technology according to claim 1, characterized in that, The clone number of the CD61 antibody is 10E5; the clone number of the PAC-1 antibody is 4O21; and the emission wavelength of the CdTe quantum dots is 580nm-650nm.

4. The reagent kit for detecting platelet aggregation function based on CRET technology according to claim 1, characterized in that, The concentration of platelets in the sample detected by the kit is 20 × 10⁻⁶. 9 / L-300×10 9 / L.

5. The detection method of the reagent kit for detecting platelet aggregation function based on CRET technology according to any one of claims 1-4, characterized in that, Includes the following steps: S1. Take a whole blood sample, add an anticoagulant containing vitamin C, mix well, and obtain a pretreated whole blood sample; or take a whole blood sample, add an anticoagulant containing vitamin C, mix well, centrifuge, and obtain a pretreated plasma sample. S2. Mix the pretreated whole blood or plasma sample with platelet aggregation inducer, donor probe and receptor probe to obtain the test solution, and then incubate at 35℃-38℃ for 1min-5min. S3. Add the chemiluminescent substrate to the solution to be tested, and use a chemiluminescence detector to detect the chemiluminescence intensity; S4. Calculate the platelet aggregation rate based on the luminescence intensity, and obtain the corrected aggregation rate using the concentration correction formula.

6. The detection method of the reagent kit for detecting platelet aggregation function based on CRET technology according to claim 5, characterized in that, In step S1, the volume ratio of whole blood sample to anticoagulant is 9:1; the anticoagulant contains 0.001% w / v-0.005% w / v vitamin C; when the anticoagulant is sodium citrate solution, the concentration of sodium citrate in the pretreated whole blood / plasma sample is 8 mmol / L-15 mmol / L; when the anticoagulant is heparin solution, the concentration of heparin in the pretreated whole blood / plasma sample is ≥1.45 IU / mL.

7. The detection method of the reagent kit for detecting platelet aggregation function based on CRET technology according to claim 5, characterized in that, When the platelet aggregation inducer mentioned in step S2 is selected from ADP, collagen, adrenaline, or arachidonic acid, the working concentrations are as follows: ADP 2μmol / L-50μmol / L, collagen 5μg / mL-50μg / mL, adrenaline 1μmol / L-10μmol / L, and arachidonic acid 50μmol / L-200μmol / L.

8. The detection method of the reagent kit for detecting platelet aggregation function based on CRET technology according to claim 5, characterized in that, In step S2, the concentration ratio of the donor probe to the acceptor probe is 1:1 to 1:3, and the working concentrations are 0.05 μg / mL to 0.5 μg / mL and 0.1 μg / mL to 1 μg / mL, respectively.

9. The detection method of the reagent kit for detecting platelet aggregation function based on CRET technology according to claim 5, characterized in that, In step S3, the working concentration of p-hydroxyphenylpropionic acid in the chemiluminescent substrate is 0.002% w / v-0.01% w / v, the working concentration of luminol is 0.1 mmol / L-1 mmol / L, and the working concentration of hydrogen peroxide is 0.01 mmol / L-0.1 mmol / L; the detection wavelength of the chemiluminescence detector is 580 nm-650 nm, and the detection time is 10 s-30 s.

10. The detection method of the reagent kit for detecting platelet aggregation function based on CRET technology according to claim 5, characterized in that, The platelet aggregation rate in step S4 is calculated using the following formula: Platelet aggregation rate = (Sample chemiluminescence intensity - Blank control chemiluminescence intensity) / (Positive control chemiluminescence intensity - Blank control chemiluminescence intensity) × 100%. The correction formula is: Corrected aggregation rate = Platelet aggregation rate × (1 + 0.001 × Platelet concentration). The blank control chemiluminescence intensity is the chemiluminescence intensity measured in the test solution prepared from platelet-free plasma or PBS instead of platelet samples and inducers. The positive control chemiluminescence intensity is the chemiluminescence intensity measured in the test solution prepared from platelet-rich plasma with normal aggregation function.

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