Reagent combination, kit and detection method for detecting platelet activation marker PAC-1

Abstract

The present disclosure relates to the field of biomedical technology, and specifically relates to a reagent combination, kit and detection method for detecting platelet activation marker PAC-1. The reagent combination comprises: a first detection probe, a second detection probe, a third detection probe and a fourth detection probe; the first detection probe is formed by at least conjugating a DNA1 and a PAC-1 antibody 1; the second detection probe is formed by at least conjugating a PAC-1 antibody 2 and a DNA2; the third detection probe is a DNA3 labeled with a fluorescent molecule; the fourth detection probe is an antioxidant bound to a carrier molecule. When the reagent combination or kit of the present disclosure is used to detect PAC-1, the cost is relatively low and whole blood can be used for detection, which allows for convenience and speed while avoiding artificial platelet activation in in vitro samples; moreover, the detection operation procedure is simple and the overall detection time is relatively short, making the invention suitable for the actual needs of clinical detection of PAC-1.

Description

A reagent combination, kit, and detection method for detecting the platelet activation marker PAC-1.

[0001] priority

[0002] The aforementioned PCT patent application claims priority to a Chinese patent with an application date of November 18, 2024, and application number 202411640047.4. This patent application incorporates the technical solution of the aforementioned patent. Technical Field

[0003] This disclosure relates to the field of biomedical technology, specifically to a reagent combination, kit, and detection method for detecting the platelet activation marker PAC-1. Background Technology

[0004] In normal blood circulation, over 90% of platelets are in a resting state. When stimulated by various physical, chemical, and biological factors, such as ADP, thrombin, or collagen, platelet morphology and structure change, leading to adhesion, aggregation, and release reactions—a process known as platelet activation. Platelet membrane glycoproteins (GPs) are the main components mediating platelet activation and can be divided into plasma membrane glycoproteins and granule membrane glycoproteins based on their distribution location. Plasma membrane glycoproteins include GPIb, GPIIb / IIIa, etc., and granule membrane glycoproteins include CD62P and CD63, etc. Among them, GPIIb / IIIa is the most abundant membrane glycoprotein on platelets and is a calcium-rich protein. 2+ Platelet-dependent dimeric complex. In the resting state of platelets, the GPⅡb / Ⅲa complex exists in an inactive form and lacks the ability to bind to ligands such as fibrinogen. Upon platelet activation, GPⅡb / Ⅲa undergoes a conformational change, transforming into an active state, becoming the activated GPⅡb / Ⅲa complex. The fibrinogen receptor exposed after GPⅡb / Ⅲa activation is PAC-1 (platelet-activating complex-1), i.e., the activated GPⅡb / Ⅲa complex. PAC-1 is a necessary condition for platelet aggregation and is the final common step in platelet aggregation induced by all platelet agonists. It is an early marker of platelet activation; therefore, detecting PAC-1 on the platelet surface can accurately reflect the early state of platelet activation.

[0005] Many diseases, especially cardiovascular and cerebrovascular diseases, are related to changes in platelet activation status. Monitoring changes in platelet activation status by detecting PAC-1 is of great significance for the diagnosis and monitoring of risk and status in thrombotic diseases such as acute coronary syndrome (ACS) and deep vein thrombosis, for guiding clinical medication for cardiovascular diseases such as coronary heart disease, and for evaluating the efficacy of antiplatelet drugs and platelet therapy. In addition to cardiovascular and cerebrovascular diseases, PAC-1 detection can also be used to assess platelet function in other disease states, such as chronic obstructive pulmonary disease (COPD). Furthermore, PAC-1 detection is important for studying the molecular mechanisms and signal transduction pathways of platelet activation, which helps in the development of new antiplatelet therapy strategies.

[0006] Traditional PAC-1 detection methods include flow cytometry (FCM) and enzyme-linked immunosorbent assay (ELISA), which are important in platelet activation research. However, these methods have certain drawbacks. For example, flow cytometry requires specialized equipment and reagents, resulting in higher testing costs. Furthermore, while platelets are relatively easy to activate in vitro, flow cytometry has a longer detection time and is more complex. Slight improper sample processing can artificially activate platelets, affecting the test results. Therefore, this method is not suitable for situations requiring rapid diagnosis and demands a high level of expertise from technicians, requiring proficiency in both instrument operation and data analysis. As for the other method, ELISA, it is currently only used for research purposes to detect PAC-1 and cannot be used for clinical diagnosis, thus limiting its application scope. In addition, the reagents in the ELISA kit cannot be mixed with reagents from other batches or sources of ELISA kits, which limits the flexibility of the experiment. Similarly, the ELISA operation steps are complex and include washing and separation steps, which can easily lead to in vitro platelet activation. Therefore, it is necessary to strictly control the experimental conditions.

[0007] Based on the above research, reagent combinations / kits that have low development costs and can conveniently, quickly and accurately detect PAC-1 have good application prospects and research significance. Summary of the Invention Technical issues

[0008] Existing kits for detecting PAC-1 are complex to operate, time-consuming, costly, and require stringent experimental conditions, which cannot meet the actual needs of clinical PAC-1 testing.

[0009] Technical solution

[0010] The first aspect of this disclosure provides a reagent combination for detecting the platelet activation marker PAC-1, the reagent combination comprising: a first detection probe, a second detection probe, a third detection probe, and a fourth detection probe; the first detection probe is composed of DNA1 and PAC-1 antibody1; the second detection probe is composed of PAC-1 antibody2 and DNA2; the third detection probe is DNA3 labeled with a fluorescent molecule; the fourth detection probe is an antioxidant bound to a carrier molecule; wherein, DNA1 contains a first pairing sequence and a second pairing sequence, DNA2 has a third pairing sequence and a fourth pairing sequence, and DNA3 contains a fifth pairing sequence and a sixth pairing sequence; the third pairing sequence is complementary to the second pairing sequence, the fifth pairing sequence is complementary to the fourth pairing sequence, and the sixth pairing sequence is complementary to the first pairing sequence.

[0011] In some embodiments, the 3' end of DNA1 is modified with NH2C7; the 5' end of DNA2 is modified with NH2C6; and the 5' end of DNA3 is modified with NH2C6.

[0012] In some embodiments, PAC-1 antibody 1 can specifically bind to the first antigenic epitope of PAC-1; and PAC-1 antibody 2 can specifically bind to the second antigenic epitope of PAC-1.

[0013] In some embodiments, the clone number of PAC-1 antibody 1 is 4O21; and the clone number of PAC-1 antibody 2 is IVA30.

[0014] In some embodiments, the 5' end of the DNA3 is coupled to the fluorescent molecule via NH2C6; the fluorescent molecule is acridinium ester (AE).

[0015] In some embodiments, the carrier molecule is graphene oxide.

[0016] In some embodiments, the DNA1 (base sequence as shown in SEQ ID NO.1) has the following sequence: 5'-ACGCTGAGTTATCAACGACTTTTTTTATCACATCAGGCTCTAGCGTATGCTATTG-NH2C7-3'; the DNA2 (base sequence as shown in SEQ ID NO.2) has the following sequence: 5'-NH2C6-TACGTCCAGAACTTTACCAAACCACACCCTTTTTTTGTCGTTGGCTGAGATTC-3'; and the DNA3 labeled with the fluorescent molecule (base sequence as shown in SEQ ID NO.3) has the following sequence: 5'-AE-NH2C6-CGATCTCAGCAACTCAGCAGCG-3'; wherein the first pairing sequence is GCTGAGTT; the second pairing sequence is CAACGAC; the third pairing sequence is GTCGTTG; the fourth pairing sequence is GCTGAGAT; the fifth pairing sequence is ATCTCAGC; and the sixth pairing sequence is AACTCAGC.

[0017] In some embodiments, the DNA1 is conjugated to PAC-1 antibody 1 via a conjugating agent, and the DNA2 is conjugated to PAC-1 antibody 2 via a conjugating agent; the conjugating agent is sodium bis(succinimide) octanoate (BS3).

[0018] In some embodiments, the antioxidant is bonded to the graphene oxide via a condensing agent; the condensing agent is a sulfoxide condensing agent and / or 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride.

[0019] In some embodiments, the carboxyl groups on the graphene oxide are bonded to the hydroxyl groups on the antioxidant via a sulfoxide condensing agent, and the carboxyl groups on the graphene oxide are bonded to the amino groups on the antioxidant via 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride.

[0020] In some embodiments, the antioxidant is selected from one or more of cannabidiol, vitamin C, vitamin E, tea polyphenols, or glutathione, with vitamin C being preferred.

[0021] A second aspect of this disclosure provides a kit for detecting the platelet activation marker PAC-1, comprising:

[0022] The first container stores the conjugate of DNA1 and PAC-1 antibody 1;

[0023] The second container stores the conjugate of DNA2 and PAC-1 antibody 2;

[0024] The third container stores DNA3 labeled with AE;

[0025] The fourth container stores antioxidants that are combined with graphene oxide.

[0026] In some embodiments, the DNA1 (base sequence as shown in SEQ ID NO.1) in the above kit has the following sequence: 5'-ACGCTGAGTTATCAACGACTTTTTTTATCACATCAGGCTCTAGCGTATGCTATTG-NH2C7-3'; the DNA2 (base sequence as shown in SEQ ID NO.2) has the following sequence: 5'-NH2C6-TACGTCCAGAACTTTACCAAACCACACCCTTTTTTTGTCGTTGGCTGAGATTC-3'; and the DNA3 labeled with fluorescent molecules (base sequence as shown in SEQ ID NO.3) has the following sequence: 5'-AE-NH2C6-CGATCTCAGCAACTCAGCAGCG-3'.

[0027] In some embodiments, the clone number of PAC-1 antibody 1 in the above kit is 4O21; and the clone number of PAC-1 antibody 2 is IVA30.

[0028] In some embodiments, the antioxidants in the above kit are one or more of cannabidiol, vitamin C, vitamin E, tea polyphenols, or glutathione.

[0029] The third aspect of this disclosure provides a method for detecting the platelet activation marker PAC-1 using any of the reagent combinations described above, the specific steps of which are as follows:

[0030] a. Mix the first detection probe, the second detection probe, the third detection probe and the fourth detection probe to obtain a detection solution, then mix the sample to be tested with the detection solution and incubate at 36℃~37℃ for 5~10min;

[0031] b. Add a chemiluminescent substrate, and collect the light signal through the PMT detection module in the chemiluminescence detector to obtain the chemiluminescence value of the sample to be tested;

[0032] c. The instrument automatically retrieves the calibration curve. By substituting the chemiluminescence value of the sample into the calibration curve, the concentration of the substance in the sample can be reported.

[0033] In some embodiments, the sample to be tested in the above method is a whole blood sample.

[0034] In some embodiments, the chemiluminescent substrate in the above method is an alkaline solution of hydrogen peroxide.

[0035] In some embodiments, the pH of the alkaline solution of hydrogen peroxide is 9.0, and the hydrogen peroxide concentration is 3% v / v.

[0036] In some embodiments, the alkaline solution is a 10 mM PBS buffer solution.

[0037] In some embodiments, the final concentration of the first detection probe in the detection solution of the above method is 1-20 nM, preferably 5-20 nM, more preferably 10 nM; the final concentration of the second detection probe is 1-20 nM, preferably 5-20 nM, more preferably 10 nM; the final concentration of the third detection probe is 0.05-0.2 μM, preferably 0.15 μM; and the final concentration of the fourth detection probe is 20-25 μg / mL, preferably 20 μg / mL.

[0038] In some embodiments, the volume ratio of the whole blood sample to the detection solution in step a in the above method is 1:10 to 20, preferably 1:20; the volume ratio of the chemiluminescent substrate to the detection solution is 0.8 to 1:1, preferably 1:1.

[0039] In some embodiments, the concentration ratio of the first detection probe, the second detection probe, and the third detection probe in the above method is 1:1:5 to 20.

[0040] Technical effect

[0041] Whole blood can be used for testing: The reagent combination / kit disclosed herein uses homogeneous chemiluminescence to directly detect whole blood samples. Because it is homogeneous, there is no need for complicated pretreatment steps, such as centrifugation to separate plasma or serum. This greatly simplifies the detection process and shortens the detection time. At the same time, it avoids the platelet activation that may occur in in vitro samples, and can reflect the true state of platelet activation in vivo, providing a more accurate assessment of platelet activation status.

[0042] Short reaction time: The detection process for PAC-1 using the reagent kit disclosed herein is very short, generally completed within 5 to 10 minutes. This makes the reagent kit disclosed herein very suitable for clinical environments that require rapid diagnosis and quick treatment decisions.

[0043] Simple to operate: The procedure for detecting PAC-1 using the reagent combination / kit disclosed herein is relatively simple and easy to implement in different medical settings, including those with limited resources.

[0044] Low cost: By eliminating complex separation and cleaning steps and potentially expensive equipment, the cost of detecting PAC-1 using the reagent combination / kit disclosed herein is relatively low, making large-scale detection more economical and practical.

[0045] The requirements for operators are generally low: using the reagent combination / kit disclosed in this publication to detect PAC-1 does not require high professional technical operation skills or data analysis skills, and the test results are easy to interpret without the need for complex data analysis. Attached Figure Description

[0046] Figure 1 shows the complementary pairing diagram of DNA3 with DNA1 and DNA2;

[0047] Figure 2 shows the detection principle diagram of PAC-1. Detailed Implementation

[0048] To facilitate the explanation of the technical solutions applied for, the following is a general explanation and definition of the terms and expressions used in this disclosure.

[0049] 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.

[0050] First, the term "an embodiment" or "embodiment" as used herein refers to a specific feature, structure, or characteristic that may be included in at least one implementation of this disclosure. The word "preferred" appearing in different places throughout this specification does not necessarily refer to the same embodiment, nor is it a single embodiment or an embodiment selectively excluded from other embodiments.

[0051] The following provides further details regarding the reagent combination, kit, and detection method for detecting the platelet activation marker PAC-1 provided in this disclosure.

[0052] The first aspect of this disclosure provides a reagent assembly for detecting the platelet activation marker PAC-1 using homogeneous chemiluminescent immunoassay, the reagent assembly comprising: a first detection probe, a second detection probe, a third detection probe, and a fourth detection probe; the first detection probe being composed of DNA1 and PAC-1 antibody1; the second detection probe being composed of PAC-1 antibody2 and DNA2; the third detection probe being DNA3 labeled with a fluorescent molecule; and the fourth detection probe being an antioxidant bound to a carrier molecule; wherein, DNA1 contains a first pairing sequence and a second pairing sequence, DNA2 has a third pairing sequence and a fourth pairing sequence, and DNA3 contains a fifth pairing sequence and a sixth pairing sequence; the third pairing sequence is complementary to the second pairing sequence, the fifth pairing sequence is complementary to the fourth pairing sequence, and the sixth pairing sequence is complementary to the first pairing sequence.

[0053] Figure 1 shows the complementary pairing diagram of DNA3 with DNA1 and DNA2: the second and third pairing sequences are each composed of 7 bases; the first, fourth, fifth, and sixth pairing sequences are each composed of 8 bases; the first and second pairing sequences are separated by 2 bases; the third and fourth pairing sequences are directly linked; and the fifth and sixth pairing sequences are directly linked. Specifically, the first pairing sequence consists of bases from the 3rd to 10th base position of the single-stranded DNA 1 starting from the 5' end; the second pairing sequence consists of bases from the 13th to 19th base position of the single-stranded DNA 1 starting from the 5' end; the third pairing sequence consists of bases from the 11th to 17th base position of the single-stranded DNA 1 starting from the 3' end; the fourth pairing sequence consists of bases from the 3rd to 10th base position of the single-stranded DNA 2 starting from the 5' end; the fifth pairing sequence consists of bases from the 3rd to 10th base position of the single-stranded DNA 3 starting from the 5' end; and the sixth pairing sequence consists of bases from the 5th to 12th base position of the single-stranded DNA 3 starting from the 3' end.

[0054] A second aspect of this disclosure provides a kit for detecting the platelet activation marker PAC-1, comprising: a first container storing a conjugate of DNA1 and PAC-1 antibody1; a second container storing a conjugate of DNA2 and PAC-1 antibody2; a third container storing DNA3 labeled with AE; and a fourth container storing an antioxidant bound to graphene oxide.

[0055] Figure 2 shows the detection principle of PAC-1. When PAC-1 is present in the sample, the conjugates of DNA1 and PAC-1 antibody 1, and DNA2 and PAC-1 antibody 2, bind to different antigenic epitopes of PAC-1 on the surface of activated platelet membranes, forming DNA1-PAC-1 antibody 1—PAC-1—DNA2-PAC-1 antibody 2 complexes. This ensures that DNA1 and DNA2 are sufficiently close to form ortho-complexes, which can hybridize with DNA3. The complexes are hardly adsorbed by graphene oxide, and luminescence occurs upon the addition of a chemiluminescent substrate. When PAC-1 is absent in the sample, the DNA1-PAC-1 antibody 1—PAC-1—DNA2-PAC-1 antibody 2 complex cannot be formed. Therefore, DNA3 is adsorbed onto the graphene oxide surface through π-π stacking. After the addition of a chemiluminescent substrate, the AE-terminal markers of DNA3 cannot oxidize and emit light due to the presence of antioxidants. Even a small portion of the chemiluminescence is quenched by CRET. Chemiluminescence (CL) signals can be obtained through automatic incubation using the portable HSCL-5000 chemiluminescence analyzer.

[0056] The third aspect of this disclosure provides a method for detecting the platelet activation marker PAC-1 using the above-described reagent combination, the specific steps of which are as follows: a. Mix the first detection probe, the second detection probe, the third detection probe, and the fourth detection probe to obtain a detection solution, then mix the sample to be tested with the detection solution and incubate at 35℃~37℃ for 5~10 min; b. Add a chemiluminescent substrate, and acquire the light signal through the PMT detection module in the chemiluminescence detector to obtain the chemiluminescence value of the sample to be tested; c. The instrument automatically calls the calibration curve, and the concentration of the substance in the sample is reported by substituting the chemiluminescence value of the sample to be tested into the calibration curve.

[0057] In the comparative experiments provided in this publication, unless otherwise specified, all experimental conditions and materials are kept consistent to ensure comparability.

[0058] Unless otherwise specified, all reagents, instruments, and experimental methods used in the embodiments of this disclosure are commercially available. Experimental methods without specific conditions are conventional methods and conditions well known in the art, or are performed according to the conditions recommended by the instrument manufacturer.

[0059] The present disclosure will be further described in detail below with reference to specific embodiments.

[0060] Example 1: Preparation of the third detection probe (DNA3 coupled with AE)

[0061] Step 1: Prepare DNA3 solution: Take 20 uM DNA3 (purchased from Genscript; sequence: 5'-AE-NH2C6-CGATCTCAGCAACTCAGCAGCG-3', base sequence as shown in SEQ ID NO.3) and add 1 mL of purified water to dissolve.

[0062] Step 2: Prepare AE solution: Weigh 4 mg acridine ester (NSP-DMAE-NHS, AE) (purchased from Suzhou Yake Technology Co., Ltd., CAS No. 194357-64-7) and dissolve it in 1 mL of purified water.

[0063] Step 3, Coupling: Add 10uL of DNA3 solution to each 1mg AE solution, mix well, and incubate at 37℃ for 30min.

[0064] Step 4, Dialysis: Aspirate the conjugated DNA3 from the EP tube and add it to a dialysis bag (5kd). Place the sealed dialysis bag into a beaker containing 2-3L of TE solution (10mM Tris, 1mM EDTA, pH=8.0) (soak the dialysis bag beforehand) and perform dialysis. Change the dialysis solution every 2-3 hours, for a total of three dialysis cycles. After dialysis, collect the liquid from the dialysis bag and place it in a centrifuge tube to obtain the prepared third detection probe (DNA3 labeled with AE). Store it at 2-8℃ for later use.

[0065] Example 2: Preparation of the first or second detection probe (PAC-1 antibody labeled DNA1 or DNA2)

[0066] Step 1: Prepare BS3 solution: Weigh 10 mg of bis(succinimide) octanoate sodium salt (BS3) (purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., item number S304724) and dissolve it in 1 mL of purified water.

[0067] Step 2, Activate the antibody: Take out the aliquoted PAC-1 antibody 1 or PAC-1 antibody 2, thaw, and centrifuge to mix. Add 3uL of BS3 solution to each 1mg of antibody, add 6.5uL of DNA1 (purchased from GenScript; sequence: 5'-ACGCTGAGTTATCAACGACTTTTTTTATCACATCAGGCTCTAGCGTATGCTATTG-NH2C7-3', base sequence as shown in SEQ ID NO.1) or DNA2 (purchased from GenScript; sequence: 5'-NH2C6-TACGTCCAGAACTTTACCAAACCACACCCTTTTTTTGTCGTTGGCTGAGATTC-3', base sequence as shown in SEQ ID NO.2), mix well, and incubate at 37℃ for 30min.

[0068] Step 3, Dialysis: Aspirate the conjugated antibody from the EP tube and add it to a dialysis bag (100kb). Tie the dialysis bag and place it in a beaker containing 2-3L of PBS solution for dialysis (soak the dialysis bag beforehand). Change the dialysis solution every 2-3 hours, for a total of three dialysis cycles. After dialysis, collect the liquid from the dialysis bag into a centrifuge tube to obtain the prepared first detection probe (a conjugate of DNA1 and PAC-1 antibody 1) or the second detection probe (a conjugate of DNA2 and PAC-1 antibody 2). Store at 2-8℃ for later use.

[0069] Example 3: Screening of PAC-1 antibodies

[0070] Seven representative antibodies for detecting human PAC-1 were selected as candidate PAC-1 antibodies conjugated with the nucleic acid disclosed in this paper. The information of the candidate PAC-1 antibodies is shown in Table 1.

[0071] Table 1 Information on candidate PAC-1 antibodies

[0072] According to Example 3 above, all 7 antibodies were labeled with DNA1 or DNA2, and a first detection probe (a conjugate of DNA1 and PAC-1 antibody 1) and a second detection probe (a conjugate of DNA2 and PAC-1 antibody 2) were prepared, resulting in a total of 42 pairs (see Table 2).

[0073] Table 2 Antibody-Nucleic Acid Pairing Combinations

[0074] The performance of 42 antibody-nucleic acid pair combinations in detecting PAC-1 in whole blood was tested, and the steps are as follows:

[0075] Step 1: Prepare the detection reagents: Mix the first detection probe (a conjugate of DNA1 and PAC-1 antibody 1), the second detection probe (a conjugate of DNA2 and PAC-1 antibody 2), the third detection probe (DNA3 labeled with AE), and the fourth detection probe (vitamin C bound to graphene oxide; purchased from Xianfeng Nano, catalog number XF248) to obtain a mixed detection solution. The final concentrations of the first, second, third, and fourth detection probes in the detection solution are 10 nM, 10 nM, 0.15 μM, and 20 μg / mL, respectively.

[0076] Step 2: Prepare ADP-activated whole blood sample: Take a whole blood sample and remove a portion of the whole blood. Add 5 μM ADP (purchased from Sigma, item number 01905) to obtain ADP-activated whole blood.

[0077] Step 3, Incubation: Add 10 μL of whole blood or ADP-activated whole blood, mix with 200 μL of the above detection solution, place in the rotor detection port of the HSCL-5000 chemiluminescence analyzer, start incubation, and incubate at 37°C for 10 min. After incubation, rotate the detection port to the detection position, aligning it with the light source module.

[0078] Step 4, Detection: After incubation, the excitation reagent injection step is initiated via the circuit board module and host computer software of the HSCL-5000 chemiluminescence analyzer. Driven by the excitation pump and passing through the heating module, 200 μL of chemiluminescence substrate (hydrogen peroxide in PBS buffer solution, pH 9.0, H2O2 concentration 3% v / v, PBS concentration 10 mM) is added to the reaction vessel. The chemiluminescence signal of the solution is immediately detected by a photomultiplier tube (PMT). Combined with the counting unit, the raw photon count (RLU) is reported. The detection time is 3 seconds, and the recorded chemiluminescence values ​​RLU(S0) and RLU are obtained. ADP激活 (S1), and the ratio (S1 / S0) between the two was calculated. The results are shown in Table 3. Pair 1 has the smallest S0 value, indicating that the other paired antibodies not only detected PAC-1 when platelets are activated, but also detected GPⅡb / Ⅲa complex when platelets are not activated. That is, they cannot identify activated and unactivated GPⅡb / Ⅲa complex. Meanwhile, pair 1 has the largest S1 / S0 ratio, indicating that pair 1 has the best performance.

[0079] Table 3. Screening of different antibody-nucleic acid pairings

[0080] Example 4: Detection of PAC-1 in whole blood

[0081] Step 1: Prepare the detection reagents: Select the first and second detection probes of Pair 1 screened in Example 3. Mix the first detection probe (a conjugate of DNA1 and PAC-1 antibody 1), the second detection probe (a conjugate of DNA2 and PAC-1 antibody 2), the third detection probe (DNA3 labeled with AE), and the fourth detection probe (vitamin C bound to graphene oxide) to obtain a mixed detection solution. The final concentrations of the first, second, third, and fourth detection probes in the detection solution are 10 nM, 10 nM, 0.15 μM, and 20 μg / mL, respectively.

[0082] Step 2, Incubation: Mix 10 μL of calibration solutions of different concentrations (0, 1, 2, 5, 10, 20, 50, 100, 200, 500 mg / L) or whole blood samples containing PAC-1 with 200 μL of the above detection solution, place the mixture in the rotor detection port of the HSCL-5000 chemiluminescence analyzer, and start the incubation at 37°C for 10 min. After incubation, rotate the detection port to the detection position, aligning it with the light source module.

[0083] Step 3, Detection: After incubation, the excitation reagent injection step is initiated via the circuit board module and host computer software of the HSCL-5000 chemiluminescence analyzer. Driven by the excitation pump and passing through the heating module, 200 μL of chemiluminescent substrate (PBS solution of hydrogen peroxide, pH 9.0, H2O2 concentration 3%) is added to the reaction vessel. The chemiluminescence signal of the solution is immediately detected by a photomultiplier tube (PMT). Combined with the counting unit, the raw photon count (RLU) is reported, with a detection time of 3 seconds. The photon count is converted to a concentration value using a calibration curve (imported from the barcode scanning module). Based on the recorded chemiluminescence value (RLU), the calibration curve for PAC-1 and the concentration of PAC-1 in the whole blood sample are obtained. Finally, the test report is selected via a menu on the host computer and printed using the printing module. The heat dissipation module remains operational throughout the entire detection process to ensure reliable temperature control.

[0084] Example 5: Detection of PAC-1 in whole blood

[0085] The difference from Example 5 is as follows: in step 1, the final concentrations of the first detection probe, the second detection probe, the third detection probe, and the fourth detection probe in the detection solution are 1 nM, 1 nM, 0.05 μM, and 20 μg / mL, respectively; in step 2, "mix with 200 μL of the above detection solution" is replaced with "mix with 100 μL of the above detection solution"; and in step 3, "add 200 μL of chemiluminescent substrate" is replaced with "add 160 μL of chemiluminescent substrate".

[0086] Example 6: Detection of PAC-1 in whole blood

[0087] The difference from Example 5 is that in step 1, the final concentrations of the first, second, third, and fourth detection probes in the detection solution are 5 nM, 5 nM, 0.1 μM, and 20 μg / mL, respectively; and in step 3, "add 200 μL of chemiluminescent substrate" is replaced with "add 160 μL of chemiluminescent substrate".

[0088] Example 7: Detection of PAC-1 in whole blood

[0089] The difference from Example 5 is that the fourth detection probe is tea polyphenols bound to graphene oxide (purchased from Xianfeng Nano, catalog number XF247); the final concentrations of the first, second, third and fourth detection probes in the detection solution in step 1 are 20 nM, 20 nM, 0.2 μM and 25 μg / mL, respectively.

[0090] Forty clinical samples (18 apparent healthy individuals and 22 patients with various cardiovascular diseases) were tested using the methods and ELISA detection methods of this disclosure. The results are shown in Table 4.

[0091] Table 4 Comparison of detection values ​​between the method of this disclosure and the ELISA detection method

[0092] The results showed that the error between the detection reagent of this embodiment and the test value of the Human (PAC-1) ELISA KIT from Newlife Biotechnology was 2.51%, indicating that the detection method of this embodiment has high accuracy. After multiple tests, the detection limit of this detection method for PAC-1 was found to be 3–480 pg / mL.

[0093] The above specific embodiments further illustrate the purpose, technical solution and beneficial effects of this disclosure. It should be understood that the above are only specific embodiments of this disclosure and are not intended to limit the scope of protection of this disclosure. Any modifications, equivalent substitutions, improvements, etc., made on the basis of the technical solution of this disclosure should be included within the scope of protection of this disclosure.

Claims

1. A reagent combination for detecting the platelet activation marker PAC-1, wherein, The reagent combination includes: a first detection probe, a second detection probe, a third detection probe, and a fourth detection probe; The first detection probe is composed of DNA1 and PAC-1 antibody1 conjugated together; the DNA1 contains a first pairing sequence and a second pairing sequence; the 3' end of the DNA1 is modified with NH2C7; the PAC-1 antibody1 can specifically bind to the first antigenic epitope of PAC-1; wherein, the clone number of the PAC-1 antibody1 is 4O21; The second detection probe is composed of PAC-1 antibody 2 and DNA 2 conjugated; the DNA 2 has a third pairing sequence and a fourth pairing sequence; the 5' end of the DNA 2 is modified with NH2C6; the third pairing sequence is complementary to the second pairing sequence; the PAC-1 antibody 2 can specifically bind to the second antigenic epitope of PAC-1; wherein, the clone number of the PAC-1 antibody 2 is IVA30; The third detection probe is DNA3 labeled with a fluorescent molecule; the DNA3 contains a fifth pairing sequence and a sixth pairing sequence; the 5' end of the DNA3 is modified with H2C6 and is coupled to the fluorescent molecule via NH2C6; the fifth pairing sequence is complementary to the fourth pairing sequence, and the sixth pairing sequence is complementary to the first pairing sequence; wherein, the fluorescent molecule is AE; The fourth detection probe is an antioxidant bound to a carrier molecule; wherein the carrier molecule is graphene oxide. The sequence of DNA1 is as follows: 5'-ACGCTGAGTTATCAACGACTTTTTTTATCACATCAGGCTCTAGCGTATGCTATTG-NH2C7-3', and the base sequence is shown in SEQ ID NO.1; The sequence of DNA2 is as follows: 5'-NH2C6-TACGTCCAGAACTTTACCAAACCACACCCTTTTTTTGTCGTTGGCTGAGATTC-3', and the base sequence is shown in SEQ ID NO.2; The DNA3 labeled with the fluorescent molecule has the following sequence: 5'-AE-NH2C6-CGATCTCAGCAACTCAGCAGCG-3', and the base sequence is shown in SEQ ID NO.3; Wherein, the first pairing sequence is GCTGAGTT; the second pairing sequence is CAACGAC; The third pairing sequence is GTCGTTG; the fourth pairing sequence is GCTGAGAT; The fifth pairing sequence is ATCTCAGC; the sixth pairing sequence is AACTCAGC.

2. The reagent combination for detecting platelet activation marker PAC-1 according to claim 1, wherein, The DNA1 is coupled to PAC-1 antibody 1 via a coupling agent, and the DNA2 is coupled to PAC-1 antibody 2 via a coupling agent; the coupling agent is sodium bis(succinimide) octanoate.

3. The reagent combination for detecting platelet activation marker PAC-1 according to claim 2, wherein, The antioxidant is selected from one or more of cannabidiol, vitamin C, vitamin E, tea polyphenols, or glutathione.

4. The reagent combination for detecting platelet activation marker PAC-1 according to claim 3, wherein, The antioxidant is vitamin C.

5. A kit for detecting the platelet activation marker PAC-1, wherein, It includes: The first container stores the conjugate of DNA1 and PAC-1 antibody 1; The second container stores the conjugate of DNA2 and PAC-1 antibody 2; The third container stores DNA3 labeled AE; The fourth container stores antioxidants that are combined with graphene oxide.

6. The kit for detecting platelet activation marker PAC-1 according to claim 5, wherein, The sequence of DNA1 is as follows: 5'-ACGCTGAGTTATCAACGACTTTTTTTATCACATCAGGCTCTAGCGTATGCTATTG-NH2C7-3', and the base sequence is shown in SEQ ID NO.1; The sequence of DNA2 is as follows: 5'-NH2C6-TACGTCCAGAACTTTACCAAACCACACCCTTTTTTTGTCGTTGGCTGAGATTC-3', and the base sequence is shown in SEQ ID NO.2; The DNA3 labeled with fluorescent molecules has the following sequence: 5'-AE-NH2C6-CGATCTCAGCAACTCAGCAGCG-3', with the base sequence shown in SEQ ID NO.3; the clone number of PAC-1 antibody 1 is 4O21; the clone number of PAC-1 antibody 2 is IVA30; the antioxidant is one or more of cannabidiol, vitamin C, vitamin E, tea polyphenols, or glutathione.

7. A method for detecting the platelet activation marker PAC-1 using the reagent combination according to any one of claims 1 to 4, wherein, The specific steps are as follows: a. Mix the first detection probe, the second detection probe, the third detection probe and the fourth detection probe to obtain a detection solution, then mix the sample to be tested with the detection solution and incubate at 36℃~37℃ for 5~10min; b. Add a chemiluminescent substrate, and collect the light signal through the PMT detection module in the chemiluminescence detector to obtain the chemiluminescence value of the sample to be tested; c. The instrument automatically retrieves the calibration curve. By substituting the chemiluminescence value of the sample into the calibration curve, the concentration of the substance in the sample can be reported.

8. The detection method according to claim 7, wherein, The sample to be tested is a whole blood sample.

9. The detection method according to claim 8, wherein, The chemiluminescent substrate is an alkaline solution of hydrogen peroxide, wherein the pH of the alkaline solution is 9.0 and the concentration of hydrogen peroxide is 3% v / v.

10. The detection method according to claim 9, wherein, The volume ratio of the whole blood sample to the detection solution in step a is 1:10 to 20; the volume ratio of the chemiluminescent substrate to the detection solution is 0.8 to 1:1 μL.

11. The detection method according to claim 7, wherein, The final concentration of the first detection probe in the detection solution is 1–20 nM; the final concentration of the second detection probe is 1–20 nM; the final concentration of the third detection probe is 0.05–0.2 μM; and the final concentration of the fourth detection probe is 20–25 μg / mL.

12. The detection method according to claim 11, wherein, The concentration ratio of the first detection probe, the second detection probe, and the third detection probe is 1:1:5 to 20.

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