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

Abstract

The present invention relates to the field of biomedicine technology, and specifically to a reagent combination, a kit and a detection method for detecting platelet activation marker PAC‑1. 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 at least formed by coupling DNA1 and PAC‑1 antibody 1; the second detection probe is at least formed by coupling PAC‑1 antibody 2 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. When using the reagent combination or kit of the present invention to detect PAC‑1, the cost is low, and whole blood can be used for detection, which is convenient and quick while avoiding artificial platelet activation in in vitro samples. In addition, the detection operation process is simple, the overall detection time is short, and it is suitable for the actual needs of clinical detection of PAC‑1.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a reagent combination, a kit and a detection method for detecting a platelet activation marker PAC-1. Background Art

[0002] More than 90% of platelets in normal blood circulation are in a resting state. When stimulated by various physical, chemical and biological factors, such as ADP, thrombin or collagen, the morphological structure of platelets changes and causes adhesion, aggregation and release reactions, which is called platelet activation. Platelet membrane glycoprotein (GP) is the main component that mediates platelet activation. It can be divided into plasma membrane glycoprotein and granular membrane glycoprotein according to the distribution site. Plasma membrane glycoproteins include GPIb, GPIæⅡa, GPⅡb / Ⅲa, etc. Granular membrane glycoproteins include CD62P and CD63, etc. Among them, GPⅡb / Ⅲa is the most abundant membrane glycoprotein on platelets and is Ca 2+ Dependent dimer complex. In the resting state of platelets, the GPⅡb / Ⅲa complex exists in an inactive form and does not have the ability to bind to ligands such as fibrinogen. After platelet activation, GPⅡb / Ⅲa undergoes conformational changes and transforms into an active state, becoming an activated GPⅡb / Ⅲa complex. The fibrinogen receptor exposed after GPⅡb / Ⅲa activation is PAC-1 (platelet activation complex-1), that is, the activated GPⅡb / Ⅲa complex. PAC-1 is a necessary condition for platelet aggregation and the final common link for all platelet agonists to cause platelet aggregation. It is an early marker of platelet activation. Therefore, detecting PAC-1 on the surface of platelets can accurately reflect the early state of platelet activation.

[0003] The occurrence and development of 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 the risk and status of thrombotic diseases such as acute coronary syndrome (ACS) and deep vein thrombosis, clinical medication guidance for cardiovascular diseases such as coronary heart disease, and evaluation of the effects of antiplatelet drugs and platelet therapy. At the same time, in addition to cardiovascular and cerebrovascular diseases, the detection of PAC-1 can also be used to evaluate platelet function in other disease states, such as chronic obstructive pulmonary disease (COPD). In addition, the detection of PAC-1 is of great significance for studying the molecular mechanisms and signal transduction pathways of platelet activation, and helps to develop new antiplatelet treatment strategies.

[0004] Traditional PAC-1 detection mainly includes flow cytometry (FCM) and enzyme-linked immunosorbent assay (ELISA), which have important applications in platelet activation research. However, these methods have certain disadvantages. For example, flow cytometry requires professional equipment and detection reagents, and the test cost is high. In addition, platelets are easier to be activated in vitro, while flow cytometry takes a long time to detect and the operation process is relatively complicated. When handling samples, any improper handling may cause artificial activation of platelets, which in turn affects the test results. Therefore, this detection method is not suitable for situations that require rapid diagnosis, and it requires high professional skills of technicians, who must master professional instrument operation and have professional data analysis capabilities. As for another method, ELISA, it is currently only used for scientific research to detect PAC-1 and cannot be used for clinical diagnosis, which limits its scope of application. In addition, the reagents in the ELISA kit cannot be mixed with ELISA kit reagents from other batches or sources, which limits the flexibility of the experiment. Similarly, the ELISA operation steps are complicated, and there are washing and separation steps, which can easily lead to in vitro activation of platelets, so the experimental conditions must be strictly controlled.

[0005] Based on the above research, a reagent combination / kit that has low development cost and can conveniently, quickly and accurately detect PAC-1 has good application prospects and research significance. Summary of the invention

[0006] Technical issues

[0007] The existing PAC-1 detection kits are complex to operate, time-consuming, costly, and have harsh experimental conditions, and cannot meet the actual needs of clinical PAC-1 detection.

[0008] Technical Solution

[0009] The first aspect of the present invention 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 formed by coupling DNA1 and PAC-1 antibody 1; the second detection probe is formed by coupling PAC-1 antibody 2 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 the DNA1 contains a first pairing sequence and a second pairing sequence, the DNA2 has a third pairing sequence and a fourth pairing sequence, and the 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.

[0010] In some embodiments, the 3' end of the DNA1 is modified with NH2C7; the 5' end of the DNA2 is modified with NH2C6; and the 5' end of the DNA3 is modified with NH2C6.

[0011] In some embodiments, the PAC-1 antibody 1 can specifically bind to a first antigenic epitope of PAC-1; and the PAC-1 antibody 2 can specifically bind to a second antigenic epitope of PAC-1.

[0012] In some embodiments, the clone number of the PAC-1 antibody 1 is 4021; the clone number of the PAC-1 antibody 2 is IVA30.

[0013] In some embodiments, the 5' end of the DNA3 is coupled to the fluorescent molecule via NH2C6; the fluorescent molecule is acridinium ester (AE).

[0014] In some embodiments, the carrier molecule is graphene oxide.

[0015] In some embodiments, the sequence of the DNA1 (the base sequence is shown in SEQ ID NO.1) is as follows: 5'-ACGCTGAGTTATCAACGACTTTTTTTATCACATCAGGCTCTAGCGTATGCTATTG-NH2C7-3'; the sequence of the DNA2 (the base sequence is shown in SEQ ID NO.2) is as follows: 5'-NH2C6-TACGTCCAGAACTTTACCAAACCACACCCTTTTTTTGTCGTTGGCTGAGATTC-3'; the sequence of the DNA3 labeled with the fluorescent molecule (the base sequence is shown in SEQ ID NO.3) is as follows: 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.

[0016] In some embodiments, the DNA1 is coupled to the PAC-1 antibody 1 via a coupling agent, and the DNA2 is coupled to the PAC-1 antibody 2 via a coupling agent; the coupling agent is disuccinimidyl suberate sodium salt (BS3).

[0017] In some embodiments, the graphene oxide is combined with the antioxidant via a condensing agent; the condensing agent is an oxide sulfoxide condensing agent and / or 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride.

[0018] In some embodiments, the carboxyl groups on the graphene oxide are bonded to the hydroxyl groups on the antioxidant via an oxidized sulfoxide condensation 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.

[0019] In some embodiments, the antioxidant is selected from one or more of cannabidiol, vitamin C, vitamin E, tea polyphenols or glutathione, preferably vitamin C.

[0020] The second aspect of the present invention provides a kit for detecting platelet activation marker PAC-1, comprising:

[0021] A first container stores a conjugate of DNA 1 and PAC-1 antibody 1;

[0022] The second container stores the conjugate of DNA2 and PAC-1 antibody 2;

[0023] The third container stores DNA3 labeled with AE;

[0024] The fourth container stores an antioxidant combined with graphene oxide.

[0025] In some embodiments, the sequence of the DNA1 (base sequence as shown in SEQ ID NO.1) in the above-mentioned kit is as follows: 5'-ACGCTGAGTTATCAACGACTTTTTTTATCACATCAGGCTCTAGCGTATGCTATTG-NH2C7-3'; the sequence of the DNA2 (base sequence as shown in SEQ ID NO.2) is as follows: 5'-NH2C6-TACGTCCAGAACTTTACCAAACCACACCCTTTTTTTGTCGTTGGCTGAGATTC-3'; the sequence of the DNA3 labeled with the fluorescent molecule (base sequence as shown in SEQ ID NO.3) is as follows: 5'-AE-NH2C6-CGATCTCAGCAACTCAGCAGCG-3'.

[0026] In some embodiments, the clone number of the PAC-1 antibody 1 in the above kit is 4021; the clone number of the PAC-1 antibody 2 is IVA30.

[0027] In some embodiments, the antioxidant in the above kit is one or more of cannabidiol, vitamin C, vitamin E, tea polyphenols or glutathione.

[0028] The third aspect of the present invention provides a method for detecting the platelet activation marker PAC-1 using any one of the above reagent combinations, the specific steps of which are as follows:

[0029] a. The first detection probe, the second detection probe, the third detection probe and the fourth detection probe are mixed to obtain a detection solution, and then the sample to be tested is mixed with the detection solution and incubated at 36 ℃ ~ 37 ℃ for 5 to 10 min;

[0030] b. Adding a chemiluminescent substrate, collecting light signals through the PMT detection module in the chemiluminescence detector to obtain the chemiluminescence value of the sample to be tested;

[0031] c. The instrument automatically calls the calibration curve, and the chemiluminescence value of the sample to be tested is substituted into the calibration curve to report the concentration of the substance in the sample.

[0032] In some embodiments, the sample to be tested in the above method is a whole blood sample.

[0033] In some embodiments, the chemiluminescent substrate in the above method is an alkaline solution of hydrogen peroxide.

[0034] In some embodiments, the alkaline solution of hydrogen peroxide has a pH of 9.0 and a hydrogen peroxide concentration of 3% v / v.

[0035] In some embodiments, the alkaline solution is a 10 mM PBS buffer solution.

[0036] In some embodiments, the final concentration of the first detection probe in the detection solution in the above method is 1-20 nM, preferably 5-20 nM, and more preferably 10 nM; the final concentration of the second detection probe is 1-20 nM, preferably 5-20 nM, and more preferably 10 nM; the final concentration of the third detection probe is 0.05-0.2 μM, preferably 0.15 μM; the final concentration of the fourth detection probe is 20-25 μg / mL, preferably 20 μg / mL.

[0037] In some embodiments, the volume ratio of the whole blood sample in the above method to the detection solution in step a is 1:10-20, preferably 1:20; the volume ratio of the chemiluminescent substrate to the detection solution is 0.8-1:1, preferably 1:1.

[0038] In some embodiments, in the above method, the concentration ratio of the first detection probe, the second detection probe, and the third detection probe is 1:1:5-20.

[0039] Technical Effects

[0040] Whole blood testing can be used: The reagent combination / kit of the present invention uses homogeneous chemiluminescence to directly detect whole blood samples. Because it is homogeneous, no complicated pretreatment steps are required, 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 be experienced by in vitro samples, and can reflect the true state of platelet activation in vivo, providing a more accurate assessment of platelet activation status.

[0041] Short reaction time: The process of the reagent combination / kit of the present invention for detecting PAC-1 takes a very short time, generally completed within 5 to 10 minutes, which makes the reagent combination / kit of the present invention very suitable for clinical environments that require rapid diagnosis and prompt treatment decisions.

[0042] Simple operation: The operation process of the reagent combination / kit for detecting PAC-1 is relatively simple and can be easily implemented in different medical environments, including those with limited resources.

[0043] Low cost: Since complex separation and washing steps, as well as expensive equipment that may be required, are omitted, the cost of detecting PAC-1 using the reagent combination / kit of the present invention is relatively low, making large-scale detection more economical and more practical.

[0044] Requirements for operators are general: using the reagent combination / kit of the present invention to detect PAC-1 does not require high professional technical operation ability and data analysis ability, and the test results are easy to interpret without complex data analysis. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 Shown is the complementary pairing diagram of DNA3 and DNA1 and DNA2;

[0046] Figure 2 Shown is a schematic diagram of the detection principle of PAC-1. DETAILED DESCRIPTION

[0047] To facilitate the technical solution of the application, the following first provides a general description and definition of the terms and expressions involved in the present invention.

[0048] The terms "comprises", "includes" or any other variation thereof are intended to cover non-exclusive inclusion, such that a process, method, article or apparatus that includes a list of elements includes not only those elements, but also other elements not explicitly listed, or elements inherent to such process, method, article or apparatus. In the absence of further limitations, an element defined by the phrase "comprises a ..." does not exclude the presence of additional identical elements in the process, method, article or apparatus that includes the element.

[0049] First, the term "one embodiment" or "embodiment" as used herein refers to a specific feature, structure or characteristic that may be included in at least one implementation of the present invention. The term "preferably" that appears in different places in this specification does not refer to the same embodiment, nor is it a separate or selective embodiment that is mutually exclusive with other embodiments.

[0050] The following further describes the reagent combination, kit and detection method for detecting the platelet activation marker PAC-1 provided by the present invention.

[0051] The first aspect of the present invention provides a reagent combination for detecting platelet activation marker PAC-1 using homogeneous chemiluminescence immunoassay, 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 formed by coupling DNA1 and PAC-1 antibody 1; the second detection probe is formed by coupling PAC-1 antibody 2 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 the DNA1 contains a first pairing sequence and a second pairing sequence, the DNA2 has a third pairing sequence and a fourth pairing sequence, and the 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.

[0052] The complementary pairing diagram of DNA3, DNA1 and DNA2 is shown in the figure Figure 1 As shown: the second pairing sequence and the third pairing sequence are both composed of 7 bases; the first pairing sequence, the fourth pairing sequence, the fifth pairing sequence and the sixth pairing sequence are all composed of 8 bases; the first pairing sequence and the second pairing sequence are separated by 2 bases, the third pairing sequence is directly connected to the fourth pairing sequence, and the fifth pairing sequence is directly connected to the sixth pairing sequence. Specifically, the first pairing sequence is composed of bases at 3-10 base sites starting from the 5' end of the single-stranded DNA1; the second pairing sequence is composed of bases at 13-19 base sites starting from the 5' end of the single-stranded DNA1; the second pairing sequence is composed of bases at 11-17 base sites starting from the 3' end of the single-stranded DNA1; the fourth pairing sequence is composed of bases at 3-10 base sites starting from the 5' end of the single-stranded DNA2; the fifth pairing sequence is composed of bases at 3-10 base sites starting from the 5' end of the single-stranded DNA3; the sixth pairing sequence is composed of bases at 5-12 base sites starting from the 3' end of the single-stranded DNA3.

[0053] The second aspect of the present invention provides a kit for detecting the platelet activation marker PAC-1, which comprises: a first container for storing a conjugate of DNA1 and PAC-1 antibody 1; a second container for storing a conjugate of DNA2 and PAC-1 antibody 2; a third container for storing DNA3 labeled with AE; and a fourth container for storing an antioxidant combined with graphene oxide.

[0054] The detection principle diagram of PAC-1 is as follows Figure 2As shown in the figure, when PAC-1 is present in the sample, the conjugate of DNA1 and PAC-1 antibody 1, and the conjugate of DNA2 and PAC-1 antibody 2 bind to different antigenic epitopes of PAC-1 on the surface of activated platelet membrane, respectively, to form a DNA1-PAC-1 antibody 1-PAC-1-DNA2-PAC-1 antibody 2 complex, so that DNA1 and DNA2 are close enough to form an adjacent complex, and can hybridize with DNA3. The complex is almost not adsorbed by graphene oxide, and can emit light by adding a chemiluminescent substrate. When there is no PAC-1 in the sample, the DNA1-PAC-1 antibody 1-PAC-1-DNA2-PAC-1 antibody 2 complex cannot be formed, and DNA3 is adsorbed on the surface of graphene oxide through π-π stacking. After adding the chemiluminescent substrate, the AE labeled at the end of DNA3 cannot be oxidized and emitted light due to the presence of antioxidants, and even a small amount of chemiluminescence is quenched due to CRET. Chemiluminescence (CL) signals can be obtained by automatic incubation with a portable HSCL-5000 chemiluminometer.

[0055] The third aspect of the present invention provides a method for detecting the platelet activation marker PAC-1 using the above-mentioned reagent combination, and 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, and then mix the sample to be tested with the detection solution, and incubate at 35°C~37°C for 5~10 min; b. Add a chemiluminescent substrate, and collect light signals 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 chemiluminescence value of the sample to be tested is substituted into the calibration curve to report the concentration of the substance in the sample.

[0056] In each group of comparative experiments provided by the present invention, unless otherwise specified, other experimental conditions, materials, etc., except for the differences indicated in each group, are kept consistent to ensure comparability.

[0057] The reagents, instruments, equipment and experimental methods used in the embodiments of the present invention, unless otherwise specified, are all commercially available. Experimental methods without specific conditions are conventional methods and conventional conditions well known in the art, or are based on the conditions recommended by the instrument manufacturer.

[0058] The present invention is further described in detail below in conjunction with specific embodiments.

[0059] Example 1: Preparation of the third detection probe (DNA3 coupled to AE)

[0060] 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), add 1 mL purified water to dissolve.

[0061] Step 2, prepare AE solution: weigh 4 mg of acridinium ester (NSP-DMAE-NHS, AE) (purchased from Suzhou Yaco Technology Co., Ltd., CAS No. 194357-64-7) and dissolve it in 1 mL of purified water.

[0062] Step 3, coupling: add 10 uL DNA3 solution to every 1 mg AE solution, mix well, and incubate at 37°C for 30 min.

[0063] Step 4, dialysis: aspirate the coupled DNA3 from the EP tube and add it to a dialysis bag (specification 5 kd), place the tied dialysis bag in a beaker containing 2-3 L TE solution (10 mM Tris, 1mM EDTA, PH=8.0) (soak the dialysis bag in advance), and perform dialysis. Change the dialysis solution every 2-3 hours, and perform dialysis three times in total. After the dialysis is completed, collect the liquid in the dialysis bag and place it in a centrifuge tube to obtain the configured third detection probe (DNA3 labeled with AE), and store it at 2-8°C for later use.

[0064] Example 2: Preparation of the first detection probe or the second detection probe (PAC-1 antibody labeled DNA1 or DNA2)

[0065] Step 1. Prepare BS3 solution: weigh 10 mg of disuccinimidyl suberate sodium salt (BS3) (purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., catalog number S304724) and dissolve it in 1 mL of purified water.

[0066] Step 2, Activate the antibody: Take out the aliquoted PAC-1 antibody 1 or PAC-1 antibody 2, thaw it, and centrifuge to mix. Add 3 uL BS3 solution to every 1 mg of antibody, add 6.5 uL 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°C for 30 min.

[0067] Step 3, dialysis: aspirate the conjugated antibody from the EP tube, add it to a dialysis bag (specification 100 kd), tie the dialysis bag and place it in a beaker containing 2~3 L PBS solution for dialysis (soak the dialysis bag in advance), replace the dialysis solution once every 2~3 hours, and dialyze three times in total. After the dialysis is completed, collect the liquid in the dialysis bag into a centrifuge tube to obtain the configured first detection probe (conjugate of DNA1 and PAC-1 antibody 1) or second detection probe (conjugate of DNA2 and PAC-1 antibody 2), and store it at 2~8℃ for use.

[0068] Example 3: Screening of PAC-1 Antibodies

[0069] Seven representative antibodies for detecting human PAC-1 were selected as candidate PAC-1 antibodies conjugated to the nucleic acid of the present invention. The information of the candidate PAC-1 antibodies is shown in Table 1.

[0070] Table 1 Information of candidate PAC-1 antibodies

[0071]

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

[0073] Table 2 Antibody-nucleic acid pairing combinations

[0074]

[0075] The performance of each of the 42 antibody-nucleic acid pairs in detecting PAC-1 in whole blood was tested as follows:

[0076] Step 1, prepare the detection reagent: mix the first detection probe (DNA1 and PAC-1 antibody 1 conjugate), the second detection probe (DNA2 and PAC-1 antibody 2 conjugate), the third detection probe (DNA3 labeled with AE) and the fourth detection probe (vitamin C conjugated with graphene oxide; purchased from Xianfeng Nano, catalog number XF248) to obtain a mixed detection solution. 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 10 nM, 10 nM, 0.15 μM and 20 μg / mL, respectively.

[0077] Step 2. Prepare ADP-activated whole blood sample: Take a whole blood sample, take out part of the whole blood, add 5 uM ADP (purchased from Sigma, catalog number 01905), and obtain ADP-activated whole blood.

[0078] Step 3, incubation: add 10 uL whole blood or ADP-activated whole blood, mix with 200 μL of the above detection solution, place in the rotor detection hole of the HSCL-5000 chemiluminescence instrument, start incubation, and incubate at 37°C for 10 min. After the incubation, the detection hole is turned to the detection position, facing the light source module.

[0079] Step 4, detection: after incubation, the step of injecting the stimulator reagent into the reaction cup is started through the circuit board module and the upper computer software control of the HSCL-5000 chemiluminescence instrument. The stimulator reagent is driven by the stimulator pump, passes through the heating module, and finally adds 200 μL of chemiluminescent substrate (PBS buffer solution of hydrogen peroxide, PH=9.0, H2O2 concentration of 3% v / v, PBS concentration of 10 mM) into the reaction cup. The chemiluminescent signal of the solution is immediately detected by the photomultiplier tube (PMT). Combined with the counting unit, the raw photon value (RLU) is reported. The detection time is 3 s, and the recorded chemiluminescence values ​​RLU (S0) and RLU are obtained. ADP激活 (S1), and calculated the ratio of the two (S1 / S0). The results are shown in Table 3. The S0 value of pairing 1 is the smallest, indicating that the other paired antibodies not only detect PAC-1 when platelets are activated, but also detect the GPⅡb / Ⅲa complex when platelets are not activated, that is, they cannot identify activated and non-activated GPⅡb / Ⅲa complexes. At the same time, the S1 / S0 ratio of pairing 1 is the largest, indicating that pairing 1 has the best performance.

[0080] Table 3 Test screening of different antibody-nucleic acid pairing combinations

[0081]

[0082] Example 4: Detection of PAC-1 in whole blood

[0083] Step 1, prepare the detection reagent: select the first detection probe and the second detection probe of the pair 1 screened in Example 3, mix the first detection probe (conjugate of DNA1 and PAC-1 antibody 1), the second detection probe (conjugate of DNA2 and PAC-1 antibody 2), the third detection probe (DNA3 labeled with AE) and the fourth detection probe (vitamin C combined with graphene oxide) to obtain a mixed detection solution. 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 10 nM, 10 nM, 0.15 μM and 20 μg / mL, respectively.

[0084] Step 2, incubation: Mix 10 μL of calibration solution of different concentrations (0, 1, 2, 5, 10, 20, 50, 100, 200, 500 mg / L) or whole blood sample containing PAC-1 with 200 μL of the above detection solution, place it in the rotor detection hole of the HSCL-5000 chemiluminescence instrument, start incubation, and incubate at 37°C for 10 min. After the incubation, the detection hole is turned to the detection position, facing the light source module.

[0085] Step 3, detection: After incubation, the step of injecting the excitation reagent into the reaction cup is started through the circuit board module of the HSCL-5000 chemiluminescence instrument and the upper computer software control. The excitation reagent is driven by the excitation pump, passes through the heating module, and finally adds 200 μL of chemiluminescent substrate (PBS solution of hydrogen peroxide, PH=9.0, H2O2 concentration of 3%) to the reaction cup, and immediately detects the chemiluminescent signal of the solution through the photomultiplier tube (PMT). Combined with the counting unit, the original photon value (RLU) is reported, and the detection time is 3 s. The photon value is converted to the concentration value through the calibration curve (imported by the code scanning module), and the calibration curve of PAC-1 and the concentration of PAC-1 in the whole blood sample to be tested are obtained according to the recorded chemiluminescence value (RLU). Finally, the test report is selected through the menu on the upper computer and printed out in combination with the printing module. The heat dissipation module is in operation throughout the entire detection process to ensure reliable temperature control.

[0086] Example 5: Detection of PAC-1 in whole blood

[0087] The difference from Example 5 is that 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, "mixed with 200 μL of the above detection solution" is replaced by "mixed with 100 μL of the above detection solution"; in step 3, "add 200 μL of chemiluminescent substrate" is replaced by "add 160 μL of chemiluminescent substrate".

[0088] Example 6: Detection of PAC-1 in whole blood

[0089] The difference from Example 5 is that 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 5 nM, 5 nM, 0.1 μM and 20 μg / mL, respectively; in step 3, "add 200 μL of chemiluminescent substrate" is replaced by "add 160 μL of chemiluminescent substrate".

[0090] Example 7: Detection of PAC-1 in whole blood

[0091] The difference from Example 5 is that the fourth detection probe is tea polyphenols combined with graphene oxide (purchased from Xianfeng Nano, product number XF247); 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 in step 1 are 20 nM, 20 nM, 0.2 μM and 25 μg / mL, respectively.

[0092] 40 clinical samples (including 18 apparently healthy subjects and 22 patients with various cardiovascular diseases) were tested using the method of the embodiment of the present invention and the ELISA detection method. The results are shown in Table 4.

[0093] Table 4 Comparison of the detection values ​​of the method of the present invention and the ELISA detection method

[0094]

[0095] The results show that the error between the detection reagent of the present invention and the test value of Human (PAC-1) ELISA KIT of Newlife Biotech is 2.51%, indicating that the detection method of this embodiment has a high accuracy. After multiple tests, the detection limit of the detection method of the present invention for PAC-1 is 3~480 pg / mL.

[0096] The above specific implementation methods further illustrate the purpose, technical solutions and beneficial effects of the present invention in detail. It should be understood that the above are only specific implementation methods of the present invention and are not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc. made on the basis of the technical solutions of the present invention should be included in the scope of protection of the present invention.

Claims

1. A reagent combination for detecting platelet activation marker PAC-1 in whole blood, characterized in that: 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 coupling DNA1 and PAC-1 antibody 1; 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 antibody 1 can specifically bind to the first antigen epitope of PAC-1; wherein the clone number of the PAC-1 antibody 1 is 4O21; The second detection probe is formed by coupling PAC-1 antibody 2 and DNA2; the DNA2 has a third pairing sequence and a fourth pairing sequence; the 5' end of the DNA2 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 antigen 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 coupled with the fluorescent molecule through 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; and the antioxidant is vitamin C; The sequence of the DNA 1 is as follows: 5'-ACGCTGAGTTATCAACGACTTTTTTTATCACATCAGGCTCTAGCGTATGCTATTG-NH2C7-3', and the base sequence is shown in SEQ ID NO.1; The sequence of the DNA2 is as follows: 5'-NH2C6-TACGTCCAGAACTTTACCAAACCACACCCTTTTTTTGTCGTTGGCTGAGATTC-3', and the base sequence is shown in SEQ ID NO.2; The sequence of the fluorescent molecule labeled DNA 3 is as follows: 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 the platelet activation marker PAC-1 in whole blood according to claim 1, characterized in that: The DNA1 is coupled to the PAC-1 antibody 1 through a coupling agent, and the DNA2 is coupled to the PAC-1 antibody 2 through a coupling agent; the coupling agent is disuccinimidyl suberate sodium salt.

3. A kit, characterized in that: The kit comprises the reagent combination for detecting the platelet activation marker PAC-1 in whole blood according to claim 1 or 2; the kit comprises: A first container stores a conjugate of DNA 1 and PAC-1 antibody 1; The second container stores the conjugate of DNA2 and PAC-1 antibody 2; The third container stores DNA3 labeled with AE; The fourth container stores an antioxidant combined with graphene oxide.

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