CN111735809A

CN111735809A - Chemiluminescence detection method and detection kit for aspirin metabolism and platelet high reactivity

Info

Publication number: CN111735809A
Application number: CN201911224764.8A
Authority: CN
Inventors: 李明勇
Original assignee: Hunan Yuanjing Biotechnology Co ltd
Current assignee: Hunan Yuanjing Biotechnology Co ltd
Priority date: 2019-06-02
Filing date: 2019-12-04
Publication date: 2020-10-02
Anticipated expiration: 2039-12-04
Also published as: CN111735809B

Abstract

The invention discloses a chemiluminescence detection method and a detection kit for aspirin metabolism and platelet high reactivity, wherein the kit comprises a monoclonal antibody combined with 11-dehydrothromboxane B2, an 11-dehydrothromboxane B2 competitor, magnetic particles and a luminescent substrate, wherein the monoclonal antibody is a murine monoclonal antibody, and the amino acid sequence of the light chain variable region of the murine monoclonal antibody is shown in a sequence table SEQ ID NO: 2, the amino acid sequence of the heavy chain variable region of the mouse single-antibody is shown as the sequence table SEQ ID NO: 4, respectively. The invention adopts the chemiluminescence detection method and the detection kit for aspirin metabolism and platelet high reactivity, can quickly detect whether a patient has aspirin resistance or resistance, provides a scientific reference basis for clinical administration, has high sensitivity and high accuracy, is not easily influenced by biochemical indexes, and reduces the problem that the anti-platelet aggregation treatment fails or has poor effect due to aspirin resistance of clinical patients.

Description

Chemiluminescence detection method and detection kit for aspirin metabolism and platelet high reactivity

Technical Field

The invention relates to a chemiluminescence detection method and a detection kit for aspirin metabolism and platelet high reactivity, and belongs to the field of biological detection.

Background

First, metabolism of TXA2 and detection of 11dhTXB2

11-dehydro-thromboxane B2 (11-hydro-thromboxane B2, 11dhTXB2) is a final metabolite of thromboxane A2(thromboxane A2, TXA2), is mainly discharged through the kidney, and plays an important role in hemostasis and cardiovascular disease occurrence. Arachidonic acid produces prostaglandin H under the action of prostaglandin H synthase 1 and 2 (also known as COX-1 and COX-2). Prostaglandin H is unstable in chemical properties and can be converted into various prostaglandins with biological activity under the action of isomerase, including TXA2, prostacyclin I2, prostaglandin D2, prostaglandin E2 and prostaglandin F2 alpha and the like. TXA2 is mainly synthesized in platelets through COX-1 pathway, and newborn platelets express COX-1 and COX-2 at the same time, while mature platelets express COX-1 only, and COX-2 is mainly expressed in nucleated cells such as monocytes and endothelial cells. TXA2 has strong vasoconstricting effect, and can also activate platelets by binding Thromboxane Platelet Receptor (TPR) to promote aggregation, thereby playing a role in promoting thrombosis. In addition to TXA2, thrombin, collagen and Adenosine Diphosphate (ADP) can activate platelets through other pathways. TXA2 is highly unstable and rapidly hydrolyzes to the more stable thromboxane B2(thromboxane B2, TXB 2). TXB2 is then converted to 11dhTXB2 with a longer half-life in the liver and excreted via the urine. TXA2 is a substance produced by arachidonic acid under the action of Cyclooxygenase (COX) and has the biological activity of promoting platelet aggregation, contracting blood vessels, etc., and plays an important role in the process of thrombosis. Aspirin inhibits platelet aggregation and its role in the development of atherosclerotic heart disease (coronary heart disease) and the development of Acute Coronary Syndrome (ACS) by irreversibly inhibiting COX-1 activity, reducing TXA2 synthesis. The level of the metabolite 11dhTXB2 of TXA2 reflects platelet activity and the antiplatelet effect of aspirin.

In vitro platelet activation significantly affected the results of the serum TXB2 assay, but did not affect the serum 11dhTXB2 levels, and urine correlated well with the serum 11dhTXB2 concentration, thus determining the urine 11dhTXB2 level more effectively reflects the in vivo TXA2 production. The detection methods of 11dhTXB2 include Radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA), and liquid chromatography-tandem mass spectrometry (LC-MS/MS). Since 11dhTXB2 has a small molecular weight and is not highly concentrated in urine, ELISA is currently used for measurement, the anti-11 dhTXB2 antibody used includes both monoclonal and polyclonal antibodies, and the measured urine 11dhTXB2 concentration needs to be corrected by the urine creatinine concentration to exclude the influence of urine concentration and renal function, so random urine samples can be used for detection. The LC-MS/MS method has shorter application time than ELISA and RIA, but can detect 11dhTXB2 more specifically, and the variation rate of the detection result is smaller.

Second, action and mechanism of aspirin

Atherosclerosis is the basis of ischemic heart disease and cerebrovascular disease, and arterial thrombosis is the leading cause of myocardial infarction and ischemic stroke. Platelets are the main cellular component of arterial thrombosis and are also involved in the development and progression of atherosclerosis. Aspirin can acetylate serine in the key part of prostaglandin H synthetase, thus inhibiting COX activity, and blocking the generation of TXA2 and thromboxane-induced platelet activation. Although the half-life of aspirin is only 15-20 min, the half-life of aspirin can irreversibly inhibit COX in platelets, and mature platelets without cell nuclei cannot synthesize new COX, so that the antiplatelet effect can last for 7-10 d (namely, the average life of platelets). The inhibition effect of aspirin on COX-1 is about 50-100 times of that of aspirin on COX-2, and under the physiological condition, low-dose aspirin (30-75 mg/d) can effectively inhibit 95% of COX-1 activity and the amount of TXA2, but has no obvious influence on prostacyclin I2 mainly generated by COX-2 and the effects of expanding blood vessels, resisting atherosclerosis, protecting gastrointestinal mucosa and the like. Aspirin plays an important role in the primary and secondary prevention of cardiovascular and cerebrovascular diseases.

Large meta-analysis shows that aspirin can reduce the risk of cardiovascular events (myocardial infarction, stroke, vascular death) of various types in 23% of high-risk people, wherein the dose of less than 75mg/d can reduce the risk of cardiovascular events by 13%, the dose of 75-150 mg/d can reduce the risk of cardiovascular events by 32%, and further increase of aspirin dose does not benefit more. Patients who have suffered myocardial infarction and stroke may also receive similar benefits.

Although the prophylactic role of aspirin in cardiovascular events has been widely demonstrated, in the case of standard aspirin therapy, some patients still develop arterial thrombotic events, and laboratory examinations for the effect of aspirin, including determination of COX-1 pathway-associated platelet aggregation rates and determination of thromboxane metabolite content in blood and urine, also suggest a large variability in the response of the population to aspirin, a condition originally referred to as Aspirin Resistance (AR). However, the metabolic disorder or genetic defect of the drug that causes the real aspirin to resist is not clear, and the description of the AR is misleading, so that the state of incomplete aspirin reaction is described by high-on-aspirin platelet reactivity (HAPR) under aspirin treatment. The mechanism of HAPR generation is not fully understood, and possible causes suggested by current research include: (1) under the conditions of diabetes, severe peripheral arterial disease and the like, the platelet renewal is accelerated, the immature platelets are increased, the aggregation is enhanced, and the COX-1 and COX-2 activities of the newborn and immature platelets cannot be inhibited in time by 1 time of low-dose aspirin every day; (2) TXA2 produced by monocyte macrophage and vascular endothelial cell COX-2 pathway is increased under inflammation and the like, and cannot be effectively inhibited by low-dose aspirin; (3) platelets are activated via the non-COX-TXA 2 pathway; (4) certain genetic polymorphisms that may affect thromboxane metabolism and platelet receptor function affect an individual's response to aspirin; (5) the combined use of certain non-steroidal anti-inflammatory drugs, such as ibuprofen, can impair the antiplatelet effect of aspirin.

Reflection of the Effect of tris, 11dhTXB2 on Aspirin

The meta-study showed that patients with HAPR who were tested by the various Platelet Function Test (PFT) were 3-4 times more at risk of cardiovascular events than non-HAPR patients (39% and 16%, OR3.85, 95% CI 3.08-4.80, respectively). Therefore, the method is especially important for reducing the incidence rate of adverse events of the high-risk population and improving the cardiovascular prognosis by determining the population with HAPR and correspondingly adjusting the anti-platelet scheme. However, under the influence of factors such as a detection mechanism, application population, aspirin dosage and the like, the proportion of HAPR detected by different PFTs is greatly different (1-60 percent), and the prompt intensity of adverse events is different, so that the determination of the platelet detection method with the most guiding significance is always a research hotspot in the field. Light Transmission Aggregation (LTA) has been used as a gold standard for platelet function detection. However, the detection method of each laboratory lacks a unified standard, and the detection result is susceptible to interference of various factors, so that the application of the method has certain limitations. The urine 11dhTXB2 method uses random urine as a detection sample, is a non-invasive platelet function detection method, is not limited by detection time, and can effectively reflect the generation of TXA2 in vivo and indirectly reflect the activity of platelets in vivo. The HOPE study and CHARISMA study (using polyclonal antibody ELISA assays) showed that high levels of 11dhTXB2 in urine from patients using aspirin were associated with increased risk of stroke, myocardial infarction, and cardiovascular death, suggesting that platelet activity following aspirin application, as reflected by the concentration of 11dhTXB2 in urine, could help identify cardiovascular high risk groups.

The thromboxane metabolism is influenced by various factors, and the detection method used in each research is different, so that the urine 11dhTXB2 level of people reported in the past documents is different, so that the transverse comparison among different researches aiming at a specific people is difficult, and the large-scale research or meta-research which is well designed is further evaluated. Comparisons between subgroups in each study showed a trend of 11dhTXB2 levels in different populations, i.e. healthy population is lower than high risk (especially diabetic) and stable coronary population, ACS is higher than stable coronary patient, and Percutaneous Coronary Intervention (PCI) further increased 11dhTXB2 levels. Although the urine 11dhTXB2 levels of different people are different, the inhibition rate after aspirin is applied is similar and is 60-80 percent mostly. Aspirin can effectively inhibit platelet function by inhibiting platelet COX-1 activity, thereby reducing the risk of cardiovascular events. The urine 11dhTXB2 concentration reflects the overall synthesis level of thromboxane and thus can directly reflect the inhibitory effect of aspirin on thromboxane synthesis and platelet function. By studying the 11dhTXB2 levels in urine and aspirin reactivity in the past literature for coronary heart disease and related populations, the following conclusions were drawn: (1) the 11dhTXB2 level of the urine of coronary heart disease dangerous population and coronary heart disease patients (especially ACS patients) is obviously higher than that of healthy population, and has positive correlation trend with the level of risk factors, which indicates that the activity of blood platelets is gradually increased; factors such as sex, age and weight also have certain influence on thromboxane metabolism; (2) by using aspirin, the level of 11dhTXB2 in urine of all people can be obviously reduced, and the inhibition rate is 60-80%; (3) the level of 11dhTXB2 in urine of high risk people and coronary heart disease after aspirin is applied is also obviously higher than that of healthy people, and the higher incidence rate of HAPR and the higher risk of cardiovascular adverse events are suggested; (4) PCI can remarkably improve the excretion of 11dhTXB2 urine, which prompts a great amount of activation of platelets, and CABG does not have the effect; (5) most prognostic studies support the association of HAPR with increased incidence of cardiovascular events suggested by high levels of urinary 11dhTXB2 following aspirin application. Measures to reduce urinary 11dhTXB2 excretion may improve aspirin response, or may reduce the risk of cardiovascular adverse events.

Disclosure of Invention

In view of the above problems in the prior art, the present invention aims to obtain a chemiluminescence detection method and a chemiluminescence detection kit for aspirin metabolism and platelet high reactivity.

In order to realize the purpose, the technical scheme of the chemiluminescence detection method and the detection kit for aspirin metabolism and platelet high reactivity is as follows:

the invention provides a chemiluminescence detection kit for aspirin metabolism and platelet high reactivity, which is used for detecting the responsiveness of aspirin by detecting the content of 11-dehydrothromboxane B2(11-DH-TXB2) in a urine sample of a subject, and comprises a monoclonal antibody combined with 11-dehydrothromboxane B2, an 11-dehydrothromboxane B2 competitive product, magnetic particles and a luminescent substrate, wherein the monoclonal antibody is a mouse monoclonal antibody, and the amino acid sequence of a light chain variable region of the mouse monoclonal antibody is shown in a sequence table SEQ ID NO: 2, the amino acid sequence of the heavy chain variable region of the mouse single-antibody is shown as the sequence table SEQ ID NO: 4, respectively.

Preferably, the amino acid sequence of the light chain variable region of the murine monoclonal antibody is shown in a sequence table SEQ ID NO: 2 is represented by a gene sequence shown in a sequence table SEQ ID NO: 1 is expressed.

Preferably, the amino acid sequence of the light chain variable region of the murine monoclonal antibody is shown in a sequence table SEQ ID NO: 4 is represented by a gene sequence shown in a sequence table SEQ ID NO: 3, and expressing the gene sequence shown in the specification.

Wherein the 11-dehydrothromboxane B2 competitive product is alkaline phosphatase-labeled 11-DH-TXB 2.

The kit also comprises a quality control product or a calibrator 11-DH-TXB 2.

The magnetic particles are streptavidin-coated magnetic particles.

The luminescent substrate is an enzymatic luminescent substrate.

The mouse monoclonal antibody is a Biotin (Biotin) marked mouse monoclonal antibody.

The 11-DH-TXB2 in the sample to be tested and the AP-marked 11-DH-TXB2 compete to bind the monoclonal antibody; the method comprises the steps of connecting an antigen-antibody complex to magnetic particles through the combination of streptavidin and biotin, directly adsorbing and precipitating in an external magnetic field, separating the complex formed by immune reaction from other unbound substances, washing the complex, adding an enzymatic chemiluminescence substrate, detecting the luminous intensity, and calculating the concentration of 11-DH-TXB2 in a sample, thereby indicating the aspirin metabolic rate in a patient.

The invention also provides a method for evaluating the aspirin metabolic capacity of a cardiovascular relative risk patient, wherein the 11-DH-TXB2 concentration judgment method comprises the following steps:

1) respectively preparing a 11-DH-TXB2 calibrator, a biotin-bound murine monoclonal antibody and an alkaline phosphatase-bound 11-DH-TXB2 competitor;

2) adding an alkaline phosphatase-bound 11-DH-TXB2 competitor and a to-be-detected 11-DH-TXB2 sample into the biotin-bound mouse monoclonal antibody prepared in the step 1) at the same time, and carrying out competitive reaction;

3) adding magnetic particles coated with streptavidin into the system after the step 2), and adsorbing and precipitating by an external magnetic field;

4) taking the precipitate obtained in the step 3), cleaning, and adding an enzymatic luminescent substrate;

5) detecting the luminous intensity, wherein the luminous intensity is inversely proportional to the 11-DH-TXB2 in the sample to be detected;

6) the concentration of 11-DH-TXB2 in the sample can be calculated by fitting a four-parameter Logistic equation.

The method can also be used for aspirin metabolism detection and platelet high reactivity detection methods, or 11-DH-TXB2 concentration detection methods in samples. The detection method detects the content of 11-DH-TXB2 to be detected by the competition of 11-DH-TXB2 to be detected and an enzyme-labeled 11-DH-TXB2 with a mouse monoclonal antibody.

Preferably, the method comprises administering a first dose of aspirin to the patient, collecting a biological fluid sample from the patient; and determining the concentration of 11-dehydrothromboxane B2(11-DH-TXB2) in the sample; thereby determining the aspirin metabolic rate in the patient.

Preferably, the patient is administered a second dose of aspirin that is a different dose from the first dose of aspirin, the difference between the second dose and the first dose being at least 50mg, and the corresponding concentration of 11-DH-TXB2 in the patient is assayed, and the difference in doses is considered to not cause a metabolic change in the aspirin when the difference between the concentration of 11-DH-TXB2 in the first dose and the concentration of 11-DH-TXB2 in the second dose is not significant.

Preferably, the biological fluid sample is a urine sample.

An in-vitro detection system for high reactivity of platelets, which adopts the aspirin metabolic capacity detection method and/or the detection kit disclosed by the invention.

The in-vitro detection system for the platelet high reactivity detects the concentration of 11-DH-TXB2 in a sample to be detected by adopting a chemiluminescence method, and further obtains the aspirin drug metabolic capacity of a person to be detected.

The invention also provides a system and a method for dynamically detecting aspirin curative effect, wherein the method detects the metabolic rate of aspirin in a patient by detecting the concentration of 11-dehydrothromboxane B2(11-DH-TXB2) in a sample, and the detection method comprises the following steps:

a) the method comprises the following steps: establishing a target drug-time curve model; the curve comprises two stages, wherein the first stage is an ascending stage at the early stage of aspirin concentration, and the second stage is a descending stage at the later stage of aspirin concentration; in the first stage, a central control unit simulates the relation between aspirin concentration and time in a curve, and calculates the metabolic rate of aspirin liquid medicine at each corresponding time point according to the aspirin concentration and the concentration of 11-DH-TXB2 in a sample; in the second stage, the central control unit calculates the dosage of aspirin to be added at corresponding time according to the aspirin concentration of each time point, the target aspirin concentration of the next time point and the metabolic rate;

b) step two: collecting samples at each time point, detecting to obtain an actually measured drug-time curve, and feeding back the actually measured drug-time curve to the central control unit;

c) step three: the central control unit changes the administration amount of aspirin according to the target drug-time curve and the actually measured drug-time curve in the step two, and obtains a corrected drug-time curve by sampling and detecting the concentration of 11-DH-TXB 2;

d) step four: and repeating the third step, and revising the revised drug-time curve until the error of the revised drug-time curve and the target drug-time curve is within 10 percent.

The invention also provides a gene edited mouse monoclonal antibody which is used for combining with 11-DH-TXB2, and the amino acid sequence of the light chain variable region of the mouse monoclonal antibody is shown in the sequence table SEQ ID NO: 2, the amino acid sequence of the heavy chain variable region of the mouse single-antibody is shown as the sequence table SEQ ID NO: 4, respectively.

In conclusion, the invention also provides a urine TXB2 detection kit, wherein the detection kit is any one of the aspirin metabolism kits.

The urine TXB2 detection kit adopts any one of the detection methods for detection.

Compared with the prior art, the invention adopts the chemiluminescence detection method and the detection kit for aspirin metabolism and platelet high reactivity, can quickly detect whether a patient has aspirin resistance or resistance, provides scientific reference basis for clinical administration, and has high sensitivity and high accuracy, and is not easily influenced by biochemical indexes such as creatinine and the like; clinical experiments show that the kit can greatly reduce the problem that anti-platelet aggregation treatment fails or the effect is poor due to aspirin resistance of clinical patients.

Drawings

FIG. 1 is a schematic diagram showing the gene sequence of the light chain variable region of a murine monoclonal antibody;

FIG. 2 is a schematic diagram showing the gene sequence of the heavy chain variable region of a murine monoclonal antibody.

Detailed Description

The present invention provides a chemiluminescence detection method and a chemiluminescence detection kit for aspirin metabolism and platelet hyperreactivity, which are described in detail and completely in the following with reference to the following embodiments. The following examples are illustrative only and are not to be construed as limiting the invention.

The experimental procedures in the following examples are conventional unless otherwise specified. The experimental materials used in the following examples were all commercially available unless otherwise specified.

The chemiluminescence detection method for aspirin curative effect disclosed in this embodiment is to detect 11-DH-TXB2 content in human urine by using chemiluminescence method. The chemiluminescence method in the embodiment adopts a competition method principle, takes magnetic particles as a solid phase of immunoreaction, and utilizes a chemiluminescence immunoassay method to be matched with a chemiluminescence determinator to detect the content of 11-DH-TXB 2.

In order to obtain more excellent detection effect, in this example, the gene editing is performed on the murine monoclonal antibody structure for detecting 11-DH-TXB2, and the gene sequence of the light chain variable region of the murine monoclonal antibody is as shown in the sequence table SEQ ID NO: 1, and the expressed amino acid sequence is shown in a sequence table SEQ ID NO: 2 is shown in the specification; the gene sequence of the heavy chain variable region of the mouse monoclonal antibody is shown in a sequence table SEQ ID NO: 3, the expressed amino acid sequence is shown in a sequence table SEQ ID NO: 4, respectively.

The light chain variable region gene sequence (SEQ ID NO: 1) of the mouse monoclonal antibody is as follows:

ATGG AGAC AGAC ACAC TCCT GTTA TGGG TACT GCTG CTCT GGGT TCCA GGTT CCACTGGT

GACA TTGT GCTG ACAC AGTC TCCT GCTT CCTT AGCT GTAT CTCT GGGG CAGA GGGCCACC

ATCT CGTG CAGG GCCA CCCC CAAC ACAC TGTG CGAC GAAT TCAA AGCC ATGC ACTGGTAC

CAAC AGAA ACCA GGAC AGTC ACCC AAAC TCCT CATC TATG ACGA CTCC AACC TAGAATCT

GGGG CCCTCCC TGCC AGGT TCAG TGGC AGTG GGTC TGGG ACAG ACTT CACC CTCAACAT CCAT

CCTG TGGA GGAG GAGG ATGC TGCA ACCT ATTA CTGT AAAG GCGC TTGC CTGC TCCCTAAA

ATCT TCGG AGGG GGGG ACCA AGCT GGAA ATAA AA

the expressed light chain amino acid sequence (SEQ ID NO: 2) is specifically as follows:

the gene sequence of the heavy chain variable region of the mouse monoclonal antibody (SEQ ID NO: 3) is specifically as follows:

ATGC TGTT GGGG CTGC AGTG GGTT TTCT TTGT TGTT TTTT ATCA AGGT GTGC ATTGT

GAGG TGCT GCTT GTTG AGTC TGGT GGAG GATT GGTG CAGC CTAG AGGG TCAT TGAAA

CTCT CATG TGCA GCCT CTGT TTTT GACA AACT TAAA CACC TTAT GAAC TGGG TCCGC

CAGG CTCC AGGA AAGG GTTT GGAA TGGG TTGC TCGC CTTG GGGA ATAC GGGT TTCAA

AACG CACT TATT ATGC CGAT TCAG TGAA AGAC AGGT TCAC CATC TCCA GAGA TGAT

TCAC ATAG CATG CTCT CTCT GCAG TTGA ACGG CTTG AAAA CTGA GGAC ACAG CCTTG

TATT ACTG TGTG TCTG TCTT GTTG GAGT TGCC GAAA GAAA TTGA AGCC TGGG GTCAA

GGAA CCTC AGTC ACCG TCTC CTCA

the expressed heavy chain amino acid sequence (SEQ ID NO: 4) is specifically as follows:

the light chain sequence of the 11-DH-TXB2 monoclonal antibody is shown in FIG. 1, and the heavy chain sequence is shown in FIG. 2. The prepared monoclonal antibody is analyzed by electrophoresis, and the size of the active fragment in the prepared monoclonal antibody is determined to be correct, wherein a lane 1 of an electrophoresis chart is the full length of mouse IgG, a lane 2 is an Fc fragment, and a lane 3 is an Fab fragment.

In the embodiment, the detection kit is prepared by adopting the monoclonal antibody with the sequence, the kit adopts a competition method principle, takes the magnetic particles as a solid phase of immunoreaction, and is matched with a chemiluminescence immunoassay analyzer by utilizing a chemiluminescence immunoassay method to measure the content of 11-DH-TXB2 in human urine.

1. Mouse monoclonal antibody kit composition

Ra: streptavidin-coated magnetic microparticles, 50mM Tris buffer containing a preservative;

rb: alkaline Phosphatase (AP) -labeled 11-DH-TXB2 competitor, 50mM PBS buffer with preservative;

rc: biotin (Biotin) -labeled murine monoclonal antibody, preservative-containing 50mM PBS buffer;

calibration (selection): adding different amounts of 11-DH-TXB2 antigen and 50mM Tris buffer solution containing preservative into 1-6 bottles of the calibrator respectively;

quality control (selection): 1-2 bottles of quality control 1-2, respectively, added with different amounts of 11-DH-TXB2 antigen and 50mM Tris buffer solution containing preservative.

2. Matching reagent

The product does not contain the following reagents but must be used for detection:

substrate solution for full-automatic immunoassay system: photons can be generated by the catalysis of the marker enzyme in the immunoreagent, and the luminescence intensity can be detected by using a chemiluminescence immunoassay instrument. The reagent is provided by far JING Biotechnology GmbH in Hunan.

Cleaning solution: diluted and used for cleaning a reaction system. The reagent is provided by far JING Biotechnology GmbH in Hunan.

Cleaning fluid for pipelines: after dilution, the sample injection needle and the pipeline are cleaned. The reagent is provided by far JING Biotechnology GmbH in Hunan.

3. Traceability of calibrator

The calibrators were traceable to a Corgenix enzyme linked immunoassay system.

4. Sample requirement

1) The sample suitable for the kit recommends using a urine sample, and the sample needs to be restored to the room temperature before detection.

2) In order to ensure the test requirement, the sample size is required to be not less than 200 mu L.

3) Storing the sample, keeping the sample completely closed, and keeping the temperature at room temperature (15-25 ℃) for no more than 8 hours.

4) If the detection can not be finished within 8 hours, the sample can be immediately placed in a refrigerator for refrigeration at the temperature of 2-8 ℃, the sample can be stored for 24 hours at the temperature of 2-8 ℃, the sample can be stored for 30 days at the temperature of-20 ℃ and can be stored for one year at the temperature of-80 ℃ for a long time, and repeated freeze thawing is avoided.

In this embodiment, the detection method using the kit is to perform competitive binding of 11-DH-TXB2 of a sample to be detected, a calibrator or a quality control variety and 11-DH-TXB2 labeled with Alkaline Phosphatase (AP) to a Biotin-labeled mouse monoclonal antibody, then add streptavidin-coated magnetic particles, connect an antigen-antibody complex to the magnetic particles by the binding of streptavidin and Biotin, directly adsorb and precipitate in an applied magnetic field, separate the complex formed by an immunoreaction from other unbound substances, wash the complex, and add an enzymatic chemiluminescent substrate. The substrate is catalytically cracked under the action of enzyme to form an unstable excited intermediate, and when the excited intermediate returns to the ground state, photons are emitted to form a luminescence reaction, namely, a chemiluminescence apparatus is used for detecting the luminescence intensity of the reaction. Within the detection range, the luminescence intensity is inversely proportional to the content of 11-DH-TXB2 in the sample, and the concentration of 11-DH-TXB2 in the sample can be calculated by using a modified four-parameter Logistic equation fitting.

The specific method comprises the following steps:

5. inspection method

Checking the requirement of working conditions: room temperature (15-35 ℃); the relative humidity is less than or equal to 80 percent.

a) Preparation before testing

i. All reagents are required to be placed at room temperature before the test, and the instrument is started to be preheated for at least 30 minutes;

and ii, loading the reaction tube (cup), adding or replacing a matched reagent, cleaning waste liquid and waste tubes and filling a liquid path according to the system operation guide.

b) Reagent, sample Loading

i. Before loading the reagent, placing the reagent to be used on mixing equipment for fully mixing, and visually checking that the components of a reagent solution are clarified without foreign matters, precipitates and floccules and a magnetic particle reagent is a uniform suspension without obvious agglutination;

scanning the bar code of the reagent according to the system operation guide to complete the loading of the magnetic particle reagent and the anti-reagent;

and iii, uniformly mixing the calibrator and the quality control product, transferring the mixture into a reaction cup special for the instrument, loading the mixture into an instrument sample position, and directly loading the urine sample obtained by centrifugation into the instrument sample position after scanning.

c) Detection step

The operation is carried out according to the operation manual of the full-automatic chemiluminescence determinator. The detection reaction process and related parameters are predefined in the instrument operating software.

d) Calibration curve and calibration

The acquisition of the calibration curve comprises the direct generation of a scanning main curve card or the direct detection of 6 calibrator point fitting generation.

Due to reagent activity drift and universal reagent batch changes, the calibrated working curve needs to be recalibrated after a certain period of use. Recalibration should occur when: 1 month later (28 days) using the same batch of reagent; using a new batch reagent whole box or luminous substrate liquid; when the quality control value is not within the quality control range; or after each maintenance of the meter.

e) Result output

The analyzer automatically calculates the 11-DH-TXB2 concentration of each sample by a working curve obtained by two-point calibration of the standard curve, the result being expressed in pg/mL.

f) Quality control

i. The quality control substances of two levels are measured simultaneously every day when the sample detection is carried out, and the quality control substances are treated as patient samples;

and ii, referring to a kit quality control list for the quality control range, the using method and the quality control period of the kit quality control product, and if the quality control result does not accord with the expectation, prompting that the detection result is unreliable and not issuing a detection report.

If the measurement result is within the quality control range acceptable by the system, the detection result is satisfactory, otherwise, the detection result is not reliable, and a detection report is not issued.

6. Reference interval

When the reference interval of the kit is determined, 400 urine samples of healthy people (or normal people) are collected from a hospital or a health examination center, wherein 110 urine samples of males and 290 urine samples of females are collected, and the age is 15-75 years. One-sided 95% (upper limit 95%) of the detection value of the normal human sample was calculated as a reference value.

Calculating the obtained reference interval of the kit:

11-Dehydroarboxane B2/creatinine > 1500pg/mg, suggesting a lack of aspirin (ASA) action

11-dehydrothromboxane B2/creatinine < 1500pg/mg, which indicates the presence of aspirin (ASA) effect

The levels of 11-DH-TXB2 measured in different regions, in different individuals, and by different methods, are different, and therefore, it is recommended that each laboratory establish its own range of normal values. The 11-DH-TXB2 value obtained by the method cannot be used for diagnosis, and other clinical data including specific conditions and treatment conditions of patients should be combined for analysis.

7. The result of the detection

By adopting the method described in the above embodiment, the results after sampling detection are as follows:

wherein the linear range (300.0-4000.0) is pg/mL, and the correlation coefficient r is more than or equal to 0.9900. The relative deviation of accuracy was within ± 10.0%. The blank limit should be no greater than 300.0 pg/mL. The coefficient of variation of repeatability, CV, should be no more than 8.0%. The coefficient of variation, CV, of the inter-batch difference should be no more than 15.0%.

Because of methodology or antibody specificity, testing the same sample using reagents from different manufacturers may yield different test results, and therefore the results obtained from testing with different reagents should not be directly compared to each other to avoid erroneous medical interpretation. The measurement result beyond the measurement range of the kit is a calculation result obtained by extending the calibration curve, and attention is required when reporting the result. If the exact measurement is desired, the sample is diluted appropriately and then tested. The quality control product can be used as a reference basis for the reliability of the current experimental result, and the measured value of the quality control product is within the range allowed by the quality control unit of the batch of products. The test result should be determined comprehensively according to the reference value range and other clinical factors and results, and when the detection result is close to the upper limit value of the reference value range, the sample can be considered to be subjected to a confirmation test.

Finally, it must be said here that: the above embodiments are only used for further detailed description of the technical solutions of the present invention, and should not be understood as limiting the scope of the present invention, and the insubstantial modifications and adaptations made by those skilled in the art according to the above descriptions of the present invention are within the scope of the present invention.

Sequence listing

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atggagacag acacactcct gttatgggta ctgctgctct gggttccagg ttccactggt 60

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atctcgtgca gggccacccc caacacactg tgcgacgaat tcaaagccat gcactggtac 180

caacagaaac caggacagtc acccaaactc ctcatctatg acgactccaa cctagaatct 240

ggggtccctg ccaggttcag tggcagtggg tctgggacag acttcaccct caacatccat 300

cctgtggagg aggaggatgc tgcaacctat tactgtaaag gcgcttgcct gctccctaaa 360

atcttcggag ggggggacca agctggaaat aaaa 394

<210>2

<211>131

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<221>UNSURE

<222>(1)..(131)

<400>2

Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro

1 5 10 15

Gly Ser Thr Gly Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala

20 25 30

Val Ser Leu Gly Gln Arg Ala Thr Ile Ser Cys Arg Ala Thr Pro Asn

35 40 45

Thr Leu Cys Asp Glu Phe Lys Ala Met His Trp Tyr Gln Gln Lys Pro

50 55 60

Gly Gln Ser Pro Lys Leu Leu Ile Tyr Asp Asp Ser Asn Leu Glu Ser

65 70 75 80

Gly Val Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr

85 90 95

Leu Asn Ile His Pro Val Glu Glu Glu Asp Ala Ala Thr Tyr Tyr Cys

100 105 110

Lys Gly Ala Cys Leu Leu Pro Lys Ile Phe Gly Gly Gly Thr Lys Leu

115 120 125

Glu Ile Lys

130

<210>3

<211>422

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221>unsure

<222>(1)..(422)

<400>3

atgctgttgg ggctgcagtg ggttttcttt gttgtttttt atcaaggtgt gcattgtgag 60

gtgctgcttg ttgagtctgg tggaggattg gtgcagccta gagggtcatt gaaactctca 120

tgtgcagcct ctgtttttga caaacttaaa caccttatga actgggtccg ccaggctcca 180

ggaaagggtt tggaatgggt tgctcgcctt ggggaatacg ggtttcaaaa cgcacttatt 240

atgccgattc agtgaaagac aggttcacca tctccagaga tgattcacat agcatgctct 300

ctctgcagtt gaacggcttg aaaactgagg acacagcctt gtattactgt gtgtctgtct 360

tgttggagtt gccgaaagaa attgaagcct ggggtcaagg aacctcagtc accgtctcct 420

ca 422

<210>4

<211>141

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<221>UNSURE

<222>(1)..(141)

<400>4

Met Leu Leu Gly Leu Gln Trp Val Phe Phe Val Val Phe Thr Gln Gly

1 5 10 15

Val His CysGlu Val Leu Leu Val Glu Ser Gly Gly Gly Leu Val Gln

20 25 30

Pro Arg Gly Ser Leu Lys Leu Ser Cys Ala Ala Ser Val Phe Asp Lys

35 40 45

Leu Lys His Leu Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu

50 55 60

Glu Trp Val Ala Arg Leu Gly Glu Tyr Gly Phe Gln Asn Ala Leu Tyr

65 70 75 80

Tyr Ala Asp Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser

85 90 95

His Ser Met Leu Ser Leu Gln Leu Asn Asn Leu Lys Thr Glu Asp Thr

100 105 110

Ala Leu Tyr Tyr Cys Val Ser Val Leu Leu Arg Leu Ala Lys Glu Tyr

115 120 125

Glu Ala Trp Phe Gln Gly Thr Ser Val Thr Val Ser Ser

130 135 140

Claims

1. A chemiluminescence detection kit for aspirin metabolism and platelet high reactivity is characterized in that: the kit is used for detecting the content of 11-dehydrothromboxane B2 in a urine sample of a subject, and comprises a monoclonal antibody combined with 11-dehydrothromboxane B2, an 11-dehydrothromboxane B2 competitor, magnetic particles and a luminescent substrate, wherein the monoclonal antibody is a murine monoclonal antibody, and the amino acid sequence of the light chain variable region of the murine monoclonal antibody is shown in a sequence table SEQ ID NO: 2, the amino acid sequence of the heavy chain variable region of the mouse single-antibody is shown as the sequence table SEQ ID NO: 4, respectively.

2. The chemiluminescent detection kit according to claim 1 wherein the amino acid sequence of the light chain variable region of the murine monoclonal antibody is as set forth in SEQ ID NO: 2 is represented by a gene sequence shown in a sequence table SEQ ID NO: 1 is expressed.

3. The chemiluminescent detection kit of claim 1 wherein the amino acid sequence of the mouse single-antibody heavy chain variable region is as set forth in SEQ ID NO: 4 is represented by a gene sequence shown in a sequence table SEQ ID NO: 3, and expressing the gene sequence shown in the specification.

4. The chemiluminescent detection kit of claim 1, wherein: the 11-dehydrothromboxane B2 competitive product is alkaline phosphatase-labeled 11-DH-TXB 2.

5. The chemiluminescent detection kit of claim 1, wherein: the mouse monoclonal antibody is a biotin-labeled mouse monoclonal antibody.

6. The chemiluminescent detection kit of claim 1 further comprising a quality control or calibrator 11-DH-TXB 2.

7. The chemiluminescent detection kit of claim 1 wherein the luminescent substrate is an enzymatic luminescent substrate.

8. The chemiluminescent detection kit of claim 1 wherein the magnetic particle is a streptavidin coated magnetic particle.

9. An aspirin metabolism detection and platelet high reactivity detection method is characterized in that the detection method obtains the metabolic capacity of aspirin by detecting the concentration of 11-DH-TXB2 in a sample to be detected.

10. The detection method according to claim 9, wherein the detection method is specifically:

i. respectively preparing a 11-DH-TXB2 calibrator, a biotin-bound murine monoclonal antibody and an alkaline phosphatase-bound 11-DH-TXB2 competitor;

ii) adding an alkaline phosphatase-bound 11-DH-TXB2 competitor and a sample to be tested 11-DH-TXB2 into the biotin-bound murine monoclonal antibody prepared in the step i) at the same time, and carrying out competitive reaction;

adding magnetic particles coated with streptavidin into the system completing the step ii), and adsorbing and precipitating by an external magnetic field;

taking the precipitate in the step iii), washing, and adding an enzymatic luminescent substrate;

and v, detecting the luminous intensity, wherein the luminous intensity is inversely proportional to the 11-DH-TXB2 in the sample to be detected.