CN111024941A

CN111024941A - Protein chip for detecting multiple thrombus markers and preparation method thereof

Info

Publication number: CN111024941A
Application number: CN201911249276.2A
Authority: CN
Inventors: 朱旭国; 泮锋纲; 丁俊杰; 施启尧
Original assignee: Sunlant Biological Engineering Co ltd
Current assignee: Sunlant Biological Engineering Co ltd
Priority date: 2019-12-09
Filing date: 2019-12-09
Publication date: 2020-04-17

Abstract

The invention discloses a protein chip for joint detection of multiple thrombus markers, which comprises the following steps of (1) pretreatment of a black glass slide, (2) spotting of an antibody solution, and (3) a sealing process, wherein the protein chip is prepared by the steps of detecting the concentrations of the multiple thrombus markers, preventing and reducing thrombus risk and guiding thrombus treatment.

Description

Protein chip for detecting multiple thrombus markers and preparation method thereof

Technical Field

The invention relates to the technical field of biology, in particular to a protein chip for detecting various thrombus markers and a preparation method thereof.

Background

The seven items of coagulation and the screening of coagulation factors belong to passive detection after thrombosis and are insensitive to the items before thrombosis. Compared with thrombolytic therapy after thrombus is discovered, it is very important to discover thrombus risk in time before thrombus formation and take measures in time. Aiming at the clinical diagnosis problems before and in the early stage of thrombosis, the Japanese Sysmex company firstly proposes four items (TAT, TM, PIC, tPAIC) of new thrombosis and performs combined detection of two indexes of FDP and DD of a fibrinolysis system, so that the thrombus can be discovered and treated early.

At present, the detection means of the four markers of the new thrombus are very limited. Although a few companies develop chemiluminescence immune products by a magnetic bead method, the chemiluminescence immune products are expensive, low in efficiency, complex to operate and multiple index detection needs independent calibration and quality control. And because the FDP and DD need to be measured by an additional blood coagulation instrument, the FDP and DD are not beneficial to the rapid, timely and frequent clinical monitoring and supervision of thrombus patients.

The existing hard matrix protein chip detection platform of the triple organism is utilized to integrate the detection of new four indexes of thrombus, FDP, DD and the like on one protein chip, and a plurality of thrombus marker detection chips are developed, so that the effective diagnosis can be carried out at the early stage of thrombus formation, and the treatment effect of the antithrombotic drug can be monitored in an auxiliary manner. Compared with the existing products, the protein chip joint inspection has obvious advantages in the aspects of improving efficiency, reducing cost, being simple and convenient to operate and the like.

(1) TAT (Thrombin-antithrombin Complex)

Thrombin (thrombin) is a multifunctional serine proteolytic enzyme having a sequence and structure similar to chymotrypsin, comprising A, B polypeptide chains linked by an interchain disulfide bond β chain is a functional chain having a typical serine proteolytic enzyme folding structure comprising 1 active center between two β folding barrels, 2 exo-binding sites, 1 Na ion binding site, a autocatalytic hydrolysis loop and a W60d loop thrombin has Na ion-induced allosteric properties, and has both Na ion-binding and Na ion-dissociating conformations in blood^[1]。

The thrombin directly acts on the last step of the blood coagulation process to promote the conversion of soluble fibrinogen in the plasma into insoluble fibrin, thereby achieving the purpose of quick-acting hemostasis. But also can promote the mitosis of epithelial cells and accelerate wound healing, and is a quick-acting local hemostatic. Thrombin is suitable for the bleeding of small blood vessels, capillaries, parenchymal organs and other various kinds of bleeding which are difficult to be stopped by ligation. It also has the effects of activating receptor, and producing cell biological effect, such as inducing inflammation, inducing cell to release cytokine, inducing cell proliferation, and regenerating tissue and organ. Thrombin in the central nervous system is also neurotoxic and can cause brain cell damage.

The thrombin has short half-life and is difficult to detect, so that the complex formed by the thrombin and the antithrombin can be detected. Indications of elevation relate to DIC, DVT, PE and other states of coagulation system activation such as atrial fibrillation^[2]。

(2) PIC (plasmin- α 2 antiplasmin complex)

Plasminogen is an inactive form of plasmin, synthesized primarily in the liver, and expressed in major organs and tissues. Under physiological conditions, plasminogen is converted to plasmin after cleavage by the active ring. Activated plasmin has a wide range of substrate properties including fibrin, fibrinogen, complement component 3, complement component 5, and the like. Plasmin can also target the key prothrombin activators, tPA and uPA, creating a positive feedback loop. Active plasmin plays an important role in fibrinolysis and hemostasis, extracellular matrix degradation, cell migration, tissue remodeling, wound healing, angiogenesis, inflammation, tumor cell migration and other physiological and pathological processes.

Full-length plasminogen includes 791 amino acid residues and 7 domains. A Pap domain, KRI-5 domain, and a serine protease domain. Plasminogen has two different conformations, closed and open. Glycosylated plasminogen is present in the blood stream in a closed conformation and cannot be activated by tPA and uPA. When glycosylated plasminogen binds fibrin or the cell surface, it exists in an open conformation^[3]。

After the free plasmin is released from the surface, almost simultaneously, the free plasmin forms an irreversible complex with the protease inhibitor α 2 antithrombin and the interaction speed of the free plasmin and the protease inhibitor α antithrombin is one of the fastest known effects (Ka: 2X 10)⁷mol^- ¹s^-1)。

The half-life of activated plasmin is extremely short and cannot be directly detected in vivo, and the plasmin can form a compound PIC with plasmin inhibitor, so the detection is relatively easy. Elevated levels are common in DIC, DVT, PE, and thrombolytic therapy^[4]。

(3) tPAIC (tissue plasminogen activator-plasminogen activation inhibiting complex)

PAI-1 is originally produced in endothelial cells and platelets and is the predominant PAI in plasma. PAI-1 is a glycoprotein with molecular weight of 45-50kDa, belonging to serine protease inhibitor, and arginine as the reaction center. The PAI-1 gene consists of 8 introns and 9 exons, and has a total length of 12.3 kb. Most cells produce both PA and PAI-1, and their release is independent and controlled by environmental conditions. PAI-1 is usually present in two forms, an active form and an inactive form, with the inactive form being the majority^[5]。

PAI-1 and PAI-2 inhibit tPA and uPA by substrate mechanisms. PAI-1 is inserted into the active site of PA to form a temporary Michaelis complex. As a protease inhibitor, PAI-1 can specifically act on tPA and uPA, and the inhibition capability of the tPA and the uPA is at least 1000 times faster than that of other proteases. PAI-1 rapidly and irreversibly inhibits PA, a major negative regulator in fibrinolytic systems. PAI-1 is a potential therapeutic target and prognostic indicator in blood coagulation diseases. High levels of PAI-1 may prevent plasmin formation.

After fibrin is formed, a fibrinolytic system is activated to form a tissue plasminogen activator (t-PA) and further to convert plasminogen into plasmin, so that DD and FDP are degraded and formed, PAI-1 is a physiological inhibitor of t-PA, and the DD and FDP are combined to form a compound tPAIC, wherein the level of the tPAIC is positively correlated with the concentration of the t-PA and vascular endothelial injury^[6]. Elevation is frequently seen in DIC, vascular endothelial injury, DVT, acute myocardial infarction, etc.

(4) TM (thrombomodulin)

Mature human TM has 557 amino acid residues encoded by the intron-free gene THBD. TM comprises 5 structural regions, the N-terminus contains 2 modules, the first is a leptin-mimetic domain of 155 amino acid residues, which plays an important role in inflammation, innate immunity, and cancer. This domain contains 2N-glycosylation sites and calcium binding sites, regulating cellular transport functions. The second module is a hydrophobic 67 amino acid module comprising 6 epidermal growth factor-like repeats^[7]。

Transmembrane glycoproteins present on the surface of cell membranes can assist in the anticoagulant effect. After being combined with thrombin, the thrombin can reduce the coagulation activity of the thrombin, and the activity of activated protein C is strengthened. Because activated protein C has an anticoagulant effect, TM is an important intravascular coagulation inhibitor for changing thrombin from procoagulant to anticoagulant, and is used as a marker of endothelial cell injury^[8]. The increase is frequently seen in DIC, autoimmune disease SLE, Acute Respiratory Distress Syndrome (ARDS), vascular endothelial injury, etc.

TM is associated with a variety of diseases such as malignancies, atherosclerosis, transplantation-related vascular disease, diabetic nephropathy, organ ischemia reperfusion injury, and pulmonary disease.

(5) FDP (fibrinogen degradation product)

Fibrin (ogen) is degraded by plasmin after its formation during coagulation to produce fibrin (ogen) degradation products (FDP), which are the general term for degradation products produced by decomposition of fibrin or fibrinogen in blood circulation during hyperfibrinolysis. In cases of primary fibrinolysis, DIC and the like, the concentration of FDP in blood is increased^[9]。

(6) DD (D-dimer)

Like FDP, DD is also formed by degradation of fibrin by plasmin. In contrast, DD cannot be formed by degradation of fibrinogen by plasmin, so DD levels are not high during primary fibrinolysis and FDP levels will increase. However, in pathological conditions such as DIC, the level groups of the two proteinsThis is at the same level^[10]. The application of FDP and DD is very wide at present and is expanded to various clinical fields, malignant diseases DD such as leukemia, liver cancer, lung cancer, ovarian cancer and the like are higher than those of normal people, and infectious diseases and myocardial ischemia DD are also obviously higher than those of the normal people; pregnancy hypertension DD, FDP is high, has primary and secondary hyperfibrinolysis, and is suggested to prevent bleeding; in diabetes, there are extensive thrombotic disorders with a markedly higher DD. The combined detection of FDP and DD has great significance, and a plurality of papers and monographs report that the sensitivity and specificity of single-detection FDP and DD are less in DIC diagnosis than those of combined detection.

For blood coagulation, PT (prothrombin time), APTT (activated partial thromboplastin time), TT (thrombin time) and Fbg (fibrinogen) are generally detected clinically, the indexes are more used for diagnosing hemorrhagic diseases, monitoring of activation of a blood coagulation system is not sensitive enough and early, patients develop to middle and late stages when abnormality occurs, and the clinical opportunity is kept passive. TAT, TM, PIC and tPAIC abnormity can indicate the occurrence of thrombus early condition, and the DD and FDP are combined to carry out early diagnosis on the thrombus and the blood coagulation abnormity condition, thereby obviously increasing the treatment time window of a clinician^[11]。

Disclosure of Invention

Aiming at the problems of high cost, low efficiency, complex operation and the like in the prior art, the applicant of the invention provides a protein chip for detecting a new thrombus marker and a preparation method thereof. The product of the invention is used for detecting the new marker of thrombus, can effectively improve the detection efficiency and reduce the detection cost; the present inventors have developed a protein chip diagnostic kit simultaneously containing six indexes of TAT, TM, PIC, tPAIC, FDP and DD, and have the advantages of rapidness, high efficiency, low cost, etc.

The technical scheme of the invention is as follows:

a protein chip for detecting multiple thrombus markers is prepared by the following steps:

(1) pretreating a black glass slide;

(2) spotting an antibody solution;

(3) and (3) sealing to obtain the protein chip.

The method for pretreating the black glass slide in the step (1) comprises the following steps:

①, soaking the black glass slide in a glass slide pretreatment solution containing NaOH for 16-24 h, and then cleaning with purified water for 2-8 times;

②, soaking the black glass slide in a silane water solution with the mass concentration of 0.05-1% for 20-60 min;

③ placing the soaked black glass slide into an oven, and baking for 0.2-0.6 h at 100-180 ℃.

The antibody solution in the step (2) comprises a TAT monoclonal antibody solution, a PIC monoclonal antibody solution, a tPAIC monoclonal antibody solution, a TM monoclonal antibody solution, an FDP monoclonal antibody solution and a DD monoclonal antibody solution.

The spotting method in the step (2) is machine-automated spotting.

The sealing process in the step (3) is as follows: and immersing the spotted black glass slide into the sealing liquid for 1-24 h, then taking out the black glass slide, and centrifuging to remove residual sealing liquid to obtain the protein chip.

The confining liquid is a buffer solution containing confining protein; the blocking protein is bovine serum albumin or ovalbumin; the buffer solution is one or more of PBS buffer solution, Tris buffer solution, HEPES buffer solution and MOPS buffer solution.

An application of the protein chip, and the protein chip is prepared into a kit.

The kit also includes a solution of a secondary antibody labeled with HRP enzyme, a chemiluminescent substrate sensitive to HRP enzyme.

The concentration of the second antibody solution marked with HRP enzyme is 1ug/ml, wherein the medium is enzyme-labeled secondary antibody diluent of an outsourced Saimer Feiko company, and the pH value is 6.0; the chemiluminescence substrate comprises a detection liquid A and a detection liquid B which respectively contain luminol and hydrogen peroxide.

The beneficial technical effects of the invention are as follows:

the protein chip is used for detecting six important thrombus markers, and joint detection can be realized by applying the protein chip technology, so that the detection efficiency can be effectively improved, and the detection cost is reduced; the protein chip diagnostic kit simultaneously comprises six indexes of TAT, TM, PIC, tPAIC, FDP and DD, and has the advantages of rapidness, high efficiency, low cost and the like. The automatic detection can be realized by matching with an automatic protein chip reader of the company. Because the six indexes are effectively integrated in one chip for detection, the simultaneous and rapid detection of the six indexes can be realized by only one patient blood sample. As a novel detection method, the product and the technology do not have like products to be marketed in the world at present.

The invention adopts a classical immunological double-antibody sandwich method. And (3) fixing capture antibodies on a chip substrate taking glass as a carrier, wherein the antibodies can capture specific antigens in the detected sample, and the captured antigens are combined with a secondary antibody labeled with HRP enzyme to form a sandwich type combined product. Adding a chemiluminescent substrate sensitive to HRP enzyme for chemiluminescence, collecting an optical signal by a CCD camera, and judging the concentration of a specific marker antigen in the detected sample according to the intensity of the optical signal.

Drawings

FIG. 1 is a schematic representation of the spotting of antibodies of the invention;

in the figure, column 1: positive quality control; column 2: blank; column 3: TAT assay column; column 4: a TM assay column; column 5: PIC measurement column; column 6: tPAIC assay column; column 7: FDP measurement column; column 8: DD measurement column; column 9: negative quality control.

Detailed Description

The present invention will be described in detail with reference to the accompanying drawings and examples.

Example 1

(1) pretreating a black glass slide;

① soaking the black glass slide in a glass slide pretreatment solution containing 2% NaOH for 16h, and then cleaning with purified water for 2-8 times;

② soaking the black glass in 0.05% silane water solution for 60 min;

③ the soaked black glass slide is put into an oven and baked for 0.2h at 180 ℃.

(2) Spotting an antibody solution;

referring to fig. 1, TAT monoclonal antibody solution, PIC monoclonal antibody solution, tpa monoclonal antibody solution, TM monoclonal antibody solution, FDP monoclonal antibody solution, DD monoclonal antibody solution are spotted automatically using a machine. The concentration of the antibody solution was 0.1mg/mL, and 20nL of the antibody was spotted per spot, and the distribution of the spotting of each antibody was as shown in FIG. 1.

(3) Sealing process;

and immersing the spotted black slide into a blocking solution (PBS buffer solution containing 1% bovine serum albumin) for 10 hours, then taking out the black slide, and centrifuging to remove residual blocking solution to obtain the protein chip.

(4) A kit;

the protein chip was packaged together with a secondary antibody solution labeled with HRP enzyme (concentration 1ug/ml, wherein the medium was an enzyme-labeled secondary antibody diluent of semelafin, purchased from outsource, pH 6.0), detection solution a (containing 1% luminol and 2% Tris), and detection solution B (containing 1% hydrogen peroxide) into a kit.

Example 2

(1) pretreating a black glass slide;

①, soaking the black glass slide in glass slide pretreatment liquid containing 2% NaOH for 24h, and then cleaning for 2-8 times by adopting pure water;

② soaking the black glass in 0.5% silane water solution for 30 min;

③ the soaked black glass slide is put into an oven and baked for 0.5h at 140 ℃.

(2) Spotting an antibody solution;

(3) Sealing process;

and immersing the spotted black slide into a blocking solution (PBS (phosphate buffer solution) containing 2% ovalbumin) for 24 hours, taking out the black slide, and centrifuging to remove residual blocking solution to obtain the protein chip.

(4) A kit;

Example 3

A protein chip for detecting a new thrombus marker is prepared by the following steps:

(1) pretreating a black glass slide;

① soaking the black glass slide in a glass slide pretreatment solution containing 2% NaOH for 20h, and then cleaning with pure water for 2-8 times;

② soaking the black glass in 1% silane water solution for 20 min;

③ the soaked black glass slide is put into an oven and baked for 0.6h at 100 ℃.

(2) Spotting an antibody solution;

(3) Sealing process;

and immersing the spotted black slide into a blocking solution (Tris buffer solution containing 3% bovine serum albumin) for 8 hours, then taking out the black slide, and centrifuging to remove residual blocking solution to obtain the protein chip.

(4) A kit;

Test example:

the SLXP-001B biochip reader manufactured by the company is used for detecting clinical serum. The working process of the SLXP-001B type biochip reader is as follows:

200ul of a serum sample to be detected is automatically sucked into a reaction cup by an instrument, the protein chip prepared by the embodiment of the invention is automatically placed into the serum to be detected by the instrument, the incubation is carried out for 40 minutes at 37 ℃, then the chip is taken out by an instrument clamping jaw, the chip is automatically washed by the instrument and then put into a second antibody solution (200ul, the instrument is automatically sucked in advance), which is marked with HRP enzyme, after the incubation is carried out for 40 minutes again, the chip is taken out again by the instrument clamping jaw, the chip is automatically washed by the instrument and then put into a luminescent substrate solution (formed by mixing 100ul of a detection solution A and 100ul of a detection solution B, the chip is automatically sucked and mixed by the instrument), and finally, the protein chip is photographed and imaged, the picture is automatically analyzed by. The results of the measurements are shown in tables 1 and 2. The reference values for TAT, TM, PIC, tPAIC are the results of the chemiluminescence immunoassay analyzer of the Hesimecon. The reference method for FDP and DD is the hemagglutination analyzer analysis of Sysmex.

TABLE 1

TABLE 2

As can be seen from the table above, the kit provided by the invention can simultaneously detect six indexes, namely TAT, TM, PIC, tPAIC, FDP and DD, can obtain results similar to those of a single-index kit (the relative error is mostly within 5%), and has no significant difference in the aspects of sensitivity, linear range and the like. The kit can realize the advantages of high efficiency, simple operation, low cost, short time and the like, is very helpful for timely and accurately finding early thrombus, and is also suitable for tracking and monitoring thrombus patients, knowing the progress of the disease and preventing the continuous thrombus.

Reference documents:

[1]Li,W.,et al.,Structure of the antithrombin-thrombin-heparinternary complex reveals the antithrombotic mechanism of heparin.NatureStructural&Molecular Biology,2004.11(9):p. 857-862

[2] permissive peaks, etc., structure of thrombin and its allosteric characteristics, application of Chinese medicine biotechnology 2004, pages 18-23

[3]Law,R.H.,D.Abu-Ssaydeh and J.C.Whisstock,New insights into thestructure and function of the plasminogen/plasmin system.Curr Opin StructBiol,2013.23(6):p.836-41

[4] Chenchong, Wuxue, application of blood coagulation and fibrinolysis markers in treating malignant tumor, Shandong medicine, 2019.59 (20), page 100-

[5]Gong,L.,et al.,Crystal Structure of the Michaelis Complex betweenTissue-type Plasminogen Activator and Plasminogen Activators Inhibitor-1.TheJournal of biological chemistry,2015. 290(43):p.25795-25804

[6] Quihongliang et al, research progress of detection of molecular markers for thrombosis and hemostasis in thrombotic diseases International journal of laboratory medicine 2017.38(17) pp 2434-2436

[7]Loghmani,H.and E.M.Conway,Exploring traditional and nontraditionalroles for thrombomodulin.Blood,2018.132(2):p.148-158

[8] Wangjian Ru, et al, change and significance of thrombomodulin content in plasma of cerebrovascular disease patients J Clin neurology 1998(04) pp 31-32

[9] Clinical application of combined detection of Pentao, DD, FDP and AT in thrombus formation risk assessment after fracture operation, medicine frontier, 2016.6 (32) page 120- & ltSUB & gt 121- & ltSUB & gt

[10] Zhanying et al, D-dimer detection of interest in the diagnosis of thrombotic disease, Jiangxi medicine 2018.53(7): 766-767, page 770

[11] Linjing et al, combined detection of TM, TAT, PIC, t-PAIC levels for DIC diagnostic value J International journal of laboratory medicine 2019.40 (12) pp 1413-1416.

Claims

1. A protein chip for detecting multiple thrombus markers is characterized in that the preparation method of the protein chip comprises the following steps:

(1) pretreating a black glass slide;

(2) spotting an antibody solution;

(3) and (3) sealing to obtain the protein chip.

2. The protein chip according to claim 1, wherein the black glass slide pretreatment method in step (1) is:

①, soaking the black glass slide in a glass slide pretreatment solution containing NaOH for 16-24 h, and then cleaning with pure water for 2-8 times;

3. The protein chip according to claim 1, wherein the antibody solution in step (2) comprises a TAT monoclonal antibody solution, a PIC monoclonal antibody solution, a tpa monoclonal antibody solution, a TM monoclonal antibody solution, an FDP monoclonal antibody solution, and a DD monoclonal antibody solution.

4. The protein chip according to claim 1, wherein the spotting method in step (2) is machine automated spotting.

5. The protein chip according to claim 1, wherein the blocking process in step (3) is: and immersing the spotted black glass slide into the sealing liquid for 1-24 h, then taking out the black glass slide, and centrifuging to remove residual sealing liquid to obtain the protein chip.

6. The protein chip according to claim 5, wherein the blocking solution is a buffer solution containing blocking protein; the blocking protein is bovine serum albumin or ovalbumin; the buffer solution is one or more of PBS buffer solution, Tris buffer solution, HEPES buffer solution and MOPS buffer solution.

7. The use of the protein chip according to any one of claims 1 to 6, wherein the protein chip is used to prepare a kit.

8. The use according to claim 7, characterized in that the kit further comprises a solution of a secondary antibody labeled with HRP enzyme, a chemiluminescent substrate sensitive to HRP enzyme.