CN105203770B - Application of STIM1 protein in risk diagnosis of related diseases - Google Patents

Abstract

The present invention relates to the use of STIM1 protein in the preparation of medicines for diagnosing the risk of diseases related to the adhesion, activation or aggregation of platelets on the exogenous matrix exposed by endothelial cells.

Description

Application of STIM1 protein in risk diagnosis of related diseases

technical field

The present invention relates to the field of medical biotechnology, in particular to the use of STIM1 protein in the preparation of drugs for diagnosing disease risks associated with platelet adhesion, activation or aggregation on the exogenous matrix exposed by endothelial cells and the enzyme-linked enzyme used for the use Immunization Kit.

Background technique

Platelets, a type of anucleated cell, are released by a complex process from the meganucleus in the bone marrow. The activation and aggregation of platelets is an important physiological process to protect the body after vascular injury, and this process is a multi-step cascade reaction process. But this physiological process is also the main cause of many vascular diseases such as thrombosis. Platelet adhesion, activation, and aggregation to the exposed extramatrix of endothelial cells induce a hemostatic response that can lead to thrombus formation. The initiating factor of platelet activation is the increase of intracellular free calcium ion concentration, and the store-operated calcium channels (SOCC) controlled by the library composed of matrix interaction molecule 1 (Stromal interaction molecules 1, STIM1) and cell membrane protein Orai1 It is one of the main pathways of platelet activation [Progress in Physiological Sciences, 2012, 43:417-421]. STIM1 is highly expressed in platelets. Studies have found that compared with wild-type mice, mice knocked out of STIM1 show higher postnatal lethality and obvious developmental delay [J Exp Med, 2008, 205, 1583-1591 ]. Under high shear stress, compared with normal mice, the platelets of STIM1-knockout mice showed decreased adhesion, decreased thrombus coverage, and decreased thrombus volume [J Exp Med, 2008, 205, 1583-1591] . STIM1 and Orai1 are jointly involved in GPVI-induced store-operated Ca ²⁺ entry SOCE, thrombin source activation and thrombus formation [J Biol Chem, 2010, 285, 23629-23638]. Platelets knocked out of STIM1 can significantly inhibit platelet collagen receptor glycoprotein VI-dependent calcium signaling, resulting in reduced exposure of phosphatidylserine (a component necessary for platelet coagulation activity), thereby inhibiting thrombus formation [J Exp Med,2008,205,1583 -1591]. Thus, STIM1 plays a crucial role in the process of thrombus formation.

Ischemic stroke (cerebral ischemic stroke) refers to cerebral blood circulation disorder, ischemic necrosis or softening of localized brain tissue caused by ischemia and hypoxia. Ischemic stroke mainly includes cerebral thrombosis and cerebral embolism, and vascular wall lesions, vascular compression, heart disease, hemodynamic changes, and changes in blood components can all cause ischemic stroke. The clinical manifestations of ischemic stroke are complex, depending on the infarct location and infarct volume, mainly characterized by symptoms and signs of focal neurological deficits, such as hemiplegia, hemisensory disturbance, aphasia, ataxia, etc. Some may manifest as whole brain symptoms such as headache, vomiting, and coma. According to the results of the 2010 Global Burden of Disease Study (GBD 2010) (N Engl J Med, 2013, 369:448-457), stroke has become a global health problem, affecting life expectancy and causing disability in the third place. reason. According to the latest statistics from relevant departments, there are 2.5 million new stroke patients in my country every year, and the rate of increase is 8.7% per year. At present, there are at least 7 million stroke patients, and the disability rate is as high as 75%. reason. Stroke is mainly divided into two categories: ischemic stroke and hemorrhagic stroke. In some developed countries, up to 67.3% to 80.5% of stroke cases are attributed to ischemic stroke. According to statistics from the Ministry of Health of my country , Ischemic stroke accounts for 45.5%-75.9% of the total number of stroke patients. At present, due to the impact of social pressure and living habits, the incidence of young people is increasing year by year. The direct and indirect economic losses caused by stroke are as high as tens of billions of dollars every year. Therefore, stroke is not only a medical problem, but also a social problem that cannot be ignored.

Therefore, there is a need in the art for diagnosing in the human population the risk of diseases associated with platelet adhesion, activation or aggregation on the exposed extramatrix of endothelial cells, i.e. there is a diagnosis of the risk of having said diseases, said diseases Diagnosis of severity and/or need for diagnosis of prognostic recovery of the disease. This has important clinical significance and social and economic benefits for the prediction and prevention of related diseases.

Contents of the invention

The purpose of the present invention is to provide a simple and rapid method for diagnosing the disease risk associated with the adhesion, activation or aggregation of platelets on the exogenous matrix exposed by endothelial cells and related drugs.

In the first aspect, the present invention provides the use of STIM1 protein in the preparation of a drug for diagnosing the risk of diseases related to the adhesion, activation or aggregation of platelets on the exogenous matrix exposed by endothelial cells.

In an embodiment of the present invention, the disease may be thrombosis, preferably cerebral thrombosis, more preferably ischemic stroke caused by cerebral thrombosis.

In an embodiment of the invention, said diagnosis may comprise a diagnosis of risk of having said disease, a diagnosis of said disease severity and/or a diagnosis of said disease prognosis recovery.

In an embodiment of the present invention, the diagnosis can be performed by measuring the level of STIM1 protein in platelets in peripheral blood, preferably by enzyme-linked immunoassay.

In an embodiment of the present invention, the drug may contain an antibody to the STIM1 protein, preferably a capture antibody A and/or an enzyme-labeled detection antibody B.

In an embodiment of the present invention, the medicament may include a kit, preferably, the kit is an enzyme-linked immunosorbent kit.

In a second aspect, the present invention provides an enzyme-linked immunosorbent kit for use as described in the first aspect, the kit comprising the capture antibody A of the STIM1 protein and the enzyme-labeled detection antibody B of the STIM1 protein .

In an embodiment of the present invention, the capture antibody A and the enzyme-labeled detection antibody B can be prepared by the following method:

(1) Preparation of monoclonal cells: Mix and emulsify the expressed and purified STIM1 protein with an equal volume of Freund's adjuvant and YOULONG adjuvant into a water-in-oil state, immunize 4 Balb/c mice, and subcutaneously immunize 3 two weeks after the last immunization, intraperitoneal injection of antigen for booster immunization, and three days later, the mice were sacrificed to obtain splenocytes, which were added to SP2/0 myeloma cells, under the action of PEG4000 Carry out cell fusion; spread the fused cells into a 96-well plate, culture them with HAT culture medium, and culture them with HT culture medium three days later. After 10 days, take the cell culture supernatant to detect positive wells; use the limited dilution method to detect positive wells. The wells were cloned, tested 10 days later, and the positive clones were cloned by limiting dilution until all clones were positive, so as to establish no less than 50 positive cell lines;

(2) Preparation and purification of monoclonal antibody: Inject mineral oil into the peritoneal cavity of the mouse, inject the obtained positive cell line into the peritoneal cavity of the mouse one week later, collect ascites about 10 days; centrifuge the ascites at 4000rpm room temperature for 15min, and take the supernatant , slowly add saturated ammonium sulfate drop by drop under stirring at 4°C to half saturation, continue to stir for 30 minutes, centrifuge at 13,000 rpm, 4°C for 30 minutes, discard the supernatant; dissolve the precipitate in 0.01M, pH7.4 PBS; stir at 4°C Slowly add saturated ammonium sulfate dropwise to 33%, continue to stir for 30 minutes, centrifuge at 13000 rpm, 4°C for 30 minutes, discard the supernatant; dissolve the precipitate in 0.01M PBS, pH7.4, and dialyze overnight at 4°C;

(3) Paired screening: use the purified monoclonal antibody as the coating antibody, dilute it 1:100 with pH 9.6 carbonate buffer, add 100 μL/well to the multi-well plate, and coat overnight at 4°C; wash Wash the coated plate 3 times with 0.2% Tween-20-containing PBS, 300uL each time for 1min, dilute 10% goat serum 1:9 times with pH9.6 carbonate buffer For blocking, 150 μL per well, at a constant temperature of 37°C for 3 hours; add STIM1 protein solution with a concentration gradient of 1000, 100, 10, and 0 ppb, and add a negative control, 100 μL/well, at 25°C, and incubate for 45 minutes; the washing solution washes the coating The plate was washed 3 times, with 300uL of washing solution each time, and the time was 1min; the obtained monoclonal antibody was routinely labeled with horseradish peroxidase as the detection antibody, diluted with diluent at a ratio of 1:1000, mixed thoroughly, and Well 100μl, 25°C, incubate for 45min; Wash the coated plate 3 times with washing solution, 300μL of washing solution for 1min, add 100μL of TMB to each well, react at 25°C for 15min; add 2M H ₂ SO ₄ , 100μL/ Well, read the absorbance value at 450nm, and select a pair of coating antibody and detection antibody with the strongest binding signal as the capture antibody A and the enzyme-labeled detection antibody B used in the kit, respectively.

In an embodiment of the present invention, the kit may also include: coating buffer, 0.1M carbonate buffer CB, pH 9.6; diluent, 0.01mM phosphate buffer PBS, pH 7.4 ; washing solution, PBS containing 0.2% Tween-20; blocking solution, CB containing 1% BSA; and stop solution, 2M H ₂ SO ₄ .

Beneficial effect:

The inventors of the present invention have found that the expression level of STIM1 protein in platelets in human peripheral blood is positively correlated with diseases related to platelet adhesion, activation or aggregation on the outer matrix exposed by endothelial cells, and thus provides a quantitative Analyzing the expression level of STIM1 protein in platelets in human peripheral blood to diagnose the risk of diseases related to the adhesion, activation or aggregation of platelets on the outer matrix exposed by endothelial cells. This method is easy to operate and can accurately diagnose many diseases such as brain The risk, disease severity, and prognosis risk of diseases such as thrombosis and ischemic stroke.

Description of drawings

Through the following description of the embodiments of the present invention with reference to the accompanying drawings, the above and other objects, features and advantages of the present invention will be more clear, in the accompanying drawings:

Fig. 1 is according to the embodiment of the present invention, the calibration curve of the measured OD value of the standard.

detailed description

The present invention is described below based on examples, but the present invention is not limited to these examples.

Example 1: Antibody Preparation

1 material

1.1 Reagents

BL21(DE3) prokaryotic expression host bacteria, LB medium, Kana antibiotics, high-affinity NI resin, agarose gel, imidazole, urea, NaCl, SDS, ammonium persulfate, TEMED, acrylamide, Coomassie brilliant blue, BCA protein concentration determination kit, Freund's complete adjuvant, Freund's incomplete adjuvant, etc.

1.2 Instruments

Autoclave, constant temperature shaker, refrigerated centrifuge, ultrasonic breaker, protein purifier, constant temperature incubator, protein electrophoresis equipment, constant temperature stirrer, vortex instrument, microplate reader, etc.

2 methods

2.1 Vector construction

According to the requirements, the STIM1 gene sequence is designed and constructed into a prokaryotic expression vector or a eukaryotic expression vector. The amino acid sequence of the expressed STIM1 protein can be found in GenBank: AFZ76986.1

2.2 Antigen preparation (prokaryotic expression)

2.2.1 Protein expression and purification

(1) Transformation: The pET30a-STIM1 positive plasmid was transformed into the prokaryotic expression host strain BL21(DE3), spread on Kanna solid medium (Kana concentration 100 μg/mL), and cultured at 37°C for 12-16h. Pick a single colony and culture it in Kanna liquid medium (Kana concentration 50 μg/mL) for 12-16 hours, and store the glycerol bacteria at -80°C.

(2) Activation of strains: resuscitate the strains, and activate them by culturing overnight at 37°C.

(3) Induced expression: the next day, the strain was expanded to 300mL at a ratio of 1:50, cultured at 37°C until OD600=0.4-0.6, 1mM IPTG was added, and the expression was induced at 37°C for 4h. Centrifuge at 8000rpm at 4°C for 5min to collect the bacteria.

(4) Strain cracking: Add 100mL disrupting solution for ultrasonic cracking. Cracking conditions: temperature ice bath, power 60%, ultrasonic 2s, interval 2s, time 15min. Centrifuge at 12000rpm at 4°C for 15min, collect supernatant and precipitate, and detect by SDS-PAGE.

(5) Purification: if it is an inclusion body, add the enrichment solution to suspend the precipitate, pre-ice for 2 hours, centrifuge at 10,000 rpm and 4°C for 15 minutes, and collect the supernatant. It is feasible to use high-affinity NI resin to purify protein, collect flow-through and eluate, and detect protein purification effect by SDS-PAGE.

(6) Protein renaturation: The purified protein obtained under denaturing conditions was renatured with gradient urea and detected by SDS-PAGE, and the protein renaturation effect was feasible. The finally obtained protein had a purity of 85%, a protein concentration of 2 mg/mL, and a protein amount of 10 mg.

2.3 Polyclonal antibody preparation

2.3.1 Immunogen preparation:

Mix and emulsify the expressed and purified protein with an equal volume of Freund's adjuvant and YOULONG adjuvant to form a water-in-oil state for rabbit immunization.

2.3.2 Immunization strategy:

Two New Zealand white rabbits were immunized subcutaneously for 3 times with an interval of one month, and finally tested by ELISA, the antiserum titer was >1:50000.

2.3.3 Taking blood:

The ear veins were boosted for immunization, and rabbit blood was collected half a month after the booster immunization (all collected).

2.3.4 Polyclonal antibody purification:

Centrifuge the rabbit blood for 15min (4000rpm, room temperature), take the supernatant, slowly add saturated ammonium sulfate drop by drop under stirring at 4°C to half saturation, continue stirring for 30min, centrifuge for 30min (13000rpm, 4°C), discard the supernatant; Appropriate amount of PBS (0.01M, pH7.4); slowly add saturated ammonium sulfate dropwise to 33% under stirring at 4°C, continue stirring for 30min, centrifuge for 30min (13000rpm, 4°C), discard the supernatant; dissolve the precipitate in an appropriate amount of PBS ( 0.01M, pH7.4), dialyzed overnight at 4°C, determined the antibody content, and stored at -20°C for later use. After the ammonium sulfate precipitation, continue to use the Protein A column for purification. The new column is first passed through the column with 5ml ultrapure water, and then 5ml of 0.4M PB buffer (pH 7.0) is used to equilibrate the purification column; column, in order to better bind the antibody protein to the binding site; continue to equilibrate the purification column with 10ml 0.4M PB buffer (pH 7.0); 5ml 0.1M glycine-hydrochloric acid buffer (pH 3.0) to elute the binding site Antibody, and add 1M Tris-HCl (pH 8.0) to neutralize glycine to keep the pH neutral for antibody storage.

2.4 Monoclonal antibody preparation

2.4.1 Immunogen preparation:

Mix and emulsify the expressed and purified protein with an equal volume of Freund's adjuvant and YOULONG adjuvant into a water-in-oil state for immunization of mice.

2.4.2 Immunization strategy:

The protein was immunized to 4 Balb/c mice, subcutaneously immunized 3 times with an interval of 4 weeks, and finally detected by ELISA.

2.4.3 Cell Fusion:

Two weeks after the last immunization, antigen was injected intraperitoneally for booster immunization, and cell fusion was performed three days later. The mice were killed by neck dislocation, sterilized by soaking in 70% ethanol for 30 minutes, and the abdominal cavity was cut open on an ultra-clean table, the spleen was taken out, ground, passed through an 80-mesh sieve, and splenocytes were obtained, and SP2/0 myeloma cells were added to the effect of PEG4000 for cell fusion,

2.4.4 Fusion screening:

Spread the fused cells into a 96-well plate, culture with HAT medium, change the medium after three days, and culture with HT medium instead. After 10 days, the cell culture supernatant was taken for detection.

2.4.5 Cloning and strain establishment:

Use the limiting dilution method to clone the positive wells, detect after 10 days, and continue to clone the positive clones by the limiting dilution method until the obtained clones are all positive, and positive cell lines can be established, no less than 50 cell lines.

2.4.6 Expansion culture: The monoclonal cells of the established strain were expanded and cultured, and frozen.

2.5 Antibody preparation and purification

2.5.1 Ascites preparation:

One week in advance, inject mineral oil into the peritoneal cavity of mice, inject a certain number of cells into the peritoneal cavity of mice, collect ascites about 10 days, centrifuge at 4000rpm, and obtain supernatant as monoclonal antibody ascites.

2.5.2 Monoclonal antibody purification:

Centrifuge the ascitic fluid for 15min (4000rpm, room temperature), take the supernatant, slowly add saturated ammonium sulfate drop by drop to half saturation under stirring at 4°C, continue stirring for 30min, centrifuge for 30min (13000rpm, 4°C), discard the supernatant; PBS (0.01M, pH7.4); slowly add saturated ammonium sulfate dropwise to 33% under stirring at 4°C, continue stirring for 30min, centrifuge for 30min (13000rpm, 4°C), discard the supernatant; dissolve the precipitate in an appropriate amount of PBS (0.01 M, pH7.4), dialyzed overnight at 4°C, determined the antibody content, and stored at -20°C for later use. After the ammonium sulfate precipitation, continue to use the Protein G column for purification. The new column is first passed through the column with 5ml ultrapure water, and then 5ml of 0.4M PB buffer (pH 7.0) is used to equilibrate the purification column; column, in order to better bind the antibody protein to the binding site; continue to equilibrate the purification column with 10ml 0.4M PB buffer (pH 7.0); 5ml 0.1M glycine-hydrochloric acid buffer (pH 2.7) to elute the binding site Antibody, and add 1M Tris-HCl (pH 8.0) to neutralize glycine to keep the pH neutral for antibody storage.

2.5.3 Potency determination:

Dilute the HPT protein with coating buffer, the coating concentration is 1ug/ml, coat 100ul per well, coat overnight at 4°C, wash the plate, block with 150ul blocking buffer, 37°C for 3h. The purified antibody was serially diluted with antibody diluent, 100 μl per well, reacted at 37°C for 1 h; washed the plate, added 100 μl enzyme-labeled secondary antibody to each well, and reacted at 37°C for 1 h; washed the plate,

Add 100 μl of chromogen to each well, react at room temperature in the dark for 15 min, and read the OD value at 450 nm. Antibody titers were obtained using 0.5 as a cutoff value.

2.6 Pair screening

2.6.1 Coating:

The purified murine monoclonal antibody was diluted 1:100 times with pH 9.6 carbonate buffer solution (coating solution, the same below), added 100 μL/well to a multi-well plate, and coated overnight at 4°C.

2.6.2 Closure:

Wash the coated plate 3 times with washing solution, 300uL each time, 1min, block with 10% goat serum (diluted 1:9 times with pH9.6 carbonate buffer), 150μL per hole, constant temperature 37 °C for 3 hours.

2.6.3 Adding samples:

STIM1 protein solution (high standard) with a concentration gradient of 1000, 100, 10, and 0 ppb was added, and a negative control was added, 100 μL/well, incubated at 25° C. for 45 minutes.

2.6.4 Add detection antibody:

The obtained monoclonal antibodies were routinely labeled with horseradish peroxidase one by one as the detection antibody, and the coated plate was washed with the washing solution for 3 times, and the amount of each washing solution was 300uL for 1min. Depends) Proportionally dilute the detection antibody, mix thoroughly, 100 μl per well, incubate at 25°C for 45min.

2.6.5 Color rendering:

The coated plate was washed with washing solution for 3 times, and the amount of washing solution was 300 μL for 1 minute. Add 100 μL of TMB to each well and react at 25°C for 15 minutes.

2.6.6 Detection:

Add 2M H ₂ SO ₄ , 100 μL/well, and read OD (absorbance) value at 450 nm.

2.6.7 Results:

In the monoclonal antibody, the strong positive monoclonal antibody is paired with the rabbit polyclonal antibody or the strong positive monoclonal antibody is paired. According to the strongest binding signal, determine the best coating antibody A and detection antibody B (enzyme-labeled antibody B)

Pairing effect detection

Antibody number positive feminine 1 2.453 0.181 2 2.497 0.197 3 2.169 0.273 4 2.38 0.239 6 2.597 0.278 7 2.66 0.45 8 2.389 0.272 11 2.463 0.246 12 2.156 0.234 13 2.218 0.229 14 2.277 0.201 17 2.38 0.239 18 2.415 0.227 19 2.3 0.155

2.6.8 Sensitivity determination:

Select a pair of coating antibody A and detection antibody B with a low negative value and a high positive value for paired screening, and add different concentrations of the standard product and enzyme-labeled antibody B according to the above method to determine the sensitivity and the concentration of enzyme-labeled antibody B used.

Embodiment 2: establish and optimize ELISA test method:

Including the determination of the composition of the reagent, the determination of the amount of capture and detection antibodies, the test of specificity and sensitivity, the test of the error between groups and within the group, and the determination of the sample recovery rate. The detection sensitivity of the detection method is 5ng/ml, and the standard curve is from 50ng/ml to 1000ng/ml; for STIM1 with a sample concentration of 110-920ng/ml, the recovery rate is >90%. For the low concentration of 200ng/ml, the medium concentration of 500ng/ml and the high concentration of 800ng/ml, the coefficients of variation within the group were 3.6%, 5.1% and 5.9%, respectively, while the coefficients of variation between groups were 6.1%, 7.9% and 9.2 %. The true positive rate of the kit is: 84.8%; the specificity (true negative rate): 85.1%; the false positive rate: 5%; the false negative rate is 16%.

Example 3: Diagnostic kits according to the invention and exemplary diagnostic applications thereof

1.1 Reagents

1.1.1 Coating buffer, 0.1M carbonate buffer CB, pH 9.6;

1.1.2 Diluent, 0.01mM phosphate buffer saline PBS, pH 7.4;

1.1.3 Washing solution, PBS containing 0.2% Tween-20;

1.1.4 CB containing 1% BSA in blocking solution;

1.1.5 Stop solution 2M H ₂ SO ₄

1.1.6 Preparation of protein standard: STIM1 prokaryotically expressed in E. coli and purified by affinity, was dried overnight in a freeze dryer to obtain a standard freeze-dried powder, which was diluted to different concentrations according to the specific sensitivity. 1000ng/ml, 500ng/ml, 250ng/ml, 50ng/ml, 0ng/ml.

1.1.7 Capture antibody A and enzyme-labeled detection antibody B:

1.1.7.1 Preparation of enzyme-labeled antibody: Take the purified antibody and couple it with HRP to obtain enzyme-labeled antibody B.

1.1.7.2 Take a certain amount of enzyme-labeled antibody B and add it to the diluent, mix well to make the final concentration 2μg/mL, and store (depending on the specific conditions) at 2-8°C in the dark.

1.2 Preparation of ELISA plate

1.2.1 Coating

Pipette a certain amount of STIM1 antibody into the required volume of coating solution to reach a certain concentration to form a coating working solution. Add 100 μL of the coating working solution to the microwells of the microtiter plate, and let it stand overnight at 4°C (note that water evaporation should be prevented).

1.2.2 Closure

Add a certain amount of calf serum, sodium salicylate, and sucrose in the CB buffer solution (pH9.6), so that the concentrations of calf serum, sodium salicylate, and sucrose are 10%, 0.05%, and 5% respectively. into a closed working solution. Plates were blotted dry in a plate washer - washed twice, and the strips were patted dry on clean absorbent paper. Add 150 μL of blocking working solution to each well and bake at 37°C for 3 hours.

1.2.3 Drain seal

Discard the blocking solution, pat dry the strips on clean absorbent paper, put them in a freeze-vacuum dryer for 3 hours, and heat-seal them in vacuum.

1.2.4 Assembly of the kit

name quantity Standard freeze-dried powder 1 bottle Enzyme-labeled antibody working solution 1 bottle Diluent 2 bottles Reagent 1 bottle Terminator 1 bottle

detergent 1 bottle

1.3 Detection of protein STIM1 in human body using the kit of the present invention

Add standards or samples of unknown concentration:

1.3.1 Add 100 μL of sample diluent (blank control)/standard substance/sample of unknown concentration to the corresponding microwell, shake gently to mix, and react in a dark environment at 25°C for 45 minutes.

1.3.2 Unknown sample processing: 5-10ml of human peripheral blood was collected in EDTA tube, 1000rpm, 10 minutes, and the supernatant was taken. The supernatant was centrifuged again at 2000 rpm for 20 minutes, the supernatant was discarded, and the precipitate was retained. Add 30ul RIPA (add 1:1000PMSF), let stand on ice for 20 minutes, and pipette up and down. Centrifuge and save the supernatant.

1.3.3 Detect the protein concentration of the supernatant (unknown sample platelet) in 1.3.2. Store at -20°C.

1.3.2 Washing the plate: Shake the liquid in the wells to dry, fully wash 3 times with 250 μL washing solution/well, with an interval of 1 min between each time, and pat dry with absorbent paper.

1.3.3 Add enzyme-labeled antibody: add 100 μL/well of enzyme-labeled antibody, shake gently to mix, react in a dark environment at 25°C for 45 minutes, remove and repeat plate washing step 2

1.3.4 Color development: Add 100 μL/well of chromogenic agent, and place in a dark environment at 25°C for 15 minutes to react.

1.3.5 Measurement: Add 100 μL of stop solution/well, shake gently to mix, set the microplate reader to 450nm, and measure the OD value of each well (preferably use dual wavelength 450/630nm for detection, and read the data within 5min). If there is no microplate reader, it can be judged by visual inspection without adding stop solution.

1.3.6 Calculation: Use statistical drawing software to draw a standard curve according to the OD value of the measured standard substance, as shown in Figure 1; the concentration of STIM1 in the unknown sample can be calculated by the standard curve and the OD value of the unknown concentration sample (Need to be divided by the protein concentration of each sample).

Table 1: Comparison of the results of STIM1 in the peripheral serum of normal unaffected people and ischemic stroke patients.

It can be seen from Table 1 that even in the low-expression group with low STIM1 protein expression in the diseased population, the average STIM1 protein expression is 2.83 times that of the normal population, so we can quantify the expression of STIM1 protein To diagnose whether the subjects have ischemic stroke, or whether they have a higher risk of stroke.

Table 2: The NIHSS scores of the STIM1 low expression group and the medium expression group were compared with the high expression group for three months after discharge. The NIHSS score of the STIM1 high expression group was significantly worse than that of the low expression and medium expression groups (p<0.005).

It can be seen from Table 2 that there are differences in the expression of STIM1 protein in peripheral blood platelets of patients with different disease severity, or that there is a positive correlation between the expression of STIM1 protein in peripheral blood platelets and the severity of the disease of patients, so we By quantifying the expression of STIM1 protein, it is possible to diagnose the severity of the subject's illness, prognosis and recovery, etc.

The above descriptions are only preferred embodiments of the present invention, and are not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and changes. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims (8)

1. The use of STIM1 protein in the preparation of drugs for diagnosing ischemic stroke, wherein the diagnosis includes the diagnosis of disease severity and/or the diagnosis of recovery of disease prognosis, and the diagnosis is carried out by measuring STIM1 in platelets in peripheral blood protein content. 2. The use according to claim 1, wherein the diagnosis is detected by an enzyme-linked immunosorbent assay kit. 3. The use according to claim 1, wherein the medicament comprises an antibody to the STIM1 protein. 4. The use according to claim 3, wherein the medicament comprises capture antibody A and/or enzyme-labeled detection antibody B. 5. The use according to claim 4, wherein the medicament comprises a kit. 6. The use according to claim 5, wherein the kit is an enzyme-linked immunosorbent assay kit. 7. The use of an enzyme-linked immunosorbent kit in the preparation of a drug for diagnosing ischemic stroke, wherein the diagnosis includes the diagnosis of the severity of the disease and/or the diagnosis of the recovery of the disease prognosis, and the diagnosis is carried out by measuring peripheral The content of STIM1 protein in platelets in blood is carried out, and the kit includes the capture antibody A of the STIM1 protein and the enzyme-labeled detection antibody B of the STIM1 protein. The kit also includes: coating buffer, 0.1M carbon Salt buffer CB, pH value is 9.6; Dilution solution, 0.01mM phosphate buffer saline solution PBS, pH value is 7.4; Washing solution, the PBS containing 0.2% Tween-20; Blocking solution contains the CB of 1%BSA; With Stop solution, 2M H ₂ SO ₄ . 8. purposes as claimed in claim 7, wherein said capture antibody A and described enzyme-labeled detection antibody B are prepared by the following method: (1) Preparation of monoclonal cells: Mix and emulsify the expressed and purified STIM1 protein with an equal volume of Freund's adjuvant and YOULONG adjuvant into a water-in-oil state, immunize 4 Balb/c mice, and subcutaneously immunize 3 two weeks after the last immunization, intraperitoneal injection of antigen for booster immunization, and three days later, the mice were sacrificed to obtain splenocytes, which were added to SP2/0 myeloma cells, under the action of PEG4000 Carry out cell fusion; spread the fused cells into a 96-well plate, culture them with HAT culture medium, and culture them with HT culture medium three days later. After 10 days, take the cell culture supernatant to detect positive wells; use the limited dilution method to detect the positive wells. The wells were cloned, tested 10 days later, and the positive clones were cloned by limiting dilution until all clones were positive, so as to establish no less than 50 positive cell lines; (2) Preparation and purification of monoclonal antibody: Inject mineral oil into the peritoneal cavity of mice, inject the obtained positive cell lines into the peritoneal cavity of mice one week later, collect ascites about 10 days later; centrifuge ascites at 4000rpm room temperature for 15 min, take Add saturated ammonium sulfate dropwise under stirring at 4°C to half saturation, continue stirring for 30 min, centrifuge at 13000 rpm, 4°C for 30 min, discard the supernatant; dissolve the precipitate in 0.01M, pH7.4 PBS; Slowly add saturated ammonium sulfate dropwise to 33% under stirring at 4°C, continue stirring for 30 min, centrifuge at 13000 rpm, 4°C for 30 min, discard the supernatant; dissolve the precipitate in 0.01M PBS, pH7.4, and dialyze overnight at 4°C; (3) Pair screening: use the purified monoclonal antibody as the coating antibody, dilute it 1:100 with pH 9.6 carbonate buffer, add 100 µL/well to the multi-well plate, and coat overnight at 4°C; 0.2% Tween-20 PBS was used as the washing solution to wash the coated plate 3 times, the amount of each washing solution was 300 uL for 1 min, and 10% sheep serum was diluted 1:9 times with pH9.6 carbonate buffer For blocking, 150 µL per well, at a constant temperature of 37°C for 3 hours; add STIM1 protein solution with a concentration gradient of 1000, 100, 10, and 0 ppb, and add a negative control, 100 µL/well, at 25°C, and incubate for 45 min; The plates were plated 3 times, with 300 uL of washing solution each time, and the time was 1 min; the obtained monoclonal antibodies were routinely labeled with horseradish peroxidase as the detection antibody, diluted with diluent at a ratio of 1:1000, and mixed thoroughly. 100 µl per well, at 25°C, incubate for 45 min; wash the coated plate 3 times with washing solution, use 300 µL of each wash for 1 min, add 100 µL of TMB to each well, react at 25°C for 15 min; add 2M H ₂ SO ₄ , 100 µL/well, read the absorbance value at 450 nm, and select a pair of coating antibody and detection antibody with the strongest binding signal as the capture antibody A and the enzyme-labeled detection of the kit respectively Antibody B.