CN114686442B - Hybridoma cell strain and application thereof - Google Patents
Hybridoma cell strain and application thereof Download PDFInfo
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- CN114686442B CN114686442B CN202011633074.0A CN202011633074A CN114686442B CN 114686442 B CN114686442 B CN 114686442B CN 202011633074 A CN202011633074 A CN 202011633074A CN 114686442 B CN114686442 B CN 114686442B
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/40—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against enzymes
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/573—Immunoassay; Biospecific binding assay; Materials therefor for enzymes or isoenzymes
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
- C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/90—Enzymes; Proenzymes
- G01N2333/914—Hydrolases (3)
- G01N2333/948—Hydrolases (3) acting on peptide bonds (3.4)
- G01N2333/972—Plasminogen activators
- G01N2333/9726—Tissue plasminogen activator
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/22—Haematology
- G01N2800/226—Thrombotic disorders, i.e. thrombo-embolism irrespective of location/organ involved, e.g. renal vein thrombosis, venous thrombosis
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/32—Cardiovascular disorders
- G01N2800/324—Coronary artery diseases, e.g. angina pectoris, myocardial infarction
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/50—Determining the risk of developing a disease
Abstract
The invention relates to a hybridoma cell strain and application thereof, and belongs to the field of biomedicine. The hybridoma cell strain is t-PAIC2#, and is preserved in China Center for Type Culture Collection (CCTCC) in the year 2020, month 4 and 18, and the preservation number is CCTCC NO: c202066, the preservation address is Wuhan, university of Wuhan in China. The invention also provides a tPAI-C monoclonal antibody secreted by the hybridoma cell strain and application of the tPAI-C monoclonal antibody. The hybridoma cell strain can secrete monoclonal antibodies with high affinity to different epitopes of the tPAI-C protein; the monoclonal antibody of the invention is used for detecting tPAI-C, is not easy to be interfered by tPA and PAI-1 monomers, and has high accuracy.
Description
Technical Field
The invention relates to a hybridoma cell strain and application thereof, and belongs to the field of biomedicine.
Background
Thrombotic disease is a disease with hidden onset, sudden onset and high mortality disability rate, and can be seen in almost every clinical department, and the pathological process of the thrombotic disease involves three systems of vascular endothelium, blood coagulation and fibrinolysis. The existing researches prove that the vascular endothelial, the blood coagulation and the fibrinolysis system are changed in the pre-thrombus state, TAT, PIC, tPAI-C and t-PAI-C are effective indexes for effectively reflecting the early change of the vascular endothelial, the blood coagulation and the fibrinolysis system of the organism, and are suitable for early diagnosis of thrombus and risk assessment of thrombus of high-level population in clinical subjects.
Tissue plasminogen activator and its inhibitor-1 complex (tPAI-C) are a combination of tissue plasminogen activator (t-PA) released by vascular endothelial cells into the blood with the physiological inhibitor plasminogen activator inhibitor (PAI-1) at a ratio of 1:1, and a complex formed by binding them. tPAI-C is a molecular marker of the fibrinolytic system and also of vascular endothelial cell damage.
Studies have shown that tPAI-C is one of the best diagnostic markers for Venous Thromboembolism (VTE), with significantly higher levels of activated PAI-1 and tPAI-C in the plasma of VTE patients than in healthy control populations, with a sensitivity of 74% (95% CI 64-82%), 69% (95% CI, 59-77%) for diagnosis of VTE, and an area under the ROC curve of 0.75 (95% CI, 0.68-0.72) when the cutoff of tPAI-C is taken to be 3.6 ng/ml.
Simultaneously, tPAI-C can also be used as a risk prediction index of Myocardial Infarction (MI). The tPAI-C concentration in plasma is significantly correlated with MI risk in either men OR women, with risk ratio (OR) of 2.4 and 2.0 for men and women, respectively. Smoking or diabetes has a synergistic effect with plasma tPAI-C in male patients. The risk ratio (OR) was 4.6 and 7.9, respectively.
tPAI-C acts as a complex of tPA and PAI-1, whose detection is susceptible to interference by tPA and PAI-1 monomers. Thus, if it is to be quantified accurately by immunology, a monoclonal antibody to a specific epitope is required.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provide a hybridoma cell strain capable of secreting the tPAI-C (tissue type plasminogen activator and inhibitor-1 complex thereof), and the tPAI-C monoclonal antibody secreted by the hybridoma cell strain can accurately measure the tPAI-C.
In order to achieve the above purpose, the technical scheme adopted by the invention is as follows:
in a first aspect, the present invention provides a hybridoma cell line, wherein the hybridoma cell line is t-paic2#, and has been preserved in the China center for type culture collection (cctccc) at 18 days in the year 2020: c202066, the preservation address is Wuhan, university of Wuhan in China.
In a second aspect, the present invention provides a tPAI-C monoclonal antibody secreted by the hybridoma cell line described above.
In a third aspect, the invention provides the use of a tPAI-C monoclonal antibody as described above in the preparation of a kit for detecting tPAI-C or venous thromboembolism.
In a fourth aspect, the invention provides the use of the tPAI-C monoclonal antibody in the preparation of a kit for predicting myocardial infarction risk.
In a fifth aspect, the present invention provides a kit for detecting tPAI-C or venous thromboembolism or predicting risk of myocardial infarction, the kit comprising the tPAI-C monoclonal antibody as described above.
As a preferred embodiment of the kit according to the invention, the kit further comprises magnetic particles, anti-tPAI-C antibodies, AP, glutaraldehyde and bovine serum albumin.
As a preferred embodiment of the kit of the present invention, the kit is used for chemiluminescent detection.
In a sixth aspect, the invention provides a polynucleotide encoding the tPAI-C monoclonal antibody described above.
In a seventh aspect, the present invention provides a vector comprising the polynucleotide described above.
Compared with the prior art, the invention has the beneficial effects that: the invention provides a novel hybridoma cell strain which can secrete monoclonal antibodies with high affinity to different epitopes of tPAI-C protein. The monoclonal antibody of the invention is used for detecting tPAI-C, is not easy to receive the interference of tPA and PAI-1 monomers, and has high accuracy.
The tPAI-C monoclonal antibody can be used as an in-vitro diagnostic reagent, for example, can be used for preparing a chemiluminescent detection kit, accurately quantifies the concentration of tPAI-C protein in blood, has huge economic and social benefits, and provides an important auxiliary detection means for accurate diagnosis, medicine application and other clinical treatments.
Drawings
FIG. 1 is a diagram showing SDS-PAGE results in preparing tPAI-C complexes according to the present invention;
FIG. 2 is a graph showing the results of tail blood titer detection after mice are immunized with tPAI-C complex according to the present invention;
FIG. 3 is a graph showing the results of the determination of antibody titer by the indirect ELISA method of the present invention;
FIG. 4 is a standard graph of the invention for detecting tPAI-C using a tPAI-C monoclonal antibody;
FIG. 5 is a standard graph of the detection of tPAI-C using the Hizikian chemiluminescent reagent.
Detailed Description
For a better description of the objects, technical solutions and advantages of the present invention, the present invention will be further described with reference to the accompanying drawings and specific examples.
tPAI-C acts as a complex of tPA and PAI-1, whose detection is susceptible to interference by tPA and PAI-1 monomers. Monoclonal antibodies to specific epitopes are required if they are to be quantified accurately by immunology. For this purpose, the invention adopts the following technical scheme:
in a first aspect, the present invention provides a hybridoma cell line, wherein the hybridoma cell line is t-paic2#, and has been preserved in the China center for type culture collection (cctccc) at 18 days in the year 2020: c202066, the preservation address is Wuhan, university of Wuhan in China.
The hybridoma cell strain is obtained by the following method: the tPAI-C is used as antigen to immunize mice, spleen cells of the mice with the immunization titer meeting the requirement are fused with myeloma cells, and the hybridoma cell strain is obtained.
The tPAI-C monoclonal antibody secreted by the hybridoma cell strain has high affinity to different epitopes of the tPAI-C protein, and the tPAI-C is detected by adopting the tPAI-C monoclonal antibody, so that the accuracy is high. Thus, the tPAI-C monoclonal antibody can be used as a diagnostic reagent (e.g., an in vitro diagnostic reagent) for detecting tPAI-C, and whether a subject has venous thromboembolism or predicting myocardial infarction risk can be judged according to the detection result of tPAI-C.
In a second aspect, the present invention provides a tPAI-C monoclonal antibody secreted by the hybridoma cell line described above.
In a third aspect, the invention provides the use of a tPAI-C monoclonal antibody as described above in the preparation of a kit for detecting tPAI-C or venous thromboembolism.
In a fourth aspect, the invention provides the use of the tPAI-C monoclonal antibody in the preparation of a kit for predicting myocardial infarction risk.
In a fifth aspect, the present invention provides a kit for detecting tPAI-C or venous thromboembolism or predicting risk of myocardial infarction, the kit comprising the tPAI-C monoclonal antibody as described above.
The tPAI-C monoclonal antibody provided by the invention has high affinity to different epitopes of the tPAI-C protein, can be favorable for the affinity of the tPAI-C protein and can be used for carrying out immunoassay on the tPAI-C protein.
As a preferred embodiment of the kit according to the invention, the kit further comprises magnetic particles, anti-tPAI-C antibodies, AP, glutaraldehyde and bovine serum albumin. Wherein the anti-tPAI-C antibody differs from the tPAI-C protein in the recognition site of the tPAI-C monoclonal antibody. The using method of the kit comprises the following steps: (1) Covalently coupling the tPAI-C monoclonal antibody to Magnetic Particles (MPs) via an N-terminal amino group; (2) Coupling an anti-tPAI-C antibody recognizing another site, AP, and glutaraldehyde; (3) Using the conjugates obtained in step (1) and step (2), the tPAI-C is detected in a sandwich reaction mode.
The chemiluminescence method has high detection sensitivity and quick detection, and the kit can be used for detection by the chemiluminescence method. Of course, the method of detecting tPAI-C using the tPAI-C monoclonal antibody is not limited to the chemiluminescent method, and other methods such as a chromogenic method and the like may be used. The person skilled in the art can determine by which method is used according to routine choice.
In a sixth aspect, the invention provides a polynucleotide encoding the tPAI-C monoclonal antibody described above.
In a seventh aspect, the present invention provides a vector comprising the polynucleotide described above.
Examples
In this example, a tPAI-C monoclonal antibody was prepared, and the tPAI-C was detected by chemiluminescence using the tPAI-C monoclonal antibody as a detection reagent. The method comprises the following specific steps:
1. preparation of tPAI-C monoclonal antibodies
1. Antigen preparation (preparation of tPAI-C Complex)
the tPAI-C complex was prepared in the following manner. PAI-1 is fully activated by 4M guanidine hydrochloride at pH5.5, fully mixed with Tpa in a molar ratio of 2:1 in a neutral environment, and then incubated at 37 ℃ for 1h to fully compound. Then separating and purifying by agarose gel column with fibrin as ligand, and SDS-PAGE result of separating and purifying is shown in figure 1. The compound with purity of more than 90% is obtained.
2. Animal immunization method and procedure
6 female BALB/C mice of 5-6 weeks are selected, and after the first immunization is completed by using the tPAI-C complex, the mice are divided into two groups, 3 mice in each group are respectively subjected to two times of boosting by using tPA and PAI-1, and the immunization process is shown in Table 1. On the seventh day after the third immunization, tail blood was taken for serum titer detection, and the results are shown in fig. 2. And selecting the mice with highest titers for impact fusion.
Table 1: protein immunity information summarization
3. Fusion of spleen cells with myeloma cells
One week before fusion, the frozen myeloma cells (SP 2/0) are recovered by adopting a conventional cell recovery method, and then the SP2/0 cells with good states are selected, and the liquid is changed the day before fusion. Spleen cells of mice with the immunization titer meeting the requirements are taken and placed on a sterile screen. The spleen was ground with a syringe core and 1640 of incomplete medium was added dropwise while grinding. Grind until only white spleen membrane remains. Spleen cells were collected into 50mL centrifuge tubes, centrifuged at 1200rpm for 5min, the supernatant discarded, collected and resuspended. SP2/0 and splenocytes were centrifuged and resuspended in 10mL of incomplete medium, respectively, and counted in dilution. The optimum ratio is 1:1 to 1:4, mixing the two cells in proportion. Centrifuge 1200rpm for 5min and discard the supernatant. Re-suspending with ECF Buffer, centrifuging at 1200r/min for 5min, and removing supernatant. The ECF Buffer wash was repeated one time. Finally, 6.4mL ECF Buffer was used for resuspension. The power supply of the electric fusion instrument is connected, the 'omega' key is pressed, and the resistance is measured, and the resistance value is larger than 2 Komega. 6.4ml of ECF Buffer resuspended cell mix was added. Note that the addition should be slow to avoid bubbles affecting the resistance. Pressing the 'omega' key confirms that the resistance value is between 0.8K and 2 Komega. Pressing the Start key after no error, and waiting for the completion of the electrofusion prompt. After the electric shock is finished, the cell mixture is transferred into 12.8mL of repairing liquid, and the cell membrane is repaired and destroyed. And (3) standing for 10min at 37 ℃ (incubator). During transfer, the suction head should be extended below the liquid level, and then slowly pumped out and centrifuged at 800rpm for 5min. The supernatant was discarded. Cells were resuspended in complete medium (containing 20% fetal bovine serum, 1% hat and appropriate proportions of feeder cells), transferred to 96-well plates and cultured in a 37 ℃ cell incubator.
4. Indirect ELISA detection
Diluting immune mice corresponding to immune antigens by using coating liquid, adding 96-well ELISA plates, adding 100 mu L of each well, coating at 4 ℃ overnight, and washing the well plates by PBST for 1 time; then blocking the coating with 3% Tween PBS at 4deg.C overnight; PBST washed the well plate 1 time later. Serum of each immunized mouse (or cell supernatant to be detected after fusion culture and positive clone cells to be screened) is diluted respectively 10 3 、10 4 、10 5 、10 6 Double, adding into a reaction hole, taking the serum of an immunized mouse as a negative control, placing into a constant temperature box at 37 ℃ for reaction for 1h, adopting goat anti-mouse marked by horseradish peroxidase (HRP) as a secondary antibody for detection, adding TMB chromogenic substrate for reaction, and reading OD450 of each reaction hole by an enzyme-labeled instrument.
5. Antibody epitope analysis
The enzyme label plates are respectively coated with tPAI-C, TPA and PAI-1, monoclonal antibodies obtained by detecting three immunogens are detected, and the antibodies are divided into three groups according to detection results: the group 1 epitope is located on tPAI-C, designated tPAI-C, the group 2 epitope is located on Tpa monomer, designated TPA, and the group three epitope is located on PAI-1 monomer, designated PAI.
6. Identification of antibody subclasses
Cell culture supernatants were taken and the prepared monoclonal antibodies were subjected to subclass analysis according to the monoclonal antibody subclass identification kit of Sigma.
The results of the antibody information and characterization are shown in Table 2.
Table 2 summary of antibody information and characterization
7. Preparation of monoclonal antibodies
Selecting a cell strain in the tPAI-C reaction, subcloning to a fixed strain, injecting the fixed strain into the abdominal cavity of a mouse at a density of 5 x 10 times 5/mouse, collecting ascites for 7-14 days, carrying out affinity purification by using Protein A, measuring the egg quality by using a micro spectrophotometer, and measuring the purity of an antibody by SDS-PAGE.
8. Determination of antibody titers
The measurement was carried out by indirect ELISA, the purified antibodies were adjusted to 1ug/Ml with PBS, and diluted to 10 with 1% Tween PBST, respectively 3 、10 4 、10 5 、10 6 Multiple times. On the ELISA detector, the values of each well were measured after zeroing with the negative control well at 450nm (410 nm if developed with ABTS), and positive if the OD was greater than 2.1 times the OD value of the prescribed negative control. See FIG. 3 (for example, the final selected tPAI-C1# and tPAI-C2#).
2. Establishment of chemiluminescent tPAI-C detection reagent
1. tPAI-C antibody coated MPs
The high affinity tPAI-C monoclonal antibodies are covalently coupled to Magnetic Particles (MPs) via an N-terminal amino group. First, 20mg/mL of MPs were placed in a 2.0mL Ep tube. MPs were washed five times with binding buffer. During the washing process, the tube was placed on a magnetic concentrator and the supernatant removed. The MPs were then resuspended in 2mL of binding buffer. The antibody solution was added to the above suspension at 37℃overnight by shaking culture. After incubation, ep tubes were placed in a magnetic concentrator to separate them from the supernatant. The residual binding sites on MPs were blocked with 3% Bovine Serum Albumin (BSA), incubated at 37 ℃ and gently shaken for 2 hours. After 5 washes, the coated immunomagnetic beads (mAb-MPs) were dispersed in 2mL buffer and stored at 4℃for further use.
2. Preparation of AP-labeled tPAI-C antibody
An anti-tPAI-C antibody recognizing another site, alkaline Phosphatase (AP), and coupling to glutaraldehyde. First, AP and anti-tPAI-C antibodies were suspended in ultrapure water and diluted to 4 and 8mg/mL, respectively. A250. Mu.L aliquot of the 4mg/mL AP solution was transferred to a 1.5mL Ep tube and mixed with 250. Mu.L of 8mg/mL anti-tPAI-C antibody solution. Next, 0.5mL of 0.1mol L-1 phosphate buffer (pH 7.4) containing 1% glutaraldehyde was added to the solution. The resulting mixture was incubated at 37℃for 4 hours with gentle shaking in the dark. In the third step, 0.1mL of 1mol/L monoethanolamine solution was added to the mixture, followed by incubation with shaking at room temperature for 2 hours. The mixture was dialyzed against PBS solution at 4℃overnight. After dialysis, the enzyme-labeled tPAI-C antibodies were transferred to Ep tubes and mixed with equal volumes of glycerol and 1% bsa. Finally, the enzyme-labeled tPAI-C antibodies (AP-mAbs) were stored at-20deg.C for use.
3. MPs-based full-automatic chemiluminescence detection of tPAI-C
tPAI-C detection was performed on a full-automatic luminometer using a sandwich reaction pattern of magnetic bead coated antibodies (mAb-MPs) and enzyme-labeled antibodies (AP-mAbs). First, 50. Mu.L of mAb-MP and CPP samples or standards (30. Mu.L) of different concentrations were separately pipetted into an instrument-matched tube and incubated for 20 minutes with gentle shaking at 37 ℃. The tube was then washed 3 times with wash solution (0.01 mol L-1PBS containing 0.05% Tween) through a wash station to remove non-specific binding. AP-mAbs were then added and incubated for 10 minutes with gentle shaking at 37 ℃. At this time, the sandwich immunocomplex MPs-tPAI-C-AP is formed. The sandwich immunocomplexes formed were magnetically separated and excess AP-mAbs were removed by washing. Subsequently, a solution containing the luminescent substrate AMPPD (200. Mu.l) was added to the sandwich complex. The resulting mixture was incubated in an immunoassay instrument and the Relative Light Unit (RLU) values were measured.
4. Optimization and performance assessment of immunoassay reagents
A series of diluted AP-mAbs (1:50, 1:100,1:200, 1:500) and mAb-MPs (1:20, 1:50,1:100,1:200, 1:500) were reacted with standard positive calibrator (S3, 10 TU/mL) and negative sample (S0, 0 TU/mL) of tPAI-C. The optimal dilution was determined as the maximum RLU ratio of the two (RLUs 3/RLUs 0). Based on the optimal combination, a standard curve (the standard curve for detecting tPAI-C by using the tPAI-C monoclonal antibody of the invention is shown in FIG. 4, the standard curve for detecting tPAI-C by using the Hissen-Messaging chemiluminescent reagent is shown in FIG. 5) is drawn, the sensitivity and linear interval of the standard curve are determined, and 30 clinical samples are compared and analyzed by using the Hissen-Messaging chemiluminescent reagent to investigate the feasibility of the newly developed immunoassay method. The results are shown in Table 3, and Table 3 shows the negative coincidence rate: 100.0%, positive compliance rate: 95.0% of total coincidence rate: 96.7%.
TABLE 3 clinical relevance
Finally, it should be noted that the above embodiments are only for illustrating the technical solution of the present invention and not for limiting the scope of the present invention, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that the technical solution of the present invention may be modified or substituted equally without departing from the spirit and scope of the technical solution of the present invention.
Claims (9)
1. A hybridoma cell line, wherein the hybridoma cell line is t-paic2#, and has been preserved in the China center for type culture collection (cctccc) NO: c202066, the preservation address is Wuhan, university of Wuhan in China.
2. A tPAI-C monoclonal antibody secreted by the hybridoma cell line of claim 1.
3. Use of a tPAI-C monoclonal antibody according to claim 2 in the preparation of a kit for detecting tPAI-C or venous thromboembolism.
4. Use of the tPAI-C monoclonal antibody of claim 2 in the preparation of a kit for predicting the risk of myocardial infarction.
5. A kit for detecting tPAI-C or venous thromboembolism or predicting risk of myocardial infarction, comprising the tPAI-C monoclonal antibody of claim 2.
6. The kit of claim 5, further comprising magnetic microparticles, anti-tPAI-C antibodies, alkaline phosphatase, glutaraldehyde and bovine serum albumin.
7. The kit of claim 5 or 6, wherein the kit is for chemiluminescent detection.
8. A polynucleotide encoding the tPAI-C monoclonal antibody of claim 2.
9. A vector comprising the polynucleotide of claim 8.
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| US5225540A (en) * | 1988-04-26 | 1993-07-06 | Du Pont Merck Pharmaceutical Company | Monoclonal antibodies to tissue plasminogen activator (t-pa) which prolong its functional half-life |
| CN105705520A (en) * | 2013-08-13 | 2016-06-22 | 赛诺菲 | Antibodies to plasminogen activator inhibitor-1 (PAI-1) and uses thereof |
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| CN111983239A (en) * | 2020-08-23 | 2020-11-24 | 深圳市巨东生物医学工程有限公司 | T-PAI-C marker detection kit and preparation method thereof |
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| US5225540A (en) * | 1988-04-26 | 1993-07-06 | Du Pont Merck Pharmaceutical Company | Monoclonal antibodies to tissue plasminogen activator (t-pa) which prolong its functional half-life |
| CN105705520A (en) * | 2013-08-13 | 2016-06-22 | 赛诺菲 | Antibodies to plasminogen activator inhibitor-1 (PAI-1) and uses thereof |
| CN111983239A (en) * | 2020-08-23 | 2020-11-24 | 深圳市巨东生物医学工程有限公司 | T-PAI-C marker detection kit and preparation method thereof |
| CN111948390A (en) * | 2020-08-31 | 2020-11-17 | 浙江盛域医疗技术有限公司 | Time-resolved fluoroimmunoassay kit for detecting t-PAI-C and application thereof |
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