CN114316042A - cTnI protein antigenic determinant polypeptide and application thereof - Google Patents
cTnI protein antigenic determinant polypeptide and application thereof Download PDFInfo
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Abstract
The invention provides cTnI protein antigenic determinant polypeptide and application thereof. Wherein the amino acid sequence of the cTnI protein antigenic determinant polypeptide is a sequence shown in SEQ ID NO. 1. The cTnI protein antigen is prepared by coupling the antigenic determinant polypeptide and carrier protein, the cTnI protein antibody is a monoclonal antibody or a polyclonal antibody prepared by the antigen, and the monoclonal antibody and the polyclonal antibody are used for preparing a cTnI protein detection kit. The cTnI protein antigenic determinant polypeptide has good antigenicity, an antigen immune animal prepared by the cTnI protein antigenic determinant polypeptide can generate a specific antibody, the prepared cTnI protein antibody can be specifically combined with cTnI in human serum, and the prepared cTnI detection kit can effectively detect the level of the cTnI protein in the human serum.
Description
Technical Field
The invention relates to the field of polypeptide chemistry, in particular to cTnI protein antigenic determinant polypeptide and application thereof.
Background
Acute Myocardial Infarction (AMI) is one of the cardiovascular diseases that are common in clinic and is considered as the leading cause of death in patients with cardiovascular diseases. 1-3 hours after the onset of AMI is the "gold" window for thrombolytic and interventional therapy, which means that rapid diagnosis of early AMI is critical to treatment. Traditional AMI diagnostics rely primarily on angina symptoms, electrocardiograms, and biomarker detection, where identification of patients with atypical myocardial infarction manifestations (i.e., no chest pain or no elevation of the ST segment of the electrocardiogram) is particularly important. Cardiac troponin i (ctni) is considered as a gold standard for early diagnosis of AMI, and has high myocardial tissue specificity and clinical sensitivity, and may even reflect myocardial ischemia or necrosis in a minute area. Healthy persons typically have cTnI concentrations below 0.4 ng/mL, while levels above 2.0 ng/mL mean an increased risk of future severe cardiac events.
Therefore, the determination of the concentration of cTnI has important clinical significance and value for clinical diagnosis, risk stratification and prognosis management of patients with cardiovascular diseases. Conventional methods for the detection of cTnI are usually immunoassays, i.e., the level of cTnI is detected using antibodies to cTnI, and therefore, the discovery and preparation of suitable cTnI protein epitope polypeptides has also become a major point in the detection of cTnI.
With the development of the medical examination field, there is a clinical need for a cTnI protein epitope polypeptide with high specificity and antigenicity, so as to be suitable for the preparation and production of highly specific antibodies. Therefore, the cTnI protein antigenic determinant polypeptide and the application thereof have good application prospect and practical significance.
Disclosure of Invention
Aiming at the defects in the background technology, the invention provides a cTnI protein antigenic determinant polypeptide and application thereof.
In order to achieve the purpose, the invention provides the following technical scheme:
in a first aspect, the invention provides a cTnI protein antigenic determinant polypeptide, wherein the amino acid sequences of antigenic determinant polypeptides I and II are shown as a sequence shown in SEQ ID NO.1 and a sequence shown in SEQ ID NO.2, and specifically comprise:
(1) Leu-Cys-Arg-Gln-Leu-His-Ala-Tyr; and
(2)Asp-Lys-Val-Asp-Glu-Glu-Arg-Tyr-Asp-Ile-Tyr。
in a second aspect, the present invention provides a cTnI protein antigen prepared by conjugating an antigenic determinant polypeptide as defined in the first aspect to a carrier protein.
In a third aspect, the present invention provides a cTnI protein antibody, which is a monoclonal antibody or a polyclonal antibody prepared from the antigen of the second aspect.
In a fourth aspect, the invention provides a use of the cTnI protein antibody according to the third aspect in the preparation of a cTnI detection kit.
In a fifth aspect, the present invention provides a cTnI protein detection kit comprising the antibody of the third aspect as a coating antibody, preferably a monoclonal antibody.
Preferably, the cTnI protein detection kit is used for quantitatively detecting the level of cTnI protein in human serum.
Compared with the prior art, the invention has the following beneficial effects:
1. the cTnI protein antigenic determinant polypeptide provided by the invention has good antigenicity, and an antigen immune animal prepared by using the polypeptide can generate a specific antibody;
2. the cTnI protein antigen provided by the invention has good immunogenicity, and can stimulate immune response of antibody immune animals;
3. the cTnI protein antibody provided by the invention can be specifically combined with cTnI in human serum;
4. the cTnI detection kit provided by the invention can effectively detect the cTnI protein level in human serum.
Drawings
Fig. 1 is a concentration and absorbance standard curve of a cTnI protein calibrator in the cTnI protein detection kit.
Detailed Description
In order that the technical contents of the invention can be more clearly understood, the following detailed description is provided for further explanation.
The terms used herein have meanings commonly understood by those of ordinary skill in the relevant art. However, for a better understanding of the present invention, some definitions and related terms are explained as follows:
the term "antigenic determinant", as used in the present invention, refers to an antigenically determining chemical group, which is localized on the surface of an antigenic substance and may consist of a contiguous sequence of amino acids or a discontinuous three-dimensional structure of a protein. As used herein, "antigenic determinant" refers specifically to antigenic determinant I and II polypeptides of the human cTnI protein.
I, cTnI protein antigenic determinant.
The cTnI proteins described in the present invention are known in the art, and their amino acid sequences are known and can be searched in professional databases.
The invention provides a cTnI protein antigenic determinant polypeptide, wherein the amino acid sequences of the antigenic determinant polypeptides I and II are shown as a sequence shown in SEQ ID NO.1 and a sequence shown in SEQ ID NO.2, and specifically comprise:
(1) Leu-Cys-Arg-Gln-Leu-His-Ala-Tyr; and
(2)Asp-Lys-Val-Asp-Glu-Glu-Arg-Tyr-Asp-Ile-Tyr。
through theoretical research and experimental exploration, the screened cTnI protein antigenic determinant polypeptide has good antigenicity and can be specifically combined with a corresponding antibody.
The epitope I is a polypeptide fragment from the 96 th position to the 102 th position of the N-terminal of the human cTnI protein (Swiss-Prot accession number: P19429.3), and is obtained by adding the amino acid Y to the C-terminal, and the total content of the amino acid Y is 8 amino acids.
The epitope II is a polypeptide fragment from the 105 th to the 114 th positions of the N-terminal of the human cTnI protein (Swiss-Prot accession number: P19429.3), and is obtained by adding amino acid Y to the C-terminal, and contains 11 amino acids in total.
The addition of amino acid Y to the C-terminus is intended to allow the antigenic determinant to be cross-linked to a carrier protein via Bis-diazotized benzidine (BDB) and thereby allow the corresponding antibody to be prepared as an antigen. The antigenic determinant has the characteristics of high hydrophilicity, strong antigenicity and easy synthesis.
The cTnI epitope polypeptide fragments described above can be synthesized using solid phase methods, see example 1.
The antigenic determinant provided by the invention has the following characteristics:
1. the antigen is strong, and can be specifically combined with a corresponding antibody;
2. after the antigenic determinant is combined with carrier protein, the antigen immune animal can be stimulated to prepare a monoclonal antibody;
3. antibodies prepared using this epitope can specifically bind to human blood cTnI.
The molecular weight of the antigenic determinant provided by the invention is 1003.18, and the molecular weight can be determined by mass spectrometry.
And II, cTnI protein antigen.
The cTnI protein antigen provided by the invention is prepared by coupling the cTnI protein antigenic determinant polypeptide provided by the invention with carrier protein.
The carrier protein includes hemocyanin (KLH), Bovine Serum Albumin (BSA), Ovalbumin (OVA), etc., wherein the preferred carrier protein is KLH, which has strong immunogenicity, good immune effect and is not easy to cause cross reaction. The preparation method adopts the existing mature technology, see example 2.
And III, cTnI protein antibody.
The cTnI protein antibody provided by the invention is a monoclonal antibody and a polyclonal antibody prepared by combining the cTnI protein antigen provided by the invention with a carrier protein antigen, the preparation method is a conventional technology in the field, and the cTnI protein antibody can be used for preparing a cTnI protein detection kit, and the kit can be used for detecting the cTnI protein in a human blood sample based on an immunization method. The preparation method adopts the existing mature technology, see example 2.
And fourthly, a cTnI protein detection kit.
The cTnI protein detection kit provided by the invention preferably adopts an ELISA double-antibody sandwich method to detect human cTnI protein, and the kit comprises necessary reagents such as a coating antibody, a binding antibody, an enzyme-labeled second antibody and the like.
Wherein, when the coating antibody is the cTnI protein monoclonal antibody I, the binding antibody is a cTnI protein polyclonal antibody II; when the coating antibody is the cTnI protein monoclonal antibody II, the binding antibody should be a cTnI protein polyclonal antibody I.
In addition, the cTnI protein detection kit may further comprise any reagents or tools required for the assay, such as pre-coated plates, washing solutions, stop solutions, and the like. See example 3 for the preparation of the kit.
The invention also provides application of the cTnI protein detection kit in quantitative determination of human serum cTnI protein. See example 3 for use of the kit.
To further illustrate the technical means and effects of the present invention, the present invention is further described with reference to the following embodiments, but the scope of the present invention is not limited by the specific embodiments, and it should be understood that the claims are only directed to the described embodiments, and not to the whole embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention. The examples do not show the specific techniques or conditions, according to the technical or conditions described in the literature in the field, or according to the product specifications. The reagents or apparatus used are conventional products commercially available from normal sources, not indicated by the manufacturer.
Example 1 preparation of cTnI protein epitopes I and II.
The preparation method of the cTnI protein antigenic determinants I and II adopts a chemical solid phase method to synthesize the antigenic determinants, and the principle is that the C tail end of amino acid is fixed on insoluble resin, and then condensation reaction is carried out in sequence and a peptide chain is prolonged until the preparation of polypeptide is finished.
The cTnI protein antigenic determinants I and II of the present invention are prepared as follows.
1. Reagents and raw materials:
HMP resin (P-hydroxymethylphenoxymethyl polyethylene resin, Wang resin);
Fmoc-AA (9-fluorenylmethoxycarbonyl protected amino acid);
NMP (nitrogen methyl pyrrolidone);
DCM (dichloromethane);
MeoH (methanol);
piperidine (Piperidine);
DMAP (dimethylaminopyridine);
HOBT (hydroxybenzotriazole);
DCC (dicyclohexylcarbodiimide);
TFA (trifluoroacetic acid);
EDT (1, 2-ethanedithiol);
thioanisole;
crystallizing phenol;
and (3) acetonitrile.
2. Using an instrument:
a polypeptide automatic synthesizer;
high performance liquid chromatograph.
3. The synthesis method and the process are as follows:
step 1, activating Fmoc-AA.
The structural formula of Fmoc-AA is shown as follows:
can be abbreviated as:
the Fmoc-AA was activated by reaction with DCC and HOBT, the reaction scheme is as follows:
Reacting the activated amino acid with HMP resin under DMAP condition to obtain the resin connected with the amino acid, wherein the reaction formula is as follows:
and 3, removing the Fmoc group of the resin connected with the amino acid.
Under the catalytic action of Piperidine, removing the Fmoc group of the resin connected with the amino acid, wherein the reaction formula is as follows:
and 4, repeating the step 1 and the step 2 to obtain another activated amino acid, coupling the obtained activated amino acid with the resin obtained in the step 3, and removing amino groups in the coupled amino component, wherein the reaction formula is as follows:
and 5, repeating the step 3 and the step 4 until the synthesis is finished to obtain the polypeptide resin connected with all the amino acids.
And 6, separating and purifying the polypeptide resin connected with all the amino acids obtained in the step 5 to obtain cTnI protein antigenic determinants I and II with the immunocompetence. The steps for the analysis and purification of both epitopes are as follows:
step 6-1, using TFA to combine a scavenging agent mixed by EDT, thioanisole and water to react with the polypeptide resin connected with all amino acids obtained in the step 5, and separating antigenic determinants I and II of the cTnI protein with the immunological activity from the polypeptide resin, wherein the reaction time is 3 hours;
6-2, removing the scavenging agent, and extracting by using ether to obtain crude products of cTnI protein antigenic determinants I and II with immune activity;
and 6-3, purifying the cTnI protein antigenic determinant I and II crude products with the immunocompetence to obtain the cTnI protein antigenic determinant I and II with the immunocompetence.
Wherein the purification is performed by high performance liquid chromatography, and the conditions are shown in the following table:
TABLE 1 separation and purification conditions of high performance liquid chromatography
Example 2 Using epitopes I and II obtained in example 1, monoclonal and polyclonal antibodies were prepared, respectively.
The cTnI protein antigenic determinants I and II obtained in example 1 were linked to a carrier protein, respectively, and cTnI protein antigens I and II were prepared, and animals were immunized with the antigens I and II, respectively, to prepare specific monoclonal and polyclonal antibodies.
The preparation steps of the cTnI protein antibody are as follows:
1. preparation of cTnI protein antigen.
Step 1. 10 mg of cTnI protein epitope I or II obtained in example 1 was dissolved in 1 mL of 0.1M PBS buffer (pH 7.4).
And 3, mixing the solutions obtained in the step 1 and the step 2, cooling to 0 ℃, taking 110 mu L of BDB coupling agent chromium chloride, reacting for 1.5 hours at room temperature, dialyzing overnight, subpackaging to obtain the cTnI protein antigen I or II, and storing at-20 ℃.
2. Monoclonal antibodies were prepared using immunized animals.
Step 1, animal immunization. Taking the cTnI protein antigen I or II prepared in the step 1 as an immunogen, taking a Balb/c mouse as an immune animal, and increasing the immunogenicity of the cTnI protein antigen by using a Freund's complete adjuvant and a Freund's incomplete adjuvant. Fully and uniformly mixing the same amount of antigen and Freund's complete adjuvant, and injecting the immune mouse subcutaneously at multiple points with the dosage of 50 mu g/mouse to complete primary immunity; after four weeks, the same amount of antigen and Freund's incomplete adjuvant are fully mixed, and then the immune mouse is injected subcutaneously with multiple points at a dose of 50 mug/mouse to complete secondary immunity; after four weeks, the mice were immunized by subcutaneous multi-point injection with 50 μ g/mouse of the antigen to complete three immunizations, and the titer was detected by blood sampling seven days later to obtain immunized mice.
And 2, fusing the cells. Opening the abdomen of the immune mouse obtained in the step 1, taking the spleen, washing, removing the surrounding connective tissues, preparing a cell suspension, and culturing by using an incomplete culture medium to prepare a single-cell suspension; the single cell suspension was used to fuse with the prepared myeloma cell SP2/0 under the mediation of 50% PEG to obtain fused cells.
And 3, screening and cloning culture of the hybridoma cells. And (3) placing the fused cells obtained in the step (2) in an incubator at 37 ℃ for 7-9 days, then generating larger cell clones, screening positive holes for cloning culture, obtaining hybridoma cells, and freezing and storing.
And 4, producing the monoclonal antibody. And (2) inoculating adult Balb/c mice with pristine inoculum of pristane or liquid paraffin, wherein each mouse is 0.3-0.5 mL, inoculating hybridoma cells 0.5 mL after 7-10 days, collecting mice with obviously expanded abdomens at five days intervals, collecting ascites, centrifuging, collecting supernatant, subpackaging and freezing to obtain the monoclonal antibody I or II prepared from the cTnI protein antigenic determinant polypeptide I or II.
And 5, measuring the titer of the antibody. The titer of the monoclonal antibody I or II prepared by using the cTnI protein antigenic determinant I or II polypeptide is measured by an ELISA method and reaches more than 1: 32000.
3. Polyclonal antibodies were prepared using immunized animals.
Step 1, animal immunization. Taking the cTnI protein antigen I or II prepared in the step 1 as an immunogen, taking a New Zealand white rabbit as an immune animal, and using a Freund's complete adjuvant and a Freund's incomplete adjuvant to increase the immunogenicity of the cTnI protein antigen. Fully and uniformly mixing the same amount of antigen and Freund's complete adjuvant, and injecting the mixture into immunized rabbits at multiple subcutaneous points with the dosage of 100 mu g/rabbit to complete primary immunization; after four weeks, the same amount of antigen and Freund's incomplete adjuvant are fully mixed, and then the mixture is injected into the immunized rabbit subcutaneously at multiple points with the dosage of 100 mu g/rabbit to complete the secondary immunization; after four weeks, the antigen is used for carrying out subcutaneous multi-point injection on the rabbits at a dose of 100 mu g/rabbit to complete three times of immunization; after four times of immunization, blood sampling is carried out at intervals of ten days to detect titer, an immune rabbit is obtained, and after carotid bleeding and serum separation, the polyclonal antibody I or II prepared from the cTnI protein antigenic determinant polypeptide I or II is obtained.
And 2, measuring the titer of the antibody. The titer of the polyclonal antibody I or II prepared by the cTnI protein antigenic determinant I or II polypeptide is over 1:16000 measured by an ELISA method.
Example 3. a cTnI protein detection kit was prepared using the monoclonal and polyclonal antibodies obtained in example 2.
In this example, monoclonal antibody i prepared using cTnI protein epitope i polypeptide in example 2 was used as the coating antibody in the cTnI protein detection kit, and polyclonal antibody ii prepared using cTnI protein epitope ii polypeptide was used as the binding antibody in the cTnI protein detection kit.
1. Reagents and buffers.
Coating buffer solution: carbonate buffer 0.05M, pH 9.6;
sample/wash buffer: 10 XPBS-Tween 20, pH7.2;
enzyme marker dilutions: 10 XPBS-Tween 20 (10 mL), FCS (calf serum) (20 mL), distilled water diluted to 1000 mL, enzyme stabilizer (1 g), biological preservative (1 mL);
color-developing agent A: citric acid (35.5 g), carbamide peroxide (10 g), diluted to 1000 mL with distilled water, Tween20 (10 mL);
and a color developing agent B: citric acid (120 g), EDTA-2Na (1 g), TMB-2HCl (2 g), distilled water diluted to 1000 mL;
stopping liquid: 2M H2SO4。
2. And preparing a pre-coated plate.
Dissolving the cTnI protein monoclonal antibody I in 0.05M carbonate buffer solution with the pH value of 9.6 to prepare pre-coating solution, adding 100 mu L of the pre-coating solution into each hole of an enzyme label plate according to 0.1 mu g/hole, standing at the temperature of 4 ℃ for 18-24 hours, throwing off the coating solution, washing, sealing by BSA for 16 hours, drying overnight, filling in an aluminum platinum bag, vacuumizing, sealing, and storing at the temperature of 4 ℃.
3. And (3) composition of the kit.
Pre-coating a plate: 48/96 holes;
cTnI calibrant: 6, 6 multiplied by 1.0 mL, the concentration is respectively 0 ng/mL, 2 ng/mL, 5 ng/mL, 10 ng/mL, 25 ng/mL and 50 ng/mL;
cTnI binding antibody: 1X 10 mL;
enzyme conjugate: 1X 10 mL;
concentrated wash (25 × PBS-Tween 20): 1X 20 mL;
color-developing agent A: 1X 6.0 mL;
and a color developing agent B: 1X 6.0 mL;
stopping liquid: 1X 6.0 mL.
4. And (3) using the kit.
Adding 100 mu L/hole of a blood sample to be detected and a standard substance into each hole of the pre-coated plate, incubating for 60 minutes at 37 ℃, washing for 5 times by using 1 multiplied by washing buffer solution, and patting dry; add cTnI binding antibody 100 u L/hole in each hole, 37 degrees C were incubated for 30 minutes, 1 x washing buffer washing 5 times, pat dry; then, 100. mu.L/well of the enzyme conjugate was added to each well, incubated at 37 ℃ for 30 minutes, washed 5 times with 1 XWash buffer, and patted dry. Adding 50 μ L of color development agent A, B solution into each well, mixing, and incubating at 37 deg.C for 15 min; adding 50 μ L of stop solution/well to stop reaction, and detecting absorbance with enzyme-linked detector at double wavelength (450 nm, 620 nm).
5. And (5) judging the detection result of the kit.
And drawing a standard curve according to the concentration and the absorbance of the cTnI calibrator in the kit, and calculating the cTnI concentration of the sample to be detected through the standard curve after the sample is detected. The following table shows the concentrations of the cTnI calibrator and the corresponding average absorbance values (OD), which are the average absorbance values of several batches, used only as a reference, and should be based on the actual absorbance values of the cTnI calibrator in actual use.
TABLE 2 soluble cTnI calibrator concentration and corresponding average absorbance (OD)
As shown in FIG. 1, the goodness of fit R of the standard curve is plotted as a function of the concentration of the cTnI calibrator in the table above and the corresponding average absorbance2=0.9938。
According to the kit using steps and the kit test results, the serum cTnI protein detection is carried out on 110 patients with acute heart failure and 138 healthy individuals, and the detection results show that the content of the serum cTnI protein of the patients with heart failure is obviously higher than that of the healthy group (2.49 V.S. 0.23) and the difference has statistical significance (P = 0.0166), as shown in Table 2.
TABLE 3 comparison of serum cTnI levels in acute Heart failure patient groups and healthy groups
From the above results, it was found that the cTnI protein detection kit of the present invention can specifically detect the cTnI content in the serum.
It is noted that, herein, relational terms such as first and second, and the like may be used solely to distinguish one entity or action from another entity or action without necessarily requiring or implying any actual such relationship or order between such entities or actions. Also, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus.
The above description is only a preferred example of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Sequence listing
<110> Tianjin Qiyunnaods biomedicine Co., Ltd
<120> cTnI protein antigenic determinant polypeptide and application thereof
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 1
Leu Cys Arg Gln Leu His Ala Tyr
1 5
<210> 2
<211> 11
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 2
Asp Lys Val Asp Glu Glu Arg Tyr Asp Ile Tyr
1 5 10
Claims (5)
1. A cTnI protein antigenic determinant polypeptide, the amino acid sequence of which is shown as SEQ ID NO.1, specifically: Leu-Cys-Arg-Gln-Leu-His-Ala-Tyr.
2. A cTnI protein antigen prepared by coupling a carrier protein with the cTnI protein determinant polypeptide of claim 1.
3. A cTnI protein antibody which is a polyclonal antibody prepared from the cTnI protein antigen of claim 2.
4. Use of the cTnI protein antibody of claim 3 in the preparation of a cTnI protein detection kit.
5. A cTnI protein detection kit comprising the antibody of claim 3 as a coating antibody.
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1982337A (en) * | 2005-12-14 | 2007-06-20 | 中国医学科学院放射医学研究所 | Synthesis of polypeptide antibody from anti-human myocardial troponin I, its production and use |
| US20110129854A1 (en) * | 2009-12-02 | 2011-06-02 | Abbott Laboratories | Assay for diagnosis of cardiac myocyte damage |
| CN105137071A (en) * | 2015-07-29 | 2015-12-09 | 安徽省煦棠医疗科技有限公司 | Cardiac troponin I homogenous enzyme immunoassay kit |
| CN111925432A (en) * | 2020-08-18 | 2020-11-13 | 杭州启泰生物技术有限公司 | Preparation method of cardiac troponin I recombinant antigen and monoclonal antibody thereof |
| CN113248590A (en) * | 2021-06-24 | 2021-08-13 | 天津奇云诺德生物医学有限公司 | NT-proBNP protein antigenic determinant polypeptide and application thereof |
-
2022
- 2022-01-19 CN CN202210058185.6A patent/CN114316042A/en active Pending
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1982337A (en) * | 2005-12-14 | 2007-06-20 | 中国医学科学院放射医学研究所 | Synthesis of polypeptide antibody from anti-human myocardial troponin I, its production and use |
| US20110129854A1 (en) * | 2009-12-02 | 2011-06-02 | Abbott Laboratories | Assay for diagnosis of cardiac myocyte damage |
| CN102725633A (en) * | 2009-12-03 | 2012-10-10 | 雅培制药有限公司 | Assay for diagnosis of cardiac myocyte damage |
| CN105137071A (en) * | 2015-07-29 | 2015-12-09 | 安徽省煦棠医疗科技有限公司 | Cardiac troponin I homogenous enzyme immunoassay kit |
| CN111925432A (en) * | 2020-08-18 | 2020-11-13 | 杭州启泰生物技术有限公司 | Preparation method of cardiac troponin I recombinant antigen and monoclonal antibody thereof |
| CN113248590A (en) * | 2021-06-24 | 2021-08-13 | 天津奇云诺德生物医学有限公司 | NT-proBNP protein antigenic determinant polypeptide and application thereof |
Non-Patent Citations (2)
| Title |
|---|
| XINGQING XIAO: "Advancing Peptide-Based Biorecognition Elements for Biosensors Using in-Silico Evolution", 《ACS SENS》, vol. 3, pages 1024 * |
| 王德芝: "心肌肌钙蛋白I（cTnI）合成肽制备、鉴定及抗体研究", 《免疫学杂志》, vol. 29, no. 9, pages 1 * |
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