CN107446040B - Human ST2 epitope peptide, antigen, antibody, kit and application - Google Patents
Human ST2 epitope peptide, antigen, antibody, kit and application Download PDFInfo
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Abstract
The invention relates to human ST2 epitope peptide, antigen, antibody, kit and application. The amino acid sequence of the human ST2 epitope peptide is one of the sequences shown in SEQ ID NO.1 and SEQ ID NO.2 of a sequence table. The ST2 antigen is prepared by coupling human ST2 epitope peptide and a protein carrier. The ST2 monoclonal antibody or polyclonal antibody of the invention is prepared from the ST2 antigen of the invention. The ST2 monoclonal antibody or polyclonal antibody is used for preparing an ST2 in-vitro diagnostic kit. The human ST2 epitope peptide has good antigenicity, and an antigen (immunogen) immune animal prepared by the human ST2 epitope peptide can generate a monoclonal antibody and a polyclonal antibody with high specificity, so that the human ST2 epitope peptide can be applied to in vitro detection of human ST 2.
Description
Technical Field
The invention belongs to the field of polypeptide chemistry and immunology, and particularly relates to an epitope peptide of human soluble growth stimulation expression gene 2 protein (ST2), an ST2 specific antigen prepared from the epitope peptide, a corresponding monoclonal antibody or polyclonal antibody, application of the antibody in preparation of a human ST2 in-vitro diagnostic kit, a human ST2 in-vitro diagnostic kit, and application of the kit in quantitative detection of human ST2 protein in serum.
Background
In 1989, soluble growth-stimulating expression gene 2 protein (ST2) was discovered as one of the members of the interleukin I receptor family, and was later shown to be involved in a variety of pathophysiological processes, playing an important role in inflammatory and allergic diseases. However, since no functional ligand was found, ST2 has long been considered to be an orphan receptor, and the study of ST2 has been advanced. ST2 is a cardiac muscle protein, and there are two ST2 proteins that directly affect the progression of heart disease, soluble ST2 and the transmembrane form of STL 2. These two ST2 proteins are directly associated with the progression of heart disease: when the concentration of soluble ST2 is low, the ligand interleukin 33(IL-33) of ST2 can bind to ST2L, which has a protective effect on the heart. When soluble ST2 levels are high, soluble ST2 can competitively bind to IL-33, resulting in reduced binding of IL-33 to ST2L, thereby disabling IL-33 from entering the cardioprotective signal transduction pathway. Thus, the heart continues to be stressed, resulting in cell death and tissue fibrosis, reducing heart function, and increasing the rate of disease progression. After mechanical stress induces cardiomyocytes to produce serum soluble ST2, it blocks the action of interleukin 33(IL-33) against myocardial hypertrophy and against myocardial fibrosis, is a marker of cardiomyocyte hypertrophy and fibrosis, and is not affected by age, renal function impairment and body mass index. Soluble ST2 is a marker of myocardial fibrosis, can predict the prognosis of patients with acute myocardial infarction and heart failure, can also predict the admission and death probability of the patients with heart failure, can also provide prognostic information on the basis of a diuretic index, and is considered to have high application value in heart failure and other cardiovascular diseases.
ST2 has repeatedly been shown to be of potential value as a biomarker, and measuring ST2 levels in the serum of acute dyspnea or myocardial infarction emergency patients may provide some useful prognostic information for stratified care. ST2 has the advantages of early risk monitoring, immediate response, easy interpretation of data, and has been approved by the professional community. The ST2 diagnosis can be used for pre-morbidity screening, pre-treatment guidance and reference, mid-treatment and post-treatment monitoring and observation.
The most ideal method for detecting the ST2 level in serum is immunoassay, and the ST2 level can be detected by conventional blood drawing by using ST2 antibody and advanced enzyme immunization and chemiluminescence technology. Therefore, the focus is to find suitable immunogenic ST2 epitope peptide and prepare specific ST2 antigen and antibody.
Disclosure of Invention
In order to solve the problems in the prior art, the invention provides a human ST2 epitope peptide, an ST2 specific antigen and a corresponding monoclonal antibody or polyclonal antibody prepared by using the epitope peptide, application of the epitope peptide in preparing a human ST2 kit, a human ST2 in-vitro diagnosis kit and application of the kit in quantitatively detecting human ST2 protein in serum.
Specifically, the present invention provides:
a human ST2 epitope peptide, wherein the amino acid sequence of the ST2 epitope peptide is one of the following:
(1)Gly-Lys-Asn-Ala-Asn-Leu-Thr-Gln-Gln-Glu-Glu-Gly-Gln-Asn-Gln-Ser-Tyr；
(2)Tyr-Lys-Asp-Glu-Thr-Arg-Val-Arg-Leu-Ser-Arg-Lys-Asn-Pro-Ser-Lys-Glu。
the invention also provides an ST2 antigen, which is prepared by coupling the human ST2 epitope peptide (1) and a carrier protein.
The invention also provides an ST2 antigen, which is prepared by coupling the human ST2 epitope peptide (2) and a carrier protein.
The invention also provides a human ST2 antibody, which is a monoclonal antibody or a polyclonal antibody prepared from the ST2 antigen, wherein the ST2 antigen is prepared by coupling the human ST2 epitope peptide (1) and a carrier protein.
The invention also provides a human ST2 antibody, which is a monoclonal antibody or a polyclonal antibody prepared from the ST2 antigen, wherein the ST2 antigen is prepared by coupling the human ST2 epitope peptide (2) with a carrier protein.
The invention also provides application of the human ST2 antibody in preparing a human ST2 in-vitro diagnostic kit.
The invention also provides an in vitro diagnostic kit of human ST2, which comprises the human ST2 antibody as a coating antibody.
Preferably, the kit further comprises a binding antibody which is the human ST2 antibody of claim 4 or 5, and when the binding antibody is derived from one of the human ST2 epitope peptides (1) and (2), the coating antibody is derived from the other of the human ST2 epitope peptides (1) and (2).
Preferably, the coating antibody is a monoclonal antibody.
Preferably, the binding antibody is a polyclonal antibody.
Preferably, the kit is used for quantitatively detecting human ST2 protein in serum.
The invention also provides application of the human ST2 in-vitro diagnostic kit in quantitative detection of human ST2 protein in serum.
Compared with the prior art, the invention has the following advantages and positive effects:
1. the human ST2 epitope peptide has good antigenicity, and the antigen (immunogen) prepared by the human ST2 epitope peptide can be used for immunizing animals to produce monoclonal antibodies and polyclonal antibodies with high specificity.
2. The ST2 monoclonal antibody and polyclonal antibody prepared by the invention can be combined with ST2 in blood (especially serum) sample with high specificity.
3. The human ST2 in-vitro diagnostic kit can effectively detect the ST2 level in a blood sample (particularly a serum sample), and can be used for prognosis and risk assessment of patients with acute myocardial infarction and heart failure.
Detailed Description
The present invention is further described in the following description of the specific embodiments, which is not intended to limit the invention, but various modifications and improvements can be made by those skilled in the art according to the basic idea of the invention, within the scope of the invention, as long as they do not depart from the basic idea of the invention.
Human ST2 antigen epitope peptide
The human ST2 protein described herein is known in the art, and its amino acid sequence is known in the art and can be found in professional databases such as NCBI.
The invention provides human ST2 epitope peptides (1) and (2), the amino acid sequences of which are respectively shown as SEQ ID No.1 and SEQ ID No.2 in sequence tables, and the peptides are as follows:
(1) G-K-N-A-N-L-T-Q-Q-E-E-G-Q-N-Q-S-Y; and
(2)Y-K-D-E-T-R-V-R-L-S-R-K-N-P-S-K-E。
through a large amount of theoretical researches and experimental groping, the inventor finally screens and obtains two antigen epitope peptides with good antigenicity.
The ST2 epitope peptide (1) is obtained by combining the 1 ST to 7 th positions of the N-terminus and the 40 th to 48 th positions of the peptide fragment of the human ST2 protein (NCBI accession number BAA20539.1), and adding amino acid Y to the C-terminus of the peptide fragment, thereby constituting the ST2 epitope peptide (1) having 17 amino acids.
The ST2 epitope peptide (2) comprises a peptide segment from 89 th to 99 th positions of C terminal of human ST2 protein (NCBI accession number BAA20539.1), and amino acid Y, K, D, E, T, R is added at the N terminal of the peptide segment, thereby constituting the ST2 epitope peptide (2) with 17 amino acids.
The conception and the design of the invention ensure that the two peptide fragments have the characteristics of good hydrophilicity, strong antigenicity and easy synthesis.
Currently, the research of the invention finds that the ST2 epitope peptide has the following functions:
1. has antigenicity; 2. after being connected with carrier protein, the carrier protein is used as immunogen to stimulate animals to produce specific antibody; 3. antibodies prepared with epitope peptides can specifically bind to human ST 2.
The preparation method of the ST2 epitope peptide can be realized by a chemical synthesis method: the epitope peptide was synthesized by a solid phase method using an ABI model 431A polypeptide automatic synthesizer in the United states. The epitope peptides (1) and (2) of the present invention have molecular weights of 2197.21 and 2394.59, respectively, and can be determined by mass spectrometry, and the sequences of the synthesized epitope peptides can be identified by polypeptide sequence determination. The purity of the peptide fragment can be evaluated by thin layer chromatography and high performance liquid chromatography, and the concentration of the epitope peptide is determined.
II, ST2 antigen
The present invention also provides ST2 antigen prepared by coupling one of the human ST2 epitope peptides (1) and (2) of the present invention with a carrier protein. Specifically, the present invention provides ST2 antigens (1) and (2), the ST2 antigen (1) being prepared by coupling the human ST2 epitope peptide (1) of the present invention with a carrier protein; the ST2 antigen (2) is prepared by coupling the human ST2 epitope peptide (2) of the invention with a carrier protein. The ST2 antigen has immunogenicity and specificity, is an immunogen, and can be used for immunizing animals so as to prepare specific ST2 antibodies. In the present invention, examples of carrier proteins that can be used include KLH (keyhole limpet hemocyanin), Bovine Serum Albumin (BSA), ovalbumin OVA, and the like. KLH (keyhole limpet hemocyanin) is preferred because it has high immunogenicity, many binding sites, good immunological effects, is distant from the immunized animal, and is less likely to cause cross-reactions when used as a carrier protein.
III, ST2 monoclonal antibody, ST2 polyclonal antibody and human ST2 in-vitro diagnostic kit
The present invention also provides human ST2 antibodies, including human ST2 monoclonal antibody and human ST2 polyclonal antibody, each of which can be prepared by immunizing an animal with ST2 antigen (1) or (2) (immunogen) of the present invention. The preparation method can adopt the conventional technology in the field, and can be specifically seen in example 2.
The ST2 monoclonal antibody and the polyclonal antibody can be used for preparing a human ST2 in-vitro diagnosis kit, and the kit can be used for detecting ST2 in human tissues, cells and body fluid based on an immunization method, and preferably can be used for detecting ST2 in blood samples, particularly serum.
Accordingly, the present invention provides an in vitro diagnostic kit for human ST2 comprising the human ST2 monoclonal or polyclonal antibody of the invention.
The currently known immunoassay methods for clinical examination mainly include the following methods: ELISA, chemiluminescence, fluorescence chromatography, colloidal gold immunoassay, etc.
While ELISA methods include several types: antigen detection by a double-antibody sandwich method, antibody detection by a double-antigen sandwich method, antibody detection by an indirect method, antibody detection by a competitive method, antigen detection by a competitive method, antibody detection by a capture coating method and the like.
The human ST2 in vitro diagnostic kit of the invention preferably adopts an ELISA double-antibody sandwich method to detect the ST2 protein. The kit may contain coated antibodies, conjugated antibodies, enzyme-labeled anti-antibodies and/or necessary tools and reagents, etc.
Preferably, the in vitro diagnostic kit for human ST2 uses the human ST2 monoclonal antibody of the present invention as a coating antibody. Herein, the term "coated antibody" refers to an antibody coated on an microplate of a solid phase. Furthermore, the human ST2 in vitro diagnostic kit preferably further comprises a human ST2 polyclonal antibody as a binding antibody, wherein, when the binding antibody is derived from one of the human ST2 epitope peptides (1) and (2) of the present invention, the coating antibody is derived from the other of the epitope peptides (1) and (2). Herein, the term "binding antibody" refers to a specific antibody in a kit that can bind to an antigen to be detected and an enzyme-labeled anti-antibody. The kit can also comprise an enzyme-labeled anti-antibody, wherein the anti-antibody can be a goat anti-rabbit IgG antibody, and the enzyme label can be horseradish peroxidase, alkaline phosphatase and the like.
The human ST2 in vitro diagnostic kit of the invention preferably detects ST2 in serum.
Any reagents or means required for detection, such as pre-coated plates, washing solutions, color developers, stop solutions, and the like, may also be included in the kit of the present invention.
The invention also provides application of the human ST2 in-vitro diagnostic kit in quantitative detection of human ST2 protein in serum.
The present invention will be further explained or illustrated below by way of examples, which should not be construed as limiting the scope of the invention.
Examples
Unless otherwise stated, the solutions described below are aqueous solutions and the percentages in the solutions are volume percentages.
Example 1: preparation of ST2 epitope peptides (1) and (2).
The preparation method comprises the following steps: ST2 epitope peptides (1) and (2) were synthesized by a solid phase method using an ABI431A type polypeptide automatic synthesizer, usa, respectively. The purity of the epitope peptide was evaluated by high performance liquid chromatography, and the concentration of the peptide fragment was measured. The epitope peptides (1) and (2) of the present invention have molecular weights of 2197.21 and 2394.59, respectively, and are determined by mass spectrometry, and the synthesized polypeptide sequences are identified by polypeptide sequence determination.
Synthesis of ST2 epitope peptides (1) and (2)
The peptide fragment is synthesized by a solid phase method. The main idea of solid phase peptide synthesis is: the carboxyl group of the carboxyl terminal amino acid of the peptide chain to be synthesized is firstly connected with an insoluble macromolecular compound (resin) in a covalent bond mode, then the amino acid combined on the solid phase carrier is taken as an amino component, and the peptide chain is lengthened by removing the amino protecting group and reacting with an excessive activated carboxyl component. Such steps may be repeated several times until the desired length of the peptide chain is reached. This synthesis is shown below.
The specific preparation steps of the ST2 epitope peptides (1) and (2) are as follows:
1. the used raw materials are as follows:
HMP resin (P-methylolphenoxymethyl polyethylene resin available from sigma company)
Fmoc-AA (9-fluorenylmethoxycarbonyl protected amino acids, commercially available from Merck)
NMP (Nitrogen methyl pyrrolidone, available from sigma company)
DCM (Dichloromethane, available from Crystal park chemical Co., Ltd.)
MeOH (methanol, available from Crystal park chemical Co., Ltd.)
Piperidine (Piperidine, available from sigma)
DMAP (dimethylaminopyridine, available from sigma)
HOBT (hydroxybenzotriazole, available from sigma)
DCC (dicyclohexylcarbodiimide, available from sigma)
TFA (trifluoroacetic acid, available from sigma company)
EDT (1, 2-ethanedithiol, commercially available from sigma)
Thioanisole available from Guangzhou Weber chemical Co Ltd
Crystalline phenol available from chemical reagents of national pharmaceutical group, Inc
Acetonitrile, commercially available from chemical reagents of the national pharmaceutical group, Inc
2. Using an instrument:
automatic polypeptide synthesizer, model 431A, available from ABI
Rotary evaporator, model R-201, available from Shanghai Shenshu
High performance liquid chromatography, Waters 600, available from Waters corporation, USA
Freeze dryer, model VFD-2000, available from Beijing Bo Yi kang Kong company
3. The synthesis method and the process are as follows:
100mg of HMP resin is weighed, the substitution equivalent is 1.0meq, namely 0.1mmol is placed in a reaction cavity of an ABI431A type polypeptide automatic synthesizer of the United states, the specific amino acids are automatically connected by the synthesizer according to different sequences, and the coupling rate reaches 99%. The reaction is as follows:
(1) activation of amino acid (HOBt/DCC method)
(2) Attaching amino acids to resins
(3) Fmoc protecting group for removing amino acid
(4) Activation of another amino acid (HOBt/DCC method)
(5) Coupling of
(6) And (5) repeating the steps (3) to (5) until the synthesis is finished.
Thus, 209mg of peptide resin of ST2 peptide fragment (1) and 196mg of peptide resin of ST2 peptide fragment (2) were obtained, respectively.
(7) Cleavage of peptide resin:
the peptide chain was cleaved with TFA (trifluoroacetic acid), reacted at room temperature for 3.0 hours using EDT (2.5 vol%), thioanisole (2.5 vol%) as scavenger, the cleavage reagent was removed, and extracted with ether to give crude ST2 peptides (1) and (2), respectively.
Secondly, purification of crude ST2 epitope peptides (1) and (2):
separating and purifying by adopting high performance liquid chromatography:
conditions are as follows: a chromatographic column: C810X 100mm, available from Waters corporation, USA
Chromatograph: waters 600, Waters corporation, USA
Mobile phase: a: 0.1% aqueous TFA (trifluoroacetic acid)
B: 0.1% TFA (trifluoroacetic acid) in 60% acetonitrile
Detection wavelength: 214nm
Flow rate: 4 ml/min
Elution gradient: 20-60% B, 30 min
HPLC (high Performance liquid chromatography) analysis
A chromatographic column: C184.6X 150mm, available from Waters corporation, USA
Mobile phase: a: 0.1% aqueous TFA (trifluoroacetic acid)
B: 0.1% TFA (trifluoroacetic acid) in acetonitrile
Detection wavelength: 214nm
Flow rate: 1 ml/min
Elution gradient: 0-60% B, 30 min
The peptide fragment analysis results show that the purity of the ST2 epitope peptides (1) and (2) is more than 95%.
Identification of ST2 epitope peptides (1) and (2)
1. The molecular weights of the purified ST2 epitope peptides (1) and (2) were determined by mass spectrometry, respectively.
(1) Raw materials of reagents
TFA (trifluoroacetic acid, available from sigma company)
HCCA (alpha-cyano-4-hydroxycinnamic acid, available from sigma company)
Acetonitrile (commercially available from national medicine group chemical reagents Co., Ltd.)
(2) Instrument for measuring the position of a moving object
Matrix-assisted laser desorption ionization time-of-flight mass spectrometer MALDI-TOF-MS (model: REFLEX III, Bruker, Germany);
(3) matrix liquid: dissolving alpha-CCA in 50% ACN solution containing 0.1% TFA to prepare saturated solution, centrifuging, and taking supernatant;
(4) the detection conditions of the instrument are as follows: a reflection detection mode; the length of the flight tube is 3 m; nitrogen laser: wavelength of 337nm, acceleration voltage of 20 KV; the reflected voltage is 23 KV.
(5) The method comprises the following operation steps: mu.L of each of the above purified polypeptides (1) and (2) was sampled, mixed with 1. mu.L of the supernatant of the saturated matrix in equal volume, and 1. mu.L of each of the samples was spotted on the sample target and sent to an ion source for detection.
As a result, the molecular weight of the ST2 epitope peptide (1) was determined to be 2197.1, and the molecular weight of the ST2 epitope peptide (2) was determined to be 2394.5, which was consistent with the theoretical molecular weights of 2197.21 and 2394.59, demonstrating that the synthesized polypeptide was the target product.
2. The sequences of the resulting ST2 epitope peptides (1) and (2) were identified by polypeptide sequence determination, respectively.
(1) The principle is as follows: the basic principle of polypeptide amino acid sequence analysis is Edman degradation, which is a cyclic chemical reaction process. It comprises three main chemical steps: (1) coupling: phenyl isothiocyanate reacts with the N-terminal residue of proteins and polypeptides to form Phenylaminothiocarbonyl (PTC) derivatives, PTC-peptides. (2) Cyclizing and cracking: PTC-peptide cyclization cleavage. (3) And (3) transformation: thiazolone Anilines (ATZ) are converted to benzethionamide (PTH-amino acid). The reaction is repeated with the remaining peptide in solution reduced by one amino acid residue, and the entire sequencing process is now automated by the sequencer.
(2) The instrument comprises the following steps: 491 model protein/polypeptide N-terminal amino acid sequence analyzer manufactured by ABI corporation of America
(3) Raw materials of reagents
Phenyl isothiocyanate PITC, available from sigma
N-heptane, available from Chemicals, Inc., of the national pharmaceutical group
Aqueous trimethylamine TMA solution available from Chemicals, Inc., national pharmaceutical group
TFA (trifluoroacetic acid, available from sigma company)
Ethyl acetate available from Chemicals of national pharmaceutical groups Ltd
Chlorobutane, commercially available from sigma
Acetonitrile, commercially available from chemical reagents of the national pharmaceutical group, Inc
(4) Measurement of
According to the instrument instruction.
As a result: the sequences of the obtained ST2 epitope peptides (1) and (2) are respectively identified as follows:
(1) G-K-N-A-N-L-T-Q-Q-E-E-G-Q-N-Q-S-Y and
(2)Y-K-D-E-T-R-V-R-L-S-R-K-N-P-S-K-E。
the results are consistent with the target synthetic peptide fragment.
Example 2: the ST2 epitope peptides (1) and (2) obtained in example 1 were linked to a carrier protein to prepare ST2 antigens (1) and (2), respectively, and animals were immunized with the obtained antigens (1) and (2), respectively, to prepare specific monoclonal and polyclonal antibodies using the antigen (1), and specific monoclonal and polyclonal antibodies using the antigen (2).
1. Preparation of antigen: ST2 antigens (1) and (2) were prepared by linking the ST2 peptides (1) and (2), respectively, to the carrier protein KLH (keyhole limpet hemocyanin) (from sigma) by the BDB (Bis-diazotizedbenzidine dichloride) method.
10.0mg of ST2 peptide fragment (1) or (2) was buffered with 1ml of 0.1M PBSDissolving the solution (pH 7.4); KLH 10mg, dissolved in 20ml of 0.2M borate buffer (pH 8.6); mixing the two solutions, cooling to 0 deg.C, and collecting BDBCl2110 μ L, reacting at room temperature for 1.5h, dialyzing overnight, packaging, and storing at-20 deg.C.
In each example, the formulation of PBS buffer (if used) was: 0.2mol/L of Na2HPO481ml of NaH added with 0.2mol/L2PO419ml of the mixture is mixed.
The formula of the borate buffer solution is as follows: 80ml of 0.05mol/L borax and 20ml of 0.2mol/L boric acid are mixed to obtain the borax.
2. Preparing monoclonal antibody by immunizing animal:
2.1. balb/c mice were immunized by mixing the ST2 antigens (1) and (2) (immunogen) prepared above with an equal volume of Freund's complete adjuvant (purchased from sigma Biotech) and injecting 50. mu.g of antigen per mouse at multiple subcutaneous points. After 4 weeks, serum titers were measured, and mice with good immunoreactivity were selected for boosting: after mixing antigen with an equal volume of incomplete Freund's adjuvant (purchased from Sigma Bio Inc.), the antigen dose was 25 μ g/mouse, and injected subcutaneously at multiple points, the number of booster immunizations was 6 times, each time at intervals of 2-3 weeks, two additional booster immunizations were continued before fusion, each time at intervals of 1-2 weeks, after which splenocytes were fused with Sp2/0 myeloma cells using 50% PEG (MW4000) (purchased from Garden chemical Co., Ltd.) mediated by a conventional method, and selectively cultured in HAT conditioned medium (purchased from Sigma Co., Ltd.). After fusing, adding CO2After culturing for 9-11 days at 37 ℃ in the incubator, larger cell clones appear in the holes. Screening with indirect ELISA was started 11 days. The primary-screened positive wells were subjected to 4 times of cloning culture (even if the selected cells were proliferated by dividing a large amount) by the limiting dilution method, and then the cells were expanded, frozen and prepared into ascites.
2.2. Balb/c mice were treated with 0.5 ml/mouse of pristane (from sigma), and one week later were inoculated with 2X 10 hybridoma cells6Ascites were collected 10 days later.
2.3. And (3) measuring the antibody titer: the titer of the monoclonal antibody (1) prepared from the ST2 antigen (1) was measured by an indirect ELISA method, and the result showed that the titer of the monoclonal antibody reached 1:32000 or more.
The titer of the monoclonal antibody (2) produced using the ST2 antigen (2) was measured by the same method, and the titer reached 1:32000 or more.
3. Preparing polyclonal antibody by immunizing animals:
3.1. new Zealand white rabbits with the age of three months and the body weight of about 2kg are selected as immune animals. In the primary immunization, 1-2mg of the ST2 antigens (1) and (2) (immunogen) prepared above were mixed with an equal volume of complete Freund's adjuvant (purchased from Sigma Bio Inc.) respectively-thoroughly emulsified and injected subcutaneously at multiple sites on the back of rabbits. The booster was administered at intervals of 4 weeks, 6 boosts, and the antigen was thoroughly emulsified with incomplete Freund's adjuvant (purchased from Sigma Bio Inc.) and injected subcutaneously at 100. mu.g/dorsal multiple sites. Carotid bleeding was performed 10 days after the last booster immunization and serum was isolated.
3.2. And (3) measuring the antibody titer: the titer of the polyclonal antibody (1) prepared from the ST2 antigen (1) was measured by an indirect ELISA method, and the result showed that the antibody titer reached 1:32000 or more.
The titer of the polyclonal antibody (2) produced using the ST2 antigen (2) was measured by the same method, and the titer reached 1:32000 or more.
3.3. Blood sampling and serum separation: blood was taken from the carotid artery by intubation and serum was isolated.
4. Separating and purifying the antibody: after ammonium sulfate precipitation, affinity purification was performed using Protein G (available from Sigma).
5. And (5) subpackaging the antibody, freeze-drying and storing at low temperature.
Example 3: specific identification of human ST2 monoclonal antibodies (1) and (2)
Detection was performed by ELISA. Human ST2 protein, GDF-15 protein (growth differentiation factor-15) (purchased from Shanghai Shuzo Co., Ltd.) and NT-proBNP protein (N-terminal pro-B-type natriuretic peptide) (purchased from Shanghai peptide Co., Ltd.) were used as detection antigen-coated ELISA plates, the specific reactions of the prepared ST2 monoclonal antibodies (1) and (2) and the human ST2 protein were respectively detected by ELISA, and normal BALB/c mouse serum was used as a negative control and PBS liquid was used as a blank control.
As a result: the ST2 monoclonal antibodies (1) and (2) reacted positively only with ST2 protein (P/N >2.1) and negatively with GDF-15 protein and NT-proBNP protein, respectively, indicating that the ST2 monoclonal antibodies (1) and (2) of the present invention have specificity, respectively.
Example 4: specific identification of human ST2 polyclonal antibodies (1) and (2)
The identification was carried out by the same method as that for the above-described identification of the specificity of the monoclonal antibody.
The results show that: the ST2 polyclonal antibodies (1) and (2) reacted positively with ST2 (P/N >2.1) and negatively with GDF-15 protein and NT-proBNP protein, respectively, indicating that the ST2 polyclonal antibodies (1) and (2) of the present invention have specificity, respectively.
Example 5: the ST2 in vitro diagnostic kit was prepared using the ST2 monoclonal antibody and the ST2 polyclonal antibody.
In this example, the monoclonal antibody (1) prepared using the ST2 epitope peptide (1) in example 2 was used as the coating antibody in the present kit; the polyclonal antibody (2) prepared using the ST2 epitope peptide (2) in example 2 was used as a binding antibody.
The preparation and operation of the ST2 in vitro diagnostic kit are as follows:
1. preparation of various buffers and reagents:
A. coating buffer solution: 0.050M, CB (carbonate buffer) at pH9.6
Na2CO3: 16.0 g
NaHCO3: 29.0 g
Dissolving in distilled water to 1000ml
B. Sample/wash buffer: 10 XPBS-Tween 20, pH7.2
Na2HPO4·12H2O: 58 g
KH2PO4: 4 g
NaCl: 100 g
KCl: 4 g
Dissolving in distilled water to 1000ml
Adding Tween 20: 20ml of
C. Enzyme marker diluent
10×PBS-Tween 20：10ml
FCS (calf serum): 20ml of
Dissolving in distilled water to 1000ml
Enzyme stabilizers (available from shanghai xibao corporation): 1 g
Biological preservatives (available from sigma corporation): 1ml of
D. Color-developing agent A:
citric acid: 35.5 g
Carbamide peroxide: 10 g
Dissolving in distilled water to 1000ml
Tween 20：10ml
E. And a color developing agent B:
citric acid: 120 g
EDTA-2 Na: 1 g
TMB.2HCl: 2 g
Dissolving in distilled water to 1000ml
F. Stopping liquid: 2M H2SO4
Concentrated sulfuric acid (95-98%): 22.2ml
Distilled water: 177.3ml
When in use, concentrated sulfuric acid is slowly dropped into distilled water and shaken up while adding.
2. Preparing a pre-coated plate:
dissolving the ST2 monoclonal antibody (1) in 0.05M carbonate buffer solution with pH of 9.6 to prepare pre-coating solution, adding 100 μ l per well of 0.1 μ g per well on an enzyme label plate (available from Shenzhen Jinlau company), standing at 4 ℃ for 18-24 hours, taking out, throwing off the coating solution, washing with sample/washing buffer solution, sealing with 1 (w/v)% BSA-0.05M ethanolamine for 16 hours, drying overnight, filling into an aluminum platinum bag, vacuumizing, sealing, and storing at 4 ℃.
3. The dilution ratio of the bound antibody (ST2 polyclonal antibody (2)) and the enzyme conjugate (horseradish peroxidase-labeled goat anti-rabbit IgG antibody) (purchased from Kyoto Sequoia jejuni Co., Ltd.) was determined by a matrix titration experiment, and the horseradish peroxidase-labeled goat anti-rabbit IgG antibody was diluted with an enzyme-labeled diluent.
4. The kit comprises the following components:
pre-coating a plate: 48/96 hole
ST2 calibrator (raw materials from Abcan): 6, the number of the cells is as follows: 6X 0.5ml (concentration of 0.0ng/ml, 8.0ng/ml, 15.0ng/ml, 50.0ng/ml, 100.0ng/ml, 200.0ng/ml respectively)
ST2 binding antibody: 1X 2.5 ml/1X 5ml (diluted 1: 5000)
Enzyme conjugate: 2X 5 ml/2X 5ml (diluted 1: 5000)
ST2 quality control: 1 × 0.5mL (ST2 serum with concentration of 60.0ng/mL, obtained from the sixth people Hospital in Shenzhen City)
Concentrated wash (25 × PBS-Tween 20): 1X 20ml
Color-developing agent A: 1X 5.0ml
And a color developing agent B: 1X 5.0ml
Stopping liquid: 1X 5.0ml
5. The kit comprises the following steps:
adding 50 mul/well of serum to be detected, calibrator and quality control material into each well of the pre-coated plate, incubating for 30 min at 37 ℃, washing for 5 times by using 1 Xwashing buffer solution 200-. 100. mu.l/well of ST 2-conjugated antibody was added to each well, incubated at 37 ℃ for 30 minutes, washed 5 times with 1 × 200. sup. th and 300. mu.l/well of washing buffer, and patted 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 Xwashing buffer 200 and 300. mu.l/well, and patted dry. Add 50. mu.l of color reagent A, B per well, mix well, incubate for 15 min at 37 ℃. The reaction was stopped by adding 50. mu.l of stop solution to each well, and absorbance was measured by using a dual wavelength (450nm, 620nm) using an enzyme-linked detector (model RT-6000, available from Redu Co.).
6. And (4) judging a result:
table 1: calibrator concentration and corresponding average absorbance (OD) value
| Concentration ng/ml | 0 | 8 | 15 | 50 | 100 | 200 |
| Average OD value | 0.014 | 0.124 | 0.201 | 0.453 | 0.769 | 1.106 |
Drawing a standard curve by using the concentration of the calibrator and the logarithmic value of the corresponding absorbance, and obtaining the R of the standard curve2＝0.977。
The results of ST2 concentration in the samples tested were calculated from the standard curve.
The serum ST2 test was performed on 63 patients with heart failure and 121 healthy patients in the above manner, and the content of ST2 in the serum of the patients with heart failure was significantly higher than that of the healthy control group, and the difference was statistically significant (P <0.01), as shown in Table 2. (ST2 value reference interval: 0.0-35.0 ng/ml)
Table 2: comparison of concentrations of ST2 in two sets of samples
From the above data, the kit of the present invention can effectively and specifically detect the content of ST2 in serum, thereby detecting the difference in ST2 content between patients with heart failure and normal persons, and thus can play a role in auxiliary diagnosis of heart failure and other cardiovascular diseases.
Claims (8)
1. A human ST2 epitope peptide, wherein the amino acid sequence of the ST2 epitope peptide is one of the following:
(1)Gly-Lys-Asn-Ala-Asn-Leu-Thr-Gln-Gln-Glu-Glu-Gly-Gln-Asn-Gln-Ser-Tyr；
(2)Tyr-Lys-Asp-Glu-Thr-Arg-Val-Arg-Leu-Ser-Arg-Lys-Asn-Pro-Ser-Lys-Glu。
2. an ST2 antigen prepared by coupling the human ST2 epitope peptide (1) of claim 1 to a carrier protein.
3. An ST2 antigen prepared by coupling the human ST2 epitope peptide (2) of claim 1 to a carrier protein.
4. A human ST2 antibody, which is a polyclonal antibody prepared from the ST2 antigen of claim 2.
5. A human ST2 antibody, which is a polyclonal antibody prepared from the ST2 antigen of claim 3.
6. Use of the human ST2 antibody of claim 4 or 5 for the preparation of a human ST2 in vitro diagnostic kit.
7. An in vitro diagnostic kit of human ST2 comprising the human ST2 antibody of claim 4 or 5 as a coating antibody.
8. The in vitro diagnostic kit of human ST2 according to claim 7, wherein the kit further comprises a binding antibody which is the human ST2 antibody of claim 4 or 5, and when the binding antibody is derived from one of the human ST2 epitope peptides (1) and (2), the coating antibody is derived from the other of the human ST2 epitope peptides (1) and (2).
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| WO2003094856A3 (en) * | 2002-05-09 | 2005-03-24 | Brigham & Womens Hospital | 1l1rl-1 as a cardiovascular disease marker and therapeutic target |
| CN102640001A (en) * | 2009-11-05 | 2012-08-15 | 诺瓦提斯公司 | Biomarkers predictive of progression of fibrosis |
| WO2013173761A3 (en) * | 2012-05-18 | 2014-02-20 | Amgen Inc. | St2 antigen binding proteins |
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| WO2003094856A3 (en) * | 2002-05-09 | 2005-03-24 | Brigham & Womens Hospital | 1l1rl-1 as a cardiovascular disease marker and therapeutic target |
| CN1668763A (en) * | 2002-05-09 | 2005-09-14 | 布赖汉姆妇女医院 | 1L1RL-1 as a cardiovascular disease marker and therapeutic target |
| CN102640001A (en) * | 2009-11-05 | 2012-08-15 | 诺瓦提斯公司 | Biomarkers predictive of progression of fibrosis |
| WO2013173761A3 (en) * | 2012-05-18 | 2014-02-20 | Amgen Inc. | St2 antigen binding proteins |
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| 可溶性 ST2 在心力衰竭诊断及预后评估中的作用;尹秋生等;《中华保健医学杂志》;20160229;第18卷(第1期);86-87 * |
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