CN112094338B - Amino-terminal brain natriuretic peptide precursor polypeptide and conjugated protein - Google Patents
Amino-terminal brain natriuretic peptide precursor polypeptide and conjugated protein Download PDFInfo
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- C07K14/575—Hormones
- C07K14/58—Atrial natriuretic factor complex; Atriopeptin; Atrial natriuretic peptide [ANP]; Cardionatrin; Cardiodilatin
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- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
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Abstract
The invention discloses an amino-terminal pro-brain natriuretic peptide polypeptide, which is formed by respectively adding a first cysteine and a second cysteine to the vicinity of an amino terminal and the vicinity of a carboxyl terminal of a specific amino acid sequence of a natural amino-terminal pro-brain natriuretic peptide, wherein the specific amino acid sequence is a polypeptide sequence shown in SEQ ID No.4, the vicinity of the amino terminal is 1-3 amino acids of the amino terminal of the specific amino acid sequence, the vicinity of the carboxyl terminal is 1-3 amino acids of the carboxyl terminal of the specific amino acid sequence, and the first cysteine and the second cysteine can form a disulfide bond, so that the amino-terminal pro-brain natriuretic peptide can form a cyclic polypeptide. The invention also discloses a coupling protein.
Description
The application is a divisional application with application date of 2019, 05 and 14, application number of 201910398843.4 and patent name of amino-terminal brain natriuretic peptide precursor polypeptide, antibody and preparation method thereof, detection kit and detection method.
Technical Field
The invention relates to the technical field of biology, in particular to an amino-terminal brain natriuretic peptide precursor polypeptide and a conjugated protein.
Background
The amino-terminal pro-brain natriuretic peptide (NT-proBNP for short) is derived from ventricular myocytes and is initially synthesized as pre-pro-brain natriuretic peptide (pre-proBNP) and is a polypeptide chain of 134 amino acids. The pro-brain natriuretic peptide is cleaved in the cardiomyocytes into a pro-brain natriuretic peptide (proBNP, 108 amino acids) and a signal peptide (26 amino acids). When myocardial cells are stimulated, proBNP is cleaved by the action of an activating enzyme into the inactive linear polypeptide NT-proBNP consisting of 76 amino acids and the active cyclic polypeptide BNP consisting of 32 amino acids, which is released into the blood circulation. NT-proBNP has a long half-life (60-120min) and is more easily detected, i.e. the sensitivity of detection is increased, compared to BNP, which more easily reflects early or slight changes in cardiac function. NT-proBNP has lower individual difference and is not influenced by individual physiological rhythm. Therefore, NT-proBNP is clinically used as a biomarker for heart failure diagnosis and clinical prognosis.
Currently, NT-proBNP concentration in a sample is mostly detected by an immunoassay, i.e., by the property of the NT-proBNP antibody to specifically bind to NT-proBNP in the sample. The NT-proBNP antibody can be prepared by immunizing animals with natural NT-proBNP protein, but the antibody produced by the immunized animals with natural NT-proBNP protein is mostly sensitive to glycosylation sites, so that the detection result is false negative. The antibody prepared by immune animals adopting linear polypeptide can avoid the influence of glycosylation sites, but the affinity between the antibody prepared by the method and the natural NT-proBNP in a sample is poor, so that the detection sensitivity is low, and the detection result is inaccurate.
Disclosure of Invention
Accordingly, there is a need for an amino-terminal pro-brain natriuretic peptide polypeptide composition and a conjugated protein that have high sensitivity and accurate detection results for detecting NT-proBNP.
An amino-terminal pro-brain natriuretic peptide polypeptide formed by adding a first cysteine and a second cysteine to the vicinity of an amino terminal and the vicinity of a carboxyl terminal of a specific amino acid sequence of a natural amino-terminal pro-brain natriuretic peptide, respectively, wherein the specific amino acid sequence is a polypeptide sequence shown in SEQ ID No.4, the vicinity of the amino terminal is 1-3 amino acids of the amino terminal of the specific amino acid sequence, the vicinity of the carboxyl terminal is 1-3 amino acids of the carboxyl terminal of the specific amino acid sequence, and the first cysteine and the second cysteine can form a disulfide bond, so that the amino-terminal pro-brain natriuretic peptide can form a cyclic polypeptide.
In one embodiment, the amino-terminal pro-brain natriuretic peptide polypeptide is the polypeptide set forth in SEQ ID No. 7.
A conjugated protein comprising said amino-terminal pro-brain natriuretic peptide polypeptide, and further comprising a carrier protein conjugated to said amino-terminal pro-brain natriuretic peptide polypeptide.
In one embodiment, the amino-terminal pro-brain natriuretic peptide polypeptide has a free carboxyl group or a group that can be carboxylated.
In one embodiment, the method for coupling the amino-terminal pro-brain natriuretic peptide polypeptide to the carrier protein is selected from one of a mixed anhydride method and a carbodiimide method.
In one embodiment, the amino-terminal pro-brain natriuretic peptide polypeptide has a free amino group or a reducible nitro group.
In one embodiment, the method for coupling the amino-terminal pro-brain natriuretic peptide polypeptide to the carrier protein is selected from one of the glutaraldehyde method and the diazotization method.
In one embodiment, the amino-terminal pro-brain natriuretic peptide polypeptide has a free hydroxyl group.
In one embodiment, the method for coupling the amino-terminal pro-brain natriuretic peptide polypeptide to the carrier protein is selected from one of a succinic anhydride method and a carbonyldiimidazole method.
In one embodiment, the carrier protein is selected from one or more of KLH, bovine serum albumin and OVA.
The amino-terminal pro-brain natriuretic peptide polypeptide of the present invention is an altered polypeptide formed by providing a first cysteine and a second cysteine near the amino-terminal and near the carboxy-terminal, respectively, of a specific amino acid sequence of a native amino-terminal pro-brain natriuretic peptide, wherein the first cysteine and the second cysteine are capable of forming a disulfide bond, thereby enabling the amino-terminal pro-brain natriuretic peptide polypeptide to form a cyclic polypeptide. Compared with linear polypeptide, the cyclic polypeptide can realize more specific recognition, the immunogenicity of the polypeptide is increased, namely the performance of the polypeptide for generating an immune response to form a specific antibody, when the cyclic amino-terminal pro-brain natriuretic peptide is used for animal immunization to prepare the amino-terminal pro-brain natriuretic peptide antibody, the formed amino-terminal pro-brain natriuretic peptide can be improved in the affinity of the target amino-terminal pro-brain natriuretic peptide, and the detection sensitivity and the detection result accuracy of the target amino-terminal pro-brain natriuretic peptide are improved. The specific amino acid sequence is selected from sequences unrelated to glycosylation, so that the influence of glycosylation on the detection result of the amino-terminal pro-brain natriuretic peptide is avoided. Depending on the length of a particular amino acid sequence, sequence differences, or the location of addition of cysteine, a variety of amino-terminal pro-brain natriuretic peptide polypeptides can be formed.
The different amino-terminal brain natriuretic peptide precursor polypeptides obtained by the invention can be immunized by animals to obtain corresponding various types of antibodies of the amino-terminal brain natriuretic peptide precursors, the various types of antibodies of the amino-terminal brain natriuretic peptide precursors respectively coat a solid phase carrier and mark chemiluminescent molecules, the concentration of the amino-terminal brain natriuretic peptide precursors in a sample can be detected by a sandwich method, the first antibodies coat the solid phase carrier, the second antibodies mark the chemiluminescent molecules, and the first antibodies and the second antibodies are respectively combined with the target amino-terminal brain natriuretic peptide precursors, so that the specificity and the accuracy of the target amino-terminal brain natriuretic peptide precursors are improved. The solid phase carrier is used as a combining place and is beneficial to transfer and cleaning of a combined product, a target amino terminal brain natriuretic peptide precursor is combined with a first antibody on the solid phase carrier and a second antibody marked with a chemiluminescent molecule, the chemiluminescent molecule is used as a detection marker, the concentration of the target amino terminal brain natriuretic peptide precursor is represented by the luminescence amount of the chemiluminescent molecule, and the luminescence amount of the chemiluminescent molecule is in direct proportion to the concentration of the target amino terminal brain natriuretic peptide precursor.
Drawings
FIG. 1 is a photograph of the glycosylation sites of a native amino-terminal pro-brain natriuretic peptide according to one embodiment of the present invention;
FIG. 2 is a photograph of a standard curve relating the concentration of the amino-terminal pro-brain natriuretic peptide to the emission value according to an embodiment of the present invention.
Detailed Description
In order to make the objects, technical schemes and advantages of the present invention more clearly understood, the amino-terminal pro-brain natriuretic peptide polypeptide, antibody, preparation method thereof, detection kit and detection method of the present invention are further described in detail by way of examples with reference to the accompanying drawings. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
The embodiment of the invention provides an amino-terminal pro-brain natriuretic peptide polypeptide, which is formed by adding a first cysteine and a second cysteine to the vicinity of an amino terminal and the vicinity of a carboxyl terminal of a specific amino acid sequence of a natural amino-terminal pro-brain natriuretic peptide, respectively, wherein the first cysteine and the second cysteine can form a disulfide bond, the specific amino acid sequence is selected from at least partial sequences in a polypeptide sequence shown in SEQ ID NO.1, and/or the specific amino acid sequence is selected from at least partial sequences in a polypeptide sequence shown in SEQ ID NO.2, the vicinity of the amino terminal is 1-3 amino acids of the amino terminal of the specific amino acid sequence, and the vicinity of the carboxyl terminal is 1-3 amino acids of the carboxyl terminal of the specific amino acid sequence.
Referring to FIG. 1, the amino acid sequence of the natural amino-terminal pro-brain natriuretic peptide has glycosylation sites, and most of the antibodies generated by animals immunized with the natural NT-proBNP protein are sensitive to the glycosylation sites, resulting in false negative detection results. The specific amino acid sequence of the embodiment of the invention is selected from sequences at two sides of the amino acid sequence corresponding to the glycosylation site, namely at least partial sequence in SEQ ID NO.1 or SEQ ID NO.2, the amino acid sequence corresponding to the glycosylation site is avoided, and the accuracy of the detection result can be improved by detecting the target amino-terminal pro-brain natriuretic peptide by using the antibody prepared from the amino-terminal pro-brain natriuretic peptide polypeptide.
The amino-terminal pro-brain natriuretic peptide polypeptide of an embodiment of the invention is formed by placing a first cysteine and a second cysteine, which are capable of forming a disulfide bond, near the amino terminus and near the carboxy terminus, respectively, of a particular amino acid sequence of a native amino-terminal pro-brain natriuretic peptide such that the amino-terminal pro-brain natriuretic peptide polypeptide is capable of forming a cyclic polypeptide. Compared with linear polypeptide, the cyclic polypeptide can realize more specific recognition, the immunogenicity of the polypeptide is increased, namely the performance of the polypeptide for generating an immune response to form a specific antibody, when the cyclic amino-terminal pro-brain natriuretic peptide is used for animal immunization to prepare the amino-terminal pro-brain natriuretic peptide antibody, the formed amino-terminal pro-brain natriuretic peptide can be improved in the affinity of the target amino-terminal pro-brain natriuretic peptide, and the detection sensitivity and the detection result accuracy of the target amino-terminal pro-brain natriuretic peptide are improved. The specific amino acid sequence is selected from sequences unrelated to glycosylation, so that the influence of glycosylation on the detection result of the amino-terminal pro-brain natriuretic peptide is avoided. The amino-terminal pro-brain natriuretic peptide polypeptide can form a variety of species depending on the length of a particular amino acid sequence, sequence differences, or the location of addition of cysteine.
In one embodiment, the amino-terminal pro-brain natriuretic peptide can have a sequence length of 10 to 35 amino acids, i.e., the specific amino acid sequence can have a length of 8 to 33 amino acids. The length of the amino acid sequence of the polypeptide has great influence on the immunogenicity of the polypeptide, the specificity of the polypeptide is poor due to too short length, the polypeptide forms nonspecific immunity, and the generated antibody is a nonspecific antibody of an amino-terminal pro-brain natriuretic peptide, so that the detection result is inaccurate. Too long a polypeptide affects recognition of the immune site, resulting in poor immunogenicity of the polypeptide and reduced antibody-forming properties of the polypeptide. Preferably, the amino-terminal brain natriuretic peptide precursor polypeptide can have a sequence length of 15-25 amino acids.
Preferably, the specific amino acid sequence can be a polypeptide sequence as shown in a first amino acid segment of SEQ ID NO.3, a second amino acid segment of SEQ ID NO.4 or a third amino acid segment of SEQ ID NO. 5. The specific amino acid sequence is a more specific amino acid sequence which is different from other proteins in the amino-terminal pro-brain natriuretic peptide, so that the antibody generated by immunization by the specific amino acid sequence has stronger recognition and affinity to the amino-terminal pro-brain natriuretic peptide, and the detection sensitivity of the antibody generated by immunization by the specific amino acid sequence to the target amino-terminal pro-brain natriuretic peptide is higher.
Cysteine is added near the amino-terminal and near the carboxy-terminal of the specific amino acid sequence, respectively, and a first cysteine near the amino-terminal and a second cysteine near the carboxy-terminal of the resulting amino-terminal pro-brain natriuretic peptide form a disulfide bond, thereby forming a cyclic polypeptide from the amino-terminal pro-brain natriuretic peptide and increasing immunogenicity. The cysteine is arranged near the amino terminal and the carboxyl terminal of the amino terminal pro-brain natriuretic peptide polypeptide, so that the immunogenicity of the polypeptide is increased and the antibody forming performance of the polypeptide immunity is improved while the specificity of the specific amino acid sequence is not basically damaged.
In one embodiment, the amino-terminal pro-brain natriuretic peptide polypeptide is a polypeptide as set forth in SEQ ID No.6, SEQ ID No.7, or SEQ ID No.8, respectively, of the first polypeptide. SEQ ID NO.6 is the addition of a first cysteine before the positive first amino acid and a second cysteine before the last first amino acid of the specific amino acid sequence SEQ ID NO. 3. SEQ ID NO.7 is the addition of a first cysteine before the positive first amino acid and a second cysteine before the last first amino acid of the specific amino acid sequence SEQ ID NO. 4. SEQ ID NO.8 is the addition of a first cysteine before the positive first amino acid and a second cysteine before the last first amino acid of the specific amino acid sequence SEQ ID NO. 5. The amino acid sequences of SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7, and SEQ ID NO.8 are shown in Table 1.
Table 1 amino acid sequence listing
| Name (R) | Sequence of |
| SEQ ID NO.1 | HPLGSPGSASDLETSGLQEQRNHLQGKLSELQVEQ |
| SEQ ID NO.2 | EGIRGHRKMVLYTLRAPR |
| SEQ ID NO.3 | HPLGSPGSASDLETSGLQEQRNHL |
| SEQ ID NO.4 | ETSGLQEQRNHLQGKLSELQVE |
| SEQ ID NO.5 | GIRGHRKMVLYTLRAPR |
| SEQ ID NO.6 | CHPLGSPGSASDLETSGLQEQRNHCL |
| SEQ ID NO.7 | CETSGLQEQRNHLQGKLSELQVCE |
| SEQ ID NO.8 | CGIRGHRKMVLYTLRAPCR |
The amino-terminal brain natriuretic peptide precursor polypeptide can be prepared by an artificial synthesis method.
The embodiment of the invention also provides an amino-terminal pro-brain natriuretic peptide detection antibody, wherein the amino-terminal pro-brain natriuretic peptide detection antibody is formed by immunizing animals with coupled protein formed by coupling the amino-terminal pro-brain natriuretic peptide polypeptide with carrier protein.
The polypeptide is used as hapten, has antigenicity and no immunogenicity, and can obtain immunogenicity when the polypeptide is combined with carrier protein to form macromolecules. The hapten polypeptide and the carrier form an immunogen, namely, the immunogen can induce the animal to be immunized to generate antibodies which can be specifically combined with the polypeptide and specific antibodies aiming at the carrier protein. Since the polypeptide is substantially identical to the amino-terminal pro-brain natriuretic peptide in terms of its specific amino acid sequence, the antibody capable of specifically binding to the polypeptide is capable of specifically binding to the amino-terminal pro-brain natriuretic peptide.
In one embodiment, the amino-terminal pro-brain natriuretic peptide is the first polypeptide, the second polypeptide, and the third polypeptide, the coupled proteins are the first coupled protein, the second coupled protein, and the third coupled protein, and the amino-terminal pro-brain natriuretic peptide antibodies are anti-first polypeptide antibody, anti-second polypeptide antibody, and anti-third polypeptide antibody, which are formed by animal immunization, and each of the anti-first polypeptide antibody, anti-second polypeptide antibody, and anti-third polypeptide antibody can specifically bind to the amino-terminal pro-brain natriuretic peptide.
The method of coupling the amino-terminal pro-brain natriuretic peptide to the carrier protein differs depending on the type of free radical that the amino-terminal pro-brain natriuretic peptide has. When the amino-terminal pro-brain natriuretic peptide polypeptide has a free carboxyl group or a carboxylable group, the coupling method may be selected from one of a mixed anhydride method and a carbodiimide method. When the amino-terminal brain natriuretic peptide precursor polypeptide has a free amino group or a reducible nitro group, the coupling method may be one selected from the glutaraldehyde method and the diazotization method. When the amino-terminal pro-brain natriuretic peptide polypeptide has a free hydroxyl group, the coupling method may be one selected from the group consisting of a succinic anhydride method and a carbonyldiimidazole method. The specific coupling method can be determined according to the type of the amino-terminal brain natriuretic peptide precursor polypeptide.
In one embodiment, the carrier protein may be selected from one or more of klh (keyhole lipnet hemocyanin), Bovine Serum Albumin (BSA), and ova (ovalbumin).
The embodiment of the invention also provides a preparation method of the antibody, which comprises the following steps:
s110, injecting the coupling protein into subcutaneous tissues of mice to obtain immunized mice;
s120, fusing myeloma cells and spleen cells of the immunized mouse to obtain hybridoma cells;
s130, screening positive hybridoma cells capable of generating the NT-proBNP antibody; and
s140, injecting the positive hybridoma cells into the abdominal cavity of other mice for culture, and obtaining the amino-terminal pro-brain natriuretic peptide antibody in the ascites of the cultured mice.
The embodiment of the invention also provides an amino-terminal pro-brain natriuretic peptide detection kit, which comprises: a first antibody coated solid support and a chemiluminescent molecule labeled second antibody, and the first antibody is different from the second antibody.
The different kinds of amino-terminal brain natriuretic peptide precursor polypeptides of the embodiment of the invention can be immunized by animals to obtain corresponding various kinds of antibodies against amino-terminal brain natriuretic peptide precursors, the different antibodies against amino-terminal brain natriuretic peptide precursors respectively coat a solid phase carrier and a labeled chemiluminescent molecule, the concentration of the amino-terminal brain natriuretic peptide precursors in a sample can be detected by a sandwich method, a first antibody coats the solid phase carrier, a second antibody labels the chemiluminescent molecule, and the first antibody and the second antibody are respectively combined with target amino-terminal brain natriuretic peptide precursors, so that the specificity and accuracy of the target amino-terminal brain natriuretic peptide precursor detection are improved. The solid phase carrier is used as a combining place and is beneficial to transfer and cleaning of a combined product, a target amino terminal brain natriuretic peptide precursor is combined with a first antibody on the solid phase carrier and a second antibody marked with a chemiluminescent molecule at the same time, the chemiluminescent molecule is used as a detection marker, the concentration of the target amino terminal brain natriuretic peptide precursor is represented by the luminous quantity of the chemiluminescent molecule, and the luminous value of the chemiluminescent molecule is in direct proportion to the concentration of the target amino terminal brain natriuretic peptide precursor. The luminous value of the target amino-terminal pro-brain natriuretic peptide obtained by the sample to be detected can be brought into a standard curve of the relation between the concentration of the amino-terminal pro-brain natriuretic peptide and the luminous value determined by the amino-terminal pro-brain natriuretic peptide calibrator, so that the concentration of the amino-terminal pro-brain natriuretic peptide in the sample to be detected can be obtained.
In one embodiment, the chemiluminescent molecule may be selected from one or more of, but not limited to, acridinium esters and acridinium amides.
In one embodiment, the test kit further comprises one or more of a calibrator, a wash, and a stimulant.
The calibrator is a natural amino-terminal brain natriuretic peptide precursor solution with a specific concentration gradient, and the solvent in the amino-terminal brain natriuretic peptide precursor solution can comprise one or more of Tris-HCl buffer solution and bovine serum albumin solution.
The washing solution is used to wash the reagent not bound to the immobilization carrier. The wash agent may comprise one or more of a tween and Tris-HCl buffer.
The activator is for reacting with the chemiluminescent molecule to cause the chemiluminescent molecule to emit light. In one embodiment, the chemiluminescent molecule is selected from one or more of acridinium ester and acridinium sulfonamide, and the exciting agent may include hydrogen peroxide, nitric acid and sodium hydroxide. In the alkaline hydrogen peroxide solution, when the chemiluminescent molecules are attacked by hydrogen peroxide ions, unstable ethylene dioxide is generated and decomposed into CO2And an electronically excited state of N-methylacridone which emits photons having a maximum emission wavelength of 430nm when it returns to the ground state. Preferably, the kit may further comprise a luminescence enhancer, which may be a surfactant, which may be selected from one or more of Triton (Triton X-100), cetyltrimethylammonium chloride (CTAC) and tween 20.
The solid phase carrier and the first antibody may be coated by binding to the first antibody through an active group on the surface of the solid phase carrier or by binding to a labeled molecule through molecular labeling of the solid phase carrier and the first antibody, respectively. The reactive group may include one or more of an amino group, a carboxyl group, and a hydroxyl group. In one embodiment, the surface of the solid support has carboxyl groups, and the solid support is capable of binding to the amino groups of the first antibody via the carboxyl groups. The molecular marker can be biotin and avidin markers which can be specifically combined. In another embodiment, the first antibody is labeled with biotin and the solid support is labeled with avidin. The avidin may be selected from streptavidin. Each streptavidin can bind 4 biotins, thereby expanding the capacity of the solid support to bind the first antibody.
In one embodiment, the solid phase carrier is a carrier for a binding reaction of the amino-terminal pro-brain natriuretic peptide detection, and the binding of the target amino-terminal pro-brain natriuretic peptide with the first antibody and the second antibody is integrated on the solid phase carrier, so that the transfer, washing and separation of the bound product are facilitated, and the operation is more convenient. The solid phase carrier can be in a particle shape, the surface area of the particle-shaped solid phase carrier is large, more binding sites are provided, so that the reaction capacity is improved, and the particle-shaped solid phase carrier is more suitable for liquid phase reaction and provides convenience for implementation of detection. The solid phase carrier particles are selected from one or more of polystyrene particles and magnetic particles. Preferably, the solid support is selected from magnetic particles. The magnetic particles as solid phase carriers have the characteristic of easy separation, and can meet the requirement of automatic production. And the good environmental resistance of the magnetic particle makes it possible to adapt to a severe reaction environment from an acid to a base, thereby making the reaction system more inclusive.
Preferably, the type of the first antibody and the type of the second antibody are obtained by respectively coating magnetic particles and labeled chemiluminescent molecules on all the types of the amino-terminal pro-brain natriuretic peptide detection antibodies, and then pairwise pairing and screening the amino-terminal pro-brain natriuretic peptide detection antibodies coated with the magnetic particles and labeled with the chemiluminescent molecules to obtain the optimal combination of the type of the first antibody and the type of the second antibody.
The embodiment of the invention also provides a detection method of the amino-terminal pro-brain natriuretic peptide, which adopts the detection kit of the amino-terminal pro-brain natriuretic peptide and comprises the following steps:
s210, mixing a detection sample, the solid phase carrier coated by the first antibody and a second antibody marked by a chemiluminescent molecule to obtain a mixture;
s220, separating the solid phase carrier from the mixture;
s230, detecting the separated luminescent signal of the solid phase carrier; and
s240, bringing the luminescence signal into a standard curve of the relation between the concentration of the amino-terminal pro-brain natriuretic peptide and the luminescence signal to obtain the concentration of the amino-terminal pro-brain natriuretic peptide in the detection sample.
Preferably, the step of mixing the detection sample, the first antibody-coated solid support and the chemiluminescent molecule-labeled second antibody to obtain a mixture may comprise:
s211, mixing the detection sample with the solid phase carrier coated by the first antibody, and incubating for a preset time to obtain a first mixture;
s212, cleaning the first mixture by using a cleaning solution;
s213, separating the solid phase carrier from the washed first mixture; and
and S214, mixing and incubating the solid phase carrier separated from the first mixture and the second antibody labeled by the chemiluminescent molecules to obtain a second mixture.
The solid phase carrier coated by the first antibody and a sample to be detected are mixed, and then the mixture is collected with a second antibody marked by a chemiluminescent molecule after being cleaned, so that the amino terminal brain natriuretic peptide precursor in the sample to be detected is combined with the first antibody and fixed on the solid phase carrier in the step S211, and then a reagent which is not combined on the solid phase carrier is cleaned through the step S212 and the step S213, and the interference of the non-combined chemiluminescent molecule is avoided.
The mass ratio of the sample to be detected, the solid phase carrier coated by the first antibody and the second antibody marked by the chemiluminescent molecules can be determined according to the approximate condition of the concentration of the amino-terminal brain natriuretic peptide precursor in the sample to be detected.
Examples
(1) Immunogen preparation (amino-terminal brain natriuretic peptide precursor polypeptide synthesis):
the following 3 sections of polypeptides are synthesized by Shanghai Jier biochemical biochemistry, and a first polypeptide SEQ ID NO.6, a second polypeptide SEQ ID NO.7 and a third polypeptide SEQ ID NO.8 are respectively coupled with carrier protein BSA to obtain a first coupling protein, a second coupling protein and a third coupling protein.
(2) Preparation of amino-terminal pro-brain natriuretic peptide monoclonal antibody:
respectively taking the first coupled protein, the second coupled protein and the third coupled protein obtained in the step (1) as immunogen immune mice, and simultaneously selecting natural protein NT-proBNP (provided by Xin Bai Nuo biology, Ltd.) immune mice as a control to prepare an anti-first polypeptide monoclonal antibody, an anti-second polypeptide monoclonal antibody, an anti-third polypeptide monoclonal antibody and an anti-natural protein monoclonal antibody.
The preparation method comprises the following steps:
the first coupling protein, the second coupling protein and the third coupling protein or the natural protein are respectively mixed with Freund's adjuvant in equal amount, fully emulsified and respectively injected into 3 Balb/c mice with each mouse being 25 mu g, and are immunized 3 times at intervals of 15 days to obtain the immunized mice.
Cell fusion, selective culture and screening: spleen cells of the immunized mice were taken out 3 days after the injection of the conjugated protein or the native protein, and 2.0X 10 cells were added7SP2/0 myeloma cells and 2.0X 108Spleen cells of each immunized Balb/c mouse were mixed well, centrifuged, the supernatant discarded, and mixed well with gentle shaking. Dropping 1mL of 50% polyethylene glycol-1500 aqueous solution in a water bath at 37 ℃ within 90 seconds, then dropping 20mL of 1640 culture medium, centrifuging, discarding the supernatant, washing once again, centrifuging, discarding the supernatant, and obtaining the hybridoma cell. Hybridoma cells were cultured on 96-well cell culture plates using HAT selection medium with a total fusion rate > 95% as detected under a microscope. Selecting a hole with a monoclonal cell from a 96-well plate, detecting the supernatant of the monoclonal cell hole by adopting a coating natural NT-proBNP protein, selecting the cell in the hole with OD450 more than 0.8 for subcloning, and finally obtaining the cell clone with the reaction positive rate more than 99 percent to the natural NT-proBNP as a positive line for secreting the anti-NT-proBNP monoclonal antibodySex hybridoma cell strain.
Cell cloning: cloning the obtained positive hybridoma cell strain, using a limiting dilution method, cloning for 3 times, finally obtaining 7 strains of the anti-NT-proBNP monoclonal antibody hybridoma cell line which can generate high titer, carrying out amplification culture, and freezing and storing.
Preparing ascites: treating 6-8 week female Balb/c mouse with paraffin for 10 days, and collecting positive hybridoma cells at 2 × 106One cell/mouse was injected intraperitoneally. Obtaining ascites rich in NT-proBNP antibody from mouse abdominal cavity after 10 days, and determining ascites titer > 105。
And (3) purification: protein G affinity chromatography was used. Firstly, preparing a protein G affinity chromatographic column, balancing the column by PBS, taking ascites to pass through the column, then washing the column by PBS, eluting by a glycine hydrochloride solution of 50nmol/L, collecting eluent, measuring the OD value of each collecting pipe, reserving the eluent in a peak area, dialyzing the eluent in the peak area and collecting the eluent. The purity of the purified monoclonal antibody is more than 98 percent after being identified by SDS-PAGE electrophoresis.
(3) Antibody titer detection
The antibodies obtained by immunizing mice with the natural protein, the first polypeptide, the second polypeptide and the third polypeptide are respectively named: an anti-natural protein monoclonal antibody, an anti-first polypeptide monoclonal antibody, an anti-second polypeptide monoclonal antibody and an anti-third polypeptide monoclonal antibody. Coating natural NT-proBNP 1ug/mL as antigen, and performing titer indirect detection on the purified anti-natural protein monoclonal antibody, anti-first polypeptide monoclonal antibody, anti-second polypeptide monoclonal antibody and anti-third polypeptide monoclonal antibody respectively. The antigen dilution concentration is 3ug/mL, 1ug/mL, 0.33ug/mL, 0.11ug/mL, 0.036ug/mL, 0.012ug/mL, the antibody titer of the multiple strains is detected by indirect method, and the detection result by enzyme-linked immunosorbent assay is shown in tables 2-5. 2 antibodies with the highest titer are selected respectively and labeled with horseradish peroxidase (HRP), and further antibody pairing screening experiments are carried out.
TABLE 2 anti-native protein monoclonal antibody titers
The first strain (1#) and the second strain (2#) of monoclonal antibodies against the first polypeptide are selected for further antibody pairing screening test.
TABLE 3 anti-first polypeptide monoclonal antibody titers
The first strain (1#) and the second strain (2#) of monoclonal antibodies against the first polypeptide are selected for further antibody pairing screening test.
TABLE 4 anti-second polypeptide monoclonal antibody titers
The first strain (1#) and the second strain (2#) monoclonal antibodies against the second polypeptide are selected for further antibody pairing screening test.
TABLE 5 anti-third polypeptide monoclonal antibody titers
And selecting the first strain (1#) and the second strain (2#) of monoclonal antibodies against the third polypeptide for further antibody pairing screening test.
(4) Antibody-labeled HRP
The workload of pairing screening is large, and the reagent usage amount is large. This example screens best-matched combinations of primary and secondary antibodies using HRP-labeled antibody instead of acridinium ester labeling.
Each antibody was 200ug in total and 200ul in volume, and packed in a dialysis bag, dialyzed against 50mM Carbonate (CB) buffer (pH9.6) overnight at 4 ℃ and replaced with CB buffer the next morning. NaIO prepared at 5mg/ml3And 5mg/ml HRP solution, stirring and mixing the two solutions uniformly, and adding glycol to terminate the reaction, thereby obtaining the oxidized HRP. 160 mu l of oxidized HRP is added into a dialysis bag for dialyzing the antibody for each antibody, the antibody is mixed up and down, and the reaction is carried out for 3 hours at the lower side of 4 ℃ to obtain the HRP-labeled antibody (the CB buffer is changed after the reaction is carried out for 1 hour). The HRP-labeled antibody was removed from the dialysis bag and its volume V was recorded. Preparing NaBH of 5mg/ml4And (3) solution. Add 8. mu.l NaBH per antibody4The solution is mixed evenly for 1h at the low temperature of 4 ℃. Adding 1/2V bovine serum and V saturated ammonium sulfate according to the volume V of the HRP-labeled antibody, mixing, and standing at 4 deg.C for 30 min. 12000rpm, centrifugation for 10min, and discarding the supernatant. First 100. mu.l (80% PBS + 20% bovine serum) was added for reconstitution, then 100. mu.l glycerol was added, and finally the concentration of the HRP-labeled antibody was 1 mg/ml.
(5) Antibody pairing screening
And (3) selecting 2 high-titer polypeptide antibodies obtained in the step (3), respectively coating magnetic particles and carrying out HRP labeling, carrying out antibody pairing screening by using a natural protein NT-proBNP as an antigen. Specific experimental data are shown in table 6. The pair with the highest detection value was selected for further experiments.
TABLE 6 polypeptide antibody pairing Titer comparison
As can be seen from Table 6, the combination of the first monoclonal antibody against the first polypeptide coated magnetic particles and the first monoclonal antibody against the second polypeptide labeled, and the combination of the second monoclonal antibody against the third polypeptide coated magnetic particles and the first monoclonal antibody against the second polypeptide labeled, have the highest potency as measured by the sandwich method.
(6) Preparation of NT-proBNP detection kit
Preparing first antibody coated magnetic particles:
taking 50mg of carboxylated magnetic particles (with the particle size of 0.05-1um), carrying out magnetic separation, leaving a precipitate, then using 20mM, pH 5.52- (N-morpholine) ethanesulfonic acid (MES) buffer for resuspension, adding 1mL of freshly prepared 10mg/mL 1-ethyl- (3-dimethylaminopropyl) carbodiimide (EDC) aqueous solution, activating the carboxyl groups on the surface of the magnetic particles, adding 4mg of a first antibody (an anti-first-polypeptide monoclonal antibody, an anti-first-polypeptide monoclonal antibody or an anti-first-polypeptide monoclonal antibody), suspending at room temperature for 6h, carrying out magnetic separation, removing a supernatant, using 100mM Tris buffer with pH8.0 containing 2% BSA for resuspension to 1mg/mL to obtain first-antibody-coated magnetic particles, subpackaging the first-antibody-coated magnetic particles according to 5 mL/bottle, and storing at 4 ℃ for later use.
Preparation of a second antibody labeled with acridinium ester:
taking 50ul of 25mg/mL second antibody (anti-first polypeptide monoclonal antibody, anti-first polypeptide monoclonal antibody or anti-first polypeptide monoclonal antibody, non-first antibody), adding 150ul of 0.1M carbonate buffer solution with pH of 9.0-9.5, mixing uniformly, then adding 1.5ul of 5mg/mL acridinium ester, mixing uniformly, reacting in a dark place at room temperature, taking out after 1.5h, Desalting with a 5mL GE desaling prepacked column, using a TBS equilibrium chromatographic column, then adding the reacted acridinium ester solution, collecting protein peak samples, subpackaging according to 5 mL/bottle, and storing at 4 ℃ for later use.
Preparation of NT-proBNP calibrator:
the prepared NT-proBNP solution was prepared with a buffer (40mM Tris-Cl, 0.5% BSA, 1% NaCl, pH8.0) to a concentration of 0pg/mL, 120pg/mL, 2000pg/mL, and 1mL per vial was lyophilized and stored at 4 ℃ for further use.
(7) The chemiluminescence immunoassay method of the human NT-proBNP comprises the following steps:
the method takes a chemiluminescence determinator as a detection tool, the kit in the step (6) as a detection reagent, and the methodology mode is a sandwich method, namely the instrument sequentially adds a detection sample, magnetic particles coated by a first antibody and a second antibody labeled with acridinium ester, performs magnetic separation after reaction for 10min, sends a reaction mixture into a darkroom, sequentially adds chemiluminescence pre-excitation liquid and chemiluminescence excitation liquid for luminescence reaction, finally records luminescence intensity, and calculates the concentration of NT-proBNP of the detection sample from a standard curve.
(8) NT-proBNP detection kit performance evaluation
And (3) detecting the NT-proBNP calibrator with the concentration of 0pg/mL, 120pg/mL or 2000pg/mL by adopting the method in the step (7), and drawing a standard curve as shown in FIG. 2.
Then, for the actual test sample to be tested, the NT-proBNP concentration of the test sample is calculated according to the luminescence value of the test sample.
Detection of sensitivity:
the sensitivity of the NT-proBNP assay kit was calculated with reference to the recommended protocol of the American Standard institute of clinical laboratory standards (CLSI EP17-A) file, and the sensitivity was found to be 5.0 pg/mL.
And (3) linear detection:
the NT-proBNP antigen with the concentration of 5pg/mL, 100pg/mL, 500pg/mL, 1000pg/mL, 5000pg/mL, 10000pg/mL, 20000pg/mL and 35000pg/mL is subjected to linear analysis by adopting the NT-proBNP detection kit, a linear correlation coefficient is calculated, and r is 0.9995, and the linear range of the detection of the NT-proBNP sample by the kit is 5.0 pg/mL-35000 pg/mL.
And (3) measuring precision:
taking two NT-proBNP samples with the concentrations of 120pg/mL and 2000pg/mL, respectively carrying out 3 parallels on each sample, detecting by using three batches of kits, and calculating the intra-batch and inter-batch differences of the kits, wherein the results show that the intra-batch and inter-batch differences of the kits are both less than 5%.
Interference experiments:
taking mixed serum to be respectively added with interferents, wherein the interferents comprise: combining one or more of bilirubin, free bilirubin, hemoglobin, ascorbic acid and glyceride, and adding the serum and the interferent according to a mass ratio of 1: 20, the measurement values of the mixed serum and the mixed serum added with various interferents were measured, and the deviation between the two was calculated to be within an acceptable range of. + -. 10%. The result shows that the interference reaches the NCCLS file standard, and can be used for accurate assessment of NT-proBNP condition in clinical laboratories.
The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the present invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.
Sequence listing
<110> Shenzhen Shenhuilong Biotech stock Co., Ltd
<120> amino-terminal brain natriuretic peptide precursor polypeptide, conjugated protein
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<211> 35
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 1
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1 5 10 15
Leu Gln Glu Gln Arg Asn His Leu Gln Gly Lys Leu Ser Glu Leu Gln
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Val Glu Gln
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<210> 2
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<212> PRT
<213> Artificial Sequence (Artificial Sequence)
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Pro Arg
<210> 3
<211> 24
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
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His Pro Leu Gly Ser Pro Gly Ser Ala Ser Asp Leu Glu Thr Ser Gly
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Leu Gln Glu Gln Arg Asn His Leu
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<210> 4
<211> 22
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
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Glu Thr Ser Gly Leu Gln Glu Gln Arg Asn His Leu Gln Gly Lys Leu
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Ser Glu Leu Gln Val Glu
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<210> 5
<211> 17
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 5
Gly Ile Arg Gly His Arg Lys Met Val Leu Tyr Thr Leu Arg Ala Pro
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<210> 6
<211> 26
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
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Cys His Pro Leu Gly Ser Pro Gly Ser Ala Ser Asp Leu Glu Thr Ser
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Gly Leu Gln Glu Gln Arg Asn His Cys Leu
20 25
<210> 7
<211> 24
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 7
Cys Glu Thr Ser Gly Leu Gln Glu Gln Arg Asn His Leu Gln Gly Lys
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Leu Ser Glu Leu Gln Val Cys Glu
20
<210> 8
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<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 8
Cys Gly Ile Arg Gly His Arg Lys Met Val Leu Tyr Thr Leu Arg Ala
1 5 10 15
Pro Cys Arg
Claims (9)
1. An amino-terminal pro-brain natriuretic peptide polypeptide, which is formed by adding a first cysteine and a second cysteine to the vicinity of an amino terminal and the vicinity of a carboxyl terminal of a specific amino acid sequence of a natural amino-terminal pro-brain natriuretic peptide, respectively, wherein the specific amino acid sequence is a polypeptide sequence shown in SEQ ID No.4, the vicinity of the amino terminal is 1-3 amino acids of the amino terminal of the specific amino acid sequence, the vicinity of the carboxyl terminal is 1-3 amino acids of the carboxyl terminal of the specific amino acid sequence, and the first cysteine and the second cysteine can form a disulfide bond, so that the amino-terminal pro-brain natriuretic peptide can form a cyclic polypeptide, and the amino-terminal pro-brain natriuretic peptide is a polypeptide shown in SEQ ID No. 7.
2. A conjugated protein comprising the amino-terminal pro-brain natriuretic peptide polypeptide of claim 1 and a carrier protein conjugated to the amino-terminal pro-brain natriuretic peptide polypeptide.
3. The conjugated protein of claim 2, wherein the amino-terminal pro-brain natriuretic peptide polypeptide has a free carboxyl group or a carboxylable group.
4. The conjugated protein of claim 3, wherein the method for coupling the amino-terminal pro-brain natriuretic peptide polypeptide to the carrier protein is selected from one of a mixed anhydride method and a carbodiimide method.
5. The conjugated protein of claim 2, wherein the amino-terminal pro-brain natriuretic peptide polypeptide has a free amino group or a reducible nitro group.
6. The conjugated protein of claim 5, wherein the conjugation of the amino-terminal pro-brain natriuretic peptide polypeptide to the carrier protein is performed by a method selected from the group consisting of glutaraldehyde method and diazotization method.
7. The conjugated protein of claim 2, wherein the amino-terminal pro-brain natriuretic peptide polypeptide has a free hydroxyl group.
8. The conjugated protein of claim 7, wherein the amino-terminal pro-brain natriuretic peptide polypeptide is conjugated to the carrier protein by a method selected from the group consisting of a succinic anhydride method and a carbonyldiimidazole method.
9. The conjugated protein of any one of claims 2 to 8, wherein the carrier protein is selected from one or more of KLH, bovine serum albumin and OVA.
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| WO2006108686A2 (en) * | 2005-04-14 | 2006-10-19 | Aic | Bnp agonists |
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| EP2292662A2 (en) * | 1996-03-04 | 2011-03-09 | Scios Inc. | Assay and reagents for quantifying hBNP |
| CN102887943A (en) * | 2012-08-30 | 2013-01-23 | 重庆业为基生物科技有限公司 | B cell epitope peptide segment of amino-terminal pro-brain natriuretic peptide and applications thereof |
| CN103601808A (en) * | 2012-12-11 | 2014-02-26 | 深圳大学 | Novel natriuretic peptide, preparation method and applications thereof in preparation of genetic engineering drugs |
| CN104262489A (en) * | 2014-09-30 | 2015-01-07 | 陈仁奋 | Cyclic chimeric citrullinated peptide antigen and application thereof |
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| EP2899544B1 (en) * | 2014-01-28 | 2018-12-26 | Roche Diagnostics GmbH | Biomarkers for risk assessment and treatment monitoring in heart failure patients who receive B-type natriuretic peptide guided therapy |
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| EP2292662A2 (en) * | 1996-03-04 | 2011-03-09 | Scios Inc. | Assay and reagents for quantifying hBNP |
| WO2006108686A2 (en) * | 2005-04-14 | 2006-10-19 | Aic | Bnp agonists |
| WO2007047614A2 (en) * | 2005-10-14 | 2007-04-26 | Scios Inc. | Glycosylated probnp |
| CN102887943A (en) * | 2012-08-30 | 2013-01-23 | 重庆业为基生物科技有限公司 | B cell epitope peptide segment of amino-terminal pro-brain natriuretic peptide and applications thereof |
| CN103601808A (en) * | 2012-12-11 | 2014-02-26 | 深圳大学 | Novel natriuretic peptide, preparation method and applications thereof in preparation of genetic engineering drugs |
| CN104262489A (en) * | 2014-09-30 | 2015-01-07 | 陈仁奋 | Cyclic chimeric citrullinated peptide antigen and application thereof |
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