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Amino terminal brain natriuretic peptide precursor polypeptide, antibody, preparation method thereof, detection kit and detection method thereof

A detection kit and amino terminal technology, applied in the biological field, can solve the problems of inaccurate detection results, poor affinity, sensitive glycosylation sites, etc., to increase immunogenicity, improve detection sensitivity and accuracy of detection results, The effect of improving affinity

Active Publication Date: 2019-08-06
SHENZHEN YHLO BIOTECH
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  • Description
  • Claims
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AI Technical Summary

Problems solved by technology

NT-proBNP antibody can be prepared by immunizing animals with natural NT-proBNP protein, but most of the antibodies produced by natural NT-proBNP protein immunized animals are sensitive to glycosylation sites, resulting in false negative test results
Using linear polypeptides to immunize animals to prepare antibodies can avoid the influence of glycosylation sites, but the antibodies prepared by this method have poor affinity with the natural NT-proBNP in the sample, resulting in low detection sensitivity and inaccurate detection results

Method used

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  • Amino terminal brain natriuretic peptide precursor polypeptide, antibody, preparation method thereof, detection kit and detection method thereof
  • Amino terminal brain natriuretic peptide precursor polypeptide, antibody, preparation method thereof, detection kit and detection method thereof
  • Amino terminal brain natriuretic peptide precursor polypeptide, antibody, preparation method thereof, detection kit and detection method thereof

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preparation example Construction

[0056] The embodiment of the present invention also provides a preparation method of the antibody, comprising:

[0057] S110, injecting the coupling protein into the subcutaneous tissue of the mouse to obtain the immunized mouse;

[0058] S120, fusing the myeloma cells with the spleen cells of the immunized mouse to obtain hybridoma cells;

[0059] S130, screening positive hybridoma cells capable of producing NT-proBNP antibody; and

[0060] S140, injecting the positive hybridoma cells into the peritoneal cavity of other mice for culture, and obtaining the amino-terminal BNP precursor antibody in the ascites of the cultured mice.

[0061] An embodiment of the present invention also provides a detection kit for N-terminal N-terminal natriuretic peptide precursor, comprising: a solid phase carrier coated with a first antibody and a second antibody labeled with a chemiluminescence molecule, and the first antibody and the second antibody Secondary antibodies are different.

[0...

Embodiment

[0084] (1) Immunogen preparation (amino-terminal BNP precursor polypeptide synthesis):

[0085] The following three polypeptides were synthesized by Shanghai Jill Biochemical Biochemistry. The first polypeptide SEQ ID NO.6, the second polypeptide SEQ ID NO.7 and the third polypeptide SEQ ID NO.8 were respectively coupled to the carrier protein BSA to obtain the first Coupling protein, secondary coupling protein and tertiary coupling protein.

[0086] (2) Preparation of N-terminal BNP precursor monoclonal antibody:

[0087] The first coupling protein, the second coupling protein and the third coupling protein obtained in step (1) were respectively used as immunogens to immunize mice, and the natural protein NT-proBNP (provided by Xinbainuo Biological Co., Ltd.) was selected to immunize mice As a control, an anti-first polypeptide monoclonal antibody, an anti-second polypeptide monoclonal antibody, an anti-third polypeptide monoclonal antibody and an anti-natural protein monocl...

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Abstract

The present invention discloses an amino terminal brain natriuretic peptide precursor polypeptide. The amino terminal brain natriuretic peptide precursor polypeptide is formed by respectively adding first cysteine and second cysteine near an amino terminal end and a carboxy terminal end of a specific amino acid sequence of a native amino terminal brain natriuretic peptide precursor. The first cysteine and the second cysteine are capable of forming a disulfide bond, the specific amino acid sequence is selected from one or more of the polypeptide sequences shown as SEQ ID NO. 1, and / or the specific amino acid sequence is selected from one or more of the polypeptide sequences shown as SEQ ID NO. 2, the vicinity of the amino terminal is 1 to 3 amino acids at the amino terminal of the specificamino acid sequence, and the vicinity of the carboxyl terminal is 1 to 3 amino acids at the carboxy terminal of the specific amino acid sequence. The invention also discloses an antibody and a preparation method thereof. The invention also discloses a detection kit. The invention also discloses a method for detecting the amino terminal brain natriuretic peptide precursor.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to an amino-terminal brain natriuretic peptide precursor polypeptide, an antibody and a preparation method thereof, a detection kit and a detection method. Background technique [0002] N-terminal pro-brain natriuretic peptide (abbreviated as NT-proBNP) comes from ventricular myocytes and is initially synthesized as pre-proBNP, which is a polypeptide chain composed of 134 amino acids. ProBNP is cleaved in cardiomyocytes into proBNP (proBNP, 108 amino acids) and signal peptide (26 amino acids). When cardiomyocytes are stimulated, proBNP is cleaved into an inactive linear polypeptide NT-proBNP consisting of 76 amino acids and an active cyclic polypeptide BNP consisting of 32 amino acids under the action of activating enzyme, and released into the blood circulation. NT-proBNP has a long half-life (60-120min), and is easier to detect than BNP, that is, the sensitivity of detection is impro...

Claims

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Application Information

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IPC IPC(8): C07K14/58C07K16/26G01N33/68G01N33/74C12N5/20
CPCC07K14/58C07K14/765C07K14/77C07K14/795C07K2319/00
Inventor 程方明邢智浩钱纯亘胡鹍辉
Owner SHENZHEN YHLO BIOTECH
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