High-affinity monoclonal antibody against human copeptin, preparation method and application thereof

A monoclonal antibody and copeptin technology, applied in the field of biomedicine, can solve the problems of high operational difficulty, uncontrollable immunization dose, and high immunization cost, and achieve the effects of promoting effective maturation, shortening detection time, and increasing immune response

Inactive Publication Date: 2019-04-26
GUANGZHOU WONDFO BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, DNA immunization has certain technical difficulties, high operatio

Method used

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  • High-affinity monoclonal antibody against human copeptin, preparation method and application thereof
  • High-affinity monoclonal antibody against human copeptin, preparation method and application thereof
  • High-affinity monoclonal antibody against human copeptin, preparation method and application thereof

Examples

Experimental program
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Example Embodiment

[0038] The preparation method of hybridoma cells secreting anti-human and copeptin monoclonal antibodies of the present invention includes the following steps: immunizing mice with human and copeptin antigens; delivering cytokine-expressing plasmids to the immunized mice; collecting The spleen cells or lymph node cells of the mouse are delivered by the plasmid, and the spleen cells or lymph node cells are fused with the mouse tumor cells to obtain hybridoma cells.

[0039] Optionally, the human and copeptin antigens are polypeptides containing the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3. Preferably, the human and copeptin antigens are human and copeptin antigens coupled with a macromolecular protein, and the macromolecular protein is at least one of KLH, OVA, and BSA. More preferably, the human and copeptin antigens are human and copeptin antigens coupled with KLH at the C terminal.

[0040] In some embodiments, the cytokine includes mFlt31, mGM-CSF and m...

Example Embodiment

[0046] Example 1 Preparation method of high-affinity anti-human and copeptin monoclonal antibody

[0047] 1.1 Peptide synthesis

[0048] Synthesize and copeptin antigen polypeptides (cppF, N-cpp, C-cpp) and couple KLH to prepare complete antigens to obtain KLH-cppF, KLH-N-cpp, KLH-C-cpp. The amino acid sequence is:

[0049] cppF: ATQLDGPAGALLLRLVQLAGAPEPFEPAQPDAY (SEQ ID NO:1),

[0050] N-cpp: CATQLDGPAGALLLRLV (SEQ ID NO: 2),

[0051] C-cpp: CLAGAPEPFEPAQPDAY (SEQ ID NO: 3),

[0052] That is, the complete antigen obtained by coupling KLH is:

[0053] KLH-cppF(KLH-C-ATQLDGPAGALLLRLVQLAGAPEPFEPAQPDAY),

[0054] KLH-N-cpp(KLH-C-ATQLDGPAGALLLRLV),

[0055] KLH-C-cpp(KLH-C-LAGAPEPFEPAQPDAY);

[0056] Polypeptide synthesis and coupling were performed by conventional methods and commissioned by Gil Biochemical Shanghai Co., Ltd. The purity determined by high performance liquid chromatography (HPLC) method was >95%.

[0057] 1.2 Plasmid construction

[0058] The pCAGGS vector was used to construct ...

Example Embodiment

[0073] Example 2 Immune effect and antibody titer determination

[0074] Cytokine group: The immunization strategy described in Example 1: That is, cytokine injection is performed via tail vein once a week after each subcutaneous immunization.

[0075] Control group 1: Compared with Example 1, no cytokine tail vein injection was performed after each immunization, and the rest of the steps were the same as in Example 1.

[0076] Control group 2: Compared with Example 1, after the initial immunization by subcutaneous injection of the site, the cytokine-expressing plasmids: pCAGGS-mFlt3L and pCAGGS-mGM-CSF were injected through the tail vein one week later, that is, compared with the experimental group, No injection of pCAGGS-mCCL20.

[0077] The comparison results of the above two groups of immune effects are shown in Table 1.

[0078] Table 1 Comparison of immune effects

[0079]

[0080]

[0081] It can be seen from Table 1 that the cytokine group, that is, the method described in Examp...

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Abstract

The invention provides a high-affinity monoclonal antibody against human copeptin, a preparation method and an application thereof. Common immune and immune adjustment molecules are combined with eachother, conventional immune is performed while cell factors Flt31, mGM-CSF and CCL20 are delivered in an auxiliary manner, so that immunoreaction of immune animal is enhanced, effective submission ofantigen molecules in organism is promoted, effective maturity of organism is boosted and the high-affinity antibody is acquired. The affinity of the acquired antibody is 10-100 times higher than the affinity of the antibody prepared according to a conventional method. The antibody can be applied to copeptin immunodetection.

Description

technical field [0001] The invention belongs to the field of biomedicine, in particular to a high-affinity anti-human copeptin monoclonal antibody and its preparation method and application. Background technique [0002] Copeptin is a polypeptide containing 39 amino acid residues that is homologous to arginine vasopressin (AVP), and is a C-terminal partial peptide of pro-arginine vasopressin (pro-VAP) . Recent studies have found that copeptin, as a new biomarker, can replace AVP, and it has a certain role in the early diagnosis and prognosis evaluation of cardiovascular and cerebrovascular diseases, diabetic nephropathy, sepsis and urinary tract infection. Clinical application value is an important clinical disease predictor. At present, the detection of copeptin mainly includes chemiluminescence and enzyme-linked immunosorbent assay. Both methods are detection techniques based on the principle of immunoassay, and the quality of the antibodies used in the detection is dir...

Claims

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Application Information

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IPC IPC(8): C12N5/20C07K16/26G01N33/68G01N33/577
CPCC07K16/26G01N33/577G01N33/68G01N2333/575
Inventor 王羽吴培钿何小维康业黄幼珍
Owner GUANGZHOU WONDFO BIOTECH
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