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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 operational difficulty, uncontrollable immunization dose, and high immunization cost

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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preparation example Construction

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

[0039] Optionally, the copeptin antigen is a polypeptide comprising the amino acid sequence of SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3. Preferably, the copeptin antigen is a copeptin antigen coupled with a macromolecular protein, and the macromolecular protein is at least one of KLH, OVA, and BSA. More preferably, the copeptin antigen is a copeptin antigen with KLH coupled to the C-terminus.

[0040] In some of these embodiments, the cytokines include mFlt31, mGM-CSF and mCCL20. Preferably, the cytokine-expressing plasmid is pCAGGS.

[0041...

Embodiment 1

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

[0047] 1.1 Polypeptide synthesis

[0048] Copeptin antigen polypeptides (cppF, N-cpp, C-cpp) were synthesized and coupled with KLH to prepare complete antigens to obtain KLH-cppF, KLH-N-cpp, KLH-C-cpp. The amino acid sequence is:

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

[0050] N-cpp: CATQLDGPAGALLLLRLV (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-ATQLDGPAGALLLLRLVQLAGAPEPFEPAQPDAY),

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

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

[0056] Peptide synthesis and coupling adopt conventional methods, entrusted to Jill Biochemical Shanghai Co., Ltd. to complete. The purity was determined by high performance liquid chromatography (HPLC), all >95%.

[0057] 1.2 Plasmid construction

[0058] The pCAGGS v...

Embodiment 2

[0073] Example 2 Immune Effect and Antibody Titer Measurement

[0074] Cytokine group: the immunization strategy described in Example 1: the tail vein injection of cytokines was performed one week after each subcutaneous immunization.

[0075] Control group 1: Compared with Example 1, cytokine tail vein injection was not performed after each immunization, and the remaining steps and methods were the same as Example 1.

[0076] Control group 2: Compared with Example 1, after the initial immunization of subcutaneous site injection, the plasmids expressing cytokines: pCAGGS-mFlt3L and pCAGGS-mGM-CSF were injected through the tail vein one week later, that is, compared with the experimental group, pCAGGS-mCCL20 was not injected.

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

[0078] Table 1 Comparison of immune effects

[0079]

[0080]

[0081] It can be seen from Table 1 that the immune effect of the cytokine group, ...

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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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