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Quick quantitative detection method and kit for human copeptin

A quantitative detection and kit technology, applied in the field of biomedicine, can solve the problems of limiting the clinical value of CPP, no clinical use, long detection time, etc., and achieve the effect of increasing clinical application value, increasing immune response, and improving effective presentation

Inactive Publication Date: 2019-04-09
GUANGZHOU WONDFO BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although their sensitivity can meet the requirements, the detection time is too long, so it is only used for scientific research, not for clinical use
Up to now, the point-of-care test (POCT) of CPP is still not reported, thus it largely limits the clinical value of CPP

Method used

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  • Quick quantitative detection method and kit for human copeptin
  • Quick quantitative detection method and kit for human copeptin
  • Quick quantitative detection method and kit for human copeptin

Examples

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preparation example Construction

[0042] The present invention provides a kit for rapid quantitative detection of human and peptidin, which is characterized by comprising: a human and peptidin monoclonal antibody, and the anti-human and peptidin monoclonal antibody is prepared by the following method:

[0043]Immunize mice with human and peptidin antigens; deliver cytokine-expressing plasmids to immunized mice; collect spleen cells or lymph node cells from the plasmid-delivered mice, and combine the spleen cells or lymph node cells with mouse bone marrow The tumor cells are fused to obtain hybridoma cells, which are cultured and secreted to obtain anti-human and peptidin monoclonal antibodies.

[0044] Optionally, the human and peptidin 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 human and peptidin antigens are human and peptidin antigens coupled with macromolecular proteins, and the macromolecular proteins are preferably at least one ...

Embodiment 1

[0060] Example 1 Preparation method of high-affinity anti-human and peptidin monoclonal antibody

[0061] 1.1 Peptide synthesis

[0062] 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, and KLH-C-cpp. The amino acid sequence is:

[0063] cppF: ATQLDGPAGALLLRLVQLAGAPEFEPAQPDAY (SEQ ID NO: 1),

[0064] N-cpp: CATQLDGPAGALLLRLV (SEQ ID NO: 2),

[0065] C-cpp: CLAGAPEPFEPAQPDAY (SEQ ID NO:3)

[0066] That is, the complete antigen obtained by coupling KLH is:

[0067]KLH-cppF(KLH-C-ATQLDGPAGALLLRLVQLAGAPEFEPAQPDAY);

[0068] KLH-N-cpp(KLH-C-ATQLDGPAGALLLRLV);

[0069] KLH-C-cpp(KLH-C-LAGAPEPFEPAQPDAY);

[0070] The peptide synthesis and coupling were carried out by conventional methods, and were entrusted to Gill Biochemical Shanghai Co., Ltd. The purity was determined by high performance liquid chromatography (HPLC), and all were >95%.

[0071] 1.2 Plasmid construction

[...

Embodiment 2

[0087] Embodiment 2 Immune effect and antibody titer determination

[0088] Cytokine group: The immunization strategy described in Example 1: that is, a cytokine tail vein injection was performed once a week after each subcutaneous immunization.

[0089] Control group 1: Compared with Example 1, no cytokine tail vein injection was performed after each immunization, and the remaining steps were the same as those of Example 1.

[0090] Control group 2: Compared with Example 1, the plasmids expressing cytokines: pCAGGS-mFlt3L and pCAGGS-mGM-CSF were injected through the tail vein one week after the initial immunization of the subcutaneous injection of the site, i.e., compared with the experimental group, pCAGGS-mCCL20 was not injected.

[0091] The comparison results of the immune effects of the above two groups are shown in Table 1.

[0092] Table 1 Comparison of immune effects

[0093]

[0094]

[0095] As can be seen from Table 1, the cytokine group was immunized by t...

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Abstract

The invention provides a method for detecting CPP (Copeptin) by a full-automatic chemiLuminescence method on the basis of MPs (magnetic particles). Common immunity and immunoregulation molecules are combined, cell factors Flt3l (Mouse fms-like tyrosinekinase 3ligand), mGM-CSF (Mouse Granulocyte Macrophage Colony Stimulating Factor) and CCL20 (Mouse CC subtype chemokine ligand 20) are assisted in delivering while conventional common immunity is carried out to obtain an antibody with high affinity, the antibody is combined with magnetic separation, a magnetic bead has a large superficial area and a large area ratio, so that the CPP with the low concentration can be effectively enriched, and therefore, CPP in human serums can be quickly and sensitively measured. The measurement time, the sensitivity and the specificity of the CPP can be obviously improved.

Description

technical field [0001] The invention belongs to the field of biomedicine, and particularly relates to a rapid quantitative detection method and kit of human and peptidin. Background technique [0002] In response to the need for more rapid diagnosis, more accurate prognostic assessment and treatment decisions of various diseases, research on new biomarkers has been developed. Arginine vasopressin (AVP) is one of the major hormones of the hypothalamic-pituitary-adrenal axis. The AVP system is sensitive to the stimuli that the body is exposed to endogenous stress, and its level can sensitively reflect the degree of stress in the body in a disease state. However, because AVP is unstable and mainly adheres to the surface of platelets, and is rapidly cleared by the body after secretion, there are doubts about the reliability of AVP detection methodology. Copeptin (CPP) is a polypeptide containing 39 amino acid residues homologous to arginine vasopressin (AVP) and is the C-termi...

Claims

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Application Information

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IPC IPC(8): G01N33/68G01N33/577G01N33/531
CPCG01N33/531G01N33/577G01N33/6803
Inventor 王羽康业崔跃吴培钿何小维王继华
Owner GUANGZHOU WONDFO BIOTECH
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