Polypeptide for preparing ELISA mouse monoclonal coating antibody and rabbit polyclonal detection antibody and application thereof

A monoclonal antibody and polyclonal antibody technology, applied in the field of biomedicine, can solve the problem of blank clinical detection kits and related targeted drugs, and achieve the effect of strong immunogenicity

Active Publication Date: 2019-09-13
于向东
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there are still gaps in the antibodies and proteins for laboratory research against

Method used

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  • Polypeptide for preparing ELISA mouse monoclonal coating antibody and rabbit polyclonal detection antibody and application thereof
  • Polypeptide for preparing ELISA mouse monoclonal coating antibody and rabbit polyclonal detection antibody and application thereof
  • Polypeptide for preparing ELISA mouse monoclonal coating antibody and rabbit polyclonal detection antibody and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Example 1 Synthesis of the Antigen Peptide of the Class 1 Anaphylaxis Specific Receptor MRGPRX2 Protein

[0030] DNAstar analysis software was used to predict and analyze the epitope of human MRGPRX2 amino acid sequence, such as protein hydrophilicity, sequence flexibility, protein surface accessibility, and protein antigenic index, and finally determined the 286-330th position as the target, amino acid The sequence is:

[0031] RKQWRLQQPILKLALQRALQDIAEVDHSEGCFRQGTPEMRSRSSLV.

[0032] The manual solid-phase Fmoc method is used to synthesize from the C-terminal to the N-terminal direction to obtain the crude product of the target polypeptide. Purify the target polypeptide by using reversed-phase high-performance liquid chromatography (HPLC) method to separate different polypeptide molecules according to the difference in hydrophobicity. After freeze-drying the solvent, the pure polypeptide in a fluffy state was obtained. Its chemical structure was characterized by MALD...

Embodiment 2

[0034] Embodiment 2 Preparation of anti-polypeptide mouse monoclonal antibody

[0035] Prepare the conjugated KLH-polypeptide into an emulsified injection for immunization, and inject subcutaneously in points, spray alcohol on the back midline of the mouse, avoid the parts with immune swellings, and inject one injection into four times, respectively. 4 different points. After five immunizations, blood was collected from the tail vein of the mice to detect the titer of antiserum by indirect ELISA. The titer of ELISA antiserum reached 1:50000, indicating that the immunity was qualified. Select the mouse with the highest titer for fusion screening, and then carry out subcloning, and screen pure monoclonal cell lines according to the results of subcloning. The supernatant of the monoclonal cell line was injected into mice to prepare ascites, and the ascites was subjected to protein G affinity purification to obtain purified monoclonal antibodies.

Embodiment 3

[0036] Example 3 Antibody identification using indirect ELISA method to detect monoclonal antibody titer

[0037] 1) ELISA plate preparation: After assembling the ELISA plate, prepare for antigen coating.

[0038] 2) Antigen dilution and coating: Add the prepared antigen working solution into the concave sample addition tank, and use a 300 μL range 8-well pipette to take 100 μL of the antigen working solution and add it to the bottom of the well of the ELISA plate. Incubate in aluminum foil for 2 h at 37°C or overnight at 4°C.

[0039] 3) Wash the plate: Take out the ELISA plate that has been coated with the antigen, remove the liquid in the well by inverting it, and then place it on a clean absorbent towel to drain; add 200ml of TBST to each well with a drain gun until it is full but not enough. Overflow, after the whole plate is added and left for 3 minutes, the TBST is thrown out, thrown 3 times, and then placed on a clean absorbent towel to drain, and each plate is patted 6...

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Abstract

The invention discloses a polypeptide for preparing an ELISA mouse monoclonal coating antibody and a rabbit polyclonal detection antibody and application thereof, and belongs to the technical field ofbiomedicine. The polypeptide is a polypeptide which has immunogenicity on an anaphylactoid reaction specific receptor MRGPRX2 protein, and the amino acid sequence of the polypeptide is as shown in SEQ ID NO:1. The polypeptide is a polypeptide which has immunogenicity on an anaphylactoid reaction specific receptor MRGPRX2 protein, and can be used for preparing an ELISA mouse monoclonal coated antibody and a rabbit polyclonal detection antibody. A double-antibody sandwich ELISA method is successfully prepared, wherein the monoclonal antibody is a coating antibody, the polyclonal antibody is a detection antibody, and the application of the method in clinic is verified. The invention is beneficial to the research work of anaphylactoid reaction specific receptor MRGPRX2, and has important significance for clinically guiding the safety of medication.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, and relates to a polypeptide for preparing ELISA mouse monoclonal coating antibody and rabbit polyclonal detection antibody and application thereof. Background technique [0002] Drug-like anaphylaxis is a serious, potentially life-threatening allergic reaction. In 1920, Hanzlik and Karsner reported for the first time that the phenomenon similar to allergic symptoms caused by intravenous injection of jelly in humans or animals was called "anaphylactoid reaction". Between 1947 and 1949, Selye et al. also called the phenomenon of cyanosis and edema of the tongue, face and paws near the tongue, face and paws after the first intravenous injection of ovalbumin and other colloidal substances to rats, which was also called "anaphylactoid reaction". During 1986-1988, Toman et al. found that the typical allergic reactions such as itching, edema, restlessness and salivation after the first injection o...

Claims

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Application Information

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IPC IPC(8): C07K14/705C07K16/28G01N33/68
CPCC07K14/705C07K16/28G01N33/68G01N2333/705
Inventor 张涛丁园园刘瑞贺浪冲王楠马维娜李超美
Owner 于向东
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