Polypeptide used for preparing anaphylactoid reaction double-antibody sandwich kit paired antibody and application thereof

A double-antibody sandwich and paired antibody technology, applied in the field of biomedicine

Active Publication Date: 2019-09-17
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 MRGPRX2, even more so than the clinical detection kits for MRGPRX2.

Method used

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  • Polypeptide used for preparing anaphylactoid reaction double-antibody sandwich kit paired antibody and application thereof
  • Polypeptide used for preparing anaphylactoid reaction double-antibody sandwich kit paired antibody and application thereof
  • Polypeptide used for preparing anaphylactoid reaction double-antibody sandwich kit paired antibody and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Example 1 Synthesis of the Antigen Peptide of the Class 1 Anaphylaxis Specific Receptor MRGPRX2 Protein

[0033] DNAstar analysis software was used to predict and analyze the epitopes of human MRGPRX2 amino acid sequence, such as protein hydrophilicity, sequence flexibility, protein surface accessibility, and protein antigenic index, and finally determined the 134-144 and 286-330 positions For the target, the amino acid sequence is (134-144) IWYRCRRPRH and (286-330) RKQWRLQQPILKLALQRALQDIAEVDHSEGCFRQGTPEMRSSLV. 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...

Embodiment 2

[0035]Embodiment 2 Preparation of anti-polypeptide mouse monoclonal antibody

[0036] 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

[0037] Example 3 Antibody identification using indirect ELISA method to detect monoclonal antibody titer

[0038] 1) Microtiter plate preparation: After assembling the microtiter plate, prepare for antigen coating.

[0039] 2) Antigen dilution and coating: Add the prepared antigen working solution into the concave sampling tank, take 100 μL of the antigen working solution with a 300 μL range 8-hole pipette and add it to the bottom of the microplate well. After adding the sample, cover it Aluminum foil paper, incubate in an oven at 37°C for 2h or overnight at 4°C.

[0040] 3) Plate washing: Take out the ELISA microplate plate coated with antigen, turn it upside down to remove the liquid in the well, and then place it on a clean absorbent towel to drain; add 200ml TBST to each well with a drain gun until it is full but not If it overflows, after adding the whole plate and let it stand for 3 minutes, shake out the TBST for 3 times, then place it on a clean absorbent towel to dra...

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Abstract

The present invention discloses a polypeptide used for preparing an anaphylactoid reaction double-antibody sandwich kit paired antibody and an application thereof, and belongs to the technical field of biomedicines. The polypeptide of the double-antibody sandwich kit paired antibody comprises a polypeptide used for preparing a mouse monoclonal antibody and a polypeptide used for preparing a rabbit polyclonal antibody; an amino acid sequence of the polypeptide used for preparing the mouse monoclonal antibody is shown in SEQ ID NO:1; and an amino acid sequence of the polypeptide used for preparing the rabbit polyclonal antibody is shown in SEQ ID NO:2. The polypeptide is an immunogenic polypeptide of an anaphylactoid reaction specific receptor MRGPRX2 protein, can be used for preparing an ELISA mouse monoclonal coating antibody and a rabbit polyclonal detection antibody, successfully prepares a double-antibody sandwich ELISA, uses the monoclonal antibody as the coating antibody and the polyclonal antibody as the detection antibody, verifies clinical applications, contributes to research work of the anaphylactoid reaction specific receptor MRGPRX2, and is of great significance for clinically guiding safety of drug use.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, and relates to a polypeptide for preparing a paired antibody of a double-antibody sandwich kit for pseudoanaphylaxis and an application thereof. Background technique [0002] Anaphylactoid reaction (anaphylactoid reaction or pseudo-allergy reaction) refers to that when the drug is used for the first time, it can cause allergic-like symptoms in the patient's body. Respiratory system symptoms such as increased blood pressure and dyspnea, digestive system symptoms such as salivation, nausea, vomiting, diarrhea, anorexia, etc., can cause respiratory and circulatory failure in severe cases, resulting in shock or even death of the patient. Because anaphylactoid reactions have similar symptoms to type I anaphylaxis, they are often mistaken for type I anaphylaxis, but they are essentially different from type I anaphylaxis. Symptoms of anaphylactoid reactions usually appear within a few minutes after...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/72G01N33/68
CPCC07K14/723G01N33/6893G01N2333/726G01N2800/7004
Inventor 贺浪冲王楠张涛丁园园刘瑞韩省力
Owner 于向东
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