H7 avian influenza virus monoclonal antibody and application thereof

A monoclonal antibody and avian influenza virus technology, applied in the field of clinical immunology technology, to achieve the effects of shortening the test time, simple operation steps, and improving specificity and sensitivity

Active Publication Date: 2017-12-15
WUHAN KEQIAN BIOLOGY CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] At present, there have been reports on the application of monoclonal antibodies to H7 subtype avian influenza virus, mainly in the application of colloidal gold test strips, indirect immunofluorescence, and immunohistochemistry. No report

Method used

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  • H7 avian influenza virus monoclonal antibody and application thereof
  • H7 avian influenza virus monoclonal antibody and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0019] Obtaining of hybridoma cell line 1H11:

[0020] Establishment of monoclonal antibody hybridoma cell lines

[0021] animal immunity

[0022] A bottle of H7 standard antigen purchased from Harvey was resuspended in 500 μL of normal saline, fully emulsified with an equal volume of Freund’s complete adjuvant, and injected subcutaneously with 200 μL / mouse of 8-week-old BALB / c mice; 2 weeks, Four weeks later, the antigens emulsified with Freund's incomplete adjuvant were used to immunize again in the same way; six weeks later, the same dose of antigen was injected intraperitoneally without adjuvant; four days later, they were fused.

[0023] cell fusion

[0024] Preparation of myeloma cells Select SP2 / 0 cells in good growth state, discard the supernatant when the density grows to 75% of the bottom of the culture bottle, wash once with incomplete DMEM medium, blow the cells gently with 10mL incomplete DMEM medium Down.

[0025] Preparation of splenic lymphocytes Take mice ...

Embodiment 2

[0033] Preparation and potency determination of ascites:

[0034] Take 10-week-old BALB / c mice, intraperitoneally inject sterilized liquid paraffin, 0.5 mL / mouse; 1 week later, intraperitoneally inject hybridoma cells 1H11 in logarithmic growth phase diluted with PBS, 5×10 5 One per mouse; when the abdomen of the mouse was obviously raised, ascites was collected from the abdominal cavity with a 16# needle, centrifuged at 2500g for 10 minutes, the fat tissue was removed, the supernatant was absorbed, and stored at -70°C for later use.

[0035] Characterization of mAbs:

[0036] Determination of ascites titer Two methods of indirect ELISA and HI test were used to detect the titer of ascites, and the results are as follows:

[0037]

[0038] Subclass identification was carried out according to the method introduced in the instruction manual of the monoclonal antibody subclass kit, and the results showed that monoclonal antibody 1H11 was IgG 1 subclass.

Embodiment 3

[0040] Stability identification of hybridoma cell 1H11

[0041] The obtained hybridoma cell line 1H11 was continuously passaged for 15 generations, and the titer of the cell supernatant was determined by HI test. The results showed that the cell line can stably secrete antibodies, and the HI antibody titers of the cell culture supernatant were all above 2 7 above.

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PUM

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Abstract

The invention belongs to the technical field of clinical immunology, and concretely discloses a H7 avian influenza virus monoclonal antibody and an application thereof. The monoclonal antibody is secreted by a hybridoma cell 1H11, and the hybridoma cell is preserved in CCTCC with the preservation number of CCTCC NO:C2017112. The monoclonal antibody has the advantages of high titer, good specificity and good blocking effect, and can be used for preparing an H7 subtype avian influenza blocking ELISA antibody detection kit. The kit is used for testing a sample to be tested, and has a coincidence rate of 98% or above with HI test; and compared with the HI, the kit has the advantages of shortening of the test time, and simple operating steps.

Description

technical field [0001] The invention belongs to the technical field of clinical immunology, relates to the technical field of detection and identification of H7 avian influenza virus, specifically relates to a monoclonal antibody of H7 avian influenza virus, and also relates to a hybridoma cell secreting a monoclonal antibody of H7 avian influenza virus , also relates to a preparation method of H7 avian influenza virus monoclonal antibody, and also relates to the application of the H7 avian influenza virus monoclonal antibody in the preparation and detection of H7 avian influenza virus kit. Background technique [0002] Avian influenza is an acute respiratory infectious disease caused by avian influenza virus, which has the characteristics of strong infectivity, fast transmission speed and easy mutation of antigen. Avian influenza virus belongs to the Orthomyxoviridae family Influenzavirus, its genome has 8 segments of single-stranded negative-sense RNA, encoding 11 known pr...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/20C07K16/10G01N33/569G01N33/577
CPCC07K16/1018C07K2317/35C07K2317/76G01N33/56983G01N33/577
Inventor 范俊青但汉并贾慧勤周云朵韦奇勇李盼盼董晓辉徐高原
Owner WUHAN KEQIAN BIOLOGY CO LTD
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