Method for rapidly preparing Zika-virus specific full human monoclonal antibodies and application

A monoclonal antibody, Zika virus technology, applied in the field of medicine and biology, can solve problems such as unfavorable large-scale promotion and application, loss, etc., and achieve the effects of simple and fast method, improved efficiency and time saving

Inactive Publication Date: 2017-03-08
GUANGZHOU EIGHTH PEOPLES HOSPITAL
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  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This method generally selects the memory cells in the peripheral blood mononuclear cells of convalescent patients. It can only screen for known antigens, but it will lose some important functional antibodies that do not bind to recombinant antigen proteins but have the effect of neutralizing viruses. , which is not conducive to the large-scale promotion and application of the method

Method used

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  • Method for rapidly preparing Zika-virus specific full human monoclonal antibodies and application
  • Method for rapidly preparing Zika-virus specific full human monoclonal antibodies and application
  • Method for rapidly preparing Zika-virus specific full human monoclonal antibodies and application

Examples

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Embodiment 1

[0030] Example 1. A method for rapidly preparing Zika virus-specific fully human monoclonal antibodies

[0031] S1 Peripheral blood mononuclear cell isolation: take EDTA anticoagulated blood from the peripheral vein of Zika-infected patients in the acute stage, and use density gradient centrifugation to separate peripheral blood mononuclear cells, and aliquot 5×10 6 / tube, placed in liquid nitrogen for freezing;

[0032] S2 Plasma cell separation: Place the peripheral blood mononuclear cells frozen in step S1 in a 37°C water bath to dissolve, wash 3 times with PBS buffer, then add fluorescent-labeled antibody for staining, incubate at room temperature for 15 minutes in the dark, use PBS buffer After washing, 400 μl of PBS buffer was added to suspend the cells on a flow cytometer, and a single plasma cell was sorted out and frozen in a 96-well PCR plate; the antibodies were IgD, CD19, CD27, CD38, IgM, and CD45, and the labeled fluorescent For FITC, ECD, PC7, APC A750, PB and ...

Embodiment 2

[0037] Example 2. A specific operation step for rapid preparation of Zika virus-specific fully human monoclonal antibody

[0038] S1 Peripheral blood mononuclear cell isolation: take EDTA anticoagulated blood from the peripheral vein of Zika-infected patients in the acute stage, and use density gradient centrifugation to separate peripheral blood mononuclear cells, and aliquot 5×10 6 / tube and stored in liquid nitrogen.

[0039] S2 Plasma cell separation: Place the peripheral blood mononuclear cells frozen in step S1 in a water bath at 37°C to dissolve, wash 3 times with PBS buffer, then add antibodies for staining, incubate at room temperature in the dark for 15 minutes, wash with PBS buffer , add 400 μl of PBS buffer to suspend the cells and put them on the flow cytometer, which is the Beckman Coulter MoFlo Astrios EQ ultra-high-speed flow cytometry sorting system, fluorescently labeled antibody staining: set up 9 analysis tubes, as shown in Table 1 , tubes 1-7 add corresp...

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Abstract

The invention belongs to the field of medical biology, and particularly relates to a method for rapidly preparing Zika-virus specific full human monoclonal antibodies and an application. The method for rapidly preparing the Zika-virus specific full human monoclonal antibodies includes the steps of peripheral blood mononuclear cell separating, plasma cell separating, antibody-variable-region-gene PCR amplification, antibody cloning, cotransfection and identifying and the like. The method for rapidly preparing the Zika-virus specific full human monoclonal antibodies is simple and fast and convenient, antigens do not need to be marked, functional antibodies of conformation structural domains which exists in vivo and is difficult to emulate in vitro can be separated, the obtained specific antibodies have important guiding significance on researching of Zika vaccine, and some antibodies can have clinic-treatment application prospects.

Description

technical field [0001] The invention belongs to the field of medicine and biology, and in particular relates to a method and application for rapidly preparing Zika virus-specific fully human monoclonal antibodies. Background technique [0002] Monoclonal antibodies (monoclonal antibodies, mAbs) are undoubtedly the most promising type of biological agents for treatment and diagnosis. Currently, monoclonal antibodies have been used in the diagnosis and treatment of tumors, autoimmune diseases and infectious diseases. With the application of monoclonal antibodies more and more widely, the preparation technology of monoclonal antibodies has also been developed rapidly. Chen Hui published a paper titled "Using Mouse Hybridoma Technology to Produce Measles Virus Antibody", in which BALB / e mouse myeloma p3x63Ags cells were immunized with measles virus LEC strain passaged by vero cells from syngeneic mice Splenocytes were hybridized under the action of PEG. The hybridized cells we...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/10C12N15/13C12N15/85G01N33/577G01N33/569A61K39/42A61P31/14
CPCA61K2039/505C07K16/1081G01N33/56983G01N33/577G01N2800/26Y02A50/30
Inventor 庾蕾张复春尹炽标管永军
Owner GUANGZHOU EIGHTH PEOPLES HOSPITAL
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