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Broad-spectrum neutralizing antibody against HIV as well as preparation method and application thereof

An antibody and broad-spectrum technology, applied in the fields of cellular immunology and genetic engineering, can solve problems such as the difficulty of producing antibodies and achieve good application prospects

Active Publication Date: 2021-01-26
SUZHOU HEALTH COLLEGE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This may be the reason why HIV broad-spectrum neutralizing antibodies are difficult to produce

Method used

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  • Broad-spectrum neutralizing antibody against HIV as well as preparation method and application thereof
  • Broad-spectrum neutralizing antibody against HIV as well as preparation method and application thereof
  • Broad-spectrum neutralizing antibody against HIV as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0086] The preparation of embodiment 1 anti-HIV antibody

[0087] 1. Sorting of mature B cells

[0088] Buffer1: 500ml 1×PBS+2ml 0.5M EDTA+25ml 10%BSA (containing 2mM EDTA, 0.5%BSA)

[0089] (1) Collect 200 μl of peripheral blood from humanized GTL mice and add 200 μl of Buffer1;

[0090] (2) Add 10 times the volume of ACK lysing buffer (Fisher / BioWhittaker), incubate at room temperature for 10 minutes, and centrifuge at 1500 rpm for 10 minutes;

[0091] (3) Discard the supernatant, add 10ml Buffer 1 to wash the cells, and centrifuge at 1500rpm for 10min;

[0092] (4) Discard the supernatant, add 10 μl Buffer 1 to resuspend the cells; add the following flow cytometry antibodies to label mature B cells:

[0093] anti-CD5 / FITC (UCHT2, eBioscience)

[0094] anti-CD19 / PE (SJ25-C1, BD Pharmingen)

[0095] anti-CD10 / APC (BC96, eBioscience)

[0096] anti-CD27 / PE-Cy7 (O323, eBioscience)

[0097] anti-IgM / PE-Cy5 (G20-127, BD Pharmingen)

[0098] (5) Sorting mature B cells (CD5-...

Embodiment 2

[0116] The detection of embodiment 2 antibody characteristics

[0117] 1. Self-reactivity detection

[0118] Antibody self-reactivity was detected using a clinical standard antinuclear antibody detection kit (QUANTA Lite TM ANAELISA, INOVA Diagnostics, Inc.) were tested, and negative reaction samples, low positive reaction samples, and high positive reaction samples were provided in the detection kit. 4E10 is an HIV broad-spectrum neutralizing antibody and also an autoreactive / polyreactive antibody. In the experiment, we prepared 4E10 scFv-Fc as a positive control antibody. The detection steps are as follows:

[0119] (1) Coating: the Elisa plate in the ANA kit has been coated with antigen;

[0120] (2) Primary antibody: Add 50 μl of 50 μg / ml scFv-Fc protein, react at room temperature for 2 hours;

[0121] (3) Secondary antibody: Discard the liquid in the well, wash 3 times with PBST, add HRP-labeled goat anti-human IgG-Fc antibody, and react at room temperature for 1 hour...

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PUM

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Abstract

The invention discloses a broad-spectrum neutralizing antibody against HIV as well as a preparation method and application thereof. Specifically, the invention relates to a heavy chain variable regions CDR-H1, CDR-H2 and CDR-H3 of amino acid sequences shown as SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3, and a light chain variable regions CDR-L1, CDR-L2 and CDR-L3 of amino acid sequences as shown asSEQ ID NO.5, SEQ ID NO.6 and SEQ ID NO.7. The invention also relates to nucleic acids encoding the antibody, vectors containing the nucleic acids, cells containing the vectors and pharmaceutical compositions. The invention also relates to the use of the antibody for the detection and treatment of HIV infection and HIV-related diseases.

Description

technical field [0001] The invention belongs to the fields of cellular immunology and genetic engineering, and relates to a broad-spectrum neutralizing antibody against HIV and its preparation method and application. Background technique [0002] Acquired Immunodeficiency Syndrome is AIDS (Acquired Immunodeficiency Syndrome, AIDS), which is caused by Human Immunodeficiency Virus (Human Immunodeficiency Virus Type, HIV) infection. HIV-1 has three main routes of transmission, namely blood transmission, mother-to-child transmission and sexual transmission. HIV mainly invades the immune system of the human body, and the immune function of the infected person will gradually weaken or even lose, which will allow various secondary infections to take advantage of the vacancy and further invade the human body, eventually leading to death. AIDS has seriously endangered the health and safety of the people, and many medical researchers all over the world are investing a lot of manpower...

Claims

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Application Information

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IPC IPC(8): C07K16/10C12N15/13A61K39/395A61P31/18
CPCA61K2039/505A61P31/18C07K16/1045C07K2317/565
Inventor 常宏刘松柏
Owner SUZHOU HEALTH COLLEGE
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