Identification of Env-specific monoclonal antibodies from Chinese HIV-1 infected person by magnetic beads separating B cells and single cell RT-PCR cloning.
- Author:
Xiang-Ying HUANG
1
;
Shuang-Qing YU
;
Zhan CHENG
;
Jing-Rong YE
;
Ke XU
;
Xia FENG
;
Yi ZENG
Author Information
- Publication Type:Journal Article
- MeSH: Amino Acid Sequence; Antibodies, Monoclonal; isolation & purification; B-Lymphocytes; immunology; HIV Antibodies; isolation & purification; HIV Envelope Protein gp120; immunology; HIV-1; immunology; Immunomagnetic Separation; methods; Molecular Sequence Data; Reverse Transcriptase Polymerase Chain Reaction; methods
- From: Chinese Journal of Experimental and Clinical Virology 2013;27(2):123-125
- CountryChina
- Language:Chinese
-
Abstract:
OBJECTIVETo establish a simple and practical method for screening of Env-specific monoclonal antibodies from HIV-1 infected individuals.
METHODSHuman B cells were purified by negative sorting from PBMCs and memory B cells were further enriched using anti-CD27 microbeads. Gp120 antigen labbled with biotin was incubated with memory B cells to specifically bind IgG on cells membrane. The memory B cells expressing the Env-specific antibody were harvested by magnetic beads separating, counted and diluted to the level of single cell in each PCR well that loading with catch buffer containing RNase inhibitor to get RNAs. The antibody genes were amplified by single cell RT-PCR and nested PCR, cloned into eukaryotic expression vectors and transfected into 293T cells. The binding activity of recombinant antibodies to Env were tested by ELISA.
RESULTSThree monocolonal Env-specific antibodies were isolated from one HIV-1 infected individual.
CONCLUSIONWe can obtain Env-specific antibody by biotin labbled antigen, magnetic beads separating technique coupled with single cell RT-PCR and expression cloning.
