A novel strategy for generating monoclonal antibodies from single, isolated lymphocytes producing antibodies of defined specificities

被引:103
作者
Babcook, JS [1 ]
Leslie, KB [1 ]
Olsen, OA [1 ]
Salmon, RA [1 ]
Schrader, JW [1 ]
机构
[1] UNIV BRITISH COLUMBIA, BIOMED RES CTR, VANCOUVER, BC V6T 1Z3, CANADA
关键词
PCR; antibody-forming cells; V-H and V-L genes; immunoglobulin; plaque assays;
D O I
10.1073/pnas.93.15.7843
中图分类号
O [数理科学和化学]; P [天文学、地球科学]; Q [生物科学]; N [自然科学总论];
学科分类号
07 ; 0710 ; 09 ;
摘要
We report a novel approach to the generation of monoclonal antibodies based on the molecular cloning and expression of immunoglobulin variable region cDNAs generated from single rabbit or murine lymphocytes that were selected for the production of specific antibodies. Single cells secreting antibodies for a specific peptide either from gp116 of the human cytomegalovirus or from gp120 of HIV-1 or for sheep red blood cells were selected using antigen-specific hemolytic plaque assays. Sheep red blood cells were coated with specific peptides in a procedure applicable to any antigen that can be biotinylated. Heavy- and light-chain variable region cDNAs were rescued from single cells by reverse transcription-PCR and expressed in the context of human immunoglobulin constant regions. These chimeric murine and rabbit monoclonal antibodies replicated the target specificities of the original antibody-forming cells. The selected lymphocyte antibody method exploits the in vivo mechanisms that generate high-affinity antibodies. This method can use lymphocytes from peripheral blood, can exploit a variety of procedures that identify individual lymphocytes producing a particular antibody, acid is applicable to the generation of monoclonal antibodies from many species, including humans.
引用
收藏
页码:7843 / 7848
页数:6
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