A novel strategy for generating monoclonal antibodies from single, isolated lymphocytes producing antibodies of defined specificities.
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, and is applicable to the generation of monoclonal antibodies from many species, including humans.
Babcook
J S
JS
The Biomedical Research Centre, The University of British Columbia, Vancouver, Canada.
Leslie
K B
KB
Olsen
O A
OA
Salmon
R A
RA
Schrader
J W
JW
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis*; D000918:Antibody Specificity; D001483:Base Sequence; D003587:Cytomegalovirus; D017931:DNA Primers; D018076:DNA, Complementary; D006023:Glycoproteins / Q000032:analysis / Q000276:immunology; D015699:HIV Envelope Protein gp120 / Q000032:analysis / Q000276:immunology; D015497:HIV-1; D006462:Hemolytic Plaque Technique; D006801:Humans; D007143:Immunoglobulin Heavy Chains / Q000096:biosynthesis; D007147:Immunoglobulin Light Chains / Q000096:biosynthesis; D007135:Immunoglobulin Variable Region / Q000096:biosynthesis; D007158:Immunologic Techniques; D051379:Mice; D008969:Molecular Sequence Data; D009101:Multiple Myeloma; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000276:immunology; D016133:Polymerase Chain Reaction / Q000379:methods; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D012756:Sheep; D014759:Viral Envelope Proteins / Q000032:analysis / Q000276:immunology
D000911:Antibodies, Monoclonal; D017931:DNA Primers; D018076:DNA, Complementary; D006023:Glycoproteins; D015699:HIV Envelope Protein gp120; D007143:Immunoglobulin Heavy Chains; D007147:Immunoglobulin Light Chains; D007135:Immunoglobulin Variable Region; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D014759:Viral Envelope Proteins; C101488:cytomegalovirus envelope protein gp116