Efficient generation of monoclonal antibodies against peptide in the context of MHCII using magnetic enrichment.
Monoclonal antibodies specific for foreign antigens, auto-antigens, allogeneic antigens and tumour neo-antigens in the context of major histocompatibility complex II (MHCII) are highly desirable as novel immunotherapeutics. However, there is no standard protocol for the efficient generation of monoclonal antibodies that recognize peptide in the context of MHCII, and only a limited number of such reagents exist. In this report, we describe an approach for the generation and screening of monoclonal antibodies specific for peptide bound to MHCII. This approach exploits the use of recombinant peptide:MHC monomers as immunogens, and subsequently relies on multimers to pre-screen and magnetically enrich the responding antigen-specific B cells before fusion and validation, thus saving significant time and reagents. Using this method, we have generated two antibodies enabling us to interrogate antigen presentation and T-cell activation. This methodology sets the standard to generate monoclonal antibodies against the peptide-MHCII complexes.
Department of Medicine, Center for Immunology, University of Minnesota Medical School, Minneapolis, Minnesota 55455, USA.
Justin A
JA
Spanier
Department of Microbiology and Immunology, Tulane University School of Medicine, New Orleans, Louisiana 70112, USA.
Daniel R
DR
Frederick
Department of Microbiology and Immunology, Center for Immunology, University of Minnesota Medical School, Minneapolis, Minnesota 55455, USA.
Justin J
JJ
Taylor
Department of Medicine, Center for Immunology, University of Minnesota Medical School, Minneapolis, Minnesota 55455, USA.
James R
JR
Heffernan
Department of Microbiology and Immunology, Center for Immunology, University of Minnesota Medical School, Minneapolis, Minnesota 55455, USA.
Dmitri I
DI
Kotov
Department of Medicine, Center for Immunology, University of Minnesota Medical School, Minneapolis, Minnesota 55455, USA.
Tijana
T
Martinov
Department of Medicine, Center for Immunology, University of Minnesota Medical School, Minneapolis, Minnesota 55455, USA.
Kevin C
KC
Osum
Department of Medicine, Center for Immunology, University of Minnesota Medical School, Minneapolis, Minnesota 55455, USA.
Jenna L
JL
Ruggiero
Department of Biochemistry, Tulane University School of Medicine, New Orleans, Louisiana 70112, USA.
Blake J
BJ
Rust
Department of Biochemistry, Tulane University School of Medicine, New Orleans, Louisiana 70112, USA.
Samuel J
SJ
Landry
Department of Microbiology and Immunology, Center for Immunology, University of Minnesota Medical School, Minneapolis, Minnesota 55455, USA.
Marc K
MK
Jenkins
Department of Microbiology and Immunology, Tulane University School of Medicine, New Orleans, Louisiana 70112, USA.
James B
JB
McLachlan
Department of Medicine, Center for Immunology, University of Minnesota Medical School, Minneapolis, Minnesota 55455, USA.
Brian T
0000-0001-9826-5637
BT
Fife
D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis*; D000915:Antibody Affinity; D049109:Cell Proliferation; D000949:Histocompatibility Antigens Class II / Q000378:metabolism*; D006801:Humans; D006825:Hybridomas / Q000378:metabolism; D008280:Magnetics / Q000379:methods*; D008810:Mice, Inbred C57BL; D010455:Peptides / Q000276:immunology*; D010641:Phenotype; D015203:Reproducibility of Results
D000911:Antibodies, Monoclonal; D000949:Histocompatibility Antigens Class II; D010455:Peptides