Rapid identification of monospecific monoclonal antibodies using a human proteome microarray.
To broaden the range of tools available for proteomic research, we generated a library of 16,368 unique full-length human ORFs that are expressible as N-terminal GST-His(6) fusion proteins. Following expression in yeast, these proteins were then individually purified and used to construct a human proteome microarray. To demonstrate the usefulness of this reagent, we developed a streamlined strategy for the production of monospecific monoclonal antibodies that used immunization with live human cells and microarray-based analysis of antibody specificity as its central components. We showed that microarray-based analysis of antibody specificity can be performed efficiently using a two-dimensional pooling strategy. We also demonstrated that our immunization and selection strategies result in a large fraction of monospecific monoclonal antibodies that are both immunoblot and immunoprecipitation grade. Our data indicate that the pipeline provides a robust platform for the generation of monoclonal antibodies of exceptional specificity.
Department of Pharmacology, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA.
Jun Seop
JS
Jeong
Lizhi
L
Jiang
Edisa
E
Albino
Josean
J
Marrero
Hee Sool
HS
Rho
Jianfei
J
Hu
Shaohui
S
Hu
Carlos
C
Vera
Diane
D
Bayron-Poueymiroy
Zully Ann
ZA
Rivera-Pacheco
Leonardo
L
Ramos
Cecil
C
Torres-Castro
Jiang
J
Qian
Joseph
J
Bonaventura
Jef D
JD
Boeke
Wendy Y
WY
Yap
Ignacio
I
Pino
Daniel J
DJ
Eichinger
Heng
H
Zhu
Seth
S
Blackshaw
D000818:Animals; D058846:Antibodies, Monoclonal, Murine-Derived / Q000276:immunology*; D000918:Antibody Specificity; D000941:Antigens / Q000737:chemistry / Q000276:immunology; D045744:Cell Line, Tumor; D006801:Humans; D006825:Hybridomas; D055767:Immobilized Proteins / Q000737:chemistry / Q000276:immunology; D051379:Mice; D008807:Mice, Inbred BALB C; D040081:Protein Array Analysis; D020543:Proteome / Q000737:chemistry / Q000276:immunology*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000276:immunology*
D058846:Antibodies, Monoclonal, Murine-Derived; D000941:Antigens; D055767:Immobilized Proteins; D020543:Proteome; D011993:Recombinant Fusion Proteins