title: Determination of complementary antibody pairs using protein A capture with the AlphaScreen assay format.
abstract: The utility of antibody reagents for the detection of specific cellular targets for both research and diagnostic applications is widespread and continually expanding. Often it is useful to develop specific antibodies as reagent pairs that distinguish different epitopes of the target such that sandwich enzyme-linked immunosorbent assay can be used for selective and specific detection. However, the identification of pairing antibodies is often cumbersome and labor-intensive even with the use of designed peptide-specific epitopes as antigens. We have developed a robust and high-throughput method for identifying pairing complementary antibodies derived either from commercial sources or during a rabbit hybridoma monoclonal screening and selection process using protein A capture with the AlphaScreen bead-based assay format. We demonstrate the value and effectiveness of this assay with three protein targets: Akt2, ATF3, and NAEβ (the β-subunit of the neddylation activation enzyme).
authors.affiliation: Millennium Pharmaceuticals, The Takeda Oncology Company, Cambridge, MA 02139, USA. michael.bembenek@mpi.com
authors.forename: Michael E
authors.initials: ME
authors.lastname: Bembenek
authors.forename: Anne
authors.initials: A
authors.lastname: Burkhardt
authors.forename: Jingya
authors.initials: J
authors.lastname: Ma
authors.forename: Zhi
authors.initials: Z
authors.lastname: Li
authors.forename: Huay-Keng
authors.initials: HK
authors.lastname: Loke
authors.forename: Dongyun
authors.initials: D
authors.lastname: Wu
authors.forename: Qing
authors.initials: Q
authors.lastname: Xu
authors.forename: Olga
authors.initials: O
authors.lastname: Tayber
authors.forename: Liying
authors.initials: L
authors.lastname: Xie
authors.forename: Ping
authors.initials: P
authors.lastname: Li
authors.forename: Li
authors.initials: L
authors.lastname: Li
mesh_terms: D051700:Activating Transcription Factor 3 / Q000737:chemistry / Q000276:immunology; D000818:Animals; D000906:Antibodies / Q000737:chemistry* / Q000276:immunology; D000911:Antibodies, Monoclonal / Q000737:chemistry / Q000276:immunology / Q000378:metabolism; D000939:Epitopes / Q000276:immunology; D006825:Hybridomas / Q000378:metabolism; D007118:Immunoassay / Q000379:methods*; D010455:Peptides / Q000276:immunology; D051057:Proto-Oncogene Proteins c-akt / Q000737:chemistry / Q000276:immunology; D011817:Rabbits; D013205:Staphylococcal Protein A / Q000737:chemistry*
chemical_list: D051700:Activating Transcription Factor 3; D000906:Antibodies; D000911:Antibodies, Monoclonal; D000939:Epitopes; D010455:Peptides; D013205:Staphylococcal Protein A; D051057:Proto-Oncogene Proteins c-akt