Isolation of cross-reacting antigen candidates by mRNA-display using a mixed cDNA library.
The identification of cross-reacting antigens is an important process in the characterization of antibodies. We describe here the application of mRNA-display technology with a cDNA library for the isolation of cross-reacting antigens using a monoclonal antibody against human tumor protein p53 (hTP53) as a model. A mixed cDNA library constructed from mRNAs prepared from several human tissues and cell-lines was used for the mRNA-display. After several rounds of panning, five annotated polypeptides, topoisomeraseII-binding protein 1 (TOBP1), RAS protein activator like 2 isoform 1 (RASAL2), endosome-associated FYVE-domain protein (ZFYVE16), and utrophin (UTRN) as well as hTP53, were identified as cross-reactive antigen candidates. All of them had a consensus motif, -S-D-L-( )-K-L-, which was included in the known epitope of the antibody. They were synthesized in vitro, and their binding was compared by conducting a pull-down assay. In cross-activity to the antibody, they ranked as follows: ZYVE16 congruent with hTP53 congruent with TOBP1 > UTRN > RASAL2.
Shibui
Tatsuro
T
Molecuence Corporation, Mitsubishi Chemical Group Yokohama Research Center, 1000 Kamoshida-cho, Aoba-ku, Yokohama, Kanagawa 227-8502, Japan. 1807766@cc.m-kagaku.co.jp
Kobayashi
Teruaki
T
Shiratori
Miwa
M
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000906:Antibodies / Q000276:immunology; D000941:Antigens / Q000737:chemistry / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification; D002352:Carrier Proteins / Q000737:chemistry / Q000276:immunology; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D003429:Cross Reactions; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000276:immunology; D000939:Epitopes / Q000235:genetics / Q000276:immunology; D020869:Gene Expression Profiling / Q000379:methods*; D015723:Gene Library; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000737:chemistry / Q000276:immunology; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000737:chemistry / Q000276:immunology; D010455:Peptides; D016133:Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D012697:Serine Endopeptidases / Q000737:chemistry / Q000276:immunology; D016159:Tumor Suppressor Protein p53 / Q000737:chemistry / Q000276:immunology*; D049411:Utrophin / Q000737:chemistry / Q000276:immunology; D020703:ras GTPase-Activating Proteins / Q000737:chemistry / Q000276:immunology
D000906:Antibodies; D000941:Antigens; D002352:Carrier Proteins; D004268:DNA-Binding Proteins; D000939:Epitopes; D047908:Intracellular Signaling Peptides and Proteins; D009687:Nuclear Proteins; D010455:Peptides; C110513:TOPBP1 protein, human; D016159:Tumor Suppressor Protein p53; C483599:UTRN protein, human; D049411:Utrophin; D020703:ras GTPase-Activating Proteins; C116420:ZFYVE16 protein, human; D012697:Serine Endopeptidases