Isolation of cross-reacting antigen candidates by mRNA-display using a mixed cDNA library

• Published in: Biotechnology letters Vol. 30; no. 12; pp. 2037 - 2043
• Main Authors: Shibui, Tatsuro, Kobayashi, Teruaki, Shiratori, Miwa
• Format: Journal Article
• Language: English
• Published: Dordrecht Dordrecht : Springer Netherlands 01.12.2008; Springer Netherlands; Springer; Springer Nature B.V
• Subjects: Amino Acid Motifs; Amino Acid Sequence; Antibodies - immunology; Antigens; Antigens - chemistry; Antigens - genetics; Antigens - immunology; Antigens - isolation & purification; Applied Microbiology; Biochemistry; Biological and medical sciences; Biomedical and Life Sciences; Biomedical engineering; Biotechnology; Carrier Proteins - chemistry; Carrier Proteins - immunology; Chromatography, Affinity; Cloning, Molecular; Cross Reactions; Deoxyribonucleic acid; DNA; DNA-Binding Proteins - chemistry; DNA-Binding Proteins - immunology; Epitopes - genetics; Epitopes - immunology; Fundamental and applied biological sciences. Psychology; Gene Expression Profiling - methods; Gene Library; Humans; Intracellular Signaling Peptides and Proteins - chemistry; Intracellular Signaling Peptides and Proteins - immunology; Life Sciences; Microbiology; Molecular Sequence Data; Nuclear Proteins - chemistry; Nuclear Proteins - immunology; Original Research Paper; Peptides; Pharmacology; Polymerase Chain Reaction; ras GTPase-Activating Proteins - chemistry; ras GTPase-Activating Proteins - immunology; Ribonucleic acid; RNA; Sequence Analysis, DNA; Serine Endopeptidases - chemistry; Serine Endopeptidases - immunology; Tumor Suppressor Protein p53 - chemistry; Tumor Suppressor Protein p53 - immunology; Utrophin - chemistry; Utrophin - immunology; Antigen; mRNA-display; Antibody; cDNA library; Messenger RNA; Complementary DNA; Antibody, Antigen, cDNA library; Cross reaction; Isolation
• ISSN: 0141-5492; 1573-6776
• DOI: 10.1007/s10529-008-9803-5

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.