title: Array-in-well binding assay for multiparameter screening of phage displayed antibodies.
abstract: Phage display is a well-established and powerful tool for the development of recombinant antibodies. In a standard phage display selection process using a high quality antibody phage library, a large number of unique antibody clones can be generated in short time. However, the pace of the antibody discovery project eventually depends on the methodologies used in the next screening phase to identify the clones with the most promising binding characteristics e.g., in terms of specificity, affinity and epitope. Here, we report an array-in-well binding assay, a miniaturized and multiplexed immunoassay that integrates the epitope mapping to the evaluation of the binding activity of phage displayed antibody fragments in a single well. The array-in-well assay design used here incorporates a set of partially overlapping 15-mer peptides covering the complete primary sequence of the target antigen, the intact antigen itself and appropriate controls printed as an array with 10×10 layout at the bottom of a well of a 96-well microtiter plate. The streptavidin-coated surface of the well facilitates the immobilization of the biotinylated analytes as well-confined spots. Phage displayed antibody fragments bound to the analyte spots are traced using anti-phage antibody labelled with horseradish peroxidase for tyramide signal amplification based highly sensitive detection. In this study, we generated scFv antibodies against HIV-1 p24 protein using a synthetic antibody phage library, evaluated the binders with array-in-well binding assay and further classified them into epitopic families based on their capacity to recognize linear epitopes. The array-in-well assay enables the integration of epitope mapping to the screening assay for early classification of antibodies with simplicity and speed of a standard ELISA procedure to advance the antibody development projects.
authors.affiliation: Department of Biochemistry/Biotechnology, University of Turku, Tykistökatu 6A, FI-20520 Turku, Finland.
authors.forename: Susan
authors.initials: S
authors.lastname: Pérez-Gamarra
authors.affiliation: Medical Biotechnology Centre, VTT Technical Research Centre of Finland, Espoo FI-02044 VTT, Finland.
authors.forename: Liisa
authors.initials: L
authors.lastname: Hattara
authors.affiliation: Centre for Biodesign and Diagnostics, Translational Health Science and Technology Institute, NCR Biotech Science Cluster, Faridabad 122001, India.
authors.forename: Gaurav
authors.initials: G
authors.lastname: Batra
authors.affiliation: Medical Biotechnology Centre, VTT Technical Research Centre of Finland, Espoo FI-02044 VTT, Finland.
authors.forename: Petri
authors.initials: P
authors.lastname: Saviranta
authors.affiliation: Department of Biochemistry/Biotechnology, University of Turku, Tykistökatu 6A, FI-20520 Turku, Finland. Electronic address: urplammi@utu.fi.
authors.forename: Urpo
authors.initials: U
authors.lastname: Lamminmäki
mesh_terms: D000595:Amino Acid Sequence; D000915:Antibody Affinity; D000918:Antibody Specificity; D000941:Antigens / Q000737:chemistry* / Q000276:immunology; D001710:Biotin / Q000737:chemistry; D004797:Enzyme-Linked Immunosorbent Assay; D018604:Epitope Mapping / Q000379:methods*; D000939:Epitopes; D016655:HIV Core Protein p24 / Q000737:chemistry* / Q000276:immunology; D006735:Horseradish Peroxidase / Q000737:chemistry; D019151:Peptide Library; D011327:Printing; D040081:Protein Array Analysis / Q000295:instrumentation*; D011485:Protein Binding; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D057127:Single-Chain Antibodies / Q000096:biosynthesis / Q000302:isolation & purification*; D013194:Staining and Labeling / Q000379:methods; D019809:Streptavidin / Q000737:chemistry
chemical_list: D000941:Antigens; D000939:Epitopes; D016655:HIV Core Protein p24; D019151:Peptide Library; D011994:Recombinant Proteins; D057127:Single-Chain Antibodies; C514799:p24 protein, Human Immunodeficiency Virus Type 1; D001710:Biotin; D019809:Streptavidin; D006735:Horseradish Peroxidase
keywords: Antibody discovery; Antibody phage library; Epitope mapping; Overlapping peptide libraries; Protein array