title: A Combinatory Antibody-Antigen Microarray Assay for High-Content Screening of Single-Chain Fragment Variable Clones from Recombinant Libraries.
abstract: We have developed a combinatory antibody-antigen microarray for direct screening of multiple single-chain fragment variable (scFv) clones with no need for pre-purification or enrichment before screening. The straightforward workflow allows for early selection of binders to predefined peptide and glycopeptide targets. A capture antibody is contact printed on microarray slides, side by side with the antigens of interest. A large number of scFv clones, in supernatants, are printed on top of the capture antibody and the antigen in a "spot-on-spot" print. The printed scFv clones, which bind to the capture antibody, are detected using biotinylated antigen, while the binding of scFv clones to the printed antigen is detected through a mouse anti-tag antibody. Two different analyses are thus performed on the same slide, generating two kinds of information: one on the ability of an individual scFv clone to bind to the soluble form of the antigen, which may favour selection for higher affinity rather than avidity, while the other allows the identification of large numbers of clones, simultaneously, due to the binding of scFv clones to densely presented antigens, thus providing an overall increased hit rate. The functionality of the new screening approach was illustrated through the generation of antibodies against peptides from the chaperone complex Ku70/Ku80 and the GalNAcα-serine/threonine epitope on the IgA1 alpha chain hinge region. In total, 659 scFv clones were screened with a hit rate of approximately 20%. This approach allowed the identification of functional antibodies in both cases, illustrating the usefulness and capacity of this combinatory microarray screening technique for efficient analysis and validation of antibodies at an early stage of antibody generation.
authors.affiliation: Chemical Glyco-Biology Laboratory, Department of Chemistry, Copenhagen University, Copenhagen, Denmark.
authors.forename: Nina
authors.identifier: http://orcid.org/0000-0003-4143-6276
authors.initials: N
authors.lastname: Persson
authors.affiliation: Division of Oncology and Pathology, Department of Clinical Sciences Lund, Lund University, Lund, Sweden.
authors.forename: Bo
authors.initials: B
authors.lastname: Jansson
authors.affiliation: Chemical Glyco-Biology Laboratory, Department of Chemistry, Copenhagen University, Copenhagen, Denmark.
authors.forename: Nicolai
authors.initials: N
authors.lastname: Stuhr-Hansen
authors.affiliation: Chemical Glyco-Biology Laboratory, Department of Chemistry, Copenhagen University, Copenhagen, Denmark.|Division of Clinical Chemistry and Pharmacology, Department of Laboratory Medicine, Lund University, Lund, Sweden.
authors.forename: András
authors.initials: A
authors.lastname: Kovács
authors.affiliation: Division of Oncology and Pathology, Department of Clinical Sciences Lund, Lund University, Lund, Sweden.|Centre of Excellence in Biological and Medical Mass Spectrometry (CEBMMS), Biomedical Centre D13, Lund University, Lund, Sweden.
authors.forename: Charlotte
authors.initials: C
authors.lastname: Welinder
authors.affiliation: Division of Clinical Chemistry and Pharmacology, Department of Laboratory Medicine, Lund University, Lund, Sweden.
authors.forename: Lena
authors.initials: L
authors.lastname: Danielsson
authors.affiliation: Chemical Glyco-Biology Laboratory, Department of Chemistry, Copenhagen University, Copenhagen, Denmark.
authors.forename: Ola
authors.initials: O
authors.lastname: Blixt
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000937:Antigen-Antibody Reactions; D004797:Enzyme-Linked Immunosorbent Assay; D051379:Mice; D008807:Mice, Inbred BALB C; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D019151:Peptide Library; D010455:Peptides / Q000138:chemical synthesis / Q000737:chemistry / Q000276:immunology; D040081:Protein Array Analysis; D057127:Single-Chain Antibodies / Q000032:analysis* / Q000276:immunology / Q000302:isolation & purification
chemical_list: D019151:Peptide Library; D010455:Peptides; D057127:Single-Chain Antibodies