title: Engineering camel single-domain antibodies and immobilization chemistry for human prostate-specific antigen sensing.
abstract: The specificity and affinity characteristics of antibodies make them excellent probes in biosensor applications. Unfortunately, their large size, unstable behavior, and random immobilization properties create numerous problems. The single-domain antigen-binding fragment derived from heavy-chain antibodies of camelids (termed VHH) offers special advantages in terms of size, stability, and ease of generating different antibody constructs. In this study, we show the potential of those VHHs in sensing human prostate-specific antigen (hPSA) by SPR technology. Different VHH constructs were immobilized onto commercial and custom-built sensor surfaces by metal chelation, biotin-streptavidin interaction, or covalent coupling. The detection of subnanogram per milliliter hPSA concentrations could be attained on a covalently coupled three-dimensional dextran surface. Moreover, the ratio of different hPSA isoform concentrations could be assessed via a sandwich assay and resulted in the detection of clinically significant antigen concentrations within 15 min. In addition, for the first time, the intrinsic protein stability is presented as an important probe design factor, since our results reveal that higher intrinsic stability offers higher resistance to harsh regeneration conditions. In conclusion, we present VHHs as a novel class of biosensor probes rivaling conventional antibodies and their derived antibody fragments.
authors.affiliation: Laboratory of Cellular and Molecular Immunology, Department of Molecular and Cellular Interactions, Vlaams Interuniversitair Instituut voor Biotechnologie, Vrije Universiteit Brussel, Belgium. dsaerens@vub.ac.be
authors.forename: Dirk
authors.initials: D
authors.lastname: Saerens
authors.forename: Filip
authors.initials: F
authors.lastname: Frederix
authors.forename: Gunter
authors.initials: G
authors.lastname: Reekmans
authors.forename: Katja
authors.initials: K
authors.lastname: Conrath
authors.forename: Karolien
authors.initials: K
authors.lastname: Jans
authors.forename: Lea
authors.initials: L
authors.lastname: Brys
authors.forename: Lieven
authors.initials: L
authors.lastname: Huang
authors.forename: Eugène
authors.initials: E
authors.lastname: Bosmans
authors.forename: Guido
authors.initials: G
authors.lastname: Maes
authors.forename: Gustaaf
authors.initials: G
authors.lastname: Borghs
authors.forename: Serge
authors.initials: S
authors.lastname: Muyldermans
mesh_terms: D000818:Animals; D000906:Antibodies / Q000235:genetics / Q000276:immunology* / Q000378:metabolism*; D015374:Biosensing Techniques / Q000379:methods*; D001710:Biotin / Q000737:chemistry; D002162:Camelus; D006639:Histidine / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008239:Lysine / Q000737:chemistry; D008297:Male; D009532:Nickel; D017430:Prostate-Specific Antigen / Q000737:chemistry* / Q000276:immunology*; D015202:Protein Engineering / Q000379:methods*; D012680:Sensitivity and Specificity; D019809:Streptavidin / Q000737:chemistry
chemical_list: D000906:Antibodies; D006639:Histidine; D001710:Biotin; D009532:Nickel; D019809:Streptavidin; D017430:Prostate-Specific Antigen; D008239:Lysine