title: Method for Sorting and Pairwise Selection of Nanobodies for the Development of Highly Sensitive Sandwich Immunoassays.
abstract: Single domain heavychain binders (nanobodies) obtained from camelid antibody libraries hold a great promise for immunoassay development. However, there is no simple method to select the most valuable nanobodies from the crowd of positive clones obtained after the initial screening. In this paper, we describe a novel nanobody-based platform that allows comparison of the reactivity of hundreds of clones with the labeled antigen, and identifies the best nanobody pairs for two-site immunoassay development. The output clones are biotinylated in vivo in 96-well culture blocks and then used to saturate the biotin binding capacity of avidin coated wells. This standardizes the amount of captured antibody allowing their sorting by ranking their reactivity with the labeled antigen. Using human soluble epoxide hydrolase (sEH) as a model antigen, we were able to classify 96 clones in four families and confirm this classification by sequencing. This provided a criterion to select a restricted panel of five capturing antibodies and to test each of them against the rest of the 96 clones. The method constitutes a powerful tool for epitope binning, and in our case allowed development of a sandwich ELISA for sEH with a detection limit of 63 pg/mL and four log dynamic range, which performed with excellent recovery in different tissue extracts. This strategy provides a systematic way to test nanobody pairwise combinations and would have a broad utility for the development of highly sensitive sandwich immunoassays.
authors.affiliation: Cátedra de Inmunología, DEPBIO, Facultad de Química, Instituto de Higiene, UDELAR , Montevideo, Uruguay.
authors.forename: Martín A
authors.initials: MA
authors.lastname: Rossotti
authors.affiliation: Cátedra de Inmunología, DEPBIO, Facultad de Química, Instituto de Higiene, UDELAR , Montevideo, Uruguay.
authors.forename: Macarena
authors.initials: M
authors.lastname: Pirez
authors.affiliation: Cátedra de Inmunología, DEPBIO, Facultad de Química, Instituto de Higiene, UDELAR , Montevideo, Uruguay.
authors.forename: Andres
authors.initials: A
authors.lastname: Gonzalez-Techera
authors.affiliation: Department of Entomology and Nematology and UC Davis Comprehensive Cancer Center, University of California , Davis, California, United States.
authors.forename: Yongliang
authors.initials: Y
authors.lastname: Cui
authors.affiliation: Department of Entomology and Nematology and UC Davis Comprehensive Cancer Center, University of California , Davis, California, United States.
authors.forename: Candace S
authors.initials: CS
authors.lastname: Bever
authors.affiliation: Department of Entomology and Nematology and UC Davis Comprehensive Cancer Center, University of California , Davis, California, United States.
authors.forename: Kin S S
authors.initials: KS
authors.lastname: Lee
authors.affiliation: Department of Entomology and Nematology and UC Davis Comprehensive Cancer Center, University of California , Davis, California, United States.
authors.forename: Christophe
authors.initials: C
authors.lastname: Morisseau
authors.affiliation: Parque Lecocq, IMM, Montevideo, Uruguay.
authors.forename: Carmen
authors.initials: C
authors.lastname: Leizagoyen
authors.affiliation: Department of Entomology and Nematology and UC Davis Comprehensive Cancer Center, University of California , Davis, California, United States.
authors.forename: Shirley
authors.initials: S
authors.lastname: Gee
authors.affiliation: Department of Entomology and Nematology and UC Davis Comprehensive Cancer Center, University of California , Davis, California, United States.
authors.forename: Bruce D
authors.initials: BD
authors.lastname: Hammock
authors.affiliation: Cátedra de Inmunología, DEPBIO, Facultad de Química, Instituto de Higiene, UDELAR , Montevideo, Uruguay.
authors.forename: Gualberto
authors.initials: G
authors.lastname: González-Sapienza
mesh_terms: D000937:Antigen-Antibody Reactions; D000941:Antigens / Q000737:chemistry / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay; D004851:Epoxide Hydrolases / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D061905:Single-Domain Antibodies / Q000276:immunology* / Q000302:isolation & purification*; D012995:Solubility
chemical_list: D000941:Antigens; D011994:Recombinant Proteins; D061905:Single-Domain Antibodies; D004851:Epoxide Hydrolases