title: Precise construction of oligonucleotide-Fab fragment conjugate for homogeneous immunoassay using HaloTag technology.
abstract: The use of oligonucleotide-protein conjugates enables the development of novel types of bioanalytical assays. However, convenient methods for producing covalent and stoichiometric oligonucleotide-protein conjugates are still rare. Here we demonstrate, for the first time, covalent conjugation of DNA oligonucleotide to Fab fragments with a 1:1 ratio using HaloTag self-labeling technology. The oligonucleotide coupling was carried out while the Fab was attached to protein G matrix, thereby enabling straightforward production of covalent conjugates. Furthermore, it allowed convenient purification of the product because the unreacted components were easily removed before the elution of the high-purity conjugate. The prepared conjugate was employed in a homogeneous immunoassay where prostate-specific antigen was used as a model analyte. Switchable lanthanide luminescence was used for detection, and the obtained limit of detection was 0.27 ng/ml. In the future, the developed method for covalent conjugation and successive purification in protein G column could also be applied for introducing other kinds of modifications to Fab fragments in a simple and site-specific manner.
authors.affiliation: Department of Biochemistry/Biotechnology, University of Turku, FI-20014 Turun yliopisto, Finland. Electronic address: henna.pakkila@utu.fi.
authors.forename: Henna
authors.initials: H
authors.lastname: Päkkilä
authors.affiliation: Department of Biochemistry/Biotechnology, University of Turku, FI-20014 Turun yliopisto, Finland.
authors.forename: Riikka
authors.initials: R
authors.lastname: Peltomaa
authors.affiliation: Department of Biochemistry/Biotechnology, University of Turku, FI-20014 Turun yliopisto, Finland.
authors.forename: Urpo
authors.initials: U
authors.lastname: Lamminmäki
authors.affiliation: Department of Biochemistry/Biotechnology, University of Turku, FI-20014 Turun yliopisto, Finland.
authors.forename: Tero
authors.initials: T
authors.lastname: Soukka
mesh_terms: D000818:Animals; D001426:Bacterial Proteins / Q000737:chemistry; D006801:Humans; D007118:Immunoassay / Q000379:methods; D007140:Immunoglobulin Fab Fragments / Q000737:chemistry*; D007610:Kallikreins / Q000032:analysis*; D009838:Oligodeoxyribonucleotides / Q000737:chemistry*; D017430:Prostate-Specific Antigen / Q000032:analysis*; D011817:Rabbits
chemical_list: D001426:Bacterial Proteins; C034284:IgG Fc-binding protein, Streptococcus; D007140:Immunoglobulin Fab Fragments; D009838:Oligodeoxyribonucleotides; C549870:KLK3 protein, human; D007610:Kallikreins; D017430:Prostate-Specific Antigen
keywords: Bioconjugation; Homogeneous detection; Immunoassay; Lanthanide chelate