title: Affinity capillary electrophoresis to evaluate the complex formation between poliovirus and nanobodies.
abstract: It was demonstrated that nanobodies with an in vitro neutralizing activity against poliovirus type 1 interact with native virions. Here, the use of capillary electrophoresis was investigated as an alternative technique for the evaluation of the formation of nanobody-poliovirus complexes, and therefore predicting the in vitro neutralizing activity of the nanobodies. The macromolecules are preincubated offline in a specific nanobody-to-virus ratio and analyzed by capillary electrophoresis with UV detection. At low nanobody-to-virus ratios, a clear shift in migration time of the viral peak was observed. A broad peak was obtained, indicating the presence of a heterogeneous population of nanobody-virion complexes, caused by the binding of different numbers of nanobodies to the virus particle. At elevated nanobody-to-virus ratios, a cluster of peaks appeared, showing an additional increase in migration times. It was shown that, at these high molar excesses, aggregates were formed. The developed capillary electrophoresis method can be used as a rapid, qualitative screening for the affinity between poliovirus and nanobodies, based on a clearly visible and measurable shift in migration time. The advantages of this technique include that there is no need for antigen immobilization as in enzyme-linked immunosorbent assays or surface plasmon resonance for the use of radiolabeled virus or for the performance of labor- and time-intensive plaque-forming neutralization assays.
authors.affiliation: Department of Pharmaceutical Biotechnology and Molecular Biology, Vrije Universiteit Brussel, Brussels, Belgium; Center for Neurosciences, Vrije Universiteit Brussel, Brussels, Belgium.
authors.forename: Hadewych
authors.initials: H
authors.lastname: Halewyck
authors.forename: Lise
authors.initials: L
authors.lastname: Schotte
authors.forename: Iuliana
authors.initials: I
authors.lastname: Oita
authors.forename: Bert
authors.initials: B
authors.lastname: Thys
authors.forename: Ann
authors.initials: A
authors.lastname: Van Eeckhaut
authors.forename: Yvan
authors.initials: Y
authors.lastname: Vander Heyden
authors.forename: Bart
authors.initials: B
authors.lastname: Rombaut
mesh_terms: D019075:Electrophoresis, Capillary; D049329:Nanostructures / Q000737:chemistry*; D017955:Poliovirus / Q000737:chemistry*
keywords: Affinity; Capillary electrophoresis; Complex formation; Nanobodies; Poliovirus