Antibodies 2013, 2(2), 168-192; doi:10.3390/antib2020168
Article

Isolation of Panels of Llama Single-Domain Antibody Fragments Binding All Nine Neuraminidase Subtypes of Influenza A Virus

Received: 16 February 2013; in revised form: 3 March 2013 / Accepted: 25 March 2013 / Published: 10 April 2013
(This article belongs to the Special Issue Single-Domain Antibody)
This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Abstract: Avian influenza A virus comprises sixteen hemagglutinin (HA) and nine neuraminidase (NA) subtypes (N1–N9). To isolate llama single-domain antibody fragments (VHHs) against all N subtypes, four llamas were immunized with mixtures of influenza viruses. Selections using influenza virus yielded predominantly VHHs binding to the highly immunogenic HA and nucleoprotein. However, selection using enzymatically active recombinant NA (rNA) protein enabled us to isolate NA binding VHHs. Some isolated VHHs cross-reacted to other N subtypes. These were subsequently used for the capture of N subtypes that could not be produced as recombinant protein (rN6) or were enzymatically inactive (rN1, rN5) in phage display selection, yielding novel VHHs. In total we isolated 188 NA binding VHHs, 64 of which were expressed in yeast. Most VHHs specifically recognize a single N subtype, but some VHHs cross-react with other N-subtypes. At least one VHH bound to all N subtypes, except N4, identifying a conserved antigenic site. Thus, this work (1) describes methods for isolating NA binding VHHs, (2) illustrates the suitability of llama immunization with multiple antigens for retrieving many binders against different antigens and (3) describes 64 novel NA binding VHHs, including a broadly reactive VHH, which can be used in various assays for influenza virus subtyping, detection or serology.
Keywords: recombinant neuraminidase; nanobody; single-domain antibody; phage display; enzymatic activity; antigenic site
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MDPI and ACS Style

Harmsen, M.M.; Blokker, J.C.; Pritz-Verschuren, S.B.; Bartelink, W.; van der Burg, H.; Koch, G. Isolation of Panels of Llama Single-Domain Antibody Fragments Binding All Nine Neuraminidase Subtypes of Influenza A Virus. Antibodies 2013, 2, 168-192.

AMA Style

Harmsen MM, Blokker JC, Pritz-Verschuren SB, Bartelink W, van der Burg H, Koch G. Isolation of Panels of Llama Single-Domain Antibody Fragments Binding All Nine Neuraminidase Subtypes of Influenza A Virus. Antibodies. 2013; 2(2):168-192.

Chicago/Turabian Style

Harmsen, Michiel M.; Blokker, Juliette C.; Pritz-Verschuren, Sylvia B.; Bartelink, Willem; van der Burg, Herman; Koch, Guus. 2013. "Isolation of Panels of Llama Single-Domain Antibody Fragments Binding All Nine Neuraminidase Subtypes of Influenza A Virus." Antibodies 2, no. 2: 168-192.

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