Llama single domain antibody fragments (VHHs) available at CVI:
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1 Llama single domain antibody fragments (VHHs) available at CVI: VHH(s) Antigen specificity Cross-reaction Remarks Sequence Foot-and-mouth disease virus (FMDV) M3 FMDV, strain O1 Manisa O, A, C and Asia1 (not SAT2) Binds specifically to 12S particles + [1,2] M8 FMDV, strain O1 Manisa O, A and Asia1 Binds trypsin sensitive linear epitope on VP1 GH-loop + [2] M23 FMDV, strain O1 Manisa O1BFS, O Taiwan Binds to antigenic site independent of M8, M3 + [2] M170 FMDV, strain O1 Manisa O1BFS Binds specifically to 146S particles + [1-3] M94 FMDV, strain Asia 1 Shamir None? Virus neutralizing - Unpublished M98 FMDV, strain Asia 1 Shamir None? Virus neutralizing - Unpublished M159 FMDV, strain Asia 1 Shamir None? Virus neutralizing - Unpublished M301 FMDV, strain Asia 1 Shamir Unknown Virus neutralization unknown - Unpublished M303 FMDV, strain Asia 1 Shamir Unknown Virus neutralization unknown - Unpublished M304 FMDV, strain Asia 1 Shamir Unknown Virus neutralization unknown - Unpublished M306 FMDV, strain Asia 1 Shamir Unknown Virus neutralization unknown - Unpublished M307 FMDV, strain Asia 1 Shamir Unknown Virus neutralization unknown - Unpublished M308 FMDV, strain Asia 1 Shamir Unknown Virus neutralization unknown - Unpublished M311 FMDV, strain SAT2 Unknown - Unpublished M314 FMDV, strain SAT2 Unknown - Unpublished M315 FMDV, strain SAT2 Unknown - Unpublished M317 FMDV, strain SAT2 Unknown - Unpublished M377 FMDV, strain SAT2 None Binds specifically to 146S particles - Unpublished M379 FMDV, strain SAT2 None Binds specifically to 146S particles - Unpublished M380 FMDV, strain SAT2 None Binds specifically to 146S particles - Unpublished Influenza A virus (IAV) IV5 IAV Haemagglutinin H7 subtype None (?) + [4] IV29 IAV Haemagglutinin H7 subtype None (?) + [4] IV146 IAV Haemagglutinin H5 subtype H1, H2 + [4] IV156 IAV Haemagglutinin H5 subtype None (?) + [4] IV169 IAV Neuraminidase N1 subtype See reference - [5] IV403 IAV Neuraminidase N1 subtype See reference - [5] IV417 IAV Neuraminidase N1 subtype See reference - [5] IV171 IAV Neuraminidase N1 subtype See reference - [5] IV414 IAV Neuraminidase N1 subtype See reference - [5] IV426 IAV Neuraminidase N1 subtype See reference - [5] IV422 IAV Neuraminidase N1 subtype See reference - [5] IV63 IAV Neuraminidase N2 subtype See reference + [5] IV82 IAV Neuraminidase N2 subtype See reference + [5] IV104 IAV Neuraminidase N2 subtype See reference + [5] 1
2 VHH(s) Antigen specificity Cross-reaction Remarks Sequence IV65 IAV Neuraminidase N2 subtype See reference + [5] IV77 IAV Neuraminidase N2 subtype See reference + [5] IV105 IAV Neuraminidase N2 subtype See reference + [5] IV75 IAV Neuraminidase N2 subtype See reference + [5] IV78 IAV Neuraminidase N2 subtype See reference + [5] IV95 IAV Neuraminidase N2 subtype See reference + [5] IV97 IAV Neuraminidase N2 subtype See reference + [5] IV107 IAV Neuraminidase N2 subtype See reference + [5] IV81 IAV Neuraminidase N2 subtype See reference N-glycosylated + [5] IV84 IAV Neuraminidase N2 subtype See reference + [5] IV185 IAV Neuraminidase N3 subtype See reference - [5] IV191 IAV Neuraminidase N3 subtype See reference - [5] IV235 IAV Neuraminidase N3 subtype See reference - [5] IV456 IAV Neuraminidase N3 subtype See reference N-glycosylated - [5] IV187 IAV Neuraminidase N3 subtype See reference - [5] IV202 IAV Neuraminidase N3 subtype See reference N-glycosylated - [5] IV540 IAV Neuraminidase N3 subtype See reference - [5] IV207 IAV Neuraminidase N4 subtype See reference - [5] IV215 IAV Neuraminidase N4 subtype See reference - [5] IV213 IAV Neuraminidase N4 subtype See reference - [5] IV297 IAV Neuraminidase N4 subtype See reference - [5] IV208 IAV Neuraminidase N4 subtype See reference - [5] IV224 IAV Neuraminidase N5 subtype See reference - [5] IV451 IAV Neuraminidase N5 subtype See reference - [5] IV442 IAV Neuraminidase N5 subtype See reference - [5] IV447 IAV Neuraminidase N5 subtype See reference - [5] IV501 IAV Neuraminidase N6 subtype See reference - [5] IV457 IAV Neuraminidase N6 subtype See reference - [5] IV512 IAV Neuraminidase N6 subtype All except N4? - [5] IV618 IAV Neuraminidase N6 subtype All except N4? - [5] IV461 IAV Neuraminidase N6 subtype See reference - [5] IV462 IAV Neuraminidase N6 subtype See reference - [5] IV467 IAV Neuraminidase N6 subtype See reference N-glycosylated - [5] IV513 IAV Neuraminidase N6 subtype See reference - [5] IV514 IAV Neuraminidase N6 subtype See reference - [5] IV619 IAV Neuraminidase N6 subtype See reference - [5] IV516 IAV Neuraminidase N6 subtype See reference - [5] IV246 IAV Neuraminidase N7 subtype See reference - [5] IV252 IAV Neuraminidase N7 subtype See reference - [5] IV242 IAV Neuraminidase N7 subtype See reference - [5] 2
3 VHH(s) Antigen specificity Cross-reaction Remarks Sequence IV237 IAV Neuraminidase N7 subtype See reference - [5] IV240 IAV Neuraminidase N7 subtype See reference - [5] IV265 IAV Neuraminidase N8 subtype See reference - [5] IV280 IAV Neuraminidase N8 subtype See reference - [5] IV268 IAV Neuraminidase N8 subtype See reference - [5] IV274 IAV Neuraminidase N8 subtype See reference - [5] IV275 IAV Neuraminidase N8 subtype See reference - [5] IV284 IAV Neuraminidase N8 subtype See reference - [5] IV525 IAV Neuraminidase N9 subtype See reference - [5] IV295 IAV Neuraminidase N9 subtype See reference - [5] IV294 IAV Neuraminidase N9 subtype See reference - [5] IV300 IAV Neuraminidase N9 subtype See reference - [5] IV530 IAV Neuraminidase N9 subtype See reference - [5] IV533 IAV Neuraminidase N9 subtype See reference - [5] IV329 IAV Haemagglutinin H1 subtype Unknown - Unpublished IV337 IAV Haemagglutinin H1 subtype Unknown - Unpublished IV345 IAV Haemagglutinin H1 subtype Unknown - Unpublished IV346 IAV Haemagglutinin H1 subtype Unknown - Unpublished IV349 IAV Haemagglutinin H1 subtype Unknown - Unpublished IV564 IAV Haemagglutinin H2 subtype None - Unpublished IV566 IAV Haemagglutinin H2 subtype None - Unpublished IV355 IAV Haemagglutinin H3 subtype Unknown - Unpublished IV357 IAV Haemagglutinin H3 subtype Unknown - Unpublished IV360 IAV Haemagglutinin H3 subtype Unknown - Unpublished IV361 IAV Haemagglutinin H3 subtype Unknown - Unpublished IV365 IAV Haemagglutinin H3 subtype Unknown - Unpublished IV369 IAV Haemagglutinin H3 subtype Unknown - Unpublished IV372 IAV Haemagglutinin H4 subtype Unknown - Unpublished IV376 IAV Haemagglutinin H4 subtype Unknown - Unpublished IV377 IAV Haemagglutinin H4 subtype Unknown - Unpublished IV387 IAV Haemagglutinin H4 subtype Unknown - Unpublished IV393 IAV Haemagglutinin H4 subtype Unknown - Unpublished IV477 IAV Haemagglutinin H5 subtype None? - Unpublished IV486 IAV Haemagglutinin H5 subtype None? - Unpublished IV576 IAV Haemagglutinin H6 subtype None - Unpublished IV650 IAV Haemagglutinin H6 subtype H2 - Unpublished IV651 IAV Haemagglutinin H6 subtype H5, H11, H13, H14 - Unpublished IV406 IAV Haemagglutinin H7 subtype None? Not yet yeast-expressed due to missing BstEII site - Unpublished IV416 IAV Haemagglutinin H7 subtype None? - Unpublished IV578 IAV Haemagglutinin H8 subtype None - Unpublished 3
4 VHH(s) Antigen specificity Cross-reaction Remarks Sequence IV581 IAV Haemagglutinin H8 subtype None - Unpublished IV309 IAV Haemagglutinin H9 subtype Unknown - Unpublished IV310 IAV Haemagglutinin H9 subtype Unknown - Unpublished IV311 IAV Haemagglutinin H9 subtype Unknown - Unpublished IV313 IAV Haemagglutinin H9 subtype Unknown - Unpublished IV323 IAV Haemagglutinin H9 subtype Unknown - Unpublished IV583 IAV Haemagglutinin H10 subtype None - Unpublished IV588 IAV Haemagglutinin H10 subtype None - Unpublished IV590 IAV Haemagglutinin H11 subtype None - Unpublished IV593 IAV Haemagglutinin H11 subtype H5, H6? - Unpublished IV596 IAV Haemagglutinin H11 subtype None - Unpublished IV599 IAV Haemagglutinin H12 subtype None - Unpublished IV602 IAV Haemagglutinin H12 subtype H6, H15? - Unpublished IV604 IAV Haemagglutinin H12 subtype None - Unpublished IV606 IAV Haemagglutinin H13 subtype None - Unpublished IV612 IAV Haemagglutinin H13 subtype None - Unpublished IV615 IAV Haemagglutinin H14 subtype None - Unpublished IV622 IAV Haemagglutinin H15 subtype H3, H7? - Unpublished IV154 IAV Nucleoprotein - Unpublished IV155 IAV Nucleoprotein - Unpublished IV482 IAV Nucleoprotein - Unpublished IV653 IAV Nonstructural Protein 1 (NS1) Allele B NS1 - Unpublished IV664 IAV Nonstructural Protein 1 (NS1) Allele A+B NS1 - Unpublished IV670 IAV Nonstructural Protein 1 (NS1) Allele B NS1 - Unpublished IV675 IAV Nonstructural Protein 1 (NS1) Allele A+B NS1 - Unpublished IV687 IAV Nonstructural Protein 1 (NS1) Allele B NS1 - Unpublished IV688 IAV Nonstructural Protein 1 (NS1) Allele A+B NS1 - Unpublished IV695 IAV Nonstructural Protein 1 (NS1) Allele B NS1 - Unpublished IV705 IAV Nonstructural Protein 1 (NS1) Allele B NS1 - Unpublished IV707 IAV Nonstructural Protein 1 (NS1) Allele A+B NS1 - Unpublished IV708 IAV Nonstructural Protein 1 (NS1) Allele A NS1 - Unpublished Rift Valley Fever Virus (RVFV) RV60 RVFV GnGc protein Specificity needs validation - Unpublished RV61 RVFV GnGc protein Specificity needs validation - Unpublished RV66 RVFV GnGc protein Specificity needs validation - Unpublished RV70 RVFV GnGc protein Specificity needs validation - Unpublished RV81 RVFV GnGc protein Specificity needs validation - Unpublished Bluetongue Virus BT15 Bluetongue Virus serotype 8, VP7 - Unpublished BT21 Bluetongue Virus serotype 8, VP7 - Unpublished 4
5 VHH(s) Antigen specificity Cross-reaction Remarks Sequence Escherichia coli F4 fimbriae 17 different VHHs See reference Well-characterized VHHs + [6,7] Escherichia coli heat-labile toxin 27 different VHHs See reference Well-characterized VHHs + [8] Campylobacter jejuni flagel 18 different VHHs Known Work in progress - Unpublished Porcine Immunoglobulin G 19 different VHHs See reference Well-characterized VHHs + [9] VHH-based sandwich ELISAs for antigen detection available at CVI: Coating Biotinylated Antigen detected Limit of detection Remarks VHH VHH (ng/ml) M3 M3 FMDV 12S particles of all strains except SAT2 ~2-10 Also suitable for measurement in vaccine [1] M8 M8 FMDV 12S and 146S particles of most strains ~2 Also suitable for measurement in vaccine Unpublished M23 M23 FMDV O serotype 12S and 146S particles ~2 Also suitable for measurement in vaccine Unpublished M170 M170 FMDV O1 Manisa and O1 BFS 146S particles ~2 Also suitable for measurement in vaccine [1] M98 M98 FMDV Asia 1 Shamir Unpublished IV156 IV146 IAV Haemagglutinin H5 subtype ~1 Unpublished IV146 IV146 IAV Haemagglutinin H1, H2, H5 ~10 Unpublished IV29 IV5 IAV Haemagglutinin H7 subtype ~1 Unpublished IV403 IV426 IAV Neuraminidase N1 subtype Unpublished IV65 IV95 IAV Neuraminidase N2 subtype Unpublished IV191 IV185 IAV Neuraminidase N3 subtype Unpublished IV215 IV215 IAV Neuraminidase N4 subtype Unpublished IV451 IV442 IAV Neuraminidase N5 subtype Unpublished IV467 IV457 IAV Neuraminidase N6 subtype Unpublished IV242 IV240 IAV Neuraminidase N7 subtype Unpublished IV275 IV280 IAV Neuraminidase N8 subtype Unpublished IV295 IV530 IAV Neuraminidase N9 subtype Unpublished IV688 IV705 IAV Nonstructural protein 1, allele A Unpublished IV675 IV653 IAV Nonstructural protein 1, allele B Unpublished 5
6 1. Harmsen, M.M.; Fijten, H.P.; Westra, D.F.; Coco-Martin, J.M., Effect of thiomersal on dissociation of intact (146S) foot-and-mouth disease virions into 12S particles as assessed by novel ELISAs specific for either 146S or 12S particles. Vaccine 2011, 29, Harmsen, M.M.; Van Solt, C.B.; Fijten, H.P.D.; Van Keulen, L.; Rosalia, R.A.; Weerdmeester, K.; Cornelissen, A.H.M.; De Bruin, M.G.M.; Eblé, P.L.; Dekker, A., Passive immunization of guinea-pigs with llama single-domain antibody fragments against foot-and-mouth disease. Vet. Microbiol. 2007, 120, Harmsen, M.M.; Jansen, J.; Westra, D.F.; Coco-Martin, J.M., Characterization of foot-and-mouth disease virus antigen by surface-enhanced laser desorption ionization time of flight mass spectrometry in aqueous and oil-emulsion formulations. Vaccine 2010, 28, Hultberg, A.; Maassen, B.; Vanlandschoot, P.; Depla, E.; Stortelers, C.; Verrips, C.T.; Van Gucht, S.; Melero, J.; Saunders, M.J.S.; De Haard, J.J.W., et al. Amino acid sequences directed against envelope proteins of a virus and polypeptides comprising the same for the treatment of viral diseases Patent application WO2009/ 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, Harmsen, M.M.; van Solt, C.B.; Hoogendoorn, A.; van Zijderveld, F.G.; Niewold, T.A.; van der Meulen, J., Escherichia coli f4 fimbriae specific llama single-domain antibody fragments effectively inhibit bacterial adhesion in vitro but poorly protect against diarrhoea. Vet. Microbiol. 2005, 111, Harmsen, M.M.; van Solt, C.B.; van Zijderveld-van Bemmel, A.M.; Niewold, T.A.; van Zijderveld, F.G., Selection and optimization of proteolytically stable llama single-domain antibody fragments for oral immunotherapy. Appl. Microbiol. Biotechnol. 2006, 72, Harmsen, M.M.; van Solt, C.B.; Fijten, H.P., Enhancement of toxin- and virus-neutralizing capacity of single-domain antibody fragments by N-glycosylation. Appl Microbiol Biotechnol 2009, 84, Harmsen, M.M.; Van Solt, C.B.; Fijten, H.P.D.; Van Setten, M.C., Prolonged in vivo residence times of llama single-domain antibody fragments in pigs by binding to porcine immunoglobulins. Vaccine 2005, 23,
Isolation of Panels of Llama Single-Domain Antibody Fragments Binding All Nine Neuraminidase Subtypes of Influenza A Virus
Antibodies 2013, 2, 168-192; doi:10.3390/antib2020168 Article OPEN ACCESS antibodies ISSN 2073-4468 www.mdpi.com/journal/antibodies Isolation of Panels of Llama Single-Domain Antibody Fragments Binding
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