Improved functional immobilization of llama single-domain antibody fragments to polystyrene surfaces using small peptides.
We studied the effect of different fusion domains on the functional immobilization of three llama single-domain antibody fragments (VHHs) after passive adsorption to polystyrene in enzyme-linked immunosorbent assays (ELISA). Three VHHs produced without any fusion domain were efficiently adsorbed to polystyrene, which, however, resulted in inefficient antigen binding. Functional VHH immobilization was improved by VHH fusion to a consecutive myc-His6-tag and was even more improved by fusion to the llama antibody long hinge region containing an additional His6-tag (LHc-His6). The partial dimerization of VHH-LHc-His6 fusion proteins through LHc-mediated disulfide-bond formation was not essential for their improved functional immobilization. VHH fusions to specific polystyrene binding peptides, hydrophobins, or other, unrelated VHH domains were less suitable for increasing functional VHH immobilization because of reduced microbial expression levels. Thus, VHH-LHc-His6 fusion proteins are most suited for functional passive adsorption in ELISA.
Central Veterinary Institute of Wageningen UR, Lelystad, The Netherlands. Michiel.Harmsen@wur.nl
Michiel M
MM
Harmsen
Helmi P D
HP
Fijten
D000327:Adsorption; D000818:Animals; D002161:Camelids, New World; D004797:Enzyme-Linked Immunosorbent Assay; D007128:Immunoglobulin Fragments / Q000737:chemistry*; D007143:Immunoglobulin Heavy Chains / Q000737:chemistry*; D007135:Immunoglobulin Variable Region / Q000737:chemistry*; D010455:Peptides / Q000737:chemistry; D011137:Polystyrenes / Q000737:chemistry*; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry
D007128:Immunoglobulin Fragments; D007143:Immunoglobulin Heavy Chains; D007135:Immunoglobulin Variable Region; D010455:Peptides; D011137:Polystyrenes; D011993:Recombinant Fusion Proteins