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...

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Bibliographic Details
Published in:Journal of immunoassay & immunochemistry 2012-01, Vol.33 (3), p.234-251
Main Authors: Harmsen, Michiel M., Fijten, Helmi P. D.
Format: Article
Language:English
Subjects:
Adsorption
Animals
bivalent
Camelids, New World
ELISA
Enzyme-Linked Immunosorbent Assay
escherichia-coli
functional immobilization
hydrophobins
Immunoglobulin Fragments - chemistry
Immunoglobulin Heavy Chains - chemistry
Immunoglobulin Variable Region - chemistry
in-vitro
linked-immunosorbent-assay
microarrays
mouth-disease
nanobody
oriented immobilization
passive-immunization
Peptides - chemistry
pigs
Polystyrenes - chemistry
protein biochip
Protein Structure, Tertiary
Recombinant Fusion Proteins - chemistry
single-domain antibody
strategies
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Summary: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.
ISSN:1532-1819
1532-4230
DOI:10.1080/15321819.2011.634473