IMPROVED FUNCTIONAL IMMOBILIZATION OF LLAMA SINGLE-DOMAIN ANTIBODY FRAGMENTS TO POLYSTYRENE SURFACES USING SMALL PEPTIDES

Abstract

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.

Keywords:

• ELISA
• functional immobilization
• nanobody
• oriented immobilization
• protein biochip
• single-domain antibody

ACKNOWLEDGMENTS

The authors thank Aart van Amerongen and Willem Norde (Agrotechnology & Food Sciences Group of Wageningen UR, The Netherlands) for valuable discussions and Karin Scholtmeijer and Han Wösten (Department of Microbiology, Utrecht University, The Netherlands) for help with hydrophobin fusion protein design.

Notes

^a His6-tags are present at the extreme C-terminus, except for hydrophobin fusion proteins, where they are located immediately N-terminal from the hydrophobin coding region. Minus and plus signs indicate absence and presence of the His6-tag, respectively.

^b This construct encodes two tandem copies of peptide 2-1.

^a Mean and standard deviation of triplicate measurements are shown.

^b ND, not determined.

^c ELISA measuring K609 or M180 moiety of VHH2.

^d ELISA measuring VI-11 moiety of VHH2.

^a Mean and standard deviation of triplicate measurements are shown.

^b ND, not determined.

^c Absorbance of 0.4 was not reached at highest VHH concentration tested (1000 ng/mL).