1 research outputs found
Method for Sorting and Pairwise Selection of Nanobodies for the Development of Highly Sensitive Sandwich Immunoassays
Single domain heavychain binders
(nanobodies) obtained from camelid
antibody libraries hold a great promise for immunoassay development.
However, there is no simple method to select the most valuable nanobodies
from the crowd of positive clones obtained after the initial screening.
In this paper, we describe a novel nanobody-based platform that allows
comparison of the reactivity of hundreds of clones with the labeled
antigen, and identifies the best nanobody pairs for two-site immunoassay
development. The output clones are biotinylated in vivo in 96-well
culture blocks and then used to saturate the biotin binding capacity
of avidin coated wells. This standardizes the amount of captured antibody
allowing their sorting by ranking their reactivity with the labeled
antigen. Using human soluble epoxide hydrolase (sEH) as a model antigen,
we were able to classify 96 clones in four families and confirm this
classification by sequencing. This provided a criterion to select
a restricted panel of five capturing antibodies and to test each of
them against the rest of the 96 clones. The method constitutes a powerful
tool for epitope binning, and in our case allowed development of a
sandwich ELISA for sEH with a detection limit of 63 pg/mL and four
log dynamic range, which performed with excellent recovery in different
tissue extracts. This strategy provides a systematic way to test nanobody
pairwise combinations and would have a broad utility for the development
of highly sensitive sandwich immunoassays