Method for Sorting and Pairwise Selection of Nanobodies for the Development of Highly Sensitive Sandwich Immunoassays

Abstract

Single domain heavy-chain 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.

Nanobody Based Immunoassay for Human Soluble Epoxide Hydrolase Detection Using Polymeric Horseradish Peroxidase (PolyHRP) for Signal Enhancement: The Rediscovery of PolyHRP?

“… a Results are average ± SD (CV, n=3); 1 nM sEH corresponds to 62.5 ng/mL sEH. b,c,d Data from ref 40, 29, 30, respectively. …”

Single-Domain Antibodies As Versatile Affinity Reagents for Analytical and Diagnostic Applications

“…For instance, in different studies we have selected both VHHs and VHs against human soluble epoxy hydrolase and tetanus toxin. In both cases, the recombinant VHs and VHHs had similar affinity (in the nanomolar to subnanomolar range) and showed also similar levels of expression and stability ( 39 , 40 ). In the following sections, we will highlight the properties of Nbs that make them a salient option for immunosensing applications.…”

“…Once the best capture antibodies are selected, they can be tested in parallel against the rest of the Nb clones using trace amounts of the antigen in a sandwich format. Using this method, we empirically found a pair of Nbs that performed with a detection limit of 63 pg/mL of human soluble epoxy hydrolase in highly complex matrices ( 40 ). The use of biotinylated Nbs for two-site immunoassay also has the advantage of the uniform orientation of the immobilized antibody, which has been show to contribute to enhance the assay sensitivity for the detection of bacterial toxins and influenza virus ( 50 , 51 ).…”

VHH antibodies: emerging reagents for the analysis of environmental chemicals

“…Another future prospect is to utilize the Avi-Tag system for incorporating a biotin moiety. The Avi-Tag system has been used with VHHs to larger protein targets [50], and more recently the first demonstration for the analysis of a small molecule, methotrexate, by immunoaffinity chromatography [51]. Thus, there are a variety of molecular tags that open the door to coupling multiple affinity and detection reagents.…”