Bispecific Single-Domain Antibodies as Highly Standardized Synthetic Calibrators for Immunodiagnosis

Abstract

Commonly, serological immunoassays and diagnostic kits include reference standard reagents (calibrators) that contain specific antibodies to be measured, which are used for the quantification of unknown antibodies present in the sample. However, in some cases, such as the diagnosis of allergies or autoimmune diseases, it is often difficult to have sufficient quantities of these reference standards, and there are limitations to their lot-to-lot reproducibility and standardization over time. To overcome this difficulty, this study introduces the use of surrogate recombinant calibrators formulated on the basis of two single-domain antibodies (nanobodies) combined through a short peptide linker to produce a recombinant bispecific construct. One of the nanobodies binds to the cognate analyte of the target antibody and the second is specific for the paratope of the secondary detecting antibody. The bispecific nanobody inherits the outstanding properties of stability and low-cost production by bacterial fermentation of the parent nanobodies, and once calibrated against the biological reference standard, it can be reproduced indefinitely from its sequence in a highly standardized manner. As a proof of concept, we present the generation and characterization of two bispecific calibrators with potential application for the diagnosis of allergy against the antibiotics aztreonam and amoxicillin in humans.

An ultra-sensitive homologous chemiluminescence immunoassay to tackle penicillin allergy

“…After subcloning into the pINQ-bis vector [23], sequencing confirmed that the clones possessed the two VHHs sequences of interest. Figure 1A depicts the structural model of rBBN using the Robetta web server and visualized in UCSF Chimera.…”

“…We recently presented the generation and characterization of standardized synthetic standards consisting of a nanobody against the paratope of the detection antibody expressed in tandem with a second nanobody against the target antigen for immunodiagnosis [23].…”

Standardizing In Vitro β-Lactam Antibiotic Allergy Testing with Synthetic IgE

“…Conversely, establishing a serum bank from a diverse range of allergic patients with substantial levels of IgE antibodies is impractical due to limitations in size, reproducibility of serum pools, and the significant quantity of IgE-positive sera required for each specificity . Previous attempts to develop artificial human sera, such as chimeric adaptor molecules and specific bi-nanobodies, have involved animal immunization and lack the functional structure of IgE. − …”

Enhancing Oriented Immobilization Efficiency: A One-for-Two Organism-Bispecific Nanobody Scaffold for Highly Sensitive Detection of Foodborne Pathogens

“…Additionally, several chemical modification methods, such as site-directed immobilization via sortase-mediated approaches, have also been explored . However, the application of BsNb in oriented immobilization has not been reported, which at present is mainly focused on virus neutralization, cell-engaged immunotherapeutics, or calibrator formulation …”