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 the specific antibodies to be measured, which are used for the quantification of the unknown antibody contained 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 also limitations to their lot-to-lot reproducibility and standardization over time. To overcome this difficulty, this work 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 antigen 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 proof of concept, we present the generation and characterization of two bispecific calibrators with potential application to the diagnosis of allergy against the antibiotics aztreonam and amoxicillin.