Bispecific Single-Domain Antibodies as Highly Standardized Synthetic Calibrators for Immunodiagnosis
| Autor: | Paula Segovia-de los Santos, Pedro Quintero-Campos, Sergi Morais, Cesar Echaides, Ángel Maquieira, Gabriel Lassabe, Gualberto Gonzalez-Sapienza |
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| Rok vydání: | 2021 |
| Předmět: | |
| Zdroj: | Analytical Chemistry. 94:1342-1349 |
| ISSN: | 1520-6882 0003-2700 |
| DOI: | 10.1021/acs.analchem.1c04603 |
| Popis: | [EN] 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. This work was supported by CSIC 2007-348, ANII FMV 2019_156321, and ANII FMV 2018_148245. P.S. is a recipient of a scholarship from ANII-Uruguay. P.Q.-C. acknowledges financial support from Generalitat Valenciana through the research staff training program (GVA ACIF/2018/173) . This research was also funded by Agencia Estatal de Investigacion (PID2019-110713RB-I00, FEDER) , program UPV-La FE 2019 (P105 VALBIOAL) , and PROMETEO/2020/094. |
| Databáze: | OpenAIRE |
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