A highly sensitive nanobody-based immunoassay detecting SARS-CoV-2 nucleocapsid protein using all-recombinant reagents

Antigen tests have been crucial for managing the COVID-19 pandemic by identifying individuals infected with SARS-CoV-2. This remains true even after immunity has been widely attained through natural infection and vaccination, since it only provides moderate protection against transmission and is highly permeable to the emergence of new virus variants. For this reason, the widespread availability of diagnostic methods is essential for health systems to manage outbreaks effectively. In this work, we generated nanobodies to the virus nucleocapsid protein (NP) and after an affinity-guided selection identified a nanobody pair that allowed the detection of NP at sub-ng/mL levels in a colorimetric two-site ELISA, demonstrating high diagnostic value with clinical samples. We further modified the assay by using a nanobody-NanoLuc luciferase chimeric tracer, resulting in increased sensitivity (detection limit = 61 pg/mL) and remarkable improvement in diagnostic performance. The luminescent assay was finally evaluated using 115 nasopharyngeal swab samples. Receiver Operating Characteristic (ROC) curve analysis revealed a sensitivity of 78.7% (95% confidence interval: 64.3%-89.3%) and specificity of 100.0% (95% confidence interval: 94.7%-100.0%). The test allows the parallel analysis of a large number of untreated samples, and fulfills our goal of producing a recombinant reagent-based test that can be reproduced at low cost by other laboratories with recombinant expression capabilities, aiding to build diagnostic capacity

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

This document is the Accepted Manuscript version of a Published Work that appeared in final form in Analytical Chemistry, copyright © American Chemical Society after peer review and technical editing by the publisher. To access the final edited and published work see https://doi.org/10.1021/acs.analchem.1c04603.[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.Segovia-De Los Santos, P.; Quintero-Campos, P.; Morais, S.; Echaides, C.; Maquieira Catala, A.; Lassabe, G.; Gonzalez-Sapienza, G. (2022). Bispecific Single-Domain Antibodies as Highly Standardized Synthetic Calibrators for Immunodiagnosis. Analytical Chemistry. 94(2):1342-1349. https://doi.org/10.1021/acs.analchem.1c046031342134994

An ultra-sensitive homologous chemiluminescence immunoassay to tackle penicillin allergy

[EN] Penicillin is one of the most widely used antibiotics to treat bacterial infections in clinical practice. The antibiotic undergoes degradation under physiological conditions to produce reactive compounds that in vivo bind self- proteins. These conjugates might elicit an immune response and trigger allergic reactions challenging to di- agnose due to the complex immunogenicity. Penicillin allergy delabeling initiatives are now part of antibiotic stewardship programs and include the use of invasive and risky in vivo tests. Instead, the in vitro quantification of specific IgE is highly useful to confirm immediate allergy to penicillins. However, discrepant results associated with the low sensitivity and accuracy of penicillin allergy in vitro tests have limited their routine diagnostic use for delabeling purposes. We aimed to develop a homologous chemiluminescence-based immunochemical method for the reliable determination of specific IgE to penicillin G, using unprecedented synthetic human-like Analytica Chimica Acta 1214 (2022) 339940 2standards. The synthetic standard targets the major antigenic determinant of penicillin G and the paratope of Omalizumab, acting as human-like specific IgE. It is a potent calibrator, highly stable, easy, and inexpensive to produce, overcoming the limitations of the pooled human serum preparations. The developed method achieved a good agreement and strong positive relationship, reaching a detection limit below 0.1 IU mLP.Q-C acknowledges financial support from Generalitat Valenciana through the research staff-training program (GVA ACIF/2018/173). P.C. awards grants Ramon y Cajal and PID2019-110630 GB-I00 funded by MCIN/AEI/10.13039/501100011033 and by ERDF A way of making Europe¿. S.M awards grants PID2019-110713RB-I00 funded by MCIN/ AEI/10.13039/501100011033 and by ERDF A way of making Europe. PROMETEO/2020/094 funded by Generalitat Valenciana, and program UPV-La FE 2019 (P105 VALBIOAL), FMV-ANII 148245, FMV-ANIII 156321 funded by Agencia Nacional de Investigacion and CSIC 2007- 348 UdelaR, Uruguay, are also acknowledged.Quintero-Campos, P.; Segovia-De Los Santos, P.; Ibáñez-Echevarría, E.; Hernández-Fernández De Rojas, D.; Casino, P.; Lassabe, G.; Gonzalez-Sapienza, G.... (2022). An ultra-sensitive homologous chemiluminescence immunoassay to tackle penicillin allergy. Analytica Chimica Acta. 1214:1-8. https://doi.org/10.1016/j.aca.2022.33994018121

La Pizarra Digital Interactiva como recurso educativo para alumnos con necesidades educativas especiales

Propuesta de utilización de la Pizarra Digital Interactiva en un centro de educación especial. Se utiliza como instrumento de adquirir y afianzar los prerrequisitos del aprendizaje, tales como atención, percepción, o memoria; se usa también como metodología en el desarrollo de los conocimientos en las áreas curriculares y en el desarrollo de las habilidades y conocimientos respecto al uso de la TIC y su habilidad en la vida diaria; y como recurso motivador y no discriminador para los alumnos con necesidades educativas especiales porque se adapta a las necesidades específicas de cada alumno. La pizarra se usa como apoyo de las explicaciones del profesorado y facilita al profesor la posibilidad de presentar sus actividades y recursos de forma adaptada a las diferentes necesidades de los alumnos. Se usa también para la realización de trabajos colaborativos en clase; para la corrección colectiva de ejercicios en clase, para la búsqueda de respuestas a preguntas no previstas; para el aprendizaje del manejo de programas informáticos o para lograr interacciones directas del alumno sobre su medio. Adjunta materiales usados para las actividades.Madrid (Comunidad Autónoma). Consejería de Educación. Dirección General de Ordenación AcadémicaMadridMadrid (Comunidad Autónoma). Subdirección General de Formación del Profesorado. CRIF Las Acacias; General Ricardos 179 - 28025 Madrid; Tel. + 34915250893ES