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.

An ultra-sensitive homologous chemiluminescence immunoassay to tackle penicillin allergy.

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 diagnose 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 standards. 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 mL-1 and excellent reproducibility (RSD <9%). The clinical sensitivity of the assay significantly increased (66%), doubling the accuracy of the reference method with an overall specificity of 100%. The new diagnostic strategy compares favorably with results obtained by the standard procedure, paving the way towards the standardization of penicillin allergy testing, and enhancing the detection sensitivity of specific IgE in serum to tackle reliably ß-lactam allergy delabeling.

Functionalization of Magnetic Beads with Biotinylated Nanobodies for MALDI-TOF/MS-Based Quantitation of Small Analytes.

Over the last two decades, the variable domains from heavy chain-only antibodies in camelids (nanobodies) have emerged as valuable immunoreagents for analytical and diagnostic applications. One prominent use of nanobodies is for the detection of small molecules due to their ease of production, resistance to solvents used in sample extraction, facile genetic manipulation, and small size. These last two properties make it possible to produce biotinylated nanobodies in vivo, which can be loaded in an orientated manner on magnetic beads covered with avidin, creating high-density immunoadsorbenpi twbch ""ts. The method described here details the use of nanobody-based adsorbents to concentrate small molecular weight analytes for subsequent quantitative analysis by MALDI-TOF mass spectrometry. Quantitation requires the inclusion of an internal standard (IS), a compound with properties similar to those of the analyte, enabling compensation for uneven distribution during crystallization of the MALDI-TOF matrix. Since nanobody generation against small compounds requires conjugation to carrier proteins, the same conjugation chemistry can be used to synthesize the IS. By design the IS cross reacts with the capture nanobody and can be preloaded in the immunoadsorbent, facilitating quantitative detection of the target compound.

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

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.

Development of anti-human IgM nanobodies as universal reagents for general immunodiagnostics.

Nanobodies are the smallest antibody fragments which bind to antigens with high affinity and specificity. Due to their outstanding physicochemical stability, simplicity and cost-effective production, nanobodies have become powerful agents in therapeutic and diagnostic applications. In this work, the advantages of nanobodies were exploited to develop generic and standardized anti-human IgM reagents for serology and IgM+ B-cell analysis. Selection of anti-IgM nanobodies was carried out by evaluating their yields, stability, binding kinetics and cross-reactivity with other Ig isotypes. High affinity nanobodies were selected with dissociation constants (KDs) in the nM range and high sensitivities for detection of total IgM by ELISA. The nanobodies also proved to be useful for capturing IgM in the serodiagnosis of an acute infection as demonstrated by detection of specific IgM in sera of dengue virus patients. Finally, due to the lack of an Fc region, the selected nanobodies do not require Fc receptor blocking steps, facilitating the immunophenotyping of IgM+ cells by flow cytometry, an important means of diagnosis of immunodeficiencies and B-cell lymphoproliferative disorders. This work describes versatile anti-IgM nanobodies that, due to their recombinant nature and ease of reproduction at low cost, may represent an advantageous alternative to conventional anti-IgM antibodies in research and diagnosis.

Noncompetitive Homogeneous Detection of Small Molecules Using Synthetic Nanopeptamer-Based Luminescent Oxygen Channeling.

Our group has previously developed immunoassays for noncompetitive detection of small molecules based on the use of phage borne anti-immunocomplex peptides. Recently, we substituted the phage particles by biotinylated synthetic anti-immunocomplex peptides complexed with streptavidin and named these constructs nanopeptamers. In this work, we report the results of combining AlphaLisa, a commercial luminescent oxygen channeling bead system, with nanopeptamers for the development of a noncompetitive homogeneous assay for the detection of small molecules. The signal generation of AlphaLisa assays relies on acceptor-donor bead proximity induced by the presence of the analyte (a macromolecule) simultaneously bound by antibodies immobilized on the surface of these beads. In the developed assay, termed as nanoAlphaLisa, bead proximity is sustained by the presence of a small model molecule (atrazine, MW = 215) using an antiatrazine antibody captured on the acceptor bead and an atrazine nanopeptamer on the donor bead. Atrazine is one of the most used pesticides worldwide, and its monitoring in water has relevant human health implications. NanoAlphaLisa allowed the homogeneous detection of atrazine down to 0.3 ng/mL in undiluted water samples in 1 h, which is 10-fold below the accepted limit in drinking water. NanoAlphaLisa has the intrinsic advantages for automation and high-throughput, simple, and fast homogeneous detection of target analytes that AlphaLisa assay provides.

Recombinant streptavidin nanopeptamer anti-immunocomplex assay for noncompetitive detection of small analytes.

Short peptide loops selected from phage libraries can specifically recognize the formation of hapten-antibody immunocomplexes and can thus be used to develop phage anti-immunocomplex assays (PHAIA) for noncompetitive detection of small molecules. In this study, we generated recombinant chimeras by fusing anti-immunocomplex peptides selected from phage libraries to the N- or C-termini of core streptavidin and used them to setup phage-free noncompetitive assays for the herbicide clomazone (MW 240 Da). The best conditions for refolding were optimized by a high throughput screening allowing to obtain tens of mg of purified protein per liter of culture. The noncompetitive assay developed with these chimeras performed with a 50% saturating concentration (SC50) of 2.2 ± 0.3 ng/mL and limit of detection (LOD) of 0.48 ng/mL. Values that are 13- and 8-fold better that those obtained for the SC50 and LOD of the competitive assay setup with the same antibody. Apart from the first demonstration that recombinant peptide-streptavidin chimeras can be used for sensitive immunodetection of small molecules with a positive readout, this new assay component is a highly standardized reagent with a defined stoichiometry, which can be used in combination with the broad option of existing biotinylated reagents offering a great versatility for the development of conventional immunoassay and biosensors. The utility of the test was demonstrated analyzing the clomazone runoff during the rice growing season in northern Uruguay.

Shiga-like toxin B subunit of Escherichia coli as scaffold for high-avidity display of anti-immunocomplex peptides.

Small compounds cannot bind simultaneously to two antibodies, and thus, their immunodetection is limited to competitive formats in which the analyte is indirectly quantitated by measuring the unoccupied antibody binding sites using a competing reporter. This limitation can be circumvented by using phage-borne peptides selected for their ability to specifically react with the analyte-antibody immunocomplex, which allows the detection of these small molecules in a noncompetitive format (PHAIA) with increased sensitivity and a positive readout. In an effort to find substitutes for the phage particles in PHAIA, we explore the use of the B subunit of the Shiga-like toxin of Escherichia coli, also known as verotoxin (VTX), as a scaffold for multivalent display of anti-immunocomplex peptides. Using the herbicides molinate and clomazone as model compounds, we built peptide-VTX recombinant chimeras that were produced in the periplasmic space of E. coli as soluble pentamers, as confirmed by multiangle light scattering analysis. These multivalent constructs, which we termed nanopeptamers, were conjugated to a tracer enzyme and used to detect the herbicide-antibody complex in an ELISA format. The VTX-nanopeptamer assays performed with over a 10-fold increased sensitivity and excellent recovery from spiked surface and mineral water samples. The carbon black-labeled peptide-VTX nanopeptamers showed great potential for the development of a lateral-flow test for small molecules with a visual positive readout that allowed the detection of up to 2.5 ng/mL of clomazone.

A monoclonal antibody-based copro-ELISA kit for canine echinococcosis to support the PAHO effort for hydatid disease control in South America.

Cystic echinococcosis is still a major concern in South America. While some regions show advances in the control of the disease, others have among the highest incidence in the world. To reverse this situation the Pan American Health Organization (PAHO) has launched a regional project on cystic echinococcosis control and surveillance. An early concern of the program was the lack of a standardized diagnostic tool to monitor infection in dogs, a key target of control programs. Under this premise, we have developed a new copro-ELISA test after extensive screening of a large panel of monoclonal antibodies (MAbs) and polyclonal sera, which performs with high standards of sensitivity (92.6%) and specificity (86.4%) as established by necropsy diagnosis of dogs. The key component of the test, MAbEg9 has a convenient IgG isotype and reacts with a periodate-resistant epitope found in high molecular weight components of the worm. Time-course analysis of experimentally infected dogs showed that even animals with a very low number of parasites could be detected as early as day 20 post infection. The test was formulated in a ready-to-use kit format with proven stability of each component for a minimum of 3 months at room temperature. This characteristic facilitates its standardized use and shipping to other laboratories, which was demonstrated by the identical results obtained by two different laboratories in Peru and our own laboratory when a large number of field samples were analyzed independently in a blind fashion.