RESUMEN
The pandemic readiness toolbox needs to be extended, targeting different biomolecules, using orthogonal experimental set-ups. Here, we build on our Cov-MS effort using LC-MS, adding SISCAPA technology to enrich proteotypic peptides of the SARS-CoV-2 nucleocapsid (N) protein from trypsin-digested patient samples. The Cov2MS assay is compatible with most matrices including nasopharyngeal swabs, saliva, and plasma and has increased sensitivity into the attomole range, a 1000-fold improvement compared to direct detection in a matrix. A strong positive correlation was observed with qPCR detection beyond a quantification cycle of 30-31, the level where no live virus can be cultured. The automatable sample preparation and reduced LC dependency allow analysis of up to 500 samples per day per instrument. Importantly, peptide enrichment allows detection of the N protein in pooled samples without sensitivity loss. Easily multiplexed, we detect variants and propose targets for Influenza A and B detection. Thus, the Cov2MS assay can be adapted to test for many different pathogens in pooled samples, providing longitudinal epidemiological monitoring of large numbers of pathogens within a population as an early warning system.
Asunto(s)
COVID-19 , SARS-CoV-2 , Humanos , Prueba de COVID-19 , Técnicas de Laboratorio Clínico/métodos , Espectrometría de Masas/métodos , Péptidos , Sensibilidad y EspecificidadRESUMEN
The World Health Organization advices the use of a quadrivalent vaccine as prophylaxis against influenza, to prevent severe influenza-associated disease and -mortality, and to keep up with influenza antigenic diversity. Different small molecule antivirals to treat influenza have become available. However, emergence of drug resistant influenza viruses has been observed upon use of these antivirals. An appealing alternative approach to prevent or treat influenza is the use of antibody-based antivirals, such as conventional monoclonal antibodies and single-domain antibodies (sdAbs). The surface of the influenza A and B virion is decorated with hemagglutinin molecules, which act as receptor-binding and membrane fusion proteins and represent the main target of neutralizing antibodies. SdAbs that target influenza A and B hemagglutinin have been described. In addition, sdAbs directed against the influenza A virus neuraminidase have been reported, whereas no sdAbs targeting influenza B neuraminidase have been described to date. SdAbs directed against influenza A matrix protein 2 or its ectodomain have been reported, while no sdAbs have been described targeting the influenza B matrix protein 2. Known for their high specificity, ease of production and formatting, sdAb-based antivirals could be a major leap forward in influenza control.