Suchergebnisse

Microscale thermophoresis (MST), and the closely related Temperature Related Intensity Change (TRIC), are synonyms for a recently developed measurement technique in the field of biophysics to quantify biomolecular interactions, using the (capillary-based) NanoTemper Monolith and (multiwell plate-based) Dianthus instruments. Although this technique has been extensively used within the scientific community due to its low sample consumption, ease of use, and ubiquitous applicability, MST/TRIC has not enjoyed the unambiguous acceptance from biophysicists afforded to other biophysical techniques like isothermal titration calorimetry (ITC) or surface plasmon resonance (SPR). This might be attributed to several facts, e.g., that various (not fully understood) effects are contributing to the signal, that the technique is licensed to only a single instrument developer, NanoTemper Technology, and that its reliability and reproducibility have never been tested independently and systematically. Thus, a working group of ARBRE-MOBIEU has set up a benchmark study on MST/TRIC to assess this technique as a method to characterize biomolecular interactions. Here we present the results of this study involving 32 scientific groups within Europe and two groups from the US, carrying out experiments on 40 Monolith instruments, employing a standard operation procedure and centrally prepared samples. A protein-small molecule interaction, a newly developed protein-protein interaction system and a pure dye were used as test systems. We characterized the instrument properties and evaluated instrument performance, reproducibility, the effect of different analysis tools, the influence of the experimenter during data analysis, and thus the overall reliability of this method. ; All authors acknowledge the COST Action project ARBRE-MOBIEU CA15126 under the auspices of whose Working Group 4 this study was carried out. ARBRE-MOBIEU COST Action CA15126 funded meetings in the Center for Protein Research, University of Copenhagen (ECOST-MEETING-CA15126-280618-098884) to organize this study and in the Vienna Biocenter Core Facilities GmbH (ECOST-MEETING-CA15126-121119-111544) to discuss the results of the benchmark. Ondřej Vaněk acknowledges support from the Ministry of Education, Youth and Sports of the Czech Republic (LTC17065 in frame of the COST Action CA15126). Jitka Holková and Josef Houser acknowledge CF Biomolecular Interactions and Crystallization of CIISB, Instruct-CZ Centre, supported by MEYS CR (LM2018127). Jasmina Rokov-Plavec acknowledges support from the Croatian Science Foundation (IP-2016-06-6272). Alexander K. Buell thanks the Novo Nordisk Foundation (grant number NNFSA170028392) for funding. Li Peng Lundgren acknowledges for preliminary MST/TRIC experiments (STMS 37745 in the frame of the COST Action CA15126) and the COST Action (CA15126) for a STMS (STMS 44783) for the data analysis. The Novo Nordisk Foundation Center for Protein Research is supported financially by the Novo Nordisk Foundation (Grant agreement NNF14CC0001). Katja Pirc and Marjetka Podobnik acknowledge the grant by the Slovenian Research Agency P1-0391 (Title: Molecular Interactions). This research was funded by Fondo de Investigaciones Sanitarias from Instituto de Salud Carlos III and European Union (ERDF/ESF, "Investing in your future" PI18/00349 and Diputación General de Aragón Digestive Pathology Group B25_17R to O.A. Spanish Ministry of Science, Innovation and Universities, FPI Predoctoral Research Contract BES-2017-080739 to D.O.A. This research was funded by project no 2015/17/B/NZ2/01160 (granted to MA)

The surge of SARS-CoV-2 has challenged health systems worldwide and efficient tests to detect viral particles, as well as antibodies generated against them, are needed. Specificity, sensitivity, promptness or scalability are the main parameters to estimate the final performance, but rarely all of them match in a single test. We have developed SCOVAM, a protein microarray with several viral antigens (spike, nucleocapsid, main protease Nsp5) as capturing probes in a fluorescence immunoassay for COVID-19 serological testing. SCOVAM depicts IgG and IgM antibody responses against each of these proteins of 22 individuals in a single microscope slide. It detects specific IgM (0.094 μg ml-1 ) and IgG (~0.017 μg ml-1 ) and is scalable and cost-effective. We validated SCOVAM by comparing with a widely used chemiluminescent commercial serological test (n = 742). SCOVAM showed twice the sensitivity and allowed following seroconversion in a single assay. By analysing the prevalence 4 months later in a subset of 76 positive sera, we still detected 93.42% of positives, almost doubling the detection of the commercial assay. The higher sensitivity of SCOVAM is especially relevant to screen sera for convalescent plasma-based treatments, high-throughput antibody response monitoring after vaccination or evaluation of vaccine efficiency. ; This work has been funded by INTA's internal budget and the Spanish Ministry of Science and Innovation grant nos. RTI2018-094368-B-I00, and MDM-2017-0737 (Excelencia 'María de Maeztu' to Centro de Astrobiología). DRG is funded by the 'Programa de Atracción de Talento' of local Government of Madrid. Centro Superior de Investigaciones Cientificas: project number 202020E079

The surge of SARS‐CoV‐2 has challenged health systems worldwide and efficient tests to detect viral particles, as well as antibodies generated against them, are needed. Specificity, sensitivity, promptness or scalability are the main parameters to estimate the final performance, but rarely all of them match in a single test. We have developed SCOVAM, a protein microarray with several viral antigens (spike, nucleocapsid, main protease Nsp5) as capturing probes in a fluorescence immunoassay for COVID‐19 serological testing. SCOVAM depicts IgG and IgM antibody responses against each of these proteins of 22 individuals in a single microscope slide. It detects specific IgM (0.094 μg ml‐1) and IgG (~0.017 μg ml‐1) and is scalable and cost‐effective. We validated SCOVAM by comparing with a widely used chemiluminescent commercial serological test (n = 742). SCOVAM showed twice the sensitivity and allowed following seroconversion in a single assay. By analysing the prevalence 4 months later in a subset of 76 positive sera, we still detected 93.42% of positives, almost doubling the detection of the commercial assay. The higher sensitivity of SCOVAM is especially relevant to screen sera for convalescent plasma‐based treatments, high‐throughput antibody response monitoring after vaccination or evaluation of vaccine efficiency. ; This work has been funded by INTA's internal budget and the Spanish Ministry of Science and Innovation grant nos. RTI2018‐094368‐B‐I00, and MDM‐2017‐0737 (Excelencia 'María de Maeztu' to Centro de Astrobiología). The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication. DRG is funded by the 'Programa de Atracción de Talento' of local Government of Madrid. Centro Superior de Investigaciones Cientificas: project number 202020E079. ; Peer reviewed