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Background: Antibody tests are essential tools to investigate humoral immunity following SARS-CoV-2 infection or vaccination. While first-generation antibody tests have primarily provided qualitative results, accurate seroprevalence studies and tracking of antibody levels over time require highly specific, sensitive and quantitative test setups., Methods: We have developed two quantitative, easy-to-implement SARS-CoV-2 antibody tests, based on the spike receptor binding domain and the nucleocapsid protein. Comprehensive evaluation of antigens from several biotechnological platforms enabled the identification of superior antigen designs for reliable serodiagnostic. Cut-off modelling based on unprecedented large and heterogeneous multicentric validation cohorts allowed us to define optimal thresholds for the tests' broad applications in different aspects of clinical use, such as seroprevalence studies and convalescent plasma donor qualification., Findings: Both developed serotests individually performed similarly-well as fully-automated CE-marked test systems. Our described sensitivity-improved orthogonal test approach assures highest specificity (99.8%); thereby enabling robust serodiagnosis in low-prevalence settings with simple test formats. The inclusion of a calibrator permits accurate quantitative monitoring of antibody concentrations in samples collected at different time points during the acute and convalescent phase of COVID-19 and disclosed antibody level thresholds that correlate well with robust neutralization of authentic SARS-CoV-2 virus., Interpretation: We demonstrate that antigen source and purity strongly impact serotest performance. Comprehensive biotechnology-assisted selection of antigens and in-depth characterisation of the assays allowed us to overcome limitations of simple ELISA-based antibody test formats based on chromometric reporters, to yield comparable assay performance as fully-automated platforms., Funding: WWTF, Project No. COV20-016; BOKU, LBI/LBG., Competing Interests: Declaration of Competing Interest Dr. Klausberger has nothing to disclose. Dr. Duerkop has nothing to disclose. Dr. Haslacher has nothing to disclose. Dr. Wozniak-Knopp has nothing to disclose. Dr. Cserjan-Puschmann has nothing to disclose. Dr. Perkmann has nothing to disclose. Dr. Lingg has nothing to disclose. Dr. Pereira Aguilar has nothing to disclose. Dr. Laurent has nothing to disclose. Dr. De Vos has nothing to disclose. Mag.rer.nat. Hofner has nothing to disclose. Dr. Holzer has nothing to disclose. Mrs. Stadler has nothing to disclose. Dipl.-Ing. Manhart has nothing to disclose. DI Vierlinger has nothing to disclose. Dr. Egger has nothing to disclose. Dipl. Ing. Milchram has nothing to disclose. Dr. Gludovacz has nothing to disclose. Dr. Marx has nothing to disclose. Dipl.-Ing. Köppl has nothing to disclose. Christopher Tauer, BSc has nothing to disclose. Jürgen Beck, MSc reports nothing to disclose. Daniel Maresch has nothing to disclose. Dr. Grünwald-Gruber has nothing to disclose. Mr. Strobl has nothing to disclose. Dr. Satzer has nothing to disclose. Dr. Stadlmayr has nothing to disclose. Ing. Vavra has nothing to disclose. Ms. Huber BSc has nothing to disclose. Dr. Wahrmann has nothing to disclose. Dr. Eskandary has nothing to disclose. Dr. Breyer has nothing to disclose. Dr. Sieghart has nothing to disclose. Dr. Quehenberger reports other from Roche Austria, personal fees from Takeda, outside the submitted work; . Dr. Leitner has nothing to disclose. Dr. Strassl has nothing to disclose. Dr. Egger has nothing to disclose. Dr. IRSARA has nothing to disclose. Dr. Griesmacher has nothing to disclose. Dr. Hoermann has nothing to disclose. Dr. Weiss has nothing to disclose. Dr. Bellmann-Weiler has nothing to disclose. Dr. Löffler-Ragg has nothing to disclose. Dr. Borth has nothing to disclose. Dr. Strasser has nothing to disclose. Dr. Jungbauer has nothing to disclose. Dr. Hahn has nothing to disclose. Dr. Mairhofer reports other from enGenes Biotech GmbH, outside the submitted work; In addition, Dr. Mairhofer has a patent PCT/EP2016/059597-Uncoupling growth and protein production issued. Dr. Hartmann has nothing to disclose. Dr. Binder reports grants from Vienna Business Agency, during the conduct of the study; and Employee of Technoclone Herstellung von Diagnostika und Arzneimitteln GmbH. Dr. Striedner reports other from enGenes GmbH, outside the submitted work; In addition, Dr. Striedner has a patent. US20180282737A1 issued to enGenes GmbH. Dr. Mach has nothing to disclose. Dr. Weinhaeusel has nothing to disclose. Dr. Dieplinger has nothing to disclose. Dr. Grebien has nothing to disclose. Dr. Gerner has nothing to disclose. Dr. Christoph Binder is board member of Technoclone GmbH. Dr. Grabherr has nothing to disclose., (Copyright © 2021 The Author(s). Published by Elsevier B.V. All rights reserved.)

Monoclonal antibodies can acquire the property of engagement of a second antigen via fusion methods or modification of their CDR loops, but also by modification of their constant domains, such as in the mAb 2 format where a set of mutated amino acid residues in the C H 3 domains enables a high-affinity specific interaction with the second antigen. We tested the possibility of introducing multiple binding sites for the second antigen by replacing the Fab C H 1/C L domain pair with a pair of antigen-binding C H 3 domains in a model scaffold with trastuzumab variable domains and VEGF-binding C H 3 domains. Such bispecific molecules were produced in a "Fab-like" format and in a full-length antibody format. Novel constructs were of expected molecular composition using mass spectrometry. They were expressed at a high level in standard laboratory conditions, purified as monomers with Protein A and gel filtration and were of high thermostability. Their high-affinity binding to both target antigens was retained. Finally, the Her2/VEGF binding domain-exchanged bispecific antibody was able to mediate a potentiated surface Her2-internalization effect on the Her2-overexpressing cell line SK-BR-3 due to improved level of cross-linking with the endogenously secreted cytokine. To conclude, bispecific antibodies with Fabs featuring exchanged antigen-binding C H 3 domains offer an alternative solution in positioning and valency of antigen binding sites., (© 2021 The Authors. Published by Elsevier B.V.)