@misc{GoethelListekMesserschmidtetal.2021, author = {G{\"o}thel, Markus and Listek, Martin and Messerschmidt, Katrin and Schl{\"o}r, Anja and H{\"o}now, Anja and Hanack, Katja}, title = {A New Workflow to Generate Monoclonal Antibodies against Microorganisms}, series = {Mathematisch-Naturwissenschaftliche Reihe}, journal = {Mathematisch-Naturwissenschaftliche Reihe}, number = {20}, issn = {1866-8372}, doi = {10.25932/publishup-52334}, url = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus4-523341}, pages = {17}, year = {2021}, abstract = {Monoclonal antibodies are used worldwide as highly potent and efficient detection reagents for research and diagnostic applications. Nevertheless, the specific targeting of complex antigens such as whole microorganisms remains a challenge. To provide a comprehensive workflow, we combined bioinformatic analyses with novel immunization and selection tools to design monoclonal antibodies for the detection of whole microorganisms. In our initial study, we used the human pathogenic strain E. coli O157:H7 as a model target and identified 53 potential protein candidates by using reverse vaccinology methodology. Five different peptide epitopes were selected for immunization using epitope-engineered viral proteins. The identification of antibody-producing hybridomas was performed by using a novel screening technology based on transgenic fusion cell lines. Using an artificial cell surface receptor expressed by all hybridomas, the desired antigen-specific cells can be sorted fast and efficiently out of the fusion cell pool. Selected antibody candidates were characterized and showed strong binding to the target strain E. coli O157:H7 with minor or no cross-reactivity to other relevant microorganisms such as Legionella pneumophila and Bacillus ssp. This approach could be useful as a highly efficient workflow for the generation of antibodies against microorganisms.}, language = {en} } @misc{RoggenbuckBorghiSommaetal.2016, author = {Roggenbuck, Dirk and Borghi, Maria Orietta and Somma, Valentina and B{\"u}ttner, Thomas and Schierack, Peter and Hanack, Katja and Grossi, Claudia and Bodio, Caterina and Macor, Paolo and von Landenberg, Philipp and Boccellato, Francesco and Mahler, Michael and Meroni, Pier Luigi}, title = {Antiphospholipid antibodies detected by line immunoassay differentiate among patients with antiphospholipid syndrome, with infections and asymptomatic carriers}, series = {Postprints der Universit{\"a}t Potsdam Mathematisch-Naturwissenschaftliche Reihe}, journal = {Postprints der Universit{\"a}t Potsdam Mathematisch-Naturwissenschaftliche Reihe}, number = {436}, issn = {1866-8372}, url = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus4-407211}, pages = {14}, year = {2016}, abstract = {Background Antiphospholipid antibodies (aPL) can be detected in asymptomatic carriers and infectious patients. The aim was to investigate whether a novel line immunoassay (LIA) differentiates between antiphospholipid syndrome (APS) and asymptomatic aPL+ carriers or patients with infectious diseases (infectious diseases controls (IDC)). Methods Sixty-one patients with APS (56 primary, 22/56 with obstetric events only, and 5 secondary), 146 controls including 24 aPL+ asymptomatic carriers and 73 IDC were tested on a novel hydrophobic solid phase coated with cardiolipin (CL), phosphatic acid, phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylinositol, phosphatidylserine, beta2-glycoprotein I (β2GPI), prothrombin, and annexin V. Samples were also tested by anti-CL and anti-β2GPI ELISAs and for lupus anticoagulant activity. Human monoclonal antibodies (humoAbs) against human β2GPI or PL alone were tested on the same LIA substrates in the absence or presence of human serum, purified human β2GPI or after CL-micelle absorption. Results Comparison of LIA with the aPL-classification assays revealed good agreement for IgG/IgM aß2GPI and aCL. Anti-CL and anti-ß2GPI IgG/IgM reactivity assessed by LIA was significantly higher in patients with APS versus healthy controls and IDCs, as detected by ELISA. IgG binding to CL and ß2GPI in the LIA was significantly lower in aPL+ carriers and Venereal Disease Research Laboratory test (VDRL) + samples than in patients with APS. HumoAb against domain 1 recognized β2GPI bound to the LIA-matrix and in anionic phospholipid (PL) complexes. Absorption with CL micelles abolished the reactivity of a PL-specific humoAb but did not affect the binding of anti-β2GPI humoAbs. Conclusions The LIA and ELISA have good agreement in detecting aPL in APS, but the LIA differentiates patients with APS from infectious patients and asymptomatic carriers, likely through the exposure of domain 1.}, language = {en} } @misc{CzarneckaWeicheltRoedigeretal.2022, author = {Czarnecka, Malgorzata and Weichelt, Ulrike and R{\"o}diger, Stefan and Hanack, Katja}, title = {Novel Anti Double-Stranded Nucleic Acids Full-Length Recombinant Camelid Heavy-Chain Antibody for the Detection of miRNA}, series = {Zweitver{\"o}ffentlichungen der Universit{\"a}t Potsdam : Mathematisch-Naturwissenschaftliche Reihe}, journal = {Zweitver{\"o}ffentlichungen der Universit{\"a}t Potsdam : Mathematisch-Naturwissenschaftliche Reihe}, issn = {1866-8372}, doi = {10.25932/publishup-56914}, url = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus4-569142}, pages = {1 -- 18}, year = {2022}, abstract = {The discovery that certain diseases have specific miRNA signatures which correspond to disease progression opens a new biomarker category. The detection of these small non-coding RNAs is performed routinely using body fluids or tissues with real-time PCR, next-generation sequencing, or amplification-based miRNA assays. Antibody-based detection systems allow an easy onset handling compared to PCR or sequencing and can be considered as alternative methods to support miRNA diagnostic in the future. In this study, we describe the generation of a camelid heavy-chain-only antibody specifically recognizing miRNAs to establish an antibody-based detection method. The generation of nucleic acid-specific binders is a challenge. We selected camelid binders via phage display, expressed them as VHH as well as full-length antibodies, and characterized the binding to several miRNAs from a signature specific for dilated cardiomyopathy. The described workflow can be used to create miRNA-specific binders and establish antibody-based detection methods to provide an additional way to analyze disease-specific miRNA signatures.}, language = {en} } @misc{SchloerHirschbergBenAmoretal.2022, author = {Schl{\"o}r, Anja and Hirschberg, Stefan and Ben Amor, Ghada and Meister, Toni Luise and Arora, Prerna and P{\"o}hlmann, Stefan and Hoffmann, Markus and Pf{\"a}nder, Stephanie and Eddin, Omar Kamal and Kamhieh-Milz, Julian and Hanack, Katja}, title = {SARS-CoV-2 neutralizing camelid heavy-chain-only antibodies as powerful tools for diagnostic and therapeutic applications}, series = {Zweitver{\"o}ffentlichungen der Universit{\"a}t Potsdam : Mathematisch-Naturwissenschaftliche Reihe}, journal = {Zweitver{\"o}ffentlichungen der Universit{\"a}t Potsdam : Mathematisch-Naturwissenschaftliche Reihe}, number = {1280}, issn = {1866-8372}, doi = {10.25932/publishup-57012}, url = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus4-570124}, pages = {14}, year = {2022}, abstract = {Introduction: The ongoing COVID-19 pandemic situation caused by SARS-CoV-2 and variants of concern such as B.1.617.2 (Delta) and recently, B.1.1.529 (Omicron) is posing multiple challenges to humanity. The rapid evolution of the virus requires adaptation of diagnostic and therapeutic applications. Objectives: In this study, we describe camelid heavy-chain-only antibodies (hcAb) as useful tools for novel in vitro diagnostic assays and for therapeutic applications due to their neutralizing capacity. Methods: Five antibody candidates were selected out of a na{\"i}ve camelid library by phage display and expressed as full length IgG2 antibodies. The antibodies were characterized by Western blot, enzyme-linked immunosorbent assays, surface plasmon resonance with regard to their specificity to the recombinant SARS-CoV-2 Spike protein and to SARS-CoV-2 virus-like particles. Neutralization assays were performed with authentic SARS-CoV-2 and pseudotyped viruses (wildtype and Omicron). Results: All antibodies efficiently detect recombinant SARS-CoV-2 Spike protein and SARS-CoV-2 virus-like particles in different ELISA setups. The best combination was shown with hcAb B10 as catcher antibody and HRP-conjugated hcAb A7.2 as the detection antibody. Further, four out of five antibodies potently neutralized authentic wildtype SARS-CoV-2 and particles pseudotyped with the SARS-CoV-2 Spike proteins of the wildtype and Omicron variant, sublineage BA.1 at concentrations between 0.1 and 0.35 ng/mL (ND50). Conclusion: Collectively, we report novel camelid hcAbs suitable for diagnostics and potential therapy.}, language = {en} }