@article{TscheuschnerKaiserLisecetal.2022, author = {Tscheuschner, Georg and Kaiser, Melanie N. and Lisec, Jan and Beslic, Denis and Muth, Thilo and Kr{\"u}ger, Maren and Mages, Hans Werner and Dorner, Brigitte G. and Knospe, Julia and Schenk, J{\"o}rg A. and Sellrie, Frank and Weller, Michael G.}, title = {MALDI-TOF-MS-based identification of monoclonal murine Anti-SARS-CoV-2 antibodies within one hour}, series = {Antibodies}, volume = {11}, journal = {Antibodies}, number = {2}, publisher = {MDPI}, address = {Basel}, issn = {2073-4468}, doi = {10.3390/antib11020027}, pages = {22}, year = {2022}, abstract = {During the SARS-CoV-2 pandemic, many virus-binding monoclonal antibodies have been developed for clinical and diagnostic purposes. This underlines the importance of antibodies as universal bioanalytical reagents. However, little attention is given to the reproducibility crisis that scientific studies are still facing to date. In a recent study, not even half of all research antibodies mentioned in publications could be identified at all. This should spark more efforts in the search for practical solutions for the traceability of antibodies. For this purpose, we used 35 monoclonal antibodies against SARS-CoV-2 to demonstrate how sequence-independent antibody identification can be achieved by simple means applied to the protein. First, we examined the intact and light chain masses of the antibodies relative to the reference material NIST-mAb 8671. Already half of the antibodies could be identified based solely on these two parameters. In addition, we developed two complementary peptide mass fingerprinting methods with MALDI-TOF-MS that can be performed in 60 min and had a combined sequence coverage of over 80\%. One method is based on the partial acidic hydrolysis of the protein by 5 mM of sulfuric acid at 99 degrees C. Furthermore, we established a fast way for a tryptic digest without an alkylation step. We were able to show that the distinction of clones is possible simply by a brief visual comparison of the mass spectra. In this work, two clones originating from the same immunization gave the same fingerprints. Later, a hybridoma sequencing confirmed the sequence identity of these sister clones. In order to automate the spectral comparison for larger libraries of antibodies, we developed the online software ABID 2.0. This open-source software determines the number of matching peptides in the fingerprint spectra. We propose that publications and other documents critically relying on monoclonal antibodies with unknown amino acid sequences should include at least one antibody fingerprint. By fingerprinting an antibody in question, its identity can be confirmed by comparison with a library spectrum at any time and context.}, language = {en} } @article{EisoldSellrieMemczaketal.2018, author = {Eisold, Ursula and Sellrie, Frank and Memczak, Henry and Andersson, Anika and Schenk, J{\"o}rg A. and Kumke, Michael Uwe}, title = {Dye tool box for a fluorescence enhancement immunoassay}, series = {Bioconjugate chemistry}, volume = {29}, journal = {Bioconjugate chemistry}, number = {1}, publisher = {American Chemical Society}, address = {Washington}, issn = {1043-1802}, doi = {10.1021/acs.bioconjchem.7b00731}, pages = {203 -- 214}, year = {2018}, abstract = {Immunochemical analytical methods are very successful in clinical diagnostics and are nowadays also emerging in the control of food as well as monitoring of environmental issues. Among the different immunoassays, luminescence based formats are characterized by their outstanding sensitivity making this format especially attractive for future applications. The need for multiparameter detection capabilities calls for a tool box of dye labels in order to transduce the biochemical reaction into an optically detectable signal. Here, in a multiparameter approach each analyte may be detected by a different dye with a unique emission color (covering the blue to red spectral range) or a unique luminescence decay kinetics. In the case of a competitive immunoassay format for each of the different dye labels an individual antibody would be needed. In the present paper a slightly modified approach is presented using a 7-aminocoumarin unit as the basic antigen against which highly specific antibodies were generated. Leaving the epitope region in the dyes unchanged but introducing a side group in positon 3 of the coumarin system allowed us to tune the optical properties of the coumarin dyes without the necessity of new antibody generation. Upon modification of the parent coumarin unit the full spectral range from blue to deep red was accessed. In the manuscript the photophysical characterization of the coumarin derivatives and their corresponding immunocomplexes with two highly specific antibodies is presented. The coumarin dyes and their immunocomplexes were characterized by steady-state and time-resolved absorption as well as emission spectroscopy. Moreover, fluorescence depolarization measurements were carried out to complement the data stressing the different binding modes of the two antibodies. The binding modes were evaluated using the photophysics of 7-aminocoumarins and how it was affected in the respective immunocomplexes, namely, the formation of the intramolecular charge transfer (ICT) as well as the twisted intramolecular charge transfer (TICT). In contrast to other antibody-dye pairs reported a distinct fluorescence enhancement upon formation of the antibody-dye complex up to a factor of SO was found. Because of the easy emission color tuning by tailoring the coumarin substitution for the antigen binding in nonrelevant position 3 of the parent molecule, a dye tool box is on hand which can be used in the construction of competitive multiparameter fluorescence enhancement immunoassays (FenIA).}, language = {en} } @article{HoangMertensWessigetal.2018, author = {Hoang, Hoa T. and Mertens, Monique and Wessig, Pablo and Sellrie, Frank and Schenk, J{\"o}rg A. and Kumke, Michael Uwe}, title = {Antibody Binding at the Liposome-Water Interface}, series = {ACS Omega}, volume = {3}, journal = {ACS Omega}, number = {12}, publisher = {American Chemical Society}, address = {Washington}, issn = {2470-1343}, doi = {10.1021/acsomega.8b03016}, pages = {18109 -- 18116}, year = {2018}, abstract = {Different signal amplification strategies to improve the detection sensitivity of immunoassays have been applied which utilize enzymatic reactions, nanomaterials, or liposomes. The latter are very attractive materials for signal amplification because liposomes can be loaded with a large amount of signaling molecules, leading to a high sensitivity. In addition, liposomes can be used as a cell-like "bioscaffold" to directly test recognition schemes aiming at cell-related processes. This study demonstrates an easy and fast approach to link the novel hydrophobic optical probe based on [1,3]dioxolo[4,5-f]-[1,3]benzodioxole (DBD dye mm239) with tunable optical properties to hydrophilic recognition elements (e.g., antibodies) using liposomes for signal amplification and as carrier of the hydrophobic dye. The fluorescence properties of mm239 (e.g., long fluorescence lifetime, large Stokes shift, high photostability, and high quantum yield), its high hydrophobicity for efficient anchoring in liposomes, and a maleimide bioreactive group were applied in a unique combination to build a concept for the coupling of antibodies or other protein markers to liposomes (coupling to membranes can be envisaged). The concept further allowed us to avoid multiple dye labeling of the antibody. Here, anti-TAMRA-antibody (DC7-Ab) was attached to the liposomes. In proof-of-concept, steady-state as well as time-resolved fluorescence measurements (e.g., fluorescence depolarization) in combination with single molecule detection (fluorescence correlation spectroscopy, FCS) were used to analyze the binding interaction between DC7-Ab and liposomes as well as the binding of the antigen rhodamine 6G (R6G) to the antibody. Here, the Forster resonance energy transfer (FRET) between mm239 and R6G was monitored. In addition to ensemble FRET data, single-molecule FRET (PIE-FRET) experiments using pulsed interleaved excitation were used to characterize in detail the binding on a single-molecule level to avoid averaging out effects.}, language = {en} } @article{DippongCarlLenzetal.2017, author = {Dippong, Martin and Carl, Peter and Lenz, Christine and Schenk, J{\"o}rg A. and Hoffmann, Katrin and Schwaar, Timm and Schneider, Rudolf J. and Kuhne, Maren}, title = {Hapten-Specific Single-Cell Selection of Hybridoma Clones by Fluorescence-Activated Cell Sorting for the Generation of Monoclonal Antibodies}, series = {Analytical chemistry}, volume = {89}, journal = {Analytical chemistry}, publisher = {American Chemical Society}, address = {Washington}, issn = {0003-2700}, doi = {10.1021/acs.analchem.6b04569}, pages = {4007 -- 4012}, year = {2017}, language = {en} } @article{EisoldSellrieSchenketal.2015, author = {Eisold, Ursula and Sellrie, Frank and Schenk, J{\"o}rg A. and Lenz, Christine and St{\"o}cklein, Walter F. M. and Kumke, Michael Uwe}, title = {Bright or dark immune complexes of anti-TAMRA antibodies for adapted fluorescence-based bioanalysis}, series = {Analytical \& bioanalytical chemistry}, volume = {407}, journal = {Analytical \& bioanalytical chemistry}, number = {12}, publisher = {Springer}, address = {Heidelberg}, issn = {1618-2642}, doi = {10.1007/s00216-015-8538-0}, pages = {3313 -- 3323}, year = {2015}, abstract = {Fluorescence labels, for example fluorescein or rhodamin derivatives, are widely used in bioanalysis applications including lateral-flow assays, PCR, and fluorescence microscopy. Depending on the layout of the particular application, fluorescence quenching or enhancement may be desired as the detection principle. Especially for multiplexed applications or high-brightness requirements, a tunable fluorescence probe can be beneficial. The alterations in the photophysics of rhodamine derivatives upon binding to two different anti-TAMRA antibodies were investigated by absorption and fluorescence-spectroscopy techniques, especially determining the fluorescence decay time and steady-state and time-resolved fluorescence anisotropy. Two monoclonal anti-TAMRA antibodies were generated by the hybridoma technique. Although surface-plasmon-resonance measurements clearly proved the high affinity of both antibodies towards 5-TAMRA, the observed effects on the fluorescence of rhodamine derivatives were very different. Depending on the anti-TAMRA antibody either a strong fluorescence quenching (G71-DC7) or a distinct fluorescence enhancement (G71-BE11) upon formation of the immune complex was observed. Additional rhodamine derivatives were used to gain further information on the binding interaction. The data reveal that such haptens as 5-TAMRA could generate different paratopes with equal binding affinities but different binding interactions, which provide the opportunity to adapt bioanalysis methods including immunoassays for optimized detection principles for the same hapten depending on the specific requirements.}, language = {en} } @article{KuhneDippongFlemigetal.2014, author = {Kuhne, Maren and Dippong, Martin and Flemig, Sabine and Hoffmann, Katrin and Petsch, Kristin and Schenk, J{\"o}rg A. and Kunte, Hans-J{\"o}rg and Schneider, Rudolf J.}, title = {Comparative characterization of mAb producing hapten-specific hybridoma cells by flow cytometric analysis and ELISA}, series = {Journal of immunological methods}, volume = {413}, journal = {Journal of immunological methods}, publisher = {Elsevier}, address = {Amsterdam}, issn = {0022-1759}, doi = {10.1016/j.jim.2014.07.004}, pages = {45 -- 56}, year = {2014}, abstract = {A novel method that optimizes the screening for antibody-secreting hapten-specific hybridoma cells by using flow cytometry is described. Cell clones specific for five different haptens were analyzed. We selectively double stained and analyzed fixed hybridoma cells with fluorophore-labeled haptens to demonstrate the target-selectivity, and with a fluorophore-labeled anti-mouse IgG antibody to characterize the level of surface expression of membrane-bound IgGs. ELISA measurements with the supernatants of the individual hybridoma clones revealed that antibodies from those cells, which showed the highest fluorescence intensities in the flow cytometric analysis, also displayed the highest affinities for the target antigens. The fluorescence intensity of antibody-producing cells corresponded well with the produced antibodies' affinities toward their respective antigens. Immunohistochemical staining verified the successful double labeling of the cells. Our method makes it possible to perform a high-throughput screening for hybridoma cells, which have both an adequate IgG production rate and a high target affinity. (C) 2014 Elsevier B.V. All rights reserved.}, language = {en} } @article{SchenkFettkeLenzetal.2012, author = {Schenk, J{\"o}rg A. and Fettke, J{\"o}rg and Lenz, Christine and Albers, Katharina and Mallwitz, Frank and Gajovic-Eichelmann, Nenad and Ehrentreich-F{\"o}rster, Eva and Kusch, Emely and Sellrie, Frank}, title = {Secretory leukocyte protease inhibitor (SLPI) might contaminate murine monoclonal antibodies after purification on protein G}, series = {Journal of biotechnology}, volume = {158}, journal = {Journal of biotechnology}, number = {1-2}, publisher = {Elsevier}, address = {Amsterdam}, issn = {0168-1656}, doi = {10.1016/j.jbiotec.2011.12.025}, pages = {34 -- 35}, year = {2012}, abstract = {The large scale production of a monoclonal anti-progesterone antibody in serum free medium followed by affinity chromatography on protein G lead to a contamination of the antibody sample with a protein of about 14 kDa. This protein was identified by mass spectrometry as secretory leukocyte protease inhibitor (SLPI). This SLPI contamination lead to a failure of the fiber-optic based competitive fluorescence assay to detect progesterone in milk. Purification of the monoclonal antibody using protein A columns circumvented this problem.}, language = {en} } @article{EttlingerSchenkMicheeletal.2012, author = {Ettlinger, Julia and Schenk, J{\"o}rg A. and Micheel, Burkhard and Ehrentreich-F{\"o}rster, Eva and Gajovic-Eichelmann, Nenad}, title = {A direct competitive homogeneous immunoassay for progesterone - the Redox Quenching Immunoassay}, series = {Electroanalysis : an international journal devoted to fundamental and practical aspects of electroanalysis}, volume = {24}, journal = {Electroanalysis : an international journal devoted to fundamental and practical aspects of electroanalysis}, number = {7}, publisher = {Wiley-VCH}, address = {Weinheim}, issn = {1040-0397}, doi = {10.1002/elan.201200107}, pages = {1567 -- 1575}, year = {2012}, abstract = {A direct competitive amperometric immunoassay format for the detection of haptens and proteins was developed. The method is based on the quenching of electroactivity of ferrocenium, which is coupled to the antigen and used as the primary reporter, upon binding to a monoclonal anti-ferrocenium antibody, which is coupled to the detection antibody and used as a secondary reporter. A separation-free progesterone immunoassay with a lower detection limit of 1 ng?mL-1 (3.18 nmol?L-1) in 1?:?2 diluted blood serum was realised by combining two bifunctional conjugates, a ferrocenium-PEG-progesterone tracer and a bioconjugate of one anti-progesterone and one anti-ferrocenium antibody. The immune complex is formed within 30 s upon addition of progesterone, resulting in a total analysis time of 1.5 min.}, language = {en} } @article{StechMerkSchenketal.2012, author = {Stech, Marlitt and Merk, Helmut and Schenk, J{\"o}rg A. and St{\"o}cklein, Walter F. M. and W{\"u}stenhagen, Doreen Anja and Micheel, Burkhard and Duschl, Claus and Bier, Frank Fabian and Kubick, Stefan}, title = {Production of functional antibody fragments in a vesicle-based eukaryotic cell-free translation system}, series = {Journal of biotechnology}, volume = {164}, journal = {Journal of biotechnology}, number = {2}, publisher = {Elsevier}, address = {Amsterdam}, issn = {0168-1656}, doi = {10.1016/j.jbiotec.2012.08.020}, pages = {220 -- 231}, year = {2012}, abstract = {Cell-free protein synthesis is of increasing interest for the rapid and high-throughput synthesis of many proteins, in particular also antibody fragments. In this study, we present a novel strategy for the production of single chain antibody fragments (scFv) in a eukaryotic in vitro translation system. This strategy comprises the cell-free expression, isolation and label-free interaction analysis of a model antibody fragment synthesized in two differently prepared insect cell lysates. These lysates contain translocationally active microsomal structures derived from the endoplasmic reticulum (ER), allowing for posttranslational modifications of cell-free synthesized proteins. Both types of these insect cell lysates enable the synthesis and translocation of scFv into ER-derived vesicles. However, only the one that has a specifically adapted redox potential yields functional active antibody fragments. We have developed a new methodology for the isolation of functional target proteins based on the translocation of cell-free produced scFv into microsomal structures and subsequent collection of protein-enriched vesicles. Antibody fragments that have been released from these vesicles are shown to be well suited for label-free binding studies. Altogether, these results show the potential of insect cell lysates for the production, purification and selection of antibody fragments in an easy-to-handle and time-saving manner.}, language = {en} } @article{InalKoelschSellrieetal.2013, author = {Inal, Sahika and K{\"o}lsch, Jonas D. and Sellrie, Frank and Schenk, J{\"o}rg A. and Wischerhoff, Erik and Laschewsky, Andr{\´e} and Neher, Dieter}, title = {A water soluble fluorescent polymer as a dual colour sensor for temperature and a specific protein}, series = {Journal of materials chemistry : B, Materials for biology and medicine}, volume = {1}, journal = {Journal of materials chemistry : B, Materials for biology and medicine}, number = {46}, publisher = {Royal Society of Chemistry}, address = {Cambridge}, issn = {2050-750X}, doi = {10.1039/c3tb21245a}, pages = {6373 -- 6381}, year = {2013}, abstract = {We present two thermoresponsive water soluble copolymers prepared via free radical statistical copolymerization of N-isopropylacrylamide (NIPAm) and of oligo(ethylene glycol) methacrylates (OEGMAs), respectively, with a solvatochromic 7-(diethylamino)-3-carboxy-coumarin (DEAC)-functionalized monomer. In aqueous solutions, the NIPAm-based copolymer exhibits characteristic changes in its fluorescence profile in response to a change in solution temperature as well as to the presence of a specific protein, namely an anti-DEAC antibody. This polymer emits only weakly at low temperatures, but exhibits a marked fluorescence enhancement accompanied by a change in its emission colour when heated above its cloud point. Such drastic changes in the fluorescence and absorbance spectra are observed also upon injection of the anti-DEAC antibody, attributed to the specific binding of the antibody to DEAC moieties. Importantly, protein binding occurs exclusively when the polymer is in the well hydrated state below the cloud point, enabling a temperature control on the molecular recognition event. On the other hand, heating of the polymer-antibody complexes releases a fraction of the bound antibody. In the presence of the DEAC-functionalized monomer in this mixture, the released antibody competitively binds to the monomer and the antibody-free chains of the polymer undergo a more effective collapse and inter-aggregation. In contrast, the emission properties of the OEGMA-based analogous copolymer are rather insensitive to the thermally induced phase transition or to antibody binding. These opposite behaviours underline the need for a carefully tailored molecular design of responsive polymers aimed at specific applications, such as biosensing.}, language = {en} }