@article{MichelchenMicheelHanack2021, author = {Michelchen, Sophia and Micheel, Burkhard and Hanack, Katja}, title = {In vitro immunization approach to generate specific murine monoclonal IgG antibodies}, series = {Journal of immunological methods : JIM}, volume = {499}, journal = {Journal of immunological methods : JIM}, publisher = {Elsevier}, address = {Amsterdam}, issn = {0022-1759}, doi = {10.1016/j.jim.2021.113149}, pages = {8}, year = {2021}, abstract = {Generating a monoclonal antibody to date is a time intense process that requires immunization of laboratory animals. The transfer of the humoral immune response into in vitro settings enables a shortening of this process and circumvents the necessity of in vivo immunization. However, to orchestrate the complex interplay of dendritic cells, T and B lymphocytes in vitro is very challenging. We therefore aimed for a simplified approach focusing on the protagonist of antibody production: the B lymphocyte. We activated purified murine B lymphocytes alone in vitro by using combinations of antigen and stimuli. We were able to induce a specific antibody response within ten days of culture against a viral coat protein as model antigen. Antibodies were of both IgM and IgG subclass. The stimulated B lymphocytes were transformed into permanently antibody-producing hybridomas by cell fusion technology. We furthermore used this method to induce a specific antibody response against L. pneumophila in vitro. We thus established a useful and effective in vitro protocol to generate monoclonal antibodies. By overcoming the necessity of in vivo immunization this protocol may be the first step towards a universal strategy to generate antibodies from various species.}, language = {en} } @article{EngelMicheelHanack2022, author = {Engel, Robert and Micheel, Burkhard and Hanack, Katja}, title = {Three-dimensional cell culture approach for in vitro immunization and the production of monoclonal antibodies}, series = {Biomedical materials : materials for tissue engineering and regenerative medicine}, volume = {17}, journal = {Biomedical materials : materials for tissue engineering and regenerative medicine}, number = {5}, publisher = {Inst. of Physics}, address = {London}, issn = {1748-6041}, doi = {10.1088/1748-605X/ac7b00}, pages = {11}, year = {2022}, abstract = {The generation of monoclonal antibodies using an in vitro immunization approach is a promising alternative to conventional hybridoma technology. As recently published, the in vitro approach enables an antigen-specific activation of B lymphocytes within 10-12 d followed by immortalization and subsequent selection of hybridomas. This in vitro process can be further improved by using a three-dimensional surrounding to stabilize the complex microenvironment required for a successful immune reaction. In this study, the suitability of Geltrex as a material for the generation of monoclonal antigen-specific antibodies by in vitro immunization was analyzed. We could show that dendritic cells, B cells, and T cells were able to travel through and interact inside of the matrix, leading to the antigen-specific activation of T and B cells. For cell recovery and subsequent hybridoma technique the suitability of dispase and Corning cell recovery solution (CRS) was compared. In our experiments, the use of dispase resulted in a severe alteration of cell surface receptor expression patterns and significantly higher cell death, while we could not detect an adverse effect of Corning CRS. Finally, an easy approach for high-density cell culture was established by printing an alginate ring inside a cell culture vessel. The ring was filled with Geltrex, cells, and medium to ensure a sufficient supply during cultivation. Using this approach, we were able to generate monoclonal hybridomas that produce antigen-specific antibodies against ovalbumin and the SARS-CoV-2 nucleocapsid protein.}, language = {en} } @article{HolzloehnerHanack2017, author = {Holzl{\"o}hner, Pamela and Hanack, Katja}, title = {Generation of murine monoclonal antibodies by hybridoma technology}, series = {JoVE : Video journal}, journal = {JoVE : Video journal}, number = {119}, publisher = {JoVE}, address = {Cambridge}, issn = {1940-087X}, doi = {10.3791/54832}, pages = {7}, year = {2017}, abstract = {Monoclonal antibodies are universal binding molecules and are widely used in biomedicine and research. Nevertheless, the generation of these binding molecules is time-consuming and laborious due to the complicated handling and lack of alternatives. The aim of this protocol is to provide one standard method for the generation of monoclonal antibodies using hybridoma technology. This technology combines two steps. Step 1 is an appropriate immunization of the animal and step 2 is the fusion of B lymphocytes with immortal myeloma cells in order to generate hybrids possessing both parental functions, such as the production of antibody molecules and immortality. The generated hybridoma cells were then recloned and diluted to obtain stable monoclonal cell cultures secreting the desired monoclonal antibody in the culture supernatant. The supernatants were tested in enzyme-linked immunosorbent assays (ELISA) for antigen specificity. After the selection of appropriate cell clones, the cells were transferred to mass cultivation in order to produce the desired antibody molecule in large amounts. The purification of the antibodies is routinely performed by affinity chromatography. After purification, the antibody molecule can be characterized and validated for the final test application. The whole process takes 8 to 12 months of development, and there is a high risk that the antibody will not work in the desired test system.}, language = {en} } @article{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 = {Frontiers in Immunology}, journal = {Frontiers in Immunology}, publisher = {Frontiers Media SA}, address = {Lausanne, Schweiz}, issn = {1664-3224}, doi = {10.3389/fimmu.2022.930975}, pages = {1 -- 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} } @article{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 = {International Journal of Molecular Sciences}, volume = {23}, journal = {International Journal of Molecular Sciences}, edition = {11}, publisher = {MDPI}, address = {Basel, Schweiz}, issn = {1422-0067}, doi = {10.3390/ijms23116275}, 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} } @article{FudickarRoderListeketal.2021, author = {Fudickar, Werner and Roder, Phillip and Listek, Martin and Hanack, Katja and Linker, Torsten}, title = {Pyridinium alkynylanthracenes as sensitizers for photodynamic therapy}, series = {Photochemistry and photobiology}, volume = {98}, journal = {Photochemistry and photobiology}, number = {1}, publisher = {Wiley}, address = {Hoboken}, issn = {0031-8655}, doi = {10.1111/php.13554}, pages = {193 -- 201}, year = {2021}, abstract = {Photodynamic therapy (PDT) is a mild but effective method to treat certain types of cancer upon irradiation with visible light. Here, three isomeric methylpyridinium alkynylanthracenes 1op were evaluated as sensitizers for PDT. Upon irradiation with blue or green light, all three compounds show the ability to initiate strand breaks of plasmid DNA. The mayor species responsible for cleavage is singlet oxygen (O-1(2)) as confirmed by scavenging reagents. Only isomers 1m and 1p can be incorporated into HeLa cells, whereas isomer 1o cannot permeate through the membrane. While isomer 1m targets the cell nucleus, isomer 1p assembles in the cellular cytoplasm and impacts the cellular integrity. This is in accordance with a moderate toxicity of 1p in the dark, whereas 1m exhibits no dark toxicity. Both isomers are suitable as PDT reagents, with a CC50 of 3 mu m and 75 nm, for 1p and 1m, respectively. Thus, derivative 1m, which can be easily synthesized, becomes an interesting candidate for cancer therapy.}, language = {en} } @article{LuetkecosmannWarsinkeTschoepeetal.2017, author = {L{\"u}tkecosmann, Steffi and Warsinke, Axel and Tsch{\"o}pe, Winfried and Eichler, R{\"u}diger and Hanack, Katja}, title = {A novel monoclonal antibody suitable for the detection of leukotriene B4}, series = {Biochemical and biophysical research communications}, volume = {482}, journal = {Biochemical and biophysical research communications}, number = {4}, publisher = {Elsevier}, address = {San Diego}, issn = {0006-291X}, doi = {10.1016/j.bbrc.2016.11.157}, pages = {1054 -- 1059}, year = {2017}, abstract = {Leukotriene B4 as an inflammatory mediator is an important biomarker for different respiratory diseases like asthma, chronic obstructive pulmonary disease or cystic lung fibrosis. Therefore the detection of LTB4 is helpful in the diagnosis of these pulmonary diseases. However, until now its determination in exhaled breath condensates suffers from problems of accuracy. Reasons for that could be improper sample collection and preparation methods of condensates and the lack of consistently assay specificity and reproducibility of the used immunoassay detection system. In this study we describe the development and the characterization of a specific monoclonal antibody (S27BC6) against LTB4, its use as molecular recognition element for the development of an enzyme-linked immunoassay to detect LTB4 and discuss possible future diagnostic applications.}, language = {en} } @article{HolzloehnerButzeMaieretal.2018, author = {Holzl{\"o}hner, Pamela and Butze, Monique and Maier, Natalia and Hebel, Nicole and Schliebs, Erik and Micheel, Burkhard and Fuener, Jonas and Heidicke, Gabriele and Hanack, Katja}, title = {Generation of murine monoclonal antibodies with specificity against conventional camelid IgG1 and heavy-chain only IgG2/3}, series = {Veterinary Immunology and Immunopathology}, volume = {197}, journal = {Veterinary Immunology and Immunopathology}, publisher = {Elsevier}, address = {Amsterdam}, issn = {0165-2427}, doi = {10.1016/j.vetimm.2018.01.006}, pages = {1 -- 6}, year = {2018}, abstract = {Camelids possess antibodies with a conventional four-chain structure consisting of two heavy and two light chains (of subclass IgG1) but further they also generate heavy-chain only antibodies (of subclass IgG2 and 3) which are fully functional in antigen binding. In this study subclass-specific murine monoclonal antibodies specific to conventional camelid IgG1 and heavy-chain only IgG2/3 were generated and validated for the use as potent secondary detection reagents. The monoclonal antibodies are able to differentiate between all camelid IgGs, conventional four-chain camelid antibodies (of subclass IgG1) and exclusively heavy chain-only antibodies (of subclasses IgG2 and IgG3). Further these antibodies were used to detect specific immune responses after vaccination of Camelids against bovine corona- and rotavirus strains and different E.coli. and Clostridia - antigens and to identify Erysipelothrix rhusiopathiae infected animals within a herd. The described antibodies are suitable as new secondary agents for the detection of different camelid subclasses and the validation of camelid immune reactions.}, language = {en} } @article{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 = {Applied Sciences}, volume = {11}, journal = {Applied Sciences}, number = {20}, publisher = {MDPI}, address = {Basel}, issn = {1454-5101}, doi = {10.3390/app11209359}, pages = {15}, 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} } @article{SchloerHolzloehnerListeketal.2018, author = {Schl{\"o}r, Anja and Holzl{\"o}hner, Pamela and Listek, Martin and Grieß, Cindy and Butze, Monique and Micheel, Burkhard and Hentschel, Christian and Sowa, Mandy and Roggenbuck, Dirk and Schierack, Peter and F{\"u}ner, Jonas and Schliebs, Erik and Goihl, Alexander and Reinhold, Dirk and Hanack, Katja}, title = {Generation and validation of murine monoclonal and camelid recombinant single domain antibodies specific for human pancreatic glycoprotein 2}, series = {New biotechnology}, volume = {45}, journal = {New biotechnology}, publisher = {Elsevier}, address = {Amsterdam}, issn = {1871-6784}, doi = {10.1016/j.nbt.2018.03.006}, pages = {60 -- 68}, year = {2018}, abstract = {Pancreatic secretory zymogen-granule membrane glycoprotein 2 (GP2) has been identified as a major autoantigenic target in Crohn's disease patients. It was reported recently that a long (GP2a) and a short (GP2b) isoform of GP2 exist and that in the outcome of inflammatory bowel diseases (IBD) GP2-specific autoantibodies probably appear as new serological markers for diagnosis and therapeutic monitoring. To investigate this further and in order to establish diagnostic tools for the discrimination of both GP2 isoforms, a set of different murine monoclonal and camelid recombinant single domain antibodies (camelid VHH) was generated and validated in various enzyme-linked immunosorbent assay (ELISA) formats, immunofluorescence on transgenic cell lines and immunohistochemistry on monkey pancreas tissue sections. Out of six binders identified, one was validated as highly specific for GP2a. This murine monoclonal antibody (mAb) was used as capture antibody in construction of a sandwich ELISA for the detection of GP2a. Camelid VHHs or a second murine mAb served as detection antibodies in this system. All antibodies were also able to stain GP2a or GP2b on transgenic cell lines as well as on pancreatic tissue in immunohistochemistry. The KD values measured for the camelid VHHs were between 7 nM and 23pM. This set of specific binders will enable the development of suitable diagnostic tools for GP2-related studies in IBD.}, language = {en} } @article{LuetkecosmannFaupelPorstmannetal.2019, author = {Luetkecosmann, Steffi and Faupel, Thomas and Porstmann, Silvia and Porstmann, Tomas and Micheel, Burkhard and Hanack, Katja}, title = {A cross-reactive monoclonal antibody as universal detection antibody in autoantibody diagnostic assays}, series = {Clinica chimica acta}, volume = {499}, journal = {Clinica chimica acta}, publisher = {Elsevier}, address = {Amsterdam}, issn = {0009-8981}, doi = {10.1016/j.cca.2019.09.003}, pages = {87 -- 92}, year = {2019}, abstract = {Diagnostics of Autoimmune Diseases involve screening of patient samples for containing autoantibodies against various antigens. To ensure quality of diagnostic assays a calibrator is needed in each assay system. Different calibrators as recombinant human monoclonal antibodies as well as chimeric antibodies against the autoantigens of interest are described. A less cost-intensive and also more representative possibility covering different targets on the antigens is the utilization of polyclonal sera from other species. Nevertheless, the detection of human autoantibodies as well as the calibration reagent containing antibodies from other species in one assay constitutes a challenge in terms of assay calibration. We therefore developed a cross-reactive monoclonal antibody which binds human as well as rabbit sera with similar affinities in the nanomolar range. We tested our monoclonal antibody S38CD11B12 successfully in the commercial Serazym (R) Anti-Cardiolipin-beta 2-GPI IgG/IgM assay and could thereby prove the eligibility of S38CD11B12 as detection antibody in autoimmune diagnostic assays using rabbit derived sera as reference material.}, language = {en} } @article{RoggenbuckGoihlHanacketal.2017, author = {Roggenbuck, Dirk and Goihl, Alexander and Hanack, Katja and Holzloehner, Pamela and Hentschel, Christian and Veiczi, Miklos and Schierack, Peter and Reinhold, Dirk and Schulz, Hans-Ulrich}, title = {Serological diagnosis and prognosis of severe acute pancreatitis by analysis of serum glycoprotein 2}, series = {Clinical chemistry and laboratory medicine : journal of the Forum of the European Societies of Clinical Chemistry - the European Branch of the International Federation of Clinical Chemistry and Laboratory Medicine}, volume = {55}, journal = {Clinical chemistry and laboratory medicine : journal of the Forum of the European Societies of Clinical Chemistry - the European Branch of the International Federation of Clinical Chemistry and Laboratory Medicine}, publisher = {De Gruyter}, address = {Berlin}, issn = {1434-6621}, doi = {10.1515/cclm-2016-0797}, pages = {854 -- 864}, year = {2017}, abstract = {To better understand emerging adults' perceptions of family interactions and value transmission to the next generation, we examined Hmong American emerging adults' reflections on their parents' parenting. Participants discussed what parenting practices they would do differently and others they hoped to emulate with their future adolescent children. Thirty Hmong American emerging adults (18-25 years; M = 21.2 years; 50\% female) participated in interviews that focused retrospectively on the parent-adolescent relationship. Results revealed that emerging adults wanted to parent differently in three ways: less pressure about education, fewer restrictions, and more open communication. Emerging adults imagined being a similar parent in four ways: promoting education, promoting life values, giving guidance, and offering love and support. The findings highlight parenting practices that Hmong American emerging adults plan on transmitting (and not transmitting) to their own children, offering a glimpse into the type of parents the emerging adults may become.}, language = {en} } @article{ListekHoenowGossenetal.2020, author = {Listek, Martin and H{\"o}now, Anja and Gossen, Manfred and Hanack, Katja}, title = {A novel selection strategy for antibody producing hybridoma cells based on a new transgenic fusion cell line}, series = {Scientific Reports}, volume = {10}, journal = {Scientific Reports}, publisher = {Macmillan Publishers Limited, part of Springer Nature}, address = {London}, issn = {2045-2322}, doi = {10.1038/s41598-020-58571-w}, pages = {12}, year = {2020}, abstract = {The use of monoclonal antibodies is ubiquitous in science and biomedicine but the generation and validation process of antibodies is nevertheless complicated and time-consuming. To address these issues we developed a novel selective technology based on an artificial cell surface construct by which secreted antibodies were connected to the corresponding hybridoma cell when they possess the desired antigen-specificity. Further the system enables the selection of desired isotypes and the screening for potential cross-reactivities in the same context. For the design of the construct we combined the transmembrane domain of the EGF-receptor with a hemagglutinin epitope and a biotin acceptor peptide and performed a transposon-mediated transfection of myeloma cell lines. The stably transfected myeloma cell line was used for the generation of hybridoma cells and an antigen- and isotype-specific screening method was established. The system has been validated for globular protein antigens as well as for haptens and enables a fast and early stage selection and validation of monoclonal antibodies in one step.}, language = {en} } @article{SchmidtRoedigerGruneretal.2016, author = {Schmidt, Carsten and Roediger, Stefan and Gruner, Melanie and Moncsek, Anja and Stohwasser, Ralf and Hanack, Katja and Schierack, Peter and Schroeder, Christian}, title = {Multiplex localization of sequential peptide epitopes by use of a planar microbead chip}, series = {Analytica chimica acta : an international journal devoted to all branches of analytical chemistry}, volume = {908}, journal = {Analytica chimica acta : an international journal devoted to all branches of analytical chemistry}, publisher = {Elsevier}, address = {Amsterdam}, issn = {0003-2670}, doi = {10.1016/j.aca.2015.12.030}, pages = {150 -- 160}, year = {2016}, abstract = {Epitope mapping is crucial for the characterization of protein-specific antibodies. Commonly, small overlapping peptides are chemically synthesized and immobilized to determine the specific peptide sequence. In this study, we report the use of a fast and inexpensive planar microbead chip for epitope mapping. We developed a generic strategy for expressing recombinant peptide libraries instead of using expensive synthetic peptide libraries. A biotin moiety was introduced in vivo at a defined peptide position using biotin ligase. Peptides in crude Escherichia coli lysate were coupled onto streptavidin-coated microbeads by incubation, thereby avoiding tedious purification procedures. For read-out we used a multiplex planar microbead chip with size- and fluorescence-encoded microbead populations. For epitope mapping, up to 18 populations of peptide-loaded microbeads (at least 20 microbeads per peptide) displaying the primary sequence of a protein were analyzed simultaneously. If an epitope was recognized by an antibody, a secondary fluorescence-labeled antibody generated a signal that was quantified, and the mean value of all microbeads in the population was calculated. We mapped the epitopes for rabbit anti-PA28 gamma (proteasome activator 28 gamma) polyclonal serum, for a murine monoclonal antibody against PA28 gamma, and for a murine monoclonal antibody against the hamster polyoma virus major capsid protein VP1 as models. In each case, the identification of one distinct peptide sequence out of up to 18 sequences was possible. Using this approach, an epitope can be mapped multiparametrically within three weeks. (C) 2016 Elsevier B.V. All rights reserved.}, language = {en} } @article{RoggenbuckBorghiSommaetal.2016, author = {Roggenbuck, Dirk and Borghi, Maria Orietta and Somma, Valentina and Buettner, 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 = {IEEE transactions on geoscience and remote sensing}, volume = {18}, journal = {IEEE transactions on geoscience and remote sensing}, publisher = {BioMed Central}, address = {London}, issn = {1478-6354}, doi = {10.1186/s13075-016-1018-x}, 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 (beta 2GPI), prothrombin, and annexin V. Samples were also tested by anti-CL and anti-beta 2GPI ELISAs and for lupus anticoagulant activity. Human monoclonal antibodies (humoAbs) against human beta 2GPI or PL alone were tested on the same LIA substrates in the absence or presence of human serum, purified human beta 2GPI or after CL-micelle absorption. Results: Comparison of LIA with the aPL-classification assays revealed good agreement for IgG/IgM a beta 2GPI and aCL. Anti-CL and anti-beta 2GPI 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 beta 2GPI 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 beta 2GPI 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-beta 2GPI 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} } @article{KabaMaierSchliebeOhleretal.2015, author = {Kaba, Hani E. J. and Maier, Natalia and Schliebe-Ohler, Nicole and Mayer, Yvonne and Mueller, Peter P. and van den Heuvel, Joop and Schuchhardt, Johannes and Hanack, Katja and Bilitewski, Ursula}, title = {Identification of whole pathogenic cells by monoclonal antibodies generated against a specific peptide from an immunogenic cell wall protein}, series = {Journal of microbiological methods}, volume = {108}, journal = {Journal of microbiological methods}, publisher = {Elsevier}, address = {Amsterdam}, issn = {0167-7012}, doi = {10.1016/j.mimet.2014.11.003}, pages = {61 -- 69}, year = {2015}, abstract = {We selected the immunogenic cell wall beta-(1,3)-glucosyltransferase Bgl2p from Candida albicans as a target protein for the production of antibodies. We identified a unique peptide sequence in the protein and generated monoclonal anti- C. albicans Bgl2p antibodies, which bound in particular to whole C. albicans cells.}, language = {en} }