TY - JOUR A1 - Neumann-Schaal, Meina A1 - Messerschmidt, Katrin A1 - Grenz, Nicole A1 - Heilmann, Katja T1 - Use of antibody gene library for the isolation of specific single chain antibodies. by ampicillin-antigen conjugates JF - Immunology letters : an international journal providing for the rapid publication of short reports in immunology N2 - Isolation of recombinant antibodies from antibody libraries is commonly performed by different molecular display formats including phage display and ribosome display or different cell-surface display formats. We describe a new method which allows the selection of Escherichia coil cells producing the required single chain antibody by cultivation in presence of ampicillin conjugated to the antigen of interest. The method utilizes the neutralization of the conjugate by the produced single chain antibody which is secreted to the periplasm. Therefore, a new expression system based on the pET26b vector was designed and a library was constructed. The method was successfully established first for the selection of E. coli BL21 Star (DE3) cells expressing a model single chain antibody (anti-fluorescein) by a simple selection assay on LB-agar plates. Using this selection assay, we could identify a new single chain antibody binding biotin by growing E. coil BL21 Star (DE3) containing the library in presence of a biotin-ampicillin conjugate. In contrast to methods as molecular or cell surface display our selection system applies the soluble single chain antibody molecule and thereby avoids undesired effects, e.g. by the phage particle or the yeast fusion protein. By selecting directly in an expression strain, production and characterization of the selected single chain antibody is possible without any further cloning or transformation steps. KW - Single chain antibody KW - Selection method KW - Anti-biotin antibody KW - Naive single chain library Y1 - 2013 U6 - https://doi.org/10.1016/j.imlet.2013.02.005 SN - 0165-2478 VL - 151 IS - 1-2 SP - 39 EP - 43 PB - Elsevier CY - Amsterdam ER - TY - JOUR A1 - Messerschmidt, Katrin A1 - Heilmann, Katja T1 - Toxin-antigen conjugates as selection tools for antibody producing cells JF - Journal of immunological methods N2 - The generation of antibodies with designated specificity requires cost-intensive and time-consuming screening procedures. Here we present a new method by which hybridoma cells can be selected based on the specificity of the produced antibody by the use of antigen-toxin-conjugates thus eliminating the need of a screening procedure. Initial experiments were done with methotrexate as low molecular weight toxin and fluorescein as model antigen. Methotrexate and a methotrexate-fluorescein conjugate were characterized regarding their toxicity. Afterwards the effect of the fluorescein-specific antibody B13-DE1 on the toxicity of the methotrexate-fluorescein conjugate was determined. Finally, first results showed that hybridoma cells that produce fluorescein specific antibodies are able to grow in the presence of fluorescein-toxin-conjugates. KW - Monoclonal antibody KW - Hybridoma technology KW - Selection of antibody producing cells Y1 - 2013 U6 - https://doi.org/10.1016/j.jim.2012.10.010 SN - 0022-1759 VL - 387 IS - 1-2 SP - 167 EP - 172 PB - Elsevier CY - Amsterdam ER - TY - JOUR A1 - Heilmann, Katja A1 - Groth, Thomas A1 - Behrsing, Olaf A1 - Wagner, Albrecht A1 - Schossig-Tiedemann, Michael A1 - Lendlein, Andreas A1 - Micheel, Burkhard T1 - The influence of the chemical composition of cell culture material on the growth and antibody production of hybridoma cells N2 - The multiplication and antibody production of murine hybridoma cells cultured on five different polymer membranes were tested and compared with conventional tissue culture polystyrene (TCPS). Membranes were prepared from polyacrylonitrile (PAN) and acrylonitrile copolymerized with N-vinylpyrrolidone (NVP20, NVP30), Na-methallylsulfonate (NaMAS) and N-(3-amino-propyl-methacrylamide-hydrochloride) (APMA). Cell number and antibody concentration were quantified as criteria for viability and productivity. Adhesion of hybridoma cells was characterized by vital and scanning electron microscopy. The results suggest that a strong adhesion of cells, observed on APMA and TCPS, increased cell growth but reduced monoclonal antibody production. In contrast membranes with lowered adhesivity such as NVP20 provided favourable conditions for monoclonal antibody production. In addition it was shown that this membrane also possessed a minor fouling as indicated by the low decrease of water flux across the membrane after protein adsorption. It was concluded that NVP20 could be a suitable material for the development of hollow fibre membranes for bioreactors. Y1 - 2005 UR - http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6T3C-4DPYNGY- 4&_coverDate=02%2F09%2F2005&_alid=268995355&_rdoc=1&_fmt=&_orig=search&_qd=1&_cdi=4943&_sort=d&view=c&_acct=C000053886&_v e ER - TY - JOUR A1 - Heilmann, Katja A1 - Groth, Thomas A1 - Schossig, Michael A1 - Lendlein, Andreas A1 - Micheel, Burkhard T1 - Modulation of hybridoma cell growth and antibody production by coating cell culture material with extracellular matrix proteins N2 - The influence of coating polystyrene tissue culture plates with different proteins on murine hybridoma cell growth and antibody production was investigated. Fibronectin, collagen I, bovine serum albumin and laminin were used to coat NUNC and COSTAR cell culture plates. Cell number and antibody concentration in culture fluids were quantified as indicators for cell viability, proliferation and productivity. Adhesive behaviour, morphology, expression of surface receptors of hybridoma cells and the presence of tyrosine-phosphorylated proteins in cell lysates were characterized by cell adhesion experiments, microscopy, flow cytometry and Western Blot analysis. It was shown that coatings with fibronectin (0.2 ;g/ml) lead to a substantial improvement of cell growth by 50-70% and an increase of monoclonal antibody production by 100-120%. Collagen I coatings showed an improvement in cell growth by 30-70% and by 60% for the production of monoclonal antibodies. Coatings with BSA and laminin had minor effects on these parameters. It was found that the hybridoma cell lines used in this study did not express the ;2-chain of the ;2;1-integrin, which is responsible for binding to collagen and laminin. However, the presence of ;1- integrin on the cell surface was shown, which should enable hybridoma cells to bind fibronectin. We propose, therefore, that fibronectin adsorption to cell culture materials may be a promising approach to enhance the production of monoclonal antibodies by cultivated hybridoma cells. Y1 - 2007 UR - http://www.sciencedirect.com/science/journal/1369703X U6 - https://doi.org/10.1016/j.bej.2007.01.035 SN - 1369-703X ER - TY - JOUR A1 - Bartel, Manuela A1 - Hartmann, Stefanie A1 - Lehmann, Karola A1 - Postel, Kai A1 - Quesada, Humberto A1 - Philipp, Eva E. R. A1 - Heilmann, Katja A1 - Micheel, Burkhard A1 - Stuckas, Heiko T1 - Identification of sperm proteins as candidate biomarkers for the analysis of reproductive isolation in Mytilus: a case study for the enkurin locus JF - Marine biology : international journal on life in oceans and coastal waters N2 - Sperm proteins of the marine sessile mussels of the Mytilus edulis species complex are models to investigate reproductive isolation and speciation. This study aimed at identifying sperm proteins and their corresponding genes. This was aided by the use of monoclonal antibodies that preferentially bind to yet unknown sperm molecules. By identifying their target molecules, this approach identified proteins with relevance to Mytilus sperm function. This procedure identified 16 proteins, for example, enkurin, laminin, porin and heat shock proteins. The potential use of these proteins as genetic markers to study reproductive isolation is exemplified by analysing the enkurin locus. Enkurin evolution is driven by purifying selection, the locus displays high levels of intraspecific variation and species-specific alleles group in distinct phylogenetic clusters. These findings characterize enkurin as informative candidate biomarker for analyses of clinal variation and differential introgression in hybrid zones, for example, to understand determinants of reproductive isolation in Baltic Mytilus populations. Y1 - 2012 U6 - https://doi.org/10.1007/s00227-012-2005-7 SN - 0025-3162 VL - 159 IS - 10 SP - 2195 EP - 2207 PB - Springer CY - New York ER - TY - JOUR A1 - Hess, Anne-Katrin A1 - Bartel, Manuela A1 - Roth, Karina A1 - Messerschmidt, Katrin A1 - Heilmann, Katja A1 - Kenchington, Ellen A1 - Micheel, Burkhard A1 - Stuckas, Heiko T1 - Expression of M6 and M7 lysin in Mytilus edulis is not restricted to sperm, but occurs also in oocytes and somatic tissue of males and females JF - Molecular reproduction and development N2 - Sperm proteins of marine sessile invertebrates have been extensively studied to understand the molecular basis of reproductive isolation. Apart from molecules such as bindin of sea urchins or lysin of abalone species, the acrosomal protein M7 lysin of Mytilus edulis has been analyzed. M7 lysin was found to be under positive selection, but mechanisms driving the evolution of this protein are not fully understood. To explore functional aspects, this study investigated the protein expression pattern of M7 and M6 lysin in gametes and somatic tissue of male and female M. edulis. The study employs a previously published monoclonal antibody (G26-AG8) to investigate M6 and M7 lysin protein expression, and explores expression of both genes. It is shown that these proteins and their encoding genes are expressed in gametes and somatic tissue of both sexes. This is in contrast to sea urchin bindin and abalone lysin, in which gene expression is strictly limited to males. Although future studies need to clarify the functional importance of both acrosomal proteins in male and female somatic tissue, new insights into the evolution of sperm proteins in marine sessile invertebrates are possible. This is because proteins with male-specific expression (bindin, lysin) might evolve differently than proteins with expression in both sexes (M6/M7 lysin), and the putative function of both proteins in females opens the possibility that the evolution of M6/M7 lysin is under sexual antagonistic selection, for example, mutations beneficial to the acrosomal function that are less beneficial the function in somatic tissue of females.Mol. Reprod. Dev. 79: 517-524, 2012. Y1 - 2012 U6 - https://doi.org/10.1002/mrd.22056 SN - 1040-452X VL - 79 IS - 8 SP - 517 EP - 524 PB - Wiley-Blackwell CY - Hoboken ER - TY - JOUR A1 - Wand, Inga A1 - Holzlöhner, Pamela A1 - Neupert, Steffi A1 - Micheel, Burkhard A1 - Heilmann, Katja T1 - Cooperation of dendritic cells with naive lymphocyte populations to induce the generation of antigen-specific antibodies in vitro JF - Journal of biotechnology N2 - The production of monoclonal antibodies by hybridoma technology is dependent on lymphocytes taken from vertebrates which have to be immunized against the corresponding antigen. We present here our first experiments which should allow the replacement of this in vivo immunization step by an in vitro immunization procedure. This work provides new possibilities for the specific activation of immune cells in order to use them for the generation of antibodies which are not of murine origin. Bone marrow-derived dendritic cells were loaded with antigen and co-cultured with naive T and B lymphocytes of non-immunized mice. The interaction and activation of the different cell types were investigated by measuring the expression of specific cell surface markers, the release of activation-dependent interleukins and the secretion of antigen-specific antibodies. We could demonstrate that dendritic cells process and present antigen fragments and activate T cells, that T cells proliferate and release activation-induced interleukins, and that B cells maturate under the influence of activated T cells and secrete antigen-specific antibodies. KW - In vitro immunization KW - Activation of dendritic cells KW - Interaction of T and B cells with antigen-presenting cells KW - Induction of antibody responses Y1 - 2011 U6 - https://doi.org/10.1016/j.jbiotec.2011.09.002 SN - 0168-1656 VL - 156 IS - 3 SP - 173 EP - 181 PB - Elsevier CY - Amsterdam ER -