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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.
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.
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.
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.
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.
Pancreatic secretory zymogen-granule membrane glycoprotein 2 (GP2) has been identified to be a major autoantigenic target in Crohn’s disease patients. It was discussed recently that a long and a short isoform of GP2 exists whereas the short isoform is often detected by GP2-specific autoantibodies. In the outcome of inflammatory bowel diseases, these GP2-specific autoantibodies are discussed as new serological markers for diagnosis and therapeutic monitoring. To investigate this further, camelid nanobodies were generated by phage display and selected against the short isoform of GP2 in order to isolate specific tools for the discrimination of both isoforms. Nanobodies are single domain antibodies derived from camelid heavy chain only antibodies and characterized by a high stability and solubility. The selected candidates were expressed, purified and validated regarding their binding properties in different enzyme-linked immunosorbent assays formats, immunofluorescence, immunohistochemistry and surface plasmon resonance spectroscopy. Four different nanobodies could be selected whereof three recognize the short isoform of GP2 very specifically and one nanobody showed a high binding capacity for both isoforms. The KD values measured for all nanobodies were between 1.3 nM and 2.3 pM indicating highly specific binders suitable for the application as diagnostic tool in inflammatory bowel disease.
The monoclonal antibody B13-DE1 binds fluorescein, several fluorescein derivatives, and three peptide mimotopes. Our results revealed that this antibody also catalyzed the redox reaction of resazurin to resorufin, which are both structurally related to fluorescein. By using sodium sulfite as a reducing agent, the antibody B13-DE1 lowered the activation energy of this reaction. The Michaelis-Menten constant and turnover number of the catalyzed reaction were determined as 4.2 mu mol/l and 0.0056 s(-1), respectively. Because the results showed that fluorescein inhibited the catalytic activity of the antibody, we assume that the antigen-binding site and the catalytic active site are identical.
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.
A direct competitive homogeneous immunoassay for progesterone - the Redox Quenching Immunoassay
(2012)
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.
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.
Insertion of artificial cell surface receptors for antigen-specific labelling of hybridoma cells
(2012)
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.
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.
A monoclonal antibody against the potential tumor suppressor kinase-enhanced protein phosphatase 1 (PP1) inhibitor KEPI (PPP1R14C) was generated and characterized. Human KEPI was expressed in Escherichia coli and used to immunize Balb/c mice. Using hybridoma technology, one clone, G18AF8, was isolated producing antibodies which bound specifically to the KEPI protein in ELISA, immunoblotting and flow cytometry. The antibody was also successfully applied to stain KEPI protein in paraffin sections of human brain. The epitope was mapped using peptide array technology and confirmed as GARVFFQSPR. This corresponds to the N-terminal region of KEPI. Amino acid substitution analysis revealed that two residues, F and Q, are essential for binding. Affinity of binding was determined by competitive ELISA as 1 mu M. In Western blot assays testing G18AF8 antibody on brain samples of several species, reactivity with hamster, rat and chicken samples was found, suggesting a broad homology of this KEPI epitope in vertebrates. This antibody could be used in expression studies at the protein level e.g. in tumor tissues.
Peripheral T-cell (TC) tolerance can be induced by tolerogenic antigen-presenting cell (APC). A prerequisite is the reduction or blockade of B7 of APC. Besides dendritic cell, B cells can be used as APC. Here, we show the generation B cells with reduced B7 expression by lentiviral transduction of endoplasmic reticulum (ER)-directed CTLA4. Vectors coding for the human CTL4-Ig were used for the human B-cell line Raji. Transduction efficiency was over 90% (MOI = 3). For the murine B-cell line A20 and for primary mouse B cells, murine CTLA4 was used. We show that B cells with reduced B7 expression reduce the antigen (Ag) specific TC proliferation in vitro. B cells expressing an ER-directed CTLA4 may reduce Ag-specific immune responses.
The mussel Mytilus edulis can be used as model to study the molecular basis of reproductive isolation because this species maintains its species integrity, despite of hybridizing in zones of contact with the closely related species M. trossulus or M. galloprovincialis. This study uses selective antibody production by means of hybridoma technology to identify molecules which are involved in sperm function of M. edulis. Fragmented sperm were injected into mice and 25 hybridoma cell clones were established to obtain monoclonal antibodies (mAb). Five clones were identified producing mAb targeting molecules putatively involved in sperm function based on enzyme immunoassays, dot and Western blotting as well as immunostaining of tissue sections. Specific localization of these mAb targets on sperm and partly also in somatic tissue suggests that all five antibodies bind to different molecules. The targets of the mAb obtained from clone G26-AG8 were identified using mass spectrometry (nano-LC-ESI-MS/MS) as M6 and M7 lysin. These acrosomal proteins have egg vitelline lyses function and are highly similar (76%) which explains the cross reactivity of mAb G26- AG8. Furthermore, M7 lysin was recently shown to be under strong positive selection suggesting a role in interspecific reproductive isolation. This study shows that M6 and M7 lysin are not only found in the sperm acrosome but also in male somatic tissue of the mantle and the posterior adductor muscle, while being completely absent in females. The monoclonal antibody G26-AG8 described here will allow elucidating M7/M6 lysin function in somatic and gonad tissue of adult and developing animals.