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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.
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.
MALDI-TOF-MS-based identification of monoclonal murine Anti-SARS-CoV-2 antibodies within one hour
(2022)
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.
Filamin c is the predominantly expressed filamin isoform in striated muscles. It is localized in myofibrillar Z- discs, where it binds FATZ and myotilin, and in myotendinous junctions and intercalated discs. Here, we identify Xin, the protein encoded by the human gene 'cardiomyopathy associated 1' (CMYA1) as filamin c binding partner at these specialized structures where the ends of myofibrils are attached to the sarcolemma. Xin directly binds the EVH1 domain proteins Mena and VASP. In the adult heart, Xin and Mena/VASP colocalize with filamin c in intercalated discs. In cultured cardiomyocytes, the proteins also localize in the nonstriated part of myofibrils, where sarcomeres are assembled and an extensive reorganization of the actin cytoskeleton occurs. Unusual intraexonic splicing events result in the existence of three Xin isoforms that associate differentially with its ligands. The identification of the complex filamin c-Xin-Mena/VASP provides a first glance on the role of Xin in the molecular mechanisms involved in developmental and adaptive remodeling of the actin cytoskeleton during cardiac morphogenesis and sarcomere assembly. (c) 2006 Elsevier Inc. All rights reserved
Over the last decade the modeling and the storage of biological data has been a topic of wide interest for scientists dealing with biological and biomedical research. Currently most data is still stored in text files which leads to data redundancies and file chaos. In this paper we show how to use relational modeling techniques and relational database technology for modeling and storing biological sequence data, i.e. for data maintained in collections like EMBL or SWISS-PROT to better serve the needs for these application domains. For this reason we propose a two step approach. First, we model the structure (and therefore the meaning of the) data using an Entity-Relationship approach. The ER model leads to a clean design of a relational database schema for storing and retrieving the DNA and protein data extracted from various sources. Our approach provides the clean basis for building complex biological applications that are more amenable to changes and software ports than their file-base counterparts.
A water soluble fluorescent polymer as a dual colour sensor for temperature and a specific protein
(2013)
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.
KEPI is a protein kinase C-potentiated inhibitory protein for type 1 Ser/Thr protein phosphatases. We found no or reduced expression of KEPI in breast cancer cell lines, breast tumors and metastases in comparison to normal breast cell lines and tissues, respectively. KEPI protein expression and ubiquitous localization was detected with a newly generated antibody. Ectopic KEPI expression in MCF7 breast cancer cells induced differential expression of 95 genes, including the up-regulation of the tumor suppressors EGR1 (early growth response 1) and PTEN (phosphatase and tensin homolog), which is regulated by EGR1. We further show that the up-regulation of EGR1 in MCF7/KEPI cells is mediated by MEK-ERK signaling. The inhibition of this pathway by the MEK inhibitor UO126 led to a strong decrease in EGR1 expression in MCF7/KEPI cells. These results reveal a novel role for KEPI in the regulation of the tumor suppressor gene EGR1 via activation of the MEK-ERK MAPK pathway.
A water soluble fluorescent polymer as a dual colour sensor for temperature and a specific protein
(2013)
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.
Tumor antigen-specific T cell clones represent a useful tool in tumor immunology; however, their long-term culture is limited. To generate an immortalized cytotoxic T cell clone against the human tumor antigen mucin, we exposed a previously generated T cell culture to Herpesvirus saimiri. We obtained an immortalized human CD4+ T cell clone, termed SITAM. Clonality of these cells was shown by analysis of the alpha/beta-T cell receptor (TCR) repertoire. Cytolytic activity was demonstrated against several mucin-expressing tumor cell lines and could not be detected against non-mucin-expressing cells. SITAM cells maintained their features stably for 2 years. Furthermore, growth of the tumor cell line Capan-2 in NOD/SCID mice was inhibited when SITAM cells were coinjected subcutaneously with tumor cells. SITAM cells provide an unlimited source of clonal T cells for analysis of tumor recognition and may be of help in TCR-targeted immunotherapy.
BACKGROUND: Dendritic cells (DC) as antigen presenting cells play an important role in immunotherapy of cancer. Mucin, encoded by the gene MUC1, is a human tumor antigen expressed in breast, pancreatic and ovarian cancers. Therefore, MUC1-transfected DC would be an attractive tool in constructing cancer vaccines. MATERIALS AND METHODS: Using two different cationic liposome preparations and, for comparison, a recombinant adenovirus expressing mucin, we tested the efficiency of mucin gene transfer into DC by flow cytometry. We investigated if these transfected DC were able to specifically stimulate autologous peripheral blood lymphocytes (PBL) from healthy donors. RESULTS: Flow cytometry revealed that 5-20% of DC transfected with liposomes Lipofectin and 20-40% of DC transduced with adenovirus expressed the relevant mucin epitopes. The expression of mucin on DC was similar to the expression of mucin found on carcinoma cells. After antigen uptake, DC specifically stimulated autologous PBL. CONCLUSION: We have shown that cationic liposomal gene transfer into human DC was feasible. We could obtain antigen specific stimulation of PBL at a similar rate as with adenoviral MUC1-transduced DC.