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International Journal of Molecular... Jun 2023It has been shown that synovial fibroblasts (SF) play a key role in the initiation of inflammation and joint destruction, leading to arthritis progression. Fibroblasts...
It has been shown that synovial fibroblasts (SF) play a key role in the initiation of inflammation and joint destruction, leading to arthritis progression. Fibroblasts may express major histocompatibility complex class II region (MHCII) molecules, and thus, they could be able to process and present antigens to immunocompetent cells. Here we examine whether different types of fibroblasts (synovial, dermal, and thymic murine fibroblasts, destructive LS48 fibroblasts, and noninvasive NIH/3T3 fibroblasts) may be involved in the initiation of rheumatoid arthritis (RA) pathogenesis and can process and present type II collagen (COL2)-an autoantigen associated with RA. Using a panel of MHCII/Aq-restricted T-cell hybridoma lines that specifically recognize an immunodominant COL2 epitope (COL2), we found that NIH/3T3 fibroblasts activate several T-cell clones that recognize the posttranslationally glycosylated or hydroxylated COL2 epitope. The HCQ.3 hybridoma, which is specific for the glycosylated immunodominant COL2 epitope 259-273 (Gal264), showed the strongest response. Interestingly, NIH/3T3 cells, but not destructive LS48 fibroblasts, synovial, dermal, or thymic fibroblasts, were able to stimulate the HCQ.3 hybridoma and other COL2-specific T-cell hybridomas. Our experiments revealed that NIH/3T3 fibroblasts are able to activate COL2-specific T-cell hybridomas even in the absence of COL2 or a posttranslationally modified COL2 peptide. The mechanism of this unusual activation is contact-dependent and involves the T-cell receptor (TCR) complex.
Topics: Mice; Animals; Collagen Type II; T-Lymphocytes; Arthritis, Rheumatoid; Fibroblasts; Epitopes; Immunodominant Epitopes; Hybridomas
PubMed: 37445989
DOI: 10.3390/ijms241310811 -
The Yale Journal of Biology and Medicine 1981The use of monoclonal antibodies produced by the hybridoma technique has had a profound impact on the biological and medical sciences. While the general technique for...
The use of monoclonal antibodies produced by the hybridoma technique has had a profound impact on the biological and medical sciences. While the general technique for the production of monoclonal antibodies is available from the numerous papers which report their use, there is a need for a source which describes the procedure and its pitfalls in detail so that an investigator who decides to make hybridomas can do so quickly and easily. This guide provides such a source.
Topics: Animals; Antibodies, Monoclonal; Clone Cells; Cytological Techniques; Hybridomas; Immunologic Techniques; Mice; Mice, Inbred BALB C; Quality Control
PubMed: 7039149
DOI: No ID Found -
Journal of Immunological Methods Nov 2018Immortalized T cells such as T cell hybridomas, transfectomas, and transductants are useful tools to study tri-molecular complexes consisting of peptide, MHC, and T cell...
Immortalized T cells such as T cell hybridomas, transfectomas, and transductants are useful tools to study tri-molecular complexes consisting of peptide, MHC, and T cell receptor (TCR) molecules. These cells have been utilized for antigen discovery studies for decades due to simplicity and rapidness of growing cells. However, responsiveness to antigen stimulation is typically less sensitive compared to primary T cells, resulting in occasional false negative outcomes especially for TCRs having low affinity to a peptide-MHC complex (pMHC). To overcome this obstacle, we genetically engineered T cell hybridomas to express additional CD3 molecules as well as CD4 with two amino acid substitutions that increase affinity to MHC class II molecules. The manipulated T cell hybridomas that were further transduced with retroviral vectors encoding TCRs of interest responded to cognate antigens more robustly than non-manipulated cells without evoking non-antigen specific reactivity. Of importance, the manipulation with CD3 and mutated human CD4 expression was effective in increasing responsiveness of T cell hybridomas to a wide variety of TCR, peptide, and MHC combinations across class II genetic loci (i.e. HLA-DR, HLA-DQ, HLA-DP, and murine H2-IA) and species (i.e. both humans and mice), and thus will be useful to identify antigen specificity of T cells.
Topics: Antigens; CD3 Complex; Cell Line, Transformed; Histocompatibility Antigens Class II; Humans; Hybridomas; Lymphocyte Activation; Receptors, Antigen, T-Cell
PubMed: 30165064
DOI: 10.1016/j.jim.2018.08.011 -
BMC Biotechnology Jan 2008The isolation and production of human monoclonal antibodies is becoming an increasingly important pursuit as biopharmaceutical companies migrate their drug pipelines... (Comparative Study)
Comparative Study
BACKGROUND
The isolation and production of human monoclonal antibodies is becoming an increasingly important pursuit as biopharmaceutical companies migrate their drug pipelines away from small organic molecules. As such, optimization of monoclonal antibody technologies is important, as this is becoming the new rate-limiting step for discovery and development of new pharmaceuticals. The major limitations of this system are the efficiency of isolating hybridoma clones, the process of stabilizing these clones and optimization of hybridoma cell secretion, especially for large-scale production. Many previous studies have demonstrated how perturbations in the aqueous environment can impact upon cell biology. In particular, radio frequency (RF) irradiation of solutions can have dramatic effects on behavior of solutions, cells and in particular membrane proteins, although this effect decays following removal of the RF. Recently, it was shown that nanoparticle doping of RF irradiated water (NPD water) produced a stabilized aqueous medium that maintained the characteristic properties of RF irradiated water for extended periods of time. Therefore, the ordering effect in water of the RF irradiation can now be studied in systems that required prolonged periods for analysis, such as eukaryotic cell culture. Since the formation of hybridoma cells involves the formation of a new membrane, a process that is affected by the surrounding aqueous environment, we tested these nanoparticle doped aqueous media formulations on hybridoma cell production.
RESULTS
In this study, we tested the entire process of isolation and production of human monoclonal antibodies in NPD water as a means for further enhancing human monoclonal antibody isolation and production. Our results indicate an overall enhancement of hybridoma yield, viability, clonability and secretion. Furthermore, we have demonstrated that immortal cells proliferate faster whereas primary human fibroblasts proliferate slower in NPD water.
CONCLUSION
Overall, these studies indicate that NPD water can enhance cell proliferation, clonability and secretion. Furthermore, the results support the hypothesis that NPD water is effectively composed of stable microenvironments.
Topics: Animals; Antibodies, Monoclonal; Biotechnology; CHO Cells; Cell Line, Tumor; Cricetinae; Cricetulus; Culture Media; Humans; Hybridomas; Nanoparticles; Radio Waves; Water
PubMed: 18194567
DOI: 10.1186/1472-6750-8-3 -
ELife Jan 2019Generating recombinant monoclonal antibodies (R-mAbs) from mAb-producing hybridomas offers numerous advantages that increase the effectiveness, reproducibility, and...
Generating recombinant monoclonal antibodies (R-mAbs) from mAb-producing hybridomas offers numerous advantages that increase the effectiveness, reproducibility, and transparent reporting of research. We report here the generation of a novel resource in the form of a library of recombinant R-mAbs validated for neuroscience research. We cloned immunoglobulin G (IgG) variable domains from cryopreserved hybridoma cells and input them into an integrated pipeline for expression and validation of functional R-mAbs. To improve efficiency over standard protocols, we eliminated aberrant Sp2/0-Ag14 hybridoma-derived variable light transcripts using restriction enzyme treatment. Further, we engineered a plasmid backbone that allows for switching of the IgG subclasses without altering target binding specificity to generate R-mAbs useful in simultaneous multiplex labeling experiments not previously possible. The method was also employed to rescue IgG variable sequences and generate functional R-mAbs from a non-viable cryopreserved hybridoma. All R-mAb sequences and plasmids will be archived and disseminated from open source suppliers.
Topics: Animals; Antibodies, Monoclonal; Antibody Specificity; Brain; Enzyme-Linked Immunosorbent Assay; Humans; Hybridomas; Immunoglobulin G; Immunohistochemistry; Mice; Neurosciences; Rats; Recombinant Proteins
PubMed: 30667360
DOI: 10.7554/eLife.43322 -
Scientific Reports Nov 2013Electrofusion is an efficient method for fusing cells using short-duration high-voltage electric pulses. However, electrofusion yields are very low when fusion partner...
Electrofusion is an efficient method for fusing cells using short-duration high-voltage electric pulses. However, electrofusion yields are very low when fusion partner cells differ considerably in their size, since the extent of electroporation (consequently membrane fusogenic state) with conventionally used microsecond pulses depends proportionally on the cell radius. We here propose a new and innovative approach to fuse cells with shorter, nanosecond (ns) pulses. Using numerical calculations we demonstrate that ns pulses can induce selective electroporation of the contact areas between cells (i.e. the target areas), regardless of the cell size. We then confirm experimentally on B16-F1 and CHO cell lines that electrofusion of cells with either equal or different size by using ns pulses is indeed feasible. Based on our results we expect that ns pulses can improve fusion yields in electrofusion of cells with different size, such as myeloma cells and B lymphocytes in hybridoma technology.
Topics: Animals; CHO Cells; Cell Fusion; Cell Size; Cricetulus; Electricity; Electroporation; Hybridomas; Melanoma, Experimental; Mice; Pulse
PubMed: 24287643
DOI: 10.1038/srep03382 -
MAbs 2014Modification of antibody class and binding properties typically requires cloning of antibody genes, antibody library construction, phage or yeast display and recombinant...
Modification of antibody class and binding properties typically requires cloning of antibody genes, antibody library construction, phage or yeast display and recombinant antibody expression. Here, we describe an alternative "cloning-free" approach to generate antibodies with altered antigen-binding and heavy chain isotype by mimicking the germinal center reaction in antibody-secreting hybridoma cells. This was accomplished by lentiviral transduction and controllable expression of activation-induced cytidine deaminase (AID) to generate somatic hypermutation and class switch recombination in antibody genes coupled with high-throughput fluorescence-activated cell sorting (FACS) of hybridoma cells to detect altered antibody binding properties. Starting from a single established hybridoma clone, we isolated mutated antibodies that bind to a low-temperature structure of polyethylene glycol (PEG), a polymer widely used in nanotechnology, biotechnology and pharmaceuticals. FACS of AID-infected hybridoma cells also facilitated rapid identification of class switched variants of monoclonal IgM to monoclonal IgG. Mimicking the germinal center reaction in hybridoma cells may offer a general method to identify and isolate antibodies with altered binding properties and class-switched heavy chains without the need to carry out DNA library construction, antibody engineering and recombinant protein expression.
Topics: Animals; Antibodies, Monoclonal, Murine-Derived; BALB 3T3 Cells; Cytidine Deaminase; Germinal Center; Humans; Hybridomas; Immunoglobulin Class Switching; Lentivirus; Mice; Polyethylene Glycols; Somatic Hypermutation, Immunoglobulin; Transduction, Genetic
PubMed: 24874693
DOI: 10.4161/mabs.29124 -
Haematologica Dec 2022
Topics: Mice; Animals; Hybridomas; Antibodies, Monoclonal
PubMed: 36453514
DOI: 10.3324/haematol.2022.281671 -
Methods (San Diego, Calif.) Mar 2016Understanding the properties and functions of complex biological systems depends upon knowing the proteins present and the interactions between them. Recent advances in...
Understanding the properties and functions of complex biological systems depends upon knowing the proteins present and the interactions between them. Recent advances in mass spectrometry have given us greater insights into the participating proteomes, however, monoclonal antibodies remain key to understanding the structures, functions, locations and macromolecular interactions of the involved proteins. The traditional single immunogen method to produce monoclonal antibodies using hybridoma technology are time, resource and cost intensive, limiting the number of reagents that are available. Using a high content analysis screening approach, we have developed a method in which a complex mixture of proteins (e.g., subproteome) is used to generate a panel of monoclonal antibodies specific to a subproteome located in a defined subcellular compartment such as the nucleus. The immunofluorescent images in the primary hybridoma screen are analyzed using an automated processing approach and classified using a recursive partitioning forest classification model derived from images obtained from the Human Protein Atlas. Using an ammonium sulfate purified nuclear matrix fraction as an example of reverse proteomics, we identified 866 hybridoma supernatants with a positive immunofluorescent signal. Of those, 402 produced a nuclear signal from which patterns similar to known nuclear matrix associated proteins were identified. Detailed here is our method, the analysis techniques, and a discussion of the application to further in vivo antibody production.
Topics: Animals; Antibodies, Monoclonal; Antibody Affinity; Antibody Specificity; Atlases as Topic; HeLa Cells; High-Throughput Screening Assays; Humans; Hybridomas; Immunization; Machine Learning; Mice; Mice, Inbred BALB C; Nuclear Matrix; Principal Component Analysis; Proteome; Vaccination
PubMed: 26521976
DOI: 10.1016/j.ymeth.2015.10.021 -
Cell Reports Feb 2021Fast-dissociating, specific antibodies are single-molecule imaging probes that transiently interact with their targets and are used in biological applications including...
Fast-dissociating, specific antibodies are single-molecule imaging probes that transiently interact with their targets and are used in biological applications including image reconstruction by integrating exchangeable single-molecule localization (IRIS), a multiplexable super-resolution microscopy technique. Here, we introduce a semi-automated screen based on single-molecule total internal reflection fluorescence (TIRF) microscopy of antibody-antigen binding, which allows for identification of fast-dissociating monoclonal antibodies directly from thousands of hybridoma cultures. We develop monoclonal antibodies against three epitope tags (FLAG-tag, S-tag, and V5-tag) and two F-actin crosslinking proteins (plastin and espin). Specific antibodies show fast dissociation with half-lives ranging from 0.98 to 2.2 s. Unexpectedly, fast-dissociating yet specific antibodies are not so rare. A combination of fluorescently labeled Fab probes synthesized from these antibodies and light-sheet microscopy, such as dual-view inverted selective plane illumination microscopy (diSPIM), reveal rapid turnover of espin within long-lived F-actin cores of inner-ear sensory hair cell stereocilia, demonstrating that fast-dissociating specific antibodies can identify novel biological phenomena.
Topics: Animals; Antibodies; Cell Culture Techniques; Humans; Hybridomas; Mice; Microscopy; Single Molecule Imaging
PubMed: 33535030
DOI: 10.1016/j.celrep.2021.108708