-
Immunotherapy Mar 2011Hybridoma technology features effective usage of innate functions of both immune cells and cancers, allowing production of hybridoma cells, which continuously generate... (Review)
Review
Hybridoma technology features effective usage of innate functions of both immune cells and cancers, allowing production of hybridoma cells, which continuously generate monoclonal antibodies specific to antigens of interest. For standard generation of hybridoma cells, B lymphocytes must be somatically fused with myeloma cells using various technologies. However, the methods generally do not necessarily result in selective fusion of target B lymphocytes with myeloma cells. To overcome this problem, we have developed a new hybridoma technology that involves preselection of B lymphocytes with target antigens based on immunoglobulin receptors and selective fusion of B cell-myeloma cell complexes with electrical pulses. The advanced methodology, termed B-cell targeting, multitargeting and stereospecific targeting, may be applicable to simultaneous production of monoclonal antibodies, selective production of stereospecific monoclonal antibodies, and also to efficient generation of human monoclonal antibodies for clinical purposes.
Topics: Animals; Antibodies, Monoclonal; Antibody Formation; Antibody Specificity; Antigens; B-Lymphocytes; Cell Fusion; Cell Line; Cross-Linking Reagents; Efficiency; Electricity; Humans; Hybridomas; Lasers; Mice; Models, Biological; Multiple Myeloma; Polyethylene Glycols
PubMed: 21395379
DOI: 10.2217/imt.11.4 -
Methods in Molecular Biology (Clifton,... 2012Hybridoma technology has long been a remarkable and indispensable platform for generating high-quality monoclonal antibodies (mAbs). Hybridoma-derived mAbs have not only...
Hybridoma technology has long been a remarkable and indispensable platform for generating high-quality monoclonal antibodies (mAbs). Hybridoma-derived mAbs have not only served as powerful tool reagents but also have emerged as the most rapidly expanding class of therapeutic biologics. With the establishment of mAb humanization and with the development of transgenic-humanized mice, hybridoma technology has opened new avenues for effectively generating humanized or fully human mAbs as therapeutics. In this chapter, an overview of hybridoma technology and the laboratory procedures used routinely for hybridoma generation are discussed and detailed in the following sections: cell fusion for hybridoma generation, antibody screening and characterization, hybridoma subcloning and mAb isotyping, as well as production of mAbs from hybridoma cells.
Topics: Animals; Antibodies, Monoclonal; Cell Fusion; Enzyme-Linked Immunosorbent Assay; Humans; Hybridomas; Immunohistochemistry
PubMed: 22723097
DOI: 10.1007/978-1-61779-931-0_7 -
Cytotechnology May 1990Energy metabolism and the production of ammonia in hybridoma cell culture and its inhibitory effects on cell growth are reviewed. The interactive roles of glucose and... (Review)
Review
Energy metabolism and the production of ammonia in hybridoma cell culture and its inhibitory effects on cell growth are reviewed. The interactive roles of glucose and glutamine metabolism affect the rate of production of ammonia, and these interactions are described. It is shown that growth inhibition usually occurs between 2-4 mM ammonia although some cell lines have been shown to adapt to much higher concentrations, particularly in continuous culture. In batch cultures cell growth appears to be particularly susceptible to increased ammonia concentrations during the early stages of growth; ammonia increased the rate of cell death in the late stage of batch growth. The specific productivity of monoclonal antibodies is much less sensitive to the released ammonia than is growth; lower volumetric productivities relate to the lower viable cell concentrations which are achieved at the high ammonia levels. Techniques to prevent ammonia accumulation or remove ammonia selectively have been relatively unsuccessful to date.
Topics: Ammonia; Animals; Cell Division; Energy Metabolism; Glucose; Glutamine; Growth Inhibitors; Hybridomas
PubMed: 1366658
DOI: 10.1007/BF00365485 -
Cold Spring Harbor Protocols Oct 2020Hybridoma and myeloma cell lines can be stored by slowly freezing cells in an appropriate solution of nutrients and a cryoprotectant such as glycerol or dimethyl...
Hybridoma and myeloma cell lines can be stored by slowly freezing cells in an appropriate solution of nutrients and a cryoprotectant such as glycerol or dimethyl sulfoxide (DMSO). In this protocol, cells are centrifuged at 4°C, resuspended in cold freezing solution (10% DMSO in FBS), and then transferred to an appropriate freezing vial. The vials are slowly frozen to -70°C in Styrofoam racks and then stored in liquid nitrogen (LN). Cells stored in LN will remain viable for years. Once a frozen vial has been removed from LN storage, it should be thawed as described, grown out into log phase, and refrozen.
Topics: Animals; Cell Line, Tumor; Cryopreservation; Cryoprotective Agents; Dimethyl Sulfoxide; Freezing; Humans; Hybridomas; Nitrogen; Reproducibility of Results
PubMed: 33004552
DOI: 10.1101/pdb.prot103267 -
Science (New York, N.Y.) May 1982
Topics: Allergy and Immunology; Animals; History, 20th Century; Hybridomas; Mice
PubMed: 7043734
DOI: 10.1126/science.216.4548.798-c -
Advances in Biochemical... 1988
Review
Topics: Animals; Antibodies, Monoclonal; Cell Division; Cytological Techniques; Hybridomas; Kinetics
PubMed: 3140607
DOI: 10.1007/BFb0009175 -
Cold Spring Harbor Protocols Nov 2019Isolating a stable clone of hybridoma cells that all secrete the correct antibody is the most time-consuming step in the production of hybridomas. Single-cell cloning...
Isolating a stable clone of hybridoma cells that all secrete the correct antibody is the most time-consuming step in the production of hybridomas. Single-cell cloning ensures that cells that produce the antibody of interest are truly monoclonal and that the secretion of this antibody can be stably maintained. The original positive well will often contain more than one clone of hybridoma cells, and many hybrid cells have an unstable assortment of chromosomes. Both of these problems may lead to the desired cells being outgrown by cells that are not producing the antibody of interest. Cloning hybridoma cells by limiting dilution is the easiest of the single-cell-cloning techniques. Two approaches are presented here, one rapid technique for generating cultures that are close to being single-cell-cloned and one for single-cell cloning directly. Even though every attempt is made to ensure that the cells are in a single-cell suspension before plating, there is no way to guarantee that the colonies do not arise from two cells that were stuck together. Therefore, limiting dilution cloning should be performed at least twice to generate a clonal population.
Topics: Animals; Clone Cells; Cloning, Molecular; Humans; Hybridomas; Indicator Dilution Techniques; Single-Cell Analysis
PubMed: 31676581
DOI: 10.1101/pdb.prot103192 -
Biotechnology & Genetic Engineering... 1994
Review
Topics: Animals; Antibodies, Monoclonal; Biotechnology; Cells, Cultured; Culture Techniques; Hybridomas; Kinetics
PubMed: 7727036
DOI: 10.1080/02648725.1994.10647920 -
Analytical Chemistry Feb 2018Using an enzyme-linked immunosorbent assay (ELISA) and limited dilution methods to screen and clone antigen-specific hybridoma cells is extremely time-consuming and...
Using an enzyme-linked immunosorbent assay (ELISA) and limited dilution methods to screen and clone antigen-specific hybridoma cells is extremely time-consuming and labor-intensive. This work features a simple and rapid cell surface fluorescence immunosorbent assay (CSFIA), designed for the detection and isolation of antigen-specific hybridoma clones. In this assay, antigens are first anchored to the hybridoma cell surface through a dual-functioning molecular Oleyl-PEG4000-NHS. Specific antibodies secreted from hybridoma cells are then captured by the antigens on the cell surface. Positive hybridoma cells are stained using a fluorescently labeled anti-mouse IgG-Fc antibody. After the addition of a methylcellulose semisolid medium, positive clones are easily picked using a pipet. These positive cell clones can be used to produce monoclonal antibodies after direct expansion. Using this method, positive hybridoma clones against both malachite green and porcine epidemic diarrhea virus are selected with high efficiency. Compared to the ELISA-based method, the CSFIA-based method achieved the capability of isolating >2-fold more hybridoma clones in <25% of the corresponding processing time. In brief, the CSFIA-based method is highly efficient and inexpensive with a simple and direct operation, which is an excellent candidate method for antigen-specific positive clone isolation in a monoclonal antibody preparation.
Topics: Animals; Antibodies, Monoclonal; Antigens; Cell Line, Tumor; Cell Separation; Hybridomas; Immunosorbent Techniques; Mice, Inbred BALB C; Porcine epidemic diarrhea virus; Rosaniline Dyes
PubMed: 29290124
DOI: 10.1021/acs.analchem.7b04595 -
Cold Spring Harbor Protocols Nov 2019Single-cell cloning during hybridoma production ensures that cells that produce the antibody of interest are truly monoclonal and that the secretion of this antibody can...
Single-cell cloning during hybridoma production ensures that cells that produce the antibody of interest are truly monoclonal and that the secretion of this antibody can be stably maintained. Cloning of hybridoma cells in semisolid medium is one of the most commonly used methods for producing single-cell clones. The technique is easy, but, because it is performed in two stages, it does take longer than other methods. Not all cells will grow in soft agar, and there may be a bias on the type of colony that appears. However, most of the commonly used myeloma fusion partners have relatively good cloning efficiencies in soft agar, and, consequently, so do most hybridomas. Even though every attempt is made to ensure that the cells are in a single-cell suspension before plating, there is no way to guarantee that the colonies do not arise from two cells that were stuck together. Therefore, single-cell cloning in soft agar should be repeated at least twice before the cells are considered clonal.
Topics: Agar; Animals; Cell Proliferation; Cloning, Molecular; Feeder Cells; Humans; Hybridomas; Single-Cell Analysis
PubMed: 31676582
DOI: 10.1101/pdb.prot103200