In 1975, German scholars Kohler and Milstein successfully fused myeloma cells with antibody-producing B lymphocytes, creating hybridoma cells. These hybridoma cells possess the characteristics of B lymphocytes that can produce monoclonal antibodies targeting a specific antigenic determinant cluster, and they also exhibit the unlimited proliferation characteristic of tumor cells. The establishment of hybridoma technology marked a new era in the preparation and use of antibodies. With advancements in new materials and technologies, such as nanoparticles, electrochemistry, nucleic acid sensors, and chips, monoclonal antibodies have found widespread applications in areas such as food safety hazard detection, early disease screening, and immunotherapy.
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Mouse monoclonal antibodies, due to their stable origin, ease of preparation in later stages, and high yield, are the most commonly used antibodies in immune detection analysis and early disease screening. The preparation of well-performing mouse monoclonal antibodies involves various technical steps, including antigen design and selection, animal immunization, cell fusion and screening techniques, and antibody purification methods. Each of these technical steps is indispensable for the successful development of monoclonal antibodies.
Fig. 1 Antibodies attack a virus.
Mouse Hybridoma Fusion Technology
Traditional Mouse Hybridoma Fusion Technology
The conventional method for generating mouse hybridomas involves antigen identification, animal immunization, serum titer determination, and fusion of spleen cells with myeloma cells. After selection using HAT and HT culture media and confirmation through indirect ELISA, hybridoma cells exhibited unlimited proliferation and the secretion of antibodies specific to the antigen. Amid the global COVID-19 crisis, neutralizing antibodies produced using mouse hybridoma technology have been utilized.
Transgenic Mouse Hybridoma Technology
Transgenic mouse hybridoma technology introduces genes encoding human antibodies into mice lacking antibody genes. This results in chimeric antibodies or fully humanized antibodies. Chimeric antibodies, like 4G4, combine mouse variable region genes with human constant region genes, reducing murine components and enhancing therapeutic efficacy. Humanized antibodies are essentially chimeric, while fully humanized antibodies involve transferring the entire gene encoding human antibodies to genetically modified mice.
Antigen Design and Screening
Hapten Design and Screening
Haptens, also known as incomplete antigens, refer to small molecules with relatively simple structures that cannot induce immune responses when immunized alone, meaning they lack immunogenicity. Common haptens include small molecule drugs, fungal toxins, pufferfish toxins, carbohydrates, nucleic acids, and lipids. These substances cannot elicit immune responses in organisms and need to be conjugated with bovine serum albumin (BSA), ovalbumin (OVA), or synthetic peptides to generate immunogenicity for the preparation of monoclonal antibodies.
Antigen Design and Selection
Selecting appropriate antigens is a key challenge in antibody development and vaccine design. To efficiently induce the host's immune response, researchers must carefully design and choose antigens with clear biological information. Relevant data can be gathered from databases, and immunogenicity and structure analysis, epitope prediction, and homology modeling contribute to constructing an immunogenicity scoring model. Finally, selected antigens are expressed using expression systems to produce corresponding antibodies, ensuring optimal outcomes with minimal effort.
B Cell Enrichment and Selection
B cell enrichment and selection are crucial for hybridoma technology. While various cell types exist in mouse lymphoid organs, only B cells expressing IgG, mainly plasma cells, can form hybridomas secreting monoclonal antibodies. Traditional fusion methods using polyethylene glycol (PEG) yield low fusion efficiency, necessitating effective enrichment of effector B cells to enhance fusion rates. Current B cell sorting relies on flow cytometry. For instance, utilizing CD45R immunomagnetic beads for enrichment and subsequent flow cytometry with fluorescence-labeled CD19 antibody and IgG+ signals allows the sorting of individual B cells. Alternatively, a combined approach using anti-mouse IgM antibodies and the Pan-B cell isolation kit (Miltenyi) enriches effector B cells by removing IgM+ cells and non-B cells, significantly improving the efficiency of hybridoma technology. This approach demonstrates increased total clone numbers, IgG-producing clone numbers, and target-specific IgG-producing clone numbers compared to PEG fusion, making it a valuable method for monoclonal antibody production.
Modification of Myeloma Cells
In the early stages of hybridoma cell culture, antibody concentration in the supernatant is typically low. To expedite hybridoma cell selection, it is beneficial to temporarily limit antibody release into the culture medium, allowing rapid sorting through antigen-antibody binding. Incorporating the fundamental principle of phage display technology into hybridoma cell technology involves capturing secreted antibodies on the surface of hybridoma cells. Listek et al. achieved this by transfecting myeloma cells with a plasmid encoding biotin receptor peptides, enabling surface expression. This method facilitates efficient selection and identification of hybridoma cells based on desired antigen specificity, allowing for subsequent characterization and applications such as fluorescence-activated cell sorting.
Positive Hybridoma Cell Selection
Conventional Methods: After hybridoma fusion, positive cells are detached, counted, and diluted in different concentrations. Microscopic examination the next day focuses on wells with single cells or small clusters. Limited dilution aims to obtain single hybridoma cells, requiring 7-10 days per subcloning cycle with indirect ELISA for confirmation. However, this process may lead to the loss of hybridoma cells.
Semisolid Method: Researchers introduced a semisolid medium using methylcellulose in 1982. This simplifies hybridoma clonal cultivation, promoting colony growth without interference. Cloning is done in a single step, but further expansion and confirmation of single hybridoma cells' antigen specificity are required.
Fluorescence Immunoadsorption Assay: In methylcellulose culture, hybridoma cells form single-cell colonies. "Oil links" are used for antibody capture, and fluorescently labeled secondary antibodies identify hybridoma cells. This method efficiently selects hybridoma clones secreting antibodies against specific antigens.
Chip Screening: Chip-based screening offers the advantages of sensitivity, small volume, and high throughput. Microarrays using PDMS facilitate the simultaneous evaluation of multiple hybridoma clones, allowing the selection of high-affinity antibody-producing hybridomas.
Microfluidic Chip Screening: Droplet-based microfluidics separates single hybridoma cells, enabling the analysis of secreted proteins. This provides a high-throughput, time-efficient method for hybridoma screening.
Rapid Methods for Monoclonal Antibody Assessment
Rapid ELISA Method: Monoclonal antibody assessment, including specificity and potency, currently relies on indirect ELISA. This involves fixing antigens on a carrier, incubating with antibodies, and using enzyme-labeled secondary antibodies for detection. However, a simpler, faster method is needed to enhance efficiency in antibody screening and preparation.
Visual Macroarray: Visual bacterial microarray technology spots inactivated bacteria on a nitrocellulose membrane. Detection antibodies labeled with horseradish peroxidase allow simultaneous screening for optimal reactivity and cross-reactivity. This visually efficient method enhances monoclonal antibody screening efficiency.
Immunochromatography: Widely used for pathogen detection, immunochromatography can detect antibodies in serum. Using gold-labeled antibodies against IgM and IgG allows visual detection of antibodies in blood samples. This technique holds the potential for rapid testing of hybridoma antibodies, ascites antibodies, and purified antibodies.
Electrochemical Methods: A label-free capacitive immunosensor for rapid detection of ultra-low concentrations of virus-specific antibodies. Immobilizing antigens on a glass slide with PDMS, this method detects changes in electrode resistance caused by antibody-antigen binding. It offers a quick and efficient way to test antibody concentrations, including subtypes like IgG1, IgG2a, IgG2b, IgG3, IgA, and IgM.
References
- Listek M., et al. A novel selection strategy for antibody producing hybridoma cells based on a new transgenic fusion cell line. Sci Rep. 2020, 10: 1664.
- Köhler G., Milstein C. Continuous cultures of fused cells secreting antibody of predefined specificity. Nature. 1975, 256(5517): 495-497.
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