Monoclonal antibodies are valuable reagents commonly used in biomedical research, diagnostic testing, and treatment of diseases, including various types of cancer. They are composed of identical antibodies derived from a single B-cell clone and specifically bind to and recognize one epitope on a target antigen.
Monoclonal antibody production consists of the following steps:
1Immunize the Host Animal and Screen for Antibody Production
Monoclonal antibody production begins with the immunization of a host animal against the target antigen to trigger a robust immune system response. Researchers provide booster injections every two to three weeks and regularly screen an animal’s blood after each injection to monitor antibody production and measure antibody titer through techniques such as ELISA and flow cytometry. After the desired antibody titer is reached, they perform a final injection of the antigen without the adjuvant two weeks after the prior immunization.
2Isolate Antibody-Producing Cells from the Spleen
Unlike polyclonal antibody production where serum is collected to obtain the antibody population, monoclonal antibody production involves collecting B-cell lymphocytes from the animal’s spleen. These lymphocytes produce monoclonal antibodies that specifically recognize and bind to target antigens. Researchers harvest these antibody-producing splenocytes and isolate them for in vitro hybridoma production.
3Fuse Isolated Splenocytes with Myeloma Cells to Form Hybridomas
Splenocytes can generate large quantities of monoclonal antibodies, but they have a limited life span and must be fused with myeloma cells (immortal cancerous B cells) to create hybrid cells referred to as hybridomas. These hybridomas will manufacture the same monoclonal antibodies as created by the original B-cell lymphocytes. Fusing these cells combines the ability of the splenocytes to produce bulk quantities of purified antibodies with the ability of myeloma to grow indefinitely at a quicker rate than normal healthy cells. This allows for fast replication and continuous, unlimited cell growth in a culture that can produce large amounts of monoclonal antibodies against target antigens while undergoing multiple passages in vitro.
Researchers can create hybridomas through multiple methods, but chemical fusion and cell electrofusion are the most common. Both methods disrupt the cell membranes of antibody-secreting splenocyte cells and adjacent myeloma fusion cell lines and allow them to merge.
After fusion, researchers place the generated hybridoma cells in a selective HAT culture medium containing hypoxanthine, aminopterin, and pyrimidine thymidine. This medium inhibits DNA synthesis and allows them to separate fused hybrids from mortal B-cells and myeloma cells. B-cells and fused hybrids possess the enzyme thymidine kinase, allowing them to overcome the HAT culture and synthesize DNA polymerase precursors from the thymidine in the medium. Myeloma cells do not possess thymidine kinase and cannot survive in the medium. B-cells will gradually die off because they have limited in vitro replication abilities and cannot grow. This leaves behind only the fused hybrids.
4Achieve Clonality with Limiting Dilution
The cell population that survives the selection in the previous step remains heterogeneous, meaning it contains both clones that are specific to the target antigen and clones that produce non-specific antibodies. Researchers perform a technique called limiting dilution in which the concentrations of this cell population are placed into microplate wells and diluted to assure clonality. The goal is for each well to contain one cell, but if some wells contain multiple cells, this procedure is repeated after each round of cell expansion until each well only contains the expansion of a single cell, resulting in the production of identical antibodies.
5Screen and Select High-Performing Clones
After achieving clonality, researchers screen the hybridoma clones for expression of the desired monoclonal antibody and select the appropriate ones based on their specificity and immunoglobulin class. The clones that produce the desired antibody are isolated and grown in tissue cultures. Functional characterization of potentially high-performing clones is conducted via ELISA or other assays to confirm and validate that the monoclonal antibodies secreted by these clones are recognized, binded to, and purify the antigen they were raised against. These clones can also be screened for specific applications at this stage. At the end of the screening and selection process, all unwanted immunoglobulins will have been removed from the culture.
6Expand and Preserve Clones for Antibody Production
When researchers have confirmed that the antibodies are monoclonal and perform properly in the screening assays, they select hybridomas from the cultures, harvest them, and purify them from the medium via Protein A/G purification to recover the pure antibodies. The hybridomas are scaled up in size to produce large batches of antibodies and then frozen to preserve and protect them for future use. Using the same monoclonal antibody stock for multiple runs of an experiment allows researchers to reduce the risk of batch-to-batch variability and ensure reliable performance from the antibodies over time.