The Principle and Methods of Hybridoma Cell Screening
During the preparation of monoclonal antibodies, there are two screening processes. The first is to screen for hybridoma cells, and the second is to further screen for hybridoma cells that produce specific antibodies.
First screening
During the fusion of B cells and tumor cells, various cell types exist, including unfused B cells, unfused tumor cells, "B cell-B cell" fused cells, "tumor cell-tumor cell" fused cells, and "B cell-tumor cell" fused cells. Our goal is to screen out the hybridoma cells formed by the fusion of B cells and tumor cells. The selective culture of hybridoma cells is the key to the first round of screening, and HAT selective medium is commonly used for this purpose.
In HAT medium, H stands for hypoxanthine, A for aminopterin, and T for thymidine. The purpose of adding aminopterin to HAT medium is to inhibit the main pathway of nucleotide synthesis. The addition of hypoxanthine and thymidine aims to provide raw materials for the alternative pathway of nucleotide synthesis, allowing cells to synthesize DNA through the salvage pathway.
Cellular DNA synthesis involves two pathways: de novo synthesis and salvage synthesis. De novo synthesis refers to the pathway where nucleotides are synthesized from amino acids and other small-molecule compounds, followed by further synthesis of DNA from these nucleotides. In this process, dihydrofolate reductase participates as an important coenzyme.
The salvage pathway is a process where hypoxanthine and thymidine are synthesized into corresponding nucleotides by hypoxanthine guanine phosphoribosyl transferase (HGPRT) and thymidine kinase (TK), followed by further DNA synthesis. Both HGPRT and TK are indispensable for this pathway.
Tumor cells lack HGPRT, a key enzyme in the salvage pathway, and thus cannot utilize hypoxanthine for DNA synthesis via this pathway. In HAT medium, they fail to survive as aminopterin inhibits the de novo synthesis pathway.
B cells can synthesize DNA using hypoxanthine and thymidine through the salvage pathway, allowing them to survive in HAT medium. However, B cells are unable to proliferate indefinitely, so they will die quickly even if they survive initially.
Successfully fused hybridoma cells inherit both the unlimited proliferation capacity of tumor cells and the salvage pathway enzyme HGPRT from B cells. Therefore, they can synthesize nucleotides through the salvage pathway in HAT medium and maintain continuous survival and proliferation.
Second screening
During the production of monoclonal antibodies, due to the distinct specificity of B cells, the antibodies produced by the hybridoma cells initially screened using HAT medium also vary. These include specific antibody-secreting cells, irrelevant antibody-secreting cells, and non-antibody-secreting cells. Therefore, a second screening of hybridoma cells is necessary to select those capable of producing the target antibody, followed by cloning—meaning culturing and propagating cells from a single cell in a culture well to form a monoclonal population, ensuring the homogeneity of the secreted antibodies.
The most commonly used method for screening hybridoma cells that secrete specific monoclonal antibodies is the limiting dilution assay. Following the principle of serial gradient dilution, the cell suspension is serially diluted to an extremely low density and inoculated into 96-well plates until single-cell clones appear in the wells. This method is simple and easy to operate but relatively time-consuming throughout the culture process. The specific steps are as follows:
Identify positive hybridoma cell wells using the ELISA method, prepare a cell suspension with complete medium, and perform cell counting.
Transfer the cell suspension to a graduated centrifuge tube for serial dilution to a concentration of 5–10 cells/mL.
Inoculate the diluted cell suspension into 96-well plates separately and incubate in an incubator.
After cell adhesion, observe the number of cells in each well under an inverted microscope, mark wells containing only one cell, and continue culturing. During incubation, decide whether to replace the medium based on changes in the pH value of the medium.
If the cells in the positive wells cannot be guaranteed to originate from a single cell, repeat the limiting dilution and the aforementioned steps until the proliferating cells in the wells are confirmed to be monoclonal.
Note
Hybridoma cells that test positive for antibody secretion should be cloned as early as possible. Otherwise, antibody-secreting cells may be inhibited by non-antibody-secreting cells—since non-antibody-secreting cells grow faster, competition between the two cell types will eventually lead to the loss of antibody-secreting cells.
Even cloned hybridoma cells require regular re-cloning to prevent mutations or chromosome loss in the cells, which could result in the loss of their antibody-producing ability.
 | Felicia Felicia is a technical support specialist at EnkiLife, with extensive professional experience in antibody development, optimization, and ELISA assay design and application. She is committed to assisting our clients in selecting suitable antibody products, optimizing ELISA experimental protocols, and resolving technical challenges encountered in the process, thereby supporting the smooth progress of their life science research projects. |