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What is Hybridoma Technology?

Brian Conrad
Tags: Bioprocess, Successful Research Tips

Before the mid-1970s, molecular biology researchers relied heavily on the use of polyclonal antibodies derived from conventional sera to identify and quantify proteins of interest in their complex, biological sample mixtures. Researchers using polyclonal antibodies were met with many challenges such as the need for/repeat use of large numbers of immunized animals to acquire them due to the lack of immortalized antibody-producing cells. Then, the high levels of background and lot-to-lot inconsistencies in experiments that came from the broad specificity afforded by these many antibodies for single antigen with different epitope. Hence, a key issue of the time was the inability to isolate and purify single antibodies of singular known specificity (monoclonal antibody) from the many different kinds produced in the body. At the Medical Research Council Laboratory of Molecular Biology in Cambridge, UK, Cesar Milstein and George Kohler inspired by the methods proposed by Cotton Lab in 1973 to generate immortalized hybrid clones of two myeloma cells sought to provide a solution by combining the immortality provided by a myeloma cell while maintaining the specificity of an individual B-cell clone producing antibody specific for a single epitope. By 1975, Kohler and Milstein with the help of Shirley Howe successfully developed a method to generate “hybridomas” capable of yielding large quantities of monoclonal antibodies by fusing myeloma cell line P3-X63-Ag8 with individual clones of normal antibody-secreting B-cells (“monoclonal”) from mouse spleen immunized with sheep red blood cells – introducing hybridoma technology to the field of molecular biology.

Monoclonal antibodies are antibodies that are highly specific for the same, single binding site on an antigen (foreign substance) that enters the body. These antibodies originate from the same, single B-cell clone (or B-cell lineage) of a unique, parent B-cell that has divided (upon stimulation by the antigen) in order to produce more B-cells to target and eliminate the antigen. B-cells can respond in multiple ways to the same, single antigen by producing multiple B-cell lineages or clones with the ability to produce multiple antibodies with differing, predetermined binding specificities to the same target antigen. This heterogeneous mixture of different antibodies to same antigen are otherwise known as polyclonal antibodies. Polyclonal responses by B-cells provide the extraordinary capacity of the humoral immune response to quickly and efficiently recognize and eliminate a perceived threat the body.

In the present day, hybridoma technology is the most commonly used method for accessing monoclonal antibodies. Monoclonal antibodies are secreted from hybridoma cells which are created by fusing a collection of short-lived antibody producing B cells with immortal myeloma cells. This process begins by injecting laboratory animals with the antigen the antibody is to be generated against via a series of injections over the course of several weeks to stimulate B-cell production of different B-cell lineages (and thus produce different antibodies for the same antigen derived from each cloned lineage). When a sufficient antibody titer is reached in the serum after several weeks of immunizations, animals are euthanized. The spleens are then removed for the source B-cells and activated B-cells are isolated via density centrifugation for cell fusion. Serum antibody titer is determined via ELISA or flow cytometry – when the end-users have a high enough titre per their experimental demands, the end-user can then move onto fusing the activated B-cells with their myeloma cell lines.

Cell fusion is performed through co-centrifuging freshly harvested spleen cells with previously prepared myelomas in polyethylene glycol (PEG) which fuses cells by their membranes or under the application of an electric field. Myelomas are incubated in 8-azaguanine a week prior to cell fusion to produce myelomas with non-functional hypoxanthine-guanine phosphoribosyltransferase (HGPRT) gene which prevents the production of purine nucleotides from the salvage pathway and makes them sensitive to HAT medium during hybridoma selection. Additionally, Ig genes are made nonfunctional in myelomas to prevent the myeloma from producing its own antibodies.

After cell fusion, there is a mixture of fused cells (B-cells fused to each other, myelomas fused to each other, and hybrid cells (both B-cells and myelomas fused)) and unfused cells (B-cells, myelomas) that are produced. This mixture of cells are then incubated and grown in a selection medium called HAT medium for 10-14 days. HAT medium contains hypoxanthine-aminopterin-thymidine; aminopterin blocks the ability of cells to synthesize by de novo synthesis while hypoxanthine and thymidine is present to be used by cells with HGPRT to survive using salvage pathways for nucleotide synthesis. Unfused B-cells typically die within a few days because of their short-life span in cell culture. Unfused myeloma cells die because they lack functional HGPRT and Aminopterin blocks their ability to perform de novo nucleotide synthesis. Therefore, the remaining cells are hybrid cells with HGPRT from the B-cell fused to the myeloma that lacks HGPRT. After incubation, HAT-selected hybridomas are transferred onto multi-well plates and diluted to the extent that each well only contains a single hybrid cell (“limiting dilution”). Once the single hybridoma is isolated in the well, since one b-cell lineage/clone produces an antibody with its own epitope, the antibodies yielded are “monoclonal”. There may be several hybridomas each producing its own monoclonal antibodies for the same antigen isolated after HAT selection. Once the different hybridomas have been isolated, a screening process selecting hybridomas that only produce antibodies of appropriate specificity is performed. After selection, Hybridomas are then transferred into larger tissue culture flasks where they are maintained in culture media and cryopreserved.

Enzo Life Sciences offers the DiSH Kit for the generation and isolation of antigen-specific hybridomas for monoclonal antibody generation. The DiSH kit comes included with a genetically-modified Sp2ab myeloma fusion partner which has been modified via a proprietary vector containing an inducible functional expression sequence for mutant, non-signaling cell-surface Iga and Igb receptors that retain their ability to bind to antibodies of all major immunoglobulin classes (IgA, IgD, IgE, IgG, and IgM). After end-users have isolated B-cells activated by an antigen of interest during immunization, they can then fuse their activated B-cells with the kit’s Sp2ab myeloma cells which will allow the formation of hybridomas that can secrete antigen-specific antibody and also express antibody on the cell surface as well. These antigen-specific hybridomas can be isolated using antigen of interest bound to a magnetic or fluorescent label that will bind to surface presented and/or secreted antibodies. Once labeled, these hybridomas can be isolated from among a pools of cells in a matter of hours using fluorescence activated cell sorting (FACS) or magnetic separation. Supernatant containing antibody secreted from antigen-specific hybridomas are analyzed for secretion after 10 days. Through providing end-users with a kit that can label secreted antibody from antigen-specific hybridomas that can then be fluorescently cell-sorted or magnetically separated, we can help circumvent lengthy hybridoma isolation steps requiring limiting dilution sub-cloning. This step is often time-consuming and problematic. Cells are repeatedly diluted out to low cell numbers and their supernatants assayed looking for secreted monoclonal antibody and may be lost in the process. Furthermore, in the case of rapidly growing hybridomas, the cells may die from lack of nutrients due to screening to isolate for specific monoclonal antibodies derived from respective specific hybridoma. Lastly, in a mixture of hybridomas, undesirable hybridomas may continuously outgrow desired ones. Collectively, these issues may extend the limit dilution step to be extended to weeks or months while losing important hybridomas; typically, the limit dilution of pure cell lines can go on for 3-4 weeks for any one hybridoma. Instead, using the DiSH kit, the labeling step makes it possible to produce a hybridoma where the total elapsed time from harvest to screening is around 14 to 18 days which directly reduces the time-cost and labor costs to our end-users. Aside from the myeloma fusion partner, the kit comes included with a protocol and the necessary reagents for your cultures, the cell fusion steps (comes included with Polyethylene Glycol (PEG) & fusion medium), selection and recovery which helps to further reduce time and costs for rapid hybridoma production. Figure 1 summarizes the workflow for the DiSH Kit.

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Figure 1: Workflow for the Direct Selection of Hybridomas (DiSH) Kit

Hybridoma-derived monoclonal antibodies are secreted in the tissue culture supernatants that hybridoma cell cultures are harvested in. Additionally, they can be produced by growing hybridomas within the peritoneal cavity of a mouse which yields a high concentration of antibody to be harvested in the ascites fluid of the abdomens of these animals. In addition to hybridoma-derived antibodies, polyclonal antibodies are often available in relatively unpurified forms from antiserum or blood from an immunized host after the removal of clotting proteins and red blood cells. Irrespective of if the antibodies are coming from polyclonal antiserum or monoclonal ascites fluid or tissue culture supernatant, they are purified in using protein A/G purification or affinity purification. Protein A/G purification uses Staphylocooccus aureus protein A or Streptococcus protein G which has high affinity to immunoglobulin Fc domain. Affinity purification separates proteins on the basis of similar characteristics within a group of proteins. An end-user will look to manipulate a reversible interaction between those proteins and a ligand coupled to chromatographic matrix. Lastly, recombinant antibodies created in vitro using synthetic genes can be be produced. At Enzo Life Sciences, we offer serum, ascites, tissue culture supernatant –derived antibodies and recombinant antibodies that can be used in a wide range of applications.

Enzo Life Sciences offers thousands of antibodies backed by thousands of peer-reviewed citations, our Worry-free Antibody Trial Program, and a comprehensive bioprocess portfolio with thoroughly validated ELISAs for contamination monitoring of Protein A and Host Cell Protein (HCP) residuals for process optimization in your biologics preparations. We also have our widely cited PROTEOSTAT® assays for monitoring protein aggregation and protein stability under systematic thermal stress conditions to enhance your manufacturing workflows. Please check out our Bioprocess Optimization platform for more information or contact our Technical Support Team for further assistance.

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