Understanding Hybridoma Technology for Monoclonal Antibody Production

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What Is Hybridoma Technology?

Hybridoma technology involves fusing short-lived antibody-producing B cells with immortal myeloma cells, creating cell lines that produce a never-ending supply of a specific monoclonal antibody. The technique was created in 1975 by Nobel prize-winning scientists Georges Kohler and Cesar Milstein.¹

Monoclonal Antibodies

Scientists create monoclonal antibodies by cloning a single antibody-producing cell line. In contrast to polyclonal antibodies, monoclonals are highly specific to an antigen. By fusing myeloma cells that can divide indefinitely with B cells that produce specific antibodies against the target of interest, researchers obtain a nearly unlimited source of identical monoclonal antibodies.²

Since 1986, over 117 monoclonal antibodies have been FDA-approved, beginning with the mouse monoclonal antibody Muromonab-CD3 for kidney transplant rejection.³ In additional to antibodies from mice and humans, researcher have produced chimeric and humanized monoclonal antibodies that are composed of sequences from both species. By replacing mouse-derived protein sequences with human ones, these monoclonal antibodies reduce the risk of triggering an immune response in humans.⁴  Using various methods, scientists have also produced monoclonal antibodies from other mammals for diverse purposes.

Hybridoma Technology Step by Step

Step 1: Immunization

Researchers inject a mammal, typically a mouse, with a target antigen, stimulating an immune response. Antigen injection may occur in a series over the course of several weeks. Then, researchers harvest the mouse's spleen to obtain B cells that produce the desired antibody.¹

Step 2: Cell Fusion

Researchers fuse antibody-producing B cells with myeloma cells in cell culture. Polyethylene glycol (PEG) facilitates fusion of both cells’ plasma membranes, forming a single hybridoma cell with two or more nuclei. Alternatively, electrofusion can merge the cells using a pulsed electrical field.⁵

Step 3: Hybridoma Cell Growth

Less than 1 percent of the initial cells fuse to form hybridoma cells. Unused B cells in the culture stop dividing naturally, while chemotherapy destroys the unfused myeloma cells. Researchers use HAT (hypoxanthine-aminopterin-thymidine) medium to allow the selective proliferation of immortal monoclonal antibody-producing cell lines. The aminopterin in the HAT medium stops nucleotide synthesis, while hypoxanthine and thymidine can be used by cells, such as B cells, carrying the HGPRT (hypoxanthine-guanine phosphoribosyl transferase) enzyme. Hybridoma cells with functional HGPRT enzyme can survive and grow, while myeloma cells lacking it eventually die.¹

Step 4: Screening

Researchers often screen hybridoma cells for the monoclonal antibody of interest using an enzyme-linked immunosorbent assay (ELISA). Indirect ELISAs identify antibodies with the appropriate specificity by immobilizing the antigen on a surface and incubating it with a hybridoma cell supernatant. Researchers also use techniques including western blot, flow cytometry, and immunoprecipitation-mass spectrometry to screen their hybridoma cultures.¹

Step 5: Hybridoma Expansion 

The final step involves cloning desired hybridoma cells to obtain a stable cell population and growing the culture to collect large amounts of monoclonal antibodies. This can be achieved through one of two methods.¹

• In vitro growth of hybrid cells in tissue culture 
• In vivo growth following inoculation of hybridoma cells into a mouse’s abdomen

     

Hybridoma Benefits and Limitations

This technology offers numerous advantages, namely^1,6

• Precise antigen targeting
• A never-ending supply of consistent antibodies 
• High sensitivity and specificity for use in biological assays
• Elimination of the need for animal models (in vitro method)
• Utilization in therapeutic and diagnostic treatments, vaccine creation, and chemotherapy

Nevertheless, the technology also has a few limitations.^1,7

• Long production time
• Resource-intensive and expensive workflow
• Not suitable for generating short peptides and fragment antigens
• Susceptibility to contamination and poor cell viability
• Risk of virus contamination and disease transmission
• Absence of stable myeloma cells for human antibody production

Hybridoma Applications

Diagnostic applications

Owing to their high specificity, the antibodies produced by hybridoma technology have a wide range of diagnostic applications, including the following.

• Enzyme-linked immunosorbent (ELISA): detecting HIV antibodies, hepatitis B surface antigen, and pregnancy hormone⁸
• Immunofluorescence assay (IFA): detecting autoimmune disorders, influenza virus, and Chlamydia trachomatis⁹
• Western blot: analyzing cancer biomarkers⁸
• Flow cytometry: assessing immune cells in HIV, leukemia, and lymphoma⁸
• Immunohistochemistry (IHC): analyzing cancer biomarkers⁸
• Rapid antigen tests: detecting malaria, dengue, Zika virus, and COVID-19^10,11 

Immunotherapy

There are various FDA-approved indications of monoclonal antibodies (see table below).³ Common indications include the following.

• Cancer treatment: anticancer immunotherapy against prostate, breast, lung, bladder, liver, gastric, colorectal, and endometrial cancer
• Autoimmune disorders: management of rheumatoid arthritis, Crohn's disease, lupus, psoriasis
• Infectious diseases: prevention and treatment of respiratory syncytial virus and COVID-19
• Organ transplant: rejection prevention of kidney, liver, lung, and heart transplants

Noteworthy FDA-approved monoclonal antibodies³