How hybridomas are created
The process of obtaining hybridomas consists of several key steps:
- Immunization: A laboratory mouse is immunized with the required antigen.
- Cell harvesting: Antibody-producing cells (splenocytes) are extracted from the animal's spleen.
- Hybridization: Splenocytes are fused with special myeloma cells. The main requirement for the myeloma line is that it must not produce its own immunoglobulins. This results in a heterokaryon (a cell with two nuclei), and subsequently a single hybrid cell.
- HAT selection: The resulting cell mixture is placed in a selective medium to weed out unfused cells.
- Cloning: Cells producing antibodies of the desired specificity are selected and propagated. Clonality is confirmed by isoelectric focusing.
The principle of selection on HAT medium
HAT medium contains hypoxanthine, aminopterin, and thymidine. Aminopterin is a poison that blocks the main pathway of nucleotide synthesis in the cell.
Only hybridomas survive in this medium, as they combine the properties of both parent cells:
- Unfused myeloma cells die. Their main pathway is blocked by aminopterin, and the enzymes for the salvage pathway are genetically defective.
- Unfused splenocytes die on their own because they are unable to live long in vitro.
- Hybridomas survive. They inherit "immortality" from the myeloma and fully functional genes for the nucleotide salvage pathway using hypoxanthine and thymidine from the normal cell.
Antibody humanization
Since creating hybridomas from human cells has proven difficult, and the demand for human monoclonal antibodies for therapy is high, genetic engineering is employed.
The "humanization" process involves replacing the framework regions of mouse genes with human genes. As a result, only the hypervariable regions responsible for antigen binding remain from the mouse antibody, and the molecule itself loses antigenicity for the human immune system.
Phage display and minimal antibodies
Phage display technology makes it possible to create antigen-binding molecules without using animals:
- A combinatorial library of Fab molecules is formed from B-lymphocyte genes.
- The genes are fused with the coat protein gene of a bacteriophage.
- Phages of the desired specificity are selected using the antigen and then propagated in bacteria.
This yields minimal antigen-binding molecules: scFv (covalently linked V domains of H and L chains) and minibodies. They are convenient for creating conjugates with drugs, toxins, or fluorophores.
Briefly: answers to topical questions
What stages does phage display technology include?
Phage display technology includes six stages of creating and selecting antigen-binding molecules of the required specificity.
- Gene isolation — V genes of H and L chains are isolated from expressed gene libraries of B lymphocytes.
- Creation of a combinatorial library — genes are combined in random combinations to obtain a set of Fab molecules of various specificities.
- Gene fusion — genes of Fab molecules are fused with the genes of the filamentous bacteriophage coat protein pIII.
- Phage propagation — phages are propagated in bacteria, forming a phage display library.
- Selection — using an immobilized antigen, phages encoding Fab domains of the required specificity are extracted.
- Infection — bacterial cells are infected with the selected phages, becoming factories for the production of the desired molecules.
Class and exam questions
What is the purpose of HAT medium?
HAT medium allows the destruction of unfused tumor cells (by blocking nucleotide synthesis pathways), leaving only hybridomas alive, which receive enzymes for the alternative pathway from the normal lymphocyte.
What is the essence of mouse antibody humanization?
Humanization is the genetic engineering replacement of the framework regions of a mouse antibody with human ones. Only the regions recognizing the antigen remain from the mouse, so that the antibody does not cause an immune response in humans.
What is an scFv?
This is a minimal antigen-binding molecule (a single chain of heavy and light chain V domains) created using phage display technology.
What else to study on this topic
- Antibody screening methods (ELISA, flow cytometry)
- Criteria for selection of hybridoma clones
- Features of storage and production of monoclonal antibodies
- Practical application of minimal antibodies in targeted therapy
- Immunological memory and secondary response
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