Monoclonal Antibodies and Hybridoma Technology
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Direct answer
Monoclonal antibodies are identical antibodies secreted by a single B-cell clone, and hybridoma technology produces them by fusing an antibody-producing B lymphocyte with a myeloma cell. Kohler and Milstein described the method in 1975, and fused cells are selected in HAT medium before cloning. Today these antibodies serve as diagnostic reagents in microbiology and as therapeutic agents such as rituximab.
What you must remember
- Hybridoma: a hybrid cell that carries the antibody-making ability of a B lymphocyte and the unlimited growth of a myeloma cell.
- Fusing agent: polyethylene glycol is the classic agent that joins the membranes of splenic B cells and myeloma cells.
- HAT selection: hypoxanthine, aminopterin and thymidine medium kills unfused myeloma cells, while unfused B cells die off on their own in culture, leaving only hybridomas that use the salvage pathway.
- HGPRT: the enzyme hypoxanthine-guanine phosphoribosyltransferase is absent from myeloma lines, so those cells die in HAT, while hybridomas inherit it from the B cell.
- Cloning: limiting dilution produces single-cell clones, and each clone secretes one antibody with a single specificity.
- Nomenclature: murine antibodies end in -omab, chimeric in -ximab, humanised in -zumab and fully human in -umab.
- Therapeutic examples: rituximab, an anti-CD20 chimeric antibody, is used in B-cell lymphoma, and trastuzumab, an anti-HER2 humanised antibody, is used in breast cancer.
A six-step route from immunised mouse to clone
The procedure combines an immune response in an animal with a cell line that divides without limit. Each step solves one problem, namely getting the right antibody, getting an immortal partner, and keeping only the fused cells.
- Immunise: a mouse receives the target antigen, so that splenic B cells make antibody against it.
- Harvest: spleen cells are isolated and mixed with myeloma cells that lack HGPRT.
- Fuse: polyethylene glycol joins the two cell types, forming hybrid cells.
- Select: the mixture is grown in HAT medium, where only hybridomas survive.
- Screen: culture supernatants are tested for antibody against the target antigen.
- Clone and expand: positive wells are cloned by limiting dilution and grown to harvest antibody.
Because each clone descends from one fused cell, its antibody is uniform from batch to batch. That uniformity is the basis of the reproducible reagents used in antigen-detection kits.
Where the exam tries to catch you
Monoclonal antibody questions often test the selection logic and the name of the antibody.
- Trap: calling antibodies from an immunised rabbit monoclonal Correct: antisera are polyclonal mixtures from many B-cell clones, whereas a hybridoma yields one specificity.
- Trap: believing myeloma cells survive HAT because they are immortal Correct: they die in aminopterin because they lack HGPRT and cannot use the salvage pathway.
- Trap: reading -mab as proof that an antibody is human Correct: the infix before -mab shows origin, so -omab is murine and -umab is fully human.
Frequently asked questions
Why is HAT medium used in hybridoma selection?
HAT medium is used because aminopterin blocks de novo nucleotide synthesis, forcing cells to rely on the salvage pathway. Only hybridomas survive, because they inherit HGPRT from the B cell, while unfused myeloma cells die in HAT and unfused B cells do not survive long in culture.
What does HGPRT deficiency in myeloma cells achieve?
HGPRT deficiency in myeloma cells means they cannot use the salvage pathway, so they die when aminopterin blocks de novo synthesis. This selective defect allows fused hybridoma cells to be picked out by growth in HAT medium.
Which monoclonal antibody is used in B-cell lymphoma therapy?
Rituximab is the monoclonal antibody used in B-cell lymphoma therapy, and it targets the CD20 antigen on B cells. It is a chimeric antibody, which is why its name carries the -xi- infix that marks chimeric antibodies.
How do monoclonal antibodies improve antigen detection tests?
Monoclonal antibodies improve antigen detection tests because each one binds a single defined epitope, which reduces cross-reactivity between organisms. This specificity suits ELISA and direct fluorescent assays used in diagnostic kits, and it gives consistent reagents from one batch to the next.