Hybridoma Technology (Part 2- mAbs)
Welcome to the 2nd part of the 19-part series on monoclonal antibodies (mAbs). You can read the first part here to understand what are monoclonal antibodies and how do they differ from polyclonal antibodies.
The hybridoma technology for the production of monoclonal antibodies was developed by Georges JF Koehler and Cesar Milstein in 1975.
The first step in producing monoclonal antibodies is the immunization of an animal, say mouse. The mouse, which is 2–4 weeks old, is immunized with the antigen against which monoclonal antibodies are to be raised. Suppose the injected antigen has four epitopes.
As a result, the four types of B cells in the mouse get activated. The activated B cells then differentiate into antibodies producing plasma cells, producing polyclonal antibodies.
After several weeks, when the B cells reach the optimal amount, the mouse is sacrificed, and its spleen is removed. Spleen is then subjected to mechanical and enzymatic disruption, resulting in the release of cells. The plasma cells are separated from the other cells by density gradient centrifugation. And as a result of this step, the four types of plasma cells are isolated that can produce antibodies against the four epitopes present on the injected antigen. In the next step, the antibody-producing plasma cells are fused with myeloma cells to generate hybrid cells known as hybridomas. Here it is essential to understand that the antibody-producing plasma cells have a definite life span, but the myeloma cells are cancerous plasma cells, which are immortal and can divide indefinitely.
The myeloma cells used in the hybridoma technology have mutations in 2 genes.
· One in hypoxanthine-guanine phosphoribosyltransferase gene abbreviated as HGPRT. Because of the mutation, the HGPRT gene is non-functional in myeloma cells.
· And the second mutation is in immunoglobulin genes, because of which myeloma cells are unable to produce antibodies.
The fusion of plasma and myeloma cells is facilitated either with polyethylene glycol, abbreviated as PEG, or by electrofusion. As a result of fusion, three types of cells are obtained: fused hybridoma cells, unfused plasma cells, and unfused myeloma cells. The fused hybridoma cells possess the immortal growth properties of the myeloma cell and secrete the antibodies produced by the plasma cells. On the other hand, the unfused antibodies producing plasma cells have a limited life span. And unfused myeloma cells, though immortal, are unable to produce antibodies because of the mutations in the immunoglobulin genes.
Since the injected antigen has four epitopes, four types of plasma cells were produced in the mouse. Thus, when the four types of plasma cells are fused with the myeloma cells, four types of hybridoma cells are obtained. The hybridoma cells are then selected out of the mixture of fused and unfused cells.
Selection of Hybridoma Cells on HAT Media
The selection of hybridoma cells from the mixture of fused and unfused cells is carried out on HAT media. HAT stands for Hypoxanthine Aminopterin Thymidine. The selection of the cells on the media is based on the fact that there are two pathways of nucleotide synthesis. In the media, the mother cells divide to form daughter cells for which DNA replication occurs, and DNA replication requires the synthesis of new nucleotides. Two pathways of nucleotide synthesis are the Salvage pathway and the De-novo pathway.
1. The salvage pathway is the pathway in which new nucleotides are synthesized from the parts of degraded nucleotides.
2. Whereas in the case of the de-novo pathway, completely new nucleotides are synthesized using small metabolites like sugars and amino acids present in the media.
When the cells are grown on HAT media, they are unable to operate the de-novo pathway because of the presence of Aminopterin (Fig 1a). Aminopterin blocks the critical enzyme dihydrofolate reductase involved in nucleotide synthesis. Once this enzyme is blocked, cells can not convert simple sugars into nucleotides and thus can’t operate the de-novo pathway. Therefore, the cells are left with the option of using the salvage pathway to synthesize nucleotides.
But for the Salvage pathway to operate, cells must possess the enzyme Hypoxanthine guanine phosphoribosyltransferase, i.e., HGPRT. HGPRT is a key enzyme that helps cells to use hypoxanthine and thymidine from the HAT medium as precursors to synthesize nucleotides (Fig 1b).
Because of this reason, the unfused myeloma cells die on the HAT media, as they have non-functional HGPRT gene; therefore, they cannot produce nucleotides by the salvage pathways.
On the contrary, unfused plasma cells and hybridoma cells can operate salvage pathways on the HAT media. The functional HGPRT gene in hybridoma cells is contributed by the plasma cells that get fused with the myeloma cells. But the unfused plasma cells die after a few cell divisions as they have a short life span. In contrast, the hybridoma cells can divide indefinitely on the HAT media, and this property is contributed by the myeloma cells. Therefore, at last, only the hybridoma cells are left in the HAT media. These hybridoma cells:
· Possess the capability to produce antibodies, a property of plasma cells
· And they also become immortal, a property of myeloma cells
The population of hybridoma cells that survive selection is heterogeneous, containing clones of 4 different types of hybridoma cells that produce antibodies with different epitope specificities. Recall that each type of hybridoma cell produces antibodies specific to an epitope on the antigen. But the aim is to select and propagate a single antibody-producing hybridoma cell. For this, these hybridomas are needed to be isolated and grown individually. The isolation of hybridomas of a single specificity is done by a method known as limiting dilution.
Limiting dilution is a technique that dilutes the concentrations of the heterogeneous population such that, on average, each well contains one cell. In practice, some wells may contain no cells, some may have a single cell, and others may contain multiple cells.
In the next step, each hybridoma cell is screened for the secretion of antibodies with the desired specificity. This screening is done by ELISA technique and selects only those hybridomas that produce antibodies of appropriate specificity.
For this, the hybridoma culture supernatant containing monoclonal antibodies (mAbs) is added to the antigen-coated microtiter well. The antigen-coated is the desired Ag against which mAbs are to be raised. The mAbs are then allowed to interact with Ag. After this, the mAbs bound to Ag are detected by adding secondary anti-isotype antibodies labeled with an enzyme, which binds with primary mAbs. Then the chromogenic substrate is added. And upon addition of this substrate, if a colored product is obtained, it indicates a positive hybridoma. After this, the desired antibody-producing hybridoma cell can be cloned to produce multiple identical daughter clones. These identical daughter cells produce monoclonal antibodies or, in other words, identical antibodies with the same antigen specificities.
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