Hybridoma technology is proposed by Kohler and Milstein in 1975.
Hybridoma technology is used for the production of monoclonal antibodies (mAbs). These antibodies are widely used in biotechnology, medicine, research, diagnostics and therapeutic purposes. This is the reason why hybridoma technology is a widely used technique in many areas of life science sector.
Antibodies (Ab) or immunoglobulins are protein molecules which are produced by a specialized group of cells called B-lymphocytes (plasma cells) in mammals
POLYCLONAL VS MONOCLONAL ANTIBODY
Polyclonal antibodies are usually produced in our body in response to an antigen (which has several different epitopes). Polyclonal antibodies are heterogenous (they have different specificity and affinity to bind with an epitope) and they lack specificity because they are produced by several different clones of plasma cells
Monoclonal antibodies (MAbs) are produced from clone of single B-lymphocyte directed against a single antigenic determinant or epitope. This improves specificity and thus monoclonal antibodies are used for therapeutic and diagnostic purposes
Hybridoma Technology
Hybridoma technology is a widely used technique for the production of Monoclonal antibodies (mABs) at large scale so that these antibodies can be used for therapeutic, diagnostic and research purposes. Hybridoma technology involves fusion of antibody producing B-cells with immortalized myeloma cells to create hybrid cells called hybridomas. These hybrid cells (hybridoma cells) are selected on HAT medium after which, only those hybrid cells (Hybridoma cells) which can secrete or produce desired monoclonal antibody are isolated and then cultured to establish hybridoma cell lines so they can be further propagated for the large scale production of monoclonal antibodies (mAbs).
Since hybridomas (hybrid cells) are produced and propagated on a large scale by this technique, its called as Hybridoma technology
Steps of Hybridoma Technology
1) Immunization of Mouse :- Mouse is injected with desired antigen (against which we have to produce our antibodies). Antibodies are then produced due to immune response elicited or stimulated by B-cells.
[Antigen + adjuvant like Freund’s complete or incomplete adjuvant is injected subcutaneously. Series of injections are given for several weeks so that high amount of antibodies can be produced in mice. We can measure the concentration of antibodies produced by Radioimmunoassay (RIA) technique]
2) Isolation of B-cells :-Spleenocytes are isolated from the mouse because Spleen tissue is rich in B-cells. Thus we will get high amount of B-cells to conduct our experiment.
3) Cultivation of myeloma cells :- Myeloma cells are grown in the serum containing complete media. When the ratio of B-cells : Myeloma cells is 2:1 or 5:1 , we will proceed for next step i.e fusion.
4) Fusion of myeloma and B-cells :- B-cells are mortal (i.e they will die after dividing for some generations) whereas Myeloma cells are immortal (i.e they can divide indefinitely i.e No Hayflick’s limit followed).
[Hayflick’s limit is the phenomenon in which it is observed that, the cells can divide maximum 40-60 times before it enter senescence or before it proceed to programmed cell death or apoptosis]
- B-cells and Myeloma cells are fused together using Polyethylene glycol (PEG) for short amount of time as PEG is toxic. B-cells and Myeloma cells when fused together, these cells will form hybrid cell lines or Hybridomas and such production of Hybridomas is referred to as Hybridoma technology.
- PEG is removed by washing and the cells are then kept in a fresh medium.
- Now, the medium will be composed of Hybridoma cells (fused cells) ; Free Myeloma cells ; Free B-cells or Lymphocytes
[But, we are only interested in Hybridoma cells (fused cells). So, we need to separate or select only Hybridoma cells (fused cells) from a mixture containing Hybridoma cells (fused cells), Free Myeloma cells and Free B-cells]
5) Selection of hybridoma or fused cells :- Hybridomas are grown on HAT medium (Hypoxanthine aminopterin-thymidine medium) which is the selective medium
There are 2 pathways for nucleotide synthesis namely Salvage Pathway and De novo Pathway.
[If nucleotide synthesis does not occur, DNA replication will not occur, No cell division will occur, No proteins will be produced and the cells will die]
[ B-cells (HGPRT+) and Myeloma cells (HGPRT-) ]
- Free Myeloma cells present in the medium will undergo death in HAT medium because aminopterin blocks the de novo pathway of nucleotide synthesis. Moreover, since they are HGPRT- , they also cannot synthesize nucleotides by Salvage pathway.
Result = Death of Free Myeloma cells
- Free B-cells will survive in the HAT medium but only for some time. Although aminopterin will block the de novo pathway of nucleotide synthesis, but B-cells are HGPRT+ . This means that Salvage pathway of nucleotide synthesis is active. Since, B-cells are mortal, they will divide finite times (40-60 divisions) and then they will undergo normal cell death
Result = Death of Free B-cells
- Hybrid cells (Fused cells) will have HGPRT+ (by B-cells) as well as they are immortal (property from Myeloma cells). Thus, only Hybrid cells will survive on HAT medium
Result = Survival of Hybrid cells (Fused cells)
6) Screening :- Hybridomas are screened for the secretion of the antibody of the desired specificity [We can check this using ELISA and RIA immunotechniques]
7) Cloning and Propagation :- Single hybrid cells (fused cells) producing the desired antibody are isolated and cloned. Medium containing IL-6 can be used to propagate Hybridoma cells and to establish hybridoma cell lines
Hybridomas are transferred to large culture flasks for large scale production of antibodies
8) Storage :- Supernatant will be collected and stored at -200 degree celsius for further usage
Applications of Monoclonal Antibody
1) Use in Laboratories for diagnosis and research :- Monoclonal antibodies are used for laboratory diagnosis of various diseases. They are also routinely used in RIA and ELISA immunotechniques in labs and research labs to conduct experiments, research and diagnosis.
2)Diagnostic Imaging :- Diagnostic imaging of diseases is referred to as immunoscinigraphy. Radioisotopes used for labeling Monoclonal antibody (mAb) are Ionine-131 (specific to breast cancer) ; and technetium-99
[Immunoscintigraphy can detect tumor because radiolabeled monoclonal antibodies (mAbs) are tumor specific]
3) Myocardial infarction :- Cardiac protein i.e myosin gets exposed wherever myocardial necrosis occurs. Antimyosin monoclonal antibody (mAb) labeled with radioisotope indium chloride (111In) is used for detecting myosin and thus the sire of myocardial infarction
4)Therapeutic applications :- E.g a) In destroying disease causing microorganisms :- Monoclonal antibodies promote opsonization of pathogenic organisms (by coating them with antibody) thereby increasing phagocytosis
E.g b) In the treatment of cancer :- Monoclonal antibodies will bind with cancer cells and these cells will be destroyed by Antibody dependent cell mediated cytotoxicity (ADCC) or phagocytosis of cancer cells (coated with monoclonal antibodies) by macrophages
5) To produce immunotoxins :- Toxins can be coupled with Monoclonal antibodies to form immunotoxins and used in therapy. E.g Diphtheria toxin
6) Drug delivery :- Drugs are less effective in vivo as compared to in vitro because of sufficient quantity of the drug does not reach target tissue. This problem can be solved by using tissue specific monoclonal antibodies. Drugs can be coupled with Monoclonal antibody (directed against a cell surface antigen of cells,say tumor) and specifically targeted to reach the site of action
- In treatment of certain diseases, pro-drug (inactive form of drug) can be used. This can be enzymatically converted to active drug in target tissue. This approach is called antibody directed enzyme pro-drug delivery (ADEPT)
Advantages of Monoclonal Antibody
1) Monoclonal antibodies are highly specific and thus used in labs for research, diagnostic and therapeutic purposes.
2) Monoclonal antibodies can be engineered for various applications. For example, toxins can be coupled with monoclonal antibodies to form immunotoxins. For example – Diphtheria toxin.
3) Some monoclonal antibodies have long half-lives due to which less frequent dosing is required.
4) Due to its high specificity, accurate diagnosis of disease is possible.
5) Monoclonal antibodies produced by hybridoma technology are homogenous in nature.
Following features make Monoclonal antibodies homogenous :-
- Single cell origin :- They are produced by a single clone of cells known as hybridomas, which are derived from a single B-cell
- Identical antigen specificity :- They are highly specific and all the antibodies will recognize the same epitope or antigenic determinant on the target molecule
- Uniform structure :- They have uniform structure including their variable regions (Fab) and constant regions (Fc) required for binding to antigen
Disadvantages of Monoclonal Antibody
1) High Labour is required for their production using Hybridoma technology
2) Time consuming steps required for their production using Hybridoma technology
3) Mice used in Monoclonal antibody (mAb) production may carry viruses like andeovirus, retrovirus etc. This may be risky as there is no guarantee that mAb will be totally virus free, despite the purification.
4) Continuous use of monoclonal antibodies can lead to the development of resistance in host (patient) thereby reducing their effectiveness over time
5) Storage of monoclonal antibodies require specific storage temperature. If not stored at specific temperature, they may lose effectiveness over a long period of time
6) Immunization of mouse is required for the production of monoclonal antibodies. This may raise ethical concern regarding animal welfare when hybridoma technology is used for their production