Steps involved in hybridoma Technology

Steps involved in hybridoma Technology:
The manufacturing process of MAbs can be done in vivo or in vitro or even it can be a complex of both the process. In hybridoma method hybrid cells are made prior to the manufacture that will give the antibodies of interest. (National Research Council, 1999)
It is beneficial to remember that the cells that will create the MAbs need the applicability of animals, mice are the perfect candidates. At the end of the final stage we will have a “cell line” that will provide a singular kind of antibody and last for a broad time. (National Research Council, 1999)
In this study the production procedures or the stages are illustrated as below:

Step 1: Immunization of Mice and Selection of Mouse Donors for Generation of Hybridoma Cells:
At first the specific antigen was injected into a healthy mice and such antigen was made by the process of emulsification with Freund’s adjuvant or it was homogenized with a gel slice. Some other sources of immunogens can be intact cells, entire membranes, and even microorganisms are used times. It a common practice for the researchers to use mice in their respective labs in order to obtain their antibodies of interest. All it takes 14-21 days for the mice to be immunized which may fluctuate from researcher to researcher. When the time comes and the mice serum attains the optimum level of antibody titer, then the spleen is taken out and cells are collected which will be fused with myeloma cells.(National Research Council, 1999)

Step 2: Screening of Mice for Antibody Production:
At this stage, when few weeks have passed by sample of blood are collected from the immunized mice and the antibody quantity is recorded. Loed and Quimbly quoted in 1999 that “several humane techniques have been developed for collection of small volumes of blood from mice.” Multiple methods can be used to identify the antibody titer and among these methods the ELISA and flow cytometry are the most popular. It is necessary for the titer to be in maximum amount in order for the fusion to be done. A boosting dose may be required in the case of insufficient titer amount. After the boosting dose blood was collected respectively until the enough repercussion is obtained. The boosting dose is also given even if the titer prevails in adequate amount. In this case the animals are treated solely by the antigen but this time no adjuvant will be used. This is done by either through intraperitonial route or IV route and must be performed 3 days prior to fusion but a couple of week followed by the initial antigen introduction. Then lastly the spleens from the immunized mice were collected in order to manufacture hybridoma cells in vitro. (National Research Council, 1999)

Step 3: Preparation of Myeloma Cells:
The cells that were collected from the spleen having the ability to produce antibody survives for a definite period of time. In stage 3 these cells are fused with immortal tumor of lymphocytes. The resulting product is known as hybridoma and its core characteristic is that it shows indefinite growth. Such tumor cells are known as myeloma cells and such cells are grown with 8-azaguanine and thus it make sure its susceptibility to the hypoxanthine-aminopterin-thymidine(HAT) medium. HAT medium is the culture medium that will be used following the fusion step. (National Research Council, 1999)

The myeloma cells are developed in 8-azaguanine one week prior to fusion. Elevated durability and enhanced development is highly desired from the cells. (National Research Council, 1999)

Step 4: Fusion of Myeloma Cells with Immune Spleen Cells:
For the production of MAbs by hybridoma technology this step is believed to be the most hardiest and vital of all. It is found that three methods are available for the fusion of cells in other words creating hybridomas. They are 1) using PEG, 2) fusogenic viruses, 3) electrical cytofusion where no. 3 is found to be the most feasible most popular one.(Smith ; Crowe, 2015)
But in this study we will only use the PEG. In this step fusion is created between the spleen cells and the myeloma cells and this is done by co-centrifuging the spleen cells with the myeloma cells in the PEG. The cells screened by performing ELISA were collected and were made to develop in the HAT medium were allocated to 96 well plate containing feeder cells. These feeder cells were obtained from saline peritoneal washes of mice. According to Quinlan and O’Kennedy’s study in 1994 they quoted that, ” feeder cells are believed to supply growth factors that promote growth of the hybridoma cells.” For commercial purposes some medias are used as a substitute of mouse-derived feeder cells. (National Research Council, 1999)

Step 5: Cloning of Hybridoma Cell Lines by “Limiting Dilution” or Expansion and Stabilization of Clones by Ascites Production:
This is the final step in hybridoma method and the purpose of this step is to collect the end product that was obtained by fusion method and also known as biological cloning.(Smith ; Crowe, 2015)
In order to obtain hybridoma clones limiting dilution cloning is used and the basis of this method is poisson distribution. It provides the superiority than other methods because of its simplicity and the equipments that used are not so costly after all. (Smith ; Crowe, 2015)
To start this step firstly the cells that were put into the 96 well plates are made to be grown into fresh small groups. After this, sorting for the antigen attachment were done or it can be further cloned by mouse ascites method. The use of “limiting dilution” guarantees that each of the wells is accompanied solely by a single clone. There are some complications too that the resulting antibodies might show toxicity. In order to prevent such phenomenon mouse ascites expansion method can be applied. What Ishaque and Al-Rubeai quoted in 1998 that “Also, it is the experience of many that a brief period of growth by the mouse ascites method produces cell lines that at later in vitro and in vivo stages show enhanced hardiness and optimal antibody production.” (National Research Council, 1999)

Effectiveness of the process:
Since we have known how the MAbs are produced from hybridoma technology now let us has a glance on how effective this method is for the production of MAbs. To know such facts a study that was done by R.V. Natalia et al. which was published on the 2nd December 2017 is discussed here. The study they did was titled as “Immunochemical assay with monoclonal antibodies for detection of staphylococcal enterotoxin H”. Now let us talk about their findings.
It was the S. enterotoxins that cause food poisoning and dairy products like milk are the most susceptible to SEH contamination. So in order to identify the SEH a very particular and effective technique had to be taken. R. V. Natalia and her fellow researchers exploited the gene that is expressed in the SEH and they were able to make a recombinant toxin. They were successful in preparing a MAbs which showed a very high- affinity by applying the hybridoma technology. What they found in their result was the most interesting part that there were no cross reactions between the antibodies and the SEH enterotoxins. So, by using such MAbs for the detection of SEH have become possible and easier by developing the sandwich enzyme immunoassay. The MAbs detected the SEH by a process named immunoblotting. By adopting this method they were able to detect the toxin upto 100% in milk. (Rudenko et al., 2018)

?
Conclusion:
It is the MAbs that have given a brand new approach for remediation towards a broad spectrum of diseases and it has been solely possible by the advancements of medicines towards the novel period of intrinsic therapy and these MAbs produced through hybridoma technologies are leading the path with domination. It has been more than 30 years since the initial MAbs were used clinically, as the time passed and through the use of cutting edge technologies now it has taken an industrial shape and the market value is calculated to be of billion dollars.(Liu, 2014)
The MAbs are being widely used in many sectors. For example in genetic sequencing it is being used as a major tool. Same thing can be said for various biomedical researches too. Though having lots of potential advantages the hybridoma technology derived MAbs are not in a stalled state, researchers from all around the globe are trying heart and soul bearing in mind that the primary focus is to improve and invent new drug targets. By doing so the new generations of MAbs are being produced having maximum efficiency and applications in the clinical field.(Liu, 2014)