Hybridoma Technique

In the process to develop the monoclonal antibody. One of the important steps is to develop Hybridoma. And as per the name of the developed cell, the process is known as the Hybridoma. At first, the antigen is inserted in the body of the mouse. Then it left for 72 hours. After 72 hours of the injection, the spleen cell of the mouse is collected. Spleen is the most important cell in the immune system. in the spleen cell, the antibody from the antigen develops. So, the spleen cells are taken for developing the monoclonal antibody.

Now, one of the major disadvantages of these cells is, they are not immortal. This means after taking out those cells for some time those cells will have died. But the intention is to create the immortal version of those cells. for that purpose, there is a need to fuse with myeloma cells. Myeloma cells are cancerous cells. This means they will always develop until there is a disconnection of the nutrients. Now, the intention is to fuse the B cells of the spleen & the myeloma cells of the mouse.

These two cells are placed in the same medium for 48 hours. They are placed in a medium named Polyethylene Glycol.  In this medium both the cells are placed to fuse. After the fusion, a new cell will be derived. The cells will have the antibody production power from the B cells of the spleen as well as they will be immortal as the myeloma cells. These cells are termed Hybridoma. This means these cells are the hybrid form of the cells. and this overall process is known as the Hybridoma process.

HAT Medium

It is one of the essential elements in the production of Monoclonal Antibodies. This medium is completely used to extract the hybridoma cells from the mixture of the medium. The HAT medium is made up of three components. They are the Hypoxanthine, Aminopterin, and Thymidine. The first letter of these elements is being used to name this medium. The H from the Hypoxanthine, the A from the Aminopterin & the T from the Thymidine. All these substances are combinedly and named the HAT medium. All these elements are proteins. And their main goal is to stop the growth of any cells in the proper medium. This medium is used in the production of the monoclonal antibody.

The HAT medium is not alone able to do the entire task. This means the cells can’t able to be placed in a single HAT medium. The cells where it is from the beginning of the experiment, there the HAT medium should be included. Hypoxanthine is the substance that deactivates purine protein in a certain medium. Purine is a type of protein that is used in the growth of cells. This is the protein that is being used as a nutrient for the growth of any cells. Aminopterin is a type of drug. That helps to stop the metabolism of any cells. This means the cells can’t able to grow more in the medium. And Thymidine creates issues with the synthesis of the DNA in any cell. This means all these elements participate to stop the growth of the cells at any cost. The influence of these substances can only recover by the Hybridoma cells.

As the HAT medium can kill natural cells, the cells that are not introduced by the nature can skip this effect of the medium. Purine is the protein that helps to make the growth of cells. now, this protein is created using some special pathway. They are the De Novo pathway. For creating some proteins, there are certain processes. They are the list of processes. Now, the Purines can able to synthesize using the De Novo pathway. And in absence of the De Novo pathway, the Purine can be produced using the Salvage pathway. Now, the components of the HAT medium create an issue in the formation of the Purine proteins.

The Aminopterin substance in the HAT medium at first blocks the De Novo pathway of all the cells present in the medium. So, the pathway can’t able to use by the cells to produce the proteins. So, by force, they have to follow the Salvage pathway. In the Salvage pathway, two essential elements are needed. One is a special type of enzyme & another is a protein. The enzyme is the Hypoxanthine Guanine Phosphoribosyl Transferase (HGPRT). And the protein is the Thymidine Kinase. The Thymidine Kinase also acts as the enzyme there.

Now, the Thymidine Kinase is blocked by the Thymidine in the HAT medium. So, the cells can’t able to access that element for life long. And unfortunately, there are no HGPRT enzymes present in the Myeloma cells. So, as a result, the Myeloma cells can’t able to live more in this medium. As there is no HGPRT key element present. So, as a result, eventually, all the Myeloma cells will be destroyed. Now, there are only the Hybridoma cells & the spleen cells present.

The Spleen cells can able to replicate here. As they have the HGPRT present in their cells. But as they are not Myeloma cells. This means they are not ever-living cells, so eventually, they have to die. So, with time, the spleen cells will be removed from the medium. Now, the Hybridoma cells will only survive there. As Hybridoma cells come from nature, they have some special features. As they have derived from the spleen cell, so they can create the HGPRT enzyme in their cells. And as they are a hybrid with Myeloma cells, they have the feature of cancerous cells. This means they will never live.

So, the HAT medium is used to remove all the unnecessary cells. The Myeloma cells need to have died as there is no option to create an HGPRT enzyme. And spleen cells need to have died as they are not ever-living cells. But they have the HGPRT enzyme. As the Hybridoma cells have all the features so they will only survive in this medium.

Purification Technique

Purification is the second most important process for developing the monoclonal antibody. After the hybridoma process, there is a mix of cells in the hybridoma solutions. As all the spleen cells & the myeloma cell will not be fused. Among the quantity of them will be fused & some of them will not be fused. So, in the solution, there will be three types of cells present. One is the newly created Hybridoma cell, another will be the B cells of the spleen and the last one will be the myeloma cells.

Now, the main goal is to only extract the hybridoma cells. for that purpose, all other cells need to be eradicated. For that purpose, all the mixture is placed into a new solution. That is the HAT solution. This is a special solution. In this solution, there are three substances present. They are the Hypoxanthine, Aminopterin, and Thymidine. These substances will act on the myeloma cells. These substances will cut down the nutrients supply for the myeloma cells. in simple terms, the myeloma cells can’t able to grow more or live more in this solution. So with time, all the cells will be destroyed.

Now, two cells are remaining. One is the hybridoma cell & another is the B cell of the spleen. There is no need to do any experiments on the B cells. As they are not immortal. With time, these cells will be destroyed automatically. So, there will be simple the Hybridoma cells will be present. And that is the goal of this process. This overall process is known as the Purification process. As the other cells will be removed from the mixture.

The human immune system is strange. it is such a complicated system that can’t be imagined. It is a well-developed process in the human body. Whenever any foreign element inserts into the human body, the immune cells start acting. It starts operations on the foreign elements & destroys them within the body. It is a complicated structure. Whenever there is any threat from the foreign element, it is considered as the antigen. The element that is not a part of the body. And the substances that help to destroy the antigen are known as the antibody. The monoclonal antibody is one type of that.