1. INTRODUCTION:Monoclonal antibodies are antibodies that have selected specificity to a single epitope of an antigen that are secreted in large quantities by immortalised Hybridoma cells. Since their first production, monoclonal antibodies have proven to be of extensive importance to the biomedical field.

They are widely used for the identification of nucleic acids, proteins and carbohydrates. Monoclonal antibodies are exquisitely specific and thus play a key role in disease diagnosis, prevention and treatment in both humans and in animals. Furthermore, they have elucidated and helped us understand the response of host to infectious disease toxins produced by certain agents relating to the disease, antigens that lead to autoimmunity, spontaneously proliferating tumour cells, etc. While monoclonal antibodies are specificity to one single epitope, they differ form polyclonal antibodies which are a collection of antibodies secreted by multiple B cells which recognise and have affinities to different epitopes of the same antigen. (Fig 1.1)The hybridoma cells which are engineered to produce the monoclonal antibody are biologically constructed thorough a technique known as Hybridoma Technology. Hybridoma Technology is a revolutionary technology that was discovered in 1975 by the scientists, Georges J. F.

Kohler (from West Germany) and Cesar Milstein (from Argentina). It involves the injection of an animal, typically a mouse with the antigen of choice and isolating the B cells from the spleens of the mouse after the immune response. Then immortal Myeloma cells which proliferate infinitely are fused with the B cells from the mice to create Hybridoma cells which exist with both the antibody producing nature of the B cells and the immortality of the myeloma cells. This technology led to a significant advancement in the biomedical field as, in principle, it furnishes a path to acquire unlimited supplies of highly specific antibodies.

In this experiment, monoclonal antibodies are produced against HIV. Human Immunodeficiency Virus (HIV) is a retrovirus that attacks and damages the body’s immune system. This leads to malnutrition which is when the cellular demand for energy and nutrient is higher than what is being provided to the body.

This causes even more immune deficiency facilitating expeditious progression of the virus infection to Acquired Immune Deficiency Syndrome (AIDS). HIV along with accounting for consequential immunosuppression in an infected individual, also affects multiple vital organs of the body like the Heart, Brain, Kidney, etc.     2. METHODS AND MATERIALS: PRACTICAL I:After sterilising the work station, the Hemocytometer slide is made ready. Cells a                                                                                                                                PRACTICAL II:The nitrocellulose paper is marked into 48 equal squares and the supernatants from each well are transferred onto the squares. After the spots have dried, blocking solution is added to block any non-specific binding.

Later the binding solution is removed, and the nitrocellulose paper is washed with a washing solution. Washing solution is then poured out and the diluted conjugate is added and incubated. Conjugate is then removed, and the paper is washed with the washing solution. Washing solution is removed and washed with tap water and poured out and substrate solution is added to allow reactions to develop and the paper is finally washed away in water a few times. The paper is finally dried and recorded. PRACTICAL III:After selecting any three hybridomas, sera from them are added to the three preblocked strips separately. The sera and blocking solution are then poured out and washed with the washing solution and that is removed as well. Diluted secondary antibody conjugate is added and washed away with the washing solution and later rinsed with tap water to add the substrate solution.

Once bands start forming, the solutions are washed away with water and the paper is dried and recorded.  3. RESULTS: D10   D3      C11*    C12   B8*      Fig 3.1 – The results of the Dot Blot Test: The antibodies produced by the Hybridoma cells bind with the conjugated anti-mouse antibody and can be visualised with the addition of a substrate solution as the dark yellow or light brown dots on the Nitrocellulose paper. We chose 3 different antibodies (marked with a *) produced by the Hybridoma cells of wells A3, B8 and C11 for further experiments. Fig 3.4   Fig 3.2     Fig 3.

3                                                                                          Fig 3.5                                                            Fig 3.6 Fig 3.5 – The Positive Control showing that the antibodies have bound to the four specific key proteins of the HIV virus.

Thus denoting that these antibodies were specific to the four proteins.Fig 3.6 –  The Negative Control showing that none of the antibodies binded with the proteins of the HIV virus.

Thus denoting that these antibodies werent specific to the four proteins. 4. DISCUSSION:PART I : FUSION OF THE CELLS TO CREATE HYBRIDOMASAs we can understand from the results, only the Hybridoma cells survived the HAT medium selection.

This is because one of the components of the HAT medium, Aminopterin which is an antibiotic blocks the De Novo pathway. Thus all the cells have to switch to the Salvage pathway in order to produce the nucleic acids for cell replication and survival. For the Salvage pathway to work, we require Hypoxanthine-guanine phosphoribosyl transferase (HGPRT) to convert the substrates Hypoxanthine and Thymidine to produce Purines and Pyrimidines. Since Myeloma cells are HGPRT deficient ( HGPRT-), they cant produce the nucleic acids for cell replication and thus don’t survive in the HAT medium.

Whereas the B cells have HGPRT (HGPRT+) and can thus replicate and survive in the medium. Hybridoma cells which retain the characteristics of the B cells are also HGPRT+ and thus survive the HAT selection media. Although in due course the B cells will die due to their limited lifespan thus leaving behind only the fused Hybridoma cells.

(fig 4.1)  PART II : ANTIBODY SCREENING BY DOT BLOT TESTOnce the cells are fused together to form the Hybridoma cells, they start producing antibodies but not all the cells produce the antibodies. As we can see from the results, only few of the cells made antibodies.

The squares containing the dark yellow or light brown dots show the antibody from the hybridomas have bound with the peroxidase anti-mouse conjugated Ig-G and the colour seen is for visual indication produced with the use of a substrate. Antibody production by the cells is unpredictable so this is precisely why we require a test to determine the productivity of a hybridoma cell and so that we don’t end up using all the hybridomas for further testing without the knowledge of whether all the cells are productive or not to avoid any sort of errors. PART III : SPECIFICITY TESTING BY WESTERN BLOTTo check for specificity, we use four key proteins to check if the antibodies are specific for any of them. If the antibodies are specific to any of these proteins, they can help fight against the virus. The key protein gp120 and gp41 glycoproteins that are a part of the HIV envelope that help the virus to attach, enter and incorporate with the host cells. p66 polymerase protein codes for a segment of reverse transcriptase polymerase which takes part in the genome replication of the virus in the host cell. While p24 Is one of the constituent of the virus capsid. From the results, we can see that there are certain bands with a dark yellow or a light brown colour.

This shows that the antibody has been bound with the HIV protein (antigen) on that particular position, thus showing its specificity to that protein. Having knowledge of a specific antibody and its corresponding specific protein can help not only understand how the virus works on its own and in the host cells, but also give us a way to strategically treat and eradicate the virus and its proliferation. Since we are dealing with monoclonal antibodies, each antibody can only be specific to one protein of the HIV which is why there aren’t any western strips from our experiment with more than one band seen. If there were, it indicates that the experiment has gone wrong. But from our results where p120 was receptive to both B8 and C11 antibodies, it showed that antigens can be specific to multiple monoclonal antibodies but a monoclonal antibody can only be specific to one antigen protein. This way monoclonal antibodies are all identical and single specific unlike polyclonal antibodies as monoclonal antibodies are produced by a single B wheres as polyclonal antibodies are from multiple B cells. Also from our results we can notice that our positive control has bands over all four antigens and the negative control has none.

These controls are to guide us by setting an ideal standard as to what the correct results should look like for us to compare our own experimental results to. The positive control has all 4 bands to show active binding for all four antigens.BIOLOGICAL ENGINEERING OF HYBRIDOMA CELLS EMPLOYING THE HYBRIDOMA TECHNOLOGY TO SYNTHESISE HUMAN IMMUNODEFICIENCY VIRUS (HIV) SPECIFIC MONOCLONAL ANTIBODIES. 1. INTRODUCTION:Monoclonal antibodies are antibodies that have selected specificity to a single epitope of an antigen that are secreted in large quantities by immortalised Hybridoma cells.

Since their first production, monoclonal antibodies have proven to be of extensive importance to the biomedical field. They are widely used for the identification of nucleic acids, proteins and carbohydrates. Monoclonal antibodies are exquisitely specific and thus play a key role in disease diagnosis, prevention and treatment in both humans and in animals. Furthermore, they have elucidated and helped us understand the response of host to infectious disease toxins produced by certain agents relating to the disease, antigens that lead to autoimmunity, spontaneously proliferating tumour cells, etc. While monoclonal antibodies are specificity to one single epitope, they differ form polyclonal antibodies which are a collection of antibodies secreted by multiple B cells which recognise and have affinities to different epitopes of the same antigen. (Fig 1.

1)The hybridoma cells which are engineered to produce the monoclonal antibody are biologically constructed thorough a technique known as Hybridoma Technology. Hybridoma Technology is a revolutionary technology that was discovered in 1975 by the scientists, Georges J. F. Kohler (from West Germany) and Cesar Milstein (from Argentina). It involves the injection of an animal, typically a mouse with the antigen of choice and isolating the B cells from the spleens of the mouse after the immune response. Then immortal Myeloma cells which proliferate infinitely are fused with the B cells from the mice to create Hybridoma cells which exist with both the antibody producing nature of the B cells and the immortality of the myeloma cells. This technology led to a significant advancement in the biomedical field as, in principle, it furnishes a path to acquire unlimited supplies of highly specific antibodies.

In this experiment, monoclonal antibodies are produced against HIV. Human Immunodeficiency Virus (HIV) is a retrovirus that attacks and damages the body’s immune system. This leads to malnutrition which is when the cellular demand for energy and nutrient is higher than what is being provided to the body. This causes even more immune deficiency facilitating expeditious progression of the virus infection to Acquired Immune Deficiency Syndrome (AIDS).

HIV along with accounting for consequential immunosuppression in an infected individual, also affects multiple vital organs of the body like the Heart, Brain, Kidney, etc.     2. METHODS AND MATERIALS: PRACTICAL I:After sterilising the work station, the Hemocytometer slide is made ready.

Cells a                                                                                                                                PRACTICAL II:The nitrocellulose paper is marked into 48 equal squares and the supernatants from each well are transferred onto the squares. After the spots have dried, blocking solution is added to block any non-specific binding. Later the binding solution is removed, and the nitrocellulose paper is washed with a washing solution. Washing solution is then poured out and the diluted conjugate is added and incubated.

Conjugate is then removed, and the paper is washed with the washing solution. Washing solution is removed and washed with tap water and poured out and substrate solution is added to allow reactions to develop and the paper is finally washed away in water a few times. The paper is finally dried and recorded. PRACTICAL III:After selecting any three hybridomas, sera from them are added to the three preblocked strips separately. The sera and blocking solution are then poured out and washed with the washing solution and that is removed as well. Diluted secondary antibody conjugate is added and washed away with the washing solution and later rinsed with tap water to add the substrate solution. Once bands start forming, the solutions are washed away with water and the paper is dried and recorded.

 3. RESULTS: D10   D3      C11*    C12   B8*      Fig 3.1 – The results of the Dot Blot Test: The antibodies produced by the Hybridoma cells bind with the conjugated anti-mouse antibody and can be visualised with the addition of a substrate solution as the dark yellow or light brown dots on the Nitrocellulose paper. We chose 3 different antibodies (marked with a *) produced by the Hybridoma cells of wells A3, B8 and C11 for further experiments. Fig 3.

4   Fig 3.2     Fig 3.3                                                                                          Fig 3.5                                                            Fig 3.6 Fig 3.5 – The Positive Control showing that the antibodies have bound to the four specific key proteins of the HIV virus. Thus denoting that these antibodies were specific to the four proteins.Fig 3.

6 –  The Negative Control showing that none of the antibodies binded with the proteins of the HIV virus. Thus denoting that these antibodies werent specific to the four proteins. 4. DISCUSSION:PART I : FUSION OF THE CELLS TO CREATE HYBRIDOMASAs we can understand from the results, only the Hybridoma cells survived the HAT medium selection. This is because one of the components of the HAT medium, Aminopterin which is an antibiotic blocks the De Novo pathway. Thus all the cells have to switch to the Salvage pathway in order to produce the nucleic acids for cell replication and survival.

For the Salvage pathway to work, we require Hypoxanthine-guanine phosphoribosyl transferase (HGPRT) to convert the substrates Hypoxanthine and Thymidine to produce Purines and Pyrimidines. Since Myeloma cells are HGPRT deficient ( HGPRT-), they cant produce the nucleic acids for cell replication and thus don’t survive in the HAT medium. Whereas the B cells have HGPRT (HGPRT+) and can thus replicate and survive in the medium. Hybridoma cells which retain the characteristics of the B cells are also HGPRT+ and thus survive the HAT selection media.

Although in due course the B cells will die due to their limited lifespan thus leaving behind only the fused Hybridoma cells. (fig 4.1)  PART II : ANTIBODY SCREENING BY DOT BLOT TESTOnce the cells are fused together to form the Hybridoma cells, they start producing antibodies but not all the cells produce the antibodies. As we can see from the results, only few of the cells made antibodies.

The squares containing the dark yellow or light brown dots show the antibody from the hybridomas have bound with the peroxidase anti-mouse conjugated Ig-G and the colour seen is for visual indication produced with the use of a substrate. Antibody production by the cells is unpredictable so this is precisely why we require a test to determine the productivity of a hybridoma cell and so that we don’t end up using all the hybridomas for further testing without the knowledge of whether all the cells are productive or not to avoid any sort of errors. PART III : SPECIFICITY TESTING BY WESTERN BLOTTo check for specificity, we use four key proteins to check if the antibodies are specific for any of them. If the antibodies are specific to any of these proteins, they can help fight against the virus. The key protein gp120 and gp41 glycoproteins that are a part of the HIV envelope that help the virus to attach, enter and incorporate with the host cells. p66 polymerase protein codes for a segment of reverse transcriptase polymerase which takes part in the genome replication of the virus in the host cell.

While p24 Is one of the constituent of the virus capsid. From the results, we can see that there are certain bands with a dark yellow or a light brown colour. This shows that the antibody has been bound with the HIV protein (antigen) on that particular position, thus showing its specificity to that protein. Having knowledge of a specific antibody and its corresponding specific protein can help not only understand how the virus works on its own and in the host cells, but also give us a way to strategically treat and eradicate the virus and its proliferation.

Since we are dealing with monoclonal antibodies, each antibody can only be specific to one protein of the HIV which is why there aren’t any western strips from our experiment with more than one band seen. If there were, it indicates that the experiment has gone wrong. But from our results where p120 was receptive to both B8 and C11 antibodies, it showed that antigens can be specific to multiple monoclonal antibodies but a monoclonal antibody can only be specific to one antigen protein. This way monoclonal antibodies are all identical and single specific unlike polyclonal antibodies as monoclonal antibodies are produced by a single B wheres as polyclonal antibodies are from multiple B cells. Also from our results we can notice that our positive control has bands over all four antigens and the negative control has none. These controls are to guide us by setting an ideal standard as to what the correct results should look like for us to compare our own experimental results to. The positive control has all 4 bands to show active binding for all four antigens.

 

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