Introduction their sensitivity to the hypoxanthine aminopterin-thymidine

Introduction
Monoclonal antibody production refers to the production of mAb-producing cells by combining myeloma cells and B cells. B cells obtained from animals (mainly from mice). After cell combination, vast quantities of clones are screened and chosen based on antigen specificity and immunoglobulin class.(Molecular-devices,online, 2018)
In this technology, immortal hybridomas are grown on artificial medium. These are further veiled for production of antibody specifically monoclonal antibodies for large scale. This technology for monoclonal antibody production opens a vast area of application specifically in medical sciences(Kumar et al., 2018)

Method
The production of hybridoma that persistently discharge the valuable monoclonal immunizer requires a few stages.
1.Immunization: Firstly immunogen is injected into the mice, before that it was mixed with suitable adjuvant and injected subcutaneously or intradermally. This injection procedure should be in repeated manner. Blood sample is taken and assayed for determining the presence of desired antibody. If the antibody is present and in desired amount then the mice is sacrificed. Its spleen is then disintegrated into spleenocytes. (Kulkarni, 2002)
2.Cell fusion to produce hybridomas: Myeloma cells are immortalized cells that are refined with 8 azaguanine to ensure their sensitivity to the hypoxanthine aminopterin-thymidine (HAT) choice medium utilized after cell combination. A week before cell fusion, myeloma cells are developed in 8-azaguanine for testing the growth ability. (Institute for Laboratory Animal Research National Research Council, 1999)
3.Selection of hybridoma: In the selection step the fused cells are incubated in HAT medium (Hypoxanthine-Aminopterine-Thymidine medium) for 10-14 days. The viable Hybridoma cells are formed in this medium, Afterwards, they are transferred to a regular culture medium. The incubated medium is then diluted into 96-well plastic culture plate in such a volume that every well contain only one cell. The medium in each well is examined periodically. (Kulkarni,. 2002)

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4.Screening of antibodies: : Hybridomas need to screened for desired antibody specificity. The cultures are tested for several times by using ELISA or RIA methods. These are done by binding to the antibody with specific antigens washed off to collect the desired cells for cloning desired antibodies(Biology Discussion, 2018).

Adapted from: Nature chemical biology. (2018). Production of monoclonal antibodies by the hybridoma method. online. Available at: https://www.nature.com/articles/nchembio0608-326/figures/1 Accessed 5 Nov. 2018
Figure: Using hybridoma technology for the production of monoclonal antibody.

5.Cloning: The B-cells that produce the desired antibodies can be cloned to produce many same daughter clones. The cloning techniques are Limiting Dilution Method and Soft Agar Method. They can be used individually but most of the cases used in combination. In limiting dilution method, 96-well plastic culture plate is used for cloning, The hybridoma cells are diluted and each cell is placed in each well. This process is repeated and it ensures that monoclonality is attained. (Kulkarni ,2002)

6.Characterization and Storage: The monoclonal antibody should be subjected to biochemical and biophysical characterization for the required specificity. The mAbs must be characterized for their capacity to resist freezing, and defrosting. (Jha, 2018)

Applications:
1. For the detection of bacterial and viral disease monoclonal antibody are applied. (Kumar, et al., 2012)
2. For the treatment of CVS diseases, Deep vein thrombosis, Atherosclerosis, Infectious diseases, Cancer etc. (Kulkarni, 2002)
3. For Biotechnological application the MAbs is used for purification of protein, antigens, drugs targeting and gene cloning & expression(Kumar et al., 2012).
4. Humanized monoclonal antibody is used to detect the infant with bronchopulmonary dysplasia. (Kumar et al, 2012)
5. In cancer treatment monoclonal antibody used as chemotherapy drug. (Kumar et al, 2012)

Conclusion: Hybridoma technology has brought a revolution by producing mAbs which can be used for the detection of diseases, treatment of diseases, purification of drugs at the same time.

Introduction
Monoclonal antibody production refers to the production of mAb-producing cells by combining myeloma cells and B cells. B cells obtained from animals (mainly from mice). After cell combination, vast quantities of clones are screened and chosen based on antigen specificity and immunoglobulin class.(Molecular-devices,online, 2018)
In this technology, immortal hybridomas are grown on artificial medium. These are further veiled for production of antibody specifically monoclonal antibodies for large scale. This technology for monoclonal antibody production opens a vast area of application specifically in medical sciences(Kumar et al., 2018)

Method
The production of hybridoma that persistently discharge the valuable monoclonal immunizer requires a few stages.
1.Immunization: Firstly immunogen is injected into the mice, before that it was mixed with suitable adjuvant and injected subcutaneously or intradermally. This injection procedure should be in repeated manner. Blood sample is taken and assayed for determining the presence of desired antibody. If the antibody is present and in desired amount then the mice is sacrificed. Its spleen is then disintegrated into spleenocytes. (Kulkarni, 2002)
2.Cell fusion to produce hybridomas: Myeloma cells are immortalized cells that are refined with 8 azaguanine to ensure their sensitivity to the hypoxanthine aminopterin-thymidine (HAT) choice medium utilized after cell combination. A week before cell fusion, myeloma cells are developed in 8-azaguanine for testing the growth ability. (Institute for Laboratory Animal Research National Research Council, 1999)
3.Selection of hybridoma: In the selection step the fused cells are incubated in HAT medium (Hypoxanthine-Aminopterine-Thymidine medium) for 10-14 days. The viable Hybridoma cells are formed in this medium, Afterwards, they are transferred to a regular culture medium. The incubated medium is then diluted into 96-well plastic culture plate in such a volume that every well contain only one cell. The medium in each well is examined periodically. (Kulkarni,. 2002)

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4.Screening of antibodies: : Hybridomas need to screened for desired antibody specificity. The cultures are tested for several times by using ELISA or RIA methods. These are done by binding to the antibody with specific antigens washed off to collect the desired cells for cloning desired antibodies(Biology Discussion, 2018).

Adapted from: Nature chemical biology. (2018). Production of monoclonal antibodies by the hybridoma method. online. Available at: https://www.nature.com/articles/nchembio0608-326/figures/1 Accessed 5 Nov. 2018
Figure: Using hybridoma technology for the production of monoclonal antibody.

5.Cloning: The B-cells that produce the desired antibodies can be cloned to produce many same daughter clones. The cloning techniques are Limiting Dilution Method and Soft Agar Method. They can be used individually but most of the cases used in combination. In limiting dilution method, 96-well plastic culture plate is used for cloning, The hybridoma cells are diluted and each cell is placed in each well. This process is repeated and it ensures that monoclonality is attained. (Kulkarni ,2002)

6.Characterization and Storage: The monoclonal antibody should be subjected to biochemical and biophysical characterization for the required specificity. The mAbs must be characterized for their capacity to resist freezing, and defrosting. (Jha, 2018)

Applications:
1. For the detection of bacterial and viral disease monoclonal antibody are applied. (Kumar, et al., 2012)
2. For the treatment of CVS diseases, Deep vein thrombosis, Atherosclerosis, Infectious diseases, Cancer etc. (Kulkarni, 2002)
3. For Biotechnological application the MAbs is used for purification of protein, antigens, drugs targeting and gene cloning & expression(Kumar et al., 2012).
4. Humanized monoclonal antibody is used to detect the infant with bronchopulmonary dysplasia. (Kumar et al, 2012)
5. In cancer treatment monoclonal antibody used as chemotherapy drug. (Kumar et al, 2012)

Conclusion: Hybridoma technology has brought a revolution by producing mAbs which can be used for the detection of diseases, treatment of diseases, purification of drugs at the same time.

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