Assignment On Production of Monoclonal Antibodies by Using Hybridoma Technology Course code

Assignment
On
Production of Monoclonal Antibodies by Using Hybridoma Technology
Course code: PHRM407
Course title: Biotechnology
Section: 01
Submitted to:
Submitted by: Susmita Chakraborty
ID: 2016-1-70-022
Index
Topic Page
Introduction
Process of hybridoma production
Reference
Introduction

Immune system produces antibodies to target foreign particles .It is called monoclonal antibody when this antibody is derived from a single B-cell clone and gives specific work by binding specific antigen .When an antigen entered into the blood ,the circulating B-cell of blood multiplies and produce clone of plasma cell which secrets identical immunoglobulin molecules . Actually this immunoglobulins are monoclonal antibodies which have a great affinity for their epitope.1

Adapted from: Lymphoma action, available at : https://lymphoma-action.org.uk/about-lymphoma-treatment-lymphoma/antibody-therapy-including-rituximab (accessed : 4 November , 2018 )
Figure: Monoclonal antibody
In 1975, Georges Köhler and César Milstein were produced monoclonal antibodies from Hybridoma by using Hybridoma Technology.

Fusion between myeloma cell lines and B-cells secret a specific antibody results in a remarkable hybrid cell .This hybrid cell can produce antibody which is derived from B-cell and it can be divided continuously with the same quality of myeloma cell . This hybrid cell is known as hybridoma.

The process of producing hybridoma is called hybridoma technic.

In hybridoma technic PEG (polyethylene glycol) is used during the fusion of myeloma cell and B-cell. Before using myeloma cells, it should be ensure that this cells never secrete antibodies and they lake of HGPRT (hypoxanthine-guanine phosphoribosyl transferase ).Because this can make the cell sensitive to the HAT medium (hypoxanthine-aminopterin-thymidine medium).Only fused cells are survived in this media . Unfused myeloma cells die and by de novo or salvage pathways they cannot produce nucleotides because they are lack of HGPRT. B cell-myeloma hybrids can survive because HGPRT gene from the B cells is active .This cells are produced antibodies which are immortal.

Adapted from: Biology today and tomorrow, available at : http://biological-discoveries.blogspot.com/2009/04/monoclonal-antibodies.html (accessed : 4 November , 2018 )
Figure : production of monoclonal antibody
Process of hybridoma production
During the production of hybridoma some necessary steps are followed to secrete efficient monoclonal antibodies .Such steps are:
Immunization and Isonization
Fusion of cells
Hybrid cells Selection
Screening of hybridoma cells
Cloning
Characterization and storage

Adapted from: Hybridoma Fusion Partners Cell Lines , available at : https://www.sigmaaldrich.com/technical-documents/protocols/biology/cell-culture/hybridoma-fusion.html , (accessed : 4 November , 2018 )
Figure: Hybridoma technology
Immunization and Isonization
A young mouse is selected as a host. Very few quantity of antigen is injected intradermally or subcutaneously. When the concentration of desired antibodies is optimum, the mouse is sacrificed. Single spleenocyte is dissociated from the spleen of the mouse. The main target of this step is the isolation of B-cell from the spleen of an immunized mouse.

Adapted from :  Medical Animations , Dr.G.Bhanu Prakash , Published on Sep 11, 2012,available at : www.youtube.com/watch?v=c_krTc9M1WU , (accessed : 4 November , 2018 )
Figure: Immunization
Fusion of cells
B-cell and the myeloma cells are mixed in an effective HAT medium.

High concentration of PEG (poly ethylene glycol) is used here for proper mixing.

A proper time is given for fusion.

Two nucleolus of two different cells are fused and produce heterokaryons.

After a period of time by normal losing of chromosome process, hybridoma is produced.
In this process human monoclonal antibody is produced and this time isolating B-cell and oncogenic virus is used.

Adapted from:  slide share, available at : https://www.slideshare.net/ARYAGEORGE1/hybridoma-technology-86161785 , (accessed : 4 November , 2018 )
Figure: Cell fusion
Hybrid cells Selection
For the selection of hybrid cells HAT (hypoxanthine-aminopterin-thymidine) media is used.

HGPRT + B-cell can survive in the HAT media but it has natural death after division.

The myeloma cells which are used has less HGPRT .Hybrid cells can survive in the HAT media due to its HGPRT enzyme which is got from the B-cells and have the quality to multiply rapidly like myeloma cells .From the medium the hybridoma cells are selected to further screening and cloning .
Screening of Hybridoma Cells
ELISA is the first technic of screening.

96 well plastic culture plates used.

In this culture plate, the antigens are absorbed .Hybridoma cells are incubated in the plate .Desire antibodies are bound to the antigens and unbound antibodies are washed off and it is detected by immunoconjugate.

In the period of second washing , a colorless product is used . Desire types of antibodies will converted to a colored product .For this procedure colorimeter which is known as plate reader or ELISA reader is used .

Adapted from:  Get quantitative data from In-Cell ELISA, available at : https://www.abcam.com/kits/get-quantitative-data-from-in-cell-elisa , (accessed : 4 November , 2018 )
Figure: Screening
Cloning
Two types of procedure is involved of multiplying the hybridoma cells.

In vitro
In vivo
In vitro:
In this procedure proper culture media is use. Soft ager is used for this process. The antibodies are allowed to re grow and the steps are repeated several times and purified.

In vivo:
It is done in the peritoneal cavity of an host animal. The antibodies are isolated from the ascetic fluid.

Characterization and Storage
The process by which monoclonality of an antibody is established ,commonly known as characterization problem .Technics that are use such as spectrometric ,electrophoretic and chromatographic .

There are several important aspect is needed to be observed that are the stability and the cell line.

The cell must be stable during freezing and storage for different time .In the period of storage chemical and physical stability are determined .https://en.wikipedia.org/wiki/Hybridoma_technologyhttps://www.slideshare.net/ARYAGEORGE1/hybridoma-technology-86161785https://en.wikipedia.org/wiki/Monoclonal_antibodyhttps://www.britannica.com/science/monoclonal-antibody
http://nicb.ie/biotechnology/what-is-a-monoclonal-antibody/