INTRODUCTION OF HYBRIDOMA TECHNOLOGY Hybridoma Technology is a process by which large number of antibodies

INTRODUCTION OF HYBRIDOMA TECHNOLOGY
Hybridoma Technology is a process by which large number of antibodies (monoclonal antibodies) can be produced. This method begins with injecting a mouse with an antigen which provokes an immune response. One type of white blood cell (B cell) can produce antibodies which binds to the injected antigen and newly introduced antibodies are then isolated from the mouse. These B cells which are isolated from mouse,then fused with immortal B cell cancer cells, a myeloma for producing a cell line (hybrid) and this is called Hybridoma. Cesar Milstein and GeorgesJ.F.Kohler invented the production of monoclonal antibodies from hybridoma technology in 1975. (Wikipedia-online)

Steps involved in hybridoma technology
1. MAKING THE PROPER MEDIA:
Chemical protocol,polyethylene glycol is used and HAT medium is also used for this purpose. In addition to it, FCS 10%, Penicillin 100 and Streptomycin 100microgram/ml are also used.

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2. MAINTAINING TEMPERATURE AND HUMIDITY:
Temperature should be controlled in incubator and temperature of water bath should be kept at 37and 56 degree Celsius. Humidity and concentration of gas should be maintained.
3.CHOOSING OF ANTIGENS
Mice strains are used and in the antibody producing hybrids, spleen cells can be utilized.
4.SELECTION OF PARENT MYELOMA
Myeloma cells are sensitive to HATmedium as they lack the hypoxanthine guaninephosphoribosyl transferase (HGPRT)gene.

ANTIBODY PRODUCTION FROM HYBRIDOMA TECHNOLOGY
Atfirst, a specific antigen is injected into a mouse and then antigen specific plasma cells are produced by mouse spleen and is intermixed with these cells. Each B cells is committed to produce antibody and after stimulation, B cells divide fast to produce memory cells and activated cells. These cells mature into plasma cells to produce antibody for the specific epitope. This way,antigen along with multiple epitopes stimulate B cells which produce antibodies of different specificities and this is known as Polyclonal Activation. One technique is needed to immortalize B cells to produce monoclonal antibodies where B cells can survive on artificial medium. This technique is known as Hybridoma Technology. B cells are fused with myeloma to make them immortal. Hybridomas are in screened to synthesize antibodies and thus we can select cells producing monoclonal antibodies .

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1.IMMUNIZATION:
Immunogen are taken to mix with suitable adjuvant and injected to mouse subcutaneously at different sites randomly and when the bleeding starts from their skin,they are taken to assay for the antibodies of required specificity. The animal should be sacrificed when antibody concentration is optimum and can be dissociated into spleenocytes.

2.FUSION:
In an appropriate medium,plasmacytoma is mixed with spleenocytes and high concentration of polyethylene glycol is exposed where fusion is done.
3.SELECTION AND SCREENING
ELISA can be a suitable method .The required antibody in the sample remains bound to antigen and detected by immune conjugate. Immune conjugate consists of two components.One component is distinct to particular antibody for an specific epitope and the other one is an alkaline phosphatase enzyme. If sample consists of required antibody ,it will be bound with antigen and as it remains in the well,unbound part will be washed off.After last washing,substrate of the enzyme which is colorless is added to wells and this will appear as colored one by the enzyme.2FHybridoma_technology

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Google.com. (2018). Redirect Notice. online Available at: https://www.google.com/url?sa=t&rct=j&q=&esrc=s&source=books&cd=1&cad=rja&uact=8&ved=0ahUKEwimyIDqj7veAhXSfH0KHeh2A30QFggmMAA&url=https%3A%2F%2Fwww.amazon.com%2FBiotechnology-Its-Applications-Pharmacy-Kulkarni%2Fdp%2F8171797768&usg=AOvVaw0CJD_3lCWLVYsmpxAJGswi from https://www.google.com/url?sa=i&rct=j&q=&esrc=s&source=images&cd=&cad=rja&uact=8&ved=2ahUKEwjX_cGk9LreAhUUeCsKHZutCuMQjRx6BAgBEAU&url=https%3A%2F%2Fslideplayer.com%2Fslide%2F6419549%2F&psig=AOvVaw3OqNnmvUvTJ5RMmIOy0A6H&ust=1541426753779881 Accessed 5 Nov. 2018.

4.CLONING:After screening,cloning can be done. One the day of assay, culture wells need to be cloned from 96 well plate.It is also important to assay subclones at a very early stage.There are many techniques by which cloning can be done. One of them is the limiting dilution method which allows the enumeration of cells. Another one is soft agar method that allows the proliferation of enormous malignant cells in semi solid medium.Google Books-Methods of Hybridoma Formation

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Google.com. (2018). online Available at: https://www.google.com/url?sa=t&rct=j&q=&esrc=s&source=web&cd=1&cad=rja&uact=8&ved=2ahUKEwij7azQubveAhUCEnIKHTHGDkoQFjAAegQIABAB&url=https%3A%2F%2Fwww.euromabnet.com%2Fprotocols%2F&usg=AOvVaw0Hp5GiHLVh4Pec5OUiwhwR Accessed 5 Nov. 2018.
Figure: Cloning process by limiting dilutions

5.CHARACTERIZATION:
Specificity is determined in this step and binding affinity can be measured.Only one subclass of antibody should be present in hybridoma and for recognition of different antibodies, sets of antisera are used.Three types of activities are involved in this step:
a) Screening: Antibody samples are identified which has specific antigen binding site
b) Tittering: Antibody concentration is measured.
c) Isotyping:Monoclonal antibody class and subclass is determined.Thermo Fisher Scientific

6.PRODUCTION, PURIFICATION AND LABELING OF MONOCLONAL ANTIBODIES
This step involves antibody isolation from serum and this method ranges from crude to highly specific. Mabs can be produced by two processes,one is tissue culture and another is maturing hybridoma. In this process, ammonium sulfate is used to precipitate antibody and for antibody purification, antigen affinity chromatography is used. A number of protein labeling kits are offered for the direct attachment of fluorescent t dyes. Direct labeling allows to utilize more than one special antibody.Thermofisher Scientific

Adopted from
Google.com. (2018). online Available at: https://www.google.com/url?sa=t&rct=j&q=&esrc=s&source=web&cd=1&cad=rja&uact=8&ved=2ahUKEwij7azQubveAhUCEnIKHTHGDkoQFjAAegQIABAB&url=https%3A%2F%2Fwww.euromabnet.com%2Fprotocols%2F&usg=AOvVaw0Hp5GiHLVh4Pec5OUiwhwR Accessed 5 Nov. 2018.

Figure: Full process of MAbs production

APPLICATIONS OF MONOCLONAL ANTIBODIES:
1.Monoclonal antibodies are used to detect infectious disease.
2.MAbs are used for diagnostic imaging and this process is known as immunoscintigraphy.
3.Antimyosin MAbs with radioisotope indium chloride can be used to detect myosin in myocardial infarction.
4.Against many human cancers, MAbs are used.
5.MAbs are used in hematopoietic malignancies.
6.MAbs are used as therapeutic agents directly. applications-of-monoclonal-antibodies-4
CONCLUSION:
In monoclonal antibody production, mouse is immunized with antigen and then blood has to be screened for producing antibody. After that, spleenocytes are isolated. Myeloma cells are fused with spleen cells. Then myeloma cells and spleenocytes which were isolated are fused to produce monoclonal antibody. Screening of clones are done on the basis of specificity of antigen. And ofcourse, characterization,purification and labeling is very important.Production, M.

REFERENCE:
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