Esprit Rock


Introduction: Normally the antigen expresses several epitopes. And finally a complex form of antibodies is synthesized. Every B cell is committed to synthesize antibody which has a single specificity. When a antigen has multiple epitopes , it can impulse a series of B cells. And at last multiple clones of plasma cell are produced . They can synthesize antibodies of multiple specificities. Those antibodies are called polyclonal Antibodies .But the scientists were trying to separate out antibodies which have a single specificity . They are called Mono clonal Antibodies. They Have monospecific epitope at a time .Monoclonal antibody is used to determine the unknown substance. It plays an important in role bio –molecular science, pharmacy .

There are many techniques applied to produce unlimited supply of monoclonal antibodies. In all those techniques, artificial medium is used where B cell should be grown on . But B cell died eventually . At last technique is produced where B cells are fused with myeloma cell and they become immortal . This technique is called Hybridoma Technology . . Kulkarni, G. (2002)

Principle : Before the production of hybridoma technology , there are many problems were arised in producing unlimited monoclonal antibodies . But in hybridoma technology , all the problems were solved by scientist Little Field .

He used an enzyme – Hypoxanthine Guanine Phosphoribosyl Transferase ( HGPRT ) and a medium containing Hypoxanthine , Aminopterin,and Thymidine( HAT Medium ) . . Kulkarni, G. (2002)

Figure:Hybridoma Technology Used in Monoclonal Antibodies

There are several steps in hybridoma production They are :-

1. Immunization
2. Cell fusion
3. Selection
4. Screening
5. Cloning
6. Characterization and storage

Immunization:A mixture is made with immunogen and adjuvant and then it is injected to the mice intra dermally and subcutaneously . It is applied repeatedly at various sites and at different times .

Cell fusion:A mixture is made with splenocytes and plasmacytoma cells . This mixture is placed in a proper medium with high concentration of PEG . After some time , fusion is occurred.

Selection and Screening: When fusion is completed ,all the substances are brought to HAT medium and incubation is occurred.The most used screening aassay is the ELISA.Here,we can give an example:p_nitrophenyl phosphate is turned into the yellow colored p_nitrophenol with the help of alkaline phosphate.When the incubation is completed,the activity of the enzyme is also finished and optical density is measured with the help of a technique.This machine is called plate reader or ELISA reader. . Kulkarni, G. (2002)

Cloning:In this method ,there is a semisolid method is used to proliferate the malignant cells.In this case,soft agar method and limiting dilution method are used.In many cases,this two methods need to combined.Then repeated dilution is occurred and transfer,frozen method are used. . Kulkarni, G. (2002)

Characterization and Storage: Biochemical and biophysical characterization are needed to subject for the desired specificity.There are used many methods.They are spectrometric,electrophoretic and chromatographic methods.It is also very important for detecting the class and epitope.
Characteristics of monoclonal antibodies are also very significant. So,for this reason
storage and freeze are very important .Here, liquid nitrogen is used. Kulkarni, G. (