True about Hybridoma : (PGI Dec 2008)
Ans : A (Immortalise myeloma cell); D (Prior I immunisation is done) & E (Produce monospecific..) I Cell Lineage in Hybridoma-Harper27th/604The myeloma ceils are immortalized, do not produce Ab & are HGPRT (Hypoxanthine-guanine phosphoribosyl transferase) rendering the salvage pathway of purine synthesis inactiveThe B cells are not immortalized, each produce a specific j Ab St they are HGPRT*The resulting hybridoma ceils are immortalized (via the j parental myeloma cells), produce Ab St HGPRT* (both latter | properties gained from the parental B cells)B celt will die because they are not immortalized. In the presence of HAT (hypoxanthine, aminopterin St thymidine), | the myeloma cells will also die, since the aminopterin in HAT suppresses purine synthesis by the de novo pathway by inhibiting reutilization of tetrahydrofolate.However, the hybridoma cells will survive, grow (because they are HGPRT*) & if cloned-produce monoclonal Ab.Hybridoma TechniqueIt was developed by Kohler & MilsteinQB celt are obtained from Spleen of mouse (or other suitable animal) previously injected with an Ag or mixture of antigens (eg. foreign cells)In this large amount of a single monoclonal Ab specific9 for one epitope can be obtainedThe method involve cell fusion in presence of polyethylene glycol St resulting permanent cell line is called a hybridomaThe hybridoma cells can be frozen & stored St subsequently thawed when more of the Ab is required, this ensures its longterm supplyThe hybridoma cell can also be grown in the abdomen of mice providing relatively large supply of Ab.Application of hybridoma technique :To determine the nature of infective agentQThey can be used to measure individual protein (eg. plasma protein)To subclassify both normal (eg lymphocyte) & tumour cell (eg leukaemia cell)Used in autoimmune ds (eg. Rheumatoid arthritis)Used for blood groupingQTo accelerate removal of drugs from circulation when they reach toxic level (eg. digoxin) * Proliferation -ve* Igs secretion+* HAT resistant* HGPRTase+ (unfused normal cell die)Splenic cells from mouse immunized with specific antigen Mouse myeloma cells* Proliferation+* Igs secretion -ve* HAT sensitive* HGPRTase-ve- - Cell fusion inPEG-1500 ,r| - Fused cells | Microtiter plates in HAT selective medium Cells are coloned and subcloned in plates containing small wells ||8 to 10 days time ||-Lymphocytes hybrid develop(hvbridoma cells) Supematent medium tested for specific desired monolconal antibody
Immortalise myeloma cell
Hybridoma cell produced by fusion of T-cell & myeloma
Cell is of human origin
Prior immunisation is done
Immunology