Hybridoma technology

Hybridoma technology

1. HYBRIDOMA TECHNOLOGY By Anita Devi P.hD 15099009

2. HYBRIDOMA TECHNOLOGY

3. HISTORY o Developed by Georges J.F. Kohler and Cesar Milstein in 1975 They shared nobel prize for this discovery in 1984 The term hybridoma was coined by Leonard Herzenberg in 1975

4. o A hybridoma is a hybrid cell obtained by fusion of B lymphocyte with usually a tumor cell of antibody forming system or B lymphocyte (these are called myelomas)

5. PRINCIPLE The hybrid cell has the capacity of antibody production derived from B cells At the same time it can divide continuously by the quality derived from Myeloma cells By combining the desired qualities of both the cells, the technology ensures large scale Antibody production of single specificity

6. o Specific hybridomas are either cultured in vitro or passed through mouse peritoneal cavity to obtain monoclonal antibodies, this is called as hybridoma technology

7. STEPWISE PROCEDURE o Isolation of B cells -Mice , 2-4 weeks old are immunized with the antigen against which monoclonal antibodies are to be raised by subcutaneous injection -Later B cells are isolated from the spleen of an immunized mouse

8. from o Isolation of myeloma cells - Myelo m a cell s are isolated bone marrow - T h e myelo m a cell s use d are HGPRT(Hypoxanthine-guanine phosphoribosyl transferase) mutant cells ( raised by mutations using 8- azaguanine)

9. o Somatic cell fusion -Electrofusion : cells are allowed to fuse with the application of an electric field -Done by using PEG medium -PEG stands for P oly E thylene G lycol

10. o Selection of hybrid cells -HAT medium is used for the selection of hybrid cells -HAT stands for H ypoxanthine A minopterine T hymidine

11. Nucleotide synthesis is essential for cell survival In HAT medium, aminopterine blocks the cellular synthesis of purines and pyramidines from simple sugars (denovo pathway) But cells can thrive by using hypoxanthine and thymidine present in the medium by salvage pathway using the enzyme HGPRT

12. o How HAT medium works in the selection of hybrid cells -B cells are HGPRT+ and can survive in the HAT medium, but they undergo normal cell death after some division - In hybridoma technology, the myeloma cells used are HGPRT deficient -So these cells can’t survive in HAT medium as Aminopterine blocks the Denovo pathway

13. Hybrid cells has HGPRT enzyme from the B cell as well as they have the ability to multiply repeatedly as myeloma cells So only hybrid cells can survive in HAT medium

14. H A T S E L E C T I O N

15. o Identification and isolation of the hybridoma cells The first screening technique used is ELISA -Done by incubating the hybridoma culture supernatant, secondary enzyme labeled conjugate and chromogenic substrate -Formation of a coloured product indicates a positive hybridoma

16. Two methods have been used for multiplying the hybridoma cells In-vivo 2.In-vitro

17. Iv-vivo procedure involves introduction of hybridoma cells into the peritoneal cavity of the animal , then ascetic fluid is isolated and then antibodies are isolated from it In-vitro method involves culturing of hybridoma cells in suitable culture media and then antibodies are isolated and purified

18. Once a hybridoma colony is established, it will continually grow in culture medium like RPMI-1640 and produce antibodies

19. Multiwell plates are used initially to grow the hybridomas After selection, they are changed to tissue culture flasks This provides enough cells for cryopreservation and supernatant for subsequent investigations The supernatant can yield 1 to 60micrograms per ml which can be maintained at lower temperatures for future use

20. RECENT ADVANCEMENTS availability of these antibodies with the unmatched ability to identify highly specific protein targets , has been extensively exploited for both in vitro diagnostics and in vivo therapeutics Monoclonal Antibody Development for Quantitative Analysis of Pancreatitis Associated Protein Immuno-PET: PET Imaging using Radiolabeled Antibodies

21. o The pancreatitis-associated protein (PAP) is a pancreatic stress protein which is not produced in a healthy pancreas but synthesized in high amounts in pancreatic cells in response to acute and chronic pancreatitis, hypoxia, toxins, diabetes and organ transplant o Strong induction of PAP observed during the early phase of pancreatic diseases suggests that PAP serum levels may be used as a valuable biological marker. Pancreatic stress is among the symptoms of Cystic Fibrosis (CF), so, PAP can also be used as a marker of CF and numerous studies emphasize the use of PAP in early diagnosis of CF in neonates

22. Positron Emission Tomography (PET) is a highly sensitive functional imaging modality that provides 3-dimensional and quantifiable visualization of biological processes. The effectiveness of PET imaging, is dependent on the specificity of the biomolecule portion of the injectable radiotracer, to the desired target. PET imaging quality can be compromised by high background signal due to unbound isotopes or nonspecifically targeted agents Monoclonal Antibodies (mAbs) are high affinity molecules that can be used for specific binding and delivery to cell surface molecules Antibody targets can be identified and generated for a variety of applications including cancer detection and staging, tumor and metastasis phenotyping, stratification of patients into treatment groups and the evaluation of tumor targeting and therapy response. Once the optimal antibody has selected, it can be labelled with a radionuclide, a combination used for Immuno-PET imaging

23. B.D. Singh, Biotechnology Expanding Horizones (3 rd Revised edition) 2010, Kalyani Publishers https://www.ncbi.nlm.nih.gov/pcm/articl es/pm428445 http://www.biologyexams4u.com http://www.biotecharticles.com/others- article/hybridoma-technology REFERENCES

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