Hybridoma Technology ( Production , Purification , and Application )

Hybridoma technology revolutionized the field of immunology by enabling the production of monoclonal antibodies with high specificity and affinity. This presentation delves into the principles of DNA hybridoma technology, highlighting its significance in antibody production, therapeutic applications, and biomedical research. Learn about the key steps involved in generating hybridomas, from immunization to antibody screening, and discover the potential of recombinant DNA techniques in enhancing antibody engineering. Whether you're a student, researcher, or industry professional, this overview will provide valuable insights into the innovative world of hybridoma technology."
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1. NAGPUR COLLGE OF PHARMACY Present By :- Ku . Sakshi R. Ghasle TOPIC NAME :- HYBRIDOMA TECHNOLOGY ( PRODUCTION , PURIFICATION , APPLICATION )

2. Hybridoma technology was discovered in 1975 by two scientists, Georges Kohler and Cesar Milstein . The conventional or polyclonal antibodies which are isolated from serum are represents a collection of antibodies from different B cells that recognize multiple epitope on the same antigen . Each of these individual antibodies recognizes a unique epitope that is located on that antigen. To overcome the limitations of the polyclonal antibodies , researchers invented new methodology to produce monoclonal antibodies . Monoclonal antibodies can be produced in specialized cells through a technique now popularly known as HYBRIDOMA TECHNOLOGY . Monoclonal antibodies are single antibodies made by fusing antibody producing cells with an immortalized cell line , resulting in cell call as HYBRIDOMA . Hybridoma Technology .

3. MONOCLONAL ANTIBODIES POLYCLONAL ANTIBODIES 1. Expensive production 1. Inexpensive production 2. Long production time 2. Rapid production 3. Large quantities of specific antibodies 3. Large quantities of nonspecific antibodies 4. Recognize a single epitope on an antigen 4. Recognize multiple epitope on an antigen 5. Production is continuous and uniform . 5. Different batches vary in composition .

4. It is used for producing hybrid cells by fusing B lymphocyte with tumor or myeloma cells. The hybrid cells produced using hybridoma technology , are either cultured in laboratory or sub – cultured using mouse peritoneal cavity . Thus , due to the presence of B lymphocyte genetic material the hybrid cells can produce antibodies. The tumour cell used to produce hybrid cells make them undergo indefinite division in the culture. The B lymphocytes involved are pre-programmed to respond to a single type of antigen or antigenic determinant , thus they produce a single type of antibody that shows specificity for a specific antigen. The reaction of antigen with B – lymphocytes receptors triggers the rapid division of the lymphocytes . As a result , a clone of B cells is produce that generate antibodies against that specific antigen. This entire process is called as the CLONAL SELECTION.

5. The myeloma cells used in hybridoma technology should not synthesise their own antibodies . The hybridoma cells are selected based on inhibiting the nucleotide ( subsequently , the DNA ) synthesising machinery . The mammalian cells can synthesise nucleotides either by De novo synthesis or salvage pathway .

6. In the de novo synthesis of nucleotides : - Tetrahydrofolate formed from dihydrofolate is required . Aminopterin ( an inhibitor ) is used to block the formation of tetrahydrofolate (and therefore nucleotides ) In the Salvage pathway :- The purines and pyrimidines are converted into the corresponding nucliotides . Key enzymes is Hypoxanthine Guanine Phosphoribosyl Transferase ( HGPRT), Which convert hypoxanthine to guanine to inosine monophosphate and guanosine monophosphate , respectively . Thymidine kinase ( TK ) is involved in the salvage pathway of pyrimidine and converts thymine to thymidine monophosphate (TMP ) When mutated cells ( deficient in HGPRT ) are grown in a medium containing Hypoxanthine , Aminopterin , Thymine (HAT medium ) , they fail to survive as the de novo synthesis of purine nucleotides is inhibited . Thus , cells deficient in HGPRT and grown in HAT medium die .

7. PRODUCTION OF MA bs Involves following steps :- Immunisation Cell fusion Selection of hybridomas Screening of products Cloning and propagation and Characterisation and storage

8. Immunisation : Immunise a mouse with a suitable antigen Antigen and Freund’s adjuvant are injection vis subcutaneous route Injection are repeated multiple times at many sites . Increases the stimulation of B lymphocytes which are responding to the antigen . Give rises to large number of immune stimulated cells for the synthesis of antibodies . The animal is sacrifices when the conc . Of the antibodies in serum becomes optimal . The spleen is removed aseptically and distrupted mechanically or enzymatically to release the cells The spleen lymphocytes are separated from the remaining cells by density gradient centrifugation

9. Cell fusion The lymphocytes are thoroughly washed and mixed with HGPRT defective myeloma cells The mixture of cells is treated with polyethylene Glycol ( PEG ) but for few minutes due to its toxicity . The cells are then washed to remove PEG and kept it in fresh medium . These cells contain a mixture of hybridomas ( fused cells ) , free myeloma cells and free lymphocytes .

10. SELECTION OF HYBRIDOMA On culturing the cells in HAT medium , the hybridoma cells grow and the remaining cells disapper slowly within 7-10 days . Selecting a single antibody producing hybrid cells is very essential , and is possible if the hybridomas are isolated and grown individually. The suspension of hybridoma cells is diluted to such intensity that the individual aliquots contain one cell each . These cells are grown in a regular culture medium to produce the desired antibody .

11. Screening of products : The hybridomas should be screened for the secretion of the antibodies of desired specificity , and the culture medium from each hybridoma culture should be tested occasionally ( by ELISA and RIA )for desired antibody specificity . In this , the antibody binds to the specific antigen (coated to plastic plates ) and the unbound antibody and other components of the medium are washed off . Thus , the hybridoma cells producing the desired antibody are identified by screening . The antibody secreted by the hybrid cells is the Monoclonal antibody .

12. Cloning and propagation The single cells producing the desired antibody are isolated and cloned by using 2 techniques :- LIMITING DILUTION TEST : In this medthod , the suspension of hybridoma cells is serially diluted and aliquots of each dilation are transferred into micro culture wells . The dilutions are made such that each aliquot in a sell contains a single hybrid cell , thus ensuring that the antibody produced is monoclonal . SOFT AGAR TEST : In this method , the hybridoma cells are cultured in soft agar . Many cells can be grown simultaneously in a semisolid medium to form monoclonal colonies .

13. Characterisation and storage The obtained monoclonal antibodies are subjected to biochemical and biophysical characterization for the desired specificity. The MAbs should also be elucidated for the immunoglobulin class or sub class , its specific epitope , and its number of binding sites . The stability of the cell lines and the MAbs is also important . Both are characterized to check their ability to withstand freezing and thawing by freezing the desired cell lines in liquid nitrogen at several stages of cloning and culture .

14. Production of MAbs : In cultured bottle is low ( 5-10 ug / ml ) , thus to increase the yield the hybrid cells are grown as ascites in the peritonial cavity of mouse . The ascites fluid contain 5-20 mg of Mabs /ml , which is much superior to the in vitro cultivation technique . Collection of Mabs is heavy risk of contamination by pathogenic organism of the animal . Worker prefer in vitro technique over using animals . )( You can simply impress your audience and add a unique zing and appeal to your Presentations.

15. PURIFICATION OF MA bs For , purification the sample is conditioned by removing cells , cell debris , lipids and clotted materials through CENTRIFUGATION AND THEN FILTERATION through a 0.45 um filter. Also , Ultra filtration or dialysis By ion exchange chromatography : 1.) At sufficiently low PH = Cation exchange chromatography 2.) AT sufficiently high Ph = Anion exchange chromatography Size exclusion chromatography : Chromatogram (analysis of final purity ) Gel electrophoresis and capillary electrophoresis

16. APPLICATION : Therapeutic application Protein purification Diagnostic application Miscellaneous application

17. DIAGNOSTIC APPLICATION : MAbs are used in Biochemical analysis , as diagnostic reagents and in imaging of diseases as diagnostic tools . MAbs in Biological analysis : Pregnancy ( by ,measuring HGC ) Cancer (estimation of plasma carcinoembryonic antigens in colorectal cancer , etc ) Hormonal Disorders (for detecting thyroid disorders ) MAbs in Diagnostic Imaging : ( Immunoscintigrapgy ) Myocardial infraction Deep vein Thrombosis Atherosclerosis Cancer Bacterial Infection

18. THERAPEUTIC APPLICATION : MAbs as Direct Therapeutic Agents : Used for direct enhancement of the immune function of the host . Minimises the toxicity to the target tissues or the host . In Destroying Disease –Causing organisms ( inc. phagocytosis ) In Cancer Treatment In Immunosuppression of organ transplantation In AIDS Treatment In treatment of Auto immune Diseases MAbs as Targeting Agents in therapy : As immunotoxin In drug delivery In the dissolution of blood clots In radio immunotheraphy

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