Hybridoma technology

Hybridoma technology is a method for producing large numbers of identical antibodies (also called monoclonal antibodies). This process starts by injecting a mouse (or other mammal) with an antigen that provokes an immune response.

1. MONOCLONAL antibody (hybridoma technology) Archana Soni Assistant Professor Shri Shankaracharya Mahavidyalaya , Junwani, Bhilai (C. G.) INDIA

2. Monoclonal Antibodies Contents 1. Definition. 2. Introduction. 3. Pharmacology. 4. Effects of MAbs. 5. Principle for creation of MAbs. 6. Production of MAbs. 7. Human monoclonal antibodies. 8. Advantages of MAbs. 9. Limitations of MAbs. 10. Applications of MAbs. 11. Antibody fingerprinting. 12. Conclusion. 13. Reference.

3. Definition MAbs are antibodies that are identical because they were produced by one of the immune cell (B cell), all clones of a single parent cell. Given any substance, it is possible to create monoclonal antibodies that specifically bind to that substance; they can then serve to detect or purify that substance. This has become an important tool in biochemistry, molecular biology and medicine.

4. Introduction Antibodies or immunoglobulins are protein molecules produced by a specialized group of cells called B-lymphocytes (plasma cells) in mammals. Monoclonal antibody (MAb) is single type of antibody that is directed against a specific antigenic determinant (epitope). In the early years, animals were immunized against a specific antigen, B-lymphocytes were isolated and cultured in vitro for producing MAbs. This approach was not successful since culturing normal B-lymphocytes is difficult, and the synthesis of MAb was short-lived and very limited. It is interesting that immortal monoclonal antibody producing cells do exists in nature. They are found in the patients suffering from a disease called multiple myeloma (a cancer of B-lymphocytes). It was in 1975. George Kohler and Cesar Milstein (Noble Prize, 1984) achieved large scale production of MAbs. They could successfully hybridize antibody—production B-lymphocytes with myeloma cells in vitro and create a hybridoma . The production of monoclonal antibodies by the hybrid cells is referred to as hybridoma technology.

5. Pharmacology A) Mechanism of action of MAbs: Blocking or steric hindrance of the function of target antigen i.e., T-lymphocytes, B-lymphocytes, tumor necrosis factor-a (TNFa) and interleukin (IL) which are capable of transducing intracellular signals. Cytotoxicity to the cell expressing target AG by ADCC or CDC. Inhibition of growth . B) Pharmacokinetics: MAbs are used by intravascular route and remain essentially intravascular. Intravenous injection is not always be appropriate for long-term treatment for a variety of reasons. Hour-long infusion require a hospital environment and are often associated with mind to very severe side effects. Continous and sustained delivery of antibodies can lead to induction of neutralizing anti- idiotypic immune responses, which sometimes develop when massive doses of purified immunoglobulins are repeatedly injected into patients. Additionally, the bioavailability of therapeutic antibodies is often detrimental to the treatment efficacy. They have small volume of distribution and limited tissue penetration. They remain in circulation for 2 days to 2 weeks. Another limitation is the high cost of recombinant proteins certified for human use.

6. Effects of MAbs 1.) Fever, chills 2.) Weakness 3.) Headache 4) Nausea 5) Diarrhea 6) Rashes 7) Changes in blood pressure 8) Flushes&faintness

7. PRINCIPLE FOR CREATION OF HYBRIDOMA CELLS The myeloma cells used in hybridoma technology must not be capable of synthesizing their own antibodies. The selection of hybridoma cells is based on inhibiting the nucleotide synthesizing machinery. The mammalian cells can synthesize nucleotides by two pathway— de novo synthesis and salvage pathway. The de novo synthesis of nucleotides require tetrahydrofolate which is formed from dihydrofolate. The formation of tetrahydrofolate can be blocked by the inhibitor aminopterin . The salvage pathway involves the direct conversion of purines & pyrimidines into the corresponding nucleotides. Hypoxanthine guanine phosphoribosyl transferase (HGPRT) is a key enzyme in the salvage pathway of purines. Thymidine kinase (TK), involved in the salvage pathway of pyrimidines converts thymidine monophosphate (TMP). Any mutation in either one of the enzymes blocks the salvage pathway. When cells deficient in HGPRT are grown in a medium containing h ypoxanthine a minopterin & t hymidine (HAT medium) , they cannot survive due to inhibition of de novo synthesis of purine nucleotiodes. (salvage pathway is not operative due to lack of HGPRT). Thus, cells lacking HGPRT, grown in HAT medium die. The hybridoma cells, possess the ability of myeloma cells to grow in vitro with a functional HGPRT gene obtained from lymphocytes. Thus, only the hybridoma cells can proliferate in HAT medium, & this procedure is successfully used for their selection.

8. Production of Mab’s The establishment of hybridomas and production of MAbs involves the following steps. 1.) Immunization. 2.) Cell fusion 3.) Selection of hybridomas. 4.) Screening the products. 5.) Cloning & propagation. 6.) Characterization & storage.

9. Human monoclonal antibodies From SCID mouse by injecting human B and T cells and grafting mouse by transplanting spleens and lymph nodes. The transplanted mouse is immunized with target antigens to produce human antibodies. Phage derived combinatorial antibody library, phage display technique-mimics in vitro affinity maturation.

10. ADVANTAGES OF MAB’S 1.) Homogeneity: Monoclonal antibody represents a single antibody molecule that binds to antigen with the same affinity and promote the same effectors functions. 2.) Specificity: The product of a single hybridoma reacts with the same epitope on antigens. 3.) Immunizing Antigen: Need not be characterized and is ultimately not needed in large quantities to produce large quantities of antibody. 4.) Selection: It is possible to select for specific epitope specificities and generate antibodies against a wider range of antigenic determinants. 5.) Antibody Production: Unlimited quantities of a single well-defined monospecific reagent.

11. Limitations of MAb’s As they are specific to a particular antigen, they cannot distinguish molecule as a whole. Some times they cannot distinguish groups of different molecules. Ex: Presence of retro viruses as a part of mammalian chromosomes is not distinguished. The presence of some of these viruses is detected in hybridomas. This poses a great danger since there is no guarantee for MAb produced is totally virus free. For this reason US food and drug administration insists that MAb for human use should be totally free all pathogenic organism including viruses.

12. APPLICATION OF MAB’S 1.) Diagnostic applications. a) MAbs in biochemical analysis b) MAbs in diagnostic imaging 2.) Therapeutic uses a) MAbs as direct therapeutic agents b) MAbs as targeting agents in therapy 3.) Protien purification 4.) Miscellaneous applications (ABZYMES).

13. ANTIBODY FINGERPRINTING The occurrence of antibodies and their involvement in certain diseases is well known (e.g., rheumatic arthritis). A new category of individual specific (IS) autoantibodies have been discovered in recent years. These IS--autoantibodies are produced after birth and reach maximum in number by 2 years, and the remain constant for the later part of life. Monoclonal antibodies produced against IS--autoantibodies can be used for their detection, and identification of individuals. This technique referred to as autoantibody fingerprinting, is particularly useful for the detection of criminals, rapists etc. The autoantibodies collected from samples such as blood, saliva, semen and tears can be used.

14. CONCLUSION Since for human health care, the subject of production and use of antibodies has become a very important area of research, not only for academic purposes but also for its relevance to industrial growth for diagnosis and therapeutics, monoclonal antibodies can be utilized for it. In near future more such monoclonal antibodies should be produced for biochemical research that will be of great commercial and medical value.

15. References http://www.searhpdfengine.com/HYBRIDOMA-TECHNOLOGY-FOR-PRODUCTION-OF-MONOCLONAL-ANTIBODIES.html grants.nih.gov/grants/policy/antibodies.pdf http://www.searchpdfengine.com/Therapeutic-monoclonal-antibodies.html Biotechnology by U satyanaryana (pages:213-226)