Hybridoma Technology & its application in fisheries

New arena for fish biotechnology

1. A SEMINER ON Hybridoma Technology Submitted by: Uday Narayan Das Fs - 27/10 FAQ-329 Submitted To: Dr. T.K.Ghosh Dept. of Aquaculture

2. Contents: Introduction History & Development Procedure Identification & Isolation Application Persisting Problems Improvements Summary Conclusion

3. Introduction Hybridoma technology is one kind of Biotechnology. It is a hybridization technique which is used to produce antibody producing hybrid cell . These hybrid cells are produced by fusing the genetic material of B-lymphocyte with tumour cell. The antibodies produced are monoclonal antibodies as they are all of a single specificity. Presence of B-lymphocyte genetic material helps in antibody production, whereas capacity to divide indefinitely in the culture due to the presence of tumour cell.

4. History & Development of Hybridoma Technology The production of monoclonal antibodies was invented by Cesar Milstein and Georges J. F. Köhler in 1975 . They pre-programmed B-lymphocytes to respond to a single type of antigen or antigenic determinant , therefore they produce single type of antibody specific to the specific antigen.  When an antigen reacts with B-lymphocyte receptors, lymphocytes divide rapidly and produce a clone of B cells, all these B cells produce antibodies against that specific antigen and this is called as clonal selection. T hat is B-lymphocytes produce only one type of antibodies which are specific to only one type of antigen or antigenic determinant. But fully differentiated antibody producing B-lymphocyte cells known as plasma cells does not divide when cultured in a laboratory.  The term hybridoma was coined by Leonard Herzenberg during his sabbatical in Cesar Milstein 's laboratory in 1976/1977.

5. Procedure of Hybridoma Technology

6. Step-I Mouse is immunized by giving antigen injection against which monoclonal antibodies have to produced along with an adjuvant. This is followed by booster doses of the antigen. After 72 hrs of immunization the spleen is collected from the mouse. Then a free cell suspension medium of the spleen is prepared using sterile serum free medium followed by centrifugation. All these processes are applied for extraction of plasma cells from the spleen.

7. Step-II Maintain the plasma cells in the cell culture medium for 16-24 hrs before fusion. It should be ensured that the cells are in early phase of growth at the time of fusion. The myeloma cells are selected based on some criteria like these cells themselves should not produce antibodies and also they should contain a genetic markers such as HGPRT (hypoxanthine-guanine phosphoribosyltransferase ) . This genetic marker helps in easy selection of the resulting hybrid cells. 

8. Step-III At the time of fusion both myeloma & spleen cells are counted & then mix in the appropriate ratio. Depending on the properties of the tumour cell, the mixing ratio of spleen to tumour cell may vary from 5:1 to 2:1 Following the mixing, the cells are centrifuged into a loose pellet. The supernatant is removed & the pellet is mixed with 1 ml of Polyethylene glycol(PEG) for 3 min. In doing so the pellet will be broken up into uniform small clumps.

9. Step-IV Following the fusion, dilute the cells in serum free medium slowly to reduce the osmotic disruption of the fused cell. Then centrifuge the cells & resuspend in HAT (Hypoxanthine Aminopetrin Thymidine ) medium. Then Hat medium containing the fused cells are allowed to incubate in a CO 2 incubator for 3-4 days.

10. Step-V This mixture of cell population is then cultured in selective media known as HAT medium along with the drug aminopterin . The HGPRT myeloma cells cannot divide in the HAT medium due to the presence of aminopterin . The Specific antibody producing B-lymphocytes are unable to divide continuously in the culture medium, therefore eventually they die. 

11. Step-V Only the hybridoma cells have got the ability to divide and proliferate on the HAT medium because genome from the B-lymphocyte makes them HGPRT positive and genome from the myeloma cells they can divide indefinitely. Thus only the hybridoma cells or fused cells are selected using selective media called as HAT medium. 

12. Identification & Isolation Of Hybridoma Cells

13. Reclone and cultivate positive clones ELISA PLATE The specific antibodies present in the each microwell are identified using one of the methods such as precipitation method or agglutination method. Most commonly used and most sensitive and rapid method is ELISA (Enzyme Linked Immunosorbant Assay)A Wells containing the hybridoma cells are identified and isolated from these wells and cultured (cloned). After these hybridoma cells are multiplied using in vitro or in vivo method. 

14. Most common screening techniques are ELISA and RIA) Low concentration (1-20 ug/ml) High concentration (1-10 mg/ml) In- Vitro In- Vivo

15. Advantages of Mouse Ascites (In-Vivo) Method:- The mouse ascites method usually produces very high mAb concentrations that often do not require further concentration procedures that can denature antibody and decrease effectiveness. The high concentration of the desired mAb in mouse ascites fluid avoids the effects of contaminants in in -vitro batch-culture fluid when comparable quantities of mAb are used. The mouse ascites method avoids the need to teach the antibody producer tissue-culture methods.

16. Disadvantages of Mouse Ascites (In-Vivo) Method:- The mouse ascites method involves the continued use of mice requiring daily observation. MAb produced by in vivo methods can contain various mouse proteins and other contaminants that might require purification. The mouse ascites method can be expensive if immuno -deficient mice in a barrier facility used. In vivo methods can cause significant pain or distress in mice.

17. In-vitro

18. ADVANTAGES OF IN VITRO METHOD In vitro methods reduce the use of mice at the antibody-production stage but can use mice as a source of feeder cells when antibody generation is under way). In vitro methods are usually the methods of choice for large-scale production by the pharmaceutical industry because of the ease of culture for production, compared with use of animals, and because of economic considerations. In vitro methods avoid the need to submit animal protocols to IACUCs. In vitro methods avoid or decrease the need for laboratory personnel experienced in animal handling. In vitro methods using semipermeable -membrane-based systems produce mAb in concentrations often as high as those found in ascitic fluid and are free of mouse ascitic fluid contaminants.

19. DISADVANTAGES OF IN VITRO METHODS It should be noted that each of the items below pertains to only a fraction (3–5%) of hybridomas , but they indicate some of the difficulties associated with in vitro methods. Some hybridomas do not grow well in culture or are lost in culture . The loss of proper glycosylation of the antibody (in contrast with in vivo production) might make the antibody product unsuitable for in vivo experiments because of increased immunogenicity, reduced binding affinity, changes in biologic functions, or accelerated clearance in vivo. In general, batch-culture supernatants contain less mAb (typically 0.002-0.01) per milliliter of medium than the mouse ascites method. Note that semipermeable -membrane-based systems have been developed that can produce concentrations of mAb comparable with concentrations observed in mouse ascites fluid.

20. Application of Mab Dose determination of a medicine To detect allergies & viral disease to detect certain type of cancer; to monitor the presence or appearance of malignant cells after surgical or radio-therapeutic treatments. For purification of complex biological mixture. Envisaged for the labelling & precise identification of some specialized cell such as neurone for gaining better knowledge of the way of association & operation. For purification of structural cell membrane protein. Mab has a great role in serotherapy

21. Used to produce Immunotoxin Used in the preparation of very specific vaccines, particularly against certain virus strain or against some parasites. Neutrilize the action of lymphocytes responsible for the rejection of grafts & destroy the auto-antibodies produced in auto-immune disease. Mab could considerably increase the effectiveness of the medicinal substances on the target cells, while avoiding the serious side-effects of cancer therapies.

22. Persisting Problem... The production of monoclonal antibodies remains inefficient and labor intensive The process of immortalizing a B cell is so inefficient that only 1 in 20,000, or sometimes 1 in 200,000, B cells forms a viable hybridoma . Generation of useful monoclonal an- tibodies to weak immunogens are very difficult as If the antigen is poorly immunogenic, available in only small amounts, or impure, the numbers of B cells making antibody to that antigen will be low and only a few positive hybridomas will be identified. The efficiency of fusion decreases greatly if one waits until the end of the immune response. when B cells are no longer proliferating rapidly and synchronously, even though this is when the highest affinity antibodies are being made.

23. IMPROVEMENTS IN THE HYBRIDOMA TECHNOLOGY Enrichment of the cells making the antibody(B- Cells) of interest using in vitro immunization would reduce the amount of screening and make it possible to start with larger populations of cells and immortalize the more number of B cells. In one version of this technique, immune cells are incubated with antigen-biotin conjugates and then mixed with biotin-coated myeloma cells. Avidin is added to bring the B cells and the fusion partner(Tumour Cell) together and they are then fused by electrofusion When antibody-forming cells are grown in culture, most of the B cells die, but those that are stimulated by antigen will proliferate and over- grow than the rest of the population.

24. Summary...

25. Harvest Ab Monoclonal antibodies Myeloma cells Grow indefinitely in cell culture but don't secrete the desired antibody FUSE Hybridoma cells Secrete antibody but don't grow in tissue culture Grow indefinitely in cell culture AND secrete antibody

26. Conclusion Many European & North-American firms are interested in the applications of Mab . In california , for-instance, certain medicine companies are preparing diagnostic kits designed for the screening of certain lethal diseases. It is anticipated that the future support of some aspects of this hybridomas based monoclonal antibody technology will be tailored to the needs of aquaculture industry of each developing country of the world.

27. REFERENCES http://www.medarex.com/Development/UltiMAb.htm http://wwwext.amgen.com/media/media_pr_detail.jsp?year=2006&releaseID=837754 http://www.regeneron.com/velocimmune.html http://www.ukbusinesspark.co.uk/cay92125.ht http://www.biotecharticles.com/Others-Article/Hybridoma-Technology-A-Biotechnology-Technique-378.html Links: Book: Fish Biotechnology by Ranga & Shammi (Pg No. 230-237)

28. Thank you