1. Applications of Biotechnological Processes Antibody Production

2. Antibody facts Antibodies fight off infections in immune systems. They are produced by β-lymphocyte cells (B cells) in the spleen. Antibodies are made in response to antigens (foreign bodies). β-lymphocyte cells divide rapidly so that they can produce many copies of the antibodies.

3. The antibodies bind with the antigen to form an antibody/antigen complex that is engulfed by phagocytosis and destroyed.

4. Specificity B cells produce specific antibodies in response to specific antigens. Because of the infinite number of antigens available, a large number of different B cells must be available.

5. Antibody-antigen recognition An antibody recognises part of an antigens structure. The recognised region is called an epitope. Antigens have more than one type of epitope. This means that a number of β-lymphocyte cells will recognise the antigen and produce antibodies. An immune response results in the production of a number of different antibodies.

6. Epitope sites

7. Antibodies can be used in diagnostic tests because they have the ability to recognise epitope structures exactly. Antibodies can also be used to give a person passive immunity against a disease. The original method of making antibodies was through the preparation of polyclonal sera. The modern method involves the preparation of monoclonal antibody sera.

8. Preparation of Polyclonal Sera Polyclonal sera is a sera that contains a mixture of antibodies. It can be injected into the patient e.g. treatment for Hepatitis B Sera – clear portion of any body fluid

9. Procedure: 1.Inject an animal e.g. mouse, with antigen 2.The animal will elicit an immune response to raise antibodies in the blood, against the antigen. 3.Inject an animal with antigens for a 2nd time to ensure a high enough concentration of antibody. 4.Take blood from the animal at intervals. 5.Remove the red blood cells, to leave only the antibody sera. The sera has a mixture of antibodies in it, due to different β-lymphocyte cells recognising different antigens.

10. Disadvantages The following disadvantages make polyclonal production inconsistent: Animal has a finite life span. Recovery time must be left between bleeds and only a limited volume of serum can be collected at any one time. The animal will raise a mixture of antibodies against the antigen (different epitopes on the antigen will raise a response from different β-lymphocyte cells). A new animal may not raise exactly the same antibodies as another animal (due to different epitopes being recognised).

11. Monoclonal Antibody Production However, it is now possible to produce antibodies with a single specificity (derived from a single B cell).

12. Procedure: 1.Inject an animal e.g. mouse, with antigen. 2.The animal will elicit an immune response to raise antibodies in the blood. The number of β- lymphocyte cells in the spleen will increase. 3.After a few weeks the spleen is removed. The spleen is then separated into individual cells. These cells will contain a mixture of β- lymphocyte cells.

13. 4.Spleen cells are fused to myeloma cells in tissue culture. Myeloma cells are cancer cells that do not have the ability to make antibodies on their own. Therefore only the spleen cells to which the myeloma are fused will produce antibodies, ensuring that only one type is made. Myeloma cells are used because they have an almost limitless lifetime in tissue culture and readily fuse with other cells.

14. Myeloma cells will have been altered (mutated) so that they cannot utilise the nutrient aminopterin 5.Spleen and fused myeloma cells are grown up on aminopterin containing medium. Only myeloma cells that are fused to spleen cells survive (spleen cells utilise the aminopterin).

15. The cells that grow are called hybridoma cells. They will have received the antibody producing genes and the aminopterin utilising genes from the spleen cells. The ability to grow indefinitely (for many years) is derived from the genes in the myeloma cells. The overall process is know as immortalisation.

16. 6.The hybridoma cells are then tested for secretion of the desired antibody. The cells are identified and cloned. The are then transferred to fermenters (200-400L) to continually produce the one type of antibody. 7.At the end of the growth period the antibody is extracted and purified.

17. Uses of Monoclonal Antibodies The antibodies can be used to treat disease and to diagnose it. For example: Pregnancy testing Food safety testing Drug testing e.g. heroin Breast cancer treatment e.g. herceptin

18. Pregnancy Testing A mouse is injected with HCG hormone. This hormone is usually only present in blood and urine of a pregnant woman. Antibodies are raised and monoclonal hybridoma cells are produced. Hybridoma cells are screened and a suitable, highly specific (good binding to the hormone) line is selected.

19. The antibody produced by these cells are purified. Small quanitites are coated onto a dipstick (can be dipped into urine).

20. An enzyme is also attached to the dipstick. When the antibody binds to HCG, the enzyme is activated and reacts to produce a blue line/product. Blue indicates that a woman is most likely pregnant. http://www.sumanasinc.com/webcontent/animations/content/pregtest.html

21. Food Safety Bacteria have surface proteins that vary between species If one of the surface proteins is injected into a mouse, it will raise an antibody response to the protein. These antibodies can be purified to make diagnostic kits. Kits can be used to test for the presence of disease causing bacteria in foods.

22. Drug Testing Consistent detection of low concentrations of drugs e.g. heroin, can be carried out using monoclonal antibodies. Herceptin The herceptin antibodies that are purified are specific to breast cancer cells. They bind to the cancer cells, preventing further growth.

23. Past Paper Questions 2006 Q2 2005 Q3