Immunology Session 2 Structure and production of a ntibodies

1. ImmunologySession 2Structure and production of antibodies

2. ImmunologyTutorial Outline 1 Antibody structure Role of antibody in defence Polyclonal antibodies Monoclonal antibodies

3. 1 revision Antibody structure (p295)

4. Antibody structure (p295) Antibody facts. They compose the gammaglobulin fraction of the blood proteins. They are also called immunoglobulins (Ig) There are 5 different classes of Ig. We will use only IgG. All Ig have the basic structure of 4 polypeptide chains linked together via disulphide bonds. TWO chains are heavy and identical TWO chains are light (short) and also identical

5. Antibody structure (p295) Antibody facts. The Fab region contains the variable regions and this region binds antigens (at the epitope). This is a highly specific interaction (like substrate-enzyme). Amino acids making up this region determine the specificity (coded for in the gene). The Fc (crystalline fragment) is called the constant region since all antibodies in the same class have identical amino acid sequence in this region.

6. Antibody structure (p295) The basic function of antibodies is to bind with their antigens and form antigen-antibody complexes. The antibodies bring about the removal of the antigen in a number of ways: OPSONISATION COMPLEMENT FIXATION NEUTRALISATION (ENHANCES PHAGOCYTOSIS) AGGLUTINATION (OR PRECIPITATION)

7. Roles of antibodies in defense

8. Polyclonal antibody production (p298) Remember immunisation can be done more than once before collection of serum ….WHY? http://kitto.cm.utexas.edu/research/Kittolabpage/Protocols/Immunology/PAb.html http://www.cytographica.com/overheads/antibody.jpg

9. Polyclonal Antibodies: Recognise multiple epitopes on any one antigen. Serum obtained will contain a heterogeneous complex mixture of antibodies of different affinity. Polyclonals are made up mainly of IgGsubclass General advantages: Recognizes multiple epitopes on any one antigen which has the following advantages:  Amplifies signal from target protein with low expression level, as the target protein will bind more than one antibody molecule on the multiple epitopes. This would not be an advantage for quantification experiments e.g. in flow cytometry, as the results would become inaccurate. More tolerant of minor changes in the antigen, e.g., polymorphism, heterogeneity of glycosylation, or slight denaturation, than monoclonal (homogenous) antibodies.

10. General advantages (continued): • Polyclonals can identify proteins with high homology to the antigen, Thus polyclonal antibodies are sometimes used when the nature of the antigen in an untested species is not known.  • Polyclonal antibodies are often the preferred choice for detection of denatured proteins. • Multiple epitopes generally provide more robust detection. • Polyclonal antibodies not useful for probing specific domains of antigen, because antiserum will usually recognize many domains. • Disadvantages: • Prone to batch to batch variability. • They produce large amounts of non-specific antibodies which can sometimes give background signal in some applications. • Multiple epitopes make it important to check immunogen sequence for any cross-reactivity

11. Monoclonal Antibody production (p299-304)

12. Monoclonal antibody production HAT Medium (hypoxanthine-aminopterin-thymidine medium) is a selection medium for mammalian cell culture Aminopterin= drug that blocks DNA de novo synthesis, required for cell division. Hypoxanthine and thymidine provide cells with the raw material to evade the blockage (the "salvage pathway"), provided that they have the right enzymes. Spleen cells have the salvage pathway but are short-lived. Myeloma cells do not have the salvage pathway but are immortal. The hybridomas have both and will survive. http://www.cellbiolabs.com/sites/default/files/AKR-160-rapid-antibody-purification-kit.pdf

13. Monoclonal Antibodies Detect only one epitope on the antigen Antibody production  High technology required. Training is required for the technology used. Time scale is long for hybridomas. Advantages: Once hybridomas are made it is a constant and renewable source and all batches will be identical – useful for consistency and standardization of experimental procedures and results

14. Advantages of Monoclonals: Detect one target epitope, thus are less likely to cross-react with other proteins. Because of their specificity, they are excellent as the primary antibody in an assay, or for detecting antigens in tissue, since give significantly less background staining than polyclonal antibodies. Compared to polyclonal antibodies, homogeneity of monoclonal antibodies is very high. If experimental conditions are kept constant, results from monoclonal antibodies are highly reproducible, between experiments. Disadvantages: They produce large amounts of specific antibodies but may be too specific (e.g. less likely to detect in across a range of species)  More vulnerable to the loss of epitope through chemical treatment of the antigen than are polyclonal antibodies. (This can be offset by pooling two or more monoclonal antibodies to the same antigen).