Structure and function of antibodies Immunoglobulin genes Immunological assays

1. Structure and function of antibodies Immunoglobulin genes Immunological assays

2. Basic structure of antibodies (immunoglobulins) Tiselius and Kabat, 1939 immunized rabbits with ovalbumin serum was electrophoresed some serum was incubated with ovalbulin and also electorphoresed

3. p. 77 Treated with ovalbumin (to absorb antibody)

4. p. 77

5. Study of antibody structure Enzyme digests Reduction and alkylation Using antibodies as probes Protein sequencing proteins from patients with multiple myeloma MOPC- mineral-oil plasmacytoma (this was long before monoclonal antibody technology!)

6. p. 80

7. Immunoglobulin domains 4 (or 5) in heavy chain, 2 in light chain. Both heavy and light chains have 1 variable domain at the N-terminus about 110 amino acids each intrachain disulfide bonds How are chains held together? disulfide bonds noncovalent interactions

8. Variable regions Site of antigen interaction Hypervariable (CDR; complementarity-determining region) site of antigen binding Rest of domain- framework

10. Fv fragment (VH and VL)

11. Constant-region domains CH1 and CL stabilize V regions contribute to antibody diversity Hinge flexibility Fab and Fc can move around it present in IgG, IgA, IgD IgE and IgM have no hinge, instead a fourth C domain

12. CH2 has conserved glycosylation sites (some Ig subclasses have additional sites) Carbohydrate is sequestered between domains “Spreads out” the CH2; these regions tend to be biologically active

13. Boxes indicate functional units

14. Carboxy-terminal domain (CH3 or CH4) Can be membrane-bound or secreted Secreted form: hydrophilic tail Membrane-bound: hydrophilic spacer transmembrane sequence cytoplasmic tail

15. Immature B cell: mIgM only Mature B cell that has not seen antigen: mIgM and mIgD Memory B cell: mIgM, mIgG, mIgA, mIgE Any one of these can be combined with the same antigen specificity How??

16. Ig isotypes differ in size, protein sequence and function (p. 91)

17. IgG- most common in serum; monomeric four subclasses p. 92 Slight differences in structure; significant differences in function

18. IgG1 and IgG3 are most active Fix complement Bind to Fc receptors on phagocytes opsonization ADCC IgG4 binds to Fc receptors; does not fix complement IgG2 fixes complement moderately; has low affinity for Fc rceptors

19. IgM pentamer (or hexamer), so 10 antigen- binding sites produced in primary response

20. IgA most common antibody in body- not serum, but in secretions. Monomer in serum, multimer elsewhere helps protect portals of entry in body main protective antibody in breast milk

21. p. 93

22. IgE Very low concentration in serum Binds to Fc receptors on basophils and mast cells; induces hypersensitivity response

23. p. 94

24. IgD Very low concentration in serum Function of sIgD is not known

25. Antibodies are good antigens (p. 95)

26. Cytoplasmic part of mIg is very short. how does it function as an antigen-specific receptor? As part of a complex

27. The immunoglobulin superfamily Many proteins have a domain-like structure similar to immunoglobulins These other proteins do not share function and do not bind antigen What is the significance of this common structure?

28. p. 98

29. Summary of antibody features Basic structure: two identical heavy chains, two identical light chains Antigen-binding and effector functions Membrane-bound and secreted forms Five heavy-chain isotypes that vary in function, serum concentration and serum stability