1. Immunology Review Part Two Antibodies and Antigens

2. Immunoglobulins Glycoprotein molecules produced by plasma cells in response to an antigen and capable of binding to that antigen. AKA Ig or antibody

3. BASIC STRUCTURE OF IMMUNOGLOBULIN  Four polypeptide chains  2 heavy chains these define the class of Ig  2 light chains  The chains are held together by disulfide bonds. NH3+ COO-

4. REGIONS  Each Ig molecule has a constant region and a variable region.  It is the variable region that confers antibody specificity. NH3+ COO-

5. DOMAINS  Within the regions, are domains. These are loops of compatible amino acids that determine the biologic properties of the immunoglobulin molecule. C C C C C C

6. THE EFFECT OF ENZYMES  Papain cleaves Ig at the hinge region, above the disulfide bonds. This leaves 3 pieces. 2 Fab pieces (fragment antigen binding) containing the antigen binding sites. 1 Fc piece (fragment crystalizable). The Fc piece plays a role in complement activation, monocyte binding, and transfer of IgG across the placenta.

7. THE IMMUNOGLOBULIN CLASSES

8. IgG  Gamma heavy chain.  Comprises 80 % of Ig.  Exists as a monomer. 160,000 Daltons 7s  Reacts best at 37 o C and the AHG phase NH3+ COO-

9. IgG  May activate complement  Enhances opsonization  Neutralizes toxins and viruses  Crosses the placenta - cause of HDFN  Considered an “incomplete” antibody

10. IgM  Mu heavy chain.  Comprises 4-6 % of Ig.  Exists as pentamer. 900,000 Daltons 19s  React best in cold temperatures (4 o C to room temp.) Y Y Y Y Y J

11. IgM  Surface receptor on B lymphocytes  Activates complement - can cause intravascular hemolysis  Increases opsonization  Neutralizes toxins  Considered a “complete” antibody

12. IgA  Alpha heavy chain.  Comprises 13% of Ig.  Exists as a monomer or a dimer. 360,000 Daltons 7s  Main Ig in secretions such as saliva and tears.  Prevents bacteria from adhering to mucosa, neutralizes toxins, and opsonization.

13. IgD  IgD - Delta heavy chain.  Comprises 1% of Ig. Greatest quantity is found attached to immunocompetent, unstimulated B lymphocytes.  Exists as monomer. 160,00 Daltons 7s  Probably involved in B cell maturation into plasma cells.

14. IgE  IgE - Epsilon heavy chain.  Present in trace amounts.  Exists as monomer. 200,000 Daltons 8s  Almost all IgE is bound to circulating basophils or tissue mast cells.  Enhances allergic reactions, and promotes inflammation.

15. Antigens

16. Antigen  A chemical substance, seen by the body as foreign, that is capable of eliciting an immune response when introduced into an individual (host) who lacks that substance.  In Immunohematology, the antigens of interest are usually on red blood cells.

17. Lock and Key Fit  Antigen combines with antibody based on complementary structure. The better the “fit” the more tight the bond between the two. AGAB AG

18. Epitope  The portion of the antigen that reacts with the antibody.  A single antigen may have multiple epitopes.  May be linear or conformational

19. SPECIFICITY  The antigen that is the “target” of the antibody produced.  A single antigen may possess many different epitopes, each capable of eliciting a specific response.  Each epitope is the target of an antibody produced by the response, yet all of these antibodies are said to have the same specificity (the antigen).

20. For Example:  The D antigen is a mosaic, comprised of 4 epitopes : D a, D b, D c, and D d.  Each epitope may stimulate antibody directed at that portion of the molecule.  All 4 antibodies would have anti-D specificity. DaDb DdDc

21. Hapten  A small molecule that alone cannot stimulate antibody production.  When a hapten is coupled with another molecule, it may stimulate production of an antibody that will bind to the hapten.  Once the antibody has been produced, the hapten no longer needs the carrier molecule in order to bind with the antibody.

22. IMMUNOGENICITY  The ability of an antigen to elicit an immune response (i.e. stimulate antibody production)  Dependant on the following factors: Antigen composition & size Route of entry Dose Degree of foreignness Host factors

23. ANTIGENICITY  The ability of the antigen to bind with its antibody.  Affected by : location size shape charge biologic properties

24. Antigen/Antibody Reactions

25. Immunology vs. Serology The study of host reactions to foreign substances. The study of serum; in particular, the study of antibodies in serum and other body fluids.

26. Definitions  Sensitivity: How well a test is able to detect minute quantities of an analyte.  Specificity: The ability of a test to detect one unique analyte.  Cross reactivity: Reactions with substances that are structurally similar to an analyte. (False positive reaction)

27. Antigen / Antibody Reactions  Antigen and antibody bind with “Lock and Key” fit.  Affinity – the total attractive force that draws antibody to antigen.  Avidity – how “tightly” antigen and antibody bind. AGAB

28. Antigen / Antibody Reactions  Antigen / antibody reactions are readily reversible.  Free Ag + Free Ab Ag-Ab complex

29. Antigen / Antibody Reactions  Visualized when lattice structures form. Sensitization: Antibody attaches to antigen Agglutination or precipitation: Antibody cross- links form between antigens from different cells, causing cells to “clump” together. Y Y Y Y Y Y

30. Zone of Equivalence Y Y Y Y Y YY Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Prozone – antibody excess Postzone- antigen excess

31. Ag/Ab binding is influenced by:  Ionic strength- shielding and zeta potential  Shielding – charges that surround the Fab portion of an antibody.  Zeta potential – the difference in electrical charge between the surface of a cell and the outer layer of the ionic cloud that surrounds the cell in an electrolyte solution.

32. - - - - - - - - - - - - - When the zeta potential is lowered, the red cells can more closely approach each other, allowing antibodies to cross- link antigens between cells.

33. Ag/Ab binding is influenced by:  pH- optimal 6.5 to 7.5  Reaction time- depends on the immunoglobulin class and test medium  Temperature- depends on the immunoglobulin class  Ag/Ab ratio – Zone of equivalence  Number and location of antigens

34. Dosage  An antibody that reacts stronger with a cell that has homozygous expression of an antigen than it does with a cell showing heterozygous antigen expression is said to show dosage.

35. If there is an anti- antibody, which of these cells will yield the stronger reaction with it? Homozygous Heterozygous

36. Visualizing Antigen/Antibody Reactions Test Methods

37. Agglutination Based Methods Antibodies cause the cross-linking of particulate antigen, usually found on a cell.

38. Direct Agglutination  The antigen is a natural part of the solid’s surface.  Often performed at room temperature.  May use centrifugation to bring antigen and antibody into closer proximity.  Can be used to detect antigen or antibody

39. Inhibition of Agglutination  Antibody reagent is combined with patient’s specimen.  If patient’s specimen contains the antigen for that antibody, they will react.  Reagent antigen is added.  A positive reaction will show no agglutination, because the antibodies were bound to the patient antigen before the reagent antigen was added. Y Y Y Y Y Y

40. Neutralization Positive Test Negative Test The presence of an antibody prevents the antigen from functioning correctly.

41. Precipitation Based Methods Soluble antigen combines with antibody to form aggregates which precipitate out of solution.

42. One example of a precipitation based method: Flocculation Negative testPositive test Uses fine particles of antigen to detect antibody in patient’s serum.

43. “Labeled” Methods Attaches a “tag” to either the antigen or antibody. This “tag” can be detected and measured.

44. One example of a “labeled” method: Competitive EIA  Enzyme labeled antigen competes with unlabeled patient antigen for binding sites on fixed antibodies.  A chromogen is added that reacts with the enzyme.  The level of color development is inversely proportional to the level of patient antigen.

45. This concludes “Immunology Review, Part Two: Antibodies and Antigens” Please complete the exercise “Comparison of Immunoglobulins”