1. IMMUNOLOGICAL METHODS IN DERMATOLOGY Shkilna M.I.

2. 1. QUALITATIVE AND QUANTITATIVE TESTS FOR ANTIGENS AND ANTIBODIES 2. Tests for cellular immune function 3. Immunofluorescence-used to detect antibodies in tissue or fluids

3. 1. Immunodiffusion-detection of antigen-antibody complex, visible as a precipitate in a medium at optimal pH and temperature: a. Single immunodiffusion-either antibody or antigen remains fixed and the other diffuses through semisolid medium, either linearly or radially, and is allowed to complex with the prescribed reactant b. Double immunodiffusion - both antibody and antigen diffuse toward each other, either linearly or radially, and are allowed to complex and precipitate; commonly used as the Ouchterlony radial immunodiffusion technique (or test) QUALITATIVE AND QUANTITATIVE TESTS FOR ANTIGENS AND ANTIBODIES

4. Single immunodiffusion a. Single immunodiffusion- either antibody or antigen remains fixed and the other diffuses through semisolid medium, either linearly or radially, and is allowed to complex with the prescribed reactant

5. Double immunodiffusion b. Double immunodiffusion - both antibody and antigen diffuse toward each other, either linearly or radially, and are allowed to complex and precipitate; commonly used as the Ouchterlony radial immunodiffusion technique (or test)

6. 2. Electrophoresis-protein separation in an electric field: a. Zone-proteins separated by surface charge; used to quantitate serum protein and immunoglobulins. b. Immunoelectrophoresis-proteins are separated by diffusion and precipitation of proteins c. Radioimmunoelectrophoresis-immunoelectrophoresis combined with radiolabeled antigens

7. Immunoelectrophoresis b. Immunoelectrophoresis- proteins are separated by diffusion and precipitation of proteins

8. 3. Electroimmunodiffusion-use of an electric field to rapidly drive reactants toward each other: a. Counterimmunoelectrophoresis (one dimensional double electroimmunodiffusion) -antigen and antibody rapidly diffuse in gel medium toward each other, leading to semiquantitative measurement of antigen in biological fluid; particularly useful in CSF and serum diagnosis of several diseases. b. Rocket electrophoresis (one-dimensional single electroimmunodiffusionl-antigens are electrophoresed from a well through gel medium with fixed antibody.

9. 4. Radioimmunoassay-very sensitive and specific technique to measure small quantities of hormones, immunoglobulins, etc 5. Agglutination-A very sensitive test of antigen reaction with insoluble antigen or antigen- coated particle which is performed in tubes or microtiter plates: a. Direct agglutination-antibody titer determined by agglutination of serially-diluted unknown quantity of antibody with fixed quantity of insoluble antigen (RBC, bacteria, fungi,)

10. Direct agglutination a. Direct agglutination- antibody titer determined by agglutination of serially- diluted unknown quantity of antibody with fixed quantity of insoluble antigen (RBC, bacteria, fungi,)

11. b. Indirect (passive) agglutination-soluble antigen coupled to RBCs or inert particles C. Hemagglutination inhibition-prevent agglutination of antigen- coated RBCs by homologous antigen d. Coombs' test (antiglobulin test)-measures nonagglutinating antibodies or antibodies too small to effectively agglutinate. Test is used in blood typing, evaluation of hemolytic diseases of newborns, and autoimmune hemolytic anemias. i. Direct Coombs' test detects serum proteins adherent to RBCs taken from a sensitized person ii. Indirect Coombs' test detects incomplete antibodies in serum. Serum is incubated with RBCs. The antibody-coated cells are then agglutinated by a Coombs' antiglobulin serum

12. Indirect (passive) agglutination b. Indirect (passive) agglutination- soluble antigen coupled to RBCs or inert particles

13. Hemagglutination inhibition C. Hemagglutination inhibition-prevent agglutination of antigen-coated RBCs by homologous antigen

14. Indirect Coombs' ii. Indirect Coombs' test detects incomplete antibodies in serum. Serum is incubated with RBCs. The antibody- coated cells are then agglutinated by a Coombs' antiglobulin serum

15. 6. Complement assays: a. Hemolytic assay (CH50)-antibody-mediated hemolysis of RBCs by complement b. Functional assays-determines the activity of individual complement components c. Immunoassays-antibodies to complement components and then immunochemical determination is made

16. Hemolytic assay (CH50) a. Hemolytic assay (CH50)-antibody- mediated hemolysis of RBCs by complement

17. Functional assays b. Functional assays- determines the activity of individual complement components

18. Immunoassays c. Immunoassays- antibodies to complement components and then immunochemical determination is made

19. 7. Complement fixation-consumption of complement used to measure antibodies and antigen. 8. Monoclonal antibodies-refers to production of identical antibodies.

20. 9. Latex fixation-latex particles are used as passive carriers for adsorbed antigen. Test detects rheumatoid factor (RF), which is a pentameric IgM directed against IgG. IgG is passively absorbed to latex particles, and determinants on IgG then react with IgM rheumatoid factor. 10.Enzyme-linked immunosorbent assay (ELISA)

21. Enzyme-linked immunosorbent assay (ELISA)