Chapter 19 Diagnostic Immunology

1. Chapter 19Diagnostic Immunology

2. Precipitation reactions • Visible soluble precipitate • mix soluble antigen and antibody • excess antigen or antibody--no precipitate • zone of equivalence--precipitate forms Figure 19.1

3. Zone of equivalence • change the amount of antigen • constant amount of antibody Figure 19.2

4. Gel precipitation • Agar dish • solid medium • One well contains antibody • Other well contains antigen • Allow diffusion • Form precipitate at zone of equivalence Figure 19.3

5. Double immunodiffusion • Two antigens and one antibody • Place in separate wells • Allow diffusion • Lines of precipitation • continuous • identical antigens • crossing lines • completely different antigens • continuous with spur • partial identity Figure 19.4

6. Single immunodiffusion • Antibody mixed into gel • specimens in well • screening for presence of antigen • precipitate forms band around well • indicate presence of antigen • size of band relative to concentration of antigen Figure 19.5

7. Immunoelectrophoresis • Separate antigens before testing • put antigen in well • expose to electrical field • antigens are separated by size and charge • add antibody and allow diffusion and precipitation • precipitation with specific antibody gives identity of antigen Figure 19.6

8. Agglutination reactions Figure 19.7 • Visible reaction because antigen or antibody is on larger molecule • cell • latex bead • Interaction of antigen and antibody • clumping of large particles • Similar to precipitation reaction

9. Agglutination reactions • Direct--detect antibodies • using cells with antigen on them • Indirect--detect antigen or antibody • coated spheres or cells • observe agglutination • Hemagglutination • Red blood cells agglutinate • certain viruses (influenza) Figure 19.8

10. Qualitative agglutination • Known antigen in fluid • Unknown specimen added • Agglutination • positive reaction • No agglutination • negative reaction Figure 19.8

11. Quantitative agglutination • Similar to qualitative • Diluted samples of antibody • Measure amount of agglutination for each dilution Figure 19.10

12. Complement fixation • Positive reaction: • Antibody present in serum • Serum added to test antigen • Bound antibody “fixes” complement • No available complement to lyse indicator cells Figure 19.11

13. Complement fixation • Negative reaction • No antibody in serum • Complement not “fixed” • Free complement lyses indicator cells Figure 19.11

14. Immunoassays • Detect antigen or antibody • use a secondary antibody • tagged with marker • radioactive • fluorescent • enzyme • Multiple samples tested at once • Great sensitivity • dependent on type of tag • much greater than other tests

15. ELISA • Example of immunoassay • Indirect ELISA • antigen coated to plastic well • protein blocks remaining plastic surface Figure 19.12

16. ELISA • Serum added • primary antibody • if antibodies • bind antigen • if no antibodies • antigen not bound • Indicator antibody • enzyme-linked anti-Ig antibody binds primary antibody Figure 19.12

17. ELISA • Substrate • specific for enzyme linked to secondary antibody • enzyme causes substrate to change color • Reactions • color change • antibody in serum • no color change • no antibody in serum Figure 19.12

18. Immunofluorescence • Antibody with fluorescent label • Bind to cell • Visualize under UV light • Purpose • detect specific proteins in cells • detect viruses in cells • identify microbial cells • identify and sort cells • fluorescent activated cell sorter