1. © 2004 Wadsworth – Thomson Learning Chapter 19 Diagnostic Immunology

2. © 2004 Wadsworth – Thomson Learning 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. © 2004 Wadsworth – Thomson Learning Zone of equivalence change the amount of antigen constant amount of antibody Figure 19.2

4. © 2004 Wadsworth – Thomson Learning 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. © 2004 Wadsworth – Thomson Learning 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. © 2004 Wadsworth – Thomson Learning 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. © 2004 Wadsworth – Thomson Learning 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. © 2004 Wadsworth – Thomson Learning Agglutination reactions 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 Figure 19.7

9. © 2004 Wadsworth – Thomson Learning 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. © 2004 Wadsworth – Thomson Learning Qualitative agglutination Known antigen in fluid Unknown specimen added Agglutination –positive reaction No agglutination –negative reaction Figure 19.8

11. © 2004 Wadsworth – Thomson Learning Quantitative agglutination Similar to qualitative Diluted samples of antibody Measure amount of agglutination for each dilution Figure 19.10

12. © 2004 Wadsworth – Thomson Learning 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. © 2004 Wadsworth – Thomson Learning Complement fixation Negative reaction –No antibody in serum –Complement not “fixed” –Free complement lyses indicator cells Figure 19.11

14. © 2004 Wadsworth – Thomson Learning 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. © 2004 Wadsworth – Thomson Learning ELISA Example of immunoassay Indirect ELISA –antigen coated to plastic well –protein blocks remaining plastic surface Figure 19.12

16. © 2004 Wadsworth – Thomson Learning 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. © 2004 Wadsworth – Thomson Learning 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. © 2004 Wadsworth – Thomson Learning 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