1. PowerPoint ® Lecture Presentations prepared by Mindy Miller-Kittrell, North Carolina State University C H A P T E R © 2014 Pearson Education, Inc. Immunization and Immune Testing 17

2. © 2014 Pearson Education, Inc. Immunization Two Artificial Methods of Immunity Active immunization Administration of antigens so patient actively mounts an adaptive immune response Passive immunization Individual acquires immunity through the transfer of antibodies formed by immune individual or animal

3. © 2014 Pearson Education, Inc. Immunization Brief History of Immunization 1796 – Edward Jenner discovered process of vaccination 1879 – Louis Pasteur developed a vaccine against Pasteurella multocida Antibody transfer developed when it was discovered vaccines protected through the action of antibodies

4. © 2014 Pearson Education, Inc. Immunization Brief History of Immunization Many developing nations do not receive vaccines Effective vaccines not developed for some pathogens Vaccine-associated risks discourage investment in developing new vaccines

5. © 2014 Pearson Education, Inc. Immunization Active Immunization Vaccine types Attenuated (modified live) vaccines Use pathogens with reduced virulence Can result in mild infections Active microbes stimulate a strong immune response Can provide contact immunity Modified microbes may retain enough residual virulence to cause disease in susceptible individuals

6. © 2014 Pearson Education, Inc. Immunization Active Immunization Vaccine types Inactivated (killed) vaccines Safer than live vaccines Whole agent vaccines Deactivated but whole microbes Subunit vaccines Antigenic fragments of microbes Often require multiple doses to achieve full immunity Chemicals added to increase effective antigenicity

7. © 2014 Pearson Education, Inc. Immunization Active Immunization Vaccine types Toxoid vaccines Chemically or thermally modified toxins used to stimulate active immunity Useful for some bacterial diseases Stimulate antibody-mediated immunity Require multiple doses because toxoids possess few antigenic determinants

8. © 2014 Pearson Education, Inc. Immunization Active Immunization Vaccine types Combination vaccines Simultaneous administration of antigens from several pathogens Vaccines using recombinant gene technology Research attempts to make vaccines more effective, cheaper, and safer

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11. Immunization Active Immunization Vaccine safety Problems associated with immunization Mild toxicity Risk of anaphylactic shock Allegations certain vaccines cause autism, diabetes, and asthma Research has not substantiated these allegations

12. © 2014 Pearson Education, Inc. Immunization Passive Immunotherapy Administration of antiserum that contains preformed antibodies Provides immediate protection against a recent infection or ongoing disease Antisera have several limitations Can trigger allergic reactions called serum sickness Antibodies of antisera are degraded relatively quickly Individual not protected from subsequent infections

13. © 2014 Pearson Education, Inc. Serological Tests That Use Antigens and Corresponding Antibodies Serology is the determination of the presence of specific antigens or antibodies in blood serum Serological tests available to identify a variety of antigens and antibodies in serum Serological tests have several uses Monitor the spread of infection within a population Establish diagnosis of disease

14. © 2014 Pearson Education, Inc. Serological Tests That Use Antigens and Corresponding Antibodies Precipitation Tests One of the simplest of serological tests Antigens and antibody mixed in the proper proportion form large complexes called precipitates Antigen-antibody complexes also called immune complexes

15. © 2014 Pearson Education, Inc. No precipitatePrecipitateNo precipitate Antibody Antigen Amount of antibody precipitated Increasing amount of antigen Antibody excess Optimal proportionsAntigen excess Figure 17.6 Characteristics of precipitation reactions.

16. © 2014 Pearson Education, Inc. Well containing antigen molecules Line of immune precipitation Well containing antibodies against the antigen Agar Zone of antigen excess Zone of optimal precipitation Zone of antibody excess Well containing four different antigens Lines of immune precipitation Well containing a mixture of antibodies, each reacting to a different antigen Figure 17.7 Immunodiffusion, a type of precipitation reaction.

17. © 2014 Pearson Education, Inc. Serological Tests That Use Antigens and Corresponding Antibodies Turbidimetric and Nephelometric Tests Turbidimetry and nephelometry measure the cloudiness of a solution Turbidimetry measures the light passing through a solution Nephelometry measures the light reflected from a solution Can be used to quantify the amounts of proteins in serum

18. © 2014 Pearson Education, Inc. Serological Tests That Use Antigens and Corresponding Antibodies Agglutination Tests Agglutination occurs due to the cross-linking of antibodies with particulate antigens Agglutination is the clumping of insoluble particles Precipitation involves the aggregation of soluble molecules Reactions can be easy to see and interpret with the unaided eye Hemagglutination Agglutination of red blood cells Can be used to determine blood type

19. © 2014 Pearson Education, Inc. Anti-A antibody addedAnti-B antibody added Blood sample Negative result: no agglutination of blood cells Positive result: agglutination of blood cells A A B B Figure 17.8 The use of hemagglutination to determine blood types in humans.

20. © 2014 Pearson Education, Inc. Serological Tests That Use Antigens and Corresponding Antibodies Neutralization Tests Viral neutralization Cytopathic effect Viruses introduced into appropriate cell cultures will kill the cells Ability of virus to kill culture cells is neutralized when virus is first mixed with antibodies against it Viral neutralization test Mixture of virus and serum added to cell culture Absence of cytopathic effect indicates presence of antibodies against the virus in the serum Identifies whether individual exposed to a particular virus

21. © 2014 Pearson Education, Inc. Serological Tests That Use Antigens and Corresponding Antibodies Labeled Antibody Test Uses antibody molecules linked to some "label" that enables them to be easily detected Used to detect either antigens or antibodies

22. © 2014 Pearson Education, Inc. Serological Tests That Use Antigens and Corresponding Antibodies Labeled Antibody Test Fluorescent antibody tests Use fluorescent dyes as labels Fluorescently labeled antibodies used in two types of tests Direct fluorescent antibody tests Indirect fluorescent antibody tests

23. © 2014 Pearson Education, Inc. Figure 17.10 A direct fluorescent antibody test.

24. © 2014 Pearson Education, Inc. Cells with antigen are attached to slide and flooded with patient’s serum. Antigen on cell bound to slide IgG from patient’s serum Fluorescent-labeled anti-Ig antiglobulin is added. Fluorescent label Anti-IgG (antiglobulin, anti-antibody) 1 2 Figure 17.11 The indirect fluorescent antibody test.

25. © 2014 Pearson Education, Inc. Serological Tests That Use Antigens and Corresponding Antibodies Labeled Antibody Test ELISAs Stands for enzyme-linked immunosorbent assay Uses an enzyme as the label Reaction of enzyme with its substrate produces a colored product indicating a positive test Commonly used to detect the presence of serum antibodies

26. © 2014 Pearson Education, Inc. Serological Tests That Use Antigens and Corresponding Antibodies Labeled Antibody Test ELISAs Advantages of the ELISA Can detect either antibody or antigen Sensitive Can quantify amounts of antigen or antibody Easy to perform and can test many samples quickly Relatively inexpensive and easy to automate Plates coated with antigen can be stored for later testing

27. © 2014 Pearson Education, Inc. Serological Tests That Use Antigens and Corresponding Antibodies Labeled Antibody Test Immunoblots Also called a western blot Technique to detect antibodies against multiple antigens Used to confirm the presence of proteins

28. © 2014 Pearson Education, Inc. Figure 17.14 Immunoblotting. Wells containing antigens Polyacrylamide gel Electrophoresis (not shown) Blotting Nitrocellulose membrane Filter paper Polyacrylamide gel Absorbant paper Nitrocellulose (blot) is cut into strips ELISA is performed on strips Polypeptides 1 2 3 4 5 6 Patient 1 2 3 4 5 6 Positive control Negative control Polyacrylamide gel 1 2 3

29. © 2014 Pearson Education, Inc. Serological Tests That Use Antigens and Corresponding Antibodies Point-of-Care Testing Simple immunoassays that give results in minutes Useful in determining a quick diagnosis Common tests Immunofiltration assay Immunochromatography assay