1. Copyright © 2009 Pearson Education Inc., publishing as Pearson Benjamin Cummings Lecture prepared by Mindy Miller-Kittrell, University of Tennessee, Knoxville M I C R O B I O L O G Y WITH DISEASES BY BODY SYSTEM SECOND EDITION Chapter 17 Immunization and Immune Testing

2. Copyright © 2009 Pearson Education Inc., publishing as Pearson Benjamin Cummings Immunization Two Artificial Methods of Immunity – Active immunization – administration of a vaccine so that the patient actively mounts a protective immune response – Passive immunization – individual acquires immunity through the transfer of antibodies formed by an immune individual or animal

3. Copyright © 2009 Pearson Education Inc., publishing as Pearson Benjamin Cummings Immunization Brief History of Immunization – The Chinese noticed that children who recovered from smallpox did not contract the disease again – They infected young children with material from a smallpox scab to induce immunity in these children, a process known as variolation – The use of variolation spread to England and America but was stopped due to risk of death

4. Copyright © 2009 Pearson Education Inc., publishing as Pearson Benjamin Cummings Immunization Brief History of Immunization – 1796 – Edward Jenner discovered process of vaccination by inoculating material from a person infected with cowpox – 1879 – Louis Pasteur developed a vaccine against Pasteurella multocida – Practice of transferring antibodies was developed when it was discovered vaccines protected through the action of antibodies

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6. Copyright © 2009 Pearson Education Inc., publishing as Pearson Benjamin Cummings Immunization Brief History of Immunization – Socioeconomic and political problems prevent many developing nations from receiving vaccines – Effective vaccines unable to be developed for some pathogens – Vaccine-associated risks discourage investment in developing new vaccines

7. Copyright © 2009 Pearson Education Inc., publishing as Pearson Benjamin Cummings Immunization Active Immunization – Vaccine types – Attenuated (live) vaccines – Use pathogens that have reduced virulence – Can result in mild infections but no serious disease – Contain active microbes that stimulate a strong immune response due to the large number of antigen molecules – Can provide contact immunity through vaccinated individuals infecting those around them – Can be hazardous because modified microbes may retain enough residual virulence to cause disease

8. Copyright © 2009 Pearson Education Inc., publishing as Pearson Benjamin Cummings Immunization Active Immunization – Vaccine types – Inactivated (killed) vaccines – Whole agent vaccines – produced with deactivated but whole microbes – Subunit vaccines – produced with antigenic fragments of microbes – Both types are safer than live vaccines because they cannot replicate or mutate to a virulent form – Antigenically weak because the microbes don’t reproduce and don’t provide many antigenic molecules to stimulate the immune response – Often contain adjuvants, chemicals added to increase the effective antigenicity

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10. Copyright © 2009 Pearson Education Inc., publishing as Pearson Benjamin Cummings 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 they possess few antigenic determinant

11. Copyright © 2009 Pearson Education Inc., publishing as Pearson Benjamin Cummings Immunization Animation: Host Defenses: Vaccines: Types Animation: Host Defenses: Vaccines: Types

12. Copyright © 2009 Pearson Education Inc., publishing as Pearson Benjamin Cummings Immunization Active Immunization – Vaccine types – Vaccines using recombinant gene technology – Research attempts to make vaccines that are more effective, cheaper, and safer – A variety of recombinant DNA techniques can be used to make improved vaccine

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16. Copyright © 2009 Pearson Education Inc., publishing as Pearson Benjamin Cummings Immunization Active Immunization – Vaccine safety – Problems associated with immunization – Mild toxicity most common – Pain at injection site or, in rare cases, general malaise or fever high enough to induce seizures – Risk of anaphylactic shock – Residual virulence from attenuated viruses – Allegations that certain vaccines against childhood diseases cause or trigger autism, diabetes, and asthma – Research has not substantiated these allegations

17. Copyright © 2009 Pearson Education Inc., publishing as Pearson Benjamin Cummings Immunization Passive Immunotherapy – Administration of antiserum containing preformed antibodies to a patient – Used to provide immediate protection against a recent infection or an ongoing disease – Antisera has several limitations, including – Contains antibodies against many antigens, not just the ones of interest – Can trigger allergic reactions called serum sickness – May be contaminated with viral pathogens – Antibodies of antisera are degraded relatively quickly – Limitations are overcome through development of hybridomas

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20. Copyright © 2009 Pearson Education Inc., publishing as Pearson Benjamin Cummings Immune Testing Uses serology – study and diagnostic use of antigen-antibody interactions in blood serum Uses immunological processes in two general diagnostic ways – Uses known antibodies to detect antigens associated with an infectious agent – Uses antigens to detect specific antibodies in a patient’s blood to determine exposure to a specific pathogen Test chosen based on the suspected diagnosis, cost to perform the test, and the speed with which a result can be obtained

21. Copyright © 2009 Pearson Education Inc., publishing as Pearson Benjamin Cummings Immune Testing Precipitation Tests – One of the easiest of serological tests – Relies on fact that antigens and antibody mixed in the proper proportion form large macromolecular complexes called precipitates – Correct proportions are important to create precipitation – Two techniques determine optimal antibody and antigen concentrations – Immunodiffusion – Immunelectrophoresis

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25. Copyright © 2009 Pearson Education Inc., publishing as Pearson Benjamin Cummings Immune Testing Precipitation Tests – Radial immunodiffusion – Used to measure the concentrations of specific antibodies or immunoglobulins in a person’s serum – Produces anti-antibodies – inject human antibodies into an individual of another species, where they will be antigenic and cause production of antibodies directed against the human antibodies – The human antibodies are the “antigen” in the test, and the anti-antibody is the antibody

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27. Copyright © 2009 Pearson Education Inc., publishing as Pearson Benjamin Cummings Immune Testing Precipitation Tests – Immunoelectrophoresis – Improves the resolution of an immunodiffusion test – Can resolve more than 30 distinct antigens at once – Commonly used to demonstrate the absence of a normal antigen or to detect the presence of excessive amounts of an antigen

28. Copyright © 2009 Pearson Education Inc., publishing as Pearson Benjamin Cummings Immune Testing Agglutination Tests – Agglutination occurs due to the cross-linking of antibodies with particulate antigens – Agglutination is the clumping of insoluble particles, whereas precipitation involves the aggregation of soluble molecules – These reactions are easy to see and interpret with the unaided eye – Hemagglutination, the agglutination of red blood cells, can be used to determine blood type

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31. Copyright © 2009 Pearson Education Inc., publishing as Pearson Benjamin Cummings Immune Testing Neutralization Tests – Viral neutralization – Cytopathic effect – viruses introduced into appropriate cell cultures will invade and kill the cells – Ability of virus to kill culture cells is neutralized when virus is first mixed with antibodies against it – Absence of cytopathic effect indicates the presence of antibodies against the virus – Test is sensitive and specific enough to identify whether an individual has been exposed to a particular virus or viral strain

32. Copyright © 2009 Pearson Education Inc., publishing as Pearson Benjamin Cummings Immune Testing Neutralization Tests – Viral hemagglutination inhibition test – Useful for viruses that aren’t cytopathic – Based on viral hemagglutination, the ability of some viral surface proteins to clump red blood cells – Serum from an individual will stop viral hemagglutination if the serum contains antibodies against the specific virus – Commonly used to detect antibodies against influenza, measles, and mumps

33. Copyright © 2009 Pearson Education Inc., publishing as Pearson Benjamin Cummings Immune Testing The Complement Fixation Test – Based on the generation during complement activation of membrane attack complexes that disrupt cytoplasmic membranes – Used to detect the presence of specific antibodies in an individual’s serum – Can detect antibody amounts too small to be detected by agglutination

34. Copyright © 2009 Pearson Education Inc., publishing as Pearson Benjamin Cummings Immune Testing

35. Copyright © 2009 Pearson Education Inc., publishing as Pearson Benjamin Cummings Immune Testing Labeled Antibody Test – Uses antibody molecules that are linked to some molecular “label” that enables them to be easily detected – Used to detect either antigens or antibodies

36. Copyright © 2009 Pearson Education Inc., publishing as Pearson Benjamin Cummings Immune Testing Labeled Antibody Test – Fluorescent antibody tests – Use fluorescent dyes as labels – Fluorescein is the most important dye used in these test – Fluorescein-labeled antibodies used in two types of tests – Direct fluorescent antibody test – Indirect fluorescent antibody tests

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39. Copyright © 2009 Pearson Education Inc., publishing as Pearson Benjamin Cummings Immune Testing Labeled Antibody Test – ELISAs – Stands for enzyme-linked immunosorbent assay – Uses an enzyme as the label – Reaction of the enzyme with its substrate produces a colored product indicative of a positive test – Most common form of ELISA is used to detect the presence of antibodies in serum

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41. Copyright © 2009 Pearson Education Inc., publishing as Pearson Benjamin Cummings Immune Testing Labeled Antibody Test – ELISAs – Antibody sandwich ELISA – Modification of the ELISA technique – Commonly used to detect antigen – Antigen being tested for is “sandwiched” between two antibody molecules

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43. Copyright © 2009 Pearson Education Inc., publishing as Pearson Benjamin Cummings Immune Testing Labeled Antibody Test – ELISAs – Advantages of the ELISA – Can detect either antibody or antigen – Can quantify amounts of antigen or antibody – Easy to perform, inexpensive, and can test many samples quickly – Plates coated with antigen and gelatin can be stored for later testing

44. Copyright © 2009 Pearson Education Inc., publishing as Pearson Benjamin Cummings Immune Testing Labeled Antibody Test – Western blot test – Technique for detecting antibodies against multiple antigens in a complex mixture – Can detect more types of antibodies and is less subject to misinterpretation than other tests

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46. Copyright © 2009 Pearson Education Inc., publishing as Pearson Benjamin Cummings Immune Testing Recent Developments in Immune Testing – Simple immunoassays that give results in minutes – Useful in determining a preliminary diagnosis – Most common are immunofiltration and immunochromatography assays – Immunofiltration – Rapid ELISA that uses antibodies bound to membrane filters rather than polystyrene plates – Membrane filters have large surface area making the assay quicker to complete

47. Copyright © 2009 Pearson Education Inc., publishing as Pearson Benjamin Cummings Immune Testing Recent Developments in Immune Testing – Immunochromatography – Very rapid and easy to read ELISAs – Antigen solution flows through a porous strip where it encounters antibody labeled with either pink colloidal gold or blue colloidal selenium – Antigen-antibody immune complexes flow through a region and encounter antibody against them, resulting in a visible pink or blue line – Used in pregnancy testing to detect human chorionic growth hormone

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