Applications of Immune Responses

1. Applications of Immune Responses Chapter 19

2. Principles of Immunization • Naturally acquired immunity is acquisition of adaptive immunity through natural events • Immunization mimics these events by inducing artificially acquired immunity • Natural or artificial immunity can be divided into • Active immunity • Passive immunity

3. Principles of Immunization • Active immunity • Results from immune response upon exposure to an antigen • Active immunity can develop naturally • Following illness • Or artificially • After immunization

4. Principles of Immunization • Passive Immunity • Occurs naturally during pregnancy • IgG from mother crosses placenta • Infers protection to the baby • Occurs naturally as result of breast feeding • IgA antibodies in breast milk given to child • Artificial passive immunity involves transfer of antibodies produced by another person or animal • Can be used to prevent disease before or after likely exposure

5. Vaccines and Immunization • Attenuated vaccines • Weakened form of pathogen • Generally unable to cause disease • Strain replicates in vaccine recipient • Causes infection with undetectable or mild symptoms • Results in long lasting immunity

6. Vaccines and Immunization • Attenuated vaccines • Advantages • Single dose usually sufficient to induce long-lasting immunity • Due to multiplication of microbe in body • Continued stimulation of immune system • Vaccine has added potential for being spread • “Disease” after immunization could be spread to un-immunized individuals inadvertently • Disadvantages • Have potential to cause disease in immunocompromised individuals • Pregnant women should also avoid immunization with attenuated vaccine • Attenuated vaccines in use include • Sabin polio vaccine • MMR • Yellow fever

7. Vaccines and Immunization • Inactivated vaccines • Unable to replicate in vaccinated individual • Retains immunogenicity of infectious agent • Immunogenic not pathogenic • Inactivated vaccines fall into two categories • Whole agents • Contain killed organisms of inactivated virus • Does not change epitopes • Cholera, plague, influenza and Salk polio are whole agents • Fragments • Portions of organisms or agents including toxins, proteins and cell wall components

8. Principles of Immunological Testing • Terms • Seronegative • Person not yet exposed to antigen and has no specific antibodies • Seropositive • Person with exposure and actively producing antibody • Titer • Concentration of antibody in serum---Indicates previous exposure

9. Principles of Immunological Testing • Obtaining antibody • Serum is fluid portion of blood with no clotting factors • Plasma is fluid portion with clotting factors • Laboratory animals are used to produce known antibodies • Animal is immunized with antigen and produces specific antibodies • Antibodies are retrieved by harvesting animal’s serum

10. Principles of Immunological Testing • Quantifying antigen-antibody reactions • Concentrations of antibody usually determined through dilution • Antigen added to dilution • Titer is taken from last dilution to give detectable reaction

11. Observing Antigen-Antibody Aggregations • Precipitation reactions • Antibodies binding to soluble antigen form insoluble complexes • Complexes precipitate out of solution • Complete aggregate formation occurs at certain concentrations • To achieve concentrations, place separate antigen and antibody suspensions side by side • Diffuse together to create zone of optimal proportion

12. Observing Antigen-Antibody Aggregations • Immunodiffusion tests • Most widely known is Ouchterlony • Antigen and antibody placed in separate wells cut in gel • Solutions diffuse and meet between the wells • Result in line of precipitation at zone of optimal proportion

13. Observing Antigen-Antibody Aggregations: Ouchterlony Technique

14. Observing Antigen-Antibody Aggregations • Immunodiffusion tests • Radial immunodiffusion test is quantitative • Antibody is added to liquid agar that is allowed to harden • Creates a uniform antibody concentration • Antigen added to wells cut in gel • Diffusion outward forms concentration gradient • Ring forms at antigen-antibody precipitation • Standards can be used to construct standard curve to establish concentration

15. Observing Antigen-Antibody Aggregations

16. Observing Antigen-Antibody Aggregations • Immunoelectrophoresis • Proteins separated using gel electrophoresis • Antibodies are placed in wells and allowed to diffuse towards separated proteins • Line of precipitation forms at antibody-protein recognition • Used to determine patient antibody levels • High levels of certain antibody classes can indicate disease

17. Observing Antigen-Antibody Aggregations • Agglutination reactions • Large insoluble particles are involved • Obvious aggregations are formed • Makes them easier to see • Direct agglutination • Specific antibody mixed with insoluble antigen • Readily visible clumping indication of positive result • Indirect agglutination • Amplifies aggregation formation • Antibody attached to latex bead • Agglutination of these beads much easier to see

18. Using Labeled Antibodies to Detect Interactions • Detectable markers can be attached to specific antibodies • Marked antibodies used to detect presence of given antigen • Tests include • Fluorescent Antibody (FA) test • Enzyme-Linked Immunosorbant Assay (ELISA) • Western blotting • Fluorescence Activates Cell Sorter (FACS)

19. Using Labeled Antibodies to Detect Interactions • Fluorescent antibody test • Relies on fluorescent microscopy to locate labeled antibodies fixed to a microscope slide • Fluorescent polarized immunoassay uses beam of polarized light to rate spin of labeled antibodies • Works under principle that bound antibodies are heavier then unbound and will spin more slowly

20. Using Labeled Antibodies to Detect Interactions • Enzyme-Linked Immunosorbant Assay (ELISA) • Employs antibody that has been labeled with detectable enzyme • Commonly horseradish peroxidase • Labels antibody bonds to antigen • Binding can be direct or indirect • Antigen location is determined using colormetric assay

21. Using Labeled Antibodies to Detect Interactions • Enzyme-Linked Immunosorbant Assay • Direct ELISA • Looks for specific antigen • Specimen placed in wells of microtiter plate • Wells treated with antibody for antigen • Indirect ELISA • Looks for antibody in patient serum • Human IgG • Wells of plate treated with known antigen

22. Using Labeled Antibodies to Detect Interactions • Western blotting • Technique used to detect antigenic proteins • Proteins are separated by size before reacting with antibody • Proteins separated by special gel electrophoresis • SDS PAGE • Makes it possible to establish which proteins are recognized by antibodies

23. Using Labeled Antibodies to Detect Interactions • Fluorescence Activated Cell Sorter (FACS) • Special version of flow cytometry counts cells labeled with fluorescent antibodies • Used to count subsets of T cells • CD4 and CD8 cells especially • Antibodies are attached to the CD4 and CD8 markers • Cells with fluorescently labeled markers are counted