Applications of Immune Responses

1. Applications of Immune Responses Chapter 17

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 • Result 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 • Inferres 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. 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 as 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 Vaccines and Immunization

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 • Includes toxoids, protein subunit vaccines and polysaccharide vaccines

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 • Obtaining antibody • Certain serological tests bind human IgG • Antibodies are termed anti-human IgG • These can be produced in animals immunized with IgG from human serum

11. 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

12. Observing Antigen-Antibody Aggregations • Antigen-antibody complexes form aggregates • Antigen-antibody binding can be seen in precipitation and agglutination reactions

13. 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

14. Agglutination - Precipitation Antibody Antigen When there is an excess of antibodies each antigen is individually bound and no agglutination occurs When there is an excess of antigen each antibody is individually bound and no agglutination occurs If there is an appropriate ratio of antigen / antibody agglutination and precipitation occur

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

16. Ouchterlony Double Diffusion Note: A line of precipitation has formed between the center well and wells 3 & 5. This indicates there is antigen/antibody specificity between the center well and these two wells. Usually a known antigen or known antibody is placed in the center and test serum is placed in the peripherial wells. Antigens and antibodies will diffuse and at some point optimal concentrations will occur and if the antigen is specific for the antibody a precipitate line will form. Example: Has this patient ever had rubella, rubeolla, or diptheria? If they have their serum will contain antibodies against the disease. Put patient serum in the center. Put the disease agents (antigens) in wells 1 – 5, and allow to diffuse. A precipitation line between wells indicates that the patient has had that disease

17. Observing Antigen-Antibody Aggregations • Immunodiffusion tests • Radial immunodiffusion test is quantitative • Antibody is added to liquid agar that is allowed to harden • Created 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

18. 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

19. Observing Antigen-Antibody Aggregations • Agglutination reactions • Large insoluble particles are involved • Obvious aggregations are formed • Makes the 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

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

21. Using Labeled Antibodies to Detect Interactions • Fluorescent antibody test • Relies on fluorescence 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

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

23. 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 of antibody in patient serum • Human IgG • Wells of plate treated with known antigen

24. Using Labeled Antibodies to Detect Interactions • Western blotting • Technique used 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

25. 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 cell especially • Antibodies are attached to the CD4 and CD8 markers • Cells with fluorescently labeled markers are counted