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