Applications of Immune Responses

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

Active immunity› Results from

immune response upon exposure to an antigen

› Active immunity can develop naturally Following illness

› Or artificially After

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

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

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

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

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

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

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

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

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

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

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

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

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)

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

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

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

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

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