Applications of Immune Responses

Chapter 17

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

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

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

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

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 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

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

Vaccines and Immunization

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

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

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

Principles of Immunological Testing

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

Observing Antigen-Antibody Aggregations Antigen-antibody complexes form aggregates Antigen-antibody binding can be seen in

precipitation and agglutination reactions

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

Agglutination - Precipitation

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

AntibodyAntigen

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

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.

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

Usually a known antigen or known antibody is placed in the center and test serum is placed in the peripherial wells.

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

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

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

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)

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

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

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

Using Labeled Antibodies to Detect Interactions

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

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