Transcript Antigen Antibody Interactions

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Antigen -Antibody Interactions
Hugh B. Fackrell
4/13/2015
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Antigen-Antibody Interactions
Assigned Reading
 Content Outline
 Performance Ojectives
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 Key
terms
 Key Concepts
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Short Answer Questions
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Assigned Reading
Chapter: 6 pp 144-164
 Janis Kuby’s Immunology 3rd Ed
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Content Outline
Radioimmunoassay (RIA)
 Enzyme Linked Immunosorbent Assay
(ELISA)
 Western Blots
 Immunofluorescence
 Immunoelectron Microscopy
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Strength of Antigen-Antibody
Interactions
affinity
 avidity
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Immunoadsorbent Assays
Enzyme Linked Immuno Sorbent Assay
 Fluorescent Immuno Sorbent Assay
 Radio Immuno Assay
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Enzyme Linked Immunosorbent
Assay (ELISA)
indirect ELISA
 sandwich ELISA
 Competitive ELISA
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Indirect ELISA
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Competitive ELISA
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Sandwich ELISA
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ELISA: Advantages
Specific & Sensitive- Wide Application
 Equipment cheap & available
 Reagents “Cheap”, long shelf life
 Assays may be rapid
 Simultaneous assays; variety of labels
 Potential for automation
 no radiation hazards
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ELISA:Disadvantages
Number of separation methods limited
 Expertise required to label and purify
conjugates
 Susceptible to interference from non
specific factors
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ELISA:
features of labeled enzymes
Stable when conjugated
 High substrate turnover number
 High Extinction coefficient of product
 Enzyme & substrate not present in test
samples
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ELISA: Enzyme Choices
Horse Radish Peroxidase
 Alkaline Phosphatase
 Glucose Oxidase
 Urease
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RIA: Advantages
Measurement simple, not affected by
composition of sample matrix
 Sensitivity & precision not dependent on the
measurement of the magnitude of the signal
 Large variety of radiolabelled compounds
 labels do not affect reaction kinetics
 Mathematically documented
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RIA: Disadvantages
Labeled reagents have short shelf life
 Potential health hazards
 Disposal of radioactive wastes
 Equipment is expensive
 Variability between batches of labels
 Dependence on duration of count time may
limit sensitivity of assays
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Western Blot
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Electrophoresis proteins to separate
 Molecular
weight, charge, pI etc
 2D electrophoresis possible
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Immobilize separated proteins
 Electrophoresis
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onto nitrocellulose
Develop as an ELISA
 Product
MUST be INSOLUBLE chromogen
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Western Blot with MABS
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Same antigen was
exposed to 6 different
MABS
 Staphylococcal
oligomer
alpha
toxin
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Each MAB reacted with
a monomer and a
oligomer form of the
toxin
Maria Sawicki 1996
monomer
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Immunofluorescent Methods
Fluoresecence Immuno Assay
 Fluorescence Quenching
 Fluorescence Enhancement
 Fluorescence Polarization
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Characteristics of Fluorescent
Molecules
Many loosely bound electrons
 Resonance of double bonds
 Hybridization
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Plasma cell function
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Antigen localization in Spleen
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Flow Cytometry
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The End
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Performance Objectives
Key terms, concepts
short answers
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Key Terms
agglutination, direct agglutination reaction, indirect
agglutination reaction
 antibody affinity, antiserum, association constant
(K), average affinity,
 average intrinsic association constant(Ka), avidity,
ELISA, equilibrium constant,
 equilibrium dialysis, fluorescein, fluorochromes,
hemagglutination,
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passive hemagglutination, passive
hemagglutination inhibition,
 reverse passive hemagglutination, immune
precipitation, immunoelectrophoresis
 immunofluorescence, Indirect fluorecent
antibody test, ring test,
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Ouchterlony methods, plasma, primary
antigen-antibody interactions,
Radioimmunoassay(RIA
 Rhodamine, secondary antigen-antibody
interactions, serology,
 serum, titer, zone phenomena (antibody
excess, antigen excess, equivalence)
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Key Concepts
Explain a primary antigen-antibody interaction
and include at least three important
characteristics.
 Describe the forces that encourage primary
antigen-Antibody interactions
 Assess the reasons for using the different gel
preciptitin reactions
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Distinguish betweeen antibody affinity and
avidity.
 Describe the strength of the primary
antigen-antibody interactions using
equilibrium dialysis. Include the terms K
and Ka
 Compare and contrast RIA and ELISA
 Describe direct and indirect fluorescent
antibody methods.
 Explain zone phenomena.
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Describe a secondary antigen-antibody
interaction in terms of lattice formation and
antigen:antibody ratios.
 Construct a table to compare the various
procedures used to determine the presence of
soluble antigen or antibody in a fluid and in a
gel.
 Distinguish between agglutination and
preciptin reactions and give the advantages
and disadvantages of each.
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Short Answer Questions
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Cross reactivity of antibodies creates
problems for their application in serology.
Explain.
 Differentiate between a primary and a
secondary antigen-antibody reaction.
 What are three important characteristics that
distinguish the two reactions?
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What kinds of noncovalent interactions are
important in antigen-antibody interactions?
What aspect of these interactions is most
important and why?
 How is equilibrium dialysis used to measure
PRIMARY antigen-antibody reactions?
 Differentiate between avidity and affinity.
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Discuss the term lattice formation.
 What are the pros and cons of RIA?
 Describe two types of immunofluorescence
tests.
 What is the advantages of the indirect
procedure over the direct procedure?
 What are some commonly used fluors?
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 What
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colour does each fluor emit?
What makes precipitin reactions visible?
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What two factors are important in the
development of precipitin reactions?
 Three patterns can be observed in the
Ouchterlony test. DRAW and LABEL
diagrams to illustrate these patterns. What does
each pattern show?
 What is the major advantage of
immunoelectrophoresis over immunodiffusion?
 What are the disadvantages?
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How does agglutination differ from
precipitation?
 Why are agglutinatin tests more sensitive that
precipitin tests?
 Differentiate between direct and indirect
agglutination reactions?
 What is a major advantage of indirect
agglutination reaction over direct reactions?
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