Antigen Antibody Interactions

Antigen -Antibody Interactions

• Hugh B. Fackrell

Antigen-Antibody Interactions

• Assigned Reading

• Content Outline

• Performance Ojectives

• Key terms
• Key Concepts

• Short Answer Questions

Assigned Reading

• Chapter: 6 pp 144-164

• Janis Kuby’s Immunology 3rd Ed

Content Outline

• Radioimmunoassay (RIA)

• Enzyme Linked Immunosorbent Assay (ELISA)

• Western Blots

• Immunofluorescence

• Immunoelectron Microscopy

Strength of Antigen-Antibody Interactions

• affinity

• avidity

Immunoadsorbent Assays

• Enzyme Linked Immuno Sorbent Assay

• Fluorescent Immuno Sorbent Assay

• Radio Immuno Assay

Enzyme Linked Immunosorbent Assay (ELISA)

• indirect ELISA

• sandwich ELISA

• Competitive ELISA

Indirect ELISA

Competitive ELISA

Sandwich ELISA

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

ELISA:Disadvantages

• Number of separation methods limited

• Expertise required to label and purify conjugates

• Susceptible to interference from non specific factors

ELISA: features of labeled enzymes

• Stable when conjugated

• High substrate turnover number

• High Extinction coefficient of product

• Enzyme & substrate not present in test samples

ELISA: Enzyme Choices

• Horse Radish Peroxidase

• Alkaline Phosphatase

• Glucose Oxidase

• Urease

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

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

Western Blot

• Electrophoresis proteins to separate

• Molecular weight, charge, pI etc
• 2D electrophoresis possible

• Immobilize separated proteins

• Electrophoresis onto nitrocellulose

• Develop as an ELISA

• Product MUST be INSOLUBLE chromogen

Western Blot with MABS

• Same antigen was exposed to 6 different MABS

• Staphylococcal alpha toxin

• Each MAB reacted with a monomer and a oligomer form of the toxin

Immunofluorescent Methods

• Fluoresecence Immuno Assay

• Fluorescence Quenching

• Fluorescence Enhancement

• Fluorescence Polarization

Characteristics of Fluorescent Molecules

• Many loosely bound electrons

• Resonance of double bonds

• Hybridization

Plasma cell function

Antigen localization in Spleen

Flow Cytometry

The End

Performance Objectives

• Key terms, concepts

• short answers

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,

• passive hemagglutination, passive hemagglutination inhibition,

• reverse passive hemagglutination, immune precipitation, immunoelectrophoresis

• immunofluorescence, Indirect fluorecent antibody test, ring test,

• Ouchterlony methods, plasma, primary antigen-antibody interactions, Radioimmunoassay(RIA

• Rhodamine, secondary antigen-antibody interactions, serology,

• serum, titer, zone phenomena (antibody excess, antigen excess, equivalence)

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

Distinguish betweeen antibody affinity and avidity.

• 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.

Describe a secondary antigen-antibody interaction in terms of lattice formation and antigen:antibody ratios.

• 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.

Short Answer Questions

• 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?

What kinds of noncovalent interactions are important in antigen-antibody interactions? What aspect of these interactions is most important and why?

• 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.

Discuss the term lattice formation.

• 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?

• What colour does each fluor emit?

• What makes precipitin reactions visible?

What two factors are important in the development of precipitin reactions?

• 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?

How does agglutination differ from precipitation?

• 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?