Immunology (elective) MLIL-101

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Immunology (elective)MLIL-101
Prepared by Dr. Mohamed S. Abdel-Latif
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Ag-Ab reactionsTests for Ag-Ab reactions
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Learning Outcome
At this time you should know the following 1.
To describe the nature of Ag-Ab reactions 2. To
compare and contrast antibody affinity and
avidity. 3. To delineate the basis for antibody
specificity and cross reactivity. 4. To discuss
the principles of commonly used tests for
antigen/antibody Reactions.
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Nature of Ag/Ab Reactions

• Lock and Key Concept

• Non-covalent Bonds

• Hydrogen bonds
• Electrostatic bonds
• Van der Waal forces
• Hydrophobic bonds

• Multiple Bonds

• Reversible

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Affinity

• Strength of the reaction between a single
  antigenic determinant and a single Ab combining
  site

Affinity ? attractive and repulsive forces
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Calculation of Affinity
Ag Ab ? Ag-Ab
Applying the Law of Mass Action
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Avidity

• The overall strength of binding between an Ag
  with many determinants and multivalent Abs

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Specificity

• The ability of an individual antibody combining
  site to react with only one antigenic
  determinant.
• The ability of a population of antibody molecules
  to react with only one antigen.

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Cross Reactivity

• The ability of an individual Ab combining site to
  react with more than one antigenic determinant.
• The ability of a population of Ab molecules to
  react with more than one Ag

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Factors Affecting Measurement of Ag/Ab Reactions

• Affinity

• Avidity

• AgAb ratio

• Physical form of Ag

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Tests Based on Ag/Ab Reactions

• All tests based on Ag/Ab reactions will have to
  depend on lattice formation or they will have to
  utilize ways to detect small immune complexes
• All tests based on Ag/Ab reactions can be used to
  detect either Ag or Ab

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Agglutination Tests
Lattice Formation
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Agglutination/Hemagglutination

• Definition - tests that have as their endpoint
  the agglutination of a particulate antigen
• Agglutinin/hemagglutinin

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Agglutination/Hemagglutination

• Quantitative agglutination test
• Titer
• Prozone

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Agglutination/Hemagglutination

• Definition
• Qualitative test
• Quantitative test

• Applications
• Blood typing
• Bacterial infections
• Fourfold rise in titer

• Practical considerations
• Easy
• Semi-quantitative

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Passive Agglutination/Hemagglutination

• Definition - agglutination test done with a
  soluble antigen coated onto a particle

• Applications
• Measurement of antibodies to soluble antigens

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Coombs (Antiglobulin)Tests

• Incomplete Ab
• Direct Coombs Test
• Detects antibodies on erythrocytes

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Coombs (Antiglobulin)Tests

• Indirect Coombs Test
• Detects anti-erythrocyte antibodies in serum

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Coombs (Antiglobulin)Tests

• Applications
• Detection of anti-Rh Ab
• Autoimmune hemolytic anemia

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Agglutination/Hemagglutination Inhibition

• Definition - test based on the inhibition of
  agglutination due to competition with a soluble
  Ag

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Agglutination/Hemagglutination Inhibition

• Definition

• Applications
• Measurement of soluble Ag
• Practical considerations
• Same as agglutination test

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Precipitation Tests

• Lattice Formation

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Radial Immunodiffusion (Mancini)

• Method
• Ab in gel
• Ag in a well

• Interpretation
• Diameter of ring is proportional to the
  concentration
• Quantitative
• Ig levels

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Immunoelectrophoresis

• Method
• Ags are separated by electrophoresis

• Ab is placed in trough cut in the agar

• Interpretation
• Precipitin arc represent individual antigens

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Immunoelectrophoresis

• Method
• Interpretation
• Qualitative
• Relative concentration

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Countercurrent electrophoresis

• Method
• Ag and Ab migrate toward each other by
  electrophoresis
• Used only when Ag and Ab have opposite charges

• Qualitative
• Rapid

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Radioimmuoassays (RIA)Enzyme-Linked
Immunosorbent Assays (ELISA)

• Lattice formation not required

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Competitive RIA/ELISA for Ag

• Method
• Determine amount of Ab needed to bind to a known
  amount of labeled Ag

• Use predetermined amounts of labeled Ag and Ab
  and add a sample containing unlabeled Ag as a
  competitor

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Competitive RIA/ELISA for Ag

• Method cont.
• Determine amount of labeled Ag bound to Ab
• ? NH4SO4
• ? anti-Ig
• Immobilize the Ab

• Concentration determined from a standard curve
  using known amounts of unlabeled Ag

• Quantitative
• Most sensitive test

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Solid Phase Non-Competitive RIA/ELISA

• Ab detection
• Immobilize Ag
• Incubate with sample
• Add labeled anti-Ig
• Amount of labeled Ab bound is proportional to
  amount of Ab in the sample

• Quantitative

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Solid Phase Non-Competitive RIA/ELISA

• Ag detection
• Immobilize Ab
• Incubate with sample
• Add labeled antibody
• Amount of labeled Ab bound is proportional to the
  amount of Ag in the sample

• Quantitative

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Tests for Cell Associated Antigens

• Lattice formation not required

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Immunofluorescence

• Direct
• Ab to tissue Ag is labeled with fluorochrome

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Immunofluorescence

• Indirect
• Ab to tissue Ag is unlabeled
• Fluorochrome-labeled anti-Ig is used to detect
  binding of the first Ab.

• Qualitative to Semi-Quantitative

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Immunofluorescence

• Flow Cytometry
• Cells in suspension are labeld with fluorescent
  tag
• Direct or Indirect Fluorescence
• Cells analyzed on a flow cytometer

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Immunofluorescence

• Flow Cytometry cont.
• Data displayed

One Parameter Histogram
Unstained cells
FITC-labeled cells
Number of Cells
Green Fluorescence Intensity
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Assays Based on Complement

• Lattice formation not required

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Complement Fixation

• Methodology

• Ag mixed with test serum to be assayed for Ab

• Standard amount of complement is added

• Erythrocytes coated with Abs is added

• Amount of erythrocyte lysis is determined

Ag
Ag

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Assignment As a part of the semester activity,
Group of students are selected every week to
prepare a short seminar about his/her point of
interest in one of the lecture topics. That to be
discussed and evaluated during the next lecture.