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Immunology (elective) MLIL-101

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Immunology (elective) MLIL-101 Prepared by: Dr. Mohamed S. Abdel-Latif – PowerPoint PPT presentation

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Title: Immunology (elective) MLIL-101


1
Immunology (elective)MLIL-101
Prepared by Dr. Mohamed S. Abdel-Latif
2
Ag-Ab reactionsTests for Ag-Ab reactions
3
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.
4
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

5
Affinity
  • Strength of the reaction between a single
    antigenic determinant and a single Ab combining
    site

Affinity ? attractive and repulsive forces
6
Calculation of Affinity
Ag Ab ? Ag-Ab
Applying the Law of Mass Action
7
Avidity
  • The overall strength of binding between an Ag
    with many determinants and multivalent Abs

8
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.

9
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

10
Factors Affecting Measurement of Ag/Ab Reactions
  • Affinity
  • Avidity
  • AgAb ratio
  • Physical form of Ag

11
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

12
Agglutination Tests
Lattice Formation
13
Agglutination/Hemagglutination
  • Definition - tests that have as their endpoint
    the agglutination of a particulate antigen
  • Agglutinin/hemagglutinin

14
Agglutination/Hemagglutination
  • Quantitative agglutination test
  • Titer
  • Prozone

15
Agglutination/Hemagglutination
  • Definition
  • Qualitative test
  • Quantitative test
  • Applications
  • Blood typing
  • Bacterial infections
  • Fourfold rise in titer
  • Practical considerations
  • Easy
  • Semi-quantitative

16
Passive Agglutination/Hemagglutination
  • Definition - agglutination test done with a
    soluble antigen coated onto a particle
  • Applications
  • Measurement of antibodies to soluble antigens

17
Coombs (Antiglobulin)Tests
  • Incomplete Ab
  • Direct Coombs Test
  • Detects antibodies on erythrocytes

18
Coombs (Antiglobulin)Tests
  • Indirect Coombs Test
  • Detects anti-erythrocyte antibodies in serum

19
Coombs (Antiglobulin)Tests
  • Applications
  • Detection of anti-Rh Ab
  • Autoimmune hemolytic anemia

20
Agglutination/Hemagglutination Inhibition
  • Definition - test based on the inhibition of
    agglutination due to competition with a soluble
    Ag

21
Agglutination/Hemagglutination Inhibition
  • Definition
  • Applications
  • Measurement of soluble Ag
  • Practical considerations
  • Same as agglutination test

22
Precipitation Tests
  • Lattice Formation

23
Radial Immunodiffusion (Mancini)
  • Method
  • Ab in gel
  • Ag in a well
  • Interpretation
  • Diameter of ring is proportional to the
    concentration
  • Quantitative
  • Ig levels

24
Immunoelectrophoresis
  • Method
  • Ags are separated by electrophoresis
  • Ab is placed in trough cut in the agar
  • Interpretation
  • Precipitin arc represent individual antigens

25
Immunoelectrophoresis
  • Method
  • Interpretation
  • Qualitative
  • Relative concentration

26
Countercurrent electrophoresis
  • Method
  • Ag and Ab migrate toward each other by
    electrophoresis
  • Used only when Ag and Ab have opposite charges
  • Qualitative
  • Rapid

27
Radioimmuoassays (RIA)Enzyme-Linked
Immunosorbent Assays (ELISA)
  • Lattice formation not required

28
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

29
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

30
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

31
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

32
Tests for Cell Associated Antigens
  • Lattice formation not required

33
Immunofluorescence
  • Direct
  • Ab to tissue Ag is labeled with fluorochrome

34
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

35
Immunofluorescence
  • Flow Cytometry
  • Cells in suspension are labeld with fluorescent
    tag
  • Direct or Indirect Fluorescence
  • Cells analyzed on a flow cytometer

36
Immunofluorescence
  • Flow Cytometry cont.
  • Data displayed

One Parameter Histogram
Unstained cells
FITC-labeled cells
Number of Cells
Green Fluorescence Intensity
37
Assays Based on Complement
  • Lattice formation not required

38
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

39
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.
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