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Antigen and Antibody Reactions1582
Antigen and Antibody Reactions1582
Advanced Clinical
Immunohematology
Antigen and Antibody
Reactions
Immune Response
Definition: Physiological mechanism to fight
disease or clear foreign substances.
Primary immune response: First exposure
of a foreign agent. IgM is the predominant
antibody produced that attaches to and fights
the foreign agent. In our case a foreign red
blood cell antigen.
Secondary immune response: Subsequent
exposure of the same foreign agent. Rapid
response in which IgG is the predominant
antibody produced.
Antigen/Antibody Reactions
1. Antigen:
Characteristics of Antigens
1. Antigenicity of an
Antigen is affected
by its:
2. Foreign:
3. Size:
4. Most common
forms of Blood
Group Antigens:
Characteristics of
Immunoglobulins (Antibodies)
RBC Immune Vs. Non-RBC Immune
RBC Immune Antibody: antibody that results
from exposure to foreign red cell antigen either
by transfusion or pregnancy (anti-D, etc.)
Non-RBC Immune Antibody: antibody that is
present without any evidence of exposure to
foreign red cell antigen (anti-A, anti-N, etc.)
1. Monomer
1. Pentamer
2. Phase of reactivity
3. Placenta
4. Complement
Activation
5. Clinical
Significance
IgG Antibody
IgM Antibody
(Monomer Structure)
(Pentamer Structure)
1. Cold reacting
IgM
Antibodys
2. Good complement
activator
3. Pentamer
4. ABO, Ii, Lewis, MN, & P
1. Warm Reacting
IgG
Antibodys
2. Poor complement
activators
3.
Monomer
2. RBC Hemolysis
3. Agglutination
Hemolysis
When antibody (IgG or IgM) has
activated Complement to completion.
The complement (C) cascade is the
mechanism that actually destroys the red
blood cell.
Antibody (immunoglobulin) by itself
cannot tear a hole in the RBC
membrane. It is the C that does that.
Seen when supernatant is CLEAR and RED.
Not seen at the AHG phase! Why??
Agglutination Reactions
Two Stage Process:
Stage 1
Stage 2
Stage 1
Stage 2
Stage 1 of Agglutination:
Sensitization
Weak Non-specific
Intermolecular Forces
Electrostatic forces
Attraction of oppositely charged or ionized molecules
Force of attraction is inversely proportional to square
of the distance between charges: closer you get the
stronger the attachment of Ag to Ab.
Hydrogen bonds
Weak, reversible hydrogen bridges between
hydrophilic groups which are stronger at lower
temperatures.
Weak Non-specific
Intermolecular Forces
Hydrophobic forces
Water hating groups (valine, leucine) come into
close contact and exclude water between them,
resulting in lower free energy system disrupting
the water envelope.
Disruption of water envelope is the result of
hydrophobic forces: requires close proximity
Weak Non-specific
Intermolecular Forces
van der Waals bonds
Temporary disruption of electrons in one
molecule effectively forms a dipole which
causes the formation of a dipole in another
molecule, the two dipoles then exhibit an
attraction for each other: Requires close
proximity.
Factors Affecting
Stage 1 of Agglutination
Temperature
Factors Affecting
Stage 1 of Agglutination
pH
Changes in pH can affect electrostatic bonds.
Optimal range is physiologic pH
Some Abs like lowered pH particularly anti-M
Incubation Time
The time needed to reach equilibrium
Saline systems: 30-60 minutes at 37oC
Enhancement media can reduce incubation time
significantly: LISS is 10-15 minutes
Factors Affecting
Stage 1 of Agglutination
Ionic Strength
In normal saline, Na+ and Cl- ions cluster around
and partially neutralize opposite charges on Ag
and Ab molecules, which hinders the association
of Ab with Ag.
If you lower the ionic strength of the test system
then you increase rate of Ag-Ab association.
Using LISS (low ionic strength solution)
decreases incubation time.
Stage 2 of Agglutination:
Lattice Formation
Lattice formation: formation of bridges
between the sensitized red cells to form the
lattice that results in agglutination (clumping)
of red blood cells.
Concentration of Ag and Ab
Factors Affecting
Stage 2 of Agglutination
Size of the Immunoglobulin
IgG: Monomer, takes two to activate C, etc.
IgM: Pentamer, takes one to activate C, etc.
Factors Affecting
Stage 2 of Agglutination
Location and Number of Antigenic
Determinants
A, B, M and N antigens: 600,000 to >1,000,000
antigen sites per RBC
Kidd: approximately 10-20,000 Ag sites/RBC
Centrifugation
Brings Abs and Ags into close proximity
Undercentrifugation: may result in false negative
Overcentrifugation: may result in false positive
Factors Affecting
Stage 2 of Agglutination
Zeta Potential
Net negative charge surrounding RBC
Now considered a minor player
Waters of Hydration
Acts as an insulating bubble around RBC
Water molecules tightly bound to
hydrophilic macromolecules on the red cell
surface.
Hemolysis
ALWAYS observe the supernatant of the test tube
after centrifugation. If the supernatant is clear and red
Hemolysis is indicated. THIS IS A POSITIVE
REACTION! And indicates a Nasty antibody.
Potentiators
We have not covered the topic of
potentiators. This is found on pages 60-62
in Harmening and is Objective #10.
We also did not cover Solid Phase and Gel
testing techniques. This is found on page
62-63 in Harmening and is Objective #6.