PRINCIPLES OF SEROLOGIC REACTIONS

Antigen- Antibody Interactions: Principles and Applications

I. AGGLUTINATION
- The interaction between antibody and a particulate antigen (bacteria, WBC, RBC, latex particle and other
compounds that appear cloudy when suspended in saline) resulting in visible clumping
- Antibodies that agglutinate antigens are referred to as agglutinins and the associated antigens are agglutinogens.

Principles of Agglutination
-  Agglutination is a visible manifestation of antigen-antibody interaction; however not all antigen-antibody
interactions lead to agglutination. Rather, agglutination occurs only when the properties and relative
concentrations of Ag and Ab allow sufficient lattice formation.

 Carrier particles may be needed to indicate visibly that an antigen-antibody reaction has taken place.
a. Artificial carriers: latex particles and colloidal charcoal
b. biologic carriers: RBC, bacteria

- Agglutination is a two-step process that results in a stable lattice formation:

1. Sensitization- involves antigen–antibody combination through single antigenic determinants on the particle
surface. The combination of antigen and antibody is a reversible chemical reaction. Altering the physical conditions
can result in the release of antibody from the antigen-binding site.

Note: sensitization is dependent on equilibrium constant.

- All antigen–antibody binding is reversible and is governed by the law of mass action. This law states that free
reactants are in equilibrium with bound reactants. The equilibrium constant represents the difference in the ratesof the
forward and reverse reactions according to the following equation:
-

Note: the higher the equilibrium constant, the higher is the rate of association and the slower the rate of
dissociation of antibody molecules.
Affinity vs. Avidity

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 2. Lattice Formation- formation of cross-links that form the visible aggregates. This represents the
stabilization of antigen–antibody complexes with the binding together of multiple antigenic determinants

Factors that Affect Agglutination

1. Nature and class of antibody molecules

2. Antigen-antibody ratio

3. Particle charge
Note: RBC, latex and bacteria have a net negative surface-zeta potential

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4. Physical conditions:
a. pH
b. temperature
c. length of incubation
d. steric hindrance/mutual blocking

5. Nature of the antigen-bearing surface

6. Electrostatic interactions between particles

Reading agglutination reactions

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Causes of False (+) and False (-) Reactions

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Types of Agglutination Reactions

1. DIRECT IMMUNE AGGLUTINATION

- aggregation of particles with the native Antigens or the antigens that are naturally present on the surface of particle
such as RBC or bacteria.
- Applications:
A. Hemagglutination
1. Detect antigens on RBCs by using known antisera (Ex: ABO, Rh forward typing)

2. Detect Antibodies to RBCs by using known Antigens (Ex: ABO reverse typing)

B. Bacterial Agglutination
1. Detect bacterial antigens using known antisera
Reagent:
Unknown:
Example:

2. Detect antibodies to bacteria by using known whole pathogens

Reagent:
Unknown:
Example:

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2. DIRECT NONIMMUNE AGGLUTINATION
A. Viral Hemagglutination

Principle:

B. Hemagglutination Inhibition
Principle:

Procedure: Patient’s serum + hemagglutinating viral antigen  expose to red cells (indicator particles)
Positive result:
Example:

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3. AGGLUTINATION INHIBITION
Principle: based on competition between particulate and soluble antigens for limited antibody combining site.
Procedure: Reagent Ab + Px sample
Result:
If Px Ag is present: No agglutination  positive result
If Px Ag is absent: Agglutination  negative result
Example:

4. INDIRECT/ PASSIVE
Principle: Abs do not attach to antigenic determinants native to the carrier but to Ags anchored to the carriers.
Application: used to detect RF, ANA, Abs to group A streptococcus, Abs to Trichinella spiralis, Abs to viruses
Types:
A. Passive Hemagglutination
Used for testing Rubella Abs

B. Latex agglutination
Used for testing Rubella Abs

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5. REVERSE PASSIVE
Principle: Latex particles coated with Ab is reacted with antigens present in patient’s sample.
Procedure: Reagent (latex coated with Ab) + patient’s sample
Result:
Example:

Methods of Enhancing Agglutination:

1. Centrifugation

2. Treatment with proteolytic enzymes

3. Addition of colloids

4. Addition of AHG
AHG:
A. DAT
Principle:
Result:

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 B. IAT
Principle:
Result:

5. Controlling physical conditions

Application of agglutination tests:

“God has a purpose for your pain, a

reason for your struggles, and a gift for
your faithfulness. Don’t give up. Be
patient even if it’s difficult, God is
not finished with you yet.” =)

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