Agglutination To Precipitation

MT309 Lec Immunology and
Serology
Antigen-Antibody Reactions
Prepared by: Nhoelyn E. Burcao RMT, MSMT© Prepared by: Nhoelyn E. Burcao RMT, MSMT©
Objectives:
At the end of the discussion, students are able to:
1. List the types of intermolecular interactions between
antigen and antibody.
2. Explain the principle of protein separation using
electrophoresis.
3. Discuss the principles of the agglutination reaction and
list the factors that affect the reaction.
Antigen-Antibody interactions
Characterized as:
• Non-covalent interaction (similar to “lock and key” fit of
enzyme-substrate)
• Does not lead to irreversible alteration of Ag or Ab
• exact and specific interaction has led to many
immunological assays used to:
» detect Ag or Ab
» diagnose disease
» measure magnitude of humoral immune response
Ag-Ab interactions
Bonds:
• Hydrogen
• Ionic
• Hydrophobic interactions
• Van der Waals forces
Each bond is weak; many are

strong
To “hold” they must be close →
requiring high amounts of
complementarity
Binding of the epitope in the
antigen binding site
GOOD FIT POOR FIT
antibody combining site
antigen determinant
high attraction high repulsion

low repulsion low attraction
Affinity
Avidity
Levels of Ab-Ag interactions
Primary Reactions Secondary Reactions
• Immunofluorescence • Precipitation
• Radioimmunoassay • Agglutination
• Enzyme immunoassay • Flocculation
• Chemiluminescence • Neutralization
• Complement Fixation
SEROLOGIC
PRINCIPLES
Reagents:
SEROLOGIC PRINCIPLES
NAMING ANTIBODIES
AGGLUTININS Antibodies that aggregate cellular antigens
PRECIPITINS Antibodies that form precipitates with soluble antigens
ANTITOXINS Antibodies that neutralize toxins

LYSINS Antibodies that cause dissolution of cell membrane
o Hemolysins Cause the destruction of RBC in the presence of the
complement
o Bacteriolysins Cause destruction of cells of gram negative bacteria under
certain conditions
o Cytolysins Cause destruction of other cell types (tumor cells) under
appropriate condition in the presence of the complement
A. Agglutination
B. Precipitation
C. Electrophoresis
D. Labelled Immunoassays
E. Neutralization
F. Complement Fixation
G. Cellular Assays
AGGLUTINATION
METHODS
Agglutination
AGGLUTINATION Assays demonstrate the
presence of antigen-antibody reactions by
the visible aggregation of antigen-antibody
complexes.
1.Latex Agglutination
2.Direct Bacterial Agglutination
3.Hemagglutination Tests
4.Agglutination Inhibition Assays
AGGLUTINATION
• Principle
– Particulate antigen + antibody →clumping
– Lattice formation (antigen binds with Fab sites of 2
antibodies forming bridges between antigens)
Sensitization Lattice Formation

Direct Agglutination
L
L
L
L
L L
Antigens Latex-bound
(in sample) Antibodies
(in reagent)
AGGLUTINATION
Direct Bacterial Agglutination
Bacterial Surface Antibodies

Antigens
Nephelometry
AGGLUTINATION
Latex Agglutination
L L
L L
L
L L L
Antigens Antibodies
Bound to latex (in sample)
(in reagent)
AGGLUTINATION
Latex Agglutination
AGGLUTINATION
Passive Agglutination
(in sample)
Reverse Passive Agglutination
(in sample)
Hemagglutination: Direct
RBC RBC RBC L

RBC
L
LRBC
RBC RBC RBC
Antigens Antibodies
(sample) (reagent)
AGGLUTINATION
AGGLUTINATION
AGGLUTINATION INHIBITION
Agglutination Inhibition Assays
POSITIVE: No Agglutination NEGATIVE: Agglutination

+ YY → Y + → Y +
Antigen
Y
Antibody Ag-Ab Ag-Ab Unbound
Particle
Complex (Indicator) Complex Particle
POSITIVE Result: No Agglutination
X + YY → Y + → Y
No
Y
Antibody Antibody Particle P-Ab
Antigen (Indicator) Complex
NEGATIVE Result: Agglutination

Y + → Y Y
Y Y Y +Y → Y +Y Y
Y
Antibody Antigen Ag-Ab Indicator Ag-Ab Unbound
(Sample) (Reagent) Complex Complex Indicator
POSITIVE Result: No Agglutination
Y
X + → +Y Y →Y Y
No Antigen Antigen Indicator Indicator
Antibody (reagent) (reagent) - Ag
Complex
NEGATIVE Result: Agglutination
Hemagglutination Inhibition
V
V
V V
V
+ →
V
Antibody (Sample)
V
+
RBC V RBC
→
RBC
RBC
+ RBC
RBC
Indicator Cells
Coagglutination
Bacteria
Bacteria
Bacteria
Antigens Antibodies
(reagent) (sample)
PRECIPITATION
METHODS
PRECIPITATION
Principle
-Soluble antigen + antibody (in proper
proportions) → visible precipitate
-Lattice formation (antigen binds with Fab sites

of 2 antibodies)
PRECIPITIN CURVE
Flocculation Test
P P P P
P
P P P
Antigens Antibodies
(in reagent) (in sample)
FLOCCULATION
Flocculation Tests
FLOCCULATION
Single Linear Diffusion
Known Antibody fixed in agar

+
Unknown Antigen (overlaid)
= Precipitin lines
Read before it reaches the maximum :

6-12 hours
Single Radial Diffusion
Known Antibody fixed in agar

+
Unknown Antigen (well cut in agar plate)
= Precipitin ring
Read at maximum size: 24-48 hours
Radial Immunodiffusion (Mancini)
• Method Ab in gel
– Ab in gel Ag Ag Ag Ag
– Ag in a well
• Interpretation
– Diameter of ring is
proportional to the
Diameter2
concentration
• Quantitative
– Ig levels
Ag Concentration
Ouchterlony Immunodiffusion
Smooth arc of Two precipitation Precipitation lines

precipitation line line cross each other meet forming a spur
Ouchterlony Immunodiffusion
Smooth arc of Precipitation lines Two precipitation

precipitation line meet forming a spur line cross each other
PRECIPITATION
ELECTROPHORESIS
Radial Immunodiffusion
ELECTROPHORESIS
Electrophoresis
Electrophoresis
ELECTROPHORESIS
IMMUNOELECTROPHORESIS
Gel diffusion + electrophoresis
Size = concentration
Contour of arc = may indicate monoclonal
gammopathy
Immunoelectrophoresis
• Method
– Ags are separated by electrophoresis
– Ab is placed in trough cut in the agar
+ -
Ag Ag
Ab
Ag
Ab
• Interpretation
– Precipitin arc represent individual antigens
ELECTROPHORESIS
ELECTROPHORESIS
IMMUNOFIXATION
ELECTROPHORESIS
Separate proteins from

serum + antisera →
precipitation → wash
→stain
ELECTROPHORESIS
IMMUNOFIXATION ELECTROPHORESIS
Protein electrophoresis + immunoprecipitation
Normal Pattern
ELECTROPHORESIS
IMMUNOFIXATION ELECTROPHORESIS
Monoclonal IgM and Kappa chain in serum

Electroimmunodiffusion
Countercurrent immunelectrophoresis
Rocket/Laurel electrophoresis

Agglutination To Precipitation

Uploaded by

Document Information

Original Title

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

Agglutination To Precipitation

Uploaded by

Copyright:

Available Formats

MT309 Lec Immunology and

Each bond is weak; many are

high attraction high repulsion

ANTITOXINS Antibodies that neutralize toxins

Sensitization Lattice Formation

Bacterial Surface Antibodies

RBC RBC RBC L

POSITIVE: No Agglutination NEGATIVE: Agglutination

NEGATIVE Result: Agglutination

-Lattice formation (antigen binds with Fab sites

Known Antibody fixed in agar

Read before it reaches the maximum :

Known Antibody fixed in agar

Read at maximum size: 24-48 hours

Smooth arc of Two precipitation Precipitation lines

Smooth arc of Precipitation lines Two precipitation

Separate proteins from

Monoclonal IgM and Kappa chain in serum

You might also like