Methods for Ag-Ab detection

• Precipitation
• Agglutination
• Hemagglutination and Hemagglutination
inhibition
• Viral neutralization test
• Radio-immunoassays
• ELISA
• Immunofluorescence
• Immmunoblotting
• Immunochromatography
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Precipitation or Agglutination?
• Precipitation
– Antibodies react with soluble substances (proteins,
carbohydrates, etc)
– Reaction visible with naked eye - particles

• Agglutination
– Antibodies react with insoluble substances (RBCs,
bacterial cells, latex particles coated with antigen)
– Reaction visible with naked eye – larger clumps
(aggregates)
– If agglutination target is RBCs, called hemagglutination

2
 DIRECT AGGLUTINATION
- Test patient serum against large, cellular
antigens to screen for the presence of
antibodies in pt. serum.
- Antigen is naturally present on the surface
of the cells.
- In this case, the Ag-Ab reaction forms an
agglutination, which is directly visible.

3
 DIRECT AGGLUTINATION
• The particle antigen may be a bacterium.
e.g.: Serotyping of E. coli, Salmonella using a
specific antiserum
• The particle antigen may be a parasite.
e.g.: Serodiagnosis of Toxoplasmosis
• The particle antigen may be a red blood cell.
e.g.: Determination of blood groups

4
Positive Negative
 Passive Agglutination
• An agglutination reaction that employs
particles that are coated with antigens not
normally found in the cell surfaces
• Particle carriers include:
– Red blood cells
– Polystyrene latex
– Bentonite
– charcoal

6
 Reverse passive agglutination
Principle
– Antigen (in serum)
binds to antibody
(from kit) coated
on carrier particles
and results in
agglutination
Example
– detecting cholera
toxin
 Serology Testing –
Agglutination Reactions
ASO, Cold Agglutinins, CRP, RF
 Streptolysin O
• Group A β-hemolytic Streptococcus
– S. pyogenes
• Streptolysin O – hemolytic factor
– Sequelae:
• Rheumatic fever
• Glomerulonephritis
• Antistreptolysin O (ASO)
– Neutralizing antibody
– 7-30 days post-onset
 ASO Serological Testing
• ASO Latex Slide Test (Qualitative)
– “Drop & Rock”

• ASO Titration Test (Quantitative)

• ASO Nephelometry (Quantitative)
 ASO Titration Test
• Dilution testing
– Streptolysin O (constant)
– Patient Serum (dilution)
– Add O- RBCs (human or rabbit)
• Examine for RBS lysis – highest dilution with
no hemolysis
• Read as “Todd Units”
 1 2 3 4 5 6 7 8 9 10

1 2 3 4 5 6 7 8 9 10

Saline - 100 100 100 100 100 100 100 100 100

100 100 100 100 100 100 100

Patient Serum 100 100
from 2 from 3 from 4 from 5 from 6 from 7 from 8
-

Streptolysin O 10 10 10 10 10 10 10 10 10 10

Rabbit RBCs 100 100 100 100 100 100 100 100 100 100
Latex Agglutination Assays

“Drop and Rock”

 Cold Agglutinin Disease
• Autoimmune disease
– Antibodies (IgM) against RBCs
– React at lower temps (28-31° C)
– Can cause hemolytic anemia in winter
• Primary - idiopathic
• Secondary - Lymphoproliferative disease
– Lymphoma, CLL, Waldenstrom’s
– Mycoplasma, mono, HIV
 Testing
• Cold agglutinins react with “I” antigen on RBCs
– Serial dilute patient serum
– Mix with RBCs
– Record titer (≥32 significant)
• Upon warming
– Agglutination goes away
– Report titer
 C-Reactive Protein
• Acute phase reactant
• Precipitation Assay
• Latex Agglutination Assay
– Particles coated with anti-CRP antibody
– Direct or Passive?
– Forward or Reverse?
 Acute Phase Reactions
SPE Band Protein Function Change following
acute inflammation
Albumin
Albumin Major serum protein Decreased
Alpha-1
α-1 antitrypsin Serine protease inhibitor 2-4x increase
Alpha-2
α-2 macroglobulin Serine protease inhibitor 0.5x increase
Ceruloplasmin Copper transport 0.5x increase
Haptoglobin Scavenges free hemoglobin 2-4x increase
Gamma
C-Reactive Protein Tags damaged cells 100x increase
Plasma
Fibrinogen Fibrin clot 2-4x increase
 Rheumatoid Factor Assay
• Rheumatoid Factor
– Antibodies (IgM) directed against IgG
– Antigenic site revealed during denaturation of IgG
• Disease Association
– Rheumatoid Arthritis (90%)
– Sjogren’s Syndrome (75%)
– Systemic Lupus Erythematosus (30%)
 Rheumatoid Factor Assay
• Dilute patient serum (RF antibody)
• Latex particles – Heat-denatured IgG
– Direct or Passive?
– Forward or Reverse?