Lecture Note
Agglutination Reactions. Principles and Reactions
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University:
Franklin University -
Course:
SCIE-254 | Health & Human Disease Academic year:
2021
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Views:
77
Pages:
30
Author:
LunarLullaby
Agglutination Reactions
Principles and Practice Sampler: Fixed and Adjustable
Mouth pipetting is forbidden in lab
Use digital or mechanical pipetting
Agglutination tests
3 Pipetting method
Agglutination tests
4 Serologic tests are based on
Ag-Ab reactions
• Previously we discussed that four items play
role in Antigen-Antibody interaction:
– Antigen and Antibody characteristics, their
concentration and ratio and finally the
environmental condition
Agglutination tests
6 Antigen properties
• Based on size, Ags could be:
– Particulate: Whole Bacteria or RBC, other
particles
– Soluble: Protein antigens
– Colloidal: Lipid antigens
• Based on cellular location,
Ags could be:
– Intercellular
– Extracellular
Agglutination tests
7 Antibody
• Abs could be from different class and subclasses
that their Ag binding capacities varies.
• IgM is produced in acute phase of infection and
has 10 Fabs, so has more binding sites and is a
better Ag agglutinating agent.
IgM was more sensitive to reducing agents (2ME)
• IgG is produced in chronic phase of infection and
has 2 Fabs, but usually have higher binding
affinity and is produced in higher amounts. So it
can be a good Ag agglutinating molecule, too.
Agglutination tests
8 Environmental factors
• In the laboratory (in vitro) we try to mimic the
similar physiologic pH (7.0 - 7.4) and thermal
(37℃) conditions found in vivo.
• Shaking and agitating can help us to obtain
better and rapid reactions.
• The optimal binding may take time, thus we
need some incubation time.
Agglutination tests
9 Classification of Ag-Ab
reactions
1- Primary assays: Labeled immunoassays
(Lattice formation is not necessary)
Used for evaluation of low amounts of Ag or Ab
2- Secondary assays: Visible results
(Lattice formation is the basics)
Used for evaluation of high amounts of Ag or Ab
3- Tertiary assays: Functional assays
Used for evaluation of the consequences following
Ag-Ab reactions Secondary serologic reactions
• Based on Ag type and size three types of
reactions are possible:
– Agglutination of particulate Ags
– Flocculation (microscopic agglutination) of
colloidal Ags
– Precipitation of soluble Ags
Agglutination tests
11 Agglutination
• Agglutination is based on aggregation of
particulate Ags (Agglutinogens) by Abs
(agglutinins) or other agents such as lectins.
• Agglutination is a simple and sensitive test
with different applications, specially in
diagnosis of febrile diseases such as
Brucellosis and Salmonellosis.
Agglutination tests
12 Classification of Agglutination
tests based on method
Slide Agglutination Test (Rapid)
Tube Agglutination Test (Standard)
Microtiter plate Agglutination Test
(Micro)
Agglutination tests
13 Slid or Rapid Agglutination Test
• Screening assays report format
• Qualitative (Pos / Neg), (Pos: 1+, 2+, 3+, 4+):
mostly used in passive agglutination tests
(Reactive/ Non-Reactive): Blood grouping
• Semi-quantitative (Titer): Brucellosis (Wright),
Sallemonellosis (Widal)
14 Serodiagnosis of Brucellosis
Brucellosis is the most common global zoonosis
Serological diagnosis is based on agglutination of B.
abortus bacterial particles with patients serum
Other species of the Brucella genus bacteria have
similar antigens to B. melitensis and B. suis.
Thus inactivated B. abortus is used in the test as the
available bacteria.
Agglutination tests
15 Agglutination steps
Agglutination takes place in two steps:
1. Sensitization or initial binding of molecules
Rapid and reversible attachment of Abs to
particulate Ags
2. Lattice formation or aggregation or stabilization
Cross linking of particles and formation of visible
enlarged aggregates
Agglutination tests
16 Agglutination advantages
•
•
•
•
•
•
Rapid
Low cost
Sensitive
May be done on slide, tube or miroplate
Easily monitored by eye
Small amount of serum and reagent needed
Agglutination tests
17 Classification of Agglutination
tests based on Ag properties
• Active agglutination: Ag is a native part or structure of
the particle
– Heamagglutination: Abs bind to RBC surface Ags
• Blood grouping: Cell type, Back type
– Microbial agglutination: Abs bind to bacteria surface Ags
• Wright, Widal
• Passive agglutination: A soluble Ag was bound on an
inert particle to make a particulate Ag
– Latex agglutination: ASO (Streptolysin O is bound to latex)
– Heamagglutination: Viruses are bound to RBC and antiviral Abs studied
Agglutination tests
18 Active Agglutination
• Screening of surface Ags via specific Abs.
• Serotyping of E. coli, Salmonella, Vibrio cholera, …
with specific antisera
• Blood groups (cell type)
• Screening of produced Abs to surface Ags.
• Wright and Widal tests
• Blood grouping (Back type)
Agglutination tests
19 Screening of Brucellosis
• Bacterial suspension of Brucella abortus
agglutinated with specific Abs in the serum
• attenuated strain of B. abortus were inactivated
and stained with rose Bengal or other stains for
better visibility of the results
Agglutination tests
20 Serologic tests for Brucellosis
•
•
•
•
Rapid Wright test (screening)
Standard Wright test in tube
2ME-Wright test
Coombs’ Wright test
Agglutination tests
21 Wright Tube Agglutination test
10
9
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2
1
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.9
Saline
0.1
Serum
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Tube
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0.5
out
0.5
←
0.5
←
0.5
←
0.5
←
0.5
←
0.5
←
0.5
←
0.5
←
0.5
0.5
0.5
0.5
0.5
0.5
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0.5
Ag
Control
1:5120
1:2560
1:1280
1:640
1:320
1:160
1:80
1:40
1:20
Titer
Agglutination tests
22 Wright Tube Agglutination test
In this case, the titre is 1/80
1/20
1/40
1/80
Agglutination
1/160
1/320
1/640
1/1280
Neg. ctrl
No agglutination
Agglutination tests
23 Interpretation of the results
• Clinical history
• Rapid tests
• Standard tests
• Follow up tests
Agglutination tests
24 Passive Agglutination
• Agglutination reaction of inert particles
coated with soluble Ags that are not normally
found on their surfaces
• Inert Ag carriers:
– Polystyrene latex
– Red blood cells (O neg)
– Charcoal
Agglutination tests
25 Common Passive Agglutination
tests
• ASO (Anti-Streptolysin O)
• RF (Rheumatoid factor)
• CRP (C-reactive protein)
• Gravindex (HCG)
Agglutination tests
26 Passive Agglutination
Agglutination tests
27 Tube Agglutination Test
• Also known as the standard or Quantitative
agglutination test, e.g. Brucellosis (Wright)
• Serum diluted in 2-fold manner
• Constant amount of Ag is added
• Incubation at RT or 37°C (overnight or hours)
• Visible agglutination at the bottom of tubes
Agglutination tests
28 Microtiter plate agglutination
Agglutination tests
29 Microscopic agglutination
or Flocculation
• VDRL (Venereal Disease Research Laboratory)
• Nowadays RPR (Rapid Plasma Reagin) is
substituted for RPR which is a agglutination
test.
• The positive results are checked by FTA-ABS
Agglutination tests
30 CRP, RF, ASO, Gravindex
• Sample + Reagent Mix and Rotate for 3
minutes, Read the results within 3 minutes
•
•
•
•
Use positive and negative controls
Could be qualitative or semiquantitative
Application of the tests
Interpretation of the results
Agglutination tests
31 CRP
Agglutination tests
32
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