Agglutination
Part 2
1
Latex agglutination
• In latex agglutination
procedures, Ag
molecules can be
bound to the surface
of latex beads.
• If Ab is present in the
test specimen, the Ag
will combine with the
Ab and form visible
aggregates.
2
Latex agglutination
• Latex particles can
be coated with Ab,
and in the
presence of Ag can
form visible
aggregates.
3
Reverse Indirect agglutination
• Involves the adherence of
Ab to inert particles which
can then be used to detect
the presence of Ag.
• Example – latex particle
coated with Ab (known) +
serum looking for
(unknown) Ag. If Ag
present, then you get
visible agglutination.
4
Reverse Indirect agglutination
• Numerous kits are available for rapid
identification of antigens on
infectious agents
• Examples:
• Staphylococcus aureus
• Neisseria maningetidis
• Mycoplasma pneumoniae
• Such tests used for organisms that
• are difficult to grow
• or when rapid identification is required
5
Reverse Indirect agglutination
• Used also to measure
• levels of therapeutic drugs,
• hormones,
• plasma proteins
6
Hemagglutination
• The agglutination of red blood cells
by either
• direct agglutination
• or indirect agglutination
• Direct agg. Ag is an intrinsic
component of RBC
• Indirect agg. Soluble Ags are
adsorbed to the RBC
7
Hemagglutination
•
There are 3 ways in which Ags may
be bound to RBCs:
1. Spontaneous adsorption of Ags by RBCs
2. Covalent binding using chemical links
3. Tannic acid treatment
8
Hemagglutination
• Qualitative agglutination test
– Ag or Ab
+
↔
9
Hemagglutination
• Quantitative agglutination test
Neg.
Pos.
1/1024
1/512
1/256
1/128
1/64
1/32
1/16
1/8
1/4
Patient
1/2
• Titer
• Prozone
Titer
1
2
3
64
8
512
4
5
<2
32
6
7
8
128
32
4
10
Indirect Hemagglutination
• Definition - agglutination test done
with a soluble antigen coated onto a
particle
+
+
↔
11
Agglutination inhibition / Hemagglutination
inhibition
• Involves interference by Ag or Ab
with an Ag-Ab reaction which would
have resulted in agglutination, if
interference had not occurred. The
technique is called hemagglutination
inhibition if the particle in the
reaction is a rbc.
12
Agglutination inhibition - pos
• Tube containing Ab
(known)
• Patient has Ag
(unknown) and
combines with Ab. Not
visible.
• Latex bead coated with
known Ag is added. It
has nothing to attach to
– no visible reaction or
agglutination inhibition
is positive.
13
Agglutination inhibition - neg
• Tube containing Ab
(known).
• Patient serum does not
contain Ag (unknown),
therefore no combination.
• Latex bead coated with
known Ag directed to
known Ab coated to tube.
Visible agglutination,
therefore agglutination
inhibition is negative
14
Agglutination inhibition
15
Hemagglutination Inhibition
• Definition - test based on the inhibition of
agglutination due to competition with a
soluble Ag
+
+
↔
Test
Patient’s sample
16
Coagglutination
• Coagglutination (CoA) is similar to the Latex
Agglutination technique for detecting antigen
• Protein A, a uniformly distributed cell wall
component of Staphylococcus aureus, is able
to bind to the Fc region of most IgG isotype
antibodies leaving the Fab region free to
interact with antigens present in the applied
specimens.
• The visible agglutination of the S. aureus
particles indicates the antigen-antibody
reaction.
17
Coagglutination
18
Coagglutination
• These particles exhibit greater
stability than Latex particles
• However, bacteria are not colored &
therefore reactions are often difficult
to read.
• Such tests are highly specific, but
the sensitivity is less than latex
particles method
19
Viral Hemagglutination
• Many viruses have nonserological
hemagglutinating properties
• ِThey can agglutinate RBCs in the
absence of Ab (nonimmune
agglutination)
20
Viral Hemagglutination
• Hemagglutination (HA) can be used to
determine titers of certain viruses.
• Mammalian reoviruses agglutinate
erythrocytes through interactions between
the viral surface protein sigma 1 and
carbohydrate groups attached to proteins
on erythrocyte membranes.
• Visually, this interaction creates a shield
of erythrocytes in a round-bottom 96 well
plate (well F1).
• In the absence of virus, the erythrocytes
form a button in the well (well F12).
21
Viral Hemagglutination
22
Slide indirect
Hemagglutination test
Exercise 4
23
Principle
• The Waaler-Rose (WR) reagent is a
suspension of stabilized sheep red cells
sensitized with anti-sheep rabbit IgG.
• The WR test reagent sensivity has been
adjusted to detect a minimum of 6
IU/mL of rheumatoid factors according
with the WHO International Standard
without previous sample dilution.
24
Sample
• Use fresh serum obtained by
centrifugation of clotted blood.
• The sample may be stored at +2 oC to
+8 oC for 48 hours before performing
the test.
• For longer periods of time the serum
must be frozen. Hemolytic, lipemic or
contaminated sera must be discarded
25
Reagents
• Waaler Rose: Suspension of
solubilized sheep erythrocytes
sensitized with rabbit IgG anti-sheep
erythrocytes
• Positive Control
• Negative Control
26
Preparation of reagents
• Shake the Reagent 1 (red cells) before use.
After that it must be uniform and without
visible clumping. The sensitivity of the test
depends of the drop volume (50 μl). Do not
use droppers than those provided and hold
the dropper perpendicular to the slide
surface.
• The reagent and controls have to be stored at
+2 oC to +8 oC. Do not freeze. In these
conditions the components will remain stable
until the expiry date printed on the label.
27
Procedure
• Before using the kit, allow the components
to reach room temperature.
• Gently shake the reagent to disperse the
particles. Check the reagent against the
positive and negative controls in the same
way as the undiluted serum.
28
1. Qualitative Determination
• Serum 1 drop (50 μl) + Reagent 1 drop
• Place a drop of UNDILUTED serum onto a circle of
the slide.
• Add a drop of the Reagent 1 (red cells) next to the
drop of serum.
• Mix both drops spreading them over the full surface
of the circle.
• Let the slide to stay on a flat surface for two minutes.
• After this time twist the slide 45 degrees once and let
again to stay for one minute.
• Read the presence or absence of visible
agglutination in this period of time.
• Unespecific agglutination could appear if the test is
read later than this time.
29
Interpretation of Results
• Examine macroscopically the presence3 or absence of
visible agglutination immediately avoiding any
movement or lifting of the slide during the observation.
• The presence of agglutination indicates a content of
RF in the sample equal or greater than 8 IU/ml.
• The lack of agglutination indicates a RF level lower than
8 IU/ml in the sample
30
Semi-Quantitative method
• This test is performed in the same way
as the qualitative test but by using
previous dilution of the serum sample in
saline (NaCl 9 g/l),
31
Semi-Quantitative method
32
Calculations
• The approximate RF concentration in
the patient sample is calculated as
follows:
8 X RF titer = IU/ml
33