Agglutination reaction
immunology Tutorial
MLT402
PREPARE BY :
1) Saad Dahesh Ali Al-Amri
2) Salem Dahesh Ali Al-Amri
3) Saleh Al-Juhani
Mr. Mohamed AL-Khatatneh
Agglutination

Is the clumping of antibody–antigen complex.

Reaction occurs between insoluble antigen and
appropriate antibody.

The reaction will result in forming visible
aggregates or agglutinate
Fig1: agglutination Rex.
Stages of agglutination reaction
Phase one
 Antibody reacts with single antigenic
determinants on or close to particle
surface.
 It is a rapid reaction.
Stages of agglutination reaction
Phase one
Stages of agglutination reaction
Secondary phase

A single antibody molecule binds to antigenic determinants
on adjacent particles.

The visible reaction occur under appropriate conditions
Stages of agglutination reaction
Secondary phase
Factors affecting the agglutination reaction in
vitro:
1. Antigen to antibody ratio:
 The ratio between antigen and antibody influences the
detection of antigen-antibody complexes.
 Antigen or antibody excess make invisible reaction.
◦ Prozone phenomenon (antibody excess):
 There are too many antibodies.
no agglutination appears (false-negative reactions).
◦ Zone of equivalence:
 Antibodies and antigens are present in an optimum ratio.
 This leads to cross-linkages between acells or particles, so
agglutination appers (positive reaction)
Factors affecting the agglutination reaction in
vitro. Cont :

Post-zone phenomenon (Antigen
excess):
 There are too many antigens

These false-negative reactions can be
detected by repeating the test at a
higher dilution of sample, which
reduces the antigen or antibody
concentration into the range that
produces visible agglutination.
Factors affecting the agglutination reaction in
vitro. Cont :
2. Number of Antigen Sites:
 The more antigen sites on a cell result in
more cross-linkages between cells.

These cross-linkages result in more visible
agglutination.
Factors affecting the agglutination reaction in vitro.
Cont :
3. Size and Structure of the Antibody:
 Larger antibody causes more cross-linkages between
different cells.
 The larger antibodies (IgM) can reach between more
antigen sites on different cells and therefore causing
stronger agglutination reactions.
4. Distance between cells:
 Centrifugation of the cells attempts to bring the cells
closer together, so enhance agglutination.
Classification of agglutination
reactions
1. Direct Agglutination
The antigen is a natural part of
the solid’s surface.
 at room temperature.
 May use centrifugation to
bring antigen and antibody
into closer proximity.
 Can be used to detect antigen
or antibody

Examples:
 ABO blood group typing
 Rh (D) Ag

2. Passive Agglutination (indirect)


Passive Agglutination is a very sensitive method for antibody
detection.
RBC, bacterial cells or inert particles such latex can be used as a
carrier for antigens.

This makes the reaction more visible.
 latex -------------- latex agg.
 RBC ----------------passive heamagg.

If Antibody is attached to the particulate carrier called Reverse
Passive Agglutination
Passive Agglutination. Con’t

Example:
1) Rheumatoid factor detection.
2) C-Reactive Protein detection (CRP)
Passive Agglutination
Antigens on a carrier molecule, such as latex, combine
with patient’s sample for antibody detection.
Reverse Passive Agglutination
•
Antibody is bound to the carrier molecule, which is then
mixed with patient’s sample to detect antigen.
•
Uses include ID of bacteria, measuring hormone and drug
levels.
Agglutination reaction
Glass slides
 Tubes
 Microtiter plates

Advantages of agglutination methods
ease of performance.
 speed of performance, usually requiring
few minutes.
 high degree of sensitivity.

Disadvantages of agglutination methods
the reaction are only semiquantitative.
 the occurrence of the prozone
phenomenon, in which agglutination is
inhibited by extreme antibody.

Limitations:
1)
2)
3)
The technique for shaking the tubes to detect
agglutination is critical. Harsh shaking may cause weak
agglutinates.
Test should be performed regularly,
Dispensing incorrect quantities of diluent or reagent
or transferring more or less than the required amount
of diluted serum will affect the outcome of this test .
False positive and false negative
agglutination :
False positives w/ agglutination :
1) contaminated glassware
2) 2) overcentrifugation of cells
3) 3) autoagglutination
 False negatives w/ agglutination :
1) improper condition for test: specimen
not prop prepared
2) expired/improperly stored reagents
3) too much Ab or Ag

Materials required :
Timer
Disposable
Tube rack
test tubes
Mechanical
slide rotator
Materials
Required
Stir Sticks
Reaction Slide or
Physiological Saline
Microtiter Plate
used 0.9% NaCl
Serological
pipettes
Procedure :
1)
2)
3)
4)
5)
6)
Keep regent to take RT
Mixing
Put one drop of serum with one drop of
test reagent
Mixing
Rotate
Observe agglutination .
Factors Affecting Agglutination
Class of Antibody.
.1
Charge of the Carrier Particle.
.2
Number of Antigenic Sites .
.3
Concentration Of Reaction .
.4
Environmental Factor.
amna alotiby 2008-2009
.5
Result
GRADING AGGLUTINATION REACTIONS
GRADE
DESCRIPTION
Negative (-)
No aggregates
1+
A few small aggregates just visible macroscopically;
2+
Medium-sized aggregates;
3+
Several large aggregates;
4+
One solid aggregate;

Rheumatoid Factor (RF) Testing by latex
agglutination :
Introduction RF :

Rheumatoid arthritis (RA) is a chronic inflammatory
disease affecting primarily the joints and tissues. For
many years it has been known that several abnormal
proteins circulate in the blood of patients with RA.
These proteins, because disease, became known as
rheumatoid factor (RF).
Principle RF test :

Rheumatoid factor (RF) is an anti-antibody, which invitro, is detected by its ability to agglutinate latex
particles (or red blood cells) coated with human IgG. RF
in patient sample, if present, will attach to the IgG
coating the latex particles. Agglutination of the latex
particles is a positive result indicating the presence of
RF.
Materials:
1. Rheumatoid factor test kit(s).
2. Patient and control serum specimens.
3. Timer
4. Other materials as directed by reagent
product insert(s).
Procedure:
See reagent product insert(s)
Interpretation &Expected Results :

Interpretation : Agglutination of latex
particles is considered a positive reaction,
indicating the presence of rheumatoid factor at
detectable level..

Expected Results : Although the diagnosis
of rheumatoid arthritis is based largely on
clinical findings .
Limitations:
1). RF is not detected in all patients diagnosed
with RA.
2) Some products may produce questionable
results from hemolyzed, lipemic or
contaminated specimens.
3) Avoid contamination of reagent or dropper.
ASO Latex Test

Introduction : ASO Latex Test is a rapid
latex agglutination test for the qualitative
and semi-quantitative determination of
antistreptolysinO antibodies (ASO) in
serum.
PRINCIPLES :

The ASO Latex Test is a stabilised buffered
suspension of polystyrene latex particles
that have been coated with Streptolysin O.
When the latex reagent is mixed with a
serum containing ASO, agglutination occurs.
The sensitivity of the latex reagent will yield
agglutination when the level of ASO is
greater than 200 IU/ml, a level determined
to be indicative of disease by epidemiological
and clinical studies.
MATERIALS :
1. ASO Latex ( kit )
2. ASO Positive Control
3. ASO Negative Control
4. Glass Reaction Slide.
5. Disposable wooden stick
PROCEDURE
1) Take test reagents and samples to room temperature.
2. Use pipette to take drop of sample into its identified
circle of the slide. Retain each pipette for mixing.
3. drop of positive and negative control into its identified
circle.
4. Mix the ASO latex reagent by gently shaking. Add one
drop of reagent to each control and sample.
5. Using the wooden stirrer thoroughly mix each sample
with reagent within the full area of the circle. Discard
the disposable stirrer.
6. Wait two (2) minutes and observe for agglutination
under a high light.
7. Record results.
RESULTS

test sample is considered to contain ASO
antibodies when agglutination is observed
when compared to the result of the
negative control .
LIMITATIONS OF THE
PROCEDURE



Serum samples showing gross hemolysis, lipemia should
not be used .
The test reaction must be read immediately following
the two (2) minute
. Only serum specimens should be used. Do not use
plasma samples as they could cause non-specific
agglutination of the latex .
Reference :
MLAB 1235 Immunology/Serology
 http://quizlet.com/7317754/immunologyagglutination-flash-cards/
 . Rantz LD, DiCaprio JM, Randall E. Am .J.
Med. Sci., 24, 1952. 2. Halbert,SP. Ann. N.Y.
Acad. Sci., 103, 1027:1051; 1963
