The document discusses the principles and methods of agglutination reactions, highlighting their relevance in serological testing for detecting antigens and antibodies. It explains various types of agglutination tests, including direct, passive, and reverse passive agglutination, with examples of their applications in diagnosing diseases. Additionally, it contrasts agglutination with precipitation reactions regarding their sensitivity and the nature of the formed complexes.

1. Ajay Subedi
Janamaitri foundation institute of
health
sciences,Balaju,Kathmandu,Nepal

2. 2
In 1896, Gruber and Durham published the
first report about the ability of antibody to
clumps cells, based on observation of
agglutination of bacterial cells by serum
ABO blood group
Typhoid fever
Brucellosis

3. • It is one of important laboratory method to detect
antigen antibody reaction.
• It provides flexible and useful method for semi
quantitating of either antigen or antibody
concentration.
• The reaction occurs between insoluble antigen
and appropriate antibody.
• The reaction will results in forming aggregate or
agglutinate.
• Antibodies that produce such reactions are
called agglutinins.

4. Primary phage- Sensitization
• Antibody reacts with single
antigenic determinants on
or close to particle surface.
• It is a rapid and reversible
reaction.
• Based on Law of Mass
Action

5. • A single antibody molecule binds to
antigenic determinants on adjacent
particles.
• The visible reaction occur under
appropriate conditions and over
time, particles remain connected
and interconnected by antibody
bridge.
• IgM is 700 times more efficient in
agglutination than IgG (Restricted
hinge region of IgG than IgM)

6. • Erythrocytes and bacterial cells have slightly
negative charge in their surface
• Like charges tend to repel
• Use of low ionic strength saline (Use of 5 to 30%
albumin neutralize the surface charge)
• Increase the viscosity (red cells)- add dextran or
polyethylene glycol (PEG)
• Agitation and centrifugation
• Temperature- (IgG- 30-37/ IgM-4-27); ABO-IgM
• pH- best 6.5-7.5; IgM- best at low pH
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7. An excess of antibody inhibits precipitation reactions, such excess can also
inhibit agglutination reactions; this inhibition is called the prozone effect.

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• Antibodies can agglutinate multivalent particulate
antigens, such as Red Blood Cells (RBCs) or bacteria or
latex particles
• Some viruses also have the ability to agglutinate with
RBCs.
• This behavior is called agglutination.
• Serological tests based on agglutination are usually more
sensitive than those based on precipitation

9. • Direct
• Passive
• Reverse Passive
• Agglutination Inhibition
• Coagglutination
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10. Active/Direct agglutination test
combination of an insoluble particulate antigen with its soluble antibody
– forms antigen-antibody complex
– particles clump/agglutinate
• used for antigen detection .
Examples
 The particle antigen may be a bacterium. e.g.: Serotyping of E. coli, Salmon
using a specific antiserum.
 The particle antigen may be a parasite. e.g.: Serodiagnosis of Toxoplasmosi
 The particle antigen may be a red blood cell. e.g.: Determination of blood gro

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• Converting a precipitating test to an agglutinating test
• Chemically link soluble antigen to inert particles such as
LATEX carbon, gelatin, silicates or RBCs.
• Particle size- 7um to 0.8um
• Addition of specific antibody will cause the particles to
agglutinate
• Examples: RA factor, antinuclear antibodies (ANA) –
detects autoimmnity, ASO, Spirochete antibody(TPHA).
+ =

12. E.g. Rose waaler test –detects rheumatoid arthritis
• RA factor in rheumatoid arthritis patients acts as antibody
to human gamma globulin.
• It can agglutinate red cells coated with globulins
• Antigen is sheep RBCs suspension sensitized with rabbit
anti sheep erythrocytes antibody (amboreceptor).

13. Examples
• Microhemagglutination test for Syphilis (MHA-TP)
• Hemagglutination treponemal test for Syphilis (HATTS)
• Passive hemagglutination tests for antibody to
extracellular antigen of Streptococci
• Rubella indirect hemagglutination test
• Hemagglutination Inhibition Test (HAI) for Avian
Influenza
• Quantitative Micro Hemagglutination Test (HA)

14. 2.Latex agglutination : latex particles used as carrier molecules
Eg: antigen coated latex particles are used in detection of ASO ,
S.agalactiae, Clostridium difficile toxins A and B , rotavirus
Antibody coated latex particles are used in detection of CRP, RA
factor, HCG.
Reaction is dependent on
• Amount and avidity of antigen bound to carrier
• Time of the incubation with specimen
• Environment of interaction (pH, Protein concentration etc)

15. 3.RPR or rapid plasma reagin:
• Used for syphillis
• Carbon is the carrier particle
• Cardiolipin antigen is coated with charcoal (carbon)
• Detect reagin – an antibody like substance in the plasma
of syphillis

16. • Antibody is attached to the particulate carrier. One of the
most commonly used laboratory tests that involves latex
particle agglutination is the procedure for C-Reactive
Protein (CRP).
• Reverse PAT: antibody linked to LATEX
e.g. Lancefield grouping in Streptococci.
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17. Antibodies against the viral protein can prevent
haemagglutination so this phenomenon is called the
haemagglutination-inhibition (HAI).

18. Positive Negative
Hemagglutination inhibition for detection of antibodies

19. Co-agglutination:
• The test is based on the presence of protein A on the
outer surface of Staphylococcus aureus produced by
most strains(Heat killed Cowan 1 strain).
• The antibody(IgG) binds by Fc portion to protein A and
the antigen combining Fab portion is free.
• Coagglutination test can be done for sterptococcus(
lancefield groups), Spneumoniae, N.gonorrhoea,
N.meningitidis

21. • When a particulate antigen is mixed with its antibody in
the presence of electolytes at a suitable temperature and
pH, the particles are clumped or agglutinated
Agglutination tests can be done :
• On slides
• In tubes
• In microtritation plates

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• Used for serotyping (e.g. Salmonella)
• Antigen: isolated Salmonella in suspension
• Antibody: specific antisera against Salmonella
Procedure:
• Place test Salmonella in a drop of saline on a
slide,
• Add a drop of antiserum, mix and rock slide for
approx. 1 minute.
• Examine for agglutination

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• Also known as the standard agglutination test or serum
agglutination test (SAT)
• Test serum is diluted in a series of tubes (doubling
dilutions)
• Constant defined amount of antigen is then added to
each tube and tubes incubated for ~20h @37°C
• Particular antigen clumps at the bottom of the test tube
• Test is read at 50% agglutination
• Quantitative
• Confirmatory test for ELISA reactors
• Example: Brucellosis screening

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Tube Agglutination Test

27. No agglutination
Agglutination
1/10 1/20 1/40 1/80 1/160 1/320 Neg. ctrl
In this case, the titre is 40

28. Negative
Positive
Positive Negative
Slide
Microtitration plate

29. • Direct Coombs Test
– Detects antibodies on erythrocytes
+ ↔
Patient’s RBCs Coombs Reagent
(Antiglobulin)

30. • Indirect Coombs Test
• Detects anti-erythrocyte antibodies in serum
Patient’s
Serum
Target
RBCs
+ ↔
Step 1
+ ↔
Coombs Reagent
(Antiglobulin)
Step 2

31. • • Weil – Felix Test or Reaction in Serodiagnosis
of typhus fevers is heterophile agglutination test
and sharing of common antigen between typhus,
Rickettsiae and some strains of Proteus bacilli.

32. • Positive in Mycoplasma ( Primary Atypical )
Pneumonia
• The patients sera agglutinated human O group
erythrocytes at 4 o c the agglutination being
reversible at 37 0 c.

33. Properties Precipitation reaction Agglutination reaction
Definition It is the antigen antibody
reaction where the antibody
reacts with the soluble antigen
to form precipitin
It is the antigen antibody
reaction where the antibody
reacts with the soluble antigen
to form agglutinin
Size of an antigen larger Comparatively smaller
Solubility of antigen Soluble form Sedimented form
Sensitivity Less sensitive More sensitive
Media used Either liquid or gel matrix Does not require
Types It is of three types:
Precipitation in solution,
precipitation in agar by
diffusion and electrophoresis
It is of two types: Active and
passive agglutination
Matrix Precipitation reaction can be
performed on glass slides,
petri plates and test tubes
Agglutination reaction can be
performed on microtitre plate,
glass slides and test tubes
Resulted compound Precipitins Agglutinins
Formation of resulted
compound
Either found as suspension or
sink to the bottom
The end product sinks to the
bottom
Appearance of end product Appear as large, insoluble
mass of visible precipitate
Appear as large visible
aggregates

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