Agglutination test

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

Typhoid fever
Brucellosis
ABO blood group

2
Agglutination test
• 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.
Stages of agglutination reaction
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
Secondary phase- Lattice formation
• 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)
Enhancement of lattice formation
• 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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An excess of antibody inhibits precipitation reactions, such excess can also inhibit
agglutination reactions; this inhibition is called the prozone effect.
Agglutination tests- type of particles
involved
• 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

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Types of agglutination Rxn
• Direct
• Passive
• Reverse Passive
• Agglutination Inhibition
• Coagglutination

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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, 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.
Passive Agglutination Test
• 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).

+ =

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Hemeagglutination
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).
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)
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)
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
Reverse Passive Agglutination test
• 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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 Agglutination Inhibition
Antibodies against the viral protein can prevent haemagglutination so
this phenomenon is called the haemagglutination-inhibition (HAI).
 Positive Negative

Hemagglutination inhibition for detection of antibodies

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
Co-agglutination reaction
Agglutination reaction
• 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
Slide Agglutination Test
• 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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Slide Agglutination Test

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Tube Agglutination Test
• 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

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Tube Agglutination Test

Agglutination No agglutination

1/10 1/20 1/40 1/80 1/160 1/320 Neg. ctrl

In this case, the titre is 40

 Positive

Negative

Positive Negative

Slide

Microtitration plate
 Coombs (Antiglobulin)Tests

• Direct Coombs Test

– Detects antibodies on erythrocytes

+ ↔

Patient’s RBCs Coombs Reagent

(Antiglobulin)
 Coombs (Antiglobulin)Tests

• Indirect Coombs Test

• Detects anti-erythrocyte antibodies in serum

Step 1
+ ↔
Patient’s Target
Serum RBCs

Step 2

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