Experiment 1:

Serial Dilution and

Agglutination Reaction
Group 6, MT III-B
Balatbat, Charlene
De Leon, Sheila Mae
Maniulit, Jinni
Santos, Ian Lawrence
• measured amount of a serum sample - used
for detection of antibodies
• Ag and Ab should be proportional to each
other

• Agglutination – visible aggregation of particles

caused by combination with specific antibody.
• Agglutinins – antibodies that produce such
agglutination
• Agglutination Process:
sensitization – initial binding
lattice formation - formation of large aggregates
 Procedure
• Macrotiter
 Procedure
• Microtiter
 Simple Dilution
• two entities
Solute – material being diluted
Diluent – medium making up the rest of the
solution

• expressed as a fraction:
denominator – total volume (solute + diluent)
Numerator – volume of solute

1/Dilution = Amount of Solute/Total Volume

 Simple Dilution
2 mL of a 1:20 dilution is needed to run a specific
serological test. How much serum and how much diluent
are needed to make this dilution?

1/20 = x/2 mL

• 20 = total number of parts in the solution

• 2 mL = total volume desired
• Do cross-multiplication
• Serum = 0.1mL

Diluent = 2mL – x mL
= 2mL – 0.1mL
= 1.9mL
 Compound Dilutions
• make very large dilution with small amounts of solute
and diluent
• plan the number and sizes of simple dilutions
necessary to reach the desired end point

• Ex: Make a 1:500 dilution

• (5)(10)(10) = 500
 Serial Dilution
• used to obtain a titer (indicator of an
antibody’s strength)

• A series of test tubes is set up with exactly the

same amount of diluent in each = same
dilution

• most common: doubling dilution = amount of

serum is cut in half with each dilution
 Serial Dilution
For example,
 Serial Dilution
• final dilution: count number of tubes and set
up a multiplication series in which the original
dilution factor is raised to a power equal to
the number of tubes
 Agglutination Reactions
• visible clumping due to interaction between
antibody and antigen
• Agglutinins - antibodies that produce such
reactions

• Prozone effect: excess of antibody causing

inhibition of agglutination reactions
high antibody concentrations: the number of antibody
binding sites may greatly exceed the number of
epitopes
most antibodies bind antigen only univalently instead
of multivalently
Antibodies that bind univalently cannot crosslink one
antigen to another
Questions
for
Research
 Write the steps that you will undertake to
prepare 1:5 dilution of Anti-A antisera.

• Transfer 4mL of NSS into a test tube.

• Transfer 1mL of anti-A antiserum into the same
tube.

1/Dilution = Amount of Solute/Total Volume

1/5 = 1mL antiserum/(4mL NSS + 1mL antiserum)
1/5 = 1/5
 List down factors which may affect
agglutination reactions.
• Class of antibody
• Number of epitopes
• Environmental factors:
Agitation
Temperature
pH
incubation time
 Give at least two uses of serial
dilution of serum.
• used to make high dilutions with a small
number of test tubes and a minimal amount
of diluents

• done to determine the strength or titer of a

particular antibody in patient serum
 If 0.3 mL serum and 3.0 mL normal saline solution
were available in the laboratory, how would you
prepare a 1:1000 dilution? You cannot use more than
3.0mL NSS.

• A diluent of 0.9 mL is added to three tubes.

0.1 mL of the serum is added to tube 1. 0.1 mL
from tube one will be transferred to tube 2,
and 0.1 mL from tube 2 will be transferred to
tube 3.

(10)(10)(10) = 1000
List down the manner of reporting for
agglutination reactions.

• Stevens, “Clinical Immunology and Serology –

A Laboratory Perspective,” 2009, page 140
 How is agglutination different from
precipitation?
Precipitation
• aggregation of soluble antigens
• combination of soluble antigen with soluble
antibody to produce a visible insoluble complex

Agglutination
• specific antigens aggregate to form larger visible
clumps when corresponding specific antibody is
present in the serum
Enumerate the immunoglobulins which participate in
agglutination reactions.

IgG
• cannot bridge distance between particles,
because of their small size and restricted
flexibility at the hinge region
• agglutinate best at 30°C to 37°C
• Valence: 2.5

IgM:
• 35nm diameter
• react best at 4°C – 27°C.
• potential valence: 10
 Enumerate the types of agglutination
reactions and differentiate one from
the other through their characteristics
and applications.
 Direct Agglutination
• agglutination occurs when antigens are found
naturally on a particle

Example:
• Hemagglutination:
 ABO blood group typing of human red blood cells
 Passive Agglutination
• employs particles coated with antigens not
normally found on their surfaces
• latex, gelatin, and silicates

Used to:
• detect rheumatoid factor
• antinuclear antibody in lupus erythematosus
• antibodies to group A streptococcus antigens
• antibodies to Trichinella spiralis, Treponema
pallidum, viruses
Passive Agglutination
 Reverse Passive Agglutination
• antibody rather than antigen is attached to a
carrier particle

• The antibody must still be reactive and is joined

in such a manner that the active sites are facing
outward

• This type of testing is often used to detect

microbial antigens
 Agglutination Inhibition
• based on competition between particulate and soluble
antigens for limited antibody-combining sites

• involves haptens (non-antigenic molecules that react with

specific antibodies ) complexed to proteins
 sample is reacted with limited amount of reagent antibody that
is specific for the hapten being tested
 Indicator particles that contain the same hapten are added
 no free hapten = reagent antibody is able to combine with
carrier particles and produce a visible agglutination.

• Agglutination is a negative reaction: patient did not have

sufficient hapten to inhibit the secondary reaction.
 Agglutination Inhibition
Hemagglutination inhibition

RBCs – indicator particles

• have naturally occurring viral receptors
• Presence of virus = spontaneous agglutination
• virus – links RBCs together
• Presence of patient antibody – inhibits
agglutination reaction
 Coagglutination
• systems using bacteria as the inert particles to
which antibody is attached

• Staphylococcus aureus: has a protein on its outer

surface (protein A) which naturally adsorbs the
fragment crystallizable (FC) portion of antibody
molecules

• The active sites face outward and are capable of

reacting with specific antigen
Coagglutination
 Define
• Agglutinogen – antigenic substance
that stimulates the formation of specific agglutinin

• Agglutinin – antibodies that produce agglutination

• Prozone – excess antibody; antigen combines with

only one or two antibody molecules and no cross-
linkages are formed

• Postzone reactions – excess antigens; small

aggregates are surrounded by excess antigen and no
lattice formation is established
 Give the two steps involved in
agglutination reactions.
• Sensitization – involves antigen–antibody
combination through single antigenic
determinants on the particle surface

• Lattice Formation – formation of cross-links that

form the visible aggregates
stabilization of antigen–antibody complexes with the
binding together of multiple antigenic determinants
Antibody must be able to bridge the gap between
cells
Give the two steps involved in agglutination reactions.
 Enhancement of Lattice Formation
• 5-30% albumin – neutralizes surface charge and allows
red cells to approach each other more closely

• Increase viscosity (dextran or polyethylene glycol) –

reduce hydration around cells and allow them to come
into closer proximity for antibody to join together

• Enzymes (romelin, papain, trypsin, ficin) - reduce

surface charge on RBCs through cleaving of chemical
groups and decreasing hydration

• Agitation and centrifugation – physical means to

increase cell–cell contact

• pH: 6.5 - 7.5

 Give at least five causes of false positive
and agglutination reactions.
Causes of False Positive
Effect
Reaction
Button is packed too tight and is difficult to
Overcentrifugation
resuspend.
Contaminated glassware, Contaminants (dust, dirt, or finger prints) may
slides, or reagents cause cells to clump.
Test cells clump without specific antibody
Autoagglutination
present.
Delay in reading a slide
Dried out antigen may look like agglutination.
test
Saline stored in glass Colloidal silica may leak out and cause
bottles agglutination.
 Give at least five causes of false
negative and agglutination reactions.
Causes of False
Effect
Negative Reaction
Under centrifugation Cells may not be close enough to interact.
Inadequate washing of Unbound immunoglobulins may neutralize the
cells antihuman globulin.
Delays in testing Antibody may be eluted from red blood cells.
procedures
Incorrect incubation Too low a temperature may result in the lack of
temperature association of antigen and antibody.
Insufficient incubation Antigen and antibody may not have time for
time association.
 Reference
• Stevens, “Clinical Immunology and Serology –
A Laboratory Perspective,” 2009, page 115-
117, 138-139, 140-142, 145

• Turgeon, “Immunology and Serology in

Laboratory Medicine,” 2014, page 139