Immunologic Methods

Part 2
Basic Methods
CLS 420 Clinical Immunology &
Molecular Diagnostics

Objectives
Explain the principle of each method presented, and give
a clinical use for each.
Contrast precipitation, agglutination and flocculation,
including:
Reaction time and conditions
Antigen state
Immunoglobulin class
Lattice formation
Describe heat inactivation of patient serum, including
method and purpose.
Discuss general reasons for performing immunologic
tests.
Precipitation Based Methods
Soluble antigen combines with
antibody to form aggregates which
precipitate out of solution.
Nephelometry
Antibody reagent is
combined with patient
sample.
If antigen is present in
the patients sample,
Ag/Ab complexes will
form and precipitate
out of solution.

Click on image at right wait for animation to begin.
Nephelometry
When light is passed
through the solution, the
precipitates cause the
light to scatter at various
angles.
The light that is scattered
at a particular angle is
measured. This
corresponds to the level
of antigen in the sample.
Light
source
Detector
Flocculation
Negative test Positive test
Uses fine particles of antigen to detect
antibody in patients serum.
Click on images above wait for animation to begin.
Double Immunodiffusion
Ouchterlony Method
Testing performed in
agar gel.
Antigen is placed in
one well.
Antibody is place in
other well.
Each diffuses
through gel.
If antibody is specific
to that antigen, a
precipitin line will
form where the 2
meet.
AG
AB
Double Immunodiffusion
Identity
Click on image above wait for animation to begin.
Double Immunodiffusion
Nonidentity
Click on image above wait for animation to begin.
Double Immunodiffusion
Partial Identity
D
AG
Anti-D
Da
AG
Da Db
Dd Dc
D antigen
Immunofixation Electrophoresis
Proteins separated by electrophoresis
Antiserum (antibody) is applied to the gel.
Ag/Ab complexes form in the gel.
The gel is stained to reveal precipitin bands.
Anode Patient serum Cathode
(+ electrode) (- electrode)
Application point
IFE stained gels
= Serum application point
SPE Anti-IgG Anti-IgA Anti-IgM Anti-Kappa Anti-Lambda
Western Blot
Negative Patient Positive
Control specimen Control

No bands Patient bands compared to Pos Control
p24
gp 41


gp120/160
Agglutination Based Methods
Antibodies cause the cross-linking
of particulate antigen, usually
found on a cell.
Direct Agglutination
The antigen is a natural
part of the solids surface.
Often performed at room
temperature.
May use centrifugation to
bring antigen and
antibody into closer
proximity.
Can be used to detect
antigen or antibody

Click on image at right wait for animation to begin.
Passive Agglutination
Antigens on a carrier molecule, such as latex,
combine with patients sample for antibody detection.
Click on image above wait for animation to begin.
Reverse Passive Agglutination
Antibody is bound to the carrier molecule, which is
then mixed with patients sample to detect antigen.
Click on image above wait for animation to begin.
Inhibition of Agglutination
Antibody reagent is combined
with patients specimen.
If patients specimen contains
the antigen for that antibody,
they will react.
Reagent antigen is added.
A positive reaction will show
no agglutination, because the
antibodies were bound to the
patient antigen before the
reagent antigen was added.
A negative reaction shows
agglutination between reagent
antibodies and antigen.
Y
Y
Y

Y

Click on images at right wait for animation to begin.
Other Methods
Neutralization
Positive Test Negative Test
The presence of an antibody prevents the
antigen from functioning correctly.
Click on images above wait for animation to begin.
Complement Fixation
The patients serum is heated at 56
o
C for 30 minutes to
inactivate any complement present.
Patients treated serum is incubated with known antigen
and a known quantity of guinea pig complement.
If the patient has an antibody to the antigen, they will
react and the complement will bind to the Fc pieces of
the antibodies.
Sheep RBCs that are coated with hemolysin are added.
The test is incubated, centrifuged and read for
hemolysis.
In a positive test, the complement will have been used
up by the patients antibody, and no hemolysis will be
present.
Complement Fixation
Negative test Positive test
Click on images above wait for animation to begin.
Labeled Methods
Attaches a tag to either the
antigen or antibody. This tag
can be detected and measured.
Parts of a labeled assay
Analyte (labeled and unlabeled)
Specific antibody
Separation of bound and free
components
Detection of label
Standards/calibrators
Classification
Heterogeneous: Method that requires a
step that separates bound analyte from
unbound analyte.
Homogeneous: Method that does not
require a separation step.
Competitive EIA
Enzyme labeled
antigen competes with
unlabeled patient
antigen for binding
sites on fixed
antibodies.
A chromogen is added
that reacts with the
enzyme.
The level of color
development is
inversely proportional
to the level of patient
antigen.
Click on image at right wait for animation to begin.
Capture (Sandwich) EIA
Patients sample is
incubated with bound
antibody.
Following a wash, a
second antibody that is
labeled with a
chromogen is added.
The level of color
development
corresponds with the
amount of antigen
captured.
Click on image at right wait for animation to begin.
Enzyme-multiplied Immunoassay
Technique (EMIT)
Y Y Y Y
This is a homogeneous competitive binding assay.
Color development is _______proportional to the
concentration of antigen.
Direct Fluorescence
Negative test Positive test
Fluorescently labeled antibody is used to detect
antigen fixed to a slide.
Click on images above wait for animation to begin.
Indirect Fluorescence
Positive Test Negative Test
Known antigen fixed to slide
Patients serum added (unknown antibody)
Incubation & wash
Fluorescently labeled anti-human globulin reagent added.
Click on images above wait for animation to begin.
Microparticle Capture
Uses microbeads coated with known
antigen or antibody.
The beads are incubated with a
fluorescently labeled analyte and the
patients sample.
The test mixture is centrifuged (or
magnetized) to collect the beads, which
are then analyzed for fluorescence.
Fluorescent Polarization
Free labeled antigen excited
by polarized light emits
unpolarized light.
Labeled antigen/antibody
complexes excited by
polarized light emit polarized
light.
FPIA is a competitive binding
assay in which labeled antigen
competes with unlabeled
(patient) antigen for antibody
binding sites.
The more labeled antigen that
is bound to antibody, the more
polarized light is emitted.
Y
Chemiluminescence
Uses chemical labels that, when
oxidized, produce a substance of a
higher energy level.
When this substance decays to its
original state, it emits energy in the form
of light.
Common label materials include:
Luminol
Acridium esters
Peroxyoxalates
Comparing Antibody
Quantities
Antibody titers
Antibody Titer
An antibody titration can help determine
antibody concentration levels.
Twofold serial dilutions of serum
containing an antibody are made, then
tested against cells possessing the target
antigen.
The titer is the reciprocal of the greatest
dilution in which agglutination is observed.
Two-fold Serial Dilutions
Tube
1 2 3 4 5 6 7 8 9 10 11 12

Saline
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Serum
0.2
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tube
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6%
BSA
RBC
Suspen
sion
0.1
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Final
Dilution
1:1 1:2 1:4 1:8 1:16 1:32 1:64
1:128 1:256 1:512
1:1024 control
Results
Titers provide more valuable information
when tested in parallel with a previous titer
specimen.
A comparison of the current specimens
results and previous specimens current
results should be made.
A change in titer of 2 or more tubes is
considered to be significant.
Reasons to perform a titer
Acute and convalescent
Prenatal
Verify past infection
Confirm vaccination
Primary vs. Secondary Humoral
Response
First
exposure
Second
exposure
IgM
IgM
IgG
IgG
Congratulations
You have finished
Immunology Student Lab
Lectures!
Good Luck on your exam!