CLS 420 Clinical Immunology &
Molecular Diagnostics
Immunologic Methods
Part 2
Basic Methods
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 patient’s 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
Uses fine particles of antigen to detect
antibody in patient’s serum.
Negative test
Positive test
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 antigen
Da
Dc
Db
Dd
Da
AG
D
AG
Anti-D
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.
Application point
Anode
(+ electrode)
Patient serum
Cathode
(- electrode)
IFE stained gels
= Serum application point
SPE
Anti-IgG
Anti-IgA
Anti-IgM Anti-Kappa Anti-Lambda
Western Blot
p24
gp 41
gp120/160
Negative
Control
Patient
specimen
Positive
Control
No bands
Patient bands compared to Pos Control
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 solid’s 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 patient’s 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 patient’s sample to detect antigen.
Click on image above wait for animation to begin.
Inhibition of Agglutination
Y
Y
Y
Y
• Antibody reagent is combined
with patient’s specimen.
• If patient’s 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.
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 patient’s serum is heated at 56oC for 30 minutes to
inactivate any complement present.
• Patient’s 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 patient’s 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
• Patient’s 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
•Patient’s 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
patient’s sample.
The test mixture is centrifuged (or
magnetized) to collect the beads, which
are then analyzed for fluorescence.
Fluorescent Polarization
Y
• 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.
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
1 2 3
4
5
6
7
8
9
10 11 12
0
0.2
ml
0.2
ml
0.2
ml
0.2
ml
0.2
ml
0.2
ml
0.2
ml
0.2
ml
0.2
ml
0.2
ml
0.2
ml
0.2
ml
0.2
ml
0.2
ml of
tube
2
0.2
ml of
tube
3
0.2
ml of
tube
4
0.2
ml of
tube
5
0.2
ml of
tube
6
0.2
ml of
tube
7
0.2
ml of
tube
8
0.2
ml of
tube
9
0.2
ml of
tube
10
0.2
ml of
6%
BSA
RBC
Suspen
sion
0.1
ml
0.1
ml
0.1
ml
0.1
ml
0.1
ml
0.1
ml
0.1
ml
0.1
ml
0.1
ml
0.1
ml
0.1
ml
0.1
ml
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
Tube
Saline
Serum
Results
• Titers provide more valuable information
when tested in parallel with a previous titer
specimen.
• A comparison of the current specimen’s
results and previous specimen’s 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
IgG
IgM
IgM
IgG
First
exposure
Second
exposure
Congratulations
You have finished
Immunology Student Lab
Lectures!
Good Luck on your exam!