2. 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.
4. 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.
Y
Y
Y
Click on image at right wait for animation to begin.
5. 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
6. Flocculation
Negative test Positive test
Uses fine particles of antigen to detect
antibody in patient’s serum.
Click on images above wait for animation to begin.
7. 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
11. 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
15. 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.
16. 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.
17. 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.
18. Inhibition of Agglutination
• 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.
Y
Y
Y
Y
Y
Y
Click on images at right wait for animation to begin.
20. 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.
21. Complement Fixation
• The patient’s serum is heated at 56o
C 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.
24. Parts of a labeled assay
• Analyte (labeled and unlabeled)
• Specific antibody
• Separation of bound and free
components
• Detection of label
• Standards/calibrators
25. Classification
• Heterogeneous: Method that requires a
step that separates bound analyte from
unbound analyte.
• Homogeneous: Method that does not
require a separation step.
26. 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.
27. 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.
29. 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.
30. 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.
31. 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.
32. 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
33. 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
35. 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.
37. Tube 1 2 3 4 5 6 7 8 9 10 11 12
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1:1 1:2 1:4 1:8 1:16 1:32 1:64 1:128 1:256 1:512 1:1024 control
38. 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.
39. Reasons to perform a titer
• Acute and convalescent
• Prenatal
• Verify past infection
• Confirm vaccination
40. Primary vs. Secondary Humoral
Response
First
exposure
Second
exposure
IgM
IgM
IgG
IgG
May use known antigen as a reagent to look for antibodies in a patient’s specimen.
Light scattered at 90 degrees in commonly measured.
Endpoint – reaction is allowed to go to completion. Problems with precipitate settling out, reducing the amount of scatter.
Rate – measures rate that scatter increases following addition of reagent.
Nephelometry can be used to quantitate serum protein levels.
Nephelometry can be used to detect serum proteins.
Most common application is testing for syphilis, detecting antibodies to reagin.
Patient’s serum is placed within a ring on a slide or card.
A measured volume of reagent containing antigen is placed in the ring.
The slide or card is rotated to mix the sample and reagent.
The reaction is examined for fine precipitates, which indicate a positive test.
Known antibody with multiple specificities is placed in center well. Known antigen is placed in an outside well. Patient specimen containing antigen is placed in other outside well. The pattern of precipitin lines is interpreted.
This test may be used to identify fungal antigens and antibodies to nuclear antigens.
Electrophoresis used to separate proteins on a gel according to size and electrical charge.
If the antibody applied to the gel is directed against a particular protein, precipitin bands form where Ag/Ab complexes have been trapped in the gel.
The gel is washed to remove any unprecipitated proteins, then stained to reveal the bands.
Example of an IgG monoclonal antibody with kappa light chains
SPE = Serum Protein Electrophoresis
Modification of IFE
Known antigens are electrophoresed to separate them.
The separated components are transferred to nitrocellulose paper by blotting the gel.
The patient’s serum is applied to the paper.
If the patient has antibodies to any of the antigens on the paper, it will form a precipitate.
Paper is washed and stained.
If antibody to more than one antigen of an organism is detected in the patient’s serum, infection with that particular organism is highly likely.
Uses include ID of bacteria, measuring hormone and drug levels, and measuring levels of some proteins.
Applications include ASO titers and anti-DNase B titers.
Hemolysin = antibodies known to activate complement and cause hemolysis.
This test is not used frequently, but has been used to detect antibodies to viruses, fungus and rickettsia.
Tests are fast, sensitive and specific.
Radioactivity, fluorescence, chemiluminous materials and enzymes have all been used as tags.
Often a wash step removes the unbound analyte. Other separation methods include adsorption coupled with centrifugation or filtration, magnets, or chemically modifying the test medium so that bound analytes remain in solution while Ag/Ab complexes fall out of solution.
When antibody binds to the labeled antigen, it blocks enzymatic activity, reducing the amount of color development.
The more patient antigen that binds to the antibody, the more enzyme-tagged antigen remains free to react with the chromogen.
Commonly used to test for drugs.
This test has been used for detection of Chlamydia, Legionella, RSV, and other antigens.
Anti-Human Globulin (AHG) is antibody to human antibodies. The Fab portion of AHG is directed at the Fc portion of the human immunoglobulin.
Clinical applications of indirect fluorescence include detection of viral, treponemal, and antinuclear antibodies.
Microbeads may be made of polysaccharides, polyacrylamide or magnetizable cellulose.
Free antigen can rotate when hit by polarized light whereas Ag/Ab complexes are too large to turn that quickly.
The degree of polarization is inversely proportional to the level of patient antigen.
Used to measure hormones and therapeutic drugs.
Requires sophisticated instrumentation that is specific to the chemical being used.
This labeling technique can be applied to heterogeneous or homogenous assays, and may detect antigens or antibodies.
Clinical applications include detection of drugs, hormones, and viral antigens.
Twofold dilutions are the most common, however other dilutions may be used.