2. Basic Immunologic
Procedures
Part 6 Labeled Immunoassays
Terry Kotrla, MS, MT(ASCP)BB
Labeled Immunoassays
Some antigen/antibody reactions not detected
by precipitation or agglutination.
 Looking for very small amounts.
 Measured indirectly using a labeled reactant.
 Referred to as receptor-ligand assays.
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Terminology
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Ligand is the substance to be measured.
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Defined as a molecule that binds to another molecule
of a complementary configuration.
Usually binds to the substance the test is trying to
detect.
The receptor is what binds the specific target
molecule.
 “Sandwich” technique is an example.
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“Sandwich” Technique ELISA
Terminology
Reactions may be competitive or noncompetitive
 Competitive – labeled known and patient
unknown are added to reaction and “compete”
for the target.
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For example, looking for an antibody.
Add labeled reagent antibody of known specificity,
patient sample and known antigen.
Patient antibody competes with reagent antibody for
the target antigen.
Concentration is inversely proportional to results.
Terminology
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Non-competitive
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Add patient sample, for example looking for antibody,
to known reagent antigen.
Reaction occurs and the concentration is directly
related to the amount of antibody in patient sample.
Terminology
Heterogeneous or homogeneous
 Heterogeneous assays called separation
assays
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Require multiple steps
Careful washing of surface to remove unbound
reagents and samples.
Homogeneous assays do NOT require a
separation step.
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Mix reagents and patient sample.
Measure the labeled product.
Competitive Binding
Add known labeled antigen
 Add unknown antigen
 Will compete with each other for sites on bound
antibody molecule.
 Must wash off unreacted substances.
 Type of label on known antigen will determine
method of detection.
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Competitive Binding
Noncompetitive Binding
Patient sample added.
 Will react with its homologous antigen or
antibody, depending upon what is being tested
for.
 The reaction is measured and the concentration
is directly related to the detected amount.
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Standards or Calibrators
Substance of exact known concentration.
 Usually run for each new lot number
 Based on results create standard curve.
 Standard curve used to “read” results or built
into machine to provide results.
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Labels
Used to detect reaction which has occurred.
 Most common are:
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Radioactive
Enzymes
Fluorescent
Chemiluminescent
Radioimmunassay (RIA)
Competitive binding
 Uses Iodine 125 (I 125) as label
 Radioactive label competes with patient for sites
 High radioactivity, small amount of patient
substance
 Low radioactivity high amount of patient
substance.
 Refer to your textbook for diagrams.
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Radioimmunoassay
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Sensitive technique used to measure small concentrations of antigens.
Known quantity of antigen is made radioactive, usually with Iodine 125.
Known labeled antigen and patient sample added to the reagent antibody.
Known antigen will compete with the unknown patient antigen for sites on
the antibody.
The bound antigens are separated from the unbound ones.
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Can measure the radioactivity of labeled free antigen in the supernatant solution.
Can measure radioactivity of fixed labeled antigen to the well.
High radioactivity indicates a low concentration of patient antigen was
bound to the reagent antibody.
Low radioactivity indicates a high concentration of patient antigen was
bound to the reagent antibody.
Thus, the results are inversely related to the label detected.
Standards are run and results read off of standard curve.
Radioimmunoassay
Radioimmunassay
Radioimmunoassay Competitive
Immunoradiometric Assay (IRMA)
Labeled antibody plus patient antigen
 Solid phase antigen added to bind excess
antibody.
 Labeled antibody binds to both patient antigen, if
present, and bound antigen.
 Spin down to separate
 Labeled antibody/antigen remain in solution.
 Measure radioactivity.
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Advantages/Disadvantages
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Advantages
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Disadvantages
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Extremely sensitive and precise
Detects trace amounts of analytes small in size.
Health hazard
Disposal problems
Short shelf life
Expensive equipment necessary
Enzyme immunoassays have largely replaced
radioimmunoassay.
Enzyme Immunoassay
Enzymes occur naturally and catalyze
biochemical reactions.
 Enzymes are
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Cheap
Readily available
Have a long shelf life
Easily adaptable to automation.
Automation relatively inexpensive.
Enzyme Immunoassay
Techniques pose no health hazards.
 Little reagent enzyme necessary.
 Can be used for qualitative or quantitative
assays.
 Enzymes selected according to
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Substrate molecules converted per molecule of
enzyme.
Ease and speed of detection.
Stability.
Availability and cost
Enzyme Immunoassay
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Enzymes used include:
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Horseradish peroxidase
Glucose-6-phosphate dehydrogenase
Alkaline phosphatase
Β-D-galactosidase
Horseradish peroxidase and alkaline
phosphatase are the most popular.
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Highest turnover
High sensitivity
Easy to detect
Heterogenous EIA
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Competitive
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Enzyme labeled antigen competes with unlabeled
patient antigen for antibody sites.
Wash to remove unbound reactants.
Add substrate which causes color change.
Results are inversely proportional to concentration.
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More patient antigen bound, less color.
If little or no patient antigen bound, dark color.
Used to measure small antigens such as insulin and
estrogen.
Competitive ELISA
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Unknown antigen competes with labeled known antigen
Enzyme produces color reaction
Heterogenous EIA
Noncompetitive are very popular.
 Often referred to as enzyme linked
immunosorbent assay – ELISA
 Enzyme labeled reagent DOES NOT participate
in the initial antigen-antibody reaction.
 Sandwich technique
 Advantages
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High sensitivity and specificity.
Relatively simple to perform.
Low cost.
Noncompetitive EIA
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Variety of solid support
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Microtiter plates
Nitrocellulose membranes
Magnetic beads
Procedure
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Antigen bound to solid phase
Add patient sample, antibody will bind if present
Wash
Add known enzyme labeled antibody
Wash
Add substrate
Measure enzyme label
Positive Reaction = Color Change
Sandwich or Capture Assays
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Antibody bound to solid phase.
If looking for antigen must have multiple epitopes, bound
antibody specific for one epitope, second labeled
antibody added specific for a different epitope.
Antigens detected can be
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Antibodies
Hormones
Proteins
Tumor markers
Microorganisms especially viruses
Enzyme label used to detect reaction
Sandwich or Capture Assays
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Add patient sample with antigen.
Antigen will bind to antibody bound to solid phase.
Add enzyme labeled antibody directed against a different
epitope on the antigen.
Add substrate, measure intensity of color.
Rapid Immunoassays
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Membrane based cassettes are rapid, easy to perform and give
reproducible results.
Popular in POCT and home use.
Designed to be single use and disposable.
Membrane coated with antigen or antibody produces color reaction.
Rapid Immunoassays
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Immunochromatography
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Apply sample to one end, migrates forward.
Sample dissolves labeled antigen or antibody to which it binds.
Migrates towards detection zone where it will bind to immobilized
antigen or antibody.
Color change occurs.
Homogeneous Enzyme Assay
Reaction which requires NO separation of
reactants.
 Less sensitive BUT rapid, easy to perform and
automate.
 Chief use is to detect low molecular weight
analytes such as:
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Hormones
Therapeutic drugs
Drugs of abuse
Can use serum or urine.
Homogeneous Enzyme Assay
Based on principle of change in enzyme activity
as specific antigen-antibody combinations occur.
 Reagent antigen labeled with enzyme tag.
 Antibody binds to specific determinant sites on
antigen, active site on enzyme blocked, causes
measurable loss of activity.
 Free antigen competes with enzyme-labeled
antigen for limited number of antibody sites.
 Enzyme activity directly related to patient
antigen.
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Fluorescent Immunoassay
Fluorescent Immunoassay Markers
Fluorophores or fluorochromes
 Ability to absorb energy and emit light
 Two most commonly used:
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Fluorescein – green
Tetramethylrhodamine – red
Tests may be qualitative or quantitative
Fluorescent Immunoassay
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Complex must form for fluorescence to occur.
Fluorescence
Fluorescent Immunoassay
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Antibodies and bacteria are fixed on a glass-plate.
The surplus i.e. non-bounded antibodies are washed out, antibodybacteria-complexes ("sandwiches") remain.
The "sandwich" becomes visible by adding fluorescent anti bovine
immunoglobulin which can be seen as green light in the
fluorescence microscope.
Fluorescent Immunoassay
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Direct immunofluorescence
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Tagged antibody added to unknown antigen fixed to
slide
If patient antigen present = fluorescence
Indirect immunofluorescence – sandwich assay
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Patient plus known fixed antigen
Allow to react and wash off unbound reactants
Add tagged anti-antibody
Fluorescence
Fluorescent Immunoassay
Positive Immunofluorescence
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Cryptosporidium parvum oocysts
Photo Credit: H.D.A Lindquist, U.S. EPA
Fluorescent Polarization
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Fluorescence polarization is a measure of the time-averaged rotational motion of fluorescent
molecules.
A fluorescent molecule, when excited by a polarized light, will emit fluorescence with its
polarization primarily determined by the rotational motion of the molecule.
Since the molecular rotation is inversely proportional to the molecular volume, the polarization is
in turn related to the molecular size.
A small molecule rotates fast in solution and exhibits low value of polarization whereas a large
molecule exhibits a higher polarization because of its slower motion under the same conditions.
Thus, changes in fluorescence polarization can reflect the association or dissociation between
molecules of interest.
Fluorescent Polarization
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Another picture to illustrate the principle.
Measure polarized light.
Chemiluminescent Immunoassays
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The process of chemiluminescence occurs when energy
in the form of light is released from matter during a
chemical reaction.
Chemiluminescent Immunoassays
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Large number of molecules capable of
chemiluminescence
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Luminol
Acridium esters
Ruthenium derivatives
Nitrophenyl oxalates
Use sodium hydroxide as a catalyst
Light emission ranges from quick burst or flash to light
which remains for a longer time.
Different types of instruments are required based on
emission.
Chemiluminescent Immunoassays
Can be used for heterogeneous or
homogeneous assays.
 Can attach label to antigen or antibody.
 Heterogeneous assays use competitive and
sandwich assay.
 Competitive assays used to measure smaller
analytes.
 Sandwich assays are used to measure larger
analytes.
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Chemiluminescent Immunoassay
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Many applications.
Can measure antigen or antibody.
Add chemiluminescently tagged analyte.
Measure light which is emitted which is directly related to
concentration although competitive binding assays are available.
Chemiluminescent Immunoassays
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Best known application of chemiluminescense is luminol
Luminol reacts with the iron in blood hemoglobin.
References
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http://web.indstate.edu/thcme/PSP/labtests/precip.htm
http://www.gla.ac.uk/departments/immunology/education/nursing/lectures/antibody.ht
m
http://www.cellsalive.com/mac.htm
http://jeeves.mmg.uci.edu/immunology/Assays/Assays.htm
http://www.medschool.lsuhsc.edu/microbiology/DMIP/dmex03.htm
http://www.tulipgroup.com/Common/html/TurbidTech.pdf
http://departments.oxy.edu/biology/Franck/Bio222/Lectures/Feb1lecture.htm
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http://www.mercodia.se/global/mainpage.asp?page_id=41 ELISA
http://www.clinprointl.com/technical.htm ELISA
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http://www.nsbri.org/HumanPhysSpace/focus4/sf-hormonal.html
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http://ccm.ucdavis.edu/cpl/Tech%20updates/TechUpdates.htm molecular
diagnostics
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References (Continued)
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http://www.liv.ac.uk/~agmclen/Medpracs/practical_5/theory_5.html
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http://www.fao.org/docrep/W0049E/w0049e06.htm
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http://www.genwaybio.com/gw_file.php?fid=6056