[MID] Chapter 10: Labeled Immunoassays

Labeled Immunoassays
Designed for antigens and antibodies that may be small in
size or present in very low concentrations
Rapid quantitative measurement of many important entities
(eg. Viral Ag in HIV patients)
Analyte
Substance to be measured
Usually bound by molecules (Antibodies)
o
Bacterial Ag
o
Hormones
o
Drugs
o
Tumor Markers
o
Immunoglobulins

Immunology and Serology

1.
2.
3.

Decantation
Centrifugation
Filtration
Ag + Ab Partitioning/Separation Washing

Detection of the Label

The last step common to all immunoassays is detection of the
labeled analye.

Characteristics of Labeled Assays

Counting radioactivity Radioimmunoassays

Change in absorbance Enzymes, Fluorescence,
Chemiluminescence

Competitive versus Noncompetitive Assays

Quality Control

Competitive
All reactants are mixed together
simultaneously

Labeled Ag competes with

unlabeled patient Ag

Running a blank tube (Phosphate buffered saline) with every

test serves as a check for:
1. Nonspecific adsorption
2. Inadequate washing between steps

Background
Any readings indicative of label in the blank

To check for quality of the reagents run:

1. Negative control
2. Low positive control

More label detected, less patient

Ag
Random interaction between Ag
and Ab molecules

Noncompetitive
Capture Ab passively absorbed to
solid phase

Ag is reacted and captured by Ab

Wash to remove unbound Ag

2nd Ab with label is added

More label detected, more patient
Ag

If any control is out of range, test values should NOT be reported.

Radioimmunoassays

Antibodies
In any assay, it is essential for the antibody used to have a high
affinity for the antigen in question

Standards/Calibrators
Unlabeled analytes that are made up in known
concentrations of the substance to be measured
Establish relationship between labeled analyted and
unlabeled analyte that might be present

Separation Methods

Affinity
Strength of the primary interaction between a single
antibody-combining site and an antigenic determinant or
epitope
Monoclonal Antibodies
Constant source of highly specific antibody that has increase
ability to detect small amounts of analyte

Standards of Calibrators

Competitive Binding Assays

Solid-phase Vehicle
Used by most immunoassays in the partitioning step
o
Polystyrene test tubes
o
Microtiter plates
o
Glass or Polystyrene beads
o
Magnetic beads
o
Celllulose membranes

Physical Means of Separating Bound and Unbound Fractions

Radioactive Immunoassay (RIA)

First type of immunoassay developed (Yalow and Berson)
Determine insulin-anti-insulin complexes levels in diabetic
RADIOACTIVE SUBSTANCE as a label
o
131 I
o
125 I most popular

half-life: 60 days

Emits gamma radiation

o
Tritiated Hydrogen or 3 H

PRINCIPLE OF RADIOIMMUNOASSAYS
Labeled antigen competes with patient antigen for a limited number of
binding sites on solid-phase antibody
Antigen = Radioactive Label
Inversely Proportional
The detection limits of competitive assays are largely determined by
the affinity of the antibody. These limits have been calculated to be as
low as 10 fmol/L or 600,000 molecules in a sample volume of 100 uL
Advantages and Disadvantages of Radioimmunoassays

Substances that are measured by RIA

1. TSH
2. Total Serum IgE

Disadvantages of RIA
1. Health hazard involved in working with radioactive
substances
2. License difficult to obtain and expensive

[MID] Chapter 10: Labeled Immunoassays

3.
4.
5.

Immunology and Serology

Disposal problems
Short shelf-life
Need for expensive equipment

1.
2.
3.

Enzyme Immunoassay

Enzymes
Naturally occurring molecules that catalyze
biochemical reactions
React with substrates to produce products
o
Chromogenic
o
Fluorogenic
o
Luminescent

Ag (bound to solid-phase) + Serum (with Ab)

Wash

Enzyme-labeled Ag (reacts with any patient Ab that is bound to slid

phase)

Wash

Enzyme substrate is added

Color development is directly proportional to the amount of patient
Ab present

certain

Advantages of EIA
1. Cheap and readily available
2. Long shelf-life
3. Easily adapted to automation
4. Can be measure using inexpensive equipment
5. Without disposal problems or health hazards

Enzyme labels can either be used qualitatively or quantitatively to

determine the actual concentration of an analyte in an unknown
specimen.

Availability & Cost

Play a role in the choice of a particular enzyme as reagent

Enzyme Labels in Colorimetric Reactions

1. Horseradish peroxidase
2. Glucose-6-phosphate dehydrogenase
3. Alkaline phosphatase
4. B-D-galactosidase

Horseradish peroxidase & G6P dehydrogenase

Highest turnover rates (conversion of substrate)
High sensitivity
East to detect
Most often used

Heterogenous Enzyme Immunoassay

Heterogenous EIA
Require a step to physically separate free from bound
analyte

Competitive EIA
First EIAs
Principle: Enzyme-labeled antigen competes with unlabeled
antigen for a limited number of binding sites on antibody
attached to solid phase
Sensitivity: 10^-9 g/mL
Detects:
o
Insulin
o
Estrogen
Antigen = Enzyme-labeled Antigen can attach
Inversely Proportional

Noncompetitive EIA
Have higher sensitivity (1 pg/mL)
Aka Indirect ELISA (Enzyme-linked Immunoassay)
o
Enzyme label does not participate in the initial AgAb binding reaction
Either Ag or AB may be bound to solid phase
Solid-phase Supports

Microtiter plates
Nitrocellulose membranse
Magnetic latex beads

Uses of Noncompetitive EIA/ELISA

1. Measure Ab production to infectious agents (difficult to
isolate)
a. HIV
b. Hepa A
c. Hepa C
d. Epstein-Barr-specific antibodies infectious
mononucleosis
2. Autoantibody testing

Viral Infections are more easily diagnosed by Noncompetitive

EIA/ELISA
Capture Assays
Aka Sandwich Assays
Ab is bound to solid phase and Ag must have multiple
epitopes

Patient Ag is incubated with solid-phase Ab

Wash

Enzyme-labeled Ig is added

Wash

Enzyme substrate is added

Color development is directly proportional to the amount of patient
antigen present
Capture assays are best suited to Ag that have multiple determinants

Antigens that have Multiple Determinants

1. Polypeptide Hormone
2. Proteins
3. Tumor markers
4. Microorganisms (esp. Viruses)

Examples of Capture Assay

1. Rotavirus
2. Respiratory Syncytial Virus

Parasites detected by ELISA

1. Giardia lamblia
2. Cryptosporidium

Fungi detected by ELISA

1. Aspergillus
2. Candida

[MID] Chapter 10: Labeled Immunoassays

3.

Cryptococcus

Immunology and Serology

Advantages and Disadvantages of Enzyme Immunoassay

Another major use of Capture Assay is in the measurement of

immunoglobulins

Immunoglobulins acts as an Ag and Ab is Antihuman

globulin

Indirect ELISA tests are more sensitive than their direct counterparts,
because all patient antigen has a change to participate in the reaction.

Hook effect
In which Sandwich assays are subjected
An unexpected fall in the amount of measured analyte when
an extremely high concentration is present

Fluorescent Immunoassays

Fluorophores/Fluorochromes
Fluorescent compounds that can absorb energy from an
incident light source and convert that energy into light of
longer wavelength
ORGANIC molecules with a ring structure
Albert Coons
o
Demonstrated Ab could be labeled with
fluorophores (1941)

Two (2) compounds most often used:

1. Fluorescein
a. Absorption 490-495 nm
b. Emission 517-520 nm Green
2. Rhodamine/ Tetramethylrhodamin
a. Absorption - 550 nm
b. Emission 580-585 nmn
3. Phycoeryrthrin
4. Europium (Beta-naphthyl trifluoroacetone)
5. Lucifer Yellow VS

Rapid Immunoassays

Membrane-Based Cassette Assays

New type of immunoassay
Rapid, easy to perform, and give reproducible results
Designed primarily for POC or home testing
Single-use, disposable in a plastic cartridge
The membrane is isually NITROCELLULOSE
o
Easily immobilize proteins and nucleic acids
Immunochromatography
Combines all the previously mentioned steps into one
Analyte is applied at one end of the strip and migrates
toward the distal end, where there is an absorbent pad to
maintain a constant capillary flow rate
Labeling and detection zones two ends
Ag or Ab immobilized in the detection zone captures
immunecomplex and forms a colored line positive test
Used to identify:
o
Strep pyogenes
o
Strep agalactiae
Used to test:
o
Pregnancy
o
Troponin
o
Hepa B surface Ag

Fluorescent tags were first used for histochemical localization of Ag in

tissues called Immunofluorescent Assay (IFA)
Presence of Ag is determined by appearance of localized color against
a dark background
Direct Immunofluorescent Assays

Homogeneous Enzyme Immunoassay

Homogenous Enzyme Immunoassay

Ag-Ab system in which no separation step is necessary
Less sensitive than heterogenous assays but rapid and
simple to perform
Chief use: Determination of low-molecular weight analytes:
o
Hormones
o
Therapeutic Drugs
o
Drugs of Abuse
Example: Enzyme Multiplied Immunoassay Technique
(EMIT) by Syva Corp.
Princple: Change in enzyme activity as specific Ag-Ab
combination occurs

Determinants of the Sensitivity of Homogenous Assays:

1. Detectability of enzymatic activity
2. Change in activity when Ab binds to Ag
3. Strength of Abs binding
4. Susceptibility of the assay to interference from endogenous
enzyme activity, cross-reacting Ag, enzyme inhibitors

Enzyme Activity may be altered by:

1. Steric exclusion
2. Changes in Conformation or shape

Advantages
1. The use of nonisotopic enzyme labels increases sensitivity
2. No need for expensive instrumentation
3. Reagents are inexpensive and have long shelf-life
Disadvantages
1. Some specimens contain natural inhibitors
2. Size of the label may be a limiting factors
3. Nonspecific protein binding

Direct Immunofluorescent Assay

Ab that is conjugated with a fluorescent tag is added directly
to unknown Ag that is fixed to a microscope slide
Ag Bright apple green or orange-yellow
Best suited for Ag detection in:
o
Tissue
o
Body fluids
Examples of Ag Detected:
o
Legionella pneumophila
o
Pneumocystis carinii
o
Chlamidia trachomatis
o
Respiratory syncytial virus

DIRECT IMMUNOFLUORESCENT ASSAY

Solid Phase Ag fixed on Microscope slide

Incubated directly with fluorescent-labeled Ab

Washing

Fluorescence (If Ag is presen)

Indirect Immunofluorescent Assays

Indirect Immunofluorescent Assay

[MID] Chapter 10: Labeled Immunoassays

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Result in increased staining because multiple molecules can

bind to each primary molecules, thus making this a more
sensitive technique
Useful in Ab identification (but can also be used to detect Ag)
o
Treponema
o
Anti-nuclear
o
Chlamydial
o
Toxoplasma Ab
o
Herpes simplex
o
Epstein-Barr
o
Cytomegalovirus

INDIRECT IMMUNOFLUORESCENT ASSAY

Solid Phase Ag fixed on Microscope slide + Ab

Wash

Labeled Antihuman Ig added

Wash to remove uncombined Antihuman Ig

Amount of Fluorescence is directly proportional to amount of Ab
present

Immunology and Serology

-

Limited to molecules under 2000 d.

Degree of polarized light reflects the amount of labeled analyte that is

bound
The more Ag is present in patient sample, less fluorescence-labeled Ag
is bound, less polarization detected
Advantages and Disadvantages of Fluorescent Immunoassay

Advantages
1. High sensitivity and versatility
2. Methodology is simple
3. No need to deal with dispose of hazardous substances
Disadvantages
1. Separation of signal on the label from autofluorescence
produced by different organic substances
2. Nonspecific binding can cause QUENCHING or diminishing
of the signal and change the amount of fluorescence
generated
Chemiluminescent Immunoassays

Principle of Chemiluminescence
Other Fluorescent Immunoassays

Solid-phase Heterogeneous Fluorescent Assay

Identification of Ab to:
o
Nuclear Ag
o
Toxoplasma Ag
o
Rubella virus
Detect biological compounds
o
Cortisol
o
Progesterone
o
Serum Thyroxine (T4)
Homogeneous Fluorescent Immunoassays
Requires no separation procedure
Rapid and simple to perform
Only 1 incubation step and no wash step
Usually competitive binding
Changes occurs in label on Ag when bound to Ab and may
be related to:
o
Wavelength emission
o
Rotation freedom
o
Polarity
o
Dielectric strength
There is a DIRECT relationship between amount of
fluorescence and amount of Ag
Fluorescence Polarization Immunoassay
Based on the change in polarization of fluorescent light
emitted from a labeled molecules when bound by Ab
Prism/ Lens polarizes incident light
Molecule is small rotates quickly = light emitted is
unpolarized
Labeled molecule is bound to Ab unable to tumble rapidly
= emits increased amount of polarized light

Chemiluminescence
Emission of light caused by a chemical reaction, typically an
oxidation reaction producing an excited molecule that
decays back to its original ground state

Most common substances that are chemiluminescent

1. Luminol and Dioxethane light remains for a long time
2. Acridinium esters emit quick burst or flash of light
3. Ruthenium derivatives
4. Nitrophenyl Oxalates

Substances oxidized by H2O2 + enzyme Intermediates with higher

energy state gives off light
Smaller analytes (therapeutic drugs, steroid hormones) for competitive
assays, and sandwich format for larger analytes (protein hormones)
Advantages and Disadvantages of Chemiluminescent Assays

Advantages
1. Excellent sensitivity [attamoles (10^-18) to zeptomoles (10^21)]
2. Reagents are stable and nontoxic
3. High speed of detection faster turnaround time

False results may be obtained if:

1. Lack of precision in injection of H2O2
2. Biological materials (urine/plasma) cause quenching of the
light emission

Detection systems consists of photomultiplier tubes