| Introduction
For over 40 years, immunoassays
have been used in hospitals, laboratory medicine, and
research to improve the health and well-being of humans
and animals. Information gained by clinical immunoassay
testing has shortened hospital stays and decreased the
severity of illness by identifying and assessing the
progression of disease, thereby leading to improved
therapeutic choices. In life science research,
immunoassays are used in the study of biological systems
by tracking different proteins, hormones, and
antibodies. In industry, immunoassays are used to detect
contaminants in food and water, and in quality control
to monitor specific molecules used during product
processing.
What
is an Immunoassay or ELISA?
Immunoassays are quick and accurate tests that can be
used on-site and in the laboratory to detect specific molecules (How
do I run an ELISA?). Immunoassays
rely on the inherent ability of an antibody to bind to the specific structure
of a molecule. Antibodies are proteins generated by animals in response to the
invasion of a foreign molecule (antigen) into the body. Antibodies are found in
blood and tissue fluids and will bind to the antigen whenever it is
encountered. Because antibodies are developed based on the specific
three-dimensional structure of an antigen, or analyte, they are highly specific
and will bind only to that structure. Once purified from the blood, monoclonal
and polyclonal antibodies are ideal assay reagents to detect and monitor
specific target molecules with limited interferences from other substances.
4 typical formats are: monoclonal-polyclonal
sandwich assays, competitive
inhibition assays, antigen-down
immunoassays, and rapid assays.
History
of Immunoassays
Evolution of diagnostic tests
began in the 1940s with colorimetric measurements of the
enzymes and metabolites found in biological fluids using
classical chemistry methods and agglutination reactions.
In the 1950s, the radio-immunoassay (RIA) was developed
by Rosalyn Yalow and Solomon Berson (this group was
later awarded the Nobel prize in 1977 for developing an
RIA to detect and measure blood glucose levels in
diabetic patients). In the 1960s, immunoassay technology
was enhanced by replacing radio-isotopes with enzymes
for color generation. The use of enzymes eliminates the
use of radioactive materials - and laboratory regulation
by the Nuclear Regulatory Commission (NRC). EIAs also
have faster reaction times, higher specificity to the
target molecule, and longer shelf-lives compared to RIAs.
Although
immunoassay techniques were first described in the
1950s, they were not readily applied outside of clinical
laboratories until the advent of economical automated
plate-reading systems and personal computers to analyze
data. Within the past 10 years, EIAs have become
increasingly popular. Scientists in fields outside of
medicine are now finding EIAs to be a convenient tool
that can be used on a daily basis to detect and monitor
specific molecules during the processing of materials.
Immunoassays make it possible for scientists to run
tests in-house with a minimal investment of time and
expense.
Although
unskilled technicians can run immunoassays, the
development of these tests requires knowledge in many
areas of immunology (such as antibody specificity) and
protein chemistry (such as binding interactions). As the
typical scientist is a specialist in a very narrow field
of study, he or she typically may not have the ability
to make his or her own rugged tests. Because Immunochemistry
Technologies develops assays with the end-user in mind,
we try to eliminate as many dilution, mixing, and
measuring steps as possible. Most of our reagents come
ready-to-use and may have a shelf life of 1 year.
The scientists at
Immunochemistry Technologies have the knowledge to
develop reliable, sensitive, and specific immunoassays.
For more information, call ICT at 1-800-829-3194.
Monoclonal-Polyclonal
Sandwich Immunoassay
In a typical microtiter plate
sandwich immunoassay, a monoclonal antibody is adsorbed
onto a plastic microtiter plate. When the test sample is
added to the plate, the antibody on the plate will bind
the target antigen from the sample, and retain it in the
plate. When a polyclonal antibody is added in the next
step, it also binds to the target antigen (already bound
to the monoclonal antibody on the plate), thereby
forming an antigen ‘sandwich’ between the two
different antibodies.
This binding reaction can then
be measured by radio-isotopes, as in a radio-immunoassay
format (RIA), or by enzymes, as in a enzyme immunoassay
format (EIA or ELISA) attached to the polyclonal
antibody. The radio-isotope or enzyme generates a color
signal proportional to the amount of target antigen
present in the original sample added to the plate.
Depending on the immunoassay format, the degree of color
can be detected and measured with the naked eye (as with
a home pregnancy test), a scintillation counter (for an
RIA), or with a spectrophotometric plate reader (for an
EIA) (for a price quote, see Sandwich
ELISA Sample Proposals).
Step
1: Monoclonal antibodies adsorbed onto the well of a
plastic microtiter plate (no sample added).
Step 2: Addition of a sample (such as human
blood) added to the well of the plate, with binding of
the target antigen to the antibody already bound to the
plate, thereby retaining the antigen in the well.
Step
3: Binding of a polyclonal antibody (with enzymes
molecules attached) to the target antigen (already bound
to the monoclonal antibody on the plate), thereby
forming an antigen ‘sandwich’ between the two
different antibodies. The enzymes on the polyclonal
antibodies will generate a color signal proportional to
the amount of target antigen present in the original
sample added to the plate.
Antigen-Down
Immunoassay
In an antigen-down immunoassay,
the analyte is coated onto a 96-well microtiter plate
(rather than an antibody) and used to bind antibodies
found in a sample. When the sample is added (such
as human serum), the antigen on the plate is bound by
antibodies (IgE for example) from the sample, which are
then retained in the well. A species-specific
antibody (anti-human IgE for example) labeled with HRP
is added next, which, binds to the antibody bound to the
antigen on the plate. The higher the signal, the
more antibodies there are in the sample.
Antigen-down
assays can be configured as rapid tests and are often
used to diagnose allergy conditions - routinely a
patient's blood is tested against different allergens to
see if the person has antibodies to that allergen (for a
price quote, see Antigen-Down
ELISA Sample Proposal).
Competitive
Inhibition Immunoassay
In addition to the MoAb-PoAb
sandwich format, many immunoassays are structured in a
competitive inhibition format. Competitive
inhibition assays are often used to measure small
analytes because competitive inhibition assays only
require the binding of 1 antibody rather than 2 as is
used in standard ELISA formats.
Because of the high probability for steric hindrance
occurring when 2 antibodies attempt to bind to a small
molecule at the same time, a sandwich assay format may
not be feasible, therefore a competitive inhibition
assay would be preferable. In a sequential
competitive inhibition assay, the sample and conjugated
analyte are added in steps like a sandwich assay, while
in a classic competitive inhibition assay, these
reagents are incubated together at the same time.
In
a sequential competitive inhibition assay format, a
monoclonal antibody is coated onto a 96-well microtiter
plate. When the sample is added, the MoAb captures
free analyte out of the sample. In the next step, a
known amount of analyte labeled with either biotin or
HRP is added. The labeled analyte will then also
attempt to bind to the MoAb adsorbed onto the plate,
however, the labeled analyte is inhibited from binding
to the MoAb by the presence of previously bound analyte
from the sample. This means that the labeled analyte
will not be bound by the monoclonal on the plate if the
monoclonal has already bound unlabeled analyte from the
sample.
The amount of unlabeled analyte
in the sample is inversely proportional to the signal
generated by the labeled analyte. The lower the signal,
the more unlabeled analyte there is in the sample. A
standard curve can be constructed using serial dilutions
of an unlabeled analyte standard. Subsequent
sample values can then be read off the standard curve as
is done in the sandwich ELISA formats.
The
classic competitive inhibition assay format requires the
simultaneous addition of labeled (conjugated analyte)
and unlabeled analyte (from the sample). Both
labeled and unlabeled analyte then compete
simultaneously for the binding site on the monoclonal
capture antibody on the plate. Like the sequential
competitive inhibition format, the colored signal is
inversely proportional to the concentration of unlabeled
target analyte in the sample.
Detection of labeled analyte
may be made by using a peroxidase substrate such as TMB,
which can be read on a microtiter plate reader (for
price quotes, see Competitive
Inhibition ELISA Sample Proposal and Rapid
Competitive Inhibition ELISA Sample Proposal).
Rapid
Immunoassay
In addition to microtiter
plates, immunoassays are also configured as rapid tests,
such as a home pregnancy test. Like microtiter
plate assays, rapid tests use antibodies to react with
antigens and can be developed as MoAb-PoAb sandwich
formats, competitive inhibition formats, and
antigen-down formats. With a rapid test, the
antibody and antigen reagents are bound to porous
membranes, which react with positive samples while
channeling excess fluids to a non-reactive part of the
membrane.
Rapid immunoassays commonly
come in 2 configurations: a lateral flow test where the
sample is simply placed in a well and the results are
read immediately; and a flow through system, which
requires placing the sample in a well, washing the well,
and then finally adding an analyte-colloidal gold
conjugate and the result is read after a few minutes.
One sample is tested per strip or cassette.
Because
rapid tests are faster than microtiter plate assays,
require little sample processing, are often cheaper, and
generate yes/no answers without using an instrument,
they often used in the field by non-laboratory people
testing whole samples. However, rapid immunoassays
are not as sensitive nor can they be used to accurately
quantitate an analyte. (Self-monitoring of blood
glucose levels by diabetics is considered quantitative
rapid testing, however, immunoassay technology is not
used for these tests.) All rapid immunoassay tests
can be converted to a microtiter plate assay, but not
all microtiter plate assays can be converted to a rapid
test (for price quotes, see Rapid
ELISA Sample Proposals). |
