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Immunochemistry
Technologies
is a custom service laboratory specializing in
immunoassay development and manufacturing. Once developed, we
will ship the finished test to you so it can be used on-site
by your staff, or in the field by your customers. We
will then manufacture the components of the test whenever you need more.
A typical 96-well microtiter plate sandwich
immunoassay kit may include the following
components (there are several different formats of an
immunoassay; only 1 is presented here):
Typical
Components of an Sandwich Immunoassay and Typical
Immunoassay Protocol
| Pre-Coated,
Stabilized 96-well Microtiter Plate:
For microtiter plate assays, the plates are
provided ready-to-use. They are pre-coated with
the capture antibody, blocked, stabilized, and
packaged in a resealable foil pouch with a
desiccant packet. Using our methods, the plates
may have a real time stability of 1 year when
stored at 2o – 8o C.
With a standard curve (1 blank and 7 standards)
and 3 controls, a 96-well microtiter plate format
can test 21 samples in triplicate and 37 samples
in duplicate (also see Plate
Coating Buffers and Blocking
Buffers for more information).
Sample
Diluent: The sample diluent is used
to ensure that the analyte in the sample matrix is
measured accurately. It is used to dilute the
sample within the target range of the assay (also
see Sample Diluents
for more information).
Standards
and controls:
A known amount of the analyte (such as 1 ng) is
included with a kit and run on every plate.
The standard is often lyophilized. Once
reconstituted, it is diluted several times to
prepare a range of known values with which to
compare and properly quantitate the unknown amount
of analyte in a sample. Controls are used to
confirm the readings of the standard and to
compare readings from different plates.
Because they contain a known amount of analyte,
they should always read within a certain optical
density (OD) value based on the standard.
Assay
Diluent:
In addition to a specimen diluent, certain
matrixes require the use of a special assay
diluent (which is applied to the plate just prior
to adding the samples, standards, and controls).
Assay diluents are often paired with a specific
type of sample (such as serum, or cell culture
media) to eliminate interference and non-specific
binding generated from the matrix of the sample
itself. These interferences are especially
noticeable when running neat samples. An
assay diluent may not be required for every assay
(also see Assay
Diluents for more information).
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Calibrator
Diluent: A calibrator diluent is
used to ensure that the standards and controls
will be measured accurately. This diluent must
compliment the target analyte, capture antibody,
and resemble the matrix of the sample (also see Sample
Diluents for more information).
Conjugated
Detection Antibody: For some
assays, the detection or ‘top’ antibody is
often an affinity purified polyclonal antibody
conjugated to HRP. The enzyme-antibody conjugate
can often be supplied ready-to-use in its working
concentration in a special conjugate diluent. The
conjugate diluent stabilizes the conjugate and
minimizes nonspecific binding of the conjugate
onto the blocked plate or matrix residue. The
working conjugate will often have a real-time
stability of 1 year when stored at 2o–8o
C (also see Conjugate
Diluents for more information).
10X
Wash Solution: This
specially formulated buffer is used to rinse the
plate after the sample and conjugate incubation
periods, just prior to the addition of the next
reagent. It minimizes matrix residue and
non-specific binding interferences of the samples
and conjugate (also see Wash
Buffer for more information).
Single
Component Substrate:
This reagent reacts with HRP to generate a
colored signal product. It can come in many
formulations (powdered, 2-component liquid, etc.).
ICT recommends a low-background,
high-signal-generating ABTS or TMB that needs no
preparation prior to use.
Stop
Solution: An appropriate stop
solution is added to the plate with the ABTS or
TMB substrate and stops its reaction with HRP. By
stopping this reaction after 20 minutes, the
plates can equilibrate before reading, which
increases the accuracy and sensitivity of the
assay.
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Typical
Immunoassay Protocol
Preparation
In this example, 1 blank (which
is simply the sample diluent), 7 standards, 3 controls,
and 37 unknown samples are being tested in duplicate in
1 microtiter plate MoAb-PoAb sandwich immunoassay.
Standards
As each kit typically comes
with only 1 vial of a high-concentrate standard which is
lyophilized (freeze dried into a powder), it must be
reconstituted, and then further diluted with sample
diluent. If the lyophilized standard was at
5000pg, when reconstituted with 5mL, it would have a
concentration of 5000pg/5mL or 1000pg/mL. To
generate a standard curve, it should be serially diluted
1:2 (500uL standard with 500uL sample diluent) to
create standards at 500pg/mL, 250pg/mL, 125pg/mL,
62.5pg/mL, 31.25pg/mL, and 15.13pg/mL.
Controls
The controls may or may not
come with a kit, but are often lyophilized and must also
be reconstituted, typically with 2mL sample diluent.
Controls are usually not diluted further.
Unknown
Samples
Any samples thought to
have a value higher than the reconstituted standard (at
1000pg/mL) should be diluted with the sample diluent so
that it falls between the high standard (at 1000pg/mL)
and the low standard (at 15.13 pg/mL). If
they do not need to be diluted, samples often can be run
neat. However, if the samples have some sort of
interfering substance, such as rheumatoid factors, or
complement, they may need to be treated prior to running
in the assay. If you do not know about your
samples, simply run them in the assay once and see where
they fall (neat and diluted samples can be run at the
same time). If the values are too high, just
dilute, and/or treat the samples and run the assay
again. Each investigator must determine their own
protocols for sample dilutions and pre-assay treatments
(see Consultation
for assistance from ICT)
Immunoassay
Procedure
Step
1: Add 50 uL per well of assay diluent into every well
of the plate.
Step 2: Add 200 uL per well of your blanks, standards,
controls, or samples onto the plate. Each item
should be tested in duplicate (in 2 wells).
Step 3: Cover with plate sealer and incubate 2 hours at
room temperature.
Step 4: Wash plate by filling wells with 400 uL wash
buffer and dumping. Wash for a total of 4 cycles.
Blot on paper towels.
Step 5: Add 200uL per well of PoAb-HRP conjugate
solution into every well of the
plate.
Step 6: Cover with plate sealer and incubate 2 hours at
room temperature.
Step 7: Wash plate by filling wells with 400 uL wash
buffer and dumping. Wash for a total of 4 cycles.
Blot on paper towels.
Step 8: Add 200uL per well of TMB substrate solution into
every well of the plate.
Step 9: Incubate 20 minutes at room temperature.
Step 10: Add 50 uL per well 2N HCl stop solution into
every well of the plate.
Step 11: Read plate at 450nm while subtracting a
reference wavelength of 540nm.
Step 12: Calculate data based on OD values of the
unknown samples compared to the known values of the
standard curve.
Typical
Plate Map
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1
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2
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3
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4
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5
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6
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7
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8
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9
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10
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11
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12
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A
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Blank
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Blank
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Lo C
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Lo C
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Sp 6
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Sp 6
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Sp 14
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Sp 14
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Sp 22
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Sp 22
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Sp 30
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Sp 30
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B
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Std 1
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Std 1
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Mid C
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Mid C
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Sp 7
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Sp 7
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Sp 15
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Sp 15
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Sp 23
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Sp 23
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Sp 31
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Sp 31
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C
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Std 2
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Std 2
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Hi C
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Hi C
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Sp 8
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Sp 8
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Sp 16
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Sp 16
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Sp 24
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Sp 24
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Sp 32
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Sp 32
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D
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Std 3
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Std 3
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Sp 1
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Sp 1
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Sp 9
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Sp 9
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Sp 17
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Sp 17
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Sp 25
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Sp 25
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Sp 33
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Sp 33
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E
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Std 4
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Std 4
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Sp 2
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Sp 2
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Sp 10
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Sp 10
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Sp 18
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Sp 18
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Sp 26
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Sp 26
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Sp 34
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Sp 34
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F
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Std 5
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Std 5
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Sp 3
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Sp 3
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Sp 11
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Sp 11
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Sp 19
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Sp 19
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Sp 27
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Sp 27
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Sp 35
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Sp 35
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G
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Std 6
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Std 6
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Sp 4
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Sp 4
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Sp 12
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Sp 12
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Sp 20
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Sp 20
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Sp 28
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Sp 28
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Sp 36
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Sp 36
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H
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Std 7
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Std 7
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Sp 5
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Sp 5
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Sp 13
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Sp 13
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Sp 21
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Sp 21
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Sp 29
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Sp 29
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Sp 37
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Sp 37
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