586 The Immunoassay Handbook
Typical Assay Protocols
For Immuno-Rate assays, both competitive and immuno-
metric assays are performed automatically starting with
the application of 11µL of a serum or plasma sample to a
dry chemistry slide into which reagents speciﬁc for each
assay have been incorporated. The slides are then incu-
bated for 5min at 37°C to allow for equilibration of
binding reactions. After incubation, 12µL of a wash solu-
tion is applied to the slide. This wash solution serves a
dual purpose. It separates bound reagents from free
reagents using the ﬂow of the ﬂuid to move mobile (non-
bound) reagents out of the detection area of the slide and
also initiates the enzymatic reaction required for the
detection reaction because it contains the enzyme sub-
strate. Following the wash step, there is a second 2.5min
incubation at 37°C during which reﬂectance densitome-
try measurements are made at regular intervals. The rates
of these reactions can be correlated to the concentration
of analyte in the original serum sample. From the appli-
cation of the ﬁrst sample to its prediction requires
MicroTip assays are performed automatically with the
application of 2–16.7µL of a serum or plasma sample to a
disposable cuvette into which reagents speciﬁc for each
test have been added. The cuvette is then incubated at
37°C to allow for thermal equilibration. After this time, a
second reagent is added to the cuvette. Following the addi-
tion of the second reagent, there is a second incubation at
37°C during which spectrophotometric measurements are
made at regular intervals. The rates or endpoints of these
reactions can be correlated to the concentration of analyte
in the original serum sample. From the application of the
ﬁrst sample to its prediction requires 8–16min, depending
on the assay requirements.
G VITROS systems are self-contained and fully auto-
mated. They are capable of performing both immuno-
assays and standard clinical chemistry tests in batch or
random access fashion.
G Less than 17µL of sample is required for each
MicroSlide- or MicroTip-based immunoassay.
G For Immuno-Rate assays, MicroSlide reagents (other
than a standard wash solution) are stored on the
instrument for up to one week.
G For MicroTip immunoassays, reagents are stored in
ready-to-use liquid reagent packs on the instrument for
up to four weeks.
G There is no risk of carryover from sample to sample or
reagent to reagent. Disposable tips are used for indi-
vidual metering steps for both Immuno-Rate and
G The integrity of patient results is maintained, and
sample repeats and redraws are minimized by the
presence of Intellicheck™ Technology which includes
liquid level sensing, sample clot and bubble detection,
sample and reagent aspirate, and dispense veriﬁcation
features on the analyzers.
G VITROS systems provide on-board dilution capability
for improved labor optimization and error reduction.
G VITROS systems do not require any plumbing to a
water source or drain to execute testing and manage
G VITROS systems allow for bidirectional interfacing
with hospital laboratory information processing sys-
tems, supporting Broadcast Download, (and Host
Query on the VITROS 4600 Chemistry System and
VITROS 5600 Integrated System), allowing for
increased laboratory productivity.
In addition, the VITROS 4600 Chemistry System and
VITROS 5600 Integrated System:
G Utilize MicroSensor™ technology to measure sample
quality indices for hemolysis, turbidity, and icterus
without using additional samples, reagents, disposables,
or slowing system throughput.
G Support remote, real-time system diagnostics using
e-Connectivity which can reduce time spent on
G Do not require the use of ﬁxed probes or reagent mix-
Immuno-Rate assays are based on heterogeneous competi-
tive and immunometric methods adapted to thin-ﬁlm for-
mats (Fig. 4). In general, the layers are formed as follows.
Buffering agents are placed in a cross-linked gelatin layer
that comprises the lowest layer of the thin-ﬁlm format.
When sample is applied, the buffer is rehydrated and helps
control the pH at which the immunoassay binding reactions
and subsequent enzyme reactions occur. Over the gelatin
layer is an isotropically porous polymeric bead-spreading
layer whose function is to accept the serum or plasma sample
and assure that it spreads to an area so that the surface den-
sity of analyte is constant. The non-porous beads that com-
prise this layer are about 30µ in diameter, leaving ample
interstitial capillary space so the sample is accepted rapidly (a
few seconds). This layer also may contain buffer, stabilizers
for enzymes and antibodies, dyes that can be developed by
the enzyme label, and other reactants as needed. Antibodies
that have been covalently immobilized on polymeric beads
(about 1µ in diameter) may also be placed in this layer or
FIGURE 3 VITROS® 5600 Integrated System (The color version of
this ﬁgure may be viewed at www.immunoassayhandbook.com).
587CHAPTER 7.14 VITROS® Immuno-Rate and MicroTip™ Assays
alternatively in a thin receptor layer which lies between the
gelatin layer and the spreading layer. For competitive assays,
the enzyme-labeled drug (horseradish peroxidase) is coated
in a thin layer at the top of the bead-spread layer in order to
prevent binding to the antibody before the serum sample is
applied. For immunometric assays, where binding cannot
occur in the absence of the analyte, the enzyme-label is
incorporated into a receptor layer.
Binding reactions are initiated upon sample addition.
Because of the small spaces between the beads of the spread
layer, diffusional distances are very small (a few microns)
so that equilibrium is reached rapidly (within a few min-
utes) with no agitation necessary. After 5min incubation at
37°C, a wash solution is applied. The wash ﬂuid ﬂows by
capillary action from the point of application through the
read region of the slide to the periphery of the slide. Wash
is efﬁcient, a result of bulk ﬂuid movement being the
primary mode of molecular movement within the porous
spreading layer and the small capillary structure within the
spreading layer which directs ﬂuid ﬂow away from the
point of application. Unbound molecules are carried away
in the direction of ﬂuid ﬂow from their immobilized coun-
terparts. There is little or no difference in movement
between high and low molecular weight materials. Thus, a
12µL volume is sufﬁcient for the bound-free separation
required by the heterogeneous assay format. The wash
removes unbound material and initiates the enzyme reac-
tion used for detection. The bound horseradish peroxidase
produces a blue color. Rate measurements are made using
reﬂectance densitometry at 670nm during a 2.5min incu-
bation (at 37°C) following the wash solution application.
Movement of an inert magenta dye in the slide is auto-
matically monitored to ensure that the wash step has been
MicroTip immunoassays are based on homogeneous
competitive and turbidimetric methods using traditional
liquid reagents. In general, two liquid reagents in an inte-
grated reagent pack are available for each of the Micro-
Tip immunoassays. The reagent packs are stored
refrigerated on the analyzer. The analyzer automatically
uncaps and recaps the reagents when required. The assays
themselves are carried out in disposable spectrophoto-
metric cuvettes that are incubated at 37°C. A typical assay
protocol uses a disposable pipette tip to add about 100µL
of the ﬁrst liquid reagent to a cuvette. The sample is
pipetted (2–16.7µL) using a specially designed disposable
MicroTip, which provides the necessary metering preci-
sion. This tip also is used to mix the reagents and sample.
After a typical 5min incubation time to allow for thermal
equilibration, the second reagent is added and mixed
using a disposable tip. Incubation continues at 37°C while
spectrophotometric or turbidimetric readings are taken at
prescribed times. Therapeutic drugs are measured using
EMIT™ technology while serum protein and other assays
(e.g. high sensitivity CRP, %A1c, and rheumatoid factor)
are measured turbidimetrically. Samples are automati-
cally diluted if required by the speciﬁc assay protocol.
Because MicroTip assays are processed using disposable
tips, cuvettes, and reagents, the maintenance, cost, and
carryover associated with the use of reusable cuvettes,
ﬁxed probes, water, wash reagents, plumbing, drains, and
mixing assemblies are eliminated. MicroTip technology
also eliminates sample and reagent carryover that may
occur with sample probes, mixing devices, and reusable
Immuno-Rate assays use a stored, lot-speciﬁc calibration
function that has been factory determined and delivered to
the instrument using a calibration disk supplied by OCD.
Wet calibrations are performed on the analyzers using a
3-level multianalyte calibration set available from OCD. A
new lot of MicroSlides is calibrated when it is ﬁrst used on
an instrument. Subsequent re-calibrations are done every
3–6 months. Assays can be purchased in 18 or 50/60 slide
cartridges. Unopened cartridges are stored refrigerated or
frozen for up to 18 months.
FIGURE 4 Principle of competitive binding VITROS® Immuno-Rate
588 The Immunoassay Handbook
MicroTip immunoassays use multiple level calibrators
to generate a calibration curve on the analyzer. Calibra-
tions are good for at least 4 weeks or until there is a change
in the lot of assay reagents. Calibrator kits have a shelf-life
of greater than 1 year. The liquid reagent packs contain
ﬂuids for 50 assays. The typical shelf-life of an unopened
refrigerated reagent pack is greater than 1 year. Opened
reagent packs can be stored on analyzer for typically
All VITROS Immuno-Rate assays use mouse monoclonal
antibodies that have been covalently immobilized to small
(1µ), uniform-sized copolymeric latex beads. The C-reactive
protein (CRP) assay contains both a monoclonal anti-CRP
antibody conjugated to horseradish peroxidase and a deriva-
tive of phosphorylcholine covalently bound to the 1µ latex
beads in place of a second monoclonal antibody.
MicroTip immunoassays use either monoclonal or poly-
clonal antibodies. Turbidimetric (agglutination) assays use
uncomplexed antibodies or antibodies coupled to latex
particles. EMIT assays use uncomplexed antibodies that
have been selected for their ability to inhibit speciﬁc drug-
enzyme conjugates in the absence of free drug.
In Immuno-Rate assays, separation of bound enzyme-label
from free enzyme-label is accomplished by application of a
wash solution as described above. No separation is required
in the homogeneous MicroTip assays.
The Immuno-Rate assays use horseradish peroxidase as
the enzyme label. Its reaction is initiated by the addition
of the wash ﬂuid, which contains hydrogen peroxide and
an electron transfer agent, 4-hydroxyacetanilide. The
reaction results in a blue color due to the oxidation of a
leuco dye that is incorporated in one of the thin-ﬁlm
layers. Detection of the rate of blue color formation is by
periodic reﬂectance densitometry reads at 670nm. Only
the color development at the center of the slide (washed
region) is read.
The MicroTip immunoassays for therapeutic drugs
(EMIT technology) use drug conjugates of glucose-6-
phosphate dehydrogenase (G6P-DH). The reaction rate is
monitored as 340nm to detect the formation of NADH
(NAD is a cofactor of G6P-DH). The turbidimetric
MicroTip assays measure the agglutination reactions at a
variety of wavelengths.
Once a rate or end-point has been calculated by internal
algorithms, it is transformed into a predicted concentra-
tion using the on-board calibrations described above. If
the predicted concentration is outside the pre-determined
calibration range either at the low or high end, the opera-
tor is alerted through an error message (ﬂag). If the sample
is beyond the high end of the concentration range, the
sample can be automatically diluted and re-processed by
the analyzer. Patient reports are ﬂagged if there is not
enough sample volume for the assay, and, for Immuno-
Rate assays, if the sample is not applied to the thin-ﬁlm, or
if the wash ﬂuid is not applied.
Quality control ﬂuids for immunoassays are also available
from OCD. They can be run daily with the results tabulated
and/or plotted in a variety of customer-speciﬁed formats.
Interfacing to Laboratory
An RS232-C serial interface with bidirectional capabil-
ity, along with the necessary software, is available to link
the VITROS chemistry systems with central laboratory
information management systems. The instruments can
be interfaced with lab automation consisting of a variety
of transport systems that automate the pre- and post-
analytical processes including centrifugation, aliquoting,
sample integrity checking, stopper removal and recap-
ping, sample sorting, and sample storage. VITROS sys-
tems can also be connected to a variety of automation
systems, including the enGen™ Automation solution
from OCD. The design of these systems is centered
around a concept in which the metering track is extended
so that the disposable sample tip can sample from a tube
while it is on the automation system transport track, thus
eliminating the need for robotics to move tubes to and
from the sample supply and for extra aliquot tubes. The
VITROS Systems retain the capability to process urgent
STAT samples in a manual mode.
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