Note: Descriptions are shown in the official language in which they were submitted.
CA 02573933 2007-01-12
WO 2006/007726 PCT/CA2005/001147
Method to measure dynamic internal calibration true dose response curves
Field of the Invention
The invention relates to assay devices and methods for constructing assay
devices for
detecting the presence of an analyte in a biological sample and the quantity
of same.
Background of the Invention
Quality standards for immunoassays have traditionally been driven by external
calibration reference standards. Current methods of analysis for typical
immunodiagnostic assays provide diagnostic test results based on generally
accepted
external standard reference measurements. A number of known discrepancies have
become apparent to be quantitation errors induced when assays are carried out,
leading to variations in test results. For example, the concept of assay
sensitivity
attempts to characterize sensitivity by classic statistical analysis based on
repeated
measurement of low concentration samples to confirm that the sample result is
not
statistically different from zero. As the standard error incurred is inversely
proportional to the square root of the number of actual measurements, this
method
does not actually measure the inherent assay sensitivity. Further refinement
has led to
some improvements. Known in the art as analytical sensitivity, the zero
standard is
measured several times and the limit of sensitivity becomes a concentration
equating
to 2-3 standard deviations (SD) from the mean (M). However, the precision for
this
theoretical determination may be incorrect by an order of magnitude. The
concomitant fitting of any derived external calibration curve(s) does not
create a true
value dynamic dose response curve that can lead to considerable error in the
actual
sensitivity.
To further measure the accuracy of such analytical measurement, accuracy is
used to
define how close the average measured value is to the true value. The
difference in"
measurement is known as the bias or degree of accuracy. Bias may vary over the
range of the assay. It is known in the art that methods for measuring this
true value
need to be developed.
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The repeatability of an assay or the estimated error in an analytical assay is
known in
the art as the percentage coefficient of variation (%CV). Automated assay
analysis
machines can be affected by variations in sample concentration, temperature,
heat and
edge effects, incomplete suspension of particles and solid phase
precipitation.
Precision effects also result from fraction separation and counting errors. In
optical
systems error is due to effects of turbidity, presence of fluorophores,
deterioration of
lamps and detectors and the deterioration, over time, of reagents. These
factors
generally lead to significant decreases in signal to noise ratio. Mechanical
manipulation errors can result from poor pipetting and instrument stand-by
periods.
As a direct result, the assessment for precision of any analytical method
requires the
measurement of resulting variability at known and relevant concentrations by
using
defined or standard control solutions to create baseline calibration
standards. Accurate
determination of such calibrators is based on measurement of known
concentrations
in dilution series at predetermined intervals, which are then interpolated.
Commercially available, as well as in-house prepared reference solutions or
reference
standards are available, but are often calibrated with standard or pooled
matrices,
which may vary considerably from actual patient test samples. Part of the
solution to
overcome these errors is to plot the precision against a wide range of
concentrations to
obtain a precision profile, or calibration, of the assay.
Cross reactivity, assay specificity, bias causing interference, alterations in
antigen,
antibody, binding sites, low dose (competitive assay) and high dose (sandwich
assay)
hook effects, heterophilic antibody interference, endogenous interfering auto-
antibodies, complement, rheumatoid factor, interference in solid phase
antibody
binding, endogenous signal generating substances, enzyme inhibitors, catalysts
and
co-factors have also been shown to express confounding activity in assays,
including
cross reactivity, matrix effects and carry over of sample in automated
immunoassay
instruments and samplers.
For diagnostic applications, the quality control samples may not reflect
actual clinical
concentrations in the patient, may not reflect the spectrum of present
analytes and
interfere with the sample matrix to no longer reflect the content of the
patient
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samples. The quality control samples may measure performance at discrepant
intervals of concentration which may not reflect clinical decision points.
There is therefore a need for an immunoassay that can be reliably calibrated.
Summary of the Invention
The invention is directed to a method for internal dynamic calibration of an
assay
device for determining the concentration of an analyte in a sample where the
assay
device has an assay surface. A plurality of calibration dots containing pre-
determined
quantities of the analyte are printed on the assay surface. A test dot
containing a
reagent for binding said analyte is also printed on the assay surface. The
analyte is
labeled with a detectable marker complex prior to introduction onto the assay
device.
The analyte - detectable marker complex binds to the reagent in the test dot.
The
amount of antigen in the sample is proportional to the intensity of detectable
marker
in the test dot. The calibration dots contain differing pre-determined
quantities of the
analyte. Any unlabeled detectable marker will bind to the calibration dots. An
internally calibrated calibration curve is thus prepared. The intensity of
detectable
marker in the test dot can be compared to the calibration curve to obtain an
absolute
value.
The method provides a quantitative analysis that is carried out rapidly using
a single
assay device with the ability to contain a known minimum volume of test fluid
and
also to have the ability for flowing fluid through the device in order to meet
a known
concentration of analyte as a function of analyte concentration per tested
volume.
Both the calibration dots and test dots are printed within a single assay
device which
then needs only the application of a single, premixed solution containing the
analyte
and an excess of detecting reagent which is preferably an antibody.
The invention further includes a method for obtaining dynamic true dose
response
curves by printing both calibrator and test samples onto a common test
platform
device. The test platform has a minimum of one test dot. Each test dot has
multiple
corresponding calibration dots. The signal obtained from the total number of
comparative concentration dynamic calibration dots, at indexed X / Y co-
ordinates is
integrated to form the dynamic internal calibration true dose response curve.
In a
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similar process, the unknown test dot label response reading is also
integrated over all
obtained readings. The use of multiple test arrays or matrices in platform
format
predicates a confidence limit approaching one hundred percent in having
obtained the
correct test result. The common test platform is exposed to the same test
fluid and
because the calibrator and test samples are exposed simultaneously to the same
test
fluid, accurate measurement of the concentration of analyte present in the
test sample
is determined by the resulting true dose response calibration curve. The
invention
provides the surprising result that the various errors, incurred using known
state of the
art methods, are not reflected when a test sample is processed with the
disclosed
method and device.
According to one aspect of the invention, there is provided a method of
determining
an amount of analyte in a sample solution comprising the following steps:
= providing an assay device having an assay surface having a plurality of
calibration dots printed thereon and a test dot printed thereon, the
calibration
dots containing pre-determined quantities of the analyte, the test dot
including a reagent for binding to said analyte;
= providing a solution having a reagent for binding to the analyte, said
reagent
being labeled with a detectable marker;
= introducing the analyte into said solution to form a sample solution;
= introducing said sample solution onto said assay device;
= measuring an intensity of detectable marker in said calibration dots;
= preparing a calibration curve correlating the amount of analyte in said
calibration dots to said intensity of detectable marker;
= measuring an intensity of detectable marker in said test dot; and
= calculating an amount of analyte present in said test dot by comparing the
intensity of detectable marker to the amount of analyte corresponding to said
intensity in said calibration curve.
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Brief Description of the Drawings
Figure 1 is a top view of an assay device of the present invention for
carrying out a
fixed array test;
Figure 2 is a plot of showing a verification that single and aggregate immuno
complexes are quantifiable;
Figure 3 is a plot showing that antigen concentration in the test sample does
not
impact fluorescence intensity in the calibration spots;
Figure 4 is an illustration of a PicoTip array printing, spot size and array
matrices;
Figure 5 is a plot showing a correlation of analyte concentration with
fluorescence
using dynamic true dose response measurement; and
Figure 6 is top view of an assay device to test for the presence of respective
antibody
response to micro-organisms having been present in human plasma.
Detailed Description of the Invention
The present method is for calibrating an assay device. A preferred assay
device is
shown in Figure 1. The assay device I has an assay surface 10 that preferably
includes a
loading area 18 and a reading area 16. The reading area 16 has printed thereon
at least
one and preferably at least two test dots 20. More preferably, a plurality of
dots for
detecting the presence of the analyte are printed on the reading area 16. The
test dots
include a reagent that specifically binds to the protein analyte. Preferably,
the
20 reagent is bound antibodies that specifically bind to the analyte. Other
reagents known
in the art to bind a specific analyte can also be used. For the balance of the
present
discussion the reagent will be referred to as an antibody.
The bound antibodies are preferably spaced apart to make each bound antibody
available for binding to the test antigen free of stearic hindrance from
adjacent antigen
complexes. Preferably, a non-reactive protein separates the bound antibodies
in the
test dots.
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The reading area 16 has calibration dots 22 printed thereon. The calibration
dots
include a pre-determined amount of said analyte for reacting with un-reacted
reagent
in a vessel, conjugated with a detectable marker. The calibration dots allow
the
intensity of the label to be correlated to the amount of the antigen present.
The
intensity of label in the test dots can then be used to derive the quantity of
antigen
present.
The calibration dots have a concentration of the analyte that corresponds to a
dynamic
range of the analyte. Dynamic range is the concentration of analyte normally
found in
the patient. The quantitation needs to be within this lowest and highest
concentration
and relate to the clinically relevant concentrations.
Many of the problems associated with current methods typical for immunoassays
derive from the assay calibration being determined by introducing external
standard
reference samples for calibration. The present method provides more accurate
results
by not using these standard external calibration samples.
In developing a platform device for measuring the quantity of a respective
analyte,
instead of using external standards to generate a calibration or base line,
both
calibration dots as well as test dots at unknown concentration are printed
onto the
same test platform. The calibration test dots are printed at known
concentrations of
analyte. The test dots are printed, also at predetermined X-Y locations,
containing
only capture reagent which is preferably an antibody specific for the analyte
under
investigation. The test sample is then conjugated with an excess of marker
reagent,
which is preferably an antibody, and analyte. The marker antibody has
previously
been conjugated with a respective fluorescent label, emitting at a suitable
wavelength
(e.g. 650 nanometers). The marker antibody/antigen complex as well as the
free,
remaining marker antibody is then flowed over the test platform using laminar
flow.
A person skilled in the art will appreciate however that other assay devices
that do not
rely on laminar flow may also be employed. For example, an assay device in the
form
of a vessel where the antibody/antigen complex as well as the remaining free
marker
antibody move through the device by diffusion limited kinetics may also be
employed
for the purposes of the present invention.
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In this fashion, marker antibody/unknown concentration of antigen complexes
are
bound by the capture antibody test dots, whereas free marker antibodies bind
to the
pre-printed antigen dots at known concentrations. Both the test dots of
unknown
concentration to be measured and the calibration dots of known concentrations
encompassing the dynamic range of the analyte are exposed to the same test
fluid
sample at the same time. The laminar flow effectively places the analyte
components
within proximity of the respective binding sites to promote optimal adhesion
kinetics
for the respective association constants.
The test platform is examined in a reader for determination of the respective
concentrations of fluorescent label attached to the dots on the platform when
activated
by suitable wavelength irradiation. The calibration dots, preferably
originally printed
at up to ten different concentrations of analyte, result in producing a
dynamic internal
true dose response curve providing very accurate calibration reference for the
assay.
The intensity reading obtained from the test dots of unknown concentration is
compared to this calibration line. The unknown test concentrations accurately
and
efficiently interpolate into the dynamic internal true dose response
calibration
obtained from the known calibration spots.
Each assay device tested provides similarly accurate and reproducible results
confirming that the present method for on-platform dynamic calibration
provides an
accurate, enhanced and sensitive determination of analyte in both quantitative
as well
as qualitative assays for diagnostic use and detection of analytes in clinical
use for
humans and animals. This immediate and significant benefit demonstrates that
these
assays, when processdd using the described method (Dynamic Internal
CalibrationTM),
do not reflect the errors described in association with current other methods
for
running these assays while using externally derived calibration standards.
The ability to calibrate and test simultaneously also enhances the confidence
level in
assuring that the obtained measurements are true. This methodology of the
present
invention, allows for several different analyte tests to be run on the same
assay device
at the same time. Several sets of test dots or test arrays for testing for
different
analytes as well as multiple sets of corresponding calibration dots or
calibration arrays
for the different analytes can be run on the same assay device at the same
time. In
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order to reach a better than 99% confidence level for a test, the test needs
to be run a
minimum of three times. Multiple panels of different tests may also be used
and run,
all at the same time, on a single test platform. The surprising results of the
present
invention prove that errors in the tests are eliminated, reproducibility is
enhanced, the
dynamic range for any test is easily extended to cover a required analyte
concentration range, sensitivity and coefficient of variance is dramatically
improved.
The combination of these advantages over prior state of the art also results
in
considerable saving in time to test results. It is an important advantage that
the format
of the present invention is device independent and may be applied by those
skilled in
1 o the art.
The assay device and method as described, effectively represent a novel,
quantitative
and fast method for the accurate determination of analyte and or marker
concentrations, typically for diagnostic clinical markers associated with
disease
processes. The immediate benefit of rapid, accurate measurement of marker
concentration allows for rapid dynamic detection of marker concentration as an
indicator of a disease process such as increasing, steady or decreasing
concentration,
as well as rapid quantitative monitoring of drug efficacy in modifying gene
expression
for the production of specific marker proteins to indicate drug efficacy.
Examples
Example 1: Quantitative Fluorescent Immuno-Assay.
The sandwich immunoassay matrix incorporates a capture antibody that is
specific for the antigen of interest and a fluorescence conjugated secondary
antibody for detection of analytes and immune complexes. Human chorionic
gonadotropin (HCG), a marker of pregnancy in humans, was used as antigen
for testing of this platform assay. Currently, the dynamic analytical range
for
this test is between 1 to 150 fmol/uL (280-37,600mlU/mL) with an assay
volume of 5 L. Comparison between the calculated HCG concentration using
the device compared with known HCG concentrations has excellent agreement
between values (Figure 2, y=1.0717x + 9.9313) and high correlation between
mean values for each concentration tested (r=0.9786). Figure 2 is a graph that
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shows confirmation that single and aggregate immuno complexes provide
quantifiable fluorescence when bound to the device platform.
Example 2: Measurement of antigen dot fluorescence at various sample antigen
concentrations.
The assay principle is based on quantitative, non-competitive, heterogeneous
immunoassays. First generation devices were printed with a series of antigen
dots at decreasing concentrations for standard curve auto-calibration;
followed
by capture antibody dots. Test sample containing the antigen was processed
with lyophilized detecting antibody already conjugated to the respective
indicator or dye. The test sample reacted with label for 2 minutes and was
then dispensed into the chip assay device to be inserted into the reader. The
fluorescent intensity of each dot was measured. The reader software compares
the fluorescence of the capture antibody dots having unknown antigen
concentration with those of the true dose response standard curve having
known concentrations and calculates the concentration of the test antigen.
Figure 3 is a graph showing data to confirm that antigen concentration in the
test sample does not affect the fluorescence intensities of antigen in the
calibration dots.
Example 3: Advanced Array Printing on the Assay Device platform.
Each of four 10 x 10 matrices containing 100 test dots as shown in Figure 4,
used 2.6
mm x 2.6 mm of platform area. The center-to-center separation from dot to dot
was
260 m. The number of dispensed droplets per spot increments from 1 to 4
droplet(s)
per dot per array matrix. Total chip assay device reading area was 8000 m x
10000
m. This printing pattern allowed 1200 dots to be printed on the platform
reading
area. With a minimum of 5 dots per test, 3 for calibration and 2 test dots,
each
platform supports 240 tests. This technology advantage allows for fmol/ml
antigen
detection sensitivity and an increasing number of multiplex test arrays for
optimal
confidence in diagnostic results by being able to multiplex required test
matrices to
optimize the calibration curves. Figure 4 provides an illustration of advanced
PicoTip
Array printing technology on the chip assay device platform.
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Example 4. Correlation of human Para Thyroid Hormone Concentration with
Fluorescent Intensity calibrated using True Dose Response Arrays measurement.
As shown in Figure 5, the concentrations of human parathyroid hormone was
measured to determine the dynamic internal true dose response calibration
curve. The
tests confirm that the true dose response was accurately plotted over the
dynamic
range as tested..
Example 6: Layout of multiplex format when testing human plasma for 12
different
antibodies in response to having been exposed to 12 micro-organisms.
As shown in Figure 6, the test arrays are printed as triplicate arrays at four
1o concentrations for each test, whereas the calibration arrays, in this case
for human IgG
immunoglobulin, are printed at 6 concentrations in triplicate. An array of 4
dots x 5
dots is used to measure the response of fluorescent label (Dy647) in a binary
coding
format to confirm the identity of the assay platform when inserted into a
reading
device.
Those skilled in the art will recognize, or be able to ascertain using no more
than
routine experimentation, many equivalents to the embodiments of the invention
described above. Such equivalents are intended to be encompassed in the scope
of the
following claims.
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