Note: Descriptions are shown in the official language in which they were submitted.
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MULTIPLEX MEASURE OF ISOTYPE ANTIGEN RESPONSE
FIELD OF INVENTION
The present invention relates to immunogenicity testing of therapeutic
biologicals with
simultaneous quantification of isotype immunoglobulin classes Ig G, A, M, E, D
and sub-
classes including IgGl, IgG2, IgG3, IgG4, IgA 1 and IgA2 within a single
containment
vessel. The invention relates to microarray methods for the simultaneous
detection and
quantification of multiple analytes in a single sample. Analytical components
may include
subunits of therapeutic proteins, including antibody fragments or fusion
partners,
metabolic products, peptide components, formulation components, bio-similars
or
potential cross reacting entities.
BACKGROUND
Current immunoassay methods detect one target per detection test cycle within
a
single reaction well. It is common for several antigenic substances or bio-
markers to be
associated with detection and diagnosis for any pathological or physiological
disorder.
To confirm the presence of multiple markers, each marker within a test sample
often
requires a separate and different immunoassay to confirm the presence of each
target
analyte to be detected. This often requires a multitude of tests and samples,
increases
delay in time to treatment, costs and possibility of analytical error. Current
immunoassay
methods do not detect antibodies that are of multiple isotypes and subclasses
in the same
test well/cycle.
Enzyme Linked Immunosorbent Assay (ELISA) was developed by Engvall et al.,
Immunochem. 8: 871 (1971) and further refined by Ljunggren et al. J. Immunol.
Meth.
88: 104 (1987) and Kemeny et al., Immunol. Today 7: 67 (1986). ELISA and its
applications are well known in the art.
A single ELISA functions to detect a single analyte or antibody using an
enzyme-
labelled antibody and a chromogenic substrate. To detect more than one analyte
in a
sample, a separate ELISA is performed to independently detect each analyte.
For
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example, to detect two analytes, two separate ELISA plates or two sets of
wells are
needed, i.e. a plate or set of wells for each analyte. Prior art chromogenic-
based ELISAs
detect only one analyte at a time. This is a major limitation for detecting
diseases with
more than one marker or transgenic organisms which express more than one
transgenic
product.
Macri, J. N., et al., Ann Clin Biochem 29: 390-396 (1992) describe an indirect
assay wherein antibodies (Reagent-1) are reacted first with the analyte and
then second
labelled anti-antibodies (Reagent-2) are reacted with the antibodies of
Reagent 1. The
result is a need for two separate washing steps which defeats the purpose of
the direct
assay.
US2007141656 to Mapes et al. measures the ratio of self-antigen and auto-
antibody by comparing to a bead set with monoclonal antibody specific for the
self-
antigen and a bead set with the self antigen. This method allows at least one
analyte to
react with a corresponding reactant, i.e. one analyte is a self-antigen and
the reactants are
auto-antibodies to the self antigen.
Another method for detecting multiple analytes is disclosed in US2005118574 to
Chandler et at which makes use of flow cytometric measurement to classify, in
real time,
sequential automated detection and interpretation of multiple biomolecules or
DNA
sequences, each biomolecule having to be separated and independently bound to
a
specific substrate particle, each particle being specific for separating and
concentrating
only a single species of analyte. This concentration of analyte is detected
when the
substrate particle is laser illuminated, as the particles flow, in single
file, past the
illuminating laser beam.
W00113120 to Chandler and Chandler determines the concentration of several
different analytes in a single sample. It is necessary only that there is a
unique
subpopulation of microparticles for each sample / analyte combination using
the flow
cytometer. These bead based systems' capability is limited to each
microparticle i.e. bead
being suspended in a volume of test fluid that contains the analyte to be
detected as a
separate entity which needs to bind freely and specifically onto the surface
of the test
bead. Each bead effectively provides a requisite detection signal for only a
specific
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analyte entity. Multiple, different entity binding events onto a single
microparticle are not
well distinguished or quantified using flow cytometry being restricted to
multiple events
of the same antibody isotype/subclass.
Simultaneous detection of more than one analyte, i.e. multiplex detection for
simultaneous measurement of proteins has been described by Haab et al.,
"Protein micro-
arrays for highly parallel detection and quantization of specific proteins and
antibodies in
complex solutions," Genome Biology 2(2): 0004.1-0004.13,( 2001), which is
incorporated herein by reference. Mixtures of different antibodies and
antigens were
prepared and labelled with a red fluorescence dye and then mixed with a green
fluorescence reference mixture containing the same antibodies and antigens.
The
observed variation between the red to green ratio was used to reflect the
variation in the
concentration of the corresponding binding partner in the mixes. This method
is not
suitable for quantitative results.
Mezzasoma et al. (Clinical Chemistry 48, 1, 121-130 (2002) published a micro-
array format method to detect analytes bound to the same capture spot in two
separate
assays, specifically different auto-antibodies reactive to the same antigen.
The results
revealed that when incubating the captured analytes with one reporter (for
example that to
detect immunoglobulin IgG), the corresponding analyte is detected. When
incubating the
captured analytes with the second reporter in an assay using a separate
microairay solid-
state substrate (for example to detect IgM), a second analyte (IgM) is
detected.
W00250537 to Damaj and Al-assaad discloses a method to detect up to three
immobilized concomitant target antigens, bound to requisite antibodies first
coated as a
mixture onto a solid substrate. A wash step occurs before the first marker is
detected. The
presence of the first marker may be detected by adding a first specific
substrate. The
reaction well is read and a color change is detectable with light microscopy.
Another
wash step occurs before the second marker is detected. The presence of the
second
marker may be detected by adding a second substrate, specific for the second
enzyme, to
the reaction well. After sufficient incubation, the reaction well may be
assayed for a color
change. Similarly, a wash step may occur before the third marker is detected.
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The presence of the third marker may be detected by adding a third substrate,
specific for the third enzyme, to the reaction well. After sufficient
incubation, the reaction
well may be assayed for a color change. Although more than one analyte may be
detected
in a single reaction or test well, each reaction is processed on an individual
basis.
W02005017485 to Geister et al. describes a method to sequentially determine at
least two different antigens in a single assay by two different enzymatic
reactions of at
least two enzyme labelled conjugates with two different chromogenic substrates
for the
enzymes in the assay (ELISA), which comprises (a) providing a first antibody
specific for
a first analyte and a second antibody specific for a second analyte
immobilized on a solid
support; (b) contacting the antibodies immobilized on the solid support with a
liquid
sample suspected of containing one or both of the antigens for a time
sufficient for the
antibodies to bind the antigens; (c) removing the solid support from the
liquid sample and
washing the solid support to remove unbound material; (d) contacting the solid
support to
a solution comprising a third antibody specific for the first antigen and a
fourth antibody
specific for the second antigen wherein the third antibody is conjugated to a
first enzyme
label and the fourth antibody is conjugated to a second enzyme label for a
time sufficient
for the third and fourth antibodies to bind the analytes bound by the first
and second
antibodies; (e) removing the solid support from the solution and washing the
solid support
to remove unbound antibodies; (f) adding a first chromogenic substrate for the
first
enzyme label wherein conversion of the first chromogenic substrate to a
detectable color
by the first enzyme label indicates that the sample contains the first
analyte; (g) removing
the first chromogenic substrate; and (h) adding a second chromogenic substrate
for the
second enzyme label wherein conversion of the second chromogenic substrate to
a
detectable color by the second enzyme label indicates that the sample contains
the second
analyte.
U.S. Patent 7,022,479, 2006 to Wagner, entitled "Sensitive, multiplexed
diagnostic assays for protein analysis", is a method for detecting multiple
different
compounds in a sample, the method involving: (a) contacting the sample with a
mixture
of binding reagents, the binding reagents being nucleic acid-protein fusions,
each having
(i) a protein portion which is known to specifically bind to one of the
compounds and (ii)
a nucleic acid portion which includes a unique identification tag and which in
one
embodiment, encodes the protein; (b) allowing the protein portions of the
binding
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reagents and the compounds to form complexes; (e) capturing the binding
reagent-
compound complexes; (d) amplifying the unique identification tags of the
nucleic acid
portions of the complex binding reagents; and (e) detecting the unique
identification tag
of each of the amplified nucleic acids, thereby detecting the corresponding
compounds in
the sample.
While methods for sequentially detecting and quantifying multiple analytes
limited to capturing isotype and subclass for a single analyte are known,
these methods
require the use of separate assaying steps for each of the analytes of
interest and as such,
can be time consuming and costly, especially in the context of a clinical
setting. There is
a need for a method of sequentially detecting and quantifying multiple
antibody isotypes
and subclasses from a single sample using a single reaction vessel.
SUMMARY OF INVENTION
The present invention provides a fast and cost effective method for
simultaneous
detection and quantifying of multiple analytes in a test sample using a single
reaction
vessel. The method disclosed herein allows for the simultaneous detection of
multiple
analytes without the need for separate assays or reaction steps for each
target analyte.
In one aspect, the present invention provides a method for simultaneously
detecting and quantifying two or more target analytes in a test sample
comprising two or
more target analytes:
a) providing a reaction vessel having a microarray printed thereon, said
microarray comprising:
i) a first calibration matrix comprising a plurality of the first calibration
spots, each calibration spot comprising a predetermined amount of a first
target
analyte, the first target analyte being an antibody isotype or an antibody sub-
class,
ii) a second calibration matrix comprising a plurality of the second
calibration spots, each calibration spot comprising a predetermined amount of
a
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second target analyte, the second target analyte being an antibody isotype or
antibody sub-class,
iii) a first capture matrix comprising a plurality of first capture spots,
each
of said first capture spots comprising a predetermined amount of a first agent
which selectively binds to the target analytes, and
iv) a second capture matrix comprising a plurality of second capture spots,
each of said second capture spots comprising a predetermined amount of a
second
agent which selectively binds to the target analytes;
b) adding a predetermined volume of the test sample to the microarray;
c) simultaneously applying at least two fluorescently labelled antibodies into
the
same well, each of the at least two fluorescently labelled antibodies being
specific for one
of the target analytes for selectively binding to one of the target analytes
for individual
identification and quantification of said target analytes, each of said
fluorescently labelled
antibodies comprising a different fluorescent dye having emission and
excitation spectra
which do not overlap with each other;
d) measuring a signal intensity value for each of the analytes within each
microarray spot;
e) generating calibration curves by fitting a curve to the measured signal
intensity
values for each analyte contained in each of the calibration spots versus a
known
concentration of the first target analyte and the second target analyte; and
0 determining the concentration for the first target analyte and the second
target
analytes using the generated calibration curves.
In a further embodiment of the present invention, the reaction vessel is a
well of a
multi-well plate where the well has the microarray printed therein.
In a further embodiment of the present invention, the test sample is a
biological
sample.
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In another aspect, the present invention provides a method for detecting and
quantifying biomarkers diagnostic for immunogenicity testing of a therapeutic
protein e.g.
insulin, comprising the following steps:
a) providing an assay device having a microarray printed thereon, said
microarray
comprising:
i) a plurality of calibration matrices, each comprising a plurality of
calibration
spots, each calibration spot comprising a predetermined amount of a target
analyte being
an antibody selected from the group consisting of anti-insulin peptide human
immunoglobulin classes Ig G, A, M, E and sub-classes including anti-insulin
human
immunoglobulin peptide sub-classes IgGl, Ig02, IgG3, IgG4 and IgA.
ii) an analyte capture matrix comprising a plurality of capture spots, each
capture
spot comprising a predetermined amount of an agent which selectively binds to
the target
analytes,
b) applying a predetermined volume of a serum sample to the assay device;
c) applying a plurality of different fluorescently labelled antibodies which
selectively bind to human immunoglobulin classes Ig G, A, M, E, D and sub-
classes
including IgG 1 , IgG2, IgG3, IgG4 and IgA 1 , IgA2 respectively, the
fluorescently
labelled antibodies including a first fluorescently labelled anti-antibody
which
specifically binds to IgA antibodies, a second fluorescently labelled anti-
antibody which
selectively binds to IgG antibodies, a third fluorescently labelled anti-
antibody which
selectively binds to IgM antibodies, a fourth fluorescently labelled anti-
antibody which
selectively binds to IgE antibodies, a fifth fluorescently labelled anti-
antibody which
selectively binds to IgD antibodies, a sixth fluorescently labelled anti-
antibody which
selectively binds to sub-class IgG1, a seventh fluorescently labelled anti-
antibody which
selectively binds to sub-class IgG2, an eighth fluorescently labelled anti-
antibody which
selectively binds to sub-class IgG3, a ninth fluorescently labelled an anti-
antibody which
selectively binds to sub-class IgG4,wherein said first, second, third fourth,
fifth, sixth,
seventh and eighth fluorescently labelled antibodies each comprise a different
fluorescent
dye having emission and excitation spectra which do not overlap with each
other;
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d) measuring a signal intensity value for each immunoglobulin contained within
each spot forming the microarray printed in each well of the assay device;
e) generating calibration curves by fitting a curve to the measured signal
intensity
values for the each of the calibration spots versus the known concentration of
the human
IgA, IgG, IgE, IgM, IgD and subclass antibodies; and
0 determining the concentration for each of captured anti-insulin human
isotype
class and subclass immunoglobulins.
In another aspect, the present invention provides a method for diagnosing
multiplex immunogenicity, antibody and insulin factor immunogenicity in a
subject,
comprising:
a) measuring the concentration levels of class and subclass anti-insulin IgA,
anti-
insulin IgG, anti-insulin IgM, anti-insulin IgE; and
b) comparing the measured concentration levels of anti-insulin IgA, anti-
insulin -
IgG, anti-insulin IgM, anti-insulin IgE, anti-insulin peptide immunoglobulins
IgM with
index normal levels of anti-insulin immunoglobulins and anti-insulin peptide
immunoglobulins wherein measured concentrations levels which exceed index
normal
levels is diagnostic for insulin immunogenicity.
In an embodiment of the present invention, the detection and quantification of
predominantly anti-insulin IgM and anti-insulin peptide-IgM antibodies is
diagnostic for
an early stage of insulin immunogenicity.
In a further embodiment of the present invention, the detection and
quantification
of anti-insulin IgA and anti-insulin peptide-IgA antibodies is diagnostic for
a transitional
stage of insulin immunogenicity.
In a further embodiment of the present invention, the detection and
quantification
of anti-insulin IgG, anti-insulin peptide-IgG antibodies as well as their
subclasses is
diagnostic for a late stage of insulin immunogenicity.
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In another aspect, the present invention provides a method for monitoring
reactions to immune stimuli by multiplex immuno testing, monitoring
development of
neutralizing antibodies, including for example, specific insulin
immunogenicity in a
subject exposed to various insulin drug immune stimuli, using the method
disclosed
herein, a plurality of times in the course of treatments.
BRIEF DESCRIPTION OF THE DRAWINGS
Preferred embodiments of the invention will now be described, by way of
example, with reference to the accompanying drawings, in which:
Figure 1 is a schematic illustration of the multiplex analyte detection method
of
the present invention;
Figure 2 is a schematic illustration of the multiplex analyte immunogenicity
detection content per spot, in a single well, including IgG, IgA, IgM, IgE and
subclasses
for method of the disclosed invention, defined as Ig_plex.
Figure 3 is a schematic illustration of a microarray printed on an assay
device of
the present invention;
Figure 4 illustrates typical 4 level multiplex microarray spots contained in a
single
well of an assay device of the present invention; and
Figure 5 is a plot to illustrate composite fluorescent intensity detection for
six
fluorophores at non-interfering wavelengths, at 457nm (nanometers), 488 nm,
575 urn,
615nm, 667 nm and 767 nm of the present invention.
DESCRIPTION
The present invention provides a method for the detection and quantification
of
multiple target analytes contained within each test spot or arrays of spots,
of a test or test
samples, within a single reaction well, per test cycle. The method disclosed
herein
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provides for the simultaneous incubation of an assay device with two or more
fluorescently labelled reporters in the same detection mixture as shown in
Figure 2. The
method disclosed herein can detect a plurality of multiplexed analytes per
test spot or
capture spot, using a single reaction vessel instead of separate reaction
vessels to detect
each analyte. The terms "test spot" and "capture spot" can be used
interchangeably for the
purposes of the present specification.
Figure 1 illustrates the capturing of six different antibodies which
selectively bind
to two different antigens. The six different antibodies fall into three
different antibody
classes. In this example, an IgG is included that specifically binds to
antigen A while a
separate IgG is included that specifically binds to antigen B. Similarly, an
IgA is
included that specifically binds to antigen A while a separate IgA is included
that
specifically binds to antigen B. Finally, an IgM is included that specifically
binds to
antigen A while a separate IgM is included that specifically binds to antigen
B. In such
embodiments, only one calibration matrix may be required for each of the three
different
classes of immunoglobulins.
For example, when the target analytes of interest are different classes of
human
antibodies e.g. hIgG, hIgA, hIgM, hIgE and their respective subclasses are
directed to the
same antigen (i.e. the Fc region of hIgG), the detection and quantification of
each of the
target antibodies requires separate assays when conventional methods are
employed.
With conventional methods, one assay is performed to detect and quantify the
amount of
hIgG present in a test sample. A second assay must be performed to detect and
quantify
the amount of hIgM and more assays must be performed to detect and quantify
the
presence of isoform classes and subclasses. In contrast, the method of the
present
invention eliminates the need for multiple detection steps thus reducing costs
and time.
Using the method of the present invention, target hIgG, hIgA, hIgM, hIgD and
hIgE
molecules contained in a test sample can be bound to a single capture spot in
an assay
device. In the disclosed method, the different classes of antibodies and
antibody sub-
classes can be detected in a single test by using a cocktail of fluorescently
labelled
antibodies directed to each of the isoform class hIgG, hIgM, hIgA, hIgE and
respective
subclass targets. As the antibodies are labelled with different optically
excited and
emitted fluorescent probes, each of the targets bound to a single capture spot
can be
detected and quantified using an appropriate calibrator. The use of multi-
channel
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detectors allows for substantially simultaneous detection of multiplex
analytes in a single
assay. The spot morphology and density of capture molecules is optimized so as
to
mitigate for steric hindrance. As shown in Figure 2, the target analytes are
IgG, IgA, IgM,
IgE, IgG I, IgG2, IgG3, and IgG4. Capture spots include an antigen that binds
to these
antibody isotypes and subclasses. Once the target analyte antibodies bind to
the capture
spots, fluorescently labeled anti-IgG, anti-IgA, anti-IgM, anti-IgE, anti-IgG
1, anti-IgG2,
anti-IgG3, and anti-IgG4 bind to the respective target analyte antibodies so
that the
amount of bound IgG, IgA, IgM. IgE, IgGl, IgG2, IgG3, and IgG4 can be
detected.
The methods disclosed herein employ assay devices useful for conducting
immunoassays. The assay devices may be microarrays in 2 or 3-dimensional
planar array
format.
In one embodiment, the method may employ the use of a multi-well plate and
wherein each well has a microarray printed therein. A single well is used as a
reaction
vessel for assaying the desired plurality of target analytes for each test
sample.
The microarray may comprise a calibration matrix comprising a series of
calibration spots for each target analyte and an analyte capture matrix
comprising one or
more of test spots or capture spots which bind the target analytes. A
representative
microarray is shown in Figure 3. The microarray 1 includes capture spots 2 and
calibration spots 4. In addition internal control spots 6 are included to
ensure that the
microarray is functioning properly.
As used herein, the term "calibration matrix" refers to a subarray of spots
printed
on and adhering to the reaction vessel, wherein each spot comprises a
predetermined
amount of a calibration standard. The term "predetermined amount" as used
herein,
refers to the amount of the calibration standard as calculated based on the
known
concentration of the spotting buffer comprising the calibration standard and
the known
volume of the spotting buffer printed on the reaction vessel.
The choice of the calibration standard will depend on the nature of the target
analyte. In such embodiments, the microarray will comprise a separate
calibration
standard for each target analyte. Alternatively, the microarray may comprise a
single
calibration matrix having calibration spots containing each of the target
analytes.
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In alternate embodiments, the calibration standard is a surrogate compound.
For
example if the target analyte is an antibody, the surrogate compound may be
another
different antibody but of the same class of immunoglobulin, as shown in Figure
4. The
calibration matrix may be printed on the base of the individual reaction
vessel in the
format of a linear, proportional dilution series. The predetermined
concentrations of
calibration standards are selected to include lower and upper expected
detection limits to
define the dynamic range. The mid-point of dynamic calibrated concentration
range
approximates the diagnostic critical concentration of the detection system
used to read the
microarray
As used herein, the term "analyte capture matrix" refers to a subarray of
spots
comprising agents which selectively bind the target analytes. In embodiments
where the
target analyte is a protein, the agent may be an analyte specific antibody or
fragment
thereof. Conversely, in embodiments wherein the target analyte is an antibody,
the agent
may be an antigen specifically bound by the antibody. For example, Figure 4
illustrates
the capturing of five different antibodies which selectively bind to two
different antigens.
A predetermined volume of a test sample is applied to the assay device. Each
of
the target analytes will bind to their specific capture spot. Thus, in a
single capture spot,
multiple target analytes may be bound. To detect each of the target analytes,
a
fluorescently labelled antibody which specifically binds to the target analyte
is used.
Each fluorescently labelled antibody is coupled to a unique fluorescent dye
with a
specific excitation and emission wavelength to obtain the desired Stokes shift
and
excitation and emission coefficients. The fluorescent dyes are chosen based on
their
respective excitation and emission spectra such that each of the labelled
antibodies
comprises a different fluorescent dye having emission and excitation spectra
which do not
overlap with each other. The fluorescently labelled antibodies can be applied
to the assay
device in a single step in the form of a cocktail.
A signal intensity value for each spot within the assay device is then
measured as
shown in Figure 5. The fluorescent signals can be read using a combination of
scanner
components such as light sources and filters. A signal detector can be used to
read one
optical channel at a time such that each spot is imaged with multiple
wavelengths, each
wavelength being specific for a target analyte. An optical channel is a
combination of an
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excitation source and an excitation filter, matched for the excitation at a
specific
wavelength. The emission filter and emission detector pass only a signal
wavelength for
a specific fluorescent dye. The optical channels used for a set of detectors
are selected
such that they do not interfere with each other, i.e. the excitation through
one channel
excites only the intended dye, not any other dyes. Alternatively, a multi-
channel detector
can be used to detect each of the differentially labelled antibodies. The use
of differential
fluorescent labels allows for substantially simultaneous detection of the
multiple target
analytes bound to a single capture spot.
The intensity of the measured signal is directly proportional to the amount of
material contained within the printed calibration spots and the amount of
analyte from the
test sample bound to the printed analyte capture spot. For each calibration
compound, a
calibration curve is generated by fitting a curve to the measured signal
intensity values
versus the known concentration of the calibration compound. The concentration
for each
target analyte in the test sample is then determined using the appropriate
calibration curve
and by plotting the measured signal intensity for the target analyte on the
calibration
curve.
The method disclosed herein can be used to detect and quantify multiple
clinically
relevant biomarkers in a biological sample for diagnostic or prognostic
purposes. The
measured concentrations for a disease related biomarker can be compared with
established index normal levels for that biomarker. The measured
concentrations levels
which exceed index normal levels may be identified as being diagnostic of the
disease.
The method disclosed herein can also be used to monitor the progress of a
disease and
also the effect of a treatment on the disease. Levels of a clinically relevant
biomarker can
be quantified using the disclosed method a plurality of times during a period
of treatment.
A trending decrease in biomarker levels may be correlated with a positive
and/or negative
patient response to treatment.
The method disclosed herein can be used to detect and quantify biomarkers
diagnostic for insulin immunogenicity. In one embodiment, the method comprises
the
provision of an assay device having a microarray printed thereon. The
microatray may
comprise: i) a calibration matrix comprising plurality of calibration spots,
each calibration
spot comprising a predetermined amount of one of: a human IgA antibody, a
human IgG
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antibody, a human IgM antibody, a human IgE antibody and respective
subclasses; ii) a
first analyte capture matrix comprising a plurality of capture spots
comprising a
predetermined amount of a compound, for example, insulin; and optionally iii)
a second
analyte capture matrix comprising a plurality of capture spots comprising a
predetermined
amount of anti-insulin peptide. A predetermined volume of a biological sample,
preferably a serum sample, is applied to the assay device. A cocktail
comprising a first
fluorescently labelled reporter compound which selectively binds to IgA
antibodies, a
second fluorescently labelled reporter compound which selectively binds to IgG
antibodies, a third fluorescently labelled reporter compound which selectively
binds to
IgM antibodies, a fourth fluorescently labelled reporter which selective binds
to IgE and
fluorescent labels which bind selectively to imtnunoglobulin subclasses, is
then applied to
the assay device. The first, second, third, fourth and selected subclass
fluorescently
labelled antibodies are chosen such that each of the antibodies comprises a
different
fluorescent dye having emission and excitation spectra which do not overlap
with each
other, as shown in Figure 5. A signal intensity value for each spot within the
assay device
is then measured using a single or multi-channel detector as discussed above.
Using the
measured signal intensity values, calibration curves are then generated by
fitting a curve
to the measured signal intensity values for each of the calibration spots
versus the
concentration of the human IgA, IgG, IgM, IgE and subclass antibodies. The
concentration for each of the captured insulin analytes is determined using
the calibration
curves.
In certain embodiments, the method disclosed herein can be used to diagnose or
monitor the progress of autoimmune diseases. In other embodiments, the method
disclosed herein can be used for monitoring the progress of treatment.
Example 1 ¨ Multiplex Immunoaenicitv Testinz
Wherein a therapeutic protein and /or its analytical components are
immobilized on a
planar microarray surface analytical components may include subunits of the.
Analytical
components include subunits of the therapeutic protein e.g. antibody fragments
or fusion
partners, metabolic products of said therapeutic protein, peptide components,
formulation
components, biosimilars, or potential cross reacting entities.
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Samples collected from untreated and therapeutic protein treated patients are
incubated
with the immobilized microarray components. Samples are most likely to be
serum or
plasma. These samples may be pre-treated or prepared in such a way as to
enrich for the
availability of any antibodies which the patient may have developed in
response to the
therapeutic protein or prior exposure to similar entities. Following sample
incubation the
microarray surface is interrogated for the presence of patient derived
antibodies which
have been captured and bound by the immobilized analytes.
The amount of and heavy chain characteristics of the captured patient
antibodies are
determined by the use of specific anti-human secondary antibodies which have
been
conjugated to fluorescent dyes.
Secondary reagents can be included to determine the immunoglobulin class Ig G,
A, M or
E or the sub- classes, including IgGl, IgG2, IgG3, and IgG4. Specific dyes are
conjugated to each of the secondary reagents to constitute a reporter and
allow
differentiation of each of the Ig classes or subclasses. A reporter aliquot is
made up of a
mix of conjugates as determined by the classes and subclasses that are of
interest in the
patient study
This assay can be configured to use (i) a three color fluorescent scanner by
including the
same patient sample in multiple interrogated wells and adding a three color
constituent
reporter blend to each of the wells; or (ii) increased to multiples of up to
six colors per
well as determined by selecting fluorescent dyes which have separable emission
peaks
used in conjunction with a scanner equipped with appropriate excitation and
emission
filters.
The intensity of the multiple fluorescent signals when compared to standard
curves
intensities will allow the qualitative and quantified measurement of a
specific immune
response to the therapeutic protein or the protein associated analytes.
Example 2: Neutralizine Antibodies
The method also interrogates neutralizing effects of a patient's antibodies,
ie; their ability
to directly affect the active mechanism of the therapeutic protein
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In cases where the therapeutic protein is a ligand that binds to a receptor,
the receptor will
be immobilized on the array surface. A fluorescently labeled derivative of the
therapeutic
protein will be incubated with patient serum in a competitive type
immunoassay. A high
fluorescent signal in this case indicates an absence of neutralizing
antibodies. As the titer
of neutralizing antibodies increases in a sample, they will interfere with the
ability of the
labeled therapeutic protein to bind the receptor and thus decrease the
florescent signal on
the array surface.
In cases where the mechanism of the therapeutic protein is to block a
ligand/receptor
interaction: the receptor is immobilized on the microarray surface. A
fluorescently
labeled derivative of the appropriate ligand, and the therapeutic protein is
incubated with
patient serum. In the absence of neutralizing antibodies the therapeutic
protein will block
the binding of the labeled ligand to the receptor and little or no fluorescent
signal will be
detected. As the titer of neutralizing antibodies increases, they will
interfere with the
ability of the therapeutic protein to block the ligand/receptor interaction,
and the
fluorescent signal will increase.
Example 3 Insulin Immunogenicity
As new insulin variants are developed the need to study the range of immune
responses in
patients requires the ability to detect, characterize and quantitate anti-
insulin antibodies.
Regardless of purity and origin, therapeutic insulins continue to be
immunogenic in
humans. Severe immunological complications rarely occur. Current human insulin
and
insulin analog therapies result in decreased anti-insulin antibodies levels.
Anti-insulin
antibody development is also affected by the mode of delivery: e.g. use of
subcutaneous
and implantable insulin pumps or inhaled insulin. Formulation also effects
immunogenic
potential with regular or semilente insulins being less immunogenic than
intermediate or
long acting preparations. Aggregation levels also affect immunogenicity.
Anti-insulin antibodies responses consisting of Ig classes and IgG subclasses
have
been reported. Primarily IgG1-4 but IgA, IgM and IgE have also been reported.
IgG is
implicated in the most severe cases of insulin resistance. Insulin delivered
or inhaled
results in a similar distribution of IgG subclasses: IgG1 >IgG4 > IgG2 and
IgG3. IgG1
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levels have been reported to decline where IgG4 rises with increased duration
of insulin
treatment.
The method disclosed is uniquely suited to detect and differentiate the range
of
anti-insulin antibodies in a single assay, as opposed to running a separate
assay for each
Ig class or subclass.
In this case the therapeutic insulin is printed as multiple replicate spots in
each well of a
96 well functionalized glass plate. The print conditions, including buffers,
concentration,
and post print processes are selected to optimize eptitope presentation and
assay
precision. Assay controls including anti-human antibodies or other variants of
insulin
could be included in each of the 96 wells.
Each well is incubated with patient serum. In cases where the patient has anti-
insulin
antibodies they are captured by the spotted insulin. Fluorescently labeled
anti-human Ig
secondary reagents are used to detect the binding anti-insulin antibodies.
Secondary
reagents include Ig Class specific ( IgG, IgA , IgM or IgE) or subclasss
specific (IgGl,
IgG2, IgG3, IgG4). A Fluorescent dye with a different emission spectrum is
conjugated to
each of the secondary reagents allowing the patient immune response to be
characterized
based on the intensity of each signal.
In the case where a commercial 3-color array scanner is used to detect the
fluorescent
signals, the same patient sample is interrogated in multiple wells and
different aliquots of
labeled reporters are used to fluorescently label each reporter in each well
with a specific
dye wavelength, e.g. in Well 1, IgA is labeled with dye Cy5 at 667 nm
(nanometers),
IgG1 with dye FITC at 488 nm and IgG3 with dye PE at 575 nm to measure the
IgA,
IgG1 and IgG3 in Well 1. In Well 2, IgM is labeled with Cy5 at 667 nm, IgG2 is
labeled
with FITC at 488 nm and IgG4 is labeled with PE to fluoresce at 575 nm to
measure
IgM, IgG2 and IgG4. The six immunoglobulins are measured using only three
fluorescing
labels.
For simultaneous detection of up to six different color fluorescent
wavelengths per well,
as illustrated in Figure 5, the excitation source and emission filters are
coordinated with a
set of dyes with compatible spectra; IGg 1 -FITC dye at 488 nm, IgG2- PE dye
at 575 nm,
IgG3 - Cy5 dye at 667 nm, IgG4 - Pe-Cy dye at 767 nm, IgA- Pac Blue dye at 457
run,
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IgM - Alexa fluor dye at 594 615 nm. If more than six fluorescent labels are
required to
report and measure multiple antigens, the identified set, or compatible sets
of six
wavelengths are applied in each separate well. It is to be understood that
more than six
different color fluorescent wavelengths per well can be detected in alternate
embodiments
of the present invention.
Various embodiments of the present invention having been thus described in
detail by
way of example, it will be apparent to those skilled in the art that
variations and
modifications may be made without departing from the invention. The invention
includes
all such variations and modifications as fall within the scope of the appended
claims.
