U.S. patent application number 16/298869 was filed with the patent office on 2020-01-09 for low density microarrays for vaccine related protein quantification, potency determination, and efficacy evaluation.
The applicant listed for this patent is InDevR, Inc.. Invention is credited to Kathy L. Rowlen.
Application Number | 20200011867 16/298869 |
Document ID | / |
Family ID | 47293655 |
Filed Date | 2020-01-09 |
United States Patent
Application |
20200011867 |
Kind Code |
A1 |
Rowlen; Kathy L. |
January 9, 2020 |
Low Density Microarrays for Vaccine Related Protein Quantification,
Potency Determination, and Efficacy Evaluation
Abstract
Methods for the quantification of influenza HA proteins and
anti-influenza antibodies for the fields of vaccine-related protein
quantification, potency determination, and efficacy evaluation are
provided. According to the technology, quantification is achieved
by providing capture agents attached to an array in a series of
decreasing concentrations. Serial dilutions of a reference material
also may be introduced. The reference material within each solution
binds to the capture agents on the array and is labeled with a
label agent capable of producing a detectable signal used to
construct a calibration curve. A target material of unknown
concentration is introduced to a separate identical array, and the
target material binds to the capture agents and also is labeled by
a label agent to produce a detectable signal. The calibration curve
based on the reference material is then utilized to determine the
concentration of the target material without the need to perform
replicate experiments.
Inventors: |
Rowlen; Kathy L.; (Boulder,
CO) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
InDevR, Inc. |
Boulder |
CO |
US |
|
|
Family ID: |
47293655 |
Appl. No.: |
16/298869 |
Filed: |
March 11, 2019 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13494802 |
Jun 12, 2012 |
10261081 |
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16298869 |
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61496223 |
Jun 13, 2011 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
G01N 33/56983 20130101;
G01N 2333/11 20130101; G01N 33/6845 20130101 |
International
Class: |
G01N 33/569 20060101
G01N033/569; G01N 33/68 20060101 G01N033/68 |
Goverment Interests
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
[0003] The U.S. Government has a paid-up license in this invention
and the right in limited circumstances to require the patent owner
to license others on reasonable terms as provided for by the terms
of Grant No. R43 All 02318 awarded by National Institutes of
Health.
Claims
1-15. (canceled)
16. A system for quantification of an amount of influenza
hemagglutinin (HA) target protein present in a sample, comprising:
a solid support; a plurality of capture agents affixed to said
solid support, wherein: said plurality of capture agents bind
influenza HA, said plurality of capture agents are affixed to the
solid support in a plurality of discrete areas in an array, said
discrete areas each containing only a single unique capture agent;
said plurality of capture agents comprise: a first anti-influenza A
H1 antibody capture agent; a second anti-influenza A H3 antibody
capture agent; and a third anti-influenza B antibody capture agent;
or sialic acid glycopeptides or proteins; at least one label agent
to produce a detectable signal indicative of binding between said
plurality of capture agents and the influenza HA target.
17. (canceled)
18. The system of claim 16, wherein the at least one label agent
binds target influenza hemagglutinin A H1N1 and A H3 and influenza
B.
19. The system of claim 16, wherein at least one of said capture
agents is a universal anti-influenza antibody and the at least one
label agent binds target influenza hemagglutinin.
20. (canceled)
21. (canceled)
22. The system of claim 16, wherein said plurality of discrete
areas are arranged in a configuration of decreasing concentration
of said capture agents.
23. The system of claim 16, wherein the label agent is a
fluorescent, colorimetric, electrical or magnetic label.
24. The system of claim 16, further comprising a calibration curve
to quantify influenza HA in the sample from the detected
signal.
25. The system of claim 24, wherein the calibration curve is based
on a magnitude of detectable signal created by binding between a
standard influenza antigen and the capture agents.
26. The system of claim 16, wherein said anti-influenza A H1
antibody capture agent is a monoclonal antibody specific to
influenza A H1.
27. The system of claim 26, wherein the influenza A H1 is influenza
A H1N1.
28. The system of claim 16, wherein said anti-influenza A H3
antibody capture agent is a monoclonal antibody specific to
influenza A H3.
29. The system of claim 28, wherein the influenza A H3 is influenza
A H3N2.
30. The system of claim 16, configured for use with a sample
comprised of material from an influenza vaccine production
process.
31. The system of claim 16, further comprising at least one
positive control and at least one negative control affixed to the
solid support.
32. The system of claim 16, wherein the capture agents comprise
.alpha.2,6 and .alpha.2,3 sialic acid glycopeptides or
proteins.
33. The system of claim 16, wherein the capture agents comprise:
the first anti-influenza A H1 antibody capture agent; the second
anti-influenza A H3 antibody capture agent; and the third
anti-influenza B antibody capture agent.
34. The system of claim 16, comprising a plurality of label agent
species, with each species specific to a unique target protein.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation application of U.S.
application Ser. No. 13/494,802, filed Jun. 12, 2018, which claim
priority to and the benefit of U.S. Provisional Patent Application
Ser. No. 61/496,223, filed Jun. 13, 2011, both of which are
incorporated herein by reference in their entirety.
ABSTRACT
[0002] The invention provides array-based methods for the
quantification of vaccine-related proteins for potency
determination and efficacy evaluation. In certain embodiments,
influenza HA proteins and anti-influenza antibodies can be
quantified by providing a plurality of appropriate capture agents
immobilized onto an array at a series of concentrations so as to
generate a complete binding curve for a reference material that can
then be utilized to determine the unknown concentration of a target
species. In some embodiments, the capture agents are
strain-specific influenza anti-HA antibodies. In other embodiments,
the capture agents are universal anti-influenza antibodies. In
other embodiments, the capture agents are sialic acid
glycoproteins. In even other embodiments, the capture agents are
influenza hemagglutinin proteins.
BACKGROUND
[0004] The FDA approved gold standard potency assay for influenza
hemagglutinin protein-based vaccines is single radial
immunodiffusion (SRID). SRID is a time and labor intensive assay,
often requiring 2-3 days to complete and a minimum of 6 hours hands
on time by well-trained analysts. In the SRID assay, viral antigen
is applied to an agarose gel matrix that contains specific
antibodies to the antigen being measured. The applied antigen
diffuses radially, and interaction between antigen and antibody
produces a zone of precipitation. The size of this precipitation
zone is directly proportional to the amount of antigen applied. The
assay is also separately performed a number of times on dilutions
of a reference standard, and the resulting calibration curve is
then used to then determine the amount of antigen present in the
unknown. Although used as the current gold standard, it is
well-known that the SRID assay for influenza potency determination
is fraught with uncertainty [Minor, P. D. "Vaccines against
seasonal and pandemic influenza and the implications of changes in
substrates for virus production", Clinical Infectious Diseases,
2010, 50, 560-565. and references therein].
[0005] While the reference standards are provided at no cost by the
Center for Biologics Evaluation and Research (CBER), additional
materials must be purchased and prepared by the entity performing
the testing. Based on a conservative operating cost of $100 per
hour for a small protein manufacturer, the minimal cost per SRID
assay is $600. At the research phase, a small-scale influenza
vaccine producer typically conducts 20-25 SRID assays per week
during the development and testing phase. Thus, the cost of SRID
even for small scale R&D can be on the order of $60,000 per
month. Perhaps even more important is the development and
production delays imposed by the slow turn-around time for the
method. Often vaccine producers must wait days for results prior to
moving forward in the process, decreasing throughput and
efficiency.
[0006] In their Fiscal Year 2010 CBER Annual Report [FY2010 CBER
Annual Report: Innovative Technology Advancing Public Health,
downloaded Apr. 19, 2012 from
http://www.fda.gov/aboutfda/centersoffices/officeofmedicalproductsandtoba-
cco/cber/ucm122930.htm], CBER summarized a 3-day workshop held to
evaluate lessons learned from potency testing of the pandemic 2009
H1N1 vaccines. The suitability of current methods (SRID) was
questioned, and a recommendation was made for improvements to the
SRID assay. In addition, a recommendation was made to continue to
explore alternative methods and technologies for potential future
application. Also, in a talk presented at the USP 2.sup.nd Bioassay
Workshop (Aug. 13, 2009), Dr. Armen Donabedian of Health and Human
Services and BARDA called for a wide range of efforts to "improve
or replace the SRID assay [to] facilitate seasonal and pandemic
influenza preparedness."
[0007] U.S. patent application Ser. No. 12/587,136, filed Oct. 2,
2009, titled Novel Assay for the Separatoin and Quantification of
Hemagglutinin Antigens, which is incorporated herein by reference
as if set out in full, describes a high performance liquid
chromatrography (HPLC) based method for quantification of influenza
HA proteins as an alternative to SRID. HPLC methods can work
reasonably well but have not been widely adopted, likely due to the
typical drift, instrument maintenance required, poor ease-of-use,
and calibration problems associated with HPLC.
[0008] US patent application number 20110201039 describes a
combined liquid chromatography mass spectrometry (LCMS) method for
the quantification of viral proteins, targeted towards the
quantification of influenza HA protein for use in vaccine
production. LCMS in general tends to be too complex for routine use
by minimally trained personnel and suffers from many of the same
maintenance, ease of use, and calibration issues previously
mentioned.
[0009] U.S. patent application Ser. No. 12/994,189, filed Nov. 23,
2010, titled Method for Virus Detection, which is incorporated
herein by reference as if set out in full, describes a sensor-based
method for determining the concentration of virus or viral antigen
based on surface plasmon resonance detection. This method offers
improved sensitivity relative to SRID, but is too expensive for
routine use by all but the largest vaccine producers and requires
large, complex instrumentation.
[0010] For steps in the vaccine development process where rapid
protein quantification is needed, many vaccine producers rely
heavily on enzyme linked immunosorbent assays (ELISA) as an
alternative to SRID. In a traditional "sandwich" ELISA assay, an
antibody specific to a particular antigen is immobilized on a solid
support (such as the bottom of a microtiter plate). An unknown
amount of antigen is then added, and binds to the
surface-immobilized antibody. After the antigen is immobilized, an
enzyme-linked detection antibody is then added and forms a complex
with the antigen. Subsequent addition of an appropriate substrate
for the enzyme ultimately results in the formation of a detectable
signal. In general, ELISA assays are more rapid than SRID (hours
versus days).
[0011] Although more rapid than SRID, the hands-on time required
for sample preparation and analysis for ELISA assays is still
costly. A research unit within a large vaccine company may conduct
upwards of .about.50 ELISA assays per week. Each assay requires 7
serial dilutions per sample and 2 replicates per dilution for both
a standard and an unknown sample. Thus, each sample requires 28
preparations. The analysis is typically performed over 2 days;
plates are prepared on day one, with 4-6 hours of
incubation/analysis on the second day. The estimated costs for
materials and labor for each protein sample quantified by ELISA is
$300-$400 USD, such that the cost for a lab that conducts 50 ELISAs
per week is $70,000 USD per month or $840,000 USD per year. During
production at a medium scale facility, the costs associated with
materials, labor, and time delays are estimated to exceed several
million USD per year.
[0012] Other technical disadvantages of ELISA methods include the
fact that the signal is generated by an enzymatic reaction as a
function of time, requiring critical timing of the reaction and
inclusion of a standard with every batch of samples. Also, the
enzymatic reaction must be stopped after a specified period time
using a "stopping agent", and quantification must be conducted
within 2 hours of the addition of the stopping agent.
[0013] After a vaccine is developed, its efficacy must be evaluated
in clinical trials whereby the immune response is evaluated
following vaccination. The current gold standard assay for the
evaluation of post-vaccination efficacy of a number of viral
vaccines, including vaccines for influenza viruses, is the
hemagglutinin inhibition (FIT) assay which involves
semi-quantification of antibodies within patient serum following
vaccination. The HI assay is based on the underlying
hemagglutination assay (`HA assay`) for semi-quantification of
hemaglutinin (HA) protein concentration. The HA surface protein on
influenza virus particles are known to bind to red blood cells and
hemagglutinate (forming an extended lattice-type structure). To
perform the HA assay, a series of dilutions of an
influenza-containing sample are prepared, and each dilution is
mixed with a known amount of red blood cells, (typically in a
microtiter plate). Red blood cells that are not bound in the
lattice structure due to the presence of influenza virus
precipitate to the bottom of the well, whereas the red blood cells
that are bound to influenza particles form a lattice that coats the
well. The HI assay used for vaccine efficacy evaluation takes
advantage of the fact that antibodies to influenza virus that
should be present after vaccination will inhibit hemagglutination
due to the binding of the antibodies to the HA surface proteins.
Serum obtained from a vaccinated individual is collected, and
serial dilutions of the serum are added to wells each containing a
known amount of red blood cells and antigen (influenza virus). The
HI titer of the serum sample is determined as the highest dilution
that prevents hemagglutination. In the absence of appropriate
anti-HA antibodies (in an unvaccinated individual, for example),
hemagglutination would be observed.
[0014] There are several practical limitations to traditional HA
and HI assays. The 2002 World Health Organization Manual on Animal
Influenza Diagnosis and Surveillance outlines the importance of
preparing fresh antigen solutions, standardization of the protocol
for red blood cell suspension, strict adherence to proper
incubation times, and prompt visual "reading" of the plates after
the assay is completed. In addition, non-antibody inhibitors can
also exist that inhibit hemagglutination and can cause incorrect
interpretation. Moreover, the HA and HI assays is limited by the
subjective nature of the visual readout. Because the detection
relies on interpretation of the hemagglutination pattern present in
the wells, abnormal hemagglutination patterns can complicate the
interpretation. In addition, a two-fold difference in HI titer is
generally considered within the variability of the test and not
considered significant.
In view of the foregoing, there is a clear and considerable need in
the art to overcome the time, cost, and complexity limitations
associated with existing protein and antibody quantification
methods including SRID, ELISA, HI assay methods in the fields of
vaccine-related protein quantification, potency determination, and
efficacy evaluation. The technology of the present application
addresses these and other limitations in the current state of the
art.
SUMMARY OF THE INVENTION
[0015] The technology of the present application provides improved
methods for the quantification of influenza HA proteins and
anti-influenza antibodies for the fields of vaccine-related protein
quantification, potency determination, and efficacy evaluation
which provides improvements over the many drawbacks associated with
the current state of the art assays. In some embodiments,
quantification according to the present application is achieved by
providing a plurality of capture agents attached to an array, said
capture agents being affixed to the array at a series of decreasing
concentrations. In some embodiments, serial dilutions of a
reference material are introduced, each dilution being contacted
with a separate identical array. The reference material within each
solution then binds to the corresponding capture agents on the
array and is subsequently labeled with an appropriate label agent
capable of producing a detectable signal. The detectable signals
from each array for each concentration of reference material are
used to construct a calibration curve. A target material of unknown
concentration is introduced to a separate identical array, and the
target material binds to the capture agents on the array and is
labeled by an appropriate label agent to produce a detectable
signal. The calibration curve based on the reference material is
then utilized to determine the concentration of target material
present without the need to perform replicate experiments on serial
dilutions of the target. In some embodiments, the capture agents
are strain-specific influenza anti-HA antibodies. In other
embodiments, the capture agents are universal anti-influenza
antibodies. In other embodiments, the capture agents are sialic
acid glycoproteins. In even other embodiments, the capture agents
are influenza hemagglutinin proteins.
The foregoing and other features, utilities, and advantages of the
technology of the present application will be apparent from the
following more particular detailed description of the technology as
illustrated in the accompanying drawings.
BRIEF DESCRIPTION OF THE DRAWINGS
[0016] A better understanding of the technology of the present
application will be had upon reference to the following detailed
description read in conjunction with the accompanying drawings. In
the accompanying drawings, like reference characters refer to like
parts throughout, and wherein:
[0017] FIG. 1 is a representative array layout of the current
invention including serial dilutions of two unique capture
sequences, a positive control, a negative control, and a spotting
control
[0018] FIG. 2 is a schematic representation of the binding curves
generated from reference material(s) and target used to construct a
calibration curve and subsequently determine the unknown
concentration of target
[0019] FIG. 3 illustrates a single capture event on an array
involving an antibody as capture agent, antigen as target or
reference material, and a fluorescently labeled antibody as a label
agent
[0020] FIG. 4 is a fluorescence image of an array containing serial
dilutions of an anti-human influenza A H3 antibody as capture
agent, purified influenza HA H3 antigen as reference material, and
a fluorescently labeled universal antibody as label agent
[0021] FIG. 5 is a binding curve obtained from the analysis of the
detectable fluorescence signals obtained after exposing the array
represented in FIG. 4 to 0.5 .mu.g/mL of H3 HA reference
material.
[0022] FIG. 6 is a linear regression obtained by plotting the
normalized maximum signal obtained for serial dilutions of HA
reference material added to identical replicate arrays
[0023] FIG. 7 illustrates a single capture event on an array
involving a glycopeptide or glycoprotein as capture agent,
influenza HA from a vaccine as target or reference material, and a
fluorescently labeled universal antibody or antibody fragment as a
label agent
[0024] FIG. 8 illustrates a single capture event on an array
involving HA antigen as capture agent, an anti-influenza antibody
as target or reference material, and a fluorescently labeled
universal antibody or antibody fragment as a label agent
DETAILED DESCRIPTION
[0025] As mentioned previously, the technology of the present
application relates to methods for improved vaccine-related protein
quantification, potency determination, and efficacy evaluation
using an array-based format in which serial dilutions of an
appropriate capture agent are printed. Detectable signals from
replicate arrays contacted with different concentrations of a
reference material are analyzed, and the maximum signal achieved
for each concentration determined from a non-linear regression to
the binding curve data is then used to construct a one-time
calibration curve; the calibration curve can then be used to
quantify a target material after a single concentration of said
target material has been contacted with a replicate array and a
detectable signal is produced. The technology of the present
application provides significant advantages over the current state
of the art methods in that a single concentration of target
material(s) can be used to obtain the concentration of said target
material(s) due to array encoded serial dilution of the capture
agent, and that the quantification of multiple targets can be
achieved in a single assay due to the multiplexing capability of an
array-based approach. Moreover, while the technology of the present
application is described in relation to influenza, one of ordinary
skill in the art will understand on reading the present application
that the technology may be used for many different viral proteins,
and other protein biomarkers. Additionally, the below detailed
description of the technology of the present application is
provided with regard to certain exemplary embodiments. The word
"exemplary" is used herein to mean serving as an example, instance,
or illustration. Any embodiment described herein is not necessarily
to be construed as preferred or advantageous over other
embodiments. If a single exemplary embodiment is provided for
completeness, it is not to be concluded that no other embodiments
are possible. Moreover, all embodiments described herein should be
considered exemplary unless otherwise stated.
[0026] For the purposes of the present application, an array refers
to any solid support onto which a specific arrangement of capture
agents can be placed onto one or more of its surfaces. The solid
support can be constructed of but is not limited to glass, plastic,
silicon-coated substrate, macromolecule-coated substrate, membrane
or filter material, metal, particles or microparticles, beads or
microbeads, magnetic or paramagnetic beads, or a variety of other
materials known to those skilled in the art. In one embodiment of
the current invention, the arrangement of capture agents upon said
array are in discrete regions or locations, with each region or
location containing only one specific capture agent, so as to
create a specific pattern or configuration of capture agents. While
provided in discrete regions or locations in the exemplary
embodiments, in certain embodiments the capture agents may be
provided in a contiguous region having delineated concentrations or
the like.
[0027] In reference to the present application, a capture agent
refers to anything that can be immobilized on the surface of said
array that will capture or bind to a target material. For the
purposes of the current invention, capture agents include any
number of biological molecules including proteins, monoclonal and
polyclonal antibodies, antisera, antigens, polypeptides, viral
epitopes, cell membrane receptors, glycoproteins, glycopeptides,
sugars, and other similar species known to those skilled in the
art. In one embodiment of the technology, capture agents are
designed to bind appropriate reference and target materials from a
solution applied to the array for the purposes of detecting the
presence and quantity of said reference and target materials.
[0028] For the purposes of the present application, reference
material refers to any material of known concentration that can be
used to construct a calibration curve based on the magnitude of
detectable signals created by the binding event between capture
agent(s) and reference material(s). As a non-limiting example, in
the present application, reference material may refer to a standard
influenza antigen from which a calibration curve of influenza
hemagglutinin (HA) concentration can be created. As another
non-limiting example, reference material may refer to standard
anti-influenza antibody/antibodies from which a calibration curve
of antibody concentration can be created.
[0029] A target material, for the purposes of the present
application, is a material from the same family as the reference
material (e.g. as a non-limiting example, both the reference and
target materials are influenza HA antigens or are both
anti-influenza antibodies) that is present at an unknown
concentration and whose concentration can be quantified by
utilizing a calibration curve constructed from a reference
material.
[0030] For the purposes of the present application, a label agent
is any species that can be utilized to detect the presence of
target material on an array by exhibiting a detectable signal. For
the purpose of the present application, a label agent could be an
antibody capable of binding to the target agent that is either
directly conjugated to a fluorescent molecule or entity, or
conjugated to another moiety capable of subsequently binding to a
fluorescent molecule or entity. In addition, a label agent could be
a fluorescent molecule designed to be incorporated directly into
the target agent by means known to those of ordinary skill in the
art. In other embodiments of the technology, the detectable signal
is based on a property other than fluorescence, including
absorbance, chemiluminescence, electrical signal, or other signal
types known to those skilled in the art.
[0031] The illustrative example shown in FIG. 1 details a typical
layout of an array 100 of the present application. In one
embodiment of the technology, a spotting control 102 is utilized to
provide spatial orientation. Advantageously, the spotting control
can also be utilized to normalize the detectable signals from the
reference and target materials. In FIG. 1, with exception of the
spotting control, all other capture agents 104 spotted on the array
are spotted in triplicate (3 contiguous spots in a row). In another
embodiment as shown in FIG. 1, the array 100 layout also contains a
positive control 106 designed to validate the assay is functioning
properly. In yet another embodiment as shown in FIG. 1, a negative
control capture agent 108 (designed not to bind to the reference or
target agents) is also included to determine if non-specific
binding is occurring. In other words, if the negative control
provides a detectable signal, the results of the array 100 are
suspect. Advantageously, each dilution within a dilution series of
a particular capture agent is spotted in triplicate in a defined
configuration. FIG. 1 shows a non-limiting example in which two
unique capture agents are spotted on the array. Each dilution
within the series is spotted in triplicate. Advantageously, the
dilution series of the capture agent(s) covers a broad
concentration range that encompasses all key regions of a binding
curve. In another embodiment of the technology, the array can be
spotted within a flat-bottomed 96 well plate or other methods
conventionally known to those of ordinary skill in the art for easy
integration into existing laboratory systems. Regardless of the
particular nature of the capture agent, reference, and target
agents, a similar array layout can be used.
[0032] In current state of the art immunoassays, such as SRID or
ELISA, the unknown target antigen concentration is determined from
the linear portion of the calibration curve constructed from a
serial dilution of a known, or standard, antigen concentration. The
key mathematical relationships are described in Equations 1 and 2
below:
S.sub.kn=m.sub.knC.sub.kn+S.sub.o (Equation 1)
C.sub.unk.apprxeq.S.sub.unk/m.sub.kn (Equation 2)
where in Equation 1, S.sub.kn is the detectable signal from a
standard as a function of known concentration (C.sub.kn), m.sub.kn
is the slope of the linear portion of the calibration curve, and
S.sub.o is the background signal. Once the calibration curve is
constructed, a serial dilution of the unknown is also analyzed to
ensure that the signal is within the linear portion of the
calibration curve. For those dilutions in the linear range, the
unknown concentration can be obtained from the relationship shown
in Equation 2, where C.sub.unk is the unknown antigen
concentration, S.sub.unk is the signal for the unknown antigen
concentration, and m.sub.kn is the slope of the linear portion of
the calibration curve.
[0033] In contrast to the above mentioned SRID and ELISA methods,
the technology of the present application provides a method for
which the analysis of a single concentration of unknown target
material results in a complete binding curve. Consider the binding
curve obtained for a single concentration of target or reference
material shown pictorially in FIG. 2A and described by Equation 3
below:
S = S o + S max 1 + ( C im ) A ( Equation 3 ) ##EQU00001##
[0034] where S is the signal from the single concentration of
target or reference material as a function of immobilized capture
agent concentration (C.sub.im), S.sub.o is the background signal,
S.sub.max is the maximum value of the signal obtained, and A is
related to the slope of the binding curve. The maximum signal
(S.sub.max) extracted from a non-linear regression to the data is
directly proportional to the target or reference concentration and
is used for quantification, rather than relying on the slope.
Several dilutions of a reference material of known concentrations
are prepared and applied to replicate arrays, and the binding
curves for the reference material (dotted, dashed, and dash-dotted
Reference material binding curves shown in FIG. 2A) are then used
to construct a calibration curve by subsequently plotting S.sub.max
versus C.sub.kn, as graphically illustrated in FIG. 2B. A single
concentration of target material is then applied to a replicate
array, a non-linear regression to the data is applied, and the
maximum value (S.sub.max) obtained (solid black Target material
binding curve in FIG. 2A). The equation of the calibration curve
can then be used to solve for the unknown concentration of target
material.
[0035] The technology of the present application is advantageous
over the state of the art methods for a number of reasons. One
reason is that all signals can be normalized to an internal
reference (S.sub.iref) that is encoded on the array. As a
non-limiting example, the spotting control shown in FIG. 1 can be
used as S.sub.iref. Specifically, the S.sub.max value is divided by
S.sub.iref (i.e., S.sub.max-norm=S.sub.max/S.sub.iref) to yield a
signal normalized for random experimental variables (e.g., chip to
chip variations). The ramification of this advantage is that the
current method does not require a complete serial dilution of the
standard for each batch of samples analyzed. In principle, for a
given set of experimental conditions (e.g., influenza HA from
recombinant baculovirus expression), a single serial dilution of
the standard could be used to determine the concentration of large
number of sample batches using S.sub.max-norm in a calibration
curve (see FIG. 2B).
[0036] In another embodiment, numerous capture agents could be
immobilized on the array to take advantage of multiplexing,
allowing multiple target antigens (for example, HA antigens from
influenza A H1N1, influenza A H3N2, and influenza B) could be
quantified simultaneously in a multiplex analysis, thereby
dramatically reducing the total number of assays required to
quantify the targets of interest. This specific example could be
utilized in potency determination in yearly trivalent influenza
vaccines to speed the development time.
[0037] Several non-limiting examples follow to further explain and
illustrate the advantages of the technology over the state of the
art.
[0038] Quantification of Influenza HA Using Strain-Specific Capture
Antibodies for Influenza Vaccine Potency Determination
[0039] In an illustrative example of the technology of the present
application, subtype-specific monoclonal antibodies against
influenza A H3 HA and influenza A H1 HA (Meridian, catalog #C01318M
and #C86304M) were printed onto aldehyde-functionalized glass
slides in the layout illustrated in FIG. 1, with the anti-H3
antibody represented by Capture Agent 1 (104, left-hand side of
array layout) and the anti-H1 antibody represented by Capture Agent
2 (104, right-hand side or array layout). The general detection
scheme for this example is schematically shown for a single binding
event is shown in FIG. 3. For reference, FIG. 3 provides an
exemplary array 300 consistent with the technology of the present
application. Array 300 having a discretely located capture agent
302. The capture agent 302 binds in this exemplary embodiment to
the HA antigen 304. An antibody label agent 306 binds the HA
antigen 304 to a fluorescent label 308, which is capable of
detection as is generally known in the art.
[0040] Serial dilutions of H3 hemagglutinin (HA) antigen from the
influenza A H3N2 strain A/Wy/3/2003 (Protein Sciences Corporation,
Meriden, Conn.) in phosphate buffer were each incubated on separate
replicate microarrays at room temperature for 1 hr. After
subsequent washing, the captured HA antigen was labeled with a
universal biotinylated anti-HA antibody fragment (Sigma Aldrich,
catalog # B9183) followed by a Cy3-labeled streptavidin at 1.2
.mu.g/mL. The universal antibody label utilized was raised against
a universal HA peptide often used as a tag in recombinant proteins,
and has demonstrated response to a conserved region of both native
and denatured HA.
[0041] A representative fluorescence image for a single H3 HA
concentration applied to the array (0.5 .mu.g/mL) is shown in FIG.
4. The normalized signal intensity from the fluorescence image as a
function of the spotted anti-H3 antibody concentration was fit to
Equation 3 using non-linear regression and the maximum signal
achieved determined, as shown in FIG. 5. Only a limited range of HA
concentrations were examined for this example, and the maximum
signals determined for these different concentrations are plotted
in FIG. 6. As expected, the extracted S.sub.norm value (plotted on
the y-axis of FIG. 6) for this limited range of H3 HA
concentrations is linear as determined by regression. It is
anticipated from extrapolation that the quantification of the
technology of the present application should be below 0.1 .mu.g/mL.
Considering that the currently used SRID assay has a quantification
limit of .about.2-5 .mu.g/mL, the technology of the present
application would have an improved limit of detection over current
state of the art methods and therefore provide benefits over the
current state of the art to vaccine manufacturers.
[0042] Quantification of Influenza HA using Universal Capture
Agent(s) for Influenza Vaccine Potency Determination
[0043] As previously mentioned, the traditional SRID assay requires
the development and use of well characterized standard reference
antibodies and antigens for each new strain of influenza produced.
Since influenza viruses continually evolve, new standards must be
produced in response to the emergence of each seasonal virus. Thus,
the generation of these standard materials (e.g., antibodies and
antigens) often represent a bottleneck in influenza vaccine
development. Vaccine manufacturers must often wait to obtain
reference materials from the Center for Biologics Evaluation and
Research (CBER).
[0044] In one embodiment of the technology described herein, stable
and universal standards used as capture agent(s) on an array for
determination of influenza vaccine potency would streamline vaccine
production by reduction of "wait time" for new reference antibodies
and antigens and allow for improved quality control and assurance
through historical trends with respect to a single reference
standard (rather than relying on new unique standards each year for
each strain of virus).
[0045] In another embodiment of the technology described herein,
.alpha.2,6 and .alpha.2,3 sialic acid glyopeptides or proteins are
used as capture agents to determine influenza vaccine potency. In
the influenza viral infection process, influenza HA on the surface
of the virus particle binds to sialic acid receptors on the host
cell surface. It is well-known that influenza viruses from
different host species vary in their affinity for sialic acid
receptors with different linkages. Specifically, influenza viruses
that replicate in humans selectively bind to the .alpha.2,6 sialic
acid found on the surface of human respiratory epithelial cells,
whereas influenza viruses that replicate in birds selectively bind
to the .alpha.2,3 sialic acid found on the surface of epithelial
cells found in the waterfowl gut.
[0046] Typical gold standard potency assays are based on the two
irresolvable experimental parameters of concentration and binding
constant. By utilizing the ratio of detectable signals produced on
an array for the two types of receptors, the concentration and
binding constant can be separated. As a non-limiting example, with
.alpha.2,6 and .alpha.2,3 sialic acid glyopeptides or proteins, the
relative binding strength of a target HA to both types of sialic
acid glycopeptides or glycoproteins could be measured in a single
assay. Mathematically, if we neglect the labeling step, the
separation of variables can be shown by:
S.sub.max,.alpha.2,6.varies.K.sub..alpha.2,6[C.sub.HA] (Equation
4)
and
S.sub.max,.alpha.2,3.varies.K.sub..alpha.2,3[C.sub.HA] (Equation
5)
where S.sub.max is the maximum signal obtained from a binding curve
as described in Equation 3, K.sub.x is the binding constant, and
C.sub.HA is the concentration of HA in solution applied to the
microarray. Since the concentration of HA for both is the same, the
relationship below follows:
S max , .alpha.2 , 6 S max , .alpha.2 , 3 .varies. K .alpha.2 , 6 K
.alpha.2 , 3 ( Equation 6 ) ##EQU00002##
[0047] This ratio may be a more direct measurement of vaccine
potency, as a theoretical quantitative value can be determined from
insight into cell receptor composition for each species (e.g.
.alpha.-2,6 for human, .alpha.-2,3 for avian, and a mixture of
receptors, therefore an intermediate ratio, for swine). As a
non-limiting example, optimal potency of a human influenza vaccine
would produce a high signal ratio in Equation 6 due to the much
higher binding constant for HA to the human .alpha.-2,6 sialic acid
(K .alpha.2,6 K .alpha.2,3).
[0048] To overcome the limitations associated with the current gold
standard SRID assay, a sialic acid glycoprotein(s) or
glycopeptide(s), in combination with a strain-independent label,
can be developed, fully characterized, and used as a universal
reference for determining influenza vaccine potency. The detection
scheme for this non-limiting example is schematically illustrated
in FIG. 7, where an immobilized glycopeptide or glycoprotein acts
as capture agent. Serial dilutions of .alpha.-2,6 and .alpha.-2,3
sialic acid glyopeptide(s) or protein(s) are configured in an
array. Influenza HA antigen is allowed to bind to the array and is
subsequently labeled with a label agent. In one embodiment of the
current invention, the label agent is a fluorescently tagged,
strain-independent anti-HA antibody fragment (such as monoclonal
anti-HA # H9037 from Sigma Aldrich). For reference, FIG. 7 provides
an array 700 consistent with the technology of the present
application. Discretely located on array 700 are a series of
glycopeptides or glycoprotein capture agents 702 that capture or
bind an associated HA antigen 704. An antibody label agent 706
binds the HA antigen 704 to a fluorescent label 708, which is
capable of detection as is generally known in the art.
[0049] Hemagglutinin Inhibition Assay on an Array for Influenza
Vaccine Efficacy Determination
[0050] As previously mentioned, the principle of the hemagglutinin
inhibition (HI) assay is that antibodies will prevent the
agglutination of red blood cells by influenza viruses. For
influenza viruses, agglutination refers to the process whereby the
virus binds to sialic acid receptors on cell surfaces and can cause
the cells to aggregate around the virus. Aggregation leads to cell
complexes that prevent red blood cells from settling to the bottom
of a well. The assay is conducted by serial dilution of patient
serum mixed with a specific concentration of virus and red blood
cells whereby the highest dilution of the patient serum that
prevents hemagglutination is the HI titer value.
[0051] To overcome the aforementioned limitations associated with
the current gold standard HI assay for influenza vaccine efficacy,
a reference standard of influenza hemagglutinin (HA) is used as a
capture agent where serial dilutions of the HA standard(s) is/are
configured onto an array under carefully controlled and
reproducible conditions. The array of HA antigen(s) is then used to
quantify antibodies in serum obtained from a patient that are
developed in response to an influenza vaccination. The detection
scheme for this non-limiting example of the current invention is
illustrated in FIG. 8, where standard HA antigen is used as a
capture agent. HA antigen is configured onto an array, antibodies
produced in response to the vaccination from patient serum are
captured on the array, and the captured antibodies are subsequently
labeled with a label agent to produce a detectable signal. In the
current non-limiting example, the label agent is a fluorescently
tagged anti-human antibody. The patient produced antibodies are
quantified by the value of S.sub.max-norm mapped to a calibration
curve generated by serial dilutions of a reference antibody,
similar to the process described previously and schematically
represented in FIG. 2. Advantages of this example of the current
invention are the quantitative nature of the assay where if the
binding constant is known, the concentration determined can be
expressed in absolute terms and compared to results from other
common measurements of protein quantification (e.g. pg/mL or
moles/mL). In addition, this example produces a rapid time to
result, is automatable, and eliminates the use of red blood cells
the assay. For reference, FIG. 8 provides an array 800 consistent
with the technology of the present application. Discretely located
on array 800 are a series of HA antigen capture agents 802 that
capture or bind an associated target material 804, which is an
antibody in this exemplary embodiment. An antibody label agent 806
binds the target material 804 to a fluorescent label 808, which is
capable of detection as is generally known in the art. Unless
otherwise indicated, all numbers or expressions, such as those
expressing dimensions, physical characteristics, etc. used in the
specification are understood as modified in all instances by the
term "approximately." At the very least, and not as an attempt to
limit the application of the doctrine of equivalents to the claims,
each numerical parameter recited in the specification or claims
which is modified by the term "approximately" should at least be
construed in light of the number of recited significant digits and
by applying ordinary rounding techniques. Moreover, all ranges
disclosed herein are to be understood to encompass and provide
support for claims that recite any and all subranges or any and all
individual values subsumed therein. For example, a stated range of
1 to 10 should be considered to include and provide support for
claims that recite any and all subranges or individual values that
are between and/or inclusive of the minimum value of 1 and the
maximum value of 10; that is, all subranges beginning with a
minimum value of 1 or more and ending with a maximum value of 10 or
less (e.g., 5.5 to 10, 2.34 to 3.56, and so forth) or any values
from 1 to 10 (e.g., 3, 5.8, 9.9994, and so forth).
* * * * *
References