U.S. patent application number 12/994189 was filed with the patent office on 2011-03-24 for method for virus detection.
This patent application is currently assigned to GE HEALTHCARE BIO-SCIENCES AB. Invention is credited to Peter Borg, Asa Frostell-Karlsson, Markku Hamalainen, Anita Larsson.
Application Number | 20110070574 12/994189 |
Document ID | / |
Family ID | 41398332 |
Filed Date | 2011-03-24 |
United States Patent
Application |
20110070574 |
Kind Code |
A1 |
Borg; Peter ; et
al. |
March 24, 2011 |
METHOD FOR VIRUS DETECTION
Abstract
A method of determining the concentration of a virus or antigen
thereof in a sample comprises the steps of: providing a sensor
surface having immobilized thereto a virus antigen or a virus
antigen analogue, mixing the sample with a known amount of antibody
to the virus antigen to obtain a predetermined concentration of
antibody to the antigen in the sample mixture, contacting the
sample mixture with the sensor surface to bind free antibody in the
mixture to the sensor surface, measuring the response of the sensor
surface to the binding of free antibody, and determining the
concentration of the virus or antigen in the sample from a
calibration curve prepared by measuring the responses obtained for
mixtures containing the predetermined concentration of antibody and
different concentrations of virus.
Inventors: |
Borg; Peter; (Uppsala,
SE) ; Frostell-Karlsson; Asa; (Uppsala, SE) ;
Hamalainen; Markku; (Uppsala, SE) ; Larsson;
Anita; (Uppsala, SE) |
Assignee: |
; GE HEALTHCARE BIO-SCIENCES
AB
Uppsala
SE
|
Family ID: |
41398332 |
Appl. No.: |
12/994189 |
Filed: |
June 1, 2009 |
PCT Filed: |
June 1, 2009 |
PCT NO: |
PCT/SE09/50637 |
371 Date: |
November 23, 2010 |
Current U.S.
Class: |
435/5 ;
436/501 |
Current CPC
Class: |
G01N 21/658 20130101;
G01N 21/553 20130101; G01N 33/54373 20130101; G01N 33/54306
20130101 |
Class at
Publication: |
435/5 ;
436/501 |
International
Class: |
C12Q 1/70 20060101
C12Q001/70; G01N 33/53 20060101 G01N033/53 |
Foreign Application Data
Date |
Code |
Application Number |
Jun 2, 2008 |
SE |
0801304-7 |
Apr 24, 2009 |
SE |
0950272-5 |
Claims
1: A method of determining the concentration of a virus or antigen
thereof in a sample comprising the steps of: providing a sensor
surface having immobilized thereto a virus antigen or a virus
antigen analogue; mixing the sample with a known amount of antibody
to the virus antigen to obtain a predetermined concentration of
antibody to the antigen in the sample mixture; contacting the
sample mixture with the sensor surface to bind free antibody in the
mixture to the sensor surface; measuring the response of the sensor
surface to the binding of free antibody; and determining the
concentration of the virus or antigen in the sample from a
calibration curve prepared by measuring the responses obtained for
mixtures containing the predetermined concentration of antibody and
different concentrations of virus or virus antigen, wherein
multiple analysis cycles are performed on the sensor surface with
intermediate regenerations and a virtual calibration curve is
calculated for each analysis cycle.
2. (canceled)
3: The method of claim 1, wherein calculating a calibration curve
comprises: fitting each of the known concentrations in the curves
to a double exponential equation using cycle number as x and
response as y; using these equations for calculation of a virtual
calibration curve for each cycle; and determining the concentration
of the virus or antigen in the sample from a virtual calibration
curve for that particular cycle.
4: The method of claim 1, wherein the concentration of at least
two, preferably at least three different viruses or virus antigens
in a sample are determined, and wherein mixtures of sample and
antibodies selective to the respective virus antigens are
successively, in separate analysis cycles, contacted with a sensor
surface having a different one of the antigens or analogues
immobilized thereto.
5: The method of claim 1, wherein the concentration of at least
two, preferably at least three different viruses or antigens in a
sample are determined, and wherein mixtures of sample and
antibodies selective to the respective virus antigens are contacted
with a sensor surface having discrete sensing areas with a
respective antigen or analogue immobilized thereto.
6: The method of claim 1 wherein the concentration of at least two,
preferably at least three different viruses or antigens in a sample
are determined, and wherein mixtures of sample and antibodies
selective to the respective virus antigens are contacted with a
sensor surface comprising a mix of respective antigen immobilized
to the same sensing area.
7: The method of claim 1, wherein the concentration of at least
two, preferably at least three different viruses or virus antigens
in a sample are determined, and wherein a mixture of sample and
antibodies selective to the respective virus antigens, in a single
analysis cycle, is contacted with a sensor surface having discrete
sensing areas with a respective antigen or analogue immobilized
thereto.
8. (canceled)
9: The method of claim 1, wherein the virus is an influenza
virus.
10: The method of claim 1, wherein the different viruses are
selected from influenza virus types and subtypes.
11: The method of claim 1, wherein the virus antigen comprises
hemagglutinin.
12: The method of claim 1, wherein the sample is derived from
influenza vaccine production.
13: The method of claim 12, wherein the sample is derived from a
multivalent influenza vaccine.
14: The method of claim 12, wherein the virus antigen comprises
hemagglutinin, and the immobilized hemagglutinin is generic for a
plurality of different strains of an influenza virus type or
subtype.
15: The method of claim 12, wherein the virus antigen comprises
hemagglutinin, and the immobilized hemagglutinin is derived from at
least 2 different strains of an influenza virus type or
subtype.
16: The method of claim 13, wherein the multivalent influenza
vaccine is trivalent and derived from influenza A/H1N1, A/H3N2 and
B strains.
17: The method of claim 1, wherein binding interactions on the
sensor surface are detected by mass-sensing, preferably evanescent
wave sensing, particularly surface plasmon resonance (SPR).
18-19. (canceled)
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a filing under 35 U.S.C. .sctn.371 and
claims priority to international patent application number
PCT/SE2009/050637 filed Jun. 1, 2009, published on Dec. 10, 2009 as
WO 2009/148395, which claims priority to application number
0801304-7 filed in Sweden on Jun. 2, 2008 and application number
0950272-5 filed in Sweden on Apr. 24, 2009.
FIELD OF THE INVENTION
[0002] The present invention relates to the detection and
quantification of virus or virus antigen in a virus-containing or
virus-derived medium, particularly determination of the virus
antigen concentration in vaccine manufacturing and process
development.
BACKGROUND OF THE INVENTION
[0003] Influenza viruses are generally divided into three types, A,
B and C, based on the antigenic differences between their
nucleoprotein and matrix protein antigens. Influenza A viruses are
further divided into subtypes on the basis of the two main surface
glycoproteins hemagglutinin (HA) and neuraminidase (NA) which
appear as spikes on the surface of the viral body.
[0004] Infection of a host cell starts with the binding of virus HA
to sialic structures on the cell causing the virus particles to
stick to the cell surface and induce uptake of the virus. RNA and
viral proteins are duplicated and assemble into new viral
particles, which bud from the cell. The progeny virus particles are
released from the cell surface by the enzyme NA on the virus
cleaving terminal sialic residues.
[0005] Currently, there are 15 different HA subtypes (H1-H15) and 9
different NA subtypes (N1-N9). Subtypes of influenza A virus are
named according to their HA and NA surface proteins, e.g. H1N1,
H1N2, H3N2, H5N1. Influenza B viruses and substypes of influenza A
are further characterized into strains, e.g. B Malaysia, H3N2
Beijing, H1N1 Taiwan.
[0006] Both HA and NA carry antigenic epitopes. Antibodies raised
against HA and NA reduce the risk of infection or illness in humans
and animals. While the first influenza vaccines contained
inactivated or killed whole virus particles, commercially available
influenza vaccines are usually of two types, "split vaccines" and
"subunit vaccines".
[0007] Split vaccines are prepared by disintegration of purified
virus particles with ether or detergents, and then removal of the
detergent with the bulk of the viral lipid material. Split vaccines
thereby contain essentially the same elements as whole virus
vaccines and in the same proportions. In subunit vaccines, on the
other hand, the surface glycoproteins HA and NA are purified
separately and then combined into a vaccine.
[0008] Influenza viruses can change in two different ways, by
"antigenic drift" and by "antigenic shift". Antigenic drift
produces new virus strains that may not be recognized by the body's
immune system, whereas antigenic shift is an abrupt, major change
in the influenza A viruses, resulting in new hemagglutinin and
neuraminidase proteins and producing a new influenza A subtype. In
most years, one or two of the three virus strains in the influenza
vaccine are updated to keep up with the changes in the circulating
flu viruses.
[0009] An influenza vaccine is usually multivalent (polyvalent),
i.e. the vaccine is prepared from cultures of two or more strains
of the same species of virus. Currently, influenza A/H1N1, A/H3N2
and influenza B strains are typically included in each year's
influenza vaccine.
[0010] The efficacy of a vaccination against influenza is largely
determined by the amount of immunogenic HA in a vaccine. The HA
concentration in vaccines has typically been determined by single
radial immuno-diffusion (SRID) assay. In SRID, influenza virions
are disrupted by detergent, and submitted to diffusion of whole
virus or purified viral antigens into agarose gel containing
specific anti-hemagglutinin (anti-HA) antibodies. The resulting
antigen/antibody reaction or zone (visible by staining) is directly
proportional to the amount of HA antigen in the preparation.
However, the SRID assay has a number of disadvantages. In addition
to having a high detection limit (about 20 .mu.g/ml) with high
variation (about 10%), it is laborious and has a low throughput.
Despite its shortcomings, SRID is still, however, the method
recommended by the European Pharmacopeia and WHO and approved by
regulatory authorities for the evaluation of influenza vaccines.
Due to the poor precision of the method, vaccine doses are usually
"overfilled" by the manufacturers.
[0011] Other methods for quantification of influenza virus include
reversed-phase high performance liquid chromatography
(RP-HPLC).
[0012] Also biosensor-based methods have been developed for the
detection of virus, including influenza virus.
[0013] EP 0276142 A1 discloses a method for detecting influenza A
virus using surface plasmon resonance (SPR) on a gold coated
diffraction grating. Monoclonal antibodies to discrete determinants
of influenza A virus were immobilized on the gold surface, and
virus was applied and incubated. Monoclonal antibodies to the
influenza A virus determinants were then incubated with the
surface, and the enhanced response obtained when the second
antibody was bound to the influenza virus particle was
detected.
[0014] JP 3054467A discloses measurement of the concentration of a
virus by a piezoelectric vibrator having its electrode coated with
an antibody to a surface antigen of a virus. When antigen binds to
the electrode, the vibration frequency of the vibrator becomes
lower.
[0015] Shofield, D. J., and Dimmock, N. J., J. Virol. Methods 62
(1996) 33-42 discloses use of an SPR biosensor instrument for
detection of influenza virus. A monoclonal antibody for capture of
influenza virus was coupled to a sensor chip coated with
carboxylated dextran. Influenza virus was then injected into the
instrument flow system to contact the sensor chip, and the binding
affinity with the immobilized antibody was monitored.
[0016] Boltovets, P. M., et al., J. Virol. Methods 121 (2004)
101-106 discloses detection of plant virus using surface plasmon
resonance (SPR) by detecting the binding of complexes between viral
antigen and antibody formed during a pre-incubation step to an SPR
sensor surface with immobilized protein A.
[0017] Jie Xu, et al., Analytical and Bioanalytical Chemistry 389,
4 (2007) 1193-1199 discloses an interferometric biosensor
immunoassay for detection of avian influenza. Whole virus particles
were captured by antigen-specific (hemagglutinin) antibodies (both
polyclonal and monoclonal) on a waveguide surface, and the
refraction changes resulting from the binding were measured. Three
influenza virus subtypes (two H7 and one H8) were tested.
[0018] All these other methods, however, also suffer from
substantial disadvantages. There is therefore a need for improved
methods and means for accurate determination of the concentration
of influenza virus, specifically HA concentration, in crude as well
as in purified samples.
SUMMARY OF THE INVENTION
[0019] One object of the present invention is to provide a method
for detection and quantification of virus, especially influenza
virus, which is devoid of the disadvantages of the prior art
methods, and which in particular has a high precision, a high
detection range, and is less laborious than the standard SRID
assay.
[0020] Another object of the present invention is to provide a
method which is suitable for quantifying virus or virus antigen in
vaccine production, both in process samples and final vaccine
samples.
[0021] These objects as well as other objects and advantages are
obtained with a method according to claim 1.
[0022] The method according to the present invention is based on
the use of biosensor-technology and an inhibition type assay
format.
[0023] Broadly, the method of determining the concentration of a
virus or virus antigen in a sample comprises the steps of:
[0024] providing a sensor surface having immobilized thereto a
virus antigen or a virus antigen analogue,
[0025] mixing the sample with a known amount of antibody to the
virus antigen to obtain a predetermined (total) concentration of
antibody to the antigen in the sample mixture,
[0026] contacting the sample mixture with the sensor surface to
bind free antibody in the mixture to the sensor surface,
[0027] measuring the response of the sensor surface to the binding
of free antibody, and
[0028] determining the concentration of the virus or antigen in the
sample from a calibration curve prepared by measuring the responses
obtained for mixtures containing the predetermined concentration of
antibody and different concentrations of virus or virus
antigen.
[0029] Preferably, multiple analysis cycles are performed on the
sensor surface with intermediate regenerations and a virtual
calibration curve is calculated for each analysis cycle. This may
be done by:
[0030] fitting each of the known concentrations in the curves to a
double exponential equation using cycle number as x and response as
y,
[0031] using these equations for calculation of a virtual
calibration curve for each cycle and,
[0032] determining the concentration of the virus or antigen in the
sample from a virtual calibration curve for that particular
cycle.
[0033] The virus antigen may be an internal antigen or, preferably,
a surface antigen of the virus. Optionally, the virus antigen is
the whole virus particle. A virus antigen to analogue may, for
example, be a synthetic peptide.
[0034] Preferably, the sample contains a plurality of different
virus or virus types which are determined simultaneously by the
method.
[0035] The term "antibody" as used herein refers to an
immunoglobulin which may be natural or partly or wholly
synthetically produced and also includes active fragments,
including Fab antigen-binding fragments, univalent fragments and
bivalent fragments. The term also covers any protein having a
binding domain which is homologous to an immunoglobulin binding
domain. Such proteins can be derived from natural sources, or
partly or wholly synthetically produced. Exemplary antibodies are
the immunoglobulin isotypes and the Fab, Fab', F(ab').sub.2, scFv,
Fv, dAb, and Fd fragments.
[0036] Typically, the antibody is a serum to the virus or virus
antigen.
[0037] In one embodiment, the sensor chip has multiple sensing
areas and virus antigens or analogues specific to a respective
virus or virus type are immobilized on different discrete sensing
areas. The sample is mixed with a fixed amount of the antibody to
one virus, and the mixture is then contacted with either only the
sensing area with the antigen specific to the antibody,
or--provided that the antibody does not cross-react with the other
antigens or analogues--all sensing areas, and the response is
detected. This is successively repeated for the other antibodies
and sensing areas.
[0038] In another embodiment, the sensor chip has a single sensing
area, or only a single sensing area of a sensor chip with multiple
sensing surfaces is used. In this case, the virus antigens or
analogues are co-immobilized on a single sensing area, the sample
is mixed with a fixed amount of a respective antibody, one at a
time, and the respective mixtures are successively contacted with
the sensing area.
[0039] In a preferred embodiment, however, provided that the
antibodies are selective lacking cross-reactivity to the other
viruses or antigens, all the different antibodies are to mixed with
the sample which is then contacted with a sensor chip with multiple
sensing areas, each with a respective immobilized virus antigen or
analogue, and the responses of the different sensing areas are
detected. The sensor surface may alternatively comprise a mix of
respective antigen immobilized to the same sensing area. In this
way, all viruses or virus antigens in a sample may be detected in a
single analytical cycle.
[0040] The viruses or virus antigens in a sample to be quantified
are preferably different influenza virus types or antigens thereof,
preferably hemagglutinins.
[0041] The immobilized hemagglutinin may be generic for several
strains of influenza virus types or subtypes or be derived from at
least 2 different strains of an influenza virus type or
subtype.
[0042] Generally, in a biosensor assay, when analytes (here free
antibodies in the sample), have bound to immobilized ligands (here
virus antigen or analogue) on a sensor surface, the bound
antibodies are released by treatment with a suitable fluid to
prepare the surface for contact with a new sample, a process
referred to as regeneration. Usually, a sensor surface can be
subjected to fairly large number of analysis cycles. Many ligands
(such as e.g. virus antigens), however, often have poor stability
making the analyte binding capacity of the surface decrease with
the number of cycles and may hamper the use of the ligand for
quantitative purposes. While minor decreases in binding capacity
can often be compensated by frequent calibrations, this
significantly decreases the throughput.
[0043] The method of the invention therefore includes a
normalization step wherein each analysis cycle is evaluated using a
virtual calibration curve (i.e. each cycle obtains a unique
calibration curve), thus minimizing the need of frequent
calibrations during drift and significantly improving the quality
of the quantitative measurements.
[0044] A more complete understanding of the present invention, as
well as further features and advantages thereof, will be obtained
by reference to the following detailed description and
drawings.
BRIEF DESCRIPTION OF THE DRAWINGS
[0045] FIG. 1 is a schematic illustration of an inhibition type
virus assay on a sensor surface for three cases (a-c) with
different virus concentrations in the sample.
[0046] FIG. 2 is a schematic illustration similar to FIG. 1 where
three different virus antigens are immobilized to respective
separate spots on a sensor surface and three different antibodies
specific for each antigen are used for quantification.
[0047] FIG. 3 is a diagram showing measured relative
response/stability versus concentration of different influenza
virus/anti-serum mixtures in an inhibition type assay with
immobilized influenza virus antigen on a sensor surface.
[0048] FIG. 4 is a diagram showing measured relative response
versus analysis cycle number for the binding of a plurality of
different influenza virus anti-sera to a sensor surface with
immobilized influenza virus antigen.
[0049] FIG. 5 is a diagram showing fitted normalisation curves
based on cycle number as x and the measured response as y for seven
concentrations of virus control samples at four ordinary
calibrations in an inhibition type assay run on a sensor surface
with immobilized virus antigen. These normalisation curves are used
for prediction of virtual concentrations for each cycle. These
virtual concentrations are then used for the construction of a
cycle specific calibration curve using the virtual concentration as
x and the known concentration as y.
[0050] FIG. 6 is a diagram showing calculated concentration versus
analysis cycle number for two different control sample
concentrations with four ordinary calibrations at different cycle
numbers in an inhibition type assay run on a sensor surface with
immobilized virus antigen.
[0051] FIG. 7 is a diagram showing application of a virtual
calibration curve (according to FIG. 5) for each cycle to the same
raw data as in FIG. 6 for two control sample concentrations.
[0052] FIG. 8 is a similar diagram as FIG. 7 showing measured
binding data for a control sample and corresponding data normalized
by a virtual calibration curve for each cycle.
[0053] FIGS. 9A-C show calibration curves prepared in an assay for
simultaneous detection of three different virus types.
DETAILED DESCRIPTION OF THE INVENTION
[0054] As mentioned above, the invention relates to a method for
the detection and quantification of at least one virus or virus
antigen in a sample medium, using biosensor technology and an
inhibition type assay format.
[0055] First, with regard to biosensor technology, a biosensor is
broadly defined as a device that uses a component for molecular
recognition (for example a layer with immobilised antibodies) in
either direct conjunction with a solid state physicochemical
transducer, or with a mobile carrier bead/particle being in
conjunction with the transducer. While such sensors are typically
based on label-free techniques detecting a change in mass,
refractive index or thickness for the immobilized layer, there are
also biosensors relying on some kind of labelling. Typical sensors
for the purposes of the present invention include, but are not
limited to, mass detection methods, such as optical methods and
piezoelectric or acoustic wave methods, including e.g. surface
acoustic wave (SAW) and quartz crystal microbalance (QCM) methods.
Representative optical detection methods include those that detect
mass surface concentration, such as reflection-optical methods,
including both external and internal reflection methods, which may
be angle, wavelength, polarization, or phase resolved, for example
evanescent wave ellipsometry to and evanescent wave spectroscopy
(EWS, or Internal Reflection Spectroscopy), both of which may
include evanescent field enhancement via surface plasmon resonance
(SPR), Brewster angle refractometry, critical angle refractometry,
frustrated total reflection (FTR), scattered total internal
reflection (STIR) (which may include scatter enhancing labels),
optical wave guide sensors, external reflection imaging, evanescent
wave-based imaging such as critical angle resolved imaging,
Brewster angle resolved imaging, SPR-angle resolved imaging, and
the like. Further, photometric and imaging/microscopy methods, "per
se" or combined with reflection methods, based on for example
surface enhanced Raman spectroscopy (SERS), surface enhanced
resonance Raman spectroscopy (SERRS), evanescent wave fluorescence
(TIRF) and phosphorescence may be mentioned, as well as waveguide
interferometers, waveguide leaking mode spectroscopy, reflective
interference spectroscopy (RIfS), transmission interferometry,
holographic spectroscopy, and atomic force microscopy (AFR).
[0056] Biosensor systems based on SPR as well as on other detection
techniques including QCM, for example, are commercially available,
both as flow-through systems having one or more flow cells and as
cuvette-based systems. Exemplary SPR-biosensors with multiple
sensing surfaces and a flow system include the BIACORE.TM. systems
(GE Healthcare, Uppsala, Sweden) and the PROTEON.TM. XPR36 system
(Bio-Rad Laboratories). These systems permit monitoring of surface
binding interactions in real time between a bound ligand and an
analyte of interest. In this context, "ligand" is a molecule that
has a known or unknown affinity for a given analyte and includes
any capturing or catching agent immobilized on the surface, whereas
"analyte" includes any specific binding partner thereto.
[0057] With regard to SPR biosensors, the phenomenon of SPR is well
known. Suffice it to say that SPR arises when light is reflected
under certain conditions at the interface to between two media of
different refractive indices, and the interface is coated by a
metal film, typically silver or gold. In the BIACORE.TM. system,
the media are the sample and the glass of a sensor chip which is
contacted with the sample by a microfluidic flow system. The metal
film is a thin layer of gold on the chip surface. SPR causes a
reduction in the intensity of the reflected light at a specific
angle of reflection. This angle of minimum reflected light
intensity varies with the refractive index close to the surface on
the side opposite from the reflected light, in the BIACORE.TM.
system the sample side.
[0058] A detailed discussion of the technical aspects of the
BIACORE.TM. instruments and the phenomenon of SPR may be found in
U.S. Pat. No. 5,313,264. More detailed information on matrix
coatings for biosensor sensing surfaces is given in, for example,
U.S. Pat. Nos. 5,242,828 and 5,436,161. In addition, a detailed
discussion of the technical aspects of the biosensor chips used in
connection with the BIACORE.TM. instrument may be found in U.S.
Pat. No. 5,492,840. The full disclosures of the above-mentioned
U.S. patents are incorporated by reference herein.
[0059] While in the Examples that follow, the present invention is
illustrated in the context of SPR spectroscopy, and more
particularly a BIACORE.TM. system, it is to be understood that the
present invention is not limited to this detection method. Rather,
any affinity-based detection method where an analyte binds to a
ligand immobilised on a sensing surface may be employed, provided
that a change at the sensing surface can be measured which is
quantitatively indicative of binding of the analyte to the
immobilised ligand thereon.
[0060] Now to the detection and quantification assay. Generally, in
an inhibition type assay (also called solution competition), a
known amount of a detecting molecule (here an antibody) is mixed
with the sample (here a virus), and the amount of free detecting
molecule in the mixture is measured. More specifically, an
inhibition type assay for to concentration measurements in the
present biosensor context may typically comprise the following
steps:
1. The analyte or a derivative thereof is attached to the sensor
surface as ligand. 2. A constant (known or unknown) concentration
of detecting molecule is added to different concentrations of the
calibrant solutions (analyte). 3. The mixtures are contacted with
the sensor surface (injected over the surface in a flow system) and
the response is measured. 4. Calibration curves are calculated. 5.
The measurements are then performed by mixing the samples (analyte)
with the constant concentration of detecting molecule, the samples
are contacted with the sensor surface (injected over the surface in
a flow system) and the response is measured. 6. The calibration
curve is used for calculation of the analyte concentration in the
sample. The amount of free detecting molecule is inversely related
to the concentration of analyte in the sample. 7. The surface is
regenerated and a new sample can be injected.
[0061] In the method of the present invention, the ligand is a
virus antigen, preferably a surface antigen (or optionally the
whole virus), whereas the analyte is an antibody to the antigen.
The antibody may be polyclonal, e.g. serum, or monoclonal. Due to
the inhibition type assay format, diffusion effects of the large
virus particles to the surface are avoided.
[0062] For purposes of illustration, without any limitation
thereto, the invention will in the following be described with
regard to the determination of the concentration of at least one
influenza virus in a sample, and more particularly of the
concentrations of hemagglutinin (HA) of three different virus types
in a trivalent flu vaccine. Reference is made to FIGS. 1 to 9 in
the accompanying drawings.
[0063] As schematically depicted in FIG. 1a, purified virus HA
designated by reference numeral 1 is immobilized on a biosensor
sensor surface 2. A mixture of virus particles 3 and anti-serum
containing antibodies 4 is made to pass as a liquid flow over the
sensor surface 2. As illustrated in FIG. 1a, the antibodies 4 can
either be bound to the virus particle or to the immobilized HA
antigen or be free in solution. Binding to the sensor surface
increases the response signal from the sensor surface.
[0064] FIG. 1b illustrates the case when no virus is present in the
sample. A maximum amount of antibodies 4 then bind to the HA
antigen 1 on the sensor surface, resulting in a high response
signal.
[0065] In FIG. 1c, on the other hand, a high concentration of virus
particles 3 results in a low amount of free antibodies 4, and a low
response signal is therefore measured. Thus, the higher the
concentration of virus in the sample, the lower is the amount of
binding antibodies to the surface HA, resulting in a lower response
level.
[0066] If the sensor surface has, or is capable of providing
multiple discrete sensing areas or "spots", such as three or more,
e.g. three different HA's may be immobilized as is schematically
illustrated in FIG. 2, where HA specific to virus types/subtypes
A/H1N1, A/H3N2 and B (which are typically used in current flu
vaccines) are immobilized to the respective spots on the sensor
surface.
[0067] As will be demonstrated below, the binding of different
virus anti-sera to HA is selective, i.e. there is no
cross-reactivity between different virus types or subtypes. Due to
this selectivity, two or more different virus components in a
sample, such as a multivalent vaccine, may be determined
simultaneously.
[0068] An exemplary method embodiment of the invention applied to a
sample containing the three above-mentioned virus types/subtypes
A/H1N1, A/H3N2 and B will now be described.
[0069] HA from the three different virus types is immobilized on
three different spots on the sensor surface.
[0070] A calibration procedure is then performed. Calibrants
consisting of a fixed concentration of a standard anti-serum for
each virus type are mixed with different known concentrations of
virus (or virus antigen) covering the concentration range to be
measured. The calibrants are then injected, either separately or
together for all three types, over the sensor surface spots and the
response is measured. From the results of the measurements,
calibration curves are then calculated.
[0071] Measurement of the sample content of virus HA is then
performed by mixing each virus with the fixed concentration of the
anti-serum, either one at a time, or, preferably, with all three
anti-sera. The sample is injected over the sensor surface and the
free anti-sera concentration is measured. The calibration curve is
used for the calculation of virus antigen concentration in the
sample.
[0072] The surface is then regenerated (i.e. bound antibodies are
dissociated from the immobilized HA by contacting the surface with
a suitable regeneration fluid), and a new sample can be passed over
the surface.
[0073] By mixing the sample with all three anti-sera and injecting
the sample over all three spots as preferred above, the
concentration of the HA from the three different virus
types/subtypes can be analyzed in a single analysis cycle. An assay
may therefore be developed which can simultaneously measure all
virus components of a multivalent, e.g. trivalent, flu vaccine.
[0074] As will be shown below, it has been found that there is
substantial cross-reactivity between different strains of influenza
virus subtypes to the hemagglutinin of a strain of the subtype. It
is therefore likely that a generic assay for each of the common
virus strains could be developed. Such an assay could thus have
general use for measuring the virus antigen content in a flu
vaccine irrespective of the yearly changing combination of virus
strains thereof.
[0075] In the above described assay, a prerequisite for high
quality analytical results is a constant binding capacity of the
ligand (HA) immobilized on the sensor surface and a high stability
of the calibrants injected over the surface. Generally, a minor
decrease in binding capacity can often be compensated by frequent
calibrations. However, frequent calibrations decrease the
throughput and increase the cost due to reagent consumption.
[0076] According to the present invention a method has been devised
where each analysis cycle is evaluated using a "virtual"
calibration curve, thus minimizing the need of frequent calibration
during drift and significantly improving the quality of
quantitative measurements using biosensor systems, such as e.g. the
above-mentioned BIACORE.TM. systems. While the method basically is
generally applicable to any ligand, and may be used in various
different assay formats including inter alia direct binding assays,
inhibition assays and sandwich assays, the method has particular
relevancy in the present virus detection context, since virus
antigens like HA usually exhibit significant instability causing
drift on sensor surfaces.
[0077] While the binding capacity of the sensor surface decreases,
the measured/calculated concentration of the controls increases as
a function of the number of analytical cycles performed (or cycle
number) until a new calibration run is performed, since in the
inhibition type assay format the calibration curve interprets the
binding capacity decrease as an increased HA concentration in the
sample. This drift increases with an increased number of analysis
cycles, and is often exponential. In the present method, an
analysis cycle includes the steps of passing the mixture of virus
and detecting antibodies over the sensor surface with immobilized
HA, and then regenerating the surface to prepare it for the next
analysis cycle.
[0078] In accordance with the invention, the new calibration
routine can be designed in different ways.
[0079] In one variant, raw data from calibration runs is used for
prediction of virtual concentrations for each analysis cycle
followed by calculation of a cycle specific calibration curve and
prediction of the concentration for the sample/control.
[0080] In another variant, calibration equations are calculated for
each of the real calibrations followed by prediction of calibration
coefficients for each cycle which are then used for prediction of
samples/controls.
[0081] The first-mentioned method above will be described in more
detail in Examples 4 and 5, and the second variant in Example 6
below.
[0082] An assay kit for carrying out the method of the present
invention for analysis of, for example, a multivalent flu vaccine
may comprise hemagglutinin (or a hemagglutinin analogue) for the
target influenza virus types/subtypes, standard sera and virus
standards for the virus types/subtype, and optionally also a sensor
chip.
[0083] In the following Examples, various aspects of the present
invention are disclosed more specifically for purposes of
illustration and not limitation.
EXAMPLES
[0084] The present examples are provided for illustrative purposes
only, and should not be construed as limiting the invention as
defined in the appended claims.
Instrumentation
[0085] A BIACORE.TM. T100 (GE Healthcare, Uppsala, Sweden) was
used. This instrument, which is based on surface plasmon resonance
(SPR) detection at a gold surface on a sensor chip, uses a
micro-fluidic system (integrated micro-fluidic cartridge--IFC) for
passing samples and running buffer through four individually
detected flow cells, designated Fc 1 to Fc 4, one by one or in
series. The IFC is pressed into contact with the sensor chip by a
docking mechanism within the BIACORE.TM. T100 instrument.
[0086] As sensor chip was used Sensor Chip CM5 (GE Healthcare,
Uppsala, Sweden) which has a gold-coated (about 50 nm) surface with
a covalently linked hydrogel matrix (about 100 nm) of
carboxymethyl-modified dextran polymer.
[0087] The output from the instrument is a "sensorgram" which is a
plot of detector response (measured in "resonance units", RU) as a
function of time. An increase of 1000 RU corresponds to an increase
of mass on the sensor surface of approximately 1 ng/mm.sup.2
Example 1
Assay for Influenza Virus A/H3N2/Wyoming, A/H3N2/New York and
B/Jilin
Materials
[0088] Hemagglutinin (HA) A/H3N2, Wyoming/3/2003, Wisconsin and New
York was from Protein Sciences Corp., Meriden, USA. HA A/H1N1, New
Caledonia/20/99 was from ProsPec, Rehovot, Israel. HB/Jilin was
from GenWay Biotech Inc., San Diego, USA. Sera as well as virus
strains were from NIBSC--National Institute for Biological
Standards and Control, Potters Bar, Hertfordshire, U.K. Assay and
sample buffer: HBS-EP+, GE Healthcare.
Surfactant P20, GE Healthcare.
Method
[0089] HA (H3N2, H1N1 and B) are immobilized to a Sensor Chip CM5
in three respective flow cells of the BIACORE.TM. T100 using amine
coupling as follows: H3N2/Wyoming and Wisconsin: 10 .mu.g/ml in 10
mM phosphate buffer, pH 7.0, 0.05% Surfactant P20, 7 min. H3N2/New
York: 10 .mu.g/ml in 10 mM maleate buffer, pH 6.5, 0.05% Surfactant
P20, 7 min. B/Jilin: 5 .mu.g/ml in 10 mM maleate buffer, pH 6.5,
0.05% Surfactant P20, 20-30 min. Immobilisation levels are
5000-10000 RU. Sera to the respective virus strains are diluted
using a dilution factor based on SRID titre, as recommended by the
supplier. E.g. 7 .mu.l serum diluted to 1 ml for SRID corresponds
to .times.200 dilution in the BIACORE.TM. T100. (Dilutions are made
to obtain approximately 500-1500 RU.) The injection time is 5 min.
Three to ten start-up cycles with serum are performed. Calibration
curves are prepared with virus antigen (HA), first diluted in MQ as
recommended by the supplier (HA is then kept frozen in aliquots)
and then further diluted in sera to typically 0.1-15 .mu.g/ml.
Standards and samples have 400 s injection time. Regeneration is
performed with 20-50 mM HCl, 0.05% Surfactant P20, 30 s followed by
30 s stabilization.
Example 2
Generality of Detection of Different Strains of the Same Virus
Subtype
[0090] H3N2 strain Wyoming HA was immobilized to a Sensor Chip CM5
and the surface was contacted with different virus/antiserum
combinations: virus/anti-serum from Wyoming (W/W); virus/anti-serum
from New York (N.Y./N.Y.), Wyoming virus and serum from New York
(W/N.Y.); New York virus and serum from Wyoming (N.Y./W).
Calibration curves with the respective combinations were run. The
results are shown in FIG. 3. From the figure, it is clear that
there is cross-reactivity between the different virus strains. The
Wyoming HA and virus/anti-serum can therefore be used for
quantification of the New York strain and vice versa.
Example 3
Selectivity in Binding of Anti-Sera to Different Influenza Virus
Types/Subtypes HA
[0091] 27 different anti-sera to different strains of influenza
virus A/H3N2, A H1N1 and B were injected over immobilized H3N2
Wyoming HA and the binding thereof was detected. The results as
well as a listing of the strains used are indicated in FIG. 4. As
apparent from the figure, all H3N2 anti-sera bind with signals
higher than 100 RU while all H1N1 and B anti-sera have signals
below 50 RU. This indicates that several virus strains may be
quantified simultaneously and that one or only a few HA's are
required for measurement of H3N2.
Example 4
Virtual Calibration Procedure
[0092] A number of assay cycles (about 100) were run on the
BIACORE.TM. T100 and a Sensor Chip CM5, during which four ordinary
calibrations were performed with seven different concentrations of
control samples (0.156, 0.31, 0.625, 1.25, 2.5, 5 and 10 .mu.g/ml).
The function y(x)=a*exp(-b*x)+c*exp(-d*x)+e, using cycle number as
x, response as y, and a, b, c, d and e as fitted parameters, was
fitted for each of the seven different concentrations. The results
are shown in FIG. 5, the top curve represents the lowest
concentration of the control (i.e. the highest response--inhibition
assay) and the bottom curve the highest (i.e. the lowest response).
The equations were then used for calculation of a virtual response
for each cycle. These responses were then used for the calculation
of a calibration curve for each cycle which were used for the
prediction of samples and controls run at exactly that cycle, as
described below with reference to FIGS. 6 and 7.
[0093] FIG. 6 illustrates the drift on the calculated concentration
of 2 controls, 1.0 .mu.g/ml and 0.5 .mu.g/ml. A large number of
assay cycles were run and four intermediate calibrations were
performed at cycle numbers indicated by the double dotted arrows.
The concentrations of the 3 (2) controls following each calibration
were calculated against the closest preceding calibration curve. As
indicated by the dotted arrows, there is a systematic increase in
calculated concentrations with increased distance to the
calibration. This increase in calculated concentration is due to a
decreased signal from the control sample. This is in turn due to a
decrease in binding capacity of the surface as a function of cycle
numbers, which the calibration curve interprets as an increased
concentration. This decrease in binding capacity is also visible in
FIG. 3 and FIG. 4.
[0094] Application of the virtual calibration method described
above to the raw data in FIG. 6 gives the concentration estimates
for shown in FIG. 7 for the 0.5 and 1.0 .mu.g/ml controls, which is
a considerable improvement of the repeatability in the prediction
of the concentration of control samples.
Example 5
Normalization of Binding Data by a Virtual Calibration
Procedure
[0095] HA recombinant proteins HB/Jilin, H1N1/New Caledonia and
H3N2/Wyoming were immobilized. Calibration curves were obtained.
Samples were diluted and concentrations between 0.5-15 .mu.g/ml
were measured and recalculated. To avoid drift of the response, the
results were normalized using the normalization procedure outlined
in Example 4 above, each cycle obtaining a unique calibration
curve. FIG. 8 shows the results before and after normalization for
control samples, 5 .mu.g/ml of Baiangsu/10/2003, giving a response
of 250 RU, CV=1.2%.
Example 6
Simultaneous Detection of Three Different Virus Types
[0096] Three flow cells were immobilized with three different
recombinant influenza virus HA proteins: H1N1/New Caledonia,
H3N2/Wisconsin and B/Jilin.
Virus standards from the three influenza strains, H1N1/New
Caledonia, H3N2/Wisconsin and B/Malaysia, were diluted and mixed
together so that the final concentration of each standard was 16
.mu.g/ml. Calibration curves were then made as 2-fold serial
dilutions from 16 .mu.g/ml to 0.5 .mu.g/ml.
[0097] The three vaccines, H1N1, H3N2 and B, to be analysed, were
diluted 8, 16, 32 and 64 times.
[0098] Three serums (H1N1/New Caledonia, H3N2/Wisconsin and
B/Malaysia from NIBSC) were diluted to concentrations giving
responses of 500-700 RU and mixed together.
TABLE-US-00001 Prior mix with ag End dilution H1N1/New Caledonia
60x dilution 180x H3N2/Wisconsin 70x dilution 210x B/Malaysia 20x
dilution 60x
[0099] To analyze the vaccines, duplicates of the standards and
vaccines were first mixed with the serum solution and then allowed
to flow through all flow cells using a method created in "Method
Builder".
[0100] The general method from "Method Builder":
Start-up (7 cycles, buffer instead of sample followed by
regeneration. 2 hours) Calibration curve 1 (14 cycles) Samples (12
cycles) Calibration curve 2 (14 cycles) Samples (12 cycles)
Calibration curve 3 (14 cycles).
[0101] The results were then normalized in respect to the three
calibration curves. This was done by performing a four parameter
fit of the calibration curves to the four-parameter regression
curve (Equation 1) conventionally used for concentration
determinations with BIACORE.TM. systems to determine the four
coefficients:
Response = R high - ( R high - R low ) 1 + ( X A 1 ) A 2 ( 1 )
##EQU00001##
[0102] where R.sub.high is the response at low virus concentration,
R.sub.low is the response at low virus concentration, A.sub.1
(EC50) and A.sub.2 (Hill slope) are fitting parameters and X is the
concentration of virus.
[0103] The values obtained for each one of the four coefficients at
the different concentrations were then plotted against analysis
cycle number, whereby an equation for each coefficient was
obtained. Using the coefficients obtained with Equation 1 above,
the normalized concentrations were calculated.
Results
TABLE-US-00002 [0104] Concentration Surface HA Sample (ug/ml) Std
dev CV % B vaccine 1 102.9 1.07 1.0 vaccine 2 30.9 0.30 1.0 vaccine
3 27.8 0.44 1.6 H1N1 vaccine 1 37.4 0.05 0.1 vaccine 2 34.9 0.04
0.1 vaccine 3 30.5 0.10 0.3 H3N2 vaccine 1 36.2 0.02 0.1 vaccine 2
27.8 0.06 0.2 vaccine 3 37.7 0.12 0.3
[0105] According to the manufacturers, the HA concentration of each
strain in the vaccine should be 30 .mu.g/ml, analyzed with
SRID.
[0106] The three calibration curves are shown in FIG. 9 (the
above-mentioned calibration curves 1 to 3 in each figure).
[0107] From the foregoing, it will be appreciated that, although
specific embodiments of this invention have been described herein
for purposes of illustration, various modifications may be made
without departing from the spirit and scope of invention.
Accordingly, the invention is not limited except by the appended
claims.
* * * * *