Note: Descriptions are shown in the official language in which they were submitted.
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IMMOBILIZED SILVER IMMUNOASSAY SYSTEM
Background of the Inve~~ion
Since the late 1950s, when the work of Rosalyn Yalow and Solomon Berson
S (Yalow et al., 1959, Nature (London) 184:1648; Yalow et al., 1960, J. Clin.
Invest
39:1157) first illuminated the possibilities of immunoassays, immunodiagnostic
techniques based on the specific interaction of antibody and antigen have
become of
paramount importance in the clinical, agricultural, food, veterinary, and
environmental
sectors. In 1996, it was estimated that the worldwide market of immunoassay
products was $10 billion in the clinical sector alone, and increasing at an
annual rate
of 10% (Deshpande, Enzyme Immunoassay: From Concept to Product Development,
New York:Chapman & Hall, 1996). This market is driven by an ever-increasing
desire for assays of greater sensitivity and specificity, at reasonable
financial costs. It
is in this environment that the specific and irreversible interaction between
avidin or
streptavidin and biotin has found use.
Streptavidin, a close relative of egg white avidin, is expressed in
Streptomyces
avidinii (Green, 1990, Methods in Enzymology 184:51), and both avidin and
streptavidin exhibit an affinity for biotin on the order of 10'S M-'. The
streptavidin-
biotin system has become a widely-used tool of molecular biology in such
applications as affinity chromatography, cytometry, nucleic acid research, and
diagnostics (Diamandis et al., 1991, Clin. Chem. 37:625; Wilchek et al., 1988,
Anal.
Bio Chem. 171:1). A common immunological procedure calls for the use of
streptavidin-coated microtiter plates, which are used to capture either
biotinylated
antibodies or antigens. Since the assay is based on the interaction of
streptavidin and
biotin, universal kit-based assay formats are possible. These universal assays
are also
the basis of many automated immunological testing systems (Char, ed., 1996,
Immunoassay Automation: An Updated Guide to Systems, San Diego: Academic
Press, S 1-308). A format utilizing a microtiter-based enzyme-linked
immunosorbent
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2
assay (ELISA) can measure a wide variety of analytes using a dual antibody or
"sandwich" immunoassay. The choice of a streptavidin-coated solid support is
made
to overcome the limitations present in the direct antibody coating of
polystyrene
supports, which can result in unreliable or nonuniform coating of the solid
support, in
addition to the steric affects of binding upon the antibody. However, while
streptavidin systems allow for universal ELISA kits and can improve assay
sensitivity,
coated plates can be costly.
Research examining the behavior of biotin has found that immobilized silver
ions will bind biotinylated compounds both strongly and, in this case,
reversibly. This
research has been done with both immobilized metal affipity chromatography
(IMAC)
(Garcia et al., 1994, Reactive Polymers 23:249; Kim et al., 1995, Art. Cells,
Blood
Subs., and Immob. Biotech. 23:555; Miles et al., 1995, J. Chromatogr. A.
702:173)
and paramagnetic particles (Ramirez-Vick, 1997, Ph.D. Dissertation, Arizona
State
University, Tempe, AZ). It has been discovered in accordance with the present
invention that immobilized silver ions can be used in an immunoassay format to
provide a sensitive and inexpensive universal assay.
Summary of the Invention
The present invention provides an immunoassay system comprising bioassay
plates having silver immobilized thereon. The present invention further
provides a
method of making bioassay plates having silver immobilized thereon.
In another embodiment, the present invention provides a method for detecting
an antigen or antibody, and a kit useful for the detection of an antigen or
antibody.
An apparatus for providing activated bioassay plates is also provided by the
present invention.
Brief Description of the Drawing
Fig. 1 is a graph depicting the sensitivity of a microtiter plate having
silver
immobilized thereon.
Fig. 2 is a schematic of an immunoassay utilizing microtiter plates having
silver immobilized thereon, and biotinylated capture antibodies.
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Fig. 3 is a graph providing a kinetic analysis of a horseradish peroxidase
immunoassay.
Fig. 4 is a schematic of a checkerboard assay.
Fig. 5 shows the results of a checkerboard assay.
Fig. 6 is a schematic depicting the arrangement of activated and control wells
of a microtiter plate.
Fig. 7 is a graph of immunoassay results comparing a streptavidin method
with the silver method of the present invention.
Fig. 8 is a flow diagram describing the apparatus of the present invention.
Fig. 9 is a side view of the apparatus of the invention.
Fig. 10 is a diagram of the liquid handling system of the invention.
Figs. 11 a-11 c are diagrams of the liquid transfer manifold of the invention.
Fig. 11 a is a side view of the reagent addition stage and vacuum stage; Fig.
11 b is a
front view of the reagent addition stage; Fig. 11 c is a front view of the
vacuum stage.
petailed Description of the Invention
The present invention provides an immunoassay system comprising bioassay
plates having silver, in particular silver ions, immobilized thereon. The
invention
further provides methods of making and using such bioassay plates. The
bioassay
plates and immunoassay of the present invention are useful for the detection
of
antibodies and antigens, and provide cost and sensitivity advantages relative
to the
streptavidin-coated bioassay plates of the prior art.
The bioassay plates used in the present invention are microwell, or
microtiter,
plates known in the art for immunoassays, and are commercially available.
Conventional microwell plates are 96-well microplates having wells arranged on
an
8 x 12 matrix on 9 mm centers. Each well holds approximately 300 microliters.
384-
well plates are also available, in which the wells are arranged in a 16 x 24
matrix on a
4.5 mm center, with each wells having a brim volume of approximately 80
microliters. Well plates defined by larger matrices, e.g. 1536 well plates,
are also
available. The number and configuration of the wells are not critical to the
present
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4
invention, and are used by way of example only. The bioassay plates used in
accordance with the present invention are plastic, and preferably polystyrene.
Bioassay plates having silver ions immobilized thereon are made by a method
comprising functionalizing a mufti-well bioassay plate to provide an amine-
containing
bioassay plate, adding polymerized glutaraldehyde to the wells of the plate
for a time
and under conditions whereby the amines are activated by glutaraldehyde,
rinsing the
plates with an aqueous solution, adding thiourea to the wells of the plate for
a time
and under conditions whereby the thiourea is reacted with a glutaraldehyde
moiety of
the glutaraldehyde-activated bioassay plate, rinsing the plate with an aqueous
solution,
and contacting the plate with silver ions for a time and under conditions
whereby the
silver ions are immobilized on said plate.
The bioassay plate may be constructed of any material that can be
functionalized to contain an amine group. In a preferred embodiment, the mufti-
well
bioassay plate is a plastic mufti-well bioassay plate. For example, the plate
may be
made of polystyrene, polyethylene, polypropylene, or other primary polymers or
composite resins. Polystyrene is particularly preferred. Methods for
functionalizing
these materials to contain an amine group are known in the art. For example, a
polystyrene bioassay plate can be functionalized to contain an amine group by
methods known in the art and disclosed for example in Immobilized Affinity
Li~and
Techniaues, Hermanson et al., eds., San Diego: Academic Press, 1992, the
disclosure
of which is incorporated herein by reference. Aminated polystyrene bioassay
plates
are also commercially available, for example from Corning {Corning, NY), NLJNC
(Denmark) and Micro Membranes (Newark, NJ). Preferably the plate is amidated
or
aminated to contain from about 1 x 10'3 to 1 x 10'4 amine sites per cm2.
Polymerized glutaraldehyde may be prepared by allowing glutaraldehyde
(25 wt %) to polymerize, for example for from 1 to 36 hours at from
23°C to 70°C.
In a preferred embodiment polymerization is at 70°C for about 24
hours. The
polymerized glutaraldehyde is added to the wells of the plate and incubated
under
conditions whereby a glutaraldehyde-activated plate is produced, for example
for from
1 to 36 hours at from 23°C to 70°C. In a preferred embodiment,
incubation is at 35 to
50 ° C for 1 to 24 hours, and more preferably at about 37 ° C
for about 24 hours. The
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plate is rinsed with an aqueous solution, for example deionized water, to
remove
unreacted glutaraldehyde. The wells of the plate are then filled with
thiourea, for
example from 0.01 M to 1 M solution, and preferably a 1 M solution, under
conditions
suitable for reaction with the glutaraldehyde moiety, for example for from 1
to 36
hours at from 23 °C to 70°C. In a preferred embodiment at,
incubation is for 1 to 24
hours at 35 to 50°C, and more preferably about 24 hours at about
37°C. The plate is
rinsed with an aqueous solution, for example deionized water, to remove
unreacted
thiourea.
Silver ions, preferably in the form of silver nitrate, are then added to the
plate
under conditions whereby silver ions are immobilized on the plate, for example
for
from 1 to 36 hours at from 23 °C to 70°C. In a preferred
embodiment, incubation is
for about 24 hours at about 37°C. The plates are then rinsed with an
aqueous
solution, for example deionized water, and may be stored until use, preferably
in an
opaque sleeve.
The bioassay plates having silver ions immobilized thereon are useful in a
method for detecting an antigen or an antibody. It has been discovered in
accordance
with the present invention that the silver ions immobilized on bioassay plates
are
capable of strong binding to biotinylated antibodies and antigens.
Accordingly, the
plates of the invention may be used in standard enzyme-linked immunosorbent
assays
(ELISAs). For example, a bioassay plate having silver ions immobilized thereon
is
incubated with biotinylated antibody to provide a bioassay plate having the
antibody
immobilized thereon. After washing with an aqueous solution, the plate is
incubated
with a solution containing the cognate antigen under conditions whereby the
antigen
binds to the immobilized antibody, followed by another washing step. The
antigen is
then detected, for example by subsequent incubation with a labeled antibody
having
specificity for the antigen. Detectable labels for antibodies are known in the
art and
include radiolabels, fluorescent tags, and enzyme conjugates. In a preferred
embodiment, the aqueous solution contains deionized water and Tween
(polyoxyethylene sorbitan monolaurate).
An antibody may be detected using the plates of the present invention in an
indirect ELISA assay. For example, a bioassay plate having silver ions
immobilized
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thereon is incubated with a biotinylated antigen to provide a bioassay plate
having the
antigen immobilized thereon. After washing with an aqueous solution, the plate
is
incubated with a solution containing the cognate primary antibody under
conditions
whereby said antibody binds to the immobilized antigen. After incubation and
washing, a labeled secondary antibody is added and incubated under conditions
whereby it binds to the primary antibody. After washing, the secondary
antibody is
detected, wherein detection thereof indicates the presence of the primary
antibody.
Detectable labels for antigens are known in the art and include radiolabels,
fluorescent
tags, and enzyme conjugates. In a preferred embodiment, the aqueous solution
contains deionized water and Tween.
Conditions for biotinylating antibodies and antigens are well known in the art
and disclosed, for example, by Bayer et al., "Protein Biotinylation" (1990)
Methods in
Enzymology 184:138 and O'Shannessy "Antibodies Biotinylated via Sugar
Moieties"
(1990) Methods in Enzymology 184:162, the disclosures of which are
incorporated
herein by reference. Conditions for performing ELISAs are well-known in the
art and
disclosed, for example, by Harlowe, et al., (1988) Antibodies: A Laboratory
Manual,
Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, the disclosure of
which is incorporated herein by reference.
The present invention further provides a kit useful for the detection of an
antigen or antibody. The kit comprises, in a first container, a bioassay plate
having
silver ions immobilized thereon. In a preferred embodiment, the bioassay plate
is a
polystyrene multi-well plate. The kit may optionally contain a second
container
containing a biotinylated antibody or a biotinylated antigen. The kit may
optionally
contain a third container containing labeled antibody, when the second
container
contains a biotinylated antibody, or a labeled secondary antibody, when the
second
container contains a biotinylated antigen.
The present invention further provides an apparatus useful for the automated
production of microplates having modified surface chemistry. As shown by the
flow
diagram in Fig. 8, the apparatus provides for filling the wells of a
microplate with a
reagent in an addition/withdrawal chamber; conveying the microplate to an
incubation
chamber in which the microplate is sealed, heated and agitated, and unsealed;
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conveying the microplate to the addition/withdrawal chamber for evacuation of
reagent, washing, and addition of a second reagent; conveying the microplate
to the
incubation chamber for sealing, heating and agitation, and unsealing;
conveying the
microplate to the addition/withdrawal chamber for evacuation of reagent and
washing;
followed by subsequent cycles of reagent addition and incubation, or
conveyance of
the microplate out of the machine.
In a preferred embodiment, and with reference to Figs. 9, 10, 11 a, 11 b and
l lc, the apparatus comprises a housing having disposed therein a reagent
addition/withdrawal chamber (1) and an incubation chamber (2). Microplates are
conveyed into and between the chambers by means of a plate holder (3) movable
horizontally by a plate holder track (4). Reagents and wash solution are
provided in
storage containers (5) connected by reagent lines (13) to the dispense portion
of a
manifold (6) which delivers reagent and wash solution by dispense lines (7) by
means
of a liquid pump (11). After reagent addition, microplates positioned on the
plate
holder (3) are conveyed via the plate holder track (4) into the incubation
chamber (2).
The microplate is sealed by a non-reactive sealing plate (13) delivered
vertically. The
incubation chamber further provides a means for heating and agitating the
microplate
(14). After a time predetermined by the user, the microplate is conveyed to
the
reagent additionlwithdrawal chamber ( 1 ) via plate holder track (4). Spent
reagent is
removed through aspirator lines (8) and withdrawn by the aspirate portion of
the same
manifold (6) by means of a vacuum pump (9) through waste lines (12) to a waste
container (10). Wash solution is added through the dispense portion of the
manifold
(6) which delivers wash solution through dispense lines (7). Wash solution is
removed through aspirator lines (8) and withdrawn by the aspirate portion of
manifold
(6) by means of a vacuum pump (9) through waste lines ( 12) to a waste
container ( 10).
The foregoing steps are carned out in an automatic programmed manner under
the control of electronic circuitry contained in the housing.
All references cited herein are incorporated in their entirety.
The following examples serve to further illustrate the present invention.
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E_ xamnle 1
A polystyrene 96 well microtiter plate aminated to provide approximately 2 x
10'3 active amine sites per cm2 was obtained from Corning (Corning, NY).
Glutaraldehyde (25 wt %) which had been allowed to polymerize at 70°C
for 24 hours
was added to each well of the microplate, which was then incubated at
37°C for 24
hours to facilitate plate activation. The plate was then rinsed with deionized
water
and wells filled with a 1M solution of thiourea, followed by an additional 24
hour
period of incubation at 37°C. After another rinsing of the plate, a 1 M
solution of
silver nitrate was allowed to contact the plate during another 24 hour
incubation at
37°C. The plate was then rinsed extensively.
The biotin-binding capability of such an immobilized silver microtiter plate
was tested as follows. A complete plate was assembled from stripwells, using
alternating strips of unactivated and silver-containing wells, where the
silver-
containing strips were in the odd-numbered rows and the unactivated strips
were in
the even-numbered rows. The test consisted of the binding of biotinylated
horseradish
peroxidase (bHRPO), which was detected using the chromogenic reaction of 2,2'-
azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) diammonium salt (ABTS~). This
was a one-step, unamplified assay; 150 pl of the bHRPO solution was added to
the
wells using doubling dilutions, and allowed to bind for one hour. Following
this, the
plates were rinsed with deionized water and the ABTS~ in citrate buffer was
added.
The developed color in the wells was read after one hour at room temperature
using a
BioRad Benchmark Microplate Reader set at 415 nm. The plate setup and moles of
bHRPO corresponding to each dilution are shown in Tables I and II,
respectively.
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TABLE .
1 2 3 4 5 6 7 8 9 10 ll 12
A no bHRPO no bHRPO no bHRPO no bHRPO no bHRPO no bHRPO
B no bHRPO I / 1 1 / 64 1 / 4096 1 / 262144no bHRPO
no bHRPO 1 / 2 1 / 128 1 / 8192 1 l 524288no bHRPO
C
D no bHRPO 1 / 4 1 / 256 1 / 16384 I / 1048576no bHRPO
E no bHRPO/ABTSl / 8 1 / 512 1 / 32768 1 / 2097152no
bHRPO/ABTS
F no bHRPO/ABTS1 / l6 1 / 1024 1 / 65536 1 / 4194304no
bHRPOIABTS
G no bHRPO/ABTS1 / 32 1 / 2048 1 / 1310721 / 8388608no
bHRPO/ABTS
no bHRPO/ABTSno no bHRPO/ABTSno bHRPO/ABTSno bHRPO/ABTSno
H bHRPO/ABTS bHRPO/ABTS
TABLE II
Dilution Grams bHRPO Moles bHRPO
1 / 1 2.40E-06 5.33E-11
1 / 2 1.20E-06 2.67E-11
1 / 4 6.OOE-07 1.33E-11
1 / 8 3.OOE-07 6.67E-12
1 / 16 1.SOE-07 3.33E-12
1 / 32 7.SOE-08 1.67E-12
1 / 64 3.75E-08 8.33E-13
i / 128 1.88E-08 4.17E-13
1 / 256 9.38E-09 2.08E-13
1 / 512 4.69E-09 1.04E-13
1 / 1024 2.34E-09 5.21E-14
1 / 2048 1.17E-09 2.60E-14
1 / 4096 5.86E-10 I.30E-14
1 / 8192 2.93E-10 6.S1E-IS
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TABLE II
Dilution Grams bHRPO Motes bHRPO
1 / 32768 7.32E-11 1.63E-15
1 / 65536 3.66E-11 8.14E-16
1 / lE+OS 1.83E-11 4.07E-16
1 / 3E+OS 9.16E-12 2.03E-16
5 1 / SE+p5 4.58E-12 1.02E-16
1 / lE+p6 2.29E-12 5.09E-17
1 / 2E+06 1.14E-12 2.54E-17
1 / 4E+06 5.72E-12 1.27E-17
1 / 8E+06 2.85E-13 6.36E-18
10 After applying statistical curve fitting techniques, a concentration
activity
curve was generated and is shown in Figure 1. Results are presented in terms
of
moles of bHRPO. As demonstrated therein, the detection limit of this system
approaches femtomolar levels, even using an unamplified system. The typical
Gaussian response curve of ELISA is also seen in Figure 1, which further
1 S demonstrates that the maximum sensitivity of this assay occurs at bHRPO
levels of 2
x 10''3 moles.
Eagle II
Example I demonstrated that immobilized silver microtiter plates are capable
of binding biotin and a biotinylated antigen. This example demonstrates that
20 biotinylated capture antibodies can be bound to immobilized silver
microtiter plates
and used for antigen capture.
Immobilized silver microtiter plates were prepared as described in Example I.
The plates were incubated with biotinylated anti-peroxidase antibodies
(Jackson
Immunological) at an antibody concentration of 1.2 mg/ml diluted 1:100, with
25 addition of 150 microliters to each well for an hour. After washing the
plates with 50
mM phosphate buffer with 0.1 % v/v Tween 20 detergent (Phosphate/Tween
buffer),
the enzyme horseradish peroxidase (Sigma) was added, in the amounts shown in
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Table III, for one hour incubation. Phosphate/Tween buffer was used as the
dilution
buffer in the assay.
The results of eight assays of the type shown in Figure 2 are presented in
Figure 3, along with 2a error limits. The data for the immunoassays is shown
in
Table III, using the eight simultaneous assay replications for a basic
statistical
analysis. Fig re 3 uses kinetic rates to determine the detection of enzyme,
which
allows for a linear fit of the data when presented on a semi-log plot using
the dilution
number of the initial enzyme solution. At the initial dilutions, the margin of
error is
rather high, due to the high amounts of enzyme in the initial solution (2.56 x
10'"
moles per well) producing extremely rapid kinetics. As the enzyme goes through
doubling dilutions, the kinetics are measured more easily and the precision of
the
assay improves at dilution number 16 (1.6 x 10''2 moles per well). The
detection limit
of this assay is in the region of dilution number 1000 {0.25 femtomoles),
which
approaches the theoretical detection limit of the enzyme substrate system
being used.
(Deshpande, Enzyme Immunoassays: From Concept to Product l~Pvelonment, New
York, Chapman & Hall (1996), 1-422.
TABLE III
Kinetic Immunoassay Data
Dilution HRPO HRPO Kinetic
Number (g/well) (mol/well) Rate 2a Limits
1 1.13E-06 2.56E-11 927.65 286.73
2 5.63E-07 1.28E-11 773.66 253.20
4 2.81E-07 6.40E-12 666.47 95.75
8 1.41E-07 3.20E-12 598.88 97.22
16 7.03E-08 1.60E-12 551.07 61.58
32 3.52E-08 8.OOE-13 452.40 25.79
64 1.76E-08 4.OOE-13 361.72 24.07
128 8.79E-09 2.OOE-13 277.00 20.58
~s~ a ~nF_n9 ~ nnF_~ ~ ~ 4z nz
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TABLE III
Kinetic Immunoassay Data
Dilution HRPO HRPO Kinetic
Number (g/well) (mol/well) Rate 2Q Limits
512 2.20E-09 S.OOE-14 128.36 49.72
1024 l.lOE-09 2.SOE-14 80.85 53.07
2048 5.49E-10 1.25E-14 65.28 62.02
4096 2.75E-10 6.25E-15 60.26 61.08
8192 1.37E-10 3.12E-15 62.54 60.02
16384 6.87E-11 1.56E-15 52.25 78.54
32768 3.43E-11 7.81E-16 46.92 83.12
65536 1.72E-11 3.91E-16 36.93 69.52
131072 8.58E-12 1.95E-16 40.52 69.90
The basis of the foregoing is the assumption that the enzyme binds only to the
antibodies, which specifically select it from the test solution. In order for
this to be
valid, background binding of the enzyme to the plate must be at insignificant
levels.
In order to determine this, a checkerboard assay was performed {Deshpande,
supra).
The assay arrangement is shown in Figure 4. When performed, the initial
concentrations of the antibody and the enzyme were the same as described
above, as
were all buffers and incubation periods. The checkerboard assay, which is
primarily
visual, is a means to determine the effect of the antibody concentration and
the
enzyme concentration on the assay results. As seen in Figure 5, the enzyme
only
binds when antibody is present in the well. As antibody concentrations
decrease
horizontally across the plate, enzyme binding falls rapidly, as shown by the
absence of
color on the right side of the plate, even at the extreme enzyme
concentrations
introduced into row A.
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The foregoing examples demonstrate that immobilized silver is capable of
binding biotin in the bHRPO assay and that effective immobilization of capture
antibodies in the immobilized silver microplate wells is possible. In the
present
example, the silver ion immunoassay format is then compared to the current
streptavidin technology used to bind biotinylated antibodies. Plates coated
with
streptavidin (Xenopore, XPS 010 00) were obtained for this purpose. These
plates
represent the best of the currently available products since they feature a
covalent
linkage between the streptavidin and the plate surface, and are also blocked
with a
proprietary nonbiotinylated protein to inhibit background binding. No blocking
agents are used on the silver plates. A side by side comparison with an
immobilized
silver plate prepared according to Example I was performed as follows.
Both the streptavidin plate and the silver plate were hydrated and rinsed with
50 mM pH 7 phosphate buffer. The wells in the plates were filled to capacity
with
1 S buffer and allowed to stand at room temperature for 10 minutes. The plates
were then
rinsed twice with the same buffer.
Biotinylated anti-peroxidase antibodies (Jackson Immunological) were used to
coat the plates. In this assay, 150 ~,l of antibody solution (1.2 mg/ml)
diluted to 1:100
was added to the wells in the odd numbered columns. The even numbered columns
were used as control wells, and were filled with 150 ~.1 of buffer. This
arrangement of
activated and control wells is shown in Figure 6. The buffer used to dilute
the
antibodies and fill the control wells was 50 mM pH 7 phosphate buffer with
0.1% v/v
Tween 20 Phosphate/Tween) added to inhibit any hydrophobic binding in the
plates.
The plates were then covered and allowed to stand at room temperature for 1.5
hours.
Both plates were thoroughly washed using a Bio-Rad Plate Washer filled with
Phosphate/Tween buffer as the wash buffer. Horseradish peroxidase (Sigma) was
used as the antigen in this test. A solution of peroxidase was created by
adding 8 x
10'4g of the enzyme to 10 ml of Phosphate/Tween. 1 ml of this solution was
then
added to 9 ml of buffer. 300 pl of the diluted enzyme solution was added to
wells
Al, A2, A5, A6, A9, and A10. The amount of enzyme added to these primary wells
is shown in Table IV. The remainder of the wells were filled with 150 pi of
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14
Phosphate/Tween. For Test 1, 150 ~,1 of solution was withdrawn from A 1 and A2
and
diluted into B 1 and B2. This procedure was repeated down the plate, with the
solution from H1 and H2 carried to wells A3 and A4. Tests 2 and 3 were
performed
in a similar fashion. After all of these doubling dilutions were complete, the
plates
were covered and allowed to stand at room temperature for 1 hour.
For enzyme detection the plates were first washed using the automated plate
washer with Phosphate/Tween. Then, 150 ~.1 of an ABTS solution was added to
each
well in the plate, and the rate of formation of the colored product read at
415 nm in a
Bio-Rad Benchmark Microplate Reader. Readings took place every 15 seconds for
5
minutes. The ABTS solution was made by adding 17 mg of ABTS to 100 ml of 50
mM pH 5 citrate buffer. Immediately before use, 100 ~1 of 3% hydrogen peroxide
was added to the solution to catalyze the enzymatic reaction.
The raw data for these experiments is presented in Table IV and Table V.
Using the average of the normalized tests, a plot comparing the streptavidin
plate to
the silver plate was prepared. The data points shown on the graph in Figs. 7
are the
average values of the normalized tests shown in Tables IV and V. The exception
to
this are the data points taken at Dilution 1, which were replaced by the
values
representing the asymptotic approach of the immunoassays as determined from
the
preceding points. From this, the maximum kinetic rate achievable in the silver
plate
is 375, versus 150 in the streptavidin plate. Due to the approach to a final
value at
high levels of enzyme, this difference is due to the amount of capture
antibody on the
plate. The silver plate binds more functional capture antibody, and thus is
able to
bind more enzyme when excess enzyme is present. Assuming that each antibody
captures an average of 1.5 enzyme molecules, it is possible to estimate that
the silver
plate has approximately twice as many biotinylated antibody binding sites
available.
The ultimate detection limits of both plates is the same, however, and
approaches
femtomolar levels. This is the limit of the enzyme/substrate detection system
being
used.
CA 02346487 2001-04-04
WO 00/21665 PCT/US99/23902
TABLE IV
Silver Plate Immunoassay Data
DilutionHRPO Tat Test Test Test Tat Test Average
I 2 3 I 2 3
Number(mol/well)
AntibodyControlAntibodyControlAntibodyControlNormalizedNormalizedNormalized
S I 2.73E-
112.83E+p22.52E+012.38E+021.83E+012.90E+022.55E+012.SBE+022.20E+022.65E+022.47E
+02
2 1.77E-
II7.87E+021.58E+017.46E+021.15E+013.94E+021.73E+013.71E+023.34E+p23.76E+p23.61E
+p2
4 6.83E-
I23.67E+029.82E+Op3.76E+027.SSE+t>03.81E+021.22E+017.58E+023.68E+p23.69E+p23.65
E+p2
B 3.41E-
123.36E+026.04E+003.44E+024.20E+pp7.67E+028.71E+003.30E+023.40E+023.54E+p23.41E
+02
16 1.71E-
123.07E+025.74E+pp2.99E+023.37E+003.43E+p27.77E+003.OIE+022.96E+027.36E+023.IIE
+02
1~32
8.53E132.67E+02LSOE+012.70E+021.77E+p02.90E+027.32E+pp2.52E+022.68E+022.83E+022
.68E+02
64 4.27E-
132.18E+022.BIE+002.16E+022.IIE+002.28E+025.53E+002.ISE+022.14E+022.22E+0.2.17E
+02
128 2.13E-
131.6IE+021.49E+pp1.57E+022.30E+OOLSOE+027.92E+0p1.60+02LSIE+p2L42E+p2LSIE+02
256 1.07E-
137.38E+011.04E+007.18E+014.99E+007.39E+012.02E+007.27E+016.68E+pl7.18E+p17.OSE
+pl
512 5.33E-
145.20E+011.35E+004.73E+012.57E+005.37E+p12.32E+pp5.06E+014.48E+pl5.14E+pl4.89E
+01
1510242.67E-
142.81E+011.49E+pp2.73E+014.83E+pp2.75E+017.26E+Op2.67E+pl1,85E+012.43E+Ol2.31+
01
20481.37E-
141.20E+plL96E+001.34E+Ot5.77E+pp1.43E+014.13E+00LOOE+pl7.68E+ppL112E+019.30E+0
0
40966.67E-
IS6.66E+001.91E+00LOSE+018.35E+Op8.61E+00S.30E+004.75E+0p2.ISE+Op3.31E+003.41E+
00
81923.33E-IS4.73E+001.49E+001.03E+018.84E+003.03E+004.65E+003.24E+001.47E+00-
1.62E+001.02E+00
163841.67E-IS2.93E+pp2.41E+pp7.07E+0p6.65E+006.78E+006.25E+005.22E-014.28E-
015.25E-014.92E-01
2~327688.73E-162.12E+pp1.86E+p05.24E+pp5.19E+006.95E+p08.48E+pp-1.74E+004.82E-
02-1.53E+00-1.07E+00
CA 02346487 2001-04-04
WO 00/21665 PCT/US99/23902
16
TABLE V
Streptavidin Plate Immunoassay Data
DilutionHlt1'OTest st 2 ~3 Teu Tat Test Average
1 I 2 3
Number(mol/well)
AntibodyControlAntibodyControlAntibodyControlN~aliudNormalizedNormalized
2.77-
IIL36E+026.SOE+pp1.37E+p2B.SSE+pp1.29+02S.SOE+pp1.29+021.24+021.24+021.26+02
2 1.37-
II1.74E+p28.6IE+00L24E4023.46E+pp1.75E+p27.26+001.65E+p21.20+021.68E+p2LSIE+02
4 6.83-
121.74+021.96+00LSIE+022.29+pp1.49+022.06+011.72+021.49+021.28+02LSOE+02
-
8 7.41-12L62E+021.47-
0I1,34+021.02+pp1.38+024.83Fr011.62+021.33+021.38+021.44402
16 1.71-121.62+02-2.36-0I1.40+027.03-02L30E+02-2.57-
OI1.62+02L40E+021.70+021.44+02
1fZ 8.53-131.49+02-8.89-011.16+026.89-OI1.31+02-
1.41+00LSOE+021.16+021.33+021.73+p2
64 4.27-I31.23+02-6.99-OI1.28+023.23-01LOSE+02-
1.26+001.24+021.28+021.06+021.19+02
IZH 2.13-171.04+02-1.47-0I1.OIE+02-B.BBE-018.99+01-
2.OSE+001.04+021.02+029.20E+ol9.93+01
256 1.07-133.86+018.42-01S.OIE+011.06+Op4.32+015.47-
017.78+014.9IE+014.26+014.32+01
512 5.73-I43.95+011.37+DO7.31+017.59-024.04+016.97-
023.81+013.31+014.07+017.7ZE+01
110242.67-142.45+011.38+002,02+01-1.67-0I2.454015.45-
012.31+012.03+Ol2.40+012.25+01
2048 1.33-141.29+016.25-0I9.56+pp-8.19-011.22+011.06-
0I1.234011.04+011.21+011.16+01
4096 6.67-IS5.97+00-3.87-0I4.76+00-1.42+006.07+00-1.69-
016.36+006.18+006.44+p06.33+00
8192 3.33-IS2.75+00-3.OtE-0I1.47+00-2.08+003.ISE+Op-6.08-
027.OSE+003.55+003.24+003.28+00
163841.67-IS1.25+00-9.64-011.64-01-1.36+00-2.02+00-9.BSE-
0I2.21+001.52+pp.LpqE+008.97-0I
