U.S. patent application number 10/720909 was filed with the patent office on 2005-05-26 for method and composition for stabilizing liquid reagents.
Invention is credited to Janzen, Roland, Schelp, Carsten Ulrich.
Application Number | 20050112586 10/720909 |
Document ID | / |
Family ID | 34591679 |
Filed Date | 2005-05-26 |
United States Patent
Application |
20050112586 |
Kind Code |
A1 |
Janzen, Roland ; et
al. |
May 26, 2005 |
Method and composition for stabilizing liquid reagents
Abstract
The invention relates to methods and compositions for removing a
dissociated species from a fluid medium solution during and after
it has detached from a solid-phase immersed in said medium, thereby
allowing the concentration of free species to remain close to zero,
and for improving the signal to noise ratio in assays. This is
achieved by employing a substrate, such as a scavenging
solid-phase, having an attached binding partner or partners
("scavenger") for the specifically binding species and which is
present during storage. This substrate may also contain regions for
binding signal generating components attached to the solid-phase.
This substrate binds any free species bleeding off the solid phase,
increasing the reliability and sensitivity of assays. A subset of
the substrates in the invention additionally forms cross-linked
networks of solid-phase particles that further increase the
sensitivity of assays.
Inventors: |
Janzen, Roland; (Landenberg,
PA) ; Schelp, Carsten Ulrich; (Hockessin,
DE) |
Correspondence
Address: |
DADE BEHRING INC.
LEGAL DEPARTMENT
1717 DEERFIELD ROAD
DEERFIELD
IL
60015
US
|
Family ID: |
34591679 |
Appl. No.: |
10/720909 |
Filed: |
November 24, 2003 |
Current U.S.
Class: |
435/6.14 |
Current CPC
Class: |
G01N 33/54333
20130101 |
Class at
Publication: |
435/006 |
International
Class: |
C12Q 001/68 |
Claims
We claim:
1. A reagent, comprising: a fluid medium containing a first
substrate having a first binding species attached thereto; and a
second substrate having binding regions characterized by their
ability to selectively bind said first binding species if
dissociated from said first substrate.
2. The reagent of claim 1, wherein the first binding species is
attached to a defined binding surface on the first substrate, and
the binding regions on the second substrate are formed on a defined
binding surface on the second substrate, and wherein the defined
binding surface on the first substrate and the defined binding
surface on the second substrate are formed of the same
materials.
3. The reagent of claim 1, wherein the first binding species is
attached to a defined binding surface on the first substrate, and
the binding regions on the second substrate are formed on a defined
binding surface on the second substrate, and wherein the defined
binding surface on the first substrate and the defined binding
surface on the second substrate are formed of different
materials.
4. The reagent of claim 1, wherein said first binding species is
selected from the group essentially consisting of biotin, avidin,
streptavidin, an antigen, an antibody, a hapten, a receptor and an
oligonucleotide, and bindable derivatives thereof.
5. The reagent of claim 4, wherein said binding partner selectively
binds species selected from the group essentially consisting of
biotin, avidin, streptavidin, an antigen, an antibody, a hapten a
receptor and an oligonucleotide, and bindable derivatives
thereof.
6. The reagent of claim 1, wherein said binding regions are
selected from the group consisting of pores, interstitial spaces,
indentations and grooves.
7. A reagent, comprising: a fluid medium including a binding
species attached to a substrate; a permeable material including an
inner surface having an affinity for said binding species; and an
outer surface permeable to the binding species when dissociated
from said substrate.
8. The reagent of claim 7, wherein the binding species is selected
from the group essentially consisting of biotin, avidin,
streptavidin, an antigen, an antibody, a hapten, a receptor and an
oligonucleotide, and bindable derivatives thereof.
9. A method of stabilizing a reagent through the inactivation of a
free species, comprising: providing a reagent storage container
including a liquid medium containing a binding species attached to
a substrate; a permeable material including an inner surface having
an affinity for said binding species; and an outer surface
permeable to the binding species when dissociated from said
substrate.
10. The reagent of claim 9, wherein the first binding species is
attached to a defined binding surface on the first substrate, and
the binding regions on the second substrate are formed on a defined
binding surface on the second substrate, and wherein the defined
binding surface on the first substrate and the defined binding
surface on the second substrate are formed of the same
materials.
11. The method of claim 10, wherein the binding species is selected
from the group essentially consisting of biotin, avidin,
streptavidin, an antigen, an antibody, a hapten, a receptor and an
oligonucleotide, and bindable derivatives thereof.
12. A reagent, comprising: a fluid medium; a first substrate in
contact with the fluid medium; a first binding species, wherein a
first portion of the first binding species is attached to the first
substrate and a second portion of the first binding species is
dissociated from the first substrate; and a second substrate having
regions adapted to selectively bind the second portion of the first
binding species, wherein the first portion of the first binding
species and the second portion of the first binding species, when
combined, make up approximately 100% of the known total amount of
the first binding species.
13. A reagent comprising: a fluid medium containing a first
substrate having a first binding species attached thereto; and a
second substrate having regions characterized by their ability to
selectively bind said first binding species, wherein binding of
first binding species to said second substrate does not impair the
binding of first binding species to a binding target.
14. A reagent, comprising: a fluid medium containing a first
substrate having a first binding species attached thereto; and a
second substrate having regions characterized by their ability to
selectively bind said first binding species if dissociated from
said first substrate and regions characterized by their ability to
selectively bind first binding species associated with said first
substrate, wherein the second substrate binds a plurality of first
binding species to a common second substrate, and wherein an
aggregate is formed comprising the plurality of first substrate
having first binding species attached thereto, cross-linked by said
second substrate.
15. The reagent of claim 14, wherein the first binding species is
attached to a defined binding surface on the first substrate, and
the binding regions on the second substrate are formed on a defined
binding surface on the second substrate, and wherein the defined
binding surface on the first substrate and the defined binding
surface on the second substrate are formed of the same
materials.
16. The reagent of claim 14, wherein the first binding species is
attached to a defined binding surface on the first substrate, and
the binding regions on the second substrate are formed on a defined
binding surface on the second substrate, and wherein the defined
binding surface on the first substrate and the defined binding
surface on the second substrate are formed of different
materials.
17. A method of reducing the signal to noise ratio amplifying a
signal in an assay comprising: providing a fluid medium containing
a first substrate having at least one first binding species; and
providing a second substrate having regions characterized by their
ability to bind said first binding species if dissociated from said
first substrate and also to bind said first substrate having said
first binding species attached.
18. The method of claim 17, wherein said first substrate comprises
a plurality of particles.
19. The method of claim 17, wherein said regions bind said first
binding species from a plurality of particles.
20. The method of claim 17, wherein said second substrate is
selected from the group consisting of latex particles, proteins
such as bovine serum albumin (BSA), human serum albumin (HSA),
antibodies, or polymers such as poly-BSA, poly-antibodies,
dextrans, and dendrimers.
21. The method of claim 20, wherein said regions characterized by
their ability to selectively bind said first binding species if
dissociated from said first substrate are adapted to exclude said
first species associated with said first substrate.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to methods and compositions
for improving the signal to noise ratio in assays and stabilizing
liquid reagents through the entrapment of free binding species in a
reagent solution or suspension. In particular, the present
invention provides a method for increasing signal strength by
forming signal generating networks as well as a method for
immobilizing free binding species in a reagent solution through the
use of a binding substrate having selectivity for binding the
species of interest.
BACKGROUND OF THE INVENTION
[0002] In modern in-vitro clinical diagnostics, a variety of
methods are utilized for the detection of analytes in a sample. In
one form of a diagnostic method, an immunoassay, one or more
specifically binding species are used. Typical examples are the
sandwich immunoassay, where two specifically binding species
(antibody or antigen) bind to the analyte of interest, and the
competitive immunoassay, where the analyte of interest and an
analog of this analyte compete for binding to a specifically
binding species. One of the specifically binding species is
commonly attached to a so-called label or tag, which may be an atom
(e.g., radioactive), molecule (e.g., an enzyme, fluorescent, or
luminescent compound) or particle (magnetic or latex). This label
allows for detection of the analyte of interest through a variety
of detection methods corresponding to the label utilized. In a
competitive assay, either the specifically binding species or the
analyte analog can carry the label.
[0003] The other specifically binding species is frequently
associated to a solid or suspendable substrate ("the solid phase")
covalently or through adsorption. Alternatively, it may be linked
to a first member of a second binding pair (e.g., biotin), while
the second member of the second binding pair (e.g., streptavidin)
is attached to the solid phase. This allows the specifically
binding species to bind to the solid phase via the second binding
pair interaction (e.g., biotin-streptavidin).
[0004] Solid phases may be macroscopic solid phases, such as
microtiter well, tube and ball in tube devices, or suspendable
solid phases, such as beads, latex beads, magnetic latex beads, as
well as other paramagnetic materials. A secondary binding species
is commonly labeled through the use of a tag. The interaction
between the tag and the solid phase allows for detection and
quantification of the analyte of interest through a variety of
detection methods corresponding to the label utilized.
[0005] It is important for the stability and reproducibility of the
diagnostic assay that the binding ability of the solid-phase bound
species for its binding partner does not become impaired over time.
This may result in decreased reliability and decreased assay
sensitivity. One mechanism that may lead to such impairment
(apparent as instability) is bleeding of a portion of the
solid-phase-attached species into the surrounding medium. The free
species competes with the solid-phase-attached species for binding
to the target and usually has a significant kinetic advantage due
to its faster diffusion. Therefore, it is advantageous to maintain
the amount of free species competing with the solid phase-attached
species in a reagent constant, preferably very close to zero. This
would allow for the sensitive detection of analytes in a stable and
reproducible manner. The preferred way of eliminating free species
is to find a binding method that will eliminate dissociation.
Covalent bonding, as opposed to adsorptive association, might be
the method of choice, due to its greater bond strength. However, in
many cases this may be impossible or impractical, for various
reasons.
[0006] For example, the material chosen for the solid-phase may
lack suitable active groups for covalent coupling. If that is the
case, due to the type of assay, or for cost considerations,
adsorption will then be the preferred choice of association. An
alternative method involves having a binding species that is
attached to the solid-phase material indirectly through an
auxiliary binding species, An example of this would be a
biotinylated antigen or antibody that is attached to an avidin
coated substrate, avidin being the auxiliary species. The auxiliary
binding species itself can be associated with the solid phase via a
covalent bond or by adsorption. This method may be utilized when
the binding species becomes inactivated if directly associated with
the solid-phase material. Again, since the affinity constant of the
auxiliary binding species is not infinite, some dissociation, in
the form of bleeding, may occur.
[0007] A further method includes binding species consisting of
subunits which are either non-covalently associated with each other
or have a covalent bond that is reversible under normal storage
conditions. Subunits that are in this way reversibly and indirectly
attached to the solid phase can disassociate from the
surface-coupled subunit(s). If the free subunit retains its binding
ability in the assay, or regains it after combining with other free
subunits, the free subunit may interfere in the assay.
[0008] If bleeding cannot be eliminated, several practices, each
with its particular disadvantages, have been employed to compensate
for bleeding, including the following:
[0009] 1) Frequent recalibration to correct for the drift caused by
bleeding of the binding species. The most obvious disadvantage is
additional cost for reagent and time spent on recalibration. The
bleeding may also limit shelf life due to assay signals dropping to
insufficient levels.
[0010] 2) Supplying the immunoassays and other diagnostic assay
reagents in a dried form, such as lyophilized or air-dried product.
This approach usually eliminates bleeding, but dry reagents
typically need to be reconstituted by addition of liquid, which
requires time and effort by a user. The drying process itself
requires additional processing by the manufacturer and introduces a
source of inhomogeneity and bottle-to-bottle variation not found in
reagents in a liquid form. The drying process may also increase
lot-to-lot variation.
[0011] 3) If the solid phase is stationary (e.g. microtiter plate)
or can be conveniently separated from the liquid (magnetic beads or
other, larger or smaller sized beads), it is possible to "wash" the
reagent with water or an appropriate liquid before beginning the
assay. A disadvantage of this approach involves the additional
time, effort, and materials required for a "pre-wash" step and the
limits imposed on flexibility in designing the assay sequence.
[0012] U.S. Pat. No. 5,212,063 discloses a ligand trap useful for
reducing or eliminating biotin interference in immunoassays that
employ biotin labeled antibodies (or other biotinylated species).
Biotin, which naturally occurs in body fluids, can compete with the
biotinylated species for binding sites on the avidin or
streptavidin conjugate employed in these assays and cause erroneous
results. Biotin is selectively removed from the immunological
reaction by incubating the sample solution with polymer particles
consisting of a streptavidin-modified core and a biopolymer
coating.
[0013] U.S. Pat. No. 5,863,740 discloses an
interference-eliminating agent through the modification and
inactivation of Streptavidin molecules by biotin saturation,
chemical modification or genetic engineering.
[0014] U.S. Pat. No. 4,256,834 discloses a fluorescent scavenger
particle immunoassay. A reagent employed in the reaction contains a
signal repressor. This signal repressor interacts with a label
connected to a member of a signal producing system in bulk
solution. Upon coupling of the label and member of the signal
producing system to the homologous member of the signal generating
system, the signal repressor no longer interferes.
[0015] If the attachment of the binding species to its solid phase
is not irreversible, and the potential remedies discussed above are
not an option, a certain amount of the binding species will be
released into the fluid medium. This may lead to impairment of the
assay, typically evidenced as instability.
[0016] The situation is further exacerbated because production of
reagents typically occurs a significant amount of time prior to
their actual use. This creates the necessity of storing the assay
reagents under a variety of conditions. During storage and
transport, there may be irregularities in temperature, which may
detrimentally affect the release of solid-phase bound species.
These variables of time, temperature and motion all contribute to
the dissociation of the binding species from the solid-phase
material.
[0017] Although dissociation will primarily take place prior to
utilization of the assay raegents, the impairment will result when
a sample, such as a bodily fluid, is added. The dissociated species
will then compete with the solid-phase attached species for binding
to the target and usually has a significant kinetic advantage due
to its faster diffusion rate. When the amount of target is limited,
even a small amount of free material can significantly reduce the
number of targets bound to the solid phase. This, in turn, can
yield false results that, even when detected, will require reruns
and recalibration.
[0018] It is therefore highly desirable to keep the amount of free
species in reagents constant, preferably very close to zero. When
permanent association of the binding species to the solid-phase is
physically impossible or economically prohibitive, a remedy must be
provided to avoid instability that can result in incorrect test
results.
SUMMARY OF THE INVENTION
[0019] The present invention offers a solution by providing methods
and compositions for improving the reliability and sensitivity of
assays. In one embodiment, this solution is achieved by stabilizing
assay reagents by immobilizing a dissociated species from a fluid
medium solution during and after the species has detached from a
solid-phase, thereby allowing the concentration of free species to
remain close to zero. This is accomplished by using a substrate,
such as a scavenging solid-phase, having an attached binding
partner or partners ("scavenger") for the specifically binding
species which is present in the reagent during storage. This
substrate binds any free species bleeding from the solid phase and
prevents such species from interfering with the assay.
[0020] In another embodiment, a solution is achieved by amplifying
the immunoassay reaction signals generated in an assay.
Amplification is produced by using a substrate including multiple
binding sites for binding pair members attached to a solid-phase.
Binding pair members bound to a solid-phase may attach to common
particles of substrate thereby cross-linking signal generating
components associated with the binding pair members of the solid
phase. This aggregation of solid phase binding pair members forms a
signal-generating network, greatly amplifying signal strength. In
order to maximize reliability and sensitivity of assays, it is
preferable to provide a single substrate that immobilizes a
dissociated species and also aggregates solid phase binding pair
members to produce a signal-generating network. This network can
also be created by introducing proteins or polymers with multiple
binding sites for the binding pair members, whereas the proteins or
polymers are conjugated with multiple binding sites for the binding
pair members. Bovine serum albumin (BSA), human serum albumin
(HSA), antibodies, or polymerized BSA, polymerized antibodies,
dextrans, and dendrimers can function as such carriers for multiple
binding sites.
[0021] The present invention therefore encompasses a reagent
including a fluid medium containing a first substrate having an
attached first binding species. Further, a second substrate is
included having regions for selectively binding the first binding
species if dissociated from the first substrate, and may also
include multiple binding sites for binding pair members attached to
the first substrate.
DETAILED DESCRIPTION OF THE INVENTION
[0022] It is essential that the scavenging substrate of the present
invention fulfill a number of criteria, including but not limited
to:
[0023] 1) If the material chosen is porous, the pores must be large
enough to allow the free species to diffuse into the pore system,
where the scavenger substrate captures it.
[0024] 2) If the solid-phase is a suspendable material, the
scavenger substrate must allow for retention of the free species,
while not attaching to the suspendable solid-phase material in a
way that detrimentally affects signal strength or binding of the
binding target to the solid-phase bound species.
[0025] 3) The number and affinity of scavengers in the scavenger
substrate must be sufficient to capture and retain free binding
species that may bleed off the solid-phase during shelf life of the
product.
[0026] 4) The scavenger substrate must not interfere with a
pipetting procedure. Therefore, it is either connected to the
reagent storage vessel, separated out before pipetting (including
settling out by sedimentation) or of a size that is small enough to
pass through the pipetting device.
[0027] 5) If the scavenger substrate is pipetted with the reagent
to be stabilizes, the scavenger substrate must not interfere with
the reactions that are part of the assay; i.e., apart from attached
scavengers, the scavenger substrate should be largely inert to all
relevant constituents of the assay.
[0028] 6) Further, if the scavenger substrate is pipetted with the
reagent it stabilizes, the scavenger substrate must not interfere
with the detection system. In cases where the scavenger substrate
is capable of interfering with the detection, the substrate must be
removable before the detection by common methods such as
sedimentation, filtration, or magnetic separation.
[0029] 7) The scavenger substrate must be sufficiently stable so
that any amount of scavenger bleeding from the scavenger substrate
will be too small to interfere with the assay.
[0030] In one embodiment of the present invention, a method and
composition is provided which can improve the stability of liquid
reagents used in immunoassays or other in-vitro diagnostic methods.
It provides means to remove or neutralize binding species that
dissociate by bleeding or leaching from a surface or other
solid-phase used in the device, which otherwise may interfere with
the assay. In an exemplary instance, the present invention was
applied to Streptavidin (SA) modified latex reagents, as used in a
luminescent oxygen channeling immunoassay (LOCI.TM.). Dyed and
hydrophilically coated latex beads, covalently modified by coupling
(SA) to their surface, are a useful generic reagent in LOCI.TM.
immunoassays.
[0031] It has been unexpectedly discovered that many reagent
compositions slowly release SA upon storage in suspension even
though it was not possible to detect physically adsorbed material
or the breakage of covalent bonds linking the SA to the beads as
well as to other materials, such as SA on chrome particles with a
hydrophilic coating or on latex with a magnetic core. Possibly the
subunit structure of SA allows for the dissociation of
non-covalently bound SA subunits from the surface and
re-association to intact SA in the solution. Since it appears that
bleeding from the surface is due to an inherent property of SA,
stabilization of the beads would be expected to be difficult.
[0032] In one example, a small amount of a porous substrate,
covalently modified with biotin, was added. A polymeric material
was chosen, such as those used for affinity chromatography
(Toyopearl AF-Amino-650M, Lot 65NHM52W), with amino functional
groups (90 .mu.mol/mL), a pore size of ca. 100 nm (1000 .ANG.), and
a particle size of ca. 65 .mu.m and biotin (sulfo-NHS-LC-biotin)
was covalently coupled to the surface. The molar ratio of amino
groups to biotin derivate was ca. 2 and the coupling medium was
0.15 M sodium bicarbonate. The material was sufficiently large to
have a very small outer surface (<0.1 m.sup.2/ml), which
minimizes potential adsorption of SA beads. The pores are large
enough (ca. 100 nm) to allow access for the SA molecule (ca. 5 nm),
even after the pore structure becomes more narrow due to biotin
modification, blocking protein and partial saturation with SA. At
the same time, the pores are sufficiently small for the material to
have a large internal surface area (>50 m.sup.2/g) and
corresponding high binding capacity, thereby minimizing the amount
of scavenger beads required. The pores are also sufficiently small
to exclude SA beads which are typically 200 nm or larger.
[0033] The parameters chosen for the scavenger substrate represent
a potential optimum in terms of economy (if one wants to use as
little scavenger as possible to stabilize the immunoassay reagent).
However, a scavenger substrate with different particle sizes (from
<100 nm to about 5 mm), different average pore size (e.g.,
between 50 and 5000 .ANG.), different functional group type and
density, would also work under appropriate circumstances.
[0034] In one example, biotin coupled scavenger substrate, such as
Toyopearl beads, were added to an SA bead reagent (with a
concentration of 1400 ug beads/ml) at a concentration of
approximately 0.350 .mu.l of settled bead volume per ml of reagent.
The beads were initially mixed by vortex and then stored in wells
within Flex.TM. reagent container, available from Dade Behring
Inc., Deerfield, Ill., without further agitation. The scavenger
beads settled to the bottom of wells and acted from there. The SA
bead reagent was prepared using a Toyopearl Bead (AF-Amino-650M,
Lot 65NHM52W) having 65 um bead size, 300 .ANG. pore size, 90
umol/mL of amino-group on the surface (a product of Tosoh Haas,
available from Sigma) and biotin-LC-NHS (Sulfo) from Pierce.
[0035] The following procedure was followed:
[0036] 1. Add 1 mL of Toyopearl bead suspension (ca. 0.7 ml settled
beads) into a small end-capped column (yellow column with caps for
wide and narrow end)
[0037] 2. Wash the beads with 15-20 mL of NaHCO3 (0.15 M).
[0038] 3. Add 1 mL of 0.15 M NaHCO3 solution and vortex to
re-suspend the beads.
[0039] 4. Dissolve 20 mg of Biotin-LC-NHS (sulfo) into 1 mL of
DI-water.
[0040] 5. Add (4) into (3) with vortex.
[0041] 6. Rotate the end-capped column at room temperature over
night (about 24 hr).
[0042] 7. Add 50 mg of BSA to the solution and rotate the column at
room temperature over night.
[0043] 8. Wash the beads with 30 mL of 50 mM HEPES buffer, pH
7.5.
[0044] 9. Add 2 mL of HEPES buffer to re-suspend the beads.
[0045] The results of the stability study (TABLES 1 and 2) show
that the release of SA at 4.degree. C. leads to a decrease in
recovery in a TSH assay. When the SA beads are centrifuged
("washed") after the 35 days to remove the free SA, the original
quantitation is obtained. The SA is therefore the cause of the
apparent instability. In the presence of scavenger beads, there is
a slight initial increase due to the scavenger beads binding a
small amount of SA that is free at the beginning of the study.
After that initial period, the recovery remains constant as the
scavenger beads maintain the amount of free SA close to zero.
[0046] In another exemplary embodiment, the scavenger substrate
comprised a non-porous material, for example a surface, modified in
a way that SA has access, but modified beads do not. A material,
having indentations, such as crevices and other unpatterned design
may also be used. Alternatively, or in conjunction with the other
textures, grooves may be used to function in a manner similar to
the porous substrate. Further, the substrate may be of a brush-like
configuration, whereby the dissociated free species may migrate
past the brush-like appendages and bind to the interior of the
substrate. The solid-phase material, due to size difference would
not enter into the binding area of the brush-like substrate.
[0047] A further embodiment may consist of a non-porous substrate
having the ability to bind the free, dissociated species due to its
weight, diffusion rate and other characteristics of the dissociated
molecule, not apparent in the solid-phase bound species.
[0048] In an even further embodiment, a substrate may have multiple
binding sites for the binding pair members attached to the
solid-phase. This substrate may preferably, although not
necessarily, possess the free-species scavenging properties
discussed above. The multiple binding sites allow binding pair
members from a plurality of solid-phase particles to attach to
common substrate particles. In this manner, the substrate allows
for cross-linking of the binding members on the solid phase. The
cross-linking creates a signal generating network that improves the
sensitivity of assays and the signal to noise ratio by forming
particle aggregates. These particle aggregates further lead to an
amplification of the assay signal through the signal generating
networks.
[0049] As previously discussed, SA bleeds from the SA-modified
reagents during storage leading to decreased assay sensitivity and
reliability. In order to solve this problem, biotin was coupled to
dyed sensitizer beads. In one example, the biotinylated sensitizer
beads were prepared by the following method: uncoated dyed
sensitizer bead lot 1053:89 was activated with EDAC followed by NHS
then reacted with biotin LC-PEO amine (+) trioxaundecanediamine
(Pierce 21347 lot DH57849) at RT for 72 hours; final bead weight is
20 mg/mL; final buffer is 100 mM TRIS, 300 mM NaCl, 1 mM EDTA, 1
mg/mL BSA, 0.1% Triton.times.405, 0.01% Proclin 300 pH 8.0.
[0050] The biotin-modified beads were added to the SA reagent at
different concentrations. LOCI.TM. TSH and FT3 assays were chosen
as model assays. The SA bead concentrations in the TSH and FT3
assays were 1400 .mu.g/mL and 400 .mu.g/mL, respectively. The
biotin-modified sensitizer beads were added in concentrations of 1%
and 5% (TSH: 14 .mu.g/mL, 70 .mu.g/mL; FT3: 4 .mu.g/mL, 20
.mu.g/mL) of the SA bead concentration. The mixtures were incubated
overnight at 4.degree. C. The TSH and FT3 calibrators were measured
and compared using the different sensitizer bead suspensions in the
respective immunoassay.
[0051] The results (TABLES 5 and 6) showed that, in the presence of
the biotin-modified sensitizer beads, there was a substantial
increase in the immunoassay response signal while background noise
for TSH remained essentially unchanged. This indicates the
formation of signal generating networks in addition to the
successful scavenging of free species. The amplified signal
strength was primarily the result of the signal generating
networks. Good reproducibility of signals over the course of a
stability study was primarily the result of the scavenging
properties of the biotin-modified sensitizer beads.
[0052] A second substrate with scavenging and/or cross-linking
properties, as described above, provides an almost universal
solution for bleeding problems of compounds that represent one part
of a specific binding pair. It provides a solution to assays where
a fluid medium is utilized in conjunction with a first substrate
having a first binding species that separates into a first portion
and a second portion, the dissociated second portion being the free
species. A second substrate is used, having regions adapted to
selectively bind the second portion of the first binding species
(free species) from the solution without interfering with the
assay. Preferably, this second substrate cross-links the first
substrate into aggregates to increase signal strength. The addition
of the second substrate puts very few limitations on the nature of
the "solid phase" and does not require any separation or washing
steps or lyophilization. There is no need for short calibration
periods to correct for the effect of free species and in many
cases, the shelf life of the device may be significantly
extended.
[0053] As previously described, TABLE 1 and TABLE 2 further
describe the results of a 35 day TSH Assay Stability study. As can
be seen from TABLE 1, the assay stability was measured in the
absence of a scavenger substrate. The quantitation was performed on
the same day using a curve generated with -70.degree. C. stored
material. As can be seen between Calibration Levels 2 through 5,
the analyte values reported by the assay degrade significantly over
time.
1TABLE 1 TSH Assay Stability (4.degree. C. Storage) in the absence
of porous scavenger Quantitation performed on the same day using
curve generated with -70.degree. C. stored material. Day 35
Calibrator Day 0 Day 7 Day 14 Day 21 Day 35 (Washed) Level 1 -0.01
-0.01 -0.01 -0.01 0.00 0.01 Level 2 0.87 0.80 0.71 0.69 0.65 0.89
Level 3 3.98 3.63 3.31 3.15 2.91 4.01 Level 4 19.39 18.01 16.84
15.31 14.52 19.66 Level 5 54.68 50.01 47.03 44.59 37.25 55.39
[0054]
2TABLE 2 TSH Assay Stability (4.degree. C. Storage) in the presence
of porous scavenger Quantitation performed for each day using same
calibration curve as TABLE 1 Calibrator Day 0 Day 7 Day 14 Day 21
Day 35 Level 1 0.00 -0.02 -0.02 -0.02 -0.02 Level 2 0.98 0.99 0.98
0.99 1.01 Level 3 4.34 4.51 4.53 4.40 4.62 Level 4 20.77 22.11
22.08 21.62 22.22 Level 5 58.62 61.49 62.56 58.90 62.39
[0055] As further illustrated by TABLE 3, on average the
quantitation decay between Calibrator Levels 2 through 5, as
measured from Day 0, were as follows:
3TABLE 3 TSH Assay Stability Decay for Calibrator Levels 2-5 In
Absence of Scavenger Calibrator Day 7 Day 14 Day 21 Day 35 Level 2
.920 .816 .793 .747 Level 3 .912 .832 .791 .737 Level 4 .929 .868
.790 .749 Level 5 .915 .860 .815 .681 Average .919 .844 .797
.729
[0056] In contrast the levels of decay in the presence of a
scavenger remained at a constant near zero, as illustrated by TABLE
4.
4TABLE 4 TSH Assay Stability Decay for Calibrator Levels 2-5 In
Presence of porous Scavenger Calibrator Day 7 Day 14 Day 21 Day 35
Level 2 1.010 1.000 1.010 1.031 Level 3 1.039 1.044 1.014 1.065
Level 4 1.065 1.063 1.041 1.070 Level 5 1.049 1.067 1.005 1.064
Average 1.041 1.044 1.018 1.058
[0057] TABLE 5 and TABLE 6 illustrate the effects of elimination of
free species in addition to the creation of signal generating
networks. As previously discussed the data show a substantial
increase in the immunoassay response signal with a stabilized
background noise in the presence of biotin-modified sensitizer
beads.
5TABLE 5 LOCI TSH Immunoassay Calibration Curve SAV Sensitizer bead
suspension at 1400 .mu.g/mL with or without biotin-modified
sensitizer Additive 0% 1% 5% Calibrator Immunoassay signal response
in photon counts Level 1 3534 3658 3433 Level 2 50252 83150 93790
Level 3 208500 338382 376341 Level 4 996960 1554725 1738055 Level 5
2556284 3818949 4082537
[0058]
6TABLE 6 LOCI FT3 Immunoassay Calibration Curve SAV Sensitizer bead
suspension at 400 .mu.g/mL with or without biotin-modified
sensitizer Additive 0% 1% 5% Calibrator Immunoassay signal response
in photon counts Level 1 1772532 2157288 2405371 Level 2 1062516
1289000 1457286 Level 3 615536 761405 874360 Level 4 196229 245013
274417 Level 5 8520 10008 10852
[0059] Based on these results, it is clear that the use of a second
substrate as described greatly enhances the stability of assays
over an extended period and enhances signal strength. The present
invention, thereby allows for the accurate testing for analytes of
interest even if the assay utilized contains a solid-phase
substrate, which leaches a free species during normal storage or
when exposed to unusual temperatures or motions.
[0060] Although the present invention has been described in detail
with particular reference to preferred embodiments thereof, it
should be understood that the invention is capable of other
different embodiments, and its details are capable of modifications
in various obvious respects. As is readily apparent to those
skilled in the art, variations and modifications can be affected
while remaining within the spirit and scope of the invention.
Accordingly, the foregoing disclosure, description, and tables are
for illustrative purposes only, and do not in any way limit the
invention, which is defined only by the claims.
* * * * *