Note: Descriptions are shown in the official language in which they were submitted.
2ii5~S~
``' ~ 93/10458 PCl/US92/09281
BINDING OF MILK ALLERGENS TO A SOLID PHASE
This is a continuation-in-part application of co-pending
U.S. Patent Application Serial No. 07/509,255 tiled April 13,
5 1990, entitled ~BINDING OF ALLERGENS TO A SOLID PHASE~,
which enjoys common ownership and is incorporated herein by
reference. This application is also a related application to
U.S. Patent Application Serial No. 07/227,272 filed August 2,
1988, entitled ~TEST CARD FOR PERFORMING ASSAYS~, which
1 0 also enjoys common ownership and is incorporated herein by
reterence.
BACKGROUND OF THE INVENTION
1 5 The pr~sent inv~ntion relates to the binding ot antigens ~ -
related to milk allergens to a solid phase material for use in a ~-
diagnostic assay for immunoglobulins that bind to said
allergens. In particular, the invention improves the :
sensitivity ot a diagnostic assay for immunoglobulins,
suspected of being present in a blood sample, that bind to
proteins found in milk.
U.S. Patent No. 3,720,760 discloses that certain
immunogenic substances, called allergens, can give rise to -
allergic reactions in the form Gf asthma, hay fever, and the
like, and that the blood of a patient in whom a given allergen -
causes an allergic reaction usually contains low ;
concentrations of immunoglobulins, called reagin-
immunoglobulins (now usually called ~IgE~, which term is -~
subsequently used herein~, which are directed specifically
30 against that allergen. The patent discloses a test for
sensitivity to allergens which involves injecting given
allergens into the skin of a patient; a skilled observer then
assesses the degree of sensitivity to each of the allergens on
the basis of the observed reaction (reddening or swelling of
3 5 the skin) caused by each allergen. The patent also discloses
an ~in vivo~ test, where the patient inhales an allergen in the
form of an aerosol, and ihe patient is deemed to be sensitive
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2i1 ~-~S~ 2
to any allergen that causes hay fever, asthma or like
symptoms.
The patent further discloses an in vitro method for
determining the presence of IgE antibodies in a body fluid. The
method involves binding an allergen to fine particles of a
copolymer, e.g., a dextran-epichlorohydrin copolymer, by -~
treating the particles with cyanogen bromide, and suspending
the particles and an allergen in an aqueous medium. A body
tluid to be tested for the presence of IgE antibodies directed
against that allergen is then contacted with the allergen
bound to the copolymer. Tho product of step (2) is then
brought into contact with radio-labeled antibodies which will
bind to IgE antibodies, if any, that has become bound to the
- allergen that is bound to the copolymer. the radiation smitted
from the solids of step (3), the liquid of step ~3), or both can
then be measured. ~:
The covalent coupling of antigens, including allergens, to
a solid phase was used to prevent or inhibit their removal
from the solid phase during the assay procedure. U.S. Patent
No. 4,597,999 describes the covalent coupling of two -
molecular species to one another, using cross-linking agents
having at least two functional groups which are subject to
independent activation. Examples of such cross-linking agents ~
include 4-methylazidobenzidimate and N- -
2 5 hydroxysuccinimidylazidobenzoate. These cross-linking -
agents couple spontaneously in the dark to available amino
groups, as in aminopropyl glass, aminophenyl glass and :
aminohexylagarose, and when activated by irradiation with
light of a suitable wavelength, these agents also couple with a
ligand such as a drug, digoxin, a steroid, or a protein.
U.S. Patent No. 4,425,434 describes the use of
-biologically active substances to fill the pores of porous
titania spheroids, porous calcium phosphate spheroids, porous
zirconia spheroids, or similar porous support material, and
that the biologically active substance can then be immobilized
in the pores by precipitation and corss-linking. The
`"O 93/10458 2 1 i 3 3 3
biologically active substance can be a proteinaceous
substance, such as an enzyme.
It has been found, however, that the covalent coupling
procedures are costly to perform and time consuming. In
5 addition, some coupling procedures can decrease the
sensitivity of the assay.
Milk comprises a variety of proteins including as-casein,
c-casein, ,B-casein, ~-casein, ,B-lactoglobulin, bovine serum
albumin~ a-lactalbumin, immunoglobulins, such as bovine
l 0 gamma globulins, and proteose peptones. Milk allergy is
defined as those reactions induced by the ingestion of milk or
its components for which an immunological pathogenesis may
be demonstrated.
- Pharmacia Diagnostics (Piscataway, New Jersey)
15 markets in vitro radioimmunoassay and enzyme immunoassay
allergy products (Phadebas RAST(E~ and Phadezym~ RAS~) that
d~tect and quantitate IgE antibodies for a variety of allergens ~-
including milk and milk proteins. The Pharmacia tests are
performed individually utilizing a disk having the allergen of
20 interest bound thereto. The disk is incubated with the patient
sample, washed, incubated with labeled goat anti-human-lgE
IgG and washed and then the amount of label on the disk is
measured. Pharmacia markets tests for milk, oc-lactalbumin,
,B-lactoglobulin, and casein.
BRIEF DESCRIPTION OF THE PRESENr INVENTION
,
The present invention involves novel allergen
compositions, using a solvent such as deionized or distilled
30 water, containing (in milligrams of protein per milliliter, as
determined by a suitable protein test): from about 0.05 to
about 4.0 of AJternaria altemata allergen; from about 0.5 to
about 50 Aspergilus ~migatus allergen; from about 0.8 to
about 81.6 of Bermuda grass (Cynodon dactylon) allergen; from
35 about 0.1 to about 6~0 of birch (13etula nigra) allergen; from
about 0.6 to about 20.6 of cat (Felis domesticus) allergen;
from about 0.04 to about 4.5 of mountain-cedar (Juniperus
WO 93/10458 PCI /US92/09281.
211~S~ 4
ashel~ allergen; from about 0.1 to about 20.5 of Japanese cedar
(Cryptomeria japonica) allergen; from about 0.05 to about 10.0 ;
of Cladosporium allergen; from about 1.3 to about 38.4 of dog
(Canis familiarus) allergen; from about 0.7 to about 22.4 of
S Dermatophagoides farinae (D. farinae) allergen; from about 0.6
to about 84.2 of D. pt~ronyssinus allergen; from about 0.1 to
about 10.0 of elm (Ulmus) allergen; from about 0.02 to about
2.0 of feather allergen; from about 0.2 to about 20.5 of giant
ragweed (Ambrosia trifida) allergen; from about 0.4 to about
100 of house dust allergen; from about 0.05 to about 10.5 of ~ I
June/Kentucky blueQrass (Poa pratensis) allergen; from about
0.2 to about 20.5 of lamb's quarters (Chenopodium album)
allergen; from about 0.1 to about 11.5 of maple (Acer) :
allergen; from about 0.3 to about 90.4 of mugwort (Artemesia
het~rophylla)allergen; from about 0.1 to about 12 of mulberry -~
(Morus) allergen; trom about 0.2 to about 25.5 of oak (Querclls) ~
albrgen; from about 0.1 to about 66.8 of olive ~O~ea europ~a) ~:
allergen; from about 1.0 to about 40.0 of Parietaria
(Parietaria officinalis) allergen; from about 1.7 to about 130.4
of plantain (Plantago lanceolata) allergen; trom about 0.1 to
about 4.8 of P~nicillium (PenicilDum notatum) allergen; from
about 0.05 to about 8.5 of perennial rye (Lolium perenne)
allergen; from about 0.2 to about 20.5 ot short ragweed
(Ambrosia elatior) allergen; trom about 0.05 to about 6.6 of
timothy ~Phleum pratense) allergen; and from about 0.5 to
about 50 mg/ml of milk protein extract supplemented with
other proteins as disclosed in the present invention. Such
allergen concentrations have been found optimal for the
preparation of immunoassay devices for the detection ot anti-
IgE antibodies specific for the allergens.
The presént invention also involves devices for
detecting the presence or amount of IgE antibodies in a test
sample. the assay devices include a solid phase and an
allergen immobilized upon the solid phase, wherein the
allergen is typically applied as one of the above allergen
compositions. In certain assay devices, the allergen
composition is combined with a pretreatment substance such
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as a denaturant, organic solvent, cross-linking agent or
concentrated salt solution. It has been unexpectedly found
that such allergen pretreatment can enhance allergen
immobilization upon the solid phase. The reaction or binding
5 area of the solid phase can be optionally modified by the
addition of a protein blocking reagent. Suitable blocking -reagents include equine serum albumin, bovine serum albumin,
fish gelatin, casein and the like.
In addition, the present invention describes allergen
10 compositions containing a solvent, an allergen solubilized in
the solvent, thereby torming an allergen solution, and a
pretreatment substance chosen from denaturants, organic 'solvents, cross-linking agents or concentrated salt solutions,
wherein the allergen solution is combined with the
pretreatment substance, and wherein the resultant -composition is used for the in vitro detection of the presence
or amount of IgE antibodies in a test sample. In particular,
the present inventîon describes a composition of milk proteins
which enhance the sensitivity of the in vitro detection of the
2V presence or amount of anti-milk-protein IgE antibodies. In
vitro detection methods can involve: providing a solid phase
prepared by applying the novel aîlergen compositions or -
pretreated allergen compositions to the solid phase;
contacting the sample to be tested to that solid phase, thereby
2 5 immobilizing allergen-specific IgE antibody from the sample
upon the solid phase by forming allergen/antibody complexes;
and detecting that immobilized allergen-specific antibody to
determine the presence or amount of the antibody in the test
sample. Generally, the solid phase is contacted with an
30 indicator reagent to determine the presence or mount of lgE~
antibodies in the test sample, wherein the indicator reagent
includes a label conjugated to a binding member that is
specific for either the allergen, IgE antibodies or an ancillary
specific binding member. The label that is selected is not
35 critical to the present invention and is typically chosen from
chromogens, catalysts, fluorescent compounds,
chemiluminescent compounds, radioactive isotopes, colloidal
WO 93/10458 PCI`/US92/0928!
5 5 ~i
metallic particles, colloidal selenium particles, dye particles,
enzymes, substrates, organic polymer latex particles and ~
liposomes or other vesicles containing signal producing ~-
components. The present invention also includes assay kits
5 containing the allergen or allergens of interest immobilized
upon the solid phase and a suitable indicator reagent.
Optionally, the kit can include assay buffers and wash
reagents.
1 0 BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is an enlarged top plan view of a preferred
embodiment of a reaction cartridge support having a well
containing a test card which has discrete test sites with
1 5 moats.
FIG. 2 is a magnified view, partially cutaway, through
lines 8-8 (FIG. 1) showing sample test sites and moats in a
preferred laminat~ structure of the test card.
FIG. 3 is a plot of anti-milk-protein IgE antibody ~-
20 containing plasma sample pools analyzed on a solid phase
containing only milk protein extract and on a solid phase
containing the milk protein composition of milk protein
extract, casein and ~-lactoglobulin. The relative color
intensities (with background subtracted) of the solid phase
25 test sites (measured by reflectance) are plotted against the
concentration of IgE antibodies (PRU/ml) in the pools.
C)ESCRlPrlON OF THE PREFERRED EMBODIMENrS
The present invention is based upon the discovery that
an allergen solution can be used to bind an allergen to a solid
phase material without the need for covalent linkages. A solid
phase so prepared can then be used in an in vitro diagnostic
35 assay for IgE antibodies. Suitable solid phase materials
include cellulose nitrate or a mixed ester cellulose. In
addition, it has been discovered that certain a!lergen
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-"O 93/10458 ~ Pcr/US92/09281
concentrations are optimum insofar as the sensitivity ot the
assay is concerned. In particular, it has been discovered that
a composition containing certain milk protein concentrations
are optimum insofar as the sensitivity of an assay for anti-
S milk-protein IgE antibodies is concerned.
The invention is also based upon the discovery that many
allergens can be pretreated to improve their adherence to the
solid phase material. The allergen pretreatment methods ot
the present invention serve to enhance the binding o~ the
10 allergen to the solid phase throughout the assay. The allergen
pretreatment compositions and methods were also
unexpectedly found to increase the amount of allergen which
can be bound to the solid phase thereby enabling the binding of
allergen in an amount that is optimal for ths assay.
The present invention involves novel allergen
compositions for the preparation of solid phase devices used
in binding assays. The allergen composi~ions have been
unexpectedly found to enhance the binding of the allergen to
the solid phase material. As a result, greater amounts of
antigen may be immobilized upon the solid phase, thereby
providing mor~ antigenic sites for binding antibody during the
assay.
The present invention also involves the pretreatment of
certain allergen compositions with substances such as
2 5 denaturants, organic solvents, cross-linking agents, and
concentrated salt solutions. Pretreatment ot an allergen
composition with one or more of these substances was
unexpectedly found to enhance the adherence of the allergen to
a solid phase throughout the assay procedure which may
include multiple washing steps or other manipulations which
could otherwise dislodge the allergen form the solid phase. In
addition, the pretreatment of the allergen improves their
binding performance at elevated temperatures often used in
binding assays.
3s Suitable denaturants include, but are not limited to:
acids such as hydrochloric acid (HCI) and acetic acid. Organic
solvents, such as tetrahydrofuran, are suitable for allergen
WO 93/10458 Pcl/us92/og28
21~1^i;.ji~ 8
pretreatment. Concentrated salt solutions, such as
concentrated solutions of sodium chloride (NaCI), are also
suitable for allergen pretreatment according to the present
invention. Suitable cross-linking agents for the pretreatment
5 of allergens include, but are not limited to: formaldehyde,
glutaraldehyde and 1-ethyl-3-(3-
dimethylaminopropyl)carbodiimide (EDAC).
Allergen compositions combined with such pretreatment
substances are then used in the production ot novel solid
10 phase assay devices. The all~rgen compositions or pretreated
allergen compositions are applied to a solid phase material
upon which the allergen composition is dried and thereby
immobilized. The solid phase devices can then be used in
binding assays which include, but are not limited to,
1 S competitive assays, sandwich assays and indirect assays, and
include both forward and reverse assay tormats.
In a preferred embodiment of the present invention, the
allergen of interest is immobilized upon a solid phase
material made of nitrocellulose or a nitrocellulose derivative
20 or compound, such as cellulose acetate/nitrate mixed ester
cellulose. The maximum binding capacity of nitrocellulose for
the protein bovine serum albumin is about 140 ~g/cm2. This
binding capacity value is converted according to the desired
size of the solid phase reaction or binding area of the present
25 invention, and a value of 2.2 mg/ml is obtained. This
concentration is used as the starting protein concentration for
all allergens, but the optimum allergen concentration may be
above or below this value. Different concentrations of
allergen solutions are pretreated, immobilized on
30 nitrocellulose and tested with a positive test sample, as
described in the specific examples which follow. The allergen
concentration is adjusted such that when concentration is
plotted against signal a parabolic curve is obtained, and the
optimum allergen concentration can be determined from the
3 5 maximum detected signal.
Tha allergen protein concentrations which were tested
ranged from about 0.05 milligrams of allergen per milliliter
2 1 i ~
--'O 93/10458 PCI/US92/09281
of solvent, prior to pretreatment, to about 170 mg/mL. The
most effective concentration ranges for each of the allergens
tested are presented in the specific examples which follow.
In another preferred embodiment ot the present
5 invention, anti-milk-protein IgE antibodies can be detected by
the methods described herein utilizing a composition
comprising a milk protein extract, casein and ~-lactoglobulin
immobilized upon a suitable solid phase material. A milk
protein extract is an extract of bovine milk comprising readily
10 water soluble proteins. A milk protein extract can be
prepared from Iyophilized milk by re-solution of`lyophilized
fat-free milk powder in water or buffered water and removal
of undissolved material. A preferred bovine milk protein
extract is commercially available from Greer Laboratories
5 (Lenoir, North Carolina) under the name ^Milk, Cow~. Casein
powder is commercially available from Greer Laboratories
(Lenoir, North Carolina). ~-Lactoglobulin is commercially
available from Sigma Chemical Company (St. Louis, Missouri).
A preferred method of immobilizing this milk protein
20 composition is as follows: combining individual solutions,
preferably aqueous solutions such as de-ionized water and the
like, of milk protein extract, casein and ~-lactoglobulin into
one solution comprising a final concentration ot milk protein
extract within the range of about 0.5 to about 2.0 mg/ml, more
25 preferably at about 0.7 mg/ml, of casein within the range of
about 0.5 to about 2.0 mg/ml, more preferably at about 1
mg/ml, and ~-lactoglobulin within the range of about 5 to
about 30 mg/ml, more preferably at about 18 mg/ml;
pretreating the solution preferably by acidification, such as
30 with 6N hydrochlorig acid and the like; and depositing the
dèsired quantity of this composition solution onto the solid
phase. An alternative method involves depositing the
individual solutions, atter separately pretreating each of the
individual solutions, sequentially onto the same location of
3 5 the solid phase.
A turther preferred embodiment of the present invention
~ involves the inclusion of bovine gamma globulin (BGG) in the
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2 1 ~ 3
milk protein composition solution disclosed herein above. BGG
is found in bovine milk and is known to be present in low
concentrations. The addition of BGG to the milk protein
composition solution improves the sensitivity and specificity
S of assays for the detection of anti-milk-protein IgE
antibodies. Preferably, the final concentration of BGG in the
milk protein composition disclosed herein is within the range
ot 0.5 to about 16 mg/ml and more preferably at about 5
mg/ml.
A preferred solid phase is illustrated in Figures 1 and 2.
A preferred support for the solid phase is test card 82, as
illustrated in FIG 1. Test card 82 preferably contains circular
depressions 89 in the binding layer material which create an
array of isolated test sites 84 preferably in close proximity
l S to each other and each composed of binding layer material
encircled by a moat 99 of air space. Test card 82 is
preferably adhered to reaction cartridge 80 using two-sided
adhesive tape on the bottom of reaction well 8B, which is
defined by well wall 88. Reaction well 86 is preferably
provided with a removable, preferably transparent well cover
90 which preferably includès a reagent port 92 to facilitate
the delivery and removal of fluids from the reaction well 86.
The reaction cartridge 80 also preferably includes code means
94, such as an optical bar code, adapted to be read by an
optical reader ~not shown) and which is attached to or printed
directly on the flat surface 91. The reaction cartridge 80 also
preferably includes a panel 96 which may include information
such as the expiration date of the particular raaction
cartridge, the lot number of the particular panel of capture
reagents or assay binding components and the like.
FIG. 2 is a magnified view, partially cutaway, through
lines 8-8 of FIG. 1 showing sample test sites and moats in a
preferred laminate structure of the test card. The test card is
preferably a laminate structure comprising a binding layer 83
adhered to a non-absorbent substrate 85 using an adhesive 87.
The porous structure of nitrocellulose has been found to have
excellent absorption and adsorption qualities for a wide
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21i~5
"l0 93tlO458 11 PCI/US92/092Xl
variety of fluid capture reagents which may be used in
connection with the invention and is therefore preferred for
binding layer 83. Polyester film such as MYLAR plastic having
a thickness of approximately 0.002 inches is suitable for non-
5 absorbent substrate 85. An adhesive backed polyester film iscommercially available from several sources, such as Flexcon
(Spencer, MA). A laminate structure suitable for use in test
cards is commercially available from Millipore (Bedford, MA). ~-
A different analyte or allergen is delivered to each test
10 site 84 so that a single sample can be simultaneously tested
tor the presence of binding components specific to each ot a
panel of different capture reagents. Some test sites 84 may
have analyte delivered thereto to serve as positive control
sites and some may have no reagent de~ivered thereto, to
15 serve as negative control or reference sites. Preferably, from
about 1.1 to about 5 ~lL and more preferably, from about 1.25
to about 4 IlL of analyte or allergen solution is delivered to
each test site 84 using any number of suitable delivery
methods including reagent jetting, metered air pulsing,
20 positive displacement pump, or by capillary tube lowered to
the surface of the test site 84.
After the test sites 84 of a test card 82 are spotted
with analyte or allergen, the test sites are allowed to dry
thoroughly at room temperature. After drying is complete, the
25 binding layer 83 ot test card 82 is preferably ~blocked~ with a
protein coating such as inactivated horse serum or fish
gelatin. Blocking masks potential non-specific binding sites
on the binding layer 83. Suitable blocking is obtained during
an incubation period of about 1 hour at approximately 37C and
30 is preferably accomplished in tanks with agitation during
incubation. Following blocking, the test card 82 is washed,
such as with 10 mM Tris buffered saline, and allowed to dry
overnight.
The invention will be more fully understood from the
35 following examples, which constitute the best modes
presently contemplated by the inventors. It is to be
understood, however, that the examples are presented solely
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2~ 555 12
for the purpose of illustration, and are not to be construed as
limiting.
Before proceeding with the description of the specific
embodiments of the present invention, a number of terms will
be defined. All allergen contents herein refer to the protein
content of the allergen solutions, determined using a suitable
protein test such as Coomasie blue or Ninhydrin as are well-
known in the art.
The term ~analyte~ refers to the substance to be -~
detected in or separated from test sample. The analyte can be
any substance for which there exists a naturally occurring
specific binding member or for which a specific binding
member can be prepared. In addUion, the analyte may bind to -~
more than one specific binding member. ~Analyte~ also `
includes any antigenic subs~ances, haptens, antibodies, and
combinations thereof. In the present invention, the main
analytes to be detected or measured are IgE antibodies.
The term ~test sample~ refsrs to virtually any liquid
sample. The test sample can be derived from any desired
source, such as a physiological fluid, for example, blood, -
saliva, ocular lens fluid, cerebral spinal fluid, sweat, urine,
milk, ascites fluid, mucous, synovial fluid, peritoneal fluid,
amniotic fluid or the like. The liquid test sample can be
pretreated prior to use, such as preparing plasma from blood,
diluting viscous liquids, or the like; methods of treatment can
also involve separation, filtration, distillation, concentration,
inactivatiôn of interfering components, and the addition of
reagents. In addition, a solid can be used once it is modified
to form a liquid medium.
The term ~specific binding member" refers to a member
ot a specific binding pair, i.e, two different molecules
wherein one of the molecules through chemical or physical
means specifically binds to the second molecule. In addition
to antigen and antibody specific binding pairs such as the
- 35 allergen and antibody pair, other specific binding pairs
include, biotin and avidin, carbohydrates and lectins,
complementary nucleotide sequences, complementary peptide
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13
sequences, effector and receptor molecules, enzyme cofactors
and enzymes, enzyme inhibitors and enzymes, a peptide
sequence and an antibody specific for the sequence protein,
polymeric acids and bases, dyes and protein binders, peptides
5 and specific protein binders (e.g., ribonuclease, S-peptide and
ribonuclease S-protein), and the like. Furthermore, speeific
binding pairs ean include members that are analogs of the
original specific binding member, for example an analyte-
analog. If the speeifie binding member is an immunoreactant
10 it ean be, for example, an antibody, antigen, hapten, or
eomplex thereof. It an antibody is used, it ean be a monoelonal
or polyelonal anffbody, a recombinant protein or antibody, a
mixture or mixtures or a fragment or fragments thereof, as
well as a mixture of an antibody and other specific binding
15 members. The details of the preparation of such antibodies
and their suitability for use as specific binding members are
well-known to those skilled-in-the-art.
An ~indieator reagent~, as used herein, refers to a label
attaehed to a speeifie binding member. The indieator reagent
20 produees a detectable signal at a level relative to the amount
of an analyte in th~ test sample. Generally, the indicator
reagent is deteeted or measured after it is eaptured on the
solid phase material, but the unbound indieator reagent can
also be measured to determine the result of an assay. The
25 specific binding member eomponent of the indicator reagent
enables the indireet binding of the label to the analyte, to an
ancillary speeific binding member, to the capture reagent or
to a complex thereof.
The term ~label~ refers to any substance whieh is
30 attached to a specific binding member and which is capable of
producing a signal that is detectable by visual or instrumental
means. Suitable labels for use in the present invention can
include chromogens; catalysts; fluorescent compounds;
chemiluminescent compounds; radioactive isotopes; direct
3 5 visual labels including colloidal metallic and non-metallic
parlieles, dye particles, enzymes or substrates, or organie
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2 ~ 5 ~ ~ 14
polymer latex particles; liposomes or other vesicles
containing signal producing substances; and the like.
Many enzymes suitable for use as labels are disclosed in `
U.S. Patent No. 4,275,149, columns 19-23, herein incorporated
5 by reference. For example, an enzyme/substrate signal
producing system useful in the present invention is the
enzyme alkaline phosphatase wherein the substrate used can
be 5-bromo-4-chloro-3-indolyl phosphate or a derivative or
analog thereof. If horseradish peroxidase is ussd, o-
1 0 Phenylenediamine or 4-chloro-naphthol is added as an enzyme
substrate to torm a colored product which can be detected
and/or measured visually or instrumentally.
In an alternative signal producing system, the label can
be a fluorescent compound where no enzymatic manipulation
1 5 of the label is required to produce a detectable signal.
F~uorescent molecules such as fluorescein, phycobiliprotein,
rhodamine and their derivatives and analogs are suitable for
use as labels in this system.
An especially preferred class of labels includes the
20 visually detectable, colored particles which enable a direct
colored readout of the presence or concentration of the
analyte in the test sample without the need for using
additional signal producing reagents. Materials for use as
such particles include colloidal metals, such as gold, and dye
~: 25 particles as disclosed in U.S. Patent Numbers 4,313,734 and
4,373,932. The preparation and use of non-metallic colloids,
such as colloidal selenium particles, are disclosed in co-
owned and copending U.S. Patent Application Serial No.
072,084, filed July 9, 1987, which is incorporated by
reference herein in its entirety. Organic polymer latex
particles for use as labels are disclosed in co-owned and
copending U.S. Patent Application Serial No. 248,858, tiled
September 23, 1988, which is incorporated by reference
herein in its entirety.
- 35 A variety of different indicator reagents can be formed
by varying either the label or the specific binding member; it
will be appreciated by one skilled-in-the-art that the choice .
3 ~
"' ~ 93/10458 PCI /US92/09281
involves consideration of the analyte to be detected and the
desired means of detection. The selection of a particular
label is not critical, so long as the label is capable of
generating a detectable signal either by itselt or in
5 conjunction with one or more additional signal producing
components. The details ot the preparation of such
label/specific binding member conjugates are well-known to
those skilled-in-the-art.
The term ~signal producing component~ refers to any
10 substance capable of reacting with another assay reagent or
the analyte to produce a reaction product or signal tha1
indicates the presence of the analyte and that is detectable by
visual or instrumental means. ~Signal production system~, as
used herein, refers to the group of assay reagents that are
15 needed to produce the desired reaction product or signal. For
example, one or more signal producing components can be used
to react with a label and generate the detectable signal, i.e.,
when. the label is an enzyme, amplification of the detectable
signal is obtained by reacting the enzyme with one or more
2~ substrates or additional enzymes to produce a detectable
reaction product.
The term ~capture reagent~ refers to a capture binding
member which is attached to a solid phase material to enable
the separation of the analyte or indicator reagent, that is
25 bound thereto, from unbound analyte and assay reagents.
Typically, the attachment of the capture binding member to
the solid phase material is substantially irreversible.
In forrning a capture reagent to be used in an assay, once
the capture binding member, e.g., aliergen, is immobilized upon
30 the solid phase, the remaining surface area of the solid phase
is generally bloGked with a suitable inactivating solution,
such as bovine or equine serum albumin, casein or other
proteinaceous material, to prevent non-specific binding of
protein to the solid phase when the reaction mixture
~5 containing a specific binding member is contacted to the solid
phase. The solid phase is then washed with an appropriate
W 0 93/10458 PC~r/US92/09281 ~
2 1 1 ~ 16
solution to remove any excess blocking solution and/or
unbound capture binding member.
Once complex formation occurs between the assay
components, the solid phase can be used ~s a separation
S mechanism. For example, the reaction mixture can be
contacted to the capture reagent, and the solid phase material
retains the newly formed reaction complex(es).
Assay devices can have many configurations, several of
which are dependent upon the material chosen for the solid
10 phase. The 1erm ~solid phase material~ refers to any suitable
chromatographic, bibulous~ porous or capillary material or
other conventional solid material, well-known to thosa
skilled-in-the-art tor use in immobilizing specific binding
members. Solid phase materials can include fiberglass, nylon
lS or cellulose or derivatives thereof, such as cellulose nitrate
or a cellulose acetate/cellulose nitrate mixed ester cellulose.
The solid phase, however, is not limited to porous materials.
The solid phase material can also include, without limitation,
polymeric or glass beads, microparticles, tubes, sheets,
20 plates, slides, magnetic beads, a microtitre plate with one or
more reaction wells or a glass or plastic test tube, or the like.
Natural, synthetic or naturally occurring materials that
are synthetically modified, can be used as a solid phase
material including polysaccharides, e.g., cellulose materials
25 including paper, cellulose and cellulose derivatives such as
cellulose acetate, nitrocellulose and cellulose acetate/nitrate
mixed ester cellulose; silica; fiberglass; inorganic materials
such as deactivated alumina, diatomaceous earth or other
inorganic finely divided material uniformly dispersed in a
30 porous polymer matrix, with polymers such as vinyl chloride,
vinyl chloride-propylene copolymer, and vinyl chloride-vinyl
acetate copolym~r; cloth, both naturally occurring (e.g.,
cotton) and synthetic (e.g., nylon~; porous gels such as silica
gel, agarose, dextran and gelatin; polymeric films such as
3 5 polyacrylamide; magnetic particles; microtitre plates;
polystyrene tubes; protein binding membranes; agarose;
Sephadex@~ ~Pharmacia Fine Chemicals, Inc~, Piscataway, New
.
2 1 ~
~vo 93/10458 PCI /USg2tO9281
17
Jersey); Trisacryl (Pointet-Girard, France); silicon particles;
porous fibrous matrixes; and the like. The solid phase
material should have a reasonable inherent strength or
strength can be provided by means of a support, and it should
5 not interfere with ehe production of a detectable signal.
Optionally, the specific binding member of the capture
reagent can be affixed lo particles, e.g., microparticles. These
microparticles can serve as the solid phase material and be
retained in a column, suspended in a mixture of soluble
10 reagents and test sample, or retained and immobilized by
another solid phase base material. By ~retained and
immobilized~ is meant that the microparticles, associated
with the solid phase base material, are not capable of
substantial movement to positions elsewhere within that
15 material. The microparticles can be selected by one skilled-
- in-the-art from any suitable type of particulate material -~
including those composed of polystyrene, polymethylacrylate,
polypropylene, polytetraf luoroethylene, polyacrylonitrile,
polycarbonate or similar materials. The size of the
2 0 microparticles is not critical, although it is preferred that
the average diameter be smaller than the average pore size of
the solid phase base material if sudl is used.
The term ~ancillary specific binding member~ refers to a
specific ~inding member used in addition to the specific
` 25 binding members of the capture reagent and the indicator
reagent. One or more ancillary specific binding members can
be used in an assay. For example, an ancillary specific binding
member can be used in an assay where the specific binding
member of the indicator reagent is capable of binding the ~-
30 ancillary specific binding member which is in turn capable of
binding the analyte.
The present invention is concarned with immunoassays~
Therefore, the following discussion of immunoassays and
definitians of terms often used with respect to immunoassays
35 are set forth to facilitate the understanding of the disclosure
and claims hereof.
~ "
WO 93/10458 PCI /USg2/09281
2 ~ 5 5 18
In accordance with one method of the present invention,
a sandwich assay can be performed wherein a capture reagent
can include an allergen which has been bound to a solid phase
material. The capture reagent is contacted with a test
sample, suspected of containing the analyte, and an indicator
reagent containing an analyte-specific binding member
conjugated to a label. The reagents can be contacted to the
sample simultaneously or added sequentially. A binding
reaction results in the formation of a capture
reagenVanalyte/indicator reagent complex. The assay may ;
als~ involve a washing step to separate the resultànt complex
from the excess reagents and test sample. Either the
unreacted indicator reagent or the complex retained upon the
solid phase is then observed to detect or measure the amount
of label associ~ted therewith. If analyte is present in the
sample, then label will be present on the solid phase mate~ial. -The amount of label on the solid phase is proportional to the
amount of analyte in tha sample.
The present invention also can be used to conduct a
20 compeUtive assay. In one example of a competitive
configuration, the capture reagent again includes a specUic ;
binding member (allergen) which has been attached to a solid .
phase material. The capture reagent is contacted with both
test sample and an indicator reagent that includes an analyte
or analyte analog which has been labeled with a signal
generating compound. The indicator reagent and analyte then
compete in binding to the capture reagent. The competitive
binding reaction results in the formation of capture `
reagentlanalyte complexes or capture reagenVindicator
reagent complexes. The capture reagenttindicator reagent
complexes can be detected via the label of the indicator
- reagent. In the competitive assay, the amount of label that
becomes associated with the solid phase is inversely
proportional to the amount of analyte in the sample.
The present invention can also be used in indirect
immunoassays involving one or more ancillary specific binding
~; members. For example, an indirect sandwich immunoassay
.
S 5
'`'O 93/10458 PCl/US9~/09281
19
with the formation of a capture reagent/analyte/anti-analyte
antibody/indicator reagent eomplex can be performed, wherein
the indieator reagent is a speeifie binding partner for the
ancillary speeifie binding member whieh is speeifie for the
S analyte. The present invention ean also be used in forward and
reverse immunoassay protoeols
The following examplss are illustrative of the invention
and are in no way to be interpreted as limiting the seope of
the invention, as defined in the elaims. It will be appreeiated
10 that one skilled in the art ean eoneeive ot many other deviees
and methods of use to whieh the present inventive eoneepts
ean be applied.
Examele 1
In this experiment, Altemaria altemata allergen was
pretreated for binding to a solid phase material. A 37%
aqueous forrnaldehyde solution (12.5 IlL) was mixed with 100 ~-
mieroliters of a solution of Alternaria altemata (28.8 ~g/mL)
20 in deionized water. The amount of formaldehyde effeetive for
pretreatment was found to range from about 10 ~L to about 20
~L when the 37% aqueous formaldehyde solution was used.
The resulting mixture was ineubated at 4C for about 10
hours, and the ineubated eomposition was allowed to stand for
25 30 to 60 minutes at about 20C. The mixture was then
centrifuged, and the resultant supernatant, a pretreated
Alternaria altemata allergen eomposition, was deeanted. The
pretreated eomposition was poured onto a dise of mieroporous
eellulose nitrate (about 140 ~m thiek and about ~ mm in
30 diameter) and allowed to dry. The allergen was thereby
immobilized upon the solid phase rnaterial. The remaining
surfaee of the disc was then blocked with a ten pereent horse
serum solution.
The solid phase bound allergen, or Alternaria alternata
35 capture reagent, was then used in an enzyme immunoassay
(~EIA7. The EIA method ineluded the following steps. The
sample to be tested (e.g., serum) was contacted to the capture
WO 93/10~58 PCI /US92/09281 ~
211 S 5 3 5 20
reagent, thereby immobilizing allergen-specific IgE antibodies
upon the solid phase. Optionally, the antibody immobilization
step was followed by a wash step to remove unbound sample.
The capture reagent was then contacted to an enzyme-labeled
5 anti-lgE antibody (indicator reagent) which bound to that IgE
antibodies from the sample, if any, which had bound to the
solid phase. The solid phase was then washed to r~move
unbound indicator reagent. The solid phase was contacted to
an enzyme substrate signal producing component such that the
10 enzyme component of the complexed indicator reagent would
react with the substrate to produce a detectabls signal. Prior
to detection, ~he solid phase may undergo a third washing to
remove unbound substrate. The signal which was detected
was directly related to the amount of allergen-specific IgE
15 antibodies in the test sample.
In one EIA procedure, the enzyme label was alkaline
phosphatase, the substrate was 5-bromo-4-chloro-3-
- indolylphosphate and the detection or measurement step was
performed with a reflectance spectrophotomQter. The disc
20 turned dark blue upon the addition of substrate to the solid
phase, i.e., a positive assay result, when the serum sample
contained IgE antibody specific to Altemaria altemata. The
assay procedure was repeated using serum from different
patients, and the results were found to correlate with the
25 results obtained for the same serum samples using alternate
tests, such as a radio-allergo-sorbent test (RAST) or a skin
prick test as are well-known in the art.
Example 2
In this experiment, the nitrocellulose disc used as the
solid phase was one of many discs on a laminate composed of
a mylar sheet to which a sheet of nitrocellulose had been
glued. A circular shape was embossed onto the nitrocellulose
3S sheet to form each of the discs. The micropores in the
nitrocellulose sheet had diameters of about 450 nanometers.
Each individual disc had a separate allergen attached thereto.
.
2 ~ 3 ~ ~
vo 93/10458 PCI/US92~09281
21
Thus, the device could be used to detect the presence of
antibodies to multiple allergens.
Examplç 3
s
The procedùre of Example 1 was repeated using 100
microliter portions of solutions containing birch allergen (137
~9) or dog allergen (280 llg) which were mixed with a 37%
aqueous formaldehyde solution (12.5 ,uL) and incubated thereby
10 forming pretreated allergen compositions. The amount of
formaldehyde effective for pretreatment was found to range
from about 10 ,uL to about 15 ~L when the 37% aqueous
forrnaldehyde solution was used. The compositions were used
substantially in accordance with the procedures described in ~-
Examples 1 and 2 to produce devices which were then used to -~
test serum samples. The assay results were found to
correlate with the results of testing the same serum samples -:
by other means: the disc turned dark blue when the serum
sample contained IgE antibodies specific for the allergen
20 immobilized upon the solid pha~e.
Example 4
T~trahydrofuran (25 ,ul~) was mixsd with 100
25 microliters of a solution containing Bermuda grass allergen
(510 1l9) in deionized water. The amount of tetrahydrofuran
effective for pretreatment was ~ound to range ~r~m about 10
~lL to about 50 IlL. The resulting mixture was incubated at
4~C for about 10 hours, and the incubated composition was
30 a!lowed to stand at about 20~C for 30 to 60 minutes. The
solution was then centrifuged, and the resultant supernatant,
a pretreated Bermuda grass allergen composition, was
decanted.
The procedure was repeated using 100 microliter-
35 portions of solutions which contained Japanese cedar allergen
(150 ~9), June/Kentucky blue grass allergen (545 ~g),
perennial rye allergen (433 ,ug) or timothy allergen (43 ,ug) in
WO 93/104~8 PC~/US92/09281 -
`2li.,SSa 22
deionized water. The pretreated allergen compositions were
used to produce solid phase discs and were used in
immunoassays substantially in accordance with the procedure
described in Example 1. The assay results were found to
S correlate with the results of testing the same serum samples
by other means: the disc turned dark blue when the serum
sample contained IgE antibodies specific for the allergen
immobilized upon the solid phase.
1 0 Example 5
A 37 percent aqueous formaldehyde solution (15.6 ~ug)
was mixed with 100 microliters of a solution containing
mountain cedar allergen (733 ~9) in deionized water. The
lS resulting mixture was incubated at about 20C for
approximately 30 minutes. Tetrahydrofuran (28.7 IlL) was
then mixed with the incubated solution. The amount of
formaldehyde effective for pretreatment was found to rangè
from about 10 ~lL to about 20 IlL, and the amount of
20 tetrahydrofuran was found to range from about 10 ~L to about
50 ~lL. . The mixture was incubated for about 10 hours at 4C
and was allowed to stand at about 20C for 30 to 60 minutes.
The mixture was then centrifuged, and the resultant
supernatant, a pretreated cedar allergen composition, was
2 S decanted.
This allergen pretreatment procedure was repeated,
using 100 microliter-portions of solutions containing oak
allergen (729 ~19) or olive allergen (1670 ,ug), in deionized
water, in place of the mountain cedar allergen. The pretreated
30 allergen compositions were then used to produce immunoassay
devices substantially in accordance with the procedures
described in Examples 1 and 2. The EIA results were found to
correlate with the results of testing of the same serum
samples by other means: the disc turned dark blue when the
35 serum sample contained IgE antibodies specific for the
allergen immobilized upon the solid phase.
21i~S~
'`/093/10458 PCI/US92/Og281
23
Example 6
Aqueous NaCI (5 M, 12 ~lL) was mixed with 100
microliters of a solution containing Cladosporium (960 ~9) in
S deionized water. The resulting mixture was incubated at
about 4C for about 10 hours, and the incubated composition
was then allowed to stand at about 20C for 30 to 60 minutes.
The mixture was then centrifuged, and the resultant
supematant, a pretreated Cladosporium allergen composition,
l O was decanted. Depending upon the molar value of the
concentrated salt solution used, which value ranged from
about O.S M to about 10 M, the amount of aqueous NaCI
effective tor pretreatment ranged from about 10 ~L to about
20 ~lL.
lS The procedure was repeated using 100 microliters of a
solution containing feather allergen (7 ~19) in deionized water.
The pretreated allergen compositions were then used to
produce assay devices and were used in immunoassays
substantially in accordance with the protocol described in
Example 1. The assay results using the compositions and
dévices of th~ present invention were tound to correlate with
the results of testing the same serum samples by other means:
the disc turned dark blue when the serum contacted thereto
contained IgE antibodies specific for the allergen immobilized
upon the solid phase.
Examele 7
An aqueous solution of 1-ethyl-3-(3-
dimethylaminopropy!)carbodiimide (EDAC, 10 ~L at 50 mg/mL)
was mixed with 100 microliters of a solution containing D.
farinae (280 ~9) in deionized water. The amount of EDAC
effective for the first stage of pretreatment ranged from
about 5.0 ~L to about 15 ,uL. The resulting mixture was
incubated at about 22C for about t5 minutes. A two
microliter portion of a solution containing sodium borohydride
- ~ (20 mg/mL, NaBH4 ) in 10 ~lM phosphate buffered saline (pH 7)
,:
WO 93/10458 PCr/US92/09281
i S ~ 24
was mixed with the incubated solution, and the mixture was
further incubated at about 4~C for 10 hours. The amount of `~
NaBH4 effective for the second stage of pretreatment ranged
from about 1.0 ~lL to about 5.0 ,uL. The mixture was then
allowed to stand at about 20C for approximately 30 to 60
minutes. The mixture was centrifuged, and the resultant
supernatant, a pretreated D. farinae allergen composition, was
decanted.
The procedure was repeated using 100 microliters of a
solution containing D. pteronyssinus (263 1l9) in deionized
water. The pretreated allergen compositions were used
substantially in accordance with the procedures described in
Example 1 to produce treated discs for immunoassays. The
EIA results were found to correlate with the results of
testing the same serum samples by other means: the disc
turned dark blue when the serum sample contacted thereto
contained IgE antibodies specific for the allergen immobilized
upon the solid phase.
Example 8
Ons hundred percent acetic acid (12.5 ,uL, with effective
amounts ranging from about 5.0 IlL to about 30 IlL) was mixed
with 100 microliters of a solution containing lamb's quarters
25 allergen (1176 ~19) in deionized water. The resultant mixture
was incubated at about 22C for approximately five minutes,
after which time 6 N aqueous NaOH was added to adjust the pH
to 7. The neutralized solution was incubated at about 4C for
10 hours, and was then allowed to stand at about 20C for 30
30 to 60 minutes. The mixture was then centrifuged, and the
resultant supernatant, a pretreated lamb's quarters allèrgen
composition, was decanted~
The procedure was repeated using 100 microliters of a
solution containing mulberry allergen (40 ~,19) in deionized
35 water. The pretreated allergen compositions were used to
produce treated discs for enzyme immunoassays substantially
in accordance with the procedures described in Example 1~
2 ~ 3 c~
'`'O 93/10458 PCr/U~i92/09281
The assay results were found to correlate with the results of
testing the same serum samples by other means: the disc
turned dark blue when the serum sample contacted thereto
contained IgE antibodies specific for the allergen immobilized
upon the solid phase.
Example 9
A solution of 6 N aqueous HCI (24 ~lL, with effective
amounts ranging from about 6.0 ~lL to about 30 !lL) was mixed
with 100 microliters of a solution containing Penicitlium (120
,ug). The resulting mixture was incubated for approximately five
minutes at about 20C, after which time 6 N aqueolls NaOH was
added to adjust the pH to 7. The neutralized solution was
incubated at 4C for 10 hours and was then allowed to stand at
about 20C for 30 to 60 minutes. The mixture was then
centrifuged, and the resultant supernatant, a pretreated
P~nicillium allergen composition, was decanted.
The allergen pretreatment procedure was repeated with
2 0 100 microliters of a solution containing Parietaria allergen
(400 1l9) in deionized water. The solutions were then used
substantially in accordance with the procedures described in
Example 1 to produce discs and to test serum samples in an
El~. The assay results were found to correlate with the
~5 results of testing the same serum samples by other means:
the disc turned dark blue when the serum contacted thereto
contained IgE antibodies specific for the allergen immobilized -~
upon the solid phase.
Example 10
Untreated allergen compositions included from about 0.5
to about 50 Aspergillus allergen; from about 0.6 to about 20.6
of cat allergen; from about 0.1 to about 10.0 of elm allergen;
35 from about 0.4 to about 100 of house dust allergen; from about
0.1 to about 11.5 of maple allergen; from about 0.3 to about
W O 93/10458 PC~r/US92/0928~-
5 5 ~ 26
90.4 of mugwort allergen and from about 1.7 to about 130.4 of
plantain allergen in deionized water.
The solutions were then used substantially in accordance
with the procedures described in Example 1 to produce discs
5 and to test serum samples in an EIA. The assay results were
found to correlate with the results of testing the same serum
samples by other means: the disc turned dark blue when the
serum sample contained IgE antibodies specific for the allergen
immobilized upon the solid phase.
Examele 11
.
Pretreated allergen compositions, which were produced
as described in Examples 1, and 3 through 9, and which
differed from one another with respect to allergen content, ~
were used to test for IgE antibodies in a series of serum -;;
samples. Upper and lower allergen concentration limits were
set by classifying a pretreated allergen composition as either
~too dilute~ if that composition failed to produce a maximum
20 positive IgE antibodies test result with a serum sample which
had tested positive with a more concentrated allergen
solution, or ~too concentrated~ if the composition failed to
produce a maximum positive IgE antibodies test result with a
serum sample which had tested positive with a less
2S concentrated allergen solution. The allergen concentrations
tested ranged from about 0.05 milligrams of allergen per
milliliter of water, prior to pretreatment, to about 170
milligrams/milliliter. The test results are presented in Table
1 and illustrate the most effective concentration ranges for
30 each of the allergens tested.
In this manner, the optimum concentration of allergen
was determined for the production of solid phase assay
devices.
J 'j ~ ~
-~o 93/10458 PCr/US92/09281
27
Table
Effective coacentration range
Allereen (Drotein content in solution)
Alternaria alternata allergen from 0.05 to 4.0 mg/mL
Aspergillus fumigatus allergen from 0.5 to 50.0 mg/mL
Bermuda grass allergen from 0.8 to 81.6 mg/mL
birch allergen from 0.1 to 6.0 mg/mL
mountain cedar allergcn from 0.04 to 4.5 mg/mL
lapancsc ccdar allcrgen from 0.1 to 20,5 mg/mL :~
Cladosporium allergcn from 0.05 to 38.4 mg/mL
cat allcrgcn from 0.6 to 20,6 mg/mL
dog allergen from 1.3 to 38.4 mg/mL
D. farinae allcrgcn from 0.7 to 22.4 mg/mL
D. pteronyssinus allcrgcn from 0.6 to 84.2 mg/mL
clm allergcn fiom 0.1 to 146.0 mglmL
fcathcr allcrgcn from 0.02 to 0.2 mg/mL
giant ragwccd allergcn from 0.2 to 148.2 mg/mL
housc dust allcrgen from 0.4 to 100 mg/mL
Junc/KentucKy blucgrass from 0.05 to 21.8 mg/mL
allcrgcn
lamb's quarters allcrgen from 0.2 tO 47.0 mg/mL
maple alltrgcn from 0.1 to 166.3 mg/mL
mugwort allcrgen from 0.3 to 90.4 mg/mL
mulberry allcrgen from 0.1 to 12 mg/mL
oak allergen from 0.2 to 29.2 mg/mL
olive allergcn fiom 0.1 to 66.8 mg/mL
Parietaria allergen from 1.0 to 40.0 mglmL
plantain allergen from 1.7 to 130.4 mg/mL
Penicillium allergen from 0.1 to 4.8 mg/mL
perennial rye allergen from O.OS to 17.3 mglmL
short ragweed allergen from 0.2 to 151.6 mg/mL
timothy allergcn from 0.05 to 6.6 mg/mL
- :-
:
WO 93/104~8 PCI`/US92/09281
5 j 3 28
Example 1 2
A milk protein composition comprising milk protein
extract, casein and ,B-lactoglobulin was prepared as tollows.
Casein powder (Greer Laboratories, Lenoir, North Carolina)
was dissolved in de-ionized water at approximately 4 mg/ml.
,B-lactoglobulin powder (Sigma Chemical Company, St. Louis,
Missouri) was dissolved in de-ionized water at approximately i~
72 mg/ml. The casein solution and the ~-lactoglobulin
solution were combined in a 1:1 volume ratio. Bovine milk
protein extract powder (~Milk, Cow~ trom Greer Làboratories,
Lenoir, North Carolina) was dissolved in de-ionized water at
approximately 1.4 mg/ml. The ,B-lactoglobulin-casein solution
was combined with the milk solution in a 1:1 volume ratio.
The ,B-lactoglobulin-casein-milk solution was acidified by the
addition of 6N HCI to make a final concentration of 0.06N HCI.
After 5 minutes, the solution was neutralized by the addition
of 6N NaOH followed by 1M HEPPS buffer, pH 8.3, to make a
final concentration of 0.1M HEPPS buffer. This solution was
stored at 2-8C for approximately 3 days. Before use, the
solution was centrifuged to provide a clear solution which
may be adsorbed onto nitrocellulose.
xamele 13
- 25
A milk protein composition comprising milk protein
extract, casein, ~-lactoglobulin and BGG was prepared as
follows. Casein powder (Greer Laboratories, Lenoir, North
Carolina) was dissolved in de-ionized water at approximately
8 mg/ml. ,B-lactoglobulin powder (Sigma Chemical Company,
St. Louis, Missouri) was dissolved in de-ionized water at
approximately 144 mg/ml. The casein solution and the ,B-
lactoglobulin solution were combined in a 1:1 volume ratio.
Bovine milk protein extract powder ("Milk, Cow~ from Greer
Laboratories, Lenoir, North Carolina) was dissolved in de-
ionized water at approximately 2.8 mg/ml. The ~-
lactoglobulin-casein solution was combined with the mi~k
:
2 ~ 5 ~
'"O 93/10458 PCI /US92/09281
29
solution in a 1:1 volume ratio. The ,B-lactoglobulin-casein-
milk solution was acidified by the addition of 6N HCI to make
a final concentration of 0.06N HCI. After 5 minutes, the
solution was neutralized by the addition of 6N NaOH followed
by lM HEPPS buffer, pH 8.3, to make a final concentration of
0.2M HEPPS buffer. Bovine gamma globulin (BGG) powder
(Sigma Chemical Company, St. Louis, Missouri) was dissolved
in de-ionized water at approximately 10 mg/ml. The ,B-
lactoglobulin-casein-milk solution was then combined with
the BGG solution in a 1:1 volume ratio. This solution was
stored at 2-8C for approximately 3 days. Before ùse, the
solution was centrifuged to provide a clear solution which
may be adsorbed onto nitrocellulose. :;
Example 14
First test cards 82 with a test site 84 ~see Figures 1
and 2) containing a milk protein composition of oniy milk
protein extract powder (~Milk, Cow~ from Greer Laboratories,
Lenoir, North Carolina) were prepared by depositing 2
microliters of approximately a 0.7 mg/ml solution (0.tM
HEPPS buffer in de-ionized water), pretreated with acid
according to the method in Examples 12 and 13, onto the test -
site and drying the test site at room temperature overnight.
Second test cards 82 with a test site 84 containing a
milk protein composition of milk protein extract, casein and
~lactoglobulin were prepared by depositing 2 microliters of
the milk protein composition solution prepared according to
Example 12 onto a test site and drying the test site at room
3 0 lemperature overnight.
Third test cards 82 with a test site 84 containing a milk
protein composition of milk protein extract, casein, ,B-
lactoglobulin and BGG were prepared by depositing 2
microliters of the milk protein composition solution prepared
according to Example 13 onto a test site and drying the test
site at room temperature overnight.
'
WO 93/10458 PCI /US92/09281
2 i i 3 j 5 v
Pools of human plasma containing high levels of anti-
milk-protein IgE antibodies were prepared as follows. Human
plasma samples were tested for anti-milk-protein IgE
antibodies using Phadebas RAST~) (Pharmacia) Milk test.
Samples were combined to produce plasma pools having at
least 1.1 PRU/ml (expressed in Pharmacia Radioimmunoassay
Units (PRU) per milliliter) of anti-milk-protein IgE antibodies.
The plasma pools were then serially diluted with about 0.1M -
TRIS buffer saline (pH 7.2) containing serum proteins to
produce pooled plasma samples containing about 0.1, 0.17,
0.22, 0.34 and 1.12 PRU/ml of anti-milk-protein IgE
antibodies.
Using an Abbott Matrix~9 Analyzer (commercially
available trom Abbott Laboratories, North Chicago, Illinois),
the anti-milk-protein IgE antibody positive plasma pools were
analyzed for the pr~sence of detectable amounts of anti-milk-
protein IgE antibodi~s using the first (milk protein extract
only) and second (milk protein extract, casein and ~-
lactoglobulin) test cards prepared above. The presence of IgE
antibodies from the sample pools captured on the solid phase
was detected by incubating the solid phase with alkaline
phosphatase labeled goat anti-human-lgE IgG antibodies,
washing the solid phase with buffer and adding the enzyme
substrate 5-bromo-4-chloro-1-indolylphophate (BCIP). The
intensity of the color developed on the solid phase was
measured with a reflectance detector. After subtracting
background intensity, the relative color intensity of the
samples analyzed on each test card were plotted against the
concentration ot IgE antibodies as measured by Phadebas
RAST(~ (PRU/ml). The plot, as shown in Figure 3, illu~strates
the significant improvement in sensitivity realized by the use
- of the milk protein composition ot Example 12 on the second
test cards above.
Using an Abbott Matrix(~) Analyzer, nine (9) human
plasma (anti-milk-protein IgE positive by Phadebas RAST(~)
Milk test) patient samples were analyzed for the presence of
detectable amounts of anti-milk-protein IgE antibodies using
~, i l ;.;) S rj ;~
'`'`O 93/10458 PCl /US92/0928
31
the second and third test cards prepared above. The second
test cards (without BGG) detected anti-milk-protein IgE
antibodies in eight (8) of the nine (9) samples tested, whereas
the third test cards (with BGG) detected anti-milk-protein IgE
S antibodies in nine (9) of the nine (9) samples tested. As
described above, the presence of IgE antibodies from the
sample captured on the solid phase was detected by incubating
the solid phase with alkaline phosphatase labeled goat anti-
human-lgE IgG followed by the addition of the enzyme
10 substrate BCIP. The color intensity was again measured by a
reflectance detector and adjusted for the background
intensity.
- It will be appreciated by one skilled-in-the-art that the
15 concepts of the present invention are equally applicable to
many different allergens (specific binding members), solid
phase materials and immunoassay protocols. It will also be
appreciated that the selection of any given label, ancillary
binding member or solid phase material is generally not
20: cr~tical to the present invention. The materials are selected
to optimke the results provided by the chosen assay
configuration. The embodiments described herein are intended
as examples rather than as limitations. Thus, the description
of the invention is not intended to limit the invention to the
25 particular embodiments described in detail, but it is intended
to encompass all equivalents and subject matter within the
spirit`and scope of the invention as described above and as set
forth in the following claims.
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