CA2115555A1 - Binding of milk allergens to a solid phase - Google Patents

Abstract

A method and composition useful for the detection of anti-milk-protein IgE antibodies patient in body fluids. A composition of milk protein extract, casein and .beta.-lactoglobulin is useful for the detection of anti-milk-protein IgE antibodies. The furher addition of bovine gamma globulin to the composition improves the detection of such IgE antibodies. The method involves applying to a solid phase an amount of the composition in solution and drying.
The solid phase is placed in contact with a patient test sample and the presence or amount of anti-milk-protein IgE antibodies is determined.

Description

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 :`,, 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 ' . ' vo ~3/10458 2 1 1 ~ 3 Pcr/US92/09281 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.

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 , ' ;
.
.

-"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 : ,, .

W O 93/10458 P ~ /US92/09281 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 ~.

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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WO 93/10458 PCI /US92/0928.' 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 ~' 2~ 3~.~3 - 'O 93/1 0458 PCI /US92/09281 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 :~

WO 93/10458 Pcr/usg2/0928 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 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 .

'`'O 93/10458 PCl/US9~/09281 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 ~

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 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 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 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 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 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.

' 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 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.

:

Claims (14)

WO 93/10458 PCT/US92/0928?

What is claimed is:

1. A milk allergen composition, comprising milk protein extract, casein and .beta.-lactoglobulin.

2. The composition of claim 1 further comprising bovine gamma globulin.

3. The composition of claims 1 and 2 wherein the composition is pretreated with aqueous acid.

4. The composition of claims 1 and 3 further comprising an aqueous solution.

5. The composition of claim 4 wherein the milk protein extract concentration is within the range of about 0.5 to about 2.0 mg/ml, the casein concentration is within the range of about 0.5 to about 2.0 mg/ml and the .beta.-lactoglobulin concentration is within the range of about 5 to about 30 mg/ml.

6. The composition of claim 2 further comprising an aqueous solution wherein the milk protein extract concentration is within the range of about 0.5 to about 2.0 mg/ml, the casein concentration is within the range of about 0.5 to about 2.0 mg/ml, the .beta.-lactoglobulin concentration is within the range of about 5 to about 30 mg/ml and the bovine gamma globulin concentration is within the range of about 0.5 to about 16 mg/ml.

7. A milk allergen composition comprising milk protein extract, casein and .beta.-lactoglobulin adsorbed onto a solid phase.

8. The composition of claim 7 wherein the solid phase is selected from the group consisting of cellulose, cellulose acetate, nitrocellulose, acetate/nitrate mixed ester cellulose, silica, fiberglass, and agarose.

9. A method of detecting anti-milk-protein antibodies in a sample suspected of having said antibodies comprising:
contacting the sample with a solid phase having immobilized thereon milk protein extract, casein and .beta.-lactoglobulin; and detecting the presence or amount of antibodies bound to said solid phase.

10. The method of claim 9 wherein the antibodies are IgE antibodies.

11. The method of claim 9 wherein the solid phase is selected from the group consisting of cellulose, cellulose acetate, nitrocellulose, acetate/nitrate mixed ester cellulose, silica, fiberglass, and agarose.

12. The method of claim 15 wherein the solid phase has immobilized thereon milk protein extract, casein, .beta.-lactoglobulin and bovine gamma globulin.

13. The method of claim 9 wherein the milk protein extract, casein and .beta.-lactoglobulin are pretreated with aqueous acid prior to immobilization on the solid phase.

14. The method of claim 10 wherein the IgE antibodies are detected by contacting the solid phase with an indicator reagent specific for IgE antibodies and measuring the amount of indicator reagent bound to the solid phase.