AU769954B2 - Method to detect IgE - Google Patents

Description

-1-

AUSTRALIA

PATENTS ACT 1990 COMPLETE SPECIFICATION FOR A STANDARD PATENT

ORIGINAL

Name of Applicant: Heska Corporation Actual Inventors: Glenn Robert Frank and James P Porter and Keith E. Rushlow and Donald L. Wassom Address for Service: BALDWIN SHELSTON WATERS 60 MARGARET STREET :SYDNEY NSW 2000 CCN: 3710000352 Invention Title: 'METHOD TO DETECT IgE' Details of Original Application No. 74114/98 dated 24 Nov 1997 The following statement is a full description of this invention, including the best method of performing it known to me/us:- File: 32561AUP00 la- METHOD TO DETECT IgE Field of the Invention The present invention relates to a novel method to detect epsilon immunoglobulin (IgE). The present invention also includes novel kits to detect IgE as well as methods to produce the detection reagent.

Background of the Invention Any discussion of the prior art throughout the specification should in no way be considered as an admission that such prior art is widely known or forms part of common general knowledge in the field.

Diagnosis of disease and determination of treatment efficacy are important tools in medicine. In particular, detection of IgE production in an animal can be indicative of disease. Such diseases include, for example, allergy, atopic disease, hyper IgE syndrome, internal parasite infections and B cell neoplasia. In addition, detection of IgE production in an animal following a treatment is indicative of the efficacy of the treatment, such as when using treatments intended to disrupt IgE production.

Until the discovery of the present invention, detection of IgE in samples obtained from non-human animals has been hindered by the absence of suitable reagents for detection of IgE. Various compounds have been used to detect IgE in IgE-containing "compositions. In particular, antibodies that bind selectively to epsilon idiotype antibodies anti-IgE antibodies) have been used to detect IgE. These anti-IgE antibodies, however, can cross-react with other antibody idiotypes, such as gamma isotype antibodies. The discovery of the present invention includes the use of an Fc epsilon receptor (FcER) molecule to detect the presence of IgE in a putative IgEcontaining composition. An FceR molecule provides an advantage over, for example anti-IgE antibodies, to detect IgE because an FcR molecule can bind to an IgE with more specificity less idiotype cross-reactivity) and more sensitivity affinity) than anti-IgE binding antibodies.

Lowenthal et al., 1993, Annals ofAllergy 71:481-484, dog serum can transfer cutaneous reactivity to a human. While it is possible that Lowenthal et al. properly teach 30 the binding of human FcR to canine IgE. Lowenthal et al., however, do not provide lbdata defining the particular cellular proteins responsible for the transfer of cutaneous reactivity. As such, a skilled artisan would conclude that the transfer of cutaneous reactivity taught by Lowenthal et al. could be due to a variety of different molecular interactions and that the conclusion drawn by Lowenthal et al. is merely an 0 interpretation. In addition, Lowenthal et al. do not teach the use of purified human FcR to detect canine IgE. The subunits of human Fc R have been known as early as 1988 and have never been used to detect canine, feline or equine IgE. Indeed, U.S. Patent No.

4,962,035, to Leder et al., issued October 9, 1990, discloses human FcR but does not disclose the use of such a human Fc,R to detect human or non-human IgE. The use of purified human Fc,R avoids complications presented by use of Fc,R bound to a cell, such as non-specific binding of the Fc,R-bearing cell due to additional molecules present on the cell membrane. That purified human FcR detects non-human IgE is unexpected because inter-species binding between a FcR and an IgE is not predictable. For example, human FcR binds to rat IgE but rat FcR does not bind to human IgE.

The high affinity FcR consists of three protein chains, alpha, beta and gamma.

Prior investigators have disclosed the nucleic acid sequence for: the alpha chain (Kochan et al, Nucleic Acids Res. 16:3584, 1988; Shimizu et al., Proc. Natl. Acad. Sci. USA 85:1907-1911, 1988; and Pang et al., J. Immunol. 151:6166-6174, 1993); the beta chain (Kuster et al., J. Biol. Chem. 267:12782-12787, 1992); and the gamma chain (Kuster et al., J. Biol. Chem. 265:6448-6452, 1990).

Thus, methods and kits are needed in the art that will provide specific_detection of non-human IgE.

Summary of the Invention The present invention includes detection methods and kits that detect IgE. One embodiment of the present invention is a method to detect IgE comprising: contacting an isolated human Fc, receptor (Fc,R) molecule with a putative IgEcontaining composition under conditions suitable for formation of a FcR molecule:IgE complex, wherein the IgE is selected from the group consisting of canine IgE, feline IgE 25- and equine IgE; and determining the presence of IgE by detecting the FcR molecule:IgE complex, the presence of the FcR molecule:IgE complex indicating the presence of IgE. A preferred Fc,R -e .ecule in which a carbohydrate group of the FcR molecule is conjugated to biotin.

Another embodiment of the present invention is a method to detect IgE comprising: contacting a recombinant cell with a putative IgE-containing composition under conditions suitable for formation of a recombinant cell:IgE complex, in which the recombinant cell includes: a recombinant cell expressing a human Fc 8

R

molecule; and a recombinant cell expressing an antibody that binds selectively to an IgE including canine IgE, feline IgE and equine IgE; and determining the presence of IgE by detecting the recombinant cell:IgE complex, the presence of the recombinant cell:IgE complex indicating the presence of IgE. A preferred recombinant cell includes an RBLhFceR cell.

Accordingly, in one embodiment, the present invention provides a method to detect IgE comprising: contacting a recombinant cell with a putative IgE-containing composition under conditions suitable for formation of a recombinant cell:IgE complex, wherein said recombinant cell is selected from the group consisting of: a recombinant cell expressing a human Fc 8 R molecule; and a recombinant cell expressing an antibody that binds selectively to an IgE selected from the group consisting of canine IgE, feline IgE and equine IgE; and determining the presence of IgE by detecting said recombinant cell:IgE complex, the presence of said recombinant cell:IgE complex indicating the presence of IgE.

Another embodiment of the present invention is a method to detect flea allergy dermatitis comprising: immobilizing a flea allergen on a substrate; contacting the flea allergen with a putative IgE-containing composition under conditions suitable for formation of an antigen:IgE complex bound to said substrate; removing non-bound S 20 material from the substrate under conditions that retain antigen:IgE complex binding to •the substrate; and determining the presence of the antigen:IgE complex by contacting the antigen:IgE complex with a Fc 6 R molecule. Preferably, the flea allergen is a flea saliva antigen and more preferably flea saliva products and/or flea saliva proteins.

The present invention also includes a kit for performing methods of the present invention. One embodiment is a kit for detecting IgE comprising a human Fce receptor (FcR) molecule and a means for detecting an IgE including canine IgE, feline IgE and equine IgE. Another embodiment is a general allergen kit comprising an allergen *o common to all regions of the United States and a human FcE receptor (FceR) molecule.

Another embodiment is a kit for detecting flea allergy dermatitis comprising a human 30 Fc, receptor (FcR) molecule and a flea allergen.

Accordingly, in one embodiment the present invention provides an allergen kit comprising an allergen and a human Fc receptor (FCR) molecule, wherein said allergen is selected from the group consisting of flea, grass, Meadow Fescue, Curly Dock, plantain, Mexican Firebush, Lamb's Quarters, pigweed, ragweed, sage, elm, cocklebur, Box Elder, walnut, cottonwood, ash, birch, cedar, oak, mulberry, cockroach, Dermataphagoides, Alternaria, Aspergillus, Cladosporium, Fusarium, Helminthosporium, Mucor, Penicillium, Pullularia, Rhizopus and Tricophyton.

In another embodiment the invention provides an allergen kit comprising an allergen and a human Fcg receptor (FcR) molecule, wherein said allergen is selected from the group consisting of Johnson Grass, Kentucky Blue Grass, Orchard Grass, Perennial Rye Grass, Redtop Grass, Timothy Grass, Bermuda Grass, Brome Grass, English Plantain, Rough Pigweed, Short Ragweed, Wormwood Sage, American Elm, Common Cocklebur, Black Walnut, Eastern Cottonwood, Green Ash, River Birch, Red Cedar, Red Oak, Red Mulberry, Dermataphagoidesfarinae, Alternaria alternata, Aspergillusfumigatus, Cladosporium herbarum, Fusarium vasinfectum, Helminthosporium sativum, Mucor recemosus, Penicillium notatum, Pullularia pullulans, Rhizopus nigricans and Tricophyton spp.

Another embodiment of the present invention is an isolated human Fc, receptor (FcR) alpha chain protein, in which a carbohydrate group of the FcgR alpha chain 20 protein is conjugated to biotin. A preferred FcgR alpha chain protein comprises PhFcgRa 72

-BIOT.

o Unless the context clearly requires otherwise, throughout the description and the claims, the words 'comprise', 'comprising', and the like are to be construed in an inclusive sense as opposed to an exclusive or exhaustive sense; that is to say, in the sense of "including, but not limited to".

""Brief Description of the Figures Fig. 1 depicts ELISA results using biotinylated alpha chain of human FcgR to detect canine IgE antibodies.

Fig. 2 depicts ELISA results using biotinylated alpha chain of human FcgR to 30 detect plant allergen-specific canine IgE antibodies.

-4- Fig. 3 depicts ELISA results using biotinylated alpha chain of human Fc,R to detect human or canine IgE antibodies.

Fig. 4 depicts ELISA results using biotinylated alpha chain of human Fc,R to detect flea allergen-specific canine IgE antibodies.

Fig. 5 depicts ELISA results using biotinylated alpha chain of human Fc,R to detect flea allergen-specific and heartworm antigen-specific canine IgE antibodies.

Fig. 6 depicts ELISA results using biotinylated alpha chain of human Fc,R to detect flea saliva-specific canine IgE antibodies.

Fig. 7 depicts ELISA results using biotinylated alpha chain of human Fc,R to detect heartworm antigen-specific feline IgE antibodies.

Fig. 8 depicts ELISA results using biotinylated alpha chain of human Fc,R to detect heartworm antigen-specific feline IgE antibodies.

Fig. 9 depicts ELISA results using biotinylated alpha chain of human FcR to detect antigen-specific equine IgE antibodies.

Fig. 10 depicts ELISA results using basophilic leukemia cells expressing alpha chain of human Fc,R to detect canine IgE antibodies in sera from heartworm-infected e dogs.

°Fig. 11 depicts ELISA results using basophilic leukemia cells expressing alpha "chain of human FcR to detect canine IgE antibodies in sera from flea saliva sensitized dogs.

*Detailed Description of the Invention The present invention relates to the discovery that purified high affinity human Fc epsilon receptor Fc,RI; referred to herein as Fc,R) can be used in certain nonhuman canine, feline or equine) epsilon immunoglobulin (referred to herein as IgE or IgE antibody)-based detection diagnostic, screening) methods and kits. The use of human FcR to detect non-human IgE is unexpected because canine, feline and equine immune systems are different from the human immune system, as well as from each other molecules important to the immune system usually are species specific).

One embodiment of the present invention is a method to detect a non-human IgE using an isolated human Fc,R molecule. It is to be noted that the term entity or "an" entity refers to one or more of that entity; for example, a protein refers to one or more proteins or at least one protein. As such, the terms (or "one or more" and "at least one" can be used interchangeably herein. It is also to be noted that the terms "comprising", "including", and "having" can be used interchangeably. It is also to be noted that the terms "comprising", "including", and "having" can be used interchangeably. Furthermore, a compound "selected from the group consisting of" refers to one or more of the compounds in the list that follows, including mixtures combinations) of two or more of the compounds.

According to the present invention, an isolated, or biologically pure, FcR molecule, is a molecule that has been removed from its natural milieu. As such, "isolated" and "biologically pure" do not necessarily reflect the extent to which the molecule has been purified. An isolated human FcR molecule of the present invention can be obtained from its natural source from a human mast cell), can be produced using recombinant DNA technology or can be produced by chemical synthesis.

A FcR molecule (also referred to herein as FcR or FcR protein) of the present invention can be a full-length protein, a portion of a full-length protein or any homolog of such a protein. As used herein, a protein can be a polypeptide or a peptide. A FcR molecule of the present invention can comprise a complete FcR alpha, beta and gamma FcR chains), an alpha FcR chain (also referred to herein as FcR a chain) or portions thereof. Preferably, a FcR molecule comprises at least a portion of a FcR a chain that binds to IgE, that is capable of forming an immunocomplex with an IgE constant region. Preferably, a FcR molecule of the present invention binds to IgE with an affinity of about KA= 10 8 more preferably with an affinity of about KA= 10 9 and even more preferably with an affinity of about KA= 100.

An isolated FcR molecule of the present invention, including a homolog, can be 25 identified in a straight-forward manner by the FcR molecule's ability to form an immunocomplex with an IgE. Examples of FcR homologs include FcR proteins in which amino acids have been deleted a truncated ve--ir.n of the protein, such as a peptide), inserted, inverted, substituted and/or derivatized by glycosylation, phosphorylation, acetylation, myristoylation, prenylation, palmitoylation, amidation and/or addition of glycerophosphatidyl inositol) such that the homolog includes at least one epitope capable of forming an immunocomplex with an IgE.

Fc,R homologs can be the result of natural allelic variation or natural mutation.

Fc,R homologs of the present invention can also be produced using techniques known in the art including, but not limited to, direct modifications to the protein or modifications to the gene encoding the protein using, for example, classic or recombinant

DNA

techniques to effect random or targeted mutagenesis.

According to the present invention, a human FcR a chain of the present invention is encoded by at least a portion of the nucleic acid sequence of the coding strand of a cDNA encoding a full-length Fc,R a chain protein represented herein as SEQ ID NO: 1, the portion at least encoding the IgE binding site of the Fc,R a chain protein.

The double-stranded nucleic acid molecule including both the coding strand having SEQ ID NO: 1 and the complementary non-coding strand the nucleic acid sequence of which can be readily determined by one skilled in the art and is shown herein as SEQ ID NO:3) is referred to herein as Fc,R nucleic acid molecule nhFc,Ra,,8. Translation of SEQ ID NO: 1 suggests that nucleic acid molecule nhFcRa,,,9 encodes a full-length Fc,R a chain protein of about 257 amino acids, referred to herein as PhFc,RcU2 7 represented by SEQ ID NO:2, assuming an open reading frame having an initiation (start) codon spanning from nucleotide 107 through nucleotide 109 of SEQ ID NO: 1 and a termination (stop) codon spanning from nucleotide 878 through nucleotide 880 of SEQ ID NO: 1. The coding region encoding PhFc,Ra2~, including the stop codon, is S 20 represented by nucleic acid molecule nhFcRa 774 having a coding strand with the nucleic acid sequence represented herein as SEQ ID NO:4. The compliment of SEQ ID NO:4 is represented herein as SEQ ID NO:5. SEQ ID NO: 1 encodes a signal peptide of about 25 amino acids as well as a mature protein of about 232 amino acids, denoted herein as PhFcRa 232 the amino acid sequence of which is represented herein as SEQ ID NO:6. The nucleic acid molecule encoding the apparent mature protein is referred to as nhFcRa 69 9 the nucleic acid sequence of the coding strand of which is denoted herein as SEQ ID NO:7. SEQ ID Nn:I also encodes a hydrophobic transmembrane domain and a cytoplasmic tail which as a group extend from amino acid 205 to amino acid 257 of SEQ ID NO:2. Knowledge of these nucleic acid and amino acid sequences allows one skilled in the art to make modifications to the respective nucleic acid molecules and proteins to, for example, develop a Fc,R a chain protein with increased solubility and/or a truncated protein a peptide) capable of detecting IgE, PhFc.Ra, 9 7 and PhFcRa, 72 Preferred FcR molecules include PhFcRa 2 5 7 PhFcRai 97 PhFcRa 2 32 and PhFc,Rca72.

Preferred nucleic acid molecules to encode a FcCR molecules include nhFcRa774, nhFcRa nhFcRa,6 2 nhFc.Ra 591 nhFcRa9, and/or Isolated FcR molecule protein of the present invention can be produced by culturing a cell capable of expressing the protein under conditions effective to produce the protein, and recovering the protein. A preferred cell to culture is a recombinant cell that is capable of expressing the protein, the recombinant cell being produced by transforming a host cell with one or more nucleic acid molecules of the present invention. Transformation of a nucleic acid molecule into a cell can be accomplished by any method by which a nucleic acid molecule can be inserted into the cell.

Transformation techniques include, but are not limited to, transfection, electroporation, microinjection, lipofection, adsorption, and protoplast fusion. A recombinant cell may remain unicellular or may grow into a tissue, organ or a multicellular organism.

Transformed nucleic acid molecules of the present invention can remain extrachromosomal or can integrate into one or more sites within a chromosome of the transformed recombinant) cell in such a manner that their ability to be expressed is retained. Suitable and preferred nucleic acid molecules with which to transform a cell are as disclosed herein for suitable and preferred FcR nucleic acid molecules per se.

Particularly preferred nucleic acid molecules to include in recombinant cells of the present invention include nhFcRa 77 4 nhFcRa,,,9,, nhFRa62, nhFcERas5 9 nhFcRac99 and/or nhFcRa 5 6 -Suitable host cells to transform include any cell that can be transformed with a nucleic acid molecule of the present invention. Host cells can be either untransformed cells or cells that are already transformed with at least one nucleic acid molecule. Host *.cells of the present invention either can be endogenously naturally) capable of producing a FcR molecule protein of the present invention or can be capable of producing such proteins after being transformed with at least one nucleic acid molecule of the present invention. Host cells of the present invention can be any cell capable of producing at least one protein of the present invention, and include bacterial, fungal -8- (including yeast), parasite (including protozoa and ectoparasite), insect, other animal and plant cells.

Preferably, a recombinant cell is transfected with a recombinant molecule of the present invention is a molecule that can include at least one of any nucleic acid molecule heretofore described operatively linked to at least one of any transcription control sequence capable of effectively regulating expression of the nucleic acid molecule(s) in the cell to be transformed, examples of which are disclosed herein. A particularly preferred recombinant molecule includes pVL-nhFcRa, 2 Details regarding the production of FcR molecule nucleic acid molecule-containing recombinant molecules are disclosed herein. Particularly preferred recombinant cell of the present invention includes Trichoplusia ni-pVL-nhFcRca 61 2 A Fc,R molecule of the present invention can include chimeric molecules comprising a portion of a Fc,R molecule that binds to an IgE and a second molecule that enables the chimeric molecule to be bound to a substrate in such a manner that the FcR portion binds to IgE in essentially the same manner as a FcR molecule that is not bound to a substrate. An example of a suitable second molecule includes a portion of an immunoglobulin molecule.

A FcR molecule of the present invention can be contained in a formulation, herein referred to as a FcR formulation. For example, a FcCR can be combined with a buffer in which the FcR is solubilized, and/or a carrier. Suitable buffers and carriers are known to those skilled in the art. Examples of suitable buffers include any buffer in which a FcR can function to selectively bind to IgE, such as, but not limited to, phosphate buffered saline, water, saline, phosphate buffer, bicarbonate buffer, HEPES buffer (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid buffered saline), TES buffer (Tris-EDTA buffered saline); Tris buffer and TAE buffer (Tris-acetate-EDTA).

Examples of carriers include, but are not limited to, polymeric matrices, toxoids, and serum albumins, such as bovine serum albumin. Carriers can be in mixed with 'c R or conjugated attached) to FcR in such a manner as to not substantially interfere with the ability of the Fc,R to selectively bind to IgE.

A FcR of the present invention can be bound to the surface of a cell expressing the FcR. A preferred Fc,R-bearing cell includes a recombinant cell expressing a nucleic acid molecule encoding a human Fc,R alpha chain of the present invention. A more preferred recombinant cell of the present invention expresses a nucleic acid molecule that encodes at least one of the following proteins: PhFcR o and PhFcRa 23 2 An even more preferred recombinant cell expresses a nucleic acid molecule including nhFcRa 612 nhFcRas 5 9 nhFcRa699 and/or nhFcRa,,, with a recombinant cell expressing a nucleic acid molecule comprising a nucleic acid sequence including SEQ ID NO: or SEQ ID NO:4, or a nucleic acid molecule comprising an allelic variant of a nucleic acid molecule comprising SEQ ID NO: 1 or SEQ ID NO:4, being even more preferred. An even more preferred recombinant cell is a RBL-hFc,R cell.

In addition, a FceR formulation of the present invention can include not only a FcR but also one or more additional antigens or antibodies useful in detecting IgE. As used herein, an antigen refers to any molecule capable of being selectively bound by an antibody. As used herein, specific binding of a first molecule to a second molecule refers to the ability of the first molecule to preferentially bind having higher affinity higher avidity) to the second molecule when compared to the ability of a first molecule to bind to a third molecule. The first molecule need not necessarily be the natural ligand of the second molecule. Examples of such antibodies include, but are not limited to, antibodies that bind selectively to the constant region of an IgE heavy anti-IgE isotype antibody) or antibodies that bind selectively to an IgE having a specific antigen specificity anti-IgE idiotypic antibody). Examples of such antigens include any antigen known to induce the production of IgE. Preferred antigens include allergens and parasite antigens. Allergens of the present invention are preferably derived from fungi, trees, weeds, shrubs, grasses, wheat, corn, soybeans, rice,.eggs, milk, cheese, bovines (or cattle), poultry, swine, sheep, yeast, fleas, flies, mosquitos, mites, midges, biting gnats, lice, bees, wasps, ants, true bugs or ticks. A suitable flea allergen includes an allergen derived from a flea, in particular flea saliva antigen. A preferred flea allergen includes a flea saliva antigen Preferred fP-a saliva antigens include antigens such as those disclosed in PCT Patent Publication No. WO 96/11271, published April 18, 1996, by Frank et al. (this publication is incorporated by reference herein in its entirety), with flea saliva products and flea saliva proteins being particularly preferred.

According to the present invention, a flea saliva protein includes a protein produced by recombinant DNA methods, as well as proteins isolated by other methods disclosed in PCT Patent Publication No. WO 96/11271.

Preferred general allergens include those derived from grass, Meadow Fescue, Curly Dock, plantain, Mexican Firebush, Lamb's Quarters, pigweed, ragweed, sage, elm, cocklebur, Box Elder, walnut, cottonwood, ash, birch, cedar, oak, mulberry, cockroach, Dermataphagoides, Alternaria, Aspergillus, Cladosporium, Fusarium, Helminthosporium, Mucor, Penicillium, Pullularia, Rhizopus and/or Tricophyton. More preferred general allergens include those derived from Johnson Grass,.Kentucky Blue Grass, Meadow Fescue, Orchard Grass, Perennial Rye Grass, Redtop Grass, Timothy Grass, Bermuda Grass, Brome Grass, Curly Dock, English Plantain, Mexican Firebush, Lamb's Quarters, Rough Pigweed Short Ragweed, Wormwood Sage, American Elm, Common Cocklebur, Box Elder, Black Walnut, Eastern Cottonwood, Green Ash, River Birch, Red Cedar, Red Oak, Red Mulberry, Cockroach, Dermataphagoidesfarinae, Alternaria alternata, Aspergillusfumigatus, Cladosporium herbarum, Fusarium vasinfectum, Helminthosporium sativum, Mucor recemosus, Penicillium notatum, "Pullularia pullulans, Rhizopus nigricans and/or Tricophyton spp. Preferred tropical allergens include those derived from Bermuda Grass, June Bluegrass, Annual Bluegrass, Orchard Grass, Perennial Rye Grass, Timothy Grass, Meadow Fescue, Common Cocklebur, Yellow Dock, Sheep Sorrel, English Plantain, Lamb's Quarters, Rough Pigweed, Russian Thistle, Short Ragweed, Red Cedar, Cat Epithelium, Arizona Cypress, Bald Cypress, Date Palm, Australian Pine, Eucalyptus, Mango, Acacia, Grama Grass, Nettle, Western Cottonwood, Saltgrass, Dermataphagoides pteronyssinus, Aureobasidium pullans, Penicillium notatum, Penicillium chrysogenum, Drechslera '.sorokiniana, Fusarium roseum, Cladosporium sphaerospermum, Aspergillusfumigatus, S 25 Alernaria tenuis Dermataphagoidesfarinae and Stemphyllium sarciniforme. Preferred desert allergens include those derived from Bahia Grass, Smooth Brome, Johnson Grass, Redtop Grass, Fa' e .agweed, Carelessweed, Greasewood, Rough Marsh Elder, Kochia, Tall Ragweed, Western Ragweed, Slender Ragweed, Common Sage, Prairie Sage, Mugwort Sage and Shadscale. Preferred parasite antigens include, but are not limited to, helminth antigens, in particular heartworm antigens, such as Di33 (described in U.S.

Patent Application Serial No. 08/715,628, filed September 18, 1996, to Grieve et al.).

-11- The term "derived from" refers to a natural allergen of such plants or organisms an allergen directly isolated from such plants or organisms), as well as, non-natural allergens of such plants or organisms that possess at least one epitope capable of eliciting an immune response against an allergen produced using recombinant DNA technology or by chemical synthesis).

The present invention also includes human Fc,R mimetopes and use thereof to detect IgE. In accordance with the present invention, a "mimetope" refers to any compound that is able to mimic the ability of a FcR molecule to bind to IgE. A mimetope can be a peptide that has been modified to decrease its susceptibility to degradation but that still retains IgE-binding activity. Other examples of mimetopes include, but are not limited to, carbohydrate-based compounds, lipid-based compounds, nucleic acid-based compounds, natural organic compounds, synthetically derived organic compounds, anti-idiotypic antibodies and/or catalytic antibodies, or fragments thereof. A mimetope can be obtained by, for example, screening libraries of synthetic compounds for compounds capable of binding to IgE. A mimetope can also be obtained by, for example, rational drug design. In a rational drug design procedure, the three- .dimensional structure of a compound of the present invention can be analyzed by, for example, nuclear magnetic resonance (NMR) or x-ray crystallography. The threedimensional structure can then be used to predict structures of potential mimetopes by, for example, computer modeling. The predicted mimetope structures can then be produced by, for example, chemical synthesis, recombinant DNA technology, or by isolating a mimetope from a natural source. Specific examples of FcR mimetopes include anti-idiotypic antibodies, oligonucleotides produced using Selex technology, Speptides identified by random screening of peptide libraries and proteins identified by phage display technology.

ne embodiment of the present invention is a method to detect non-human IgE which includes the steps of: contacting an isolated human Fc receptor (FceR) molecule with a putative IgE-containing composition under conditions suitable for formation of an FceR molecule:IgE complex; and determining levels of IgE by detecting said FcR molecule:IgE complex. Presence of such a FcR molecule:IgE complex indicates that the animal is producing IgE. Preferred non-human IgE to detect -12using a human FcR molecule include canine IgE, feline IgE and equine IgE. The present method can further include the step of determining whether an IgE complexed with a Fc,R molecule is heat labile. Methods to determine heat lability of IgE are disclosed in the Examples section. Preferably, an IgE is heat labile when incubated at about 56°C for about 4 hours. Without being bound by theory, Applicants believe that heat labile forms of IgE bind to certain allergens and non-heat labile forms of IgE bind to other types of allergens. As such, detection of heat labile IgE compared with non-heat labile IgE can be used to discriminate between allergen sensitivities. For example, Applicants believe that IgE antibodies that bind to certain flea allergens and heartworm allergens are heat labile while IgE antibodies that bind to certain plant allergens are not heat labile. Thus, the presence of non-heat labile IgE can indicate that an animal is sensitive to certain plant allergens but not to certain flea or heartworm allergens.

Moreover, Applicants believe that identification of heat labile IgE and non-heat labile IgE using a human FcR suggests the presence of different sub-populations of IgE that 15 may or may not have substantially similar structures to known IgE. As such, a FceR molecule of the present invention may be useful for detecting molecules bound by the .*.FceR molecule but not identical to a known IgE.

As used herein, canine refers to any member of the dog family, including domestic dogs, wild dogs and zoo dogs. Examples of dogs include, but are not limited to, domestic dogs, wild dogs, foxes, wolves, jackals and coyotes. As used herein, a feline refers to any member of the cat family, including domestic cats, wild cats and zoo cats. Examples of cats include, but are not limited to, domestic cats, lions, tigers, leopards, panthers, cougars, bobcats, lynx, jaguars, cheetahs, and servals. As used herein, equine refers to any member of the horse family, including horses, donkeys, mules and zebras.

As used herein, the term "contacting" refers to combining or mixing, in this case a putative IgE-containing composition with a human Fc,R molecule. For nation of a complex between a Fc,R and an IgE refers to the ability of the FcR to selectively bind to the IgE in order to form a stable complex that can be measured detected). As used herein, the term selectively binds to an IgE refers to the ability of a Fc,R of the present invention to preferentially bind to IgE, without being able to substantially bind to other antibody isotypes. Binding between a FcR and an IgE is effected under conditions suitable to form a complex; such conditions appropriate concentrations, buffers, temperatures, reaction times) as well as methods to optimize such conditions are known to those skilled in the art, and examples are disclosed herein. Examples of complex formation conditions are also disclosed in, for example, in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Labs Press, 1989, the reference Sambrook et al., ibid., is incorporated by reference herein in its entirety.

As used herein, the term "detecting complex formation" refers to determining if any complex is formed, assaying for the presence existence) of a complex. If complexes are formed, the amount of complexes formed can, but need not be, determined. Complex formation, or selective binding, between FcR and any IgE in the composition can be measured detected, determined) using a variety of methods standard in the art (see, for example, Sambrook et al. ibid.), examples of which are disclosed herein.

In one embodiment, a putative IgE-containing composition of the present method includes a biological sample from an animal. A suitable biological sample includes, but is not limited to, a bodily fluid composition or a cellular composition. A bodily fluid refers to any fluid that can be collected obtained) from an animal, examples of which include, but are not limited to, blood, serum, plasma, urine, tears, aqueous humor, central nervous system fluid (CNF), saliva, lymph, nasal secretions, milk and feces.

o* oS Such a composition of the present method can, but need not be, pretreated to remove at least some of the non-IgE isotypes of immunoglobulin and/or other proteins, such as albumin, present in the fluid. Such removal can include, but is not limited to, contacting the bodily fluid with a material, s-uch as Protein G, to remove IgG antibodies and/or affinity purifying IgE antibodies from other components of the body fluid by exposing the fluid to, for example, Concanavalin A. In another embodiment, a composition includes collected bodily fluid that is pr'tr,-ated to concentrate immunoglobulin contained in the fluid. For example, immunoglobulin contained in a bodily fluid can be precipitated from other proteins using ammonium sulfate. A preferred composition of the present method is serum.

In another embodiment, a composition of the present method includes an IgEproducing cell. Such a cell can have IgE bound to the surface of the cell and/or can secrete IgE. Examples of such cells include basophil cells and myeloma cells. IgE can be bound to the surface of a cell either directly to the membrane of a cells or bound to a molecule an antigen) bound to the surface of the cell.

A complex can be detected in a variety of ways including, but not limited to use of one or more of the following assays: an enzyme-linked immunoassay, a radioimmunoassay, a fluorescence immunoassay, a chemiluminescent assay, a lateral flow assay, an agglutination assay, a particulate-based assay using particulates such as, but not limited to, magnetic particles or plastic polymers, such as latex or polystyrene beads), an immunoprecipitation assay, a BioCoreTM assay using colloidal gold) and an immunoblotting assay a western blot). Such assays are well known to those skilled in the art. Assays can be used to give qualitative or quantitative results depending on how they are used. Some assays, such as agglutination, particulate separation, and immunoprecipitation, can be observed visually either by eye or by a machines, such as a densitometer or spectrophotometer) without the need for a detectable marker. In other assays, conjugation attachment) of a detectable marker to the FccR or to a reagent that selectively binds to the FcR or to the IgE being detected (described in more detail below) aids in detecting complex formation. Examples of detectable markers include, but are not limited to, a radioactive label, a fluorescent label, S a chemiluminescent label, a chromophoric label or a ligand. A ligand refers to a molecule that binds selectively to another molecule. Preferred detectable markers include, but are not limited to, fluorescein, a radioisotope, a phosphatase alkaline phosphatase), biotin, avidin, a peroxidase horseradish peroxidase) and biotinrelated compounds or avidin-related compounds streptavidin or ImmunoPure® NeutrAvidin). Preferably, biotin is conjugated to an alpha chain of a FcR. Preferably a carbohytra'e group of the FcR alpha chain is conjugated to biotin. A preferred FcR molecule conjugated to biotin comprises PhFc,Rat,2-BIOT (the production of which is described in the Examples section).

In one embodiment, a complex is detected by contacting a putative IgEcontaining composition with a FceR molecule that is conjugated to a detectable marker.

A suitable detectable marker to conjugate to a Fc,R molecule includes, but is not limited to, a radioactive label, a fluorescent label, a chemiluminescent label or a chromophoric label. A detectable marker is conjugated to a Fc,R molecule or a reagent in such a manner as not to block the ability of the FcR or reagent to bind to the IgE being detected. Preferably, a carbohydrate group of a FceR is conjugated to biotin.

In another embodiment, a FcR molecule:IgE complex is detected by contacting a putative IgE-containing composition with a Fc,R molecule and then contacting the complex with an indicator molecule. Suitable indicator molecules of the present invention include molecules that can bind to either the Fc,R molecule or to the IgE antibody. As such, an indicator molecule can comprise, for example, a FcR molecule, an antigen, an antibody and a lectin, depending upon which portion of the FcR molecule:IgE complex being detected. Preferred identifying labeled compounds that are antibodies include, for example, anti-IgE antibodies and anti-FcR antibodies. Preferred lectins include those lectins that bind to high-mannose groups. More preferred lectins bind to high-mannose groups present on a FcR molecule of the present invention produced in insect cells. An indicator molecule itself can be attached to a detectable marker of the present invention. For example, an antibody can be conjugated to biotin, horseradish peroxidase, alkaline phosphatase or fluorescein.

In one preferred embodiment, a FcR molecule:IgE complex is detected by contacting the complex with a reagent that selectively binds to a FcR molecule of the present invention. Examples of such a reagent includes, but are not limited to, an antibody that selectively binds to a Fc,R molecule (referred to herein as an anti-Fc,R antibody) or a compound that selectively binds to a detectable marker conjugated to a FcR molecule. FcR molecules conjugated to biotin are preferably detected using streptavidin, more preferably using7ImmunoPure® NeutrAvidin (available from Pierce, Rockford, IL).

In another preferred embodiment, a FcR molecule:IgE complex is detected by contacting the complex with a reagent that selectively binds to an IgE antibody (referred to herein as an anti-IgE reagent). Examples of such an anti-IgE reagent include, but are not limited to, a secondary antibody that is an anti-isotype antibody an antibody that selectively binds to the constant region of an IgE), an antibody-binding bacterial -16surface protein Protein A or Protein an antibody-binding cell a B cell, a T cell, a natural killer cell, a polymorphonuclear leukocyte cell, a monocyte cell or a macrophage cell), an antibody-binding eukaryotic cell surface protein an Fc receptor), and an antibody-binding complement protein. Preferred anti-IgE reagents include, but are not limited to, D9, and CMI antibody CMI antibody #19, CMI antibody #59 and CMI antibody #71 (available from Custom Monoclonal International, West Sacramento, CA). In particular, as used herein, an anti-IgE antibody includes not only a complete antibody but also any subunit or portion thereof that is capable of selectively binding to an IgE heavy chain constant region. For example, a portion of an anti-IgE reagent can include an Fab fragment or a F(ab') 2 fragment, which are described in detail in Janeway et al., in Immunobiology, the Immune System in Health and Disease, Garland Publishing, Inc., NY, 1996 (which is incorporated herein by this reference in its entirety).

In one embodiment a complex can be formed and detected in solution. In another embodiment, a complex can be formed in which one or more members of the complex are immobilized on coated onto) a substrate. Immobilization techniques are known to those skilled in the art. Suitable substrate materials include, but are not limited to, plastic, glass, gel, celluloid, paper, PVDF (poly-vinylidene-fluoride), nylon, nitrocellulose, and particulate materials such as latex, polystyrene, nylon, nitrocellulose, 20 agarose and magnetic resin. Suitable shapes for substrate material include, but are not limited to, a well microtiter dish well), a plate, a dipstick, a bead, a lateral flow apparatus, a membrane, a filter, a tube, a dish, a celluloid-type matrix, a magnetic particle, and other particulates. A particularly preferred substrate comprises an ELISA plate, a dipstick, a radioimmunoassay plate, agarose beads, plastic beads, latex beads, immunoblot membranes and immunoblot papers. In one embodiment, a substrate, such as a particulate, can include a detectable marker.

A preferred method to detect IgE is an immunosorber' a, say. An immunoabsorbent assay of the present invention comprises a capture molecule and an indicator molecule. A capture molecule of the present invention binds to an IgE in such a manner that the IgE is immobilized to a substrate. As such, a capture molecule is preferably immobilized to a substrate of the present invention prior to exposure of the capture molecule to a putative IgE-containing composition. An indicator molecule of the present invention detects the presence of an IgE bound to a capture molecule. As such, an indicator molecule preferably is not immobilized to the same substrate as a capture molecule prior to exposure of the capture molecule to a putative IgE-containing composition.

A preferred immunoabsorbent assay method includes a step of either: binding an FcR molecule to a substrate prior to contacting a FcR molecule with a putative IgEcontaining composition to form a FceR molecule-coated substrate; or binding a putative IgE-containing composition to a substrate prior to contacting a FcR molecule with a putative IgE-containing composition to form a putative IgE-containing composition-coated substrate. Preferably, the substrate includes of a non-coated substrate, a FcR molecule-coated substrate, an antigen-coated substrate or an anti-IgE antibody-coated substrate.

Both a capture molecule and an indicator molecule of the present invention are capable of binding to an IgE. Preferably, a capture molecule binds to a different region of an IgE than an indicator molecule, thereby allowing a capture molecule to be bound to an IgE at the same time as an itidicator molecule. The use of a reagent as a capture molecule or an indicator molecule depends upon whether the molecule is immobilized to a substrate when the molecule is exposed to an IgE. For example, a Fc,R molecule of the present invention is used as a capture molecule when the FcR molecule is bound to a substrate. Alternatively, a Fc,R molecule is used as an indicator molecule when the Fc,R molecule is not bound to a substrate. Suitable molecule for use as capture molecules or indicator molecules include, but are not limited to, a FcER molecule of the present invention, an antigen reagent or an anti-IgE antibody reagent of the present S 25 invention.

9 An immunoabsorbent assay of the present invention can further comprise one or more layers and/or types of secin' iary molecules or other binding molecules capable of detecting the presence of an indicator molecule. For example, an untagged not conjugated to a detectable marker) secondary antibody that selectively binds to an indicator molecule can be bound to a tagged conjugated to a detectable marker) tertiary antibody that selectively binds to the secondary antibody. Suitable secondary -18antibodies, tertiary antibodies and other secondary or tertiary molecules can be selected by those of skill in the art. Preferred secondary molecules of the present invention include, an antigen, an anti-IgE idiotypic antibody and an anti-IgE isotypic. Preferred tertiary molecules can be selected by a skilled artisan based upon the characteristics of the secondary molecule. The same strategy is applied for subsequent layers.

In one embodiment, a desired antigen is used as a capture molecule by being immobilized on a substrate, such as a microtiter dish well or a dipstick. Preferred antigens include those disclosed herein. A biological sample collected from an animal is applied to the substrate and incubated under conditions suitable sufficient) to allow for antigen:IgE complex formation bound to the substrate IgE in a sample binds to an antigen immobilized on a substrate). Excess non-bound material material from the biological sample that has not bound to the antigen), if any, is removed from the substrate under conditions that retain antigen:IgE complex binding to the substrate.

Preferred conditions are disclosed herein in the Examples section and generally in Sambrook et al., ibid. An indicator molecule that can selectively bind to an IgE bound to the antigen, the indicator molecule can be conjugated to a detectable marker (preferably to an enzyme label, to a colorimetric label, to a fluorescent label, to a radioisotope, or to a ligand such as of the biotin or avidin family), is added to the substrate and incubated to :o allow formation of a complex between the indicator molecule and the antigen:IgE 20 complex. Excess indicator molecule is removed, a developing agent is added if required, and the substrate is submitted to a detection device for analysis. A preferred indicator molecule for this embodiment is a FcR molecule, preferably conjugated to biotin, to a fluorescent label or to an enzyme label.

In one embodiment, a FcR molecule is used as a capture molecule by being 25 immobilized on a substrate, such as-a microtiter dish well or a dipstick. A biological sample collected from an animal is applied to the substrate and incubated under 'orditions suitable to allow for FceR molecule:IgE complex formation bound to the substrate. Excess non-bound material, if any, is removed from the substrate under conditions that retain FcR molecule:IgE complex binding to the substrate. An indicator molecule that can selectively bind to an IgE bound to the FcR is added to the substrate and incubated to allow formation of a complex between the indicator molecule and the FcR molecule:IgE complex. Preferably, the indicator molecule is conjugated to a detectable marker (preferably to an enzyme label, to a colorimetric label, to a fluorescent label, to a radioisotope, or to a ligand such as of the biotin or avidin family). Excess indicator molecule is removed, a developing agent is added if required, and the substrate is submitted to a detection device for analysis. A preferred indicator molecule for this embodiment is an antigen that will bind to IgE in the biological sample or an anti-IgE isotype or idiotype antibody, either preferably being conjugated to fluorescein or biotin.

In one embodiment, an anti-IgE antibody isotype or idiotype specific antibody) is used as a capture molecule by being immobilized on a substrate, such as a microtiter dish well or a dipstick. A biological sample collected from an animal is applied to the substrate and incubated under conditions suitable to allow for anti-IgE antibody:IgE complex formation bound to the substrate. Excess non-bound material, if any, is removed from the substrate under conditions that retain anti-IgE antibody:IgE complex binding to the substrate. A FcR molecule is added to the substrate and incubated to allow formation of a complex between the FcR molecule and the anti-IgE antibody:IgE complex. Preferably, the FcR molecule is conjugated to a detectable marker (preferably to biotin, an enzyme label or a fluorescent label). Excess FcR molecule is removed, a developing agent is added if required, and the substrate is submitted to a detection device for analysis.

In one embodiment, an immunosorbent assay of the present invention does not *utilize a capture molecule. In this embodiment, a biological sample collected from an animal is applied to a substrate, such as a microtiter dish well or a dipstick, and incubated under conditions suitable to allow for IgE binding to the substrate. Any IgE present in the bodily fluid is immobilized on the substrate. Excess non-bound material, if any, is removed from the substrate under conditions that retain IgE binding to the o substrate. A FcR molecule is added to the substrate and incubated to allow formation of a complex between the FcCR molecule and the IgE. Preferably, the FcR molecule iconjugated to a detectable marker (preferably to biotin, an enzyme label or a fluorescent label). Excess FcR molecule is removed, a developing agent is added if required, and the substrate is submitted to a detection device for analysis.

Another preferred method to detect IgE is a lateral flow assay, examples of which are disclosed in U.S. Patent No. 5,424,193, issued June 13, 1995, by Pronovost et al.; U.S. Patent No. 5,415,994, issued May 16, 1995, by Imrich et al; WO 94/29696, published December22, 1994, by Miller et al.; and WO 94/01775, published January 1994, by Pawlak et al.; each of these patent publications is incorporated by reference herein in its entirety. In one embodiment, a biological sample is placed in a lateral flow apparatus that includes the following components: a support structure defining a flow path; a labeling reagent comprising a bead conjugated to an antigen, the labeling reagent being impregnated within the support structure in a labeling zone; and a capture reagent comprising an IgE-binding composition. Preferred antigens include those disclosed herein. The capture reagent is located downstream of the labeling reagent within a capture zone fluidly connected to the labeling zone in such a manner that the labeling reagent can flow from the labeling zone into the capture zone. The support structure comprises a material that does not impede the flow of the beads from the labeling zone to the capture zone. Suitable materials for use as a support structure include ionic anionic or cationic) material. Examples of such a material include, but are not limited to, nitrocellulose PVDF, carboxymethylcellulose The support structure defines a flow path that is lateral and is divided into zones, namely a labeling zone and a capture zone. The apparatus can further comprise a sample 20 receiving zone located along the flow path, more preferably upstream of the labeling reagent. The flow path in the support structure is created by contacting a portion of the support structure downstream of the capture zone, preferably at the end of the flow path, to an absorbent capable of absorbing excess liquid from the labeling and capture zones.

In this embodiment, the biological sample is applied to the sample receiving zone 25 which includes a portion of the sup-port structure. The labeling zone receives the sample from the sample receiving zone which is directed downstream by the flow path. The S labeling zone comprises the labeling reagent that bin s 'o IgE. A preferred labeling reagent is an antigen conjugated, either directly or through a linker, to a plastic bead substrate, such as to a latex bead. The substrate also includes a detectable marker, preferably a colorimetric marker. Typically, the labeling reagent is impregnated to the support structure by drying or lyophilization. The sample structure also comprises a capture zone downstream of the labeling zone. The capture zone receives labeling reagent from the labeling zone which is directed downstream by the flow path. The capture zone contains the capture reagent, in this case a Fc,R molecule, as disclosed above, that immobilizes the IgE complexed to the antigen in the capture zone. The capture reagent is preferably fixed to the support structure by drying or lyophilizing.

The labeling reagent accumulates in the capture zone and the accumulation is assessed visually or by an optical detection device.

In another embodiment, a lateral flow apparatus used to detect IgE includes: a support structure defining a flow path; a labeling reagent comprising a Fc,R molecule as described above, the labeling reagent impregnated within the support structure in a labeling zone; and a capture reagent comprising an antigen, the capture reagent being located downstream of the labeling reagent within a capture zone fluidly connected to the labeling zone in such a manner that the labeling reagent can flow from the labeling zone into the capture zone. The apparatus preferably also includes a sample receiving zone located along the flow path, preferably upstream of the labeling reagent.

The apparatus preferably also includes an absorbent located at the end of the flow path.

One embodiment of the present invention is an inhibition assay in which the V presence of IgE in a putative IgE-containing composition is determined by adding such composition to a FcR molecule of the present invention and an isolated IgE known to 20 bind to the Fc,R molecule. The absence of binding of the Fc,R molecule to the known SIgE indicating the presence of IgE in the putative IgE-containing composition.

The present invention also includes kits to detect IgE based on each of the disclosed detection methods. One embodiment is a kit to detect IgE comprising a human -0 *Fc receptor (FcR) molecule and a means for detecting an IgE including canine IgE, 25 feline IgE and/or equine IgE. Suitable and preferred Fc,R molecules are disclosed herein. Suitable means of detection include compounds disclosed herein that bind to either the Fc,R molecrle or to an IgE. A preferred kit of the present invention further comprises a detection means including one or more antigens disclosed herein, an antibody capable of selectively binding to an IgE disclosed herein and/or a compound capable of binding to a detectable marker conjugated to a Fc,R molecule avidin, streptavidin and ImmunoPure® NeutrAvidin when the detectable marker is biotin).

-22- Such antigens preferably induce IgE antibody production in animals including canines, felines and/or equines.

A preferred embodiment of a kit of the present invention is a flea allergen kit comprising a flea allergen such as those disclosed herein. A particularly preferred flea allergen for use with a flea allergen kit includes a flea saliva product or a flea saliva protein.

Another preferred kit of the present invention is a general allergen kit comprising an allergen common to all regions of the United States and a human FcR molecule of the present invention. As used herein, a "general allergen" kit refers to a kit comprising allergens that are found substantially throughout the United States essentially not limited to certain regions of the United States). A general allergen kit provides an advantage over regional allergen kits because a single kit can be used to test an animal located in most geographical locations on the United States. Suitable and preferred general allergens for use with a general allergen kit of the present invention include those general allergens disclosed herein.

Another preferred kit of the present invention is a food allergen kit comprising a o food allergen including beef, chicken, pork, a mixture of fish, such as cod, halibut or and tuna, egg, milk, Brewer's yeast, whole wheat, corn, soybean, cheese and rice, and a human Fc R molecule of the present invention. Preferably, the beef, chicken, pork, fish, 20 corn and rice, are cooked.

A preferred kit of the present invention includes those in which the allergen is immobilized to a substrate. If a kit comprises two or more antigens; the kit can comprise one or more compositions, each composition comprising one antigen. As such, each antigen can be tested separately. A kit can also contain two or more diagnostic reagents 25 for IgE, additional isolated IgE antigens and/or antibodies as disclosed herein.

Particularly preferred are kits used in a lateral flow assay format. It is within the scope S.of the present invention that a lateral flow assay kit can include one or more lateral flow assay apparatuses. Multiple lateral flow apparatuses can be attached to each other at one end of each apparatus, thereby creating a fan-like structure.

In particular, a method and kit of the present invention are useful for diagnosing abnormal conditions in animals that are associated with changing levels of IgE.

-23- Particularly preferred conditions to diagnose include allergies, parasitic infections and neoplasia. For example, a method and kit of the present invention are particularly useful for detecting flea allergy dermatitis (FAD), when such method or kit includes the use of a flea saliva antigen. FAD is defined as a hypersensitive response to fleabites.

Preferably, a putative IgE-containing composition is obtained from an animal suspected of having FAD. Preferred animals include those disclosed herein, with dogs and cats being more preferred. In addition, methods and kits of the present invention are particularly useful for detecting helminth infection, in particular heartworm infection, when such methods or kits include the use of a helminth antigen, such as Di33.

Preferably, a putative IgE-containing composition is obtained from an animal suspected of having a helminth infection. Preferred animals include those disclosed herein, with dogs and cats being more preferred.

The following examples are provided for the purposes of illustration and are not intended to limit the scope of the present invention.

Examples Example 1.

This example describes the construction of a recombinant baculovirus expressing a truncated portion of the a-chain of the human Fc, receptor.

Recombinant molecule pVL-nhFcRa 6 12 containing a nucleic acid molecule 20 encoding the extracellular domain of the FcR a chain, operatively linked to baculovirus polyhedron transcription control sequences was produced in the following manner. A cDNA clone encoding the full-length alpha chain (a chain) of the human Fc, receptor was obtained from Dr. Jean-Pierre Kinet (Harvard University, Cambridge, MA). The cDNA clone included an about 1198 nucleotide insert, referred to herein as nhFcRa,,g.

The nucleic acid sequence of the-coding strand of nhFcRa,, 98 is denoted herein as SEQ ID NO: 1. Translation of SEQ ID NO:1 indicates that nucleic acid molecule nhFcRaI ,g encodes a full-length human Fc. receptor a chain protein of about 257 am;no acids, referred to herein as PhFcRa2z 7 having amino acid sequence SEQ ID NO:2, assuming an open reading frame in which the initiation codon spans from nucleotide 107 through nucleotide 109 of SEQ ID NO:1 and the termination codon spans from nucleotide 878 through nucleotide 880 of SEQ ID NO: 1. The complement of SEQ ID NO: 1 is -24represented herein by SEQ ID NO:3. The proposed mature protein FcRa chain from which the signal sequence has been cleaved), denoted herein as PhFcRa 2 32 contains about 232 amino acids which is represented herein as SEQ ID NO:6. The nucleic acid molecule encoding PhFcRa 23 2 is denoted herein as nhFc,Ra,,, the coding strand of which is represented by SEQ ID NO:7.

To produce a secreted form of the extracellular domain of the Fc,R a chain, the hydrophobic transmembrane domain and the cytoplasmic tail of the FcER a chain encoded by nhFcRao 1 9 were removed as follows. A FcR a chain extracellular domain nucleic acid molecule-containing fragment of about 612 nucleotides was PCR amplified from nhFcRai 9 using a forward primer EJH 040 containing a BamHI site, having the nucleic acid sequence 5' CGC GGA TCC TAT AAA TAT GGC TCC TGC CAT GG 3' (denoted SEQ ID NO:8) and a reverse primer IgE ANTI-SENSE containing an EcoRI site, having the nucleic acid sequence 5' GGC GAA TTC TTA AGC TTT TAT TAC AG 3' (denoted herein as SEQ ID NO:9). The resulting PCR product was digested with BamHI and EcoRI to produce nhFceRa 6 Nucleic acid molecule nhFcRa 61 2 contained an about 591 nucleotide fragment encoding the extracellular domain of the human FcR a chain, extending from nucleotide 107 to nucleotide 697 of SEQ ID NO 1, denoted herein as nucleic acid molecule nhFcRa,,,, the coding strand of which has a nucleic acid sequence denoted SEQ ID NO: 10. Translation of SEQ ID NO: 10 indicates that 20 nucleic acid molecule nhFcRa 6 2 encodes a Fc,R protein of about 197 amino acids, Sreferred to herein as PhFc.Ra197, having amino acid sequence SEQ ID NO: 11. Nucleic acid molecule nhFcRa61 2 encodes a secretable form of the human FcR a chain which does not possess a leader sequence, which is denoted herein as PhFcRaz 7 2 having amino acid sequence SEQ ID NO:13. The coding region for PhFcRa,,, is denoted 25 nhFcRRasi 6 the coding strand of which has a nucleic acid sequence denoted SEQ ID NO: 12. The complement of SEQ ID NO:12 is represented herein by SEQ ID NO: 14.

In order to produce a baculovirus recrnrbinant molecule capable of directing the production of PhFcRa,97, the nucleic acid molecule nhFcRa 6 1 2 was subcloned into unique BamHI and EcoRI sites of pVL1392 baculovirus shuttle plasmid (available from Pharmingen, San Diego, CA) to produce a recombinant molecule referred to herein as pVL-nhFcRa 61 2 The resultant recombinant molecule pVL-nhFcRa 6 12 was verified for proper insert orientation by restriction mapping.

Example 2.

This example describes the production of PhFceRa 72 protein.

The recombinant molecule pVL-nhFcRa 2 was co-transfected with a linear Baculogold baculovirus DNA (available from Pharmingen) into Trichoplusia ni cells (available from Invitrogen Corp., San Diego, CA; High FiveTM cells) using the following method. About 1.5 liter cultures of serum-free ex-Cell Medium (available from Invitrogen) were seeded with about I x 106 cells per ml of medium. The Trichoplusia ni cells were infected with recombinant molecule pVL-nhFcRa6i 2 at a multiplicity of infection (MOI) of about 2 to about 5 particle forming units (pfu) per cell to produce recombinant cell Trichoplusia ni-pVL-nhFc,Ra 6 2 The infection was allowed to proceed at a controlled temperature of 27 °C for 48 hours, to produce recombinant protein PhFcRa,,2. Following infection, cells were separated from the medium by centrifugation, and the medium was frozen at -70 C.

PhFcRca 72 was purified from the culture medium described immediately above by affinity chromatography using an IgE antibody produced by the myeloma cell line U266DI (American Tissue Type Catalogue No. TIB 196) linked to sepharose 4B. The amino acid composition and N-terminal amino acid sequence of the affinity purified 20 PhFcRa, 7 were determined using methods standard in the art. The results indicated that PhFcRa 7was properly processed by the Trichoplusia ni cells.

Example 3.

This example describes the biotinylation of a recombinant human Fc,Ralpha *chain protein.

S 25 Affinity purified recombinant protein PhFcRa, 7 2 prepared as described above in Example 2, was biotinylated as follows. About 440 micrograms (pg) of PhFcRa 72 were diluted n oout 1.5 milliliter (ml) of acetate buffer (0.1 M NaAc, pH containing about 200 microliter of 0.1 M NaIO 4 The mixture was incubated for about 20 minutes, on ice, and about 2 pl of glycerol was added following the incubation.

The mixture was then dialyzed against about 2 liters of acetate buffer in a 3 ml Slide-A- Lyzer cassette (available from Pierce, Rockford, IL), 2 times for about 2 hours each -26time. About 3.72 pg of biotin-LC-hydrazide (available from Pierce) was dissolved in about 200 pl of dimethylsulfoxide (DMSO) and injected into the cassette. The cassette was then rocked at room temperature for about 2 hours. Following the incubation, the mixture containing recombinant protein and biotin dialyzed 3 times, a first time for about 18 hours and two times for about 2 hours, each time at 5 0 C against phosphate buffered saline. The biotinylated protein was recovered from the dialysis, and is referred to herein as PhFccRal 72

-BIOT.

Example 4.

This example describes detection of canine IgE in a solid-phase ELISA using PhFcRa, 72

-BIOT.

Wells of two Immulon II microtiter plates (available from Dynatech, Alexandria, VA) were coated with duplicate samples of about 100 pl/well of various concentrations of purified canine IgE as denoted in Fig. 1. The canine IgE was obtained from a canine IgE producing hybridoma, such as heterohybridoma 2.39 (described in Gebhard et al., Immunology 85:429-434, 1995) and was diluted in a CBC buffer (15 mM Na 2 CO and 34.8 mM NaHCO 3 pH 9.6. The coated plates were incubated overnight at 4 C.

Following incubation, the canine IgE-containing solution was removed from each plate, S. and the plates were blotted dry. The plates were then blocked using about 200 pl/well of 0.25% bovine serum albumin (BSA) contained in phosphate buffered saline (PBSB) for 20 about 1 hour at room temperature. The plates were then washed four times with 0.05% in PBS (PBST) using an automatic washer (available from Dynatech).

Experimental samples consisting of about 100 pl/well of a 1:4000 dilution of 40 pg/ml PhFcRa, 72 -BIOT (about 145 pg/ml; described in Example contained in PBSB with 0.05%Tween-20 (PBSBT) were added to each well of one plate coated with canine IgE.

S 25 Control samples consisting of about 100 pV of biotinylated anti-canine IgE monoclonal antibody D9 (supplied by Dr. DeBoer, U. of Wisconsin, Madison, WI) were added to each well of the other plate coated with canine IgE. The plates were incubated for 1 hour at room temperature and then washed four times with PBST. About 100 pl of about 0.25 ug/ml streptavidin conjugated to horseradish peroxidase (available from Kirkegaard and Perry Laboratories (KPL), Gaithersburg, MD; diluted in PBST) was added to each well that received experimental or control samples. The plates were then -27incubated for 1 hour at room temperature and washed four times with PBST. About 100 pI of TMB substrate (available from available from KPL), that had been pre-warmed to room temperature, was added. Plates were then incubated for 10 minutes at room temperature and then about 100 pl/well of Stop Solution (available from KPL) was added. Optical densities of wells were read on a Spectramax Microtiter Plate (available from Molecular Devices Inc.) reader at 450 nm within 10 minutes of adding the stop solution.

The results shown in Fig. I indicate that the alpha chain of human Fc 6 R detects the presence of canine IgE (closed circles) in a solid-phase assay in a similar manner as the control antibody that binds specifically to canine IgE (D9; open circles).

Example This example describes detection of plant allergen-specific canine IgE using PhFcRa, 7 2

-BIOT.

Multiple wells of an Immulon II microtiter plate (available from Dynatech) were coated with either about 100 pl/well of 1 pg/ml of Kentucky Blue Grass allergen or about 100 pl/well of about 1 pg/ml of Green Ash allergen (both available from Greer Inc., Lenoir, NC) both diluted in CBC buffer. The plate was incubated overnight at 4°C.

The plate was blocked and washed as described in Example 4. Two different pools of canine sera were then added to the antigen-coated wells. The first pool consisted of sera 20 isolated from 8 dogs reported to be allergen reactive. The second pool consisted of sera isolated from 8 dogs reported to be allergen non-reactive. Each pool of sera was diluted S. 1:10 or 1:100 in PBST. About 100 pl of each concentration of each diluted sera sample was added to the wells and incubated for 1 hour at room temperature. The plate was then washed four times with PBST. About 100 pl/well of a 1:4000 dilution of 40 pg/ml 25 PhFcRa~ 7 2 -BIOT (described in Example contained in PBSBT was added to the antigen-coated wells. The plate was incubated for 1 hour at room temperature. The plate was then washed four times with PBST. About 100 pl/well of about 0.25 pg/ml of neutravidin conjugated to horseradish peroxidase (available from Pierce) contained in PBSBT, was added. The plate was incubated for 1 hour at room temperature. The plate was then washed and the presence of neutravidin bound to the plate detected using the method described in Example 4.

-28- The results shown in Fig. 2 indicate that the alpha chain of human Fc,R detects the presence of canine IgE antibodies that bind specifically to a common grass allergen or to a.common tree allergen. In addition, detection of canine IgE antibodies is dose dependent.

Example 6.

This example describes detection of total canine IgE using PhFcRaz,7-BIOT.

Multiple wells of an Immulon I microtiter plate (available from Dynatech) were coated with about 100 pl/well of about 1 pg/ml CMI anti-canine IgE antibody #6 (available from Custom Monoclonals International, West Scramento, CA) diluted in CBC buffer. The plate was incubated overnight at 4 0 C. The plate was blocked and washed as described in Example 4. About 100 pl/well of a 1:60 dilution in PBSBT of sera samples from a variety of sources were then added to multiple wells coated with anti-IgE antibody. The samples included:(1) serum from a dog known to be allergic to flea saliva; serum from dogs infected with D. immitis; and a pool of dog sera from defined as canine allergy calibrators (available from BioProducts DVM, Tempe, AZ); pools of dog sera containing antibodies that have low binding to Kentucky Blue Grass allergen; pools of dog sera that have high binding to Kentucky Blue Grass allergen; a pool of dog sera from dogs known to be allergic to flea saliva, the sample was heat inactivated (at 56"C for 4 hours); a pool of dog sera from dogs 20 known to be allergic to flea saliva; or a pool of dog sera from dogs raised in a barrier facility negative control). A set of positive control samples consisting of IgE derived from the canine heterohybridoma described in Example 4 were also added to the plate to generate a standard curve. The plate was incubated for 1 hour at room temperature and then washed four times with PBST. The presence of canine IgE was 25 detected using either about 100 pl/well of a 1:4000 dilution of 40 pg/ml PhFcRcz 72 BIOT (described in Example 3) or about 100 pl/well of about-l- g/ml CMI anti-canine IgE antibody #19 (available from Cls'om Monoclonals International), both contained in PBSBT. The plate was incubated for 1 hour at room temperature. The plate was then washed, contacted with about 0.25 ug/ml streptavidin conjugated to horseradish peroxidase, washed again, and the presence of streptavidin bound to the plate was detected using the method described in Example 4. The optical density readings -29obtained for the control samples were used to generate a standard curve that was used to determine the total IgE bound to wells that had received test samples.

The results shown in Fig. 3 indicate that canine IgE from a variety of dog sera are detected using the alpha chain of human Fc,R in a manner similar to using an antibody that binds specifically to canine IgE. The absence of detectable amounts of IgE in the heat treated sample (Sample 7) indicates that the antibody detected by PhFcRa 72

-BIOT

is IgE. In addition, the results indicate that PhFcRa 7 2 -BIOT is an effective reagent for detecting IgE that binds to allergen Kentucky Blue Grass, Samples 5 and as well as a parasite antigen Immitis, Sample 2).

Example 7.

This example describes detection of canine IgE in dog sera isolated from dogs known to be allergic to flea saliva, using PhFcRcI 7 2

-BIOT.

Multiple wells of an Immulon II microtiter plate were coated with about 100 pl/well of varying concentrations of flea saliva recombinant protein fspN (described in PCT Patent Publication No. WO 96/11271, ibid.; concentrations shown in Fig. 4) diluted in CBC buffer. The plate was incubated overnight at 4 0 C. The plate was then blocked and washed as described in Example 4. About 100 pll/well of a 1:10 dilution in PBSBT of a pool of sera isolated from dogs known to produce IgE that binds specifically to flea saliva. Some wells did not receive dog sera so that background binding levels could be 20 determined. The plate was incubated for 1 hour at room temperature and then washed four times with PBST. About 100 pl/well of a 1:4000 dilution of 40 pg/ml PhFcRa, 72 BIOT (described in Example 3) contained in PBSBT was added. The plate was incubated for 1 hour at room temperature. The plate was then washed, contacted with about 0.25 ug/ml streptavidin-conjugated to horseradish peroxidase, washed again, and 25 the presence of streptavidin bound to the plate was detected using the method described in Example 4.

o°.

I'he results shown in Fig. 4 indicate that canine IgE that binds specifically to a flea saliva antigen is detected using the alpha chain of human Fc,R.

Example 8.

This example describes detection of total capine IgE in dog sera isolated from dogs known to be allergic to flea saliva, heartworm-infected dogs and specific pathogen free (SPF) dogs, using PhFcRam-BIOT.

Multiple wells of an Immulon II microtiter plate were coated with about 100 pi/well of about 1 pg/ml CMI anti-canine IgE antibody #6 (available from Custom Monoclonals International) in CBC buffer. The plate was incubated overnight at 4 0

C.

The plate was blocked and washed as described in Example 4. About 100 pl/well of different samples of IgE-containing fluids in PBSBT were added to multiple wells coated with the anti-canine IgE antibody. The samples included: 100 pg/ml of canine IgE purified from the heterohybridoma described in Example 4; a 1:10 dilution of a pool of sera from dogs known to be allergic to flea saliva, a 1:10 dilution of the same sera pool as in but heat inactivated; a 1:10 dilution of serum from a dog known to have clinical flea allergy dermatitis (dog CPO2); a 1:10 dilution of heat inactivated CPO2 serum; a 1:10 dilution of serum from a heartworm-infected dog (dog 417); a 1:10 dilution of heat inactivated 417 serum; a 1:10 dilution of a pool of sera from heartworm-infected dogs; a 1:10 dilution of the same sera pool as Sin but heat inactivated; and (10) a pool of sera from dogs raised in a barrier facility.

Each sample was diluted in PBSBT. The plate was incubated for 1 hour at room 20 temperature and then washed four times with PBST. About 100 pl/well of a 1:4000 dilution of 40 pg/ml PhFcRam-BIOT (described in Example 3) in PBSBT was added.

The plate was incubated for 1 hour at room temperature. The plate was then washed, contacted with about 0.25 ug/ml streptavidin-conjugated to horseradish peroxidase, washed again, and the presence of streptavidin bound to the plate was detected using the 25 method described in Example 4. The results shown in Fig. 5 indicate that canine IgE from dogs allergic to flea saliva and from dogs infected with heartworm are detected using the alpha chain of human Fe.R. In addition, the absence of colorimetric signal in samples of heat inactivated sera indicates that antibody bound to the anti-IgE antibody and detected by Fc,R alpha chain is an epsilon isotype antibody and not another isotype.

-31- Example 9.

This example describes detection of IgE that specifically binds to flea saliva, using PhFc,Raz 72

-BIOT.

Multiple wells of an Immulon II microtiter plate were coated with about 100 pl/well of about 0.1 pg/ml of flea saliva collected using the method described in PCT Patent Publication No. WO 96/11271, ibid., in CBC buffer. The plate was incubated, blocked and washed as described in Example 4. The IgE-containing samples described in Example 8 were then applied to the flea saliva coated plate. The plate was then treated using the method described in Example 8.

The results shown in Fig. 6 indicate that canine IgE that binds specifically to flea saliva, contained in serum, is detected using the alpha chain of human FcR. In addition, the absence of colorimetric signal in samples of heat inactivated serum indicates that antibody bound to the flea saliva protein and detected by Fc,R alpha chain is an epsilon isotype antibody.

Example This example describes the detection of feline IgE using PhFcRa 72

-BIOT.

Multiple wells of an Immulon II microtiter plate were coated with about 100 pl/well of about 10 pg/ml Di33 protein (described in U.S. Patent Application Serial No.

08/715,628, ibid.) or 10 pg/ml crude homogenate of heartworm, both in CBC buffer.

20 Crude homogenate of heartworm is the clarafied supernatant of adult heartworms homogenized in PBS. The plate was incubated overnight at 4 0 C. The plate was blocked and washed as described in Example 4. Serum samples from 2 heartworm infected cats were then added to Di33-coated wells and to heartworm antigen-coated wells. About 100 pl/well of a 1:10 dilution in PBSBT of sera from heartworm-infected cat AXH3 or from cat #MGC2 were added to the plate. Negative control samples consisting of serum from pre-infection bleeds of cat #AXH3 and cat# MGC2 were also added to the plate at a dilution of 1:10 in PBSBT. A positive control sample rc' listing of a pool of sera from heartworm-infected dogs-was also added to the plate at a dilution of 1:10 in PBSBT.

The plate was incubated for 1 hour at room temperature and then washed four times with PBST. About 100 pl/well of a 1:4000 dilution of 40 pg/ml PhFcRa 1 7 -BIOT (described in Eiample 3) in PBSBT was added. The plate was incubated for 1 hour at room -32temperature. The plate was then washed, contacted with 1:4000 dilution of a 0.5 mg/ml solution of streptavidin-conjugated to horseradish peroxidase, washed again, and the presence of streptavidin bound to the plate was detected using the method described in Example 4.

The results shown in Fig. 7 indicate that feline IgE that binds specifically to crude homogenate of heartworm or Di33 protein is detected using the alpha chain of human FcR.

Example 11.

This example describes detection of feline IgE using PhFcRal 72

-BIOT.

Multiple wells of an Immulon II microtiter plate were coated with Di33 as described in Example 10, in CBC buffer. The plate was incubated overnight at 4 0

C.

The plate was blocked and washed as described in Example 4. Serum samples from 2 heartworm infected cats were then added to Di33-coated wells. About 100 pl/well of a 1:10 dilution in PBSBT of serum from heartworm-infected cat MGC2 and a pool of sera from heartworm-infected cats, as well as heat inactivated samples of each of these sera, were added to the plate. A positive control sample consisting of a pool of sera from heartworm-infected dogs was also added to the plate at a dilution of 1:10 in PBSBT. The plate was incubated for 1 hour at room temperature and then washed four times with PBST. About 100 pl/well of a 1:4000 dilution of 40 pg/ml PhFcRal 72

-BIOT

20 (described in Example 3) in PBSBT was added. The plate was incubated for 1 hour at room temperature. The plate was then washed, contacted with streptavidin-conjugated to horseradish peroxidase, washed again, and the presence of streptavidin bound to the plate was detected using the method described in Example 4.

The results shown in Fig. 8 indicate that feline IgE from heartworm-infected cats 25 that specifically binds to the heartworm antigen Di33 is detected using the alpha chain of human FcR. In addition, the absence of colorimetric signal in samples of heat inactivated sera indicates that antibody bound to the Di33 protein and detected by FcR alpha chain is an epsilon isotype antibody.

Example 12 This example describes detection of equine IgE in a solid-phase ELISA using PhFcRcxa 72

-BIOT.

-33- Horse sera from a horse known to be allergic to certain allergens and horse sera from a horse known not to be allergic the same allergens, were assayed for the presence of IgE using PhFcRa, 7 2 -BIOT as follows. A North Atlantic/Ohio Valley Regional Panel plate of a CanitecTM Allergen-Specific IgE Kit (available from BioProducts DVM) was blocked and washed as described in Example 4. Two samples of about 1:10 dilutions of the two horse sera were prepared using PBSBT. The two samples were added to the blocked plate and the plate was incubated for 1 hour at room temperature.

The plate was washed as described in Example 4. About 100 Vl/well of a 1:4000 dilution of 40 pg/ml PhFc.Ral 7 2 -BIOT (described in Example contained in PBSBT was added to each well. The plate was then washed, contacted with 1:4000 dilution of a mg/ml solution of streptavidin-conjugated to horseradish peroxidase, washed again, and the presence of streptavidin bound to the plate was detected using the method described in Example 4.

The results shown in Fig. 9 indicate that equine IgE from a horse known to be 15 allergic to certain allergens specifically binds to certain plant and mite allergens is detected using the alpha chain of human FceR.

S.Example 13 This example describes detection of canine IgE in a solid-phase ELISA using basophilic cells transfected with human FcR alpha chain.

20 Rat basophilic leukemia (RBL) cells transfected with a nucleic acid molecule encoding a human FcR alpha chain (referred to herein as RBL-hFc,R cells; described in Miller et al., Science 244:334-337, 1989) were used to detect canine IgE as follows.

About 4 x 104 RBL-hFcR cells contained in Earles Modified Eagles Medium containing 10% fetal bovine serum (EMEM-FBS) were added to each well of 96-well flat bottom tissue culture plates. The RBL-hFcR cells were incubated overnight at 37 0

C.

Following the incubation the plates were washed 4 times with-PBST. The cells were then fixed for about 2 minutes using about 200 pl per well of absolute alcohol at room temperature. The plates were then washed 8 times with PBST to remove residual alcohol.

Serial dilutions in EMEM-FBS (concentrations shown in Fig. 10) were prepared using a pool of sera from dogs infected with heartworm. Serial dilutions in EMEM-FBS -34- (concentrations shown in Fig. 11) were prepared using a pool of sera from dogs sensitized to flea saliva. Additional samples were prepared in which both pools of sera were heat inactivated for about 4 hours at 56°C. The heat treated samples were diluted as described above.

About 100 pl of each dilution of each serum sample was added to separate wells containing fixed RBL-hFc,R cells and the plates were incubated at 37 0 C for about 1 hour. Following the incubation, the plates were washed 4 times with PBST. About 5 pg of a murine IgG monoclonal antibody anit-canine IgE antibody Custom Monoclonal Antibody #71; available from Custom Monoclonal International) in 100 pl of EMEM-FBS was added to each well. The plates were incubated for about 30 minutes at 37"C. Following the incubation, the plates were washed 4 times with PBST. About 100 ng of horseradish peroxidase labelled donkey anti-murine IgG (available from Jackson Laboratories, Westgrove, PA) in 100 pl of EMEM-FBS was added to each well, i and the plates were incubated for about 30 minutes at room temperature. Following the 1: 5 incubation, the plates were washed 4 times with PBST. The presence of anti-murine IgG bound to the plates thereby indicating the ability of RBL-hFcR cells to bind to canine IgE was detected using the method described in Example 4.

The results shown in Fig. 10 indicate that canine IgE from heartworm-infected dogs is detected using RBL-h FcR cells expressing the alpha chain of human FcR.

20 In addition, the absence of colorimetric signal in samples of heat inactivated samples of such sera indicates that antibody detected by the FcR alpha chain on the RBL-h FcR cells is an epsilon isotype antibody. Similarly, the results shown in Fig. 11 indicate Sthat canine IgE from dogs sensitized with flea saliva is detected using RBL-h FcR cells expressing the alpha chain of human FcR. In addition, the absence of colorimetric 25 signal in samples of heat inactivated samples of such sera indicates that antibody detected by the FcR alpha chain on the RBL-h FcR cells is an epsilon isotype antibody.

SEQUENCE LISTING GENERAL INFORMATION:

APPLICANT:

NAME: Heska Corporation STREET: 1825 Sharp Point Drive CITY: Fort Collins STATE: CO COUNTRY: US POSTAL CODE (ZIP): 80525 TELEPHONE: (970) 493-7272 TELEFAX: (970) 484-9505 (ii) TITLE OF INVENTION: METHOD TO DETECT IGE (iii) NUMBER OF SEQUENCES: 13 (iv) CORRESPONDENCE ADDRESS: ADDRESSEE: LAHIVE COCKFIELD, LLP STREET: 28 STATE STREET CITY: BOSTON STATE: MA COUNTRY: US ZIP: 02109 COMPUTER READABLE FORM: MEDIUM TYPE: Floppy disk COMPUTER: IBM PC compatible OPERATING SYSTEM: Windows 25 SOFTWARE: ASCII DOS TEXT (vi) CURRENT APPLICATION DATA: APPLICATION NUMBER: 08/756,327 FILING DATE: CLASSIFICATION: Noveber 26, 1996 (vii) PRIOR APPLICATION DATA: APPLICATION NUMBER: FILING DATE: (viii) ATTORNEY/AGENT

INFORMATION:

NAME: Rothenberger, Scott D.

REGISTRATION NUMBER: 41,277 REFERENCE/DOCKET NUMBER: (ix) TELECOMMUNICATION

INFORMATION:

TELEPHONE: (617) 227-7400 TELEFAX: (617) 742-4214 40 INFORMATION FOR SEQ ID NO:1: SEQUENCE CHARACTERISTICS: LENGTH: 1198 nucleotides TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) FEATURE: NAME/KEY: CDS LOCATION: 107..877 -36- (iv) SEQUENCE DESCRIPTION: SEQ ID NO:i TACTAAGAGT CTCCAGCATC CTCCACCTGT CTACCACCGA GCATGGGCCT

ATATTTGAAG

CCTTAGATCT CTCCAGCACA GTAAGCACCA GGAGTCCATG AAGAAG ATG GCT CCT Met Ala Pro 1.

GCC ATG GAA TCC CCT ACT CTA CTG TGT GTA GCC TTA CTG TTC TTC GCT Ala Met Glu Ser Pro Thr Leu Leu Cys Val Ala Leu Leu Phe Phe Ala 10 CCA GAT GGC GTG TTA GCA GTC CCT CAG AAA CCT AAG GTC TCC TTG AAC Pro Asp Gly Val Leu Ala Val Pro Gln Lys Pro Lys Val Ser Leu Asn 25-4n1

S.

S

S

S

CCT

Pro

AAT

Asn

GGC

Gly

AAA

Lys

GAG

Glu 100

CAG

Gin 30 -TOC Cys

GAT

Aspi

ACA

Thr

GTG

Val' 180

AAA(

LYS

GTGC

Val CCA TG Pro Trp GGG AAC Gly Asn

AA'I

Asn

AAT

Asn AGC CTT TCA Ser

TTT

Phe 85

AGT

Ser

GCC

Ala

CAT

His

GGT

Gly

.AAT

165

ITGG

rrp 3CT

'TG

lal Leu Ser 70 GAA GAC Glu Asp GAA CCT Glu Pro TCT GCT Ser Ala GOT TGG Gly Trp 135 GAA GCT Glu Ala 150 GCC ACA Ala Thr CAG CTG Gin Leu CCG CG"' Pro Arg ATT CTG Ile. Leu 215

AGA

Arg

TTC

Phe

GAA

Glu

AGT

Ser

GTG

Val

GAG

Giu 120

AGG

Arg

CTC

Leu

GTT

Val

GAC

Asp CAG1 Glu 200

TTT

Phe

ATP.

Ile

TTT

Phe

GAG

Giu

GGA

Gly

TAC

Tyr 105

GTG

Val

AAC

Asn Ljys

GAA

3iu rAT Pyr ys

;CT

kla TTT AAA GGA GAG AAT Phe Lys GAA GTC Giu Val ACA AAT Thr Asn 75 GAA TAC Glu Tyr 90 CTG GAA Leu Giu GTG ATG Val Met TGG GAT Trp Asp TAC TGG Tyr Trp 155 GAC AGT Asp Ser 17.0

GAG-TCT

Giu Ser TAC TGG Tyr Trp GTG GAC Val Asp AGT TCC ACC AAA TGG Ser 60

TCA

Ser

AAA

LYS

GTC

Val

GAG

Glu

GTG

Vai 140

TAT

Tyr

GGA

Gly

GAG

Giu

CTA

Leu AiCA rhr 220 Ser

AGT

Ser

TGT

Cys

TTC

Phe

GGC-

Gly 125

TAC

Tyr

GAG

Giu

ACC

Thr

CCC

Pro

CAA

Gin 205

GGA'

Gly Thr

TTG

Leu

CAG

Gin

AGT

Ser 110

CAG

Gin

AAG

Lys

AAC

Asn

TAC

Tyr

CTC

Leu 190

ITT

Phe

TTA

Leu Lys

AAT

Asn

CAC

His

GAC

Asp

CCC

Pro

GTG

Val

CAC

His

TAC

Tyr 175

AAC.

Asn

TTT

Phe

TTT

Phe Trp

ATT

Ile

CAA

Gin

TGG

Trp

CTC

Leu

ATC

le

AAC

Asn 160

TGT

Cys

ATT.

le AiTC Ile PiTC Ile

TTC

Phe

GTG

Val

CAA

Gin

CTG

Leu

TTC

Phe

TAT

Tyr 145

ATC

le

ACG

Thr

ACT

Thr

CCA

Pro

TCA

Ser 225 Gly Giu Asn 45 GTG ACT CTI Val Thr Leu ACA

TGT

Thr

CAC

His

AAT

Asn

GTT

Val

CTC

Leu

CTC

Leu 130

TAT

Tyr

TO-C

Ser

GGC

Gly

GTA

Val

TTG

Leu 210

ACT

Thr Cys

AAT

Asn

GCC

Ala

AAT

Asn

CTT

Leu 115

AGG

Arg

AAG

Lys

PATT

Ile

AA

Lys

W.A

Ile 19 rTG jeu

:AG

;ln 115 163 211 259 307 355 403 451 499 547 595 643 691 739 787 CAG CAG GTC ACA TTT CTC TTG Gin Gin Val Thr Phe Leu Leu 230 AAG ATT AAG AGA ACC AGG AAA GGC TTC LYS Ile Lys Arg Thr 235 Arg LYS Gly Phe 240 -37- AGA CTT CTG AAC CCA CAT CCT AAG CCA AAC CCC AAA AAC AAC TGA Arg Leu Leu Asn Pro His Pro Lys Pro Asn Pro Lys Asn Asn 245 250 255 TATAATTACT CAAGAAATAT TTGCAACATT AGTTTTTTTC CAGCATCAGC

AATTGCTACT

CAATTGTCAA ACACAGCTTG CAATATACAT AGAAACGTCT GTGCTCAAGG

ATTTATAGAA

ATGCTTCATT AAACTGAGTG AAACTGGTTA AGTGGCATGT AATAGTAAGT

GCTCAATTA

CATTGGTTGA ATAAATGAGA GAATGAATAG ATTCATTTAT TAGCATTTGT

AAAAGAGATG

TTCAATTTCA ATAAAATAAA TATAAIAACCA TGTAACAGAA TGCTTCTGAG TAAAAAA

AAAAAAAAAAA

INFORMATION FOR SEQ ID NO:2: SEQUENCE CHARACTERISTICS: LENGTH: 257 amino acids TYPE: amino acid 940 1000 1060 1120 1180 1198

TOPOLOGY:

linear protein .5*s

S

S

.5.5 S

S

S

Met 1 Phe Ser Leu 25 Phe 65 Val1 Gin Leu Phe 35 Tyr 145 Ile Thr Thr Pro (ii) MOLECU (iii) SEQUEN Ala Pro Ala Met 5 Phe Ala Pro Asp 20 Leu Asn Pro Pro 35 Thr Cys Asn.Gly His Asn Gly Ser Asn Ala Lys Phe 85 Val Asn Giu Ser 100 Leu Leu Gin Ala 115 Leu Arg Cys His 130 Tyr Lys Asp Gly Ser Ile Thr Asn 165 Gly Lys Val Trp 180 Val Ile Lys Ala 195 Leu Leu Val Val CE DESCRIPTION: LE TYPE: Glu Ser Pro Gly Val Leu Trp Asn Arg 40 Asn Asn Phe 55 Leu Ser Glu 70 Giu Asp Ser Giu Pro Vai Ser Ala Giu 120 Gly Trp Arg 135 Giu Ala Leu 150 Ala Thr Val Gin Leu Asp Pro Arg Giu 200 Ile Leu Phe 215 Thr Ala 25 Ile Phe Glu Gly Tyr 105 Val Asn Lys Glu Tyr 185 Lys Ala 10 Val Phe Glu Thr Giu 90 Leu Val Trp Ty'r Asp 170 Giu Tyr Val Leu Leu Cys Vai Ala Leu Leu Pro Lys Val Asn 75 Tyr Giu Met Asp Trp 155 Ser Ser Trp Asp Gin Gly Ser Ser Lys Val Glu Val 140 Tyr Gly Glu Leu Thr 220 Lys Giu Ser Ser Cys Phe Gly 125 Tyr Giu Thr Pro Gin 205 Gly Pro Asn Thr Leu Gin Ser 110 Gin Lys Asn Tyr Leu 190 Phe Leu Lys Val Lys Asn His Asp Pro- Vai His Tyr 175 Asn Phe Phe Val Thr Trp Ile Gin Trp Leu Ile Asn 160 Cys Ile Ile Ile SEQ ID NO:2: 210 Ser 225 Thr Gin Gin Gin Val 230 Thr Phe Leu Leu Lys li e 235 Lys Arg Thr -38- Lys Gly Phe Arg Leu Leu Asn Pro His Pro Lys Pro Asn Pro Lys Asn.

245 250 255 Asn INFORMATION FOR SEQ ID NO:3: SEQUENCE CHARACTERISTICS: LENGTH: 1198 nucleotides TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) SEQUENCE DESCRIPTION: SEQ ID NO:3:

TTTTTTTTTT

TATTTTATTG

TCATTTATTC

CTCAGTTTAA

AGCTGTGTTT

ATTTCTTGAG

TCTGAAGCCT

GATAAATAAT

TAGCCAGTAC

GTCCAGCTGC

TGTAATGGAG

GATCACCTTG

CTCCATCACC

25 CACAGGTTCA

TTTGGCATTC

CCATTTGGTG

TCCTTTAAAT

TAACACGCCA

30 GGCAGGAGCC

TCAAATATAG

TTTTTTTTTT

AAATTGAACA

AACCAATGTT

TGAAGCATTT

GACAATTGAG

TAATTATATC

TTCCTGGTTC

CCTGTGTCCA

TTCTCACGCG

CACACTTTGC

ATGTTGTGGT

TACACATCCC

ACCTCAGCAG

CTCTCATTAA

ACAATATTCA

GAACTGACTT

ATTCTATTCC

TCTGGAGCGA

ATCTTCTTCA

GCCCATGCTC

TTTTTTTACT

TCTCTTTTAC

AATTGAGCAC

CTATAAATCC

TAGCAATTGC

AGTTGTTTTT

TCTTAATCTT

CAGCAAACAG

GAGCTTTTAT

CCGTACAGTA

TCTCATACCA

AGTTCCTCCA

AGGCCTGAAG

CTTGTTGGTG

AACTTGAATT

CAAAGAAATT

ATGGAGGGTT

AGAACAGTAA

TGGACTCCTG

GGTGGT.AGAC

CAGAAGCATT

AAATGCTAAT

TTACTATTAC

TTGAGCACAG

TGATGCTGGA

GGGGTTTGGC

CAAGAGAAAT

AATCACCACC

TACAGTAATG

GTAGGTTCCA

GTACTTGAGA

ACCATGGCAC

GAGCAGCCAG

CTGACATTTG

TGTCTCTTCT

GTTCCCATTA

CAAGGAGACC

GGCTACACAC

GTGCTTACTG

AGGTGG;AGGA

CTGTTACATG

AAATGAATCT

ATGCCACTTA

ACGTTTCTAT

AAAAAACTAA

TTAGGATGTG

GTGACCTGCT

AACAATGGGA

TTGAGGGGCT

CTGTCTTCAA

GCTTCACCAT

CTGAGGAAGA

TCACTGAAGA

TATTCTCCAC

GAAAGGCTGC

CATGTAAGAG

TTAGGTTTCT

AGTAGAGTAG

TGCTGGAGAG

TGCTGGAGAC

GTTTTATATT

ACATTCTC

ACCAGTTTCA

GTATATTGCA

TGTTGCAAAT

GGTTCAGAAG

GCTGAGTTGA

TAAAAAATTG

CAGACTCATA,

CTGTGGCATT

CCTTATAATA

GGGGCTGGCC

CTTCCAGGTA

TGTCTTCAAA

CATTGTGGAA

TCACATTCTC

GAGGGACTGC

GGGATTCCAT

ATCTAAGGCT

TCTTAGTA

120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1198 INFORMATION FOR SEQ ID NO:4: i) SEQUENCE CHARACTERISTICS: LENGTH: 774 nucleotides TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) FEATURE: NAME/KEY:

CDS

LOCATION: 1. .774 (iv) SEQUENCE DESCRIPTION: SEQ ID NO:4:

ATG

Met 1 GCT CCT GCC Ala Pro Ala

ATG

Met 5 GAA TCC CCT ACT Glu Ser Pro Thr

CTA

Leu CTG TGT GTA GCC Leu Cys, Val Ala TTA CTG T-e. Leu TTC TTC GCT Phe Phe Ala TCC TTG AAC Ser Leu Asn

CCA

Pro GAT GGC GTG TTA Asp Gly Val Leu

GCA

Ala 25 GTC CCT CAG AAA Val Pro Gln Lys CCT AAG GTC Pro Lys Val AAT GTG ACT Asn Val Thr CCT CCA TGG AAT Pro Pro Trp Asn

AGA

Arg 40 ATA TTT AAA GGA Ile Phe LYS Gly 144 -39- TTT GAA Phe Glu CTT ACA TGT AAT GGG AAC AAT TTC Leu Thr Cys Asn Gly Asn Asn Phe 55 GTC AGT Val Ser TCC ACC AAA TGG Ser Thr Lys Trp 192 a a *aa.

a

TTC

Phe

GTG

Val

CAA

Gin

CTG

Leu

TTC

Phe

TAT

Tyr '145

ATC

Ile

ACG

Thr

ACT

Thr

CCA

Pro

TCA

35 Ser 225

AAA

LYS

AAC

Asn (2)

CAC

His

AAT

Asn

GTT

Val

CTC

Leu

CTC

Leu 130

TAT

Tyr

TCC

Ser

GGC

Gly

GTA

Val

TTG

Leu 210

ACT

Thr

GGC

Gly

TGA

AAT

Asn

GCC

Ala

AAT

Asn

CTT

Leu 115

AGG,

-Arg

AAG

Lys

ATT

Ile

AAA

Lys

ATA

Ile 195

TTG

Leu

CAG

Gin

TTC.

Phe Gly

AAA

Lys

GAG

GiU 100

CAG

Gin

TGC

Cys

GAT

Asp

ACA

Thr

GTG

Val 180

AAA

Lys

GTG

Val

CAG

Gin

AGA

Arg *Ser Leu 70 TTT GAA Phe Glu AGT GAA Ser Giu GCC TCT Ala Ser CAT GGT His Gly GGT GAA Gly Giu 150 AAT GCC Asn Ala 165 TGG CAG Trp Gin GCT CCG Ala Pro GTG ATT Val le CAG GTC Gin Val 230 CTT CTG Leu Leu 245 Ser Giu Giu Thr Asn Ser Ser GGC AGC CTT TCA GAA GAG ACA AAT TCA AGT GAC AGT GGA GAA TAC AAA TGT CAG CAC CAA Asp

CCT

Pro

GCT

Ala

TGG

Trp 135

GCT

Ala

ACA

Thr

CTG

Leu

CGT

Arg

CTG

Leu 215

ACA

Thr

AAC

Asn Ser

GTG

Vai

GAG

Glu 120

AGG

Arg

CTC

Leu

GTT

Val

GAC

Asp

GAG

Glu 200

TTT

Phe

TTT

Phe

CCA

Pro Gly

TAC

Tyr 105

GTG

Val

AAC

Asn

AAG

Lys

GAA

Glu

TAT

Tyr 185

AAG

Lys

GCT

Ala

CTC

Leu

CAT

His *Giu 90

CTG

Leu

GTG

Val

TGG

Trp

TAC

Tyr

GAC

Asp 170

GAG

Giu

TAC

Tyr

GTG

Val

TTG

Leu

CCT

Pro 250 Tyr

GAA

Glu

ATG

Met

GAT

Asp

TGG

Trp 155

AGT

Ser

TCT

Ser rGG rrp

GAC

Asp

AAG

Lys 235

AAG

L~ys LYS Cys Gin

GTC

Val

GAG

Giu

GTG

Val 140

TAT

Tyr

GGA

Gly

GAG

Glu

CTA

Leu

ACA

Thr 220

ATT

Ile

CCA

Pro

TTC

Phe

GGC

Gly 125

TAC

Tyr

GAG

Giu

ACC

Thr

CCC

Pro

CAA

Gin 205

GGA

Gly

AAG

Lys

AAC

Asn

AG

T

I

Ser 110

CAG

Gin

AAG

Lys

AAC

Asn

TAC

Tyr

CTC

Leu 190

TTT

Phe

TTA

Leu

AGA

Arg

CCC

Pro His Gin GAC TGG Asp Trp CCC CTC Pro Leu GTG ATC Vai Ile CAC AAC His Asn 160 TAC TGT Tyr Cys 175 AAC ATT Asn Ile TTT ATC Phe Ile TTT ATC Phe Ile AC AGG, Thr Arg 240 AAA -AAC Lys Asn 255 288 336 384 432 480 528 576 624 672 720 768 TTG AAT ATT Leu Asn Ile 774 INFORMATION FOR SEQ ID i) SEQUENCE CHARACTERISTICS: LENGTH: 774 nucleotides TYPE: nucleic acid STRAN~DEDNESS: single TOPOLOGY: linear MOLECULE TYPE: CDNA (iii) SEQUENCE DESCRIPTION: SEQ ID

TCAGTTGTTT

TCTCTTAATC

CACAGCAAAC

CGGAGCTTTT

GCCCGTACAG

GTTCTCATAC

CCAGTTCCTC.

AGAGGCCTGA

AACTTGTTGG

CAAACTTGAA

TTCAAAGAAA

CCATGGAGGG

GAAGAACAGT

TTGGGGTTTG

TTCAAGAGAA

AGAATCACCA

ATTACAGTAA

TAGTAGGTTC

CAGTACTTGA

CAACCATGGC

AGGAGCAGCC

TGCTGACATT

TTTGTCTCTT

TTGTTCCCAT

TTCAAGGAGA

AAGGCTACAC

GCTTAGGATG

ATGTGACCTG

CCAACAATGG

TGTTGAGGGG

CACTGTCTTC

GAGCTTCACC

ACCTGAGGAA

AGTCACTGAA

TGTATTCTCC

CTGAAAGGCT

TACATGTAAG

CCTTAGGTTT

ACAGTAGAGT

TGGGTTCAGA

CTGCTGAGTT

GATAAAAAAT

CTCAGACTCA

AACTGTGGCA

ATCCTTATAA

GAGGGGCTGG

GACTTCCAGG

ACTGTCTTCA

GCCATTGTGG

AGTCACATTC

CTGAGGGACT

AGGGGATTCC

AGTCTGAAGC

GAGATAAATA

TGTAGCCAGT

TAGTCCAGCT

TTTGTAATGG

TAGATCACCT

CCCTCCATCA

TACACAGGTT

AATTTGGCAT

AACCATTTGG

TCTCCTTTA

GCTAACACGC

ATGGCAGGAG

CTTTCCTGGT

ATCCTGTGTC

ACTTCTCACG

GCCACACTTT

AGATGTTGTG

TGTACACATC

CCACCTCAGC

CACTCTCATT

TCACAATATT

TGGAACTGAC

ATATTCTATT

CATCTGGAGC

CCAT

120 180 240 300 360 420 480 540 600 660 720 774 INFORMATION FOR SEQ ID NO:6: Wi SEQUENCE CHARACTERISTICS: LENGTH: 23 2 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein' (iii) SEQUENCE DESCRIPTION: SEQ ID NO:6: Val 1 Pro Gin Lys Pro Lys Val Ser Leu Asn Pro Pro Trp, 5 10 *n* Phe Lys Gly Glu Val Ser 35 Thr Asn Ser Giu Asn Val Thr Leu Thr Cys 20 25 Asn Gly Asn Asn Arg Ile Asn Phe Phe Ser Glu Giu Ser Thr Lys Trp Phe 40 His Asn Gly Ser Leu Ser Leu Asn Ile 55 Val Asn Ala Lys Phe Giu Asp Ser Giy 30 Giu Tyr Lys Cys Gin His 70 Gin Gin Val Asn Glu 75 Ser Giu Pro Val Tyr Leu Giu Vai Phe Ser Asp Trp Leu Leu* Gin Ala Ser Ala Giu Vai Arg Asn Vai Met Giu Trp Asp Val 115 Gly 100 Gin Pro Leu Phe Leu 105 Arg Cys His Gly Tyr Lys*Vai Ile Tyr 120 Tyr Lys Asp Gly Giu 125 Ala Leu Lys Tyr Trp 130 Tyr Giu Asn His Asn-I1e 135 SerIle Thr Asn 140 Ala Thr Vai Glu Asp 145 Ser Gliv Thr Tyr Tyr 150 Cys Thr Giy Lys Val 155 Trp, Gin Leu Asp Tyr Glu Ser Giu Pro Leu 165 Asn Ile Thr Val Lys Ala Pro Arg Glu Lys 175 Tyr Trp Leu Gin 180 Phe Phe Ile Pro Leu 185 Leu Val Val Ile Leu Phe Ala 190 Vai Asp Thr Gly Leu 195 Phe Ile Ser 200 Thr Gin Gin Gin Val Thr Phe Leu 205 Leu Lys Ile Lys Arg Thr Arg. Lys Gly Phe Arg Leu Leu Asn Pro His 210 215 220 Pro Lys Pro Asn Pro Lys Asn Asn 225 230 INFORMATION FOR SEQ ID NO:7: SEQUENCE CHARACTERISTICS: LENGTH: 699 nUcleotides TYPE: nucleic acid STRANDEDNESS: single

TOPOLOGY:

linear (ii) (iii) MOLECULE TYPE: cDNA

FEATURE:

NAME/KEY: CDS LOCATION: 1. .699 SEQUENCE DESCRIPTION: SEQ ID NO:7: ebb...

be

C

a.

C

GTC

Val 1

TTT

Lys

GAA

Giu 25 ACA Thr

GAA

Olu 65

CTG

Leu

GTG

Val

TGG

Trp,

TAC

GAC

Asp 1.45 (iii) CCT CAG Pro Gin AAA GGA Gly Glu GTC AGT Val Ser AAT TCA Asn Ser TAC AAA Tyr Lys GAA GTC Glu Val ATO GAG Met Giu OAT GTG Asp Val.

115 TOG TAT Trp Tyr 130 AGT GOA Ser Gly AAA CCT AAG Lys Pro Lys 5 GAG AAT GTG Asn Val Thr 20 TCC ACC AAA Ser Thr Lys AGT TTG AAT Ser Leu Asn TOT CAG CAC Cys Gin His 70 TTC AGT GAC Phe Ser Asp GOC CAG CCC- Gly Gin Pro 100 TAC AAG GTG Tyr Lys Val GAG AJAC CAC Giu Asn His ACC TAC TAC Thr Tyr Tyr 150

GTC

Val

ACT

Leu

TG

Trp

ATT

Ile 55

CAA

Gin

TGG

Trp,

CTC

Leu

ATC

I 1e

AAC

Asn 135

TOT

Cys TCC TTG AAC CCT CCA TGG AAT AGA ATA Ser Leu Asn Pro Pro Trp Asn

CTT

Thr

TTC

Phe 40

GTG

Val

CAA

Gin

CTG

Leu

TTC

Phe

TAT

Tyr 120

ATC

Ile

ACG

Thr .10 ACA TGT Cys -Phe 25 CAC A.AT His Asn AAT 0CC Asn Ala GTT AAT Val Asn CTC CTT Leu Leu 90 CTC AGG Leu Arg 105 TAT AAG Tyr Lys TCC ATT Ser Ile GGC AAA Oly Lys OTA ATA Val Ile 170 Arg Ile

AAT

Asn

GGC

Gly

AAA

Lys

GAG

Giu 75

CAG

Gin

TGC

Cys

OAT

Asp

ACA

Thr

OTG

Val 155

GG

Oly

AGC

Ser

TTT

Phe

AOT

Ser

GCC

Ala

CAT

His

GOT

Gly

AAT

Asn 140

TGG

Trp AAC AAT Asn Asn CTT TCA Leu Ser GAA GAC Olu Asp GAA CCT.

Glu Pro TCT GCT Ser Ala GGT TG Gly Trp 110 GAA OCT Giu Ala 125 0CC ACA Ala Thr CAG CTG Gin Leu TTC TTT Phe Phe GAA GAG Olu Giu AGT GGA Ser Gly OTO TAC Val Tyr GAG GTG 0Th Val AGO AAC Arg Asn CTC AAG Leu Lys OTT GAA Val Giu GAC TAT Asp Tyr 160 96 144 192.

240 288 336 384 432 480 528 GAG TCT GAG Glu Ser Glu CCC CTC AAC ATT ACT Pro Leu 165 Asn Ile Thr AAA OCT CCG COT Lys Ala Pro Arg GAG AAG Glu Lys -42- TAC TGG CTA Tyr Trp Leu GTG GAC ACA Val Asp Thr 195

CAA

Gin 180 TTT TTT ATC CCA Phe Phe Ile Pro

TTG

Leu 185 TTG GTG GTG ATT Leu Val Val Ile CTG TTT GCT Leu Phe Ala 190 ACA TTT CTC Thr Phe Leu 576 624 GGA TTA TTT ATC Gly Leu Phe Ile

TCA

Ser 200 ACT CAG CAG CAG Thr Gin Gin Gin

GTC

Val 205 TTG AAG Leu Lys 210 ATT AAG AGA ACC Ile Lys Arg Thr

AGG

Arg 215 AAA GGC TTC AGA Lys Gly Phe Arg

CTT

Leu 220 CTG AAC CCA CAT Leu Asn Pro His

CCT

Pro 225 AAG CCA AAC CCC Lys Pro Asn Pro AAA AAC AAC TGA Lys Asn Asn 230 699 a INFORMATION FOR SEQ ID NO:8: SEQUENCE CHARACTERISTICS: LENGTH: 32 bases TYPE: nucleic-acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: primer (iii) SEQUENCE DESCRIPTION: SEQ ID NO:8: CGCGGATCCT ATAAATATGG CTCCTGCCAT

GG

INFORMATION FOR SEQ ID NO:9: SEQUENCE CHARACTERISTICS: LENGTH: 26 bases 25 TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: primer (iii) SEQUENCE DESCRIPTION: SEQ ID NO:9: GGCGAATTCT TAAGCTTTTA

TTACAG

INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 591 nucleotides TYPE: nucleic acid 35 STRANDEDNESS: single.

TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) FEATURE: NAME/KEY: CDS LOCATION: 1..591 (iv) SEQUENCE DESCRIPTION: SEQ ID o ATG GCT CCT Met Ala Pro 1 GCC ATG GAA TCC CCT ACT CTA CTG TGT GTA GCC TTA CTG_ Ala Met Glu Ser Pro Thr Leu Leu Cys Val Ala Leu Leu 5 10 TTC TTC GCT CCA GAT GGC GTG TTA Phe Phe Ala Pro Asp Gly Val Leu -43- GCA GTC CCT Ala Val Pro 25 CAG AAA CCT AAG GTC Gin Lys Pro Lys Val TCC TTG AAC CCT CCA TGG AAT AGA ATA Ser

CTT

Leu

TTC

Phe 65

GTG

Val

CAA

Gin

CTG

Leu

TTC

Phe 25 TAT Tyr 145

ATC

Ile

ACG

Thr Leu

ACA

Thr

CAC

His

AAT

Asn

GTT

Val

CTC

Leu

CTC

Leu 130

TAT

Tyr

TCC

Ser

GGC

Gly Asn Pro Pro TGT AAT GGG Cys Asn Gly AAT GGC AGC Asn Gly Ser GCC AAA TTT Ala Lys Phe 85 AAT GAG AGT Asn Giu Ser 100- CTT CAG GCC Leu Gin Ala 115 AGG TGC CAT Arg Cyg His AAG GAT GGT Lys Asp Gly ATT ACA AAT Ile Thr Asn 165 AAA GTG TGG Lys Val Trp 180 Ti-p Asn Arg Ile

AAC

Asn

CTT

Leu 70

GAA

Glu

GAA

Glu

TCT

Ser

GGT

Giy

GAA

Glu 150

GCC

Ala

AAT

Asn 55

TCA

Ser

GAC

Asp

CCT

Pro

GCT

Ala

TGG

Trp 135

GCT

Ala

ACA

Thr

TTC

Phe

GAA

Giu

AGT

Ser

GTG

Val

GAG

Giu 120

AGG

Arg

CTC

Leu

GTT

Val

TTT

Phe

GAG

Glu

GGA

Gly

TAC

Tyr 105

GTG

Val

AAC

Asn

AAG

Lys

GAA

Glu

TAT

Tyr 185

*TTT

Phe

GAA

Glu

ACA

Thr

GAA

Glu 90

CTG

Leu

GTG

Val

TGG

Ti-p

TAC

Tyr

GAC.

Asp 170 AAA GGA GAG AAT Lys Gly Glu Asn GTG ACT Val Thr

GTC

Val

AAT

Asn 75

TAC

Tyr

GAA

Glu

ATG

Met

GAT

Asp

TGG

Ti-p 155

AGT

Ser

AGT

Ser

TCA

Ser

AAA

Lys

GTC

Val1

GAG

Glu

GTG

Val 140

TAT

Tyr

GGA

Gly TCC ACC AAA TGG Ser Thr Lys Ti-p AGT TTG AAT ATT Ser Leu Asn Ile TGT CAG CAC CAA Cys Gin His Gin TTC AGT GAC TGG Phe Ser Asp Trp 110 GGC CAG CCC CTC Gly Gin Pro Leu 125 TAC AAG GTG ATC Tyr Lys Val Ile GAG AAC CAC AAC Glu Asn His Asn 16 0- ACC TAC TAC TGT 144 192 240 288 336 384 432 480 528 576 591 a.

a a a.

a a Thr Tyr Tyr Cys 175 CAG CTG GAC Gin LeU ASP GAG TCT GAG CCC Glu Ser Glu Pro CTC AAC ATT Leu Asn Ile 190 ACT GTA ATA AAA GCT 35 Thr Val Ile Lys Ala 195 INFORMATION FOR SEQ ID.NO:li: i) SEQUENCE CHARACTERISTICS: LENGTH: 197 amino acids TYPE:, amino acid TOPOLOGY: linear MOLECULE protein Ci) SEQUENCE-DESCRIPTION: SEQ ID NO:i1: Met Ala Pro Ala Met Glu Ser Pro Th- Leu Leu Cys Val Ala Leu Leu *i1 5 10 Phe Phe Ala Pro Asp Gly Val Leu Ala Val Pro Gin Lys Pro Lys Val 25 S.

S*

S.

S.

-S

S

*SSSSS

S

S.

S S .5 S S *5 S Ser Leu Phe Val Gi-n Leu Phe TIyr 145 Ile Thr 20 Thr (2) 25 30

GTC

35 Val 1

TTT.

Phe.

GAA

Giu

ACA.

Thr.

Leu Asn Pro Pro Trp Asn Arg Ile Phe L, 40 Thr Cys Asn Giy Asn Asn Phe Phe Glu Vi 55 His Asri Gly Ser Leu Ser Glu Glu Thr A 70 Asn Ala Lys Phe Glu Asp Ser Gly Glu Tj 90 Val Asn Giu Ser Glu Pro Val Tyr Leu GI 100 105 Leu Leu Gin Ala Ser Ala Glu Val Val Me 115 120 Leu Arg Cys His Gly Trp, Arg Asn Trp As 130 135 Tyr Lys Asp Gly Glu Ala Leu Lys Tyr Tr 150 .15 Ser Ile Thr Asn Ala Thr Val Giu Asp Se 165 170 Gly Lys Val Trp Gin Leu Asp Tyr Glu. Se 180 185 Val Ile Lys A1d- 195 INFORMATION FOR SEQ ID NO:12: Wi SEQUENCE CHARACTERISTICS: LENGTH: 516 nucleotides TYPE: nucleic acid STIIANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) FEATURE: NAME/KEY: CDS LOCATION: 1. .516 (xi) SEQUENCE DESCRIPTION: SEQ ID CCT CAG AAA CCT AAG GTC TCC TTG AAC CC' Pro Gin Lys Pro Lys Val -Ser Leu Asn Pr4 5 10 AAA GGA GAG AAT GTG ACT CTT ACA TGT AAk Lys Gly Glu Asn Val Thr Leu Thr Cys As2 25 GTC AGT TCC ACC AAA TGG TTC CAC AAT GG( Val Ser Ser Thr Lys Trp Phe His Asn G1: 40 AAT TCA AGT TTG AAT ATT GTG AAT GCC AA; Asn Ser Ser Leu Asn Ile Val Asn Ala Ly 50 55 5n 75 r r Giy Ser Ser

LYS

Val Glu Val 140 Tyr Gly Giu *Glu Asn *Ser Thr Ser Leu Cys Gln Phe Ser .110 Gly Gin 125 Tyr Lys Glu Asn Thr Tyr Pro Leu2 190 Val Lys Asn His Asp Pro Val His ryr 175 ksn Thr Trp Ile Gin Trp Leu Ile Asn 160 Cys Ile NO: 12: T CCA TGG o Pro Trp rGGG AAC .1 Gly Asn CAGC CTT Fr Ser Leu !TTT GAA s Phe Glu AAT AGA ATA Asn Arg le AAT TTC TTT Asn Phe Phe TCA GAA GAG Ser Giu Glu GAC AGT GGA Asp Ser Gly 48 96 144 192 *5 9*

GAA

Gilu

CTG

Leu

GTG

Val.

TGG

Trp

TAC

y

GAC

Asp 145

GAG

Glu (2) 25 Val 30 1 Phe Glu Thr Giu Leu Val Trp Tyr TAC AAA TGT CAG CAC CAA CAA GTT AAT GA Tyr Lys Cys Gin His Gin Gin Val Asn G1 70 7 GAA GTC TTC AGT GAC TGG CTG CTC CTT CA Glu Val Phe Ser Asp Trp Leu Leu Leu Gi 90 ATG GAG GGC CAG CCC CTC TTC CTC AGG TG Met Giu Gly Gin Pro-Leu Phe Leu Arg Cy 100 105 GAT GTG TAC AAG GTG ATC TAT TAT AAG GA Asp Val Tyr Lys Val Ile Tyr Tyr Lys As 115 120 TGG TAT GAG AAC CAC AAC ATC TCC ATT AC Trp Tyr Giu Asn His Asn Ile Ser Ile Th 130 135 ACT GGA ACC TAC TAC TGT ACG GGC AAA CT Ser Gly Thr Tyr Tyr Cys Thr Giy Lys Va 150 15 TCT GAG CCC CTC AAC ATT ACT GTA ATA A" Ser Giu Pro Leu Asn Ile Thr Val Ile Ly 165 170 INFORMATION FOR SEQ ID NO:i3: SEQUENCE CHARACTERISTICS: LENGTH: 172 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID Pro Gin Lys Pro Lys Val Ser Leu Asn Pr 5 10 Lys Gly Giu Asn Val Thr Leu Thr Cys Asi 20 Val Ser Ser Thr Lys Trp Phe His Asn G1~ 40 Asn Ser Ser Leu Asn Ile Val Asn Ala Ly 50 55 Tyr Lys Cys Gin His Gln-Gin Val Asn G1~ 70 7! Glu Val Phe Ser Asp Trp Leu Leu Leu Gl1 85 90 Met Glu Giy Gin Pro Leu Phe Leu Arg Cy 100 105 Asp Val Tyr Lys Val Ile Tyr Tyr Lys Asi 115 120 Trp Tyr Giu Asn His Asn Ile Ser Ile Th 130 135 '5 n

C

s p

A

G

1 5

AGT

Ser

GCC

Ala

CAT

His

GGT

Gly

AAT

Asn 140

TGG

Trp

GCT

Ala GAA CCT Giu Pro TCT GCT Ser Ala GGT TGG Gly Trp 110 GAA GCT Giu Ala 125 GCC ACA Ala Thr CAG CTG Gin Leu GTC TAC Val Tyr GAG GTG Giu Val.

AGG AAC Arg Asn CTC AAG Leu Lys GTT GAA Val Glu GAC TAT Asp Tyr 160 240 288 336 384 -432 480 516 NO: 13: DPro Trp a Gly Asn r Ser Leu s Phie Glu .1 Ser Glu 5 ai Ala Ser 3.His Gly p Gly Glu 125 r Asn Ala 140 Arg Ile Phe Phe Giu Giu Ser Gly Val Tyr Giu Val Arg Asn Leu Lys Val Glu -46- Asp Ser Gly Thr Ty Tyr Cys Thr Gly Lys Val Trp Gin Leu Asp Tyr 145 150 155 160 Glu Ser Glu Pro Leu Asn Ile Thr Val Ile Lys Ala 165 170 0 6 1 &see* -47- While various embodiments of the present invention have been described in detail, it is apparent that modifications and adaptations of those embodiments will occur to those skilled in the art. It is to be expressly understood, however, that such modifications and adaptations are within the scope of the-present invention, as set forth in the following claims.

oo*

*V

0 0

Claims (57)

1. A method to detect IgE comprising: contacting an isolated human FcE receptor (FcR) molecule with a putative IgE-containing composition under conditions suitable for formation of a FcER molecule:IgE complex, wherein said IgE is selected from the group consisting of canine IgE, feline IgE and equine IgE; and determining the presence of IgE by detecting said FcR molecule:IgE complex, the presence of said FcER molecule:IgE complex indicating the presence of IgE.

2. A method to detect IgE comprising: contacting a recombinant cell with a putative IgE-containing composition under conditions suitable for formation of a recombinant cell:IgE complex, wherein said recombinant cell is selected from the group consisting of: a recombinant cell expressing a human FcER molecule; and a recombinant cell expressing an antibody that binds selectively to an IgE selected from the group consisting of canine IgE, feline IgE and equine IgE; and determining the presence of IgE by detecting said recombinant cell:IgE complex, the presence of said recombinant cell:IgE complex indicating the presence of IgE. 20 3. A kit for detecting IgE comprising a human Fc receptor (Fc 8 R) molecule and a means for detecting an IgE selected from the group consisting of canine IgE, feline IgE and equine IgE.

4. A method to detect flea allergy dermatitis comprising: immobilizing a flea allergen on a substrate; 25 contacting said flea allergen with a putative IgE-containing composition under conditions suitable for formation of an allergen:IgE complex bound to said substrate; removing non-bound material from said substrate under conditions that retain allergen:IgE complex binding to said substrate; and -49- determining the presence of said allergen:IgE complex by contacting said allergen:IgE complex with a FcR molecule, wherein said IgE is selected from the group consisting of canine IgE, feline IgE and equine IgE.

5. A kit for detecting flea allergy dermatitis comprising a human Fc, receptor (Fc 8 R) molecule and a flea allergen.

6. An isolated human Fc, receptor (Fc 6 R) alpha chain protein, wherein a carbohydrate group of said FcR alpha chain protein is conjugated to biotin.

7. The invention of Claim 1, 2, 3, 4, 5 or 6, wherein said FcR molecule comprises at least a portion of a FcR alpha chain that binds to IgE.

8. The invention of Claim 1, 3, 4, 5 or 6, wherein said FcER molecule comprises a protein selected from the group consisting of PhFcERa 257 PhFceRa1 97 PhFceRa 232 and PhFcRa172.

9. The invention of Claim 1, 3, 4, 5 or 6, wherein said FcR molecule is encoded by a nucleic acid molecule selected from the group consisting of nhFceR 77 4 nhFceRa1l98, nhFceRa61 2 nhFcERcl 59 1, nhFceRa 6 99 and nhFcRa51 6 *O 9 The invention of Claim 1, 3, 4, 5 or 6, wherein said Fc 8 R molecule is encoded by a nucleic acid molecule selected from the group consisting of a nucleic acid molecule comprising a nucleic acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO:10 and SEQ ID NO:12, and a nucleic acid molecule comprising an allelic variant of a nucleic acid molecule comprising any of said nucleic acid sequences. •11. The invention of Claim 1, 3, 4 or 5, wherein said FcER molecule is conjugated to a detectable marker.

12. The invention of Claim 1, 3, 4 or 5, wherein said FcER molecule is conjugated to a S- detectable marker selected from the group consisting of a radioactive label, a fluorescent label, a chemiluminescent label, a chromophoric label and a ligand.

13. The invention of Claim 1, 3, 4 or 5, wherein a carbohydrate group of said FcR molecule is conjugated to biotin.

14. The method of Claim 1, 2 or 4, wherein said putative IgE-containing composition comprises a composition selected from the group consisting of blood, serum, plasma, urine, tears, aqueous humor, central nervous system fluid (CNF), saliva, lymph, nasal secretions, milk and feces. The method of Claim 1, 2 or 4, wherein said putative IgE-containing composition comprises serum.

16. The method of Claim 1, 2 or 4, wherein said putative IgE-containing composition comprises a cell that produces IgE.

17. The method of Claim 1, 2 or 4, wherein said putative IgE-containing composition comprises a cell selected from the group consisting of a myeloma cell and a basophil cell.

18. The method of Claim 1, further comprising the step selected from the group consisting of binding said FcR molecule to a substrate prior to performing step to form a Fc 8 R molecule-coated substrate; and binding said putative IgE-containing •composition to a substrate prior to performing step to form a putative IgE-containing composition-coated substrate, wherein said substrate is selected from the group consisting of a non-coated substrate, a FceR molecule-coated substrate, an antigen- coated substrate and an anti-IgE antibody-coated substrate.

19. The method of Claim 18, wherein said antigen is selected from the group consisting of an allergen and a parasitic antigen. The method of Claim 18, further comprising removing non-bound material from said antigen-coated substrate or said antibody-coated substrate under conditions that 25 retain antigen or antibody binding to said substrate.

21. The method of Claim 4 or 18, wherein said substrate comprises a material selected from the group consisting of plastic, glass, gel, celluloid, paper and particulate material. -51

22. The method of Claim 1, 2 or 4, wherein said step of determining comprises performing assays selected from the group consisting of enzyme-linked immunoassays, radioimmunoassays, immunoprecipitations, fluorescence immunoassays, chemiluminescent assay, immunoblot assays, lateral flow assays, agglutination assays and particulate-based assays.

23. The method of Claim 1, wherein said step of determining comprises: contacting said FcR molecule:IgE complex with an indicator molecule that binds selectively to said FcER molecule:IgE complex; removing substantially all of said indicator molecule that does not selectively bind to FcR molecule:IgE complex; and detecting said indicator molecule, wherein the presence of said indicator molecule is indicative of the presence of IgE.

24. The method of Claim 23, wherein said indicator molecule comprises a compound selected from the group consisting of a Fc 8 R molecule, an antigen, an antibody and a lectin. The method of Claim 1, said method comprising the steps of: immobilizing said FceR molecule on a substrate; contacting said Fc 6 R molecule with said putative IgE-containing composition under conditions suitable for formation of an FcR molecule:IgE complex 20 bound to said substrate; removing non-bound material from said substrate under conditions that retain FceR molecule:IgE complex binding to said substrate; and determining the presence of said FceR molecule:IgE complex.

26. The method of Claim 25, wherein the presence of said FcER molecule:IgE complex is detected by contacting said FceR molecule:IgE complex with a compound selected from the group consisting of an antigen and an antibody that binds selectively to IgE.

27. The method of Claim 26, wherein said compound comprises a detectable marker.

28. The method of Claim 1, said method comprising the steps of 28. The method of Claim 1, said method comprising the steps of: -52- immobilizing a desired antigen on a substrate; contacting said antigen with said putative IgE-containing composition under conditions suitable for formation of an antigen:IgE complex bound to said substrate; removing non-bound material from said substrate under conditions that retain antigen:IgE complex binding to said substrate; and determining the presence of said antigen:IgE complex by contacting said antigen:IgE complex with said FceR molecule.

29. The method of Claim 1, said method comprising the steps of: immobilizing an antibody that binds selectively to IgE on a substrate; contacting said antibody with said putative IgE-containing composition under conditions suitable for formation of an antibody:IgE complex bound to said substrate; removing non-bound material from said substrate under conditions that retain antibody:IgE complex binding to said substrate; and determining the presence of said antibody:IgE complex by contacting said antibody:IgE complex with said FceR molecule.

30. The method of Claim 1, said method comprising the steps of: immobilizing said putative IgE-containing composition on a substrate; contacting said composition with said FcR molecule under conditions suitable for formation of an FcR molecule:IgE complex bound to said substrate; removing non-bound material from said substrate under conditions that retain FcR molecule:IgE complex binding to said substrate; and determining the presence of said FceR molecule:IgE complex. 25 31. The invention of Claim 1, 3,4, 5, 28, 29 or 30, wherein said FcR molecule is conjugated to a detectable marker selected from the group consisting of fluorescein, a radioisotope, a phosphatase, biotin, biotin-related compounds, avidin, avidin-related compounds and a peroxidase. -53-

32. The method of Claim 31, wherein the presence of said Fc 6 R molecule:IgE complex is determined by contacting said FcER molecule:IgE complex with an indicator molecule selected from the group consisting of an antibody, an antigen and a lectin.

33. The method of Claim 31, wherein said FcER molecule comprises a detectable marker.

34. The method of Claim 1, wherein said putative IgE-containing composition is obtained from an animal, wherein said animal is selected from the group consisting of a dog and a cat. The method of Claim 1, wherein said method is performed in solution.

36. The method of Claim 2, wherein said recombinant cell expresses a Fc'R molecule comprising a protein selected from the group consisting of PhFcERca 257 and PhFcER 232

37. The method of Claim 2, wherein said recombinant cell expresses a FcR molecule encoded by a nucleic acid molecule selected from the group consisting of nhFceRa 6 12 nhFcRa 59 1 nhFceRa 69 9 and nhFceRa516.

38. The method of Claim 2, wherein said recombinant cell expresses a FcR molecule :1'I encoded by a nucleic acid molecule selected from the group consisting of a nucleic acid molecule comprising a nucleic acid sequence selected from the group consisting of SEQ ID NO:1 and SEQ ID NO:4, and a nucleic acid molecule comprising an allelic variant of S. a nucleic acid molecule comprising SEQ ID NO:1 and SEQ ID NO:4.

39. The method of Claim 2, wherein said recombinant cell is a RBL-hFcR cell. The kit of Claim 3, wherein said detection means further comprises an antigen selected from the group consisting of an allergen and a parasite antigen, wherein said antigen induces IgE antibody production in animals selected from the group consisting of canines, felines and equines.

41. The kit of Claim 3, wherein said detection means comprises an antibody that S selectively binds to an IgE. -54-

42. The kit of Claim 3, wherein said detection means detects said FcR molecule.

43. The kit of Claim 3, wherein said FcR molecule is on the surface of a recombinant cell that expresses said Fc 8 R molecule.

44. The kit of Claim 40, wherein said antigen is immobilized on a substrate.

45. The kit of Claim 44, wherein said substrate comprises a material selected from the group consisting of plastic, glass, gel, celluloid, paper, magnetic resin, poly-vinylidene- fluoride, nylon, nitrocellulose and particulate material.

46. The invention of Claim 4, 18 or 44, wherein said substrate material is selected from the group consisting of latex, polystyrene, nylon, nitrocellulose, agarose and magnetic resin.

47. The invention of Claim 18 or 44, wherein said substrate comprises a shape selected from the group consisting of a well, a plate, a dipstick, a bead, a lateral flow apparatus, a membrane, a filter, a tube, a dish, a celluloid-type matrix and a magnetic particle.

48. The invention of Claim 4, 18 or 44, wherein said substrate comprises an ELISA •i plate, a dipstick, a radioimmunoassay plate, agarose beads, plastic beads, latex beads, 0 immunoblot membranes and immunoblot papers.

49. The kit of Claim 44, wherein said substrate is latex beads. The kit of Claim 40, wherein said allergen is derived from material selected from the group consisting of fungi, trees, weeds, shrubs, grasses, wheat, corn, soybean, rice, eggs, milk, cheese, bovine, poultry, swine, sheep, yeast, fleas, flies, mosquitos, mites, midges, biting gnats, lice, bees, wasps, ants, true bugs and ticks.

51. The invention of Claim 4 or 50, wherein said flea allergen is a flea saliva antigen. *9go

52. The kit of Claim 3, wherein said parasite antigen is a heartworm antigen.

53. The kit of Claim 3, further comprising an apparatus comprising: a support structure defining a flow path; a labeling reagent comprising a bead conjugated to said antigen, wherein said labeling reagent is impregnated within the support structure in a labeling zone; and a capture reagent comprising said Fc 6 R molecule, wherein said capture reagent is located downstream of said labeling reagent within a capture zone fluidly connected to said labeling zone in such a manner that said labeling reagent can flow from said labeling zone into said capture zone.

54. The kit of Claim 53, wherein said apparatus further comprises a sample receiving zone located along said flow path.

55. The kit of Claim 53, wherein said apparatus further comprises an absorbent located at the end of said flow path.

56. The kit of Claim 54, wherein said sample receiving zone is located upstream of said labeling reagent.

57. The kit of Claim 53, wherein said support structure comprises a material that does not impede the flow of said bead from said labeling zone to said capture zone. Goo" 58. The kit of Claim 53, wherein said support structure comprises an ionic material. *e

59. The kit of Claim 53, wherein said support structure comprises a material selected from the group consisting of nitrocellulose, PVDF and carboxymethylcellulose. The kit of Claim 53, wherein said bead comprises a latex bead.

61. The kit of Claim 53, wherein said labeling reagent is dried within said labeling zone and said capture reagent is dried within said capture zone. C. So re a e.g. 62. An allergen kit comprising an allergen and a human Fc, receptor (FCER) molecule, wherein said allergen is selected from the group consisting of flea, grass, Meadow 4 Fescue, Curly Dock, plantain, Mexican Firebush, Lamb's Quarters, pigweed, ragweed, logo*, 25 sage, elm, cocklebur, Box Elder, walnut, cottonwood, ash, birch, cedar, oak, mulberry, .00. a Go -56- cockroach, Dermataphagoides, Alternaria, Aspergillus, Cladosporium, Fusarium, Helminthosporium, Mucor, Penicillium, Pullularia, Rhizopus and Tricophyton.

63. An allergen kit comprising an allergen and a human Fce receptor (FceR) molecule, wherein said allergen is selected from the group consisting of Johnson Grass, Kentucky Blue Grass, Orchard Grass, Perennial Rye Grass, Redtop Grass, Timothy Grass, Bermuda Grass, Brome Grass, English Plantain, Rough Pigweed, Short Ragweed, Wormwood Sage, American Elm, Common Cocklebur, Black Walnut, Eastern Cottonwood, Green Ash, River Birch, Red Cedar, Red Oak, Red Mulberry, Dermataphagoidesfarinae, Alternaria alternata, Aspergillusfumigatus, Cladosporium herbarum, Fusarium vasinfectum, Helminthosporium sativum, Mucor recemosus, Penicillium notatum, Pullularia pullulans, Rhizopus nigricans and Tricophyton spp.

64. The kit of Claim 62 or Claim 63, wherein said kit comprises one or more compositions, each composition comprising one allergen. The kit of Claim 62 or Claim 63, wherein said allergen is immobilized to a substrate.

66. The invention of Claim 5 or Claim 62, wherein said flea allergen is selected from the group consisting of flea saliva products and flea saliva proteins. 00

67. The FcR alpha chain protein of Claim 6, wherein said FcR alpha chain protein is encoded by a nucleic acid molecule selected from the group consisting of a nucleic acid 0• 20 molecule comprising a nucleic acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO:10 and SEQ ID NO:12, and a nucleic acid molecule comprising an allelic variant of a nucleic acid molecule comprising any of said nucleic acid sequences. v* 68. The FcR alpha chain protein of Claim 6, wherein said Fc 8 R alpha chain protein Gs 25 comprises PhFceRa172-BIOT. *ooS

69. A method to detect IgE, substantially as herein described with reference to any one or more of the examples but excluding comparative examples. -57- A kit for detecting IgE, substantially as herein described with reference to any one or more of the examples but excluding comparative examples.

71. A method to detect flea allergy dermatitis, substantially as herein described with reference to any one or more of the examples but excluding comparative examples.

72. A kit for detecting flea allergy dermatitis, substantially as herein described with reference to any one or more of the examples but excluding comparative examples.

73. An isolated human Fc 6 receptor (FcR) alpha chain protein, substantially as herein described with reference to any one or more of the examples but excluding comparative examples. DATED this 16 th day of October 2003 BALDWIN SHELSTON WATERS Attorneys for: Heska Corporation ooooe °o V. V