WO2001004310A1 - Fc EPSILON RECEPTOR-LUMINESCENCE INDUCING PROTEIN CHIMERIC NUCLEIC ACID MOLECULES, FUSION PROTEINS AND USES THEREOF - Google Patents

Fc EPSILON RECEPTOR-LUMINESCENCE INDUCING PROTEIN CHIMERIC NUCLEIC ACID MOLECULES, FUSION PROTEINS AND USES THEREOF Download PDF

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Publication number
WO2001004310A1
WO2001004310A1 PCT/US2000/019070 US0019070W WO0104310A1 WO 2001004310 A1 WO2001004310 A1 WO 2001004310A1 US 0019070 W US0019070 W US 0019070W WO 0104310 A1 WO0104310 A1 WO 0104310A1
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Prior art keywords
nucleic acid
seq
acid sequence
protein
fusion protein
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PCT/US2000/019070
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French (fr)
Inventor
Eric R. Weber
Keith V. Wood
Mary P. Hall
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Heska Corporation
Promega Corporation
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Priority to AU60941/00A priority Critical patent/AU6094100A/en
Publication of WO2001004310A1 publication Critical patent/WO2001004310A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0069—Oxidoreductases (1.) acting on single donors with incorporation of molecular oxygen, i.e. oxygenases (1.13)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/70535—Fc-receptors, e.g. CD16, CD32, CD64 (CD2314/705F)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/32—Fusion polypeptide fusions with soluble part of a cell surface receptor, "decoy receptors"
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/61—Fusion polypeptide containing an enzyme fusion for detection (lacZ, luciferase)

Definitions

  • the present invention relates to genetic chimeras incorporating Fc epsilon receptor genes and genes encoding bioluminescence or chemiluminescence inducing proteins, fusion proteins encoded by such nucleic acid molecules and methods of using such proteins and nucleic acid molecules for the detection of IgE.
  • IgE antibody production in an animal can be indicative of disease including, for example, allergy, atopic disease, hyper IgE syndrome, internal parasite infections and B cell neoplasia.
  • 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.
  • An allergic response can be mediated by IgE antibodies when IgE complexes with Fc epsilon receptors. Fc epsilon receptors are found on the surface of certain cell types, such as mast cells and basophils.
  • Mast cells store biological mediators including histamine, prostaglandins and proteases. Release of these biological mediators is triggered when IgE antibodies complex with Fc epsilon receptors on the surface of a cell. Clinical symptoms result from the release of the biological mediators into the tissue of an animal.
  • the discovery of the present invention includes a novel genetic chimera that incorporates sequence encoding a Fc epsilon receptor (Fc e R) and sequence encoding a biolummescence or chemiluminescence inducing protein to express a fusion protein that possesses the IgE binding activity of Fc receptor and luminescence inducing activity
  • the present invention also includes the use of such a fusion protein to detect the presence of IgE in a putative IgE-contaimng composition by measu ⁇ ng or recording the presence of luminescence
  • the present invention relates to a novel product and process for detecting IgE
  • the present invention provides fusion proteins having a Fc c R domain and a luminescence inducing protein domain; genetic chimeras, including those that encode such fusion proteins; methods to obtain such proteins and nucleic acid molecules; and methods and kits comprising such proteins and nucleic acid molecules useful for detecting IgE
  • the present invention includes an isolated genetic chimera that encodes a Fc ⁇ R-
  • a prefened Fc e R-LP genetic chimera comp ⁇ ses (l) a Fc e R domain nucleic acid molecule that hyb ⁇ dizes to a nucleic acid molecule selected from the group consisting of SEQ ID NO 3, SEQ ID NO 6, SEQ ID NO 9, SEQ ID NO: 12, an additional human Fc e R nucleic acid sequence, as described m U.S. Patent No 5,945,294, issued August 31, 1999, a felme Fc c R nucleic acid sequence, as described in U.S. Patent No. 5,958,880, issued September 28, 1999, a canme Fc ⁇ R nucleic acid sequence, as described in U.S. Patent No.
  • Particularly prefened Fc e R-LP genetic chimeras include: nucleic acid molecules comprising nucleic acid sequences SEQ ID NO.17, SEQ DD NO: 19, SEQ DD NO:20, SEQ DD NO.22, SEQ ID NO:26, SEQ ID NO:28, SEQ DD NO:29 or SEQ ID NO:31.
  • the present invention also includes Fc e R-LP fusion proteins encoded by such genetic chimeras.
  • Particularly prefened Fc e R-LP fusion proteins include at least one of the following ammo acid sequences: SEQ DD NO.18, SEQ DD NO.21, SEQ DD NO:27, and SEQ ID NO:30.
  • the present invention also relates to recombinant molecules, recombinant viruses and recombinant cells that include Fc ⁇ R-LP chimeric nucleic acid molecules of the present invention. Also included are methods to produce such nucleic acid molecules, recombinant molecules, recombinant viruses and recombinant cells.
  • the present invention also includes detection methods and kits that detect IgE.
  • One embodiment of the present invention is a method to detect IgE compnsing- (a) contacting an isolated Fc e R-LP molecule with a putative IgE-contaming composition under conditions suitable for formation of a Fc e R-LP molecule:IgE complex; and (b) determining the presence of IgE by detecting the Fc e R-LP molecule:IgE complex, the presence of the Fc € R molecule:IgE complex indicating the presence of IgE.
  • Another embodiment of the present invention is a method to detect IgE comprising: (a) contacting a recombinant cell with a putative IgE-containing composition under conditions suitable for formation of a recombinant cel lgE complex, in which the recombinant cell comprises a Fc e R-LP molecule; and (b) determining the presence of IgE by detecting the recombinant celhlgE complex, the presence of the recombinant cell:IgE complex indicating the presence of IgE.
  • Another prefened method to detect IgE comprises: (a) immobilizing a specific antigen on a support substrate; (b) contacting the antigen with the putative IgE-containing composition under conditions suitable for formation of an antigemlgE complex bound to the support substrate; (c) removing non-bound material from the support substrate under conditions that retain antigemlgE complex binding to said support substrate; and (d) detecting the presence of the antigemlgE complex by contacting the antige lgE complex with said Fc e R-LP molecule.
  • Another prefened method to detect IgE comprises: (a) immobilizing an antibody that binds selectively to IgE on a support substrate; (b) contacting the antibody with the putative IgE-containing composition under conditions suitable for formation of an antibody:IgE complex bound to the support substrate; (c) removing non-bound material from the support substrate under conditions that retain antibodyTgE complex binding to the support substrate; and (d) detecting the presence of the antibodyTgE complex by contacting the antibodyTgE complex with said Fc e R-LP molecule.
  • Another prefened method to detect IgE comprises: (a) immobilizing a putative IgE-containing composition on a support substrate; (b) contacting the composition with the Fc e R-LP molecule under conditions suitable for formation of a Fc e R-LP molecule:IgE complex bound to the support substrate; (c) removing non-bound material from the support substrate under conditions that retain Fc e R-LP molecule:IgE complex binding to the support substrate; and (d) detecting the presence of the Fc € R-LP moleculeTgE complex.
  • the present invention also includes a kit for performing methods of the present invention.
  • One embodiment is a kit for detecting IgE comprising a Fc e R-LP protein, LP substrate and a means for detecting Fc e R-LP:IgE complex.
  • the present invention provides for a genetic chimera incorporating both a nucleic acid molecule encoding a high affinity Fc epsilon receptor (Fc e R) and a nucleic acid molecule encoding a bioluminescence or chemiluminescence inducing protein (collectively refened to herein as a "luminescence inducing protein" or "LP").
  • the present invention also provides a fusion protein expressed by such a genetic chimera (also refened to as chimeric nucleic acid molecules), wherein such a fusion protein possesses IgE binding activity and LP activity.
  • a genetic chimera also refened to as chimeric nucleic acid molecules
  • isolated Fc e R proteins and isolated Fc e R nucleic acid molecules refers to Fc e R proteins and Fc e R nucleic acid molecules derived from mammals and, as such, can be obtained from their natural source or can be produced using, for example, recombinant nucleic acid technology or chemical synthesis.
  • the term luminescence inducing protein refers to a protein that chemically or physically interacts with a substrate to cause the substrate to emit light when the LP is contacted with the substrate (refened to herein as a LP substrate). Also included in the present invention is the use of a fusion protein of the present invention in a method to detect epsilon immunoglobulin (refened to herein as IgE or IgE antibody) as well as in other applications, such as those disclosed below.
  • IgE or IgE antibody epsilon immunoglobulin
  • a 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.
  • a protein refers to one or more proteins or at least one protein.
  • the terms “a” (or “an”), “one or more” and “at least one” can be used interchangeably herein.
  • the terms “comprising”, “including”, and “having” can be used interchangeably.
  • a compound “selected from the group consisting of refers to one or more of the compounds in the list that follows, including mixtures (i.e., combinations) of two or more of the compounds.
  • an isolated, or biologically pure, protein is a protein that has been removed from its natural milieu.
  • isolated and isolated Fc e R nucleic acid molecules refers to Fc e R proteins and Fc e R nucleic acid molecules derived from mammals and, as such, can be obtained from their natural source or can be produced using, for example, recombinant nucleic acid technology or chemical synthesis.
  • One embodiment of the present invention is a fusion protein comprising a Fc e R protein domain and an LP domain.
  • a fusion protein refers to a protein produced by expression of a chimeric nucleic acid molecule containing nucleic acid sequences from two or more different organisms and/or synthetically created nucleic acid sequences.
  • a Fc e R-luminescence inducing protein fusion protein (refened to herein as a "Fc e R-LP" fusion protein) of the present invention preferably includes a Fc e R domain that binds to IgE with an affinity similar to that of a free (i.e.
  • Fc e R domain and an LP domain, that interacts with a LP substrate in such a manner so as to give a detectable signal, preferably to a level similar to that of a free LP domain.
  • suitable Fc e R domain include, but are not limited to, a human Fc e R domain, a canine Fc e R domain, a feline Fc e R domain, an equine Fc e R domain, a rat Fc e R domain, and a mouse Fc e R domain.
  • Suitable LP domains include, but are not limited to, luciferase, alkaline phosphatase, ⁇ -galactosidase, glucose oxidase, galactose dehydroginase, urease, catalase and galactokinase, with luciferase and alkaline phosphatase being prefened, and luciferase being particularly prefened.
  • a Fc e R-LP fusion protein of the present invention comprises a protein encoded by a genetic chimera comprising a nucleic acid molecule that encodes a Fc e R protein domain that binds to an IgE, linked to a nucleic acid molecule that encodes an LP domain in such a manner that the chimera encodes a single fused protein having both domains.
  • a genetic chimera comprising a nucleic acid molecule that encodes a Fc e R protein domain that binds to an IgE, linked to a nucleic acid molecule that encodes an LP domain in such a manner that the chimera encodes a single fused protein having both domains.
  • a Fc € R protein domain for use in a fusion protein of the present invention can be a full-length protein or any homolog of such a protein.
  • a protein domain can be a polypeptide or a peptide.
  • a Fc e R protein comprises at least a portion of a Fc e R protein that binds to IgE, i.e., that is capable of forming a complex with an IgE.
  • a Fc e R protein domain of the present invention comprises at least a portion of Fc e R alpha chain.
  • a Fc e R molecule of the present invention can be a full-length protein, a portion of a full-length protein or any homolog of such a protein.
  • a protein can be a polypeptide or a peptide.
  • a Fc e R molecule of the present invention can comprise a complete Fc e R (i.e., alpha, beta and gamma Fc e R chains), an alpha Fc e R chain (also refened to herein as Fc e R chain) or portions thereof.
  • a Fc ⁇ R molecule comprises at least a portion of a Fc ⁇ R chain that binds to IgE, i.e., that is capable of forming an immunocomplex with an IgE constant region.
  • a Fc e R protein for use in a fusion protein of the present invention, including a homolog can be identified in a straight-forward manner by the protein's ability to bind to IgE. Such methods are well known to those of skill in the art.
  • Fc e R protein homologs include Fc e R proteins in which amino acids have been deleted (e.g., a truncated version of the protein, such as a peptide), inserted, inverted, substituted and/or derivatized (e.g., by glycosylation, phosphorylation, acetylation, myristoylation, prenylation, palmitoylation, amidation and/or addition of glycerophosphatidyl inositol) such that the homolog retains the ability of binding to IgE.
  • amino acids e.g., a truncated version of the protein, such as a peptide
  • derivatized e.g., by glycosylation, phosphorylation, acetylation, myristoylation, prenylation, palmitoylation, amidation and/or addition of glycerophosphatidyl inositol
  • Fc ⁇ R protein homologs suitable for use in a fusion protein of the present invention can be the result of natural allelic variation or natural mutation.
  • Fc c R protein 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 a nucleic acid molecule encoding the protein using, for example, classic or recombinant nucleic acid techniques to effect random or targeted mutagenesis.
  • Fc e R homolog amino acid sequences suitable for use in a fusion protein of the present invention have the further characteristic of being encoded by nucleic acid molecules that hybridize under stringent hybridization conditions to Fc c R genes or other nucleic acid molecules encoding a Fc ⁇ R protein.
  • a Fc e R gene includes all nucleic acid sequences related to a natural Fc ⁇ R gene such as regulatory regions that control production of the Fc ⁇ R protein encoded by that gene (such as, but not limited to, transcription, translation or post-translation control regions) as well as the coding region itself.
  • a Fc e R gene of the present invention includes nucleic acid sequence SEQ ID NO 1, SEQ ID NO 3, SEQ ID NO 4, SEQ ID NO.6, SEQ ID NO 7, SEQ ID NO:9, SEQ ID NO: 10, and or SEQ ID NO.12
  • Nucleic acid sequence SEQ ID NO 1 represents the deduced sequence of the coding strand of a complementary DNA (cDNA) nucleic acid molecule denoted herein as nhFc ⁇ R n98 .
  • the complement of SEQ DD NO.1, represented herein by SEQ ID NO:3, refers to the nucleic acid sequence of the strand complementary to the strand having SEQ DD NO 1, which can easily be determined by those skilled in the art.
  • nucleic acid sequence complement of any nucleic acid sequence of the present invention refers to the nucleic acid sequence of the nucleic acid strand that is complementary to (l e., can form a complete double helix with) the strand for which the sequence is cited
  • SEQ DD NO.l and SEQ DD NO: 3 represent apparent nucleic acid sequences of certain nucleic acid molecules encoding Fc ⁇ R proteins of the present invention, including, but not limited to sequences encoding a human Fc ⁇ R domain, a canine Fc e R domain, a feline Fc e R domain, an equine Fc e R domain, a rat Fc ⁇ R domain, and a mouse Fc ⁇ R domain
  • a Fc ⁇ R gene can be an allelic variant that includes a similar but not identical sequence to SEQ DD NO.l, SEQ ID NO:3, SEQ DD NO 4, SEQ DD NO:6, SEQ DD NO.7, SEQ DD NO:9, SEQ DD NO: 10, SEQ ID NO.12, an additional human Fc e R nucleic
  • allelic vanant of a Fc ⁇ R gene is a gene that occurs at essentially the same locus (or loci) in the genome as any of the listed sequences, but which, due to natural vanations caused by, for example, mutation or recombination, has a similar but not identical sequence.
  • Allelic va ⁇ ants typically encode proteins having similar activity to that of the protein encoded by the gene to which they are being compared. Allelic va ⁇ ants can also comp ⁇ se alterations m the 5' or 3' untranslated regions of the gene (e.g., in regulatory control regions). Allelic variants are well known to those skilled in the art and would be expected to be found naturally within a given mammal since the genome is diploid or may be created through laboratory manipulation, such as, but not limited to, variants produced during polymerase chain reaction amplification.
  • the minimal size of a Fc ⁇ R protein homolog suitable for use in a fusion protein of the present invention is a size sufficient to be encoded by a nucleic acid molecule capable of forming a stable hybrid (i.e., hybridize under stringent hybridization conditions) with the complementary sequence of a nucleic acid molecule encoding the conesponding natural protein.
  • the size of the nucleic acid molecule encoding such a protein homolog is dependent on nucleic acid composition and percent homology between the nucleic acid molecule and complementary sequence.
  • the extent of homology required to form a stable hybrid can vary depending on whether the homologous sequences are interspersed throughout the nucleic acid molecules or are clustered (i.e., localized) in distinct regions on the nucleic acid molecules.
  • the minimal size of such nucleic acid molecules is typically at least about 12 to about 15 nucleotides in length if the nucleic acid molecules are GC-rich and at least about 15 to about 17 bases in length if they are AT-rich.
  • the minimal size of a nucleic acid molecule used to encode a Fc e R protein homolog suitable for use in a fusion protein of the present invention is from about 12 to about 18 nucleotides in length.
  • the minimal size of a Fc ⁇ R protein homolog suitable for use in a fusion protein of the present invention is from about 4 to about 6 amino acids in length.
  • the prefened size of a protein suitable for use in a fusion protein of the present invention is a portion of the protein that binds to IgE which is about 20 amino acids, more preferably about 25 amino acids, more preferably about 30 amino acids, more preferably about 35 amino acids, more preferably about 40 amino acids and even more preferably about 45 amino acids in length.
  • Stringent hybridization conditions are determined based on defined physical properties of the gene or other nucleic acid molecule to which the nucleic acid molecule to be tested is being hybridized, and can be defined mathematically. Stringent hybridization conditions are those experimental parameters that allow an individual skilled in the art to identify significant similarities between heterologous nucleic acid molecules These conditions are well known to those skilled m the art.
  • the determination of hybridization conditions involves the manipulation of a set of variables including the ionic strength (M, in moles/liter), the hybridization temperature (°C), the concentration of nucleic acid helix destabilizing agents (such as formamide), the average length of the shortest hybrid duplex (n), and the percent G + C composition of the fragment to which an unknown nucleic acid molecule is being hyb ⁇ dized.
  • T m melting temperature
  • T m 81.5°C + 16.6 log M + 0.41(%G + C) - 500/n - 0.61(%formam ⁇ de).
  • T d dissociation temperature
  • T d 4(G + C) + 2(A + T).
  • a temperature of 5°C below T d is used to detect hybridization between perfectly matched molecules.
  • base pair mismatch i.e. differences between two nucleic acid molecules being compared, including non- complementa ⁇ ty of bases at a given location, and gaps due to insertion or deletion of one or more bases at a given location on either of the nucleic acid molecules being compared, will affect T m or T d for nucleic acid molecules of different sizes.
  • T m decreases about 1°C for each 1% of mismatched base pairs for hyb ⁇ ds greater than about 150 bp
  • T d decreases about 5°C for each mismatched base pair for hyb ⁇ ds below about 50 bp
  • Conditions for hybrids between about 50 and about 150 base pairs can be determined empirically and without undue expe ⁇ mentation using standard laboratory procedures well known to those skilled m the art These simple procedures allow one skilled m the art to set the hybridization conditions (by altering, for example, the salt concentration, the formamide concentration or the temperature) so that only nucleic acid hybrids with greater than a specified % base pair mismatch will hybridize.
  • Stringent hybridization conditions are commonly understood by those skilled in the art to be those experimental conditions that will allow less than or equal to about 30% base pair mismatch (i.e., at least about 70% identity) Because one skilled in the art can easily determine whether a given nucleic acid molecule to be tested is less than or greater than about 50 nucleotides, and can therefore choose the approp ⁇ ate formula for determining hybridization conditions, he or she can determine whether the nucleic acid molecule will hyb ⁇ dize with a given gene under stringent hybridization conditions and similarly whether the nucleic acid molecule will hyb ⁇ dize under conditions designed to allow a desired amount of base pair mismatch.
  • Hybridization reactions are often earned out by attaching the nucleic acid molecule to be hyb ⁇ dized to a solid support such as a membrane, and then hybridizing with a labeled nucleic acid molecule, typically refened to as a probe, suspended in a hybridization solution.
  • Examples of common hybridization reaction techniques include, but are not limited to, the well-known Southern and northern blotting procedures.
  • the actual hybridization reaction is done under non-stringent conditions, i.e., at a lower temperature and/or a higher salt concentration, and then high st ⁇ ngency is achieved by washing the membrane in a solution with a higher temperature and/or lower salt concentration m order to achieve the desired stringency.
  • non-stringent conditions i.e., at a lower temperature and/or a higher salt concentration
  • high st ⁇ ngency is achieved by washing the membrane in a solution with a higher temperature and/or lower salt concentration m order to achieve the desired stringency.
  • the skilled artisan wished to identify a nucleic acid molecule that hybridizes under conditions that would allow less than or equal to 30% pair mismatch with a nucleic acid molecule of about 150 bp m length or greater, the following conditions could preferably be used.
  • the unknown nucleic acid molecules would be attached to a support membrane, and the 150 bp probe would be labeled, e.g.
  • the hybridization reaction could be earned out in a solution comprising 2X SSC and 0% formamide, at a temperature of about 37°C (low stringency conditions)
  • Solutions of differing concentrations of SSC can be made by one of skill in the art by diluting a stock solution of 20X SSC (175.3 gram NaCl and about 88.2 gram sodium citrate in 1 liter of water, pH 7) to obtain the desired concentration of SSC.
  • the skilled artisan would calculate the washing conditions required to allow up to 30% base pair mismatch. For example, assuming an average G + C content of the nucleic acid molecule to be hybridized of about 50%, and a wash solution comprising IX SSC and 0% fo ⁇ riamide, the T m of perfect hybrids would be about 85 ° C:
  • hybridization washes would be canied out at a temperature of less than or equal to 55 ° C. It is thus within the skill of one in the art to calculate additional hybridization temperatures based on the desired percentage base pair mismatch, formulae and G/C content disclosed herein.
  • nucleic acid molecule to be tested for hybridization against nucleic acid molecules of the present invention having sequences specified herein becomes longer than 150 nucleotides, the T m for a hybridization reaction allowing up to 30% base pair mismatch will not vary significantly from 55 °C.
  • Such software programs represent a collection of algorithms paired with a graphical user interface for using the algorithms.
  • the DNAsis version 2.0 software and SeqLab Wisconsin Package Version 10.0-UNLX software employ a particular algorithm, the Needleman-Wunsch algorithm to perform pair-wise comparisons between two sequences to yield a percentage identity score, see Needleman, S.B. and Wunch, CD., 1970, J. Mol. Biol, 48, 443, which is incorporated herein by reference in its entirety.
  • Such algorithms including the Needleman-Wunsch algorithm, are commonly used by those skilled in the nucleic acid and amino acid sequencing art to compare sequences.
  • a prefened method to determine percent identity among amino acid sequences and also among nucleic acid sequences includes using the Needleman- Wunsch algorithm, available in the SeqLab Wisconsin Package Version 10.0-UNLX software (hereinafter "SeqLab”), using the Pairwise Comparison/Gap function with the nwsgapdna.cmp scoring matrix, the gap creation penalty and the gap extension penalties set at default values, and the gap shift limits set at maximum (hereinafter refened to as "SeqLab default parameters").
  • An additional prefened method to determine percent identity among amino acid sequences and also among nucleic acid sequences includes using the Higgins-Sharp algorithm, available in the DNAsis version 2.0 software (hereinafter "DNAsis"), with the gap penalty set at 5, the number of top diagonals set at 5, the fixed gap penalty set at 10, the k-tuple set at 2, the window size set at 5, and the floating gap penalty set at 10.
  • a particularly prefened method to determine percent identity among amino acid sequences and also among nucleic acid sequences includes using the Needleman-Wunsch algorithm available in the DNAsis version 2.0 software, using the GCG default parameter function.
  • a prefened Fc ⁇ R protein domain suitable for use in a fusion protein of the present invention is encoded by at least a portion of SEQ ID NO: 1, SEQ DD NO:3, SEQ ID NO:4, SEQ DD NO:6, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:10, SEQ DD NO: 12, an additional human Fc ⁇ R nucleic acid sequence, as described in U.S. Patent No. 5,945,294, issued August 31, 1999, a feline Fc ⁇ R nucleic acid sequence, as described in U.S. Patent No. 5,958,880, issued September 28, 1999, a canine Fc ⁇ R nucleic acid sequence, as described in U.S. Patent No.
  • Additional prefened Fc ⁇ R proteins suitable for use m a fusion protein of the present invention include a protein encoded by a nucleic acid molecule that hybridizes to a nucleic acid molecule selected from the group consisting of SEQ ID NO:3, SEQ ID NO:6, SEQ ID NO:9, SEQ ID NO: 12, an additional human Fc ⁇ R nucleic acid sequence, as described in U.S Patent No. 5,945,294, issued August 31, 1999, a feline Fc ⁇ R nucleic acid sequence, as described in U.S. Patent No. 5,958,880, issued September 28, 1999, a canme Fc c R nucleic acid sequence, as described in U S. Patent No.
  • Additional prefened Fc ⁇ R proteins suitable for use in a fusion protein of the present invention include proteins compnsing ammo acid sequences that are at least about 65%, preferably at least about 70%, more preferably at least about 75%, more preferably at least about 80%, more preferably at least about 85%, more preferably at least about 90%) and even more preferably about 95%, identical to ammo acid sequence SEQ ID NO:l, SEQ DD NO:3, SEQ ID NO.4, SEQ DD NO:6, SEQ DD NO:7, SEQ DD NO:9, SEQ ID NO: 10, SEQ ID NO.12, an additional human Fc ⁇ R nucleic acid sequence, as described in U.S. Patent No.
  • Amino acid sequence analysis can preferably be performed using either the DNAsisTM program (available from Hitachi Software, San Bruno, CA) using default stringency parameters.
  • Particularly prefened Fc ⁇ R proteins suitable for use in a fusion protein of the present invention include a protein having an ammo acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO:3, SEQ DD NO:4, SEQ ID NO:6, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 12, an additional human Fc ⁇ R nucleic acid sequence, as described in U.S. Patent No. 5,945,294, issued August 31, 1999, a feline Fc ⁇ R nucleic acid sequence, as described in U.S. Patent No.
  • An LP domain for use in a fusion protein of the present invention can be a full- length LP protein or any homolog of such a protein.
  • An LP comprises at least a portion of an LP that interacts with a LP substrate to cause the LP substrate to emit light when the LP is contacted with the LP substrate.
  • Prefened LP domains for use in a fusion protein of the present invention include luciferases, alkaline phosphatases, ⁇ - galactosidases, glucose oxidases, galactose dehydroginases, ureases, catalases and galactokinases, with luciferases, alkaline phosphatases being particularly prefened.
  • Prefened luciferase domains include sequences from Photuris pennsylvanica, Vibrio cholera, Vibrio fischeri, Chesapeake Bay bacterium, Vibrio harveyi, Luciola later alis, Vargula hilgenforfii, Luciola cruciata, Photinus pyralis, Luciola mingrelica, Renilla reniformis, Photobacterium phosphoreum, and Xenorhabdus luminescens.
  • An LP domain for use in a fusion protein of the present invention can be identified in a straight-forward manner by the protein's ability to chemically or physically interact with a LP substrate to cause the LP substrate to emit light when the LP is contacted with the LP substrate. Such methods are well known to those of skill in the art.
  • LP protein homologs include LP proteins in which amino acids have been deleted (e.g., a truncated version of the protein, such as a peptide), inserted, inverted, substituted and/or derivatized (e.g., by glycosylation, phosphorylation, acetylation, myristoylation, prenylation, palmitoylation, amidation and/or addition of glycerophosphatidyl inositol) such that the homolog retains LP activity.
  • an LP homolog of the present invention includes, for example, a thermostable LP domain.
  • a thermostable LP domain means a protein which retains biological activity following incubation at 65°C for at least about 1 hour and/or following incubation at room temperature for at least about 1 week.
  • LP homologs suitable for use in a fusion protein of the present invention can be the result of natural allelic variation or natural mutation.
  • LP 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 nucleic acid techniques to effect random or targeted mutagenesis.
  • LP homolog amino acid sequences suitable for use in a fusion protein of the present invention have the further characteristic of being encoded by nucleic acid molecules that hybridize under stringent hybridization conditions to LP genes or other nucleic acid molecules encoding an LP.
  • an LP gene includes all nucleic acid sequences related to a natural LP gene such as regulatory regions that control production of the LP encoded by that gene (such as, but not limited to, transcription, translation or post-translation control regions) as well as the coding region itself.
  • an LP gene can be an allelic variant that includes a similar but not identical sequence to SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO:23 and/or SEQ DD NO:25.
  • Additional prefened LPs suitable for use in a fusion protein of the present invention include proteins comprising amino acid sequences that are at least about 65%), preferably at least about 70%, more preferably at least about 75%, more preferably at least about 80%o, more preferably at least about 85%, more preferably at least about 90% and even more preferably about 95%>, identical to amino acid sequence SEQ ID NO: 15 and/or SEQ ID NO:24.
  • Amino acid sequence analysis is preferably performed using the DNAsisTM program (available from Hitachi Software, San Bruno, CA), using default stringency parameters.
  • Additional prefened LPs suitable for use in a fusion protein of the present invention include a protein encoded by a nucleic acid molecule that hybridizes to a nucleic acid molecule selected from the group consisting of SEQ ID NO: 16, and/or SEQ ID NO:25, under conditions comprising (a) hybridization in a solution comprising 2X SSC and 0% formamide, at a temperature of 37°C, and (b) washing in IX SSC and 0% fonriamide at a temperature of 55°C
  • Particularly prefened LPs suitable for use in a fusion protein of the present invention include a protein having an ammo acid sequence selected from the group consisting of SEQ ID NO 15 and/or SEQ ID NO.24, or a fragment thereof having LP activity
  • One embodiment of the present invention is a Fc ⁇ R-LP fusion protein that includes a Fc ⁇ R protein-contammg domain and an LP-contammg domain attached to one or more fusion segments
  • Suitable fusion segments for use with the present invention include, but are not limited to, segments that can: enhance a protein's stability; act as an immunopotentiator to enhance an immune response against a Fc ⁇ R protein; and/or assist purification of a Fc ⁇ R-LP fusion protein (e.g , by affinity chromatography).
  • a suitable fusion segment can be a domain of any size that has the desired function (e.g., imparts increased stability, imparts structural or spacial mobility, imparts increased immunogenicity to a protein, and/or simplifies pu ⁇ fication of a protein).
  • Fusion segments can be joined to ammo and/or carboxyl termini of the Fc ⁇ R-contammg domain and/or the LP-contaming domain of the fusion protein and can be susceptible to cleavage m order to enable straight-forward recovery of the fusion protein.
  • Fusion proteins are preferably produced by cultu ⁇ ng a recombinant cell transformed with a fusion nucleic acid molecule that encodes a protein including the fusion segment attached to either the carboxyl and/or amino terminal end of a Fc ⁇ R-contammg domain or the carboxyl and/or ammo terminal end of an LP-contammg domain.
  • Prefened fusion segments for use in a genetic chimera of the present invention include linker sequences described by Berg , Proc Natl Acad Sci. 85:99-102, 1998, and Pomerantz et al., Science 263:671-673, 1995, and the linker sequence (Gly4Serl) 2 , i.e SEQ ID NO.13, the production of which is described in the Examples.
  • a prefened fusion protein of the present invention includes at least an IgE- binding portion of a Fc ⁇ R domain compnsing SEQ ID NO 1, SEQ DD NO 3, SEQ ID NO 4, SEQ ID NO:6, SEQ DD NO.7, SEQ ID NO 9, SEQ ID NO: 10, SEQ ID NO: 12, or an allelic variant thereof and an LP-active portion of an LP domain compnsing SEQ DD NO.14, SEQ ID NO: 16, SEQ ID NO.23 or SEQ DD NO:25, or an allelic variant thereof, and preferably also includes a linker between said domains.
  • a more prefened Fc ⁇ R-LP fusion protein of the present invention includes a protein encoded by a nucleic acid molecule comprising a nucleic acid sequence selected from the group consisting of SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:26, SEQ ID NO:28, SEQ DD NO:29 or SEQ ID NO:31. More prefened is a Fc ⁇ R-LP protein having an amino acid sequence selected from the group consisting of SEQ DD NO: 18, SEQ ID NO:22, SEQ ID NO:27, or SEQ ID NO:30.
  • Particularly prefened Fc ⁇ R-LP fusion proteins are PhFc ⁇ R-Ppluc 756 , PhFc ⁇ R-P ⁇ luc 73 impart PhFc ⁇ R-bAP 660 , and/or PhFc ⁇ R-bAP 635 .
  • Prefened Fc e R-LP fusion proteins also include amino acid sequences SEQ ID NO: 18, SEQ ID NO:21, SEQ ID NO:27, and SEQ ID NO:30.
  • a Fc ⁇ R-LP fusion protein of the present invention can also include a domain that enables the fusion protein to be bound to a support substrate in such a manner that the Fc ⁇ R domain of the fusion protein binds to IgE in essentially the same manner as a Fc ⁇ R molecule that is not bound to a support substrate.
  • An example of a suitable binding domain includes a portion of an immunoglobulin molecule or another ligand that has a suitable binding partner that can be immobilized on a support substrate, e.g., biotin and avidin, or a metal-binding protein and a metal (e.g., His), or a sugar-binding protein and a sugar (e.g., maltose).
  • Another embodiment of the present invention is a genetic chimera comprising a nucleic acid molecule encoding a Fc ⁇ R (Fc e R nucleic acid molecule) and a nucleic acid molecule encoding an LP (a LP nucleic acid molecule) such that the chimera, refened to herein as a Fc ⁇ R-LP nucleic acid molecule or a Fc ⁇ R-LP genetic chimera, encodes a single fusion protein comprising a Fc ⁇ R domain and a LP domain.
  • a nucleic acid molecule of the present invention can include an isolated natural Fc e R gene or cDNA plus an isolated natural LP gene or cDNA or homologs thereof, the latter of which are described in more detail below.
  • a nucleic acid molecule of the present invention can include one or more regulatory regions, full-length or partial coding regions, or combinations thereof.
  • an isolated nucleic acid molecule is a nucleic acid molecule that has been removed from its natural milieu (i.e., that has been subject to human manipulation) and can include DNA, RNA, or derivatives of either DNA or RNA.
  • isolated does not reflect the extent to which the nucleic acid molecule has been purified.
  • An isolated Fc e R and/or LP nucleic acid molecule of the present invention can be isolated from its natural source or can be produced using recombinant DNA technology (e.g., polymerase chain reaction (PCR) amplification, cloning) or chemical synthesis.
  • PCR polymerase chain reaction
  • isolated LP proteins and isolated LP nucleic acid molecules refers to LP proteins and LP nucleic acid molecules derived from living organisms and, as such, can be obtained from their natural source or can be produced using, for example, recombinant nucleic acid technology or chemical synthesis.
  • Isolated Fc e R and/or LP nucleic acid molecules can include, for example, natural allelic variants as well as other nucleic acid molecule homologs that are modified by one or more nucleotide insertions, deletions, substitutions, and/or inversions in a manner such that the modifications do not substantially interfere with the nucleic acid molecule's ability to encode Fc e R protein or LP proteins of the present invention, respectively, or to form stable hybrids under stringent conditions with natural gene isolates.
  • a Fc ⁇ R and/or LP nucleic acid molecule homolog for use in a genetic chimera of the present invention can be produced using a number of methods l ⁇ iown to those skilled in the art (see, for example, Sambrook et al., ibid.).
  • nucleic acid molecules can be modified using a variety of techniques including, but not limited to, classic mutagenesis and recombinant DNA techniques (e.g., site-directed mutagenesis, chemical treatment, restriction enzyme cleavage, ligation of nucleic acid fragments and/or PCR amplification), synthesis of oligonucleotide mixtures and ligation of mixture groups to "build" a mixture of nucleic acid molecules and combinations thereof.
  • Nucleic acid molecule homologs can be selected by hybridization with a Fc ⁇ R or LP gene or cDNA or by screening for function of a protein encoded by the nucleic acid molecule, e.g., ability of a Fc ⁇ R protein to bind IgE and ability of an LP protein to induce luminescence when contacted with a suitable LP substrate, respectively.
  • An isolated Fc ⁇ R-LP nucleic acid molecule of the present invention includes a nucleic acid sequence that encodes a Fc ⁇ R-LP fusion protein comprising at least one Fc ⁇ R domain and at least one LP domain of the present invention, examples of such domains and proteins being disclosed herein.
  • nucleic acid molecule primarily refers to the physical nucleic acid molecule and the phrase “nucleic acid sequence” primarily refers to the sequence of nucleotides on the nucleic acid molecule, the two phrases can be used interchangeably, especially with respect to a nucleic acid molecule, or a nucleic acid sequence, being capable of encoding a Fc ⁇ R domain or a LP domain or a Fc e R-LP fusion protein of the present invention.
  • Prefened Fc ⁇ R nucleic acid molecules for use in a genetic chimera of the present invention include nucleic acid molecules having a nucleic acid sequence that is at least about 70%), preferably at least about 75%o, preferably at least about 80%, preferably at least about 85%, more preferably at least about 90%, and even more preferably at least about 95% identical to nucleic acid sequence SEQ ID NO:l, SEQ DD NO:3, SEQ ID NO:4, SEQ DD NO:6, SEQ ID NO:7, SEQ ID NO:9, SEQ DD NO: 10, SEQ ID NO: 12, an additional human Fc ⁇ R nucleic acid sequence, as described in U.S. Patent No.
  • Another prefened Fc e R nucleic acid molecule for use in a genetic chimera of the present invention includes a nucleic acid molecule that hybridizes to a nucleic acid molecule selected from the group consisting of SEQ DD NO:3, SEQ DD NO:6, SEQ DD NO:9, SEQ DD NO: 12, an additional human Fc ⁇ R nucleic acid sequence, as described in U.S. Patent No. 5,945,294, issued August 31, 1999, a feline Fc ⁇ R nucleic acid sequence, as described in U.S. Patent No. 5,958,880, issued September 28, 1999, a canine Fc ⁇ R nucleic acid sequence, as described in U.S. Patent No.
  • Another prefened Fc ⁇ R nucleic acid molecule for use in a genetic chimera of the present invention includes at least a portion of nucleic acid sequence SEQ DD NO: 1 , SEQ ID NO:3, SEQ DD NO:4, SEQ ID NO:6, SEQ DD N0:7, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO: 12, an additional human Fc ⁇ R nucleic acid sequence, as described in U.S. Patent No. 5,945,294, issued August 31, 1999, a feline Fc ⁇ R nucleic acid sequence, as described in U.S. Patent No. 5,958,880, issued September 28, 1999, a canine Fc ⁇ R nucleic acid sequence, as described in U.S. Patent No.
  • a more prefened Fc ⁇ R nucleic acid molecule for use in a genetic chimera of the present invention includes nucleic acid sequence SEQ ID NO:l, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:9, SEQ DD NO: 10, SEQ DD NO: 12, an additional human Fc ⁇ R nucleic acid sequence, as described in U.S. Patent No. 5,945,294, issued August 31, 1999, a feline Fc ⁇ R nucleic acid sequence, as described in U.S. Patent No. 5,958,880, issued September 28, 1999, a canine Fc c R nucleic acid sequence, as described in U.S. Patent No.
  • Fc ⁇ R nucleic acid molecules can include nucleotides in addition to those included in the sequences described immediately above, such as, but not limited to, a full-length gene or a full-length coding region.
  • a prefened Fc ⁇ R nucleic acid molecule suitable for use in a genetic chimera of the present invention also includes a nucleic acid molecule that encodes a protein having at least a portion of SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO: 12, an additional human Fc e R nucleic acid sequence, as described in U.S. Patent No. 5,945,294, issued August 31, 1999, a feline Fc e R nucleic acid sequence, as described in U.S. Patent No. 5,958,880, issued September 28, 1999, a canine Fc ⁇ R nucleic acid sequence, as described in U.S. Patent No.
  • Prefened LP nucleic acid molecules for use in a genetic chimera of the present invention include nucleic acid molecules having a nucleic acid sequence that is at least about 70%, preferably at least about 75%, preferably at least about 80%, preferably at least about 85%, more preferably at least about 90%, and even more preferably at least about 95% identical to nucleic acid sequence SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO:23 and/or SEQ DD NO:25.
  • DNA sequence analysis is preferably performed using the DNAsisTM program using default stringency parameters.
  • Another prefened LP nucleic acid molecule for use in a genetic chimera of the present invention includes a nucleic acid molecule that hybridizes to a nucleic acid molecule selected from the group consisting of SEQ ID NO: 16, and/or SEQ ID NO:25, under conditions comprising (a) hybridization in a solution comprising 2X SSC and 0% formamide, at a temperature of 37°C, and (b) washing in IX SSC and 0%> formamide at a temperature of 55°C.
  • Another prefened LP nucleic acid molecule for use in a genetic chimera of the present invention includes a nucleic acid molecule having nucleic acid sequence SEQ DD NO: 14, SEQ ID NO: 16, SEQ DD NO:23 and/or SEQ DD NO:25, with SEQ ID NO: 14 and SEQ ID NO: 16, or portions thereof having LP activity, as well as allelic variants thereof being more prefened.
  • Such LP nucleic acid molecules can include nucleotides in addition to those included in the SEQ DD NOs, such as, but not limited to, a full-length gene or a full-length coding region.
  • a prefened LP nucleic acid molecule suitable for use in a genetic chimera of the present invention also includes a nucleic acid molecule that encodes a protein having at least a portion of SEQ ID NO: 15 and/or SEQ DD NO:25, including nucleic acid molecules that have been modified to accommodate codon usage properties of the cells in which such nucleic acid molecules are to be expressed.
  • Prefened genetic chimeras of the present invention include at least a Fc e R nucleic acid molecule portion which encodes a Fc ⁇ R protein which binds to IgE and a LP nucleic acid molecule portion which encodes a LP protein having LP activity, and preferably also includes a linker nucleic acid molecule.
  • More prefened genetic chimeras of the present invention include nucleic acid molecule constructs having a nucleic acid sequence that is at least about 70%, preferably at least about 75%, preferably at least about 80%) preferably at least about 85%, more preferably at least about 90%, more preferably at least about 95%, and even more preferably at least about 100% identical to nucleic acid molecules nhFc e R-npluc 2268 , nhFc ⁇ R-npluc 2]93 , nhFc ⁇ R-nbAP I9g3 , and/or nhFc ⁇ R-nbAP ]908 .
  • a particularly prefened genetic chimera of the present invention includes a nucleic acid molecule having nucleic acid sequence SEQ DD NO: 17, SEQ ID NO: 19, SEQ DD NO:20, SEQ DD NO:22, SEQ DD NO:26, SEQ DD NO:28, SEQ ID NO:29 or SEQ ID NO:31.
  • nucleic acid sequences of certain Fc ⁇ R and LP nucleic acid molecules of the present invention allows one skilled in the art to, for example, (a) make copies of those nucleic acid molecules, (b) obtain nucleic acid molecules including at least a portion of such nucleic acid molecules (e.g., nucleic acid molecules including full-length genes, full-length coding regions, regulatory control sequences, truncated coding regions), and (c) obtain Fc ⁇ R and LP nucleic acid molecules suitable for use in a genetic chimera of the present invention from other organisms.
  • nucleic acid molecules can be obtained in a variety of ways including screening appropriate expression libraries with antibodies of the present invention; traditional cloning techniques using oligonucleotide probes of the present invention to screen appropriate libraries or DNA; and PCR amplification of appropriate libraries or DNA using oligonucleotide primers of the present invention.
  • Prefened libraries to screen or from which to amplify a Fc ⁇ R nucleic acid molecule include basophil cell, mast cell, mastocytoma cell, dendritic cell, B lymphocyte, macrophage, eosinophil, and/or monocyte cDNA libraries as well as genomic DNA libraries.
  • prefened DNA sources to screen or from which to amplify a Fc ⁇ R nucleic acid molecule include basophil cells, mast cells, mastocytoma cells, dendritic cells, B lymphocytes, macrophages, eosinophils, and/or monocytes cDNA and genomic DNA.
  • Prefened sources for LP nucleic acid molecules include libraries or DNA of organisms that have luminescent proteins. Techniques to clone and amplify genes are disclosed, for example, in Sambrook et al, ibid.
  • One embodiment of the present invention includes a recombinant vector, which includes at least one isolated nucleic acid molecule comprising a genetic chimera of the present invention, inserted into any vector capable of delivering the nucleic acid molecule into a host cell
  • a vector contains heterologous nucleic acid sequences, that is nucleic acid sequences that are not naturally found adjacent to nucleic acid molecules of the present invention and that preferably are denved from a species other than the species from which the nucleic acid molecule(s) are de ⁇ ved.
  • the vector can be either RNA or DNA, either prokaryotic or eukaryotic, and typically is a virus or a plasmid.
  • Recombinant vectors can be used in the cloning, sequencing, and/or otherwise manipulation of Fc ⁇ R, LP and Fc ⁇ R-LP nucleic acid molecules of the present invention
  • One type of recombinant vector, refened to herein as a recombinant molecule comprises a nucleic acid molecule comprising a genetic chimera of the present invention operatively linked to an expression vector.
  • the phrase operatively linked refers to insertion of a nucleic acid molecule into an expression vector in a manner such that the molecule is able to be expressed when transformed into a host cell.
  • an expression vector is a DNA or RNA vector that is capable of transforming a host cell and of effecting expression of a specified nucleic acid molecule
  • the expression vector is also capable of replicating within the host cell
  • Expression vectors can be either prokaryotic or eukaryotic, and are typically viruses or plasmids.
  • Expression vectors of the present invention include any vectors that function (i.e., direct gene expression) in recombinant cells of the present invention, including in bacte ⁇ al, fungal, insect, other animal, and plant cells.
  • Prefened expression vectors of the present invention can direct gene expression in bacterial, yeast, insect and mammalian cells and more preferably in the cell types disclosed herein.
  • expression vectors of the present invention contain regulatory sequences such as transc ⁇ ption control sequences, translation control sequences, origins of replication, and other regulatory sequences that are compatible with the recombinant cell and that control the expression of nucleic acid molecules of the present invention.
  • recombinant molecules of the present invention include transcnption control sequences.
  • Transc ⁇ ption control sequences are sequences that control the initiation, elongation, and termination of transc ⁇ ption.
  • Particularly important transcription control sequences are those which control transcription initiation, such as promoter, enhancer, operator and repressor sequences.
  • Suitable transcription control sequences include any transcription control sequence that can function in at least one of the recombinant cells of the present invention.
  • Prefened transcription control sequences include those which function in bacterial, yeast, insect and mammalian cells, such as, but not limited to, tac, lac, trp, trc, oxy-pro, omp/lpp, rrnB, bacteriophage lambda (such as lambda p L and lambda p R and fusions that include such promoters), bacteriophage T7, ⁇ llac, bacteriophage T3, bacteriophage SP6, bacteriophage SP01, metallothionein, alpha-mating factor, Pichia alcohol oxidase, alphavirus subgenomic promoters (such as Sindbis virus subgenomic promoters), antibiotic resistance gene, baculovirus, Heliothis zea insect virus, vaccinia virus, herpesvirus, raccoon poxvirus, other poxvirus, adenovirus, cytomegalovirus
  • transcription control sequences include tissue-specific promoters and enhancers as well as lymphokine-inducible promoters (e.g., promoters inducible by interferons or interleukins). Transcription control sequences of the present invention can also include naturally occurring transcription control sequences naturally associated with the Fc ⁇ R or LP nucleic acid molecule being expressed.
  • Suitable and prefened Fc ⁇ R-LP nucleic acid molecules to include in recombinant vectors of the present invention are as disclosed herein.
  • Prefened nucleic acid molecules to include in recombinant vectors, and particularly in recombinant molecules include SEQ DD NO: 17, SEQ ID NO: 19, SEQ DD NO:20, SEQ DD NO:22, SEQ DD
  • a particularly prefened recombinant molecule of the present invention includes SEQ DD NO: 17, SEQ DD NO: 19, SEQ ID NO: 20, and SEQ DD NO: 22, the production of which is described in the Examples section.
  • Recombinant molecules of the present invention can and preferably do, contain secretory signals (i.e., signal segment nucleic acid sequences) to enable an expressed fusion protein of the present invention to be secreted from the cell that produces the protein
  • suitable signal segments include any signal segment capable of directing the secretion of a protein of the present invention.
  • Prefened signal segments include, but are not limited to, Fc ⁇ R alpha chain, tissue plasminogen activator (t-PA), interferon, mterleukm, growth hormone, histocompatibility and viral envelope glycoprotem signal segments, as well as natural signal segments.
  • t-PA tissue plasminogen activator
  • a nucleic acid molecule of the present invention can be joined to a fusion segment that directs the encoded protein to the proteosome, such as a ubiquitin fusion segment.
  • Recombinant molecules may also include intervening and/or untranslated sequences surrounding and/or withm the nucleic acid sequences of nucleic acid molecules of the present invention.
  • Another embodiment of the present invention includes a recombinant cell comprising a host cell transformed with one or more recombinant 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 (i.e , recombinant) cell in such a manner that their ability to be expressed is retained.
  • Prefened nucleic acid molecules with which to transform a cell include Fc ⁇ R-LP chimeric nucleic acid molecules disclosed herein.
  • Particularly prefened nucleic acid molecules with which to transform a cell include SEQ ID NO 17, SEQ ID NO- 19, SEQ DD NO.20, SEQ ID NO.22, SEQ DD NO.26, SEQ ID NO.28, SEQ ID NO.29 and/or SEQ ID NO.31
  • 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 (e.g , nucleic acid molecules encoding one or more proteins of the present invention and/or other proteins).
  • 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 (including yeast), other insect, other animal and plant cells.
  • Prefened host cells include bacterial, mycobacteria, yeast, parasite, insect and mammalian cells.
  • More prefened host cells include Salmonella, Escherichia, Bacillus, Listeria, Saccharomyces , Spodoptera, Mycobacteria, Trichoplusia, BHK (baby hamster kidney) cells, MDCK cells (normal dog kidney cell line for canine herpesvirus cultivation), CRFK cells (normal cat kidney cell line for feline herpesvirus cultivation), CV-1 cells (African monkey kidney cell line used, for example, to culture raccoon poxvirus), COS (e.g., COS-7) cells, and Vero cells.
  • Particularly prefened host cells are Escherichia coli, including E.
  • coli K-12 derivatives Salmonella typhi; Salmonella typhimurium, including attenuated strains such as UK-1 x 3987 and SR-11 x 4072; Spodoptera frugiperda; Trichoplusia ni; BHK cells; MDCK cells; CRFK cells; CV-1 cells; COS cells; Vero cells; and non-tumorigenic mouse myoblast G8 cells (e.g., ATCC CRL 1246).
  • Additional appropriate mammalian cell hosts include other kidney cell lines, other fibroblast cell lines (e.g., human, murine or chicken embryo fibroblast cell lines), myeloma cell lines, Chinese hamster ovary cells, mouse NDT/3T3 cells, LMTK 31 cells and/or HeLa cells.
  • a recombinant cell is preferably produced by transforming a host cell with one or more recombinant molecules, each comprising one or more nucleic acid molecules of the present invention operatively linked to an expression vector containing one or more transcription control sequences.
  • the phrase operatively linked refers to insertion of a nucleic acid molecule into an expression vector in a manner such that the molecule is able to be expressed when transformed into a host cell.
  • 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 prefened recombinant molecule includes nhFc ⁇ R-npluc 2268 , nhFc ⁇ R-npluc 2]93 , nhFc ⁇ R-nbAP 1983 , and/or nhFc ⁇ R-nbAP 1908 ..
  • a recombinant cell of the present invention includes any cell transformed with at least one of any nucleic acid molecule of the present invention. Suitable and prefened nucleic acid molecules as well as suitable and prefened recombinant molecules with which to transform cells are disclosed herein.
  • a particularly prefened recombinant cell includes nhFc ⁇ R-npluc 2268 , nhFc ⁇ R-npluc 2193 , nhFc ⁇ R-nbAP 1983 , and/or nhFc ⁇ R-nbAP l908 . Details regarding the production of this recombinant cell is disclosed herein.
  • Recombinant DNA technologies can be used to improve expression of transformed nucleic acid molecules by manipulating, for example, the number of copies of the nucleic acid molecules within a host cell, the efficiency with which those nucleic acid molecules are transcribed, the efficiency with which the resultant transcripts are translated, and the efficiency of post-translational modifications.
  • Recombinant techniques useful for increasing the expression of nucleic acid molecules of the present invention include, but are not limited to, operatively linking nucleic acid molecules to high-copy number plasmids, integration of the nucleic acid molecules into one or more host cell chromosomes, addition of vector stability sequences to plasmids, substitutions or modifications of transcription control signals (e.g., promoters, operators, enhancers), substitutions or modifications of translational control signals (e.g., ribosome binding sites, Shine-Dalgarno sequences), modification of nucleic acid molecules of the present invention to conespond to the codon usage of the host cell, deletion of sequences that destabilize transcripts, and use of control signals that temporally separate recombinant cell growth from recombinant enzyme production during fermentation.
  • transcription control signals e.g., promoters, operators, enhancers
  • translational control signals e.g., ribosome binding sites, Shine-Dalgarno sequences
  • Isolated Fc ⁇ R-LP fusion proteins of the present invention can be produced in a variety of ways, including production and recovery of natural proteins, production and recovery of recombinant proteins, and chemical synthesis of the proteins.
  • an isolated protein of the present invention is produced by culturing a cell capable of expressing the protein under conditions effective to produce the protein, and recovering the protein.
  • a prefened cell to culture is a recombinant cell of the present invention.
  • Effective culture conditions include, but are not limited to, effective media, bioreactor, temperature, pH and oxygen conditions that permit protein production.
  • An effective medium refers to any medium in which a cell is cultured to produce a Fc ⁇ R-LP fusion protein of the present invention.
  • a medium typically comprises an aqueous medium having assimilable carbon, nitrogen and phosphate sources, and appropriate salts, minerals, metals and other nut ⁇ ents, such as vitamins.
  • Cells of the present invention can be cultured in conventional fermentation bioreactors, shake flasks, test tubes, microtiter dishes, and pet ⁇ plates. Culturmg can be earned out at a temperature, pH and oxygen content appropriate for a recombinant cell Such cultu ⁇ ng conditions are within the expertise of one of ordinary skill in the art
  • resultant proteins of the present invention may either remain within the recombinant cell; be secreted into the fermentation medium; be secreted into a space between two cellular membranes, such as the pe ⁇ plasmic space in E coli, or be retained on the outer surface of a cell or viral membrane
  • the phrase "recovering the protein”, as well as similar phrases, refers to collecting the whole fermentation medium containing the protein and need not imply additional steps of separation or purification
  • Proteins of the present invention can be purified using a variety of standard protein pu ⁇ fication techniques, such as, but not limited to, affinity chromatography, ion exchange chromatography, filtration, electrophoresis, hydrophobic interaction chromatography, gel filtration chromatography, reverse phase chromatography, concanavahn A chromatography, chromatofocusmg and differential solubihzation.
  • Proteins of the present invention are preferably retrieved m "substantially pure” form.
  • substantially pure refers to a pu ⁇ ty that allows for the effective use of the protein as a therapeutic composition or diagnostic
  • a Fc ⁇ R-LP fusion protein of the present invention can be bound to the surface of a cell comprising the Fc ⁇ R-LP fusion protein
  • a prefened fusion protem-bearmg cell includes a recombinant cell comprising a nucleic acid molecule encoding a Fc ⁇ R-LP fusion protein of the present invention
  • a more prefened recombinant cell of the present invention compnses a nucleic acid molecule that encodes at least one of the following proteins- PhFc ⁇ R-Ppluc 756 , PhFc ⁇ R-Ppluc 73] , PhFc ⁇ R-bAP 660 , and/or PhFc ⁇ R-bAP 635
  • a recombinant cell comprising a nucleic acid molecule compnsing a nucleic acid sequence including SEQ DD NO:17, SEQ DD NO: 19, SEQ ID NO:20, SEQ ID NO:22, SEQ DD NO:26, SEQ ID NO:28, SEQ ID NO:29 and/or SEQ DD NO:31, or a nucleic acid molecule comprising an allelic va ⁇ ant of a nucleic acid molecule comprising SEQ ID NO: 17, SEQ ID NO: 19, SEQ DD NO:20, SEQ ID NO:22, SEQ ID NO:26, SEQ DD NO:28, SEQ ID NO:29 and/or SEQ ID NO:31, being even more prefened.
  • a Fc ⁇ R-LP fusion protein-containing composition of the present invention can include not only a Fc ⁇ R-LP fusion protein but also one or more additional antigens or antibodies useful in detecting IgE.
  • an antigen refers to any molecule capable of being selectively bound by an antibody.
  • selective binding of a first molecule to a second molecule refers to the ability of the first molecule to preferentially bind (e.g., 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.
  • Anti-IgE antibodies for use in a formulation of the present invention preferably are not capable of cross-linking two or more IgE antibodies.
  • prefened anti-IgE antibodies include Fab fragments of the antibodies (as defined in Janeway et al., ibid.). Examples of such antigens include any antigen known to induce the production of IgE. Prefened antigens include allergens and parasite antigens.
  • Allergens include, but are not limited to allergens ingested, inhaled or contacted by an organism of interest. Allergens of the present invention are preferably derived from fungi, rusts, smuts, bacteria, trees, weeds, shrubs, grasses, wheat, corn, grains, hays, straws, oats, alfalfa, clovers, soybeans, yeasts, 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 prefened flea allergen includes a flea saliva antigen.
  • Prefened flea saliva antigens include antigens such as those disclosed in PCT Patent Publication No. WO 96/28469, published September 19, 1996, by Stiegler et al., PCT Patent Publication No. WO 96/11271, published April 18, 1996, by Frank et al, and PCT Patent Publication No. WO 97/37676, published October 16, 1997, by Hunter et al., with flea saliva products and flea saliva proteins being particularly prefened.
  • 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.
  • Prefened general allergens include those derived from grass, Meadow Fescue, curly dock, plantain, Mexican firebush, lamb's quarters, pigweed, ragweed, goldenrod, sonel, legumes, dandelion, sage, elm, cocklebur, elder, walnut, maple, sycamore, hickory, aspen, pine, cottonwood, ash, birch, cedar, oak, mulberry, cockroach, Dermataphagoides , Alternaria, Aspergillus, Cladosporium, Fusarium, Helminthosporium, Mucor, Curvularia, Candida, Penicillium, Pullularia, Rhizopus and/or Tricophyton.
  • More prefened general allergens include those derived from Johnson grass, Kentucky blue grass, meadow fescue, orchard grass, perennial rye grass, red top grass, timothy grass, Bermuda grass, salt grass, brome grass, curly dock, yellow dock, English plantain, Mexican firebush, lamb's quarters, rough pigweed, short ragweed, goldenrod, sheep sonel, red clover, dandelion, wormwood sage, American elm, common cocklebur, box elder, marsh elder, black walnut, red maple, eastern sycamore, white pine, eastern cottonwood, green ash, river birch, red cedar, red oak, red mulberry, cockroach, grain smut, oat stem rust, wheat stem rust, Dermataphagoides farinae, Alternaria alternata, Alternaria tenuis, Curvularia spicifera, Aspergillus fumigatus, Cladosporium herbarum, Fusa
  • the term "derived from” refers to a natural allergen of such plants or organisms (i.e., an allergen directly isolated from such plants or organisms), as well as, non-natural allergens of such plants or organisms that posses at least one epitope capable of eliciting an immune response against an allergen (e.g., produced using recombinant DNA technology or by chemical synthesis).
  • Prefened allergens include those that cause allergic respiratory diseases in equines, including, for example, chronic obstructive pulmonary disease, exercise induced pulmonary hemonhage and inhalant-induced urticaria.
  • allergens include, but are not limited to, molds, components of dust and components of feed.
  • One embodiment of the present invention is a method to detect IgE which includes the steps of: (a) contacting an isolated Fc ⁇ R-LP fusion protein with a putative IgE-containing composition under conditions suitable for formation of a Fc e R-LP:IgE complex; and (b) detecting the presence of IgE by detecting the Fc ⁇ R-LP:IgE complex
  • Presence of such a Fc e R-LP:IgE complex i.e a complex between IgE and a Fc ⁇ R-LP fusion protein of the present invention indicates that the animal is producing IgE
  • Prefened IgE to detect using a Fc ⁇ R-LP fusion protein include any mammalian IgE, with human IgE, canine IgE, feline IgE, equme IgE, murine IgE and rat IgE being more prefened, with human, canme IgE, felme IgE and equme IgE being
  • a heat labile IgE is determined by mcubatmg an IgE at about 56 °C for about 3 or about 4 hours
  • the inventors believe that heat labile forms of IgE bind to certain allergens and non-heat labile forms of IgE bind to other types of allergens.
  • detection of heat labile IgE compared with non-heat labile IgE can be used to discriminate between allergen sensitivities
  • Animals in which to detect IgE include mammals and birds, with humans, dogs, cats, horses and other pets, work and/or economic food animals being prefened.
  • canme refers to any member of the dog family, including domestic dogs, wild dogs and zoo dogs.
  • dogs include, but are not limited to, domestic dogs, wild dogs, foxes, wolves, jackals and coyotes
  • felme refers to any member of the cat family, including domestic cats, wild cats and zoo cats.
  • cats include, but are not limited to, domestic cats, wild cats, lions, tigers, leopards, panthers, cougars, bobcats, lynx, jaguars, cheetahs, and servals.
  • equme refers to any member of the horse family, including, but are not limited to, domestic horses, wild horses and zoo horses.
  • the term "contacting" refers to combining or mixing, in this case a putative IgE-contammg composition with a Fc e R-LP molecule.
  • Formation of a complex between a Fc ⁇ R-LP molecule and an IgE refers to the ability of the Fc ⁇ R-LP molecule to selectively bind to the IgE in order to form a stable complex that can be measured (i.e., detected).
  • the term selectively binds to an IgE refers to the ability of a Fc ⁇ R-LP molecule of the present invention to preferentially bind to IgE, without being able to substantially bind to other antibody lsotypes. Binding between a Fc ⁇ R-LP molecule and an IgE is effected under conditions suitable to form a complex; such conditions (e.g., 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, Sambrook et al., ibid.
  • detecting complex formation refers to determining if any complex is formed, i.e., assaying for the presence (i.e., 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 Fc e R-LP molecule and any IgE in the composition can be measured (i.e., detected, determined) using a variety of methods standard in the art (see, for example, Sambrook et al. ibid.), examples of which are disclosed herein.
  • 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 (i.e., obtained) from an animal, examples of which include, but are not limited to, blood, serum, plasma, urine, tears, aqueous humor, cerebrospinal fluid (CSF), saliva, lymph, nasal secretions, tracheobronchial aspirates, milk, feces and fluids obtained through bronchial alveolar lavage.
  • CSF cerebrospinal fluid
  • 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.
  • removal can include, but is not limited to, contacting the bodily fluid with a material, such 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.
  • a composition includes collected bodily fluid that is pretreated to concentrate immunoglobulin contained in the fluid.
  • immunoglobulin contained in a bodily fluid can be precipitated from other proteins using ammonium sulfate.
  • a prefened composition of the present method is serum.
  • a complex can be formed and detected in solution.
  • a complex can be formed in which one or more members of the complex are immobilized on (e.g , coated onto) a support substrate. Immobilization techniques are known to those skilled m the art Suitable support substrate materials include, but are not limited to, plastic, glass, gel, celluloid, paper, PVDF (poly- vmylidene-fluo ⁇ de), nylon, nitrocellulose, and particulate materials such as latex, polystyrene, nylon, nitrocellulose, agarose and magnetic resm.
  • Suitable shapes for support substrate material include, but are not limited to, a well (e.g., microtiter dish well), a plate, a dipstick, a bead, a lateral flow apparatus, a membrane, a filter, a tube, a dish, a celluloid-type mat ⁇ x, a magnetic particle, and other particulates.
  • a particularly prefened support substrate comprises an ELISA plate, a dipstick, a radioimmunoassay plate, agarose beads, plastic beads, latex beads, immunoblot membranes and immunoblot papers.
  • a support substrate, such as a particulate can include a detectable marker
  • a prefened method to detect IgE is an immunosorbent assay.
  • 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 m such a manner that the IgE is immobilized to a support substrate.
  • a capture molecule is preferably immobilized to a support substrate of the present invention pnor to exposure of the capture molecule to a putative IgE-containmg composition.
  • An Fc ⁇ R-LP of the present invention detects the presence of an IgE bound to a capture molecule.
  • an indicator molecule preferably is not immobilized to the same support substrate as a capture molecule pnor to exposure of the capture molecule to a putative IgE- containing composition.
  • a specific antigen is used as a capture molecule by being immobilized on a support substrate, such as a microtiter dish well or a dipstick.
  • a support substrate such as a microtiter dish well or a dipstick.
  • Prefened antigens include those disclosed herein
  • a biological sample collected from an animal is applied to the support substrate and incubated under conditions suitable (i.e , sufficient) to allow for ant ⁇ gen:IgE complex formation bound to the support substrate (i.e., IgE in a sample binds to an antigen immobilized on a support substrate).
  • Excess non-bound material i.e., mate ⁇ al from the biological sample that has not bound to the antigen
  • Prefened conditions are generally disclosed in Sambrook et al., ibid.
  • a Fc ⁇ R-LP is added to the LP substrate and incubated to allow formation of a complex between the Fc ⁇ R-LP and the antigemlgE complex. Excess Fc ⁇ R-LP is removed, a substrate for LP is added, and the complex is submitted to a detection device for analysis.
  • the Fc c R-LP molecule is a Fc c R-luciferase fusion protein.
  • the Fc ⁇ R-LP molecule is nhFc ⁇ R-npluc 2l93 .
  • Prefened detection devices include luminometers and photographic film.
  • an anti-IgE antibody e.g., isotype or idiotype specific antibody
  • a support substrate such as a microtiter dish well or a dipstick.
  • a biological sample collected from an animal is applied to the support substrate and incubated under conditions suitable to allow for anti-IgE antibody:IgE complex formation bound to the support substrate. Excess non-bound material, if any, is removed from the support substrate under conditions that retain anti-IgE antibodyTgE complex binding to the support substrate.
  • a Fc ⁇ R-LP molecule is added to the support substrate and incubated to allow formation of a complex between the Fc ⁇ R-LP molecule and the anti-IgE antibodyTgE complex.
  • the Fc ⁇ R-LP molecule is a Fc c R-luciferase fusion protein.
  • the Fc ⁇ R-LP molecule is nhFc ⁇ R-npluc 2193 .
  • Prefened detection devices include luminometers and photographic film.
  • an immunosorbent assay of the present invention does not utilize a capture molecule.
  • a biological sample collected from an animal is applied to a support substrate, such as a microtiter dish well or a dipstick, and incubated under conditions suitable to allow for IgE binding to the support substrate.
  • any IgE present in the bodily fluid is immobilized on the support substrate.
  • Excess non- bound material, if any, is removed from the support substrate under conditions that retain IgE binding to the support substrate.
  • a Fc ⁇ R-LP molecule is added to the support substrate and incubated to allow formation of a complex between the Fc ⁇ R molecule and the IgE.
  • Excess Fc ⁇ R molecule is removed, a LP substrate is added, and the sample is submitted to a detection device for analysis.
  • the Fc e R-LP molecule is a Fc ⁇ R-luciferase fusion protein.
  • the Fc ⁇ R-LP molecule is nhFc ⁇ R-npluc 2193 .
  • the Fc ⁇ R-LP molecule is a Fc ⁇ R-luciferase fusion protein.
  • the Fc ⁇ R-LP molecule is nhFc ⁇ R-npluc 2 , 93 .
  • Prefened detection devices include luminometers and photographic film.
  • Another prefened 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 December 22, 1994, by Miller et al.; and WO 94/01775, published January 20, 1994, by Pawlak et al.
  • a biological sample is placed in a lateral flow apparatus that includes the following components: (a) a support structure defining a flow path; and (b) a capture reagent comprising an antigen or and anti-IgE antibody, the capture reagent being impregnated within the support structure in a labeling zone.
  • a labeling reagent comprising a Fc ⁇ R-LP is added to the lateral flow apparatus and a detecting means is used to detect bound complex.
  • Prefened antigens include those disclosed herein.
  • the support structure comprises a material that does not impede the flow of IgE contained in a biological sample to the capture reagent.
  • Suitable materials for use as a support structure include ionic (i.e., anionic or cationic) material. Examples of such a material include, but are not limited to, nitrocellulose (NC), PVDF, carboxymethylcellulose (CM).
  • the support structure defines a flow path that is lateral and includes a capture zone.
  • the apparatus can further comprise a sample receiving zone located along the flow path, more preferably upstream of the capture 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.
  • a biological sample is placed in a flow through apparatus that includes the following components: (a) a support structure defining a flow path; and (b) a capture reagent comprising an antigen or and anti-IgE antibody, the capture reagent being impregnated within the support structure in a labeling zone.
  • a labeling reagent comprising a Fc ⁇ R-LP is added to the lateral flow apparatus and a detecting means is used to detect bound complex.
  • Prefened antigens include those disclosed herein.
  • the present invention also includes kits to detect IgE based on each of the disclosed detection methods. Suitable and prefened Fc ⁇ R-LP fusion proteins are disclosed herein.
  • a prefened kit of the present invention further comprises a detection means including one or more antigens disclosed herein, a Fc e R-LP of the present invention and a LP substrate therefor.. Such antigens preferably induce IgE antibody production in animals including humans, canines, equines and/or felines.
  • kits of the present invention are a general allergen kit comprising an allergen common to all regions of the United States and a Fc ⁇ R protein of the present invention.
  • a "general allergen" kit refers to a kit comprising allergens that are found substantially throughout the United States (i.e., 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 prefened general allergens for use with a general allergen kit of the present invention include those general allergens disclosed herein.
  • a prefened kit of the present invention includes those in which the allergen is immobilized on a support 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. In one embodiment, multiple lateral flow apparatuses can be attached to each other at one end of each apparatus, thereby creating a fan-like structure or can comprise a single device with multiple capture lines. In another embodiment, a prefened kit comprises a flow through apparatus or an ELISA. 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. Particularly prefened conditions to diagnose include allergies, parasitic infections and neoplasia.
  • a method and kit of the present invention are particularly useful for detecting hypersensitivity to the bite of insects.
  • Particularly prefened is a method and kit useful for detecting flea allergy dermatitis (FAD), when such method or kit includes the use of flea antigens, preferably flea saliva antigens.
  • FAD is defined as a hypersensitive response to fleabites.
  • a putative IgE-containing composition is obtained from an animal suspected of having FAD.
  • Prefened animals include those disclosed herein, with humans, dogs, cats and horses being more prefened.
  • One embodiment of the present invention is a method to identify a compound capable of inhibiting Fc ⁇ R protein activity.
  • Such a method includes the steps of (a) contacting (e.g., combining, mixing) an isolated Fc ⁇ R-LP protein of the present invention, with a putative inhibitory compound under conditions in which, in the absence of the compound, the protein has Fc ⁇ R protein activity, and (b) determining if the putative inhibitory compound inhibits the activity.
  • Fc ⁇ R protein activity can be determined in a variety of ways known in the art, including but not limited to determining the ability of Fc ⁇ R protein to bind to or otherwise interact with IgE.
  • Such conditions under which a Fc e R protein or a Fc ⁇ R-LP fusion protein has Fc ⁇ R protein activity include conditions in which a Fc ⁇ R protein or a Fc e R-LP fusion has a conect three-dimensionally folded structure under physiologic conditions, i.e. physiologic pH, physiologic ionic concentrations, and physiologic temperatures.
  • Putative inhibitory compounds to screen include antibodies (including fragments and mimetopes thereof), putative substrate analogs, and other, preferably small, organic or inorganic molecules.
  • Methods to determine Fc ⁇ R protein activity are known to those skilled in the art.
  • Methods to determine binding of a putative inhibitory compound to a Fc ⁇ R-LP protein of the present invention are known to those of skill in the art and include, for example, determining changes in molecular mass using surface plasmon resonance (e.g., determining light scatter by an inhibitor of a Fc ⁇ R-LP fusion protein, before and after contacting the inhibitor or protein with a Fc ⁇ R-LP fusion protein or inhibitor, respectively) or screening for compounds that inhibit interaction between a Fc ⁇ R protein and IgE.
  • a prefened method to identify a compound capable of inhibiting Fc ⁇ R protein activity includes contacting an isolated Fc ⁇ R-LP fusion protein with a putative inhibitory compound under conditions in which, in the absence of the compound, the Fc ⁇ R-LP fusion protein has Fc ⁇ R protein activity; and determining if the putative inhibitory compound inhibits the activity; wherein such Fc ⁇ R-LP fusion protein has an LP domain comprising an amino acid sequence encoded by a nucleic acid molecule that hybridizes to an isolated nucleic acid molecule selected from the group consisting of SEQ ID NO: 16, and SEQ DD NO:25 under conditions comprising: (a) hybridizing in a solution comprising 2X SSC and 0% formamide, at a temperature of 37°C; and (b) washing in IX SSC and 0%> formamide at a temperature of 55°C and the Fc ⁇ R-LP fusion protein has a Fc epsilon receptor domain comprising an amino acid sequence encoded by
  • kits to identify an inhibitor of Fc ⁇ R protein activity comprises an isolated Fc ⁇ R-LP fusion protein of the present invention, and a means for determining inhibition of Fc ⁇ R activity, where the means enables detection of inhibition. Detection of inhibition of Fc e R-LP fusion protein activity identifies a putative inhibitor to be an inhibitor of Fc ⁇ R.
  • Means for determining inhibition of a Fc ⁇ R-LP fusion protein include an assay system that detects binding of a putative inhibitor to a Fc ⁇ R-LP fusion protein, and an assay system that detects interference by a putative inhibitor of the ability of a Fc ⁇ R-LP fusion protein to bind IgE. Means and methods are described herein and are known to those skilled in the art.
  • This example describes the construction of a recombinant baculovirus expressing a fusion protein including a firefly luciferase, fused to the carboxyl terminus of a soluble portion of the alpha-chain of an Fc epsilon receptor.
  • Recombmant molecule pFB-nhFc ⁇ R-npluc 2268 containing a nucleic acid molecule encoding the extracellular domain of a human Fc ⁇ R , fused to sequences encoding Photuris pennsylvanica firefly luciferase, and operatively linked to baculovirus polyhedron transcription control sequences was produced in the following manner A cDNA molecule encoding the full-length alpha chain of the human Fc epsilon receptor was obtained from Dr.
  • the cDNA molecule included an about 1198 nucleotide insert, refened to herein as nhFc ⁇ R ] ]9g .
  • the nucleic acid sequence of the coding strand of nhFc ⁇ R U9g is denoted herein as SEQ ID NO.1
  • Translation of SEQ ID NO: 1 indicates that nucleic acid molecule nhFc e R, 198 encodes a full-length human Fc ⁇ R alpha-chain protein of about 257 ammo acids, refened to herein as PhFc ⁇ R 257 , having ammo 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 DD NO.
  • the complement of SEQ ID NO: 1 is represented herein by SEQ DD NO:3.
  • the proposed mature protein i.e., Fc ⁇ R ⁇ chain from which the signal sequence has been cleaved
  • PhFc ⁇ R 232 contains about 232 ammo acids which is represented herein as SEQ ID NO:5.
  • the nucleic acid molecule encoding PhFc ⁇ R 232 is denoted herein as having a coding strand represented by SEQ ID NO'4 and a complementary strand represented by SEQ ID NO:6
  • a Fc ⁇ R alpha-chain extracellular domain nucleic acid molecule-containing fragment of about 591 nucleotides was PCR amplified from nhFc ⁇ R, ]98 using a forward p ⁇ mer Fc/+, having the nucleic acid sequence 5' GGC CGG ATC CTA TAA ATA TGG CTC CTG CCA TGG AAT CC 3' and containing a BamHl site indicated in bold, denoted SEQ ID NO: 32, and a reverse p ⁇ mer Fc/-, having the nucleic acid sequence 5' GGC CGA ATT CAG CTT TTA TTA CAG TAA TGT TGA G 3' and containing an EcoRI site indicated m bold, denoted herein as SEQ DD NO:33.
  • nucleic acid molecule nhFc e R 60 contamed an about 591 nucleotide fragment encoding the extracellular domain of the human Fc ⁇ R alpha-chain, extending from nucleotide 107 to nucleotide 697 of SEQ ID NO.l, denoted herein as nucleic acid molecule nhFc ⁇ R 59] , having a coding strand of which has a nucleic acid sequence denoted SEQ ID NO 7 and a complementary strand denoted SEQ ID NO: 9
  • Translation of SEQ ID NO -7 indicates that nucleic acid molecule nhFc ⁇ R 59 , encodes a Fc ⁇ R protein of about 197 ammo acids, refened to herein as PhFc ⁇ R 197 , having amino acid sequence SEQ
  • SEQ ID NO.10 The complement of SEQ ID NO.10 is represented herein by SEQ DD NO.12.
  • the nucleic acid molecule nhFc ⁇ R ⁇ 59l was subcloned into unique BamHl and EcoRI sites of pFASTBACl baculovirus shuttle plasmid, available from Pharmingen, San Diego, CA, to produce a recombinant molecule refened to herein as pFB-nhFc ⁇ R 591
  • the resultant recombinant molecule pFB-nhFc ⁇ R ⁇ 59] was ve ⁇ fied for proper insert orientation by restriction mapping.
  • DNA sequences encoding a GlyGlyGlyGlySerGlyGlyGlyGlyGlyGlySer linker peptide hereinafter refened to as (Gly4Serl) 2 , denoted S ⁇ Q DD NO: 13, were inserted between sequences encoding the 3' end of the Fc ⁇ R alpha-chain cDNA and sequences encoding the 5' end of the firefly luciferase cDNA, in the following manner.
  • Two complementary oligonucleotides containing sequences encoding the (Gly4Serl) 2 linker peptide, were prepared.
  • a sense oligonucleotide pnmer denoted as (Gly4Serl) 2 /+, having the sequence 5' AA TTC GGT GGT GGC GGT TCT GGT GGC GGT GGC TCT T 3', and containing an EcoRI sticky end site at the 5 ' end and a Xbal sticky end site at the 3 ' end, denoted S ⁇ Q ID NO:34
  • a complementary olrgonucleotide primer denoted as (Gly4Serl) 2 /-, having the sequence 5' CT AGA AGA GCC ACC GCC ACC AGA ACC GCC ACC ACC G 3 ', and containing an Xbal sticky end site at the 5 ' end and a EcoRI sticky end site at the 3' end, denoted S ⁇ Q DD NO:35 were prepared These two
  • the cDNA molecule included an about 1638 nucleotide insert, refened to herein as npluc, 638 having a coding strand denoted herein as SEQ ID NO: 14 and a complementary strand denoted SEQ ID NO: 16.
  • nucleic acid molecule npluc 1638 encodes a full-length firefly luciferase protein of about 545 ammo acids, refened to herein as Ppluc 545 , having ammo acid sequence SEQ ID NO: 15, assuming an open reading frame in which the initiation codon spans from about nucleotide 1 through about nucleotide 3 of SEQ DD NO: 14 and the termination codon spans from about nucleotide 1636 through about nucleotide 1638 of SEQ ID NO: 14.
  • a firefly luciferase nucleic acid molecule-containing fragment of about 1638 nucleotides was PCR amplified from npluc ]638 using forward primer Ppluc/+, having nucleic acid sequence 5' GGG GCC CCT CTA GAA TGG CAG ATA AGA ATA TTT TAT ATG GG 3' containing a Xbal site indicated in bold, denoted SEQ DD NO:36 and reverse primer Ppluc/-, having nucleic acid sequence 5' GCG CGC GCA AGC TTT TAC CCA TTG GTG TGT TTT TC 3' containing a HindlH.
  • the resulting recombinant molecule refened to as pFB-nhFc ⁇ R-npluc 2268 , contained an about 2268-nucleot ⁇ de chime ⁇ c nucleic acid molecule, namely nhFc ⁇ R- npluc 2268 , encoding the extracellular domain of the human Fc ⁇ R alpha-chain, fused to the 5' end of nucleic acid sequences encoding luciferase, from the firefly Photuris pennsylvanica, with these two nucleic acid sequences being linked by nucleic acid sequences encoding a GlyGlyGlySerGlyGlyGlyGlySer linker peptide.
  • the coding strand of nhFc ⁇ R-npluc 2268 has a nucleic acid sequence denoted SEQ DD NO: 17 and a complementary strand denoted SEQ ID NO: 19.
  • Translation of SEQ ID NO: 17 indicates that nucleic acid molecule nhFc ⁇ R-npluc 2268 encodes an Fc ⁇ R-luc ⁇ ferase fusion protein of about 756 amino acids, refened to herein as PhFc ⁇ R-Ppluc 756 , having ammo acid sequence SEQ ID NO: 18.
  • Recombinant molecule pFB-nhFc e R-npluc 2268 was verified for proper insert orientation by restnction mapping.
  • the nucleic acid molecule encoding PhFc ⁇ R-Ppluc 731 refened to herein as nhFc e R-npluc 2193 , has a coding strand with a nucleic acid sequence denoted SEQ ID NO: 19 and a complementary strand with a nucleic acid sequence denoted SEQ DD NO:21.
  • This example descnbes the construction of a recombinant baculovirus expressing a fusion protein including bacterial alkaline phosphatase, fused to the carboxyl terminus of a truncated, soluble portion of an alpha-chain of Fc receptor as well as production and testing of that fusion protein
  • A. Recombinant molecule pFB-nhFc e R-bAP ]983 containing a nucleic acid molecule encoding the extracellular domain of a human Fc e R alpha-chain, fused to sequences encoding bacte ⁇ al alkaline phosphatase, and operatively linked to baculovirus polyhedron transcnption control sequences was produced in the following manner.
  • Nucleic acid molecule pFB-nhFc ⁇ R 591 and the (Gly4Serl) 2 linker were used to generate the construct pFB-nhFc ⁇ R 591 /(Gly4Serl) 2 as described in Example 1.
  • a PCR fragment containing nucleic acid sequences encoding the mature form of bacterial alkaline phosphatase was generated in the following manner.
  • a bacte ⁇ al alkaline phosphatase mature domain nucleic acid molecule-contammg fragment of about 1347 nucleotides was PCR amplified from E coli XLl-Blue using forward pnmer bAP/+, having the nucleic acid sequence 5' GGG CCC TCT AGA AC A CCA GAA ATG CCT GTT CTG GAA AAC CGG 3' containing a Xbal site indicated in bold, denoted SEQ DD NO:38 and reverse primer bAP/-, having the nucleic acid sequence 5' GCG CGC AAG CTT TTA YTT MAG CCC CAG AGC GGC 3' containing an Hindm site indicated in bold, denoted herein as SEQ ID NO:39.
  • the resulting PCR product was blunt end ligated into TOPO vector (available from InVitrogen, Carlsbad, CA) for further manipulation.
  • the PCR clone included an about 1347 nucleotide insert, refened to herein as nbAP 1347 having a coding strand denoted herein as SEQ ID NO:23 and a complementary strand denoted SEQ DD NO:25.
  • nucleic acid nbAP 1347 encodes a mamre bacterial alkaline phosphatase protein of about 449 amino acids, refened to herein as PbAP 449 , having amino acid sequence SEQ ID NO:24, assuming an open reading frame in which the first codon spans from about nucleotide 1 through about nucleotide 3 of SEQ DD NO:23 and the last codon spans from about nucleotide 1345 through about nucleotide 1347 of SEQ ID NO:23.
  • nbAP 1347 was digested with Xbal and HindHI to produce nbAP 1347 , which was ligated into the unique Xbal and Hindm restriction sites of pFB-nhFc ⁇ R 591 /(Gly4Serl) 2 .
  • the resulting recombinant molecule refened to as pFB-nhFc e R-nbAP ]983 contains an about 1983 nucleotide chimeric nucleic acid molecule, namely nhFc ⁇ R- nbAP 19g3 encoding the extracellular domain of the human Fc ⁇ R alpha-chain, fused to the 5' end of nucleic acid sequences encoding alkaline phosphatase from E. coli XLl-Blue, with these two nucleic acid sequences being linked by nucleic acid sequences encoding a Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser linker peptide.
  • the coding strand of nhFc ⁇ R- nbAP 1983 has a nucleic acid sequence denoted SEQ DD NO:26 and the complementary strand denoted SEQ ID NO:28.
  • Translation of SEQ ID NO:26 indicates that nucleic acid molecule nhFc ⁇ R-bAP 1983 encodes a Fc ⁇ R/bacterial alkaline phosphatase fusion protein of about 660 amino acids, refened to herein as PhFc ⁇ R-bAP 660 , having amino acid sequence SEQ ID NO:27.
  • Recombinant molecule pFB-nhFc ⁇ R-bAP 1983
  • Positive plaques containing recombinant molecule pFB-nhFc ⁇ R-bAP, 983 were individually co-transfected with a linear Baculogold baculovirus DNA (available from Pharmingen) into separate cultures of S. frugiperda Sf9 cells (available from InVitrogen, Carlsbad, CA) to form cultures each containing recombinant cells denoted S. frugiperda:p ⁇ B-nhF c e R-b AY ⁇ 9S3 .
  • S. frugiperda:p ⁇ B-nhF c e R-b AY ⁇ 9S3 S. frugiperda:p ⁇ B-nhF c e R-b AY ⁇ 9S3 .
  • frugiperda:p ⁇ B-n ⁇ c R-bAP, 9g3 cultures were grown using conditions known to those skilled in the art in order to produce the fusion protein, PhFc ⁇ R-bAP 660 .
  • Supernatants containing the secreted form of the fusion protein, refened to as PhFc e R-bAP 635 , denoted SEQ DD NO:30 was prepared from the cultures by centrifuging for 20 minutes at 5000 x g and discarding the cell pellet.
  • nhFc ⁇ R-nbAP 190g has a coding strand with a nucleic acid sequence denoted SEQ ID NO:29 and a complementary strand with a nucleic acid sequence denoted SEQ ID NO:31.
  • PhFc ⁇ R-bAP 635 was tested for its ability to detect IgE in the following manner. Polystyrene, opaque, microtiter wells (Immunolon 96 well plate) were coated with a recombinant flea saliva antigen using the same protocols used for coating polystyrene beads described in Example 3. The wells were then blocked for 30 minutes with 100 ⁇ L of IX Tris buffered saline (TBS) (50 mM Tris-HCl, 150 mM NaCl, 2 mM MgCl 2 , containing 1%> bovine serum albumin).
  • TBS IX Tris buffered saline
  • Antigen-coated wells were incubated with either flea-allergy positive or negative pooled dog serum overnight using 250 ⁇ l serum. Following serum incubations, all sample wells were washed three times in of 300 ⁇ L IX TBS containing 0.25% Tween-20 and incubated with an PhFc ⁇ R-bAP 660 fusion protein, prepared as described in section 2, as follows. Specifically, two samples of positive serum incubations were paired with two samples of negative serum incubations and each pair was incubated with supernatant from a different transfection clone expressing PhFc ⁇ R-bAP 660 . Two wells were incubated with supernatant from a "cells only" mock transfection, i.e. supernatants from cell cultures which were not transfected with a clone expressing PhFc e R-bAP 660 . All supernatant incubations were conducted in 250 ⁇ l of supernatant for 90 minutes at room temperature.
  • a 50- ⁇ l aliquot of control beads was prepared m an identical manner except that no antigen was added to the incubation.
  • the aliquot of antigen-coated beads was divided into 10- ⁇ l samples for incubation as follows. Four samples were incubated with flea-allergy positive pooled dog sera, i.e. sera collected from dogs shown to produce IgE against FS-I; and five samples were incubated with flea-allergy negative pooled dog sera. Incubations were conducted overnight in 250 ⁇ l sera supplemented with Tween-20 to a concentration of 0.025%.
  • sample 1 was incubated with flea-allergy negative dog serum followed by incubation with non-transfected expression supernatant; sample 2 was incubated with flea-allergy negative dog serum followed by incubation with PhFc ⁇ R-Ppluc 756 expression supernatant; and sample 3 was incubated with flea-allergy positive dog serum and non- transfected expression supernatant. Incubations were performed as described above for antigen-coated beads.
  • luciferase substrate available from Promega Corporation, Madison, WI
  • Fc e RIa-luciferase fusion-incubated wells and luminescence expressed in relative light units (RLU) was measured for 5 seconds/well in a TopCount NXT TM Microplate Scintillation and Luminescence Counter, available from Packard Instrument Company Meriden, CT.
  • Streptavidin-conjugated alkaline phosphatase available from Jackson ImmunoResearch Labs, Westgrove, PA, was added to biotinylated Fc e RIa-incubated wells and incubated for one hour at room temperamre, then washed with TBST and 100 ml p-NPP substrate, available from Moss Inc., Pasadena, MD was added to the wells. After a development time of twenty minutes, the reaction was stopped with 20 mM cysteine and the optical density was read at an absorbance (A) of 405 nm in a 96-well spectrophotometer. The optical density measurement was multiplied by a factor of 1000.
  • luciferase substrate available from Promega Corportaion
  • Fc e RIa-luciferase fusion-incubated wells 100 ml luciferase substrate, available from Promega Corportaion, was added to Fc e RIa-luciferase fusion-incubated wells and luminescence was measured for 5 seconds/well in a TopCount NXT TM Microplate Scintillation and Luminescence Counter, available from Packard Instrument Company. Streptavidin-conjugated alkaline phosphatase, available from Jackson

Abstract

The present invention relates to genetic chimeras incorporating Fc epsilon receptor genes and genes encoding bioluminescence or chemiluminescence inducing proteins, fusion proteins encoded by such nucleic acid molecules and methods of using such proteins and nucleic acid molecules for the detection of IgE and identifying compounds capable of inhibiting Fc epsilon receptor activity.

Description

Fc EPSILON RECEPTOR-LUMINESCENCE INDUCING PROTEIN CHIMERIC NUCLEIC ACID MOLECULES, FUSION PROTEINS AND USES THEREOF
FIELD OF THE INVENTION The present invention relates to genetic chimeras incorporating Fc epsilon receptor genes and genes encoding bioluminescence or chemiluminescence inducing proteins, fusion proteins encoded by such nucleic acid molecules and methods of using such proteins and nucleic acid molecules for the detection of IgE.
BACKGROUND OF THE INVENTION Diagnosis of disease and determination of treatment efficacy are important tools in medicine. IgE antibody production in an animal can be indicative of disease including, 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. An allergic response can be mediated by IgE antibodies when IgE complexes with Fc epsilon receptors. Fc epsilon receptors are found on the surface of certain cell types, such as mast cells and basophils. Mast cells store biological mediators including histamine, prostaglandins and proteases. Release of these biological mediators is triggered when IgE antibodies complex with Fc epsilon receptors on the surface of a cell. Clinical symptoms result from the release of the biological mediators into the tissue of an animal.
Prior investigators have disclosed the nucleic acid sequence for: the human FceR 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 human FceR beta chain (Kuster et al, J. Biol. Chem. 267: 12782-12787, 1992); the human FcεR gamma chain (Kuster et al., J. Biol. Chem. 265:6448-6452, 1990); the canine FceR alpha chain (GenBank™ accession number D 16413); the feline FceR alpha chain (PCT Publication Number WO 98/27208), and the murine and rat FceR alpha chains (Blank et al., J. Biol. Chem, 266(4):2639-2646, 1991). Investigators have also described the construction of a few genetic chimeras having sequences from luminescent proteins, Oker-Blom et al, Biotechmques 14(5) 800-809, 1993 and Khol et al., N Y Acad Sci 646' 106-115, 1991 However, the determination of human and canme Fc epsilon receptor sequences does not indicate, suggest or predict genetic chimeras expressing a fusion protein possessing IgE binding activity and luminescence inducing activity.
Cunent methods for IgE detection use anti-IgE antibody, which can be chemically conjugated to biotin, or Fc6R chemically conjugated to biotin, Savage et al. Avidin-Biotin Chemistry: A Handbook, Pierce Chemical Company 1992, in order to create a color change upon the addition of steptavidm to indicate the presence of IgE. This strategy is disadvantageous that it requires chemical conjugation of biotm to anti- IgE or Fc epsilon receptor proteins and the test itself includes repeated washing steps Additionally, it is often difficult to measure the number of biotms present m the reaction, making it difficult to quantify the amount of IgE present in a sample.
Thus, products and processes of the present invention are needed in the art that will provide highly sensitive detection of IgE.
SUMMARY OF THE INVENTION The discovery of the present invention includes a novel genetic chimera that incorporates sequence encoding a Fc epsilon receptor (FceR) and sequence encoding a biolummescence or chemiluminescence inducing protein to express a fusion protein that possesses the IgE binding activity of Fc receptor and luminescence inducing activity The present invention also includes the use of such a fusion protein to detect the presence of IgE in a putative IgE-contaimng composition by measuπng or recording the presence of luminescence
The present invention relates to a novel product and process for detecting IgE The present invention provides fusion proteins having a FccR domain and a luminescence inducing protein domain; genetic chimeras, including those that encode such fusion proteins; methods to obtain such proteins and nucleic acid molecules; and methods and kits comprising such proteins and nucleic acid molecules useful for detecting IgE The present invention includes an isolated genetic chimera that encodes a FcεR-
LP fusion protein. A prefened FceR-LP genetic chimera compπses (l) a FceR domain nucleic acid molecule that hybπdizes to a nucleic acid molecule selected from the group consisting of SEQ ID NO 3, SEQ ID NO 6, SEQ ID NO 9, SEQ ID NO: 12, an additional human FceR nucleic acid sequence, as described m U.S. Patent No 5,945,294, issued August 31, 1999, a felme FccR nucleic acid sequence, as described in U.S. Patent No. 5,958,880, issued September 28, 1999, a canme FcεR nucleic acid sequence, as described in U.S. Patent No. 6,060,326, issued May 9, 2000, an equine FceR nucleic acid sequence, as descπbed m U.S Patent No. 6,057,127, issued May 2, 2000, and the muπne and rat FceR nucleic acid sequences as described in Blank et al., J. Biological Chemistry, 266(4):2639-2646, 1991, under conditions comprising (a) hybridization in a solution compnsing 2X SSC and 0% formamide, at a temperature of 37°C, and (b) washing in IX SSC and 0% formamide at a temperature of 55°C and (n) an LP domain encoded by a nucleic acid molecule that hybπdizes to a nucleic acid molecule selected from the group consisting of SEQ ID NO.14, SEQ DD NO 16, SEQ ID NO:23 and/or SEQ DD NO:25, under conditions comprising (a) hybridization in a solution comprising 2X SSC and 0% formamide, at a temperature of 37°C, and (b) washing m IX SSC and 0% formamide at a temperature of 55°C. Particularly prefened FceR-LP genetic chimeras include: nucleic acid molecules comprising nucleic acid sequences SEQ ID NO.17, SEQ DD NO: 19, SEQ DD NO:20, SEQ DD NO.22, SEQ ID NO:26, SEQ ID NO:28, SEQ DD NO:29 or SEQ ID NO:31. The present invention also includes FceR-LP fusion proteins encoded by such genetic chimeras. Particularly prefened FceR-LP fusion proteins include at least one of the following ammo acid sequences: SEQ DD NO.18, SEQ DD NO.21, SEQ DD NO:27, and SEQ ID NO:30.
The present invention also relates to recombinant molecules, recombinant viruses and recombinant cells that include FcεR-LP chimeric nucleic acid molecules of the present invention. Also included are methods to produce such nucleic acid molecules, recombinant molecules, recombinant viruses and recombinant cells.
The present invention also includes detection methods and kits that detect IgE. One embodiment of the present invention is a method to detect IgE compnsing- (a) contacting an isolated FceR-LP molecule with a putative IgE-contaming composition under conditions suitable for formation of a FceR-LP molecule:IgE complex; and (b) determining the presence of IgE by detecting the FceR-LP molecule:IgE complex, the presence of the Fc€R molecule:IgE complex indicating the presence of IgE.
Another embodiment of the present invention is a method to detect IgE comprising: (a) contacting a recombinant cell with a putative IgE-containing composition under conditions suitable for formation of a recombinant cel lgE complex, in which the recombinant cell comprises a FceR-LP molecule; and (b) determining the presence of IgE by detecting the recombinant celhlgE complex, the presence of the recombinant cell:IgE complex indicating the presence of IgE. Another prefened method to detect IgE comprises: (a) immobilizing a specific antigen on a support substrate; (b) contacting the antigen with the putative IgE-containing composition under conditions suitable for formation of an antigemlgE complex bound to the support substrate; (c) removing non-bound material from the support substrate under conditions that retain antigemlgE complex binding to said support substrate; and (d) detecting the presence of the antigemlgE complex by contacting the antige lgE complex with said FceR-LP molecule. Another prefened method to detect IgE comprises: (a) immobilizing an antibody that binds selectively to IgE on a support substrate; (b) contacting the antibody with the putative IgE-containing composition under conditions suitable for formation of an antibody:IgE complex bound to the support substrate; (c) removing non-bound material from the support substrate under conditions that retain antibodyTgE complex binding to the support substrate; and (d) detecting the presence of the antibodyTgE complex by contacting the antibodyTgE complex with said FceR-LP molecule. Another prefened method to detect IgE comprises: (a) immobilizing a putative IgE-containing composition on a support substrate; (b) contacting the composition with the FceR-LP molecule under conditions suitable for formation of a FceR-LP molecule:IgE complex bound to the support substrate; (c) removing non-bound material from the support substrate under conditions that retain FceR-LP molecule:IgE complex binding to the support substrate; and (d) detecting the presence of the Fc€R-LP moleculeTgE complex. The present invention also includes a kit for performing methods of the present invention. One embodiment is a kit for detecting IgE comprising a FceR-LP protein, LP substrate and a means for detecting FceR-LP:IgE complex. DETAILED DESCRIPTION OF THE INVENTION The present invention provides for a genetic chimera incorporating both a nucleic acid molecule encoding a high affinity Fc epsilon receptor (FceR) and a nucleic acid molecule encoding a bioluminescence or chemiluminescence inducing protein (collectively refened to herein as a "luminescence inducing protein" or "LP"). The present invention also provides a fusion protein expressed by such a genetic chimera (also refened to as chimeric nucleic acid molecules), wherein such a fusion protein possesses IgE binding activity and LP activity. As used herein, the terms isolated FceR proteins and isolated FceR nucleic acid molecules refers to FceR proteins and FceR nucleic acid molecules derived from mammals and, as such, can be obtained from their natural source or can be produced using, for example, recombinant nucleic acid technology or chemical synthesis. As used herein, the term luminescence inducing protein refers to a protein that chemically or physically interacts with a substrate to cause the substrate to emit light when the LP is contacted with the substrate (refened to herein as a LP substrate). Also included in the present invention is the use of a fusion protein of the present invention in a method to detect epsilon immunoglobulin (refened to herein as IgE or IgE antibody) as well as in other applications, such as those disclosed below. The products and processes of the present invention are advantageous because they enable highly sensitive detection of IgE. It is to be noted that the term "a" 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 "a" (or "an"), "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. Furthermore, a compound "selected from the group consisting of refers to one or more of the compounds in the list that follows, including mixtures (i.e., combinations) of two or more of the compounds. According to the present invention, an isolated, or biologically pure, protein, is a protein that has been removed from its natural milieu. As such, "isolated" and "biologically pure" do not necessarily reflect the extent to which the protein has been purified. An isolated protein of the present invention can be produced using recombinant DNA technology or can be produced by chemical synthesis. As such, as used herein, the terms isolated FceR proteins and isolated FceR nucleic acid molecules refers to FceR proteins and FceR nucleic acid molecules derived from mammals and, as such, can be obtained from their natural source or can be produced using, for example, recombinant nucleic acid technology or chemical synthesis. One embodiment of the present invention is a fusion protein comprising a FceR protein domain and an LP domain. As used herein, a fusion protein refers to a protein produced by expression of a chimeric nucleic acid molecule containing nucleic acid sequences from two or more different organisms and/or synthetically created nucleic acid sequences. A FceR-luminescence inducing protein fusion protein (refened to herein as a "FceR-LP" fusion protein) of the present invention preferably includes a FceR domain that binds to IgE with an affinity similar to that of a free (i.e. non-fused) FceR domain and an LP domain, that interacts with a LP substrate in such a manner so as to give a detectable signal, preferably to a level similar to that of a free LP domain. Examples of suitable FceR domain include, but are not limited to, a human FceR domain, a canine FceR domain, a feline FceR domain, an equine FceR domain, a rat FceR domain, and a mouse FceR domain. Examples of suitable LP domains include, but are not limited to, luciferase, alkaline phosphatase, β-galactosidase, glucose oxidase, galactose dehydroginase, urease, catalase and galactokinase, with luciferase and alkaline phosphatase being prefened, and luciferase being particularly prefened. In one embodiment a FceR-LP fusion protein of the present invention comprises a protein encoded by a genetic chimera comprising a nucleic acid molecule that encodes a FceR protein domain that binds to an IgE, linked to a nucleic acid molecule that encodes an LP domain in such a manner that the chimera encodes a single fused protein having both domains. Production of FceR-LP genetic chimeras and fusion proteins are described herein in the Examples.
A Fc€R protein domain for use in a fusion protein of the present invention can be a full-length protein or any homolog of such a protein. As used herein, a protein domain can be a polypeptide or a peptide. A FceR protein comprises at least a portion of a FceR protein that binds to IgE, i.e., that is capable of forming a complex with an IgE. Preferably, a FceR protein domain of the present invention comprises at least a portion of FceR alpha chain. A FceR molecule 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 FceR molecule of the present invention can comprise a complete FceR (i.e., alpha, beta and gamma FceR chains), an alpha FceR chain (also refened to herein as FceR chain) or portions thereof. Preferably, a FcεR molecule comprises at least a portion of a FcεR chain that binds to IgE, i.e., that is capable of forming an immunocomplex with an IgE constant region. Preferably, a FceR molecule of the present invention binds to IgE with an affinity of about KA~ 108, more preferably with an affinity of about KA= 109 and even more preferably with an affinity of about KA= 10 10 A FceR protein for use in a fusion protein of the present invention, including a homolog, can be identified in a straight-forward manner by the protein's ability to bind to IgE. Such methods are well known to those of skill in the art. Examples of FceR protein homologs include FceR proteins in which amino acids have been deleted (e.g., a truncated version of the protein, such as a peptide), inserted, inverted, substituted and/or derivatized (e.g., by glycosylation, phosphorylation, acetylation, myristoylation, prenylation, palmitoylation, amidation and/or addition of glycerophosphatidyl inositol) such that the homolog retains the ability of binding to IgE.
FcεR protein homologs suitable for use in a fusion protein of the present invention can be the result of natural allelic variation or natural mutation. FccR protein 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 a nucleic acid molecule encoding the protein using, for example, classic or recombinant nucleic acid techniques to effect random or targeted mutagenesis.
In one embodiment, FceR homolog amino acid sequences suitable for use in a fusion protein of the present invention have the further characteristic of being encoded by nucleic acid molecules that hybridize under stringent hybridization conditions to FccR genes or other nucleic acid molecules encoding a FcεR protein. As used herein, a FceR gene includes all nucleic acid sequences related to a natural FcεR gene such as regulatory regions that control production of the FcεR protein encoded by that gene (such as, but not limited to, transcription, translation or post-translation control regions) as well as the coding region itself. In one embodiment, a FceR gene of the present invention includes nucleic acid sequence SEQ ID NO 1, SEQ ID NO 3, SEQ ID NO 4, SEQ ID NO.6, SEQ ID NO 7, SEQ ID NO:9, SEQ ID NO: 10, and or SEQ ID NO.12 Nucleic acid sequence SEQ ID NO 1 represents the deduced sequence of the coding strand of a complementary DNA (cDNA) nucleic acid molecule denoted herein as nhFcεRn98. The complement of SEQ DD NO.1, represented herein by SEQ ID NO:3, refers to the nucleic acid sequence of the strand complementary to the strand having SEQ DD NO 1, which can easily be determined by those skilled in the art. Likewise, a nucleic acid sequence complement of any nucleic acid sequence of the present invention refers to the nucleic acid sequence of the nucleic acid strand that is complementary to (l e., can form a complete double helix with) the strand for which the sequence is cited
It should be noted that since nucleic acid sequencing technology is not entirely enor-free, SEQ DD NO.l and SEQ DD NO: 3 (as well as other nucleic acid and protein sequences presented herein) represent apparent nucleic acid sequences of certain nucleic acid molecules encoding FcεR proteins of the present invention, including, but not limited to sequences encoding a human FcεR domain, a canine FceR domain, a feline FceR domain, an equine FceR domain, a rat FcεR domain, and a mouse FcεR domain In another embodiment, a FcεR gene can be an allelic variant that includes a similar but not identical sequence to SEQ DD NO.l, SEQ ID NO:3, SEQ DD NO 4, SEQ DD NO:6, SEQ DD NO.7, SEQ DD NO:9, SEQ DD NO: 10, SEQ ID NO.12, an additional human FceR nucleic acid sequence, as descπbed in U.S. Patent No. 5,945,294, issued August 31, 1999, a feline FcεR nucleic acid sequence, as descπbed in U.S. Patent No. 5,958,880, issued September 28, 1999, a canme FcεR nucleic acid sequence, as described in U.S. Patent No. 6,060,326, issued May 9, 2000, an equine FcεR nucleic acid sequence, as described in U.S. Patent No. 6,057,127, issued May 2, 2000, and the muπne and rat FceR sequences as descπbed in Blank et al., J. Biological Chemistry, 266(4):2639-2646, 1991. An allelic vanant of a FcεR gene is a gene that occurs at essentially the same locus (or loci) in the genome as any of the listed sequences, but which, due to natural vanations caused by, for example, mutation or recombination, has a similar but not identical sequence. Allelic vaπants typically encode proteins having similar activity to that of the protein encoded by the gene to which they are being compared. Allelic vaπants can also compπse alterations m the 5' or 3' untranslated regions of the gene (e.g., in regulatory control regions). Allelic variants are well known to those skilled in the art and would be expected to be found naturally within a given mammal since the genome is diploid or may be created through laboratory manipulation, such as, but not limited to, variants produced during polymerase chain reaction amplification.
The minimal size of a FcεR protein homolog suitable for use in a fusion protein of the present invention is a size sufficient to be encoded by a nucleic acid molecule capable of forming a stable hybrid (i.e., hybridize under stringent hybridization conditions) with the complementary sequence of a nucleic acid molecule encoding the conesponding natural protein. As such, the size of the nucleic acid molecule encoding such a protein homolog is dependent on nucleic acid composition and percent homology between the nucleic acid molecule and complementary sequence. It should also be noted that the extent of homology required to form a stable hybrid can vary depending on whether the homologous sequences are interspersed throughout the nucleic acid molecules or are clustered (i.e., localized) in distinct regions on the nucleic acid molecules. The minimal size of such nucleic acid molecules is typically at least about 12 to about 15 nucleotides in length if the nucleic acid molecules are GC-rich and at least about 15 to about 17 bases in length if they are AT-rich. As such, the minimal size of a nucleic acid molecule used to encode a FceR protein homolog suitable for use in a fusion protein of the present invention is from about 12 to about 18 nucleotides in length. Thus, the minimal size of a FcεR protein homolog suitable for use in a fusion protein of the present invention is from about 4 to about 6 amino acids in length. There is no limit, other than a practical limit, on the maximal size of such a nucleic acid molecule in that the nucleic acid molecule can include a portion of a gene, an entire gene, multiple genes, or portions thereof. The prefened size of a protein suitable for use in a fusion protein of the present invention is a portion of the protein that binds to IgE which is about 20 amino acids, more preferably about 25 amino acids, more preferably about 30 amino acids, more preferably about 35 amino acids, more preferably about 40 amino acids and even more preferably about 45 amino acids in length. Stringent hybridization conditions are determined based on defined physical properties of the gene or other nucleic acid molecule to which the nucleic acid molecule to be tested is being hybridized, and can be defined mathematically. Stringent hybridization conditions are those experimental parameters that allow an individual skilled in the art to identify significant similarities between heterologous nucleic acid molecules These conditions are well known to those skilled m the art. See, for example, Sambrook, et al , 1989, Molecular Cloning A Laboratory Manual, Cold Spπng Harbor Labs Press, and Meinkoth, et al , 1984, Anal Bwchem 138, 267-284 The determination of hybridization conditions involves the manipulation of a set of variables including the ionic strength (M, in moles/liter), the hybridization temperature (°C), the concentration of nucleic acid helix destabilizing agents (such as formamide), the average length of the shortest hybrid duplex (n), and the percent G + C composition of the fragment to which an unknown nucleic acid molecule is being hybπdized. For nucleic acid molecules of at least about 150 nucleotides, these vaπables are inserted into a standard mathematical formula to calculate the melting temperature, or Tm, of a given nucleic acid molecule. As defined in the formula below, Tm is the temperature at which two complementary nucleic acid molecule strands will disassociate, assuming 100% complementarity between the two strands
Tm= 81.5°C + 16.6 log M + 0.41(%G + C) - 500/n - 0.61(%formamιde). For nucleic acid molecules smaller than about 50 nucleotides, hybrid stability is defined by the dissociation temperature (Td), which is defined as the temperature at which 50% of the duplexes dissociate. For these smaller molecules, the stability at a standard ionic strength is defined by the following equation:
Td = 4(G + C) + 2(A + T). A temperature of 5°C below Td is used to detect hybridization between perfectly matched molecules. Also well known to those skilled in the art is how base pair mismatch, i.e. differences between two nucleic acid molecules being compared, including non- complementaπty of bases at a given location, and gaps due to insertion or deletion of one or more bases at a given location on either of the nucleic acid molecules being compared, will affect Tm or Td for nucleic acid molecules of different sizes. For example, Tm decreases about 1°C for each 1% of mismatched base pairs for hybπds greater than about 150 bp, and Td decreases about 5°C for each mismatched base pair for hybπds below about 50 bp Conditions for hybrids between about 50 and about 150 base pairs can be determined empirically and without undue expeπmentation using standard laboratory procedures well known to those skilled m the art These simple procedures allow one skilled m the art to set the hybridization conditions (by altering, for example, the salt concentration, the formamide concentration or the temperature) so that only nucleic acid hybrids with greater than a specified % base pair mismatch will hybridize. Stringent hybridization conditions are commonly understood by those skilled in the art to be those experimental conditions that will allow less than or equal to about 30% base pair mismatch (i.e., at least about 70% identity) Because one skilled in the art can easily determine whether a given nucleic acid molecule to be tested is less than or greater than about 50 nucleotides, and can therefore choose the appropπate formula for determining hybridization conditions, he or she can determine whether the nucleic acid molecule will hybπdize with a given gene under stringent hybridization conditions and similarly whether the nucleic acid molecule will hybπdize under conditions designed to allow a desired amount of base pair mismatch.
Hybridization reactions are often earned out by attaching the nucleic acid molecule to be hybπdized to a solid support such as a membrane, and then hybridizing with a labeled nucleic acid molecule, typically refened to as a probe, suspended in a hybridization solution. Examples of common hybridization reaction techniques include, but are not limited to, the well-known Southern and northern blotting procedures.
Typically, the actual hybridization reaction is done under non-stringent conditions, i.e., at a lower temperature and/or a higher salt concentration, and then high stπngency is achieved by washing the membrane in a solution with a higher temperature and/or lower salt concentration m order to achieve the desired stringency. For example, if the skilled artisan wished to identify a nucleic acid molecule that hybridizes under conditions that would allow less than or equal to 30% pair mismatch with a nucleic acid molecule of about 150 bp m length or greater, the following conditions could preferably be used. The unknown nucleic acid molecules would be attached to a support membrane, and the 150 bp probe would be labeled, e.g. with a radioactive tag The hybridization reaction could be earned out in a solution comprising 2X SSC and 0% formamide, at a temperature of about 37°C (low stringency conditions) Solutions of differing concentrations of SSC can be made by one of skill in the art by diluting a stock solution of 20X SSC (175.3 gram NaCl and about 88.2 gram sodium citrate in 1 liter of water, pH 7) to obtain the desired concentration of SSC. The skilled artisan would calculate the washing conditions required to allow up to 30% base pair mismatch. For example, assuming an average G + C content of the nucleic acid molecule to be hybridized of about 50%, and a wash solution comprising IX SSC and 0% foπriamide, the Tm of perfect hybrids would be about 85°C:
81.5°C + 16.6 log (.15M) + (0.41 x 0.37) - (500/150) - (0.61 x 0) = 85°C. Thus, to achieve hybridization with nucleic acid molecules having about 30% base pair mismatch, hybridization washes would be canied out at a temperature of less than or equal to 55°C. It is thus within the skill of one in the art to calculate additional hybridization temperatures based on the desired percentage base pair mismatch, formulae and G/C content disclosed herein. For example, it is appreciated by one skilled in the art that as the nucleic acid molecule to be tested for hybridization against nucleic acid molecules of the present invention having sequences specified herein becomes longer than 150 nucleotides, the Tm for a hybridization reaction allowing up to 30% base pair mismatch will not vary significantly from 55 °C.
Furthermore, it is known in the art that there are commercially available computer programs for determining the degree of similarity between two nucleic acid or protein sequences. These computer programs include various known methods to determine the percentage identity and the number and length of gaps between hybrid nucleic acid molecules or proteins. Prefened methods to determine the percent identity among amino acid sequences and also among nucleic acid sequences include analysis using one or more of the commercially available computer programs designed to compare and analyze nucleic acid or amino acid sequences. These computer programs include, but are not limited to, the SeqLab® Wisconsin Package™ Version 10.0-UNLX sequence analysis software, available from Genetics Computer Group, Madison, WI; and DNAsis® sequence analysis software, version 2.0, available from Hitachi Software, San Bruno, CA. Such software programs represent a collection of algorithms paired with a graphical user interface for using the algorithms. The DNAsis version 2.0 software and SeqLab Wisconsin Package Version 10.0-UNLX software, for example, employ a particular algorithm, the Needleman-Wunsch algorithm to perform pair-wise comparisons between two sequences to yield a percentage identity score, see Needleman, S.B. and Wunch, CD., 1970, J. Mol. Biol, 48, 443, which is incorporated herein by reference in its entirety. Such algorithms, including the Needleman-Wunsch algorithm, are commonly used by those skilled in the nucleic acid and amino acid sequencing art to compare sequences. A prefened method to determine percent identity among amino acid sequences and also among nucleic acid sequences includes using the Needleman- Wunsch algorithm, available in the SeqLab Wisconsin Package Version 10.0-UNLX software (hereinafter "SeqLab"), using the Pairwise Comparison/Gap function with the nwsgapdna.cmp scoring matrix, the gap creation penalty and the gap extension penalties set at default values, and the gap shift limits set at maximum (hereinafter refened to as "SeqLab default parameters"). An additional prefened method to determine percent identity among amino acid sequences and also among nucleic acid sequences includes using the Higgins-Sharp algorithm, available in the DNAsis version 2.0 software (hereinafter "DNAsis"), with the gap penalty set at 5, the number of top diagonals set at 5, the fixed gap penalty set at 10, the k-tuple set at 2, the window size set at 5, and the floating gap penalty set at 10. A particularly prefened method to determine percent identity among amino acid sequences and also among nucleic acid sequences includes using the Needleman-Wunsch algorithm available in the DNAsis version 2.0 software, using the GCG default parameter function.
In one embodiment, a prefened FcεR protein domain suitable for use in a fusion protein of the present invention is encoded by at least a portion of SEQ ID NO: 1, SEQ DD NO:3, SEQ ID NO:4, SEQ DD NO:6, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:10, SEQ DD NO: 12, an additional human FcεR nucleic acid sequence, as described in U.S. Patent No. 5,945,294, issued August 31, 1999, a feline FcεR nucleic acid sequence, as described in U.S. Patent No. 5,958,880, issued September 28, 1999, a canine FcεR nucleic acid sequence, as described in U.S. Patent No. 6,060,326, issued May 9, 2000, an equine FceR nucleic acid sequence, as described in U.S. Patent No. 6,057,127, issued May 2, 2000, and the murine and rat FcεR nucleic acid sequences as described in Blank et al., J. Biological Chemistry, 266(4):2639-2646, 1991, i.e. a domain including an entire sequences or a fragment thereof having IgE binding activity. Additional prefened FcεR proteins suitable for use m a fusion protein of the present invention include a protein encoded by a nucleic acid molecule that hybridizes to a nucleic acid molecule selected from the group consisting of SEQ ID NO:3, SEQ ID NO:6, SEQ ID NO:9, SEQ ID NO: 12, an additional human FcεR nucleic acid sequence, as described in U.S Patent No. 5,945,294, issued August 31, 1999, a feline FcεR nucleic acid sequence, as described in U.S. Patent No. 5,958,880, issued September 28, 1999, a canme FccR nucleic acid sequence, as described in U S. Patent No. 6,060,326, issued May 9, 2000, an equine FcεR nucleic acid sequence, as described m U.S. Patent No. 6,057,127, issued May 2, 2000, and the muπne and rat FcεR nucleic acid sequences as described in Blank et al, J. Biological Chemistry, 266(4):2639-2646, 1991., under conditions compnsing (a) hybπdization in a solution comprising 2X SSC and 0% formamide, at a temperature of 37°C, and (b) washing in IX SSC and 0%> formamide at a temperature of 55°C.
Additional prefened FcεR proteins suitable for use in a fusion protein of the present invention include proteins compnsing ammo acid sequences that are at least about 65%, preferably at least about 70%, more preferably at least about 75%, more preferably at least about 80%, more preferably at least about 85%, more preferably at least about 90%) and even more preferably about 95%, identical to ammo acid sequence SEQ ID NO:l, SEQ DD NO:3, SEQ ID NO.4, SEQ DD NO:6, SEQ DD NO:7, SEQ DD NO:9, SEQ ID NO: 10, SEQ ID NO.12, an additional human FcεR nucleic acid sequence, as described in U.S. Patent No. 5,945,294, issued August 31, 1999, a feline Fc£R nucleic acid sequence, as described in U.S. Patent No. 5,958,880, issued September 28, 1999, a canme FcεR nucleic acid sequence, as descπbed in U.S. Patent No. 6,060,326, issued May 9, 2000, an equme FcεR nucleic acid sequence, as described in U.S. Patent No. 6,057,127, issued May 2, 2000, and muπne and rat FcεR amino acid sequences as described in Blank et al, J. Biological Chemistry, 266(4):2639-2646, 1991. Amino acid sequence analysis can preferably be performed using either the DNAsis™ program (available from Hitachi Software, San Bruno, CA) using default stringency parameters. Particularly prefened FcεR proteins suitable for use in a fusion protein of the present invention include a protein having an ammo acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO:3, SEQ DD NO:4, SEQ ID NO:6, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 12, an additional human FcεR nucleic acid sequence, as described in U.S. Patent No. 5,945,294, issued August 31, 1999, a feline FcεR nucleic acid sequence, as described in U.S. Patent No. 5,958,880, issued September 28, 1999, a canine FcεR nucleic acid sequence, as described in U.S. Patent No. 6,060,326, issued May 9, 2000, an equine FceR nucleic acid sequence, as described in U.S. Patent No. 6,057,127, issued May 2, 2000, and murine and rat FceR amino acid sequences as described in Blank et al., J. Biological Chemistry, 266(4):2639- 2646, 1991.
An LP domain for use in a fusion protein of the present invention can be a full- length LP protein or any homolog of such a protein. An LP comprises at least a portion of an LP that interacts with a LP substrate to cause the LP substrate to emit light when the LP is contacted with the LP substrate. Prefened LP domains for use in a fusion protein of the present invention include luciferases, alkaline phosphatases, β- galactosidases, glucose oxidases, galactose dehydroginases, ureases, catalases and galactokinases, with luciferases, alkaline phosphatases being particularly prefened. Prefened luciferase domains include sequences from Photuris pennsylvanica, Vibrio cholera, Vibrio fischeri, Chesapeake Bay bacterium, Vibrio harveyi, Luciola later alis, Vargula hilgenforfii, Luciola cruciata, Photinus pyralis, Luciola mingrelica, Renilla reniformis, Photobacterium phosphoreum, and Xenorhabdus luminescens. An LP domain for use in a fusion protein of the present invention, including a homolog, can be identified in a straight-forward manner by the protein's ability to chemically or physically interact with a LP substrate to cause the LP substrate to emit light when the LP is contacted with the LP substrate. Such methods are well known to those of skill in the art. Examples of LP protein homologs include LP proteins in which amino acids have been deleted (e.g., a truncated version of the protein, such as a peptide), inserted, inverted, substituted and/or derivatized (e.g., by glycosylation, phosphorylation, acetylation, myristoylation, prenylation, palmitoylation, amidation and/or addition of glycerophosphatidyl inositol) such that the homolog retains LP activity. As such, an LP homolog of the present invention includes, for example, a thermostable LP domain. As used herein, a thermostable LP domain means a protein which retains biological activity following incubation at 65°C for at least about 1 hour and/or following incubation at room temperature for at least about 1 week.
LP homologs suitable for use in a fusion protein of the present invention can be the result of natural allelic variation or natural mutation. LP 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 nucleic acid techniques to effect random or targeted mutagenesis.
In one embodiment, LP homolog amino acid sequences suitable for use in a fusion protein of the present invention have the further characteristic of being encoded by nucleic acid molecules that hybridize under stringent hybridization conditions to LP genes or other nucleic acid molecules encoding an LP. As used herein, an LP gene includes all nucleic acid sequences related to a natural LP gene such as regulatory regions that control production of the LP encoded by that gene (such as, but not limited to, transcription, translation or post-translation control regions) as well as the coding region itself. In another embodiment, an LP gene can be an allelic variant that includes a similar but not identical sequence to SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO:23 and/or SEQ DD NO:25.
Additional prefened LPs suitable for use in a fusion protein of the present invention include proteins comprising amino acid sequences that are at least about 65%), preferably at least about 70%, more preferably at least about 75%, more preferably at least about 80%o, more preferably at least about 85%, more preferably at least about 90% and even more preferably about 95%>, identical to amino acid sequence SEQ ID NO: 15 and/or SEQ ID NO:24. Amino acid sequence analysis is preferably performed using the DNAsis™ program (available from Hitachi Software, San Bruno, CA), using default stringency parameters.
Additional prefened LPs suitable for use in a fusion protein of the present invention include a protein encoded by a nucleic acid molecule that hybridizes to a nucleic acid molecule selected from the group consisting of SEQ ID NO: 16, and/or SEQ ID NO:25, under conditions comprising (a) hybridization in a solution comprising 2X SSC and 0% formamide, at a temperature of 37°C, and (b) washing in IX SSC and 0% fonriamide at a temperature of 55°C Particularly prefened LPs suitable for use in a fusion protein of the present invention include a protein having an ammo acid sequence selected from the group consisting of SEQ ID NO 15 and/or SEQ ID NO.24, or a fragment thereof having LP activity One embodiment of the present invention is a FcεR-LP fusion protein that includes a FcεR protein-contammg domain and an LP-contammg domain attached to one or more fusion segments. Suitable fusion segments for use with the present invention include, but are not limited to, segments that can: enhance a protein's stability; act as an immunopotentiator to enhance an immune response against a FcεR protein; and/or assist purification of a FcεR-LP fusion protein (e.g , by affinity chromatography). A suitable fusion segment can be a domain of any size that has the desired function (e.g., imparts increased stability, imparts structural or spacial mobility, imparts increased immunogenicity to a protein, and/or simplifies puπfication of a protein). Fusion segments can be joined to ammo and/or carboxyl termini of the FcεR-contammg domain and/or the LP-contaming domain of the fusion protein and can be susceptible to cleavage m order to enable straight-forward recovery of the fusion protein. Fusion proteins are preferably produced by cultuπng a recombinant cell transformed with a fusion nucleic acid molecule that encodes a protein including the fusion segment attached to either the carboxyl and/or amino terminal end of a FcεR-contammg domain or the carboxyl and/or ammo terminal end of an LP-contammg domain. Prefened fusion segments for use in a genetic chimera of the present invention include linker sequences described by Berg , Proc Natl Acad Sci. 85:99-102, 1998, and Pomerantz et al., Science 263:671-673, 1995, and the linker sequence (Gly4Serl)2, i.e SEQ ID NO.13, the production of which is described in the Examples. A prefened fusion protein of the present invention includes at least an IgE- binding portion of a FcεR domain compnsing SEQ ID NO 1, SEQ DD NO 3, SEQ ID NO 4, SEQ ID NO:6, SEQ DD NO.7, SEQ ID NO 9, SEQ ID NO: 10, SEQ ID NO: 12, or an allelic variant thereof and an LP-active portion of an LP domain compnsing SEQ DD NO.14, SEQ ID NO: 16, SEQ ID NO.23 or SEQ DD NO:25, or an allelic variant thereof, and preferably also includes a linker between said domains. A more prefened FcεR-LP fusion protein of the present invention includes a protein encoded by a nucleic acid molecule comprising a nucleic acid sequence selected from the group consisting of SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:26, SEQ ID NO:28, SEQ DD NO:29 or SEQ ID NO:31. More prefened is a FcεR-LP protein having an amino acid sequence selected from the group consisting of SEQ DD NO: 18, SEQ ID NO:22, SEQ ID NO:27, or SEQ ID NO:30. Particularly prefened FcεR-LP fusion proteins are PhFcεR-Ppluc756, PhFcεR-Pρluc73„ PhFcεR-bAP660, and/or PhFcεR-bAP635. Prefened FceR-LP fusion proteins also include amino acid sequences SEQ ID NO: 18, SEQ ID NO:21, SEQ ID NO:27, and SEQ ID NO:30.
A FcεR-LP fusion protein of the present invention can also include a domain that enables the fusion protein to be bound to a support substrate in such a manner that the FcεR domain of the fusion protein binds to IgE in essentially the same manner as a FcεR molecule that is not bound to a support substrate. An example of a suitable binding domain includes a portion of an immunoglobulin molecule or another ligand that has a suitable binding partner that can be immobilized on a support substrate, e.g., biotin and avidin, or a metal-binding protein and a metal (e.g., His), or a sugar-binding protein and a sugar (e.g., maltose).
Another embodiment of the present invention is a genetic chimera comprising a nucleic acid molecule encoding a FcεR (FceR nucleic acid molecule) and a nucleic acid molecule encoding an LP (a LP nucleic acid molecule) such that the chimera, refened to herein as a FcεR-LP nucleic acid molecule or a FcεR-LP genetic chimera, encodes a single fusion protein comprising a FcεR domain and a LP domain. A nucleic acid molecule of the present invention can include an isolated natural FceR gene or cDNA plus an isolated natural LP gene or cDNA or homologs thereof, the latter of which are described in more detail below. A nucleic acid molecule of the present invention can include one or more regulatory regions, full-length or partial coding regions, or combinations thereof.
In accordance with the present invention, an isolated nucleic acid molecule is a nucleic acid molecule that has been removed from its natural milieu (i.e., that has been subject to human manipulation) and can include DNA, RNA, or derivatives of either DNA or RNA. As such, "isolated" does not reflect the extent to which the nucleic acid molecule has been purified. An isolated FceR and/or LP nucleic acid molecule of the present invention can be isolated from its natural source or can be produced using recombinant DNA technology (e.g., polymerase chain reaction (PCR) amplification, cloning) or chemical synthesis. As such, as used herein, the terms isolated LP proteins and isolated LP nucleic acid molecules refers to LP proteins and LP nucleic acid molecules derived from living organisms and, as such, can be obtained from their natural source or can be produced using, for example, recombinant nucleic acid technology or chemical synthesis. Isolated FceR and/or LP nucleic acid molecules can include, for example, natural allelic variants as well as other nucleic acid molecule homologs that are modified by one or more nucleotide insertions, deletions, substitutions, and/or inversions in a manner such that the modifications do not substantially interfere with the nucleic acid molecule's ability to encode FceR protein or LP proteins of the present invention, respectively, or to form stable hybrids under stringent conditions with natural gene isolates.
A FcεR and/or LP nucleic acid molecule homolog for use in a genetic chimera of the present invention can be produced using a number of methods lαiown to those skilled in the art (see, for example, Sambrook et al., ibid.). For example, nucleic acid molecules can be modified using a variety of techniques including, but not limited to, classic mutagenesis and recombinant DNA techniques (e.g., site-directed mutagenesis, chemical treatment, restriction enzyme cleavage, ligation of nucleic acid fragments and/or PCR amplification), synthesis of oligonucleotide mixtures and ligation of mixture groups to "build" a mixture of nucleic acid molecules and combinations thereof. Nucleic acid molecule homologs can be selected by hybridization with a FcεR or LP gene or cDNA or by screening for function of a protein encoded by the nucleic acid molecule, e.g., ability of a FcεR protein to bind IgE and ability of an LP protein to induce luminescence when contacted with a suitable LP substrate, respectively.
An isolated FcεR-LP nucleic acid molecule of the present invention includes a nucleic acid sequence that encodes a FcεR-LP fusion protein comprising at least one FcεR domain and at least one LP domain of the present invention, examples of such domains and proteins being disclosed herein. Although the phrase "nucleic acid molecule" primarily refers to the physical nucleic acid molecule and the phrase "nucleic acid sequence" primarily refers to the sequence of nucleotides on the nucleic acid molecule, the two phrases can be used interchangeably, especially with respect to a nucleic acid molecule, or a nucleic acid sequence, being capable of encoding a FcεR domain or a LP domain or a FceR-LP fusion protein of the present invention.
Prefened FcεR nucleic acid molecules for use in a genetic chimera of the present invention include nucleic acid molecules having a nucleic acid sequence that is at least about 70%), preferably at least about 75%o, preferably at least about 80%, preferably at least about 85%, more preferably at least about 90%, and even more preferably at least about 95% identical to nucleic acid sequence SEQ ID NO:l, SEQ DD NO:3, SEQ ID NO:4, SEQ DD NO:6, SEQ ID NO:7, SEQ ID NO:9, SEQ DD NO: 10, SEQ ID NO: 12, an additional human FcεR nucleic acid sequence, as described in U.S. Patent No. 5,945,294, issued August 31, 1999, a feline FcεR nucleic acid sequence, as described in U.S. Patent No. 5,958,880, issued September 28, 1999, a canine FcεR nucleic acid sequence, as described in U.S. Patent No. 6,060,326, issued May 9, 2000, an equine FcεR nucleic acid sequence, as described in U.S. Patent No. 6,057,127, issued May 2, 2000, and murine and rat FcεR amino acid sequences as described in Blank et al., J. Biological Chemistry, 266(4):2639-2646, 1991. DNA sequence analysis is preferably performed using the DNAsis™ program using default stringency parameters.
Another prefened FceR nucleic acid molecule for use in a genetic chimera of the present invention includes a nucleic acid molecule that hybridizes to a nucleic acid molecule selected from the group consisting of SEQ DD NO:3, SEQ DD NO:6, SEQ DD NO:9, SEQ DD NO: 12, an additional human FcεR nucleic acid sequence, as described in U.S. Patent No. 5,945,294, issued August 31, 1999, a feline FcεR nucleic acid sequence, as described in U.S. Patent No. 5,958,880, issued September 28, 1999, a canine FcεR nucleic acid sequence, as described in U.S. Patent No. 6,060,326, issued May 9, 2000, an equine FcεR nucleic acid sequence, as described in U.S. Patent No. 6,057,127, issued May 2, 2000, and the murine and rat FcεR nucleic acid sequences as described in Blank et al., J. Biological Chemistry, 266(4):2639-2646, 1991., under conditions comprising (a) hybridization in a solution comprising 2X SSC and 0% formamide, at a temperature of 37°C, and (b) washing in IX SSC and 0%o formamide at a temperature of 55°C. Another prefened FcεR nucleic acid molecule for use in a genetic chimera of the present invention includes at least a portion of nucleic acid sequence SEQ DD NO: 1 , SEQ ID NO:3, SEQ DD NO:4, SEQ ID NO:6, SEQ DD N0:7, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO: 12, an additional human FcεR nucleic acid sequence, as described in U.S. Patent No. 5,945,294, issued August 31, 1999, a feline FcεR nucleic acid sequence, as described in U.S. Patent No. 5,958,880, issued September 28, 1999, a canine FcεR nucleic acid sequence, as described in U.S. Patent No. 6,060,326, issued May 9, 2000, an equine FcεR nucleic acid sequence, as described in U.S. Patent No. 6,057,127, issued May 2, 2000, and the murine and rat FcεR nucleic acid sequences as described in Blank et al, J. Biological Chemistry, 266(4):2639-2646, 1991., that binds to IgE, as well as allelic variants thereof. A more prefened FcεR nucleic acid molecule for use in a genetic chimera of the present invention includes nucleic acid sequence SEQ ID NO:l, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:9, SEQ DD NO: 10, SEQ DD NO: 12, an additional human FcεR nucleic acid sequence, as described in U.S. Patent No. 5,945,294, issued August 31, 1999, a feline FcεR nucleic acid sequence, as described in U.S. Patent No. 5,958,880, issued September 28, 1999, a canine FccR nucleic acid sequence, as described in U.S. Patent No. 6,060,326, issued May 9, 2000, an equine FcεR nucleic acid sequence, as described in U.S. Patent No. 6,057,127, issued May 2, 2000, and the murine and rat FceR nucleic acid sequences as described in Blank et al., J. Biological Chemistry, 266(4):2639-2646, 1991, as well as allelic variants of such a nucleic acid molecule. Such FcεR nucleic acid molecules can include nucleotides in addition to those included in the sequences described immediately above, such as, but not limited to, a full-length gene or a full-length coding region.
A prefened FcεR nucleic acid molecule suitable for use in a genetic chimera of the present invention also includes a nucleic acid molecule that encodes a protein having at least a portion of SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO: 12, an additional human FceR nucleic acid sequence, as described in U.S. Patent No. 5,945,294, issued August 31, 1999, a feline FceR nucleic acid sequence, as described in U.S. Patent No. 5,958,880, issued September 28, 1999, a canine FcεR nucleic acid sequence, as described in U.S. Patent No. 6,060,326, issued May 9, 2000, an equine FcεR nucleic acid sequence, as described in U.S. Patent No. 6,057,127, issued May 2, 2000, including nucleic acid molecules that have been modified to accommodate codon usage properties of the cells in which such nucleic acid molecules are to be expressed.
Prefened LP nucleic acid molecules for use in a genetic chimera of the present invention include nucleic acid molecules having a nucleic acid sequence that is at least about 70%, preferably at least about 75%, preferably at least about 80%, preferably at least about 85%, more preferably at least about 90%, and even more preferably at least about 95% identical to nucleic acid sequence SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO:23 and/or SEQ DD NO:25. DNA sequence analysis is preferably performed using the DNAsis™ program using default stringency parameters. Another prefened LP nucleic acid molecule for use in a genetic chimera of the present invention includes a nucleic acid molecule that hybridizes to a nucleic acid molecule selected from the group consisting of SEQ ID NO: 16, and/or SEQ ID NO:25, under conditions comprising (a) hybridization in a solution comprising 2X SSC and 0% formamide, at a temperature of 37°C, and (b) washing in IX SSC and 0%> formamide at a temperature of 55°C.
Another prefened LP nucleic acid molecule for use in a genetic chimera of the present invention includes a nucleic acid molecule having nucleic acid sequence SEQ DD NO: 14, SEQ ID NO: 16, SEQ DD NO:23 and/or SEQ DD NO:25, with SEQ ID NO: 14 and SEQ ID NO: 16, or portions thereof having LP activity, as well as allelic variants thereof being more prefened. Such LP nucleic acid molecules can include nucleotides in addition to those included in the SEQ DD NOs, such as, but not limited to, a full-length gene or a full-length coding region.
A prefened LP nucleic acid molecule suitable for use in a genetic chimera of the present invention also includes a nucleic acid molecule that encodes a protein having at least a portion of SEQ ID NO: 15 and/or SEQ DD NO:25, including nucleic acid molecules that have been modified to accommodate codon usage properties of the cells in which such nucleic acid molecules are to be expressed.
Prefened genetic chimeras of the present invention include at least a FceR nucleic acid molecule portion which encodes a FcεR protein which binds to IgE and a LP nucleic acid molecule portion which encodes a LP protein having LP activity, and preferably also includes a linker nucleic acid molecule. More prefened genetic chimeras of the present invention include nucleic acid molecule constructs having a nucleic acid sequence that is at least about 70%, preferably at least about 75%, preferably at least about 80%) preferably at least about 85%, more preferably at least about 90%, more preferably at least about 95%, and even more preferably at least about 100% identical to nucleic acid molecules nhFceR-npluc2268, nhFcεR-npluc2]93, nhFcεR-nbAPI9g3, and/or nhFcεR-nbAP]908. A particularly prefened genetic chimera of the present invention includes a nucleic acid molecule having nucleic acid sequence SEQ DD NO: 17, SEQ ID NO: 19, SEQ DD NO:20, SEQ DD NO:22, SEQ DD NO:26, SEQ DD NO:28, SEQ ID NO:29 or SEQ ID NO:31. Knowing the nucleic acid sequences of certain FcεR and LP nucleic acid molecules of the present invention allows one skilled in the art to, for example, (a) make copies of those nucleic acid molecules, (b) obtain nucleic acid molecules including at least a portion of such nucleic acid molecules (e.g., nucleic acid molecules including full-length genes, full-length coding regions, regulatory control sequences, truncated coding regions), and (c) obtain FcεR and LP nucleic acid molecules suitable for use in a genetic chimera of the present invention from other organisms. Such nucleic acid molecules can be obtained in a variety of ways including screening appropriate expression libraries with antibodies of the present invention; traditional cloning techniques using oligonucleotide probes of the present invention to screen appropriate libraries or DNA; and PCR amplification of appropriate libraries or DNA using oligonucleotide primers of the present invention. Prefened libraries to screen or from which to amplify a FcεR nucleic acid molecule include basophil cell, mast cell, mastocytoma cell, dendritic cell, B lymphocyte, macrophage, eosinophil, and/or monocyte cDNA libraries as well as genomic DNA libraries. Similarly, prefened DNA sources to screen or from which to amplify a FcεR nucleic acid molecule include basophil cells, mast cells, mastocytoma cells, dendritic cells, B lymphocytes, macrophages, eosinophils, and/or monocytes cDNA and genomic DNA. Prefened sources for LP nucleic acid molecules include libraries or DNA of organisms that have luminescent proteins. Techniques to clone and amplify genes are disclosed, for example, in Sambrook et al, ibid. One embodiment of the present invention includes a recombinant vector, which includes at least one isolated nucleic acid molecule comprising a genetic chimera of the present invention, inserted into any vector capable of delivering the nucleic acid molecule into a host cell Such a vector contains heterologous nucleic acid sequences, that is nucleic acid sequences that are not naturally found adjacent to nucleic acid molecules of the present invention and that preferably are denved from a species other than the species from which the nucleic acid molecule(s) are deπved. The vector can be either RNA or DNA, either prokaryotic or eukaryotic, and typically is a virus or a plasmid. Recombinant vectors can be used in the cloning, sequencing, and/or otherwise manipulation of FcεR, LP and FcεR-LP nucleic acid molecules of the present invention One type of recombinant vector, refened to herein as a recombinant molecule, comprises a nucleic acid molecule comprising a genetic chimera of the present invention operatively linked to an expression vector. The phrase operatively linked refers to insertion of a nucleic acid molecule into an expression vector in a manner such that the molecule is able to be expressed when transformed into a host cell. As used herein, an expression vector is a DNA or RNA vector that is capable of transforming a host cell and of effecting expression of a specified nucleic acid molecule Preferably, the expression vector is also capable of replicating within the host cell Expression vectors can be either prokaryotic or eukaryotic, and are typically viruses or plasmids. Expression vectors of the present invention include any vectors that function (i.e., direct gene expression) in recombinant cells of the present invention, including in bacteπal, fungal, insect, other animal, and plant cells. Prefened expression vectors of the present invention can direct gene expression in bacterial, yeast, insect and mammalian cells and more preferably in the cell types disclosed herein. In particular, expression vectors of the present invention contain regulatory sequences such as transcπption control sequences, translation control sequences, origins of replication, and other regulatory sequences that are compatible with the recombinant cell and that control the expression of nucleic acid molecules of the present invention. In particular, recombinant molecules of the present invention include transcnption control sequences. Transcπption control sequences are sequences that control the initiation, elongation, and termination of transcπption. Particularly important transcription control sequences are those which control transcription initiation, such as promoter, enhancer, operator and repressor sequences. Suitable transcription control sequences include any transcription control sequence that can function in at least one of the recombinant cells of the present invention. A variety of such transcription control sequences are known to those skilled in the art. Prefened transcription control sequences include those which function in bacterial, yeast, insect and mammalian cells, such as, but not limited to, tac, lac, trp, trc, oxy-pro, omp/lpp, rrnB, bacteriophage lambda (such as lambda pL and lambda pR and fusions that include such promoters), bacteriophage T7, Υllac, bacteriophage T3, bacteriophage SP6, bacteriophage SP01, metallothionein, alpha-mating factor, Pichia alcohol oxidase, alphavirus subgenomic promoters (such as Sindbis virus subgenomic promoters), antibiotic resistance gene, baculovirus, Heliothis zea insect virus, vaccinia virus, herpesvirus, raccoon poxvirus, other poxvirus, adenovirus, cytomegalovirus (such as intermediate early promoters), simian virus 40, retrovirus, actin, retroviral long terminal repeat, Rous sarcoma virus, heat shock, phosphate and nitrate transcription control sequences as well as other sequences capable of controlling gene expression in prokaryotic or eukaryotic cells. Additional suitable transcription control sequences include tissue-specific promoters and enhancers as well as lymphokine-inducible promoters (e.g., promoters inducible by interferons or interleukins). Transcription control sequences of the present invention can also include naturally occurring transcription control sequences naturally associated with the FcεR or LP nucleic acid molecule being expressed.
Suitable and prefened FcεR-LP nucleic acid molecules to include in recombinant vectors of the present invention are as disclosed herein. Prefened nucleic acid molecules to include in recombinant vectors, and particularly in recombinant molecules, include SEQ DD NO: 17, SEQ ID NO: 19, SEQ DD NO:20, SEQ DD NO:22, SEQ DD
NO:26, SEQ DD NO:28, SEQ DD NO:29 and/or SEQ ID NO:31. A particularly prefened recombinant molecule of the present invention includes SEQ DD NO: 17, SEQ DD NO: 19, SEQ ID NO: 20, and SEQ DD NO: 22, the production of which is described in the Examples section. Recombinant molecules of the present invention can and preferably do, contain secretory signals (i.e., signal segment nucleic acid sequences) to enable an expressed fusion protein of the present invention to be secreted from the cell that produces the protein Examples of suitable signal segments include any signal segment capable of directing the secretion of a protein of the present invention. Prefened signal segments include, but are not limited to, FcεR alpha chain, tissue plasminogen activator (t-PA), interferon, mterleukm, growth hormone, histocompatibility and viral envelope glycoprotem signal segments, as well as natural signal segments. In addition, a nucleic acid molecule of the present invention can be joined to a fusion segment that directs the encoded protein to the proteosome, such as a ubiquitin fusion segment. Recombinant molecules may also include intervening and/or untranslated sequences surrounding and/or withm the nucleic acid sequences of nucleic acid molecules of the present invention.
Another embodiment of the present invention includes a recombinant cell comprising a host cell transformed with one or more recombinant 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 (i.e , recombinant) cell in such a manner that their ability to be expressed is retained. Prefened nucleic acid molecules with which to transform a cell include FcεR-LP chimeric nucleic acid molecules disclosed herein. Particularly prefened nucleic acid molecules with which to transform a cell include SEQ ID NO 17, SEQ ID NO- 19, SEQ DD NO.20, SEQ ID NO.22, SEQ DD NO.26, SEQ ID NO.28, SEQ ID NO.29 and/or SEQ ID NO.31
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 (e.g , nucleic acid molecules encoding one or more proteins of the present invention and/or other proteins). 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 (including yeast), other insect, other animal and plant cells. Prefened host cells include bacterial, mycobacteria, yeast, parasite, insect and mammalian cells. More prefened host cells include Salmonella, Escherichia, Bacillus, Listeria, Saccharomyces , Spodoptera, Mycobacteria, Trichoplusia, BHK (baby hamster kidney) cells, MDCK cells (normal dog kidney cell line for canine herpesvirus cultivation), CRFK cells (normal cat kidney cell line for feline herpesvirus cultivation), CV-1 cells (African monkey kidney cell line used, for example, to culture raccoon poxvirus), COS (e.g., COS-7) cells, and Vero cells. Particularly prefened host cells are Escherichia coli, including E. coli K-12 derivatives; Salmonella typhi; Salmonella typhimurium, including attenuated strains such as UK-1 x3987 and SR-11 x4072; Spodoptera frugiperda; Trichoplusia ni; BHK cells; MDCK cells; CRFK cells; CV-1 cells; COS cells; Vero cells; and non-tumorigenic mouse myoblast G8 cells (e.g., ATCC CRL 1246). Additional appropriate mammalian cell hosts include other kidney cell lines, other fibroblast cell lines (e.g., human, murine or chicken embryo fibroblast cell lines), myeloma cell lines, Chinese hamster ovary cells, mouse NDT/3T3 cells, LMTK31 cells and/or HeLa cells.
A recombinant cell is preferably produced by transforming a host cell with one or more recombinant molecules, each comprising one or more nucleic acid molecules of the present invention operatively linked to an expression vector containing one or more transcription control sequences. The phrase operatively linked refers to insertion of a nucleic acid molecule into an expression vector in a manner such that the molecule is able to be expressed when transformed into a host cell.
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 prefened recombinant molecule includes nhFcεR-npluc2268, nhFcεR-npluc2]93, nhFcεR-nbAP1983, and/or nhFcεR-nbAP1908..
A recombinant cell of the present invention includes any cell transformed with at least one of any nucleic acid molecule of the present invention. Suitable and prefened nucleic acid molecules as well as suitable and prefened recombinant molecules with which to transform cells are disclosed herein. A particularly prefened recombinant cell includes nhFcεR-npluc2268, nhFcεR-npluc2193, nhFcεR-nbAP1983, and/or nhFcεR-nbAPl908. Details regarding the production of this recombinant cell is disclosed herein. Recombinant DNA technologies can be used to improve expression of transformed nucleic acid molecules by manipulating, for example, the number of copies of the nucleic acid molecules within a host cell, the efficiency with which those nucleic acid molecules are transcribed, the efficiency with which the resultant transcripts are translated, and the efficiency of post-translational modifications. Recombinant techniques useful for increasing the expression of nucleic acid molecules of the present invention include, but are not limited to, operatively linking nucleic acid molecules to high-copy number plasmids, integration of the nucleic acid molecules into one or more host cell chromosomes, addition of vector stability sequences to plasmids, substitutions or modifications of transcription control signals (e.g., promoters, operators, enhancers), substitutions or modifications of translational control signals (e.g., ribosome binding sites, Shine-Dalgarno sequences), modification of nucleic acid molecules of the present invention to conespond to the codon usage of the host cell, deletion of sequences that destabilize transcripts, and use of control signals that temporally separate recombinant cell growth from recombinant enzyme production during fermentation. The activity of an expressed recombinant protein of the present invention may be improved by fragmenting, modifying, or derivatizing nucleic acid molecules encoding such a protein. Isolated FcεR-LP fusion proteins of the present invention can be produced in a variety of ways, including production and recovery of natural proteins, production and recovery of recombinant proteins, and chemical synthesis of the proteins. In one embodiment, an isolated protein of the present invention is produced by culturing a cell capable of expressing the protein under conditions effective to produce the protein, and recovering the protein. A prefened cell to culture is a recombinant cell of the present invention. Effective culture conditions include, but are not limited to, effective media, bioreactor, temperature, pH and oxygen conditions that permit protein production. An effective medium refers to any medium in which a cell is cultured to produce a FcεR-LP fusion protein of the present invention. Such a medium typically comprises an aqueous medium having assimilable carbon, nitrogen and phosphate sources, and appropriate salts, minerals, metals and other nutπents, such as vitamins. Cells of the present invention can be cultured in conventional fermentation bioreactors, shake flasks, test tubes, microtiter dishes, and petπ plates. Culturmg can be earned out at a temperature, pH and oxygen content appropriate for a recombinant cell Such cultuπng conditions are within the expertise of one of ordinary skill in the art
Depending on the vector and host system used for production, resultant proteins of the present invention may either remain within the recombinant cell; be secreted into the fermentation medium; be secreted into a space between two cellular membranes, such as the peπplasmic space in E coli, or be retained on the outer surface of a cell or viral membrane The phrase "recovering the protein", as well as similar phrases, refers to collecting the whole fermentation medium containing the protein and need not imply additional steps of separation or purification Proteins of the present invention can be purified using a variety of standard protein puπfication techniques, such as, but not limited to, affinity chromatography, ion exchange chromatography, filtration, electrophoresis, hydrophobic interaction chromatography, gel filtration chromatography, reverse phase chromatography, concanavahn A chromatography, chromatofocusmg and differential solubihzation. Proteins of the present invention are preferably retrieved m "substantially pure" form. As used herein, "substantially pure" refers to a puπty that allows for the effective use of the protein as a therapeutic composition or diagnostic A FcεR-LP fusion protein of the present invention can be bound to the surface of a cell comprising the FcεR-LP fusion protein A prefened fusion protem-bearmg cell includes a recombinant cell comprising a nucleic acid molecule encoding a FcεR-LP fusion protein of the present invention A more prefened recombinant cell of the present invention compnses a nucleic acid molecule that encodes at least one of the following proteins- PhFcεR-Ppluc756, PhFcεR-Ppluc73], PhFcεR-bAP660, and/or PhFcεR-bAP635 An even more prefened recombinant cell compnses a nucleic acid molecule including nhFcεR-npluc2268, nhFcεR-npluc2193, nhFcεR-nbAP1983, and/or nhFceR-nbAP,908. with a recombinant cell comprising a nucleic acid molecule compnsing a nucleic acid sequence including SEQ DD NO:17, SEQ DD NO: 19, SEQ ID NO:20, SEQ ID NO:22, SEQ DD NO:26, SEQ ID NO:28, SEQ ID NO:29 and/or SEQ DD NO:31, or a nucleic acid molecule comprising an allelic vaπant of a nucleic acid molecule comprising SEQ ID NO: 17, SEQ ID NO: 19, SEQ DD NO:20, SEQ ID NO:22, SEQ ID NO:26, SEQ DD NO:28, SEQ ID NO:29 and/or SEQ ID NO:31, being even more prefened.
In addition, a FcεR-LP fusion protein-containing composition of the present invention can include not only a FcεR-LP fusion protein 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, selective binding of a first molecule to a second molecule refers to the ability of the first molecule to preferentially bind (e.g., 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 (i.e., anti-IgE isotype antibody) or antibodies that bind selectively to an IgE having a specific antigen specificity (i.e., anti-IgE idiotypic antibody). Anti-IgE antibodies for use in a formulation of the present invention preferably are not capable of cross-linking two or more IgE antibodies. In one embodiment, prefened anti-IgE antibodies include Fab fragments of the antibodies (as defined in Janeway et al., ibid.). Examples of such antigens include any antigen known to induce the production of IgE. Prefened antigens include allergens and parasite antigens. Allergens include, but are not limited to allergens ingested, inhaled or contacted by an organism of interest. Allergens of the present invention are preferably derived from fungi, rusts, smuts, bacteria, trees, weeds, shrubs, grasses, wheat, corn, grains, hays, straws, oats, alfalfa, clovers, soybeans, yeasts, 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 prefened flea allergen includes a flea saliva antigen. Prefened flea saliva antigens include antigens such as those disclosed in PCT Patent Publication No. WO 96/28469, published September 19, 1996, by Stiegler et al., PCT Patent Publication No. WO 96/11271, published April 18, 1996, by Frank et al, and PCT Patent Publication No. WO 97/37676, published October 16, 1997, by Hunter et al., with flea saliva products and flea saliva proteins being particularly prefened. 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.
Prefened general allergens include those derived from grass, Meadow Fescue, curly dock, plantain, Mexican firebush, lamb's quarters, pigweed, ragweed, goldenrod, sonel, legumes, dandelion, sage, elm, cocklebur, elder, walnut, maple, sycamore, hickory, aspen, pine, cottonwood, ash, birch, cedar, oak, mulberry, cockroach, Dermataphagoides , Alternaria, Aspergillus, Cladosporium, Fusarium, Helminthosporium, Mucor, Curvularia, Candida, Penicillium, Pullularia, Rhizopus and/or Tricophyton. More prefened general allergens include those derived from Johnson grass, Kentucky blue grass, meadow fescue, orchard grass, perennial rye grass, red top grass, timothy grass, Bermuda grass, salt grass, brome grass, curly dock, yellow dock, English plantain, Mexican firebush, lamb's quarters, rough pigweed, short ragweed, goldenrod, sheep sonel, red clover, dandelion, wormwood sage, American elm, common cocklebur, box elder, marsh elder, black walnut, red maple, eastern sycamore, white pine, eastern cottonwood, green ash, river birch, red cedar, red oak, red mulberry, cockroach, grain smut, oat stem rust, wheat stem rust, Dermataphagoides farinae, Alternaria alternata, Alternaria tenuis, Curvularia spicifera, Aspergillus fumigatus, Cladosporium herbarum, Fusarium vasinfectum, Helminthosporium sativum, Mucor recemosus, Penicillium notatum, Pullularia pullulans, Rhizopus nigricans and/or Tricophyton spp. The term "derived from" refers to a natural allergen of such plants or organisms (i.e., an allergen directly isolated from such plants or organisms), as well as, non-natural allergens of such plants or organisms that posses at least one epitope capable of eliciting an immune response against an allergen (e.g., produced using recombinant DNA technology or by chemical synthesis). Prefened allergens include those that cause allergic respiratory diseases in equines, including, for example, chronic obstructive pulmonary disease, exercise induced pulmonary hemonhage and inhalant-induced urticaria. Such allergens include, but are not limited to, molds, components of dust and components of feed.
One embodiment of the present invention is a method to detect IgE which includes the steps of: (a) contacting an isolated FcεR-LP fusion protein with a putative IgE-containing composition under conditions suitable for formation of a FceR-LP:IgE complex; and (b) detecting the presence of IgE by detecting the FcεR-LP:IgE complex Presence of such a FceR-LP:IgE complex (i.e a complex between IgE and a FcεR-LP fusion protein of the present invention) indicates that the animal is producing IgE Prefened IgE to detect using a FcεR-LP fusion protein include any mammalian IgE, with human IgE, canine IgE, feline IgE, equme IgE, murine IgE and rat IgE being more prefened, with human, canme IgE, felme IgE and equme IgE being particularly prefened The present method can further include the step of determining whether an IgE complexed with a FcεR-LP fusion protein is heat labile. Preferably, a heat labile IgE is determined by mcubatmg an IgE at about 56 °C for about 3 or about 4 hours Without being bound by theory, the inventors 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
Animals in which to detect IgE include mammals and birds, with humans, dogs, cats, horses and other pets, work and/or economic food animals being prefened.
Particularly prefened m which to detect IgE are humans, dogs, cats and horses. As used herein, canme 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, felme 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, wild cats, lions, tigers, leopards, panthers, cougars, bobcats, lynx, jaguars, cheetahs, and servals. As used herein, equme refers to any member of the horse family, including, but are not limited to, domestic horses, wild horses and zoo horses. As used herein, the term "contacting" refers to combining or mixing, in this case a putative IgE-contammg composition with a FceR-LP molecule. Formation of a complex between a FcεR-LP molecule and an IgE refers to the ability of the FcεR-LP molecule to selectively bind to the IgE in order to form a stable complex that can be measured (i.e., detected). As used herein, the term selectively binds to an IgE refers to the ability of a FcεR-LP molecule of the present invention to preferentially bind to IgE, without being able to substantially bind to other antibody lsotypes. Binding between a FcεR-LP molecule and an IgE is effected under conditions suitable to form a complex; such conditions (e.g., 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, Sambrook et al., ibid.
As used herein, the term "detecting complex formation" refers to determining if any complex is formed, i.e., assaying for the presence (i.e., 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 FceR-LP molecule and any IgE in the composition can be measured (i.e., 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 (i.e., obtained) from an animal, examples of which include, but are not limited to, blood, serum, plasma, urine, tears, aqueous humor, cerebrospinal fluid (CSF), saliva, lymph, nasal secretions, tracheobronchial aspirates, milk, feces and fluids obtained through bronchial alveolar lavage. 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, such 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 pretreated 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 prefened composition of the present method is serum. 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 (e.g , coated onto) a support substrate. Immobilization techniques are known to those skilled m the art Suitable support substrate materials include, but are not limited to, plastic, glass, gel, celluloid, paper, PVDF (poly- vmylidene-fluoπde), nylon, nitrocellulose, and particulate materials such as latex, polystyrene, nylon, nitrocellulose, agarose and magnetic resm. Suitable shapes for support substrate material include, but are not limited to, a well (e.g., microtiter dish well), a plate, a dipstick, a bead, a lateral flow apparatus, a membrane, a filter, a tube, a dish, a celluloid-type matπx, a magnetic particle, and other particulates. A particularly prefened support 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 support substrate, such as a particulate, can include a detectable marker
A prefened method to detect IgE is an immunosorbent assay. 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 m such a manner that the IgE is immobilized to a support substrate. As such, a capture molecule is preferably immobilized to a support substrate of the present invention pnor to exposure of the capture molecule to a putative IgE-containmg composition. An FcεR-LP 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 support substrate as a capture molecule pnor to exposure of the capture molecule to a putative IgE- containing composition.
In one embodiment, a specific antigen is used as a capture molecule by being immobilized on a support substrate, such as a microtiter dish well or a dipstick. Prefened antigens include those disclosed herein A biological sample collected from an animal is applied to the support substrate and incubated under conditions suitable (i.e , sufficient) to allow for antιgen:IgE complex formation bound to the support substrate (i.e., IgE in a sample binds to an antigen immobilized on a support substrate). Excess non-bound material (i.e., mateπal from the biological sample that has not bound to the antigen), if any, is removed from the support substrate under conditions that retain antιgen:IgE complex binding to the support substrate. Prefened conditions are generally disclosed in Sambrook et al., ibid. A FcεR-LP is added to the LP substrate and incubated to allow formation of a complex between the FcεR-LP and the antigemlgE complex. Excess FcεR-LP is removed, a substrate for LP is added, and the complex is submitted to a detection device for analysis. In a prefened embodiment, the FccR-LP molecule is a FccR-luciferase fusion protein. In a particularly prefened embodiment, the FcεR-LP molecule is nhFcεR-npluc2l93. Prefened detection devices include luminometers and photographic film.
In one embodiment, an anti-IgE antibody (e.g., isotype or idiotype specific antibody) is used as a capture molecule by being immobilized on a support substrate, such as a microtiter dish well or a dipstick. A biological sample collected from an animal is applied to the support substrate and incubated under conditions suitable to allow for anti-IgE antibody:IgE complex formation bound to the support substrate. Excess non-bound material, if any, is removed from the support substrate under conditions that retain anti-IgE antibodyTgE complex binding to the support substrate. A FcεR-LP molecule is added to the support substrate and incubated to allow formation of a complex between the FcεR-LP molecule and the anti-IgE antibodyTgE complex. Excess FcεR-LP molecule is removed, a LP substrate is added, and the sample is submitted to a detection device for analysis. In a prefened embodiment, the FcεR-LP molecule is a FccR-luciferase fusion protein. In a particularly prefened embodiment, the FcεR-LP molecule is nhFcεR-npluc2193. Prefened detection devices include luminometers and photographic film.
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 support substrate, such as a microtiter dish well or a dipstick, and incubated under conditions suitable to allow for IgE binding to the support substrate.
Any IgE present in the bodily fluid is immobilized on the support substrate. Excess non- bound material, if any, is removed from the support substrate under conditions that retain IgE binding to the support substrate. A FcεR-LP molecule is added to the support substrate and incubated to allow formation of a complex between the FcεR molecule and the IgE. Excess FcεR molecule is removed, a LP substrate is added, and the sample is submitted to a detection device for analysis. In a prefened embodiment, the FceR-LP molecule is a FcεR-luciferase fusion protein. In a particularly prefened embodiment, the FcεR-LP molecule is nhFcεR-npluc2193. In a prefened embodiment, the FcεR-LP molecule is a FcεR-luciferase fusion protein. In a particularly prefened embodiment, the FcεR-LP molecule is nhFcεR-npluc2,93. Prefened detection devices include luminometers and photographic film.
Another prefened 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 December 22, 1994, by Miller et al.; and WO 94/01775, published January 20, 1994, by Pawlak et al. In one embodiment, a biological sample is placed in a lateral flow apparatus that includes the following components: (a) a support structure defining a flow path; and (b) a capture reagent comprising an antigen or and anti-IgE antibody, the capture reagent being impregnated within the support structure in a labeling zone. Preferably a labeling reagent comprising a FcεR-LP is added to the lateral flow apparatus and a detecting means is used to detect bound complex. Prefened antigens include those disclosed herein. The support structure comprises a material that does not impede the flow of IgE contained in a biological sample to the capture reagent. Suitable materials for use as a support structure include ionic (i.e., anionic or cationic) material. Examples of such a material include, but are not limited to, nitrocellulose (NC), PVDF, carboxymethylcellulose (CM). The support structure defines a flow path that is lateral and includes a capture zone. The apparatus can further comprise a sample receiving zone located along the flow path, more preferably upstream of the capture 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.
Another prefened method to detect IgE is a lateral flow assay, examples of which are disclosed in U.S. Patent No. 4,727,019. In one embodiment, a biological sample is placed in a flow through apparatus that includes the following components: (a) a support structure defining a flow path; and (b) a capture reagent comprising an antigen or and anti-IgE antibody, the capture reagent being impregnated within the support structure in a labeling zone. Preferably a labeling reagent comprising a FcεR-LP is added to the lateral flow apparatus and a detecting means is used to detect bound complex. Prefened antigens include those disclosed herein.
The present invention also includes kits to detect IgE based on each of the disclosed detection methods. Suitable and prefened FcεR-LP fusion proteins are disclosed herein. A prefened kit of the present invention further comprises a detection means including one or more antigens disclosed herein, a FceR-LP of the present invention and a LP substrate therefor.. Such antigens preferably induce IgE antibody production in animals including humans, canines, equines and/or felines.
Another prefened kit of the present invention is a general allergen kit comprising an allergen common to all regions of the United States and a FcεR protein 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 (i.e., 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 prefened general allergens for use with a general allergen kit of the present invention include those general allergens disclosed herein.
A prefened kit of the present invention includes those in which the allergen is immobilized on a support 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. In one embodiment, multiple lateral flow apparatuses can be attached to each other at one end of each apparatus, thereby creating a fan-like structure or can comprise a single device with multiple capture lines. In another embodiment, a prefened kit comprises a flow through apparatus or an ELISA. 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. Particularly prefened conditions to diagnose include allergies, parasitic infections and neoplasia. For example, a method and kit of the present invention are particularly useful for detecting hypersensitivity to the bite of insects. Particularly prefened is a method and kit useful for detecting flea allergy dermatitis (FAD), when such method or kit includes the use of flea antigens, preferably flea saliva antigens. FAD is defined as a hypersensitive response to fleabites. Preferably, a putative IgE-containing composition is obtained from an animal suspected of having FAD. Prefened animals include those disclosed herein, with humans, dogs, cats and horses being more prefened.
One embodiment of the present invention is a method to identify a compound capable of inhibiting FcεR protein activity. Such a method includes the steps of (a) contacting (e.g., combining, mixing) an isolated FcεR-LP protein of the present invention, with a putative inhibitory compound under conditions in which, in the absence of the compound, the protein has FcεR protein activity, and (b) determining if the putative inhibitory compound inhibits the activity. FcεR protein activity can be determined in a variety of ways known in the art, including but not limited to determining the ability of FcεR protein to bind to or otherwise interact with IgE. Such conditions under which a FceR protein or a FcεR-LP fusion protein has FcεR protein activity include conditions in which a FcεR protein or a FceR-LP fusion has a conect three-dimensionally folded structure under physiologic conditions, i.e. physiologic pH, physiologic ionic concentrations, and physiologic temperatures.
Putative inhibitory compounds to screen include antibodies (including fragments and mimetopes thereof), putative substrate analogs, and other, preferably small, organic or inorganic molecules. Methods to determine FcεR protein activity are known to those skilled in the art. Methods to determine binding of a putative inhibitory compound to a FcεR-LP protein of the present invention are known to those of skill in the art and include, for example, determining changes in molecular mass using surface plasmon resonance (e.g., determining light scatter by an inhibitor of a FcεR-LP fusion protein, before and after contacting the inhibitor or protein with a FcεR-LP fusion protein or inhibitor, respectively) or screening for compounds that inhibit interaction between a FcεR protein and IgE.
A prefened method to identify a compound capable of inhibiting FcεR protein activity includes contacting an isolated FcεR-LP fusion protein with a putative inhibitory compound under conditions in which, in the absence of the compound, the FcεR-LP fusion protein has FcεR protein activity; and determining if the putative inhibitory compound inhibits the activity; wherein such FcεR-LP fusion protein has an LP domain comprising an amino acid sequence encoded by a nucleic acid molecule that hybridizes to an isolated nucleic acid molecule selected from the group consisting of SEQ ID NO: 16, and SEQ DD NO:25 under conditions comprising: (a) hybridizing in a solution comprising 2X SSC and 0% formamide, at a temperature of 37°C; and (b) washing in IX SSC and 0%> formamide at a temperature of 55°C and the FcεR-LP fusion protein has a Fc epsilon receptor domain comprising an amino acid sequence encoded by a nucleic acid molecule that hybridizes to an isolated nucleic acid molecule selected from the group consisting of SEQ ID NO:3, SEQ ID NO:6, SEQ ID NO:9 and SEQ DD NO: 12 under conditions comprising: (a) hybridizing in a solution comprising 2X SSC and 0% formamide, at a temperature of 37°C; and (b) washing in IX SSC and 0% formamide at a temperature of 55°C.
Another embodiment of the present invention is an assay kit to identify an inhibitor of FcεR protein activity. This kit comprises an isolated FcεR-LP fusion protein of the present invention, and a means for determining inhibition of FcεR activity, where the means enables detection of inhibition. Detection of inhibition of FceR-LP fusion protein activity identifies a putative inhibitor to be an inhibitor of FcεR. Means for determining inhibition of a FcεR-LP fusion protein include an assay system that detects binding of a putative inhibitor to a FcεR-LP fusion protein, and an assay system that detects interference by a putative inhibitor of the ability of a FcεR-LP fusion protein to bind IgE. Means and methods are described herein and are known to those skilled in the art.
The following examples are provided for the purposes of illustration and are not intended to limit the scope of the present invention.
EXAMPLES It is to be noted that the Examples include a number of molecular biology, microbiology, immunology and biochemistry techniques considered to be known to those skilled in the art. Disclosure of such techniques can be found, for example, in Sambrook et al., ibid., and related references. Example 1
This example describes the construction of a recombinant baculovirus expressing a fusion protein including a firefly luciferase, fused to the carboxyl terminus of a soluble portion of the alpha-chain of an Fc epsilon receptor. Recombmant molecule pFB-nhFcεR-npluc2268, containing a nucleic acid molecule encoding the extracellular domain of a human FcεR , fused to sequences encoding Photuris pennsylvanica firefly luciferase, and operatively linked to baculovirus polyhedron transcription control sequences was produced in the following manner A cDNA molecule encoding the full-length alpha chain of the human Fc epsilon receptor was obtained from Dr. Jean-Piene Kinet (Harvard University, Cambπdge, MA) The cDNA molecule included an about 1198 nucleotide insert, refened to herein as nhFcεR] ]9g. The nucleic acid sequence of the coding strand of nhFcεRU9g is denoted herein as SEQ ID NO.1 Translation of SEQ ID NO: 1 indicates that nucleic acid molecule nhFceR, 198 encodes a full-length human FcεR alpha-chain protein of about 257 ammo acids, refened to herein as PhFcεR257, having ammo 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 DD NO. l and the termination codon spans from nucleotide 875 through nucleotide 877 of SEQ DD NO: 1 The complement of SEQ ID NO: 1 is represented herein by SEQ DD NO:3. The proposed mature protein (i.e., FcεRα chain from which the signal sequence has been cleaved), denoted herein as PhFcεR232, contains about 232 ammo acids which is represented herein as SEQ ID NO:5. The nucleic acid molecule encoding PhFcεR232 is denoted herein as
Figure imgf000041_0001
having a coding strand represented by SEQ ID NO'4 and a complementary strand represented by SEQ ID NO:6
To produce a secreted form of the extracellular domain of the FceR alpha-chain, the hydrophobic transmembrane domain and the cytoplasmic tail of the FcεR alpha-chain encoded by nhFcεRn98 were removed as follows. A FcεR alpha-chain extracellular domain nucleic acid molecule-containing fragment of about 591 nucleotides was PCR amplified from nhFcεR, ]98 using a forward pπmer Fc/+, having the nucleic acid sequence 5' GGC CGG ATC CTA TAA ATA TGG CTC CTG CCA TGG AAT CC 3' and containing a BamHl site indicated in bold, denoted SEQ ID NO: 32, and a reverse pπmer Fc/-, having the nucleic acid sequence 5' GGC CGA ATT CAG CTT TTA TTA CAG TAA TGT TGA G 3' and containing an EcoRI site indicated m bold, denoted herein as SEQ DD NO:33. The resulting PCR product was digested with BamHl and EcoRI to produce a 601 nucleotide fragment, denoted nhFcεR60l. Nucleic acid molecule nhFceR60] contamed an about 591 nucleotide fragment encoding the extracellular domain of the human FcεR alpha-chain, extending from nucleotide 107 to nucleotide 697 of SEQ ID NO.l, denoted herein as nucleic acid molecule nhFcεR59], having a coding strand of which has a nucleic acid sequence denoted SEQ ID NO 7 and a complementary strand denoted SEQ ID NO: 9 Translation of SEQ ID NO -7 indicates that nucleic acid molecule nhFcεR59, encodes a FcεR protein of about 197 ammo acids, refened to herein as PhFcεR197, having amino acid sequence SEQ ID NO- 8 Nucleic acid molecule nhFcεR6]2 encodes a secretable form of the human FcεR alpha chain which does not possess a leader sequence, which is denoted herein as PhFcεR172, having ammo acid sequence SEQ ID NO.11 The coding region for PhFcεR172 is denoted nhFccR5,6, the coding strand of which has a nucleic acid sequence denoted SEQ ID NO.10. The complement of SEQ ID NO.10 is represented herein by SEQ DD NO.12. The nucleic acid molecule nhFcεRα59l was subcloned into unique BamHl and EcoRI sites of pFASTBACl baculovirus shuttle plasmid, available from Pharmingen, San Diego, CA, to produce a recombinant molecule refened to herein as pFB-nhFcεR 591 The resultant recombinant molecule pFB-nhFcεRα59] was veπfied for proper insert orientation by restriction mapping.
In order to permit for rotational freedom between the FceR alpha-chain and firefly luciferase domains, which were genetically linked to generate the fusion protein, DNA sequences encoding a GlyGlyGlyGlySerGlyGlyGlyGlySer linker peptide, hereinafter refened to as (Gly4Serl)2, denoted SΕQ DD NO: 13, were inserted between sequences encoding the 3' end of the FcεR alpha-chain cDNA and sequences encoding the 5' end of the firefly luciferase cDNA, in the following manner. Two complementary oligonucleotides, containing sequences encoding the (Gly4Serl)2 linker peptide, were prepared. A sense oligonucleotide pnmer, denoted as (Gly4Serl)2 /+, having the sequence 5' AA TTC GGT GGT GGC GGT TCT GGT GGC GGT GGC TCT T 3', and containing an EcoRI sticky end site at the 5 ' end and a Xbal sticky end site at the 3 ' end, denoted SΕQ ID NO:34, and a complementary olrgonucleotide primer, denoted as (Gly4Serl)2 /-, having the sequence 5' CT AGA AGA GCC ACC GCC ACC AGA ACC GCC ACC ACC G 3 ', and containing an Xbal sticky end site at the 5 ' end and a EcoRI sticky end site at the 3' end, denoted SΕQ DD NO:35 were prepared These two pπmers were annealed to each other using techniques lαiown to those skilled m the art and subcloned into unique EcoRI and Xbal restriction sites m pFB-nhFcεR591 to generate pFB-nhFcεR591/(Gly4Serl)2
A cDNA molecule encoding the full-length luciferase protein, from the firefly Photuris pennsylvanica, was obtained from Promega Corporation, Madison, WI. The cDNA molecule included an about 1638 nucleotide insert, refened to herein as npluc,638 having a coding strand denoted herein as SEQ ID NO: 14 and a complementary strand denoted SEQ ID NO: 16. Translation of SEQ ID NO- 14 indicates that nucleic acid molecule npluc1638 encodes a full-length firefly luciferase protein of about 545 ammo acids, refened to herein as Ppluc545, having ammo acid sequence SEQ ID NO: 15, assuming an open reading frame in which the initiation codon spans from about nucleotide 1 through about nucleotide 3 of SEQ DD NO: 14 and the termination codon spans from about nucleotide 1636 through about nucleotide 1638 of SEQ ID NO: 14. A firefly luciferase nucleic acid molecule-containing fragment of about 1638 nucleotides was PCR amplified from npluc]638 using forward primer Ppluc/+, having nucleic acid sequence 5' GGG GCC CCT CTA GAA TGG CAG ATA AGA ATA TTT TAT ATG GG 3' containing a Xbal site indicated in bold, denoted SEQ DD NO:36 and reverse primer Ppluc/-, having nucleic acid sequence 5' GCG CGC GCA AGC TTT TAC CCA TTG GTG TGT TTT TC 3' containing a HindlH. restriction site indicated in bold, denoted herein as SEQ DD NO:37 The resulting PCR product was digested with Xbal and Hind ll to produce npluc1638, which was ligated into the unique Xbal and HindlR restriction sites of pFB-nhFcεR591/(Gly4Serl)2.
The resulting recombinant molecule, refened to as pFB-nhFcεR-npluc2268, contained an about 2268-nucleotιde chimeπc nucleic acid molecule, namely nhFcεR- npluc2268, encoding the extracellular domain of the human FcεR alpha-chain, fused to the 5' end of nucleic acid sequences encoding luciferase, from the firefly Photuris pennsylvanica, with these two nucleic acid sequences being linked by nucleic acid sequences encoding a GlyGlyGlyGlySerGlyGlyGlyGlySer linker peptide. The coding strand of nhFcεR-npluc2268 has a nucleic acid sequence denoted SEQ DD NO: 17 and a complementary strand denoted SEQ ID NO: 19. Translation of SEQ ID NO: 17 indicates that nucleic acid molecule nhFcεR-npluc2268 encodes an FcεR-lucιferase fusion protein of about 756 amino acids, refened to herein as PhFcεR-Ppluc756, having ammo acid sequence SEQ ID NO: 18.
Recombinant molecule pFB-nhFceR-npluc2268 was verified for proper insert orientation by restnction mapping. Four positive plaques containing recombinant molecule pFB-nhFcεR-npluc226g, refened to as Clones A-D, were individually co- transfected with a linear Baculogold baculovirus DNA (available from Pharmingen) into separate cultures of S frugiperda Sf9 cells (available from InVitrogen, Carlsbad, CA) to form cultures each containing recombinant cells denoted S frugiperda:p¥ -n ¥ceR- npluc2268. S yr«gz/?erύ?α:pFB-nhFcεR-npluc226g cultures were grown using conditions known to those skilled in the art m order to produce the fusion protein, PhFcεR-Ppluc756 Supernatants containing the secreted form of the fusion protein, refened to as PhFceR- Ppluc73], denoted SEQ ID NO.20 was prepared from the cultures by centnfuging for 20 minutes at 5000 x g and discarding the cell pellet. The nucleic acid molecule encoding PhFcεR-Ppluc731, refened to herein as nhFceR-npluc2193, has a coding strand with a nucleic acid sequence denoted SEQ ID NO: 19 and a complementary strand with a nucleic acid sequence denoted SEQ DD NO:21. Example 2
This example descnbes the construction of a recombinant baculovirus expressing a fusion protein including bacterial alkaline phosphatase, fused to the carboxyl terminus of a truncated, soluble portion of an alpha-chain of Fc receptor as well as production and testing of that fusion protein
A. Recombinant molecule pFB-nhFceR-bAP]983, containing a nucleic acid molecule encoding the extracellular domain of a human FceR alpha-chain, fused to sequences encoding bacteπal alkaline phosphatase, and operatively linked to baculovirus polyhedron transcnption control sequences was produced in the following manner.
Nucleic acid molecule pFB-nhFcεR591 and the (Gly4Serl)2 linker were used to generate the construct pFB-nhFcεR591/(Gly4Serl)2 as described in Example 1. A PCR fragment containing nucleic acid sequences encoding the mature form of bacterial alkaline phosphatase was generated in the following manner. A bacteπal alkaline phosphatase mature domain nucleic acid molecule-contammg fragment of about 1347 nucleotides was PCR amplified from E coli XLl-Blue using forward pnmer bAP/+, having the nucleic acid sequence 5' GGG CCC TCT AGA AC A CCA GAA ATG CCT GTT CTG GAA AAC CGG 3' containing a Xbal site indicated in bold, denoted SEQ DD NO:38 and reverse primer bAP/-, having the nucleic acid sequence 5' GCG CGC AAG CTT TTA YTT MAG CCC CAG AGC GGC 3' containing an Hindm site indicated in bold, denoted herein as SEQ ID NO:39. The resulting PCR product was blunt end ligated into TOPO vector (available from InVitrogen, Carlsbad, CA) for further manipulation. The PCR clone included an about 1347 nucleotide insert, refened to herein as nbAP1347 having a coding strand denoted herein as SEQ ID NO:23 and a complementary strand denoted SEQ DD NO:25. Translation of SEQ ID NO:23 indicates that nucleic acid nbAP1347 encodes a mamre bacterial alkaline phosphatase protein of about 449 amino acids, refened to herein as PbAP449, having amino acid sequence SEQ ID NO:24, assuming an open reading frame in which the first codon spans from about nucleotide 1 through about nucleotide 3 of SEQ DD NO:23 and the last codon spans from about nucleotide 1345 through about nucleotide 1347 of SEQ ID NO:23. The resulting PCR product was digested with Xbal and HindHI to produce nbAP1347, which was ligated into the unique Xbal and Hindm restriction sites of pFB-nhFcεR591/(Gly4Serl)2.
The resulting recombinant molecule, refened to as pFB-nhFceR-nbAP]983 contains an about 1983 nucleotide chimeric nucleic acid molecule, namely nhFcεR- nbAP19g3 encoding the extracellular domain of the human FcεR alpha-chain, fused to the 5' end of nucleic acid sequences encoding alkaline phosphatase from E. coli XLl-Blue, with these two nucleic acid sequences being linked by nucleic acid sequences encoding a Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser linker peptide. The coding strand of nhFcεR- nbAP1983 has a nucleic acid sequence denoted SEQ DD NO:26 and the complementary strand denoted SEQ ID NO:28. Translation of SEQ ID NO:26 indicates that nucleic acid molecule nhFcεR-bAP1983 encodes a FcεR/bacterial alkaline phosphatase fusion protein of about 660 amino acids, refened to herein as PhFcεR-bAP660, having amino acid sequence SEQ ID NO:27.
Recombinant molecule, pFB-nhFcεR-bAP1983, was verified for proper insert orientation by restriction mapping. Positive plaques containing recombinant molecule pFB-nhFcεR-bAP,983, were individually co-transfected with a linear Baculogold baculovirus DNA (available from Pharmingen) into separate cultures of S. frugiperda Sf9 cells (available from InVitrogen, Carlsbad, CA) to form cultures each containing recombinant cells denoted S. frugiperda:pΕB-nhF ceR-b AY {9S3. S. frugiperda:pΕB-n ¥c R-bAP,9g3 cultures were grown using conditions known to those skilled in the art in order to produce the fusion protein, PhFcεR-bAP660. Supernatants containing the secreted form of the fusion protein, refened to as PhFceR-bAP635, denoted SEQ DD NO:30 was prepared from the cultures by centrifuging for 20 minutes at 5000 x g and discarding the cell pellet. The nucleic acid molecule encoding PhFcεR-bAP635, refened to herein as nhFcεR-nbAP190g has a coding strand with a nucleic acid sequence denoted SEQ ID NO:29 and a complementary strand with a nucleic acid sequence denoted SEQ ID NO:31.
B. PhFcεR-bAP635 was tested for its ability to detect IgE in the following manner. Polystyrene, opaque, microtiter wells (Immunolon 96 well plate) were coated with a recombinant flea saliva antigen using the same protocols used for coating polystyrene beads described in Example 3. The wells were then blocked for 30 minutes with 100 μL of IX Tris buffered saline (TBS) (50 mM Tris-HCl, 150 mM NaCl, 2 mM MgCl2, containing 1%> bovine serum albumin). Antigen-coated wells were incubated with either flea-allergy positive or negative pooled dog serum overnight using 250 μl serum. Following serum incubations, all sample wells were washed three times in of 300 μL IX TBS containing 0.25% Tween-20 and incubated with an PhFcεR-bAP660 fusion protein, prepared as described in section 2, as follows. Specifically, two samples of positive serum incubations were paired with two samples of negative serum incubations and each pair was incubated with supernatant from a different transfection clone expressing PhFcεR-bAP660. Two wells were incubated with supernatant from a "cells only" mock transfection, i.e. supernatants from cell cultures which were not transfected with a clone expressing PhFceR-bAP660. All supernatant incubations were conducted in 250 μl of supernatant for 90 minutes at room temperature.
Following supernatant incubations, all bead samples were washed 3 times in 100 μL of IX TBS containing 0.25%> Tween-20 followed by one wash in IX TBS without Tween-20. One-hundred μl of Luciglo™ chemiluminescent phosphatase substrate, available from KPL, Gaithersburg, MD was added to each experimental and control well, mixed briefly by pipetting and the plate was read in a Packard 96 well lummometer. There was no detectable signal above background using this assay. Example 3
This example demonstrates the use of an FcεRα-luciferase fusion protein of the present invention for detection of IgE
A A 50 microhter (μl) aliquot of 3.3 micron polystyrene beads, available from Bangs Laboratories, Fisher, IN, was coated with a recombinant flea saliva antigen, namely FS-I, by overnight incubation at room temperature in a total volume of 1 milhhter (ml) of carbonate coating buffer (50 mM Na2CO3, 50 mM NaHCO3, pH 9 6) containing 100 micrograms (μg) of FS-I, produced as described in U.S. Patent No. 5,646,115, issued July 8, 1997. A 50-μl aliquot of control beads was prepared m an identical manner except that no antigen was added to the incubation. Each aliquot of beads was centπfuged at 4000 x g for 10 minutes, the supernatant was removed and the pellet then blocked for 30 minutes with 1 ml of IX phosphate buffered saline (PBS) containing 1% bovine serum albumin (BSA) and 0.25%o Tween-20.
The aliquot of antigen-coated beads was divided into 10-μl samples for incubation as follows. Four samples were incubated with flea-allergy positive pooled dog sera, i.e. sera collected from dogs shown to produce IgE against FS-I; and five samples were incubated with flea-allergy negative pooled dog sera. Incubations were conducted overnight in 250 μl sera supplemented with Tween-20 to a concentration of 0.025%.
Following serum incubations, all bead samples were washed three times in 1 ml each of IX PBS containing 0.025%> Tween-20 and incubated with an FcεRoc-lucιferase fusion protein, prepared as described in Example 1, as follows. The four samples incubated with flea-allergy positive sera were paired with four of the samples incubated with flea-allergy negative sera such that each pair was incubated separately with supernatant prepared as described in Example 1, from a different transfection clone expressing PhFcεR-Ppluc756. The remaining sample of beads incubated with negative serum was incubated with supernatant from a "cells only" mock transfection, i.e. supernatant from S. frugiperda cell cultures which were not transfected with a clone expressing PhFceR-Ppluc756. All supernatant incubations were conducted in 250 μl of supernatant for 90 minutes at room temperature.
Three samples of 10 μl of non-antigen coated beads were prepared as follows as controls. Sample 1 was incubated with flea-allergy negative dog serum followed by incubation with non-transfected expression supernatant; sample 2 was incubated with flea-allergy negative dog serum followed by incubation with PhFcεR-Ppluc756 expression supernatant; and sample 3 was incubated with flea-allergy positive dog serum and non- transfected expression supernatant. Incubations were performed as described above for antigen-coated beads. Following supernatant incubations, all bead samples were centrifuged at 4000 x g for 10 minutes and washed 3 times in 1 ml of IX PBS containing 0.025% Tween-20 followed by one wash in IX PBS without Tween-20. One-hundred μl of luciferin substrate, available from Promega Corporation, was added to each experimental and control sample, mixed briefly by pipeting and placed in a TD-2-/20 luminometer for a 30 second readout. The results, which indicate relative light units (RLU), and which also appear in Table 1, may be summarized as follows. The 3 non-allergen coated samples and the allergen coated sample which was incubated in negative serum and cell only supernatant showed no significant luminescence. Among each allergen-coated sample pair incubated with PhFcεR-Ppluc756, the samples within each pair which were incubated with allergy-negative dog serum showed no significant difference compared to control samples and the samples within each pair which were incubated with allergy-positive dog serum showed luminescence several thousand times greater than background, thus demonstrating that fusion proteins of the present invention may be used for highly sensitive, highly specific detection of IgE. Table 1. Luminescence Assay results
Figure imgf000049_0001
B. An FcεRα-luciferase fusion protein was shown to detect IgE over a dilution range of three orders of magnitude as follows. Microlite 2 ninety-six-well plates, available from Dynex Technologies, Chantilly, VA, were coated with 200 ng/well of Dermatophagoides pteronyssinus whole mite extract, available from Center Laboratories, Port Washington, NY and blocked in the presence of 1%> monoethanolamine, pH 7.5. A 100-ml sample of human sera (available from Plasma Labs International, Everett, WA) obtained from individuals allergic to house dust mite or sera obtained from control individuals was added to each well at a dilution of 1 :20, titrating across the plate in two-fold serial dilutions. The sera were incubated overnight at 4°C. Following incubation, the plates to be incubated with a FceRIa-luciferase fusion protein were washed with PBST (7 mM KHP04, 1.5 mM KH2P04, 0.15M NaCl, 0.1% Tween 20) and plates to be incubated with biotinylated FceRIa were washed with TBST (50 mM Tris-HCl, 2mM MgCl2, 0.15M NaCl, .005% Tween 20); 100 ml of purified PhFceR-Ppluc731 prepared as described in Example 1 or biotinylated FceRIa (200 ng/ml), prepared as described in Example 3 of U.S. Patent No. 5,945,294, issued August 31,
1999 was individually added to wells of each serum dilution and incubated for two hours at room temperature. The plates were then washed with either PBST or TBST as described above.
Following the wash, 100 ml luciferase substrate, available from Promega Corporation, Madison, WI,was added to FceRIa-luciferase fusion-incubated wells and luminescence expressed in relative light units (RLU) was measured for 5 seconds/well in a TopCount NXT ™ Microplate Scintillation and Luminescence Counter, available from Packard Instrument Company Meriden, CT.
Streptavidin-conjugated alkaline phosphatase, available from Jackson ImmunoResearch Labs, Westgrove, PA, was added to biotinylated FceRIa-incubated wells and incubated for one hour at room temperamre, then washed with TBST and 100 ml p-NPP substrate, available from Moss Inc., Pasadena, MD was added to the wells. After a development time of twenty minutes, the reaction was stopped with 20 mM cysteine and the optical density was read at an absorbance (A) of 405 nm in a 96-well spectrophotometer. The optical density measurement was multiplied by a factor of 1000.
Background readings were determined by negative control wells and subtracted from each value. The results, which are shown in Table 2 indicate that FcεR -luciferase fusion protein can detect IgE over a dilution range of three orders of magnitude. Table 2. IgE Detection over Three Orders of Magnitude
PhFcεRα-Ppluc731 Biotinylated FceRI
Serum Dilution RLU (^f'A=405 nm
1 1:20 41526 2241
2 1:40 18745 1078
3 1 :80 8790 507
4 1:160 5576 254
5 1:320 2902 178
6 1:640 1654 54
7 1 : 1280 834 7
8 1:2560 473
9 1:5120 219
10 1: 10240 39 C. A comparative analysis of house dust mite allergic human sera using the FceRIa- luciferase fusion protein and biotinylated FceRIa was conducted as follows. Maxisorp ninety-six well plates, available from Nalge Nunc International Corporation, Rochester, NY, and Microlite 2 ninety-six well plates, available from Dynex Technologies, were coated with whole dust mite extract from Dermatophagoides pteronyssinus for analysis of FcεRα-luciferase fusion protein and biotinylated FceRIa respectively, and blocked with 1% monoethanolamine, pH 7.5. Human sera, available from Plasma Labs International, obtained from individuals with varying levels of hypersensitivity to house dust mite were diluted 1 :31, added to the wells, and allowed to incubate overnight at 4°C. Following incubation, the plates to be incubated with a FceRIa- luciferase fusion were washed with PBST and plates to be incubated with biotinylated FceRIa were washed with TBST as described in Example 3B; 100 ml of purified PhFcεR-Ppluc731 or biotinylated FceRIa (200 ng/ml) was individually added to wells and incubated for two hours at room temperature. The plates were then washed with either PBST or TBST as described in Example 3B.
Following the wash, 100 ml luciferase substrate, available from Promega Corportaion, was added to FceRIa-luciferase fusion-incubated wells and luminescence was measured for 5 seconds/well in a TopCount NXT ™ Microplate Scintillation and Luminescence Counter, available from Packard Instrument Company. Streptavidin-conjugated alkaline phosphatase, available from Jackson
ImmunoResearch Labs, was added to biotinylated FceRIa-incubated wells and incubated for one hour at room temperature, then washed with TBST and 100 ml p-NPP substrate, available from Moss Inc., was added to the wells. After a development time of twenty minutes, the reaction was stopped with 20mM cysteine and the optical density was read at A= 05 nm in a 96-well spectrophotometer. The optical density measurement was multiplied by a factor of 1000.
Background readings were determined by negative control wells and subtracted from each value. The results, which are shown in Table 3 indicate that the PhFcεR- Ppluc731 -based assay and the biotinylated FceRIα-based assay are each able detect a range of IgE levels and are comparable in being able to differentiate IgE-positive samples from IgE-negative samples. ble 3. Comparative Assay results
PhFcεRα-Ppluc73 Biotinylated FceRIa
Sample RLU OD A=405 nm
Number
1 9524-SL 9884 2215
2 9733-DW 14362 2864
3 9735-RE 15976 3119
4 9777-LB 351 76
5 9832-CF 266 100
6 12886-EP 24054 3352
7 14457-JJ 4780 1187
8 15742-JT 5783 1094
9 15920-CK 521 87
10 16060-BE 4202 895
11 16578-SR 3718 442
12 16826-TC 6690 1372
13 17369-AJ 5222 1431
14 17694-SS 7694 2134
15 18514-CC 17193 3070
16 18704-TG 50747 3967
17 18718-HM 8201 1391
18 18744-MH 12785 1983
19 18792-CB 14151 2159
20 18859-CW 41515 4016
21 Negative Pool 120 24
22 Positive Pool 200890 4029
While the 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 are adaptations are within the scope of the present invention, as set forth in the following claims.

Claims

What is claimed is:
1. A Fc€R-LP fusion protein comprising a Fc epsilon receptor (FcεR) domain that binds to IgE and a luminescence inducing protein (LP) domain that induces a LP substrate to emit light when contacted with said LP domain.
2. A chimeric nucleic acid molecule comprising a FcεR protein-encoding nucleic acid sequence and an LP-encoding nucleic acid sequence.
3. A method for detecting IgE comprising: a. contacting a FcεR-LP fusion protein with a putative IgE-containing composition under conditions suitable for formation of a FcεR-LP:IgE complex; and b. determining the presence of IgE by detecting said FceR-LP:IgE complex, the presence of said FcεR-LP:IgE complex indicating the presence of IgE.
4. A method of producing a FcεR-LP fusion protein, said method comprising culturing a cell transformed with a nucleic acid molecule encoding said FcεR-LP fusion protein.
5. A kit for detecting IgE comprising a FcεR-LP fusion protein, a LP substrate and a means for detecting emitted light.
6. A method to identify a compound capable of inhibiting FcεR protein activity, said method comprising the steps of: (a) contacting an isolated FcεR-LP fusion protein of the present invention with a putative inhibitory compound under conditions in which, in the absence of said compound, said FcεR-LP fusion protein has FcεR protein activity; and (b) determining if said putative inhibitory compound inhibits said activity.
7. The fusion protein of Claims 1, 3, 4, 5, and 6, wherein said LP domain comprises a luciferase protein.
8. The fusion protein of Claims 1, 3, 4, 5, and 6, wherein said LP domain comprises a thermostable luciferase protein.
9. The fusion protein of Claims 1, 3, 4, 5, and 6, wherein said LP domain comprises an alkaline phosphatase protein.
10. The fusion protein of Claims 1, 3, 4, 5, and 6, wherein said LP domain comprises an amino acid sequence encoded by a nucleic acid molecule that hybridizes to an isolated nucleic acid molecule selected from the group consisting of SEQ DD NO.16, and SEQ ID NO:25 under conditions compnsing: (a) hybridizing m a solution comprising 2X SSC and 0% formamide, at a temperamre of 37°C, and (b) washing in IX SSC and 0% formamide at a temperature of 55°C
11 The fusion protein of Claims 1, 3, 4, 5, and 6, wherein said LP domain compnses an amino acid sequence selected from the group consisting of SEQ DD NO : 15 and SEQ ID NO:24
12 The fusion protein of Claim 1, wherein said LP domain comprises an ammo acid sequence encoded by a nucleic acid molecule selected from the group consisting of SEQ DD NOT4 and SEQ DD NO:23.
13 The fusion protein of Claims 1, 3, 4, 5, and 6, wherein said Fc epsilon receptor domain compnses FcεR alpha chain
14 The fusion protein of Claims 1, 3, 4, 5, and 6, wherein said Fc epsilon receptor domain is selected from the group consisting of a human FcεR domain, a canme FcεR domain, a felme FcεR domain, an equine FcεR domain, a rat FcεR domain and a mouse FcεR domain.
15. The fusion protein of Claims 1, 3, 4, 5, and 6, wherein said Fc epsilon receptor domain compnses an amino acid sequence encoded by a nucleic acid molecule that hybπdizes to an isolated nucleic acid molecule selected from the group consisting of SEQ ID NO.3, SEQ DD NO: 6, SEQ DD NO: 9 and SEQ DD NO: 12 under conditions compnsing: (a) hybridizing in a solution compnsing 2X SSC and 0% formamide, at a temperamre of 37°C; and (b) washing in IX SSC and 0%> formamide at a temperature of 55°C.
16. The fusion protein of Claims 1, 3, 4, 5, and 6, wherein said Fc epsilon receptor domain compnses a protein having an ammo acid sequence selected from the list consisting of SEQ DD NO.2, SEQ ID NO:5, SEQ DD NO:8 and SEQ ID NO: 11.
17 The fusion protein of Claims 1, 3, 4, 5, and 6, wherein said Fc epsilon receptor domain compnses a protein encoded by a nucleic acid sequence selected from the list consisting of SEQ ID NO: l, SEQ DD NO.4, SEQ ID NO:7 and SEQ DD NO:10.
18. The fusion protein of Claims 1, 3, 4, 5, and 6, wherein said FcεR-LP fusion protein compnses an amino acid sequence selected from the group consisting of SEQ DD NO:18, SEQ DD NO.21, SEQ DD NO:27 and SEQ DD NO:30.
19. The fusion protein of Claims 1, 3, 4, 5, and 6, wherein said FcεR-LP fusion protein further comprises a linker sequence.
20. The fusion protein of Claim 19, wherein said linker sequence comprises SEQ D NO: 13.
21. The nucleic acid molecule of Claim 2, wherein said LP-encoding nucleic acid sequence comprises a luciferase nucleic acid sequence.
22. The nucleic acid molecule of Claim 2, wherein said LP-encoding nucleic acid sequence comprises a thermostable luciferase nucleic acid sequence.
23. The nucleic acid molecule of Claim 2, wherein said LP-encoding nucleic acid sequence comprises an alkaline phosphatase nucleic acid sequence.
24. The nucleic acid molecule of Claim 2, wherein said LP-encoding nucleic acid sequence comprises a nucleic acid sequence that hybridizes to an isolated nucleic acid molecule selected from the group consisting of SEQ ID NO: 16, and SEQ DD NO:25 under conditions comprising (a) hybridizing in a solution comprising 2X SSC and 0% formamide, at a temperature of 37°C, and (b) washing in IX SSC and 0% formamide at a temperamre of 55°C.
25. The nucleic acid molecule of Claim 2, wherein said LP-encoding nucleic acid sequence encodes a protein comprising an amino acid sequence selected from the group consisting of SEQ DD NO: 15 and SEQ ID NO:24.
26. The nucleic acid molecule of Claim 2, wherein said LP-encoding nucleic acid sequence comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO: 14 and SEQ DD NO:23.
27. The nucleic acid molecule of Claim 2, wherein said Fc epsilon receptor nucleic acid sequence comprises a Fc epsilon receptor alpha chain nucleic acid molecule.
28. The nucleic acid molecule of Claim 2, wherein said FcεR nucleic acid sequence is selected from the group consisting of a human FcεR nucleic acid sequence, a canine FcεR nucleic acid sequence, a feline FcεR nucleic acid sequence, an equine FcεR nucleic acid sequence, a rat FcεR nucleic acid sequence and a mouse FcεR nucleic acid sequence.
29. The nucleic acid molecule of Claim 2, wherein said FcεR nucleic acid sequence comprises a nucleic acid sequence that hybridizes to an isolated nucleic acid molecule selected from the group consisting of SEQ ID NO:3, SEQ DD NO: 6, SEQ ID NO: 9 and SEQ DD NO: 12 under conditions comprising (a) hybridizing in a solution comprising 2X SSC and 0% formamide, at a temperamre of 37°C, and (b) washing in IX SSC and 0% formamide at a temperamre of 55°C.
30. The nucleic acid molecule of Claim 2, wherein said Fc epsilon receptor nucleic acid sequence comprises a nucleic acid sequence selected from the group consisting of SEQ DD NO: l, SEQ ID NO:4, SEQ DD NO:7, and SEQ DD NO: 10.
31. The nucleic acid molecule of Claim 2, wherein said genetic chimera comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO: 17, SEQ ID NO:20, SEQ DD NO:26, and SEQ DD NO:29.
32. The nucleic acid molecule of Claim 2, wherein said genetic chimera further comprises a linker nucleic acid sequence.
33. The nucleic acid molecule of Claim 32, wherein said linker nucleic acid sequence encodes a protein comprising SEQ DD NO: 13.
34. A recombinant molecule comprising a chimeric nucleic acid molecule of Claim 2.
35. A recombinant cell comprising a chimeric nucleic acid molecule of Claim 2.
PCT/US2000/019070 1999-07-13 2000-07-13 Fc EPSILON RECEPTOR-LUMINESCENCE INDUCING PROTEIN CHIMERIC NUCLEIC ACID MOLECULES, FUSION PROTEINS AND USES THEREOF WO2001004310A1 (en)

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