WO2007081777A2

WO2007081777A2 - METHODS FOR ACCURATELY MEASURING FREE IgE IN THE PRESENCE OF AN ANTI-IgE THERAPEUTIC

Info

Publication number: WO2007081777A2
Application number: PCT/US2007/000233
Authority: WO
Inventors: Mengyuan Patrick Liu
Original assignee: Tanox, Inc.
Priority date: 2006-01-06
Filing date: 2007-01-05
Publication date: 2007-07-19
Also published as: WO2007081777A3

Abstract

The invention provides methods and kits for accurately measuring the amount of IgE in a subject in the presence of an anti-IgE therapeutic, for use, for example in monitoring subjects undergoing treatment with anti-IgE therapeutics.

Description

Methods for Accurately Measuring Free IgE in the Presence of an Anti-IgE Therapeutic

BACKGROUND OF THE INVENTION

[Para 1 ] The immune system ideally functions to maintain health. Unfortunately, the immune system can also exhibit an allergic reaction to an innocuous agent such as pollen, eliciting a mild allergic rhinitis (hay fever) through to a potentially life threatening condition (systemic anaphylaxis). These allergies arise, because antibodies of the IgE class are generated against an allergen, for example pollen, giving rise to an allergic reaction. When the IgE antibody binds to an allergen, it is able to activate various effector cells, including mast cells (tissue) and basophils (blood). Activation of these effector cells, in turn, results in the release of a variety of pre-formed and newly synthesized compounds that lead to complex downstream inflammatory cascades. This culminates in the specific symptoms of the allergic reaction.

[Para 2] These allergic reactions are not simply problematic for the individual. Each year more than 50 million Americans suffer from some kind of allergic disease. Hence, allergic reactions have economic consequences. In fact, allergies are the sixth leading cause of chronic disease in the United States, costing the health care system approximately $ 18 billion annually (The American Academy of Allergy, Asthma and Immunology's The Allergy Report: Science Based Findings on the Diagnosis & Treatment of Allergic Disorders, 1996-2001). More importantly, the prevalence of the IgE-mediated disorders appears to be increasing.

[Para 3] Antibody-based therapeutics that target IgE antibodies (for example, XOLAIR^®), 'neutralize' IgE antibodies, preventing them from activating mast cells and their downstream inflammatory cascade. However, the efficacy of such drugs depends on accurate dosing and monitoring.

[Para 4] IgE is the most important biomarker for an allergic reaction upon re- exposure to the allergen. A radioallergosorbent test (RAST) was introduced in 1968 for the detection of IgE with defined allergen specificity. (Wide, L, et al., Lancet 2: 1105- 1107 (1967). This noncompetitive, heterogenous, immunoradiometric assay employs an allergen immobilized on paper discs (allegro-sorbent) to bind specific antibodies of all isotypes from serum, a separation step of free and bound human antibody, and the use of radiolabeled anti-human IgE to detect bound IgE. Many commercial variants based on the original RAST design have been developed, such as UniCAP system. (Paganelli, R et al., Allergy 50: Suppl 26:248 (1995)).

[Para 5] However, these allergen-immobilized RAST assays are only designed to measure IgE without considering the presence of allergen or the presence of new anti-IgE therapeutics in tested specimens. As a result, if the RAST assay is used to measure the level of IgE in a subject undergoing treatment with an anti-IgE therapeutic (such as XO LAIR^®), the therapeutic can interfere with the ability of the RAST antibody to bind IgE and thereby interfere with accurate measurement of the levels of IgE in the subject. [Para 6] New methods for measuring IgE in the presence of anti-IgE therapeutics are needed.

Summary of the Invention

[Para 7] In contrast to prior methods and kits, the instant disclosed invention provides an accurate measure of free IgE in a sample, thereby providing an accurate indication of the relative efficacy of anti-IgE therapeutics. [Para 8] In one aspect, the present invention provides accurate methods for quantitating the level of free IgE present in a subject undergoing treatment with an anti- IgE therapeutic, e.g., an anti-IgE antibody or an FcεRI peptide. In certain embodiments, the method is comprised of the following steps: (a) obtaining a sample, such as serum, from a subject treated with an anti-IgE therapeutic; (b) contacting an aliquot of the sample with the anti-IgE therapeutic under appropriate conditions and for a sufficient period of time, so that free IgE present in the sample binds to the anti-IgE therapeutic; (c) contacting a separate aliquot of the sample from the subject with an anti-IgE test antibody that binds to a different epitope of IgE than the epitope that is bound by the anti-IgE therapeutic, under appropriate conditions and for a sufficient period of time for free IgE and IgE bound to an anti-IgE therapeutic (i.e., total IgE) to bind to the anti-IgE test antibody; and (d) comparing the relative amount of free IgE bound to the anti-IgE therapeutic in step (b) with the relative amount of total IgE bound to anti-IgE test antibody in step (c) to provide a measure of the actual amount of free IgE in the subject. Relatively low levels of free IgE present in the subject would indicate efficacy of the anti-IgE therapeutic. [Para 9] In a second aspect, the invention provides kits for accurately measuring free-IgE in a sample, as well as measuring the allergen-specific IgE profile of a subject. [Para 10] Other features and advantages of the invention will be apparent from the following detailed description and claims. Brief Description of the Figures

[Para 1 1 ] Figure 1 shows a schematic diagram of methods for measuring free (a) and total (b) allergen-specific IgE.

[Para 1 2] Figure 2 shows reference standard curves of enzyme-linked immunosorbent assays (ELISAs) for free (a) and total (b) peanut-specific.IgE.

[Para 1 3] Figure 3 shows the levels of total and free peanut-specific IgE in serum samples of patients treated with a single subcutaneous injection of a humanized anti- human IgE monoclonal antibody (therapeutic antibody).

Detailed Description of the Invention

[Para 1 4] For convenience, certain terms and phrases employed in the specification, examples, and appended claims are defined herein. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. All of the patents and publications cited herein are hereby incorporated by reference. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the invention.

1. Definitions

[Para 1 5] The articles "a" and "an" are used herein to refer to one or to more than one

(i.e., to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

[Para 1 6] "Aliquot" refers to a specific volume separated from a total volume.

[Para 1 7] "Allergen" refers to a substance that may illicit an immune reaction

(hypersensitive response such as antibody formation) in a subject. Common examples include, but are not limited to: grasses, pollen, seafood (such as shrimp), nuts, bee venom, drugs (such as penicillin), and natural rubber latex proteins.

[Para 1 8] "Allergen-specific IgE antibody" refers to an IgE molecule that is produced in a subject in response to the presence of a specific allergen. [Para 1 9] "Allergy" or "allergic condition" refers to any disease or disorder resulting from an immunological cascade (including Type I and Type IV hypersensitivity reactions) and which is typically triggered by a stimuli. Examples of allergic conditions include, but are not limited to: allergic rhinitis, hay fever, perennial rhinitis, seasonal/perennial allergic conjunctivitis, vernal keratoconjunctivitis, giant papillary conjunctivitis, perennial allergic conjunctivitis and atopic keratoconjunctivitis, atopic dermatitis, and allergic [extrinsic] asthma, food reactions, systemic anaphylaxis, allergic pulmonary disease, anaphylaxis, urticaria and angioedema (hives; giant urticaria; angioneurotic edema), hereditary angioedema, mastocytosis, physical allergy to physical stimuli, e.g., cold, sunlight, heat; mild trauma, contact dermatitis, hypersensitivity pneumonitis, allograft rejection, granulomas due to intracellular organisms, drug sensitivity, thyroiditis, encephalomyelitis after rabies vaccination, cryoglobulinemia, cryoglobulinemic glomerulonephritis, histiocytic lymphomas, Severe Combined Immunodeficiency (SCID) and tonsillitis. [Para 20] "Antibody" refers to a protein that recognizes and binds to a particular antigen.

[Para 21 ] "Anti-IgE therapeutic" refers to a molecule, including an antibody, a small molecule or a peptide, which can bind to circulating IgE antibodies preventing these IgE antibodies from binding to mast cells or basophils, and thereby inhibiting an allergic reaction. Since IgE binds to mast cells and basophils through the FcεRI receptor, most anti-IgE therapeutics will bind IgE in such a way as to block binding to this receptor. [Para 22] "Autoimmune disease" refers to a condition, which is characterized by a specific humoral or cell-mediated immune response against constituents of the body's own tissues, such as self-antigens or auto-antigens. Examples of autoimmune diseases include but are not limited to Active Chronic Hepatitis, Addison's Disease, Anti-phospholipid Syndrome, Atopic Allergy, Autoimmune Atrophic Gastritis, Achlorhydra Autoimmune, Celiac Disease, Crohn's Disease, Cushing's Syndrome, Dermatomyositis, Diabetes (type I), Discoid Lupus, Erythematosis, Goodpasture's Syndrome, Grave's Disease, Hashimoto's Thyroiditis, Idiopathic Adrenal Atrophy, Idiopathic Thrombocytopenia, Insulin-dependent Diabetes, Lambert-Eaton Syndrome, Lupoid Hepatitis, some cases of Lymphopenia, Mixed Connective Tissue Disease, Multiple Sclerosis, Pemphigoid, Pemphigus Vulgaris, Pernicious Anema, Phacogenic Uveitis, Polyarteritis Nodosa, Polyglandular Auto. Syndromes, Primary Biliary Cirrhosis, Primary Sclerosing Cholangitis, Psoriasis, Raynaud's Syndrome, Reiter's Syndrome, Relapsing Polychondritis, Rheumatoid Arthritis, Schmidt's Syndrome, Limited Scleroderma (or CREST Syndrome), Severe Combined Immunodeficiency Syndrome (SCID), Sjogren's Syndrome, Sympathetic Ophthalmia, Systemic Lupus Erythematosis, Takayasu's Arteritis, Temporal Arteritis, Thyrotoxicosis, Type B Insulin Resistance, Ulcerative Colitis and Wegener's Granulomatosis. [Para 23] "Control" or "reference standard" refers to known quantities of a target that have been quantitated and added to or 'spiked' in a sample. Reference standards are used to generate a standard curve.

[Para 24] "Fluorophore" refers to a substance or a portion thereof which is capable of exhibiting fluorescence in the detectable range. Fluorophores include, but are not limited to, Fluorescein, Rhodamine, Texas Red, Cy2, Cy3, Cy5, VECTOR Red, ELF.TM. (Enzyme-Labeled Fluorescence), CyO, CyO.5, CyI, CyI.5, Cy3, Cy3.5, Cy5, Cy7,FluorX, Calcein, Calcein-AM, CRYPTOFLUOR.TM.'S, Orange (42 kDa), Tangerine (35 kDa), Gold (31 kDa), Red (42 kDa), Crimson (40 kDa), BHMP, BHDMAP, Br-Oregon, Lucifer Yellow, Alexa dye family, N-[6-(7-nitrobenz-2-oxa-l, 3-diazol-4-yl)amino]caproyl] (NBD), BODIPY.TM., boron dipyrromethene difluoride, Oregon Green, MITOTRACKER.TM. Red, DiOC.sub.7 (3), DiIC.sub.18, Phycoerythrin, Phycooycanin, Allophycocyanin, o-phthaldehyde, Phycobiliproteins BPE (240 kDa) RPE (240 kDa) CPC (264 kDa) APC (104 kDa), Spectrum Blue, Spectrum Aqua, Spectrum Green, Spectrum Gold, Spectrum Orange, Spectrum Red, NADH, NADPH, FAD, Infra-Red (IR) Dyes, Cyclic GDP-Ribose (cGDPR), Calcofluor White, Lissamine, Umbelliferone, Tyrosine and Tryptophan. A wide variety of other fluorescent probes are available from and/or extensively described in the Handbook of Fluorescent Probes and Research Products 8^th Ed. (2001), available from Molecular Probes, Eugene, OR., as well as many other manufacturers.

[Para 25] "IgE" or immunoglobulin E refers to an immunoglobulin of class E₅ which may be involved in a hypersensitivity response.

[Para 26] "Immobilized" refers to an object being attached or bound to a substrate. The binding can be either a direct linkage (e.g. a chemical bond) or via a linking group [Para 27] The term "immunoassay" refers to any assay that utilizes an antibody to specifically bind a target protein. Examples of immunoassays include, but are not limited to, immunoblot assays, enzyme linked immunosorbent assay ("ELISA"), enzyme immunoassay (EIA). In an ELISA or EIA assay, a labeled enzyme and a substrate are used to produce an amplified signal. [Para 28] "Label" and "detectable label" refer to a detectable compound or composition that which can be conjugated directly or indirectly to a molecule or protein, e.g., an antibody. The label itself may be detectable (e.g., radioisotope labels or fluorescent labels) or, in the case of an enzymatic label, may catalyze a chemical alteration of a substrate compound or composition, which is detectable. A label may include, but is not limited to radioactive isotopes, fluorophores, chemiluminescent moieties, enzymes, enzyme substrates, enzyme cofactors, enzyme inhibitors, dyes, metal ions, ligands (e.g., biotin or haptens) and the like. Enzymes which can be used to label an antibody include, but are not limited to, horseradish peroxidase, alkaline phosphatase, beta-galactosidase, glucose oxidase, malate dehydrogenase, staphylococcal nuclease, delta-5-steroid isomerase, yeast alcohol dehydrogenase, alpha-glycerophosphate, dehydrogenase, triose phosphate isomerase, asparaginase, ribonuclease, urease, catalase, glucose-6-phosphate dehydrogenase, glucoamylase and acetylcholinesterase.

[Para 29] The enzyme can also be directed at catalyzing a luminescence reaction of a substrate, such as, but not limited to, luciferase and aequorin, having a substantially non- soluble reaction product capable of luminescencing or of directing a second reaction of a second substrate, such as but not limited to, luciferine and ATP or coelenterazine and Ca.sup.++, having a luminescencing product.

[Para 30] The term "sample," as used herein, refers to biological material containing analytes to be assayed. Biological material may be obtained from a subject and may include, for example, tissue, cells, or bodily fluid. In exemplary embodiments, the sample is a bodily fluid. "Sample" can refer to any fluid that may be obtained from a subject, including, but not limited to saliva, blood, plasma, serum, urine, interstitial fluid, lymph, gastric juices, bile, sweat, and spinal and brain fluids, including fluids that are processed or present in their natural form. An "appropriate sample" from a subject is one that may contain circulating IgE.

[Para 31 ] The terms "solid support" refer to a material to which antibodies and other molecules may be bound. Well-known supports include glass (e.g., controlled pore glass), polysaccharides (e.g., agarose), polyacrylamides, polystyrene, polypropylene, polyethylene, dextran, nylon, amylases, natural and modified celluloses, gabbros, magnetite, polyvinyl alcohol, and silicones. The nature of the support can be either soluble to some extent or insoluble. The support material can have virtually any possible structural configuration. Thus, the support configuration can be spherical, as in a bead, or cylindrical, as in the inside of surface of a test tube, or the external surface of a rod.

Alternatively, the surface can be flat, such as a sheet, test strip or well of a microtiter plate.

In exemplary embodiments, a substrate or support is the well of a microtiter plate. In alternate embodiments, a solid phase is a cellulose or nylon membrane. In other embodiments, a solid phase may comprise a purification column (e.g., an affinity chromatography column).

[Para 32] "Specific binding" of an antibody to an antigen means that the antibody has less than about 30%, or less than 20%, or less than 10%, or less than 1% cross-reactivity with another molecule.

[Para 33] A "subject" refers to a human or a non-human animal.

[Para 34] "Treatment" refers to a specific therapeutic regimen that a subject is undergoing.

2. Assay

[Para 35] The present invention features methods and kits for accurately measuring the IgE profile of a subject (including allergen-specific IgEs), who may be undergoing treatment with an anti-IgE therapeutic.

[Para 36] The methods and kits rely on use of the therapeutic antibody being used for treatment, which binds IgE (including allergen-specific IgEs), and a second anti-IgE antibody recognizing a different epitope of IgE than the epitope that the therapeutic antibody binds allowing for the measurement of total IgE present in the sample.

According to the method of the invention, as illustrated in Figure 1, a first aliquot of a sample from a subject is incubated with a therapeutic antibody, which recognizes and binds to free IgE, (i.e., allergen-specific IgE which is not already bound to therapeutic antibody present in the sample).

[Para 37] A second aliquot of the sample is incubated with an anti-IgE antibody that recognizes a different epitope on allergen-specific IgE than the epitope recognized by the therapeutic antibody. This second antibody therefore recognizes total IgE (i.e. free IgE plus IgE that is bound to therapeutic antibody present in the patient sample). By comparing the two values obtained by incubating separate aliquots of the sample with the therapeutic antibody and second anti-IgE antibody, the IgE profile of the subject can be determined.

[Para 38] A therapeutic or anti-IgE antibody may be directly or indirectly linked to a solid support. For direct linkage to a solid support, such as an ELISA-based assay format using a microliter plate, the antibody may be diluted in a buffered solution, such as phosphate buffered saline, and spread on each well of an assay plate. [Para 39] In an alternate embodiment, an antibody may be indirectly linked to a solid support. For example, a solid support such as microliter plates, microparticles or membranes may be coated with avidin or streptavidin. An antibody can be labeled with biotin to facilitate linkage to an avidin or streptavidin coated solid support. Biotin conjugation of antibodies is well-known in the art and biotin is typically conjugated to proteins through primary amines (i.e., lysines). Biotin may be obtained from many commercial sources (such as Pierce EZ link Sulfo-NHS-LC biotin or Pierce NHS-LC biotin II) and conjugated to an antibody according to the manufacturer's instructions. Additionally, kits for biotin conjugation may be obtained from companies such as Sigma Aldrich, Alpha Diagnostic International, or Amersham Pharmacia Biotech. [Para 40] In addition, the anti-IgE antibody can be indirectly immobilized onto a solid support by using a secondary antibody, protein A or protein G via the binding of Fc of anti-IgE antibody. For example, a solid support may be coated with protein A and then the anti-IgE antibody is immobilized through the binding of protein A to its Fc. One of skill in the art would recognize other methods for immobilizing an antibody to a solid support. [Para 41 ] In one aspect of the invention, the IgE from the sample is immobilized by binding to an anti-IgE antibody immobilized on the solid support, and is detected using a labeled allergen. The free IgE and total IgE may also be detected using an indirect labeling method.

[Para 42] In exemplary embodiments, the label is an enzyme that catalyzes a chemical reaction in the presence of a substrate compound. Exemplary substrate compounds will generate a detectable reaction product, e.g., a chromogenic product, which may be detected by colorimetric, fluorimetric, or visual means. Other exemplary substrate compounds include those that utilize chemiluminescent moieties that generate a light reaction.

[Para 43] In an exemplary embodiment, the allergen is labeled with horseradish peroxidase (HRP) enzyme. Methods to label substances with HRP are well-known in the art. HRP-allergen conjugates may be prepared using activated peroxidase according to the manufacturer's instructions. HRP conjugation kits for labeling substances may be obtained from Alpha Diagnostic International or Zymed Laboratories. Chromogenic substrates that are reactive with horseradish peroxidase enzyme include, but are not limited to, 3,3', 5,5' tetramethylbenzidine (TMB), 2,2-azino-di-(3-ethylbenzthiazoline sulfonic acid) (ABTS), o-phenylenediamine dihydrochloride (OPD), aminoethyl carbazole, 3-amino, 9-ethylcarbazole AEC (3A9EC). Alpha-naphthol pyronin, 4-chloro-l- naphthol (4C1N), 3,3'-diaminobenzidine tetrahydrochloride (DAB), ortho-dianisidine, TACS Blue, TACS Red, TRUE BLUE™, VECTOR^®VIP, VECTOR^®SG and Zymed Blue HRP substrate. Chromogenic reaction products can be measured using methods that are well-known in the art. In an exemplary embodiment, using an ELISA-based assay format, 3',5,5' tetramethylbenzidine (TMB), 2,2-azino-di-(3-ethylbenzthiazoline sulfonic acid) (ABTS), o-phenylenediamine dihydrochloride (OPD) may be the preferred chromogenic substrate.

[Para 44] In another exemplary embodiment, the allergen is labeled with alkaline phosphatase. Methods to label substances with alkaline phosphatase are well-known in the art. Alkaline phosphatase-allergen conjugates may be prepared using activated alkaline phosphatase according to the manufacturer's instructions. Alkaline phosphatase conjugation kits for labeling allergen may be obtained from Zymed Laboratories, Merck or Roche. Substrates for Alkaline phosphatase (AP) include, but are not limited to, AP-Blue substrate (Zymed); AP-Orange substrate (Zymed), AP-Red substrate (Zymed), 5-bromo, 4-chloro, 3-indolyphosphate (BCIP), BCIP/nitroblue tetrazolium/iodonitrotetrazolium (BCIP/NBT/INT), BCIP/NBT, DAKO, Fast Red, Magenta-phosphate, Naphthol AS-BI- phosphate (NABP)/Fast Red TR, NABP/New Fuchsin, Naphthol AS-MX-phosphate (NAMP)ZNew Fuchsin, New Fuchsin AP substrate, p-Nitrophenyl phosphate (PNPP), VECTOR^®Black, VECTOR^®Blue, VECTOR^®Red, Vega Red. In an exemplary embodiment, using an ELISA-based assay format, p-Nitrophenyl phosphatase (PNPP) may a chromogenic substrate.

[Para 45] Enzyme-labeled allergen can also react with chemiluminescent moieties to generate light. Examples of particularly useful chemiluminescent labeling compounds are luminol, isoluminol, theromatic acridinium ester, imidazole, acridinium salt, and oxalate ester. Likewise, a bioluminescent compound can be used to label the allergen. Bioluminescence is a type of chemiluminescence found in biological systems in which a catalytic protein increases the efficiency of the chemiluminescent reaction. The presence of a bioluminescent protein is determined by detecting the presence of luminescence. Important bioluminescent compounds for purposes of labeling are luciferin, luciferase and aequorin. In an exemplary embodiment, horseradish peroxidase enzyme reacts with a chemiluminescent compound such as luminol to generate light. Emitted light can be measured in an ELISA based assay format or in an immunoblot based assay format using methods that are well-known in the art.

[Para 46] Detection may also be accomplished using a variety of other approaches. For example, the allergen may be radioactively labeled. The radioactive isotope (e.g., ¹²⁵I,

¹³¹1, ³⁵S or ³H) may be detected by such means as the use of a gamma counter, a scintillation counter or by autoradiography.

[Para 47] It is also possible to label the allergen with a fluorescent compound. When the fluorescently labeled antibody is exposed to light of the proper wavelength, its presence can then be detected. Commonly used fluorescent labeling compounds that may be used are presented above.

[Para 48] Sample may be incubated with antibodies and labeled allergen at suitable temperatures for an appropriate period. After incubation, a solid support can be washed to remove unbound substances. Exemplary wash solutions are well-known in the art and may include phosphate buffered saline or TRIS-buffered saline containing detergent. The wash step may be repeated 1, 2, 3 or more times. Following the last wash, a substrate may be added and a color change measured.

[Para 49] A colored reaction product may be read using a spectrophotometer or an

ELISA plate reader. The amount of color reaction product generated is proportional to the amount of bound allergen, which is proportional to allergen-specific IgE levels in the sample.

[Para 50] Quantification may be performed by a comparison of absorbance readings of the samples to a standard curve. A standard curve may be created by measuring known amounts of allergen-specific IgE in the assay and measuring the absorbance of each test concentration. In an exemplary embodiment, at least about 5, 6, 7, or 8 concentrations of allergen-specific IgE are be measured to generate a standard curve. The concentration of allergen-specific IgE in the sample may be measured using linear regression between concentrations versus absorbance of the standards.

[Para 51 ] If high levels of allergen-specific IgE are expected or present in the sample, the sample may be diluted, e.g., in IgE-free serum, prior to the assay. For samples requiring dilution, the linearity and accuracy of dilution should be determined to ensure that the results will be reliable after dilution. Linearity of dilution refers to the ability of the analytical method, within the assay range, to obtain test results that are close to the expected concentration of the analyte in the diluted sample. Linearity is measured by the R-squared (R²) value for the linear regression of the expected versus observed concentration while accuracy is measured by the percent recovery. 3. Kits

[Para 52] Also provided herein are kits for detecting free and total IgE in a sample from a subject undergoing anti-IgE therapeutic treatment. The kit may comprise one or more reagents for detecting IgE in a sample. The one or more reagents may comprise a therapeutic and/or an anti-IgE antibody that recognizes a binding site on IgE that is distinct from the therapeutic antibody binding site. The therapeutic and anti-IgE antibody may be immobilized onto a solid support. The kit may also contain allergen that is labeled with a marker such as chemiluminescent, enzymatic, fluorescent, or radioactive moiety labels. The kit may also contain a substrate for the labeled moiety to allow detection. [Para 53] In certain embodiments, a kit for detecting free and total IgE in a sample may further consist of a reference standard, wherein the reference standard is purified target antibody (IgE). The kit may additionally comprise a solid support such as a reagent strip that again contains immobilized capture substances (therapeutic and anti-IgE antibody). Alternatively, the kit may comprise a microtiter plate or strip upon which the capture substances may be immobilized. Further, a kit for detecting free and total IgE antibody in a sample may contain instructions for detecting the level of a free and total IgE in a sample. Additional reagents may be included in the kit, including wash buffer, and blocking buffer. These may be provided in individual containers. Exemplification

[Para 54] The invention now being generally described, will be more readily understood by reference to the following examples, which are included merely for purposes of illustration of certain aspects and embodiments of the present invention, and are not intended to limit the invention.

Example 1: Enzyme-Linked Immunosorbent Assay to determine the level of Peanut Specific Free IgE in human serum.

[Para 55] Buffers and Solutions: Coating Buffer: the solution used to coat the 96-well plates was phosphate buffered saline (PBS), pH 7.4, with 1 Dg/ml therapeutic antibody. Blocking buffer: the solution used to block the coated plates was 5OmM phosphate buffer, pH 7.4, containing 0.5% ProClin-300, 1% BSA, and 2.5% sucrose. Assay buffer: the solution used for assays was 5OmM TRIS buffer, pH 7.4, containing 1% BSA, 0.05% TWEEN 20^®, and 0.5% ProClin-300. Wash buffer: the solution used to wash between assay steps was a 5OmM TRIS buffer, pH 7.4, containing 0.9% NaCl, 0.05% TWEEN 20^®, and 0.5% ProClin-300.

[Para 56] Therapeutic Antibody: an anti-IgE antibody was used as the therapeutic antibody for testing at a concentration of lμg/ml for coating plates in a 1.42mg/ml solution of PBS. Peanut Allergen HRP: peanut allergen was conjugated to HRP by obtaining peanut allergen (Antigen Laboratories, Inc.) and conjugating it to HRP using a Zymed HRP conjugation kit, following the manufacturer's instructions. IgE free human serum: IgE free human serum was purchased from Scantibodies Laboratory, Inc. and 0.05% ProClin-300 was added as a preservative. Peanut Specific IgE Reference Material: peanut allergen sensitive plasma containing 74 IU/ml peanut-specific IgE was obtained from Teragenix. The concentration of the peanut-specific IgE was measured by Pharmacia ImmunoCap 100 and provided by Teragenix. This material was used to make references B - G (2.3 IU/ml to 74 IU/ml) by serially diluting the stock with IgE free human serum. To obtain a more concentrated reference (H), aliquots from several pre-treatment patient samples from a therapeutic anti-IgE clinical study were pooled. Reference H was determined to be 174.3 IU/ml based on dilutions run with a standard curve containing references A- G. Reference A was IgE free human serum. Serum Samples: serum samples used in the qualification of the assay were either pre-treatment patient samples, or a dilution of reference material with IgE free human serum. Patient samples were used for testing linearity of dilution, spike recovery, freeze/thaw stability, and specificity. Diluted reference material was used to make Control samples I, II, and III, and for testing analytical and functional sensitivity.

[Para 57] Coating Strip Plates: Therapeutic Ab coated Costar EIA/RIA strip plates were used for the solid support of this ELISA. Plate coating was done using lμg/ml of an anti-IgE therapeutic antibody. Two hundred μl of coating solution was added to each well and plates were incubated overnight at room temperature in a humidified chamber. Excess coating solution was aspirated and 200μl of blocking solution was added to each well. Plates were incubated overnight and then the blocking solution was aspirated from the wells. For long-term storage, plates were dried at room temperature for 4 hours, sealed in plastic bags, and stored at 4⁰C.

[Para 58] Assay Procedure: The appropriate number of therapeutic Ab coated strips was removed from the refrigerator. Samples were thawed and mixed well before use. Twenty μl of references A-H and controls (I, II and III) were added to the coated strip plate, in duplicate. Twenty μl of sample was added in duplicate to the remaining wells. One hundred μl of assay buffer was added to each well. The strips were incubated at room temperature on a plate shaker at 700 rpm for 60 minutes. The plates were washed using wash buffer in a BioRad ImmunoWash plate washer. When the plates had finished washing, each plate was slapped and opened wells down, and placed on a stack of paper towels to remove any remaining wash buffer. One hundred μl of peanut allergen-HRP conjugate working solution was added to each well. The strips were incubated for 30 minutes at room temperature on a plate shaker at 700 rpm. The plates were washed and dried as above. One hundred μl of TMB was added to each well and incubated on a plate shaker at 700 rpm for 10 minutes. One hundred μl of stopping solution was added to the wells, and the wells shaken briefly to mix. The absorbance was measured at 450nm with 590nm as a reference on a Dynex plate reader.

[Para 59] Data Evaluation: Plates were read on the Dynex MRX plate reader. The standard curve was then plotted using a sigmoidal curve fit. Sample data were processed by the Dynex Revelation 4.02 software immediately after the plate was read. [Para 60] Results: Standard Curve: In order to determine the reproducibility of this ELISA, 16 assays were run during a 9-day period. The ODs for the references in all the assays during this 9-day period showed a coefficient variation (CV) for each reference of less than 17% for a range of 2.3 - 174.3 IU/ml of peanut specific free IgE, thus showing adequate inter-assay reproducibility.

[Para 61 ] Analytical Sensitivity: Analytical sensitivity (lower limit of detection, LLOD) was determined by interpolating the mean of 20 wells of reference A (0 IU/ml) and 4 wells of reference B (2.3 IU/ml). Three separate assays were performed and the LLOD was determined as the average LLOD of the 3 assays plus 2 standard deviations. Based on this calculation, the LLOD of this ELISA was determined to be 0.29 IU/ml. [Para 62] Functional Sensitivity: Functional sensitivity, or lower limit of quantitation, was determined by analyzing several concentrations of peanut specific IgE ranging from 0.25 to 7.0 IU/ml. The average CV for each concentration from 10 assays was plotted as a function of concentration. The functional sensitivity is the lowest concentration at which the CV = 20% based on the equation for the curve fit. The functional sensitivity for this ELISA was determined to be 0.55 IU/ml.

[Para 63] Intra -assay Precision: Intra-assay precision was determined by comparing the concentrations of 3 samples containing low, medium, and high levels of peanut specific free IgE. Twenty replicates (20 wells) of each sample were measured in one assay and the average and standard deviation were calculated for each sample. The CVs for samples A, B, and C were 4%, 5%, and 7%, respectively, indicating adequate intra- assay precision for this ELISA.

[Para 64] Inter-assay Precision: Inter-assay precision was determined by comparing the concentrations of 3 control samples containing low, medium, or high levels of peanut specific free IgE in 16 assays over a 9-day period. CVs were less than 7% for all three controls.

[Para 65] Specificity: Several compounds were tested for their ability to interfere with the quantitation of peanut specific free IgE in human serum. Hemoglobin, human IgG, and a chimeric IgG, were added as 5% volume to 3 pre-dose samples collected from 3 different patients from an anti-IgE therapeutic clinical study. PBS was used as a control, and interference from each of the substances was determined. Recoveries ranged from 91% to 123%. Human IgG and a non-anti-IgE antibody recognizing a different target did not interfere at all with quantitation of peanut specific free IgE, with recoveries ranging from 94% to 105%. In the presence of hemoglobin, peanut specific free IgE quantitation was slightly higher, from 114% to 123%. However, only 1 of the 3 samples was out of the accepted 80 - 120% range.

[Para 66] Edge Effect: To determine if there was an edge effect from the location of a sample in the 96-well plate, 3 samples were set up in a series of columns across the plate, and in a separate assay, in a series of rows down the plate. The results of these assays showed that samples A and B measured the same from the top to bottom of the 96-well plate. A small decrease in optical density (OD) was observed for the center rows compared to the top and bottom of the plate for Sample 3. However, the CV for the column of samples was 6.7% and the lowest and highest values in the column were within 20% of each other.

[Para 67] Sample data was quantified from the left to the right of a 96-well plate. This assay was done twice because of the variation seen in the values. While the values for a given sample vary from well to well, there is no clear trend indicating an effect from the position of a sample across the plate.

[Para 68] Linearity: Linearity and accuracy were evaluated. The linear dilution of samples in this ELISA was tested using 7 pre-treatment patient samples from an anti-IgE therapeutic antibody clinical study with peanut specific IgE concentrations ranging from 6.9 to 148.12 IU/ml. Samples were diluted from 1:2 to 1:32 with IgE free human serum. None of the samples diluted linearly. Calculated concentrations increased as the samples are diluted and recoveries ranged from 114% to 194%.

[Para 69] Spike Recovery: To determine if the sample matrix affects the quantitation of known amounts of peanut specific IgE, 3 pre-treatment patient samples (A, B, and C) containing low, medium, and high concentrations of peanut specific IgE were analyzed without added peanut specific IgE and with 3 levels of peanut specific IgE added (spiking solutions I₃ II, and III). In the first assay, spiking solution I was Reference H from the total peanut specific IgE assay. Spiking solutions II and III were pre-treatment patient samples from an anti-IgE therapeutic antibody clinical study. Spiking solutions were added as 5% volume. The amount spiked in from each solution was determined by spiking 5% into IgE free human serum. Unspiked sample concentration was determined by^" adding 5% IgE free human serum to the samples. Recovery % was calculated according to the equation [(observed IU/ml - sample IU/ml)/amount spiked in] x 100. In the initial assay, recoveries for samples A and C were within the 80% to 120% range, while recoveries for sample B were less than 50%. Another spike recovery assay was set up spiking 20% volume of the Teragenix peanut sensitive plasma (74 IU/ml) into the samples. In this assay the recoveries were more acceptable. If we calculated recovery as (observed/expected) x 100, the recoveries were within the acceptable 80% to 120% range. The first spike recovery assay suggested there was an effect from the sample matrix because only sample B showed low recoveries. However, when the spiking matrix was changed from patient sample to the Teragenix material, recovery from sample B was within the acceptable range. This indicated the spiking matrix can also affect quantitation. Because there is no pure peanut specific IgE available we cannot eliminate these matrix effects.

[Para 70] Time Shift Study: The sample incubation time for this assay started as soon as the sample was added to the well, when IgE began binding to the anti-IgE therapeutic antibody. To determine if there was a difference in quantitation between the first and the last sample added to a plate, a time shifting study was performed. The 3 control samples were added to a plate in duplicate from 0 to 30 minutes before the standard curve. Recoveries ranged from 92% to 108%, indicating no difference in quantitation for samples added at least 30 minutes before the standard curve. [Para 71 ] Sample Stability: The stability of 5 patient samples (A - E) during 4 freeze/thaw cycles was investigated. The samples ranged from 11 to 262 IU/ml of IgE. The samples were divided into 5 aliquots, 4 of which were frozen at -7O⁰C and thawed to room temperature 1 to 4 times over a 2 day period. The fifth aliquot remained at 4°C until analysis. Sample A was diluted 1:2 with IgE free serum before analysis. Samples remained stable through all 4 freeze/thaw cycles, with recoveries ranging from 89% to 113%.

[Para 72] Total and Free Peanut Specific IgE Comparison: Ten pre-treatment patient samples from the anti-IgE therapeutic antibody clinical study were tested in both the peanut specific total IgE ELISA and the peanut specific free IgE ELISA. Because these were pre-treatment samples, no anti-IgE therapeutic antibody should be present and both total and free peanut specific IgE values should be similar. Agreement between free and total peanut specific IgE was variable. Free peanut specific IgE concentrations range from 56% to 109% of the total peanut specific IgE concentrations.

Example 2 Development of a Sandwich ELISA for Quantitative Measurement of Peanut Specific Total IgE in Human Serum.

[Para 73] Assay Procedure: The appropriate number of anti-IgE test antibody coated strips was removed from the refrigerator. The samples were thawed and dilutions made, if necessary, with IgE free human serum. Ten μl of references (A-H), controls (I₅ II, and III), or samples was added to each well, in duplicate. Fifty μl of assay buffer T was next added to each well. The plate was incubated at room temperature on a plate shaker at 700 rpm for 60 minutes. The plate was washed using the BioRad ImmunoWash plate washer. When the plate has finished washing, remaining wash buffer was removed by slapping the plates against a stack of paper towels. Peanut allergen-HRP was prepared by diluting the stock in assay buffer. Fifty μl of HRP conjugate solution was then added to each well. The plate was incubated for 30 minutes at room temperature on a plate shaker at 700 rpm. Next, the plate was washed using the BioRad ImmunoWash plate washer. Remaining wash buffer was removed. One hundred μl of TMB was added to each well, and the plate was allowed to incubate on a plate shaker at 700 rpm for 10 minutes. One hundred Dl of stopping solution was added to each well and the plate shaken briefly to mix. The wells were measured and the absorbance recorded at 450nm with 590nm as a reference on a Dynex plate reader.

[Para 74] Data Evaluation: Plates were read on the Dynex MRX plate reader. The standard curve was obtained using the sigmoidal curve fit. The sample data being processed by Dynex Revelation 4.02 software immediately after the plate was read. [Para 75] Results: Standard Curve: In order to determine the reproducibility of this assay, 18 assays were run during a 7 day period for a range of 2.3 - 181 IU/ml of peanut specific IgE (in the format of a standard curve). Data of the ODs showed that for the references in all the assays, the CVs for each reference were less than 13%, showing adequate inter-assay reproducibility.

[Para 76] Analytical Sensitivity: Analytical sensitivity (lower limit of detection, LLOD) was determined by interpolating the mean of 20 wells of reference A (0 IU/ml) and 4 wells of reference B (2.3 IU/ml). Three separate assays were performed and the LLOD was determined as the average LLOD of the 3 assays plus 2 standard deviations. Based on this calculation, the LLOD of this ELISA was 0.51 IU/ml. [Para 77] Functional Sensitivity: Functional sensitivity or the lower limit of quantitation, was determined by analyzing several concentrations of peanut specific IgE ranging from 0.5 — 4.0 IU/ml, plotting the CVs as a function of concentration, and obtaining a satisfactory curve fit. The functional sensitivity was the lowest concentration at which the CV = 20% based on the equation for the curve fit. In total, 10 assays were run, with 6 of them having satisfactory curve fits (R²>0.55). The functional sensitivity, or lower limit of quantitation (LLOQ), for this assay was the average LLOQ + 2 standard deviations of the 6 assays. The LLOQ for this ELISA was 2.1 IU/ml. [Para 78] Intra-assay Precision: Intra-assay precision was determined by comparing the concentrations of 3 controls containing low, medium, and high levels of peanut specific IgE. Twenty replicates (20 wells) of each sample were measured in one assay and the mean and standard deviation were determined for each sample. The CVs for control samples I, II, and III were 4.9%, 4.5%, and 2.5%, respectively.

[Para 79] Inter-assay Precision: Inter-assay precision was determined by comparing the concentrations of 3 controls containing low, medium, and high levels of peanut specific IgE in 18 assays over a 7 day period. The CVs were less than 7% for all three samples.

[Para 80] Specificity: Several compounds were tested for their ability to interfere with the quantitation of peanut specific IgE in this assay. Hemoglobin, human IgG, and TNX-454,224 were added as 5% volume to reference A and 3 pre-treatment patient samples (A, B, and C) from the TNX-901.01 clinical study. PBS was used as a control, and interference from each of the substances was determined. The recoveries ranged from 88 — 112%, indicating that, at these concentrations, the compounds did not interfere with peanut specific IgE quantitation.

[Para 81 ] Linearity: The linear dilution of samples in this ELISA was tested using 3 pre-treatment patient samples from the TNX-901.01 clinical study, with peanut specific IgE concentrations of 18, 41, and 158 IU/ml. Samples were diluted from 1:2 up to 1:32 with IgE free human serum. All 3 samples diluted linearly, with recoveries ranging from 85 - 123%.

[Para 82] Recovery: To determine if the sample matrix affects the quantitation of known amounts of peanut specific IgE, 3 samples (A, B, and C) containing low, medium, and high concentrations of peanut specific IgE were analyzed without added peanut specific IgE and with 3 levels of peanut specific IgE added (spiking solutions I, II, and III). Samples A, B, and C were made by diluting reference H with IgE free human serum to approximate concentrations of 10, 25, and 50 IU/ml. The 3 spiking solutions were 3 pre-treatment patient samples from the TNX-901.01 clinical study. Spiking solutions were added as 5% volume. The amount spiked in from each solution was determined by spiking 5% into IgE free human serum. Unspiked sample concentration was determined by adding 5% IgE free human serum to the samples. Recovery % was calculated according to the equation [(observed IU/ml - sample IU/ml)/amount spike in] x 100. Recoveries ranged from 87 — 110%, showing that the matrix did not affect quantitation. [Para 83] Time Shift Study: The sample incubation time for this assay was started as soon as the sample was added to the well, when IgE can begin binding to the allergen- specific anti-IgE test antibody. To determine if there was a difference in quantitation between the first and the last samples to be added to the plate, a time shifting study was done. The 3 control samples were added to a plate, in duplicate, from 0 - 30 minutes before the standard curve. In the first shifting study recoveries for controls I and II were between 92 — 102%, while recoveries for control HI were 76 — 86%. Two more studies were performed to clarify the shifting issue. Recoveries for all controls in the second and third shifting studies were between 88 - 103%.

[Para 84] Sample Stability: The stability of 5 patient samples (A - E) during 4 freeze/thaw cycles was investigated. The samples were pre-treatment serum from the TNX-901.01 clinical study, ranging from 17 - 190 IU/ml. The samples were divided into 5 aliquots, 4 of which were frozen at -70⁰C and thawed to room temperature 1 —4 times. The fifth aliquot remained at 4⁰C until analysis. Sample A was diluted 1 :2 with IgE free human serum before analysis. Samples remained very stable through the freeze/thaw cycles, with recoveries ranging from 94 — 101%.

[Para 85] Edge Effect Study: To determine if there was an effect of the location of a sample in the 96-well plate, 8 references were set up in a series of columns across the plate, and in a separate assay, in a series of rows down the plate. The results of these assays showed a small decrease in OD for the center wells compared to the top and bottom wells. However, for all non-zero references there was never more than an 11% difference between the highest and lowest OD of a given reference, and CVs were below 4%. Further, the results showed no trend in OD from the right to left of the plate. For all nonzero references the highest and lowest OD for a row were never more than 15% different, and CVs were less than 6%.

[Para 86] Conclusion: A reliable ELISA has been developed and qualified for the measurement of IgE in human serum in thhe presence of an anti-IgE therapeutic. This sandwich ELISA used an anti-human anti-IgE test antibody for the capture antibody and peanut allergen-HRP conjugate as the detection protein, however, any allergen conjugate would be useful, as would other forms of detection.

[Para 87] This ELISA had good intra-assay and inter-assay precision and patient serum samples diluted linearly within the range of the assay. There were no shifting effects or edge effects in this ELISA. Peanut specific IgE was stable through 4 freeze/thaw cycles. Hemoglobin, human IgG, and an anti-Factor D antibody (negative control) at high concentrations did not interfere with the quantitation of allergen-specific free IgE.

Claims

What is Claimed:

1. A method for accurately measuring the IgE profile of a subject undergoing treatment with an anti-IgE therapeutic, comprising: a. contacting an aliquot of a sample from the subject with an anti-IgE therapeutic antibody, under appropriate conditions and for a sufficient period of time for free IgE to bind to the therapeutic antibody; b. contacting a second aliquot of the sample with an anti-IgE test antibody, that binds to a different epitope of IgE than the epitope that is bound by the anti-IgE therapeutic antibody, under appropriate conditions and for a sufficient period of time for free IgE and total IgE bound to a therapeutic antibody to bind to the anti-IgE test antibody; and c. comparing the relative amount of free IgE bound to therapeutic antibody in step (a) with the relative amount of total IgE bound to anti-IgE test antibody in step (b) to obtain the IgE profile of the subject.

2. The method of claim 1, wherein the anti-IgE therapeutic antibody is immobilized on a substrate.

3. The method of claim 1, wherein the anti-IgE test antibody is immobilized on a substrate.

4. The method of claim 1, wherein step (a) is performed before step (b).

5. The method of claim 1, wherein step (b) is performed before step (a).

6. The method of claim 1, wherein step (a) and step (b) are performed at the same time.

7. The method of claim 1 , wherein the amount of IgE is determined by using a labeled allergen.

8. The method of claim 7, wherein the allergen is labeled with an enzyme.

9. The method of claim 8, further comprising adding a chromogenic substrate that reacts with the enzyme.

10. The method of claim 7, wherein a labeled allergen is added in step (a) and the amount of label is quantitated to determine the amount of free IgE bound by the therapeutic antibody.

11. The method of claim 1, wherein a labeled allergen is added in step (b) and the amount of label is quantitated to determine the amount of total IgE bound by the anti-IgE test antibody.

X 2. The method of claim 7, wherein labeled allergen is added in step (a) and (b) and the amount of label is quantitated to determine the amount of free allergen-specific IgE bound by the therapeutic antibody and the amount of total allergen-specific IgE bound by the anti-IgE test antibody.

13. The method of claim 1, wherein the sample obtained from the subject is selected from the group consisting of: blood, plasma, urine, interstitial fluid, lymph, gastric juices, bile, serum, saliva, sweat, spinal fluid and brain fluid.

14. The method of claim 1, wherein the anti-IgE therapeutic antibody is linked to a solid support.

15. The method of claim 1 , wherein the anti-IgE test antibody is linked to a solid support.

16. A kit comprising a therapeutic antibody, an anti-IgE test antibody and a labeled allergen.

17. The kit of claim 16, further comprising a solid support.

18. The kit of claim 17, wherein the solid support is a microliter plate.

19. The kit of claim 17, wherein the solid support is a nylon or cellulose membrane.